Biochemistry for Anesthesiologists and Intensivists [1 ed.] 9783030267216, 3030267210

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Biochemistry for Anesthesiologists and Intensivists Fernando Alemanno  Editor

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Biochemistry for Anesthesiologists and Intensivists

Fernando Alemanno Editor

Biochemistry for Anesthesiologists and Intensivists

Editor Fernando Alemanno Anesthesia Resuscitation and Pain Therapy Brescia Nosocomial Institutes Brescia Italy

ISBN 978-3-030-26720-9    ISBN 978-3-030-26721-6 (eBook) https://doi.org/10.1007/978-3-030-26721-6 © Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are reserved 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 Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

This book is dedicated to the memory of my mother, Amelia Nicolì, and my father, Francesco Alemanno

Foreword

...“si jeunesse savait, si vieillesse pouvait”...

According to Marc Augé (Everyone Dies Young: Time Without Age), there are two kinds of nostalgia: nostalgia for the past we have experienced and nostalgia for a past we could have experienced. The former, deduced from our not always completely trustworthy memory, is countered brutally by the irreversibility of time; the second not only wants to go back to a previous time but also wishes to subvert history itself. The same considerations were suggested to me in reading the new book by Fernando Alemanno, who I was fortunate enough to know and admire during my 20 years in Padova. To write this book Biochemistry for Anaesthesiologists and Intensivists, he has let himself be seduced by the magic of a theme which my group merely touched on in 1968 (“The Biochemical Bases of Anaesthesia”), considering it to be an inextricable aporia; but, supported by the knowledge acquired during a life of work and study, the author has laid bare the subject with methodological consistency equalled only by the clarity of his argument, obtaining results which I openly envy and, which I now comment, results which (in another of my roles) I would have wished to achieve myself. In the complex history of medicine, a very important role—even decisive, in some ways—has been played by biological chemistry or biochemistry. The further we go back in time, the more it is evident that from the earliest days, man has worked to improve his condition, with—as Pascal said—“the typical attitude of a King who has lost his throne”, with the clear conviction of having a certain dignity and of being destined to exercise dominion over others, an attitude which gives rise to the human ambition of desiring knowledge and wanting to use all his faculties and means to dominate and exploit the rest of the world for his own advantage. No progress made by man for his own emancipation has been more decisive than those which have enabled him to better know himself and to grow his own intellectual potential and power to protect himself in a disproportionate manner. This is the basis for the gains made by modern medicine, as evidenced in its unstoppable growth in knowledge and practical application. And, if medicine is the most potent weapon acquired by man in protecting himself against his own vulnerabilities, biochemistry—which studies and evaluates the component elements of living vii

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organisms, both structurally (static biochemistry) and in their role in the life processes (dynamic biochemistry)—is without doubt its foundation as the essential scientific basis for biology in general and physiology in particular. It is essential to recall that biochemistry (the term was coined by Carl Neuberg in 1903, in place of that used at the time, physiological chemistry) is “the chemistry of the living world, including plants, animals and unicellular organisms”. Initially, it availed itself of the research methods typical of physics and chemistry, as well as biology, but in the aggressive advance of its claim to be an autonomous science, it acquired specific techniques (developed by eminent scientists, of whom many tens have won the Nobel Prize), such as paper chromatography, the use of molecules marked with radioactive isotopes, ionophoresis, X-ray diffraction spectrograph, double polarization interferometry, electron microscope, simulated molecular dynamics and many others, all of which came together in the discovery of genes and the role they play in transferring endocellular information. One result is that the enormous expansion of its areas of investigation has given rise to new disciplines, focused on matters of the most wide-ranging theoretical and practical importance, so that its role, understood as essential support for clinical medicine, is by now substantially acknowledged in all research working into the relationship between structure and function and is able to delucidate the physiopathological relationships at the molecular level in order to act, ever more correctly and precisely, to prevent, diagnose and cure disease. It was almost self-evident that a versatile, polyvalent researcher like Fernando Alemanno would be seduced by the fascinating field of medical science: a field which is as vast as it is full of promise, both theoretical and practical, so vast and complicated that most people would recoil at the prospect of analysing it or even summing up its various sectors. Fernando Alemanno has the ambition and is daring to take on this enormous challenge, and while I envy his courage, I must congratulate him: he has taken on a titanic challenge and won it brilliantly! Alessandro Gasparetto Professor Emeritus of the Roman University “La Sapienza” Rome, Italy

Preface

Padua University, Lecture Room of the General Pathology Institute, March 1963 Professor Massimo Aloisi, head of the Institute, somewhat bent from his many hours spent over books, the microscope and the anatomic table, with completely white hair and with incredibly sky blue eyes behind a pair of lightly framed glasses, was starting his lesson perfectly on time. I on the other hand had arrived out of breath, 20 min late; the train from Venice that morning had been delayed; besides, there was more than 1 km to go on foot from the railway station to the Biological Institutes. Entering the room, not wanting to disturb, I took a seat in the last row. I had no idea what the Professor was saying and how he was approaching the subject, but I managed to catch the conclusion to the first part of the lecture: nature has no fantasy—tissue is either inflamed or degenerated. I jotted this down immediately, a simple sentence, a great rule, a revelation, a transmission of wisdom. Every disease follows this rule. He moved on to his next subject. Somebody in the first row asked something regarding biochemistry. Professor Aloisi went up to the large blackboard, perfectly cleaned by the caretaker before the lesson, and, with a sweeping movement of his right palm, said “Consider this blackboard as all we know about biochemistry”; then, he took a new perfectly tipped piece of chalk, which when it screeches against the blackboard makes one shiver, and marked a small dot in the centre, adding “This dot shows how much is actually useful”. There was a murmur of disappointment or satisfaction from those who had just taken the biochemistry exam with Professor Noris Siliprandi, some of them having just learned by heart one formula after another without understanding a great deal, like me: “…21”, declared Professor Siliprandi “…taking into consideration your marks in previous exams”. Ten years later, however, I came to understand that things were not exactly so. In the meantime, my understanding of the mechanism of some pathologies, such as cerebral oedema or shock, had grown, and I had come to the conclusion that biochemistry is not a matter of formulas but of concepts. I have tried to give a logical sequence to the 16 chapters of this book. Perhaps, I was not always able to do so, probably because it mostly derives from notes made about various topics, written or recalled to mind, over 50 years of my professional activity. Some subjects may appear obsolete (thiamine, dihydroxyacetone), especially to younger readers. After an initial period of experimentation and despite positive results, these drugs were abandoned in practice over the years. It was not possible to find a pharmaceutical firm willing to produce thiamine at the necessary ix

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high doses, due also to a warning issued by the Ministry of Health, which reported two cases of allergic reaction to the administration of vitamin B1 i.v.. Actually, these could have been two cases of idiosyncrasy; however, let’s not forget that thiamine was too cheap (ITL 2000, little more than 1 euro/kg) to have a good margin of profit, when compared with halogenated anaesthetics, whose price was and still is much higher. As far as dihydroxyacetone is concerned, it was little known because most publications about its use in cerebral oedema were in French or Italian and not in the English language. In any case, I have reported on my experience with these two molecules in the hope that it may stimulate curiosity and, possibly, some interest in further research. The book you are reading is not a textbook of biochemistry, but it deals with the biochemistry that has been of aid to me in understanding a variety of topics in the fields of anaesthesia, intensive care and pain medicine. My hope is that it may be a starting point for the development of future applications; but that will depend on the reader’s initiative and imagination. Brescia, Italy

Fernando Alemanno

Acknowledgments

I am very grateful to my invaluable “supporters” in putting into writing some chapters of this book. Particularly, I would like to thank: Dr. Francesca Alemanno, Graduate in Psychology, University of Padova, for her contribution to Chaps. 6 and 10 Dr. Mario Bosco, Director UOC Anaesthesia and Resuscitation, S. Spirito and Ophthalmic Hospitals, ASL Rome 1, Rome, for his contribution to Chap. 8 Dr. Paolo Grossi, Director, Anaesthesia and Pain Medicine Department, ASST Pini-CTO Traumatology and Orthopedics Centre, Milan, for his contribution to Chap. 16 I would also like to thank Rinaldo Cosio, Laura Lamberti, Donatella Romano and Frediano Tezzon for their expert reviews of several chapters; Rita Bertani, Angela Bettoni, Daniele Genco and Emilia Pati Chica for their contributions to the bibliography; Alessio Maule, for its support in all IT matters; and Valerio Rasi for the revision of Chap. 1.

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Contents

1 Enzymes and Coenzymes (A Brief, Boring but Necessary Preamble)����������������������������������������������������������������������������������������������������   1 Fernando Alemanno 2 Anaerobic Glycolysis or Embden–Meyerhof Pathway��������������������������   7 Fernando Alemanno 3 Tricarboxylic Acids Cycle or Krebs Cycle����������������������������������������������  15 Fernando Alemanno 4 Glutamate – GABA Collateral Cycle ������������������������������������������������������  23 Fernando Alemanno 5 Hexose-Monophosphate Shunt or Horecker Cycle (The Sleep Pathway)����������������������������������������������������������������������������������  35 Fernando Alemanno 6 Neurotransmitters��������������������������������������������������������������������������������������  49 Francesca Alemanno and Fernando Alemanno 7 Blood–Brain Barrier����������������������������������������������������������������������������������  71 Fernando Alemanno 8 Shock ����������������������������������������������������������������������������������������������������������  75 Mario Bosco and Fernando Alemanno 9 Cerebral Oedema ��������������������������������������������������������������������������������������  97 Fernando Alemanno 10 Pulmonary Surfactant ������������������������������������������������������������������������������ 119 Fernando Alemanno 11 When the Mind Does Not Work as It Should������������������������������������������ 123 Francesca Alemanno and Fernando Alemanno 12 Thiamine (Vitamin B1)������������������������������������������������������������������������������ 139 Fernando Alemanno

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13 Normobaric Oxygen Therapy ������������������������������������������������������������������ 161 Fernando Alemanno 14 Acid–Base Equilibrium ���������������������������������������������������������������������������� 167 Fernando Alemanno 15 Theories of Narcosis���������������������������������������������������������������������������������� 189 Fernando Alemanno 16 Local Anaesthetics�������������������������������������������������������������������������������������� 207 Paolo Grossi and Fernando Alemanno 17 Perineural Adjuvants�������������������������������������������������������������������������������� 225 Fernando Alemanno 18 The Grand Design�������������������������������������������������������������������������������������� 239 Fernando Alemanno 19 Epilogue and Farewell ������������������������������������������������������������������������������ 249 Fernando Alemanno

1

Enzymes and Coenzymes (A Brief, Boring but Necessary Preamble) Fernando Alemanno

In biochemistry, though, oxidation is not just a case of the simple and straightforward addition of oxygen, as this would result in combustion at temperatures incompatible with life. All oxidation occurs by dehydrogenation or the subtraction of electrons, with a slowing down of energy production, not unlike what happens in an atomic power plant when cadmium bars are introduced to prevent a chain reaction and resulting explosion. It might be worthwhile here just to pause and refresh our jaded notions of general chemistry and dwell for a moment on the concepts of oxidation and reduction. There are three different types of oxidative reaction in nature: –– The first occurs by the direct addition of O2, as in, for example: –– 2H2 + O2 = 2H2O (hydrogen representing the most environmentally friendly fuel) or C  +  O2  =  CO2 (both highly exothermic). Sometimes the reaction is not ­exothermic, as in the case of rusting iron: 2Fe + O2 + 2H2O = 2Fe(OH)2, where oxidation probably happens so slowly, the heat is given time to disperse. –– The second kind happens as a result of dehydrogenation, like when sulphuric acid comes into contact with chlorine, H2SO3 + Cl2 = 2HCl + SO3 and becomes dehydrogenated as sulphuric trioxide or, as we will see in the next chapter: glyceraldehyde 3-phosphate + ATP + NAD = 1,3 diphosphoglyceric acid + NADH2. –– The third comes about by the subtraction of electrons or rather by increasing positive valences (the loss of an electron), as in the case of 2KI + Cl2 = I2 + 2KCl. Here, there is no movement of either oxygen or hydrogen as the ion I- of potassium iodide (K + I) (K+I−) has lost an electron, under the influence of the Cl2, and oxidized to elementary iodine (I2).

F. Alemanno (*) Anaesthesia and Intensive Care, Brescia Clinical Institutes, Brescia, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 F. Alemanno (ed.), Biochemistry for Anesthesiologists and Intensivists, https://doi.org/10.1007/978-3-030-26721-6_1

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The concept undergoes a reversal when we refer to reductions, where, for the same reaction, Cl2 has acquired an electron and become KCl. Reductions may occur: 1. Through oxygen loss, for example, at high temperatures 2CO2 = 2CO + O2, as we have already seen, where the direct addition or subtraction of oxygen sometimes produces or requires high temperatures 2. By the addition of hydrogen, Cl2 + H2 → 2HCl 3. And lastly, by the acquisition of electrons, that is a decrease in positive valences or increase in negative ones, as in the example, Cl2 + Zn → ZnCl2, whereby chlorine takes on a negative valence through zinc action. To sum up, any type of oxidation will correspond to a loss of electrons (loss of negative charges or gain of positive valences), while a reduction will correspond to a gain of electrons, or gain of negative charge or loss of positive valence. And since no one substance is able to accept electrons without another one losing them and vice versa, oxidation will not occur in the absence of contemporaneous reduction. In the reaction C + O2 = CO2, carbon oxidizes, while oxygen reduces. The English acronym OIL-RIG, which stands for Oxidation Is Loss—Reduction Is Gain of electrons, may serve as a very useful reminder. But now back to biochemistry. In living matter, oxidation reactions cannot happen directly as they do in the inorganic world. In order to effect dehydrogenation reactions or electron subtractions in a controlled environment, at a constant pH and temperature, these reactions require important catalysts, namely enzymes. These on their own, and unaided by coenzymes, cannot fulfil their role, as the latter also catalyse the reaction and/or accept and transport hydrogen, electrons, or other groups. Firstly, the following big distinction needs to be made clear, that enzymes are proteins [1] with a heavy molecular weight and catalysing functions, which do not activate a reaction in their own right, but accelerate it in such a way that the reagents can proceed towards a state of reduced energy (or increased entropy). They are very numerous and it can safely be said that almost every biochemical reaction requires its own specific and particular enzyme. Coenzymes to the contrary, are not proteins, but relatively small molecules that often represent an active form of vitamins, and play a fundamental role in the enzymatic process. Enzymes, in fact, more so than catalysts help to recognize the substrate so leaving the coenzyme to perform the actual catalytic activity. Some coenzymes are represented by metal ions (Mg, Fe, Zn, Mn, Cu, K, Na), but which remain indispensable nonetheless in enzymatic reactions. Let us look at some examples: • Mg2+ is indispensable for: –– Hexokinase (Glucose → Glucose-6-phosphate) –– Phosphofructokinase (fructose-6-phosphate → fructose-1,6-diphosphate)

1  Enzymes and Coenzymes (A Brief, Boring but Necessary Preamble)

• • • • • •

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–– Phosphoglycerate-kinase (1,3 diphosphoglyceric acid  →  3-phosphoglyceric acid) –– Phosphoglycerate-mutase (3-phosphoglyceric acid  →  2 phosphoglyceric acid) –– Enolase (dehydration of 2 phosphoglyceric acid to 2-phosphoenolpiruvic acid, which sub-sequentially is the only glycolytic reaction to produce ATP by anhydration) –– And also needed for oxidative decarboxylation Mg2+, Mn2+ and Co2+: phosphoglucomutase (glucose-1-phosphate  →  glucose-6-phosphate) K+ and Mg2+: pyruvate kinase (2 phosphoenolpyruvic acid → pyruvic acid) Zn2+: carbonic anhydrase; alcohol dehydrogenase Cu2+, Fe2+, and Fe3+: Cytochrome oxidase Na+: ATPase Mn2+: Arginase (arginine + H2O → ornithine + urea)

While for almost each biochemical reaction there is a specific enzyme, coenzymes are not so well assigned and may be divided into: • H+ ions and electrons Acceptors and Transporters: –– NAD and NADP (derived from pyrimidine) –– FMN and FAD (derived from vitamin B2) –– Coenzyme Q (derived from benzoquinone) –– Cytochromes (derived from haem) Groups Acceptors and Transporters –– ATP, UTP, and CDP (transporters of phosphorylated bases) –– Coenzyme A (acceptor and transporter of acyl groups) made up of pantothenic acid, pyrophosphoric acid, and adenosine-3′-monophosphate (AMP) –– Cocarboxylase (or diphosphothymine, an active form of vitamin B1) –– Pirydoxal-phosphate (active form of vitamin B6) –– Coenzymes originating in vitamin B12 (5,6-dimethylbenzimidazole, benzimidazole, adenine), and playing a vital role in the: ◦◦ Metabolism of fatty acids (methyl-malonyl-CoA → succinyl-CoA) ◦◦ Isomerization of glutamic acid to Ac-β-methyl aspartate ◦◦ Dehydratase and dehydrogenase reactions ◦◦ Conversion of homocysteine to methionine –– Biotin, or more precisely also tetrafolic acid and s-adenosil methionine, active in transcarboxylations –– Finally, ATP (adenosine-triphosphate), acceptor of phosphate groups, representing the most common form of energy storage

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This derives from AMP (adenosine-monophosphate) by way of the addition of two phosphate groups with high-energy bonds (≈). Such energy bonds are produced by glycolysis, lipids, and, in a more complex way, by protein metabolisms. The two radical groups (P) (P), added to AMP are slightly instable due to the presence (according to a theory) of four negative charges that tend to violently repel each other [2], breaking the phosphate bonds, and releasing the sizable energy previously stored, now able to be used for the specific functions of the cell or for its survival. We could compare the AMP molecule to a relaxed spring that charges itself on the addition of high-energy phosphate bonds to become first ADP and then ATP. This spring is then ready to snap in case of necessity, therefore releasing all of its energy. In practice, if not an extreme necessity, the organism does not release all of the energy from the spring, but regulates this release by expelling the first phosphate bond, which is the one with the greatest energy yield and thus striking an equilibrium between ATP and ADP:

ATP « ADP + Pi +12, 000 calories

The second phosphate bond produces 7500 calories, the third, being a low energy bond, produces only 2000 cal. The reason for this limitation can be better understood if we compare the system to a car battery. If the battery is running low, driving the car will quickly recharge it but if the battery is completely flat, then the task is going to be more troublesome. If we stick with the example of the car, one way of understanding how we harvest energy is by comparing it to a battery which is rechargeable by an engine that uses gasoline (hydrocarbons) or diesel (lipids) indifferently. At this point we might be asking ourselves where all this energy that is being released by these enzymatic reactions (dehydrogenation, oxidative decarboxylation, etc.) is coming from. The answer is that this energy, stored in the ATP, derives basically from the breaking of a covalent bond. A bond defined by Gilbert Newton Lewis (1875–1946), chair of the Chemistry Department at the University of California Berkley, as being a chemical bond due to its ubiquity, and is the one represented by a dash when we write a chemical structure [3]. For example, the C–H has a bond energy of 411 kJ/ mol (4.18 kJ = 1 k-calories), whereas a C–C bond has 346 kJ/mol.

References 1. Moruzzi G, Rossi CA, Rabbi A.  Principi di chimica biologica. Bologna: Tinarelli Editore; 1984. p. 221. 2. Moruzzi G, Rossi CA, Rabbi A.  Principi di chimica biologica. Bologna: Tinarelli Editore; 1984. p. 365. 3. Pauling L. Chimica Generale. Milano: Longanesi Editore; 1967. p. 193.

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Further Reading Devlin TM. Biochimica. Napoli: EDISES; 2012. Galzigna L. Elementi di enzimologia. Padova: Piccin Editore; 1996. Garret RH, Grisham CM. Biochimica. Padova: Piccin Editore; 2014. Harper HA. Chimica Fisiologica e Patologica. Padova: Piccin Editore; 1965. Jevons FR. Le basi biochimiche della vita. Milano: Mondadori Editore; 1972. Nelson DL, Cox MM.  Introduzione alla biochimica di Lehninger. Bologna: Zanichelli Editore; 2015. Nelson DL, Cox MM. Lehninger principles of biochemistry. 7th ed. New York: Freman and Company; 2017. Pauling L. Chimica generale. Milano: Longanesi Editore; 1967. Siliprandi N. Chimica biologica. Roma: Edizioni Ricerche; 1975. Smith CUM. Biologia molecolare. Milano: Mondadori Editore; 1971.

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Anaerobic Glycolysis or Embden–Meyerhof Pathway Fernando Alemanno

To better understand the environment in which energy-producing metabolic reactions come about, we have to split the body’s cells into two main categories: those completely lacking in mitochondria like red cells, or poor in them like neuroglial cells as opposed to those that have them like neurons, muscle cells, glandular epithelia, etc. Anaerobic glycolysis exists in the red blood cells, while being prevalent in the neuroglial cells where it occurs in the cytoplasm. Glycolysis is performed in mitochondria-rich cells in both an anaerobic and aerobic way, in the last case through the Krebs cycle. The direct oxidative pathway (D.O.P.) or pentose phosphate pathway (PPP) (cytoplasmic) is a preserve of both (Fig. 2.1). Let us now try to explain things one step at a time. Firstly, glucose needs insulin to enter the cells, the exceptions being nerve cells, erythrocytes, the intestinal epithelium, renal tubules, the cells of the mammary gland during lactation and the islands of Langerhans, which also produce it. The reaction is as follows: Glucose + ATP « ADP + 6 ( P )　 Glucose

( hexokinase - Mg ) 2+





The four metabolic fates of glucose-6 (P), namely: 1. Glycogen synthesis 2. Pentose pathway 3. Having been de-phosphorylated and recirculated via the glucose-6-phosphatase enzyme and adrenaline action 4. Anaerobic glycolysis

F. Alemanno (*) Anaesthesia and Intensive Care, Brescia Clinical Institutes, Brescia, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 F. Alemanno (ed.), Biochemistry for Anesthesiologists and Intensivists, https://doi.org/10.1007/978-3-030-26721-6_2

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F. Alemanno Glycogen

Glucose 1(P)

Glucose+ ATP

Glucose 6(P) +NADP

6(P) Gluconic Acid

P.P.P.

Fructose 6(P) + ATP Fructose 1-6(P)(P) Dihydroxyacetone 1(P) DHA

3(P)-glyceraldehyde (3PG) + ATP + NAD 1-3(P)(P)-diphosphoglyceric acid + NADH2 + ADP 3(P)-phosphoglyceric acid + 2ATP (1 from DHA and 1 from 3PG)

2(P)-glyceric acid − H2O 2(P)-phosphoenolpyruvic acid + ADP Pyruvic acid + 2ATP (1 from DHA and 1 from 3PG) + NADH2 Lactic acid + NAD ATP reacted: 2 (prior to the halving of Fructose 1-6 (P) (P) into two tri-carbonic trunks). ATP formed: 4 (2 from each truncated tri-carbonic branch, ie from DHA and 3PG).

Fig. 2.1  Diagram showing anaerobic glycolysis. ATP worn-out: 2 (prior to the halving of Fructose 1-6 (P) (P) into two tri-carbonic trunks). ATP gained: 4 (2 from each truncated tri-carbonic branch, i.e. from DHA and 3PG)

We will now consider the various stages in point 4 above. 1. Transformation of glucose-6 (P) into fructose-6 (P) via the reaction: glucose　 6 ( P ) « fructose　 6 ( P ) ( phosphohexose　 isomerase )

2. Phosphorylation of Fructose-6 (P) via the reaction:



Fructose　 6 ( P ) + ATP « Fructose1　 6　 ( P )( P ) + ADP ( phosphofructokinasee )

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So far instead of producing energy we have consumed it, using 2 molecules of ATP. 3. Splitting of Fructose 1-6-(P)(P) into two triose-phosphates via the reaction: Fructose1　 6　 ( P )( P ) « dihydroxyacetone　 ( P ) + 3 ( P )　 glyceraldehyde ( aldolase )

4. Oxidation of 3(P)-glyceraldehyde:

3 ( P )　 glyceraldehyde + Pi ( H 3 PO 4 ) + NAD « 1　 3 ( P )( P )　 diphosphoglyceric acid + NADH 2 ( phophoglycerhaldeyde dehydrogenase ) perhaps glycolysis’ most important reaction due to a highly energetic phosphoric radical/carbon bond situated on carbon 1. 1　 3　 ( P )( P )　 diphosphoglyceric acid + ADP « 3　 ( P )　 glyceric acid + ATP 5. ( phosphoglycerate kinase + Mg 2+ ) 5 bis. Rapoport–Luebering Shunt In the red blood cell 1-3-diphosphoglyceric acid, instead of supplying 3-­phosphoglyceric acid, passes through 2-3-diphosphoglycerate (2-3-DPG) via the enzyme diphosphoglycero-mutase on its way from the lungs to the tissues whereby this important compound decreases the haemoglobin’s affinity for O2 and helps its release towards the tissues [1–3]. But the 2-3-DPG acts as a strong inhibitor of this enzyme and results in a negative feedback both along the lung ↔ tissues path and vice versa, perpetuating the reaction.



Lung ® tissues pathway : 1　 3　 DPG ® 2　 3　 DPG ( diphosphoglycero mutase )



Tissue ® lungpathway : 2　 3　 DPG + H 2 O ® 3　 PG + Pi ( diphosphoglycero　 2　 phhosphatase )

The 2-3-DPG accumulated at the end of the lung-tissue pathway is also transformed into 3-PG by phosphatase action that only detaches the phosphorus in position 2 without forming any ATP. An outline of the 2-3-DPG’s mode of action was outlined by Perutz in Nature magazine in 1970 [4] whereby he explained how the molecule is likely to create four salt bridges with β haemoglobin chains, thus weakening its bond with the O2; consequently, on reaching the tissues the Bohr effect finds an easy target (curve of dissociation of Hb depending on the pH, PO2, and CO2) [4].

Hb ( O 2 )4 + 2　 3　 DPG ® Hb　 2　 3　 DPG + 4O 2

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In patients undergoing extra-body circulation (CEC) the values of 2,3 DPG show a reduction of 34% after 2 h and 68% after 6 h. Candiani et al. [5] report how “… this effect has been attributed to damage induced by the oxygenator walls on the erythrocytes, and that manifests itself in the form of marked alterations in morphology and exalted haemolysis”. That ATP plays a fundamental role in the maintenance of red blood cell morphology is well-established [6], and it may be that, during CEC, the Embden–Meyerhof cycle gets diverted into a more intense product of ATP so moving towards an ATP-producing reaction 1,3 diphosphoglycerate → 3 phosphoglycerate, rather than the shunt of Rapoport–Luebering (thus decreasing oxyphoresis on the tissues), and continuing through to step n° 6: 6. The phosphoric radical of 3-(P)-glyceric acid passes on carbon 2: 3　 ( P )　 glyceric acid « 2 ( P )　 glyceric acid

( phosphoglyceromutase　　 Mg ) 2+





The 2-(P)-glyceric acid undergoes a process of dehydration with loss of a water molecule transforming a low energy bond into a high energy one:



2 ( P )　 Glyceric acid　 H 2 O « 2　 ( P )　 Enol　 pyruvic acid ( enolase )



2　 ( P )　 Enolpyruvic acid + ADP « Pyruvic acid + ATP ( pyruvickinase )

7.

8.





9. Pyruvic acid accepts 2H ions and turns into lactic acid. +

CH3 C=O COOH

CH3 +

NADH2

↔

(lactic dehydrogenase)

H-C-OH

+

NAD

COOH

Clearly, pyruvic acid with its carbon-oxygen double bond on carbon 2 is prepared to accept the two hydrogen ions of NADH2 and thus regenerate NAD. As we said, all energy-producing reactions occur by dehydrogenation (apart from that of 2-phosphoglyceric acid, which occurs by dehydration), so at the end of the energy-producing chain there must be a terminal acceptor of H+ ions. If that did not happen, tissue acidosis proving incompatible with life would rapidly occur. However, in aerobic glycolysis (Krebs cycle) the greatest acceptor is oxygen. This, at the end of the respiratory chain by accepting the two electrons subtracted from the H2, taken away from the substrate (oxidation), becomes electronegative to combine with 2H+ (become such due to the loss of two electrons) to form H2O and thus neutralize them. O2 can be looked on as the great universal base, this being its only function in living animal organisms.

2  Anaerobic Glycolysis or Embden–Meyerhof Pathway

11

In anaerobic glycolysis, pyruvic acid performs the function of O2, which by accepting two H+ hydrogenations from aldehydes oxidation (by dehydrogenation), with a reduction process, turns into lactic acid. The two H+ ions are supplied by NADH2 who stripped 3(P)-glyceraldehyde of them to oxidize it to 1-3-(P)(P)-diphosphoglyceric acid (reaction 4) and then obtaining 3-(P)-glyceric acid + ATP (reaction (5)). Pyruvic acid can therefore be considered the vicar of oxygen in anaerobic glycolysis. Furthermore the last reaction (reaction (9)),

Pyruvic acid + NADH 2 = Lactic acid + NAD

regenerates the coenzyme NADH2, transforming it back into NAD and thus restoring its function. The then regenerated NAD can return to its function of coenzyme acceptor of H+ ions, in the oxidation of 3P-glyceraldehyde, turning back into NADH2, thus perpetuating the cycle. Lactic acid, once expelled from the cell as waste, is disposed of by the liver, which is rich in mitochondria, and reconverts it into pyruvic acid to then burn it in the Krebs cycle and so produce ATP, CO2, and H2O. The heart too, in a similar way, is perfectly capable of using lactic acid as fuel. Anaerobic glycolysis produces only 2 ATP molecules for each glucose molecule metabolized. Actually it produces 4 ATP but uses 2, one for glucose-6-(P) phosphorylation and the other for the transformation of fructose-6-(P) into fructose-1-6 (P) (P), resulting in a total number of 2 ATP molecules. This might appear a small amount, but is all that it is required, in the case, for example, of the red blood cells maintaining cellular polarization, or for the neuroglia cells to perform their duties, for example, as the constituent elements, with endothelial cells, which make up the haemato-encephalic barrier.

2.1

The Origins of ATP

ATP comes about in three different ways: 1. By the oxidation of 3(P)-glyceraldehyde (and dehydrogenation) in the anaerobic glycolysis. The aldehydic functional group of 3(P)-glyceraldehyde is oxidized by dehydrogenation to acid and it is the carboxylic group, which combines with the inorganic phosphate, to accumulate the energy released by the oxidation of the aldehydic group. At this point the phosphate, which has accumulated the energy produced by oxidation, can be sent to the ADP to form ATP. The phosphate instead, which is bound to carbon in position 3, remains inert. 2. For the dehydration of 2-phosphoglyceric acid to phosphoenolpyruvic acid; it is the only example of ATP formation for dehydration rather than oxidation (dehydrogenation) of the substrate; phosphoenolpyruvic acid transforms into a molecule with a highly energetic phosphoric bond.

12

F. Alemanno

One might ask at this point what are the structural conditions that distinguish a simply phosphorylated molecule from a phosphorylated one with a high energy bond. Well, if we look at the two molecules, namely the 1-3(P)(P)-glyceric and then phosphoenolpyruvic acid, we realize that the high-energy bond binding the phosphorus is always positioned two atoms away from a double link. In particular in 1-3-(P)(P) glyceric acid, while the phosphoric bond in position 1 (that of P captured from the aldehyde oxidizing to acid) has gained a high energy status and is two atoms away from a double bond, the one in position 3 lies more than two atoms away from the double bond. 1-3(P)(P)-Glyceric acid O C

OH

CH2

COOH

O ≈ P = O

HCOH

C

OH OH O

Phosphoenolpyruvic acid

CH2

OH

O ≈ P=O OH

P = O OH

3. In the Krebs cycle, where the glucose, lipidic (via the β-oxidation of Knoop) and proteic (i.e. gluconeogenesis) metabolisms come together, converge, as we shall see, in the respiratory chains closely linked to the cycle. But to hear more about this third point you will have to hang on until the next chapter.

2.2

What ATP Is for

Animal organisms have the onerous task of keeping their cells’ internal environment constant to ensure constant efficiency of their bodies’ various apparatuses and which are continually exposed to external environmental variations in which they live. Such needs very enormously, for example, the brain, even though representing only 2% of total body weight, consumes at rest 20% of the oxygen used by the whole organism. This entails energy consumption and consequently the need for an energy supply, used both for active purposes (working, feeding, thinking, etc.) and anabolic ones, including growth, repairs to damage during activity, the production of molecules (hormones, neuro-mediators, antibodies and proteins of all kinds, fatty acids, phospholipids, etc.) with a continuous movement of substances from one part of the body to another. Obviously not all such movements require energy. Some substances spread by osmosis until a balance is reached between two compartments; others, sometimes the same, require energy particularly if they have to be moved against a

2  Anaerobic Glycolysis or Embden–Meyerhof Pathway

13

concentration gradient. Their ionic composition is very different, so while the Na and the K in the plasma are, respectively, 140 and 4.5 mEq/L, at an intracellular level, sodium is present at levels of 20  mEq/L, where the potassium oscillates between 100 and 160 mEq/L. Each nerve impulse is followed by a depolarization of the membrane with sodium coming into the cell and potassium escaping from it. A pump is activated which to restore the membrane potential works against the gradient, to expel the sodium, thus allowing the potassium to return. This determines the release of three Na+ ions for every two K+ ions that enter, thus restoring the membrane potential, which normally stands at −70 mV. The ATP supplies the energy to make this pump work by yielding a phosphoric group to a heterodimer (protein molecule formed by the union of two sub-units called monomers) weighing about 270 Dalton, and equipped with two α and β sub-­units whose ends are located inside the cell. The phosphoric group thus modifies its structure by twisting the two terminals carrying the sodium ions outwards towards the exterior of the cellular membrane, but the heterodimer while accepting the three Na+ ions is, until phosphorylated, unable to rotate. This affinity for sodium or potassium is due to the amino acids’ different steric conformation making up the bonding site. Once dephosphorylated, the heterodimer resumes its original form by dragging potassium inwards. The two binding sites for sodium and potassium are partly superimposed so that the two ions are inhibited from both binding to the protein at the same time. After dephosphorylation with the protein turned outwards, the protein’s affinity towards the sodium ions changes, so that two potassium ions take the place of the three sodium ions and the protein goes back to its original arrangement, releasing the potassium ions inside of the cell. At this point the sodium affinity site is restored and the cycle begins again. This is the most important function for us anaesthesiologists-resuscitators. I will not continue to bore you with all the biochemical sites of ATP employment that you will find in every book, true, of biochemistry.

References 1. Rapoport TA, Heinrich R, Rapoport SM. The regulatory principles of glycolysis in erythrocytes in  vivo and in  vitro. A minimal comprehensive model describing steady states, quasi steady states and time-dependent processes. Biochem J. 1976;154:449–69. 2. Rapoport I, Berger H, Elsner R, Rapoport S. pH dependent changes of 2-3 bisphosphoglycerate in human red cells during transitional and steady states in vitro. Eur J Biochem. 1977;73:421–7. 3. Astrup P, Rorth M, Thorohange C.  Dependency of acid-base status of oxyhemoglobin dissociation and 2,3-diphosphoglycerate level in human erythrocytes. Scand J Clin Lab Invest. 1970;26:47–52. 4. Perutz MF. Stereochemistry of cooperative effects in haemoglobin: Haem–Haem interaction and the problem of allostery. Nature. 1970;228(21):726–34. 5. Candiani A, Moschetta C, Papalia U, Sacco S, Franceschini G. Alterazioni della capacità di cessione dell’ossigeno da parte dell’ossiemoglobina nel periodo postoperatorio e postperfusionale in cardiochirurgia. Acta Anaesthesiol Ital. 1978;29(I):3. 6. Nakao M, Nakao T, Yamazoe S, Yoshikawa H. Adenosine triphosphate and shape of erythrocytes. J Biochem. 1961;49:487.

14

F. Alemanno

Further Reading Devlin TM. Biochimica. Napoli: EDISES; 2012. Harper HA. Chimica fisiologica e patologica. Padova: Piccin Editore; 1965. Jevons FR. Le basi biochimiche della vita. Milano: Mondadori Editore; 1972. Moruzzi G, Rossi CA, Rabbi A. Principi di chimica biologica. Bologna: Tinarelli Editore; 1984. Nelson DL, Cox MM.  Introduzione alla biochimica di Lehninger. Bologna: Zanichelli Editore; 2015. Nelson DL, Cox MM. Lehninger principles of biochemistry. 7th edn. USA:Freman and Company; 2017. Pauling L. Chimica generale. Milano: Longanesi Editore; 1967. Siliprandi N. Chimica biologica. Roma: Edizioni Ricerche; 1975. Smith Christopher UM. Biologia molecolare. Milano: Mondadori Editore; 1971.

3

Tricarboxylic Acids Cycle or Krebs Cycle Fernando Alemanno

More than of formulas, biochemistry is made of concepts. As we have already said, all oxidation happens by means of dehydrogenation in the cytoplasm or oxidative decarboxylation in the mitochondria (here too, the oxidation happens by means of dehydrogenation) or by subtraction of electrons in the respiratory chain (in the mitochondria). The Krebs cycle has three functions: (a) It degrades metabolites derived from carbohydrates, lipids and proteins into CO2 and H2O (b) It produces energy for ATP synthesis (c) It provides the metabolites required by the various processes of biosynthesis, such as acetylcholine The cycle substantially develops in the four basic stages: 1. Pyruvic acid (Three atoms of C), the terminal milestone of anaerobic glycolysis, is transformed by oxidative decarboxylation into acetic acid (two atoms of C) within the mitochondria in the form of acetyl-coenzyme A or active acetate, with the aid of coenzyme A and vitamin B1 (in its active form: cocarboxylase). CH3

CH3

CO + CoA-SH + NAD

CO

S-CoA + CO2 + NADH + H+

(pyruvic-dehydrogenase + cocarboxylase) COOH Pyruvic acid

Acetyl–CoA

F. Alemanno (*) Anaesthesia and Intensive Care, Brescia Clinical Institutes, Brescia, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 F. Alemanno (ed.), Biochemistry for Anesthesiologists and Intensivists, https://doi.org/10.1007/978-3-030-26721-6_3

15

16

F. Alemanno

The matter is more complicated than it appears on first sight: in reality, the enzyme pyruvic-dehydrogenase is a collective of which two other enzymes and their coenzymes form part: dehydrolipoyl-transacetylase (coenzyme: lipoic acid) and dehydrolipoyl-dehydrogenase (coenzyme: FAD). On the other hand, pyruvic acid itself (three atoms of C) can take on a molecule of CO2, thus turning into oxaloacetic acid (four atoms of C). In the past, it was believed that the assimilation of CO2 was a process typical of the plant world. The importance of this reaction lies in the fact that oxaloacetic acid is a key element in the Krebs cycle. 2. Oxaloacetic acid (4C) reacts with acetic acid (2C) to form citric acid (6C). 3. Citric acid (6C) undergoes oxidative decarboxylation via a number of intermediate compounds, and loses a carbon (in the form of CO2), thus turning into α-ketoglutaric acid (5C). 4. α-Ketoglutaric acid (5C), in turn, in a process similar to the above, but catalysed by the active form of vitamin B1 (cocarboxylase) loses a carbon and thus returns, via a series of intermediate compounds, to oxaloacetic acid (4C), which starts the cycle again. Memorising these four stages makes it easy to understand the Krebs cycle. The supply of oxaloacetic acid deserves special attention. This important metabolite of the cycle has three sources of production: 1. The first derives from aspartic acid. This mono amine dicarboxylic amino acid, known to all as a salt of K (aspartate of K), was proposed by the pharmaceutical industry as a prudent donor of potassium as an alternative to potassium chloride, the inorganic acid salt, hence almost completely dissociated and as such a higher risk to the heart, in case of excessively rapid infusion. Moreover, K aspartate, due its moderate dissociation, as an organic acid salt, easily crosses the blood brain barrier, and is thus particularly valuable in the treatment of cerebral oedema (Chap. 9). Very well then, aspartic acid can produce oxaloacetic acid in the following transamination reaction: Aspartic acid + a　 ketoglutaric acid « oxaloacetic acid + glutamic acid ( transaminase )

2 . The second source, as before mentioned, derives from pyruvic acid. This is a so-­ called anaplerotic reaction, which has the purpose of guaranteeing the supply to the Krebs cycle of this intermediate molecule which is consumed by other metabolic processes (e.g. conversion into phosphoenolpyruvic acid) [1]. The pyruvic acid is carboxylated into oxaloacetic acid in the reaction: COOH-CO-CH 3 + CO 2 + ATP + H 2 O ® HOOC-CO-CH 2 -COOH + ADP + H 3 PO 4 pyruvic acid ( 3C ) + CO 2 (1C ) + ATP + H 2 O = oxaloacetic acid ( 4C ) + ADP + H 3 PO 4

( pyrruvic　 carboxylase + Acetyl　 CoA + biotin + Mg ) 2+



3  Tricarboxylic Acids Cycle or Krebs Cycle

17

3. The third source is similar to the second, but has one more step and involves the reductive carboxylation of pyruvic acid into malic acid, using NADPH2 as the donor of H+ ions. The malic acid is then dehydrogenated into oxaloacetic acid by NAD, in the natural sequence of the Krebs cycle (Fig. 3.1). The second and third reactions are anaplerotic, and thus need a certain PaCO2 to react. This explains why one should not fall below 25 mmHg of PaCO2 when ventilating a patient, since doing so excludes the most direct pathway for the

GLICOGEN

LIPIDES

B6 NADP NADPH2 6P-GLUCONIC ACID

GLUCOSE 6 P

NADP

B1

FRUCTOSE B1

NADPH2

PENTOSE

−CO2

PYRUVIC ACID

ALANINE

NADP

NADPH2

+CO2

BUTIRYL-COA

B1

LIPOLYSIS −2H (n times)

GLUCOSE

LIPOGENESIS +2H (n times)

PROTEINS

−2H ACETYL-COA

Oxaloacetic Acid

ASPARTIC ACID

+CO2

Cholesterol Ketone Bodies

−2H

Citric Acid

Malic Acid

Cis-Aconitic Acid NAD

Fumaric Acid

FLAVOPROTiDES

−2H

Isocitric Acid

O Succinic Acid

−2H

−2H

−CO2

Oxalosuccinic Acid

ATP

CYTOCHROMES

−−

H2O

B1 α-Ketoglutaric Acid +NH2 −NH2 GABA

−CO2

Glutamic Acid +NH2 B6 Glutamine

Fig. 3.1  The diagram of the Krebs cycle. The diagram outlines the process of anaerobic glycolysis; more in detail, the Krebs cycle

18

F. Alemanno

formation of oxalacetic acid. It also explains why metabolic acidosis sets in after prolonged respiratory alkalosis (hyperventilation), not as a compensatory effect (as an optimist might be tempted to believe), but rather as a complication. On the one hand, due to the low PaCO2, the pyruvate is not able to generate oxaloacetic acid directly by carboxylation (or indirectly by reductive carboxylation, to malic acid), and on the other hand, since it cannot take part in the Krebs cycle, following oxidative decarboxylation to Acetyl-CoA, due to the lack of reaction substrate (oxaloacetic acid), it accepts the two hydrogen ions offered by NADH2, produced by the oxidation of 3-P-glyceraldehyde, and converts into lactic acid. Lipolysis too with the Knoop β-oxidation detaches two-carbon groups from the long chains of fatty acids which are then transformed by coenzyme A into acetyl-­ CoA, thus starting their combustion in the Krebs cycle. We should pay particular attention (at 6 o’clock of the cycle) to the collateral GABA cycle with the passage glutamate  →  GABA driven by vitamin B6, which transforms an excitatory neurotransmitter into an inhibitory one (see Chap. 4).

3.1

The Respiratory Chain

The most important mechanism, which forms ATP, is the electrons transport chain to oxygen, or the respiratory chain; it is located in the internal membrane of the mitochondria and the sarcosomes of the myocardium. It all starts with the transfer of a pair of hydrogen atoms (H2) from the substrate to the pyridine coenzyme NAD. The potential energy is not released when the substrate transfers H2 to the NAD, which is reduced to NADH2, but rather when the reduced forms of the pyridine coenzymes, via the flavoproteins (FAD), reach coenzyme Q: this distributes the electrons to the cytochromes, and the two hydrogen ions (2H+) to the oxygen (O2−). There are obviously also enzymatic mechanisms in the mitochondria, which couple the availability of energy to the phosphorylation of ATP; this mechanism calls oxidative phosphorylation. The cascade which transports the two electrons of the H2 and hence also the two H+ ions is founded on the redox potentials of the various components. NAD–NADH2 has a redox potential of −0.32 V; the next step in the cascade, FAD–FADH2, has a potential of −0.06 V, that is less electronegative than the preceding step and thus, in a certain sense, more electropositive, hence the two electrons are more attracted to it, followed naturally by the two H+ ions. In the same way, coenzyme Q, with its electrical potential of −0.04 V, attracts the two electrons last, followed by the two H+ ions. At this point something extraordinary happens: when they come into contact with the sequence of positive cytochromes, the two H+ ions are repelled and sorted to the enzyme ATP-synthetase; only the electrons travel along the chain of the cytochromes up to the oxygen, attracted by their gradually increasing redox potentials: +0.22 V (cytochrome b), +0.27 V (cytochrome c), +0.29 V (cytochrome a) followed by oxygen itself. The latter, with its very strong electropositive potential

3  Tricarboxylic Acids Cycle or Krebs Cycle

19

(+0.81 V), irresistibly attracts the two electrons, becoming itself electronegative and coupling to the two hydrogen ions (H+H+), thus finally forming H2O.

2H + ions + O 2 - = H 2 O

This buffers a pH, which would otherwise become ever more acid if the hydrogen ions detached from the substrate were not neutralised by the O2. As I have said above, but I am always happy to repeat it, we could call O2 the great universal base, because this is its sole function in living animal organisms. Figure 3.2 shows the respiratory chain, with the various redox potentials, which transport the H2 first, and then its two electrons alone, after the step from coenzyme Q, to the cytochrome chain, located in the internal membrane of the mitochondria. Coenzyme Q is a crucial link in the respiratory chain. This coenzyme transports the atom of hydrogen (already dissociated into H+ and its electron), sorting the electron to the cytochrome chain and the hydrogen ion to the enzyme ATP-synthetase, creating an electrochemical gradient, i.e. a protonic force, which is essential to the enzyme’s proper functioning. It is at this point, and with this mechanism, that ADP transforms into ATP by oxidative phosphorylation.

3.2

The Cytochromes

As you can see from their role in the respiratory chain, the cytochromes are mitochondrial proteins with the EME prosthetic group (like haemoglobin) [2], whose function is to transport electrons from a higher to a lower energy level, and then transfer them to the oxygen. When the substrate (e.g. pyruvic acid, isocitric acid, succynic acid, malic acid) is oxidised with the intervention of the coenzymes, which accept and transport the hydrogen ions (NAD, FAD), the transport of the electrons starts with coenzyme Q, which distributes the electrons to cytochrome b. In passing from cytochrome b to c and from cytochrome a3 to oxygen, the energy released by the drop from higher to lower energy levels is stored as ATP, due to the action of the enzyme ATP-synthetase.

3.2.1 Cytochrome P-450 When we study a given category of drugs, such as the benzodiazepines, we often encounter declarations like the following: “The drug is then eliminated in the liver by the action of cytochrome P-450”. We know that drugs are metabolised in the liver, but what role does this cytochrome P-450 play? It represents a large family of enzymes belonging to the monoamine oxidases [3, 4]. They catalyse the oxidation of both endogenous molecules produced by the catabolism of organic molecules, bilirubin, modulation of hormonal levels (oestrogens, progesterone, etc.), and of exogenous molecules (drugs, toxins). In this oxidative process, catalysed by the enzyme NADPH2-cytochrome P450-reductase, the electrons are supplied by the

E’0

Substr.

Substr. H2

H3PO4 + ADP

H3PO4 + ADP ATP

3+

- 0,04 V

2Fe

~

- 0,06 V

Q

2Fe2+ Cyochrome b

2H+

~

- 0,32 V

FMN

Flavoprotein 1 NADH dehydrogenase

Primary dehydrogenases

NAD+

2

Coenzyme Q

FMNH2

FAD

Sucinatedehydrogenase QH

NADH+H+

Fumaric Acid

FADH2 Flavoprotein 2

3+

ATP

+ 0,22 V

2Fe

Cyochrome c1

2Fe2+

+ 0,27 V

3+

2Fe

Cyochrome c

2Fe2+

H2O

~

H3PO4 + ADP

+ 0,29 V

3+

2Fe

Cyochrome a

2Fe2+

ATP

3+

2Fe

Cyochrome a3

2Fe2+

+ 0,81 V

1 2

O2

O..

Fig. 3.2  The diagram of the respiratory chain with the respective redox potentials. The figure outlines the respiratory chain, located inside the mitochondrion, in a linear fashion. It shows the various redox potentials, which transport the H2 first, and then the two electrons after the step from coenzyme Q to the cytochrome chain (Moruzzi, Rossi, Rabbi. Principi di Chimica Biologica. Courtesy of Tinarelli Editore, Bologna 1984)

Piruvic acid (FP3) Isocitric acid α-Ketoglutaric acid Malic acid Glutamic acid β-hydroxyacyl-CoA etc.

Succinic Acid

20 F. Alemanno

3  Tricarboxylic Acids Cycle or Krebs Cycle

21

coenzymes NADPH2 and NADH2. The purpose of the enzyme is to introduce a molecule of oxygen into the molecule being degraded, so as to render it more polar:

RH + O 2 + NADPH 2 ( 2H + + 2e - ) ® ROH + H 2 O + NADP

Assuming several drugs together can induce phenomena of inhibition or induction, which increase or diminish the metabolism [5]. Thus, for example, taking ranitidine and benzodiazepines together prolongs the action of the latter. On the other hand, a drug may induce an increase in a specific form of cytochrome P450, thus increasing the degradation and hence lowering the effect of other drugs, which form the substrate of the action at that time. This phenomenon of induction naturally takes effect over a longer time (3–4 weeks), i.e. the time span of genetic transcription, for example, of antibodies and proteins in general.

References 1. www.federica.unina.it/farmacia/biochimica-far/gluconeogenesi/ 2. Perutz MF. Stereochemistry of cooperative effects in haemoglobin: Haem–Haem interaction and the problem of allostery. Nature. 1970;228(21):726–34. 3. Nelson DR, Kamataki T, et al. The p450 superfamily: update on new sequences gene mapping, accession numbers, early trivial names of enzymes and nomenclature. DNA Cell Biol. 1993;12:1–51. 4. Danielson P. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans. Curr Drug Metab. 2002;3(6):561–97. 5. Ulbricht C. Risks of mixing drugs and herbal supplements: what doctors and patients need to know. Altern Complement Ther. 2012;18(2):67. https://doi.org/10.1089/act.2012.18202.

Further Reading Devlin TM. Biochimica. Naples: EDISES; 2012. Garret RH, Grisham CM. Biochimica. Padua: Piccin Editore; 2014. Garfinkel D.  Studies on pig liver microsomes. Enzymic and pigment composition of different microsomial fractions. Arch Biochem Biophys. 1958;77:493–509. Harper HA. Chimica Fisiologica e Patologica. Padua: Piccin Editore; 1965. Lombardo R. La fosforilazione ossidativa. March 2014. www.scienzeascuola.it/lezioni/biologia Moruzzi G, Rossi CA, Rabbi A. Principi di chimica biologica. Bologna: Tinarelli Editore; 1984. Nelson DL, Cox MM.  Introduzione alla biochimica di lehninger. Bologna: Zanichelli Editore; 2015. Nelson DL, Cox MM. Lehninger principles of biochemistry. 7th ed. USA:Freman and Company; 2017. Pauling L. Chimica generale. Milan: Longanesi Editore; 1967. Sartor G. www.gsartor.org/pro/didattica/pdf_files/F04.pdf Siliprandi N. Chimica biologica. Rome: Edizioni Ricerche; 1975. Smith Christopher UM. Biologia molecolare. Milan: Mondadori Editore; 1971. Tom M, Myers CR, Waterman MR. Evaluating molar CYP1A level in Fish hepatic microsomes by competitive ELISA using recombinant membrane-free CYP1A standard protein. Aquat Toxicol. 2002;59:101–14.

4

Glutamate – GABA Collateral Cycle Fernando Alemanno

Glutamate – GABA cycle is collateral to the Krebs cycle, it triggers the transamination of alpha-ketoglutaric acid (five atoms of C) into glutamic acid and then its decarboxylation into gamma-aminobutyric acid (four atoms of C), better known as GABA. The latter is, for the anaesthetist, the most interesting site of action of vitamin B6. These two neurotransmitters highlight once more the continuous play of neuronal activity in the central nervous system, between excitation and inhibition, the former due to external and internal stimuli, the latter due to the need to modulate the response and promote repolarisation. We now proceed in order. α-Ketoglutaric acid receives the NH2 group of the amino acid alanine (α-aminopropionic acid, a close relative of pyruvic acid) by means of transamination. COOH C

O

COOH H2N – C – H

CH2

CH2

CH2

CH2

COOH

COOH +NH2 α-ketoglutaric acid glutamic acid → (transaminase)

F. Alemanno (*) Anaesthesia and Intensive Care, Brescia Clinical Institutes, Brescia, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 F. Alemanno (ed.), Biochemistry for Anesthesiologists and Intensivists, https://doi.org/10.1007/978-3-030-26721-6_4

23

24

F. Alemanno CH3 H2N – C – H COOH Alanine

CH3 – NH2 → (transaminase)

C

O

COOH pyruvic acid



a-ketoglutaric acid + alanine + NADPH 2 ® glutamic acid + NADP + pyruvic acid ( transaminase )



Glutamine + H 2 O ® glutamic acid + NH 4 ( glutaminase )

The transamination of alanine at the ketone group of α-ketoglutaric acid transforms the latter into glutamic acid while the alanine transforms into pyruvic acid. The astrocytes are among the most diligent recyclers of glutamic acid, which they recover from the synapses, where it is released, and convert it into glutamine. The glutamine then diffuses into the neurons, which, using the glutaminase enzyme, reconvert it into glutamate and release it into the synaptic space when it is needed:

Among the many interesting functions of vitamin B6 there is thus also that of transforming the most important excitatory neurotransmitter into one of the most important inhibitory neurotransmitters, γ-aminobutyric acid (GABA); the reaction is irreversible. It is true that GABA is then transaminated, due to the presence of vitamin B6 as coenzyme, into succinic semialdehyde, forming first glutamate and then GABA again, and regenerating itself at the end. The succinic semialdehyde, on the other hand, enters the Krebs cycle (Fig. 4.1).



Glutamic acid ( -CO 2 ) ® g -aminobutyric acid ( + CO 2 ) ( glutamic-decarrboxylase + vitamin B6 )

GABA acts on specific sites in the brain and spinal cord, to produce postsynaptic hyper-polarisation. Once again, as for glutamate, the glial cells are responsible for removing it from the synapses and recycling it. The catabolism of GABA, as we have said, involves its deamination by a transaminase, which transforms it into succinic semialdehyde, transferring the NH2 amine group to the α-ketoglutaric acid to form glutamate and then generating GABA again (Fig. 4.1). The succinic semialdehyde is then oxidised into succinic acid and is returned to the Krebs cycle.

4  Glutamate – GABA Collateral Cycle

25 α-Ketoglutaric Acid + Alanine + NADH2 ↓ (Transaminase) Glutamic Acid + Pyruvic Acid + NAD

Citric Acid

Oxaloacetic Acid ↑ Malic Acid ↑ Fumaric Acid

(Krebs)

Glutamate Decarboxylase + Vitamin B6

α-Ketoglutaric Acid Succinic Acid Succinate Semialdehyde

+ NH2

GABA (+ CO2)

Transaminase + Vitamin B6

Fig. 4.1  The Glutamate—GABA cycle

Valproic acid (the name is taken from the abbreviation of 2-propyl valeric acid) inhibits the enzyme GABA-α-ketoglutarate-transaminase, thus blocking the degradation of GABA, while providing an antiepileptic action. Vitamin B6 (pyridoxine, active form: pyridoxal-5-phosphate) It is now time to discuss this very interesting vitamin. It is a derivative of pyridine.

N

The structural formula of pyridine The organism can get vitamin B6: from pyridoxine CH2OH CH2OH

HO

H3C

N

26

F. Alemanno

From pyridoxamine CH2NH2 CH2OH

HO

N

H3C

And from pyridoxal CHO CH2OH

HO

N

H3C

The active form is pyridoxal-5(P); hence both pyridoxine and pyridoxamine must first be transformed into pyridoxal: the former by oxidising its alcohol group (CH2OH in position 4, in front of the N) into aldehyde, the latter by first deaminating and then oxidising it. As a side note, how do we establish the numbering of a heterocyclic benzene ring, i.e. a benzene ring in which a carbon is replaced by a heteroatom (O, S or N)? The numbering starts with the heteroatom as number 1, and continues in a clockwise or counterclockwise direction, all depending on whether the second radical has the lowest possible location number. If there are two different heteroatoms in the ring, oxygen takes priority over sulphur and sulphur takes priority over nitrogen in assigning position 1. OH

CHO CH2O

HO 4

3 2

H3C

P

5

O

OH

1 6

N

Pyridoxal-5(P) Pyridoxal is activated in the liver as follows:

CH2OH

HO

OH

CHO

CHO + ATP

CH2O

HO

P

O + ADP

OH H3C

N

H3C

N

Pyridoxal + ATP = pyridoxal 5 ( P ) + ADP



4  Glutamate – GABA Collateral Cycle

27

Pyridoxal 5(P) is the coenzyme which catalyses all non-oxidative enzymatic reactions of the amino acids (transamination, decarboxylation, racemisation). The reactions activated by the transaminases obey the law of mass action, i.e. they follow the direction imposed by the quantity of the reagents, thus rebalancing the amine nitrogen content of the cell. Vitamin B6 is responsible for the oxidative deamination of the amines and the metabolism of tryptophan, transforming the xanthurenic acid (it has an emetic action, its dosage is very high during pregnancy) into nicotinic acid; this action makes the vitamin indicated as an antiemetic. But, as we have said, the reaction of greatest interest to the anaesthetist is the decarboxylation of glutamic acid into gamma-aminobutyric acid, without however forgetting that vitamin B6 also plays a role: 1 . In glycogenolysis, as the coenzyme of glycogen-phosphorylase 2. In the decarboxylation and transamination of the amino acids 3. In the synthesis of neurotransmitters like dopamine, noradrenaline and tyramine 4. In the synthesis of the nucleic acids 5. In lipid metabolism with the transformation of linoleic acid into arachidonic acid 6. In the synthesis of the sphingolipids, which are essential to the synthesis of myelin Vitamin B6 has a long and complicated history; it was first discovered in 1926 by Joseph Goldberger (Hungarian) as a pellagra-preventive factor, indeed low levels of the vitamin in rats caused a dermatitis similar to that of pellagra in man. These symptoms were cured by adding brewer’s yeast to the animals’ food, in which vitamin B1 had been destroyed by heating in the autoclave, and the dermatitis was thus ascribed to the lack of another vitamin, called B2. In the rat, however, the dermatitis was accompanied by a paresis of the rear limbs, which Paul György called acrodynia (1934), the cause of which was ascribed not to vitamin B2, but to another non-thermolabile factor in the yeast, which he called vitamin B6 [1]. It was soon noted that dermatitis of rats is different to that in man, and that the action of vitamin B6 is different from that of vitamin PP, also present in brewer’s yeast and isolated by Elvehyem in 1938. Vitamin B6 was so-called because it was the sixth B group vitamin to be discovered. Vitamin B6 can be antagonised: by isoniazid (anti-tubercular) which, with its amine group (–NH2) blocks the aldehyde group (CH=O) of pyridoxal-phosphate; by hydralazine (anti-hypertensive) and by penicillamine. The latter is used in cystinuria, in patients affected by urinary stones resistant to alkalinising therapy; pyridoxine is added to the penicillamine, 50 mg/day, precisely to avoid this low level syndrome. Since vitamin B6, as we have seen, lowers the values of xanthurenic acid, the secondary metabolite of tryptophan and an emetic, the level of which in the blood increases during pregnancy, it is successfully administered to prevent vomiting (morning sickness) in the first quarter.

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The concept of using not a drug but rather a vitamin as an antiemetic was adopted by anaesthetists worldwide in attempting to remedy one of the most unpleasant side effects of anaesthesia. It is worth including, at this point, a brief excerpt from a note submitted by W. Bergman from Yuma (Colorado) in 1947 to the Canad. M. A. J.: “In small hospitals ether is still widely used as the anesthetic of choice… The drawback of ether is that in almost every case postoperatively the patient goes through a more or less prolonged phase in which he is nauseated and vomits. Several combinations of preanesthetic medication were tried…Finally…injections of pyridoxine were given before operation was started and an immediate effect was noted: no more vomiting occurred, and nausea was slight… It seemed that patients receiving pyridoxine, got on very much better than the ones not receiving pyridoxine and the patients were ambulatory usually on the second and third day postoperatively, with full bathroom facilities, while the control series was only ambulatory around the third and fourth day” [2]. In the following years, many Italian and foreign anaesthetists adopted pyridoxine as a prophylactic against postoperative nausea and vomiting. In this practice, Acquaviva and Magrini, from the University of Padua, highlighted a second very interesting fact, in line with the mechanism of action of this important coenzyme: it facilitates the induction of narcosis with thiopental sodium (TPS) and subsequently reduces (by 20–35%) the dose of barbiturate commonly used to maintain anaesthesia, as well inducing as a certain neurovegetative stabilisation [3]. This reduction in the dose of barbiturate induced by vitamin B6 is, in our opinion, to be ascribed to the important role played by the transformation of glutamate, one of the most important nociceptive neurotransmitters, into GABA, one of the most important inhibitory neurotransmitters. According to Boschetti and Trapletti, vitamin B6 has a regulating action on magnesaemia [4].

4.1

Magnesium

We must now look deeper into the role played by this element, a member, like calcium, of the alkaline earth metals. As you can see, correlations in biochemistry are anything but flights of fancy. The entire group of alkaline earth metals (Group II, subgroup A: beryllium, magnesium, calcium, strontium, barium, radium) have two electrons in their outermost orbit (see the periodic system of the elements at the end of Chap. 16); this group can thus provide ions with a 2+ charge. The sequence of ions in the group is related to how many electronic orbits they have: Be 2, Mg 3, Ca 4, Sr 5, Ba 6, Ra 7. As the number of orbits increases, so does their atomic radius, so that the attraction exercised by the two electrons in the outermost orbit diminishes as we progress from beryllium to radium; it follows that the energy needed to dislodge the two electrons from their orbits is ever smaller. It is precisely for this, their ability to release electrons, that the alkaline earth metals have reducing properties and therefore are easily oxidisable. In human plasma, magnesium is present to the amount of 1.8–3.6 mg/100 mL, while in the red blood cells this rises

4  Glutamate – GABA Collateral Cycle

29

to 5.4–7.8 mg/100 mL. These numbers increase during pregnancy, due to the necessary relaxation of the uterine musculature. In normal musculature, we encounter levels of magnesium close to 20 mg/100 g. Another interesting detail is supplied by comparative physiology: hibernating animals progressively increase the tenor of Mg in their blood. If we inject Mg intravenously, we induce a state of somnolence when the dosage of Mg reaches around 10 mg/mL, and loss of consciousness when it exceeds 17 mg/ mL. At the same time, we can observe a state of muscular relaxation which only minimally affects the respiratory muscles, combined with a bronchodilating action. Intravenous administration must be accompanied by close ECG monitoring; any cardio-depressant effects can be antagonised with small doses of Ca-gluconate. The dose of competitive curare used in abdominal surgery is half that required when not also using magnesium (Emanuelli) [5]. The drug used was Mg ascorbate at 10% (Magnorbin, 5 mL phial). In comparison with Mg sulphate, this has the advantage of being a salt of an organic acid, and hence little dissociated and more suited to crossing the blood brain barrier. Mg sulphate, for the opposite reason, is more indicated for use in cardiology. The administration in anaesthesia of both the organic and the inorganic salt induces constant sinus bradycardia. Following administration of 5 mL of a solution of Mg ascorbate 10%, the average magnesaemia is around 11 mg/mL after 20 min, and around 5 mg/mL after 50 min. In the immediate postoperative period, there seems to be a certain level of analgesia, more extended than with other types of anaesthesia in which the drug has not been administered. Another interesting observation concerns the good condition of the patient immediately after operation, probably due also to the good peripheral perfusion caused by the most natural Ca antagonists. On the other hand, a study by Frassanito et al. [6] on postoperative analgesia, after installation of a full knee prosthesis, an operation characterised by considerable postoperative pain, concludes that the perioperative infusion of magnesium sulphate does not reduce the postoperative consumption of analgesics. But evidently in this case they have asked magnesium to do something beyond its powers: analgesia only achieved with a continuous infusion of local anaesthetics at the femoral nerve or with the administration of morphine or equipotent analgesic combinations. Moreover, there is a considerable difference between the administration of an organic salt (Mg ascorbate, little dissociated) which is easily able to cross the blood brain barrier, and an inorganic salt like magnesium sulphate (highly dissociated) which has great difficulty in crossing it. The use of magnesium is also indicated in other areas than anaesthesia. Magnesium is a coenzyme in numerous biochemical reactions, of which we can take the example of the formation of glucose-6(P), both by glycogenolysis: glucose1(P) ↔ glucose-6(P), as the coenzyme of phosphoglucomutase, and in the normal phosphorylation of glucose to glucose-6(P), as the coenzyme of hexokinase; and in many other reactions. For this reason one must bear in mind its role in the parenteral nutrition of patients in intensive care. Well, we’ve discussed GABA and vitamin B6, the important enzyme of its formation, and we’ve also looked at magnesium, correlated to it in some way; it seems only a natural progression now to discuss sodium gamma-hydroxybutyrate.

30

4.2

F. Alemanno

Sodium Gamma Hydroxybutyrate

γ-OH to French anaesthetists, GHB to English speakers. (a natural drug, nowadays poorly used) In the 50s, gamma-aminobutyric acid (GABA) was isolated in the brain of mammals, an inhibitor of the CNS, able to activate sleep if applied directly, in open surgery, to the cerebral cortex, but without any effect when administered intravenously, since it could not cross the blood brain barrier. The salts of butyric acid, with their considerable hypnotic power, were tried, but were revealed to be immediately toxic due to the induction of ketoacidosis. In 1960, Laborit et al. [7] synthesised sodium γ-hydroxybutyrate, by hydroxylating butyrolactone: CH2

CH2

CH2

® ¬

CO

O

CH2

CH2

CH2

COOH

OH γ-hydroxybutyric acid

butyrolactone

In solution, γ-hydroxybutyric acid remains in equilibrium with the butyrolactone, while its sodium salt is stable. CH2 OH

CH2

CH2

COONa γ-hydroxybutyrate of Na

Gamma-OH is normally present in the brain of mammals, and acts as a neuro-­ transmitter or -modulator. It acts on different receptors than GABA, and also acts by activating the direct oxidative pathway (DOP): through this pathway, it repairs the damage caused by the free radicals of oxygen during the waking hours. For these two reasons it should be useful in treating coma in intubated and artificially respirated patients. According to Gessa, gamma-OH should also have a direct action on dopamine, increasing its production (via the DOP) and inhibiting its release during the sleep induced by the drug, followed obviously by a tonic effect on the CNS on awakening. In expounding his ideas, Henri Laborit highlights the fact that glucose-­ 6(P), originated by glycogen lysis or the hematic circle, is used in two distinct metabolic pathways: by day, the anaerobic Embden–Meyerhof pathway, followed by the Krebs cycle in mitochondrial cells (in this phase, the orthosympathetic system is dominant), and by night, the pentose pathway or DOP (also called the sleep pathway or synthesis pathway, in which the vagal system is predominant). Now, its orientation towards one or another pathway depends on the availability of NADP which oxidates glucose-6(P) into 6(P)-gluconic acid and thus reduces to NADPH2, which provides hydrogen for synthesis (for instance, of long fatty acid chains). Here lies the importance of the NADP/NADPH2 ratio, that determines which of the two metabolic pathways (anaerobic glycolysis or DOP) the glucose-6(P) has to follow. Narcotics, by interfering with the normal activity of the Krebs cycle, according to Quastel’s theory (Chap. 14), diminish the production of NADP. Indeed, NADPH2 is

4  Glutamate – GABA Collateral Cycle

31

then no longer required for the reductive carboxylation of pyruvic acid into malic acid, from which oxaloacetic acid is formed (Chap. 3, Fig. 3.1); this last, due to the blockage of the Krebs cycle, soon goes into overproduction, blocking the reaction:

pyruvate + CO 2 + NADPH 2 = malic acid ® oxaloacetic acid.

Gamma-OH, which is itself hypnogenic, moreover, as a short chain fatty acid, inserts itself as butyryl-CoA into the syntheses in which NADPH2 acts as a donor of H2 groups, indirectly activating the DOP and with it the synthesis of higher fatty acids. This synthesis decrements the denominator of the NADP/NADPH2 ratio, increasing the prevalence of NADP, so that glucose-6(P) is further directed towards the pentose pathway. I include a statement by Gualtiero Bellucci at the First Italian Convention on Gamma-OH held in Florence, 4 October 1964 [8]: “This interpretation agrees with the potentiation of Gamma-OH by all the adrenolytics… and with the antagonism exercised by adrenaline, even at relatively moderate doses. Sleep is thus facilitated by agents, which diminish the intensity of the EMK metabolic pathway, oxidation and the adaptation reactions, and is inhibited by those which increase them. Furthermore, all agents which, according to this hypothesis, direct towards the direct oxidative pathway, induce hypokalemia, as if the cellular use of K+ were bound to a preferential function of this pathway. Gamma-OH generally induce hypokalemia, as are insulin combined with glucose, the adrenolytics, and the neuroplegics”. When used as an oral hypnotic by patients suffering from insomnia, gamma-OH has a brief duration of action. Although it provides complete neuronal recovery, it is not tranquillising. For thus reason it has enjoyed little success as an oral hypnotic, because in general, people suffering from insomnia, also usually display a neurotic component, so that if the subject at night awakes, despite a feeling of well-being due to the quick neuronal recovery, gamma-OH does not give that sensation of tranquillity given by other drugs (such as benzodiazepine), so that the subject finds himself wrestling with his everyday worries and preoccupations in the middle of the night—precisely what with sleep he was attempting to escape. In anaesthesia, used as a narcotic at a dosage of 50 mg/kg of body weight, in place of sodium thiopental, it manifests a certain delay between injection and effect (5–10 min), an interval which should be covered in normal conditions (also to prevent the querulous protests of the surgeon), with a pre-induction dose of neuroleptoanalgesia (dehydrobenzoperidol + fentanyl) taking into account the fact that gamma-OH has no analgesic effect. Induction with gamma-OH, for those who have had the good luck to have it available in the hospital pharmacy, is particularly indicated in aged and debilitated patients. In this case, it is good to reduce or eliminate the presence of dehydrobenzoperidol, also because, according to Gessa et al. [9], gamma-OH itself inhibits the release of dopamine, while increasing its production and hence storage in the presynaptic vesicles. Due to its complex mechanism of action (nothing is straightforward or simple in biochemistry), like the activation of specific receptors and of the pentose pathway, a single dose of 50 mg/kg has a duration of around 90 min, and it should thus be used in surgeries which exceed that period. It seems that the drug acts on the

32

F. Alemanno

thalamocortical system; it has no protective action on the neurovegetative system; nor does it have any myorelaxant action: “Intubation using Gamma-OH alone, at the above doses, is so inelegant and difficult, that it should be formally advised against” (Gasparetto) [10]. It has an interesting action on respiration, resulting in bradypnea compensated by an increase in the depth of each breath, so that the actual volume/minute and the pCO2 remain unchanged. From the metabolic point of view, it does not change the consumption of O2 nor the acid base equilibrium, probably because it does not interfere with the cytochromoxidase system. It is interesting that, although the loss of potassium in the urine does not increase, one may encounter a slight hypokalaemia caused by increased cellular repolarisation. A reduction of the postoperative nitrogen catabolism has been noted, along with an unincreased tenor of free fatty acids, characteristics which are normally constant in the immediate postoperative period following conventional anaesthesia. Gamma-OH has also been widely used for analgesia during labour, thanks to its inotropic action on the uterine musculature, leading to increased frequency and amplitude of the contractions. This means that the use of oxytocics may not be necessary or their dosage reduced, to avoid uterine hypertonia. The Apgar test is normally elevated even if the newborn child is asleep during birth. This latter circumstance is considered to be positive by Laborit [11]: “…to pass long moments in the genital tract of the woman, at least in this initial phase of life, must certainly be anything but a pleasant experience…”. In clinical practice, the drug is available in 10 mL phials, each of which contains 2 g of sodium gamma-hydroxybutyrate in distilled water. It is as well to bear in mind, in order to maintain the electrolytic balance when administered to stay patients in intensive therapy, that each phial contains 16  mEq of Na. Despite its alkaline pH (9.5), it has no irritating action on the peripheral veins. It is a good idea to premedicate with atropine (0.01 mg/kg) during induction, as normal, to prevent bradycardia, followed by a first dose of dehydrobenzperidol and fentanyl (for instance 5 mg + 0.1 mg). The gamma-OH must be injected normally at a dose of 30–40 mg/kg in adults and children, and 25–30 mg/kg in old people. This is followed by curarisation and maintenance of anaesthesia with neuroleptoanalgesia, or balanced anaesthesia (analgesic + low dose inhalation anaesthetic). In our experience, as we will see in Chap. 11, we have used gamma-OH at doses of approximately 2 g (25–30 mg/kg) solely to induce anaesthesia with synaptoanalgesia in 140 cases of major surgery [12]. To prevent the exclusively anti-aesthetic twitches, which may manifest on induction, 100 mg of sodium thiopental (TPS) or even a modest dose of pyridoxine (Vitamin B6), are sufficient, prior to the injection. As already noted, the drug induces sleep some 5–10 min after injection and reaches its maximum effectiveness after 15–30 min. The maintenance dose is around 20 mg/kg/h, bearing in mind that the patient awakens about 1 h after the last administration. In general, the supplementary doses act more quickly than the first. There are few counter indications, mostly precautionary; these regard eclampsia and epilepsy, due to the possibility of triggering tonic-clonic and epileptic convulsions, respectively, which can in any

4  Glutamate – GABA Collateral Cycle

33

case easily be handled or prevented with 100 mg of TPS, as well as chronic alcoholism, which makes the patient particularly resistant to induction. Reawakening is obviously affected by the different techniques used for maintenance of anaesthesia. In brief surgeries (less than an hour), in which a single dose is used for induction, reawakening depends on the dosage of the analgesics and other anaesthetics employed. If, on the other hand, the surgery is lengthy with more abundant use of analgesics and neuroleptics, with intervening lighter maintenance doses of the drug, awakening is slower. In such cases, the patient’s vital parameters, particularly respiration, must be monitored, in the immediate postoperative period, by qualified nursing staff. As for the oral assumption of gamma-OH, the drug may be used in the form of a syrup to treat alcoholism. So long as the indicated dose is observed, it does not have the collateral effects of either psychopharmaceutical or light drugs, is non-­addictive, and only gives a sensation of well-being. This may be explained by its mechanism of action as an activator of the pentose pathway (DOP), the synthesis pathway, so that, along with the other components required to repair cellular damage incurred during the day, it also optimises the production of endorphins. In contrast, both hard and light drugs displace the functionality of the endorphins, occupying their receptors, and thus induce a negative feedback, which, since the receptor is occupied, diminishes or terminates the production of natural opioids, resulting in the well-­ known withdrawal symptoms when use of the drug is suspended.

References 1. György P. Vitamin B2 and the Pellagra-like dermatitis in rats. Nature. 1934;133:498–9. 2. Bergmann W.  Relief of postanesthetic vomiting trough pyridoxine. Can Med Assoc J. 1947;56(May):554. 3. Acquaviva ES, Magrini M. Uso della vitamina B6 (piridossina) in anestesia. Acta Anaesthesiol. 1961;XII(II):137–44. 4. Boschetti C, Trapletti A. Relazione Clin Scient. 1957;9:53. Cited in: Acquaviva S, Magrini M. Uso della vitamina B6 (piridossina) in anestesia. Acta Anaesthesiol. 1961;XII(II):137–44. 5. Emanuelli H. La magnesio-narcosi. Acta Anaesthesiol. 1958;IX(II):121–36. 6. Frassanito L, Messina A, et al. Intravenous infusion of magnesium sulfate and postoperative analgesia in total knee arthroplasty. Minerva Anestesiol. 2015;81(11):1184–91. 7. Laborit H, Jouany JM, Gerard J, Fagiani F. Résumé d’une étude esperimentale et clinique sur un substrat métabolique à action centrale inhibitrice, le 4-hydroxybutirate de Na. Presse Med. 1960;68:1867–9. 8. Bellucci G, et al. Considerazioni sull’impiego dell’idrossibutirrato di sodio nell’attività anestesiologica. Atti del primo convegno italiano sul Gamma-OH. Florence, 4 October 1964. Acta Anestesiol. 1964;XV(Suppl. N. 3):103–23. 9. Gessa GL, Vargiu L, Spano PF, Crabai F, Camba R. Effect of gamma-hydroxybutyrate of Na (gamma-OH) on cerebral amines: comparison with the effect of MAO inhibitors. Boll Soc Ital Biol Sper. 1967;43(23):1611–4. 10. Gasparetto A, Torelli L. Il 4-idrossibutirrato di sodio come “farmaco base” in anestesie cliniche plurimedicamentose. Acta Anestesiol. 1964;XV(Fascicolo 1):27–51. 11. Laborit H. Les regulations metaboliques. Paris: Masson & Cie; 1965. 12. Alemanno F, Busato G, Massera G.  L’anestesia tiaminica nella pratica clinica. Acta Anaesthesiol Ital. 1973;XXIV(Fascicolo VI):725–34.

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Further Reading Agabio R, Gessa GL.  Therapeutic uses of γ-hydroxybutirate. In: Tunnicliff G, Cash CD, editors. Gamma-Hydroxybutirate, molecular functional and clinical aspects. London: Taylor & Francis; 2002. alimentazione-naturale.blogspot.com/.../lievito-integratore-naturale-o.ht...18 dic 2007. Appleton PJ, Burn JM. A neuro-inhibitory substance: gamma-hydroxybutyric acid. Preliminary report of first clinical trial in Britain. Anesth Analg. 1968;47:164–70. Bernard S. Metabolismo del magnesio. In: Vourch G, editor. Trattato di Anestesia e Rianimazione, vol. II. Verduci Editore: Rome; 1976. Blumenfeld M, Suntay RG, Harmel MH. Sodium gamma–hydroxybutiric acid: a new anesthetic adjuvant. Anesth Analg. 1962;41:721–6. Calvario M. Carenza di piridossina ed epilessia. Acta Vitaminol. 1958;12:23–6. De Cailar J, Herail J. Le 4-hydroxybtyrate de sodium en chirurgie cardio-vasculaire. Agressologie. 1962;3:209–16. Devlin TM. Biochimica. Naples: EDISES; 2012. Dickens F, Randle PJ, Whelan WJ. Carbohydrate metabolism. London: Academic Press; 1968. Du Bouchet N, Passelecq J. Anesthesie. Paris: Flammarion Médicine-Sciences; 1981. Galzigna L. Introduzione alla biochimica patologica e clinica. Padua: Piccin Editore; 1997. Garret RH, Grisham CM. Biochimica. Padua: Piccin Editore; 2014. Grosu I, Lavand’homme P, Thienpont E. Pain after knee arthroplasty an unresolved issue. Knee Surg Sports Traumatol Arthrosc. 2014;22:1744–58. Grosu I, Thienpont E, De Koch M, Scoltes JL, Lavand’homme P. Dynamic view of postoperative pain evolution after total knee arthroplasty a prospective observational study. Minerva Anestesiol. 2016;82(3):274–83. Harper HA. Chimica fisiologica e patologica. Padua: Piccin Editore; 1965. Jevons FR. Le basi biochimiche della vita. Milan: Mondadori Editore; 1972. Lund LG, Humphries JH, Virtue RW. Sodium gamma hydroxybutyrate. Laboratory and clinical studies. Can Anaesth Soc J. 1965;12:379–85. Moruzzi G, Rossi CA, Rabbi A. Principi di chimica biologica. Bologna: Tinarelli Editore; 1984. Nelson DL, Cox MM.  Introduzione alla biochimica di Lehninger. Bologna: Zanichelli Editore; 2015. Nelson DL, Cox MM. Lehninger principles of biochemistry. 7th edn. USA:Freman and Company; 2017. Siliprandi N. Chimica biologica. Rome: Edizioni Ricerche; 1975. Smith Christopher UM. Biologia molecolare. Milan: Mondadori Editore; 1971. Schneider J, et  al. Le comportement EEG de l’homme et de l’animal soumis à l’action du 4-­hydroxibutyrate de sodium. Agressologie. 1963;4:55–70. Solway J, Sadove MS. 4-Hydroxybutyrate: a clinical study. Anesth Analg Curr Res. 1965;44:532–9. Tunnicliff G, Cash CD.  Gamma- Hydroxybutirate, molecular functional and clinical aspects. London: Taylor & Francis; 2002. Vourch G. Trattato di anestesia e rianimazione. Rome: Verduci Editore; 1976. www.treccani.it/.../vitamine_res-174a9092-87e7-11dc-8e9d-0016357eee

5

Hexose-Monophosphate Shunt or Horecker Cycle (The Sleep Pathway) Fernando Alemanno

The direct oxidative pathway, also called the hexose-monophosphate shunt or Horecker cycle [1], acts in the cytoplasm like the anaerobic glycolytic pathway, hence it is present in all cells, both in those with mitochondria and those which have no or few mitochondria (red blood cells, neuroglia). It is the night-time metabolic alternative to the action pathway (Embden–Meyerhof–Krebs), when the mechanism of sleep is triggered in the ventrolateral pre-optic area of the brain, i.e. when external factors (darkness) or internal factors (fatigue) activate the GABA mechanism. This consists in opening the chlorine channels, which increases the membrane potential so as to increase the excitability threshold and render the cell insensible to normal stimuli, a mechanism that extends, like a resonance phenomenon, to the entire encephalon. Glucose-6(P) is oxidated into 6(P)gluconic acid, thus deviating the metabolism towards the pentose pathway, the pathway of synthesis in which the damages incurred during the day (wear and tear, free radicals, etc.) are repaired. The direct oxidative pathway produces: 1. Above all pentoses, required to synthesise nucleotides, the fundamental components of DNA and RNA. (a) NADPH2, used as a donor of H2 in the synthesis of the long fatty acid chains in the liver, the membrane phospholipids (sphingosine, sphingomyeline), and steroids in the adrenal gland. (b) Monosaccharides with four C atoms (erythrose) and seven C atoms (sedoheptulose). (c) Energy in the form of ATP.

F. Alemanno (*) Anaesthesia and Intensive Care, Brescia Clinical Institutes, Brescia, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 F. Alemanno (ed.), Biochemistry for Anesthesiologists and Intensivists, https://doi.org/10.1007/978-3-030-26721-6_5

35

36

F. Alemanno

Note that glucose-6(P) is an important crossroads in the metabolism of the glucides. Glycogen Glucose-1(P) Glucose

cose-6(P)

6(P)-gluconic acid (DOP)

Fructose-6(P) Anaerobic glycolysis

Once formed from glucose by hexokinase, glucose-6(P) has four different metabolic destinies: 1. It can be transformed into fructose-6(P), follow the anaerobic glycolysis pathway and then, if the cell has mitochondria, enter the Krebs cycle. (a) It can be transformed into glucose-1(P) and then into glycogen. (b) It can be oxidated by NADP and glucose-6(P)-dehydrogenase (the enzyme lacking in favism) into 6-(P)-gluconic acid and enter the pentose pathway (DOP). (c) Finally, in emergency situations, under the action of adrenalin which activates the enzyme glucose-(6)phosphatase, it may be dephosphorylated into glucose which exits the cell to be used where energy is required. Let us now consider the third way, the direct oxidative pathway (but oxidative only to a certain point, as we shall see). 1.

Glucose －　　6 ( P ) + NADP « 6 ( P ) －　　gluconic acid + NADPH 2 ( G －　　6 ( P ) －　　dehydrogenase )



The glucose-6(P) is oxidated by dehydrogenation by the enzyme glucose-6-(P)dehydrogenase and its coenzyme NADP, which accepts the two hydrogen ions, turning into NADPH2. It must be clear that things can’t be so simple, even here there is an intermediate product (gluconolactone), which produces the 6(P)-gluconic acid, whose acid valency is in carbon

5  Hexose-Monophosphate Shunt or Horecker Cycle (The Sleep Pathway) O=CH

37

O=C

HC-OH

COOH

C-OH

HCOH

NADP → NADPH2

HOCH

HO-CH

→

O

(glucose 6(P) dehydrogenase)

HOCH

→ (gluconolactonase) + H2O

HC-OH

HC-OH

HCOH

HCOH

HC

HCOH

CH2O-(P) Glucose . 1 6(P)

CH2O-(P) δ-lactone of 6(P)-gluconic acid

CH2O-(P) 6(P)-gluconic acid

Indeed the glucose, which like all monosaccharides is a polyvalent alcohol, is easy to oxidate at carbon 1 because the latter, by its nature, has an aldehyde group and has thus already taken one step along the transformation from alcohol to acid. Note, by the way, that in organic chemistry the oxidative progression of an alcohol is: alcohol → aldehyde → acid where aldehyde is simply the acronym of alcohol dehydrogenate. 6(P)-gluconic acid, preceded, as mentioned previously, in its formation by phosphogluconolactone, undergoes oxidative decarboxylation by the enzyme 6(P)-gluconic-­dehydrogenase, and is then transformed into d-ribulose-5(P), with further production of NADPH2, in the reaction 2.

6 ( P ) －　　gluconic acid + NADP « D －　　ribulose －　　5 ( P ) + NADPH 2 + CO 2 ( 6 ( P ) －　　gluconate dehydrogenase )



This completes the oxidative stage, which gives its name to the pathway (DOP), and matters get more complicated with the d-ribulose-5(P) which gives rise to two different pentoses: xylulose-5(P) and d-ribose-5(P) 3.

D －　　xylulose －　　5 ( P ) « D －　　ribulose －　　5 ( P ) « D －　　ribose －　　5 ( P ) ( phhospho ketopentose isomerase ) ( phosphoriboisomerase )



These two pentoses interact to form two other compounds: 3(P)glyceraldehyde, an old acquaintance, and another strange monosaccharide with seven carbon atoms, sedoheptulose-7(P), which is contained in celery (after which it is named); the enzyme is transketolase and the coenzyme, as acceptor and transporter of carboxyl groups, is the active form of vitamin B1: cocarboxylase or diphosphothiamine (DPT):

38

F. Alemanno

. 4 D －　　ribose －　　5 ( P ) + D －　　xylulose －　　5 ( P ) « 3( P ) －　　glyceraldehyde + Sedoheptulose －　　7 ( P )

( transketolase + vit.B1 )



Now, sedoheptulose and 3(P)-glyceraldehyde interact with each other to yield erythrose-4(P) and fructose-6(P); 5. Sedoheptulose + 3 ( P ) －　　glyceraldehyde « Erythrose －　　4 ( P ) + Fructose －　　6 ( P ) ( transaldolase ) In effect, sedoheptulose, with its 7C atoms, transfers 3 to the 3(P)-glyceraldehyde, transforming it (3  +  3  =  6) into fructose-6(P). As for the sedoheptulose: 7 – 3 = 4 = erythrose-4(P). erythrose-4(P) and xylulose-5(P) react together to form 3(P)-glyceraldehyde and fructose-6(P): 6.

Erythrose －　4 ( P ) + Xylulose －　5 ( P ) « 3 ( P ) －　glyceraldehyde + Fructose －　6 ( P ) ( transketolase + vit B1 )



Finally:

7.

Fructose －　　6 ( P ) « Glucose －　　6 ( P ) ( phosphohexoisomerase )

This reaction is called “isomerisation of fructose-6-phosphate”. It’s like playing the goose game! “go back to square one”—but that’s not really the whole story. In reality, if six molecules of glucose-6(P) enter the DOP and six molecules of CO2 exit it, then one molecule of glucose-6(P) has been completely oxidated into CO2 and H2O. Hence, if six molecules of glucose-6(P) are oxidated by 12 NADP molecules (reactions 1 and 2), 12 molecules of NADPH2 are formed, along with 6 molecules of CO2. Now, we must know that NAD is a more potent acceptor of hydrogen ions than NADP, and since the two molecules act in the cytoplasm in this case, we have the following reaction:

NADPH 2 + NAD = NADP + NADH 2

We also know that, in oxidative phosphorylation, each reoxidated NADH2 produces three molecules of ATP, hence our hypothetical six molecules of glucose-6(P) entering the pentose cycle will produce 12 × 3 = 36 molecules of ATP, which is a fine source of energy for (above all) synthesis processes.

5  Hexose-Monophosphate Shunt or Horecker Cycle (The Sleep Pathway)

39

Of the hereditary congenital recessive defects, we should mention pentosuria, which is not an illness but an anomaly characterised fundamentally by the elimination of l-xylulose in the urine; if mistaken for glucose, this can lead to an incorrect diagnosis of diabetes mellitus. Pentosuric individuals have a deficit of the enzyme l-xylulose-reductase which reduces the xylulose to xylitol, so that they are unable to close the pentose cycle. Xylitol is a fundamental step in the transformation of l-xylulose into d-xylulose before the latter is phosphorylated into xylulose-5(P): L －　　xylulose ® Xylitol ® D －　　xylulose ® D －xylulose - 5 ( P )



5.1

( NADPH ® NADPP ) ( NAD ® NADH ) ( ATP ® ADP ) ( L －　　xylulose －　　reductase )



Glucuronic Acid

This molecule, closely bound to the pentose pathway, is important for all disintoxication processes, especially linked to the administration of drugs. The process of glucuronation is catalysed at the level of carbon 1 by the enzyme glucuronyl-­ transferase. Carbon 1, due to its aldehyde group, has a reducing activity; this enables it to react easily with hydroxyl and carboxyl groups, for instance with the derivatives of benzoic acid (local anaesthetics, salicylates, etc.) and amine and thiol groups. The products of glucuronation are eliminated in the urine since they cannot be reabsorbed by the Henle loop; if their molecular weight is greater than 500 Dalton they are eliminated in the bile and not reabsorbed in the intestine, unless bacterial glucuronidases are present, which can provoke partial reabsorption. A lack of glucuronyl-transferase is, among other things, responsible for familial nonhaemolytic jaundice (Gilbert’s syndrome) in which indirect bilirubin is not transformed into direct bilirubin, and thus accumulates resulting in the symptoms of jaundice or sub-jaundice. It is interesting that the chronic administration of barbiturates like phenobarbital (Luminal, Gardenal), with an enzymatic induction mechanism, produces an increase of the enzyme and resolves the jaundice. CHO

CHO

HCOH

HCOH

HOCH

HOCH

HCOH

HCOH

HCOH

HCOH

*CH2OH

*COOH

The formula or D-glucose The formula of glucuronic acid

40

F. Alemanno

The glucuronic acid is formed by oxidation of the alcohol group of carbon 6 of d-glucose, transformed into a carboxyl group. Once it has completed its disintoxicating function, it returns to the pentose cycle via gluconic acid → 3-­ketogluconic → l-xylulose → xylitol → d-xylulose, etc. Glucuronic acid, which is a component of a large number of mucopolysaccharides, is also the precursor of hyaluronic acid and ascorbic acid (vitamin C). Unfortunately man, the primates and guinea pigs do not have the enzymes required to synthesise vitamin C, and must take in the food they eat.

5.2

Vitamin C

l-Ascorbic acid is a monosaccharide, a hexose, as shown by its formula. We include in this chapter on the pentose pathway due to its close connection with glucuronic acid. It differs structurally from 6(P)-gluconolactone (which precedes the formation of 6(P)-gluconic acid, the first stage in the pentose cycle) for the presence of a double bond between C2 and C3 (easily oxidisable) and the oxygen bridge C1–C4 (γ-gluconolactone), while 6(P)-gluconolactone has an oxygen bridge C1–C5 (δ-gluconolactone). For the reader’s convenience, we show the first stage of the DOP. O=CH

O=C

HC-OH

COOH

C-OH

HCOH

NADP → NADPH2

HOCH

HO-CH

→

O

(glucosio 6(P) deidrogenasi)

→

HOCH

(gluconolattonasi) + H2O

HC-OH

HC-OH

HCOH

HCOH

HC

HCOH

CH2O-(P) Glucose 6(P)

CH2O-(P) δ-lactone of 6(P)-gluconic acid

CH2O-(P) 6(P)-gluconic acid

5  Hexose-Monophosphate Shunt or Horecker Cycle (The Sleep Pathway) O

C

O

C

HO

C

O

C

HO

C

O

C

HCOH

H

C

H

C

HCOH

HO

C

HO

C

COOH HCOH

O HOCH

CH2OH Gluconic acid

H

−2H

O

CH2OH L-ascorbic acid

41

H

CH2OH dehydroascorbic acid

(L-gluconolactone-oxidase) this enzyme is missing in guinea pigs, primates and man.

Mammals normally synthesis ascorbic acid (hence for them it is not a vitamin). We show the stages of its synthesis below for curiosity’s sake, although it is absent in man: D －　　glucose ® D －　　glucuronic acid ® L －　　gluconic acid ® L －　　gluconic acid g －　　lactone ( ascorbic acid )

( L －　　gluconolactone - oxiddase )



Its lack in the diet causes, in guinea pigs, man and the primates, who do not possess the enzyme l-gulonolactone-oxidase, the illness known as scurvy. This first manifests with a hyperkeratotic perifolliculitis, which, in contrast with avitaminosis A, has a haemorrhagic character. It is precisely this haemorrhagic character that identifies this avitaminosis, starting with the gums bleeding when biting on his food, bleeding lips, joints and muscles, with bruising in response to even the slightest trauma. Scurvy was the disease of sailors embarked on long voyages, from the days of ancient Greece onwards [2]. In 1747 James Lind, the British Naval surgeon, tried out a variety of diets on 12 sailors divided into 6 pairs, and found that a diet containing citrus fruits was able to prevent scurvy. The results of his study were published in 1753 [3] and the British Navy adopted the use of lemons and limes in their on-­ board fare in 1795. The vitamin was isolated around 1930 by Svirbely, Szent-­ Gyorgyi (Nobel Prize 1937 for medicine) and by Glen King. It was then synthesised in 1934 by Tadeusz Reichstein and by Norman Haworth (Nobel Prize 1937 for chemistry). l-Ascorbic acid is easily oxidated to dehydroascorbic acid (an equally effective anti-scorbutic metabolyte), and this is why ascorbic acid is widely used in industry in place of bisulphate as an anti-oxidating agent in the conservation of food products. As such it is labelled E 300, while its equally active salts are labelled E 301

42

F. Alemanno

(sodium ascorbate) and E 302 (calcium ascorbate). One of its functions in this field is to prevent the transformation of nitrates (added to cured meats and some cheeses to prevent the development of botulinum clostridium) into nitrites, substances which, once ingested, transform in the gut into carcinogenic nitrosamines. The esters of ascorbic acid (ascorbyl palmitate or stearate), strongly lipophilic, labelled as E 304, are used as additives for oils and margarines to prevent them going rancid. As a coenzyme, it is an important co-factor in many hydroxylations. Collagen, for example, is an atypical protein; its amino acid sequence consists of triplets, and each triplet contains glycine, proline and lysine; the latter two are hydroxylated, and the substrates for the reaction are molecular oxygen. Hypoxia slows down the hydroxylation of proline. Studies by Uìtto and Prockop [4] conclude that synthesis in temporary hypoxia produces poorly stable collagen. This also occurs in smokers, in which the continuous formation of carbon monoxide, which binds stably to haemoglobin, added to the vasoconstrictive action of nicotine, generates a condition of relative hypoxia of the subcutaneous tissue, forming low quality collagen and precocious wrinkles. The lack of vitamin C is another factor which limits the hydroxylation of lysine and proline, and hence the synthesis of stable collagen. –

H2C

4

CH2

3

H2C5

2CH

N H Proline

– 1COOH

HO – HC – 4

CH2

3

H 2C 5

2CH

– 1COOH

N H Hydroxyproline

(proline - hydroxylase + ascorbics acid + a-ketoglutaric acid + Fe++ + O2 )

Proline does not have a primary amine group, its N is inserted into a pyrrolidine ring. The hydroxylation occurs on the carbon in position 4 (relative to the carboxyl). The α-ketoglutaric acid is the donor of electrons, while the ascorbic acid maintains the coenzyme Fe2+ in the ferrous state. Alterations to the amorphous substance, composed of proteoglycans and collagen, which supports the endothelial cells and holds them together, are responsible for the primary symptom of scurvy: the haemorrhages. Another site in which the action of ascorbic acid is essential involves the transformation of dopamine into noradrenaline. In this case too, this involves the hydroxylation of the carbon-β of the ethyl amine radical of dopamine. The enzyme is dopamine β-hydroxylase, a cuproprotein enzyme, which uses ascorbic acid as a second reducing agent. This is why the adrenal glands are rich in vitamin C: to handle the continuous process of hydroxylation.

5  Hexose-Monophosphate Shunt or Horecker Cycle (The Sleep Pathway)

HO

C in the β position ↓ NH2

HO

43

OH HO NH2

HO

Dopamine → (dopamine b-hydrozylase) → Noradrenaline (ascorbic acid )

Vitamin C is also essential in the synthesis of carnitine (trimethylbetaine), which carries the acyl groups across the mitochondrial membrane where the Knoop β-ossidazione can then take place. Indeed, acyl-CoA cannot cross the mitochondrial membrane. (CH3)3N+−CH2−CH−CH2−COO− (β-hydroxy-γ-aminobutyric acid), OH

The formula of carnitine, which makes clear the hydroxylating action of vitamin C on carbon β. Among its many other actions, we note its reactivation of vitamin E, with the loss of an electron to the radical α-tocopheroxyl, to keep the Fe2+ in the ferrous state.

5.3

Hyaluronic Acid

Hyaluronic acid is a mucopolysaccharide unsulphured acid. It is a polymer composed of the nth repetition with a glycosydic bond 1-4 of a disaccharide formed of d-glucuronic acid and N-acetylglucosamine bound by a glycosydic bond 1-3. HC1 HCOH HOCH O 4CH

HC COOH Glucuronic acid

O O

HC1

HC1

H3C-CH-N-COH 3CH

O

O

HCOH HC CH2OH N-acetylglucosamine

HCOH HOCH O 4CH

HC COOH Glucuronic acid

O O

HC1

H3C-CH-N-COH 3CH

O

HCOH HC CH2OH N-acetylglucosamine

Hyaluronic acid. The linear formula of the constituting polymer of hyaluronic acid. Each disaccharide with a 1-3 bond is itself bound to the next by a β-glycosydic 1-4 bond.

44

F. Alemanno

The mucopolysaccharides are divided into two groups: Neutral and Acids. –– Neutral, containing hexosamines, hexoses or pentoses (polysaccharides of the blood groups and antibodies). –– Acids, containing hexosamines (e.g. glucosamine), uronic acid and sialic acid. The acid mucopolysaccharides, in turn, may be sulphurated (e.g. heparin, chondroitin sulphuric acid) and unsulphurated (e.g. hyaluronic acid). Hyaluronic acid is thus a glucosamine-glycan, whose carboxyl groups are completely dissociated; their negative charges repel each other to space the lateral branches of the polymer, thus occupying a larger volume, which is important for its hydration. Indeed, the molecule, which is strongly polarised (like glucose), attracts molecules of H2O, thus undergoing hydration and swelling up. Furthermore, the negative charges, which are apparent in the dissociation of the carboxyl groups, attract a variety of cations such as Na, K and Ca, and above all sodium, which further contributes the hydration of the structure. Hyaluronic acid is practically present everywhere: it is found in the skin, in the cartilage, the synovial fluid, the tendons, the humor vitreus, and the walls of the aorta. In the joints, it is the most important component of the synovial fluid; it is synthesised by the chondrocytes and synovial cells, and acts both as a lubricant and as a shock absorber for loads and traumas. It is synthesised inside the cells of the various organs, which use it, and then released into the extracellular space; the enzyme is hyaluronic acid-synthetase, while the enzyme responsible for depolymerising the 1-4-beta-glycosyde bonds by hydrolysis is hyaluronidase. This enzyme was often added, in the 50s and 60s, to local anaesthetics to promote their diffusion. The electro-neurostimulator and ecograph were not available at that time, and nerves were localised by inducing paraesthesia. If however the anaesthetic mixture was not accompanied by adrenaline as a vasoconstrictor, surgical anaesthesia was considerably diminished. Its use was recommended in the blockade of the inguinal region and the supraclavicular blockade of the brachial plexus. Daniel C. Moore, in his famous book Regional Block [4], published in several editions (1953, 1957, 1961, 1965) by Charles C. Thomas Publisher, Sprigfield, Illinois and its Italian translation by Domenico Cecconello for Piccin Editore, Padua 1969, expressed himself as follows on its use in the brachial plexus block: “The use of 150 U of hyaluronidase in the anaesthetic solution does not increase the number of perfectly effective brachial plexus blocks; however it does accelerate the reabsorption of blood from the haematoma caused by the puncture of the subclavian artery”. Returning to our hyaluronic acid, it is basically used for articular infiltrations (gonarthrosis and coxarthrosis) and in plastic surgery. The orthopaedic indication is not only due to its lubricating effect, which itself would last no more than a few months, but to the fact that hyaluronic acid stimulates the anabolic activity of the CD 44 receptors, located on the surface of the chondrocytes, thus inducing the production of new cartilage.

5  Hexose-Monophosphate Shunt or Horecker Cycle (The Sleep Pathway)

5.4

45

Sleep

For an anaesthetist–resuscitator, sleep has always been a common denominator for both functions. I am not referring to the malevolent definition given by some surgeons, irritated by the fact that the patient often “pushes”, of “a sleepy doctor next to a half-awake patient”, during the age of ether administration with the Esmarch or Ombredanne mask. In those days the unfortunate anaesthetist (would-be surgeon), taking the place of the “ecclesiastical” anaesthesia (administered by the nun in the operating theatre), before he passed to the other side of the barricade (the sheet separating the head of the patient from the site of the operation itself) had to complete an apprenticeship while waiting to grasp, if not the scalpel, at least the Farabeuf retractors. In this unwelcome position it was inevitable that he would inhale part of the vapour intended for the patient. I am obviously referring to the fact that as an anaesthetist, we must put a fully awake patient to sleep, while as an intensivist, we must awaken a patient out of his coma (caused by the most varied factors). We have subtitled this chapter “The sleep pathway”, but we have hardly even mentioned this important aspect, which covers around a quarter of our lifetime. Sleep is as important, to make a comparison, as (although at shorter intervals) having our car serviced at the times prescribed by the manufacturer. I realise that the comparison is rough, but it should convey the idea; if we do not service our car, the small defects which accumulate during its use have long-term effects which make it first unreliable, and then no longer road worthy—and we find ourselves without a vehicle at exactly the wrong time and place. But our organism is not composed of an assembly of metal components, our biological membranes, damaged during the waking hours, are far more complex than an internal combustion engine and demand service every day—or at least every night. Our time on this earth follows the cadence day/night, light/dark, which drives us to be active when it is daytime, and to sleep when it is dark and we can no longer see. At night, our organism must repair, for instance, the damage done by the free radicals of oxygen, or synthesize proteins, antibodies, fatty acids, phospholipids, cholesterol, hormones and so on. The fact that a newborn baby sleeps for around 80% of the time is related to its need to grow—and indeed, adolescents tend to sleep longer than adults. During the day, biological processes are primarily oxidative, generating the energy required for activity, while during the night they are reductive, with the purpose of synthesizing organic material. We will not go into the electroencephalographic (EEG) details of the various phases of sleep, we will simply mention the two classic phases non-REM and REM (Rapid Eye Movement). The non-REM phase is characterised by a deep sleep with slow EEG waves; the muscles are relaxed. Sporadic movements are due to the need to change an uncomfortable position, while breathing, the heart rate, renal function and the consumption of oxygen diminish. The REM phase, on the other hand, is characterised not only by the rapid eye movement that gives it its name but also by

46

F. Alemanno

dreaming, increased heart rate and breathing, and erection of the penis/clitoris, regardless of the erotic or otherwise content of the dream. Adults generally sleep for 6–8 h. The non-REM phases alternate with the REM phases in cycles of an hour and a half on average, during which the non-REM phases account for around three quarters of the cycle, with the REM phase taking up the remaining quarter. In any case, the first period of sleep starts with a non-REM phase. In my view, this alternation of non-REM with REM sleep has a relatively simple explanation. Sleep starts with a non-REM phase due to the prevalence of GABA which, opening the chlorine channels, hyperpolarises the nerve cells, making them insensitive to external stimuli. The glucose metabolism is oriented towards the DOP and this starts its anabolic activity, activating the synthesis of proteins, phospholipids, fatty acids, hormones and neurotransmitters. This includes those which determine and are active during the waking hours (dopamine, noradrenaline, serotonin). It is probable that in this process of production and storage, small amounts of the products are released into the synapses and, although in a general situation of hyperpolarisation, activate the REM phase for a short time, resulting in a “fiction” of virtual activity, selecting episodic fragments from the memory and organising them into a collage, which is seldom logical, often without head or tail, on the verge of absurdity. The start of non-REM sleep is activated when a centre, formed of cholinergic neurons and located at the base of the prosencephalon, which is normally quiescent during the waking hours, activates its discharge frequency; to this is associated the suprachiasmatic nucleus, situated bilaterally, whose GABA-ergic neurons are sensitive to varying luminous stimuli. REM sleep, on the other hand, is characterised by a certain activity of the cortical neurons, the impulses of which activate exclusively the ocular muscles and accelerate the rhythm of the respiratory muscles. A strong emotional component may be stimulated by the activation of the limbic system. The fact that an infection results in increased activation of sleep should make us meditate on the need to produce antibodies via the DOP. Interleukin 1, produced by the macrophages and the neuroglial cells, which stimulates the production of antibodies, also facilitates sleep. Another factor would seem to be adenosine, fundamental in the synthesis of cyclic AMP; this substance increases during the waking hours and diminishes during sleep. It is known that cyclic 3′5′AMP is synthesised by the enzyme adenylcyclase and destroyed by the enzyme phosphodiesterase; the majority of hormonal and neuromediator signals would be weak and ineffective if not amplified by this signal amplifier. Now, it is logical that during the waking hours there is an increase of adenosine, a fundamental factor in the synthesis of cyclic 3′5′AMP by the enzyme adenylcyclase, to assure that all activating signals be properly expressed. All drugs that depress adenylcyclase (e.g. opioids) induce sleep, while those which block the phosphodiesterase enzyme (e.g. xanthine) promote wakefulness. Other important factors in the regular induction of sleep are magnesium, vitamin B6, essential to the production of GABA, and melatonine (see Chaps. 4 and 6 for details).

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47

References 1. Horecker BL.  Pentose phosphate pathway, uronic acid pathway, interconversion of sugars. In: Dickens F, Randle PJ, Whelan WJ, editors. Carbohydrate metabolism and its disorders. New York: Academic Press Inc. (London) Ltd; 1968. 2. Beaglehole JC. The journal of Captain James Cook on his voyages of discovery. Vol 1: The voyage of the endeavour 1768–1771. Cambridge: Cambridge University Press; 1968. p. 613. 3. Uitto J, Prockop DJ. Synthesis and secretion of underhydroxylated precollagen at varius temperatures by cells subject to temporary anoxia. Biochem Biophys Res Commun. 1974;60:414. 4. Moore DC. Regional block. Springfield: Charles C. Thomas Publisher; 1965.

Further Reading Bear MF, Connors BW, Paradiso MA.  Neuroscienze. Esplorando il cervello. Paris: Elsevier Masson; 2009. Coccagna G, Smirne S. Medicina del Sonno. Milano: UTET; 1993. Devlin TM. Biochimica. Napoli: EDISES; 2012. Didattica.uniroma2.it_assets_uploads_corsi_141858_Lezione_4_22_03_2012_did_web Frizziero L, Govoni E, Bacchini P. Intra-articular hjaluronic acid in the tretment of ostheoarthritis of the knee: clinical and morfological study. Clin Exp Rheumatol. 1998;16:441–9. Galzigna L. Introduzione alla biochimica patologica e clinica. Padova: Piccin Editore; 1997. Garret RH, Grisham CM. Biochimica. Padova: Piccin Editore; 2014. Harper HA. Chimica fisiologica e patologica. Padova: Piccin Editore; 1965. http://it.wikipedia.org/wiki/Acido_ialuronico Jevons FR. Le basi biochimiche della vita. Milano: Mondadori Editore; 1972. Littarru GP, Onder L. Energia vitale. Roma: C.E.S.I. Editore; 1991. Nelson DL, Cox MM.  Introduzione alla biochimica di Lehninger. Bologna: Zanichelli Editore; 2015. Nelson DL, Cox MM. Lehninger principles of biochemistry. 7th edn. USA:Freman and Company; 2017. Reiter RJ, Robinson J. Melatonina. Milano: Mondadori Editore; 1996. Siliprandi N. Chimica biologica. Roma: Edizioni Ricerche; 1975. Smith Christopher UM. Biologia molecolare. Milano: Mondadori Editore; 1971. www.my-personaltrainer.it_acido_ascorbico_antiossidanti. www.uniurb.it/MedChem/Farma1/Testi/Cap%2008%20Metabolismo.doc

6

Neurotransmitters Francesca Alemanno and Fernando Alemanno

A neurotransmitter is a substance which, stored in the presynaptic vesicles, is released into the synapse in response to a nerve impulse to stimulate the receptors of the successive cell and thus trigger a depolarising or hyperpolarising event, for instance a muscular contraction, a new nerve impulse or the inhibition of the latter. After being released into the synapse and having thus fulfilled its function, the neurotransmitter must be removed by means of reuptake, primarily to avoid prolonging the stimulus it produces and, even if it is metabolised by a specific enzyme, to prevent its encumbering the synapse with the products of degradation. This removal involves some neurotransporters included both in the presynaptic membrane and in that of the astrocytes, which in some cases (glutamate, GABA, serotonin) themselves remove the greater part of the waste products from the synapse, and then deliver them to the presynaptic terminal where they are re-used for synthesising and storing the new neurotransmitter.

6.1

Ionotropic and Metabotropic Receptors

When talking about neuromediators, we must first cover some basic notions about receptors. These are divided into two major categories: ionotropic receptors and metabotropic receptors. The ionotropic receptors are also called ionic channels, the opening of which is ligand dependent, in the sense that they react to the transmitter, with which they bind, by opening a channel, normally closed, which permits the ionic flow across

F. Alemanno (*) Psychologist, Graduated at University of Padua, FISPPA Department, Padova, Italy F. Alemanno Anaesthesia and Intensive Care, Brescia Clinical Institutes, Brescia, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 F. Alemanno (ed.), Biochemistry for Anesthesiologists and Intensivists, https://doi.org/10.1007/978-3-030-26721-6_6

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50

F. Alemanno and F. Alemanno

the cellular membrane. An example of an ionotropic receptor is given by the nicotinic receptor, the ionic channel of which is activated by acetylcholine when this neurotransmitter is in the closed configuration, and which is called nicotinic precisely for its similarity to the alkaloid. By the way, the alkaloids are, pharmacologically defined to be plant extracts with amine groups, which give them their basic characteristics (nicotine, caffeine, strychnine, morphine, etc.). Ionotropic transmitters have a low molecular weight and are synthesised in the cytoplasm, after which they are stored into the vesicles and released when needed. Their action on the receptor lasts for around 1  ms. The ionotropic transmitters include: aspartate, glutamate, GABA, glycine (all amino acids) and acetylcholine, which cannot be called an amino acid but rather a biogenic amine. Metabotropic receptors, on the other hand, do not open a channel, but activate signal amplifiers in the membrane called second messengers (Sutherland, Nobel Prize 1971), which induce a series of chemical reactions in response to the signal. Three types of second messenger are known. 1. The first type includes three hydrophilic molecules: (a) 3′5′AMP-cyclic, synthesised by the enzyme adenylyl cyclase and hydrolysed by the enzyme phosphodiesterase. (b) Cyclic guanosine-monophosphate (cGMP), synthesised by the enzyme guanylate cyclase (contained in the membrane) and located in the cytosol, after which it is hydrolysed by the enzyme phosphodiesterase (like the 3′-5′AMP-­ cyclic). cGMP is important for its myocardial function, since it activates a number of proteins, including Ca-ATPase of the sarcoplasmic reticulum, which regulate the flow of calcium. (c) Inositol triphosphate (IP3), obtained from the action of the enzyme phospholipase on phosphatidylinositol 4-5-bisphosphate, which activates a specific protein in the sarcoplasmic reticulum, which in turn permits entrance into the cytoplasm of the Ca2+ ion, with all its coenzymatic and depolarising functions. 2. The second type includes two hydrophobic molecules: diacylglycerol (DAG) and phosphatidylinositol, both activators of the calcium channels. 3. The third type includes a gaseous molecule: nitrogen oxide (NO), with its vasodilating and anti-aggregating function; the vasodilating action of nitroglycerine can be imputed to the formation of NO. This is produced by the enzyme nitrogen oxide synthetase, which oxidises the guanidine group of l-arginine:



arginine + NADPH + 2O 2 ® NO + citrulline + 2H 2 O + NADP ( NO - synthetase )

Nitrogen oxide has no direct effect on any membrane receptors, but rather produces, via the guanilcyclase enzyme, cyclic guanosine-monophosphate (GMPc), thus falling under the mechanism of the first type. GMPc, by phosphorylating the inositol-triphosphate (IP3) receptor, blocks the release of Ca2+ from the

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sarcoplasmic reticulum of the smooth muscle cells, thus causing vasodilation. Due to its oxidising action (it is a free radical), it is one of the causes of tissue damage due to post-ischemic cardiac or cerebral reperfusion. The various receptors mentioned above do not act independently, but often act together with a protein G (or GTP). This is a protein which is contiguous with and closely related to the membrane receptors, which are called protein G coupled or serpentine receptors. These include the muscarinic cholinergic, dopaminergic, adrenergic, serotonergic and opioid receptors. This structure consists of 7 alpha transmembrane helices, from which comes the name “serpentine”. The various neurotransmitters, once they have bound to their receptors, change, thanks to this bond, the form of protein G, in which the guanosine-diphosphate (GDP) is replaced by guanosine-triphoshate (GTP). This initiates an action on the ionic channel protein, or on enzymes such as adenylcyclase, which activates them (protein Gs) or inhibits them (protein Gi), like the enzymes phosphodiesterase and phospholipase. Is that clear? Of course it is! There are numerous neurotransmitters of the central nervous system (SNC) and the peripheral nervous system (SNP). In general, they belong to three chemical categories: the amines (acetylcholine, dopamine, noradrenaline, serotonin, histamine), the amino acids (glutamate, GABA, glycine) and the peptides (endorphins, substance P, TSH, somatostatin). We will consider those which in our opinion are the most important or which, for a variety of reasons, have been called to our attention.

6.2

Acetylcholine

This is a very common neuromediator in the CNS and PNS. The other neuromediators are just as important, but their synapses are almost always preceded or followed by an acetylcholine synapse. A typical example is that of the adrenal medulla, the chromaffin cells of which have numerous nicotinic cholinergic receptors; stimulating these provokes depolarisation of the cells and the secretion of adrenaline. Acetylcholine is synthesised in the cytoplasm. The pyruvic acid, on the other hand, is decarboxylated inside the mitochondrion, but the acetyl-CoA, which forms in this manner is unable to cross the mitochondrial membrane and must therefore exploit the permeability of the citrate formed in the classic first reaction of the Krebs cycle:

acetyl - CoA + oxaloacetic acid = citric acid + CoA

Once it enters the cytoplasm, the citrate can react once more with the CoA to yield oxaloacetic acid and acetyl-CoA, which can finally react with the choline:



acetyl - CoA + choline ® acetylcholine + CoA ( cholinacetylase )

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Well, we know what the acetylic radical is, but what is this “choline”? Biochemistry Textbooks (the real ones) state that it is a nitrogen-containing compound, but not an amino acid. Its formula is hydroxy-trimethyl-ethyl ammonium: [(CH3)3-N+-CH2-CH2-OH] OH−. This is a vitamin factor (vitamin J) contained in the egg yolk, soya seeds, wholegrain rice, wheat germ, etc. It is a component of the phospholipids of the cellular membranes and a donor of methyl via betaine (trimethylglycine), the amino acid derived from choline by oxidation of the alcohol group into an acyl group. OH (CH3)3-N-CH2-CH2-OH choline

OH →

(CH3)3-N-CH2-CH2-COOH betaine (trymethilglycine)

The acetylcholine, once it has been produced, is enclosed at the presynaptic level in 520 A vesicles, each of which contains around 62,000 molecules of the substance [1]. The concentration in the vesicle can reach 1 molar, almost twice the concentration of NaCl in sea water, which is 0.599 mol/L: Na (PM 23) + Cl (PM 35) = 58 (PM NaCl); 1 L of sea water contains 35 g of NaCl, so that 35 (g/L) divided by 58 (PM of sodium chloride) = 0.599 mol/L (~0.6 molar). The energy required to work against a formidable gradient must be supplied, not only directly by the ATP but also by the potential difference, due to the transmembrane concentration of H+ or Na+ cations, which, as they return through the membrane they were displaced across by the consumption of ATP, release this energy to the transporter which uses it to insert the neurotransmitter into the vesicle. It is as if in a hydroelectric power plant, water were to be pumped from a lower lying lake (with its low potential energy) to a lake at a higher elevation which then drives the turbine with its greater potential energy and with a greater availability over time, thanks to its reserves. This is also the principle of a current accumulator or battery. Storage in the vesicles prevents the neurotransmitter being dispersed, or hydrolysed in an uncontrolled manner, while ensuring that it is available when needed and freed into the synaptic space in small amounts (200–300 vesicles) when a nerve impulse arrives. In reality, however, there is a small continuous release of vesicles, which, by generating a mini/potential of around 0.5 mV in a programmed manner, originate the muscular tone present even at rest. The transmission of the impulse is also linked to the presence of Ca2+ and Mg2+ ions: the former promotes the breakage of the vesicles and the liberation of the mediator into the synapse, while the latter impedes it. In this way, when the Ca2+ enters the cell at the postsynaptic level, to contribute to depolarisation, it provokes a crisis of the ion at the presynaptic level, thus interrupting the breakage of the vesicles in a feedback loop (Galzigna) [1]. Acetylcholinesterase immediately hydrolyses acetylcholine, thus interrupting the duration of the depolarisation. A rigorous reuptake at the presynaptic level of the products of hydrolysis ensures perfect recycling of the choline and the acetyl radical, which are thus ready to be resynthesised and returned to the system. Any losses are reintegrated by the axon.

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The acetylcholine molecule has two different configurations, an open one (muscarinic) and a closed one (nicotinic); the latter recalls the configuration of the alkaloid. O

H2

H2

CH3-C – O – C – C – N (CH3) Acetylcholine: muscarinic configuration O O

C

CH2

CH3

CH2

N CH3 CH3 CH3 Acetylcholine: nicotonic configuration

The open form is prevalent in the central nervous system and the peripheral vagal terminals, while the closed form is prevalent in the ganglia of the paravertebral sympathetic chain and the neuromuscular plate. The intensity of synthesis varies according to the zone of the CNS. If we look at the formula of acetylcholine, an acetyl radical + choline, and the formula of acetylcholine-esterase, we can see that the latter has an esterase site and an anionic site. acetyl radical

CH3-COO - CH2-CH2-N-(CH3)3 choline

Esterase site

Anionic site (acetylcholine-esterase)

The first binds to the carbonyl carbon of the acetyl, the latter to the choline’s nitrogen molecule. The attachment of the esterase site to the carbonyl destabilises its electronic equilibrium, thus enabling the insertion of a molecule of H2O, which results in the hydrolysis of the ester bond between the acetyl and the choline: CH3–COO [H

CH2–CH2–N–(CH3)

OH] the H2O molecule is shown in bold between square brackets

There are two types of cholinesterase: true cholinesterase and pseudocholinesterase. The first is present in the red blood cells and muscles, while the second is found in the plasma, and both in the CNS, where the true one, fast-acting cholinesterase

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(1  ms), is present in the neuronal synapses, and pseudocholinesterase, which is slower acting, is found in the neuroglia synapses; moreover, it hydrolyses many choline compounds as succinylcholine. As mentioned above, there are two different forms of acetylcholine: one is called muscarinic, with an open molecule, and the other nicotinic, with a cyclic form, because it resembles the formula of nicotine. In the peripheral parasympathetic effectors, the muscarinic action is: inhibitory, hyperpolarising in the heart (bradycardia) with inhibition of the adenylcyclase, the Ca2+ channels and activation of the K+ channels; excitatory in the smooth visceral musculature (sialorrhea, bronchospasm, diarrhoea). In the CNS it is excitatory, and hence depolarising. It is not so in the spinal cord, where Abelson and Höglund have shown that an increase of acetylcholine is associated with a high pain threshold, and its reduction with hyperalgesia [2]. Now, the open form (muscarinic) is prevalent in the CNS and in the peripheral effectors, while the nicotinic form is typical of the neuromuscular plate and of the ganglia synapses. At this level the muscarinic form is also present, even if to a much lesser extent. In the paraverterbral sympathetic chain ganglia, the nicotinic form mediates the synaptic junction between the myelin pre-ganglionic fibres (of type B), coming from the neurons of the lateral column of the spinal cord, and the large neurons typical of the ganglion, from which the non-myelin post-ganglionic fibres (of type C) start. These synapses are of the excitatory type. The large neurons are accompanied by smaller interneurons, which receive pre-ganglionic fibres from the spinal cord, which produce open chain (muscarinic) acetylcholine with an inhibitory action. These small interneurons in turn maintain synaptic contact with the principal neurons; in this latter case, these are dopaminergic synapses which activate the adenylcyclase, thus producing hyperpolarisation. To sum up, at the ganglionic level, the second neuron has two types of synapse: a principal nicotinic synapse with an excitatory function, and a second dopamine synapse with an inhibitory function, starting from the small interneuron, which in turn receives an upline muscarinic synapse, itself inhibitory. The dopaminergic interneuron is that in turn triggered by a muscarinic synapse, which inhibits its dopaminergic inhibition. This is not a play of words—it really works like this. When an impulse arrives at the large post-ganglionic neuron with its nicotinic synapse, an impulse also arrives at the interneuron by a muscarinic synapse, which inhibits or moderates the inhibiting release of dopamine, to enable the principal impulse to pass through. This is the familiar concept of a driving force opposed by a brake: …adelante, Pedro, con juicio (go on Pedro, if you can), as Antonio Ferrer said to his coachman driving through the crowd in revolt (Manzoni) [3]. Not all the sympathetic nervous fibres, starting from the lateral column of the spinal cord, when they arrive at the ganglion of the paravertebral chain, are interrupted in the ganglion itself to give rise to a synapse with a second neuron. The nerve fibres of the adrenal medulla pass through the ganglion without a break and directly stimulate the production of noradrenalin and adrenalin in the adrenal gland by releasing acetylcholine; the same is true of those, which go to the celiac and

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mesenteric ganglia, only inside these ganglia do they make synaptic contact with a second neuron. The depolarisation induced by the acetylcholine seems to be correlated with the positive charge of the ammonium group, even though the presence of an oxygen bridge, capable of binding a hydrogen, would seem equally essential to the action of the choline; furthermore, the distance between the positive nitrogen and the acetonic oxygen, adjacent to the oxygen bridge, must be equal to two carbon atoms (around 5.3 Ǻ). (O O

5.3 A˚

N+)

H2 H2

C–H3–C–O–C–C–N+(CH3)

Acetylcholine in the open configuration (muscarinic). The distance between the positive nitrogen and the acetonic oxygen, adjacent to the oxygen bridge, must be equal to two carbon atoms, i.e. around 5.3 Ǻ (1 Ǻ = 1/100,000,000 cm).

6.2.1 The Muscarinic Effects of Acetylcholine In the central nervous system, acetylcholine acts on the neurons, which control movement and memory; synthesis is very high here. In the posterior horn of the spinal cord, synthesis is relatively low, with the exception of the inhibitor interneurons, where an increase of acetylcholine is associated with a high pain threshold [2]. In the peripheral nervous system, acetylcholine acts: –– On secretion of the saliva, gastric juice and, in the intestine, the endocrine and exocrine glands –– Increasing peristalsis –– On induction of vomiting –– In the heart: negative chronotropism, inotropism and dromotropism –– In the vascular system: vasodilation The muscarinic effects are suppressed by atropine.

6.2.2 The Nicotinic Effects of Acetylcholine –– In the neuro-muscular plate –– In the ganglia of the paravertebral sympathetic chain –– In the peripheral ganglia

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It is only too clear that acetylcholine has a depolarising action in the peripheral synapses; what seems less clear is its hyperpolarising action at the pacemaker level. The question is a difficult one to resolve. Initially, it was hypothesised (Vassalle) [4] that, since the automatic action of the heart is guaranteed by a pacemaker current (if) which interrupts the diastole, acetylcholine inhibits the current and thus prolongs the diastole, while the catecholamines activate the current and thus abbreviate it. Subsequent studies (DiFrancesco) [5–8] have shown that the muscarinic action on the pacemaker represents the only case of voltage-dependent activation in hyperpolarisation, which justifies the acronym “f” (funny) attributed to it [9, 10] and which, on the other hand, is very similar to the current ik2 of the Purkinje fibres, located in contact with the terminal part of the bundle of His, which generate an internal current auto-activated by repolarisation below the threshold of −50 to −60  mV.  An important role in this scheme is also played by cyclic 3′-5′-AMP, activated by noradrenaline and inhibited by acetylcholine by the inhibition of the adenylcyclase.

6.3

Catecholamines

The name derives from the fact that they are composed of a common denominator (Cathecol), a di-hydroxylated benzene ring (Fig. 6.1) and by a variously modified amine. O HO OH NH2

HO

Phenylalanine

Catechol OH

OH

HO

HO NH2

HO Dopamine

HO NH2

HO Noradrenaline

NH

HO

CH3

Adrenaline

Fig. 6.1  The diagram shows: at the top, the precursors of the catecholamines, catechol and phenylalanine (both inactive as neurotransmitters). The first, while it is not one of them (characterised by the presence of two hydroxyls on the benzene ring), gives its name to this family of neurotransmitters; phenylalanine, via the variation of the alanine radical, differentiates the three successive catecholamines. In the benzene ring, the alternation of a double with a single bond is purely symbolic, since there is no difference in the benzene molecule between the apical carbon-carbon bonds; these have an intermediate length between that of a single and a double bond. Indeed, the length of a single C–C bond is 0.154 nm (1 nm = 10─7 cm), while that of a double bond C=C is 0.134  nm; while for the apical C–C bonds of the benzene ring, the length is always the same, 0.140 nm. Furthermore, the 4 electrons of the outermost orbit of carbon do not orbit around the nucleus of their carbon atom, but are delocalised over the entire crown of the molecule. These delocalised electrons are called π electrons, and the new type of bond they form is also called π

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In saying this we have already written the formula for dopamine: 3,4-hydroxyphenyl1-ethylamine carbon C3 and C1

HO NH2

HO carbon C4

The formula of dopamine

This too, the numbering of the carbons in a complete benzene ring (not hetero-­ substituted), is a nomenclature problem, which we should go over. If more than one radical substituting the hydrogen of two or more different C atoms is present, the earlier one in alphabetic order assigns the number 1 to its carbon, followed in numerical order by the other carbon atoms with radicals (clockwise or counter-­ clockwise), so long as the second one has the lowest localisation number possible. Dopamine in turn produces noradrenaline via the enzyme dopamine-β-hydroxylase: Dopamine + ascorbic acid + O 2 ® Noradrenaline + dehydroascorbic acid + H 2 O ( dopamine - b - hydroxylase ) But this is not the origin of the formation sequence. The head of the catecholamine series is phenylalanine, an essential amino acid whose benzene ring is not hydroxylated and whose first hydroxylation at its carbon 4 forms another amino acid: tyrosine. O

O OH

NH2 Phenylalanine

OH HO

→ (hydroxylase)

NH2 Tyrosine

This, when hydroxylated at the carbon 3, forms di-hydroxyphenylalanine (l-DOPA)

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O HO OH

OH

NH2

HO

NH2

HO

Tyrosine (hydroxyphenylalanine)

→ L-DOPA (di-hydroxyphenylalanine) (tyrosine hydroxylase)

Here the di-hydroxylated benzene ring takes on the aspect of catechol, which gives its name to the family of the catecholamines. The enzyme tyrosine-hydroxylase is inhibited in the presence of an excess production of di-hydroxyphenylalanine (l-DOPA), thus limiting the production of the other catecholamines as well. Phenylalanine and l-DOPA are both inactive. O HO

HO OH NH2

HO

→

L-DOPA

NH2

HO (DOPA-decarboxylase)

→

Dopamine OH

HO

HO NH2

HO Dopamine

→

NH2

HO (dopamine-b-hydroxylase) (ascorbic acid)

→

Noradrenaline

Dopamine then forms noradrenaline by hydroxylation of the carbon-β of the ethylamine radical due to the action of the enzyme dopamine-β-hydroxylase, located in the synaptic vesicles, where hydroxylation takes place, requiring the presence of vitamin C as a coenzyme. Noradrenaline (NA) is then transformed into adrenaline by the enzyme phenylethanolamine-­N-methyltransferase. The methyl donor is methionine, an ordinary sulphurated amino acid, monoamine-monocarboxylic, derived from butyric acid COOH H2N-C-H C-H2 CH2-S-CH3

The formula of methionine

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OH HO

HO NH2

HO Noradrenaline

NH

HO

methionine + → (Phenylethanolamine N-methyltransferase)

CH3

Adrenaline

Catecholamines are produced and located not only in the adrenal medulla but also in the sympathetic nerves and the brain, as well as in many other tissues, especially in the myocardium. They are contained in special vesicles, where they are associated with ATP in a 4:1 ratio, probably because ATP has four negative charges which stabilize the catecholamines, whose ammonium group has a positive charge. They can be produced both in the cytoplasm (decarboxylation of DOPA to dopamine, methylation of NA to adrenaline) and in the vesicles themselves (dopamine → NA). The vesicles also contain the enzyme dopamine-β-hydroxylase, the ATPase enzyme, and calcium and magnesium ions. These vesicles are produced in the neurosome and then transported by the axonal flow to the nerve terminals. The catecholamines are degraded metabolically by the enzyme monoamine-­ oxydase (MAO) in the mitochondria and by catecholamine-oxymethyltransferase (COMT) in the synapse. The MAO enzyme induces oxidative deamination, forming 3-4-­hydroxymandelic acid. The COMT inserts a methyl group into the hydroxyl of the benzene ring in position 3, transforming it into 3-methoxy-4-hydroxymandelic acid (vanilmandelic acid) normally excreted with the urine, the dosage of which gives the (indirect) title of the level of catecholamines in circulation (Marzotti-Alemanno) [11]. The inhibitors of the MAO enzyme (MAOI) were long used in the Sixties and Seventies as antidepressants. Their action is due more to the increase of serotonin than of noradrenaline. It sometimes happened that a depressed patient would take drugs to commit suicide, and on arrival at the emergency room in a coma, be transferred directly to intensive care. The shift anaesthetist, after the initial attempts to resuscitate the patient, would have to ask himself what drug or cocktail of drugs the patient had taken. At that time, an oxybarbiturate (Nembutal) was often used as an oral hypnotic, for which an infusion of bicarbonate was indicated to accelerate its elimination in the urine. One had to be sure, however, that the patient had not also swallowed MAOI (e.g. Marplan), because alkalinisation would hyper-sensitise the peripheral catecholamine receptors, leading to dangerously severe arterial hypertension. The tricyclic antidepressants (imipramine) act on the reuptake in the presynaptic membrane; the MAOI, as we have seen, block the catabolism of the catecholamines. The stimulating effect of the amphetamine is determined by the increased release of catecholamines from the vesicles and blockage of the reuptake, which is also provoked by cocaine. Cocaine, the first historic local anaesthetic, was suggested in 1884 by Sigmund Freud (who used it personally) to his friend Karl Köller (the oculist) as a surface

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anaesthetic for minor surgeries. The drug, even at systemic level, probably acts by blocking—as is to be expected for a local anaesthetic—the Na channels and with them the source of cationic release energy, which is essential for the reuptake and concentration, even counter-gradient, of the neurotransmitters by the various transports. Hence, under its action, the neurotransmitters are not reabsorbed by the ­synaptic space. Tyramine, which is present in wine (especially white wine) and in some fermented cheeses, acts by promoting the release of the catecholamines from the vesicles, which leads to hypertension. On the other hand, the phenothiazines (chlorpromazine, promazine, promethazine, etc.) and the butyrophenones (haloperidol and dehydrobenzoperidol), used in psychiatry as antipsychotics and in general anaesthesia (neuroleptoanalgesia), act by blocking the dopamine receptor (Fig. 6.1).

6.3.1 The Catecholamine Receptors Two types of catecholamine receptors have been identified, the α-receptors and the β-receptors. The α-receptors have primarily excitatory effects: vasoconstriction, splenoconstriction, bronchoconstriction, contraction of the arrectores pilorum (horripilation), contraction of the uterus, mydriasis, contraction of the sphincters and relaxation of the smooth intestinal musculature, activation of glycogenolysis. The β receptors fundamentally produce bronchodilation, vasodilation, relaxation of the uterus, positive cardiac inotropism with increased heart rate (γ-receptors, specific to glycogenolysis, have also been hypothesised to exist). What is certain is that two subspecies of β have been identified: β1 and β2. Isoprotenerol (Isoprenaline) activates both types, but certain amines, like salbutamol (Ventolin), activate only the β2 receptors, while practolol (Eraldin) selectively blocks the β1 receptors. This is useful in the treatment of bronchial asthma, so long as the administration of salbutamol does not cause tachycardia as a collateral effect; on the other hand, practolol can selectively block the cardiac β1 receptors without inducing bronchoconstriction (Vincenti) [12]. Adrenaline, in contrast with the other catecholamines, does not act synaptically, but rather in the peripheral sympathetic effectors. Adrenaline and noradrenaline, after incretion by the adrenal gland, use a feedback loop to regulate the pressure. It is well known that they cannot cross the blood brain barrier, but it seems that they are able to pass it in the hypothalamus and the brainstem, thus blocking the visceral motor nuclei of these formations. In the end, it is the same mechanism as that of clonidine, the alpha2 agonist normally used as an anti-hypertensive medication. This drug acts by interfering in the visceral motor nucleus cells of the hypothalamus and brainstem, the receptors of which regulate the pressure. The route is: visceral motor nuclei of the hypothalamus and autonomous nuclei of the brainstem → lateral horn of the spinal cord  →  ganglia (where the axons are not interrupted to yield ­synapses)  →  adrenal gland (muscarinic receptor). Hence these nuclei normally ­activate a diastaltic arch which, passing via the paravertebral sympathetic chain,

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regulates the production of catecholamines by the adrenal medulla. Clonidine deceives the brain cells about the level of catecholamines in circulation, so that it appears higher than it really is, thus reducing the signal to the adrenal gland. A similar mechanism is that of α-methyl-dopamine, transformed by the enzyme dopamineβ-hydroxylase, in the same manner as the biosynthesis of the catecholamines, into α-methyl-­noradrenaline, which stimulates the receptors of the visceral motor nuclei and thus inhibits their activity (like clonidine). What is more, α-methyl-noradrenaline is not easily metabolised by MAO and, taking the place of noradrenaline in the presynaptic vesicles, brakes their ingress into them, leaving them in the cytoplasm or in the synapse, where they are easily degraded by MAO and COMT. Once synthesised, stored into the presynaptic vesicles and then released into the synapse, after having completed their action, the catecholamines must be removed from the receptors and recovered in the presynapse for recycling. Two enzymes are involved in this, as we have already mentioned: COMT (catecholamine-­ oxymethyltransferase) and MAO (monoamine oxidase), of which the first is present in the synapse and the second in the presynaptic mitochondria. Dopamine is located primarily in the substantia nigra, the basal ganglia, and also in the amygdala, the nucleus accumbens and the frontal cortex. It acts as a motor neuromediator (extrapyramidal system), as well as a precursor of NA. Noradrenaline, on the other hand, is primarily located in the hypothalamus, the locus ceruleus (43%), the lateral tegmental system (motor nucleus of the vagus, nucleus of the solitary tract, lateral tegmental nucleus) and in the spinal cord. The post-ganglionic sympathetic neurons terminate in the smooth muscles in an unusual way; they present, along their path through the muscle itself, what appear to be swellings, varicosities from which noradrenaline is released, so that multiple muscle fibres are innervated by a single neuron. This very particular way of innervating muscle fibre is called “en passant” (Ganong) [14]. In the periphery, the liberation of catecholamines from the adrenal glands is accomplished, as noted above, by the sympathetic fibres of the splanchnic nerves, which release acetylcholine onto their target. We will see in later chapters the role played by the catecholamines in the pathogenesis of shock (Chap. 8) and in behavioural anomalies (Chap. 10).

6.4

Substance P

Discovered by Uif von Euler and John Gaddum in 1931, who isolated it in the horse brain and rabbit intestine, it is so-called due to the waves of contraction of the intestinal muscles, called P, recorded after its application [15]. It is a member of the family of the tachykinins. Its receptor is called NK1 to distinguish it from the NK2 and NK3 receptors of neurokinins A and B, respectively. These receptors are linked to protein G, and thus have a transmembranal location which is not exclusively synaptic, but also identifiable in the dendrites and the membrane of the cellular body. This not exclusively synaptic location is also valid for the catecholamine receptors of the CNS.

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Substance P is located everywhere throughout the catecholamine stations, in the ganglia of the dopaminergic bases, thus in the globus pallidus, the striatum (caudate, putamen and nucleus accumbens), in the subthalamic nucleus (involved in the hyperkinetic syndromes due to the loss of the inhibitory function of GABA) and in the substantia nigra (mostly also involved in the Parkinson’s disease, where the dopaminergic impulses from the substantia nigra to the striatum are lacking). It should be noted that, while the subthalamic nucleus and the substantia nigra are located in the mesencephalon, they are functionally to be considered basal nuclei. Substance P is also present in the locus ceruleus (noradrenergic neurons), in the limbic system and in the amygdala, hypothalamus, the basal nuclei of the prosencephalon (cholinergic neurons) and in the pontine raphe (serotoninergic neurons). It is also located in the spinal cord where, when a nociceptive signal is received, it activates the respective synapses and can extend its influence into the first two or three lamina. Brown et al. [16] state that, following activation of an inflammatory process, substance P diffuses into the first five lamina. The authors, on eliminating in rats the receptors of substance P from the neurons of the amygdala, found that this made them less anxious in response to specific tests. The only site in which substance P seems not to be present is the cerebellum. When substance P interacts with its receptor NK1, via protein G, it activates phospholipase C, a Ca2+-dependent enzyme, which transforms the phosphatidylinositol into phosphatidylinositol 4-5 biphosphate (IP2) and the latter into phosphatidylinositol 1-4-5 triphosphate (IP3), which with diacylglycerol acts as a signal modulator; the effect is to open the calcium channels, releasing Ca from the mitochondria and the sarcoplasmic reticulum. In the spinal cord, substance P is responsible for facilitating the transmission of nociceptive signals. Subarachnoid injection of capsaicin (an alkaloid extract of chili peppers) results in depletion of substance P, which is correlated with the spinal terminals of the small C fibres. More than a neurotransmitter as such, substance P seems to act as a signal modulator activated by the various neurotransmitters charged with reacting to aggressive factors, from pain to stress in the widest sense. It seems that under psychic stress, as we will see in Chap. 10, the cause can be partly imputed to negative feedback with a diminution of the NK1 receptors consequent on an increased release of substance P. Indeed, the above-mentioned study by Brown et al. shows that the chemical elimination of the receptors of substance P from the amygdala (where receptors equipped with the substance make up for around 10% of the total) has a minimal effect on motility, but significantly reduces the anxiety induced by laboratory tests.

6.5

Glutamate and GABA

How these two neurotransmitters are formed can be seen in the first paragraph of Chap. 4. Glutamate is a monoamino-dicarboxylic amino acid, and as a neurotransmitter it recognizes the same receptor as aspartate: the NMDA (N-methyl-d-aspartate)

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receptor. In the central nervous system, glutamate seems to be one of the most common neurotransmitters, present also in the sympathetic lateral column of the spinal cord, after stimulation of the primary afferent fibres, and in the cerebellum, where it is not stored in vesicles, but in granules. The activation of the NMDA receptor also involves substance P, which induces a slow depolarisation, resolving its blockade by an Mg2+ ion, and activating the flow of Ca2+. An action, concomitant to substance P, is also due to the neuromodulator Calcitonin Gene Related Peptide (CGRP). But we should not view glutamate as only the neurotransmitter of unpleasant functions, we must also note its role in learning and in memory, since it is present in the pyramidal neurons and those of the hippocampus; an alteration of its function seems to be connected to cerebral ageing. The effect of glutamate is primarily excitatory, and opens ionic channels for Ca2+ and Na+. Few drugs are able to usefully interfere with glutamate on the NMDA receptor; one of these is the ketamine an antagonist used in anaesthesia. Memantine, used to slow down the progress of Alzheimer’s disease, is a non-competitive antagonist and occupies a site contiguous with that of glutamate on the NMDA receptor. It seems attenuate the action of glutamate on neurons, preventing the exhaustion of their activity. Astrocytes play an important role in the synaptic metabolism of glutamate, they clean the glutamate out of the synaptic space, transforming it into glutamine with the glutamine synthetase enzyme. The glutamine diffuses through the neuronal membrane and is recycled by the glutaminase enzyme into glutamate; this recycling by the astrocyte accounts for 20% of the glutamate released by the presynaptic vesicles; the rest is recovered directly by the neurotransporters of the neuronal membrane. In contrast with the acetylcholine vesicles, which contain up to 60,000 molecules of neurotransporters, the glutamate vesicles contain no more than 10,000 molecules each. The membrane transporters are closely linked to the efficient production of energy, which keeps the Na/K transmembrane ratio in order. If the cellular battery is not charged, the transporter is not able to do its job. This is what happens in a tissue (cardiac, cerebral) in a state of hypoxia, with consequent loss of the electrochemical gradient, which enables the transporter to function. The continuous presence of glutamate in the synapse simply worsens the ischaemic damage. By the way, it is interesting to talk about glutamate referring a fake story, which fifty years ago became to turn the press: the Chinese Restaurant Syndrome. In 1968 Dr. R.H. Kwok wrote a letter to New England Journal Medicine [17] where referred a syndrome of which he suffered after eating at a Chinese restaurant, especially with a menu of Nord China: he complained headache, hot flashes and asthma; but it was a letter, not a scientific article. Anyway, since then often the press wrote about the syndrome attributing it to an excess of glutamate. In 1970 they come the first scientific denials [18, 19]. But everyone knows that “a lie can travel halfway around the world while truth is putting on its shoes” [20]. Gamma-amino-butyric acid (GABA), derived from glutamate by the action of the enzyme glutamic acid decarboxylase (GAD) and its coenzyme pyridoxal-­5-­ phosphate (vitamin B6), is the principal inhibitory mediator in the central nervous system.

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Two principal types of GABA receptors are known: GABAA and GABAB. GABAA is an ionotropic receptor, belonging to the same category as the receptors for acetylcholine, nicotine, glycine and serotonin (5-hydroxytryptamine or 5-HT) and acts by selectively opening the chlorine channels. This produces a strong post-synaptic electronegativity in the neuronal membrane with an elevated internal/ external potential differential, which raises the excitability threshold. It is antagonised by picrotoxin, a poison extracted from the seeds of Anamirta paniculata, a climbing shrub from Asia Minor, and from bicuculline, a substance extracted from Dicentra cucullaria, a North American member of the family of ranunculaceae. The barbiturates and benzodiazepine act on a subclass of this receptor (GABAA-ρ). The GABAB receptor, on the other hand, is a metabotropic receptor linked to protein G, the action of which consists in inhibiting other excitatory neurotransmitters like substance P and glutamate; it is blocked neither by picrotoxin nor by bicuculline. Baclofen (β-parachlorophenyl-GABA), a synthetic derivate of GABA (Ciba 34.647), is used as a central myorelaxant in the treatment of spasticity. Both GABA and baclofen have an agonist action on an inhibitory protein G (Gi) which inhibits the enzyme adenylcyclase thus reducing the formation of cyclic 3′-5′-AMP.  GABA is stored in the presynaptic vesicles by a specific transporter, as is glutamate. The glia cells participate in its removal from the synapse and its return to the presynaptic terminal. Glutamate, GABA and the conversion enzyme (GAD) have all been located in the cerebral cortex, globus pallidus, mesencephalon (brainstem, substantia nigra), corpora quadrigemina, hippocampus, hypothalamus, in the cerebellum and spinal cord. As already mentioned in Chap. 4, the catabolism of GABA involves its deamination by the enzyme GABA-α-ketoglutarate-transaminase, which transforms it into succinic semialdehyde by transferring the amine group to α-ketoglutaric acid to form glutamate and then GABA once more. Valproic acid (the name is taken from the abbreviation of 2-propyl valeric acid) inhibits the enzyme GABA-α-­ ketoglutarate-transaminase, thus blocking the degradation of GABA, while providing an antiepileptic action. Hence glutamate and GABA, with their opposing actions, work together synergetically to modulate certain signals, such as nociceptive signals. What is the logic behind this mechanism? It’s the old principle—still valid—according to which, if we want to make limited and controlled movements, in this case transmit signals which are effective, but not excessively so, we must use the combination of a driving force and a brake on its action; for instance, they taught us in driving school that in order to invert the direction of travel of a car on a narrow mountain road on the edge of a precipice, we should pull the handbrake and only then use the clutch and accelerator—and with great prudence. In the same way, when we insert a Tuohy needle into the epidural space, we use our left hand on the patient’s back to act as a brake, while the right hand applies the driving force, in very small increments. Indeed, the synapse dominated by the NMDA receptor (glutamatergic) is modulated by glycine, an inhibitor neurotransmitter which acts on an ionotropic receptor for Cl− and which is hence, like GABAA, hyperpolarising. It is present above all in the spinal cord and the mesencephalon; it plays an important role in the spinal cord on the motoneurons activated for action and their subsequent inhibition by the interneurons, which release it, thus preventing prolonged action and a consequent spastic

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contraction. It can thus be seen how strychnine is one of the most powerful antagonists of glycine. On the other hand, the tetanus toxin impedes the release of glycine by the interneurons, thus provoking the notorious symptomatology.

6.6

Serotonin

Serotonin is a neurotransmitter not very widely distributed in the nervous system, it plays a very important role in relation to affect, the wake/sleep cycle and elsewhere. It seems that the leucocytes and macrophages are also affected by its absence; a low level of serotonin depresses them, so that they are less aggressive towards pathogens. It is well known that depressed individuals tend to get sick more easily. Tryptophan is the molecule which initiates the synthesis of serotonin or 5-hydroxytryptamine (5-HT). It is an amino acid, monoamino (-NH2) monocarboxylic (-COOH), and a member of a group of “essential” amino acids that the organism is not able to synthesize. We list them below for completeness and to relieve the reader of the vain attempt to recall them to mind or search for them in some dusty biochemistry textbook (which, to be quite honest, we had to do ourselves in writing this chapter). They are: phenylalanine (as we have seen the initial molecule in the formation of the catecholamines), leucin, isoleucine, histidine, lysin, methionine, threonine, valine and finally tryptophan itself. The l enantiomer of tryptophan constitutes the animal proteins, while the d enantiomer is relatively rare in nature, being present in the poison of certain marine gastropods. Tryptophan is important in the synthesis of vitamin PP (pellagra-­ preventing), NAD (nicotinamide-adenine dinucleotide), melatonin and serotonin. Serotonin is derived from tryptophan, from which it differs by one extra hydroxyl and one less carboxyl (Laborit) [21]. COOH CH2

CH

COOH OH

NH2

N H

CH2

CH

NH2

N H →

tryptophan

5-hydroxytryptophan

tryptophan hydroxylase COOH OH

CH2

CH

OH

NH2

CH2

CH2

N H

N H 5-hydroxytryptophan

→

5-hydroxytryptamine

(5-hydroxytryptophan-decarboxylase)

NH2

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The decarboxylase enzyme is the same one that transforms l-DOPA into dopamine. Serotonin, once it is released in the synapse and has acted as a neurotransmitter, is taken up again by a specific transporter, which returns it to the axonal terminal. Here it is stored in the presynaptic vesicles again or degraded by the monoamino oxidase (in a process of oxidative deamination occurring in the lungs) into 5-hydroxyindoleacetic acid and as such eliminated in the urine. Serotonin is localised in the organism: in the chromaffin cells of the supra-renal gland; in the intestinal wall (around 90% of the total, in Auerbach’s myenteric plexus and the Meissner submucosal plexus), where it increases the motility and secretion in response to vagal stimulation; in the platelets, from which it is released into the serum (hence its name) during the formation of white coagulate; in the mastocytes; in the arteries and veins (contractile action). In the central nervous system it is primarily located in the mesencephalon, the hypothalamus and the nuclei of the raphe of the medulla oblongata. It acts at these levels as an inhibitor, in a manner, which recalls the action of acetylcholine in the Renshaw interneurons on the motor neurons. Its production is at a peak during the day, and minimal at night. After it has been released at the synaptic level, a dedicated transporter effects its reuptake. It is on this reuptake that many anti-­depressants act. On the other hand, lysergic acid diethylamide (LSD), the well-­known hallucinogen, acts also as an inhibitor of serotonin at presynaptic release, so that the synaptic receptors are free and employable without competition.

6.7

Synthesis of Melatonin

We will now look at how serotonin is transformed into melatonin in the epiphysis. OH

CH2 N H

CH2

NH2 CH3

OH

CH2

CH2

N H Serotonin

→

N-acetyl-serotonin

(N-acetyltransferase + Acetyl-CoA)

NH

C

O

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67 CH3

O-CH3

CH2

CH2

NH

C

O

N H

CH3 OH

CH2

CH2

NH

C

O

N H N-acetyl-serotonin

→ Melatonin (N-acetyl-5-methoxytryptamine)

(Hydroxy-indol-oxymethyltransferase + S-adenosyl-methionine)

As you can see, methionine also in this case (like Noradrenaline → Adrenaline) is the methyl donor. Melatonin is produced at night, with a peak around 2 and 4 o’clock, regulated by a suprachiasmatic optic nucleus; its production is interrupted by daylight and substituted by the release of noradrenaline. In older people, there is a diminished secretion of serotonin, which explains the difficulty they experience in falling asleep and their relatively short sleep time. On the other hand, older people tend to depression due to the limited production of serotonin, which, as we have seen, is also the precursor of melatonin. To conclude, if serotonin is similar to tryptophan, with one additional hydroxyl and a one less carboxyl, melatonin is similar to serotonin, with an additional acetyl in the amine group and a methyl on the hydroxyl of the benzene ring.

6.8

Memory

In the conclusion to this chapter, having briefly mentioned one of the functions of glutamate, we will quickly review one of the most important functions of the brain. Memory is the basis of intelligence, and obviously also of artificial intelligence—try imagining a computer without a memory! It should be noted that human intelligence is composed of four basic elements: memory, analytical capacity, synthetic capacity (decision making) and ideation (fantasy). These elements are strongly present in a good chess player. Computers at this time have only the first three, but they are more than sufficient (since they use them to perfection) to beat any human chess player. The human organism, with its sense organs, receives a variety of impulses from its environment (sound, touch, light, pain, heat, etc.) which it memorises by means of protein changes in the synapses. Sometimes a single impulse is not sufficient to create a lasting memory; much depends on the attention and concentration we apply to it, which is why, for instance, we have to read a poem many times to commit it to memory. An impulse, which we receive many times in our lives is the encounter of our immune system with a microorganism; in this case the protein molecules,

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modified and synthesised specifically, in response to the microorganism in question by the immune system, are the antibodies. Memory, like every human function, must be exercised; remember the principle of Lamarck? “Use creates the organ, disuse atrophies it”. Thus, to develop our mnemonic capacity, we must exercise it from an early age. In the same way, when the immune system encounters a given microbial agent or its toxin after vaccination, it has a memory of it and can react more effectively. The tetanus vaccine is the classic demonstration, in which a vaccinated individual may experience the illness anyway (depending on the virulence of the type of tetanus), but in an attenuated form. In the first days of military service, all recruits were given the TABTE vaccination (typhus, paratyphoid A, paratyphoid B, tetanus). If the soldier remained in the military permanently, the vaccination was repeated every two years, thus exercising the memory of the immune system. Unfortunately, with the abolition of obligatory military service, a large part of the population has been deprived of this important defensive function. The few lucky ones are professional soldiers who enrol of their own accord. Furthermore, in recent decades, a very dangerous school of thought has arisen, convincing young parents to deny the utility of infantile vaccination, thus exposing their children to the risk of serious illness. But let’s get back to memory. This fundamental function (on which spatial orientation also depends) is located in the hippocampus, an anatomic structure in the medial temporal lobe, i.e. the part of the temporal lobe, which is folded medially at the bottom. The hippocampus itself is part of the limbic system. This complex system is charged with processing emotion, survival reactions (including neurovegetative) and memory, three functions which are only apparently distinct but which are on the contrary closely interlinked with each other. The limbic system, anatomically located in the medial part of the encephalon, includes: the hippocampus, the parahippocampal gyrus, the amygdala, the thalamus, the hypothalamus and the basal nuclei. The hippocampus consists of three nuclei: the dentate gyrus, the horn of Ammon and the entorhinal cortex, the latter of which provides the connection with the cerebral cortex. It is in this connection, of which the Shaffer collateral pathway1 is part, that the plastic modifications, establishing long-term memory, are made. In particular, the NMDA receptor and glutamate are protagonists on the Shaffer collateral pathway—horn of Ammon connection, although their activation involves numerous membrane proteins. At the postsynaptic level, along with the ionotropic NMDA receptor for Ca2+, there is also the AMPA (amino methyl propionic acid) receptor, also ionotropic, for Na+ and K+, which also recognises glutamate as a neurotransmitter. Following the ingress of calcium, due to the stimulus of glutamate on the NMDA and AMPA receptors, a long-lasting excitatory postsynaptic current is established, which initiates the long-term memory mechanism. The biochemical signals also induce a modification of the genome in the nucleus and prolong the neuronal plasticity resulting on the signal over time. 1  The collateral pathway of the hippocampus, which connects the CA3 region to the CA1 region, whose neurons connect with the neurons of the cortex via yet another intermediate centre, the subiculum.

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References 1. Vincenti E.  In: Galzigna L, Vincenti E, editors. Lezioni di biochimica applicata. Padova: Cortina Editore; 1981. p. 29. 2. Abelson KSP, Höglund AU. Intravenously administered oxotremorine and atropine, in doses known to affect pain threshold, affect the intraspinal release of acetylcholine in rats. Pharmacol Toxicol. 2002;90:187–92. 3. Manzoni A. The Bettrothed. Middlesex: Penguin Books (Classics); 1972. p. 256. 4. Vassalle M. Analysis of cardiac pacemaker potential using a “voltage clamp” technique. Am J Physiol. 1966;210:1335–41. 5. DiFrancesco D, Tortora P.  Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. Nature. 1991;351:145–7. 6. DiFrancesco D.  A new interpretation of the pace-maker current in calf Purkinje fibres. J Physiol. 1981;314:359–76. 7. DiFrancesco D. A study of the ionic nature of the pace-maker current in calf Purkinje fibres. J Physiol. 1981;314:377–93. 8. DiFrancesco D. Funny channels in the control of cardiac rhythm and mode of action of selective blockers. Pharmacol Res. 2006;53:399–406. 9. Brogioni S, Cerbai E, Mugelli A. Canali If nell’ attività di pacemaker del nodo seno-atriale. G Ital Cardiol. 2006;7(7. Suppl. 1):20S–8S. 10. Zaza A, Rocchetti M, DiFrancesco D. Modulation of hyperpolarization activated current (If) by adenosine in rabbit sino-atrial myocytes. Circulation. 1996;94:734–41. 11. Marzotti A, Alemanno F, et al. Valutazione di alcuni parametri bioumorali rilevati su sangue e linfa di animali sottoposti a shock sperimentale in condizioni diverse di anestesia. Acta Anaesth It. 1976;XXVII(7):199–210. 12. Vincenti E. In: Galzigna L, Vincenti E, editors. Lezioni di biochimica applicata: 25. Padova: Cortina Editore; 1981. 13. Alemanno F. Clonidine mechanism of action. In: Alemanno F, Bosco M, Barbati A, editors. Anesthesia of the upper limb. Milan: Springer Verlag; 2014. p. 254–5. 14. Ganong WF. Fisiologia medica. Padova: Piccin Editore; 1973. 15. Von Euler US, Gaddum JH. An unidentified depressor substance in certain tissue extracts. J Physiol. 1931;72(6):74–87. 16. Brown JL, Liu H, Maggio JE, et al. Morphological characterization of substance P receptor-­ immunoreactive neurons in the rat spinal cord and trigeminal nucleus caudalis. J Comp Neurol. 1995;356:327–44. 17. Kwok RH. Chinese restaurant syndrome (letter). New Engl J Med. 1968;278:796. 18. Maerselli PL, Garattini S. Monosodium glutamate and Chinese restaurant syndrome. Nature. 1970;227:611–2. 19. Freeman MJ.  Reconsidering the effects of monosodium glutamate: a literature review. Am Acad Nurse Pract. 2006;18(10):482–6. 20. Ayres A. The Wit & Wisdom of Mark Twain. New York: Harper & Row Edit; 1987. p. 139. 21. Laborit H. Les Régulations metaboliques. Paris: Masson & Cie Editeurs; 1965.

Further Reading Aiazzi Mancini M, Donatelli L. Trattato di farmacologia. Milano: Vallardi Editore; 1969. Barbato C, Canu N. www.treccani.it/enciclopedia/neurotrasmettitori. 2010. Bonhoeffer T, Yuste R.  Spine motility. Phenomenology, mechanisms, and function. Neuron. 2002;35(6):1019–27. D’Anna G.  La neurotrasmissione glutammatergica—INBIOCHEM—inside Biomolecular Chemistry Genn. 2012.

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Devlin TM. Biochimica. Napoli: EDISES; 2012. Dong Y, Green T, Saal D, et al. CREB modulates excitability of nucleus accumbens neurons. Nat Neurosci. 2006;9(4):475–7. Garret RH, Grisham CM. Biochimica. Padova: Piccin Editore; 2014. Gasparini L. L’ossido di azoto. www.informasalus.it/it/articoli/ossido-azoto.php http://it.wikipedia.org/wiki/Neurotrasmettitore Ialenti A. trasmissione-colinergica. www.federica.unina.it/farmacia/farmacologia it.wikipedia.org/wiki/Proteina G it.wikipedia.org/wiki/Recettori_accoppiati_a_proteine_G it.wikipedia.org/wiki/Secondo_messaggero Jorgensen EM. GABA. Worm Book Editor. 2005. http://www.wormbook.org Kim SJ, Linden DJ. Ubiquitous plasticity and memory storage. Neuron. 2007;56(4):582–92. Lynch G.  Memory enhancement. The search for mechanism-based drugs. Nat Neurosci. 2002;5(Suppl):1035–8. Malenka RC, Bear MF. LTP and LTD. An embarrassment of riches. Neuron. 2004;44(1):5–21. Marie H, Morishita W, Yu X, et al. Generation of silent synapses by acute in vivo expression of CaMKIV and CREB. Neuron. 2005;45(5):741–52. Marie H. www.treccani.it/.../meccanismi-molecolari-della-memoria Metildopa. https://it.wikipedia.org/wiki/Metildopa Mora F, Segovia G, del Arco A. Aging, plasticity and environmental enrichment. Structural changes and neurotransmitter dynamics in several areas of the brain. Brain Res Rev. 2007;55(1):78–88. Nelson DL, Cox MM.  Introduzione alla biochimica di Lehninger. Bologna: Zanichelli Editore; 2015. Nelson DL, Cox MM. Lehninger principles of biochemistry. 7th edn. USA:Freman and Company; 2017. Rittà E.  I Neurotrasmettitori: glutammato, GABA, dopamina, serotonina, melatonina. http:// mente-attiva.blogspot.it/2010/11/i-neurotrasmettitori-glutammato-gaba.html

7

Blood–Brain Barrier Fernando Alemanno

In the first year of specialization school in anaesthesia and intensive care, one question we often asked ourselves was why thiopental sodium worked on the encephalon, while curare did not, since with all those acetylcholine synapses, by using a single anaesthetic we felt we might possibly hold the solution to every problem of central and peripheral functional blocks. Then it was explained to us that generally muscarinic synapses differed from nicotinic ones in that it was more a matter of blood brain barrier (BBB), where some molecules managed to pass through, and others, like curare, did not. At that moment, it seemed a satisfactory answer. Often there was not enough time to pursue the case further, because this question and answer took place within the short time between the insertion of a Guedel cannula and orotracheal intubation. There was all the anaesthesia to set up and monitor, and in the mere blink of an eye, the tutor had already gone on to an adjoining room to check on the next specializing junior doctor. Over a distance of time, this strange barrier stuck in your mind, a kind of Great China wall, clearly protective, whose outlines, doors and mechanisms were somewhat difficult to pin down. In 1882, Ehrlich [1] noticed that on injecting trypan blue, a vital dyestuff derived from aniline, intravenously, it stained all body tissues with the exception of the encephalon, which could, however, be dyed, by injecting trypan blue into the cerebrospinal fluid. The explanation for this phenomenon lies in the fact that trypan blue when injected in such a way binds to albumin to become a complex with too high molecular weight to cross through the barrier, which is effectively impermeable to molecules above 2000 Dalton (1 Dalton corresponds to a part one-twelfth of the mass of a carbon atom). One of the causes for the troublesome passage of the various substances is the compactness of the endothelial cells in the cerebral blood capillaries. But that may well be an oversimplification. Various types of transport

F. Alemanno (*) Anaesthesia and Intensive Care, Brescia Clinical Institutes, Brescia, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 F. Alemanno (ed.), Biochemistry for Anesthesiologists and Intensivists, https://doi.org/10.1007/978-3-030-26721-6_7

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exist other than diffusion (typical of water, ethanol, oxygen, carbon dioxide, anaesthetic gases and urea) like pinocytosis for large molecules or the use of carriers, and also energy-consuming active transport. The most diffusible molecule of all is ethanol. Some amino acids such as tryptophan, phenylalanine and tyrosine pass through, while others like GABA and glutamate are not quite so lucky. Dopamine also fails, instead its precursor l-DOPA gets through the barrier. But the splitting of the molecule is what largely matters here; hence the associated fraction enters more easily, while the dissociated part does not, with salicylates only passing in their non-dissociated form. The endothelium of capillaries, that irrigate the brain is continuous and lacks the fenestrations present in the rest of the body. To the contrary, junctions between the endothelial cells, known as occluding cell joints or tight junctions, are, as the name suggests, very narrow. A further reinforcement to this already solid barrier is provided by the astroglia cells, which with their projections, or glial limitans, surround the endothelial cells. Generally the blood–brain barrier is impervious to hydrophilic substances that are blocked both by endothelial cells and by astrocytes. It is equally also impervious to hormones and substances of high molecular weight (>2000 Dalton). One example is noradrenaline, secreted by the adrenal glands, whose blood level is controlled by cells of the vasomotor centre in the central nervous system. This is the site where the blood–brain barrier possesses a window for noradrenaline to act upon the nerve receptors and where in the case of a drop in blood levels, normally activates a diastaltic arc which, via the paravertebral sympathetic chain, regulates the production of catecholamines by the adrenal medulla. A site that may also be exploited for therapeutic purposes. To illustrate this point, clonidine tricks the brain cells into overestimating the level of catecholamines in circulation above those they really are, and so induces a reduced signal to the adrenal gland [2]. Another relatively permeable spot in the barrier is to be found at the level of the “chemoreceptors trigger zone”, where this may fortunately be overcome by the use of antiemetic drugs. Apart from these two particular points, limits in the barrier’s impenetrability surface appear when, in the case of infections, antibodies or protein molecules of a certain size, fail to pass. Nonetheless in the case of infection the permeability of the barrier may modify itself to effectively promote the diffusion of molecules of various kinds, like antibiotics. According to Laborit [3], the blood–brain barrier’s basic constituent is the neuroglia, which has to be seen in a way, remotely dissimilar to any run-of-the-mill supportive connective tissue. Many of the biological and pharmacological substances present and circulating in the blood are unable to reach the neuron on account of not having crossed the neuroglia, which interposes itself between it and the capillary. Each neuron has a capillary assigned to it, and so with 100 billion neurons present in the brain of man, it follows there are equally 100 billion capillaries, whose total combined length adds up to around 600 km and which serve a surface of roughly 20 m2 [4]. In addition, the neuroglial part of the barrier is separated from the endothelium’s basal membrane by a thin layer of collagen, thus further reinforcing the barrier’s structure [5].

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Many molecules including O2, CO2, glucose, vitamins, nucleosides and liposoluble drugs generally pass the barrier via a simple diffusion mechanism or active transport (Fig. 7.1). Transport carried out by “carrier” proteins is known by the term CMT (carrier-­ mediated transport) and comes about in a centripetal mode. Another type of active transport occurs centrifugally and serves to eliminate products resulting from cerebral catabolism; this kind of transport, from the brain to capillary lumen, is known by the acronym AET (Active Efflux transport). CMT and AET carry molecules of LIPOPHILIC MOLECULES PASSIVE DIFFUSION ABC Pgp BCRP

IDROPHILIC MOLECULES TRANSCYTOSIS MONONUCLEARCELLS CARRIERS MIGRATION (SLC) AMT RMT TJ Rc

+ve

MRP1-5*

MRP1-5*

Non polar Non polar RMT conjugates Transferrin Melanotransferrin Lipoproteins B-Amyloid Glycosylated proteins IgG Insuline Leptin TNFα EGF

AMT cationic Albumin Histones Avidin TAT SynB1 (peptides penetrating the cell)

Glucose Aminoacids Nucleosides Monocarboxylates Small peptides FFAs Organic Anions Organic Cations

Fig. 7.1  The way to pass through the blood–brain barrier. By Passive Diffusion: Solutes with enough lipid solubility may enter passively through the cellular membranes of the endothelial cells and thus pass into the brain. Active efflux transporters (ABC conveyors): Are able to capture and pump out of the cell a wide range of solutes that passively penetrate through it. Trans-cytosis: mediated by RMT (receptor-mediated transport) or AMT (active-mediated transport). In RMT transcytosis processes a ligand binds to a membrane receptor cell to trigger an endocytic event that transports macromolecules through the endothelium. In AMT processes a cationic solute containing a number of positive charges similarly induces transcytosis, directly. Leucocytes: cross the BBB of endothelial cells through a diapedesis process and a previous interaction phase with the superficial cellular adhesion molecules, followed by migration. Carrier-mediated transport: This consists of the transportation of polar molecules by inertia vectors in the luminal and abluminal membrane. These may be two-way vectors that operate in the direction of the concentration gradient, unidirectional inside or outside the cell (2/3) or exchangers/co-conveyors for the exchange or shared transport of other solutes or ions travelling in the same or opposite direction (Begley et al., 2008, Modified) [6] (From: Maurizio Scarpa, Cinzia Maria Bellettato, Rosella Tomanin, Alessandra Zanetti: Barriera Emato-Encefalica e terapie farmacologiche. Prospettive in Pediatria 2012; Vol 42; N 167: 176-184 [7]. Pacini Editore, courtesy, (modified caption))

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reduced size. Both an interesting research article by Jungner et al. [8] and a previous editorial by Stocchetti and Magnoni [9] verify the permeability of BEE to a traumatic injury. The authors noted that in the first few days following the trauma, changes in BEE permeability represents above all a local rather than general phenomenon. They thus conclude that the development of oedema may be influenced by hydrostatic and osmotic pressure of the capillaries.

References 1. Saunders NR, Dreifuss JJ, Dziegielewska KM, et al. The rights and wrong of blood-brain barrier permeability studies: a walk through 100 years of history. Front Neurosci. 2014;8:1–26. 2. Alemanno F. Clonidine mechanism of action. In: Alemanno F, Bosco M, Barbati A, editors. Anesthesia of the upper limb. Milan: Springer; 2014. p. 254–5. 3. Laborit H.  Barrière hémato-encéphalique et neuroglia. In: Les régulations Métaboliques. Paris: Masson & Cie; 1965. p. 143–8. 4. Nag S, Begley DJ. Blood-brain barrier, exchange of metabolities and gases. In: Kalimo H, editor. Pathology and genetics. Cerebrovascular diseases. Basel: ISN Neuropath Press; 2005. 5. Abbot NJ, et al. Structure and function of the blood-brain barrier. Neurobiol Dis. 2010;37:13–25. 6. Begley DJ, Pontikis CC, Scarpa M. Lysosomal storage diseases and blood-brain barrier. Curr Pharm Des. 2008;14:1566–80. 7. Scarpa M, Bellettato CM, Tomanin R, Zanetti A. Barriera Emato-Encefalica e terapie farmacologiche. Prospettive in Pediatria. 2012;42(167):176–84. 8. Jungner M, Siemund R, Venturoli D, Reinstrup P, Shalén W, Bentzer P. Blook-brain barrier permeability following traumatic brain injury. Minerva Anestesiol. 2014;82(5):525–33. 9. Stocchetti N, Magnoni S. Blood brain as a target for traumatic brain injury therapy. Minerva Anestesiol. 2016;82(5):499–500.

Further Reading Barriera ematoencefalica. http://www.mypersonaltrainer.it/farmacologia/barrieraematoencefalica-16.html Begley DJ, Kahn EU, Rollinson C, Abbott NJ, Regina A, Roux F. The role of extracellular brain fluid production and efflux mechanism in drug transport to the brain. In: Begley DJ, Brandburi MW, Kreuter J, editors. The blood-brain barrier and drug delivery to the CNS.  New  York: Marcel Dekker; 2000. Begley DJ. Delivery of therapeutic agents to the central nervous system: the problems and the possibilities. Pharmacol Ther. 2004;104:29–45. Spatz H.  Die Bedeutung der vitalen färbung für die Lehre vom Stoffaustausch zwischen dem Zentralnervensystem und dem übrigen Körper. Arch Psychiatr Nervenkr. 1934;101(1):267–358.

8

Shock Mario Bosco and Fernando Alemanno

The term itself, which indicates a blow aggression or intense disequilibrium of the entire organism, is considered by Rushmer and Coll. more “a semantic enigma” than a well-defined concept. Col. Med. A. Amato, School of Military Medicine, Florence 1967

8.1

The Origins

8.1.1 Pathogenesis Hans Selye (Vienna 1907—Montreal 1982) described the General Adaptation Syndrome in 1936 [1], when he was working as a researcher at McGill University, Montreal. Selye himself remarked, when talking about “stress”, that “everyone knows what the word means, but no-one knows what it actually is”. The syndrome, due to a repetitive aggression, which is not solely physical, but also or even exclusively psychosocial, has three classic phases: 1. Alarm In this first phase, the vegetative nervous system (VNS) responds to the aggression, immediately followed by the activation of the ­hypothalamic-­pituitary-­adrenal axis, which releases catecholamines into circulation to promote fight or flight.

M. Bosco (*) UOC Anaesthesia and Resuscitation, S. Spirito and Ophthalmic Hospitals, Rome, Italy F. Alemanno Anaesthesia and Intensive Care, Brescia Clinical Institutes, Brescia, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 F. Alemanno (ed.), Biochemistry for Anesthesiologists and Intensivists, https://doi.org/10.1007/978-3-030-26721-6_8

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2. Resistance If the aggression persists, the organism continues to react, which leads to collateral effects such as adrenal hypertrophy, or somatic effects like gastric and intestinal ulcers. 3. Exhaustion When the intensity or duration of the aggression increases, the organism “exhausts its defensive capacities”, resulting in irreversible damage and even death. This latter phase is the point (as we will see) that led to the equivocation on which therapy for shock was based at that time. Unfortunately, to follow the development of the theory of the etiopathogenetic mechanism of shock means we must review the events of the first and second world wars, events, which had much to teach us about the treatment of this grave pathology. Shock is very common in war: traumatic, haemorrhagic, psychic, septic, it presents in a variety of combinations, even if not all are always present at the same time. Alfred Blalock (1899–1964), surgeon, became famous for having discovered a remedy to the tetralogy of Fallot, by means of anastomosis of the subclavian artery with the pulmonary artery, thus increasing the flow in the congenitally restricted artery. Blalock, together with Harrison (the author of a treatise of internal medicine which is still used today), ran studies of cardiac output in a variety of pathological conditions (haemorrhage, anaemia, anoxia, thyroid disease and general anaesthesia). In the years after the First World War, Blalock published his observations, defining shock as “a syndrome due to a disproportion between the circulatory bed and the mass of blood circulating in it” [2]. The definition was coherent with the various types of shock [3, 4], able to adapt, on a case-by-case basis, with one or another of its components, to vasogenic shock (due to the increased capacity of the vascular bed), haemorrhagic shock (reduced volume of blood in circulation) [5] and even cardiogenic shock. In the latter case, the disproportion is due to a relative, not absolute, diminution of the circulating volume, which, due to insufficiency of the cardiac pump, is disproportionately elevated in the venous section. This latter possibility coincides with what treatises of cardiology and internal medicine call cardiac failure. In the meantime, however, in 1923, Walter Cannon (1871–1945), a physiologist at Harvard University (Cambridge, Massachusetts), published the book Traumatic Shock [6]. Canon had experience of war and attributed the triggering of the syndrome to the release into circulation of endogenous toxins. He then demonstrated experimentally [7, 8] that the syndrome was triggered after reperfusion of the limb in which circulation had been interrupted (tourniquet shock), a phenomenon which did not occur if the limb was amputated. Among other things, we owe to Cannon the term “homeostasis”, a word first used in his popular book The Wisdom of the Body [9], published in 1932. The theory of bacterial etiopathogenesis was thus originated by this author, and is based on the hypothesis that splanchnic vasoconstriction, followed by hypoxia of the enterocolic mucous membranes, even as far as necrosis, damages the blood intestine barrier and releases bacterial endotoxins into circulation, which aggravate the tissue anoxia and result in irreversible shock. Thus,

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according to this theory, the irreversibility of any type of shock is imputable to a septic state. The bacterial endotoxins of Gram-negative germs (particularly E. coli) can induce an irreversible shock. This is why for major surgeries, it is important to treat patients in a state of shock (or at risk thereof) with antibiotics. Another theory, which imputes shock to a failure of the defensive effectiveness of the reticuloendothelial system, seems more a consequence than a cause, or at most a concomitant cause to hypoxia, which is co-responsible for irreversibility. To conclude, the concept that the adrenergic reaction is at the basis of the mechanism, which triggers the syndrome, is supported by an unpublished clinical observation by Professor Giorgio Massera, primary emeritus of the Anesthesia and Intensive Care Department of Treviso Regional Hospital (the first independent anaesthesia department in Italy, 1956): “Following a serious bleeding, a young subject is more exposed to the syndrome of shock than an older one; the latter, a poor producer of catecholamine (a potential Parkinson’s patient) has a moderate tendency to vasoconstriction; he might die of exemia, because the haematic content of the circulation is exhausted, but in comparison with the young subject he is less likely to suffer from shock itself.” Immediately after the Second World War, Henri Laborit (Hanoi 1914—Paris 1995), doctor of the French navy, served as a surgeon at the overseas military hospital of Sidi-Abdallah, near Biserta, Tunisia. In that area, with its many minefields, many patients were admitted to the military hospital in a state of shock following accidents or as a consequence of demining operations. The sample was homogeneous: the patients, all men aged from 20 to 50, had been wounded by anti-­personnel mines of a constant explosive charge. Every type of shock, pain, haemorrhage, fear and sepsis, contributed to their condition. Laborit felt that the problem consisted in the vasoconstriction of large regions like the splanchnic area (as a surgeon, he confirmed this with every exploratory or surgical laparotomy), as well as the skin and the extremities. Severe vasoconstriction means hypoxia and, when prolonged, acidosis resulting in desensitisation of the peripheral receptors to the action of the catecholamines, and the possibility of passing from reversible to irreversible shock. In this situation, continuous infusions of fluids and blood had little effect, nor did the administration of sympathomimetics. “At the time, we believed that death was caused by an “exhaustion of the defensive systems“, even when haemostasis and surgical treatment of the wound had been handled correctly. For this reason, once the volume of blood had been restored by transfusion, if the arterial pressure remained low, we attempted to raise it by using drugs to induce vasoconstriction, thus reducing the capacity of the circulatory system. The results were disappointing…” [10]. This therapy was certainly effective in some cases of cardio-circulatory collapse, but not in cases of shock, where vasoconstriction centralises the circulation at the cost of the periphery, thus causing progressive anoxic damage. This semantic confusion between collapse and shock was not without its negative therapeutic consequences! “Collapse is a symptom, shock is a syndrome… The therapeutic error, supported by the semantic confusion, was that of applying a hypertensive or transfusional symptomatic therapy to processes which respond favourably only to pathogenic therapy, which is not necessarily the same” [10].

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In the case of shock it is important, once the volume of blood has been restored, to resolve the peripheral spasm of the arterioles and meta-arterioles so as to get the volume of blood, trapped in the vasoconstricted regions, into movement and restore adequate oxygenation of organs like the liver, kidneys and intestine, i.e. the splanchnic area in general. This, by oxygenating the tissues and organs, buffers a worsening acidosis which otherwise would lead not so much to the exhaustion of the defensive systems but rather to the desensitisation of the peripheral adrenergic receptors to the action of the circulating catecholamines, passing from reversible to irreversible shock. Indeed, in shock left to its own devices, the so-called “circle of death” sets in, a term which is not exactly scientific but which is nonetheless appropriate (Fig. 8.1). To interrupt this vicious circle, Laborit invented the “lytic cocktail”, composed of: chloropromazine (Largactil) as an antiadrenergic, promethazine (Fargan) as an anti-exudative and meperidine (Dolantin) as an analgesic [11–13]. This cocktail was naturally administered together with a suitable volume of infusion fluid. The recognition of the importance of the vegetative nervous system (VNS) in the genesis of the phenomena of post-aggressive response was an important step towards understanding the pathogenesis of shock. One of the fundamental Traumatic shock Arterial pressure Venous return

Sympathetic reaction + Vasoconstriction adrenergic respone (aggravated by pain)

Hypovolemia Haemorrhagic shock

Capillary permeability (Edema) Capillary lesions Release of chemical mediators (protease, hydrolase) Septic shock

Adrenergic shock Adrenaline 16 mcg/kg/min i.v. (2 hours)

Peripheral anoxia

Capillary atony + tissue acidosis

Desensitisation of the catecholamine receptors Release of vasoactive substances (cytokines, kinins, histamines, free radicals of oxygen) Anaphylactic shock

Fig. 8.1  The diagram shows a series of reactions, initially paraphysiological, but which may become pathological, and which occur following a damaging event of a certain severity, which either itself persists, or the consequences of which persist. This series of reactions, with a lack of scientific-sounding term, but easy mnemonic content, is called the “circle of death”. The circle can start at various levels, depending on whether the shock is haemorrhagic (10 h), traumatic (12 h), septic (7 h), anaphylactic (8 h), or due to excessive administration of catecholamine (2 h) as in experimental shock (Lillehei et al.) [34]

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characteristics of the excitability of the neurovegetative centres is their sensitivity to the intensity, rather than the nature, of a stimulus, its frequency and duration. The autonomous nervous system opposes traumatic aggression by initiating diverse defence mechanisms depending on the cause of the damaging event, but always, initially, aimed at maintaining the humoral and metabolic constants required for homeostasis, i.e. the balance of oxygenation and nutrition of the parenchymas in general, and of the heart and brain in particular. Given an attack or damaging stimulus of any kind, the sympathetic nervous system responds immediately, followed directly by the hypothalamic-pituitary-adrenal humoral system, with vasoconstriction and increased heart rate. The adrenal glands produce adrenaline to prolong the duration of the nervous reaction and activate the metabolic response (activation of glycogenolysis with increased supply of nutrition to face the aggression with flight or flight). Hence, the VNS responds to aggression with two types of reaction [14]: –– An immediate, non-specific reaction, with the same nervous and hormonal characteristics no matter what the aggression, although the intensity of the reaction may not be proportional to the intensity of the stimulus (young people) and may be an excessive response. This reaction, if prolonged over time, may not always be good for the maintenance of homeostasis. This demonstrates that the VNS does not have definite plans for every battle. –– A mediated, specific reaction, following the initial reaction, which aims to directly resolve the damage caused by the aggression (repair injury, activate haematopoiesis, etc.). Thus, an unexpected loss of blood, violent pain, large burn, all immediately trigger a response of the nervous and hormonal systems to maintain sufficient supply of blood to the centres. Only later do they initiate processes aimed at repairing the organic damage—if at all possible. But if the violence of the aggression (haemorrhagic, traumatic, toxic, burn) lasts over time, the immediate, non-specific response of the VNS exhausts its effectiveness after a variable amount of time, following the setting in of bio-humoral alterations in the periphery, and especially in the splanchnic region. The persistence of the non-specific reaction to surgery was identified with what the old surgeons and anaesthetists called “post-operative illness”, the final crisis of which could sometimes coincide with shock. It is interesting at this point to take a look at how this post-operative illness, frequent in surgical wards up to the 50s, presented. The symptoms of the post-­operative illness showed up a parasympathetic inhibition and a more or less evident and long-­ lasting prevalence of the sympathetic system depending on the intensity and duration of the abnormal, dolorous and visceral stimuli, starting at the site of the surgery itself. This was the immediate reaction which, persisting over time, maintained the syndrome. This was followed, after a variable period of time, by the weakening of the aggressive phenomena: the heart rate normalised, the pallor disappeared, the temperature fell, and intestinal and vesicular function was restored. If instead of

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attenuating the situation persisted, the following phenomena could be observed: the heart rate tended to increase, cold sweating and cyanosis appeared, the temperature might fall even below normal values, respiration was superficial and frequent, intestinal transport and vesicular contraction ceased (even after having returned temporarily), and arterial pressure dropped, sometimes drastically, to the point of no longer being measurable. This was the condition of secondary shock, which could lead to the death of the patient. Barbiturate and ether narcosis were unable, unless at dangerously high doses, to inhibit the vegetative centres, as was possible, on the other hand, for the communication centres. It is now time to return to therapy for shock (note the indivisible parallelism between anaesthesia and intensive care). We noted that infusion therapy must be accompanied with blockage of the VNS with the lytic cocktail. This was a completely new therapeutic concept, also applicable as a prophylactic measure, which considered it indispensable to correct the peripheral circulatory imbalances, understood as the post-aggression vasomotor reaction, by means of pharmacological ­neurovegetative block.

8.2

The Lytic Cocktail

Proposed by Henri Laborit and Pierre Huguenard (1924–2006, director of the Henri-Mondor Hospital, Paris), it is composed of chlorpromazine (Largactil) 50 mg, promethazine (Fargan) 50  mg, and meperidine (Dolantin) 100  mg [12, 13]. “Chlorpromazine and prometazine are anti-adrenaline-depositor drugs. Chlorpromazine in particular inhibits the deposition of catecholamine and is also able to block the action of the monoamine-oxidase inhibitors (IMAO). Its action at the cerebral level is proportional to the hypothermia induced by this drug; on the other hand, its action attenuates and disappears when the ambient temperature is raised and body temperature is restored to 37°C. The explanation for this is to be found in the fact that the liberation and synaptic fixation of catecholamines at the cerebral level is an active process which is interfered with by the hypothermalizing action of chlorpromazine” (Aiazzi Mancini—Donatelli) [15]. Phenothiazine in general also partially inhibits the cytochrome oxidase system and consequently the ATPase system; thus, by decoupling oxidative phosphorylation, it acts like 2–4 dinitrophenol (a paradigmatic experimental decoupler), which would explain the pathogenesis of the malignant neuroleptic syndrome. Chlorpromazine, under the name of “4560 RP”, was synthesised at Laborit’s suggestion by chemists Paul Charpantier and Nicole Courvoisier in the Rhône-Poulene-­ Spécia laboratories. The action of chlorpromazine on the cardiovascular apparatus is due to its adrenolytic effect, with an increase in the peripheral flow: the heart rate increases although output may be unchanged, while peripheral vasodilation, with reduction in the total peripheral resistance, provokes a drop in pressure, which may be marked if there is not an adequate infusion of fluids. Promethazine (Fargan), which has an anti-histaminic and hence anti-exudative action, not only potentiates the central effects of chlorpromazine but also acts by

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opening the precapillary sphincters and, diminishing the capillary hyper-­ permeability, collaborates effectively to re-establish peripheral perfusion and restore normal micro-circulatory flow. Meperidine or pethidine (Dolantin) is a centrally acting analgesic, a synthetic derivative of morphine; its 4-phenyl piperidine structure is curiously similar, chemically and pharmacologically, to that of methylphenidate (Ritalin), which is a neurostimulant (they both have the effect of suppressing shivering). Meperidine, while less potent and with a shorter half-life than morphine, blocks incoming pain stimuli in the spinal cord and thalamus, and has the function of attenuating the sympathetic response (vasoconstriction) to pain. These drugs should not be used without contemporary infusion or transfusion, because the sudden increase of the vascular bed in relation to the exiguous amount of blood in circulation can provoke a fatal collapse due to reduced oxygenation of the centres which, even with the volume of blood greatly reduced by vasoconstriction, still receive a relatively sufficient supply of nutrients and oxygen. It was a short step from using the lytic cocktail in the treatment of shock to using it in the operating theatre to apply so-called “potentiated anaesthesia”. In 1951, Henri Laborit published L’Anesthesie facilitée par le synergies medicamentoses, Masson et Cie (no longer available) [11]. French anaesthetists immediately embraced the concept and thus Huguenard himself, Boissier, Viars and other illustrious French anaesthetists made it their own and further investigated the concept, quickly followed by their Italian colleagues: Carlo Carlon [14] (later a renowned surgeon, first at Udine Hospital and later at Padua) and Enrico Ciocatto [16], Professor of Anaesthesia and Intensive care at the University of Turin. In the light of these new approaches, it was evident that anaesthetic methods, considered as prophylaxis against both surgical shock and the post-operative illness, were in need of improvement. The drugs previously used for anaesthesia interfered with communicatory activity, but provided little protection as analgesics or to the VNS.  The drugs in the lytic cocktail started to make their appearance in pre-­ anaesthetic prescriptions (Fargan) and intravenous infusion (Largactil), while diluted meperidine was injected intravenously, in small doses, during the most painful moments of surgery. Potentiated anaesthesia made it possible to implement plans of deeper anaesthesia, thus preventing the post-operative syndrome, which was a creeping state of shock, which compromised the post-operative period of many operations, even technically successful ones. The lytic cocktail reinforced analgesia and the protection of the organs against surgical aggression, while inhalatory anaesthesia (N2O, ether or trichloroethylene at that time) was used to ensure sleep. This created so the experimental chemical and pharmacological conditions, which in 1959 led two Belgian anaesthetists, De Castro and Mundeleer, and the chemist Jahnsen, to replace the drugs, in the lytic cocktail, with their long-lasting and hence inflexible effect, with more flexible drugs, better suited to anaesthesia: the two phenothiazines (chlorpromazine and promethazine) were replaced by a butyrophenone, haloperidol (Serenase), while meperidine was replaced by the more potent phenoperidine [17, 18]. This was the birth of neuroleptanalgesia, today known worldwide, although not

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many know that it originated in an idea conceived just after the war in a French Navy hospital in Tunisia. This first form of neuroleptoanalgesia (NLA type 1) was primarily neuroleptic; the patient was most left in spontaneous or assisted respiration, and sleep was induced with barbiturate and maintained with nitrous oxide. Given the potency of the combination haloperidol + phenoperidine, it was not always necessary to also administer, even at very low concentrations, ether, trichloroethylene or halothane, which were just starting to make their appearance at the time. However, phenoperidine soon demonstrated a certain hepatic toxicity and within 2 years, it was replaced by fentanyl, with its very low toxicity at the excretory level and much higher analgesic potency, but which, for this very reason, resulted in respiratory depression, thus demanding the use of controlled respiration. Its duration of action of 30–60 min made it very easy to use. Haloperidol was later replaced by dehydrobenzoperidol, which with its duration of action of 120 min was also easier to use than its predecessor. This was the birth of NLA type 2, prevalently analgesic [19], which was pioneered in Italy by Alessandro Gasparetto, Gianpiero Giron and Enrico Ciocatto. Its introduction made intra-operative shock a rare event, always due to non-­anaesthesiological causes; the post-operative illness, on the hand, almost completely disappeared. The above considerations clearly demonstrate—if there were any need to do so!— the indissoluble link between anaesthesia and intensive care. To understand anaesthesia, one must work in intensive care, while a good intensive care physician must be an anaesthetist. Unfortunately there is a tendency to separate the two branches of specialization, so that we now find specialist physicians in hospitals and universities who are “contemptuous of anaesthesiological qualifications” (Gasparetto) [20]. To be completely honest, we must note that in the past not all authors agreed in attributing the pathogenesis of shock to the catecholamine response, often excessive or prolonged. There were also some, like Imperati et al. or Tonelli and Pesce, who considered shock to be caused by the capillary dilating effect of acetylcholine, as an expression of vagal hypertonus [21, 22]. This theory was supported by the collapse of cholinesterase in the serum and the fact that acetylcholine antagonist drugs, like atropine and tubocurarine, helped to resolve the crisis. According to Schachter, one of the benefits of transfusion was attributable to the concomitant infusion of cholinesterase [23]. In relation to this theory, one should note that in a state of shock, the liver, as a splanchnic organ, is severely affected, so that even the production of albumin is compromised: albumin and cholinesterase are controlled by the same gene. To confirm this latter statement, we recall that in cases of tubular nephrosis with albuminuria, the albumin in the blood is low but the tenor of cholinesterase increases, because the liver, in attempting to produce more albumin also produces a greater amount of cholinesterase, which is not lost into the urine. As for the action of the anti-acetylcholine drugs, which promote the resolution of the syndrome of shock, atropine by increasing the heart rate, may also aid in temporarily raising the arterial pressure; the competitive curares, due to their characteristic of acting on the nicotonic synapses, therefore also have a ganglioplegic action, and are thus aligned with the logic of preventing or attenuating, if not resolving, the vasospasm. Having reviewed the history of the pathogenesis of shock, and hence its treatment, it is time to deal with the subject in order.

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The State of Shock

The state of shock is an acute insufficiency of the peripheral circulation due to a primitive or secondary diminution—always fast and significant—of the cardio-­ circulatory flow, accompanied by vasoconstriction, progressive tissue anoxia and concomitant damage to the various organs and systems (Amato) [24]. Shock is not always hypotension Hypotension is not always shock Shock is always and only tissue hypoxia. (G.P. Novelli [25])

There are two basic types of shock: –– Hypovolemic shock, which destabilises the entire cell, and also covers primary shock of cardiac origin, in which the hypovolemia is relative and limited to the arterial system in combination with venous hypervolemia. –– Septic shock, which initially, due to the bacterial toxins, acts on the cellular membranes, after which it enters the same vicious circle as the preceding type due to the degraded function of the membranes. Nothing prevents this second type from complicating the first type, when the protracted ischaemia in the splanchnic area compromises the blood intestine barrier. –– In the first stage of septic shock, the peripheral functional hypoxia induced by the action of the toxins on the cellular membranes normally results in vascular atony and increased cardiac output. This pathogenic aspect justifies the use of the name “hyperdynamic shock” or “warm shock”, although after a certain time the pathology develops into the common denominator of vasoconstriction. –– In haemorrhagic shock, the first reaction is generalised vasoconstriction accompanied by venoconstriction and constriction of the spleen, with the introduction into circulation of the available venous reserves, followed by tachycardia. The purpose of this is to protect the heart and brain at the expense of the periphery and the splanchnic area, by opening a number of arteriovenous shunts in the periphery to centralise the circulation of blood. –– If the cause persists or is not resolved, however, a generalised haemodynamic syndrome appears, followed by disturbance of the microcirculation and biochemical alterations.

8.4

General Haemodynamic Syndrome

1 . Reduced cardiac output. 2. Modification of peripheral resistances

( vasoconstriction ® vasoplegia )

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Disturbances of the Microcirculation

(a) Vasoconstriction with: closure of the precapillary sphincters. Opening of the arteriovenous shunts Metabolic disturbances and damage to the cells Modified pH (b) Desensitisation of the peripheral receptors (vasoplegia) and sensitisation of the central receptors (arrhythmia) to the circulating catecholamines. (c) Vasoplegia with: opening of the precapillary sphincters (less resistant to hypoxia) while the venous sphincters (more resistant to hypoxia) remain closed; sequestration of the circulating volume of blood; appearance of platelet ­aggregation and stacking of the erythrocytes. This latter phenomenon, also called sludging, has an interesting mechanism which is worth considering for a moment. First off, it is a direct result of the thickening of the blood, with the consequent increase of plasma viscosity and failure of the equilibrium holding the red blood cells in suspension. There is however a second explanation, an electrostatic one: the red blood cell, which arrives well polarised, hence with its positive charge outside the membrane, finds an anoxic and hence depolarised capillary endothelium with a negative charge outside the cell, endoluminal. The electrostatic attraction is inevitable, resulting in the stacking and aggregation which, obstructing the capillaries, aggravates the tissue anoxia.

8.6

Metabolic Disturbances

The appearance of metabolic acidosis, which marks the passage from the stage of compensated to that of decompensated shock, is the direct consequence of cellular hypoxia. Recall that oxygen, in the cellular metabolism, is the great acceptor of hydrogen ions, and that in our organism oxidation reactions, almost always occur by means of dehydrogenation. When oxygen becomes scarce, the Krebs cycle immediately slows down and hence large amounts of pyruvate turn into lactate. If the latter, normally present in the blood at 8–16 mg/mL and derived mostly from the metabolism of the red blood cells and the kidneys, increases, it means that the subject is an oxygen debit and that other tissues as well are contributing to its formation by means of anaerobic glycolysis. The molecules of acetyl-CoA deriving from the oxidation of fats, rather than supplying the malfunctioning metabolic engine of the Krebs cycle, give rise to ketone bodies (acetoacetic acid, beta-oxybutyric acid and acetone) which aggravate the acidosis. In other words, the stove is smoking. However, if the hypoxia does not last for long, it only involves the cells with mitochondria and hence only affects the aerobic glycolysis pathway; if, on the other hand, the hypoxia persist, the amount of lactate in the blood continues to rise and at values of around 70–100 mg/mL the pyruvate → lactate reaction stops. When it is, the precious mechanism of reactivation of dehydrogenating (i.e. oxidizing)

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coenzyme NAD fails, which plays a fundamental role in the oxidation of the aldehydes in anaerobic glycolysis. In anaerobic glycolysis, pyruvic acid plays the role played by oxygen in the aerobic pathway, representing like the latter the sole terminal acceptor of hydrogen ions. The pyruvate → lactate reaction enables reoxidation of the coenzyme NADH2, which had accepted the hydrogen ions derived from the oxidation of the aldehydes, thus closing the cycle of the coenzyme which in its oxidised form (NAD) may be recycled to oxidize a new molecule of 3(P)-glyceraldehyde. Now, if the pyruvate  →  lactate reaction slows down, the upline reactions “are flooded” and the production of cytoplasm ATP diminishes even in cells without mitochondria, whose sole supply of energy is anaerobic glycolysis (red blood cells, neuroglia). But there’s more: an alternative to the anaerobic glycolysis pathway is the direct oxidative pathway (DOP, Chap. 5), the important pathway of sleep and the synthesis of the most diverse biological molecules. This pathway is conditioned by the availability of another coenzyme, NADP, which transforms into NADPH2, oxidising glucose-6(P) into 6-phosphogluconic acid. The opening of this pathway is conditioned by the NADP/NADPH2 ratio, and this ratio is itself dependent on the availability of NAD which, as a more powerful acceptor of H+ ions than NADP, displaces the NADP/NADPH2 ratio in favour of its numerator, as follows: NAD +  NADPH2 = NADH2 + NADP. Hence, if NAD in the oxidised form is lacking (as a result of a reduced pyruvate → lactate reaction), the DOP itself is seriously compromised. The resulting energetic crisis diminishes the membrane potential and hence sodium enters the cell (and water with it) and potassium is driven out; the sodium also enters the mitochondria, the ATP is further diminished and the cell swells up, as do the mitochondria. Finally, the ingress of calcium can seriously compromise the life of the cell. All these metabolic alterations are included in the reduced consumption of oxygen. According to Shoemaker, if the consumption of O2 falls below 75 mL/min, the prognosis is severe, while if it remains higher than 200 mL/min, there are good chances of recovery [26]. With the diminution of the levels of cyclic AMP, the receptor signal of diverse hormones also diminishes, including that of adrenaline and the response to insulin. The metabolic activity is thus further limited and the lysosomal membranes may rupture, liberating acid hydrolase from the lysosomes, followed by destruction of the cell. This is because the lysosomal enzymes are more active in acidic pH conditions. The sensitisation and activation of these enzymes is accelerated due to the concomitant progressive metabolic acidosis. It is easy to see how this mechanism increases the initial damage to the tissues and contributes substantially to its development towards irreversibility. Furthermore, the release into the circulating blood of active lysosomal protease and other enzymes may contribute to spreading the damage from one cell to another and acts as a potential factor in the development of shock to its fatal stage [27]. The formation has also been proposed, due to the lysosomal hydrolases, of an MDF (Myocardial Depressant Factor), described as a low molecular weight peptide which depresses cardiac function. In the pancreas, in a state of shock, the lysosomal hydrolases probably act together with other pancreatic enzymes on an endogenous substrate to form an MDF type peptide which, when it is released into circulation, decreases cardiac output. For the purposes of potential

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dialysis, the low molecular weight (500 Dalton) of the MDF is of significance. Another depressive factor is the PTLF (Passively Transferable Lethal Factor). This factor would have a strong negative inotropic action. In septic shock, the hyperdynamic state worsens the hypo-perfusional failure of the myocardium. As for this endotoxic shock, already proposed by Cannon, or the interference of the endotoxins in an otherwise originated state of shock, we can say that they may alter the structure of the various membranes, modifying the hydrogen bonds and the disulphide bridges of the polypeptide chains, including with the formation of free radicals. These latter react with apolar groups of the intermediate layer of the membranes, giving rise to endoperoxides and hydroperoxides, which, interfering with the fatty acid chains, phospholipids and proteins, change the nature of the membrane itself; thus seriously compromising its function, resulting in loss of ATPasic activity and hence of active transport; this promotes the ingress by diffusion of sodium, water and calcium, first into the cytoplasm, then also into the mitochondria, which are eventually definitively damaged by the lysosomal enzymes. The antigen-antibody reactions, with the release of histamine, activation of the complement and disturbance of coagulation, only aggravate the seriousness of the condition. The ATPase crisis, already in its initial stage, impacts the formation of cyclic AMP and thus diminishes all signals, both synaptic and hormonal.

8.7

Free Radicals

Among the metabolic malfunctions we must also mention those caused by the free radicals of oxygen. The orbits of a molecule of O2 normally have paired electrons, which spin in opposite directions, hence with a neutral magnetic effect. A free radical of oxygen is a molecule or atom of oxygen, which contains a single electron in its outermost orbit; this electron makes the radical highly reactive and able to bond to or subtract an electron from nearby molecules. In the biochemical earthquake, which in shock undermines the entire cellular biochemical equilibrium, it is probable that the oxidative system itself is compromised [28, 29]. Thus, it may happen that, at the end of the respiratory chain, a sole electron arrives to the O2, thus producing the highly unstable and reactive superoxide anion radical O2¯, which can cause cellular damage even at low concentrations. Not that this does not happen, to a reduced extent, even in normal conditions: indeed our organism has produced the superoxide dismutase enzyme specifically to transform the superoxide anion into hydrogen peroxide H2O2 (oxygenated water), which is then transformed into water by the catalase and peroxidase enzymes. But, if the synthesis of these enzymes is malfunctioning, the H2O2 produces two strongly reactive hydroxyl radicals OH¯. The hydroxyl radical OH¯ can trigger, even at a distance from the site of its formation, a chain reaction in an impotent organism, since it does not have a scavenging defence system against it. Furthermore, the superoxide radical, by interacting with the hydroxyl radical or H2O2, can produce singlet oxygen (1O2), which is not a free radical because it has two paired electrons in the same orbit, one of which however has been bumped up from a lower orbit due to an excess

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of energy. Although this is not strictly a free radical, it is still strongly oxidising, having a redox potential of 0.79 V in water; singlet oxygen can react with proteins and polypeptides in their cysteine, methionine, histidine and tryptophan amino acid components, with the double bonds of the unsaturated fatty acids and the carotenes producing endoperoxides and hydroperoxides. If it comes into contact with the purine bases, it can damage the DNA. Hence, the danger of the superoxide radical, apart from its own properties, lies in its ability to generate singlet oxygen, against which the organism has no scavenger defence mechanisms. In any case, to promote fully fledged reactivation of the free radicals, the anoxic situation must last for a certain amount of time. In a study by the school of Gasparetto [30], which analysed the dosage of vitamin C, vitamin E and ceruloplasmin, on admission of six patients in a state of shock (four cardiogenic and two septic) up to its resolution with the recovery or death of the patient, no decrement in the dosage of the three scavengers in question was found. The authors conclude that the period of observation, regardless of the outcome, had not been long enough. In a recent article published in Minerva Anestesiologica, Vaschetto, Navalesi, et al. propose osteopontin, a plasminogen activator urokinase, as an early warning indicator (4 days) of the passage from a state of sepsis to one of septic shock [31, 32].

8.8

Therapy

Restoration of blood volume, resolution of vasospasm, correction of metabolic acidosis. There are three pathogenic factors, which should be borne in mind when treating a patient in a state of shock: hypovolemia, and the vasoconstriction, which causes tissue hypoxia. The treatments used initially include restoration of blood volume and administration of oxygen, followed in cases of metabolic acidosis, which is most likely always present, by correction of the pH by administering bicarbonate. Sometimes 100 mEq of bicarbonate raises the arterial pressure more than a bag of plasma expander; in any case it is advisable to administer both.

8.9

Plasma Expanders

Regarding plasma expander, we must recall that gelatins give an expansion of around 75% of the infused volume, dextrans 50%, and hydroxyethyl amide 35%. As for Ringer’s solutions, on the other hand, whether lactate or acetate, the latter is to be preferred, because there is already more than enough lactate in the presence of metabolic acidosis. If a gelatin is first administered, remember not to use the same intravenous line to infuse bicarbonate and vice-versa, because the flask of gelatin often contains calcium salts, which transform the sodium bicarbonate into calcium carbonate (marble!) which precipitates. Industry produces gelatins from collagen by hydrolysis, but the hydrolytic process is hard to control and it is thus difficult to obtain the mean molecular weight of 35,000  Daltons, which is the desired figure. The process also forms portions of

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lower molecular weight, which are recovered by binding them each other with urea, succinic acid or an oxygen bridge; this yields the urea gelatins (Emagel), succinic gelatins (Eufusin) and oxygelatins (Gelinfundol). The dextrans are polysaccharides produced in the presence of sugar from cocci aspect Leuconostoc mesenteroides (Gram+); they are widespread in nature (as an opportunist, it may cause hospital infections, epsecially in patients who have been treated with vancomycin or immunodepressants). Two formulations of dextran are available commercially: dextran 40 at 10% (pm 40,000 Dalton) and 70 at 6% (pm 70,000 Dalton). Dextran 40 is more effective (it is at 10% and hence has a greater osmostic pressure) but its effect is less long lasting; after 2 h 60% is eliminated, and after 6 h 80%; it produces osmotic diuresis. Dextran 70 is longer lasting: after 24 h, 50% is still in circulation. As for hydroxyethyl amide (Voluven), a part from its low index of expansion (35%), it must be used with caution due to its effect on the kidneys. It is a synthetic polysaccharide of high molecular weight, formed from amylopectin (naturally present in the outer part of the starch granule), composed of molecules of glucose united by 1–4 α-glycosidic and 1–6 linkages at the branching points. The chemical structure of hydroxyethyl amide is very similar to that of glycogen. It is hydrolised by the α-amylase, but the products of the hydrolysis are still very osmotically active, and extend its therapeutic action, this is why we must talk of its molecular weight in vitro (130,000 Dalton) and in vivo, during post-infusional hydrolysis (~70,000 Dalton), of which the latter is still above renal elimination. In general, the colloids, further to their function as plasma expanders, improve peripheral rheology by exercising an effective anti-sludge action. Historically, one of the first plasma expanders, commercialised in the 50s, was polyvinylpyrrolidone (Periston). Professor Giorgio Massera used to tell us that the label of the bottle, due to its potentially allergenic and nephrotoxic characteristics, bore the text: “Use only once in a lifetime”, so that the bottle made a fine show in pharmacy display cases as an emergency drug, but no-one dared to use it, thinking that the patient might well have more urgent need of it on another occasion! By the way, polyvinylpyrrolidone is nowadays used as a stable excipient in the well-known 10% iodine disinfectant for external use povidone iodine (Betadine), which has replaced the old tincture of iodine (iodine 7% + 5% potassium iodide in a water/alcohol solution); the latter had a very short shelf life (1 month), due to the formation of iodohydric acid, which is still a disinfectant, but highly irritating. Invented by Antonio Grossich (Trieste) in 1908, it resolved the problem of surgical cutaneous asepsis in preparing the site of the operation. The tincture of iodine was eventually put the test in the aid stations and field hospitals of the Italian-Turkish war of 1911–1912. In restoring blood volume and oxygenating the tissues, great importance has naturally been given to transfusions of blood, plasma and albumin, as well as the above-mentioned plasma expanders, and electrolytic and glucose solutions. In the early stages, any fluid is valuable in increasing the blood volume, but one must proceed with care since every infusion of fluid can have adverse effects in the long run,

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especially when exaggerated. Indeed, an excessive supply of fluids may be useless or even damaging, since it can flood the alveolar-capillary septa, thus creating a further barrier to the diffusion of oxygen, which is already, of its own account, not as easily diffusible as CO2. Even massive blood transfusions have their attendant risks and should be limited to cases in which a loss of blood must be made up. It is thus important to oxygenate the tissues, but this depends on a variety of factors: pulmonary (shock lung) or vascular, extrinsic (more or less constricted vessel walls) or intrinsic, related to the efficiency of the red blood cells. When talking of blood transfusions, one should not forget that stored red blood cells may not lose their ability to bind oxygen, but may lose their capacity to release it when they arrive at the point of delivery, i.e. in the tissues. Now, if the amount of blood lost in a haemorrhage is reintegrated with a transfusion drawn from the blood bank (as is normally the case), various problems may arise, including problems linked to the time of storage. The amount of O2 dissolved in the plasma, which is what is directly used by the tissues, is normally quite low; when we breathe air, the dissolved O2 is 0.3% mL of blood, while if we breathe O2 100%, the amount dissolved in the blood increases to 2.1%. This source of supply must itself be continuously resupplied by the haemoglobin. It is clear that the affinity of haemoglobin for O2 will be different in the lungs, where it takes it up, than in the periphery, where it releases it. Various factors affect this oscillation in the characteristics of the haemoglobin: the pH is alkaline in the lungs, which favours the assumption of O2, and acid in the tissues, which favours its release; the PCO2, the elimination of which in the lungs favours the assumption of O2, while its assumption in the tissues has the opposite effect. The variation in the affinity of haemoglobin for O2 as a function of the pH and PCO2 constitutes the so-called “Bohr effect”. Another important factor in the regulation of the exchange of O2 in the lungs and tissue is tenor of 2-3-diphosphoglycerate (2-3-DPG); in anaerobic glycolysis we have described the Rapoport–Luebering shunt (Chap. 2). This shunt is effectively virtual in normal tissue, but very present and decisive in the red blood cells: 2-3-diphosphoglycerate is derived from 1-3-diphosphoglyceric acid, a normal intermediate product of anaerobic glycolysis, which is normally transformed into 2-3-diphosphoglycerate by the diphosphoglycerate mutase enzyme. But 2-3-DPG is itself a strong inhibitor of this enzyme, which creates a negative feedback system in the lung/tissue path and vice-versa, thus perpetuating the reaction. In abnormal conditions (storage in the bag) the anaerobic metabolism of the red blood cell excludes the Rapoport-Luebering shunt to maintain the production of ATP and with it the morphology of the membrane. Another factor which influences the oxyphoric efficiency of the red blood cell is temperature. Bags of blood are stored at 4 °C, and this low temperature displaces the haemoglobin’s dissociation curve to the left; the contrary occurs when the temperature returns to 37 °C (or beyond, due to fever). In any case, the red blood cell, once it has been transfused, takes time to recover its normal biochemical function; this time is a direct, although variable, function of the time for which the bag of blood has been stored.

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8.10 Cortisones A disputed therapy, it is effective in treating endotoxic shock, but more as a prophylactic than a therapy. At high doses, the cortisones inhibit the vasoconstrictive action of endotoxins, not via a hormonal mechanism, but rather with a pharmacological one. The cortisones are less effective in treating traumatic or haemorrhagic shock, unless used as a prophylatic measure against an endotoxic stage, secondary to the alteration of the function of the entero-haematic barrier. Cortisones may be useful to block the antigen-antibody reactions with the release of histamine, activation of the complement and disturbance of coagulation.

8.11 Catecholamines “Malgré le nombre des publications indiquant le rôle néfast d’une telle therapeutique dans les états de chock, celle-ci est encore utilisée par certains” (Laborit) [33] (Despite the number of publications reporting the negative role of this therapy in states of shock, it is still practised by some physicians). The catecholamines are administered to maintain the systemic arterial pressure at a level that assures the most important parenchymas, such as the heart, brain and kidneys, a sufficient supply of blood. But maintaining a good measurable pressure does not always correspond to an acceptable state of health for other parenchymas, such as those of the splanchnic area. One must never forget that shock is not so much hypotension, but much more hypoperfusion. There are indeed situations in which hypotension, sometimes even accentuated as in the anaesthetic technique of controlled hypotension, is accompanied by a strong increase in tissue perfusion. Without neglecting the value of measuring the arterial pressure, it must always be considered in the context of other subjective and objective parameters. Even today, the practice of administering large amounts of catecholamines is generally accepted, due to a preoccupation with the need to maintain good pressure. But in reality this risks centralising the circulation, which can be the starting point for anoxic damage to the tissues. While the prudent administration of dopamine has its justification in the attempt to reactivate compromised renal function, we should not forget that this drug, also used in higher doses to improve the inotropic function of the myocardium, turns into noradrenaline and then adrenaline after a certain time, resulting in vasospasm especially in the target areas most affected by the pathogenetic mechanism of shock. The same reasoning applies to noradrenaline, although this, in contrast with dopamine, induces a lower number of arrhythmias. In any case, we must always remember that dopamine transforms into noradrenaline, that the latter turns into adrenaline, and that there is an experimental model of shock, which can be obtaining by infusing 16 mcg/kg/min of adrenaline intravenously for at least 2 h (Lillehei et al.) [34]. In a study by De Backer et al., published in 2003 in Critical Care Med [35], which compared dopamine, noradrenaline and adrenaline in septic shock, the authors conclude that “dopamine and noradrenaline have similar haemodynamic effects, but adrenaline can worsen the splanchnic circulation in severe

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septic shock.” An editorial by B. Levy [36], published in the same number, is entitled “Epinephrine in septic shock: Dr. Jekyll or Mr. Hyde?”. However that may be, catecholamines are still recommended in treating septic shock, indeed the 2012 guidelines for the campaign against severe sepsis and septic shock, authored by Dellinger, Levy and another 20 illustrious contributors [37], make the following observations on the use of the catecholamines under the heading “Vasopressors”: 1 . Vasopressor therapy initially to target a mean arterial pressure of 65 mmHg. 2. Norepinephrine as the first choice vasopressor. 3. Epinephrine (added and potentially substituted for norepinephrine) when an additional agent is needed to maintain adequate blood pressure.

8.12 Vasodilators These have the purpose of affecting the microcirculation. If, as we have seen, the pathogenic fulcrum of shock is prolonged vasoconstriction leading to tissue hypoxia, then logic would suggest that we eliminate the spasm of the arterioles and the sphincters of the arterial and venous capillaries. It is well known that polytraumatised patients with a high medullary lesion, once the vascular lesions have been repaired and blood volume restored, even though they present considerable hypotension, are not subject to shock (due to the total blockage of the sympathetic nervous system). In the 30s, Freeman showed that sympathectomy eliminated the stage of irreversible shock [38]; as did Levy et al. in 1954 [39], Ross and Herczeg in 1956 [40], Overton and De Bakey in 1956 using ganglioplegics [41], and finally Lillehei and MacLean in 1957 with adrenolytics [42]. In 1962, Nickerson used radioactive rubidium [43] to highlight the presence of arteriolar-venular shunts in shock (which by-pass the capillaries) and showed that administering adrenolytic drugs resulted in mobilisation of the sequestrated mass of blood. These studies were followed by those of Inglis et al. [44]. who reported zero mortality in animals subjected to haemorrhagic shock when treated with chlorpromazine. A study at the University of Padua [45] evaluated a number of bio-humoral parameters of the blood and lymphatic fluid of animals subjected to experimental haemorrhagic or tourniquet shock, in a variety of conditions of anaesthesia. The study of the lymphatic fluid (thoracic duct) had the purpose of exploring the function of the extracellular sector. The following were compared: barbiturate anaesthesia, NLA type 2 and a variety of halogenated anaesthetics. The results demonstrated the validity of NLA type 2  in guaranteeing energy savings and assuring the homeostasis of the neurovegetative system in conditions of stress: the lactate/pyruvate ratio, the modification of the acid-base and electrolytic balance, the tenor of enzymes in the blood and lymph, were little changed from their initial values. This therapeutic principle, of curing a hypotensive state with drugs which themselves provoke hypotension, may seem paradoxical, and the research into the

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subject somewhat dated, but any scientific discovery remains valid until it is disputed or disproved. In any case, our experience in the field of controlled hypotension confirms every day that the circulatory flow and oxygenation, for instance in the splanchnic area, do not vary much even when the maximum arterial pressure is reduced to 80  mmHg; the rosy aspect of the viscera and the visible presence of peristalsis show this to be so.

8.13 Plasma Filtration In 2002 Ronco et al. [46] proposed filtering the plasma in septic shock (coupled plasma filtration and adsorption, CPFA) with the aim of liberating the plasma of endotoxins and all pro- and anti-inflammatory elements. Recently (2014) Livigni et al. [47] published a multi-centre study in which they express doubt that CPFA reduces mortality in septic shock. A most recent article published by Monti et al. (May 2015) [48] states that plasma filtration which a polystyrene cartridge combined with polymyxin B improves the haemodynamics and the function of various organs as well as reducing mortality at 30 days compared with preceding studies from 47 to 29%. An editorial by Donadello and Taccone [49], published in the same number of Minerva Anestesiologica (May 2015), expresses appreciation for the reported results, but observes that randomised studies would be required to further confirm the value of plasma filtration.

8.14 Gram-Positive and Gram-Negative Germs As an appendix to this chapter, we consider an issue which has mostly to do with microbiological nomenclature. It may seem out of place among the matters we have considered so far, but the anaesthetist often comes into contact with this terminology when working in intensive care, especially when dealing with a septic patient. Do you remember the colours of Gram+ and Gram− germs? You certainly knew the answer when you sat your microbiology exam, but the majority of you have no doubt forgotten it since. Gram+ germs have a fine violet colour, while Gram− germs are yellow [50]. But independently of their colour, when we are looking at a laboratory analysis which indicates the germ that is afflicting our patient, the first question we ask ourselves is whether it is Gram+ or Gram−. Either we already know the answer or we look it up in a book on microbiology (rarely available to hand), call up the laboratory (embarrassing…) or finally an ignominious search for it on the internet. The reason why germs react differently to the Gram colours is not yet clear. What is certain is these are pathological germs with quite different characteristics: Gram+ germs basically produce exotoxins, while Gram− germs produce endotoxins.

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8.15 Gram+ and Gram− (Alemanno, unpublished remark) A simple rule for determining at first sight whether a germ is Gram+ or Gram− consists in the strange coincidence of the nomenclature according to which germs with masculine scientific names are generally positive (Gram+), while those with female scientific names are generally negative (Gram−). There are some exceptions: The Gram+ germs include the Listeria. The Gram− germs include the Haemophilus, Enterobacter, Proteus, and Vibrio. Menigococcus and Gonococcus are no exception, because their scientific names are Neisseria meningitidis and Neisseria gonorrhoeae, so that they are Gram−.

References 1. Selye H. A syndrome produced by diverse nocuous agents. Nature. 1936;138(3479):32. 2. Blalock A. Mechanism and treatment of experimental shock, I: shock following hemorrhage. Arch Surg. 1927;15(5):762–98. 3. Blalock A.  Experimental shock: the cause of the low blood pressure produced by muscle injury. Arch Surg. 1930;20(6):959–96. 4. Blalock A. The Alfred Blalock Papers: letter dated June 9, 1930. Located at: The Alan Mason Chesney Medical Archives of the Johns Hopkins Medical Institutions, Baltimore, MD. 5. Blalock A. Shock: further studies with particular reference to the effects of hemorrhage. Arch Surg. 1934;29(5):837–57. 6. Cannon WB. Traumatic shock. New York: D. Appleton & Co; 1923. 7. Cannon WB. Studies in experimental traumatic shock, IV: evidence of a toxic factor in wound shock. Arch Surg. 1922;4(1):1–22. 8. Cannon WB. The Walter B. Cannon Papers: letter dated May 1, 1934. Located at: The Countway Library Rare Books and Special Collections of the Harvard Medical School, Boston. 9. Cannon WB. The wisdom of the body, vol. 184. New York: W.W. Norton & Co; 1932. p. 864. 10. Laborit H. Le vie antérieure. Paris: Grasset; 1989. 11. Laborit H. L’Anesthesie facilitée par le synergies medicamentoses. Paris: Masson et Cie; 1951. 12. Laborit H, Huguenard P. L’hibernation artificielle par moyens pharmacodynamiques et physiques. Presse Med. 1951;59:1329. 13. Laborit H, Huguenard P, Allaume R. Un nouvou stabilisateur vegetatif : le 4560 RP (chloropromazine). Presse méd. 1952;60(10):206–8. 14. Carlon CA, Cavalloni L. Importanza dei riflessi neurovegetativi durante narcosi e nella genesi della malattia postoperatoria. Comunicato alla “Società Triveneta di Chirurgia” ed alla “Sez. Alta Italia della Soc. Ital. di Anestesiologia”, il 21/12/1952. Acta Anaesth. IV(1) Gennaio-­ Febbraio 1953. 15. Aiazzi Mancini M, Donatelli L. Trattato di farmacologia. Milano: Vallardi Editore; 1968. 16. Ciocatto E.  La Neuroleptoanalgesia. In: Ciocatto E, editor. Lezioni di Anestesiologia e Rianimazione. Cap. 7. Torino: Cortina Editore; 1977. p. 147–54. 17. De Castro G, Mundeleer P. Anesthésie sans sommeil: “Neuroleptoanalgesie”. Acta Chir Belg. 1959;58:689–93.

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18. De Castro G, Mundeleer P.  Anesthésie sans barbituriques. La neuroleptoanalgésie. Anesth Analg. 1959;16:1022. 19. De Castro G, Mundeleer P. Dehydrobenzoperidol et phentanyl: Due anesthésiques novoux qui appartent de nouvelle possibilities à la neuroleptonalgésie. In: Symposium sur la neuroleptoanalgésie dans le cadre du Congrès Europeen d’Anesthesiologie à Vienne le 5 septembre 1962. 20. Gasparetto A. Dalla rianimazione alle terapie intensive. In: Gasparetto A, Gritti G, editors. La rianimazione polivalente. Padova: Tipografia “La Garangola”. p. 1976. 21. Imperati L, D’Errico G, Ruggiero A. Ricerche sul trauma operatorio quale fattore di squasso: il potere colinesterasico del siero sullo squasso nervoso. G Ital Chir. 1946;2:493. 22. Tonelli L, Pesce A. Le variazioni della colinesterasi serica nel decorso postoperatorio ed il loro significato nel problema patogenetico dello shock. Policlinico-Sez Chir. 1954;61:13. 23. Schachter RJ. Use of cholinestherase in shock. Am J Phys. 1945;143:552. 24. Amato A.  Basi anatomiche ed elementi fisiopatogenetici e terapeutici degli stati di Shock. Supplemeto alla Sinossi di Traumatologia di Guerra e Chirurgia d’urgenza. Firenze: Scuola di Sanità Militare; 1966. 25. Novelli GP.  Novelle vedute sulla etiopatogenesi dello shock. Acta Anaesthesiol. 1964;XV:625–74. 26. Shoemaker WC, Montgomery ES, Kapla E, Elwyn DH. Physiologic patterns in surviving and non surviving shock patients. Use of sequentials cardiorespiratory variables in defining criteria for therapeutics goals and early warning of death. Arch Surg. 1973;106:630. 27. Gasparetto A, Novelli GP. Aspetti bioumorali dello shock. Acta Anaesth Ital. 1979;30:943–9. 28. Candiani A, Corbucci GC, Montanari G, Crimi G, Gasparetto A.  Sistemi mitocondriali e shock. Atti del simposio “Le nuove frontiere della rianimazione”. Roma 4–5 ottobre 1982. 29. Candiani A. Aspetti emodinamici ed ossiforici dello shock (settico e cardiogeno). Min Anest. 1979;45:536. 30. Corbucci GC, Candiani A, Brancadoro D, Forastiere EMA, Gasparetto A.  Antiossidanti e shock. Acta Anaes It. 1983;34(5):667–70. 31. Vaschetto R, Navalesi P, Clemente N, et al. Osteopontin induces soluble urokinase-type plasminogen activator receptor production and release. Minerva Anestesiol. 2015;81(2):157–65. 32. Vaschetto R, Nicola S, et al. Serun levels of Osteopontin are increased in SIRS and sepsis. Intensive Care Med. 2008;34:2186–4. 33. Laborit H. Les regulations metaboliques. Paris: Masson & Cie; 1965. p. 409. 34. Lillehei RC, Longerbeam JK, Rosenberg JC. The nature of irreversible shock. In: Bock: shock pathogenesis and therapy. Berlin: Springer; 1962. p. 106–33. 35. De Backer D, Creteur J, Silva E, Vincent JL.  Effects of dopamine, norepinephrine, and epinephrine on the splanchnic circulation in septic shock: which is best? Crit Care Med. 2003;31(6):1659–67. 36. Levy B. Epinephrine in septic shock: Dr. Jekyll or Mr. Hyde? Crit Care Med. 2003;31(6):1866–7. 37. Dellinger RP, Levy MM, et al. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. 41(2); February 2013. www.comjournal.org 38. Freeman NE, et al. Effect of total sympathectomy on occurrence of shock from hemorrhage. J Clin Invest. 1938;17:359. 39. Levy EZ, North W, Wells JA. Modification of traumatic shock by adrenergic blocking agents. J Pharmacol Exp Ther. 1954;112:151–7. 40. Ross CA, Herczeg S. Effect of ganglionic blocking agents on traumatic shock in rat. Proc Soc Exp Biol Med. 1956;91:196. 41. Overton RC, De Backey ME. Experimental observation on the influence of hypothermia and autonomic blocking agents on hemorrhagic shock. Ann Surg. 1956;143:439. 42. Lillehei RC, Mc Lean LD. Physiological approach to successful treatment of endotoxin shock in experimental animal. Arch Surg. 1957;78:464. 43. Nickerson M. Drug therapy of shock. In: Von Euler US, Bock KD, editors. Shock pathogenesis and therapy. An international symposium. Springer; 1962. p. 356–70. 44. Inglis FG, Hampson LG, Gurd FN. Effect of chloropromazine on survival time and mesenteric blood flow in experimental shock. Ann Surg. 1959;149(1):43–52.

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45. Marzotti A, Alemanno F, Pierri A, Costa PP, Sammartano G. Valutazione di alcuni parametri bioumorali rilevati su sangue e linfa di animali sottoposti a shock sperimentale in condizioni diverse di anestesia. Acta Anaes Ital. 1976;27(2):199–210. 46. Ronco C, Brendolan A. E Coll. A pilot study of coupled plasma filtration with adsorption in septic shock. Crit Care Med. 2002;30(6):1250–5. 47. Livigni S, Bertolini G et al. Efficacy of coupled plasma filtration adsorption (CPFA) in patients with septic shock: a multicenter randomized controlled clinical trial. Downloaded from http:// bmjopen.bmj.com/ on November 10, 2015—Published by group.bmj. 48. Monti G, Terzi V, Calini A, et al. Rescue therapy with polymixin B hemoperfusion in high-­ dose vasopressor therapy refractory shock. Minerva Anestesiol. 2015;81(5):516–25. 49. Donadello K, Taccone FS. Refractory septic shock: who and how should be purify? Minerva Anestesiol. 2015;81(5):475–7. 50. Bendinelli M, Chezzi C, Fumarola D, Pitzurra M. Microbiologia Medica. Bologna: Monduzzi Editore; 1996. p. 99.

Further Reading Aub JC. Toxic factor in experimental traumatic shock. New England J Med. 1944;231:71–5. Benison S, Barger AC, Wolfe EL. Walter B. Cannon and the mystery of shock: a study of Anglo-­ American co-operation in world war I. Med Hist. 1991;35(2):216–49. Buchman TG. Blalock and Cannon. Arch Surg. 2010;145(4):393–4. Chambers NK, Buchman TG. Shock at the millennium. In Walter B. Cannon and Alfred Blalock. Shock. 2000;13(6):497–504. Chaudry IH, Wichterman KA, Baue AE.  Effect of sepsis on tissue adenine nucleotide levels. Surgery. 1979;85:205–11. Cortes OD, Santacruz C, Donadello K, Nobile L, Taccone FS. Colloids for fluid resuscitation: what is the role in patients with shock? Minerva Anestesiol. 2014;80(8):963–9. Dalton ML. Blalock and Harrison—a rare friendship. Pharos Alpha Omega Alpha Honor Med Soc. 1998;61(3):26–31. Eger W. Allgemeinreaktionen des Organismus und Organverȁnderungen durch die Plasmaexpander Periston, Macrodex, Haemaccel, Physiogel und Plasmagel. In: Horatz K, et al., editors. Shock un Plasmaexpander. Springer Verlag OHG. Berlin. Heidelberg: Gȍttingen; 1964. Garcia Barrebo P, Belibrea JL. Metabolic response in shock. Surg Gyn Obst. 1978;146:182. Gasparetto A, Gritti G. La rianimazione polivalente. Padua: La Garangola; 1976. Luzzani A, Polati E, Danese S.  Colloidi sintetici. In: Gullo A.  Medicina perioperatoria Terapia intensiva Emergenza. Springer-Verlag Italia, Milan; 2003. p. 137–41. Montanari G, Corbucci GG, Gasparetto A. Cellula e shock. Minerva Anestesiol. 1979;45:519. Nagler AL, Levenson SM. The nature of the toxic material in the blood of rats subjected to irreversible hemorrhagic shock. Circ Shock. 1974;1:251. Nielsen VG. Old mineshaft, new canary: can circulating osteopontin concentrations predict septic shock? Minerva Anestesiol. 2015;81(2):116–8. Rushmer RF Van Citters RL, Franklin DL. Shock: semantic enigma. Circulation. 1962;26:445–59. Saleta JL, Nieto JAS, et al. Nosocomial outbreaks caused by leuconostoc mesenteroides. Emerg Infect Dis J. 2008;14(6). http://wwwnc.cdc.gov/eid/article/14/6/07-0581 Shorr E, Zweifach BW. Furchgott. On the occurrence, sites and modes of origin and destruction, of priciples affecting the compensatory vascular mechanism in experimental shock. Science. 1945;102:489–98. Owen W, Thomas O. Transcript of discussion at the meeting. Trans Am Surg Assoc. 1934;52:123–68.

9

Cerebral Oedema Fernando Alemanno

9.1

Pathogenesis

Brain is a powerful consumer of glucose. Brain cells can be divided into two major categories: those which have no or very few mitochondria, in which the only source of energy production is anaerobic glycolysis (neuroglia), and those (neurons) which have mitochondria and hence the Krebs cycle. Anaerobic glycolysis and the Krebs cycle must both function perfectly to keep our computer (brain) working efficiently. If something goes wrong, due to a lack of oxygen, the system turns to its backup supply and the only source of energy is anaerobic glycolysis, which at least is able to assure repolarisation and hence cellular survival; but this incurs the penalty of lactic acid production, which must be dealt to avoid acidosis. The pathogenetic fulcrum of cerebral oedema is very probably tissue acidosis, which may be generically primitive (asphyxia) or correlated with general disturbances of the circulation (cardiac arrest) or secondary to disturbances of the circulation limited to the endocranial area (acute or chronic lesions occupying space). As early as 1970, Berman and Rogers [1] demonstrated the linear correlation between increased endocranial pressure and reduced cerebral intracellular pH. The concomitant variations in arterial pH are naturally relatively minor. In 1971, Bachelard showed that the brain’s energy reserves are sufficient for a few minutes, since the store of glycogen in the nerve cells are minimal [2]. A drop in cerebral pH—as of any poorly perfused tissue—can be imputed to an increase in lactic acid resulting from a deviation of the metabolism, especially in the neurons, from the normal aerobic pathway to the anaerobic pathway alone. Pyruvic

F. Alemanno (*) Anaesthesia and Intensive Care, Brescia Clinical Institutes, Brescia, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 F. Alemanno (ed.), Biochemistry for Anesthesiologists and Intensivists, https://doi.org/10.1007/978-3-030-26721-6_9

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acid must be considered the cornerstone of glucose metabolism (Moruzzi) [3] since, starting from this compound, glycolytic degradation may follow two distinct metabolic pathways, depending on whether oxygen is available or not. In normal aerobiotic conditions, the pyruvic acid enters the cycle of the tricarboxylic acids with the complete oxidation to CO2 and H2O (Chap. 3), while in anaerobiotic conditions it is transformed into lactic acid as follows: CH 3 －　　CO －　　COOH + NADH 2

pyruvic acid

®

( lactic dehydrogenase )

CH 3 －　　CHOH －　　COOH + NAD lactic acid



However, at a certain point the reaction slows down resulting in poor energy production and accumulation of the acidic metabolite. What is more, acidosis conditions the efficiency of the lactic dehydrogenase enzyme, so that as the pH decreases, the reaction stops as a result of feedback. This accumulation, for values of lactate of 70–100 mg/100 mL (normal value: 12–14 mg/100 mL), slows down the upline biochemical processes of energy production in the anaerobic pathway; although the latter, before being slowed, only had a net production of two molecules of ATP for each molecule of glucose metabolised; it was sufficient to keep the cellular membrane of the neurons repolarised at rest and satisfy the metabolic requirements of the neuroglia cells, which, with their lack of mitochondria, normally use the Embden–Mayerhof pathway as their primary energy source. The membrane pump is among the fundamental vital processes which exploit high energy bonds. It follows that a serious consequence of tissue acidosis is a slow down of the processes which maintain the potential difference, moving the Na+ out of the cell through the cellular membrane. The sodium remains trapped inside the cell, and with it the water. An increase in intracellular sodium is thus synonymous with cellular hydrops, which is the distinguishing characteristic of cytotoxic cerebral oedema. The endocranial hypertension may also be contributed to a varying extent, by interstitial oedema caused by ventricular hypertension of the liquor and, in the case of chronic space-occupying lesions (such as tumours), by so-called vasogenic oedema due to increased endothelial plasma exudation resulting in an accumulation of fluid in the extracellular space, especially in the white matter. Cerebral oedema can thus be classified as: –– Cytotoxic oedema, characterised by cellular hydrops due to the shutting down of the sodium pump, whether resulting from systemic anoxia, circumscribed anoxia (space-occupying lesion) or toxic anoxia –– Interstitial oedema, due to a failure to reabsorb CSF, primarily in the periventricular zones –– Vasogenic oedema, caused by a variety of pathological situations which increase the endothelial permeability, resulting in invasion of the interstice by water, electrolytes, proteins, etc. (the most common causes being meningitis, ictus, space-­ occupying lesions including abscesses, haemorrhages and neoplasms)

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Treatment of Cerebral Oedema

At the present time, the treatment of cerebral oedema, whether cytotoxic, interstitial or vasogenic, involves therapeutic measures which can be called “generic”, since they apply to each type of oedema: 1 . Raising the head and trunk by 30° 2. Correct water/electrolyte balance 3. Correction of blood chemistry parameters. Important among these are haematocrit (which must be kept in the range 30–35%) and glycaemia (range 100–170 mg/100 mL) 4. Maintenance of mean arterial pressure in a range of 70–90 mmHg 5. Maintenance of normothermia 6. If necessary, correct mechanical ventilation with PaCO2 ~30 mmHg, minimum necessary PEEP, PaO2 150 mmHg 7. Sufficient analgesia and sedation 8. In the first 24–48 h, administer low sodium infusions, such as repolarising solution (glucose, insulin, potassium), hold the natremia from 140 to 145 mEq, to prevent the sodium pumps, with their lack of energy (ATP), working against the gradient. N.B. As regards point 6, we must call the reader’s attention to the fact that hyperventilation causes vasoconstriction favourable to a reduction in intracranial pressure. Furthermore, since the vasoconstriction is limited to the part of the tissue not affected by the lesion, cerebral flow is facilitated in the injured areas. In any case, one should not overdo ventilation, keeping the PaCO2 around 30 mmHg. Indeed, we have seen in Chap. 3 that over-ventilation, with PaCO2  1. For example: HCl is a strong acid because its K is ~103. CH3COOH is a weak acid because its K is ~ 1.7 · 10−5 Another variant is given by Ostwald’s law [3, 4], which states that dissociation varies as a direct function of the dilution: the more dilute the electrolyte, the more it is dissociated. Vice versa, the more it is concentrated, the less it is dissociated. The upper limit of concentration is the undiluted substance, so that in its pure state the substance is not at all dissociated. The same can be said of a saturated solution. At a given temperature, a solution is said to be saturated if, when more solute is added it does not dissolve but, if it is a salt, for instance, it precipitates.

14.2.3 Dissociation of Water Pure water is electrically conductive, even if only very poorly, and is thus dissociated. But given the strong bond energy, the number of dissociated molecules is minimal.

H 2 O  H   OH －

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In water at 25  °C, the quantity of H+ and OH─ is equal, but only 10−7 gram-­ molecules are dissociated  H    OH ¯  K  H 2 O



The various molecules of water, at a given temperature, when equilibrium is reached, are a (very diluted) mixture of acids and bases. The level of dissociation is given by α, which is the fraction of gram-molecule broken up into ions; α varies with the dilution, and at infinite dilution is equal to 1. The level of dissociation α of pure water, determined experimentally by measuring the electrical conductivity, is:

  1.81 109 Since the total concentration of water in a litre is: 1000 g  55.5 mol / L 18g  molecular weight of O 2  16  H 2  18 

the concentration of water which has been dissociated, i.e. the concentration of [H+] and [OH¯] ions, is 0.00000001 gram-ions/L. Given the semantic impracticability of the number, one uses base logarithms  H     OH ¯   55.5 1.81 109  1 107 gram － ions / L



From the above we find: –– If [H+] = [OH¯] = 10−7 the solution is neutral; –– If [H+] > [OH¯] so that [H+] > 10−7 the solution is acid –– If [H+] ASA 2, coagulation disorders, cardiac, neurological, respiratory, hepatic or renal disease, pregnant women or patients with a personal history of opioid abuse. Moreover, it is especially to avoid the use, as adjuvants, of opiates in asthmatic patients or those affected by chronic obstructive pneumopathy, of epinephrine and bicarbonate in patients suffering from hypertension or of α2-agonists in patients suffering from hypotension.

17.3 Epinephrine Greatly used by surgeons and dentists as it delays the absorption of the local anesthetic and reduces the bleeding of the surgical wound. Its cardio-stimulating action balances the cardio-depressant one resulting from the local anesthetic. It is above all useful in prolonging anesthesia and therefore analgesia of short-acting local anesthetics, but not of long-acting ones. However, adrenaline specifically added to ropivacaine does not prolong either anesthesia or analgesia [5], probably because its acid pH (5.5) desensitizes adrenaline receptors in the tissue in which it has been injected. Adrenaline has a peripheral neurotoxic action especially when in the presence of a nerve lesion. It can be already mixed to local anesthetics in various concentrations: 1:50.000, 1:100,000, 1:200,000. The last concentration is the one most used and is more then adequate for its purpose. If we do not have a ready-made solution of an anesthetic with adrenaline, it is easy to prepare it. One phial of adrenaline contains 1 mg of adrenaline in 1 mL of H2O; 1 mL of H2O weighs 1 g; therefore we have 1 mg of adrenaline in 1000 mg of water: the dilution will be 1:1000. Well, if we take a syringe of 10 mL and draw up the adrenaline phial watering it down with 9 mL of physiological sodium chloride solution, we have a 1: 10,000 dilution. After having shaken it, to distribute the drug in the saline solution, we will throw away 9 mL. Only 1 mL will remain in the syringe with a dilution of 1:10,000. If now we fill up the syringe with a local anesthetic, we have an adrenaline dilution of 1:100,000. If instead of a syringe of 10 mL we use a 20 mL one, after having left inside 1 mL of the previous solution 1:10,000, filling it to 20 mL we will obtain an adrenaline dilution 1:200,000. The use of a local anesthetic with adrenaline may be useful as a marker of an intravascular injection also if the block is echo-guided.

17.4 Sodium Bicarbonate In spite of its wide use in local anesthesia with direct infiltration of tissues (aesthetic surgery) and for epidural anesthesia, it is less used as a perineural adjuvant to abbreviate the onset and to prolong anesthesia and analgesia; it is not clear if it produces any clinical result.

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Literature about the efficacy of sodium bicarbonate is vast and contradictory. Here are some relative articles: –– Tetzlaff et  al. Alkalanization of mepivacaine accelerates onset of interscalene BPB for shoulder surgery. Reg Anesth. 1990;15:242. –– Tetzlaff et al. Alkalanization of mepivacaine improves the quality of motor block associated with BPB for shoulder surgery. Reg Anesth 1995;20:128. –– Chow et  al. Alkalinization of lidocaine does not hasten the onset of axillary Brachial Plexus Block. Anesthe Analg 1998;86:566. –– Sinnott & Garfield et al. Addition of sodium bicarbonate to lidocaine decreases the duration of peripheral nerve block in the rat. Anesthesiology 2000;93:1045. Curiously, adding sodium bicarbonate to mepivacaine together with clonidine, the first offsets the analgesic effect of the last [6]. Chad Brummet and Brian Williams in their review article “Additives to local anesthetic for peripheral nerve blockade” [7], conclude so: “Given the lack of significant efficacy, the authors do not recommend the use of sodium bicarbonate in peripheral nerve blocks”.

17.5 Alpha2-Agonists 17.5.1  Clonidine This classic α2-agonist, normally used in general medicine as an antihypertensive drug, acts in this sense by interfering with some cells of the central nervous system whose receptors are responsible for blood pressure regulation. They normally activate a diastaltic arc, which uses the paravertebral sympathetic chain to regulate the production of circulatory catecholamines by the adrenal medulla. Clonidine deceives the brain cells regarding the level of circulatory catecholamines, inducing the cells to estimate them at a higher level than they actually are, thus causing a reduction of the signal arriving to the adrenal gland. The posterior horn of the spinal cord is endowed with α2-receptors that perform an analgesic function by inhibiting the presynaptic release of excitatory neurotransmitters such as glutamate (an NMDA agonist) and substance P. If administered at the subarachnoid level, clonidine induces an increase of acetylcholine. This neuromediator presents itself mainly, in the closed chain state, in what are called the nicotinic synapses (it also resembles nicotine in shape), located in the peripheral muscles and ganglia; or mainly, in the open chain state, at the level of the muscarinic receptors located in the peripheral endings of the vegetative nervous system and in the synapses of the central nervous system. The central stimulation of the muscarinic receptors induces and gives rise to an increase in GABA at the level of the primary afferent fibers, which results in suppression of the release of glutamate and substance P, two neurotransmitters with an excitatory action, and in hyperpolarization due to the opening of chloride channels. Many studies have been conducted in which clonidine has been used as a perineural adjuvant. The most interesting of these are the studies by Singelyn et al. [8,

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9], who, by adding a 150 mcg dose of clonidine to 40  mL of 1% mepivacaine, obtained an analgesia lasting twice as long as that achieved with the local anesthetic alone. With the long-lasting local anesthetics (levobupivacaine and ropivacaine) the addition of clonidine would not appear to be particularly useful because the duration of the analgesia induced does not exceed that of the typical analgesic aftereffect of the local anesthetic. In the opinion of Culebras et al. [10], it is also believed to cause problems of hemodynamic instability. According to Hutschala et al. [11] and Casati et al. [12], however, the drug will in any case prolong the analgesia induced by the local anesthetics.

17.5.2 Dexmedetomidine It is the most recent among the alpha2 agonists. It is mainly used in intensive care as a sedative by continuous intravenous infusion. It has the advantage of being a fairly good and no addictive analgesic, allowing an easy waking up after its suspension. It does not provoke neurologists or neurosurgeons because, unlike opioids, it does not result in punctiform miosis. Applied at perineural level according to Fritsch et  al. [13] it has given good results: 18 h of analgesia versus 14 h with the anesthetic alone (ropivacaine 0.5%). The authors performed a perfect triple blind prospective study, preceded by a precise preoperative assessment. It is a pity that such a perfect study should be complicated by adding a general anesthesia with a variety of other drugs, inserting therefore an unpredictable variant of analgesic and non-analgesic drug interactions and the singular patient’s reaction to them. Anyway nothing is perfect. In the successive issue of RAPM, Akiko Yabuki, Hitoshi Higuchi et  al. [14] published an article, which highlights an angiospastic action of dexmedetomidine via alpha2A adrenergic receptors. This points out that the drug could be dangerous in the case of neural lesion or intraneural injection. According to McCartney [15], “Stimulation of the α2-receptor produces hypotension, bradycardia, and sedation at higher doses, and these effects may outweigh any analgesic benefits produced by the use of these agents”. Moreover Abdallah and Brull [16] suggest “that dexmedetomidine prolongs both sensory and motor block, a difference that may be disadvantageous by delaying rehabilitation and/or discharge”.

17.6 Dexamethasone Adding 8 mg of dexamethasone to 30 mL of 0.5% ropivacaine or bupivacaine prolonged, respectively, analgesia of ropivacaine from 12 to 22 h and of bupivacaine from 15 to 22 h [17]. The action of dexamethasone to prolong analgesia has still not been clearly understood. It might be due to a local hyperpolarizing action on Kalium channels of little C fibers (via glucocorticoid-3 receptors), besides its systemic action. However, some studies, where the drug is i.v. injected, have shown a similar

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protracting of postoperative analgesia after loco-regional anesthesia [18, 19]. According to other authors [20], whose opinion is shared by the writer, the difference in prolonging analgesia between intravenous and perineural injection is around 20% more for the latter. Anyhow, being the perineural prescription of dexamethasone “off label”, according to Zhao et al. [21], we advise its parenteral use. The use of dexamethasone is best avoided in diabetic patients [22].

17.7 Midazolam According to Saffica I Shaikh and Veena K [23], 50  mcg/kg added to 30  mL of bupivacaine 0.5% for supraclavicular brachial plexus block prolonged sensory blockade and postoperative analgesia: 805 min (13 h,41) versus 502 min (8 h,36), without increasing the risk of adverse effects. But, applying midazolam to rabbits [24] provoked vascular and tissular lesions, so we advise against its intrathecal use in humans. Once again, Chad Brummet and Brian Williams [7] “do not recommend the clinical use of perineural midazolam combined with local anesthetics in peripheral nerve blocks”.

17.8 N-methyl-d-Aspartate (NMDA) Antagonists Normal nociceptive neuotransmitters of NMDA receptors are aspartate and glutamate, which normally act together with substance P, to open Ca channels. Dextromorphan and ketamine are the antagonists. Gorgias et al. [25] used 0.1 mg/ kg of ketamine added to 40 mL of lidocaine 1% to attenuate the pain caused by the tourniquet during intravenous regional anesthesia (IVRA). At this dosage, a good tolerance to the tourniquet, without any collateral effect, has been referred. But if added to 0.5% ropivacaine (30 mg in 30 mL) for interscalene brachial plexus block, ketamine does not improve the duration of sensory block [26]. Following the same logic, magnesium sulphate has also been utilized to the same purpose. It acts like a plug against the opening of calcium channels provoked by aspartate and/or glutamate. The results were not always the same in prolonging the postoperative analgesia time, but they seemed to depend on the local anesthetic to which magnesium was added. When added to bupivacaine [27] it does not prolong the analgesia time, but produces only a decreased visual analogue score (VAS); when added to ropivacaine it prolongs the duration of analgesia and of motor block [28].

17.9 Opiates After the disappointing results, as perineural adjuvants, of classic opiates, i.e. morphine, meperidine, fentanyl, alfentanil and sufentanil [15], even if used for continuous nerve blocks [29], only two opioids, according to Mc Cartney, are efficacious at perineural level: buprenorphine and tramadol [15].

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17.9.1 Rationale According to Scott Young [30], opioid receptors are produced in the ganglion of the posterior root and follow the axoplasm flow. The two molecules, naloxone and enkephalin, known for their high affinity towards opioid receptors, were labelled with iodine −125, to trace their movements. In the same year Fields asserted: “... there is evidence that both the exogenous opioids and the enkephalins act both at the presynaptic level blocking the release of neurotransmitters from the primary afferent nerve endings and at the level of the post-synaptic receptors” [31]. Moreover, as reported by Stein [32], “…Opioids bind to receptors on dorsal-root ganglia, the central terminal of primary afferent neurons, and peripheral sensory-nerve fibers and their terminals”. Opioids act on a signal amplifier of the membrane of the postsynaptic receptors, whose name is 3′–5′-cyclic AMP. This molecule is synthetized by the enzyme adenylcyclase and destroyed by the enzyme phosphodiesterase. Opioids act by depressing adenyl cyclase and therefore attenuating the efficacy of the nociceptive signal. Other drugs, on the contrary, depressing phosphodiesterase, such as xanthines (caffeine, teine and theobromine), amplify the role of 3′–5′-cyclic AMP. This is why, when we drink a coffee we may subjectively note the augmented action of epinephrine, but unfortunately we cannot note that the action of other neurotransmitters and hormones is also stressed.

17.9.2 Buprenorphine The mechanism of action of buprenorphine, a thebaine derivative, is rather complicated [33]. It is a μ-receptor agonist and its receptor affinity is 24 times that of fentanyl and 50 times that of morphine. It also binds to δ and κ receptors, but the affinity for the δ receptors is ten times less. Most receptors of the posterior horn are of the μ-type; μ and δ agonists, inhibiting adenylcyclase, opening K+ channels and closing the Ca2+ channels, inhibit pain; controversial is the role of the kappa receptors. These actions reduce the release of the pain transmitters, substance P and glutamate. After the discovery of the μ, δ and κ receptors, a fourth receptor, called the opioid receptor-like1 (ORL1) has been identified. Its neurotransmitter is called nociceptin, it has a hyperalgesic effect and differs from the other antalgic neurotransmitters for the lack of thyrosine in its constituent amino acid sequence; therefore some authors give it the name orphanin. Normally the opioids dose-effect curve is a hyperbole; buprenorphine, despite its potency unlike the other opioids does not reach a maximal response; the dose-effect curve of buprenorphine at a certain dose becomes bell-shaped, due to the activation of opioid receptors-like1 neurotransmitter and therefore attenuating its potent analgesic action. According to Lutfy and Cowan [33] buprenorphine potency is due to its lipophilia, with consequent high receptor affinity; moreover its low molecular weight facilitates its blood-brain barrier and axonal penetration. The action of buprenorphine is not antagonized by naloxone; according to Barbara Pleuring [34], after high doses of the drug, naloxone

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detaches buprenorphine only from ORL1 receptors, this way reinforcing its analgesic action. Norbuprenorphine, a metabolite of buprenorphine, active on the μ and weakly on the ORL1 receptors, further complicates matters.

17.9.3 Perineural Application Candido et al. applied it either at subclavian perivascular brachial plexus block [35] or at axillary level [36], excluding with this latter way, the suspect of a transdural spread of the drug. We too have applied buprenorphine as an adjuvant to prolong postoperative analgesia [37]. To have a homogeneous population we decided to limit the study to a population undergoing arthroscopic rotator cuff repair, always performed by the same surgical team. We anesthetized the patients, subdivided into three groups, applying the Middle Interscalene Block (MIB) [38]. The local anesthetic used was levobupivacaine (40 mL of 0.75%) added to half a phial of buprenorphine (0.15 mg) in one of the three groups. The second group received the same dose of buprenorphine intramuscularly. In the third group, half a ml of saline was added to the local anesthetic. In the first group (perineural buprenorphine), the analgesia time, calculated from the time of anesthesia injection, was 17 h and 29 min; in the second group it was 13 h and 40 min, and in the third group 10 h and 37 min. Candido et al. [35] with 0.3 mg of buprenorphine added to local anesthetic obtained the same duration of analgesia time 17.4 h, as we did with 0.15 mg. In my opinion, besides the use of different local anesthetics, the cause of this incongruity is due to the dose-response curve of buprenorphine, which, unlike the hyperbolic curve of other opioids, over a certain dose, presents itself as a bell-shaped curve, due to activation of ORL1 receptors. One might wonder how it is that 0.3 mg of buprenorphine can activate ORL1 receptors. But, we are operating via the axonal flow, which directly leads to the posterior horn of spinal cord, and not through the parenteral route. We have to consider that 0.1 mg of morphine, injected into the subarachnoidal space, can give an excellent analgesia. According to Julius Kosel et  al. [39], buprenorphine is the unique opioid adjuvant in regional anesthesia.

17.9.4 Tramadol Although structurally similar to morphine and codeine, tramadol has a prevalent α2-agonistic action. It is a synthetic molecule, composed of two enantiomers: one clockwise rotated (+tramadol) (1R-2R-2-[(dimethylaminomethyl)methyl]-1-3(methoxyphenyl) cyclohexanol hydrochloride; the other counterclockwise rotated (−tramadol) (1  L,2  L-2-[(dimethylaminomethyl)-methyl]-1-3-(methoxyphenyl) cyclohexanol hydrochloride). (+)Tramadol has a weak affinity for μ receptors (6000 times less than morphine, 10 times less than codeine). Weaker is its affinity for δ and κ receptors; moreover, it inhibits serotonin reuptake. On the contrary, (−)tramadol inhibits noradrenaline reuptake [40]. The result is analgesia obtained by the synergistic action of the two enantiomers. The incidence of nausea is due to (+)tramadol,

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but there is no advantage by using only one of the two enantiomers. Tramadol is about 30% antagonized by naloxone (very likely, the clockwise rotated). Yohimbine, a synaptic antagonist of noradrenaline, partially blocks (67%) the analgesic efficacy of the racemic mixture [40] (likely the counterclockwise rotated tramadol). Unfortunately also ondansetron, an antinausea drug, according to Arcioni et al. [41], has a similar blocking effect. The α2-adrenoreceptors are present in the spinal cord, as well as in the periaqueductal gray matter and in the periventricular diencephalon. Noradrenaline acts as an inhibitor on the cells of the posterior horn, blocking nociceptive signals with an effect imitated by clonidine. As a matter of fact, tramadol binding to the α2-receptors has never been demonstrated. According to Desmeules et al. [40], tramadol acts indirectly activating the postsynaptic α2-receptors. Also the spinal descending pathway makes use of norepinephrine to control the pain at the level of the posterior horn of the spinal cord. Therefore, tramadol acts both as an opioid on the μ receptors with its (+)enantiomer, whose effect is antagonized by naloxone and with its (−)enantiomer reuptaking noradrenaline, like clonidine.

17.9.5 Perineural Application Kapral and coworkers were among the first to apply tramadol at the perineural level [42]. They added 100 mg of the drug to 1% mepivacaine for an axillary brachial plexus block, noting a remarkable difference versus a second group of patients, treated with intravenous administration of the analgesic, and versus a third group in whom 2 mL of saline were added, instead of tramadol, to mepivacaine. Two years later, in Minerva Anestesiologica, Antonucci published a paper [43] where the addition of tramadol to the local anesthetic (ropivacaine 0.75%) in the axillary block was compared to the addition of clonidine and sufentanil. The author concludes tramadol is just as efficient as the two compared adjuvants with a minor presence of side effects such as bradycardia, hypotension, itch.Robaux et al. [44] in 2004 conducted a study where progressive doses of tramadol had been added to mepivacaine 1.5%. The most effective dose was 200 mg, with an acceptable incidence of side effects (nausea and vomit). Alemanno, Danelli et  al. in 2012 [45, 46] studied 120 patients undergoing arthroscopic rotatory cuff repair. They performed the MIB using 0.5% levobupivacaine (0.4 mL/kg). The patients, subdivided into three groups, received in the first group 1.5 mg/kg of tramadol (maximum limit 100 mg) added to the levobupivacaine and an equivalent volume of saline into the deltoid; in the second group an equivalent volume of saline added to local anesthetic, while the tramadol was injected into the deltoid muscle; the third group received saline solution added both to the local anesthetic and injected intramuscularly. The first request of analgesia by the patients in the first group was made on average 14.5 h after the injection of the anesthetic mixture, after 10.1 h by the patients of the second group (intramuscular tramadol) and after 7.6 h by the third group. In the two studies (both with buprenorphine and with tramadol) the side effects amounted to a few cases of nausea and vomiting easily resolved with 25 mg of levosulpiride i.v.

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However, when tramadol is added to a longer-acting anesthetic (l-bupivacaine 0.75%) its efficacy poorly overcomes the analgesic lingering effect of the local anesthetic. In a recent study, published in Minerva Anestesiologica, November 2014 [47], we compared buprenorphine versus tramadol, each added to L-bupivacaine 0.75%. Buprenorphine confirmed a good efficacy, tramadol less than in the previous study when added to l-bupivacaine at lower concentration (0.5%).

17.10 Perspectives 17.10.1  Thiamine In the spinal cord, at the level of the dorsal horn, acetylcholine is very important in modulating the nociceptive signals [48]. Both M2 presynaptic receptors modulating acetylcholine release and M1 postsynaptic receptors play a role in pain control [49, 50]. Following this logic, many authors have used neostigmine as an analgesic adjuvant either injecting into the subarachnoidal space [51], or for brachial plexus block [52] obtaining analgesia in the first case, but also nausea and vomiting, nausea and vomiting only in the second case. Thiamine is the coenzyme that allows the oxidative decarboxylation of the pyruvic acid to obtain acetyl-coA, a fundamental compound to produce, in the cytoplasm, acetylcholine. Unfortunately, acetyl-coA is produced at the mitochondrial level and it is unable to pass through the mitochondrial membrane. So it has to exploit the diffusibility of the citrate (obtained in the Krebs cycle by its synthesis with the oxaloacetic acid) to realize the synthesis of the neurotransmitter, according to the well-known reaction (Chap. 11):



citrate + coA + vit.B1 = oxaloacetate + acetyl - coA and finally : acetyl - coA + choline = acetylcholine

As we can see, thiamine is twice present in order to obtain the synthesis of acetylcholine. Therefore, we decided to apply, at the perineural level, vit B1, blended with the local anesthetic with the purpose of optimizing, at metameric level, the synthesis of acetylcholine [53]. The dose of vit. B1 was that suggested for intramuscular application (2 mg/kg). The results, observed with a retrospective study, were interesting; we recorded 17.6 ± 3 ± h of analgesia in the thiamine group, versus 11 ± 3 h in the group treated with the anesthetic alone; the same results obtained by adding 0.15 mg of buprenorphine [37]. Anyway, prospective studies are necessary to confirm the efficacy of thiamine as a perineural analgesic adjuvant. Is the use of analgesic adjuvants applicable to other types of block? We added tramadol and buprenorphine, in the same proportions, to the single shot femoral nerve block, but we obtained the same results as injecting the drugs intramuscularly. Moreover, Candido et al. [54] using buprenorphine for single shot sciatic nerve block failed to achieve the same results as in the brachial plexus blocks. Probably the success of applying analgesic adjuvants to brachial plexus is due to its

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topographical situation, where deep cervical fascia, fusing with the vertebral prolongation of the superficial cervical fascia, surrounds the neurovascular bundle [46]. Therefore injecting the anesthetic mixture into the sheath that envelops the brachial plexus, it remains longer in contact with the nervous tissue, before being reabsorbed. In this way the adjuvant drugs have more time to pass through the axonal membrane. Perhaps, only the paravertebral space has somehow a similar anatomic characteristic.

References 1. Finucane BT. Complications of brachial plexus block. In Finucane BT. Complications of Regional Anesthesia. Churchil Livingsone; 1999. p. 56. 2. Chelly JE, Casati A, Fanelli G.  Peripheral nerve blocks techniques. London: Mosby International Limited; 2001. 3. Capdevilla X, Jaber S, Personen P, Borgeat A, Eledjam JJ. Acute neck cellulitis and mediastinitis complicating a continuous interscalene block. Anesth Analg. 2008;107:1419–21. 4. Denise JW, Hoerlocker TT.  Infectious complications. In: Neal JM, Rathmel JP, editors. Complications in regional anesthesia and pain medicine. Philadelphia: Saunders Elsevier; 2007. 5. Weber A, Fournier R, Van Gessel E, Riand N, Gamulin Z. Epinephrine does not prolong the analgesia of 20 mL ropivacaine 0.5% or 0.2% in a femoral three-in-one block. Anesth Analg. 2001;93(5):1327–31. 6. Contreras-Domínguez V, Carbonell-Bellolio P, Sanzana-Salamanca E, Ojeda-Greciet A.  Adición de bicarbonato de sodio y/o clonidina a la mepivacaína. Influencia sobre las características del bloqueo de plexo braquial por vía axilar. Rev Esp Anestesiol Reanim. 2006;53:532–7. 7. Brummet C, Williams B.  Additives to local anesthetic for peripheral nerve blockade. Int Anestesiol Clin. 2011;49:104–16. 8. Singelyn FJ, et al. Adding clonidine to mepivacaine prolongs the duration of anethesia and analgesia after axillary brachial plexus block. Reg Anesth. 1992;17:148–50. 9. Singelyn FJ, et al. A minimum dose of clonidine added to mepivacaine prolongs the duration of anesthesia and analgesia after axillary brachial plexus block. Anesth Analg. 1996;83:1046–50. 10. Culebras X, et al. Clonidine combined with a long acting local anesthetic does not prolong postoperative analgesia after brachial plexus block but does induce hemodynamic changes. Anesth Analg. 2001;92:199–204. 11. Hutschala D, et  al. Clonidine added to bupivacaine enhances and prolongs analgesia after brachial plexus block via a local mechanism in healthy volunteers. Eur J Anaesthesiol. 2004;21(3):198–204. 12. Casati A, Magistris L, Fanelli G, Beccaria P, Cappelleri G, Aldegheri G, Torri G. Small-dose clonidine prolongs postoperative analgesia after sciatic-femoral nerve block with 0.75% ropivacaine for foot surgery. Anesth Analg. 2000;91(2):388–92. 13. Fritsch G, Danninger T, et al. Dexmedetomidine added to ropivacaine extends the duration of Interscalene brachial plexus blocks for effective for elective shoulder surgery when compared with ropivacaine alone. Reg Anesth Pain Med. 2014;39(1):37–47. 14. Yabuki A, Higuchi H, et  al. Locally injected dexmedetomidine induces vasoconstric tion via peripheral α-2A adrenoceptors subtype in Guinea pigs. Reg Anesth Pain Med. 2014;39(2):133–6. 15. Mc Cartney Colin JL, Analgesic adjuvants in the peripheral nervous system, in: Admir Hadzic. Textbook of regional anesthesia. Mc-Graw Hill 2007, page 151.

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16. Abdallah FW, Brull R.  Facilitatory effects of perineural dexmedetomidine on neur axial and peripheral nerve block: a systematic review an meta-analysis. Br J Anaesth. 2013;110(6):915–25. 17. Cummings KC, Napierkowski DE, et al. Effect of dexamethasone on the duration of interscalene nerve blocks with ropivacaine or bupivacaine. Br J Anaesth. 2011;107(3):446–53. 18. Desmet M, Braems H, et al. I.V. and perineural dexamethasone are equivalent in increasing the analgesic duration of a single shot Interscalene block with ropivacaine for shoulder surgery: a prospective, randomized, placebo-controlled study. Br J Anaesth. 2013;111(3):445–52. 19. Abdallah FW, Johnson J, Chan V, et al. Intravenous dexamethasone and perineural dexamethasone similarly prolong the duration of analgesia after supraclavicular brachial plexus block. Reg Anesth Pain Med. 2015;40(2):125–32. 20. Leurcharusmee P, Aleste I, et al. A multicentre randomized comparison between intravenous and perineural dexamethasone for ultrasound-guided infraclavicular block. Reg Anesth Pain Med. 2016;41(3):328–33. 21. Zhao WL, Ou XF, Zhang WS. Perineural versus intravenous dexamethasone as an adjuvant in regional anesthesia: a systemic review and meta-analysis. J Pain Res. 2017;10:1529–43. 22. Williams AB, Murinson BB, Grable BR, Orebough SL. Future considerations for pharmacologic adjuvants in single injection peripheral nerve blocks for patients with diabetes mellitus. Reg Anesth Pain Med. 2009;34(5):445–557. 23. Shaikh SI, Veena K.  Midazolam as an adjuvant in supraclavicular brachial plexus block. Anaesthesia Pain Intensive Care. 2012;16(1):7–11. 24. Erdine S, Yűcel A, Ozyalcin S, et  al. Neurotoxicity of midazolam in the rabbit. Pain. 1999;80(1–2):419–23. 25. Gorgias NK, et al. Clonidine versus ketamine to prevent tourniquet pain during intravenous regional anesthesia with lidocaine. Reg Anesth Pain Med. 2001;26:512–7. 26. Lee IO, et al. No enhancement of sensory and motor blockade by ketamine added to ropivacaine interscalene brachial plexus blockade. Acta Anaesth Scand. 2002;46:821–6. 27. Lee AR, Yi HW, Chung IS, et al. Magnesium added to bupivacaine prolongs the duration of analgesia after Interscalene nerve block. Can J Anesth. 2012;59(1):21–7. 28. Mukherjee K, Das A, et al. Evaluation of magnesium as an adjuvant in ropivacaine-induced supraclavicular brachial plexus block: a prospective, double-blinded randomized controlled study. J Res Pharm Pract. 2014;Oct–Dec(4):123–9. 29. Putzu M, Casati A. Local anesthetic solutions for continuous nerve blocks, in Admir Hadzic: textbook of Regional Anesthesia. New York: McGraw Hill; 2007. p. 163. 30. Young S, et al. Opioid receptors undergo axonal flow. Science. 1980;210:76–7. 31. Fields HL, et  al. Multiple opiate receptor sites on primary afferent fibers. Nature. 1980;284:351–3. 32. Stein C.: The control of pain in peripheral tissues by opioids. N Engl J Med 1995, Vol 332, N° 25: 1685–1690. 33. Lutfy K, Cowan A.  Buprenorphine: a unique drug with complex pharmacology. Curr Neuropharmacol. 2004;2(4):395–402. 34. Pleuvry B. Opioid mechanisms and opioid drugs. Anaesth Intensive Care Med. 2005;6(1):30–4. 35. Candido KD, Franco CD, Kahn MA, Winnie AP, Raja DS. Buprenorfine added to the local anesthetic for brachial plexus block to provide postoperative analgesia in outpatients. Reg Anesth Pain Med. 2001;26:352–6. 36. Candido KD, Winnie AP, Ghaleb AH, Fattouh MW, Franco CD.  Buprenorphine added to the local anesthetic for axillary brachial plexus block prolongs postoperative analgesia. Reg Anesth Pain Med. 2002;27:162–7. 37. Behr A, Freo U, Ori C, Westermann B, Alemanno F.  Buprenorphine added to levobupivacaine enhances postoperative analgesia of middle interscalene brachial plexus block. J Anesth. 2012;26(5):746–51. 38. Alemanno F, Capozzoli G, Egarter-Vigl E, Gottin L, Bartoloni A.  The middle interscalene block: cadaver study and clinical assessment. Reg Anesth Pain Med. 2006;31:563–8.

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39. Kosel J, Bobik P, Tomczyk M. Buprenorphine—the unique opioid adjuvant in regional anesthesia. Expert Rev Clin Pharmacol. 2016;9(3):375–83. https://doi.org/10.1586/17512433.201 6.1141047. 40. Desmeules JA, et  al. Contribution of monoaminergic modulation to the analgesic effect of tramadol. Br J Clin Pharmacol. 1996;41(1):7–12. 41. Arcioni R, et  al. Ondansetron inhibits the analgesic effects of tramadol: a possible 5-HT3 spinal receptor involvement in acute pain in humans. Anesth Analg. 2002;94:1553–7. 42. Kapral S, Gollmab G, et al. Tramadol added to mepivacaine prolongs the duration of an axillary brachial plexus blockade. Anesth Analg. 1999;88:853–6. 43. Antonucci S. Adiuvanti nel blocco del plesso brachiale per via ascellare: confronto tra clonidina, sufentanil e tramadolo. Minerva Anestesiol. 2001;67:23–7. 44. Robaux S, et  al. Tramadol added to 1.5% mepivacaine for axillary brachial plexus block improves postoperative analgesia dose-dependently. Anesth Analg. 2004;98:1172–7. 45. Alemanno F, Danelli G, Fanelli A, Ghisi D, Bizzarri F, Fanelli G. Tramadol and 0.5% levobupivacaine for brachial plexus interscalene block: effects on postoperative analgesia in patients undergoing shoulder artthroplsty. Minerva Anestesiol. 2012;78:291–6. 46. Alemanno F, Bosco M, Barbati A. Anesthesia of the upper limb (A state of the art guide). Springer Italia 2014. p. 257. www.springer.com/medicine/anesthesiology/book/978-88-470-5417-2 47. Alemanno F, Westermann B, Bettoni A, Candiani A, Cesana BM. Buprenorphine versus tramadol as perineural adjuvants for postoperative analgesia in patients undergoing arthroscopic cuff repair under middle interscalene block: a retrospective study. Minerva Anestesiol. 2014;80(11):1198–204. 48. Abelson KSP, Höglund AU. Intravenously administered oxotremorine and atropine, in doses known to affect pain threshold, affect the intraspinal release of acetylcholine in rats. Pharmacol Toxicol. 2002;90:187–92. 49. Bartolini A, Ghelardini C, Fantetti L, et  al. Role of muscrinic receptor subtypes in central antinoception. Br J Pharmacol. 1992;105:77–82. 50. Baba H, et al. Muscarinic facilitation of GABA release in substantia gelatinosa of the rat spinal dorsal horn. J Physiol. 1998;508:83–93. 51. Yegin A, Yilmaz M, Karsli B, Erman M. Analgesic effects of intrathecal neostigmine in perianal surgery. Eur J Anaesthesiol. 2003;20:404–8. 52. Buaziz H, Paqueron X, Bur ML, Merle M, Laxenaire MC, Benhamou D.  No enhancement of sensory and motor blockade by neostigmine added to mepivacaine axillary plexus. Anesthesiology. 1999;91(1):78–83. 53. Alemanno F, Ghisi D, Westermann B, et al. The use of vitamin B1 as a perineural adjuvant to middle interscalene block for postoperative analgesia after shoulder surgery. Acta Biomed. 2016;87(1):22–7. 54. Candido KD, Hennes J, et al. Buprenorphine enhances and prolongs the postoperative analgesic effect of bupivacaine in patients receiving infragluteal sciatic nerve block. Anesthesiology. 2010;113(6):1419–26.

Further Reading Arend I, et al. Tramadol und Pentazocin im Klinischen-Doppelblind-Crossover Vergleich. Arzneim Forsch. 1978;28:199–208. Fleetwood-Walker SM, et al. An alpha 2 receptor mediates the selective inhibition by noradrenaline of nociceptive responses of identified dorsal horn neurones. Brain Res. 1985;334:243–54. Flohe L, Arend I, et al. Klinische Prüfung der Abhängigkeitsentwicklung nach Langzeitapplikation von Tramadol. Arzneim Forsch. 1978;28:213–7. http://en.wikipedia.org/wiki/clonidine Carrol I, et al. The role of adrenergic receptors and pain: the good, the bad, and the unknown. Semin Anesth Perioperat Med Pain. 2007;26:17–21.

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Kaiser V., et  al. Effects of analgesic agent Tramadol in normal and arthritic rats: comparison with effects of different opioids, including tolerance and cross-tolerance to morphine. Eur J Pharmacol. 1991;195(1):37-45 Kuraishi Y, et al. Noradrenergic inhibition of the release of substance P from the primary afferents in the rabbit spinal dorsal horn. Brain Res. 1985;359:177–82. Pertovaara A. Noradrenergic pain modulation. Prog Neurobiol. 2006;80:53–83. Raffa RB, et al. Complementary and synergistic antinociceptive interaction between the enantiomers of tramadol. J Pharmacol Exp Ther. 1993;267(1):331–40. Reimann W., et al. Does an non-opioid component contribute to the efficacy of the central analgesic tramadol? In: Tenth European winter conference on brain research, March 3–10, 1990. Jana S. Topical and peripherally acting analgesics. Pharmacol Rev. 2003;55:1–20. Scott Lesley J, Perry Caroline M.  Tramadol a review of its use in perioperative pain. Drugs. 2000;60(1):139–76. Stein C. Peripheral mechanism of opioid analgesia. Anesth Analg. 1993;76:182–91. Stoffregen J.  Kombinationnarkose mit tramadol-infusion. Anaesthesiol Intensive Med. 1980;29:673–4. Turan A, et  al. Intravenous regional anesthesia using lidocaine and magnesium. Anesth Analg. 2005;100:1189–92. Viel EJ, Eledjam JJ, de la Coussaye J, D’Athis F. Brachial plexus block with opioids for postoperative pain relief: comparison between buprenorphine and morphine. Reg Anesth. 1989;14:274–8.

The Grand Design

18

Fernando Alemanno

And the long street—rolls on around like an enormous choochoo train—chugging oround the world—with its bawling passengers… and all of them wondering—just who is up—in the cab ahead—driving the train—if anybody…and some of them leaning out—and peering ahead—and trying to catch—a look at the driver—in his one—eye ca—, trying to see him—to glimpse his face—to catch his eye—as they whirl around a bend—but they never do - although once in a while—it looks as if—they’re going to. And the street goes rocking on—the train goes bowling on… Lawrence Ferlinghetti, The long street

Having re-read these notes, it is clear that in the end, all we are talking about is energy. In the general imagination, the production of energy is strictly bound to the phenomenon of combustion, a chemical reaction consisting in the oxidation of a substrate (fuel) by a comburent, normally atmospheric O2, which generates energy (heat, light, motion, etc.). In biochemistry, however, oxidation is not always the result of the direct addition of O2, since this would result in a combustion at temperatures, which are not compatible with life. All oxidation reactions involve dehydrogenization or the subtraction of electrons, resulting in the controlled production of energy, similar to what happens in a nuclear power plant when the cadmium bars are inserted to prevent a chain reaction and hence an explosion.

F. Alemanno (*) Anaesthesia and Intensive Care, Brescia Clinical Institutes, Brescia, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 F. Alemanno (ed.), Biochemistry for Anesthesiologists and Intensivists, https://doi.org/10.1007/978-3-030-26721-6_18

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As we have seen (Chap. 1), nature has three types of oxidating reaction: • The first involves the direct addition of O2, and is normally strongly exothermic: 2H2 + O2 → 2H2O; • The second involves dehydrogenisation (breaking a covalent bond), and is ­common in biological chemistry; • The third involves the subtraction of electrons (respiratory chains). The concept is inverted in the case of reduction. Reduction may occur: 1. By loss of oxygen, for example at high temperatures: CO2 → CO + O (as we have seen, direct addition or subtraction of oxygen generally generates or requires high temperatures); 2. By taking on hydrogen; 3. By acquiring electrons. We will simplify the discussion by considering the second mode of oxidation and reduction, since it is the easiest to understand. To obtain energy, we must dehydrogenize a substrate (suppose, vegetables) by breaking the covalent bond of hydrogen with carbon, thus liberating its energy. Hence all the energy which we use to survive and for our actions is of solar origin, being produced and stored by chlorophyllic photosynthesis.

18.1 Photosynthesis Solar energy, which is substantially electromagnetic, acts during the day by striking an electron; the struck electron acquires a high energy state, being effectively pushed in towards the nucleus and then rebounding, and after 10−8 s it returns to its original orbit, releasing its excess energy. Szent-Györgyi (Hungarian, 1893–1986, Nobel Prize for medicine in 1937 for his discovery of vitamin C and the intermediate reactions of the Krebs cycle) explains the phenomenon [1] as follows: “If a photon ejected by the sun interacts with an electron of a molecule on our globe, the electron is raised to a higher energy level to drop back as a rule within 10-8 - 10-9 sec, to its ground state. Life has shoved itself between the two processes, catches the electron in its high-energy state and lets it drop back to the ground level within its machinery, using the energy thus released for its maintenance. In order to do this efficiently, life has to meet a photon with a specially suited substance (mostly chlorophyll) and couple this substance to a system, which converts the unstable energy of excitation into a stable potential energy.” Christopher U.M. Smith [2] expresses it with the following beautiful simile: “Everyone has seen how heavy rain striking the tarmac creates an ever changing scene of activity as the drops rebound from the surface. In the same way, the rain of solar photons creates an ever changing scene of activity on the surface of the Earth.” We can express a similar idea imagining the start of a rainstorm on a dusty road; the drops of water (the photons) propel the grains of dust (the electrons) upwards off the tarmac, after which they fall back

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down, transferring part of the energy to the ground. The outermost orbits are high-­ energy states, so that when an electron moves into a higher orbit it absorbs energy (that of the photon, in this case), whereas when it returns to its original orbit it emits energy. This is the energy which is immediately used to produce NADPH and ATP during the day. NADPH and ATP then supply hydrogen and energy for the synthesis of carbohydrates, starting with CO2 captured from the atmosphere and H2O supplied by the roots of the plant. Chlorophyll, with its structure composed of the four pentagonal pyrolitic nuclei of porphin, connected by methine bridges (─CH═), is strangely similar to the haem in haemoglobin, myoglobin and cytochromes (Fig. 18.1). The substantial difference lies in the metallic central nucleus, which is magnesium in chlorophyll, while for haem it is iron. The porphin molecule has 26 electrons, so that it is particularly suited to absorbing and using photonic energy. To sum up, the electron, struck by the photon, is compressed towards the nucleus, from which it rebounds under the action of the negative charge of the electrons of the lower orbits, which it has attempted to invade; or, depending on the impact of the photon with the angle of incidence of the electron’s vector, it may increase its energy up to a maximum in which the angle of impact is equal to 0 and hence the two vectors (of the photon and the electron) coincide. In this case, the transmitted energy sums to a varying amount with that of the electron, which may also step up an orbit C

C

C

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Fig. 18.1  A comparison of haemoglobin and chlorophyll (from Wikipedia): in practice, the substantial difference lies in the central atom, which is Fe and Mg, respectively (Perutz’s article does not give the two formulas). As can be seen from the two formulas, the “Grand Design” could not be more unitary

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tangentially. If this happens to a chlorophyll electron, it oxidizes and loses an electron, and thus takes on a positive charge. Meanwhile (in a period of less than 10−8 sec) its place is temporarily occupied by the electron of the hydroxyl OH−, normally present due to the disassociation of H2O. When the electron returns to its usual orbit, the OH− electron is displaced and thus sent, by the energy released by the returning electron, into the cytochrome chain. When it returns to its home orbit in the chlorophyll, the energy of the electron is divided into a series of successive cascades represented by vitamin K or flavin-mononucleotide (FMN) and the cytochromes, so as to optimize the capture of energy for oxidative phosphorylation, i.e. for the synthesis of ATP. Porphin is an ancient molecule, and one of its modifications is protoporphyrin 9, which gave rise to haem and chlorophyll, the latter due to the insertion of an atom of magnesium. Only later, with the insertion of an iron atom, were haemoglobin, myoglobin and the haem of the cytochromes formed. Indeed, the Bible also speaks of the creation of vegetation on the third day of Genesis [3], while animals appear only on the sixth day. And God said, Let there be a firmament in the midst of the waters, and let it divide the waters from the dry land. And it was so. And God called the dry land Earth; and the gathering together of the waters called he Seas: and God saw that it was good. And God said, Let the earth bring forth grass, the herb yielding seed, and the fruit tree yielding fruit after his kind, whose seed is in itself, upon the earth: and it was so. And the earth brought forth grass, and herb yielding seed after his kind, and the tree yielding fruit, whose seed was in itself, after his kind: and God saw that it was good. And the evening and the morning were the third day. And God said, Let the earth bring forth the living creature after his kind, cattle, and creeping thing, and beast of the earth after his kind: and it was so. And God made the beast of the earth after his kind, and cattle after their kind, and every thing that creepeth upon the earth after his kind: and God saw that it was good. And God said, Let us make man in our image, after our likeness: and let them have dominion over the fish of the sea, and over the fowl of the air, and over the cattle, and over all the earth, and over every creeping thing that creepeth upon the earth.So God created man in his own image, in the image of God created he him; male and female created he them. And God blessed them, and God said unto them: Be fruitful, and multiply, and replenish the earth, and subdue it: and have dominion over the fish of the sea, and over the fowl of the air, and over every living thing that moves upon the earth. And God said, Behold, I have given you every herb bearing seed, which is upon the face of all the earth, and every tree, in the which is the fruit of a tree yielding seed; to you it shall be for meat. And to every beast of the earth, and to every fowl of the air, and to every thing that crowls upon the earth, wherein there is life, I have given every green herb for meat: and It was so. And God saw every thing that he had made, and, behold, it was very good. And the evening and the morning were the sixth day. The chlorophyll photosynthesis reaction is as follows: 6CO2 + 6H2O → C6H12O6 + 6O2, producing glucose which is converted into cellulose as a supporting material or starch as a reserve. But it’s no simple matter. As indicated above, the solar photons move the electrons from inner, lower energy orbits to outer, higher energy orbits. Their place is occupied by the H2O which is partly broken up into H+ and OH−. Once the electron returns to its original orbit, the hydroxyl electron uses the energy, released by the return of the first electron, to move

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along the cytochrome chain, thus following an inverse route relative to that of the mitochondrial respiratory chain of the animal organism; finally reaching the ferredoxin, the co-enzyme that accepts and transports electrons, which reduces NADP to NADP2− and then to NADPH2. The electrons that reach NADP come, as mentioned previously, from the water whose molecules have been partly ionised into H+ and OH−. It is the hydroxyl ion that donates the electron, which accelerated by another photon reaches a higher orbit where it is accepted by the plastoquinone (acceptor of electrons) and thus enters the cytochrome chain, at the end of which, after having been accelerated into an even higher energy orbit by yet another photon, is transferred, via the ferredoxin, to the NADP. Only at this point does the H+ ion fall prey to electrostatic attraction and transform the NADP− into NADPH. The hydrogen ions H+, passing through a membrane in the direction of the gradient, liberate the energy required to synthesize ATP, which expresses the maximum charge of the biochemical battery which, when needed, produces ADP + Pi +12,000 calories by hydrolysis (Moruzzi) [4]. This source of energy can be used to form carbohydrates, for instance by reducing CO2 by NADPH. It seems that the terrestrial atmosphere was originally poor in O2 and that it was chlorophyll photosynthesis that enriched it, while the H2 molecule derived from the disassociation of water under the action of solar photons was used to build the –CH2 group required to synthesize crbohydrates, lipids and protein. When oxygen appeared in the atmosphere, some primitive life forms learned to use it, generating much more ATP. One interesting hypothesis [5] is that mitochondrials are the same new organisms, which with their ability to use oxygen as the final acceptor of the oxidative processes of the respiratory chain, colonized the primitive single-cell organisms three billion years ago, the latter being only able to ferment sugars from alcohol (glucose) into acid (lactic) via the intermediate stage of aldehyde (glyceraldehyde). This led to the formation of the cell as we know it today, with its ability to oxidise and decarboxylate the substrates down to water and CO2. This would seem science fiction if there were not clues in mitochondrial DNA, equipped with a genetic system of protein production, which does not derive from that of the nucleus of the cell itself. Another proof is the action of certain antibiotics, which act on the DNA of bacteria (tetracycline, quinolones) and damage that of our mitochondria, while leaving the DNA of the nucleus unaffected. Now for a curious fact which will no doubt delight the feminists [5]. The spermatozoon is very poor in mitochondria, and when it reaches the ovum it impregnates it with its nuclear DNA alone, leaving the tail and mitochondria outside. Mitochondrial heredity is thus exclusively maternal. Moreover even the Judaic Law (the Torah) of matrilinearity states: “One can only be a Jew by descent from the mother”.

18.2 Considerations on the Periodic Table of the Elements A quick glance at the table of the elements (Fig. 18.2) shows that the orbits increase with the number in the periodic system of elements. In the first period, hydrogen has just one electron and hence only one orbit, followed by helium with two electrons,

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Fig. 18.2  The periodic table of the elements (Dmitrij Ivanovič Mendeleev, 1869). The table details the periodic system of the elements. It was constructed with the stencil, at a time in which computers did not exist and we used the Olivetti Lettera 44, held to the desktop by its weight, or the Lettera 22, equal to today’s laptop PC, for writing

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and this concludes the first period. The radius of a given orbit depends on the energy of its electrons, which is equal for all electrons in that orbit. At this point the Pauli exclusion principle comes into play, according to which the lowest energy level, i.e. the orbit closest the nucleus (K) cannot have more than two electrons, and the subsequent orbits (L, M, N, O, P and Q) can have at most 8, thus reaching the configuration of the noble gases. The first period, therefore, has only two elements: hydrogen (H), with 1 electron, and helium (He), with 2 electrons in the K shell. The second period starts with lithium (Li) which has 2 electrons in the K shell and 1 in the L shell. The following elements (Be, B, C, N, O, F and Ne) add 1 electron at a time to the L shell, until we reach to neon, which has 8 (octet) and closes the second period. The second period reserves a big surprise starting with lithium, which with 3 electrons must transfer one to the an orbit of the second shell (L); followed by beryllium with 4 electrons (2 in the first and 2 in the second shell) and boron with 5 electrons (2 in the first shell and 3 in the second); after which we have: carbon (2 in the first shell and 4 in the second, tetravalent), nitrogen (2 in the first and 5 in the second, trivalent), oxygen (2 in the first shell and 6 in the second, bivalent)—thus forming, together with hydrogen, the four basic elements of living matter! They have a low atomic number and thus low atomic weight, but they are able to form complex molecules often composed of a very large number of atoms. Carbon, with its 4 electrons in the peripheral shell, and hence 4 electrons absent from the complete octet, is the element most adapted to bind with other atoms: carbon itself, hydrogen, oxygen and nitrogen, to synthesise organic molecules. To complete our recital, the next element is fluorine (7 electrons in the second shell, making it monovalent). Fluorine (F) is the head of the halogen group (penultimate column of the periodic system, Fig. 18.2), with a normal potential (pn) of 2.65 V, followed by Cl (pn = 1.36), Br (pn = 1.09), I (pn = 0.54), for which the rule applies that the one with the highest normal potential moves the successive ones and cannot be moved by those of lower normal potential (this principle led researchers to synthesize halogenated anaesthetics with ever greater fluorine content). Neon (8 electrons) completes the electronic sequence of the second period, and is one of the noble gases: helium, neon, argon, krypton, xenon, radon (the last column of the periodic table, Fig. 18.2). When the octet is closed with neon, an additional electron in the M shell starts the third period with Na, followed by, one electron at a time, Mg, Al, Si, P, S, Cl and Ar (argon) which has 8 and is a noble gas. The fourth period starts with potassium (K), which inaugurates the N shell, followed by Ca and other elements of great biological significance: Mn (photosynthesis, catalysis), Fe, Co, Cu and Zn. Finally we have Br with 7 electrons in the N shell and krypton which completes the period. But, I don’t want to bore you with a pedantic recital of the periodic table, which once the principle is understood, is self-explanatory in relation to the elements of interest to us.

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To sum up we have seen that living matter is composed of four basic chemical elements (H, O, C, N), belonging to the first two periods of the system, plus another 14 important ones (Na, Mg, P, S, Cl, K, Ca, Mn, Fe, Co, Cu, Zn, I), all belonging to the third and fourth periods, with the exception of iodine, which is in the fifth period.

18.3 Conclusions If we return to the concept of a “grand design”, in the first stage of synthesis of organic matter, which occurs in the vegetable world thanks to solar energy, CO2 and H2O, in a purely chemically reductive process, synthesize the substrate (the fuel). This is followed by a process of oxidation (dehydrogenation or oxidative decarbolisation) in which the syntesized matter is used by animals to generate the energy required for action, in the anaerobic glycolysis path and the Krebs cycle, or for the synthesis of organic matter with the pentose path (during sleep). A perfect example of a complex mechanism, which is indubitably redox. The Great Bioengineer who created it certainly knew what he was trying to achieve! I quote an interesting fragment of an article by M. F. Perutz on the structure of haemoglobin, published in Le Scienze in 1979 [6]. The description is of an exemplary clarity. The article starts with a quote from “Elegy on Mistress Elizabeth Drury” by John Donne (1601): Why grass is green, or why our blood is red, Are mysteries which none have reached unto; In this low form, poor soul, what will you do? He then goes on: A molecule of haemoglobin consists of four polypeptide chains: two alpha chains of 141 amino acid residues and two beta chains of 146 amino acid residues. The alpha and beta chains have different sequences of amino acids, but are folded to form similar three-­dimensional structures. Each chain also receives a haem, which gives the blood its red colour, and consists of a run of carbon, nitrogen and hydrogen atoms, called porphirin, with an iron atom lodged at its centre like a precious stone (Fig. 18.1). A single polypeptide chain combined with its own haem is called a sub-unit of the haemoglobin or monomer. In the complete molecule, the four subunits are closed conjoined in a three-dimensional pattern, forming a tetramer…” “…Normally, the ferrous haem reacts irreversibly with oxygen to produce the ferric haem (something similar to rust, ed.), but when it is included in the folds of the globin chain it is protected and the reaction with oxygen is then reversible. The effect of globin on the chemistry of the haem has only recently been explained thanks to the discovery that the irreversible oxidation (in theory, ed.) of the latter proceeds via an intermediate compound, in which a molecule of oxygen forms a bond between the iron atoms of the two haems. In myoglobin and haemoglobin, the folds of the polypeptide chain prevent this formation, isolating each haem in a separate compartment. In the protein, ­furthermore, the iron is bound to an atom of nitrogen of the histidine amino acid, which transfers a fraction of negative charge, thus enabling the iron to form a weak bond with the oxygen.

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“All this cannot be up to chance. To say that everything is governed by chance is itself an act of faith equal to that of those who believe in a Creator. Future discoveries may provide us with more matter for reflection, but at the present time we cannot but think that the subtle structure involving the various mechanisms described so far are the outcome of a design.” (Rinaldo Cosio) [7]. As for us, “…we are in the position of a child entering a vast library full of books in many languages. The child knows that someone must have written the books. But he doesn’t know how. He doesn’t understand the languages in which they are written. He vaguely intuits a mysterious order in the arrangement of books on the shelves, but he doesn’t understand it. This, in my opinion, is the attitude of the most intelligent of human beings towards God. We see a wonderfully organized universe which obeys certain laws, but we only vaguely understand them” (Albert Einstein) [8].

References 1. Szent-Györgyi Albert. Discussions of the Faraday Society. 1959;XXVII:111–4. 2. Smith Christopher UM. Molecular biology, A structural approach (Chapter XI). London: Faber Paper Covered Editions; 1971. 3. The Holy Bible. C.E.I. text of the Holy Scriptures. Santarcangelo di Romagna: © RL Gruppo Editoiale Srl; 2011. 4. Moruzzi G, Rossi CA, Rabbi A.  Principi di chimica biologica. Bologna: Tinarelli Editore; 1984. 5. Henri L. Dieu ne joue pas aux dés (Chapitre V). Paris: Editions Grasset & Fasquelle; 1987. 6. Perutz M.F. Hemoglobin structure and respiratory transport. Sci. Am. 1979; 239 (6):92–125. 7. Rinaldo C. Anesthesiologist. Comment on re-reading this chapter (in the text); March 2016. 8. Viereck GS. What life means to Einstein. The Saturday Evening Post, 26 October 1929. p. 17.

Further Reading Cann RL, Stoneking M, Wilson AC.  Mitochondrial DNA and human evolution. Nature. 1987;325:31–6. Devlin TM. Biochimica. Naples: EdiSES; 2012. Lauro G. Introduzione alla biochimica patologica e clinica. Padova: Piccin Editore; 1997. Garret RH, Grisham CM. Biochimica. Padova: Piccin Editore; 2014. Ievons Frederic R. Le basi biochimiche della vita. Milano: Mondadori Editore; 1972. Paolo LG, Luciano O. Energia vitale. Roma: C.E.S.I.; 1991. Moruzzi G, Rossi CA, Rabbi A. Principi di chimica biologica. Bologna: Tinarelli Editore; 1979. Nelson DL, Cox MM.  Introduzione alla biochimica di Lehninger. Bologna: Zanichelli Editore; 2015. Pauling L. Chimica generale. Milano: Longanesi Editore; 1967. Perutz M.F. Stereochemical mechanism of oxygen transport by haemoglobin. Proc R Soc Lond Sci. 1980;208:135–62. Taiz L, Zeiger E. Elementi di fisiologia vegetale. Padova: Piccin Editore; 2013. Zibalsc.blogspot.com/2012/01/93-clorofilla-ed-emoglobina.html 08 gen 2012—http:// it.wikipedia.org/wiki/Clorofilla. Abstract—Chlorophyll, Hemoglobin and the elements that compose the Universe. 2012.

Epilogue and Farewell

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Fernando Alemanno

So, what is biochemistry for? It enables us to understand what we are doing to patients during anaesthesia and in intensive care. Let us look at some random examples. A curare, apart from blocking neuromuscular synapsis, may have also, for better or for worse, a ganglion-blocking action because the ganglionic synapses are also nicotinic ones. By hyperventilating long a patient with a PaCO2 far below 25 mmHg, we risk blocking the carboxylation of pyruvic acid into oxaloacetic acid, thus knocking out the connecting molecule between anaerobic glycolysis and the Krebs circle, followed by a possible tendency towards metabolic acidosis, which does not compensate respiratory alkalosis, but rather complicates it. Biochemistry needs to understand that vitamins are not those little toys that we are led to underestimate and to classify as supplements, but molecules of considerable power; just think of the hydroxylating action of ascorbic acid that affects the synthesis of collagen and the conversion of dopamine to norepinephrine; or the multiple sites of action of vitamin B1, or of vitamin B6, which transforms one of most potent excitatory neurotransmitters (glutamate) in one of the most potent inhibitors (GABA). Practising anaesthesia without a deep knowledge of its cultural foundations may be at times boring, but the only alternative to boredom can be something unpredictable happens, which must be dealt with readiness and rationally as it arises. A dear friend, with whom I studied at Padua and who was then my colleague for 2 years after our specialization at the Anaesthesia and Intensive Care Institute directed by Professor Alessandro Gasparetto, went on to work in a big and prestigious foreign hospital, famous around the world. After 4 years, his chief consultant retired and with great surprise, wonder, admiration and in some cases envy, we, his ex-colleagues, saw him promoted to chief consultant.

F. Alemanno (*) Anaesthesia and Intensive Care, Brescia Clinical Institutes, Brescia, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 F. Alemanno (ed.), Biochemistry for Anesthesiologists and Intensivists, https://doi.org/10.1007/978-3-030-26721-6_19

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Some 20 years later at a congress in his city, I paid him a visit. We met with pleasure, which shows that friendship does not vanish over the years. He had invited me for lunch in the exclusive club of his Institution where I just had to ask him: “how did you do it?” After a short pause for reflection, he answered: “it was relatively easy, as you know in Padua we were well trained, with a sound base in biochemistry and pharmacology. When I got here, I knew more or less do what they did other colleagues, mind you they were all excellent, but I knew why—and they didn’t!”

19.1 Farewell Well, it is time to finish—often harder to do than starting! The meanders of biochemistry are fascinating indeed—just as our world of anaesthesia, analgesia and intensive care is fascinating. We complete one chapter only to start another and then another; true enough, when we have finished one we wonder if it has expressed everything we wanted to say, and such doubts are never laid to rest, but at a certain point one has to say: enough. If even just one chapter of this collection of notes—I hardly dare to call it a book—or even a single paragraph has stimulated your interest in some way, then I am happy to have done something of value; I appeal especially to young colleagues. I have accompanied my ideas and knowledge, even in a practical manner, from one century—one millennium!—to the next. I can assure you that things have changed from when our monitor was the patient’s wrist, the saturimeter was the colour of her/his lips or the roots of nails, when arterial pressure was read off the column of mercury in a Riva-Rocci arm cuff, which went manually up and down continuously, especially in angio-neurosurgery conducted in hypotension controlled by nitroprusside; when we had just one monitor in the operating group, which we fought over and, once we had it in our possession, we would enter the operating theatre only to hear the surgeon say: “don’t you feel sure of yourself today?” Well, progress marches on, even if sometimes what we call progress may seem to be no more than a costly stratagem foisted on us by the SACB (Simple Affairs Complications Bureau). Industry and the market have their own logic and reasons, which we attempt to make our own, even unconsciously, so as not to appear backwards or in order to make up our own deficits with the technologies they offer us. In this context, in the suspicion that our professional conduct is sometimes conditioned by others, we publish this “collection of notes” taken from a life trying to discover the answer to “why”. Which, if it has given you any pleasure, think kindly of the man who wrote it,…But if on the other hand we have only succeeded in boring you, please believe that we did not do so on purpose. (Alessandro Manzoni: The Betrothed).