Homocysteine Metabolism in Health and Disease 9811668663, 9789811668661

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Govind Prasad Dubey Krishna Misra Rajesh K. Kesharwani Rudra P. Ojha   Editors

Homocysteine Metabolism in Health and Disease

Homocysteine Metabolism in Health and Disease

Govind Prasad Dubey • Krishna Misra • Rajesh K. Kesharwani • Rudra P. Ojha Editors

Homocysteine Metabolism in Health and Disease

Editors Govind Prasad Dubey Kriya Sharira and Kaya Chikitsa Banaras Hindu University Varanasi, Uttar Pradesh, India Rajesh K. Kesharwani Department of Computer Application Nehru Gram Bharati (Deemed to be University) Prayagraj, Uttar Pradesh, India

Krishna Misra Department of Applied Sciences Indian Institute of Information Technology (IIITA) Prayagraj, Uttar Pradesh, India Rudra P. Ojha Department of Zoology Nehru Gram Bharati (Deemed to be University) Prayagraj, Uttar Pradesh, India

ISBN 978-981-16-6866-1 ISBN 978-981-16-6867-8 https://doi.org/10.1007/978-981-16-6867-8

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Foreword

I am extremely happy to write the foreword for this book entitled Homocysteine Metabolism in Health and Disease. The book provides a plethora of information pertaining to frontier research on homocysteine metabolism, nutritional index reflecting human health culminating in substantial contribution at the need of the hour. The major focus of the book is to establish intriguing relationship between homocysteine metabolism, nutrition, and its impact on human health. The chapters of the book comprise various aspects of homocysteine metabolism with reference to the synergistic role of vitamins and nutrients and with different aspects of human health. Among all the nutritional components, homocysteine metabolism plays a significant role in the growth and development of children. It would be interesting to correlate bone health and stem cell-mediated homocysteine metabolism in developmental biology. Taken together, book embodies great innovative ideas to showcase frontier research on homocysteine metabolism and its health impact to date. It is noteworthy to mention the authors’ excellent contribution and rich information, which would provide valuable insights and lead scientists, medical researchers, faculty, and students working in the field of nutrition, health, and homocysteine metabolism. I congratulate the editors for the effort to compile frontier research on homocysteine metabolism and health. Institute of Medical Sciences, Banaras Hindu University Varanasi, Uttar Pradesh, India

D. Dash

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Preface

The book Homocysteine Metabolism in Health and Disease is a compilation of the state-of-the-art research and advanced empirical knowledge of medical science. Cumulative research findings and various advanced database available on diversified medical and biotechnological strategies acknowledge the recent development and understanding of the molecular nexus behind the malnutrition and homocysteine metabolism cascade. The major thrust in recent days is the lack of coherence between academic researchers with real-time clinical applications; this book attempts to acknowledge the current thrust. The book comprises three parts; Part I (Nutrition and Health) contains five chapters, and Part II (Homocysteine Impairment and Various Disorders) and Part III (Advanced Research in Homocysteine Metabolism) include four and five chapters, respectively.

Part I: Nutrition and Health Chapter 1 entitled “Homocysteine and Folic Acid Metabolism” by Hem Chandra Jha and associates explains that homocysteine (Hcy) is a non-proteinogenic amino acid, synthesized from methionine along with some methyl group donor intermediates. The metabolism of homocysteine involves three pivotal processes: S-adenosyl-Lmethionine (SAM)-dependent transmethylation, folate-dependent or -independent re-methylation cycles, and trans-sulfuration reactions. The entire metabolic pathway is governed by various endogenous and exogenous factors including the genetic composition (polymorphism in MTHFR, GCP2, RFC1, and TCN2 genes) and the diet intake (methionine and cysteine richness of constituents) of an organism. Additionally, the concentrations of intermediates (AdoMet, AdoHcy, and methylTHF) and enzymes (GAMT, PEMT, GNMT, BHMT, MS, and CBS) involved in these reactions regulate the process at the cellular level. Folate, an intermediate produced during the homocysteine metabolism, is involved in numerous other metabolic pathways, which are associated with neural development and reproductive, renal, and cardiovascular health of humans. The aberrantly altered level of homocysteine (commonly hyperhomocysteinemia) triggers various pathological symptoms and subsequently Hcy-related diseases. Increased plasma homocysteine concentrations could lead to hyperhomocysteinemia, which is a risk factor for several pathologies such as cardiovascular diseases (CVD) and is also related vii

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with early atherosclerosis symptoms and venous thrombosis. Further, the elevated level of homocysteine is associated with other pathologies like autoimmune responses (diabetes I and diabetes II), neural development (neural tube defects), neurodegenerative diseases, and reproductive health as male and female infertility. Chapter 2 entitled “Nutritional Determinants in Hyperhomocysteinemia” by Rajesh Dubey and his coworker emphasizes that currently a significant emphasis has been given to establish the role of malnutrition in prevention and management of coronary heart disease among individuals who are more prone to develop coronary heart disease (CHD). Folic acid deficiency is a major cause of hyperhomocysteinemia leading to CHD. The present study has been deigned to investigate the role of low level of homocysteine in different age and sex groups. Further, it was also investigated that nutritional deficiency, particularly folic acid, increases the level of homocysteine and is also responsible for elevated level of homocysteine and ultimately responsible for cardiac event. The present series strongly demonstrates a significant association between folic acid deficiencies and hyperhomocysteinemia in both age and sex groups, and also more studies are required on a large number of cases to validate the hypothesis proposed. Professor G. P. Dubey and his collogues describe the significant evidence for atherosclerosis-impaired endothelial function including hyperhomocysteinemia in Chap. 3 entitled “Role of Homocysteine in Impaired Endothelial Function in Metabolic Syndrome.” The chapter describes that the microcalcium deposition is majorly responsible for thickening of the endothelium in both age and sex groups. Further, pro-inflammatory cytokines, IL-6 and TNF-α, are also linked behind endothelial dysfunction. Chapter 4 entitled “Homocysteine and Bone Health” by Rupesh K. Srivastava and coauthors explains that homocysteine is a sulfur-containing intermediary amino acid synthesized during methionine metabolism. Homocysteine plays a significant role in the regulation of cell homeostasis, but elevated levels of plasma homocysteine (hyperhomocysteinemia) are associated with vascular and various age-related pathologies. The chapter discusses the role of homocysteine-induced dysbiosis in bone resorption and the potential of probiotics as therapy for the prevention of homocysteine-induced bone loss along with other possible therapies. Chapter 5 entitled “Biofortification: A Remedial Approach Against Malnutrition in Rural and Tribal Population” by Suneha et al. explains that malnutrition is common in rural and tribal communities due to a lack of information, availability of balanced food, and low literacy. Biofortification of essential micronutrients in staple crops is a successful technique for alleviating micronutrient insufficiency in such populations. Plant breeding, transgenic technology, and genome editing can help to increase the density of essential minerals and vitamins in staple foods. The amino acid homocysteine is found in the blood. Homocysteine levels in the blood are normally less than 15 micromoles per liter (μmol/L). Hyperhomocysteinemia is a high level of this amino acid that causes arterial damage and blood clots in the blood vessels, making it a risk factor for heart disease. A high amount of homocysteine is linked to a lack of folate. In the presence of the enzyme methionine synthases,

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5-methyltetrahydrofolate and cofactor vitamin B12 convert homocysteine to methionine. As a result, a lack of folate and vitamin B12 is a factor in the formation of increased levels of homocysteine, which is linked to cardiovascular disease (CVD). Several studies have linked malnutrition to raised homocysteine levels and, as a result, an increased risk of CVD. The main cause of folate insufficiency, and thus hyperhomocysteinemia, is a diet that is low in fresh fruits, vegetables, and fortified cereals.

Part II: Homocysteine Impairment and Various Disorders Chapter 6 entitled “Homocysteine Metabolism Pathway Genes and Risk of Type2 Diabetes Mellitus/Metabolic Disorders” by Kesharwani and his coworker describes that whole body inflammation is the principal characteristic of type-2diabetes mellitus. Those people who are suffering from type-2 diabetes mellitus always suffer from an elevated level of cytokines in both age and sex groups. Several risk factors have been identified showing marked elevated levels of IL-1β, TNF-α, and hsCRP. The elevated level of pro-inflammatory cytokines produces high level of all the above inflammatory markers including other biological parameters. The present study is based on the investigation of various pro-inflammatory cytokines in different types of diabetes mellitus. In Chapter 7 entitled “Genetic Polymorphism in Homocysteine Metabolism” compiled by Ojha and his associates, ample evidence is available to demonstrate genetic polymorphism in homocysteine metabolism. Low folic acid is another cause of hyperhomocysteinemia in both age and sex groups. This study is based on the evaluation of homocysteine metabolism in different gender. Chapter 8 entitled “Homocysteine Metabolism as a Biomarker for Cancer” is well documented by Mohapatra and associates that homocysteine balance, particularly hyperhomocysteinemia, appears to be a common hallmark of a variety of diseases. It is uncertain if high homocysteine levels contribute directly to ailment pathogenesis or serve as a biomarker for metabolic abnormalities such as abnormal methyl group metabolism. HHcy has been linked to several polymorphisms in genes implicated in the homocysteine methionine pathway, signaling that these variants may have a role in several multifactorial illnesses with a high incidence in the general population. As a result, Hcy-elevating medications should be used with prudence in cancer patients, and Hcy levels should be regularly monitored following chemotherapy or surgery. Because hyperhomocysteinemia is linked to various pathological disorders, including vascular disease, it is evident that homocysteine management should be a primary concern for nutrition and health. Chapter 9 entitled “Homocysteine Metabolism and Risk of Breast Cancer in Women” by Rinki Kumari and coauthors describes that breast cancer is the second leading cause of women’s death in economically developed countries, India, and other developing countries. Recent studies demonstrate the association between homocysteine, folic acid, vitamin D, and higher BMI in breast cancer.

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Hyperhomocysteinemia is an independent risk factor for oncogenesis and further it may also alter the cysteine metabolism.

Part III: Advanced Research in Homocysteine Metabolism Chapter 10 entitled “Genetic Susceptibility to Neural Tube Defect (NTD) and Hyperhomocysteinemia” by Dubey and coresearcher explains that increased level of homocysteine is typically caused by a genetic defect in the enzyme involved in the homocysteine metabolism. Several genetic enzyme defects like methionine synthase (MS), cystathionine beta synthase (CbS), and 5,10 methylenetetrahydrofolate reductase (MTHFR) involve the genetic defect in both males and females. Neural tube defect is another major cognitive factor of hyperhomocysteinemia. In the present chapter, attempt has been made to provide a correlation between neural tube defect and hyperhomocysteinemia. Chapter 11 entitled “Homocysteine Determinants as Risk Markers for Neurological Diseases” by Rudra P. Ojha et al. describes that the elevated level of homocysteine is one of the important risk markers for neurological disorders. Cognitive impairment and increased level of homocysteine are significantly associated with both age and sex groups. It is one of the important markers for neurodegenerative disorders where cognitive decline is present. This chapter describes homocysteine is the most important significant parameter responsible for cognitive decline in both sex groups. Chapter 12 entitled “Management of Neurogenic Hyperhomocysteinemia (HHcy) by a Plant-Based Formulation” by Kesharwani et al. describes the role of Hcy and its metabolism in neurodegeneration and the positive effect of HHcy in relation to age, sex, inflammation, stress, and memory in SDAT patients. Thus, it can be concluded that Hcy is one of the major risk factors for neurodegeneration and that supplementation with folate and vitamin B12 and B6 may reduce the decline of disease progression. Chapter 13 entitled “Homocysteinemia and Viral Infection with Special Emphasis on COVID-19” by Tripathi and Misra describes that Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-Cov-2) is one of the highly pathogenic virus causing Coronavirus pandemic 2019 (COVID-19), which has tremendously increased mortality rate across the world. COVID-19 has been recognized as a pandemic in 2020 and 2021. Different genomic mutations of this virus are closely associated with cardiovascular diseases such as ischemic heart disease, stroke, and venous thromboembolism (VTE). The importance of homocysteine lies in the potential prediction of cardiovascular risk in COVID-19 patients. This chapter summarizes the critical role of homocysteine as a key factor of coagulation in the prognosis of COVID-19-infected patients and also highlights the clinical features of SARS-Cov-2 infected patients. Chapter 14 entitled “Role of Homocysteine Metabolism in Cardiovascular Diseases (CVD)” by Kumar and associates explains that hyperhomocysteinemia is linked to vascular disease risk and other diseases. Various studies involving vitamin

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B and placebo treatment of patients suffering from vascular disease look if therapy, including homocysteine decrease, cuts cardiovascular risk. On the other hand, B-vitamin treatment has successfully restored average homocysteine concentrations without corresponding decreases in disease risk. The biochemical variability of homocysteine is responsible for many clinical conditions including the high prevalence of cardiovascular diseases. Varanasi, Uttar Pradesh, India Prayagraj, Uttar Pradesh, India Prayagraj, Uttar Pradesh, India Prayagraj, Uttar Pradesh, India

Govind Prasad Dubey Krishna Misra Rajesh K. Kesharwani Rudra P. Ojha

Acknowledgments

We are highly grateful to Prof. G. P. Dubey, Distinguished Professor, IMS, BHU, Varanasi, for his encouragement to achieve academic excellence. We are heartily thankful to Prof. (Mrs.) Krishna Misra, Honorary Professor, IIIT-Allahabad, Prayagraj, a renowned scientist and educator for her continuous support, guidance, and motivation. We will always be highly thankful to Shri J. N. Misra, Chancellor, NGB(DU), Prayagraj, and Shree Manish Mishra, Secretory, Nehru Gram Society, Prayagraj, for his consistent support and encouragement. We are highly thankful to Dr. Rajesh Dubey, Principal Consultant and Partner, Hawkins Point Partners, Boston, USA, for his consistent support throughout the compilation of scattered information in the form of a book. We are highly grateful to our contributors of this book for their extensive labor, vision, and planning in writing the chapters. We thank the reviewers whose critical comments improved the book in substantial ways. We are highly grateful to our parents, wife, and other family members for their wishes, valuable support, and encouragement. We are also thankful to our collaborators especially Prof. U. P. Shahi, Prof. V.N. Mishra, Dr. D. Jain, Dr. Rupesh K. Srivastava, Dr. Hem Chandra Jha, Dr. Suneha Goswami, and Mr. Sunil Dubey for their support. We would like to appreciate the support of Dr. Bhavik Sawhney Bhavik, Mr. Selvakumar Rajendran, and the entire team of Springer Nature for their continuous support and cooperation during the entire process of publication.

Govind Prasad Dubey Krishna Misra Rajesh K. Kesharwani Rudra P. Ojha

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Contents

Part I

Nutrition and Health

1

Homocysteine and Folic Acid Metabolism . . . . . . . . . . . . . . . . . . . . Deeksha Tiwari, Annu Rani, and Hem Chandra Jha

3

2

Nutritional Determinants in Hyperhomocysteinemia . . . . . . . . . . . . Rajesh Dubey, U. P. Shahi, V. N. Mishra, D. Jain, Akanksha Mishra, Govind Prasad Dubey, and Rudra P. Ojha

37

3

Homocysteine-Mediated Endothelial Dysfunction in Metabolic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Govind Prasad Dubey, D. Jain, V. N. Mishra, Sunil Dubey, Arti Ojha, and Rajesh K. Kesharwani

4

Homocysteine and Bone Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . Asha Bhardwaj, Leena Sapra, Bhupendra Verma, and Rupesh K. Srivastava

5

Biofortification: A Remedial Approach Against Malnutrition in Rural and Tribal Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amit Kumar Goswami, Suneha Goswami, T. Vinutha, Sanjay Kumar Singh, and Shelly Praveen

Part II

51

71

97

Homocysteine Impairment and Various Disorders

6

Homocysteine Metabolism Pathway Genes and Risk of Type 2 Diabetes Mellitus/Metabolic Disorders . . . . . . . . . . . . . . . . . . . . . 115 Rajesh K. Kesharwani, Govind Prasad Dubey, D. Jain, V. N. Mishra, Rajesh Dubey, and Rudra P. Ojha

7

Genetic Polymorphism in Homocysteine Metabolism . . . . . . . . . . . 135 Rudra P. Ojha, Govind Prasad Dubey, U. P. Shahi, V. N. Mishra, D. Jain, and Pradeep Upadhyay

8

Homocysteine Metabolism as a Biomarker for Cancer . . . . . . . . . . 159 Meghavi Kathpalia, Prashant Kumar, and Swati Mohapatra

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9

Contents

Homocysteine Metabolism and Risk of Breast Cancer in Women . . 173 Rinki Kumari, Vandana Yadav, Simon Agongo Azure, Disha Sharma, Sudhanshu Mishra, Sneh Shalini, Rudra P. Ojha, and Anita Venaik

Part III

Advanced Research in Homocysteine Metabolism

10

Genetic Susceptibility to Neural Tube Defect (NTD) and Hyperhomocysteinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Govind Prasad Dubey, V. N. Mishra, D. Jain, Sunil Dubey, and Rudra P. Ojha

11

Homocysteine Determinants as Risk Markers for Neurological Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 Rudra P. Ojha, Govind Prasad Dubey, V. N. Mishra, D. Jain, Sunil Dubey, Rajesh Dubey, and Rajesh K. Kesharwani

12

Management of Neurogenic Hyperhomocysteinemia (HHcy) by a Plant-Based Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 Rajesh K. Kesharwani, Govind Prasad Dubey, V. N. Mishra, D. Jain, Rajesh Dubey, and Rudra P. Ojha

13

Homocysteinemia and Viral Infection with Special Emphasis on COVID-19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 Anushree Tripathi and Krishna Misra

14

Role of Homocysteine Metabolism in Cardiovascular Diseases . . . . 257 Prashant Kumar, Sunil Kumar Verma, Sweta Rai, P. Shakti Prakash, and Dheeraj Chitara

About the Editors

Govind Prasad Dubey is designated as a Lifelong Distinguished Professor in the Department of Kriya Sharira and has profound expertise in scientific validation of Indigenous System of Medicine. He also bears the responsibility of Study Director and Coordinator, DST Tribal Medicine Project, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh. Prof. Dubey has more than 50 years of teaching, research, and administrative experience and has been felicitated with various posts of international and national significance. He is a distinguished member of several scientific committees including Indian delegation led by the Prime Minister of India to the USA. Prof. Dubey has received several international and national funds/grants including Department of AYUSH, Department of Science and Technology (DST), Department of Biotechnology (DBT), Indian Council of Medical Research (ICMR), New Delhi, and other government bodies in collaboration with multinational companies including Jinvator BioMed GmbH, Germany, in different areas of Herbal/Ayurveda drug development. He has published more than 250 research articles, 05 books, and holds 24 patents of the USA, European and Indian continent. Krishna Misra is an Honorary Professor, Indian Institute of Information Technology, Prayagraj, U.P. She superannuated as a Professor of Chemistry at the University of Allahabad, where she occupied the chair of head, Biochemistry department, and was the first coordinator of the Centre for Biotechnology. At present, she is fellow of the National Academy of Sciences, Allahabad, and also worked as General Secretary. Besides, she is chief advisor at India Pesticides Ltd., Lucknow, and had been a member of the advisory board of Biotech Park, Lucknow. She was awarded NASI Platinum Jubilee senior scientist Fellowship. She is chairperson of STEM program of DST and Chemistry Advisory Board of UPCST. She had been expert in many selection committees including KGMU and SGPGIMS Lucknow and is also on the editorial boards of many national and international journals. She published 250 papers, four books, and has Indian and US patents to her credit. She had been a task force member of the Department of Biotechnology, Govt of India, and fellow and founder member of BRSI (Biotechnology Research Society of India). She is a member of the advisory board of Molecular Medicine, SGPGI, Lucknow. She has written a dozen book chapters, presented papers in about 100 national and international conferences, and has Indian and US patents. She visited Japan (UNESCO xvii

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About the Editors

fellow), UK (Sponsor British Council), and USA (Invited papers/talks/chair) a number of times to deliver lectures and participate in scientific discussions. Prof. Misra has been awarded a large number of research projects including three prestigious international projects from the USA. Rajesh K. Kesharwani is an Associate Professor in the Department of Computer Application, Nehru Gram Bharati, Deemed University, Prayagraj, U.P., India. He has more than 12 years of research and 9 years of teaching experience in various institutes of India. He has received several awards, including the NASI-Swarna Jayanti Puruskar by the National Academy of Sciences of India. He has authored over 55 peer-reviewed articles, 25 book chapters, and 13 edited books with international publishers. He has been a member of many scientific communities as well as a reviewer for many international journals. His research fields of interest are Medical Informatics, Protein Structure and Function Prediction, Computer-Aided Drug Designing, Herbal Drug Development, Cancer Biology, Nano-Biotechnology, and Biomedical Sciences. Rudra P. Ojha is designated as Director of Research Centre and Associate Professor in the Department of Zoology, Faculty of Science, Nehru Gram Bharati, Deemed University, Prayagraj, U.P. India. He has more than 16 years of teaching, research, and administrative experience from various national and international reputed institutes including Institute of Pathology, ICMR, AIIMS-New Delhi, NIMHANS, Bangalore, Banaras Hindu University, Varanasi, and University of California, Davis, CA, USA. He has authored 36 research articles, 09 book chapters, and 03 edited books of national and international repute. Dr. Ojha has been conferred with various prestigious international and national awards: notably Young Scientist Award from the Department of Science & Technology (DST), New Delhi, and Full Travel support grant from the Department of AYUSH, New Delhi, for overseas presentation at World Congress of Oxygen, Club of California, Santa Barbara, California, USA. Dr. Ojha is a lifetime member of many national and international societies including Society for Neurosciences (SfN), USA, Society for Free Radical Research (SFRR), Europe, and Indian Academy of Neurosciences (IAN), India. His field of research interest is Herbal Drug Development, Cancer Biology, Neuroscience, and Biomedical Sciences.

Abbreviations

AKT BMD Ca CBS cGMP DSS eNOS ERK ERT ERα FOXO1 G-CSF H H 2S HCU Hcy HHcy IFN IL iNOS Lox MAPK MCSF MIP MMP MTRR NAHS NMDA-R NO OA OPG Plod2 RA

Protein kinase B Bone mineral density Calcium Cystathionine β synthase Guanosine monophosphate Dextran sodium sulfate Endothelial NO synthase Extracellular-signal-regulated kinase Enzyme replacement therapy Estrogen receptor alpha Forkhead box protein O1 Granulocyte colony-stimulating factor Hydrogen Hydrogen sulfide Homocystinuria Homocysteine Hyperhomocysteinemia Interferon Interleukin Inducible NO synthase Lysyl oxidase p38 mitogen-activated protein kinase Macrophage colony-stimulating factor Macrophage inflammatory protein Matrix metalloproteinase Methionine synthase reductase H2S donor N-methyl-D-aspartate receptor Nitic oxide Osteoarthritis Osteoprotegerin Lysyl hydroxylase Rheumatoid arthritis xix

xx

RANKL ROS SAA3 SERM TIMP TNF

Abbreviations

Receptor activator of the nuclear factor kappa B ligand Reactive oxygen species Serum amyloid A3 Selective estrogen modulator Tissue inhibitor matrix metalloproteinase 1 Tumor necrosis factor

Part I Nutrition and Health

1

Homocysteine and Folic Acid Metabolism Deeksha Tiwari, Annu Rani, and Hem Chandra Jha

Abstract

Homocysteine (Hcy) and folate metabolism plays a crucial role in maintaining the overall human health. Hcy is a non-proteinogenic amino acid synthesized from methionine with the involvement of some methyl group donor intermediates. The metabolism of homocysteine involves three pivotal processes: S-adenosyl-Lmethionine (SAM)-dependent transmethylation, folate-dependent or -independent remethylation cycles, and transsulfuration reactions. The entire metabolic pathway is governed by various endogenous and exogenous factors including the genetic composition (polymorphism in MTHFR, GCP2, RFC1, and TCN2 genes) and the diet intake (methionine and cysteine richness of constituents) of an organism. Additionally, the concentrations of intermediates (AdoMet, AdoHcy, and methylTHF) and enzymes (GAMT, PEMT, GNMT, BHMT, MS, and CBS) involved in these reactions regulate the process at the cellular level. Folate, an intermediate produced during the homocysteine metabolism, is involved in numerous other metabolic pathways, which are associated with neural development, reproductive, renal and cardiovascular health of humans. The aberrantly altered level of homocysteine (commonly hyperhomocysteinemia) triggers various pathological symptoms and subsequently the Hcy-related diseases. Increased plasma homocysteine concentrations could lead to hyperhomocysteinemia which is a risk factor for several pathologies such as cardiovascular diseases (CVD), and is also related to early atherosclerosis symptoms and venous thrombosis. Further, the elevated level of homocysteine is associated with other pathologies like

Deeksha Tiwari and Annu Rani contributed equally with all other contributors. D. Tiwari · A. Rani · H. C. Jha (*) Infection Bioengineering Group, Department of Biosciences and Biomedical Engineering, Indian Institute of Technology, Indore, Madhya Pradesh, India e-mail: [email protected] # The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2022 G. P. Dubey et al. (eds.), Homocysteine Metabolism in Health and Disease, https://doi.org/10.1007/978-981-16-6867-8_1

3

4

D. Tiwari et al.

autoimmune responses (diabetes I and diabetes II), neural development (neural tube defects), neurodegenerative diseases, and reproductive health as male and female infertility. Keywords

Homocysteine · Folic acid · Homocysteine metabolism · Cardiovascular diseases (CVD)

1.1

Introduction

Homocysteine (Hcy) is a non-essential, sulfur-containing, non-proteinogenic amino acid (Kumar et al. 2017). The biosynthesis of the Hcy molecule starts from amino acid methionine, which plays a key role between the folate and activated methyl cycles (Kumar et al. 2017). Initial evaluation of the Hcy biochemistry was done in the period of 1930s to the 1950s by Vincent Du Vigneaud and colleagues (Jarvis et al. 1961). Later in 1960s, Hcy metabolism and its role in physiology was further explored, and several studies evaluated various disorders related to imbalanced Hcy metabolism; including, the inborn error of Hcy metabolism, homocystinuria, and levels of Hcy as an independent risk factor for vascular disease (Al Mutairi 2020). After being synthesized, Hcy has several fates, such as to getting re-methylated to methionine, acting as precursor for cysteine biosynthetic pathway, or to getting released into the extracellular medium (Kumar et al. 2017). Hcy release into the extracellular matrix is directly responsible for increased concentrations of total Hcy (i.e., hyperhomocysteinemia) in body fluids such as urine and plasma (Medina et al. 2001). Commonly, the high level of Hcy has been strongly associated with folate and cobalamin deficiencies. Hyperhomocysteinemia (HHcy) triggers complications in the body including risk of cardiovascular disease (atherosclerosis and thrombosis), pregnancy complications, neural tube defects, mental disorders, cognitive impairment in the elderly, psoriasis, and some tumors (Medina et al. 2001). The regulation of Hcy metabolic pathway is attributed to several endogenous factors such as gene polymorphism of Hcy metabolism-involved enzymes (GAMT, PEMT, GNMT, CBS, MS, and MTHFR) and exogenous factors (dietary paucity of vitamins B6 or B12, folate, and also the intake of Met and Cys-rich protein) (Škovierová et al. 2016). Additionally, the congenital or sporadic mutation in the crucial enzymes leads to accelerated perturbation into the Hcy metabolism. The genetic polymorphism of enzymes like MTHFR, RFC1, GCP2, and TCN2 was also reported to have a substantial role in Hcy metabolism and, finally, the inter connection with the several pathologies (Sunder-Plassmann and Födinger 2003). Further, the regulation of Hcy metabolism is controlled by multiple factors like diet (Verhoef and de Groot 2005), molecular-genetic changes in the genes responsible for metabolism, and the concentration of pathway intermediates (Sunder-Plassmann and Födinger 2003).

1

Homocysteine and Folic Acid Metabolism

5

The current chapter aims to sum up the essential information of Hcy biochemistry and metabolism. We have broadly focused on the regulation of Hcy biochemistry at the genetic, enzymatic, and substrate-level by a feedback mechanism. Further, pathology associated with the Hcy metabolism and detailed elaboration of most commonly reported pathological conditions are briefly explained.

1.2

Homocysteine (Hcy) Metabolism

1.2.1

Biosynthesis and Metabolism of Hcy

As mentioned earlier, “methionine” (Met) is the exclusive source of Hcy, which can either undergo re-methylation to form Met or metabolize into cysteine (Cys) by the transsulfuration pathway. Hcy can also cyclize to form an intramolecular thioester of Hcy (homocysteine thiolactone/HTL) or can be utilized as an intermediate in the S-adenosyl-L-methionine (SAM) cycle (Jakubowski 2004). SAM, a universal methyl group donor, is generated from the SN2 reaction of Met with ATP in the presence of methionine adenosyltransferase (Niu et al. 2017). After transferring the methyl group with the help of methyltransferase to acceptor entities such as DNA, RNA, amino acids, proteins, and phospholipids, the SAM molecule converts into the demethylated compound: S-adenosyl homocysteine (SAH) (Chiang et al. 1996). SAH then undergoes S-adenosylhomocysteine hydrolase-mediated deadenylation and emerges in the form of Hcy. Otherwise, the decarboxylation of SAM molecules initiates a process named aminopropylation (Altintas and Sezgin 2004). Decarboxylated SAM is coupled to putrescine to generate spermidine and spermine, which are critical for cell growth, differentiation, and DNA/RNA stability (Pegg and Casero 2011) (Fig. 1.1b). In this polyamine synthesis, methylthioadenosine (MTA) is the by-product having potent analgesic and antiinflammatory properties. The metabolic fate of MTA and its intermediates are explained in more detail in the next section (Pegg and Casero 2011). Further, the Hcy can further be re-methylated to Met by a folate-dependent and/or -independent mechanism. In folate-dependent mechanism, Hcy (methionine synthase; vitamin B12 dependent) utilizes the methyl group from 5-N-methyl tetrahydrofolate (5-methylTHF), which is known as the re-methylation pathway (Kumar et al. 2017). The folate-independent mechanism, which is mainly restricted to the liver, involves betaine which is converted into dimethylglycine and catalyzed by betaine homocysteine-S-methyltransferase (BHMT) (Neidhart 2016) (Fig. 1.1a). Although 5-methylTHF is the major source for methyl groups in the re-methylation of Hcy to Met, the betaine and choline molecules can also serve as methyl donors (Mahmoud and Ali 2019). Nonetheless, the Hcy, in combination with another amino acid, serine, can form cystathionine. This route of Hcy metabolism is commonly called the transsulfuration pathway and is catalyzed by cystathionine-β synthase (CBS), an enzyme-dependent vitamin B6 as a cofactor (Jhee and Kruger 2005a). The transsulfuration pathway leads to the generation of hydrogen sulfide (H2S) during the metabolism of the

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mixed disulfide (Hcy-Cys) to cystathionine by the action of CBS (McBean 2017; Giuffrè and Vicente 2018). The cystathionine-γ-lyase (CSE) action on cysteine results in the production of H2S, thiocysteine, and pyruvate (liberation of NH3) (Chiku et al. 2009; Nagy 2015). The thiocysteine is subsequently cleaved to H2S and Cys. Functionally, the H2S synthesis mediated by CβS has not only renal protection role; instead, it also has a role in the induction of long-term hippocampal potentiation, development of the brain, and facilitates blood pressure regulation (Kamat et al. 2015a). The Cbs and Cse gene deletion in mice results in reduction in H2S level in

Fig. 1.1 (a) Outline of Hcy metabolism; Hcy is synthesized from SAH molecule, and its fate is to regenerate methionine, cysteine, and homocysteine thiolactone molecules in different proportions (b). Steps involved in the conversion of MTA to methionine

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Fig. 1.1 (continued)

the serum, and aorta (ultimately heart), which has a profound effect on hypertension and diminished endothelium-dependent vasorelaxation (Yang et al. 2008). Besides re-methylation and transsulfuration pathways, the Hcy has potential to undergo cyclization to form HTL, which is determined to be the toxic intermediate of Hcy (Moretti and Caruso 2019). HTL is formed due to an error-editing reaction in protein biosynthesis (Martinelli et al. 2013). Owing to the structural similarity between Met and Hcy, the methionyl-tRNA synthetase is sometimes primed to

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take up Hcy instead of Met (Serre et al. 2001). This error is detected by cellular machinery, which then tries to resolve it immediately by off-tracking the AMP from the adenylated Hcy (not S-adenosyl-Hcy), resulting in the cyclization of Hcy (Tehlivets et al. 2013) (Fig. 1.1a). Further, the accumulated levels of Hcy and/ or defects in the metabolism of Hcy also potentially trigger the formation of HTL (Kumar et al. 2017). Additionally, HTL is also associated with the atherothrombosis process. HTL reportedly forms isopeptide bonds (non-α-amino and an α-carboxyl group or a non-α-carboxyl and an α amino group of amino acids) with protein lysine (Lys) residues (Kumar et al. 2017). This isopeptide bond jeopardizes protein function, with pathophysiological effects including autoimmune responses and enhanced thrombosis response (Kumar et al. 2017).

1.2.2

Important Enzymes and Intermediates

Hcy metabolism involves several intermediate molecules, and their interconversion is dependent on a number of cofactors in the form of nutritional B vitamins, and critically regulated enzymes (Kennedy 2016). Different intermediates and enzymes involved in the generation of these intermediates are as follows:

1.2.2.1 Methionine In cellular metabolism, methionine plays a central role in protein synthesis, methyl group transfer (via SAM), polyamines and ethylene synthesis (Gao et al. 2018; Parkhitko et al. 2019), among other processes. Among all the pathways methionine is said to be involved in, the protein synthesis pathway alone consumes the maximum amount of the entire methionine concentration. Alternatively, the methyl group of methionine could also be transferred to several acceptors inside the cell, such as choline and its derivatives, including phosphatidylcholine (the major polar lipid) (Fontecave et al. 2004). The SAM synthesis is the utmost route of methionine metabolism, 80% methionine is consumed on this reaction (Parkhitko et al. 2019). Whereas, SAM (>90%) is consecutively consumed in the transmethylation reactions. Subsequently, SAM is converted into a SAH molecule which is recycled into Hcy and ultimately into methionine. The conversion is catalyzed by S-adenosylhomocysteine hydrolase (Ravanel et al. 1998). The last reaction in recycling the methionine synthesis pathway is catalyzed by methionine synthase (MS), which also serves to regenerate the methyl group of SAM (Shane 2013). In addition to the recycling pathway of methionine synthesis, a salvage pathway is also there, where SAM converts into MTA and ultimately into methionine (Sekowska et al. 2019) (Fig. 1.1a). 1.2.2.2 SAM and SAH SAM moiety has been widely utilized for polyamines synthesis, methyl group transfer, and aids in recycling methylthioadenosine moiety and regeneration of methionine (Sekowska et al. 2019). During the conversion of SAM, 5-methylthioadenosine (MTA) is generated as a by-product. The MTA is then converted into

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methionine by a series of reactions involving the intermediates S-methyl-5thioribose, S-methyl-5-thioribulose-1-phosphate, 2,3-diketo-5-methylthiopentyl-1phosphate, 2-hydroxy-3-keto-5-methylthiopentyl-1-phosphate, 1, 2-dihydroxy-3keto-5-methylthiopentene, and 4-methylthio-2-oxobutanoic acid (Wakabayashi et al. 2013; Ashida et al. 2008). This course is also called the salvage pathway of methionine regeneration, accounting for about one-third of the amount of methionine accumulating in protein (Ravanel et al. 1998) (Fig. 1.1b). Alternatively, SAM, after transferring the methyl group to acceptor molecules such as DNA, RNA, proteins, and lipids, can form a SAH molecule. Subsequently, SAH converts back to Hcy under a reaction catalyzed by hydrolase enzymes (Lennard 2010).

1.2.2.3 Cystathionine It is an intermediate of cysteine biosynthesis. Two pathways majorly produce cystathionine, one of them is during the transsulfuration pathway which converts Hcy to cystathionine under the influence of the CBS enzyme (Sbodio et al. 2019; Zuhra et al. 2020). Further, the enzymes like cystathionine gammalyase (CTH), sulfinoalanine decarboxylase, and cysteine dioxygenase (CDO) act on cystathionine and produce cysteine, hypotaurine, and then taurine like molecules (Jurkowska et al. 2015). The cysteine generated through the cystathionine gammalyase reaction can be used by the enzymes glutamate-cysteine ligase (GCL) and glutathione synthetase (GSS) to produce glutathione (Griffith and Mulcahy 2006; Chen et al. 2018) (Fig. 1.1a). Otherwise, cystathionine is generated biosynthetically from cysteine and homoserine by cystathionine-γ-synthase (Krömer et al. 2006).

1.2.3

Effects of Altered Hcy Metabolism on Human Physiology

The different forms of Hcy such as free Hcy, protein-bound Hcy (S-linked and N-linked), oxidized forms of Hcy, and Hcy-thiolactone, are considered under “total Hcy” (tHcy) (Kumar et al. 2017). Physiologically, less than 1% of tHcy is present in a free reduced form (SH group), and nearly 10%–20% of tHcy has been found in different oxidized forms, i.e., Hcy-Cys and Hcy (the Hcy dimer) in plasma (Škovierová et al. 2016). However, most of plasma tHcy (80%–90%) is N-linked and S-linked to γ-globulins or serum albumin (Škovierová et al. 2016). The tHcy has a remarkable role as a supplementary test for evaluating the vitamin B12 deficiency and predicting the risk of cardiovascular disorders, stroke progression, the inborn fallacy in Met metabolism (Markišić et al. 2017). The reference range of tHcy in plasma lies between 5 and 10 μM for humans (Škovierová et al. 2016). Under normal conditions, plasma Hcy concentrations do not exceed 15 μM. However, the elevation of plasma Hcy manifests as HHcy (Krömer et al. 2006; Škovierová et al. 2016). The condition of HHcy is classified under several types based on the tHcy concentration: moderate (16–30 μM), intermediate (31–100 μM), and severe (higher than 100 μM) (Ji 2004). Severe HHcy occurs in homocystinuria, an innate metabolic disorder characterized by a deficiency of CBS enzyme activity. Generally, the accumulation of Hcy results from the inability to regulate its metabolism and can be attributed to

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endogenous factors (polymorphisms of the genes coding enzymes involved in Hcy metabolism such as CBS, MS, and MTHFR) and/or exogenous factors (dietary deficiency of folate, vitamins B6 or B12 and also the intake of proteins rich in Met and Cys) (George et al. 2020; Azzini et al. 2020). Further, the utilization of Hcy molecules by the transsulfuration and remethylation pathways is nutritionally regulated. Dietary intervention to reduce circulating Hcy concentrations represents a tangible way to reduce cardiovascular disease risk (Martí-Carvajal et al. 2009). Several investigations have also shown the impact of Hcy lowering treatments with B vitamins to prevent vascular disease (Lonn et al. 2006). The perturbations in Hcy metabolism, particularly elevation in intracellular and subsequently circulating levels of Hcy, i.e., HHcy occur more commonly than hypo or low levels of Hcy (Schalinske and Smazal 2012). Severe HHcy occurs due to rare genetic defects resulting in deficiencies in CBS, MTHFR, or enzymes involved in methyl-B12 synthesis and Hcy methylation (Hoss et al. 2019; Al Mutairi 2020). The mild HHcy is commonly observed in the fasting conditions where mild impairment occurs in the methylation pathway (i.e., folate or B12 deficiencies or methylenetetrahydrofolate reductase thermolability) (Selhub 1999; Durand et al. 2001). The high-level Hcy showed a non-physiological role in a variety of deleterious effects on endothelial or smooth muscle cells in vitro (Ganguly and Alam 2015). Likewise, animal model studies also indicated the possibility of high Hcy levels having a deteriorating effect on the vascular wall structure and the blood coagulation system (Ganguly and Alam 2015). Imbalance in Hcy metabolism is also related to the pathogenesis of diseases, such as reproductive diseases, psychiatric disorders and neurological diseases, kidney disease (chronic), damages in bone tissues, gastrointestinal disorders, and congenital deffects (neural tube formation) (Škovierová et al. 2016). The specific pathologies associated with altered Hcy levels are explained in more detail in a later section of this chapter.

1.3

Regulation of Homocysteine Metabolism

In general, the regulation of an entire metabolic pathway is often controlled by multiple factors; thus, it is difficult to pinpoint a single element that is crucial for the regulation. Similarly, the regulation of Hcy metabolism occurs via an interplay of several exogenous and endogenous factors. Nonetheless, the primary factors responsible for governing the Hcy metabolism apart from the diet are (1) molecular-genetic changes in the genes that are responsible for different biochemical processes in the cell (Sunder-Plassmann and Födinger 2003); (2) the enzymatic modulations responsible for governing the rate of reaction at each step of the metabolic pathway (Williams and Schalinske 2007); and (3) the feedback regulation mediated by various intermediary metabolites produced during the process (Jhee and Kruger 2005b; Finkelstein 2007).

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Genetics of Hcy Metabolism

As discussed previously, folate and vitamin B-complex play a crucial role in the metabolism of Hcy. The imbalance in Hcy homeostasis has been linked with disorders of the cardiovascular, neural, and reproductive systems of the human body (Škovierová et al. 2016). Elevated levels of the tHcy in blood plasma could give rise to a condition known as HHcy; which could result from folate and/or vitamin B12 deficiency (Scazzone et al. 2014; Levy et al. 2021). Evidence has accumulated over the decade to indicate that the genetics of a cell directly governs the Hcy metabolism along with that of folate and vitamin-B12. In particular, the MTHFR (5,10-methylene tetra hydro folate reductase), GCP2 (glutamate carboxypeptidase II), RFC1 (reduced folate carrier I), and TCN2 (transcobalamin II) genes are primarily implicated in the regulation of Hcy metabolism (Sunder-Plassmann and Födinger 2003). The MTHFR 677C>T genotype influences folate and tHcy concentrations (Födingeer et al. 2001). Recent reports have suggested polymorphic forms of the GCP2 gene (1561C>T; H475Y) and RFC1 (80G>A; R27H) to be associated with alterations in Hcy and folate metabolism (Devlin 2000; LeyvaVázquez et al. 2012). Moreover, polymorphisms present on the TCN2 gene (776C>G; P259R) are also implicated in the Hcy metabolism (Namour et al. 2001). The TCN2 gene is responsible for regulating plasma concentrations of transcobalamin and thereby affects the cellular availability of vitamin B12, thus, governing the Hcy metabolism indirectly (Stanisławska-Sachadyn et al. 2010). This section of the chapter will briefly focus on the polymorphisms of MTHFR, GPC2, RFC1, and TCN2 genes and how that may affect the Hcy and folate metabolism.

1.3.1.1 MTHFR: The 5,10-Methylene Tetra Hydro Folate Reductase Gene The 5,10-methylenetetrahydrofolate reductase gene (MTHFR) in humans is present on the short arm of chromosome 1 at the locus 36.3 (i.e., 1p36.3). The product of this gene is a 656 amino acid long protein that acts as a key enzyme in the folate cycle. It catalyzes the reduction reaction of 5,10methylenetetrahydrofolate to 5-methylenetetrahydrofolate, which is the sole source of 5-methylenetetrahydrofolate (the biologically active form of folate) in a cell (Botto and Yang 2000). The MTHFR gene can exhibit three polymorphic forms located at nucleotide positions 677, 1298, and 1317 (Moll and Varga 2015). The polymorphism at nucleotide position 677C>T occurs at the folate binding site (exon 4) of the MTHFR gene and results in the conversion of alanine to valine (A222V) (Rozen 1997; Frosst et al. 1995). The MTHFR1298A>C occurs in exon 7 within the region of the presumptive regulatory domain. It converts the glutamic acid residue into an alanine (E429A) (Viel et al. 1997; van der Put et al. 1998; Weisberg et al. 1998). The exon 7 of the MTHFR gene also hosts the third polymorph, i.e., MTHFR 1317T>C, resulting in a silent mutation (Allen et al. 2007). The enzyme activity is reported to be reduced to 45%, 68%, and 42%, respectively, due to MTHFR 677T>C, MTHFR 1298A>C, along with heterozygosity for 1298A>C and 677C>T (Moll and Varga 2015).

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In erythrocytes, the mutation 677T>C in the MTHFR gene is linked with the accumulation of formylated tetrahydrofolate polyglutamates and decreased formation of 5-methylenetetrahydrofolate (Botto and Yang 2000). Furthermore, the mutation is also associated with higher plasma levels of tHcy in people who are homozygous for the mutant form of the allele (Botto and Yang 2000). According to demographic studies, this effect is universal irrespective of the person’s geographical location (Castro et al. 2003). This mutation shows a significant impact on the tHcy plasma concentration in patients suffering from renal failure (Födinger et al. 2001). However, the mutant 1298A>C alone cannot influence tHcy or folate levels (Sunder-Plassmann and Födinger 2003). On the contrary, heterozygosity for 677T and 1298C alleles could be responsible for decreased plasma folate and increased tHcy concentration (Födinger et al. 2000). The double mutant form consisting of 677T>C and 1298A>C heterozygote is strongly implicated in renal failure and several other diseases (Födinger et al. 2000). The MTHFR 677C>T/ 1298A>C genotype can also modulate the efficiency of oral or intravenous folic- or folinic-acid therapy (Födinger et al. 2000). Additionally, the MTHFR 677C>T also interferes with the effect of drugs that influence the folate status of the cells (Girelli et al. 2003). A recent study demonstrated the effect of vitamin B2 availability to regulate the plasma concentrations of tHcy in renal failure patients, which might indicate epigenetic regulation of tHcy levels through gene-nutrient interaction (McNulty et al. 2006).

1.3.1.2 RFC1: The Reduced Folate Carrier 1 Gene In humans, the reduced folate carrier I (RFC1) gene is located on the long arm of the 21st chromosome at locus 22.3 (i.e., 21q22.3) (Moscow et al. 1995). The gene product, known as human folate transporter (FOLT), is a member of the SLC19A family of transporters and is composed of 591 amino acid residues. The FOLT is crucial in embryo development due to its possible role in facilitating folate transport across the placenta (Gelineau-van Waes et al. 2008). Additionally, in murine models, it has been detected to mediate intestinal folate transport. The RFC1 gene is reported to display one polymorphic form: RFC1 80G>A. The RFC1 80G>A mutation occurs at the nucleotide position 80 in exon 2 and converts arginine into a histidine residue (R27H) (Yee et al. 2010). The mutant form does not influence plasma folate or red blood cell (RBCs) folate concentrations. However, when present as RFC1 80GG along with MTHFR 677TT, significantly lower levels of RBC folate and higher tHcy were observed (Chango et al. 2000). Another study reported a decrease in the frequency of RFC1 80A allele in the children affected by neural tube defect and their parents (De Marco et al. 2001). On the contrary, another group observed no positive correlation between the two groups (Vehaskari et al. 2001). 1.3.1.3 GCP2: The Glutamate Carboxypeptidase 2 Gene The human glutamate carboxypeptidase II gene (GPC2) is located on the short arm of the 11th chromosome at 11.2 loci (i.e., 11p11.2). The product of this gene is a 750 amino acid long protein and is known as folylpoly-γ-glutamate

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carboxypeptidase (FGCP) (Devlin 2000). It is an exopeptidase attached to the apical brush border membrane with folate hydrolase and N-acetylated α-linked acidic dipeptidase activity (Winkelmayer et al. 2003). Before their absorption, it catalyzes the hydrolysis of terminal glutamate residues of dietary folylpoly-γ-glutamates. Thereafter, the folate transporter facilitates the movement of monoglutamyl folate derivatives through the membrane. Thus, FGCP is probably responsible for regulating the availability of dietary folates (Winkelmayer et al. 2003). The polymorphism reported in GCP2, 1561C>T occurs in the exon 13 on the presumptive catalytic domain of the enzyme and results in ~53% reduction of enzyme activity (Nithya et al. 2019). The mutant form is linked with low serum folate levels and higher serum tHcy concentrations, whereas the tHcy and folate levels in RBCs remain unaffected by the mutation (Winkelmayer et al. 2003). Moreover, no correlation was observed between the mutant form of GCP2 and MTHFR genes in regulating tHcy and folate levels (Winkelmayer et al. 2003). The GPC2 1561C>T mutation was shown to be associated with increased RBC and plasma folate levels in patients suffering from vascular diseases, whereas vitamin B12 levels remain unaffected. However, the study failed to observe any correlation between the mutation and cardiovascular diseases or plasma tHcy (Sunder-Plassmann et al. 2000).

1.3.1.4 TCN2: The Transcobalamin 2 Gene The location of transcobalamin II gene has been mapped to the short arm of the 22nd chromosome at 11.2 loci (i.e., 22q11.2-qter). The gene product known as transcobalamin II (TC2) is a 409 amino acid long protein that acts as plasma globulin. It is one of the three B12 binding human proteins (Hall and Finkler 1966; Quadros et al. 2009). The holo-TC2 (apo TC2 + vitamin B12) is promptly usurped from plasma through receptor-mediated endocytosis. In humans, the holoenzyme, Cobalamin (Cbl, vitamin B12) catalyzes at least two of the reactions mediated by Cbl-dependent enzymes: L-methylmalonic-CoA mutase and MS. The L-methylmalonic-CoA mutase catalyzes the conversion of L-methylmalonyl-CoA to succinyl-CoA with the help of adenosyl-Cbl. While MS converts 5-CH3-tetrahydrofolate and Hcy to tetrahydrofolate and methionine with the help of CH3-Cbl, thus acting as an important participant in one-carbon metabolism (Wuerges et al. 2006). Deficiency of vitamin-B12 can lead to Hcy and/or methylmalonic acid accumulation in the plasma (Jarquin Campos et al. 2020). Therefore, holo-TC2 levels are sensitive and can act as an early indicator of vitamin-B12 deficiency since they depict biologically available vitamin-B12 (Jarquin Campos et al. 2020). The presence of several polymorphic forms of TCN2 has been described. One of the mutants, 776G>C P259R, is responsible for regulating the cellular availability of vitamin-B12 and Hcy metabolism by modulating TC2 plasma concentrations (McCaddon et al. 2001). Furthermore, homozygosity for GG genotype could cause an elevation in blood apo-TC2 concentrations compared to in heterozygotes or individuals with wild-type alleles.

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Higher tHcy was observed in individuals with heterozygous TCN2 compared to the homozygous genotypes. On the contrary, plasma folate, B12, and tHcy levels were unaffected in healthy individuals and dementia patients (Miller et al. 2002; Cascalheira et al. 2015). However, TCN2 776 C>G mutation was associated with increased levels of methylmalonic acid in plasma and decreased holo-TC2 levels, whereas plasma tHcy and vitamin B12 levels remain unaffected (Jarquin Campos et al. 2020). A recent study explored the link between five polymorphic forms of TCN2 gene with holo-TC2, total TC2, and RBC vitamin B12 levels (Afman et al. 2002). The investigation revealed that polymorphisms in the TCN2 gene do not affect the tHcy levels, nor were they linked with an increased risk of neural tube defect. However, the mutation 776C>G resulted in elevated concentration of tHcy and decreased levels of holo-TC2 and total TC2 concentrations. However, vitamin B12 levels remained unaffected. Yet another study comparing the genotype distribution of TCN2 776 C>G revealed no distinction between cardiovascular disease patients and healthy individuals (Lievers et al. 2002). In individuals with the highest quartile levels of vitamin B12 distribution (>299 pmol/L) and TCN 776C>G mutation, tHcy concentration was lower. Hence, individuals with GG genotype would especially benefit from high vitamin B12 with respect to the tHcy concentration, indicating epigenetic interactions between gene and environment. In the case of dialysis patients, no effect of TCN 776C>G mutation was observed on tHcy, B12, and holo-TC2 plasma concentrations (Sunder-Plassmann and Födinger 2003).

1.3.2

Enzymatic Regulation of Hcy Metabolism

The Hcy metabolism is intricately linked with folate and the transfer of the methyl group. The whole network is greatly affected by the enzymes regulating the process at each step. S-adenosyl-L-methionine (SAM), the principal intermediate formed in Hcy metabolism, is a universal methyl group donor and thereby acts as a substrate for many methyltransferases. However, three major methyltransferases that utilize SAM as a substrate and are a part of Hcy metabolism include GAMT (guanidinoacetate methyltransferase), PEMT (phosphatidylethanolamine N-methyltransferase), and GNMT (glycine N-methyltransferase). Out of these three, the largest consumers of SAM are GAMT and PEMT. Historically, GAMT is known to consume about ~70% of methyl groups derived from SAM for the production of creatine. However, a recent study suggested that PEMT might act as a primary consumer of SAM-derived methyl groups for phosphatidylcholine (PC) production and impact Hcy levels the most (Stead et al. 2006).

1.3.2.1 GAMT Guanidinoacetate methyltransferase, often abbreviated as GAMT, is a hepatic enzyme responsible for the consumption of guanidinoacetate, a compound produced in the kidney by arginine and guanine metabolism. GAMT catalyzes the conversion of guanidinoacetate to creatine with the help of SAM and produces SAH (S-adenosine-L-Hcy) as a byproduct; therefore, it plays a significant role in maintaining the

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homeostatic concentrations of Hcy. Creatine biosynthesis in the liver by utilizing SAM-derived methyl group is a major source of muscle creatine phosphate. Therefore, the reaction also serves as a principal source for the intracellular Hcy in the liver. A recent study done by Stead et al. demonstrated that adequate dietary supply of creatine mitigates the need of hepatic creatine biosynthesis and thereby decreases the Hcy production. Furthermore, dietary provision of guanidinoacetate elevated the hepatic levels of Hcy, probably due to the metabolic need of a liver to methylate it to creatine. Therefore, levels of GAMT, its substrate, and its product are important in regulating the levels of Hcy.

1.3.2.2 PEMT Phosphatidylethanolamine N-methyltransferase, which is commonly abbreviated as PEMT, catalyzes the production of PC and Hcy. In particular, the series of reactions utilizes 3 SAM-derived methyl groups to convert phosphatidylethanolamine to produce PC and 3 molecules of Hcy. The reaction is an essential source of PC production, a molecule that is crucial for cell signaling and the synthesis of membrane, lipoproteins, and bile. Studies suggest that one-third of the demand for PC in a cell is accomplished through its biosynthesis from the SAM-dependent PEMT pathway; whereas the CDP-choline pathway fulfills the remaining need. Recent evidence suggests that PEMT plays a much more significant role in maintaining the Hcy homeostasis, than previously presumed. A murine study was performed to investigate the role of hepatic PEMT regulation on plasma Hcy using a liver-specific CTP knockout mouse revealed that in the absence of CDP-choline pathway, methylation of phosphatidylethanolamine by PEMT was increased 100% (Jacobs et al. 2005). The positive correlation of PEMT activity with Hcy levels was also observed in cell culture models. However, some studies do not agree with the notion and state otherwise. For instance, PEMT expression was elevated upon glucocorticoid administration and in streptozotocin (STZ)-induced type I diabetes model (Geelen et al. 1979; Hartz et al. 2006). This increase in Hcy levels is supposed to have an immense effect on Hcy levels; however, it was observed that Hcy catabolism was enhanced in the early stages of type I diabetes resulting in the condition of HHcy. Therefore, the PEMT dependence of Hcy concentrations needs to be explored further to determine the type of correlation between the two. However, it can be said with certainty that PEMT concentrations do affect the tHcy levels. 1.3.2.3 GNMT Guanidine methyltransferase, often abbreviated as GNMT, is responsible for maintaining the SAM: SAH ratio (i.e., methylation capacity of a cell). It is the principal regulator of methyl group supply and consumption methyltransferases that SAH inhibitsare inhibited by SAH, GNMT most abundant hepatic methyltransferases. Regulation of GNMT activity and therefore methylation capacity of a cell comes under the authority of allosteric mechanisms. Unlike most of the methyltransferases that SAH inhibits, GNMT is less sensitive to inhibition by SAH (Kerr 1972; Wagner et al. 1985). It is, in fact, allosterically inhibited by 5-CH3-THF. This regulatory system ensures that when methyl group availability is compromised,

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they are conserved for biologically important SAM-dependent transmethylation reactions. Whereas, if the methyl group supply is in excess, they are disposed of conveniently. Expression of GNMT is primarily reported in a specific tissue of the liver, pancreas, kidney, and intestine (Yeo and Wagner 1994). Due to its importance in regulating the Hcy concentrations, various studies have tried to address the effect of hormonal factors regulating the expression and functions of GNMT. Similar to PEMT, the GNMT levels were observed to be elevated upon administration of glucocorticoids in STZ-induced diabetic rat (type I) and the Zucker diabetic fatty (ZDF) rat (type II) (Rowling and Schalinske 2003; Nieman et al. 2004). Furthermore, insulin treatment has been shown to prevent GNMT upregulation in these diabetic rats (Nieman and Schalinske 2011). GNMT expression has also been affected by growth hormone (GH) levels (Brown-Borg et al. 2005). In a study performed on Ames dwarf mouse, it was observed that GNMT activity and mRNA levels were elevated (Aida et al. 1997). The Ames mouse is characterized by diminished production of GH due to a lack of differentiation of portions of the pituitary gland responsible for its production. However, exogenous supply of GH to 3- or 12-month-old Ames dwarf mice lowered the GNMT activity. In rats the status of thyroid modulates the MTHFR activity which in turn could allosterically alter the GNMT activity through 5-CH3-THF (Finkelstein et al. 1978). A study done by Schalinske et al. corroborates the aforementioned notion by demonstrating the post-translational regulation of GNMT activity in the hyperthyroid state (Tanghe et al. 2004). It was observed that triiodothyronine alleviates the retinoic acid-induced increase in GNMT activity, without affecting its abundance. Further studies utilizing knock-out mice models have elaborated more on methyl group homeostasis and GNMT functions. A research group led by Luka et al. recently tried to explain the correlation between GNMT and Hcy levels, and published their study done on GNMT-deficient mice (Luka et al. 2006). They reported that abrogation of hepatic GNMT supply in homozygous mice led to ~100-fold increase in SAM: SAH ratio. However, in heterozygous or wild-type mice, the levels of hepatic methionine, SAM, and SAH were found to be unaffected. The study advocates previous findings of Wagner et al. that GNMT is partly responsible for maintaining a cell’s transmethylation potential (Wagner et al. 1985). The role of GNMT in regulating methyl group homeostasis is further asserted by the report, which states that GNMT activity and expression are, in fact, elevated in the brain of PEMT-deficient mice. On the contrary, the activity of the rest of the methyltransferases examined was diminished. In wild-type mice, the levels of GNMT were observed to be very low; however, as PEMT is a major utilizer of methyl groups, it was supposed that GNMT was probably upregulated to compensate its absence, and maintain the balance of methyl group availability in pemt
/
mice.

1.3.2.4 BHMT Hcy and methyl group metabolism is also proposed to be affected by folateindependent Hcy remethylation by BHMT (betaine Hcy methyltransferase). As discussed previously, BHMT utilizes the betaine-derived methyl group for

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remethylation of Hcy to methionine. The methionine molecule thus generated could be reactivated to SAM. Therefore, BHMT seems to be playing a regulatory role in maintaining the cellular balance of Hcy. The hepatic expression and activity of BHMT and its transcripts have been observed to increase in diabetic rats and upon administration of glucocorticoids to rat hepatoma cells (Nieman et al. 2004; Tanghe et al. 2004; Wijekoon et al. 2005). As mentioned earlier, under diabetic conditions, Hcy synthesis is presumed to be elevated owing to the upregulation of multiple transmethylases (i.e., GNMT, PEMT). However, increased BHMT activity plays a role in increasing remethylation of Hcy, leading to lower plasma Hcy levels (Ratnam et al. 2006). Elevated BHMT levels in diabetic conditions could be obviated by insulin administration, with the simultaneous restoration of SAM: SAH ratio (Ratnam et al. 2006). This reversal by insulin seems result from decreased availability and de novo transcription of BHMT mRNA. The activity of BHMT was also upregulated in the Ames dwarf mouse, which, like diabetic rats, exhibit enhanced transmethylation and diminished Hcy level (Uthus and Brown-Borg 2006). BHMT activity was also elevated in CTα knockout mice; however, it wasn’t enough to bring plasma Hcy levels back to normal (Jacobs et al. 2005). Though it was unable to normalize the plasma Hcy levels, a group led by Collinsova et al. claimed that BHMT might play a key role in modulating plasma Hcy. They observed elevated plasma Hcy concentrations in mice injected with 6 injections of a specific inhibitor of BHMT, i.e., S-(δ-carboxybutyl)-DL-Hcy. The authors noted that decreased levels of cystathionine beta-synthase (CBS) conduce to the increase, but most strikingly, significant elevation was observed in BHMT protein expression. Decreased levels of SAH might accompany this, thus alleviating potential inhibition of BHMT transcription mediated by SAM (Collinsova et al. 2006). Further experiments revealed that administration of a single injection of S-(δ-carboxybutyl)-DL-Hcy to mice caused a decrease in BHMT activity by 90% and a significant rise in Hcy levels. However, the activity of other enzymes involved in Hcy metabolism remained unaffected.

1.3.2.5 MS and MTHFR The effect of hormonal homeostasis on folate-dependent remethylation is ambiguous to a certain extent. A study done by Wijekoon et al. on 11-week-old ZDF rats demonstrated no effect on the hepatic levels of MS or MTHFR activity (Wijekoon et al. 2005). Interestingly, in prediabetic conditions, insulin-resistant stage at 5 weeks of age, transient increase in MTHFR, and decreased MS activity were observed. Contrastingly, in STZ-induced diabetic mice, the activity of MS was observed to be downregulated (Nieman et al. 2004). However, some reports suggest these effects might be tissue-specific. For instance, a report by Jacobs et al. states that activity of both MS and MTHFR remained unaffected in the liver, whereas it was found to be decreased in the kidney of STZ-induced diabetic rats (Jacobs et al. 1998). Insulin administration restored the renal MS activity; however, renal MTHFR activity remained low. The above-mentioned data suggest that MS-mediated remethylation is not a primary element maintaining the Hcy balance under diabetic conditions. However, diminished availability of methyl groups derived from folatedependent 1-carbon pools seems to be a characteristic of the diabetic state.

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1.3.2.6 CBS and g-cystathionase Hcy can be irreversibly catabolized through transsulfuration reaction, which is catalyzed by CBS and γ-cystathionase. A study done by Wang et al. revealed the capability of CBS to regulate serum Hcy levels (Wang et al. 2004). The group used a transgenic mice model in which the human CBS gene was controlled by a metallothionein promoter. When these mice were administered with zinc supplemented water, their renal and hepatic CBS activity was observed to be increased, accompanied by a decrease in the serum levels of Hcy. Furthermore, the above-mentioned strategy to enhance CBS activity and loweri Hcy was effective even upon administring high methionine-low folate diet in mice. Based on these observations, it can be concluded that elevated activity of CBS and/or γ-cystathionase plays a vital role in determining the plasma concentrations of Hcy. The activity of CBS γ-cystathionase was also observed to be elevated in the liver of Ames dwarf mouse (Uthus 2003). The effect of increased levels of these enzymes has further been validated in Ames dwarf mice, showing increased traffic through transsulfuration reaction in the kidney, liver, and brain along with low plasma Hcy levels (Uthus and Brown-Borg 2006). The activity of CBS and γ-cystathionase was also observed to be increased in the liver of ZDF and STZ-diabetic rats (Nieman et al. 2004; Wijekoon et al. 2005). In STZ-diabetic rats, treatment with insulin averted the changes in the plasma levels and activity of Hcy (Jacobs et al. 1998). HHcy observed in type I diabetes patients is thought to result from transsulfuration reaction at least in part (Abu-Lebdeh et al. 2006). However, plasma levels of Hcy seem to be directly related to the severity of renal dysfunction in both types of diabetes (type I and II). Recent kinetic studies on patients who have type II diabetes with nephropathy provided a link between the condition of HHcy and decreased clearance of Hcy (Tessari et al. 2005). Therefore, the difference in the Hcy disposal mechanism between diabetes with and without renal dysfunction might explain the transition from hypo- to HHcy in diabetics. In summary, methyl group, folate, and Hcy metabolism are involved in a wide variety of metabolic reactions. These reactions intricately connect numerous biological compounds, epigenetics of the system, regulation of gene expression, and maintenance of redox potential of the system. Given the association of homeostatic disturbances in these metabolic pathways with varied pathological conditions, understanding the factors, such as enzymes, involved regulating of these pathways is crucial for strategizing to prevent or minimize metabolic dysfunctions. Thus far, studies have explored the impact of individual enzymes using specific inhibitors and knockout mice models; further in vivo studies and network analysis by mathematical modeling may provide a clearer picture (Reed et al. 2006).

1.3.3

Metabolic Regulation of Hcy Metabolism

Framework for metabolic regulation of a pathway is established primarily by the factors such as alterations in enzymatic content in tissue, relative affinities of enzymes to their substrate, and the variation in substrate concentration. However,

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additional immediate adaptability is provided by modulations in the metabolic effectors of the pathway. In case of Hcy metabolism, three compounds, AdoMet, AdoHcy, and methylTHF could be considered the principal metabolic effectors (Finkelstein 1998).

1.3.3.1 AdoMet The hepatic levels of AdoMet are a direct indicator of the bioavailability of methionine (Mato et al. 2002). AdoMet is reported to be responsible for inhibiting MTHFR, which could result in decreased concentration of methylTHF and dysregulated MFMT reaction (Yamada et al. 2005). Later, it was found that AdoMet could activate cystathionine and inactivate BHMT (Janošík et al. 2001). Based on these observations, AdoMet was described to be the metabolic “switch,” which facilitates transsulfuration upon high levels and puts a limit to the re-synthesis of methionine. This regulatory role could be supported by the positive effector activity of AdoMet toward MAT III and glycine methyltransferases, which are enzymes responsible for the catabolism of excess methionine. 1.3.3.2 AdoHcy AdoHcy is likely to play a much more significant role in extrahepatic tissue since the expression of AdoMet is tissue-specific to a large extent (Torres et al. 2000). AdoHcy levels are a direct indicator of Hcy concentration in tissue, because of the thermodynamics of the adenosyl Hcy reaction. AdoHcy plays a significant role in several metabolic pathways. It acts as a potent inhibitor of most of the AdoMetdependent trans methylases in addition to both Hcy methyltransferases: MFMT and BHMT (Szegedi et al. 2008; Chen et al. 2013). AdoHcy also releases the AdoMet mediated inhibition of MTHFR (Kutzbach and Stokstad 1971). However, both the molecules act agonistically in one condition: which is AdoHcy-mediated activation of cystathionine synthase, similar to AdoMet. Given such diverse metabolic effects of AdoHcy, it is difficult to conclude the net impact of elevated AdoHcy concentrations. In an in vitro study model, AdoHcy was observed to have minimal impact effect on the three Hcy utilizing enzymes until the maximum concentration, at which point synthesis of cystathionine was elevated (Finkelstein and Martin 1984). However, the model does not determine alteration in MTHFR. Therefore, the accumulation of AdoHcy is most likely to enhance Hcy availability for MFMT and transsulfuration reactions and thereby increase its consumption. 1.3.3.3 MethylTHF A study done by Wagner et al. indicated that methylTHF could also play a role in the modulation of Hcy metabolism by inhibiting glycine methyltransferase (Wagner et al. 1985). As previously mentioned, AdoMet hinders the synthesis of methylTHF (Finkelstein 1998). Hence, with the increased availability of methionine, concentrations of methylTHF tend to decrease (Brody et al. 1982). Therefore, glycine methyltransferase may act as another “switch” by being the major transmethylase associated with methionine catabolism and transsulfuration. The concentrations of AdoMet could be directly related to the availability of methionine. In turn, it would

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result in downregulation of methylTHF synthesis and facilitation of glycine methylation. The concentrations of methylTHF would act as the limiting factor for glycine methyltransferase and transsulfuration reactions. This regulatory mechanism could be used to subclassify HHcy based on the levels of methylTHF. In the case of folate and MTHFR deficiency, MFMT is impaired and decreases in the levels of methylTHF. The condition would lead to a reduction in Hcy methylation and with a possible increase in AdoHcy and Hcy synthesis. Contrarily, in the case of vitamin B12 deficiency, cobalamin metabolism disorders, and toxicity due to nitrous oxide result in decreased MFMT along with increased methylTHF. The condition is supposed to cause downregulation of methionine resynthesis and with a probable reduction in the Hcy synthesis due to limitation in glycine methyltransferase. The clinical implications of these conditions are yet to be defined.

1.4

Role of Folic Acid in Hcy Metabolism

Folate is a member of the vitamin B-complex family along with thiamine and niacin. These are small, water-soluble molecules that are easily absorbed from the diet (exception: vitamin B) (Kennedy 2016). These B vitamins generally facilitate the metabolic functions of specific enzymes by acting as their cofactor. In particular, folate in its active form, tetrahydrofolate, helps the enzyme to pass on the “onecarbon groups” such as methyl. The folate generally exists in its reduced “tetrahydrofolate” form surrounded by various additional carbon groups attached to it (Zheng and Cantley 2019). The attached group varies according to the requirement of the specific pathway it is intended for. For example, purine biosynthesis for DNA requires two of the carbon atoms provided in the form of 10-formyltetrahydrofolate, to be inserted in the purine ring (C2 and C8) (Baggott and Tamura 2015). However, pyrimidine biosynthesis requires the supply of carbon groups in the form of 5,10-methylenetetrahydrofolate (Huennekens 1969). These carbon groups, including formyl, methyl, methylene, etc., are collectively called the “one-carbon group” and are supplied by the enzymes supplemented by folate as a cofactor, in the form of tetrahydrofolate. After the transfer of a “one-carbon group” to the pyrimidine/purine biosynthesis and to methylation cycles, the tetrahydrofolate molecule is regenerated. During pyrimidine biosynthesis, dihydrofolate is formed, which is reduced to form tetrahydrofolate by dihydrofolate reductase enzyme. The regenerated tetrahydrofolate molecules are again available to act as a porter for “onecarbon groups” by accepting them from either amino acid serine or formate.

1.4.1

Folate Cycles

5-methylTHF acts as an intracellular methyl donor for Hcy metabolism (Blom et al. 2006). Serine hydroxymethyltransferase (SHMT) can then directly convert the THF formed to 5,10-methyleneTHF. SHMT is vitamin B6-dependent enzyme and uses serine as a source of “one-carbon group”. Human SHMT exists in various isoforms

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in cytosol and mitochondria (Garrow et al. 1993). Methylenetetrahydrofolate dehydrogenase (MTHFD1) catalyzes the conversion of THF into 5,10-methyleneTHF, via 10-formylTHF and 5,10-methenylTHF. The enzyme MTHFD1 has the trifunctional activity of methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase (Hum et al. 1988). The “one-carbon group” donated by 10-formylTHF can be utilized in purine biosynthesis and the one donated by 5,10methylenetetrahydrofolate can be utilized as a cofactor in the conversion of dUMP to dTMP (Ducker and Rabinowitz 2017). TYMS (thymidylate synthase) catalyzes the conversion of dUMP to dTMP, to produce DHF (dihydrofolate). It is further reduced back to THF by the action of DHFR (dihydrofolate reductase). Additionally, 5,10-methyleneTHF can be reduced to 5-methylTHF by MTHFR, i.e., riboflavin (vitamin B2)-dependent enzyme that competes for 10-methyleneTHF with TYMS. The function of the MTHFR enzyme is highly crucial for regulating 5-methylTHF availability for Hcy remethylation.

1.4.2

Folate Uptake and Transport

The prime source of essential and water-soluble B vitamin folate for humans is diet, especially consisting of fruits and vegetables. Chemically, folate and folic acid differ just by one proton. However, the most stable form of these B vitamins is commonly referred to as folic acid (Blom et al. 2006). For ease of transportation, dietary folates, which are found as polyglutamate, are to be hydrolyzed to mono glutamates. This hydrolysis reaction in the gut is catalyzed by FGCP (folylpoly-γ-glutamate carboxypeptidase), which is anchored to intestinal apical brush border epithelium and is encoded by GCPII (glutamate carboxypeptidase) gene (Halsted et al. 1998). Subsequent absorption of mono glutamylated folates takes place in the duodenum and upper jejunum through PCFT1, a high-affinity proton-coupled folate receptor (Qiu et al. 2006). Predominantly, folate circulating in the plasma exists as 5-MethylTHF and can be transported through a carrier or receptor-mediated transport systems. A glycosylphosphatidylinositol-linked glycoprotein known as folate receptor α (FR-α) has a high affinity for the monoglutamate 5-methylTHF. However, the expression of FR-α is limited to certain epithelial cells of proximal convoluted tubule in the kidney, choroid plexus in the brain and placenta (Xin et al. 1992; Barber et al. 1999; Kamen 2004). The rest of the folate receptors, β and γ, show comparatively low affinity toward 5-methylTHF. Ubiquitously expressed RFCs (reduced folate carriers) execute the carrier-mediated transport of the 5-methylTHF. However, the affinity of RFCs for 5-methylTHF is significantly lowered as compared to FR-α. Further research needs to be done to ascertain the role of PCFT1 in cellular uptake and transport of 5-methylTHF.

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Disorders Associated with Altered Hcy Metabolism

The elevated level of the Hcy is a risk factor for several pathologies such as cardiovascular diseases, autoimmune responses, neurodegenerative diseases, neural tube defects, and reproductive health (Azzini et al. 2020). Since, folate moiety is involved in a different biochemical process, particularly in Hcy metabolism. Folate deficiency initiates mild hyperhomocysteinemia and results in various pathologies (Maron and Loscalzo 2009). Folate as well as Hcy has been fabricated in several developmental abnormalities, pregnancy complications, and ageing-related diseases (Forges et al. 2007). Additionally, the molecular mechanisms of Hcy instigated cellular dysfunction to involve high inflammatory cytokines responses, altered level of NO, alteration in oxidative stress, apoptosis activation, and defective methylation (Forges et al. 2007). Most commonly reported pathologies with Hcy metabolism are explained in more detail in the subsequent sections (Fig. 1.2).

1.5.1

Effects on Reproductive Health

1.5.1.1 Effect on Male Fertility Folate has a key effect on the reproductive health of both sexes (male and female). Several genetic polymorphisms of folate metabolic genes have a profound effect on the pervasiveness of reproductive problems (Gupta et al. 2013; Gong et al. 2015). For instance; the 677 genotypes of MTHFR showed prevalence among infertile male patients. Hereby, the mutant TT homozygotes prevalence was profoundly higher among patients relative to controls (18.8% vs. 9.5%) (Ebisch et al. 2003). Whereas, another study reported that there was no difference in the prevalence of MTHFR polymorphism in 77 subfertile patients (with moderate oligospermia) relative to the 113 healthy fertile men. The homozygosity in the patients and control was 9.1% and 13.3% respectively (Ebisch et al. 2003). Yet, the prevalence of homozygosity was even higher in Italian men also (20.4% and 27.6%), but it was not significantly different in fertile and infertile patients (Stuppia et al. 2003). In contrast, 151 Indian patients were having severe male infertility (severe oligozoospermia or with azoospermia), had significantly more homozygous carriers (4% vs. 0%) along with heterozygous carriers (26.5% vs. 18.5%) relative to the fertile control (200 control) (Singh et al. 2005). Park et al. study demonstrated that the MTHFR gene homozygous (T/T) C677T polymorphism was present at a substantially high significance with unexplained infertile men (with normal karyotype). Whereas, they did not report any statistical significance of MTHFR gene polymorphism site A1298C variation in infertile males (Park et al. 2005). Reports also suggested that the MTHFR polymorphism-related infertility could be fended off by improving the nutritional value in these populations (Forges et al. 2007). Therapeutic trials investigation tried to observe the effect of folate on the fertility of males, e.g., 10 mg folic acid per day for 30 days to 40 patients (normozoospermic or oligozoospermia). Consequently, they did not observe any association among fluid folate concentrations (serum as well as seminal) and total sperm count and other sperm

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Fig. 1.2 The factors affecting homocysteine metabolism and its effect on human health. Various endogenous and exogenous factors manipulate the homocysteine (Hcy) homeostasis which results in imbalanced Hcy metabolism. The dysregulated Hcy metabolism could have varied effects on general human health ranging from disorders of skeletal, digestive, circulatory, nervous, sensory, reproductive, excretory, and endocrine systems listed in figure

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parameters (Landau et al. 1978). On the contrary, another study reported significant changes in (with excessive round cell count in their ejaculate) with 15 mg of folinic acid for 3 months. Authors have observed significant modification with the proceeding of the study, particularly in sperm density (from 15.02  1.95  106/mL to 22.58  2.61  106/mL), motility (from 17.7% to 27.8%), and reduction in the count of round cells (from 9.67  0.65  106/mL to 6.36  0.49  106/mL). Among those 65 patients treated with folinic acid (all female partners were evaluated with normal fertility), 24 couples have conceived within 6 months and 17 women delivered (Bentivoglio et al. 1993). Hence, the supplementation of folate had an advantageous effect on spermatogenesis, possibly by enhancing the cohesion among cells within the seminiferous epithelium, thus preventing the abnormal release of immature germ cells into the lumen (Forges et al. 2007).

1.5.1.2 Effect of Female Fertility The serum Hcy concentration is lower in females compared to males, even though in females it is lower in premenopausal than in post menopausal women (Xu et al. 2020). The Hcy concentration was measured in follicular fluid of 40 patients (20 patients were on folic acid supplementation) undergoing IVF where authors have observed that patients with folate supplements had lower Hcy levels in the follicular fluids (Szymański and Kazdepka-Ziemińska 2003). Hereby, they observed a negative correlation between follicular fluid homocysteine concentration and the degree of maturity of the retrieved oocytes (Szymański and Kazdepka-Ziemińska 2003). For investigating the impact of Hcy metabolism on the ovarian response to ovulation induction, a total of 105 IVF patients were involved in a prospective study and MTHFR gene polymorphism (677 sites) was determined. Out of 269 cycles of IVF cycles, 245 led to oocyte retrieval. After evaluation of these cycles, it was observed that MTHFR 677 CT or TT genotype patients required significantly higher doses of FSH for induction of ovulation compared to homozygous wild-type patients (Thaler et al. 2006). Likewise, the patients with mutated T allele (including both homozygous and heterozygous) have significantly lower serum estradiol concentration and the number of oocytes collected. The MTHFR C677T polymorphism had a negative impact on follicular growth and its maturation as well. The significantly lower prevalence of the MTHFR 677T allele in women who spontaneously conceived dichorionic twins relative to spontaneous singleton pregnancies (Hasbargen et al. 2000). In 156 singleton pregnancies, the homozygous frequency of wild-type (48.7%), heterozygous (41.7%), and homozygous mutation carriers (9.6%) whereas in 40 dichorionic twin pregnancies the frequencies were 72.5, 22.5, and 5.0%, respectively. Hence, a reduced MTHFR activity subsequently reduced SAM availability and Hcy level increase which lead to HHcy. HHcy could inhibit poly ovulation, which is a condition for spontaneous dichorionic pregnancies (Hall 2003). In addition, the homozygous for the MTHFR gene 677C> T mutation reduced Hcy concentration after supplementation of folic acid (0.5 mg) in females with a history of unexplained frequent miscarriages (Nelen et al. 1998). Therefore, these studies demonstrate the importance of folate nutritional supplementation, specifically in infertile patients (with MTHFR polymorphisms).

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Effects on Neural System

Epidemiological studies have continuously hinted toward a plausible connection of HHcy with CNS neurodegenerative disorders (Kamat et al. 2015b; Moretti and Caruso 2019). Several in vitro and in vivo observations evaluated the HHcy potentially triggering damage in the neuronal system via generating oxidative stress, DNA damage (ss or ds), and activation of pro-apoptotic factors (Kruman et al. 2000; Méndez-Armenta et al. 2014). The brain tissue maintains a low level of Hcy through well-organized recycling of methionine by B12-dependent methionine synthase, catabolism into cystathionine, or by export to external circulation (Moretti and Caruso 2019). The neurotoxic effect of Hcy has been observed so far, patients with neurodegenerative disorders have Hcy concentration 100 μM which is much higher than homocysteine mean plasma levels (20 μM) (Currò et al. 2014). Hcy (~20 μM) prolonged exposure (5 days) to neuronal-like differentiated SH-SY5Y cells which showed 35% loss of cell viability and a fourfold increase in reactive oxygen species levels (Currò et al. 2014). Hcy exposure has a profound effect on DNA damage indexes which involves up-regulation of Bax, Bcl-2 (not caspase-3), p21 (p53 independent), p16 levels, and several cyclins (Currò et al. 2014). HHcy level in brain cells induces oxidative and genotoxic stress involving an early induction of cyclins which is later repressed by G1-S check-point regulators. Furthermore, Hcy and its related compounds possibly have a neurotransmitter role (excitatory agonist) on the N-methyl-D-aspartate receptor (NMDA) subtype of glutamate receptors (Shaw 1993). The elevated Hcy levels in long terms are associated with changes in mental health like cognitive impairment, depression, dementia, Alzheimer’s and Parkinson’s diseases (Rozycka et al. 2014). Patients with Alzheimer-type dementia and other dementing illnesses commonly had low serum levels of folate levels as well as vitamin B12, and this association grew stronger when HHcy was used as a substitute for vitamin B12 deficiency (Werder 2010).

1.5.3

Effects on Cardiovascular System

The level of Hcy is significantly (p < 0.001) higher in coronary artery disease (CAD) patients relative to control individuals. The serum homocysteine levels correlated well with the severity of CAD (Shenoy et al. 2014). Hcy acts as an individualistic risk factor for atherosclerosis (inflammatory injury in the arterial intima along with increased permeability to plasma) that leads to cardiovascular diseases (failure of heart, myocardial infarction, and stroke) (Ganguly and Alam 2015). In vascular smooth muscle cells (VSMCs), Hcy augmented NR1 subunit NMDA expression, while MK-801 (noncompetitive antagonist of NAMD) reduced homocysteineinduced CRP expression (Moshal et al. 2009). Thus, Hcy is likely to set off an inflammatory response in VSMCs by triggering the production of CRP that is attributed through the NMDAr-ROS-ERK1/2/p38-NF-κB signal pathway (Ganguly and Alam 2015). A total of 70 participants (Kasturba Hospital, Manipal University)

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showed Hcy is involved as an early atherosclerotic promoter where fasting serum homocysteine levels in CAD patients were significantly higher relative to the control ( p < 0.001) (Shenoy et al. 2014). Hcy is also shown as a trigger for VSMCs proliferation in vitro (Ganguly and Alam 2015). Hcy also alters the activity of HMG CoA reductase which subsequently increases cholesterol synthesis (a risk factor for atherosclerosis and CAD) (Hirche et al. 2006). Endothelial dysfunction mediated by Hcy could be mediated by mechanisms like oxidative stress, nuclear factor-kb (NF-kb) activation, inflammation, and inhibition of endothelial nitric oxide synthase (eNOS) (Pushpakumar et al. 2014). Furthermore, HHcy has also been shown to be associated with a higher risk of venous thrombosis (Ospina-Romero et al. 2018). Elevated Hcy level showed a connection toward platelet adhesion to endothelial cells, higher levels of prothrombotic factors such as β-thromboglobulin, tissue plasminogen activator, and factor VIIc (Ganguly and Alam 2015). The erythrocytes from healthy adults were exposed to Hcy (8, 20, 80, 200, 800 μmol/ L) for 24 h in a dose-dependent manner which elevate the phosphatidylserine exposure and subsequently the pro-coagulant activity of RBCs.

1.5.4

Effects on Immune Repertoire

There are various mechanisms through which Hcy modulates inflammatory responses that were evaluated by in vitro studies. For instance, Hcy triggers the production of inflammatory cytokine or chemokine in monocyte and endothelial cells (Wang et al. 2000; Poddar et al. 2001; Wang 2013; Ospina-Romero et al. 2018). In the aforementioned studies, HHcy mediates its effects concomitantly through reactive oxygen species generation (ROS), PPAR-γ and NFκB transcription factor activation. Indeed, HHcy is shown to aggravate colon inflammation through the enhancement in the IL-17 cytokine levels in animal models (Flannigan et al. 2014; Zhu et al. 2015). IL-17 was also observed during hypertension with abnormally activated T cell presence (Kirabo et al. 2014; Zhu et al. 2015). Nonetheless, the T cells also rely on cysteine to supply their metabolic needs (Gmunder et al. 1991). HHcy is either stimulatory or inhibitory toward activated T-cell proliferation. The inhibition of CBS/CSE enzymes inhibits cysteine transport by C-cystine transporter along with it also compromises cysteine synthesis (de novo) through the transsulfuration pathway. Thereby, it is inhibiting the proliferation of activated T cells and subsequently prevention of hypertension development. In addition, H2S also plays a vital role in regulating the T-cell immune responses by enhancing T-cell proliferation (Miller et al. 2012; Kaur et al. 2015). Vitamin B12 and folic acid deficiencies have also shown an independent connection with declined immune function, hematopoietic progenitor cell apoptosis, and the presence of leukocytes with hypomethylated DNA in the peripheral circulation (Dawson et al. 2004). Studies also found the pro-apoptotic effect of Hcy leads to the activation of the caspases and ultimately the cleavage of the key cellular proteins (PARP) and eventually triggering the typical morphological changes (Veeranki et al. 2017). In addition to the pro-apoptotic effects of Hcy, it also influences T-cell activation and

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cytokine secretion (type 1 cytokines including IL-2, IFN-, TNF-, and IL-10 but not type 2 cytokines IL-4 or IL-5) (Veeranki et al. 2017). Moreover, the abnormal methyl group metabolism and Hcy imbalance have been characterized in several autoimmune diseases such as both types 1 and 2 diabetes which advanced to HHcy and deteriorate renal function (Schalinske and Smazal 2012). In vivo studies, the commencement of HHcy results in increased re-methylation and catabolism of Hcy by the induction of BHMT and CBS genes in chemically induced type 1 diabetes and genetic-basis type 2 diabetes models. Diabetes is also characterized by the induction of specific methyltransferases (GNMT and PEMT) that should result in increased homocysteine production (Schalinske and Smazal 2012). Hereby, we highlight possible relationships between homocysteine with infertility (male and female), neurological symptoms, and cardiovascular diseases, and summarize the evidence that suggested these factors act together in increasing the risk for autoimmune diseases. Conflicts of Interest The authors declare no conflicts of interest.

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Thaler CJ, Budiman H, Ruebsamen H, Nagel D, Lohse P (2006) Effects of the common 677C>T mutation of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene on ovarian responsiveness to recombinant follicle-stimulating hormone. Am J Reprod Immunol 55(4): 251–258 Torres L, Ávila Mat A, Carretero MV, Latasa MU, Caballería J, López-Rodas G, Boukaba A, Lu SC, Franco L, Mato Jos M (2000) Liver-specific methionine adenosyltransferase MAT1A gene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications for MAT1A silencing during transformation. FASEB J 14(1):95–102 Uthus E (2003) Altered methionine metabolism in long living Ames dwarf mice. Exp Gerontol 38(5):491–498 Uthus EO, Brown-Borg HM (2006) Methionine flux to transsulfuration is enhanced in the long living Ames dwarf mouse. Mech Ageing Dev 127(5):444–450 van der Put NMJ, Gabreëls F, Stevens EMB, Smeitink JAM, Trijbels FJM, Eskes TKAB, van den Heuvel LP, Blom HJ (1998) A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet 62(5): 1044–1051 Veeranki S, Gandhapudi SK, Tyagi SC (2017) Interactions of hyperhomocysteinemia and T cell immunity in causation of hypertension. Can J Physiol Pharmacol 95(3):239–246 Vehaskari VM, Aviles DH, Manning J (2001) Prenatal programming of adult hypertension in the rat. Kidney Int 59(1):238–245 Verhoef P, de Groot LCPGM (2005) Dietary determinants of plasma homocysteine concentrations. Semin Vasc Med 5(02):110–123 Viel A, Dall’Agnese L, Simone F, Canzonieri V, Capozzi E, Visentin M, Valle R, Boiocchi M (1997) Loss of heterozygosity at the 5,10-methylenetetrahydrofolate reductase locus in human ovarian carcinomas. Br J Cancer 75(8):1105–1110 Wagner C, Briggs WT, Cook RJ (1985) Inhibition of glycine N-methyltransferase activity by folate derivatives: implications for regulation of methyl group metabolism. Biochem Biophys Res Commun 127(3):746–752 Wakabayashi K, Isogai A, Watanabe D, Fujita A, Sudo S (2013) Involvement of methionine salvage pathway genes of Saccharomyces cerevisiae in the production of precursor compounds of dimethyl trisulfide (DMTS). J Biosci Bioeng 116(4):475–479 Wang H (2013) Homocysteine induces inflammatory transcriptional signaling in monocytes. Front Biosci 18(2):685 Wang G, Siow YL, O K (2000) Homocysteine stimulates nuclear factor kappaB activity and monocyte chemoattractant protein-1 expression in vascular smooth-muscle cells: a possible role for protein kinase C. Biochem J 352(Pt 3):817–826 Wang L, Jhee K-H, Hua X, DiBello PM, Jacobsen DW, Kruger WD (2004) Modulation of cystathionine β-synthase level regulates total serum homocysteine in mice. Circ Res 94(10): 1318–1324 Weisberg I, Tran P, Christensen B, Sibani S, Rozen R (1998) A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol Genet Metab 64(3):169–172 Werder SF (2010) Cobalamin deficiency, hyperhomocysteinemia, and dementia. Neuropsychiatr Dis Treat 6:159 Wijekoon EP, Hall B, Ratnam S, Brosnan ME, Zeisel SH, Brosnan JT (2005) Homocysteine metabolism in ZDF (Type 2) diabetic rats. Diabetes 54(11):3245–3251 Williams KT, Schalinske KL (2007) New insights into the regulation of methyl group and homocysteine metabolism. J Nutr 137(2):311–314 Winkelmayer WC, Eberle C, Sunder-Plassmann G, Födinger M (2003) Effects of the glutamate carboxypeptidase II (GCP2 1561C>T) and reduced folate carrier (RFC1 80G>A) allelic variants on folate and total homocysteine levels in kidney transplant patients. Kidney Int 63(6):2280–2285

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Nutritional Determinants in Hyperhomocysteinemia Rajesh Dubey, U. P. Shahi, V. N. Mishra, D. Jain, Akanksha Mishra, Govind Prasad Dubey, and Rudra P. Ojha

Abstract

Recently, global attention is focused to establish the impact of malnutrition in individuals susceptible to develop coronary heart disease (CHD), and the pertinent remedial strategies in the prevention and management of CHD are of utmost importance in the present scenario. Cumulative research findings suggested that folic acid deficiency is a major cause of hyperhomocysteinemia-mediated inflammatory cascade intended to lead to coronary heart disease. The present study has been designed to investigate the homocysteine and inflammatory cytokines in different age and sex groups of the selected population. Further, it was also investigated that nutritional deficiency, particularly folic acid, is responsible for

R. Dubey Adesh University, Bathinda, Punjab, India U. P. Shahi Deptarment of Radiotherapy and Radiation Medicine, IMS, Banaras Hindu University, Varanasi, Uttar Pradesh, India V. N. Mishra Department of Neurology, IMS, Banaras Hindu University, Varanasi, Uttar Pradesh, India D. Jain Department of Cardiology, IMS, Banaras Hindu University, Varanasi, Uttar Pradesh, India A. Mishra Department of Education, Nehru Gram Bharati (Deemed to be University), Prayagraj, Uttar Pradesh, India G. P. Dubey (*) Kriya Sharira and Kaya Chikitsa, Banaras Hindu University, Varanasi, Uttar Pradesh, India R. P. Ojha Department of Zoology, Nehru Gram Bharati (Deemed to be University), Prayagraj, Uttar Pradesh, India # The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2022 G. P. Dubey et al. (eds.), Homocysteine Metabolism in Health and Disease, https://doi.org/10.1007/978-981-16-6867-8_2

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an elevated level of homocysteine which may cause detrimental cardiac events. The present series of studies strongly demonstrate a significant association between folic acid deficiency and hyperhomocysteinemia in both age and sex groups. Further extensive, multicentric studies are required with a large number of cases to validate the present hypothesis. Keywords

Homocysteine · Malnutrition · Coronary heart disease (CHD) · Folic acid

2.1

Introduction

2.1.1

Homocysteine and Nutrition

In the last few decades, dietary indices have been used to study the relationship between food intake and onset of various diseases (Kant 1996; Arvaniti and Panagiotakos 2008). The dietary index approach tries to account for the complex contribution of the human diet to health. Literature concerning the association between dietary indices and biomarkers, which are used as indicators of the current health status, is lacking which needs to be prepared for a healthy diet based on food, based dietary guidelines (Hargreaves et al. 1989;). The plasma concentration of total homocysteine (tHcy) is reported to increase the risk for cardiovascular events by 20% for each increase by 5 μmol/L of its concentration. Several recent studies reported that 12–15% increase in the intake of saturated fat was associated with a 6% rise in the tHcy (Humphrey et al. 2008; Berstad et al. 2007). The association between plasma homocysteine and phospholipids metabolism is supported by various studies suggesting that an increase in saturated fatty acids in the diet is responsible for the increase of synthesis of phosphatidylcholine (PC) through the phosphatidylethanolamine methyltransferase (PEMT) pathway (Hargreaves et al. 1989). Vasculopathic effects of Hcy are medicated through decreased expression of Apo-A-I, resulting in a reduction in high-density lipoprotein cholesterol and increasing vascular inflammation (Devlin and Lentz 2006). Thus elevated Hcy is considered as a risk factor for atherosclerosis including thrombotic and fibrotic vascular disease (McCully and Wilson 1975). Further, it is reported that inflammatory cytokines like interleukin-1β-6 and 8 and TNF-α affect lipid metabolism and cause atherosclerotic lesions (Kaul 2001; Klein et al. 2001; Niemann-Jonsson et al. 2000). The mechanisms involved demonstrate that these cytokines modulate the acute phase reactant, hsCRP secretion, and the expression of cell adhesion molecules (Verma et al. 2005). Recently, hsCRP has been considered as a major key factor in the pathogenesis of the development of atherosclerotic lesions and cardiovascular disorders (Libby 2002; Hansson et al. 2006), affected by dietary indices (Clifton 2003). One of the review articles made by Galli and Calder (2009) suggested an inverse relationship between dietary intake and inflammatory process.

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The studies conducted on dietary deficiency have shown that lack of folate intake, vitamin B12, or vitamin B6 reduces the peripheral docosahexaenoic acid (DHA) status (Durand et al. 1996; Pfeiffer and Lewis 1979; Delorme and Lupien 1976), by influencing the synthesis of PC in the liver which is synthesized through different metabolic pathways like cytidine diphosphate (CDP)-choline pathway and PEMT pathway (Kennedy and Weiss 1956) (Fig. 2.1). The CDP-choline uses 1,2-diacylglycerol, and is responsible for the synthesis of PC, whereas PEMT catalyzes the sequential methylation of phosphatidylethanolamine (PE) to PC (Vance et al. 1997). Methylation of PE to PC by PEMT plays a key role in the transport of poly-saturated fatty acids (Selley 2007; Watkins et al. 2003; Pynn et al. 2011). Secretion of very low-density lipoprotein (VLDL) from liver cells depends on PC synthesis (Yao and Vance 1988), and diminished PC synthesis restricts this secretion (Noga et al. 2002). Thus impairment of PEMT pathway has a direct impact on the transport of these components from the liver (Noga and Vance 2003). Thus PEMT activities affect DNA and its transport to the brain (Watkins et al. 2003; Pynn et al. 2011). Dietary methionine is demethylated to form homocysteine as a metabolic intermediary. Homocysteine reacts with serine in the presence of enzyme cystathionin. Homocysteine reacts with serine in the presence of enzyme cystathionine β-synthase that forms cystathionine, which is then cleaved to form homoserine plus cysteine. This series of reactions, by which the four-carbon dietary amino acid methionine is converted into the three-carbon amino acid cysteine, is called the transsulfuration pathway. In this pathway the critical step is cystathionine from homocysteine, as this step is irreversible in human beings. Thus the deficiency of the cystathionine β-synthase, which requires pyridoxine as a vitamin cofactor, results in the abnormal accumulation of homocysteine. Homocysteine can be remethylated to reform methionine by means of reactions that require the enzymes methyltransferase and methylenetetrahydrofolate reductase and the vitamins folate, cobalamin, betaine, or choline. A deficiency of these vitamins or enzymes thus also results in an abnormal accumulation of homocysteine (Kang et al. 1986). As defined that folate and vitamin B are the important determinants of methionine, S-adenosyl-methionine (SAM), S-adenosyl homocysteine (SAH) synthesis, and homocysteine clearance. The availability of vitamin B has a direct activity on liver PEMT which depends upon poly-saturated fatty acids (PUFAS) secretion. Thus the dietary intake of B-vitamins is essential to maintain the level of DHA and secretion of VLDL from the liver.

2.2

Materials and Methods

The field survey study was conducted by the Institute of Medical Sciences, Banaras Hindu University. The normal potential volunteers, aged between 35 and 50 years were registered to assess their folate, B6, B12 level along with total homocysteine concentration. As Hcy is one of the major biomarkers contributing to the

Fig. 2.1 The intriguing biochemical link between folate, vitamin B12, choline, CDP-choline pathway, and PEMT pathways

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development of various neurological and cardiovascular disorders therefore, identification of tHcy in normal population will be helpful in the prevention and management of CVD and other vascular events. This study was ethically approved by medical institutions of the Institute of Medical Sciences, Banaras Hindu University, NGB (DU), and SRM University.

2.2.1

Experimental Design (Clinical Studies)

The study was divided into two groups.

2.2.1.1 Group-I A total of 82 normal volunteers between the age group of 35 and 50 years of both sexes were screened for their health status and selected to assess their nutritional status and subsequently the serum folate, B vitamins along with total homocysteine levels were determined. Further, various inflammatory cytokines including IL-6, IL-8, and TNF-α in the inflammatory marker hsC-reactive proteins were also estimated. The lipids and lipoprotein profile were tested in order to establish the relationship between homocysteine, atherosclerotic changes, and risk of cardiovascular disease. After preliminary screening, these groups of potential normal subjects were treated with 5 mg folic acid per day for a period of 3 months and its effect was assessed on the bio-markers under investigation. 2.2.1.2 Group-II In this group, 109 healthy subjects were included. The exclusion criteria used for this study in both the groups were BMI >30 or 150 mg/dL were also not selected. Further, the volunteers who were alcoholics, smokers, or women who were pregnant or the subjects showing inability to follow guideline of the present study were discarded from the series. These groups of cases were not treated with folic acid or kept on any other medication.

2.2.2

Methods

After 12 h, fasting blood samples (10 mL each) were collected from selected subjects of both the groups. Total Hcy was measured by HPLC fluorescence method of Cornwell et al. (1993). IL-6 and IL-8, TNF-α were estimated by using an enzymelinked immunosorbent assay kits (R & D system). Serum TC, HDL-c, LDL-c, triglyceride, ApoA-I, Lp(a), and hsCRP were tested by enzymatic assays by using clinical chemical auto-analyzer (Transasia, ERBA360, Mumbai). Vitamin B12 and folate were estimated by radioimmunoassay method using commercially available kits. In order to evaluate the per day intake of B6, B12, and folate contents of the selected subjects, a 24 h dietary intake was estimated through the information

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provided by the subjects, by listening all food products, meals, and drinks consumed within 24 h before performing the study. The album which included the most common food products prepared by the National Food and Nutrition Institute was utilized to determine the amount of B vitamins including folate taken by the volunteers. The total intake of B6, B12, and folate was calculated on the basis of food consumed by the subject. The calculated vitamin intake with food taken by the study subjects was divided into three major quartiles as per mg/day intake, i.e. B6 (mg/day) 130 mmHg systolic or >85 mmHg diastolic or use of blood pressure-lowering agents. 3. Hypertriglyceridemia: serum triglycerides >1.70 mmol/L (150 mg/dL). 4. Low HDL-cholesterol: serum HDL-cholesterol, 15 μmol/L or an increase of 10 μmol/L above to normal limits. C-reactive protein levels were measured by the immune turbidimetric method, the reference limit is less than 6 mg/L and lowest levels of CRP in the present study of patient comprised of 3 mg/L. Endothelin estimations were preferred by the ELISA kit method. Levels

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of lipoprotein(a) were determined by the immunoturbidimetric method using an auto-analyser. As reported earlier, patients with MetS have a greater risk of developing coronary artery disease (The Expert Panel 2002; Grundy et al. 2004). Although HHcy and MetS both are associated with cardiovascular diseases, the association between homocysteine with MetS is also important to check the future CVD risk among patients. Various Cholesterol Education Program identified low-density lipoprotein cholesterol lowering pharmacotherapy among MetS patients is needed. However, every component of lipoproteins associated with this syndrome is considered to be a risk marker for atherosclerotic vascular disease. Similarly, the importance of novel coronary risk markers like tHcy, C-reactive protein, lipoprotein(a) which is a genetic marker, endothelin, etc. shall also be given due considerations for diagnosis, prevention and management programs for metabolic syndrome, Insulin resistance has also been accepted as a major causative factor for MetS which demonstrates the risk for development of type 2 diabetes mellitus in such patients (Ninomiya et al. 2004). The systematic inflammatory marker CRP has been identified to be a higher risk marker of several age-related diseases (Tucker 2005). Further, chronic inflammatory process is significantly found to be associated with diabetes, obesity, dyslipidemia, HHcy and ultimately CVD; therefore, patients with HHcy associated with this clinical conditions have vitamin B6 and B12 deficiency and are also involved with altered oxidative stress markers particularly reduced glutathione and high lipid peroxidation activity. From Table 3.1, it can be concluded that the age limit for developing Mets begins from 55 years and elderly women are more prone to CAD in comparison to men. High BMI leads to Mets and it has been reported that the patients having Mets with CAD involvement will have high BMI. Waist circumference is a component used to define metabolic syndrome and it is having a role as an independent predictor of coronary artery disease (CAD). The present findings validate that high waist circumference in males is independently associated with an increased prevalence of CAD in patients with metabolic syndrome. The elevated levels of systolic and diastolic blood pressure have a corelation with metabolic syndrome patients with and without CAD specifically in male population. Smokers have abnormalities in lipoprotein metabolism and endothelial function leading to metabolic syndrome and CAD. The data suggest that male smokers with metabolic syndrome are much prone to develop CAD. Patients having vegetarian diet are much safer to CAD in comparison to the non-vegetarian diet as vegetarians will have high serum folate and lower level of Hcy and it shows that nutrition is having an important role in the development of CAD in case of metabolic syndrome cases. The patients are having various changes in the metabolic conditions receiving different medications. It is well proved that ACE inhibitors are most commonly used for the treatment of CAD and according to Table 3.2, it is concluded that the Mets patients having CAD are using ACE inhibitors mostly. Other inhibitors like statin, diuretic and CABG have also shown the effects but in case of beta-blockers only marginal effects have been observed over CAD with metabolic syndrome patients.

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Table 3.1 Patient’s characteristics determined in metabolic syndrome patients with CAD involvement and with CAD involvement

Factors Age (years) BMI Waist circumference (cm) Systolic BP (mmHg) Diastolic BP (mmHg) Smoking (%) Vegetarian (%) Non-vege (%)

Sex M F M F M F

Metabolic syndrome with CAD involvement N ¼ 108 (male 62; female 46) 62.48  9.73 66.45  8.72 31.64  5.64 28.42  4.93 114.64  15.82 102.85  18.97

M F M F M F M F M F

156.90  11.72 142.73  9.62 89.42  4.01 87.68  3.96 21 6 68 76 32 24

95% confidence interval of limits of the mean 60.00–64.95 63.88–69.02 30.21–33.07 26.97–29.87 110.62–118.66 97.26–108.44 153.92–159.88 139.89–145.57 88.40–90.44 86.51–88.85

Metabolic syndrome without CAD involvement N ¼ 197 (male 116; female 81) 57.38  8.77 62.04  10.62 30.73  4.87 28.22  6.01 108.52  8.02 96.73  7.89 151.58  13.72 143.45  9.28 91.60  5.13 88.71  3.88 23 5 61 65 39 35

95% confidence interval of limits of the mean 55.75–59.00 59.75–64.33 29.83–31.63 26.92–29.52 107.03–110.00 95.03–97.12 149.03–154.13 141.45–145.45 90.65–92.55 87.87–89.55

Table 3.2 Percentage of metabolic conditions associated with MetS patients with CAD and without CAD group Medication receiving ACE/ARB (%) Beta blocker (%) Diuretic (%) CABG (%) Statin (%)

MetS with CAD N ¼ 108 (male 62; female 46) 44.9 27.3 23.7 24.2 36.8

MetS without CAD N ¼ 197 (male 116; female 81) 19.8 21.4 12.8 19.7 15.9

ACE angiotensin-converting enzyme inhibitor, ARB angiotensin receptor blocker, CABG coronary artery bypass grafting

According to the results obtained, Mets patients (male) with hypertension are at high risk of developing CAD in comparison to other clinical complications. It has been proven that hypertension induces endothelial dysfunction, exacerbates the atherosclerotic process and it contributes to CAD in most of the cases. Various evidences have proved that elevated levels of lipids (LDL, HDL, TG, TC) are

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Table 3.3 Percentage of metabolic conditions associated with MetS patients with CAD and without CAD group

Clinical conditions MetS with diabetes (%) MetS with obesity (%) MetS with abnormal lipids (%) MetS with hypertension (%)

Sex M F M F M F M F

MetS with CAD N ¼ 108 (male 62; female 46) 39 34 48 72 69 72 81 69

MetS without CAD N ¼ 197 (male 116; female 81) 33 31 53 67 78 80 78 72

Table 3.4 Blood glucose level and lipoprotein profile status associated with metabolic syndrome patients with CAD and without CAD involvement

Factors Blood glucose (mg/dL) Total cholesterol (mg/dL) LDL-c (mg/dL) HDL-c (mg/dL) Triglycerides (mg/dL)

Sex M F

Metabolic syndrome with CAD involvement N ¼ 108 (male 62; female 46) 103.45  9.78 98.52  12.73

95% confidence interval of limits of the mean 101.57–105.33 96.03–98.97

Metabolic syndrome without CAD involvement N ¼ 197 (male 116; female 81) 118.82  10.68 107.39  12.04

95% confidence interval of limits of the mean 117.30–120.34 105.68–109.11

M F

204.71  31.90 197.68  29.85

198.57–210.87 191.94–203.42

228.32  38.45 214.87  28.55

222.84–233.78 210.80–218.94

M F M F M F

136.48  24.96 128.69  26.04 44.73  8.22 45.22  7.11 196.42  73.85 182.69  66.83

131.68–141.28 123.68–133.70 43.15–46.31 43.85–46.59 182.21–187.63 169.83–195.55

131.42  30.77 129.73  23.91 42.91  6.88 43.86  7.02 247.98  76.85 221.83  81.03

127.04–135.80 126.83–132.63 41.93–43.89 42.86–44.86 237.03–258.93 210.28–233.38

strongly predictive of the risk of CAD. According to Table 3.3, females are more prone to CAD in comparison to males with dyslipidemia and obesity in metabolic syndrome cases whereas in case of diabetic condition with Mets the percentage is low. In Table 3.4, the metabolic factors were analysed in Mets patients with and without CAD involvement and it is concluded that the factors like blood glucose

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Table 3.5 Hyperhomocysteinemia in association with other biomarkers among metabolic syndrome patients with CAD and without CAD involvement

Factors Homocysteine (μmol/L) CRP (mg/L) Lipoprotein (a) (cm) Endothelin (pg/mL)

Sex M F M F M F M F

Metabolic syndrome with CAD involvement N ¼ 108 (male 62; female 46) 23.90  8.45 19.45  7.35 7.11  1.35 6.04  1.21 34.96  5.02 30.77  3.87 7.80  0.98 6.11  0.69

95% confidence interval of limits of the mean 21.75–26.05 17.28–21.62 6.77–7.45 5.68–6.40 33.69–36.24 29.63–32.18 7.55–8.05 59.07–6.31

Metabolic syndrome without CAD involvement N ¼ 197 (male 116; female 81) 18.73  9.82 16.22  8.45 6.45  2.11 5.85  1.84 29.75  8.04 26.86  10.11 5.36  1.02 6.02  0.86

95% confidence interval of limits of the mean 16.91–20.55 13.73–18.71 6.06–6.84 5.31–6.40 28.26–31.24 23.88–29.84 5.17–5.55 5.77–6.27

level, total cholesterol, LDL-c and triglycerides have higher levels whereas HDL-c is low in case of metabolic syndrome patients without the involvement of CAD in comparison to the patients with CAD because the patients are already on the medication for CAD. In Table 3.5, the Mets patients with and without the involvement of CAD were compared with the level of important biomarkers like homocysteine, CRP, lipoprotein and endothelin. It is concluded that homocysteine, CRP, lipoprotein and endothelin which are the prominent markers for the assessment of CAD values are high in case of Mets patients with CAD rather than the patients without CAD. From Table 3.6, the patients were divided into four major groups of different clinical complications with an elevated level of Hcy. The factors such as BMI, tHcy, serum folate, Vit B6 and VitB12 were observed in all the four groups. In case of patients with obesity, hypertension and dyslipidemia a higher BMI and Vit B12 level has been seen but tHcy, serum folate and Vit B12 levels have shown a decrease in comparison to the other groups. The other group having clinical conditions like obesity, dyslipidemia and insulin resistance has shown a significant rise in BMI, tHcy, serum folate, VitB6 and VitB12. Out of all factors, serum folate and VitB12 levels were much higher in comparison to other groups. In case of obese patients with hypertension, dyslipidemia and insulin resistance the factors like BMI, tHcy and VitB12 values were much higher than the serum folate and VitB6. In this condition, the serum folate has the minimum whereas VitB12 has the maximum values. The last group includes obese population with hypertension has greater BMI and highest level of tHcy and VitB6 comparatively. Serum folate and VitB12 have given moderate effects in this condition. Table 3.7 indicates a comparative study of different clinical conditions with higher levels of Hcy and their treatment with normal diet including folic acid and

34.63–37.59

33.60–36.56

36.11  4.91

35.08  3.80

44

29

Obesity with hypertension, dyslipidemia insulin resistance and elevated level of tHcy

Obesity, hypertension, with elevated level of tHcy

34.41–37.55

35.98  4.87

37

Obesity, dyslipidemia, insulin resistance and elevated level of tHcy

34.97–37.87

36.42  3.83

28

BMI (index)

Obesity, hypertension, dyslipidemia and elevated level of tHcy

Groups

No. of cases

95% confidence interval of limits of the mean

47.32  13.92

41.04  11.04

25.93  7.82

23.87  6.91

tHcy (μmol/L

44.74–49.90

37.71–44.37

23.36–28.49

21.26–26.48

95% confidence interval of limits of the mean

7.15  1.62

6.85  0.97

8.04  2.13

7.93  1.06

Serum folate (ng/mL)

6.55–7.75

6.59–7.14

7.34–8.74

7.53–8.33

95% confidence interval of limits of the mean

Table 3.6 Groupwise distribution of various factors involved with MetS patients showing HHcy

12.94  4.02

10.68  3.82

11.94  4.17

12.82  3.91

Vitamin B6 (ng/mL)

11.45–14.43

9.53–11.83

10.57–13.31

11.34–14.30

95% confidence interval of limits of the mean

294.82  78.85

314.95  96.72

311.78  78.90

281.82  108.94

Vitamin B12 (pg/mL)

265.54–324.10

284.99–344.11

285.83–337.72

240.64–322.99

95% confidence interval of limits of the mean

64 G. P. Dubey et al.

Obesity, Initial 22 hypertension, dyslipidemia After and elevated 3 months level of tHcy Comp. initial vs. after 3 months Obesity, Initial dyslipidemia, 26 insulin After resistance and 3 months elevated level of tHcy Comp. initial vs. after 3 months Initial Obesity with hypertension, 30 dyslipidemia After insulin 3 months resistance and elevated level of tHcy Comp. initial vs. after 3 months Obesity, Initial hypertension, 18 with elevated After level of tHcy 3 months Comp. initial vs. after 3 months

Groups

No. of cases

39.90  5.11

P < 0.01

28.13  4.06

25.64  6.13

P > 0.05

31.74  5.98

28.68  4.79

P < 0.001

30.85  4.93

27.16  5.13

P > 0.05

33.98  4.77

P > 0.05

34.51  4.02

33.82  3.79

P > 0.05

35.03  3.78

32.56  4.10

P > 0.05

34.85  5.06

32.62  4.90

P > 0.05

P < 0.01

12.14  2.62

8.02  3.17

P < 0.001

11.02  1.97

6.91  2.66

P < 0.001

10.89  3.04

7.91  2.28

P > 0.05

10.46  3.14

P > 0.05

349.62  92.45

287.84  112.96

P > 0.05

362.94  88.45

298.62  108.79

P > 0.05

382.91  91.32

326.14  78.99

P < 0.001

401.26  102.56

304.92  82.04

27.82  4.11

35.24  5.02

8.02  2.16

Vitamin B12 (pg/mL)

Normal diet with 5 mg folic acid tHcy (μmol/ Serum folate BMI (index) L) (ng/mL)

21

33

31

29

No. of cases

P < 0.01

30.14 3 .84

34.30  5.16

P < 0.01

32.85  5.17

36.02  3.45

P < 0.001

29.52  3.78

33.87  4.13

P < 0.01

29.64  5.13

34.16  6.02

P < 0.001

21.28  3.90

29.85  5.13

P < 0.001

25.12  6.02

31.04  5.02

P < 0.001

21.04  4.98

27.85  5.04

P < 0.001

18.72  3.94

24.10  5.43

Treated with test formulation tHcy (μmol/ BMI (index) L

Table 3.7 Reduction in tHcy concentration following folic acid and test formulation treatment in MetS patients

P < 0.01

11.03  3.16

P < 0.001

562.91  81.35

314.88  75.14

P < 0.001

P < 0.001 8.02  2.98

552.11  87.14

331.82  98.85

P < 0.001

521.64  101.45

288.91  86.69

P < 0.01

504.97  96.22

316.98  101.85

Vitamin B12 (pg/mL)

12.08  3.01

28.69  2.48

P > 0.05

11.04  3.78

8.06  4.92

P < 0.001

11.62  4.16

7.85  3.14

Serum folate (ng/mL)

3 Homocysteine-Mediated Endothelial Dysfunction in Metabolic Syndrome 65

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test formulation. The first group includes obese patients with hypertension and dyslipidemia and factors such as BMI, tHcy and serum folate have shown elevated effects when treated with test formulation whereas VitB12 has a significant rise on the treatment of test formulation in comparison to folic acid treatment after an interval of 3 months. The BMI is not significant whereas it is moderately significant on test formulation treatment, tHcy is moderately significant but it is highly significant with test formulation, serum folate values are not significant with folic acid but with formulation it is statistically significant and in case of Vit.B12 the values are significant with folic acid whereas it is less significant with the formulation. The obese population with dyslipidemia and insulin resistance has reported no significance in BMI and tHcy with normal diet whereas it is highly significant with test formulation; serum folate level is significant with normal diet but not with the test formulation whereas Vit.B12 values are significant with test formulation in comparison to normal diet including folic acid. After an interval of 3 months, another group of obese population with hypertension, dyslipidemia and insulin resistance has not shown any significance in BMI and slight significance can be seen with test formulation; both tHcy and serum folate level have shown good significance in both the conditions, and Vit. B12 level with test formulation has shown good significance in comparison to the normal diet. The last group including obesity and hypertension patients indicates that there is a slight significance with test formulation rather than normal diet, tHcy has shown good significance with test formulation and serum folate is moderately significant in both conditions. Vit.B12 has shown significant results with test formulation whereas there is no significance with normal diet in this condition.

3.11

Results and Discussion

The data of the present study suggested that patients of both groups, i.e. with CAD and without CAD, are involved with inflammatory conditions. Plasma CRP concentration, which is an important downstream inflammatory marker, that integrates the action of several activated cytokines particularly interleukin-6 and tumour necrosis factor alpha, predicts the future CVD progression, severity of CVD and also the future development of CVD/CAD/atherosclerosis event (de Maat and Trion 2004). Various studies are available showing that low B6 content has an increased CVD risk (Friso et al. 2004). The observations made in the present study indicates an inverse relationship between plasma tHcy, CRP, and lipoprotein (a) supporting the fact that inflammation has a common link between low vitamin B6 and B12 resulting in the development of CVD. Further, it may also be hypothesised that during the process of inflammation due to the production of cytokines and other inflammatory biomarkers, inflammatory response might be demanding higher vitamins B particularly in conditions of diabetes, obesity, dyslipidemia, etc. which are commonly associated with MetS.

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Various studies have reported that supplement of pyridoxine treatment decreases pancreatic and circulating insulin improves glucose tolerance and restores β-cell function (Rogers and Mohan 1994; Toyota et al. 1981; Takatori et al. 2004). Further, pyridoxine lowers blood glucose and HbA1C in diabetic conditions (Cohen et al. 1984; Jain 2007). Further, it is also studied that inadequate vitamin B6 status may decrease glutathione and impairs the antioxidant defence system. This oxidative stress may affect the mechanistic pathway through which low vitamin B6 may lead to cardiovascular involvement. Thus, there is a strong correlation between plasma Hcy, CRP and lipoprotein profile with MetS patients. Our findings suggest a potential between HHcy and CAD among MetS cases, independent of the homocysteine-mediated pathway. Thus nutritional status is significantly associated with life stress, physiological responses and ultimately resulting in chronic clinical conditions. Therefore, proper dietary recommendations and metabolic control will be helpful in the prevention and management of metabolic syndrome and associated complications particularly CAD. According to our data, the Mets patients with CAD and without CAD involvement were compared for their percentage of medication received. It is well proved that ACE inhibitors are primarily used for the management of CAD due to their antiischemic action and our data also support the same as the percentage of ACE inhibitor’s usage is high or in comparison to other therapy groups. Next is statin which is used widely for the treatment of CAD and it has been supported by our study also. Out of all, the diuretic has been used less frequently for CAD treatment in Mets patients. A group-wise distributed population with a higher level of Hcy has been seen along with various factors and our data suggest that clinical complications such as obesity, hypertension, insulin resistance and dyslipidemia influence the ranges of parameters like BMI, tHcy, serum folate, VitB6 and VitB12. It is well established that condition like obesity, hypertension, dyslipidemia and insulin resistance contributes to metabolic syndrome and is strongly correlated with BMI. Our data indicate that there is potential relation between HHcy and factors like serum folate, VitB12 and VitB6. It has been proven that low serum folate level contributes to high level of tHcy and low VitB6 and VitB12 values. Our data include the comparative study of different clinical groups and elevated Hcy with a treatment of normal diet containing folic acid and our test formulation for Mets. The formulation includes an Ayurveda composition of four plants, i.e. Hippophae rhamnoides, Terminalia chebla,Terminalia arjuna, Dioscorea bulbifera and Nardostachys jatamansi with several properties as anti-obesity, antiatherosclerotic, cardio-protective and homocysteine-lowering properties. It is proven that out of all Hippophae rhamnoides is rich in folic acid, vit. B6, B12 content and helps in lowering Hcy levels (Suomela et al. 2006). In the first group of patients with obesity, hypertension and dyslipidemia factors like BMI and serum folate have not shown any significance with normal diet but with formulation serum folate is much significant rather than BMI. tHcy is moderately significant whereas Vit.B12 is highly significant with normal diet but it is just reverse when treated with test formulation.

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The other obese group with dyslipidemia and insulin resistance indicates no significance with BMI, tHcy and Vit.B12 in normal diet of folic acid but the level of significance is higher with test formulation. The serum folate values are significant with the folic acid diet but it is reverse with test formulation.

3.12

Conclusion

The population of patients with obesity, hypertension and dyslipidemia shows a significant corelation with tHcy and serum folate with folic acid diet but there is no corelation between BMI and Vit.B12. On treatment with formulation, except BMI all the other factors have shown good significance. In the last group of patients with obesity and hypertension there is no corelation in BMI, tHcy and Vit.B12 but serum folate has shown moderate significance with normal diet containing folic acid. On the treatment with test formulation in this group, the tHcy and Vit. B12 has given better significance in comparison to BMI and serum folate. Conflicts of Interest The authors declare no conflicts of interest.

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4

Homocysteine and Bone Health Asha Bhardwaj, Leena Sapra, Bhupendra Verma, and Rupesh K. Srivastava

Abstract

Homocysteine is a sulfur-containing intermediary amino acid synthesized during methionine metabolism. Homocysteine has a significant role in the regulation of cell homeostasis but an elevated level of plasma homocysteine (hyperhomocysteinemia) is associated with vascular and various age-related pathologies. There are evidences from various laboratories and clinical studies that a high level of homocysteine shows deleterious effects on bone. Homocysteine is now considered as an independent risk factor for osteoporosis. Homocysteine exerts detrimental effects on both osteoclasts and osteoblasts. Homocysteine also promotes oxidative stress resulting in the generation of reactive oxygen species and disrupts cross-linking of collagen molecules. Thus, homocysteine impairs bone quality and reduces bone mass in several ways. In this book chapter, we reviewed all the known mechanisms responsible for hyperhomocysteinemiainduced osteoporosis. We also discuss the role of homocysteine-induced dysbiosis in bone resorption and the potential role of probiotics as a potent therapy for the prevention of homocysteine-induced bone loss along with other available therapies. Keywords

Homocysteine · Bone · Osteoclasts · Osteoblasts · Collagen cross-linking · Mitochondria · Probiotics · Estrogen · Gut microbiota

A. Bhardwaj · L. Sapra · B. Verma · R. K. Srivastava (*) Translational Osteoimmunology & Immunoporosis Lab (TOIL), Department of Biotechnology, All India Institute of Medical Sciences (AIIMS), New Delhi, Delhi, India e-mail: [email protected] # The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2022 G. P. Dubey et al. (eds.), Homocysteine Metabolism in Health and Disease, https://doi.org/10.1007/978-981-16-6867-8_4

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A. Bhardwaj et al.

Introduction

Osteoporosis is a bone disorder that results in low bone mineral density (BMD) and weakening of bone microarchitecture (Tu et al. 2018). In both men and women, osteoporosis increases the risk of fracture and the fragility of bones (Srivastava et al. 2018). Osteoporosis is mostly diagnosed by measuring BMD using the technique dual-energy X-ray absorptiometry (DEXA) also called as bone densitometry (Tu et al. 2018). World Health Organization (WHO) defined osteoporosis with BMD T-score of 2.5 or less and osteopenia with BMD T-score between 1 and 2.5 (Compston et al. 2019). According to International Osteoporosis Foundation (IOF), there is an osteoporotic fracture every 3 s resulting in more than 8.9 million fractures annually. It is estimated worldwide that 1 in 3 women and 1 in 5 men will suffer an osteoporotic fracture in their lifetime. Osteoporosis-related fractures result in extreme pain, infirmity, and increase in health care costs along with significant rise in morbidity and mortality (Srivastava et al. 2018; Tu et al. 2018). Osteoporosis causes huge economic burden and it has been projected that in the USA alone the cost for osteoporosis treatment will reach $ 25.3 billion by the end of 2025 (Burge et al. 2007). Homocysteine (Hcy) is a sulfur-containing toxic amino acid produced during methionine metabolism (Ansari et al. 2014). Hcy level is maintained by two pathways: transsulfuration and methylation. These pathways are governed by vitamin B6, vitamin B12, folate, and enzyme cystathione β synthase (CBS). Hcy regulates DNA metabolism via methylation. Hcy concentration is mainly determined by the level of dietary intake and lifestyle conditions. Too much alcohol and coffee consumption, physical inactivity, smoking, deficiency of B vitamins, and folate alter the level of Hcy (Ansari et al. 2014). An elevated level of Hcy is associated with various heart, liver, brain, and kidney diseases (Behera et al. 2017). Increased level of Hcy is now considered as an independent risk factor for osteoporotic fractures. Malfunctioning in the metabolism of Hcy promotes bone loss. Homocystinuria (HCU) is a recessive autosomal disease characterized by the strikingly increased level of circulating Hcy. HCU is associated with various diseases related to central nervous system, eyes, and vasculature and is also found to be responsible for the early onset of osteoporosis (van Meurs et al. 2004). The mechanism behind the early onset of osteoporosis due to HCU is not completely known but it is observed that Hcy-induced disruption of collagen cross-linking might be the reason (van Meurs et al. 2004). Moderate increase in the level of circulating Hcy is characterized as hyperhomocysteinemia (HHcy). HHcy is responsible for various clinical manifestations such as cardiovascular and neurodegenerative diseases (Herrmann et al. 2006). HHcy is significantly correlated with osteoporosis and osteopenia (Saoji et al. 2018). Thus, both HCU and HHcy are major risk factors for osteoporosis. High concentration of Hcy stimulates bone resorption through several mechanisms. Hcy promotes osteoclast activity, inhibits osteoblast formation, degrades collagen matrix, generates reactive oxygen species (ROS), decreases blood flow in bone, induces oxidative stress, and activates the matrix metalloproteinases (MMPs) which destroy bone matrix (Vacek et al. 2013). In this chapter, we summarized the current

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knowledge regarding the role of Hcy in the progression of bone disease and the different mechanisms through which Hcy regulates bone loss. Next, we further discuss the unexplored role of gut microbiota in Hcy metabolism and the therapeutic potential of probiotics in downregulating the Hcy level along with various treatment options for preventing Hcy-induced bone loss.

4.2

Homocysteine Metabolism

Hcy is a toxic sulfur-containing non-protein amino acid produced during interconversion pathway of methionine and cysteine. Hcy is metabolized at the intersection of two pathways: remethylation and transsulfuration. In remethylation, a methyl group is acquired by homocysteine from N-5-methyltetrahydrafolate or from betaine to produce methionine. N-5-methyltetrahydrafolate acts as methyl donor in all the tissues and donation is dependent on vitamin B12 whereas betaine acts as methyl donor only in liver and donation is independent of vitamin B12. Methionine is then converted into S-adenosylmethionine with the help of Adenosine triphosphate (ATP) (Selhub 1999). S-adenosylmethionine donates its methyl group and gets converted into a demethylated compound S-adenosyl homocysteine (SAH). SAH is then hydrolyzed resulting in the formation of Hcy which can be converted again into methionine (Kumar et al. 2017). In transsulfuration pathway Hcy combines with serine to produce cystathionine. This is an irreversible reaction catabolized by the enzyme, Cystathionine-β-synthase (CBS). Another enzyme γ-cystathionase catabolized cystathionine into cysteine and α-ketobutyrate. Excess cysteine is then oxidized into taurine and inorganic sulfates which are excreted through the urine (Fig. 4.1). In the transsulfuration pathway basically excess Hcy that is not converted into methionine is catabolized into cysteine (Selhub 1999). Usually with proper regulation of remethylation and transsulfuration pathway, there is a very low level of Hcy in the plasma. But sometimes due to metabolic malfunctions, these pathways get disrupted resulting in excess of Hcy in blood which then get accumulated in the cells. This condition is termed as HHcy. HHcy is generally caused either by a defect in one of the enzymes or deficiency of the vitamins involved in Hcy metabolism (Selhub 1999). HHcy is responsible for various diseases like atherosclerosis, age-related macular degeneration, congestive heart failure, hearing loss, and Alzheimer’s disease (Kim et al. 2018). HHcy is responsible for numerous other diseases. The association between HHcy and bone fragilities is shown by several studies. HHcy is found to modulate the complex process of remodeling through various mechanisms resulting in disruption of bone homeostasis. HHcy is recognized as an independent risk factor for osteoporosis and is considered as a pathological marker for bone diseases (Behera et al. 2017). In this chapter, we reviewed various mechanisms through which HHcy induces bone resorption.

Fig. 4.1 Schematic representation of Hcy metabolism. Hcy is produced at the intersection of two pathways: remethylation and transsulfuration. In remethylation, a methyl group is acquired by Hcy either from N-5-methyltetrahydrofolate or from betaine to produce methionine. In transsulfuration pathway excess homocysteine is converted into cysteine which is excreted out through the urine. MAT methionine adinosyltransferase, SAHH S-adenosylhomocysteine hydrolase, CBS cystathionine β-synthase, CGL cystathioine γ-lyase, BHMT betaine-homocysteine methlytransferase, SHMT serine hydroxymethyltransferase, MTHFR methylenetetrahydrofolate, MS methione synthase

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4.3

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Bone Remodeling

Bone is a dynamic organ undergoing remodeling throughout the life of the organism resulting in the replacement of low-performing bone with fully functional new bone. This task of bone remodeling is fulfilled by coordinated synergism between the bone cells. There are three different types of bone cells, viz. osteoclasts, osteoblasts, and osteocytes that are responsible for remodeling, maintenance, and matrix formation. Osteoblasts are bone-forming cells differentiated from mesenchymal stem cells in bone marrow primarily with the help of runt-related transcription factor 2 and its target Sp7 transcription factor gene. Wnt signaling also has a very imperative role in osteoblastogenesis (Srivastava et al. 2018). Osteoblasts form the osteoid matrix that later on get calcified. Osteoid matrix mainly consists of type 1 collagen that provides resistance to fractures and a large number of non-collagenous proteins that regulate various important functions of bone (Walsh et al. 2006). Osteoclasts are multinucleated bone cells having specialized bone-resorbing capability. Osteoclasts are derived from monocytic progenitors that also give rise to dendritic cells, granulocytes, macrophages, and microglia. Differentiation of osteoclasts is mainly mediated by two important cytokines: macrophage colony-stimulating factor (MCSF) and receptor activator of the nuclear factor kappa B ligand (RANKL). MCSF is required for the survival and proliferation of the monocytic progenitors, and RANKL which acts via its receptor RANK promotes differentiation and commitment of the progenitors into osteoclasts (Schett and David 2010). Osteoclasts attach to the surface of the bone and form a specialized structure called sealing zone. Sealing zone allows the osteoclasts to form resorption space that is separated from the extracellular space. Osteoclasts acidify the resorption space and degrade the mineral and organic components of bone by releasing various lysozymal enzymelike cathepsin K in the resorption space. To mediate bone resorption osteoclasts form the unique structure termed as ruffled border which increases the surface area availability to allow the active transport of hydrogen ions (H+) through a proton pump. Osteoclasts are highly motile and can resorb the larger parts of the bone (Walsh et al. 2006). Osteocytes are unique osteoblasts that are entrapped in calcified matrix formed by the differentiating osteoblasts. Osteocytes sense mechanical force on the bone and send signals to other osteocytes and osteoblasts on the surface of the bone via cellular processes called canaliculi (Walsh et al. 2006). A proper coupling between the activity of osteoblasts and osteoclasts is essential for maintaining bone integrity. Multiple interactions take place between bone-forming osteoblasts and bone-resorbing osteoclasts to regulate bone homeostasis. Osteoclasts differentiation is positively regulated by the preosteoblasts that secrete cytokines MCSF and RANKL and negatively by the mature osteoblasts that secrete anti-osteoclastogenic RANKL decoy receptor osteoprotegerin (OPG) (Schett and David 2010). Remodeling restores microdamage, maintains mechanical integrity of bone, and ensures release of calcium (Ca) and phosphorus (P) in the normal host physiology (Dar et al. 2018). Bone remodeling is divided into four phases, viz. activation, resorption, reversal, and formation (Srivastava et al. 2018) (Fig. 4.2). Bone remodeling is regulated by various factors like hormones such as estrogen and

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Fig. 4.2 Schematic representation of bone remodeling cycle. Bone remodeling occurs in four phases viz. (1) Activation phase: MCSF and RANKL induce the differentiation of osteoclast progenitors into osteoclasts. (2) Resorption phase: mature osteoclast with unique ruffled border starts resorption of bone by secreting cathepsin K, and H+ in resorption space. After resorption osteoclasts detach from the surface of the bone and undergo apoptosis. (3) Reversal phase: During the reversal phase osteoblasts precursor get differentiated into mature osteoblasts and are recruited to the resorption site. (4) Formation phase: osteoblasts get occupied in the resorbed lacuna and start depositing the bone matrix. After the formation phase osteoid gets mineralized and bone surface returns to resting phase with bone lining cells. (Figure illustrated with the help of https://smart. servier.com/)

parathyroid hormone, immune cells like T and B cells, and genetics. Estrogen is a very important regulator of bone remodeling. The deficiency of estrogen is associated with enhanced bone loss. Postmenopausal women are at increased risk of osteoporosis because of the lack of estrogen in them. Estrogen inhibits osteoclastogenesis and promotes osteoblast formation (Riggs 2000). Proper regulation of bone remodeling is required for repairing of various micro cracks. Hcy also affects the process of bone remodeling and leads to bone resorption.

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Homocysteine and Osteoporosis

The elevated level of Hcy disturbs the normal physiology of several organs and bone is one of them. Various studies have shown the association between Hcy and bone health. It is observed that a high level of Hcy increases the risk of osteoporotic fractures (Cashman 2005). In elderly women, a high level of Hcy has been found to be correlated with greater hip BMD loss (Zhu et al. 2009). Bahtiri et al. reported that HHcy is an independent risk factor for osteoporosis. Osteoporotic women have a significantly higher level of Hcy which is inversely associated with femur neck and lumbar spine BMD (Bahtiri et al. 2015). In Moroccan healthy postmenopausal women it is observed that plasma total Hcy is an independent risk factor for osteoporosis (Ouzzif et al. 2012). Similarly, Su et al. reported that a higher level of Hcy in serum is independently associated with BMD decline (Su et al. 2019). It is observed that HHcy is considerably correlated with osteopenia and osteoporosis in young northeastern Indian women (Saoji et al. 2018). In children with recurrent fractures, enhanced level of Hcy has been found to be negatively correlated with bone formation (Rehackova et al. 2013). It is observed that in healthy elderly people HHcy enhances the fracture risk and is associated with high markers of bone turnover (Dhonukshe-Rutten et al. 2005). HHcy is found to be responsible for poor physical performance, higher bone turnover, and lower BMD in elderly women. Moreover, it is observed that HHcy elevates the mortality rate among women (Gerdhem et al. 2007). It is observed that generally HCU is associated with low BMD in children and adults (Weber et al. 2016). When experimental HHcy is induced in rats it is observed that HHcy disturbed the bone homeostasis and shifted the balance of bone formation and bone resorption toward enhanced bone resorption (Ozdem et al. 2007). In a study by Azizi et al., it was reported that supplementation of Hcy to the adult female Sprague-Dawley rats for 3 weeks before mating and throughout the complete period of pregnancy induced osteopenia in newborn rats (Azizi et al. 2010). Induction of moderate HHcy in rats by supplementation of diet containing higher level of methionine for short term altered bone microarchitecture (Milovanovic et al. 2017). In Turkish postmenopausal women, it is reported that high plasma Hcy is associated with low femur and lumbar spine BMD (Bozkurt et al. 2009). It is observed that polymorphism in the methionine synthase reductase (MTRR) enzyme results in HHcy which further leads to bone loss. MTRR is required for the conversion of Hcy to methionine (Kim et al. 2006). HHcy not only increases the chances of osteoporosis but also affects the process of bone repairing. After fixation of closed femoral fracture in mice fed with high Hcy supplemented diet for 4 weeks it is observed that as compared to controls Hcy fed animals had a significant decrease in bone repair resulting in impaired fracture healing (Claes et al. 2009). Hcy promotes bone loss through various mechanisms such as it affects the process of osteoclastogenesis, osteoblastogenesis, disrupts collagen cross-linking, inhibits nitric oxide (NO) synthesis, and induces oxidative stress. We next discuss in detail various mechanisms that lead to Hcy-induced bone loss (Fig. 4.3).

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Fig. 4.3 Different mechanisms through which Hcy induces bone loss. Hcy increases osteoclastogenesis by stimulating the generation of ROS and inflammatory cytokines, induces apoptosis of osteoblasts, decreases blood flow to bone tissue, degrades collagen matrix, activates MMPs, and promotes dysbiosis. ROS reactive oxygen species, NOX NADPH oxidase, NO Nitric oxide, Lox Lysyl oxidase, Plod 2 Lysyl hydroxylase, Hcy Homocysteine, MMP Metalloproteinase, NMDA-R N-methyl-D-aspartate receptor, IL Interleukin, G-CSF Granulocyte colony-stimulating factor, MIP-1α Macrophage inflammatory protein-1α, IFN-γ Interferon-γ, TNF-α Tumor necrosis factor-α. (Figure illustrated with the help of https://smart.servier.com/)

4.4.1

Homocysteine Enhances Osteoclastogenesis

Various studies have shown that an increased level of Hcy stimulates bone resorption via inducing osteoclastogenesis. Vaes et al. have shown that Hcy promotes osteoclast formation in a dose-dependent manner (Vaes et al. 2009). Similarly, Hermann et al. have shown that Hcy increases the osteoclastogenesis of human

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peripheral blood mononuclear cells with increasing concentrations. Hcy also enhances the cathepsin-K activity in osteoclasts (Herrmann et al. 2005). One of the mechanisms through which Hcy stimulates osteoclastogenesis is by inducing oxidative stress. Oxidative stress promotes the generation of ROS which can effectively stimulate osteoclast formation (Garrett et al. 1990; Bax et al. 1992; Koh et al. 2006). HHcy stimulates the production of ROS via activation of protease-activated receptor-4 (PAR-4) that increases nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase (Nox) and decreases thioredoxin expression (Tyagi et al. 2005). Induction of osteoclastogenesis by ROS can be reversed by using antioxidant, N-acetyl cysteine (Koh et al. 2006). Estrogen inhibits osteoclastogenesis by suppressing the formation of ROS via upregulating the expression of glutathione and glutathione reductases in osteoclasts. Thus estrogen is also recommended as a therapy for preventing Hcy-induced ROS generation (Lean et al. 2003). Hcy also increases the expression of integrin beta-3 mRNA and promotes the p38 mitogenactivated protein kinase (MAPK) activity along with increasing the number of nuclei per cell in osteoclasts (Koh et al. 2006). Glutamate is an important regulator of bone health. In vitro studies have shown that polyglutamate peptide induces bone resorption. Glutamate promotes bone loss through its receptor N-methyl-D-aspartate receptor (NMDA-R). NMDAR-1 is highly expressed by osteoclasts. It is observed in vitro that monoclonal antibody against NMDAR-1 inhibited bone resorption (Chenu et al. 1998). Hcy can bind to the NMDA-R leading to the increase in levels of intracellular calcium (Ca) followed by the generation of ROS thereby inducing osteoclastogenesis (Vacek et al. 2013). HHcy also induces the production of various inflammatory cytokines viz. MCSF, interleukin (IL)-1α, IL-1β, granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1α, interferon (IFN)-γ, IL-17, and tumor necrosis factor (TNF)-α. These inflammatory cytokines promote osteoclasts formation which leads to bone loss (Vijayan et al. 2013). These studies thereby establish that Hcy can promote osteoclastogenesis through several mechanisms.

4.4.2

Homocysteine Suppresses Osteoblastogenesis

One of the mechanisms through which Hcy promotes bone loss is by affecting the process of osteoblastogenesis. Hcy promotes apoptosis of osteoblasts and osteocytes. It is observed that Hcy enhances apoptosis of these cells by inducing the expression of Nox1 and Nox2 and inflammatory cytokines IL-1β and IL-6 (Takeno et al. 2015; Notsu et al. 2019). Kanazava et al. showed that Hcy induces apoptosis of osteoblasts by promoting oxidative stress. Hcy at high concentrations increases the activity of caspases 3, 8, and 9 and also enhances the generation of intracellular ROS in a dose-dependent manner (Kanazawa et al. 2017). Hcy also suppresses osteoblastogenesis by promoting methylation of promoter A region in the estrogen receptor alpha (ERα) gene. It is reported that methylation of promoter A region in the ERα gene is associated with post-menopausal osteoporosis. The degree of hypermethylation in ERα gene is also found to be associated with a high level of Hcy. It is observed that in vitro treatment of human bone marrow stromal cells with

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Hcy inhibits proliferation and differentiation of these cells in a dose-dependent manner with time by promoting the methylation of promoter A region in the ERα gene (Lv et al. 2011). Thus Hcy induces hypermethylation of ERα gene thereby preventing osteoblast differentiation. At low concentration, Hcy downregulates the expression of lysyl oxidase (Lox) in osteoblasts by epigenetic CpG methylation and enhanced accumulation of pentosidine. Lox is required for the formation of stable matrix (Thaler et al. 2013; Kanazawa et al. 2017). Pentosidine is a glycoxidative end product which inhibits the formation of bone nodules and induces functional alteration of osteoblasts (Sanguineti et al. 2008). HHcy can promote bone loss by inducing the serum amyloid A3 (SAA3) in osteoblasts. When MC3T3-E1 cells were cultured in the presence of Hcy for 21 days and then reseeded on an extracellular matrix it was observed that it led to reduced collagen cross-linking and unlocking of arginine-glycine-aspartic acid motifs. These events resulted in the activation of SAA3 in osteoblasts along with the production of matrix metalloproteinase (MMP)-13 that degrade bone matrix (Thaler et al. 2013). HHcy downregulates the forkhead box protein O1 (FOXO1) and MAPK signaling cascades by phosphorylation of protein phosphatase 2A resulting in increased RANKL and decreased OPG synthesis in osteoblasts. Thus HHcy shifted the OPG:RANKL ratio toward osteoclastogenesis (Vijayan et al. 2013). Hcy also dysregulates the functioning of osteoblasts by attenuating the synthesis of osteocalcin and enhancing the synthesis of osteopontin (Sakamoto et al. 2005). In conclusion, these studies clearly point out that Hcy alters the activity of osteoblasts and induces their apoptosis which further leads to bone deterioration.

4.4.3

Homocysteine Promotes Collagen Matrix Degradation

Collagen matrix has a very important role in providing protection against fractures. Hcy can cause bone loss by directly degrading the bone matrix. There are various studies which have shown the correlation between level of Hcy and bone matrix degradation. Lox and lysyl hydroxylase (Plod 2) expression is required for stable matrix production. Thaler et al. have shown that Hcy upregulates the expression of genes required for epigenetic DNA methylation which leads to increased CpG methylation of Lox promoter region resulting in its repression. This means that Hcy affects the bone matrix quality in a negative way by decreasing the Lox expression via epigenetic CpG methylation (Thaler et al. 2011). Another study by the same group has shown that Hcy also decreases the expression of Plod 2 gene along with Lox gene (Thaler et al. 2010). When HHcy was induced in rats it was observed that 65% of the bone Hcy was found bounded with the collagen of extracellular matrix which was positively correlated with a distinct reduction in cancellous bone and bone strength (Herrmann et al. 2009). Holstein et al. also reported that 48% of the bone Hcy was bounded with the collagen of the extracellular matrix (Holstein et al. 2011). From histomorphometry studies, it was further observed that a high concentration of bone Hcy is associated with an increase in trabecular separation and a decrease in trabecular thickness, trabecular number, and

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trabecular area (Holstein et al. 2011). Osteoblasts treatment with Hcy leads to decreased collagen cross-linking (Thaler et al. 2013). Hcy treatment not only causes the collagen matrix disorganization but also decreases its ability to support mineralization (Khan et al. 2001). Fluorescent spectroscopy of bone matrix revealed homocysteine accumulation in the bone led to broken collagenous cross-links (Milovanovic et al. 2017).

4.4.4

Homocysteine Effect on Vasculature

Proper blood flow is required for normal physiological functions of the bone (Fleming et al. 2001). 10% of the total cardiac output is received by the bone which is used for maintaining bone remodeling, cellularity, and repair. In bone, blood is first supplied to the endosteal cavity by nutrient arteries. From nutrient arteries blood then flows through marrow sinusoids and finally takes exit into the small vessels that branched through the cortex. Various vascular niches are present in the marrow cavity which regulate the differentiation and growth of several hematopoietic and stromal cells. Various hormones like parathyroid hormone and other regulatory factors exert their osteogenic effect through the vasculature (Marenzana and Arnett 2013). Blood flow to bone is affected by various mechanisms like vasoconstriction/relaxation, sympathetic activity, and through various metabolites like NO and hormones (Vacek et al. 2013). Inadequate blood supply to bone is associated with bone loss. A study by Tyagi et al. has shown that elevated levels of Hcy in plasma can cause osteoporosis by altering the blood supply to bone. They treated Sprague-Dawley rats with Hcy and at the end of the experiment they observed that Hcy levels were significantly higher in the treated rats as compared to control groups. There was no difference in the blood pressure of the control and treated rats but the tibial blood flow of the control group was much higher than the Hcy-treated group. This study thus shows that Hcy decreases blood flow to bone which might be the reason for compromised bone biomechanical properties and osteoporosis (Neetu et al. 2011). Blood flow rate is responsible for changes in BMD which is already proven through observed bone loss in space flights. In space flights BMD is reduced at a rate of 1% and 1.5% per month from the lumbar spine and hip respectively. It is observed that tissue fluid shifts due to microgravity which is responsible for changes in BMD in space (McCarthy 2005). Thus shifts in tissue fluid supply like blood can modulate the BMD. NO is a very important factor in the regulation of bone. NO modulates the process of osteoblastogenesis and osteoclastogenesis and provides an environment for bone growth. It is observed that NO in an autocrine and paracrine manner suppresses the osteoclast-mediated bone resorption both in vitro and in vivo. In osteoclasts inducible isoform of NO synthase (iNOS) is responsible for autocrine production of NO. mRNA levels of iNOS are regulated by Ca2+ and phorbol 12-myristate 13-acetate (PMA). NO produced in osteoclasts by iNOS acts as a Ca2+ sensing signal to prevent osteoclast-mediated bone resorption (Sunyer et al. 1997). Hcy increases the oxidative stress by inducing generation of ROS resulting in decreased bioavailability of NO (Banfi et al. 2008). The decrease in NO availability due to Hcy-induced

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oxidative stress leads to decrease in blood flow and thus increases the risk of osteoporosis (Sánchez-Rodríguez et al. 2007). When HHcy is induced in heterozygous CBS+/ mice it was observed that the CBS+/ mice had decreased tibial blood flow rate. The mice also have increased levels of Nox4 and MMP-9 proteins and had reduced levels of thioredoxin and endothelial NOS (eNOS) proteins which resulted in decreased bioavailability of NO in CBS+/- mice. This study thus shows that HHcy reduces the bioavailability of NO in bone tissues which further decreases the blood flow resulting in bone resorption (Tyagi et al. 2011a, b). As HHcy decreases the NO bioavailability, NO donor therapy is considered for preventing bone loss (Wimalawansa 2010). Estrogen can also diminish the effect of HHcy on vasculature Premenopausal women have low risks of coronary artery diseases and atherosclerosis as compared to the post-menopausal women as estrogen exerts its beneficial effects on vasculature through NO/cyclic guanosine monophosphate (cGMP) pathway (Dimitrova et al. 2002).

4.4.5

Homocysteine Promotes Activation of Matrix Metalloproteinases

In mitochondria, ROS is produced which regulates the activity of MMPs (Fu et al. 2001). MMPs have a very important role in the bone remodeling and repair. Impaired activity of MMPs leads to various complications in bone healing. It is observed that the level of MMPs (MMPs-7 and 12) is significantly enhanced at the site of hypertrophic fracture non-union tissue as compared to the mineralized fracture callus (Fajardo et al. 2010). It is observed that mice having the null mutation in MMP-9 gene have abnormal pattern of skeleton growth plate vascularization and ossification resulting in lengthening of the growth plate at about eight times of the normal (Vu et al. 1998). Bone resorption due to MMPs is observed in rheumatoid arthritis (RA) and osteoarthritis (OA). Elevated levels of MMPs like MMP-1, MMP-2, MMP-3, MMP-9, and MMP-13 are reported in both RA and OA. These MMPs result in degradation of non-collagen matrix components of the joints (Burrage et al. 2006). Thus MMPs have an essential role in skeleton development. HHcy is also found to be associated with an increase in activation of MMPs (Moshal et al. 2006). HHcy increases the activation of MMPs by preventing the stimulation of tissue inhibitor matrix metalloproteinase 1 (Timp-1) which inhibits MMP-9. HHcy also alters the methylation-hydroxymethylation of MMP-9 resulting in increased MMP-9 transcription (Mohammad and Kowluru 2020). Lee et al. showed that Hcy induces the MMP-9 activation by separately stimulating the extracellular-signalregulated kinase (ERK) and protein kinase B (AKT) signaling pathways (Lee et al. 2012). Solini et al. reported that Hcy activates the MMP-TIMP pathway (Solini et al. 2006). Moshal et al. showed that Hcy activates the MMP-9 by stimulating the calpain-1. In Ca-dependent manner Hcy induces calpain-1 and then translocates the active calpain from cytosol to mitochondria. Further in mitochondria calpain-1 activates the MMP-9 (Moshal et al. 2006). It is observed in cardiomyocytes and microvascular endothelial cells that increase in Ca overload and oxidative stress via

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activation of NMDA-R results in generation of ROS which then activate the MMPs (Moshal et al. 2008). NMDA-R is also expressed by the bone cells (Itzstein et al. 2001) and Hcy can also activate the NMDA-R (Poddar and Paul 2009). Thus it can be possible that Hcy by agonizing the NMDAR in osteocytes increases the intracellular Ca and calpain 1 expression which thereby results in activation of MMPs. As MMPs have a prominent role in bone resorption it is considered that MMP inhibitors can provide a treatment option for bone healing (Pasternak and Aspenberg 2009).

4.4.6

Effect of Homocysteine and Deficiency of Vitamin B12 and Folate on Bone

Vitamins and Hcy have interconnected metabolism. Hcy is derived from amino acid methionine. Methionine is first metabolized into S-adenosylmethionine which then acts as a methyl donor. After donation of the methyl group S-adenosylmethionine becomes S-adenosylhomocysteine which later on is converted into Hcy. Vitamin B6 can convert Hcy into cystathionine and then further into cysteine. Alternatively, B12 can remethylate the Hcy to methionine with the help of folic acid which as methyltetrahydrofolate becomes the methyl donor. The resulting tetrahydrofolate again gets reconverted into methyltetrahydrofolate in various steps using the enzyme methylenetetrahydrofolate reductase (Swart et al. 2013). As vitamins are an important part of Hcy metabolism their deficiency leads to the increase in Hcy level which then further promotes bone loss. It is observed that HHCy is associated with decreased levels of vitamins. Vaes et al. showed that deficiency of vitamin B12 induces bone loss by enhancing the levels of Hcy and methylmalonic acid (Vaes et al. 2009). There are various other studies that have shown the correlation between folate, vitamin B12, Hcy, and BMD. It is reported that post-menopausal women with low BMD have increased level of Hcy and decreased levels of vitamin B12 and folate (De Martinis et al. 2020). Decreased levels of vitamin B12, B6, and folic acid with HHcy are also observed in periodontitis (Stanisic et al. 2021). Rutten et al. demonstrated that a high level of Hcy and a decreased level of vitamin B12 are significantly correlated with an increased risk of fracture (Dhonukshe-Rutten et al. 2005). Morris et al. also concluded that Hcy and vitamin B12 are associated with BMD in older Americans (Morris et al. 2005). Another study reported that elevated level of Hcy and decreased level of folate are associated with low BMD in women but not in men (Gjesdal et al. 2006). However, Cagnacci et al. reported that low folate level but not enhanced Hcy and decreased vitamin B12 is associated with low vertebral BMD in postmenopausal women (Cagnacci et al. 2008). In line with this, a similar study has shown that dietary intake of folate but not vitamin B2 and B12 is positively correlated with BMD (Rejnmark et al. 2008). The above studies have shown that vitamins deficiency results in Hcy-mediated bone loss. Thus, to overcome the effect of vitamin deficiency on bone loss vitamins supplementation is considered by various researchers. But it is observed that the results of vitamins supplementation are not fruitful. It is reported that supplementation of folate, vitamin B6 and B12 for 2 years reduced plasma Hcy level but had no positive effect on the

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bone turnover (Green et al. 2007). Hermann et al. reported that short-term folate supplementation in non-osteoporotic patients having low folate does not affect bone markers but decreases the Hcy (Herrmann et al. 2006). In osteoporotic patients also it is observed that vitamin B supplementation lowers Hcy but has no persistent effect on bone turnover or BMD (Herrmann et al. 2007). Similarly in elderly women dietary supplementation of vitamin B12 and folate decreases Hcy but has no beneficial effect on bone turnover (Keser et al. 2013). Similar results were also observed by Ferdows et al. (Shahab-Ferdows et al. 2012). But Hermann et al. showed that vitamin B12 supplementation along with vitamin D3 for 1 year improved metabolic bone markers. Vitamin D3 with or without or vitamin B12 improves bone markers. Vitamin B12 does not affect the bone markers but decreases Hcy (Herrmann et al. 2013). Thus supplementation of vitamin D3 along with vitamin B12 can be considered for enhancing bone health and decreasing Hcy.

4.4.7

Estrogen and Homocysteine

Estrogen deficiency leads to post-menopausal bone loss and thus estrogen is required for maintaining bone health. One of the mechanisms by which estrogen prevents bone loss is by regulating Hcy. It is observed that oral administration of estrogen decreased Hcy in elderly men (Giri et al. 1998). Estrogen replacement therapy considerably decreased the total Hcy in postmenopausal women (Mijatovic et al. 1998; van Baal et al. 1999). Premenopausal women have low serum concentration of Hcy with respect to men of the same age. Hcy concentration also gets decreased in men (aged more than 55) administered with estrogen (Morris et al. 2000). HHcy inhibits the beneficial activity of NO (Pruefer et al. 1999). NO is required for normal functioning of the osteoblasts (van’t Hof et al. 2004). Estrogen induces NOS activity in osteoblasts by upregulating the expression of eNOS. It is reported that estrogen replacement therapy enhanced the level of NO in postmenopausal women (Best et al. 1998). This means estrogen mediates its function on bone via NO (Armour and Ralston 1998). There are various other studies which have shown that estrogen enhances NO oxide synthesis (Giri et al. 1998; McNeill et al. 2002). Estrogen increases the production of hydrogen sulfide (H2S) by inducing the activity of cystathionine γ-lyase enzyme which synthesizes H2S (Panza et al. 2015). H2S has antioxidant properties and has been found to prevent Hcy-induced oxidation which leads to bone loss (Zhai et al. 2019). Estrogen modulators (SERMs) like bazedoxifine are considered for the treatment of HHcy and have been found to prevent Hcy-mediated apoptosis of osteocytes (Notsu et al. 2019). It is observed that administration of intranasal 17beta-estradiol reduced plasma homocysteine levels after 6 months of the treatment (Harma et al. 2005). As estrogen has such an important role in regulating the concentration of Hcy, deficiency of estrogen leads to HHcy. Thus one of the mechanisms through which Hcy induces bone loss is by impairing the activity of estrogen. It is observed that a high level of Hcy is correlated with the increased methylation of ER-α gene (Huang

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et al. 2009; Lv et al. 2011; Sun and Qu 2019) and because of the hypermethylation of the ER-α, estrogen is unable to exert its beneficial effects on the bone.

4.4.8

Homocysteine and Gut Microbiota

Gut microbiota consists of almost 100 trillion microbes that are evolved to symbiotically habituate in the human gut (Yatsonsky Ii et al. 2019). Gut microbiota has very important contribution in the regulation of metabolic health of human hosts. Gut microbiota regulates immune system, digestive system, intestinal permeability, gut endocrine function, neurological signaling, etc. Generally, gut microbiota regulates various body functions but alteration in gut microbiota composition can lead to several clinical manifestations such as obesity, malnutrition, type 2 diabetes, non-alcoholic liver disease, and cardiometabolic diseases (Fan and Pedersen 2021). Modification in gut microbiota composition is termed as dysbiosis. Dysbiosis is also reported in osteoporosis (Xu et al. 2020). HHcy is also found to cause dysbiosis through several mechanisms. HHcy causes destruction in the epithelial barrier by inducing inflammatory and oxidative damage. Damage in the epithelial barrier leads to an increase in intestinal permeability (Liang et al. 2018). It is observed that an increase in intestinal permeability promotes dysbiosis which enhances the production of various inflammatory cytokines (TNF, RANKL, and IL-17) in the small intestine and bone marrow. These inflammatory cytokines then cause osteoporosis (Li et al. 2016). Bone loss due to HHcy-induced dysbiosis is also observed in periodontitis (Stanisic et al. 2021). Induction of vitamin B12 deficiency is another mechanism by which Hcy alters gut microbiota. HHcy inversely affects the level of vitamin B12 (McMullin et al. 2001). High levels of Hcy induce vitamin B12 deficiency. In dextran sodium sulfate (DSS)-induced colitis it is observed that vitamin B12 deficiency significantly alters the gut microbiota (Lurz et al. 2020). Thus we can conclude that HHcy promotes dysbiosis and HHcy-induced dysbiosis might be one of the reasons through which Hcy causes bone loss in osteoporosis. Thus, further studies are still needed to determine the exact role of Hcy-induced dysbiosis in bone loss.

4.5

Treatment Options for Hyperhomocysteinemia-Induced Bone Loss

Bone loss in HHcy is a major problem and requires effective treatment options. Various strategies are used for preventing bone resorption in HHcy. Enzyme replacement therapy (ERT) is one of them. Administration of ERT inhibits bone loss and prevents modifications in bone composition in mice knockout for CBS. CBS enzyme is required for catalyzing the conversion of homocysteine into cystathionine (Majtan et al. 2018). Curcumin which has antioxidant property also inhibits HHcy-induced osteoclastogenesis. Curcumin suppresses osteoclastogenesis by inhibiting the stimulatory effect of Hcy on osteoclasts formation (Kim et al.

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2011). Antioxidants like H2S can also reverse the effect of Hcy on bone resorption. H2S prevents Hcy-facilitated suppression of osteoblastogenesis. Treatment of MC3T3 osteoblastic cells with H2S donor (NaHS) prevents Hcy-mediated mitochondrial toxicity, restore ATP production, oxygen consumption, and mitochondrial copy numbers in osteoblasts (Zhai et al. 2019). It is also reported that treatment with NaHS regularizes plasma Hcy level and further attenuates Hcy-induced bone loss. Hcy causes epigenetic DNA hypermethylation of OPG gene which results in stimulation of RANKL-expedited osteoclastogenesis. But NaHS treatment suppresses osteoclastogenesis and further bone resorption and thus H2S can be a potential therapy for the management of HHcy (Behera et al. 2018). SERMs have an antioxidative property and can mitigate Hcy-induced bone fragility. Bazedoxifene which is an estrogen modulator significantly prevents Hcy-induced apoptosis of osteocytes by inhibiting the expression of IL-1β, IL-6, Nox1, and Nox2 via estrogen receptors in osteocytes (Notsu et al. 2019). Statins which are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase are rate-limiting enzymes required for endogenous cholesterol synthesis which are also considered for preventing Hcy-induced bone fragility. Statins stimulate bone morphogenetic protein-2, induce bone formation and mineralization as well as suppress apoptosis of osteoblasts. Simvastatin which is a statin is found to prevent Hcy-induced apoptosis of osteocytes by inhibiting stimulation of Hcy-mediated stimulation of Nox1 and Nox2 and thus can be considered for preventing damaging effects of Hcy on the apoptosis of osteocytes and resulting bone pathologies (Takeno et al. 2016) (Fig. 4.4).

4.6

Probiotics as Treatment Option for Homocystinuria

There is no cure available for HCU and HHcy till date. HCU patients are dependent on vitamin B6 supplements but vitamin B6 supplement is not an effective treatment of HCU as most of the people don’t respond to them. Thus there is an urgent requirement of potential therapy for the control and management of HCU and HHcy. Probiotics are used for the treatment of various diseases like osteoporosis, inflammatory bowel disease, diabetes, etc. Many studies have also tested the potential of probiotics in the treatment of HHcy. It is observed that the consumption of probiotic Lactobacillus acidophillus in yogurt matrix by Egyptian children for 42 days significantly enhanced the levels of vitamin B12 and folate and decreased the level of plasma Hcy and urinary methylmalonic acid (Mohammad et al. 2006). Supplementation of milk fermented with Lactobacillus plantarum in postmenopausal women with metabolic syndrome significantly decreased the glucose and Hcy levels (Barreto et al. 2014). Probiotic fermented milk (Kefir) is also found to reduce the Hcy concentration (Alihosseini et al. 2017). Probiotics administration can also prevent the Hcy-mediated induction of inflammation and oxidation which leads to an increase in gut permeability (Liang et al. 2018). Similarly in obese women supplementation of multispecies probiotics consisting of nine probiotics for 12 weeks significantly reduced the Hcy (Majewska et al. 2020). Probiotics can

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Fig. 4.4 Potential therapies for the treatment of HHcy

prevent the Hcy-mediated cardiac dysfunction (George et al. 2020). Probiotics are suggested for the treatment of HHcy-induced dysbiosis which can be one of the potential reasons for periodontitis (Stanisic et al. 2021). Probiotics capability in reducing the levels of Hcy is also shown by various other studies (Anwar et al. 2012; Wang et al. 2017; Tillmann et al. 2018; Skrypnik et al. 2020). There are several mechanisms through which probiotics prevent HHcy. Roth et al. reported that 2 carbon folate cycle of probiotic Lactobacillus reuteri is able of transferring 2 carbon atoms to Hcy to form the amino acid ethionine which is a known immunomodulator (Röth et al. 2019). Thus converting the Hcy into ethionine, probiotics can reduce the Hcy level. Brasili et al. reported that Lactobacillus acidophilous La5 and Bifidobacterium lactis Bb12 treatment in aged and adult mice enhanced dimethylglycine in adult and aged mice. This study showed that probiotics can decrease the Hcy by modulating the Hcy pathway. Probiotics are able to synthesize and secrete folates in the intestinal environment. Increased level of folic acid decreases the Hcy which can be another mechanism by which probiotics can prevent HHcy (Taki et al. 2005; Strozzi and Mogna 2008). Valentini et al. showed that supplementation of probiotic mixture VSL#3 to healthy older people

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improved vitamin B12 and folate concentrations and reduced Hcy in people having low-grade inflammation (Valentini et al. 2015). Probiotics have antioxidant property (Wang et al. 2017) and thus they can also prevent HHcy by reducing the oxidative stress. Thus, probiotics can be a potential therapeutic option for the treatment and management of HCU.

4.7

Conclusion

An elevated level of Hcy results in various clinical manifestations leading to the impairment of normal physiological functions. Data from various retrospective and prospective studies have shown a positive correlation between a higher concentration of Hcy and bone pathologies. Several studies have evidenced that Hcy promotes osteoclastogenesis, inhibits osteoblastogenesis, degrades collagen matrix, activates MMPs, and decreases blood supply to bone tissue but still various aspects of Hcy-mediated bone resorption are unrevealed. There is no possible cure for HCU and HHcy. B-vitamins and folate supplements are the generally recommended treatments. Although vitamin supplements decrease the Hcy level, they are not found to be successful in preventing Hcy-induced bone fragilities. Recently, it is observed that probiotics can effectively reduce the Hcy concentration and thus can be the potential therapy for HHcy. Results from our lab have also shown that probiotics attenuate bone loss and thus further research for the same is warranted. Acknowledgments This work was financially supported by projects: DST-SERB (EMR/2016/ 007158), Govt. of India, Intramural project from All India Institute of Medical Sciences (AIIMS), New Delhi-India (A-596), and AIIMS-IITD (AI-15) collaborative project sanctioned to RKS. AB, LS, BV, and RKS acknowledge the Department of Biotechnology, AIIMS, New Delhi-India for providing infrastructural facilities. AB thanks DST SERB and LS thanks UGC for research fellowships. Figure are created with the help of https://smart.servier.com. Author Contributions RKS contributed in conceptualization and writing of the manuscript. AB and LS participated in writing and editing of the review. BV provided valuable inputs in the preparation of the manuscript. AB created the illustrations. Conflicts of Interest The authors declare no conflicts of interest.

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Biofortification: A Remedial Approach Against Malnutrition in Rural and Tribal Population Amit Kumar Goswami, Suneha Goswami, T. Vinutha, Sanjay Kumar Singh, and Shelly Praveen

Abstract

Stunting, wasting, being underweight, mineral and vitamin deficiency, obesity, overweight, and other diet-related noncommunicable diseases are all examples of malnutrition. A deficiency of critical micronutrients such as iron, zinc, folate, and vitamins causes malnutrition or hidden hunger. In the undernourished population, a lack of folate and vitamin B12 elevates homocysteine levels in the blood, leading to hyper-homocysteinemia, which causes a range of heart problems. Malnutrition is common in rural and tribal communities due to a lack of information, availability to healthy food, and illiteracy. Biofortification of essential micronutrients in staple crops is a successful technique for alleviating micronutrient insufficiency in such populations. Plant breeding, transgenic technology, and genome editing can help to increase the density of essential minerals and vitamins in staple foods. Keywords

Biofortification · Essential micronutrients · Genome editing · Hidden hunger Homocysteine · Hyperhomocysteinemia · Malnutrition · Undernutrition

A. K. Goswami · S. K. Singh Fruits and Horticultural Technology, ICAR-Indian Agricultural Research Institute, New Delhi, India S. Goswami (*) · T. Vinutha · S. Praveen Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi, India # The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2022 G. P. Dubey et al. (eds.), Homocysteine Metabolism in Health and Disease, https://doi.org/10.1007/978-981-16-6867-8_5

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Introduction

Nutrition is our first line of defense against disease as well as a source of energy for living a healthy lifestyle. Unfortunately, most poor countries face subtle issues such as an expanding population, insufficient food and nutrition, and lack of vital micronutrients and vitamins. Globally, more than 2 billion people, or one in every three people, suffer from micronutrient insufficiency or hidden hunger (FAO 2013). Its consequences can be grave, resulting in poor human health, mental impairment, low productivity, and even death. It has serious effects on the health and survival of a child, especially in the first 1000 days of life, from conception to the age of two, resulting in serious repercussions such as morbidity, mortality, physical, and mental impairments. Due to the chronicity of energy-protein malnutrition, which affects around one-third of all children worldwide, the rate of childhood stunting and wasting is highest in India’s rural and tribal population (International Institute for Population Sciences 2016). According to the Indian Government’s National Health and Family Survey (2015–2016), roughly 27% of women and 23% of men in rural India are malnourished (Verma and Kumar 2019). According to data from the Comprehensive National Nutrition Survey (2016–2018), 34.7% of children under the age of five are still short for their age (stunted growth), and 33.4% are underweight (Kumar and Kumar 2020). Anemia affects 72–80% of pre-school children (6–35 months) and 51–59% of women.

5.1.1

Malnutrition and Nutritional Imbalance

5.1.1.1 Iron Anemia in women and children is thought to be caused by iron deficiency in roughly half of the cases. It is the single most important determinant in the development of anemia.

5.1.1.2 Zinc Zinc is another essential element that is often lacking in underdeveloped countries, particularly in rural and tribal areas (UNICEF 2015). Zinc insufficiency has been linked to poor maternal health, gastrointestinal problems, and immune system problems, particularly in children and women.

5.1.1.3 Folate In addition to Fe and Zn deficiencies, folate deficiency is also a common micronutrient malnutrition. Folate deficiency is diagnosed by measuring the amount of folate in blood plasma (3 ng ml 1) and erythrocytes (140 ng ml 1) (Blount et al. 1997). Folate deficiency is linked to a variety of diseases and conditions, including hyperhomocysteinemia.

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5.1.1.4 Homocysteine The amino acid homocysteine is found in the blood. Homocysteine levels in the blood are normally less than 15 micromoles per liter (μmol/L). Hyperhomocysteinemia is a high level of this amino acid that causes arterial damage and blood clots in the blood vessels, making it a risk factor for heart disease. A high amount of homocysteine is linked to a lack of folate. In the presence of the enzyme methionine synthases, 5-methyltetrahydrofolate and cofactor vitamin B12 convert homocysteine to methionine. As a result, a lack of folate and vitamin B12 is a factor in the formation of increased levels of homocysteine, which is linked to cardiovascular disease (CVD). Several studies have linked malnutrition to raised homocysteine levels and, as a result, an increased risk of CVD. The main cause of folate insufficiency, and thus hyper-homocysteinemia, is a diet that is low in fresh fruits, vegetables, and fortified cereals. Due to micronutrient deficiencies, almost two billion people, especially in underdeveloped nations, are suffering from hidden hunger (FAO 2013). In India, tribal people account for around 8% of the overall population. Because of their geographical seclusion, socioeconomic disadvantages, and inadequate health services, they are particularly vulnerable to malnutrition. Undernutrition in the tribal population is mostly due to the uncertainty in the food supply, which leads to a variety of healthrelated concerns as well as major long-term effects for child development, which in turn influences the nation’s development. Around 44% of tribal children under the age of five are stunted (their height is below average for their age), 45% are underweight (their weight is below average for their age), and 27% are wasted (low weight for height). Hidden hunger in the adult population has serious economic consequences in terms of health and productivity. In India, the economic impact of micronutrient insufficiency is 2.4% of GDP, or $15–46 billion dollars (Ritchie et al. 2018). Micronutrient deficiency causes hidden hunger, which affects persons who appear to be eating a sufficient amount of food but of poor nutritional quality. Micronutrient deficiencies such as vitamins (A, B6, B12, and folate), iodine, iron, zinc, and selenium are common in malnourished people. Undernutrition has a variety of reasons, including immediate (inadequate diet and disease), underlying (family food insecurity, poverty, and limited access to health care), and basic factors (overall social, political, and economic environment). Additional problems such as prejudice, limited access to public services, cultural differences, and others contribute to indigenous people’s poor health. Aside from government provision of critical services, further efforts are needed to address some of the issues that the indigenous people face, as well as to improve their access to these services in nutrition-related areas. Recognizing the problem of malnutrition among tribal people, the Indian government launched a number of programs to help them. The Indian government has initiated various measures in the last decade or so to reach out to tribal people and offer them with public health, education, and nutrition programs, all of which are critical for addressing the primary causes of malnutrition. Setting up Anganwadi and mini-Anganwadi centers under the Integrated Child Development Services (ICDS) scheme, or health centers under the National Health Mission (NHM) are examples. Some states have also introduced state-specific tribal-specific schemes, such as

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Maharashtra’s APJ Abdul Kalam Amrut Aahaar Yojana, which includes a full-meal scheme for pregnant and lactating women, a Village Child Development Centre for severely undernourished children, a mid-day meal scheme in schools, and the POSHAN Abhiyaan, among others. However, a lack of basic infrastructure and human resources impedes the successful implementation of these programs, and undernutrition remains a serious problem in the country.

5.2

Biofortification as a Strategy to Address Malnutrition and Hidden Hunger

Due to a lack of nutrient-dense foods and low dietary diversity, a huge portion of the world’s population is deficient in micronutrients. The greatest technique for preventing micronutrient deficiency is to use food-based approaches, such as dietary improvement or modification and fortification. The delivery of micronutrient supplements via pharmaceutical preparations and food fortification are the most extensively used ways for preventing hidden hunger. The process of intentionally boosting critical micronutrient content, such as minerals and vitamins, in a portion of food to improve the nutritional quality of the food supply and give a public health benefit with low risk to health, is known as fortification. Iodized salt, vitamin A and D fortified milk, and omega-3 fatty acid enhanced eggs are just a few examples. Furthermore, agronomic approaches, conventional breeding, or advanced biotechnological tools such as genetic engineering and genome editing can be used to biofortify food crops to improve their nutritional quality. The goal of biofortification varies from that of conventional fortification in that it improves nutrient levels in crops during plant growth rather than by manual means during agricultural processing. Supplementation and traditional fortification initiatives may be difficult to undertake and/or limited in populations where biofortification may be an option. As a result, biofortification is a food-based technique for addressing widespread deficits of critical micronutrients including vitamin A, iron, and zinc, which are most prevalent in impoverished nations. Biofortification is largely aimed at the rural and tribal poor, who rely significantly on locally produced staple foods as their primary source of nourishment and have limited access to commercially processed fortified foods due to financial or market constraints. Cereals, as a staple food, provide up to 55% of dietary energy to rural and tribal people. Rice, wheat, maize, and millets like pearl millet, ragi, and others are regularly consumed by this community. These cereal grains are usually low in important micronutrients, resulting in hidden hunger and malnutrition among populations who rely entirely on them. Several studies are underway to create biofortified crops to supplement these cereal crops with critical micronutrients. For example, pearl millet, rice, and legumes can be biofortified with iron; wheat, rice, pearl millet, beans, and maize can be biofortified with zinc; rice, sorghum, and maize can be biofortified with pro-vitamin A carotenoid; and sorghum and maize can be biofortified with amino acids and proteins. Golden rice, which is high in pro-vitamin A, quality protein maize, iron and zinc biofortified pearl millet, zinc

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Table 5.1 Cereal crops biofortification by transgenic approach S No. 1

2.

3.

a

Cereal crop Rice

Wheat

Maize

Biofortification Beta-carotene Phytoene (precursor of beta-carotene Folate (Vit. B9) Phytic acids Iron

Zinc High amino acids and protein content Alpha-linolenic acid Flavonoids and antioxidants Resistant starch Provitamin A Carotenoids Iron Phytase or phytic acid Amino acid composition Anthocyanin Provitamin A carotenoids Vitamin C Vitamin E Multivitamin Lysine Lysine and tryptophan Phytase, ferritin (iron bioavailability) Phytate degradation BVLA4 30,101 (China))

References Ye et al. (2000); Beyer et al. (2002); Datta et al. (2003); Paine et al. (2005); Burkhardt et al. (1997) Storozhenko et al. (2007) Hurrell and Egli (2010) Takahashi et al. (2001); Lee and An (2009); Zheng et al. (2010); Lee et al. (2012); Trijatmiko et al. (2016); Goto et al. (1999); Vasconcelos et al. (2003); Lucca et al. (2002); Wirth et al. (2009); Masuda et al. (2012); Masuda et al. (2013) Lee and An (2009); Masuda et al. (2008) Zheng et al. (1995); Sindhu et al. (1997); Lee et al. (2003) Anai et al. (2003) Shin et al. (2006); Ogo et al. (2013) Liu et al. (2003); Itoh et al. (2003); Wei et al. (2010) Wang et al. (2014) Sui et al. (2012) Brinch-Pederson et al. (2000); Bhati et al. (2016) Tamas et al. (2009) Doshi et al. (2006) Aluru et al. (2008); Decourcelle et al. (2015) Levine et al. (88); Chen et al. (2003) Cahoon et al. (2003) Naqvi et al. (2009) Tang et al. (2013); Lai and Messing (2002) Aluru et al. (2011); Chen et al. (2008); Origin Agritech (China

Table derived from various sources

biofortified wheat and rice, and others are examples of effective biofortified cereal crops. Biofortification of cereal crops using a transgenic method is depicted in Table 5.1.

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Fig. 5.1 Biofortified horticultural crops for different essential nutrients such as pro-vitamin A, anthocyanin, and essential micronutrients like iron, zinc, and selenium

Other natural sources of micronutrients necessary for a healthy human body include fruits and vegetables, and horticulture production has the potential to provide nutritional security. Biofortification via breeding procedures or gene editing in transgenic crops can be used to develop nutrient-dense horticultural crops. Plant breeding programs today are primarily focused on high agronomic yields, and traditional methods such as diet diversification, supplementation, and industrial fortification are unable to address the problem. To meet the basic nutritional needs, an effective and comprehensive food system is required. Biofortification is a realistic and cost-effective way to provide micronutrients to people who may not have access to a variety diet or other micronutrient therapies. By boosting the proportion of dietary vitamin A, iron, and zinc – important micronutrients of health significance, biofortification of crop plants has significant potential in minimizing the gap between micronutrient requirements and intake. Biofortified horticulture crops have been created for several vital nutrients such as pro-vitamin A, anthocyanin, and essential micronutrients such as iron, zinc, and selenium, as shown in Fig. 5.1. While biofortified crops have the potential to improve the lives and health of millions of people, particularly the micronutrient-deficient rural and tribal populations, the program’s success is dependent on farmer and consumer acceptance as well as future policy interventions. Because of their nutritional importance, biofortification programs have been carried out in a variety of horticultural crops, including potato, sweet potato, cassava, sweet orange, bananas, cowpea, beans, pumpkin, and others (Table 5.2). The timeline of vegetable and fruit crops for biofortification of nutrients developed through breeding, transgenic, and genome editing is shown in Fig. 5.2.

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Table 5.2 Biofortified horticultural crops Crop name Potato Cowpea Lentils Tomato Lettuce Beans Onion Pumpkin Cabbage Brinjal Cauliflower) Cassava Banana Sweet potato a

Biofortified for the nutrient(s) Iron, zinc Iron, zinc Iron, zinc Iodine Folate Iodine Iron, zinc Selenium Pro-vitamin A, carotenoids Anthocyanin β-Carotene β-Carotene Pro-vitamin A, carotenoids, iron Carotenoid, iron β-Carotene

Release year 2007 2008 2009 2011 2004, 2007 2011 2012 2012 2015 2016 2016 2016 2017 2019 2007, 2002, 2009, 2010

Table derived from various sources

5.2.1

Breeding Approaches

Biofortification of vegetable crops can be a realistic and cost-effective way to combat malnutrition/hidden hunger. In developing countries around the world, vegetable crops form a significant element of daily nutrition. Some mineral elements are frequently insufficient in vegetable crops. Thus, increasing the bioavailable concentration of micronutrients in vegetable crops via biofortification is a promising strategy in modern agriculture, as it can effectively contribute to alleviating micronutrient deficiency by providing more nutritious foods to more people with the use of fewer lands. Biofortification of vegetable crops is being developed in a number of locations throughout the world. The following are some successful examples of biofortified vegetable crops developed through a breeding technique.

5.2.1.1 Potato By using a traditional biofortification approach, the International Centre for Potato (CIP) in Lima, Peru, developed iron-rich potato cultivars in 2017, which had 11-30 ppm iron with low phenolic compounds and hence have greater iron bioavailability to the human body. 5.2.1.2 Orange Sweet Potato In 2002, CIP, Uganda (Harvest Plus), and the National Agriculture Research and Extension System (NARES) collaborated to produce and market a pro-vitamin A biofortified sweet potato in South Africa. On fresh weight, this biofortified orange sweet potato variety provides up to 7.4 mg/100 g β-carotene.

Fig. 5.2 Timeline of vegetable and fruit crops for biofortification of nutrients developed through breeding, transgenic, and genome editing approach

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5.2.1.3 Bio Cassava+ To combat vitamin A malnutrition in Nigeria and Kenya, the Donald Danforth Plant Science Center in Nigeria created high β-carotene cassava (7.6 ppm) through hybridization and selective breeding procedures, which was released in 2017.

5.2.1.4 Cowpea Pant Lobia-1 (82 ppm Fe and 40 ppm Zn) and Pant Lobia-2 (100 ppm Fe and 37 ppm Zn) are two early maturing high iron and zinc fortified varieties produced by G.B. Pant University of Agriculture and Technology, Pantnagar, India, and introduced in 2008 and 2010, respectively. In 2013 and 2014, Pant Lobia-3 (67 ppm Fe and 38 ppm Zn) and Pant Lobia-4 (51 ppm Fe and 36 ppm Zn) were released, respectively. In 2008 and 2009, EMBRAPA created three high-iron cowpea cultivars, which were released for commercial production in Brazil.

5.2.1.5 Beans Colombia’s International Center for Tropical Agriculture (CIAT) developed biofortified beans with high iron and zinc levels. The iron level of a common bean is typically around 50 ppm, whereas the biofortified beans created in 2012 and 2014 contained 83 ppm iron and 44 ppm zinc, respectively.

5.2.1.6 Cauliflower Biofortified β-carotene rich cauliflower (Pusa Beta kesari: 1000 g/100 g-carotene) was produced by the Indian Agricultural Research Institute, New Delhi and released in 2016. Its curds are small and yellow/orange in color. With a yield potential of 42.0 to 46.0 t/ha, the average marketable curds weight is 1.250 kg.

5.2.2

Transgenic Approach

Fruit biofortification has had rather limited success. Bananas (Musa spp.) are an economically important fruit crop farmed throughout the world’s tropical and subtropical climates, where vitamin A deficiency is the most common. Because some banana genotypes are high in pro-vitamin A carotenoids, bananas can be used as a convenient vehicle for pro-vitamin A administration.

5.2.2.1 Nutri Banana Queensland University of Technology (QUT) in Australia used a transgenic technique to generate pro-vitamin A (β-carotene), α-tocopherol, and iron-rich banana varieties, which were released in 2019. These biofortified bananas contain up to 20 ppm β-carotene, 2.6 mg/100 g iron, and higher levels of α-tocopherol.

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Biofortification Through Genome Editing

Where conventional approaches cannot considerably improve micronutrient content in crop plants, the application of modern biotechnological technologies provides an alternate strategy with a better success rate. Traditional breeding requires several generations and can’t be utilized to improve a single attribute. Genetic engineering and gene editing are precision technologies that can speed up the breeding process and improve agricultural traits dramatically. Recent advancements in genome editing research have accelerated and opened up potential strategies for improving crop yields and quality. Transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) are two genome editing approaches that can target any gene of interest. These systems, however, have several disadvantages, as they are expensive, time-consuming, and labor-intensive protocols. Clustered regularly interspaced short palindromic repeats (CRISPR)- associated protein9 (Cas9) is a new era of genome editing technology that uses an RNA-based genome editing technique that is straightforward, user-friendly, and cost-effective. Because of its ease of manipulation, great effectiveness, and wide use in functional studies of genes and genetic crop development, this technology is revolutionary in a variety of plant species. The CRISPR/Cas9 tool consists of a nonspecific Cas9 nuclease and a singleguide RNA that is unique to the target gene and leads Cas9 to a specific genomic site, where it creates double-strand breaks and then repairs them, resulting in insertion or deletion changes. In a wide range of agricultural crops, this is currently the most extensively used technology for reverse genetics and crop enhancement.

5.2.3.1 Vegetable Crops In 2014, the first CRISPR/Cas9-mediated genome editing was reported in a vegetable crop, tomato, targeting the ARGONAUTE7 (SlAGO7) gene involved in leaf formation (Brooks et al. 2014). Later, CRISPR/Cas9 altered genes for anthocyanin biosynthetic pathways in tomato, including anthocyanin 1 (ANT1) (Cermak et al. 2015), phytochrome interacting factor (SlPIF4), phytoene desaturase (SlPDS) (Pan et al. 2016), and phytoene synthase (PSY1) (Hayut et. 2017). The quality of potato starch is critical for food and many commercial product developments. The “waxy genotype” of potato, which produces exclusively amylopectin-containing starch, was produced by utilizing CRSIPR/Cas9 to mutate the granule bound starch synthase (GBSS) gene (Andersson et al. 2017). The starch analysis of genomeedited lines yielded only amylopectin and no amylose, indicating that all four alleles of the GBSS gene had been knocked out. Parthenocarpy is an important component in horticulture crop plants with agricultural value for many industrial reasons as well as direct eating quality, and the creation of parthenocarpic fruits is considered as a useful goal in the context of climate change and for keeping sustainable farming. Ueta et al. (2017) effectively introduced mutations in SlIAA9, a key gene driving parthenocarpy, and found that the regenerated mutants possessed seedless fruit, which is a characteristic of parthenocarpic tomatoes. Klap et al. (2016) employed the CRISPR/Cas9 gene – SlAGAMOUS-LIKE 6 (SlAGL6) deletion to develop

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parthenocarpic tomato and confirmed that the parthenocarpic phenotype was caused by a mutation in SlAGL6.

5.2.3.2 Fruit Crops CRISPR/Cas9 genome editing has been successfully developed in a variety of fruit crops, including apple, banana, orange, and kiwi for biotic and abiotic stress tolerance. CRISPR/Cas9 genome editing for biofortification of fruit crops is a new field with limited findings to date. The majority of the changes made with this method in fruit crops were for genes involved in the carotenoid synthesis pathway (Table 5.3). Wang et al. (2014) demonstrated gene editing in citrus by altering the phytoene desaturase (CsPDS) gene in the carotenoid production pathway. Apart from citrus, grape (Nakajima et al. 2017) and watermelon (Tian et al. 2017) have both been found to have CRISPR/Cas9 genome editing by targeting the phytoene desaturase gene (Table 5.3).

5.2.4

Advantage of Biofortification

Biofortification of basic and horticultural crops is a viable method for reaching huge populations of rural and tribal people. In the manufacture of biofortified foods, the repeated expenses are small after the initial outlay of capital. Biofortification of staple/horticultural crops is a low-cost, long-term solution for reaching tens of millions of people. There are numerous advantages to biofortification: Table 5.3 Biofortification through genome editing in horticultural crops Crop Tomato

a

Trait(s) Anthocyanin

Target gene(s) Anthocyanin 1 (ANT1)

Carotenoid

Phytoene desaturase (SIPDS)& Phytochrome interacting factor (SIPIF4) Phytoene synthase (PSY1) Granule bound starch synthase (GBSS) Phytoene desaturase (PDS)

Potato

Starch quality

Apple

Carotenoid

PDS and TFL1

Grape

Carotenoid & early flowering Carotenoid

Citrus Watermelon Kiwi fruit Strawberry

Carotenoid Carotenoid Carotenoid Carotenoid

Phytoene desaturase (Cs-PDS) Phytoene desaturase (C-PDS) Phytoene desaturase (Cs-PDS) Phytoene desaturase (Cs-PDS)

Table adapted from various sources

Phytoene desaturase (Vv-PDS)

References Cermak et al. (2015) Pan et al. (2016) Hayut et al. (2017) Andersson et al. (2017) Nishitani et al. (2016)

Nakajima et al. (2017) Wang et al. (2014) Tian et al. (2017) Wang et al. (2018) Wilson et al. (2019)

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1. Biofortification is focused on the staple and horticultural crops that rural/tribal inhabitants raise and eat. 2. There is only one-time investment to develop fortified seeds and latter maintain the germplasm and seeds from which nutrient-dense food produce. It is incredibly cost-effective and may be shared globally. 3. Biofortification is a viable strategy. 4. Biofortification has the potential to reach the country’s most vulnerable population, even if they live in a distant rural location with little resources and no access to commercially marketed fortified foods. 5. The biofortified crops produce higher yield, are environment friendly, and have the potential to provide nutrient-dense food to the world’s growing population.

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Part II Homocysteine Impairment and Various Disorders

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Homocysteine Metabolism Pathway Genes and Risk of Type 2 Diabetes Mellitus/Metabolic Disorders Rajesh K. Kesharwani, Govind Prasad Dubey, D. Jain, V. N. Mishra, Rajesh Dubey, and Rudra P. Ojha

Abstract

It became very important to assess the association of gene variants involved in homocysteine metabolism in susceptibility to metabolic disorders particularly type 2 diabetes mellitus, obesity, and other related traits. Whole-body inflammation is the major event of type 2 diabetes mellitus. Those people who are suffering from type 2 diabetes mellitus always suffer from the elevated level of cytokines in both age and sex groups. Several risk factors have been identified showing marked elevated levels of IL-1β, TNF-α, and hsCRP. The elevated level of pro-inflammatory cytokines produces high level of all the above inflammatory markers including other biological parameters. The present study is based on the investigation of various pro-inflammatory cytokines in different types of diabetes mellitus. Considering the various factors like age, sex, rural and urbal differences including dyslipidemia have shown strong correlation with hyperhomocysteinemia. The present study is a part of a comprehensive R. K. Kesharwani Department of Computer Application, Nehru Gram Bharati (Deemed to be University), Prayagraj, Uttar Pradesh, India G. P. Dubey (*) Faculty of Ayurveda, IMS, Banaras Hindu University, Varanasi, India D. Jain Department of Neurology, IMS, Banaras Hindu University, Varanasi, India V. N. Mishra Department of Cardiology, IMS, Banaras Hindu University, Varanasi, India R. Dubey Hawkins Point Partners, Boston, USA R. P. Ojha Department of Zoology, Nehru Gram Bharati (Deemed to be University), Prayagraj, India # The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2022 G. P. Dubey et al. (eds.), Homocysteine Metabolism in Health and Disease, https://doi.org/10.1007/978-981-16-6867-8_6

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collaborative program between various institutions considering the ethnic variation and biochemical markers. Keywords

Diabetes · Homocysteine · Insulin · Hyperglycemia · hsCRP · IL-6 · TNF-α · IL1β

6.1

Introduction

Diabetes mellitus is characterized by hyperglycemia, reduced level of insulin, and elevated level of counter-regulatory hormones (Stubbs et al. 1991; Consoli et al. 1990). Due to these conditions, there is a disturbance in homocysteine and methyl group metabolism (Fonseca et al. 1999; Nieman et al. 2011), where homocysteine (Hcy) is a sulfur-containing amino acid synthesized during methionine metabolism which is not a part of diet (Mudd et al. 1972; Mudd et al. 1979; Mudd et al. 1975). Both the disturbances in methyl group metabolism and patients who are insulin resistance and hyperinsulinemic presume to develop higher concentrations of homocysteine (Drzewoski et al. 2000). It is found to be significantly associated with various metabolic disorders. Increased level of homocysteine in blood is termed as hyperhomocysteinemia (HHcy) which is a well-known independent risk factor for cardiovascular diseases (McCully et al., 1969), diabetic complications (Okada et al. 1999; Meigs et al. 2001; Brazionis et al. 2008; Looker et al. 2003; Buysschaert et al. 2000), obesity (Jacques et al. 2001, Martos et al. 2006), and metabolic syndrome (Ntaios et al. 2011). HHcy is markedly involved with the above clinical groups through oxidative stress (Weiss et al. 2003), systemic inflammation (Hofmann et al. 2001), and endothelial dysfunction (Stamler et al. 1993). Methylenetetrahydrofolate reductase (MTHFR) synergistically acts with angiotensin-I-converting enzyme (ACE) to modulate type 2 diabetes mellitus risk (Mehri et al. 2010). As pointed out C677T polymorphism of MTHFR is the most important genetic variation associated with HHcy (Soinio et al. 2004). However, a little work has been done showing the association of variants of genes in homocysteine metabolism in the Indian population (Kumar et al. 2009; Misra et al. 2010). Limited studies have shown the association of MTHFR-rs 1801133 with type 2 diabetes mellitus and low-density lipoprotein (LDL-c) levels. Variation in genetic makeup and Hcy pattern due to ethnic differences (Helfenstein et al. 2005) may be the important reason for inconsistency in the association of MTHFR-rs 1801133 with type 2 diabetes mellitus. It is also pointed out that dietary habit can influence levels of homocysteine, and the Indian population with vegetarian diet have been shown to have higher levels of homocysteine (Kumar et al. 2005). Thus, the genes involved in homocysteine metabolism showed association with type 2 diabetes, obesity, and other related traits.

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Homocysteine Metabolism Pathway Genes and Risk of Type 2 Diabetes. . .

6.1.1

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Relationship Between Hyperhomocysteinemia and Type 2 Diabetes Mellitus

Individuals with type 2 diabetes mellitus may be prone to vascular complications or CHD since hyperglycemia leads to vascular dysfunction (Hurst et al. 2003; Fox et al. 2004). Cardiovascular disease is increased by 2–four fold, and more prevalent in type 2 diabetic patients than non-diabetics. Recently, in various studies it was shown that homocysteine is an independent risk factor for cardiovascular disease (Tarkun et al. 2003). Homocysteine metabolism is affected by various factors like renal damage, plasma vitamin levels that are related to diabetes. The use of some oral anti-diabetic drugs also lead to hyperhomocysteinemia. It was first shown by Wilcken and Wilcken in 1976 in a clinical study that coronary artery disease is directly correlated with higher levels of homocysteine. Condition in which there is an increased level of homocysteine in urine is called homocystinuria (BolanderGouaille 2005). Abnormal homocysteine levels appear to contribute to atherosclerosis in various ways, where there is a direct toxic effect that damages the cells lining the inside of the arteries, interference with clotting factors, and oxidation. It has been estimated that the cardiovascular disorders will surpass various diseases and will be the world’s leading cause of morbidity and mortality. Various biomarkers associated with coronary heart disease are high sensitive C-reactive protein (hsCRP), fibrinogen, platelet activator inhibitor (PAI-1), and homocysteine which have all shown evidence of predictive abilities. It has been proven in multiple case-controlled studies that homocysteine level is higher among patients with proven ischemic heart disease and premature coronary artery disease. Experimental studies suggest that Hcy is probably the first agent that interferes with endothelial function and forms cell generation that comes at a later stage of atherosclerosis (Welch et al. 1998a, b). Elevated levels of Hcy increase IL-6 production in monocytes, upregulate vascular cell adhesion molecules, and enhance monocyte adhesion (Silverman 2002). Chronic increase in the level of homocysteine leads to oxidative damage of vascular endothelial cells, increased proliferation of smooth muscle cells, and high oxidized low-density lipoprotein leading to atherosclerosis. A decrease in the vascular nitric oxide bioactivity due to an increase in oxidative stress was caused by hyperhomocysteinemia. A lot of studies have shown that homocysteine is associated with an increased risk of stroke (Coull et al. 1990; Perry et al. 1996; Verhoef et al. 1994), carotid artery atherosclerosis (Selhub et al. 1996), and coronary heart disease (Arnesen et al. 1995; Stampfer et al. 1992).

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Relationship Between Hyperhomocysteinemia in Diabetes Mellitus Leading to CHD

Oxidation is a basic part of our metabolism. During oxidation, many free radicals are produced which have an unpaired, nascent electron. Atoms of oxygen or nitrogen having central unpaired electrons are called reactive oxygen or nitrogen species (Finkel and Holbrook 2000). This may be harmful to the body and may cause peroxidation of membrane lipids, aggression of tissue membranes and proteins, or damage to DNA and enzyme (Husain). Due to an increase in ROS there may be related pathology, such as arthritis, hemorrhagic shock, coronary artery diseases, cataract, cancer, AIDS as well as age-related degenerative brain diseases (Parr and Bolwell 2000). Several mechanisms are responsible for the atherogenic processes triggered by hyperhomocysteinemia. The atherogenic process in hyperhomocysteinemia is mostly seen by oxidative stress through the formation of reactive oxygen species (ROS) due to which modification of the metabolic functions of endothelial and smooth muscle cells occurs (Perna et al. 2003). Homocysteine thiolactone, a very reactive internal thioester of HCY, forms atherogenic adducts with proteins and LDL (Jakubowski 1987; McCully et al. 1989). These adducts are avidly taken up by macrophages of the vascular wall, which leads to the formation of foam cells. The foam cell-induced formation of ROS maintains the oxidative stress and therefore the atherosclerotic alteration of the vessel wall. Furthermore, the generation of ROS supports the deactivation of nitric oxide (NO) which accelerates the atherogenic process (Stanger et al. 2001). HHcy is particularly a cardiovascular risk factor for diabetics. The prognosis for diabetics with HCY level >14 μmol/l is significantly worse than for diabetics with Hcy level 14 μmol/l than in diabetics with Hcy lower than this level The prevalence of diabetic retinopathy is also more than twice as high in patients with simultaneous Hhcy (>16 μmol/l) compared to normohomocysteinemic patients (Hoogeveen et al. 2000a, b).

6.1.3

Hyperhomocysteinemia and Nephropathy

It was estimated that about 20–30% of patients with type 1 or type 2 diabetes mellitus develop evidence of nephropathy, but in type 2 diabetes mellitus, a considerably smaller number of type 2 diabetic patients develop end-stage renal disorder. Impaired renal function is associated with higher plasma homocysteine concentration. It has been suggested in one recent study that homocysteine is an indicator of nephrosclerosis and is elevated because of insufficient renal excretion of homocysteine. The significance of hyperhomocysteinemia in type 2 diabetes is further complicated by multiple ways of considering impaired renal function and vitamin

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status (Luis et al. 2005). There is evidence saying that as the age increases homocysteine level increases (Selhub et al. 1993). Hyperhomocysteinemia in diabetic patients correlates the change in glomerular filtration rate (GFR) as well as the presence of microalbuminuria. Homocysteine concentrations have, however, been shown to correlate with the presence of diabetic peripheral neuropathy (Cohen et al. 2001) and have also been associated with the presence of autonomic neuropathy in patients with type 1 diabetes. The relationship between homocysteine levels and the presence or absence of macro and microvascular disease in patients with diabetes shows that, for the most part, homocysteine elevation in patients with diabetes mellitus only occurs when renal function deteriorates. Buysschaert et al. in 2000 have found a higher incidence of complications such as macroangiopathy and nephropathy in the population of 122 diabetics in the group with hyperhomocysteinemia in comparison to the patients with normal Hcy level.

6.1.4

Type 1 Diabetic Children with HHcy Level

Hyperhomocysteinemia is also one of the independent risk factors of CHD. Classical CHD risk factors are smoking, hypercholesterolemic arterial by perfusion, and diabetes (Mayer et al. 1996; Barton et al. 1998; Welch et al. 1998a, b; Genest et al. 1991). Several studies have been conducted showing elevated homocysteine both in type 1 and type 2 diabetic patients (Hultberg et al. 1991; Hofmann et al. 1998; Okada et al. 1999) associated with vascular complications. Few studies have been conducted among children population with evidence of type 1 diabetes and homocysteine metabolism (Reddy 1997; Osganian et al. 1999). Conflicting results are given indicating the association of Hcy with diabetic children (Pavia et al. 2000; Wiltshire et al. 2001; Mutus et al. 2001; Agardh et al. 2000; Targher et al. 2000). However, the level of Hcy is mainly associated with the level of serum folate and vitamin B12, in the blood. It is reported that plasma level of folate below 20–25 nmol/ L is responsible for a gradual increase in Hcy concentrations, thus there is direct involvement of Hcy with folate level (Okada et al. 1999; Pavia et al. 2000). Frosst et al. (1995) observed that initially in diabetic children including adolescents the level of Hcy concentration in normal can be increased in adulthoods particularly among those with renal impairment and having decreased blood folate levels mainly among individuals with specific genes having C677T mutation in MTHFR gene (Hultberg et al. 1997; Neugebauer et al. 1997). Thus children with diabetes may require folate supplements in order to prevent future cardiovascular diseases. Increased plasma total homocysteine concentration has been independently associated with an increased risk for cardiovascular events in type 2 diabetes mellitus patients (Soedamah-Muthu et al. 2005). The cardiovascular involvement in diabetes attributes to fluctuations in glycemic levels and risk factors such as systemic arterial hypertension, smoking, obesity, insulin resistance, microalbuminuria, and altered lipids and lipoprotein profile (DECODE Study Group, 2003). The excess generation

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of reactive oxygen species, platelet aggregation, smooth muscle cell proliferation, and thrombotic phenomena are linked with elevated Hcy which is responsible for endothelial dysfunction among diabetic patients (Vannucchi et al. 2009; Hofmann et al. 1998). Recently, it has been demonstrated that atherosclerosis develops even in mild impairment of glucose tolerance (Stehouwer et al. 1999). Studies have linked elevated plasma homocysteine with endothelial dysfunction, renal failure, and cardiovascular disorder (CVD) (Nourooz-Zadeh et al. 1995; Eckardstein 1998). In addition, studies are also available showing a strong association between homocysteine and pathophysiology of diabetes mellitus (Eckardstein 1998; Harker et al. 1974). Workers have reported that the status of plasma homocysteine level can predict future cardiovascular events, particularly in diabetics (Ebesunun et al. 2008). The present study has been designed to evaluate the level of plasma homocysteine, vitamin B12, folic acid, lipids, and lipoprotein profile including clinical and physical characteristics of type 2 diabetic patients belonging to different localities of Chennai (Tamil Nadu), Varanasi, and Mirzapur city for an early prediction of cardiovascular events among type 2 diabetes patients so that prevention and management program may be launched.

6.1.4.1 Methods Anthropometric measurement of all selected diabetic and non-diabetic subjects was done. Blood samples were collected in the morning after an overnight fast of 10–12 h into EDTA and fluoride oxalate bottle and these were immediately placed in an icepack bag. All the general biochemical parameters like fasting and postprandial glucose levels, LDL, HDL, cholesterol, and triglycerides were assessed using a fully automated biochemical analyzer. The blood samples were centrifuged using a centrifuge manufactured by REMI. The samples were stored at
20  C until analyzed for total homocysteine and lipoproteins. Serum folate, vitamin B6, vitamin B12, and other routine biochemical tests are particularly related to liver function and renal function. Plasma total homocysteine was estimated with the help of the homocysteine enzyme immunoassay kit protocol (Frantzer et al. 1998). Vitamin B6 & B12 and folate were estimated using HPLC. Twenty four hours urine sample was collected in patients to check the renal function (Tables 6.1, 6.2, and 6.3). Reduction in glycemic index and glycated hemoglobin is the main target in treating diabetes mellitus. The quantity of HbA1c reflects the mean blood glucose concentration over the two or three preceding months and is therefore an independent parameter of carbohydrate metabolism. Table 6.4 shows the glycemic index and homocysteine levels of 168 diabetic men and 194 non-diabetic subjects. It was observed that the level of fasting glucose was 141.68  39.62 and 125.73  28.90 when compared to non-diabetic men and women. And the plasma homocysteine levels in diabetic cases are elevated compared to non-diabetic subjects suggesting that poor metabolic control might be associated with type 2 diabetic cases and decrease in the glucose level can reduce the elevated homocysteine level.

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Table 6.1 Socio-cultural demographic characteristics of type 2 diabetic patients including duration of onset of diabetes Factors Age (years) Vegetarian (%) Non-vegetarian (%) Folate supplement (%) Alcohol consumption (%) Tobacco uses (%) Belonging to rural population (%) Belonging to urban population (%) Duration of diabetes (years)

Men (N ¼ 428) 52.98  9.42 61.34 38.66 12

95% (interval of limit of the mean) 52.07–53.89 – – –

Women (N ¼ 293) 59.64  8.75 81.62 18.38 9

95% (interval of limit of the mean) 58.62–60.66 – – –

43

–

4

–

58 17

– –

38 21

– –

63

–

79

–

12–16

–

10–13

–

Table 6.2 Socio-cultural demographic characteristics of non-diabetic patients including duration of onset of diabetes

Factors Age (years) Vegetarian (%) Non-vegetarian (%) Folate supplement (%) Alcohol consumption (%) Tobacco uses (%) Belonging to rural population (%) Belonging to urban population (%) Duration of diabetes (years)

Men (N ¼ 242) 60.85  8.04 68 32

95% confidence (interval of limit of the mean) 59.81–61.88 – –

Women (N ¼ 179) 63.78  5.36 84 16

95% (interval of limit of the mean) 63.09–64.47 – –

–

–

2

–

41

–

5

–

46

–

28

–

20

–

24

–

80

–

76

–

–

–

–

–

Table 6.5 shows the mean blood pressure in 428 diabetic men (143.75  24.22) and women (138.75  19.83) was elevated compared to non-diabetic men and women. This may suggest that there is a strong correlation between diabetic patients

Groups BMI Waist circumference (cm) Waist hip ratio

95% confidence interval of limit of the mean 26.96–32.72 97.43–100.31

1.03–1.04

Diabetic men (N ¼ 428) 29.84  5.01 98.87  14.93

1.04  0.08

0.96  0.06

Diabetic women (N ¼ 293) 27.11  4.39 87.23  9.82

0.95–0.97

95% confidence interval of limit of the mean 26.60–27.62 86.08–88.38

0.89  0.04

Non-diabetic men (N ¼ 242) 24.06  3.11 87.31  10.98

Table 6.3 Anthropometric indices determined in type 2 diabetes and non-diabetic patients

0.88–0.90

95% confidence interval of limit of the mean 23.67–24.46 85.90–88.72

0.83  0.06

Non-diabetic women (N ¼ 179) 21.64  3.78 81.64  6.32

0.82–0.83

95% confidence interval of limit of the mean 21.08–22.21 80.70–82.58

122 R. K. Kesharwani et al.

Groups Fasting blood glucose (mg/dl) Postprandial blood glucose (mg/dl) HbA1C (%) Plasma tHcy (μmol/L)

95% confidence interval of limit of the mean 133.42–149.94

245.43–267.42

7.04–8.25 16.68–17.95

Diabetic men (N ¼ 92) 141.68  39.62

256.42  52.73

7.61  2.73 17.31  3.08

8.15  2.13 15.93  4.11

228.73  49.84

Diabetic women (N ¼ 76) 125.73  28.90

7.66–8.64 14.98–16.99

217.29–240.17

95% confidence interval of limit of the mean 119.10–132.26

5.05  1.32 10.61  4.19

133.01  28.98

Non-diabetic men (N ¼ 108) 88.73  9.42

4.79–5.30 9.23–11.99

127.43–138.59

95% confidence interval of limit of the mean 86.92–90.54

4.84  2.01 8.93  3.68

129.84  31.09

Non-diabetic women (N ¼ 86) 78.35  7.82

Table 6.4 Blood glucose level, glycosylated hemoglobin in relation to tHcy measured in type 2 diabetes and non-diabetic patients

4.41–5.27 8.14–9.72

123.14–136.55

95% confidence interval of limit of the mean 76.66–80.04

6 Homocysteine Metabolism Pathway Genes and Risk of Type 2 Diabetes. . . 123

Groups Systolic BP (mmHg) Diastolic BP (mmHg) Hypertension (%)

Women (N ¼ 293) 138.75  19.83

92.75  3.94

52

Men (N ¼ 428) 143.75  24.22

93.45  4.11

68

Diabetic Men 141.41– 146.09 93.05– 93.85 –

Women 136.43– 141.07 92.29– 93.21 –

95% confidence interval of limit of the mean

46

85.26  3.71

Non-diabetic Men (N ¼ 242) 128.42  8.23

32

86.31  2.87

Women (N ¼ 179) 131.95  6.84

Table 6.5 Level of blood pressure and percentage hypertension determined in type 2 diabetes and non-diabetic patients

Men 127.36– 129.48 84.78– 87.37 –

Women 130.93– 132.97 85.88– 86.74 –

95% confidence interval of limit of the mean

124 R. K. Kesharwani et al.

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Homocysteine Metabolism Pathway Genes and Risk of Type 2 Diabetes. . .

125

Table 6.6 Status of vitamin B, folate, and different fractions of lipids in type 2 diabetes patients Biochemical factors Serum folate (nmol/L) Vitamin B6 (nmol/L) Vitamin B12 (pmol/L) S. Creatinine (mg/dL) TC (mg/dL) LDL-c (mg/dL) HDL-c (mg/dL) Triglyceride (mg/dL)

Men (N ¼ 208) 14.29  5.04

95% confidence interval of limit of the mean 13.591–14.989

Women (N ¼ 173) 15.38  3.75

95% confidence interval of limit of the mean 14.89–15.95

28.45  6.01

27.62–29.28

37.69  8.11

36.46–38.92

276.98  61.34

268.47–285.49

302.73  57.44

293.996–311.46

0.72  0.12

0.70–0.74

0.68  0.09

0.67–0.69

189.458  84.731 126.93  24.16

177.71–201.21 123.79–130.28

176.487  73.82 113.84  26.24

165.26–187.71 109.85–117.83

47.13  5.91

46.31–47.95

49.06  8.13

47.82–50.30

168.86  104.73

154.34–183.38

181.04  93.75

167.68–196.20

with hyperhomocysteinemia and elevated level of blood pressure. Studies have revealed that supplementation of folic acid helped in the reduction of blood pressure, and in our study we can presume a low level of folate in a diabetic patient may be a cause for the reason of elevated level of blood pressure. As shown in Tables 6.6 and 6.7, serum folate, vitamin B6, and vitamin B12 levels were higher in non-diabetic patients than in diabetic patients (28.46  8.01 vs. 14.29  5.04, 17.85  3.26 vs. 28.45  6.01, and 593.86  104.93 respectively). Folic acid and vitamin B12 acts as substrate and enzyme respectively which influences the homocysteine metabolism through various intracellular metabolic reactions. So we can presume that the reduced level of folate and vitamins will enhance the level of homocysteine in diabetic patients. As shown in Table 6.8, it was observed that there was a decline in GFR in type 2 diabetes subjects compared to non-diabetic subjects showing impaired kidney function. As mentioned above by Buysschaert et al. in 2000, it was observed that hyperhomocysteinemia leads to nephropathy. From the table, we can suggest that insulin resistance causes elevated level of homocysteine in diabetic patients. It was seen that hyperhomocysteinemia correlates with a change in glomerular filtration rate. Out of total 428 cases of diabetes, 92 diabetic males and 76 diabetic females were tested for the level of serum creatinine and percentage of microalbuminuria. It was observed from Table 6.9 that the level of homocysteine was 17.31  3.08 in males and 15.93 and 4.11 in females and was elevated compared to the normal level. The prevalent of renal dysfunction was evidenced with an elevated level of serum creatinine (1.84  0.26) and percentage of microalbuminuria. This clearly demonstrates that in type 2 diabetic cases increased level of homocysteine leads to

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Table 6.7 Status of vitamin B, folate, and different fractions of lipids in non-diabetic patients

Factors Serum folate (ng/mL) Vitamin B6 (nmol/L) Vitamin B12 (pg/mL) Serum creatinine (mg %) Total cholesterol (mg/dL) LDL-c (mg/dL) HDL-c (mg/dL) Triglycerides (mg/dL)

Men (N ¼ 186) 28.46  8.01

95% confidence interval of limit of the mean 27.29–29.63

Women (N ¼ 115) 30.11  5.85

95% confidence interval of limit of the mean 29.02–31.20

17.85  3.26

17.37–18.33

16.83  5.08

15.88–17.78

593.86  104.93

578.47–609.25

612.73  118.35

590.66–634.80

0.75  0.08

0.74–0.76

0.68  0.09

0.66–0.70

169.78  52.80

162.04–177.52

159.96  61.01

148.58–171.34

81.22  11.46

79.51–82.93

77.35  10.68

75.36–79.34

53.45  6.82

52.45–54.45

61.04  5.13

60.08–61.80

88.42  31.64

83.78–93.06

79.98  29.87

74.41–85.55

renal dysfunction, defined by creatinine clearance and microalbuminuria (Table 6.10). The values given in tables are incorporated without any medication taken by the patients (gap of 24 h).

6.2

Discussion

Insulin resistance is considered as an independent risk factor (Savage et al. 1996) and a possible relationship between total homocysteine levels and features of insulin resistance has been suggested. In recent years there has been interest in detecting the level of homocysteine as an important indicator of cardiovascular and cerebrovascular disease. It was suggested that hyperhomocysteinemia contributes to the accelerated atherosclerotic process in diabetes (Okada et al. 1999). Insulin and blood glucose act as key regulators of homocysteine and methyl group metabolism and stress the importance of glycemic control in diabetics, as changes in blood glucose lead to an increase in subsequent epigenetic marks. Importantly, similar disturbances have been reported in type 2 diabetic rat models (Ratnam et al. 2006) and humans with insulin resistance (Rosolová et al. 2002). In our study, patients with type 2 diabetes mellitus had an elevated level of homocysteine which was increased than the normal level. In this study, patients with higher level of HbA1c (7.16  2.73) in men and women (8.15  2.13) showed higher level of homocysteine which suggests that poor metabolic control of diabetes is related to

Groups 24 h urinary protein (mg%) GFR (mL/min)

95% confidence interval of limit of the mean 227.02–284.84

88.35–91.11

Diabetic men (N ¼ 92) 255.93  138.64

89.73  6.62

88.44  7.45

Diabetic women (N ¼ 76) 149.83  188.40

86.73–90.15

95% confidence interval of limit of the mean 106.60–193.06

96.45  8.73

Non-diabetic men (N ¼ 108) Nil

94.47–98.13

95% confidence interval of limit of the mean

95.36  5.16

Non-diabetic women (N ¼ 86) Nil

Table 6.8 Total urinary protein and glomerular filtration rate in relation to HHcy measured in type 2 diabetes and non-diabetic patients

94.25–96.47

95% confidence interval of limit of the mean

6 Homocysteine Metabolism Pathway Genes and Risk of Type 2 Diabetes. . . 127

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Table 6.9 Determination of total homocysteine in relation to renal function status, i.e., serum creatinine and micro-albuminuria in type 2 diabetes patients

Groups N ¼ 92 N ¼ 76

Sex M F

Hcy (mmol/ L) 17.31  3.08 15.93  4.11

Table 6.10 Percentage of medication received by type 2 diabetes mellitus patients

95% confidence interval of limit of the mean 16.67–17.95 14.92–16.94

Serum creatinine (mg%) 1.84  0.26 1.64  0.19

95% confidence interval of limit of the mean 1.79–1.89 1.60–1.68

Groups of medicine received (%) Only life style management Life style management along with drugs Metformin Alpha-glucosidase inhibitors Sulfonylureas Dipeptidyl peptidase-4 (DPP-4) inhibitors Anti-hypertensive Statin Anti-platelet therapy (low-dose asprin)

Microalbuminuria (%) 88 76

Men 11 (%) 84 (%) 48 (%) 04 (%) 21 (%) 16 (%)

Women 12 (%) 86 (%) 52 (%) 08 (%) 16 (%) 12 (%)

61 (%) 23 (%) 42 (%)

48 (%) 14 (%) 28 (%)

hyperhomocysteinemia. This can be confirmed with work conducted by Drzewoski and Czupryniak and colleagues in 2000 that metabolic control of diabetes may influence Hcy levels. Those authors also have shown that patients with bad metabolic control of diabetes had significantly higher Hcy levels in comparison to diabetics with normal HbA1c levels. Atif et al. 2008 have concluded that homocysteine appears to be raised in patients with hypertension and this is in line with our study where diabetic patients having hyperhomocysteinemia are found to have elevated levels of blood pressure. The European Union Concerted Action Project, “Homocysteinaemia and vascular disease”, indicated that a plasma homocysteine level above 0.162 mg% accelerates the risk of myocardial infarction, cerebral, or peripheral vascular disease in both men and women. There are studies suggesting that an elevated level of homocysteine in poorly controlled type 2 diabetes mellitus is related to increased risk of atherosclerosis and cardiovascular disease. The mechanism by which homocysteine promotes cardiovascular disease is not clear. Endothelial cells when exposed to advanced glycated end products initiate an increase in thrombomodulin secretion when they get in contact with homocysteine, which is a possible mechanism showing association of homocysteine initiates thrombosis of endothelial cells. This may suggest how diabetic patients with hyperhomocysteinemia in our study might be prone to cardiovascular disease (Hoffman et al. 1997). Several studies suggest that elevated level of homocysteine has both atherogenic and thrombogenic effects. Hyperhomocysteinemia causes endothelial dysfunction by increasing oxidative

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stress (Starkebaum and Harlan 1986) and decreases the release of nitric oxide impairing vasodilation (Tawakol et al. 1997; Stuhlinger et al. 2001). Excess of homocysteine stimulates smooth muscle cell proliferation and collagen synthesis promoting intima-media thickening and the probable mechanism proposed by De Bree et al. 2002 is that homocysteine on oxidation forms homocysteine thiolactone that is complexed with LDL particles and this complex aggregates and deposits in the form of thio and then thioretinamide. These intermediates promote proliferation and fibrosis of smooth muscle. During the conversion of thio to thioretinamide highly reactive oxygen species are generated which cause several changes in the intima of blood vessels and endothelial dysfunction leading to atherosclerotic plaque. A positive correlation of homocysteine level with increasing age was found in various studies. It has been shown that the in vitro activity of rate-limiting enzyme for homocysteine metabolism, cystathione b synthase declines with age, thus declining cystathione b synthase activity and glomerular filtration rate (Nygard et al. 1995; Bostom et al. 1999a, b; Kang et al. 1986). In a study conducted by Alev Eroglu Altinov in 2003, it was concluded that homocysteine in diabetic patients is associated with increased complications especially CAD and nephropathy. The present study data demonstrated that there is kidney dysfunction, defined by urinary protein and glomerular filtration and renal dysfunction defined by serum creatinine and microalbuminuria in diabetic patients with hyperhomocysteinemia which may suggest that there is a strong relation between diabetic nephropathy and homocysteine. Our study shows similar results to another study showing a strong relationship between creatinine clearance (Ccr) and tHcy has been demonstrated by some investigators, both in patients with renal disease and in those with diabetes (Bostom et al. 1999a, b; Wollesen et al. 1999; Arnadottir et al. 1996). It has also been clearly documented that increased levels of homocysteine occur in association with marked degrees of renal dysfunction (Bostom et al. 1997; Bostom et al. 1997). Various works were related to the association of tHcy and milder renal dysfunction (Hoogeveen et al. 1998a, b; Lanfredini et al. 1998; Chico et al. 1998; Bostom et al. 1999a, b). Aghamohammadi et al. 2011 has observed the effect of folic acid supplementation in homocysteine level in type 2 diabetes mellitus and have concluded that folate supplementation lowered plasma homocysteinemia in diabetic patients and in our study diabetic patients with hyperhomocysteinemia had lower level of folate which may suggest that these patients are deficient in folic acid. The impaired metabolism of homocysteine produces the condition of hyperhomocysteinemia which is an independent risk factor for cardiovascular disease because it exerts a negative role on the endothelial membrane. Type 2 diabetes mellitus and microalbuminuria increase the cardiovascular risk significantly so in these patients, plasma homocysteine levels must be measured and if hyperhomocysteinemia was established it must be treated appropriately in order to decrease the risk. Conflicts of Interest The authors declare no conflicts of interest.

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Stamler JS, Osborne JA, Jaraki O, Rabbani LE, Mullins M, Singel D, Loscalzo J (1993) Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen. J Clin Invest 91(1):308–318 Stampfer MJ, Malinow MR, Willett WC, Newcomer LM, Upson B, Ullmann D, Tishler PV, Hennekens CH (1992) A prospective study of plasma homocyst (e) ine and risk of myocardial infarction in US physicians. JAMA 268(7):877–881 Stanger O, Weger M, Renner W, Konetschny R (2001) Vascular dysfunction in hyperhomocyst (e) inemia. Implications for atherothrombotic disease, pp 725–733 Starkebaum G, Harlan JM (1986) Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest 77(4):1370–1376 Stehouwer CD, Gall MA, Hougaard P, Jakobs C, Parving HH (1999) Plasma homocysteine concentration predicts mortality in non-insulin-dependent diabetic patients with and without albuminuria. Kidney Int 55(1):308–314 Stubbs M, York DA (1991) Central glucocorticoid regulation of parasympathetic drive to pancreatic βcells in the obese fa/fa rat. Int J Obes 15:547–553 Stuhlinger MC, Tsao PS, Her JH, Kimoto M, Balint RF, Cooke JP (2001) Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine. Circulation 104:2579– 2575 Targher G, Bertolini L, Zenari L, Cacciatori V, Muggeo M, Faccini G, Zoppini G (2000) Cigarette smoking and plasma total homocysteine levels in young adults with type 1 diabetes. Diabetes Care 23(4):524–528 Tarkun I, Arslan BC, Cantürk Z, Tarkun P, Kozdağ G, Topsever P (2003) Homocysteine concentrations in type 2 diabetes mellitus patients without cardiovascular disease: relationship to metabolic parameters and diabetic complications. Turk J Endocrinol Metab 1:11–17 Tawakol A, Omland T, Gerhand M, Wu JT, Creager MA (1997) Hyperhomocysteinemia is associated with impaired endothelium-dependent vasodilation in humans. Circulation 95: 1119–1121 Vannucchi H, Melo SS (2009) Hiper-homocisteinemia e riscocardiometabólico. Arq Bras Endocrinol Metab 53(5):540–549 Verhoef P, Hennekens CH, Malinow MR, Kok FJ, Willett WC, Stampfer MJ (1994) A prospective study of plasma homocyst (e) ine and risk of ischemic stroke. Stroke 25(10):1924–1930 Weiss N, Heydrick SJ, Postea O, Keller C, Keaney JF Jr, Loscalzo J (2003) Influence of hyperhomocysteinemia on the cellular redox state—impact on homocysteine-induced endothelial dysfunction. Clin Chem Lab Med 41(11):1455–1461 Welch GN, Loscalzo J (1998a) Homocysteine and atherothrombosis. N Engl J Med 338:1042–1050 Welch GN, Loscalzo J (1998b) Mechanisms of disease: homocysteine and Atherothrombosis. N Engl J Med 338(15):1042–1050 Wilcken DE, Wilcken B (1976) The pathogenesis of coronary artery disease. A possible role for methionine metabolism. J Clin Invest 57:1079–1082 Wiltshire E, Thomas DW, Baghurst P, Couper J (2001) Reduced total plasma homocysteine in children and adolescents with type 1 diabetes. J Pediatr 138:888–893 Wollesen F, Brattstrom L, Refsum H, Ueland PM, Berglund L, Berne C (1999) Plasma total homocysteine and cysteine in relation to glomerular filtration rate in diabetes mellitus. Kidney Int 55:1028–1035

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Genetic Polymorphism in Homocysteine Metabolism Rudra P. Ojha, Govind Prasad Dubey, U. P. Shahi, V. N. Mishra, D. Jain, and Pradeep Upadhyay

Abstract

Ample evidence is available to demonstrate genetic polymorphism in homocysteine metabolism. Low folic acid is another cause of hyperhomocysteinemia in both age and sex groups. The present study is based on the evaluation of homocysteine metabolism in different gender. The biochemical variability of homocysteine is responsible for many clinical conditions including the high prevalence of coronary heart disease. The present study has been planned to investigate the role of genetic polymorphism in the prevention and management of coronary artery disease. The study provides a beneficial role in the prevention of CHD in hyperhomocysteinemic cases.

R. P. Ojha Department of Zoology, Nehru Gram Bharati (Deemed to be University), Prayagraj, Uttar Pradesh, India G. P. Dubey (*) Kriya Sharira and Kaya Chikitsa, Banaras Hindu University, Varanasi, Uttar Pradesh, India U. P. Shahi Department of Radiotherapy & Radiation Medicine, IMS, Banaras Hindu University, Varanasi, India V. N. Mishra Department of Neurology, IMS, Banaras Hindu University, Varanasi, India D. Jain Department of Cardiology, IMS, BHU, Varanasi, India P. Upadhyay Department of Botany, NGB (DU), Prayagraj, Uttar Pradesh, India # The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2022 G. P. Dubey et al. (eds.), Homocysteine Metabolism in Health and Disease, https://doi.org/10.1007/978-981-16-6867-8_7

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Keywords

Homocysteine · CHD · CAD · MTHFR · Methionine · Polymorphisms

7.1

Introduction

7.1.1

Molecular Genetics and its Association with Homocysteine

Recently, maximum time and attention have been focused toward the association of molecular genetics of 5,10-methylenetetrahydrofolate reductase (MTHFR 677C-T) and C4BN, and their relationships to the metabolism of amino acid homocysteine. The studies conducted by various workers demonstrated that the individual homocysteine plasma level is determined by polymorphisms in genes coding for enzymes involved in its metabolism (Fodinger et al. 1999). The MTHFR 677C!T is responsible for low folate status thus enhancing Hcy level (Fodinger et al. 2000). Apart from MTHFR 677C!T, another polymorphism 1298A!C is identified which is located in coexon 7 (van der Put et al. 1998; Weisberg et al. 1998). This gene converts a glutamic acid into an alanine residue and leads to a decreased enzyme activity among homozygous individuals (60%) of control values. However, it did not exert any change in either folate status or total Hcy concentration. Whereas heterozygous individuals with 677T and 1298C MTHFR allele presented with decreased enzyme activity, increased Hcy concentrations, and decreased folate plasma level. These types of involvements are mostly observed among families showing evidence of neural tube defect (van der Put et al. 1998). Based on observations made on gene products of MTHFR FOLR1 and CUBN in the metabolism of Hcy, these findings suggest to investigate the mutations that play role in the metabolism of total Hcy among patients with renal replacement therapy. Thus the role of above-mentioned gene mutations in the metabolism of Hcy requires further investigations.

7.1.2

Homocysteine Associated with Metabolic Syndrome

To investigate the relationship between tHcy and different components of metabolic syndrome is of clinical significance as a high value of tHcy is an independent risk factor for cardiovascular disease. It is well established that elevated tHcy is toxic to vascular endothelium and is responsible for the occurrence of atherosclerosis, an independent risk factor for cardiovascular disease particularly among diabetic and non-diabetic subjects (Hoogeveen et al. 1998, 2000; Boushey et al. 1995; Eikelboom et al. 1999). Homocysteine levels have been found to be elevated in type 2 diabetes patients, both in fasting as well as following methionine administration, and are correlated with microalbuminuria (Hoogeveen et al. 2000; Araki et al. 1993; Munshi et al. 1996).

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Fonseca et al. (1998) demonstrated that Hcy levels rise with insulin resistance with changes in critical enzymes of Hcy metabolism. These workers reported that in hyperinsulinemic-euglycemic clamp Hcy level decreased in non-diabetics, but not in diabetic patients. Further, elevated tHcy is found to be associated with patients showing evidence of hyperhomocystinuria. This is a rare onset and found among those showing tHcy much higher, i.e., as high as 400 μmol/L, and this elevation of Hcy is responsible for thrombotic episodes (Sebastio et al. 1995; Selhub 1999b, c; Mudd et al. 1985). It is also well documented that mild elevation in plasma tHcy is mainly associated with an increased risk of vascular diseases. A higher level of tHcy may affect the coagulation system (Woo et al. 1997; Tawakol et al. 1997; Majors et al. 1997; Al-Obaidi et al. 2000). A number of previous studies have evaluated the tHcy levels in patients with metabolic syndrome. The Framingham offspring study mentioned that increased tHcy levels enhance the risk of cardiovascular diseases only when there is microalbuminuria (Meigs et al. 2001).

7.1.3

Genetic Basis of Plasma Homocysteine Content in Relation to Health and Disease

The gene functioning involved with folate-mediated one-carbon metabolism causes changes in homocysteine and DNA methylation, resulting in proneness to coronary heart disease. The role of folate, vitamin B6 and B12 contribute significantly to health and disease including DNA synthesis and the generation of cellular methylation process. Dietary intake as well as genes encoding folate-related enzymes are responsible for the regulation of folate content. Any alteration in folate status may cause a number of diseases particularly birth defects, cardiovascular disorders, and even cancer like a dread disease (Fox and Stover 2008). Detection of hyperhomocysteinemia (HHcy) is a marker suggesting an abnormality in folate metabolism which enhances the risk of cardiovascular disorders (Lewis et al. 2005; Wald et al. 2002). As pointed out normalcy of homocysteine concentration depends upon nutrition and genetic variants. Jamaluddin et al. (2007) described the association of Hcy with CVD and emphasized its role in DNA methylation. According to these workers, DNA methylation depends on folate-mediated one-carbon metabolism through various enzymes involved with methylation reactions (Ulrey et al. 2005; Pogribny and Beland 2009). Wernimont et al. (2011) made a conclusion that genes involved in absorption and transport have shown 30–40 percent relationship with Hcy phenotype which is mediated through plasma folate and vitamin B6 and B12 levels. Importantly, phenotype of the top single-nucleotide polymorphisms (SNP) was in a gene in the methylation/homocysteine pathway, whereas they also reported that nutrition has no association of SNPs with the methylation phenotypes. The gene pleiotropy is identified for plasma homocysteine. The 5 region SLC19AIrs 1131596 SNP is supposed to be associated with reduced RBC levels

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in coronary artery disease patients, and decreased SLC19AI influences homocysteine levels mediated by changes in nutrient biomarkers. The most important gene FTCDrs2277820 SNP is known to be associated with total homocysteine. The CT genotype group was found to be 7.2 percent higher on plasma total homocysteine vs the CC/TT group. FTCDincodes is a Golgi-associated enzyme responsible for the production of 5,10-methenyl-tetrahydrofolate (THF) (Fox & Stover 2008). Mutations in FTCD are associated with inherited disorders of folate metabolism (Hilton et al. 2003). 29 percent association between rs 2277820 and Hcy mediates through total plasma folate and vitamins B6 & B12. The gene SIC19A3 belonging to the folate transporting family is closely related to plasma total homocysteine. Similarly, total plasma Hcy was markedly found higher in CT genotype group of individuals in comparison to CC/TT group (Ganapathy et al. 2004). In the light of the above concept the association of MTHFR with Hcy was stronger if the level of folate is reduced enough, and interaction between vitamin B6 and genetic variants in the SHMT1 and CBS gene may have significance only when the level of vitamin B6 is reduced significantly (Perry et al. 2007; Taoka et al. 1999). Thus the genes play an important role in the regulation of Hcy and global genomic DNA methylation phenotypes. Further, Hcy has also beenassociated with the nutritional status of vitamins B. Thus the genetic basis for Hcy shall be evaluated for both genetic (one-carbon network) and nutritional variation in different population and extend their findings for cardiovascular biomarkers up to cardiovascular phenotypes. A high concentration of homocysteine (Hcy) of genetic origin in relation to health and disease was described in 1962 first time (1,2). Various metabolic defects were characterized by two common factors, i.e., homocystinuria and premature thromboembolic disease. Elevated Hcy and its association with arteriosclerosis were defined by various workers (4) which suggested that a moderately elevated plasma homocysteine level may be a cardiovascular risk factor in the general population. Hcy levels are affected both by genetic as well as nutritional factors. At the genetic level, Hcy elevation is caused due to defects in enzymes responsible for Hcy metabolism. The nutritional cause of elevated Hcy level is deficiencies of folate, vitamin B12 or vitamin B6 playing a role in the remethylation of homocysteine to methionine. An association between elevated plasma homocysteine level and cardiovascular disease is reported (Refsum et al. 1998; Wilcken 1998; Boushey et al. 1995; Danesh Lewington 1998). The association between genetic hyperhomocysteinemia and vascular disease indicates the elevation of Hcy level manifesting the disease process. Various studies have reported ten percent cardiovascular mortality, due to HHcy exceeding level 14 mmol/L. Similarly out of 22 studies conducted in Danesh and Lewington’s meta-analysis, 20 studies concluded that elevated plasma Hcy level has a significant association with coronary heart disease. In one of the studies, the European concerted Action Project suggested on elevated plasma homocysteine level interacts with conventional risk factors and further increases vascular disease risk (Graham et al. 1997). Under this group of studies, Selhub and Colleagues

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(Selhub et al. 1999) demonstrated that nutrition, Hcy, and cardiovascular risk are significantly associated. Further, Selhub and Colleagues attributed the risk for increased homocysteine levels that is serum folate or vitamin B12-deficient persons have high Hcy levels. Low folate and vitamin B12 is defined in elderly people. Thus it is defined that measurement of plasma Hcy will be helpful in risk prediction of neurodegeneration and cardiovascular diseases.

7.1.4

A Study on Genetic Polymorphisms in Down’s Syndrome and Associated Cognitive Deficit

One of the most important causative factors of mental retardation is Down’s syndrome resulting mainly from gene expression of chromosomal abnormality. The genetic defect is associated with elevated transsulfuration of homocysteine (Hcy) and chromosomal instability (Gericke et al. 1977; MacGregor et al. 1997; Hassold et al. 2001). Patients with Down’s syndrome develop dementia related to neocortex lesions similar to those with Alzheimer’s disease (AD). In Down’s syndrome the plasma homocysteine level is affected by folate, vitamin B12, and by genetic polymorphism of methylenetetrahydrofolate reductase, methionine synthase, and transfer from methyltetrahydrofolate. The nutritional and genetic risk factors related to vascular diseases are linked with AD. E4 allele of apolipoprotein E (ApoE) is found to be markedly associated with total Hcy and vitamin B12 (Clarke et al. 1998), but not with the MTHFR gene polymorphism (Clarke et al. 1998; Pollak et al. 2000; Nishiyama et al. 2000). According to considered Bosco et al. (2003) and James et al. (1999), MTHFR 677 C!I and MTRR 66 A!G polymorphism are responsible for giving birth to a child with Down’s syndrome. However, many evidences are needed to prove that nutritional determinants influence the intelligence quotient in patients with Down’s syndrome. Though there is no direct reference indicating the association of MTHFR677 T allele with the risk of cognitive deficits, the specific genotype increases the risk of poor cognitive function associated with hyperhomocysteinemia approximately threefold. It is observed that there is a considerable increased Hcy level among patients showing poor cognitive function particularly intelligence quotient among mental retardation cases. One of the studies conducted by Gueant et al. (2005) investigated the role of genetic markers of homocysteine in association with cognitive function/intelligence quotient. These workers reported relatively high total homocysteine levels in sub-groups of patients showing low intelligence quotient could be an indirect consequence of mental retardation, followed by reduced autonomy and deficient dietary intake. Age is another factor showing association with the IQ and t-Hcy and cognitive function (Miller et al. 2003). However, the influence of age on IQ was observed in only half the patients, those who had the lowers level of mental retardation, and IQ was associated with t-Hcy independently of age.

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Mechanism of Action

Regarding the mechanism, the increase in the activity of the transsulfuration pathway of t-Hcy, resulting from the overexpression of cystathionine β-synthase on chromosome 21 (Chadefaux et al. 1988; Tassone et al. 1999; Hattori et al. 2000), may promote a folate trap by decreasing the cellular concentration of homocysteine and subsequently methyl-tetrahydrofolate and vitamin B12-dependent remethylation and cellular synthesis of tetrahydrofolate (Pogribna et al. 2001; Chadefaux et al. 1988). In the present series of studiies, the supplement with test formulation has shown a beneficial effect in increasing the defective cellular level of methionine and s-adenosylmethonine in lymphoblastoid cells of trisomy (Wechsler and Kodama 1949; Pogribna et al. 2001). The metabolic process involved in this mechanism can be explained as a decreased activity of MTHFR resulting from a677-TT genotype, which may act as an aggravating factor of the folate by decreasing the remethylation of Hcy and synthesis of tetra-folate. The reduced activity of MTHFR is responsible for the decrease in the level of s-adenosylmethionine (Friso et al. 2002), often involved with Down’s syndrome pathogenesis (Ueland et al. 2001; Pogribna et al. 2001). This mechanism indicates the key genetic determinant of a folate imbalance between DNA synthesis and remethylation of homocysteine and DNA. There is a relationship between DNA methylation and cognitive dysfunction of genetic markers. The present study has been conducted in light of the role of t-Hcy with cognitive function. Another phenotypic abnormality related to chromosome-21 genes is the overexpression of amyloid precursor protein which plays a major role in amyloid precursor protein in neurodegeneration related to Down’s syndrome cognitive deficits (Hyman et al. 1995; Hattori et al. 2000). Based on the present study results, it is apparent that there is an association between homocysteine and β-amyloid fragment metabolism in the pathogenesis of Down’s syndrome cognitive deficits particularly dementia and intelligence quotient. Homocysteine influences through multiple mechanisms of actions with neurodegenerative disorders. Hcy is the factor which repairs the impaired DNA in hippocampal neurons, ameliorates apoptosis, reduces excitotoxicity of glutamatergic receptors, regulates oxidative stress markers, and reduces the neurons toxicity of β-amyloid peptide.

7.3

Genetics of Homocysteine

A number of references are available indicating that folate and vitamin B12 deficiency are associated with cardiovascular diseases and neural tube defects. Folate, vitamin B12 status, and Hcy metabolism all are under genetic control. MTHFR677C>T, GCP21561C>T, RFCI80G>A and TCN2776G>C may enhance the Hcy level in patients with CVD and neural tube defect. In chronic renal failure condition also there is impairment of Hcy metabolism. A polymorphism in the

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transcobalamin II gene affects transcobalamin plasma concentration and thus may interfere with cellular vitamin B12 and Hcy concentration (Namour et al. 2001). The human 5,10-methylenetetrahydrofolate reductase gene (MTHFR OMIM 607093) is located in chromosome 1p36-3. The MTHFR gene product consists of 656 amino acid residues and represents a key enzyme in the folate cycle. Further, it is reported that three types of polymorphism exist, i.e., MTHFR which is located at nucleotide protein 677 position 1298 and protein 1317; MTHFR677C>T located in exon 4 at the folate-binding site and MTHFR 1298A>C located in exon 7 is responsible for the change in a glutamic acid into an alanine residue. The MTHFR6777TT genotype is present in about 12% of the general population having heterogeneous distribution among ethnic groups. In patients with neural tube defect, a decreased frequency of the GCP2156IT allele has been identified (Brancaccio et al. 2001) which has been shown associated with elevated red blood cell folate and plasma folate level whereas no effect on vitamin B12 levels (Lievers et al. 2002). Heterozygotes showed higher tHcy concentrations as compared to homozygous genotypes (Namour et al. 2001). It is obvious that genetic variants influence tHcy levels, human diseases, the genetic nutrient interactions, as well as the pharmacogenetic consequences in Hcy including vitamin metabolism. However, further comprehensive studies are required in this area.

7.4

Maternal Genes Involved in Homocysteine and Risk of Down’s Syndrome

As reported, impairment of folate metabolism has been causally related to both DNA hypomethylation and abnormal purine synthesis (Zhou et al. 2001), and is responsible for chromosomal breakage and deficient DNA repair, leading to genomic instability. The polymorphisms of genes involved with homocysteine act through folate homeostasis and cellular methylations. The MTHFR, a regular of cellular methylations, catalyzes the conversion of 5,10-methylanetetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor for remethylation of Hcy to methionine (Frosst et al. 1995). The MTHFR 677I variant may cause reduced activity of MTHFR thus demanding an increase in folic acid content in order to the remethylation of Hcy to methionine. Two polymorphisms have been found to be associated with MTR and MTRR genes, i.e., A2756G and A66G respectively (Paz et al. 2002). It is reported that 10% of the general population carrying 844ins68 polymorphism may represent a low level of tHcy even after methionine administration. Several studies have indicated the role of polymorphisms of genes Hcy/folate metabolism known to be a maternal risk factor for Down’s syndrome. The maternal MTHFR C6771 polymorphism was found to be associated with an increased risk of Down’s syndrome (James et al. 1999; Hobbs et al. 2000; Grillo et al. 2002; Silva et al. 2005). In one of the studies conducted by Brody (2002), the gene MTHFD1

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A1958 variant associated with increased risk of neural tube defect is not been investigated as a risk factor for down syndrome point of view.. The first report prepared by James et al. (1999) reported the role of Hcy/folate metabolism in the pathogenesis of Down’s syndrome (Hobbs et al. 2000; Grillo et al. 2002; Silva et al. 2005; O’Leary et al. 2002, Chadefaux et al. 2002; Stuppia et al. 2002; Boduroglu et al. 2004; Takamura et al. 2004; Bosco et al. 2003, Chango et al. 2005). The study conducted by Iris Scala et al. (2006) demonstrated an association between polymorphisms of genes of Hcy/folate pathway and increased risk of Down’s syndrome. These workers observed that MTHFR1298C and the RFCI80G alleles were associated with an increased risk of having Down’s syndrome. The chromosome 21 non-dysfunctional process occurs more frequently with age progression as a consequence of an age-dependent derangement of meiosis-specific events (Hassold and Sherman 2000). Such type of studies provides evidence for the role of gene to gene interactions in the assessment of genetic susceptibility to Down’s syndrome. It is speculated that blood folate and Hcy levels influence the impact of genotype. Further, in this study, the worker could not correlate much influence on certain genotypes as risk factors for Down’s syndrome, and tHcy could not demonstrate a significant difference between Down’s syndrome mothers and normal control, in the light of a direct effect of certain genotypes as a risk factor for Down’s syndrome. However, it is speculated that blood folate level and tHcy concentration influence the impact of the genotype. Further, it is hypothesized that polymorphism of gene of Hcy/folate pathways may predispose to chromosome 21 (Hobbs et al. 2000; Brody et al. 2002). An impaired homocysteine-folate pathway associated with birth defects like neural tube defect and congenital heart abnormalities has warranted to supplement folic acid to involved person. It is important to furnish here that the genomic instability is kept at the minimum level when plasma folate exceeds 34 nmol/L and plasma homocysteine is 100 μmol/L) (Weiss et al. 2002). There is a difference in cause prevalence and severity of the elevated level of Hcy in relation to individuals. More severity in the level is mainly due to homozygous defects in genes as well as Hcy metabolism. The prevalence rate of hyperhomocysteinemia observed in the general population is reported to be 5% which is also associated with various types of disorders like neuronal tube defects, non-syndromic oral clefts, congenital heart defects, cardiovascular disorders, atherosclerosis, Down’s syndrome, pregnancy, Alzheimer’s disease and vascular dementia, breast cancer, depression, diabetes, various drugs, etc. Neural tube defects (NTD) are the most common defects that mainly manifest during the first 28 days of pregnancy. The use of folic acid in pregnancy is prescribed for the prevention of NTD. Several studies have reported that mothers with HHcy have an increased risk of NTD (Steegers et al. 1994; Mills et al. 1995; van der Put et al. 1997; Wenstrom et al. 2000; Ueland et al. 2001; Epeldegui et al. 2002; Ratan et al. 2008). Similarly in one of the recent studies, it has been suggested that a supplement of folic acid during the first trimester of pregnancy may prevent the occurrence of cleft lip and palate. Verkleij-Hagoort et al. (2007) made the observation that two MTHFR polymorphisms were independent risk factors for cleft lip and palate. Further, these workers also demonstrated that mothers associated with high Hcy concentration had a much higher risk of having a child with coronary heart disease. Trabetti (2008) studied the relationship between MTHFR polymorphism and the severity of CAD in patients undergoing coronary artery bypass surgery showed that Hcy levels were significantly raised in these patients, and significantly they were associated with MTHFR 677C>T genes. Similarly, various studies also concluded that elevated Hcy is an independent risk factor for atherosclerotic disease (Weiss et al. 2002). A possible role of HHcy in accelerating the atherosclerosis has been reported by various workers (Matthias et al. 1996; Zhou et al. 2008).

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Hyperhomocysteinemia and Cognitive Impairment

Elevated Hcy and low folate level is responsible for cognitive impairment and results in neurotoxic and vasotoxic effects in dementia and Alzheimer’s disease. Therefore homocysteine has been considered as one of the important biomarkers for neurodegenerative disorders, particularly Alzheimer’s disease (Köseoglu et al. 2007). Folate supplement has shown a beneficial role in the improvement of cognitive function, particularly in elderly people (Durga et al. 2007).

7.14

Hcy with Diabetes

Wotherspoon et al. (2008) postulated oral supplement with folic acid, significantly reduces Hcy level, improves endothelial dysfunction, reduces oxidative injury in patients with type I diabetes and microalbuminuria (Martinez et al. 2008; Wotherspoon et al. 2008). Several references are available indicating that some group of drugs that are in clinical practice enhances the Hcy level like anti-epileptic drugs which 10–40% enhances Hcy concentration (Apeland et al. 2002; Sener et al. 2006). At the genetic level, MTHFR 1298C allele was observed in epileptic patients (Belcastro et al. 2007).

7.15

Management of Homocysteine

Hcy metabolism requires supplementation of folate as well as vitamin B12 and B6 (Selhub 1999b, c). The daily requirement of folic acid is 50 μg and the current recommended dose is 400 μg/day for an average adult and 600 μg/day in the pregnancy period (McCully 2007). However, long-term and larger sample sizes are required in order to prove the prevention and management of various disorders by reducing elevated Hcy that will also result in better health status. We are lacking the community-based data on plasma homocysteine concentration. However, some of the case--control and prospective studies have shown an association between hyperhomocysteinemia and atherothrombotic vascular disease (Gheye et al. 1999, Bhargava et al. 2007). The individuals with a nutritional deficiency are at risk of developing HHcy and two-thirds of HHcy in the elderly population is because of vitamin B deficiency. As illustrated through various studies apart from conventional CHD risk factors HHcy is also considered as one of the key factors for CHD and stroke (Yoo et al. 1998; Graham et al. 1997). Similarly, lifestyle factors like alcohol, diet, obesity, smoking, etc., are also found to be associated with elevated Hcy concentration. One of the studies conducted by indicated that plasma homocysteine level in specific ethnic group were from 27 to 79.1 μmol/L. These workers reported that almost 55% of the population shows HHcy where males have shown a higher level of Hcy than the females suggesting a correlation between the genders. Further, these workers reported 56.8% HHcy in the age group of

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Table 7.1 Status of biological parameters in C677T genotype subjects

Parameters BMI (kg/m2) Systolic BP (mmHg) Diastolic BP (mmHg) Total cholesterol (mg/dL) HDL-c (mg/dL) LDL-c (mg/dL) TG (mg/dL Serum folate Vitamin B6 (μg/L) Vitamin B12 (ng/L) Hcy (μmol/ L)

Age (years) 15–30 (years) N ¼ 24 26.48  4.11 124.82  5.39

31–45 (years) N ¼ 21 26.98  2.75 126.87  6.93

46–60 (years) N ¼ 27 28.04  3.11 139.45  8.42

61–75 (years) N ¼ 17 28.96  3.17 148.90  10.13

78.46  2.93

84.80  3.71

88.11  3.85

93.64  3.98

168.46  22.65

192.55  31.73

223.98  33.90

219.45  32.77

48.96  4.12

51.16  6.81

48.04  4.93

43.86  5.17

98.73  9.28

106.45  13.46

117.32  12.90

118.85  10.68

86.42  8.94 5.73  1.59 6.48  1.88

93.66  7.32 5.80  2.13 6.51  2.04

146.40  9.08 5.62  1.85 5.73  2.13

152.35  7.80 5.50  1.93 5.68  1.75

581.90  241.81

611.52  310.94

589.65  261.35

633.90  258.73

8.26  2.16

10.42  2.81

11.93  3.14

10.77  3.12

41–60 years. Seshadri et al. (2002) reported that blood, Hcy level was high in the elderly population than in the younger people. In females, the HHcy was noticed maximum in the age group of 41–50 years (Hak et al. 2000). Apart from the association of lifestyle practices like alcoholics, smokers, and fatty eating habits, particularly obese persons are having higher mean Hcy concentration in comparison to apposite group. In one of the studies, it is noticed that abnormal distribution of upper body fat causes HHcy and more oxidative injuries ultimately resulting in cardiovascular disease (Chrysohoou 2004). One of the most important findings is that vegetarians have a more higher level of Hcy than non-vegetarians. The hypothesis behind that facts may be that vegetarians may have deficiency of B6, B12, and folate in their diet causing HHcy than the non-vegetarians who are having a considerably better intake of these contents in their meals (Bissoli et al. 2002, Kumar et al. 2005, Nair et al. 2002; Chandalia et al. 2003; Kumar et al. 2009; Misra et al. 2002) (Table 7.1).

7.16

Discussion

The present study is based on the relationship between various biochemical parameters and their association with lipid profiles. The body mass index (BMI) is strongly associated with C6771 genotype subjects. Such people are more prone to

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develop CHD due to genetic predisposition. A comprehensive study has been carried out to evaluate various biochemical parameters particularly lipid profile in relation to coronary heart disease. A low level of serum folic acid is a major causative factor of homocysteine deficiency leading to coronary heart disease. Further, lipid profile particularly elevated level of LDL-c showed a significant relationship with high triglycerides. Serum folate is the most important factor in the regulation of homocysteine metabolism in both age and sex groups. It is evident that low folic acid and high level of hyperhomocysteine play a major role in the occurrence of CHD. A series of studies conducted in different ethnic groups also demonstrated the role of hyperhomocysteinemia in different age and sex groups. It is proposed to carry out a bigger sample size with various parameters in a multi-site study. The present study is part of a comprehensive study where we are conducting ethnic variation in different age and sex groups.

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Sener U, Zorlu Y, Karaguzel O, Ozdamar O, Coker I, Topbas M (2006) Effects of common antiepileptic drup monotherapy on serum levels of homocysteine, vitamin B12, folic acid and vitamin B6. Seizure 2:79–85 Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D'Agostino RB, Wilson PW, Wolf PA (2002) Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. N Engl J Med 346(7):476–483 Stampfer MJ, Malinow MR, Willett WC, Newcomer LM, Upson B, Ullmann D, Tishler PV, Hennekens CH (1992) A prospective study of plasma homocyst (e) ine and risk of myocardial infarction in US physicians. JAMA 268(7):877–881 Steegers-Theunissen RP, Boers GH, Trijbels FJ, Finkelstein JD, Blom HJ, Thomas CM, Borm GF, Wouters MG, Eskes TK (1994) Maternal hyperhomocysteinemia: a risk factor for neural-tube defects? Metabolism 43(12):1475–1480 Stehouwer CD, Weijenberg MP, van den Berg M, Jakobs C, Feskens EJ, Kromhout D (1998) Serum homocysteine and risk of coronary heart disease and cerebrovascular disease in elderly men: a 10-year follow-up. Arterioscler Thromb Vasc Biol 18(12):1895–1901 Stuppia L, Gatta V, Gaspari AR, Antonucci I, Morizio E, Calabrese G, Palka G (2002) C677T mutation in the 5, 10-MTHFR gene and risk of down syndrome in Italy. Eur J Hum Genet 10(6): 388–390 Takamura N, Kondoh T, Ohgi S, Arisawa K et al (2004) Abnormal folic acid homocysteine metabolism as maternal risk factors for down syndrome in Japan. Eur J Nutr 43:285–287 Taoka S, West M, Banerjee R (1999) Characterization of the heme and pyridoxal phosphate cofactors of human cystathionine β-synthase reveals nonequivalent active sites. Biochemistry 38(9):2738–2744 Tassone F, Lucas R, Slavov D, Kavsan V, Crnic L, Gardiner K (1999) Gene expression relevant to down syndrome: problems and approaches. In: The molecular biology of down syndrome. Springer, Vienna, pp 179–195 Tawakol A, Omland T, Gerhard M, Wu JT (1997) Hyperhomocyst(e)inemia: is associated with impaired endothelial-dependent vasodilatation in humans. Circulation 95:1119–1121 Trabetti E (2008) Homocysteine, MTHFR gene polymorphisms, and cardiocerebrovascular risk. J Appl Genet 49(3):267–282 Ueland PM, Hustad S, Schneede J, Refsum H, Vollset SE (2001) Biological and clinical implications of the MTHFR C677T polymorphism. Trends Pharmacol Sci 22(4):195–201 Ueland PM, Refsum H (1989) Plasma homocysteine, a risk factor for vascular disease: plasma levels in health, disease and drug therapy. J Lab Clin Med 114:473–501 Ulrey CL, Liu L, Andrews LG, Tollefsbol TO (2005) The impact of metabolism on DNA methylation. Hum Mol Genet 14(suppl_1):R139–R147 van der Dijs FPL, Schnog J-JB, Brouwer DAJ, HJR V, van den Berg GA, Bakker AJ, Duits AJ, Muskiet FD, FAJ M (1998) Elevated homocysteine levels indicate suboptimal folate status in pediatric sickle cell patients. Am J Hematol 59:192–198 van der Put NM, van der Molen EF, Kluijtmans LA, Heil SG, Trijbels JM, Eskes TK, Van Oppenraaij-Emmerzaal D, Banerjee R, Blom HJ (1997) Sequence analysis of the coding region of human methionine synthase: relevance to hyperhomocysteinaemia in neural-tube defects and vascular disease. Q J Med 90:511–517 van der Put NM, Gabreels F, Stevens EMB et al (1998) A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects. Am J Hum Genet 62:1044–1051 Verhoef P, Hennekens CH, Malinow MR, Kok FJ, Willett WC, Stampfer MJ (1994) A prospective study of plasma homocysteine and risk of ischemic stroke. Stroke 25:1924–1930 Verkleij-Hagoort A, Bliek J, Sayed-Tabatabaei F, Ursem N, Steegers E, Steegers-Theunissen R (2007) Hyperhomocysteinemia and MTHFR polymorphisms in association with orofacial clefts and congenital heart defects: a meta-analysis. Am J Med Genet A 143(9):952–960 Wald DS, Law M, Morris JK (2002) Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 325(7374):1202

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Wechsler D, Kodama H (1949) Wechsler intelligence scale for children, vol 1. Psychological Corporation, New York Weisberg I, Tran P, Christensen B, Sibani S, Rozen R (1998) A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol Genet Metab 64(3):169–172 Weiss N, Keller C, Hoffmann U, Loscalzo J (2002) Endothelial dysfunction and atherothrombosis in mild hyperhomocysteinemia. Vasc Med 7(3):227–239 Wenstrom KD, Johanning GL, Owen J, Johnston KE, Acton S, Cliver S, Tamura T (2000) Amniotic fluid homocysteine levels, 5, 10-methylenetetrahydrafolate reductase genotypes, and neural tube closure sites. Am J Med Genet 90(1):6–11 Wernimont SM, Clark AG, Stover PJ, Wells MT, Litonjua AA, Weiss ST, Gaziano JM, Tucker KL, Baccarelli A, Schwartz J, Bollati V (2011) Folate network genetic variation, plasma homocysteine, and global genomic methylation content: a genetic association study. BMC Med Genet 12(1):1–1 Wilcken DE (1998) Novel risk factors for vascular disease: the homocysteine hypothesis of cardiovascular disease. J Cardiovasc Risk 5:217–221 Woo KS, Chook P, Lolin YI, Cheung AS, Chan LT, Sun YY, Sanderson JE, Metreweli C, Celermajer DS (1997) Hyperhomocyst(e)inemia is a risk factor for arterial endothelial dysfunction in humans. Circulation 96:2542–2544 Wotherspoon F, Laight DW, Turner C, Meeking DR, Allard SE, Munday LJ, Shaw KM, Cummings MH (2008) The effect of oral folic acid upon plasma homocysteine, endothelial function and oxidative stress in patients with type 1 diabetes and microalbuminuria. Int J Clin Pract 62(4): 569–574 Yoo JH, Chung CS, Kang SS (1998) Relation of plasma homocysteine to cerebral infarction and cerebral atherosclerosis. Stroke 29(12):2478–2483 Yoo JH, Park JE, Hong K, Lee S, Kim DK, Lee WR, Park SC (1999) Moderate hyperhomocysteinemia is associated with the presence of coronary artery disease and the severity of coronary atherosclerosis in Koreans. Thromb Res 94:45–52 Zee Robert YL, Mora S, Cheng S, Erlich H, Lindpaintner RN, Buring J, Ridker P (2007) Homocysteine, 5, 10, methylenetetrahydrofolate reductase 677 C-T polymorphism, nutrient intake, and incident cardiovascular disease in 24,968 initially healthy women. Clin Chem 53(5): 1–7 Zhou J, Werstuck GH, Lhoták Š, Shi YY, Tedesco V, Trigatti B, Dickhout J, Majors AK, DiBello PM, Jacobsen DW, Austin RC (2008) Hyperhomocysteinemia induced by methionine supplementation does not independently cause atherosclerosis in C57BL/6J mice. FASEB J 22(7): 2569–2578 Zhou ZS, Zhao G, Wan W (2001) Current developments in biological methylations. Front Biotech Pharm 2:199–217

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Homocysteine Metabolism as a Biomarker for Cancer Meghavi Kathpalia, Prashant Kumar, and Swati Mohapatra

Abstract

Changes in methyl group metabolism and homocysteine balance have been implicated in various clinical disorders in recent decades. Elevated circulating homocysteine concentrations are linked to various dietary, hormonal, and genetic variables, cancer development, and autoimmunity. Here in this chapter, we have explained detailed links about homocysteine and cancer. Homocysteine is a sulfur-containing amino acid that is metabolized via one of the two pathways: remethylation or transsulfuration. It is produced from the metabolism of methionine, an essential amino acid. Abnormalities cause hyperhomocysteinemia in this mechanism. It is linked to a higher risk of vascular and neurological illnesses, auto immunological disorders, birth abnormalities, diabetes, renal disease, osteoporosis, neuropsychiatric disorders, and cancer. Hcy appears to be a vital predictor of overall health condition, according to a wealth of evidence. Recent research has begun to uncover the cellular and molecular pathways through which folate prevents age-related illness. Even though much remains to be learned, our understanding of the relationship between disease, methyl balance, and

Meghavi Kathpalia and Prashant Kumar contributed equally with all other contributors. M. Kathpalia Department of Centre for Clinical and Translational Research, Jamia Hamdard, Delhi, India P. Kumar Department of Biotechnology, Motilal Nehru National Institute of Technology, Allahabad, India S. Mohapatra (*) Department of Infection Biology, School of Medicine, Wonkwang University, Iksan, South Korea School of Science, Gujarat State Fertilizers and Chemicals University (GSFCU), Vadodara, Gujarat, India # The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2022 G. P. Dubey et al. (eds.), Homocysteine Metabolism in Health and Disease, https://doi.org/10.1007/978-981-16-6867-8_8

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epigenetic control of gene expression has continuously progressed. However, the role of homocysteine balance in health and disease seems to be well known. Keywords

Hyperhomocysteinemia · Biomarker · Cancer · Plasma Hcy · Remethylation · Folate

8.1

Introduction

The metabolism of the methyl group and homocysteine has emerged as a metabolic process with major health and disease implications, particularly when it is interrupted. Although the link between methyl groups and epigenetic gene regulation is relatively obvious, the link between impaired homocysteine metabolism and numerous clinical illnesses is unknown (Schalinske and Smazal 2012). Homocysteine is a sulfur-containing amino acid obtained from metabolism of essential aminoacid and obtained via diet in our body. Intracellular homocysteine is secreted at high concentrations extracellularly and metabolized by one of the two pathways: remethylation or transsulfuration (Fig. 8.1). Homocysteine (Hcy) receives an alkyl radical from N-5-methyltetrahydrofolate or alkaloid throughout remethylation, leading to the formation of essential amino

Fig. 8.1 Homocysteine mechanism of breakdown (Hasan et al. 2019)

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acids. N-5-methyltetrahydrofolate reaction (anemia factor-dependent) occurs in tissues while alkaloid reaction (anti-anemia factor) occurs within the liver. Pteroylglutamic acid from the diet is converted to 5-10-methyltetrahydrofolate, which is converted to 5-methyltetrahydrofolate by the protein 5,10methylenetetrahydrofolate reductase (MTHFR) enzyme (Brustolin et al. 2010). SAdenosylmethionine is created once a substantial quantity of essential amino acid is activated by ATP (SAM) and principally functions as a universal alkyl radical donor for an enormous variety of acceptors. S-Adenosylhomocysteine (SAH), a by-product of those methylation processes, is subsequently hydrolyzed, yielding homocysteine, which can start a new methyl group transfer cycle (Hashimoto et al. 2007). Homocysteine is converted into cystathionine via cystathionine synthetase enzyme followed by an amino acid, cysteine, and ultimately, leading to glutathione within the transsulfuration pathway (Brustolin et al. 2010).

8.2

Homocysteine Metabolism Nutritional Regulation

According to research on homocysteine metabolism, the transsulfuration and remethylation mechanisms regulate homocysteine molecules nutritionally. SAM is an allosteric inhibitor of the methylenetetrahydrofolate reductase (MTHFR) enzyme and a cystathionine-synthase activator. The regulation of intracellular SAM concentration is the second mechanism by which remethylation and transsulfuration are coordinated (Fig. 8.2).

Fig. 8.2 Metabolism of homocysteine and its two regulatory pathways with combined action

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1. The low-molecular-weight SAM synthetase converts methionine to SAM rapidly when dietary methionine levels are high. Increased intracellular SAM concentration leads to inhibition of methylenetetrahydrofolate reductase which will result in suppression of N-5-methyltetrahydrofolate synthesis, thus, allowing GNMT enzymes to operate at near-total capacity. Conversely, if dietary essential amino acid levels are insufficient, SAM levels are inadequate to inhibit MTHFR, leading to an inflated rate of N-5-methyltetrahydrofolate development. Increased substrate accessibility for homocysteine remethylation is associated with GNMT inhibition and SAM conservation, as well as an increase in intracellular N-5methyltetrahydrofolate concentrations (Williams and Schalinske 2010). Hyperhomocysteinemia is a condition in which the body produces too much homocysteine. In healthy persons, hyperhomocysteinemia (HHcy) levels in the blood are related to age and gender. Men’s plasma Hcy levels are more significant than women’s, increasing from 10.8 mmol/L at 40–42 to 12.4 mmol/L at 65–67. The usual range of plasma tHcy for “healthy persons” is 5–15 Amol/L, moderate hyperhomocysteinemia is 15–25 Amol/L, intermediate hyperhomocysteinemia is 25–50 Amol/L, and extreme hyperhomocysteinemia is >50 Amol/L, according to Jacobsen. According to the guidelines, plasma tHcy should be kept below 10 Amol/L (Wu and Wu 2002). 2. HHcy is found in about 5% of the population and is associated with various illnesses (Brustolin et al. 2010).

8.3

Relationship Between Homocysteine Regulation and Disease in Hyperhomocysteinemia

Augmented DNA mutations in tumor suppressor genes are probably concerned with vitamin B deficiency and subsequently increase homocysteine levels which promote cancer formation. MTHFR polymorphisms, for instance, are shown to influence the chance of a spread of cancers, with the correlation being that MTHFR isoforms that raise homocysteine levels typically raise the risk of cancer (Mattson et al. 2002) (Fig. 8.3). It is evident that elevated homocysteine levels within the blood are related to a range of diseases; thus, homocysteine management could be an important target for health (Hasan et al. 2019). Research has shown a strong correlation between hyper homocystinuria and cancer: 1. Cancer patients have higher plasma homocysteine levels, and venous thromboembolism (VTE) is one of the leading causes of mortality in people with cancer. 2. Clinical investigations have linked polymorphisms in enzymes implicated in the Hcy detoxification pathways (transsulfuration and remethylation) to various cancer types. 3. Folate, which is required for cell growth, is antagonistic to Hcy.

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Fig. 8.3 Carbon metabolism and control of homocysteine levels (Mattson et al. 2002)

Finally, Hcy has been postulated as a possible cancer biomarker for many cancers (Hasan et al. 2019).

8.4

Hyperhomocysteinemia and Homocysteinemia

Homocysteine is primarily found in three forms: free homocysteine (less than 1%), free altered homocysteine (10–20%—cysteine and homocysteine (homocysteine dimer)), and macromolecules interacted with homocysteine, such as primarily albumen binding (80–90%) (Burdennyy et al. 2017) (Fig. 8.4). Other genetic (CBS-independent) and environmental factors may cause elevated Hcy serum levels, referred to as “hyperhomocysteinemia.” This hyperhomocysteinemic syndrome is linked to a variety of diseases. Increased plasma Hcy levels have conjointly been connected to neurodegeneration, diabetes, mongolism, exoderm defects, and pernicious anemia, consistent with further analysis (Hasan et al. 2019). Each environmental and molecular-genetic effect influences the development of complicated disorders, making it difficult to determine the source of the pathology’s origin. However, molecular changes within the genes accountable for varied organic chemistry processes within the cell tend to be the first cause, with one-carbon metabolism genes being notably involved (Islam 2015). This mechanism involves the metabolisms of vitamin Bc, methionine, and homocysteine, which are all interconnected (Table 8.1). On the other hand, alterations in homocysteine levels may indicate pathological changes and put the patient in a high-risk category for developing pathology (Fig. 8.5).

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Fig. 8.4 Forms of homocysteine in the blood Table 8.1 Gene responsible for causing the disease

Methionine synthase reductase (MSR)

Type of polymorphism 677C ! T 1298A ! C 1793G ! A 66A ! G

Methionine synthase (MTR)

2756A ! G

Methylenetetrahydrofolate dehydrogenase (MTHFD)

1958G ! A 401G ! A

Betaine-homocysteine methyltransferase (BHMT)

742G ! A

TCN2

776 G ! C

TYMS

TS 30 -UTR TSER

Cystathionine beta-synthase

595G ! A

Gene name Methylenetetrahydrofolate reductase (MTHFR)

Cancer type Cancer of endometrium Cancer of prostatic glands Blood cancer Blood cancer Cancer of inflammatory bowel syndrome Cancer of gastric cells Cancer of head and neck Cancer of inflammatory bowel syndrome Lung cancer Blood cancer Cancer of ovaries Cancer of gastric cells Cancer of breast Cancer of squamous cell Uterine carcinoma Brain tumor Blood cancer Cancer of ovaries Cancer of stomach Oral cancer Cancer of breast Cancer of liver

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Fig. 8.5 Folate, methionine, and homocysteine metabolism (Burdennyy et al. 2017)

8.5

Hyperhomocysteinemia Pathogenesis and Metabolism

The little homocysteine seen in plasma is due to a cellular export process that complements homocysteine catabolism transsulfuration and helps maintain low intracellular concentrations of potentially lethal sulfur amino acid. Hyperhomocysteinemia indicates that homocysteine metabolism has been interrupted, and the export mechanism takes extra homocysteine from the cell and into the bloodstream (Hashimoto et al. 2007). In the homocysteine metabolism, the MTHFR enzyme catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the most prevalent circulating type of folate. 677C > T and 1298A > C are two functional polymorphisms in the MTHFR gene. Reduced enzymatic activity; reduced folate concentrations in serum, plasma, and red blood cells; and slightly higher plasma total homocysteine (tHcy) concentrations are all connected to the MTHFR 677T allele. The polymorphism 1298A > C affects MTHFR activity, although there are no biochemical alterations. MTHFR activity is essential to keep the folate and methionine circulating pool balanced and prevent Hcy buildup (Brustolin et al. 2010). Changes in homocysteine and folic acid metabolism, as an example, will cause abnormal deoxyribonucleic acid methylation, with oncogenes being demethylated and tumor suppressor genes being hypermethylated (Burdennyy et al. 2017). The majority of homocysteine in circulating blood forms a disulfide bond with albumin. Homocysteine is found as a mixed disulfide with cysteine or as homocysteine alone in about 10% to 20% of the population. The amount of Hcy in the blood is relatively low (approx. 1%). Homocysteine metabolism necessitates the involvement of vitamin Bc, vitamin B complex (cobalamin), and vitamin B

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complex (pyridoxal phosphate) coenzymes; lowering homocysteine levels in plasma necessitates supplementation of all three vitamins Several studies on hyperhomocysteinemia recommend that elevated tHcy within the blood will increase the danger of cancer. Moreover hyperhomocysteinemia is possibly a risk for carcinogenesis (Wu and Wu 2002). Because functional polymorphisms in folate-related genes affect folate metabolism, it is possible that polymorphisms or gene-environment interactions, rather than only folate intake, increase the risk of breast cancer. As a result, MTHFR polymorphisms in breast cancer have been extensively studied, with diversified results (Brustolin et al. 2010).

8.6

Role of Homocysteine Metabolism in Cancer

A lack of specific essential nutritional components, like vitamin B family, could play a vital role in producing hormone-dependent tumors. Polymorphisms in genes concerned with folate-homocysteine and essential amino acid metabolism, methylation in their promoter regions, and the microRNA on gene expression leads to genetic risk factors (Burdennyy et al. 2017). The failure of malignant cells to remodel homocysteine to essential amino acids causes them to be methioninedependent. In cases of female internal reproductive organ, breast, pancreatic, and carcinoma, changes the level of homocysteine. The implying that growth markers in homocysteine, expressed growth cell activity and propagated fleetly. Because of enhanced proteosynthesis and transmethylation processes, malignant cells have a high growth rate and a higher Met requirement. Hcy remethylation will cover Met consumption in normal cells. Hcy builds up in organs like the lung, kidney, breast, colon, and bladder because malignant cells in these organs can’t convert Hcy to Met. Hcy levels that are higher are also connected to increased folate levels (Talib et al. 2021). Folate cofactors are required for Hcy to Met remethylation, SAM synthesis, and the generation of nitrogenous bases for DNA/RNA synthesis (Škovierová et al. 2016). MTHFR polymorphism can reduce the output of the enzyme’s product, 5-MTHF, and raise cancer risk. The methyl group for DNA methylation is provided by 5-MTHF, which is the most common source of folate in serum. Reduced 5-MTHF causes global genomic hypomethylation, a common and early event in carcinogenesis (Lauinger and Kaiser 2021). According to many reports, patients with acute lymphoblastic leukemia and colorectal, ovarian, pancreatic, and head and neck squamous cell carcinomas exhibited higher plasma tHcy levels. Methionineindependent cells have a lower SAM/SAH ratio (Lauinger and Kaiser 2021). Reduced intracellular SAM levels may affect cytosine methylation in CpG islands of DNA, causing tumor suppressor genes to be repressed, proto-oncogenes to be activated, and malignant transformation to be induced. Hypomethylation of coding and noncoding areas, as well as demethylation of repetitive DNA sequences, may contribute to cancer formation via the following mechanisms: chromosomal

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instability, mutations, reactivation of intragenomic parasite sequences that could be transcribed and transferred to other places, and disruption of normal cellular genes, loss of heterozygosity, aneuploidy, and upregulation of proto-oncogenes are all consequences of mitotic recombination (Keshteli 2015). Several studies studied the epigenetic changes that are associated to cancer progression and located the next Hcy level and an unchanged plasma level of Cys in cancer patients.

8.6.1

Deficiency in Folate

A deficiency of folate is frequently connected to hyperhomocysteinemia. Subjects with below-median blood folate and above-median total alcohol use, for example, were nearly twice as likely as those with above-median serum folate and belowmedian alcohol consumption to develop colorectal cancer. Hyperhomocysteinemia is frequently linked to this condition. Another reason why a lack of folate increases cancer risk is because it prevents the conversion of deoxyuridylate to thymidylate and leads to extensive uracil incorporation into human DNA, resulting in chromosome breakage (Tinelli et al. 2019).

8.6.2

Stress Due to Oxidation

The overproduction of oxygen free radicals produced by the oxidation of homocysteine can cause endothelial injury and DNA damage. When homocysteine is oxidized, a free sulfhydryl group in reduced free homocysteine forms a disulfide bond with the free sulfhydryl group of albumin, cysteine, or homocysteine, resulting in free radicals such as hydrogen peroxide (Škovierová et al. 2016). DNA oxidation can produce gene mutations, such as those in the p53 and ras genes, which lead to carcinogenesis. Oxidative DNA damage, such as 8-hydroxyguanine, has also accumulated in malignant tissue in studies (Perillo et al. 2020).

8.6.3

Methylation of DNA that Is Not Appropriate

DNA methylation appears to have a role in DNA repair and genomic integrity, according to new studies. Reduced levels of S-adenosyl methionine (SAM) and Sadenosylhomocysteine (SAH) have been discovered in children with acute lymphoblastic leukemia (ALL), and leukoencephalopathy (LE) treated with methotrexate, resulting in hypomethylation (Kishi et al. 2000). As a result, inhibiting DNA repair mechanisms, which leads to increased mutation rates and chromosomal instability, has been proposed to initiate and speed up the neoplastic process. DNA methylation patterns (epigenetic changes) are typically detected early in carcinogenesis,

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including local hypermethylation and genome-wide hypomethylation, in addition to genetic changes.

8.6.4

Thiolactone of Homocysteine

Hyperhomocysteinemia patients had greater homocysteine thiolactone concentrations. Under normal circumstances, the thiolactone can acylate with the principal amine of protein lysine residues. On the other hand, malignant cells cannot convert homocysteine thiolactone to sulfate in the same way normal cells do. Homocysteine thiolactone accumulates in malignant cells; as a result, it causes cellular macromolecule damage. Increased synthesis of Hcy thiolactone has been linked to the neoplastic transformation of human cells (Wu and Wu 2002). Because of region-specific hypomethylation, homocysteine can also predispose to cancer by activating pro-inflammatory genes. Homocysteine can cause intestinal mucosal injury via changing TNF-mediated cytotoxicity, according to in vitro and in vivo studies. Indeed, plasma homocysteine has been found as a predictor of TNF in clinical circumstances defined by low-grade inflammation, and decreasing the TNF pathway can considerably reduce homocysteine levels, showing that this cytokine is involved in homocysteine production (Bogdanski et al. 2008). Hyperhomocysteinemia significance in developing hormone-dependent malignant tumors like breast and ovarian cancer will be discussed below.

8.6.5

Cancers of Breast and Ovary

Breast and sex gland cancers are the most common cancers, with sex gland cancer (OC) having the best morbidity. Multiple studies recommend that polymorphisms in genes concerned with pteroylglutamic acid, homocysteine, and essential amino acid metabolism may well be risk factors for the event of carcinoma; however, the findings are mixed. The A66G polymorphism of the MTRR cistron and the T/T genotype of the MTHFR cistron is joined to a hyperbolic risk of carcinoma, still as an affiliation between these polymorphisms and pteroylmonoglutamic acid supplementation (Hasan et al. 2019). MTHFR C677T and MTR A2756G are coupled to the event of luminal-B and HER+ histotypes, still because of the development of the luminal-A carcinoma histotype. Patients with luminal-B and thrice harmful histologic carcinoma possessed hyperhomocysteinemia (Plazar and Jurdana 2010). Finally, the C/C genotype of the SHMT1 gene’s C1420T polymorphism was shown to possess a protecting result, leading to the lower organic phenomenon and a lower risk of developing thrice negative carcinoma. At the same time, few studies investigated the results of alternative polymorphisms in genes concerned with pteroylglutamic acid, homocysteine, and essential amino acid metabolism within carcinoma development (Burdennyy et al. 2017).

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Inflammatory Bowel Disease and Colorectal Cancer

There is a well-established link between inflammation and cancer in the gastrointestinal tract. Toll-like receptors and tumor necrosis factor (TNF) have been linked to the activation of nuclear factor B, which causes the transcription of genes involved in carcinogenesis, including COX-2, in colitis-associated cancer (Liu et al. 2017). In patients with colitis-induced dysplasia, faulty p53 signaling could be a prelude to cancer. Cancer arises when abnormal cells do not die due to p53-induced apoptosis (Keshteli 2015).

8.7

Control of Homocysteine Metabolism

There are several strategies for lowering homocysteine levels and, as a result, reducing the risk of developing various illnesses as people age. • The most prevalent approach now is dietary supplementation with folate (usually 400 g per day). • Supplementing the diet with vitamins B12 and B6 can help lower homocysteine levels and increase the effects of folate. Dietary restriction, which is used to extend the life and reduce the risk of a range of age-related disorders, can also help to reduce homocysteine levels (Barroso et al. 2017). • Vegetarian diets and physical activity have lower homocysteine levels (Mattson et al. 2002). • In most situations, vitamin supplementation leads to a near-normalization of plasma homocysteine. A diet rich in folate, found in vegetables and fruits, has been associated with a reduced risk of various malignancies (Brustolin et al. 2010). Homocysteine levels in the body are kept low by two important mechanisms. • Homocysteine is remethylated into methionine, which requires the utilization of folate and vitamin B12. Changes in methionine expression or functional activity are therefore required (Kumar et al. 2017). • Genetic mutations in MTHFR diminish enzyme function, resulting in decreased folate and higher homocysteine, increasing disease risk, and CBS can impact homocysteine levels, emphasizing the relevance of folate (MATTSON et al. 2002). • As mentioned above, we can say that tHcy within the blood tends to be a more potent tumor marker for trailing cancer patients throughout treatment. It faithfully represents tumor cell proliferation rates similarly as a response to tumor necrobiosis. It is flexible to be a sensitive marker for detecting repetition, similar to an early marker for carcinogenesis. Hyperhomocysteinemia association to vitamin Bc deficiency, aerobic stress, abnormal DNA methylation, and Hcy thiolactone adds to the case for hyperhomocysteinemia as a cancer risk issue (Wu and Wu 2002).

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Conclusion

Homocysteine balance, particularly hyperhomocysteinemia, appears to be a common hallmark of a variety of diseases. It is uncertain if high homocysteine levels contribute directly to ailment pathogenesis or serve as a biomarker for metabolic abnormalities such as abnormal methyl group metabolism. HHcy has been linked to several polymorphisms in genes implicated in the homocysteine methionine pathway, signaling that these variants may have a role in several multifactorial illnesses with a high incidence in the general population. As a result, Hcy-elevating medications should be used with prudence in cancer patients, and Hcy levels should be regularly monitored following chemotherapy or surgery. Because hyperhomocysteinemia is linked to various pathological disorders, including vascular disease, it is evident that homocysteine management should be a primary concern for nutrition and health. Most studies have found that hyperhomocysteinemia influence on carcinogenesis is linked to low folate levels and other vitamin B deficiencies caused by the exact metabolic mechanisms that cause hyperhomocysteinemia. Vitamin supplementation is a cost-effective way to reduce HHcy, and widespread vitamin supplementation would prevent these health issues. However, more efforts are still needed to fully comprehend the mechanistic relationship between homocysteine imbalance and disease and the best way to maintain homocysteine balance to lower disease risk. As a result, more sophisticated studies of all potential combinations of gene-gene interactions of the specified tripartite exchange are required to diagnose effectively, make a prognosis, and establish a treatment approach.

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Keshteli AH (2015) Hyperhomocysteinemia as a potential contributor of colorectal cancer development in inflammatory bowel diseases: a review. World J Gastroenterol 21(4):1081. https:// doi.org/10.3748/wjg.v21.i4.1081 Kishi T, Tanaka Y, Ueda K (2000) Evidence for hypomethylation in two children with acute lymphoblastic leukemia and leukoencephalopathy. Cancer 89(4):925–931. https://doi.org/10. 1002/1097-0142(20000815)89:43.0.CO;2-W Kumar A, Palfrey HA, Pathak R, Kadowitz PJ, Gettys TW, Murthy SN (2017) The metabolism and significance of homocysteine in nutrition and health. Nutr Metab (Lond) 14(1):78. https://doi. org/10.1186/s12986-017-0233-z Lauinger L, Kaiser P (2021) Sensing and signaling of methionine metabolism. Meta 11(2):83. https://doi.org/10.3390/metabo11020083 Liu T, Zhang L, Joo D, Sun S-C (2017) NF-κB signaling in inflammation. Signal Transduct Target Ther 2(1):17023. https://doi.org/10.1038/sigtrans.2017.23 Mattson M, Kruman I, Duan W (2002) Folic acid and homocysteine in age-related disease. Ageing Res Rev 1(1):95–111. https://doi.org/10.1016/S0047-6374(01)00365-7 Perillo B, Di Donato M, Pezone A, Di Zazzo E, Giovannelli P, Galasso G, Castoria G, Migliaccio A (2020) ROS in cancer therapy: the bright side of the moon. Exp Mol Med 52(2):192–203. https://doi.org/10.1038/s12276-020-0384-2 Plazar N, Jurdana M (2010) Hyperhomocysteinemia and the role of B vitamins in cancer. Radiol Oncol 44(2):79–85. https://doi.org/10.2478/v10019-010-0022-z Schalinske KL, Smazal AL (2012) Homocysteine imbalance: a pathological metabolic marker. Adv Nutr 3(6):755–762. https://doi.org/10.3945/an.112.002758 Škovierová H, Vidomanová E, Mahmood S, Sopková J, Drgová A, Červeňová T, Halašová E, Lehotský J (2016) The molecular and cellular effect of homocysteine metabolism imbalance on human health. Int J Mol Sci 17(10):1733. https://doi.org/10.3390/ijms17101733 Talib WH, Barakat M, Al Kury LT (2021) Hyperhomocysteinemia and cancer: the role of natural products and nutritional interventions. In: Nutritional management and metabolic aspects of Hyperhomocysteinemia. Springer International Publishing, New York, pp 9–32. https://doi.org/ 10.1007/978-3-030-57839-8_2 Tinelli C, Di Pino A, Ficulle E, Marcelli S, Feligioni M (2019) Hyperhomocysteinemia as a risk factor and potential nutraceutical target for certain pathologies. Front Nutr 6. https://doi.org/10. 3389/fnut.2019.00049 Williams KT, Schalinske KL (2010) Homocysteine metabolism and its relation to health and disease. Biofactors 36(1):19–24. https://doi.org/10.1002/biof.71 Wu LL, Wu JT (2002) Hyperhomocysteinemia is a risk factor for cancer and a new potential tumor marker. Clin Chim Acta 322(1–2):21–28. https://doi.org/10.1016/S0009-8981(02)00174-2

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Homocysteine Metabolism and Risk of Breast Cancer in Women Rinki Kumari, Vandana Yadav, Simon Agongo Azure, Disha Sharma, Sudhanshu Mishra, Sneh Shalini, Rudra P. Ojha, and Anita Venaik

Abstract

Despite advanced medical/clinical facilities providing “screening, diagnosis, prevention, and treatment” for breast tumor, still “breast tumor-related death” among women is high, which is influenced by poor nutritional condition in less developed countries (economic-deprived countries) such as India. Homocysteine, folic acid, and vitamin D (Vit-D) deficiency, as well as other variables associated with a higher BMI, all contribute to a higher mortality rate. Although a few scientific research have proven that “hyper-homocysteinemia” (Hhcy) is a risk factor for tumorigenesis and tumour progression, and that it can impair cysteine metabolic activity. Additionally, it is in charge of altering the gene expression and R. Kumari (*) Department of Microbiology, Hind Institute of Medical Sciences, Mau Ataria, Uttar Pradesh, India V. Yadav Department of Biotechnology, Rama University, Kanpur, Uttar Pradesh, India S. A. Azure Department of Community Health, College of Health, Yamfo, Ghana D. Sharma Department of Pharmacy, Dr. MC Saxena College, Lucknow, India S. Mishra School of Pharmaceutical Science Rajiv Gandhi Technical University, Bhopal, India S. Shalini Division of ECD-1 ICMR Hqrs, New Delhi, India R. P. Ojha Department of Zoology, Nehru Gram Bharati (Deemed to be University), Prayagraj, India A. Venaik Department of General Management, Amity Business School, Amity University Noida, Noida, Uttar Pradesh, India # The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2022 G. P. Dubey et al. (eds.), Homocysteine Metabolism in Health and Disease, https://doi.org/10.1007/978-981-16-6867-8_9

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enzymatic function of the homocysteine metabolism enzyme. Furthermore, “25hydroxyvitamin D [25(OH)D]” may affect gene expression in the homocysteine metabolism of “breast cancer patients.” Folic acid is the most commonly prescribed supplement for these patients. It may also play a role in vitamin D synthesis and “BMI” regulation. To summarize, adequate folate/folic acid levels are linked to amplification of “breast cancer” progression; however, folate/folic acid is especially important for females who are at a higher risk of “breast cancer” progression due to higher alcohol intake; as a result, the goals/aims of this chapter are to elucidate and define the effects of Vit-D, folic acid, “BMI,” and homocysteine in the prevention and management of “breast tumor.” Keywords

Hyper-homocysteinemia · Vitamin D · Breast tumor · BMI · Mortality · Body mass index

9.1

Introduction

9.1.1

Cancer

Cancer is the most frequent cause of death in the world. According to the cancer stage, the majority of research explained two models that might be used to understand cancer progression: one that is hierarchical and the other that is stochastic. Expect a variety of inevitable, hereditary, and environmental factors to influence the development of cancer. Other factors, most of which are related to our personal lifestyles, such as diets, influence our behavior, which we may control to reduce our risk of developing this frequently fatal disease (Christensen et al. 1999; COHF 2001; Clegg et al. 2002). Cancer refers to multi-clinical disorders, which continue to divide rapidly and propagate abnormal cells throughout the body via the lymphoid system. According to scientific evidence, the “human body comprises trillions of cells” (Daily Mirror – Sri Lanka Latest Breaking News and Headlines). In a normal cell division process, new cells emerge when old cells die, whereas cancer disturbs the cell division process, causing old cells to survive. Tumors grow when new cells proliferate unnecessarily (Berry et al. 2005). Some malignancies, such as leukemia, can be found in the bloodstream. Onco-cells had formed themselves as a covering over malignance cells. As the world’s most significant cause of death by the early twenty-first century, “breast cancer” plays a substantial role in this context; around two million new occurrences of breast cancer and half a million pathology-related deaths are reported per year (Coleman et al. 2008).

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The Proliferation of Cancer Cells

The invasion-metastasis cascade consists of five stages like “invasion of local tissue; intravasation into the stroma and blood vessels (survival in the vascular circulation); extravasation into the parenchyma of distant tissues; survival in a new microenvironment; and colonization to form micro- and macro” (Fig. 9.1) (Gizem CalibasiKocal and Yasemin Basbinar 2018). The steps in the invasion-metastasis cascade are described below (Berry et al. 2005; Ries et al. 2005; Coleman et al. 2008; https:// www.nutrinfo.com). Alongside skin cancer, breast cancer is the most frequent cancer in women, followed by lung cancer as the second leading cause of cancer death in women. In 2008, there were 1.38 million new occurrences of “breast cancer,” with lowerincome countries accounting for nearly half of all patients and almost 60% of deaths. Survival rates for “breast cancer” vary dramatically worldwide, with estimates ranging from 80% in high-income countries to fewer than 40% in low-income ones (Talmadge and Fidler 2010; Nicole et al. 2021). One in every 9 women in high-income countries, such as the United Kingdom, may acquire breast cancer throughout her lifetime; in 2013, approximately 232,340 women will be diagnosed with diabetes, and 39,620 will die from it (Anderson et al. 2008; Valastyan and Weinberg 2011). From 1975 to 2000, continued mammography screening and treatment are credited with a significant reduction in “breast cancer”-related mortality in the United States (Siegel et al. 2013; onlinelibrary. wiley.com).

Fig. 9.1 Steps of metastasis. During the metastatic process, cells invade local tissue (1), intravasate into the stroma and blood vessels (2), survive in the vascular circulation (3), extravasate into the distant tissues (4), survive and colonize to form micrometastasis (5), and finally form a clinically detectable macro-metastasis (6) (Source-https://www.intechopen.com/books/cancer-metastasis/ introductory-chapter-cancer-metastasis)

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Fig. 9.2 Various risk factors associated with development of breast cancer

World Health Organization declared that increasing “Breast cancer” outcomes and survival is still the backbone of “Breast cancer” prevention via early detection. Early identification, diagnosis, and treatment of “breast cancer” are complicated by resource and infrastructure constraints, just as it is in poor and middle-income nations (Anderson et al. 2008). In certain research other factors like age, gender, diet, alcohol use, body mobility, family history, lifestyle, and endocrine (Fig. 9.2) are all connected to the spread of “breast cancer” (Anderson et al. 2008). As a result, genetic and adjustable possible factors impact the development of cancerous lesions. As a result, modifiable risk factors such as environmental pollutants and stress, as well as an unhealthy lifestyle, including poor eating habits, can be avoided, all of which contribute to tumorigenesis and the clinical onset of cancer (Christensen et al. 1999; Tiwari et al. 2015). Breast malignant tissue conversion is a highly complex process that also causes severe intension, leading to a genetic material lesion (Rahman and Stratton 1998; Clegg et al. 2002; Kao et al. 2009). An estimated 5–10% of breast cancer arises due to manipulation in hereditary material, known as hereditary breast cancer, and other types, specifically, linked to BRCA1 and BRCA2 mutations and another gene (Almanza), are caused by inborn cancer predisposition (Siegel et al. 2019; Navya Ajitkumar et al. 2020). Advanced molecular biology technologies have significantly contributed to explaining multifaceted cancer development or progression mechanisms including several factors. Recent research study observed that several significant genes are suspected to be involved in the pathogenesis and their deregulation is implicated in or caused development of “breast tumor.” On the other hand, it could be a cocktail of gene alterations contributing to “breast tumor” onset and progression, worldwide (Kennedy et al. 2014; Ishita et al. 2018). Multiple genetic variations have indeed been associated with the development or progression of breast tumors. In contrast, the frequency of numerous gene aberrations remains very short due to oncogene amplification and maybe “tumor suppressor gene (TSG)” mutations or deletions (Ishita et al. 2018; Navya Ajitkumar

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et al. 2020). Other genes like “ataxia telangiectasia mutated (ATM),” “breast cancer gene BRCA1/BRCA2,” “BRCA1 associated RING domain” (Navya Ajitkumar et al. 2020), “BARD1” (Navya Ajitkumar et al. 2020), cadherin 1 (CDH1), “checkpoint kinase 2 (CHEK2),” nibrin (NBN), neurofibromin 1 (NF1), partner and localizer of BRCA2 (PALB2), phosphatase and tensin homolog (PTEN), RAD51 paralog C and RAD51D, serine/threonine kinase 11 (STK11), tumor protein p53 (TP53, p53), Circadian Locomotor Output Cycles Kaput (CLOCK), and “methylenetetrahydrofolate reductase (MTHFR)” are connected to “breast tumor” progression (Ishita et al. 2018; Navya Ajitkumar et al. 2020).). Therefore, for breast cancer screening, the germline mutation genes, especially BRCA1, stand out as the most commonly used “breast tumor” genes for diagnosis (Kennedy et al. 2014; Ishita et al. 2018). Therefore, this chapter aims to determine the role of vitamin D, folic acid, body mass index (BMI), and homocysteine in breast tumors or cancer progression.

9.2

Elevated Level of Homocysteine and Breast Tumor Cell Proliferation

Recent research has defined a few other imperative influences associated with the development of “breast tumor,” such as “homocysteine (Hcy) – a thiol-containing amino acid.” It frequently plays a vital role in the thiol compound’s metabolic pathway and is a precursor to methionine epigenetic modification. Additionally, it is produced by the catabolism of methionine, a multistep process that most likely occurs in each body cell (Froese et al. 2019). Further, high concentrations of “methionine” convert homocysteine to cysteine, and “vitamins B6 and B12” and folate are essential cofactors for metabolism (Froese et al. 2019). Hcy is also concerned with methylation and the distribution of “nucleotides for replication.” The occurrence of methionine synthase makes “vitamin B12 and methyltetrahydrofolate” a co-substrate. Then homocysteine is remethylated and converted to methionine. Methionine is also involved in producing cysteine via Hcy and provides a predecessor amino acid for synthesizing “proteins, glutathione, coenzyme A, and γ-glutamyl-cysteinyl-glycine.” The cellular process must strictly regulate the excessive quantities of homocysteine (Hcy) and cysteine in the tissues/cells. Disrupted Hcy metabolism increased the level of Hcy due to recompense in the remethylation pathways, causing hyperhomocysteinemia (Hhcy).When the catabolism was disrupted by diminutive cysteine dioxygenase, it increased the cysteine concentration in the cells or tissue. When methionine levels increase, homocysteine is converted to cysteine via the transsulfuration pathway. In the presence of a low methionine level, Hcy remethylates to methionine, a process that demands the presence of methionine synthase. This enzyme requires the cofactor methyltetrahydrofolate, which is found in vitamin B12 (co-substrate). Homocysteine produces cysteine from methionine. Glutathione, CoA, and γ-glutamyl-cysteinyl-glycine are all precursor amino acids for protein synthesis (Froese et al. 2019).

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Hyperhomocysteinemia and elevated-cysteine are retained at low levels by strict regulation. They are linked to low folate levels in tissues and cells, causing Hcy metabolism to be disrupted and defects in the transsulfuration remethylation pathways. For example, insufficient concentration of vitamin B can cause homocysteine accumulation and impaired catabolism due to low cysteine dioxygenase. It is linked to various biological processes in the cell, such as DNA repair, replication, and transcription, and can lead to tumorigenesis (Lin et al. 2010). A high level of cysteine have indeed been linked to several clinical metabolic disorders, including. According to numerous studies, raised levels of “BMI” and plasma triglyceride cause hypertension and improper oxidation of low-density lipoproteins and may be a factor in the advancement of multiple malignancies like “breast cancer” (Zhang et al. 2015). In various in vitro studies, Hhcy has been linked to tumor cell proliferation in cancer patients and causes oxidative damage in cells. Further, in vivo and in vitro studies have recently revealed that cysteine acts as a prooxidant, damages DNA, and produces a vast selection of radicals and hydrogen peroxide. In addition, several shreds of evidence have also shown the linkage between Hhcy and overall risk for emergent “breast cancer” among women. However, elevated or raised concentration of Hcy was associated with an increased risk of cancer among women with a lower grade of folic acid. Besides that, higher plasma cysteine was linked to a low folate level and an increased risk of “breast tumor” (Lin et al. 2010). Furthermore, the association between cysteine levels and risk of “breast tumor” was more vital in “ER+ and PR+” “breast tumor.” As a result, various studies have found that women with higher levels of Hhcy and cysteine along with low folate levels have a higher risk of developing “breast tumor.” Several researchers have associated high levels of Hhcy and cysteine with an increased risk of “breast tumor”; thus, both Hhcy and cysteine are linked to oxidative damage and metabolic disorders, which cause carcinogenesis (Lin et al. 2010).

9.2.1

Folate and Breast Tumor

Intake of folic acid/folate produces protective shield against a few cancers among women; in addition higher folic acid/folate intake can also reduce the risk of “breast tumor.” Folic acid is a B vitamin (water-soluble) that is required for good health, and its dihydrofolate reductase (DHFR) enzyme converts various food products to tetrahydrofolate in the liver, such as dark-green leafy vegetables and legumes. It is just a vitamin produced and found in fortified complementary foods. Various reports claim that dietary folate (DF) is present in a reduced form with significant chains that must be oxidized and hydrolyzed for proper absorption, but folic acid is present in the oxidized pteroylmonoglutamate form, which is readily bioavailable (Gropper and Smith 2013). The studies have shown the range of dietary folate Dietary folate, i.e., varying from 10% to 98%; it is also influenced by some other factors like “food matrix, intestinal enzyme, acidity, pH, alcohol, and inhibitors” as well as malabsorption/

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malnutrition disorders (Gropper and Smith 2013). The dietary allowance for adults was recommended to be 400 micrograms (mcg) of dietary folate equivalents (DFE; to account for the differences in absorption between folate and folic acid) (note: 1 microgram of DFE ¼ 1 mcg dietary folate, 0.6 mcg folic acid consumed with food, or 0.5 mcg supplemental folic acids) according to nutritional guidelines. 1000 mcg/ day is the daily requirement, one-fifth of the minimum B12 deficiency (Bailey et al. 2010; Crider et al. 2011). In the United States, through dietary supplements, about 5% of the population consumes more than the maximum limit (Bailey et al. 2010). According to nutritional standards, a level of less than 7 nmol/L to less than 10 nmol/L is considered deficient. There are various ways for determining folic acid/folate levels; red blood cell (RBCs) folate reflects folate levels over months, with folic acid levels less than 315 to 363 nmol/L considered deficiency (Benoist 2008; Crider et al. 2011). Other clinical procedures for measuring folate levels include elevated urine formiminoglutamate excretion and deoxyuridine suppression diagnosis tests. A high homocysteine level is a crucial indicator of insufficiency (Gropper and Smith 2013). Folic acid is involved in one-carbon metabolism, which is heavily involved in various cancers. “5-Methyltetrahydrofolate (5-MTHF)” and “cobalamin” are other names for folate. Both are necessary for the methionine pathway to produce methionine from homocysteine. According to the studies, methionine converted to S-adenosylmethionine (SAM) is a major methyl donor for a variety of body reactions as well as nucleic acid (DNA and RNA) methylation (Rai 2014a, 2014b). If folic acid intake low, SAM synthesis is insufficient/inadequate, leading to reduced methylation mechanism of “CpG” islands in genetic material/DNA, affecting gene-transcription activity and also modifying the expression of both “tumor suppressor genes and proto-oncogenes” (Rai 2014a, 2014b). Furthermore, several experimental studies defined that lack or insufficient concentration of folic acid creates problem in the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) (Gropper and Smith 2013). Folate deficiency associated with uracil misincorporation, resulting in unstable DNA, strand breaks, and insufficient DNA repair (Rai 2014a, 2014b). A few studies have found that low folic acid intake, as well as excessive folic acid intake, can interfere with cell bioactivity, such as cell replication, as well as gene expression and function and cell survival (Chan et al. 2010; Jorde et al. 2010; Millen et al. 2010). It also causes decreased enzyme activity or efficiency, which can interfere with nutrient metabolism; it’s also linked to the development of various diseases, including cancer (Rai 2014a, 2014b). Methylenetetrahydrofolate reductase (MTHFR) is a critical flavoenzyme that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (5-MTHF) according to research (Bistulfi et al. 2010; Varela-Rey et al. 2013; Froese et al. 2019). MTHFR is encoded by the MTHFR gene, which has two alleles: C677T (which may affect 20–40% of the population) and A1298C. Both variants and alleles reduce the enzyme efficiency. Because of the

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change in enzyme efficiency has a lower affinity for its cofactor, flavin adenine dinucleotide (Rai 2014a, 2014b; Zhang et al. 2015). Yet, the role of folate/folic acid (FA) in cancer progression is controversial, and the level of folic acid/folate is inversely proportional to the risk of developing “breast cancer” (CPO). Recent studies have suggested that high levels of FA may increase the risk of “breast cancer,” whereas other findings have supported that an adequate level of folate/folic acid (FA) may reduce the risk of “breast cancer,” and who (Varela-Rey et al. 2013; CPO). Cofactor (vitamin B12) involved the methyl group transfer from 5-MTHF to homocysteine to form methionine and THF (Ishita et al. 2017, 2018). It may not effectively transfer the methyl groups if they are not present with an adequate or low intake, affecting DNA methylation, synthesis, and repair (Frakes 1996; Dein 2005; Renee et al. 2018; Prakash and Dwivedi 2019; Paola et al. 2020). As a consequence, normal methionine level and high concentration of folate may be available for use in thymidylate synthesis, reducing the likelihood of uracil misincorporation and leading to proper DNA replication and repair (Chan et al. 2010; Jorde et al. 2010; Millen et al. 2010). As a result, the potential modifying effects of nutrients combined with a DF supplement decreased the risk of “breast cancer.” However, some studies found evidence that a high folate intake reduces the risk of “breast cancer” in women who do not drink alcohol regularly (Frakes 1996; Dein 2005; Renee et al. 2018; Prakash and Dwivedi 2019; Paola et al. 2020). Women consumed more “methionine and vitamin B12/vitamin B6,” and this association indicates that nutrient-nutrient interactions may play a role in breast cancer etiology. Furthermore, research with extensive prospective cohort studies has reported that folic acid/folate intake plays a significant role in the prevention of “breast tumor” and also helps those females who regularly and moderately consume alcohol and cigarette (Chan et al. 2010; Jorde et al. 2010; Millen et al. 2010). However, alcohol or other alcoholic beverages like beer, wine, and spirits can increase estrogen levels – estrogen associated with hormone-receptor-positive “breast tumor” (Zhang et al. 1999; Ronco et al. 1999; Rohan et al. 2000). By disrupting DNA in cells, alcohol and other alcoholic beverages may raise the chance of “breast cancer” (Frakes 1996; Dein 2005; Renee et al. 2018; Prakash and Dwivedi 2019; Paola et al. 2020). According to many studies, women who have three alcoholic drinks per week have a 15% higher risk of “breast cancer” than those who do not consume alcohol (Negri et al. 2000; Sellers et al. 2001). Furthermore, alcohol is a folate antagonist that increases the fading of alcohol. As a result, it’s possible that it’ll raise a person’s folate requirement. Other research has linked low vitamin B12 levels in the blood to an increased risk of “breast tumor” in postmenopausal women. As a result, appropriate folic acid intake controls Hcy levels and prevents “breast tumor” progression (Hillman and Steinberg 1982; Weir et al. 1985).

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Vit-D and Progression of Breast Cancer

Vitamin D (Vit-D) is a bioactive steroid derivative that regulates calcium and phosphorus levels in the body and bone mineralization. It is critical in promoting bone development (Okazaki 2014). It is produced in two forms: D2 and D3 by two-mode vitamin D (Okazaki 2014; Dunn et al. 2019). Various cell processes like “proliferation, apoptosis, differentiation, inflammation, invasion, and angiogenesis” are regulated by vitamin D (Frakes 1996; Dein 2005; Renee et al. 2018; Prakash and Dwivedi 2019; Paola et al. 2020). Furthermore, after hydroxylation in hepatic cells by mitochondrial and microsomal 24-hydroxylase to create 25-hydroxyvitamin D (25(OH)D), known as “calcidiol,” tumor metastasis is regulated through numerous signaling pathways. It has the potential to influence the tumor’s formation, amplification, and growth. Serum Vit-D has a half-life of 2 to 3 weeks, which is commonly evaluated by measuring 25(OH)D biomarkers (Dunn et al. 2019). Atoum and Alzoughool have reported that Vit-D receptor or VDR genes were found to increase “breast tumor” possibility and also low serum Vit-D levels have been linked to a higher risk of various clinical issues such as colon and bladder cancer (Dunn et al. 2019) and different biological activities in the body. In addition, low Vit-D levels are linked with various clinical issues, including other oncological sicknesses (breast, colorectal, and prostate cancer) (Frakes 1996; Dein 2005; Renee et al. 2018; Prakash and Dwivedi 2019; Paola et al. 2020). In contrast, such a link has been the subject of debate in the literature. The many action modes of Vit-D have been discovered through research (Dunn et al. 2019). Through experimental studies resecher has shown the various action modes of Vit-D, which can protectively against different cancers and also ‘breast tumor’, with another way - induced apoptosis, and act as an anti-inflammatory, antiproliferative effect (Chan et al. 2010; Jorde et al. 2010; Millen et al. 2010). It can also stimulate cell differentiation and inhibit angiogenesis, invasion, and metastasis (Feldman et al. 2014). Various factors, such as obesity, low physical activity, age, race, skin type, smoking, and living at high latitudes, are also linked to reducing the levels of socializing Vit-D (Chan et al. 2010; Jorde et al. 2010; Millen et al. 2010). In addition, Vit-D acts as a modulator and modulates calcium homeostasis along with osteosynthesis (Frakes 1996; Dein 2005; Renee et al. 2018; Prakash and Dwivedi 2019; Paola et al. 2020). Therefore, numerous researches have supported that its adequate concentration improves the body’s immune or defense system and muscular and nervous systems (Shao and Klein 2012). According to a report, Vit-D is a hydrophobic biomolecule. Its primary form is Vit-D3, called “cholecalciferol,” formed by human skin from 7-dehydrocholesterol in response to “ultraviolet radiation B” (“UVB”). Several studies have reported that vitamins D2 and D3 are metabolized in the liver into 25-hydroxyvitamin D (25 (OH)D) (Renee et al. 2018; Prakash and Dwivedi 2019; Paola et al. 2020). That form of Vit-D is the primary circulating metabolite (Chen et al. 2013; Engel et al. 2014; Shaukat et al. 2017). Later in the kidney to 1,25-dihydroxyVit-D(1,25OH2D), the

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Decreases cell proliferation Increases cell maturation and apoptosis

Suppresses inflammation Reduces the accumulation of inflammatory cells

Vitamin -D

Inhibits angiogenesis Regulates insulin secretion and action

Inhibits the renin-angiotensin system Restores the glomerular filtration barrier

Fig. 9.3 The mechanism of Vit-D in cancer proliferation through various modes of action

bioactive form of Vit-D and the Vit-D receptor ligand (VDR), through CYP27B1 (1/ hydroxylase) is a mitochondrial enzyme found in proximal renal tubules (Feldman et al. 2014) and is clear that epithelial breast cells possess the same enzyme arrangement as the kidney while the effect of Vit-D on ‘breast tumor’ is biologically probable (Shao and Klein 2012). The mechanism of Vit-D in cancer proliferation through various modes of action is summarized in Fig. 9.3. Although a low level of Vit-D has a higher risk of “breast tumor” among females, numerous findings have supported that Vit-D may play a significant role in the growth of normal breast cells and prevent “breast cancerous cell” proliferation (Khan et al. 2013; Shaukat et al. 2017). However, other research published in 2007 had not supported that association between “breast tumor” risk and Vit-D levels (Chlebowski et al. 2007) because low Vit-D levels are commonly present in the population (Shaukat et al. 2017). The finding is slightly different from further research and strongly supports the association between low Vit-D and risk of cancer in the breast (Chen et al. 2013; Engel et al. 2014; Shaukat et al. 2017). According to Narvaez et al. (2014), vitamin D deficiency is most common in “breast tumor” patients (2014), and vitamin D supplementation minimizes progression of “breast cancerous cell” proliferation (Narvaez et al. 2014; Imtiaz and Siddiqui 2014) (Fig. 9.3). Some other findings have supported that sun-induced dermal Vit-D production reduces the possibility of “breast tumor” (Frakes 1996; Dein 2005; Renee et al. 2018; Prakash and Dwivedi 2019; Paola et al. 2020). As a

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result, Vit-D deficiency is linked to a higher risk of “breast tumor,” and many aspects need to be investigated further (Narvaez et al. 2014; Imtiaz and Siddiqui 2014).

9.3

“Body Mass Index” and “Breast Cancerous Cell” Proliferation

Overweight is defined as a “BMI” of 25 to