Processes & lipids in metabolic biochemistry 9788132347118, 8132347110, 1283508699, 9781283508698

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First Edition, 2012

ISBN 978-81-323-4711-8

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Published by: The English Press 4735/22 Prakashdeep Bldg, Ansari Road, Darya Ganj, Delhi - 110002 Email: [email protected] 

Table of Contents Chapter 1 - Carbohydrate Metabolism Chapter 2 - Photosynthesis Chapter 3 - Cellular Respiration Chapter 4 - Cholesterol Chapter 5 - Eicosanoid Chapter 6 - Fatty Acid Chapter 7 - Phospholipid Chapter 8 - Sphingolipid and Steroid Chapter 9 - Triglyceride Chapter 10 - Bile Acid

Chapter- 1

Carbohydrate Metabolism

Carbohydrate metabolism denotes the various biochemical processes responsible for the formation, breakdown and interconversion of carbohydrates in living organisms. The most important carbohydrate is glucose, a simple sugar (monosaccharide) that is metabolized by nearly all known organisms. Glucose and other carbohydrates are part of a wide variety of metabolic pathways across species: plants synthesize carbohydrates from atmospheric gases by photosynthesis storing the absorbed energy internally, often in the form of starch or lipids. Plant components are eaten by animals and fungi, and used as fuel for cellular respiration. Oxidation of one gram of carbohydrate yields approximately 4 kcal of energy and from lipids about 9 kcal. Energy obtained from metabolism (e.g. oxidation of glucose) is usually stored temporarily within cells in the form of ATP. Organisms capable of aerobic respiration metabolize glucose and oxygen to release energy with carbon dioxide and water as byproducts. Carbohydrates are a superior short-term fuel for organisms because they are simpler to metabolize than fats or those amino acid portions of proteins that are used for fuel. In animals, the most important carbohydrate is glucose; so much so, that the level of glucose is used as the main control for the central metabolic hormone, insulin. Starch, and cellulose in a few organisms (eg, termites, ruminants, and some bacteria), both being glucose polymers, are disassembled during digestion and absorbed as glucose. Some simple carbohydrates have their own enzymatic oxidation pathways, as do only a few of the more complex carbohydrates. The disaccharide lactose, for instance, requires the enzyme lactase to be broken into its monosaccharides components; many animals lack this enzyme in adulthood. Carbohydrates are typically stored as long polymers of glucose molecules with glycosidic bonds for structural support (e.g. chitin, cellulose) or for energy storage (e.g. glycogen, starch). However, the strong affinity of most carbohydrates for water makes storage of large quantities of carbohydrates inefficient due to the large molecular weight of the solvated water-carbohydrate complex. In most organisms, excess carbohydrates are regularly catabolised to form acetyl-CoA, which is a feed stock for the fatty acid synthesis pathway; fatty acids, triglycerides, and other lipids are commonly used for long-term energy storage. The hydrophobic character of lipids makes them a much more compact form of energy storage than hydrophilic carbohydrates. However, animals,

including humans, lack the necessary enzymatic machinery and so do not synthesize glucose from lipids. All carbohydrates share a general formula of approximately CnH2nOn; glucose is C6H12O6. Monosaccharides may be chemically bonded together to form disaccharides such as sucrose and longer polysaccharides such as starch and cellulose.

Catabolism Carbohydrate catabolism is the breakdown of carbohydrates into smaller units. Carbohydrates literally undergo combustion to retrieve the large amounts of energy in their bonds. Energy is secured by mitochondria in the form of ATP. There exist different types of carbohydrates; these are polysaccharides (e.g., starch, amylopectin, glycogen, cellulose), monosaccharides (e.g., glucose, galactose, fructose, ribose) and the disaccharides (e.g., maltose, lactose). The genetic makeup of carbohydrate (s) can be altered with steroids which cause muscle hypertrophy when ingested. Glucose reacts with oxygen in the following redox reaction, C6H12O6 + 6O2 → 6CO2 + 6H2O, the carbon dioxide and water is a waste product and the chemical reaction is exothermic. The breakdown of glucose into energy in the form of molecules of ATP is therefore one of the most important biochemical pathways found in living organisms. Anaerobic respiration is the metabolic pathway where glucose is broken down in the absence of oxygen. Aerobic respiration is the pathway where glucose is broken down in the presence of oxygen.

Glycolysis The six-carbon glucose molecule is broken down into two three-carbon pyruvate molecules yielding two ATP molecules and two high energy NADH molecules.

Anaerobic respiration Without oxidative phosphorylation, the NADH molecules cannot be converted to ATP. When all the NAD+ molecules have been converted to NADH, glycolysis will stop unless the NAD+ is regenerated by fermentation.

Aerobic respiration Pyruvate oxidation The three carbon pyruvate molecule loses a carbon atom and is shepherded into the citric acid cycle by coenzyme A.

The Citric acid cycle (also known as the Krebs cycle) The acetyl group that came from pyruvate enters this biochemical cycle, releasing carbon dioxide, water, and the high energy molecules ATP, NADH, and FADH2.

Oxidative phosphorylation The high energy molecules NADH and FADH2 are converted into usable ATP molecules in the mitochondria by the mitochondrial electron transport chain.

Metabolic pathways • •

• • • •

Carbon fixation, or photosynthesis, in which CO2 is reduced to carbohydrate. Glycolysis - the oxidation metabolism of glucose molecules to obtain ATP and pyruvate o Pyruvate from glycolysis enters the Krebs cycle, also known as the citric acid cycle, in aerobic organisms after moving through pyruvate dehydrogenase complex. The pentose phosphate pathway, which acts in the conversion of hexoses into pentoses and in NADPH regeneration. Glycogenesis - the conversion of excess glucose into glycogen as a cellular storage mechanism; this prevents excessive osmotic pressure buildup inside the cell Glycogenolysis - the breakdown of glycogen into glucose, which provides a glucose supply for glucose-dependent tissues. Gluconeogenesis - de novo synthesis of glucose molecules from simple organic compounds. an example in humans is the conversion of a few amino acids in cellular protein to glucose.

Metabolic use of glucose is highly important as an energy source for muscle cells and in the brain, and red blood cells.

Glucoregulation Glucoregulation is the maintenance of steady levels of glucose in the body; it is part of homeostasis, and so keeps a constant internal environment around cells in the body.

The hormone insulin is the primary regulatory signal in animals, suggesting that the basic mechanism is very old and very central to animal life. When present, it causes many tissue cells to take up glucose from the circulation, causes some cells to store glucose internally in the form of glycogen, causes some cells to take in and hold lipids, and in many cases controls cellular electrolyte balances and amino acid uptake as well. Its absence turns off glucose uptake into cells, reverses electrolyte adjustments, begins glycogen breakdown and glucose release into the circulation by some cells, begins lipid release from lipid storage cells, etc. The level of circulatory glucose (known informally as "blood sugar") is the most important signal to the insulin producing cells. Because the level of circulatory glucose is largely determined by the intake of dietary carbohydrates, diet controls major aspects of metabolism via insulin. In humans, insulin is made by beta cells in the pancreas, fat is stored in adipose tissue cells, and glycogen is both stored and released as needed by liver cells. Regardless of insulin levels, no glucose is released to the blood from internal glycogen stores from muscle cells. The hormone glucagon, on the other hand, has an effect opposite to that of insulin, forcing the conversion of glycogen in liver cells to glucose, which is then released into the blood. Muscle cells, however, lack the ability to export glucose into the blood. The release of glucagon is precipitated by low levels of blood glucose. Other hormones, notably growth hormone, cortisol, and certain catecholamines (such as epinepherine) have glucoregulatory actions similar to glucagon.

Human diseases of carbohydrate metabolism • • • • •

Diabetes mellitus Lactose intolerance Fructose intolerance Galactosemia Glycogen storage disease

Chapter- 2

Photosynthesis

Composite image showing the global distribution of photosynthesis, including both oceanic phytoplankton and land vegetation.

Overall equation for the type of photosynthesis that occurs in plants Photosynthesis (from the Greek φώτο- [photo-], "light," and σύνθεσις [synthesis], "putting together", "composition") is a process that converts carbon dioxide into organic

compounds, especially sugars, using the energy from sunlight. Photosynthesis occurs in plants, algae, and many species of bacteria, but not in archaea. Photosynthetic organisms are called photoautotrophs, since they can create their own food. In plants, algae, and cyanobacteria, photosynthesis uses carbon dioxide and water, releasing oxygen as a waste product. Photosynthesis is vital for all aerobic life on Earth. As well as maintaining the normal level of oxygen in the atmosphere, nearly all life either depends on it directly as a source of energy, or indirectly as the ultimate source of the energy in their food (the exceptions are chemoautotrophs that live in rocks or around deep sea hydrothermal vents). The rate of energy capture by photosynthesis is immense, approximately 100 terawatts, which is about six times larger than the power consumption of human civilization. As well as energy, photosynthesis is also the source of the carbon in all the organic compounds within organisms' bodies. In all, photosynthetic organisms convert around 100–115 teragrams of carbon into biomass per year. Although photosynthesis can happen in different ways in different species, some features are always the same. For example, the process always begins when energy from light is absorbed by proteins called photosynthetic reaction centers that contain chlorophylls. In plants, these proteins are held inside organelles called chloroplasts, while in bacteria they are embedded in the plasma membrane. Some of the light energy gathered by chlorophylls is stored in the form of adenosine triphosphate (ATP). The rest of the energy is used to remove electrons from a substance such as water. These electrons are then used in the reactions that turn carbon dioxide into organic compounds. In plants, algae and cyanobacteria, this is done by a sequence of reactions called the Calvin cycle, but different sets of reactions are found in some bacteria, such as the reverse Krebs cycle in Chlorobium. Many photosynthetic organisms have adaptations that concentrate or store carbon dioxide. This helps reduce a wasteful process called photorespiration that can consume part of the sugar produced during photosynthesis.

Overview of cycle between autotrophs and heterotrophs. Photosynthesis is the main means by which plants, algae and many bacteria produce organic compounds and oxygen from carbon dioxide and water (green arrow). The first photosynthetic organisms probably evolved about 3,500 million years ago, early in the evolutionary history of life, when all forms of life on Earth were microorganisms and the atmosphere had much more carbon dioxide. They most likely used hydrogen or hydrogen sulfide as sources of electrons, rather than water. Cyanobacteria appeared later, around 3,000 million years ago, and drastically changed the Earth when they began to oxygenate the atmosphere, beginning about 2,400 million years ago. This new atmosphere allowed the evolution of complex life such as protists. Eventually, no later than a billion years ago, one of these protists formed a symbiotic relationship with a

cyanobacterium, producing the ancestor of many plants and algae. The chloroplasts in modern plants are the descendants of these ancient symbiotic cyanobacteria.

Overview

Photosynthesis changes the energy from the sun into chemical energy and splits water to liberate O2 and fixes CO2 into sugar Photosynthetic organisms are photoautotrophs, which means that they are able to synthesize food directly from carbon dioxide using energy from light. However, not all organisms that use light as a source of energy carry out photosynthesis, since photoheterotrophs use organic compounds, rather than carbon dioxide, as a source of carbon. In plants, algae and cyanobacteria, photosynthesis releases oxygen. This is called oxygenic photosynthesis. Although there are some differences between oxygenic photosynthesis in plants, algae and cyanobacteria, the overall process is quite similar in these organisms. However, there are some types of bacteria that carry out anoxygenic photosynthesis, which consumes carbon dioxide but does not release oxygen.

Carbon dioxide is converted into sugars in a process called carbon fixation. Carbon fixation is a redox reaction, so photosynthesis needs to supply both a source of energy to drive this process, and the electrons needed to convert carbon dioxide into carbohydrate, which is a reduction reaction. In general outline, photosynthesis is the opposite of cellular respiration, where glucose and other compounds are oxidized to produce carbon dioxide, water, and release chemical energy. However, the two processes take place through a different sequence of chemical reactions and in different cellular compartments. The general equation for photosynthesis is therefore: 2n CO2 + 2n H2O + photons → 2(CH2O)n + n O2 + 2n A Carbon dioxide + electron donor + light energy → carbohydrate + oxygen + oxidized electron donor Since water is used as the electron donor in oxygenic photosynthesis, the equation for this process is: 2n CO2 + 2n H2O + photons → 2(CH2O)n + 2n O2 carbon dioxide + water + light energy → carbohydrate + oxygen Other processes substitute other compounds (such as arsenite) for water in the electronsupply role; the microbes use sunlight to oxidize arsenite to arsenate: The equation for this reaction is: (AsO33–) + CO2 + photons → CO + (AsO43–) carbon dioxide + arsenite + light energy → arsenate + carbon monoxide (used to build other compounds in subsequent reactions) Photosynthesis occurs in two stages. In the first stage, light-dependent reactions or light reactions capture the energy of light and use it to make the energy-storage molecules ATP and NADPH. During the second stage, the light-independent reactions use these products to capture and reduce carbon dioxide. Most organisms that utilize photosynthesis to produce oxygen use visible light to do so, although at least three use infrared radiation.

Photosynthetic membranes and organelles

Chloroplast ultrastructure: 1. outer membrane 2. intermembrane space 3. inner membrane (1+2+3: envelope) 4. stroma (aqueous fluid) 5. thylakoid lumen (inside of thylakoid) 6. thylakoid membrane 7. granum (stack of thylakoids) 8. thylakoid (lamella) 9. starch 10. ribosome 11. plastidial DNA 12. plastoglobule (drop of lipids) The proteins that gather light for photosynthesis are embedded within cell membranes. The simplest way these are arranged is in photosynthetic bacteria, where these proteins are held within the plasma membrane. However, this membrane may be tightly folded into cylindrical sheets called thylakoids, or bunched up into round vesicles called intracytoplasmic membranes. These structures can fill most of the interior of a cell, giving the membrane a very large surface area and therefore increasing the amount of light that the bacteria can absorb. In plants and algae, photosynthesis takes place in organelles called chloroplasts. A typical plant cell contains about 10 to 100 chloroplasts. The chloroplast is enclosed by a membrane. This membrane is composed of a phospholipid inner membrane, a phospholipid outer membrane, and an intermembrane space between them. Within the membrane is an aqueous fluid called the stroma. The stroma contains stacks (grana) of thylakoids, which are the site of photosynthesis. The thylakoids are flattened disks,

bounded by a membrane with a lumen or thylakoid space within it. The site of photosynthesis is the thylakoid membrane, which contains integral and peripheral membrane protein complexes, including the pigments that absorb light energy, which form the photosystems. Plants absorb light primarily using the pigment chlorophyll, which is the reason that most plants have a green color. Besides chlorophyll, plants also use pigments such as carotenes and xanthophylls. Algae also use chlorophyll, but various other pigments are present as phycocyanin, carotenes, and xanthophylls in green algae, phycoerythrin in red algae (rhodophytes) and fucoxanthin in brown algae and diatoms resulting in a wide variety of colors. These pigments are embedded in plants and algae in special antenna-proteins. In such proteins all the pigments are ordered to work well together. Such a protein is also called a light-harvesting complex. Although all cells in the green parts of a plant have chloroplasts, most of the energy is captured in the leaves. The cells in the interior tissues of a leaf, called the mesophyll, can contain between 450,000 and 800,000 chloroplasts for every square millimeter of leaf. The surface of the leaf is uniformly coated with a water-resistant waxy cuticle that protects the leaf from excessive evaporation of water and decreases the absorption of ultraviolet or blue light to reduce heating. The transparent epidermis layer allows light to pass through to the palisade mesophyll cells where most of the photosynthesis takes place.

Light reactions

Light-dependent reactions of photosynthesis at the thylakoid membrane

In the light reactions, one molecule of the pigment chlorophyll absorbs one photon and loses one electron. This electron is passed to a modified form of chlorophyll called pheophytin, which passes the electron to a quinone molecule, allowing the start of a flow of electrons down an electron transport chain that leads to the ultimate reduction of NADP to NADPH. In addition, this creates a proton gradient across the chloroplast membrane; its dissipation is used by ATP synthase for the concomitant synthesis of ATP. The chlorophyll molecule regains the lost electron from a water molecule through a process called photolysis, which releases a dioxygen (O2) molecule. The overall equation for the light-dependent reactions under the conditions of non-cyclic electron flow in green plants is: 2 H2O + 2 NADP+ + 3 ADP + 3 Pi + light → 2 NADPH + 2 H+ + 3 ATP + O2 Not all wavelengths of light can support photosynthesis. The photosynthetic action spectrum depends on the type of accessory pigments present. For example, in green plants, the action spectrum resembles the absorption spectrum for chlorophylls and carotenoids with peaks for violet-blue and red light. In red algae, the action spectrum overlaps with the absorption spectrum of phycobilins for blue-green light, which allows these algae to grow in deeper waters that filter out the longer wavelengths used by green plants. The non-absorbed part of the light spectrum is what gives photosynthetic organisms their color (e.g., green plants, red algae, purple bacteria) and is the least effective for photosynthesis in the respective organisms.

Z scheme

The "Z scheme" In plants, light-dependent reactions occur in the thylakoid membranes of the chloroplasts and use light energy to synthesize ATP and NADPH. The light-dependent reaction has two forms: cyclic and non-cyclic. In the non-cyclic reaction, the photons are captured in the light-harvesting antenna complexes of photosystem II by chlorophyll and other accessory pigments (see diagram at right). When a chlorophyll molecule at the core of the photosystem II reaction center obtains sufficient excitation energy from the adjacent antenna pigments, an electron is transferred to the primary electron-acceptor molecule, pheophytin, through a process called photoinduced charge separation. These electrons are shuttled through an electron transport chain, the so called Z-scheme shown in the

diagram, that initially functions to generate a chemiosmotic potential across the membrane. An ATP synthase enzyme uses the chemiosmotic potential to make ATP during photophosphorylation, whereas NADPH is a product of the terminal redox reaction in the Z-scheme. The electron enters a chlorophyll molecule in Photosystem I. The electron is excited due to the light absorbed by the photosystem. A second electron carrier accepts the electron, which again is passed down lowering energies of electron acceptors. The energy created by the electron acceptors is used to move hydrogen ions across the thylakoid membrane into the lumen. The electron is used to reduce the coenzyme NADP, which has functions in the light-independent reaction. The cyclic reaction is similar to that of the non-cyclic, but differs in the form that it generates only ATP, and no reduced NADP (NADPH) is created. The cyclic reaction takes place only at photosystem I. Once the electron is displaced from the photosystem, the electron is passed down the electron acceptor molecules and returns back to photosystem I, from where it was emitted, hence the name cyclic reaction.

Water photolysis The NADPH is the main reducing agent in chloroplasts, providing a source of energetic electrons to other reactions. Its production leaves chlorophyll with a deficit of electrons (oxidized), which must be obtained from some other reducing agent. The excited electrons lost from chlorophyll in photosystem I are replaced from the electron transport chain by plastocyanin. However, since photosystem II includes the first steps of the Zscheme, an external source of electrons is required to reduce its oxidized chlorophyll a molecules. The source of electrons in green-plant and cyanobacterial photosynthesis is water. Two water molecules are oxidized by four successive charge-separation reactions by photosystem II to yield a molecule of diatomic oxygen and four hydrogen ions; the electron yielded in each step is transferred to a redox-active tyrosine residue that then reduces the photoxidized paired-chlorophyll a species called P680 that serves as the primary (light-driven) electron donor in the photosystem II reaction center. The oxidation of water is catalyzed in photosystem II by a redox-active structure that contains four manganese ions and a calcium ion; this oxygen-evolving complex binds two water molecules and stores the four oxidizing equivalents that are required to drive the wateroxidizing reaction. Photosystem II is the only known biological enzyme that carries out this oxidation of water. The hydrogen ions contribute to the transmembrane chemiosmotic potential that leads to ATP synthesis. Oxygen is a waste product of lightdependent reactions, but the majority of organisms on Earth use oxygen for cellular respiration, including photosynthetic organisms.

Light-independent reactions The Calvin Cycle In the Light-independent or dark reactions the enzyme RuBisCO captures CO2 from the atmosphere and in a process that requires the newly formed NADPH, called the CalvinBenson Cycle, releases three-carbon sugars, which are later combined to form sucrose and starch. The overall equation for the light-independent reactions in green plants is:

3 CO2 + 9 ATP + 6 NADPH + 6 H+ → C3H6O3-phosphate + 9 ADP + 8 Pi + 6 NADP+ + 3 H2O

Overview of the Calvin cycle and carbon fixation To be more specific, carbon fixation produces an intermediate product, which is then converted to the final carbohydrate products. The carbon skeletons produced by photosynthesis are then variously used to form other organic compounds, such as the building material cellulose, as precursors for lipid and amino acid biosynthesis, or as a fuel in cellular respiration. The latter occurs not only in plants but also in animals when the energy from plants gets passed through a food chain. The fixation or reduction of carbon dioxide is a process in which carbon dioxide combines with a five-carbon sugar, ribulose 1,5-bisphosphate (RuBP), to yield two molecules of a three-carbon compound, glycerate 3-phosphate (GP), also known as 3phosphoglycerate (PGA). GP, in the presence of ATP and NADPH from the lightdependent stages, is reduced to glyceraldehyde 3-phosphate (G3P). This product is also referred to as 3-phosphoglyceraldehyde (PGAL) or even as triose phosphate. Triose is a

3-carbon sugar. Most (5 out of 6 molecules) of the G3P produced is used to regenerate RuBP so the process can continue. The 1 out of 6 molecules of the triose phosphates not "recycled" often condense to form hexose phosphates, which ultimately yield sucrose, starch and cellulose. The sugars produced during carbon metabolism yield carbon skeletons that can be used for other metabolic reactions like the production of amino acids and lipids.

Carbon concentrating mechanisms On land

Overview of C4 carbon fixation

In hot and dry conditions, plants close their stomata to prevent the loss of water. Under these conditions, CO2 will decrease, and oxygen gas, produced by the light reactions of photosynthesis, will decrease in the stem, not leaves, causing an increase of photorespiration by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and decrease in carbon fixation. Some plants have evolved mechanisms to increase the CO2 concentration in the leaves under these conditions. C4 plants chemically fix carbon dioxide in the cells of the mesophyll by adding it to the three-carbon molecule phosphoenolpyruvate (PEP), a reaction catalyzed by an enzyme called PEP carboxylase and which creates the four-carbon organic acid, oxaloacetic acid. Oxaloacetic acid or malate synthesized by this process is then translocated to specialized bundle sheath cells where the enzyme, rubisco, and other Calvin cycle enzymes are located, and where CO2 released by decarboxylation of the four-carbon acids is then fixed by rubisco activity to the three-carbon sugar 3-phosphoglyceric acids. The physical separation of rubisco from the oxygen-generating light reactions reduces photorespiration and increases CO2 fixation and thus photosynthetic capacity of the leaf. C4 plants can produce more sugar than C3 plants in conditions of high light and temperature. Many important crop plants are C4 plants, including maize, sorghum, sugarcane, and millet. Plants that do not use PEP-carboxylase in carbon fixation are called C3 plants because the primary carboxylation reaction, catalyzed by rubisco, produces the three-carbon sugar 3phosphoglyceric acids directly in the Calvin-Benson cycle. Over 90% of plants use C3 carbon fixation, compared to 3% that use C4 carbon fixation. Xerophytes, such as cacti and most succulents, also use PEP carboxylase to capture carbon dioxide in a process called Crassulacean acid metabolism (CAM). In contrast to C4 metabolism, which physically separates the CO2 fixation to PEP from the Calvin cycle, CAM temporally separates these two processes. CAM plants have a different leaf anatomy from C3 plants, and fix the CO2 at night, when their stomata are open. CAM plants store the CO2 mostly in the form of malic acid via carboxylation of phosphoenolpyruvate to oxaloacetate, which is then reduced to malate. Decarboxylation of malate during the day releases CO2 inside the leaves, thus allowing carbon fixation to 3-phosphoglycerate by rubisco. 16,000 species of plants use CAM. In water Cyanobacteria possess carboxysomes which increase the concentration of CO2 around rubisco to increase the rate of photosynthesis. This operates by carbonic anhydrase producing hydrocarbonate ions (HCO3-) which are then pumped into the carboxysome, before being processed by a different carbonic anhydrase to produce CO2. Pyrenoids in algae and hornworts also act to concentrate CO2 around rubisco.

Order and kinetics The overall process of photosynthesis takes place in four stages. The first, energy transfer in antenna chlorophyll takes place in the femtosecond (1 femtosecond (fs) = 10−15 s) to picosecond (1 picosecond (ps) = 10−12 s) time scale. The next phase, the transfer of

electrons in photochemical reactions, takes place in the picosecond to nanosecond time scale (1 nanosecond (ns) = 10−9 s). The third phase, the electron transport chain and ATP synthesis, takes place on the microsecond (1 microsecond (μs) = 10−6 s) to millisecond (1 millisecond (ms) = 10−3 s) time scale. The final phase is carbon fixation and export of stable products and takes place in the millisecond to second time scale. The first three stages occur in the thylakoid membranes.

Efficiency Plants usually convert light into chemical energy with a photosynthetic efficiency of 3– 6%. Actual plants' photosynthetic efficiency varies with the frequency of the light being converted, light intensity, temperature and proportion of carbon dioxide in the atmosphere, and can vary from 0.1% to 8%. By comparison, solar panels convert light into electric energy at an efficiency of approximately 6–20% for mass-produced panels, and up to 41% in a research laboratory.

Evolution

Plant cells with visible chloroplasts (from a moss, Plagiomnium affine)

Early photosynthetic systems, such as those from green and purple sulfur and green and purple nonsulfur bacteria, are thought to have been anoxygenic, using various molecules as electron donors. Green and purple sulfur bacteria are thought to have used hydrogen and sulfur as an electron donor. Green nonsulfur bacteria used various amino and other organic acids. Purple nonsulfur bacteria used a variety of nonspecific organic molecules. The use of these molecules is consistent with the geological evidence that the atmosphere was highly reduced at that time. Fossils of what are thought to be filamentous photosynthetic organisms have been dated at 3.4 billion years old. The main source of oxygen in the atmosphere is oxygenic photosynthesis, and its first appearance is sometimes referred to as the oxygen catastrophe. Geological evidence suggests that oxygenic photosynthesis, such as that in cyanobacteria, became important during the Paleoproterozoic era around 2 billion years ago. Modern photosynthesis in plants and most photosynthetic prokaryotes is oxygenic. Oxygenic photosynthesis uses water as an electron donor, which is oxidized to molecular oxygen (O2) in the photosynthetic reaction center.

Symbiosis and the origin of chloroplasts Several groups of animals have formed symbiotic relationships with photosynthetic algae. These are most common in corals, sponges and sea anemones, possibly due to these animals having particularly simple body plans and large surface areas compared to their volumes. In addition, a few marine mollusks Elysia viridis and Elysia chlorotica also maintain a symbiotic relationship with chloroplasts they capture from the algae in their diet and then store in their bodies. This allows the molluscs to survive solely by photosynthesis for several months at a time. Some of the genes from the plant cell nucleus have even been transferred to the slugs, so that the chloroplasts can be supplied with proteins that they need to survive. An even closer form of symbiosis may explain the origin of chloroplasts. Chloroplasts have many similarities with photosynthetic bacteria, including a circular chromosome, prokaryotic-type ribosomes, and similar proteins in the photosynthetic reaction center. The endosymbiotic theory suggests that photosynthetic bacteria were acquired (by endocytosis) by early eukaryotic cells to form the first plant cells. Therefore, chloroplasts may be photosynthetic bacteria that adapted to life inside plant cells. Like mitochondria, chloroplasts still possess their own DNA, separate from the nuclear DNA of their plant host cells and the genes in this chloroplast DNA resemble those in cyanobacteria. DNA in chloroplasts codes for redox proteins such as photosynthetic reaction centers. The CoRR Hypothesis proposes that this Co-location is required for Redox Regulation.

Cyanobacteria and the evolution of photosynthesis The biochemical capacity to use water as the source for electrons in photosynthesis evolved once, in a common ancestor of extant cyanobacteria. The geological record

indicates that this transforming event took place early in Earth's history, at least 2450– 2320 million years ago (Ma), and possibly much earlier. Available evidence from geobiological studies of Archean (>2500 Ma) sedimentary rocks indicates that life existed 3500 Ma, but the question of when oxygenic photosynthesis evolved is still unanswered. A clear paleontological window on cyanobacterial evolution opened about 2000 Ma, revealing an already-diverse biota of blue-greens. Cyanobacteria remained principal primary producers throughout the Proterozoic Eon (2500–543 Ma), in part because the redox structure of the oceans favored photoautotrophs capable of nitrogen fixation. Green algae joined blue-greens as major primary producers on continental shelves near the end of the Proterozoic, but only with the Mesozoic (251–65 Ma) radiations of dinoflagellates, coccolithophorids, and diatoms did primary production in marine shelf waters take modern form. Cyanobacteria remain critical to marine ecosystems as primary producers in oceanic gyres, as agents of biological nitrogen fixation, and, in modified form, as the plastids of marine algae.

Discovery Although some of the steps in photosynthesis are still not completely understood, the overall photosynthetic equation has been known since the 19th century. Jan van Helmont began the research of the process in the mid-17th century when he carefully measured the mass of the soil used by a plant and the mass of the plant as it grew. After noticing that the soil mass changed very little, he hypothesized that the mass of the growing plant must come from the water, the only substance he added to the potted plant. His hypothesis was partially accurate—much of the gained mass also comes from carbon dioxide as well as water. However, this was a signaling point to the idea that the bulk of a plant's biomass comes from the inputs of photosynthesis, not the soil itself. Joseph Priestley, a chemist and minister, discovered that when he isolated a volume of air under an inverted jar, and burned a candle in it, the candle would burn out very quickly, much before it ran out of wax. He further discovered that a mouse could similarly "injure" air. He then showed that the air that had been "injured" by the candle and the mouse could be restored by a plant. In 1778, Jan Ingenhousz, court physician to the Austrian Empress, repeated Priestley's experiments. He discovered that it was the influence of sunlight on the plant that could cause it to revive a mouse in a matter of hours. In 1796, Jean Senebier, a Swiss pastor, botanist, and naturalist, demonstrated that green plants consume carbon dioxide and release oxygen under the influence of light. Soon afterwards, Nicolas-Théodore de Saussure showed that the increase in mass of the plant as it grows could not be due only to uptake of CO2, but also to the incorporation of water. Thus the basic reaction by which photosynthesis is used to produce food (such as glucose) was outlined.

Cornelis Van Niel made key discoveries explaining the chemistry of photosynthesis. By studying purple sulfur bacteria and green bacteria he was the first scientist to demonstrate that photosynthesis is a light-dependent redox reaction, in which hydrogen reduces carbon dioxide. Robert Emerson discovered two light reactions by testing plant productivity using different wavelengths of light. With the red alone, the light reactions were suppressed. When blue and red were combined, the output was much more substantial. Thus, there were two photosystems, one aborbing up to 600 nm wavelengths, the other up to 700. The former is known as PSII, the latter is PSI. PSI contains only chlorophyll a, PSII contains primarily chlorophyll a with most of the available chlorophyll b, among other pigments. Further experiments to prove that the oxygen developed during the photosynthesis of green plants came from water, were performed by Robert Hill in 1937 and 1939. He showed that isolated chloroplasts give off oxygen in the presence of unnatural reducing agents like iron oxalate, ferricyanide or benzoquinone after exposure to light. The Hill reaction is as follows: 2 H2O + 2 A + (light, chloroplasts) → 2 AH2 + O2 where A is the electron acceptor. Therefore, in light the electron acceptor is reduced and oxygen is evolved. Cyt b6, now known as a plastoquinone, is one electron acceptor. Samuel Ruben and Martin Kamen used radioactive isotopes to determine that the oxygen liberated in photosynthesis came from the water. Melvin Calvin and Andrew Benson, along with James Bassham, elucidated the path of carbon assimilation (the photosynthetic carbon reduction cycle) in plants. The carbon reduction cycle is known as the Calvin cycle, which inappropriately ignores the contribution of Bassham and Benson. Many scientists refer to the cycle as the CalvinBenson Cycle, Benson-Calvin, and some even call it the Calvin-Benson-Bassham (or CBB) Cycle. A Nobel Prize winning scientist, Rudolph A. Marcus, was able to discover the function and significance of the electron transport chain. Otto Heinrich Warburg and Dean Burk discovered the I-quantum photosynthesis reaction that splits the CO2, activated by the respiration.

Factors

The leaf is the primary site of photosynthesis in plants There are three main factors affecting photosynthesis and several corollary factors. The three main are: • • •

Light irradiance and wavelength Carbon dioxide concentration Temperature.

Light intensity (irradiance), wavelength and temperature In the early 20th century Frederick Frost Blackman along with Albert Einstein investigated the effects of light intensity (irradiance) and temperature on the rate of carbon assimilation. •

At constant temperature, the rate of carbon assimilation varies with irradiance, initially increasing as the irradiance increases. However at higher irradiance this relationship no longer holds and the rate of carbon assimilation reaches a plateau.

•

At constant irradiance, the rate of carbon assimilation increases as the temperature is increased over a limited range. This effect is only seen at high irradiance levels. At low irradiance, increasing the temperature has little influence on the rate of carbon assimilation.

These two experiments illustrate vital points: firstly, from research it is known that photochemical reactions are not generally affected by temperature. However, these experiments clearly show that temperature affects the rate of carbon assimilation, so there must be two sets of reactions in the full process of carbon assimilation. These are of course the light-dependent 'photochemical' stage and the light-independent, temperaturedependent stage. Second, Blackman's experiments illustrate the concept of limiting factors. Another limiting factor is the wavelength of light. Cyanobacteria, which reside several meters underwater, cannot receive the correct wavelengths required to cause photoinduced charge separation in conventional photosynthetic pigments. To combat this problem, a series of proteins with different pigments surround the reaction center.This unit is called a phycobilisome.

Carbon dioxide levels and photorespiration As carbon dioxide concentrations rise, the rate at which sugars are made by the lightindependent reactions increases until limited by other factors. RuBisCO, the enzyme that captures carbon dioxide in the light-independent reactions, has a binding affinity for both carbon dioxide and oxygen. When the concentration of carbon dioxide is high, RuBisCO will fix carbon dioxide. However, if the carbon dioxide concentration is low, RuBisCO will bind oxygen instead of carbon dioxide. This process, called photorespiration, uses energy, but does not produce sugars. RuBisCO oxygenase activity is disadvantageous to plants for several reasons: 1. One product of oxygenase activity is phosphoglycolate (2 carbon) instead of 3phosphoglycerate (3 carbon). Phosphoglycolate cannot be metabolized by the Calvin-Benson cycle and represents carbon lost from the cycle. A high oxygenase activity, therefore, drains the sugars that are required to recycle ribulose 5bisphosphate and for the continuation of the Calvin-Benson cycle. 2. Phosphoglycolate is quickly metabolized to glycolate that is toxic to a plant at a high concentration; it inhibits photosynthesis. 3. Salvaging glycolate is an energetically expensive process that uses the glycolate pathway and only 75% of the carbon is returned to the Calvin-Benson cycle as 3phosphoglycerate. The reactions also produce ammonia (NH3) which is able to diffuse out of the plant leading to a loss of nitrogen. A highly simplified summary is: 2 glycolate + ATP → 3-phosphoglycerate + carbon dioxide + ADP +NH3

The salvaging pathway for the products of RuBisCO oxygenase activity is more commonly known as photorespiration, since it is characterized by light-dependent oxygen consumption and the release of carbon dioxide.

Chapter- 3

Cellular Respiration

Cellular respiration in a typical eukaryotic cell Cellular respiration (also known as oxidative metabolism) is the set of the metabolic reactions and processes that take place in the cells of organisms to convert biochemical energy from nutrients into adenosine triphosphate (ATP), and then release waste products. The reactions involved in respiration are catabolic reactions that involve the oxidation of one molecule and the reduction of another. Respiration is one of the key ways a cell gains useful energy to fuel cellular reformations. Nutrients commonly used by animal and plant cells in respiration include glucose, amino acids and fatty acids, and a common oxidizing agent (electron acceptor) is molecular oxygen (O2). Bacteria and archaea can also be lithotrophs and these organisms may

respire using a broad range of inorganic molecules as electron donors and acceptors, such as sulfur, metal ions, methane or hydrogen. Organisms that use oxygen as a final electron acceptor in respiration are described as aerobic, while those that do not are referred to as anaerobic. The energy released in respiration is used to synthesize ATP to store this energy. The energy stored in ATP can then be used to drive processes requiring energy, including biosynthesis, locomotion or transportation of molecules across cell membranes.

Aerobic respiration

Aerobic respiration (red arrows) is the main means by which both plants and animals utilize energy in the form of organic compounds that was previously created through photosynthesis (green arrow).

Aerobic respiration requires oxygen in order to generate energy (ATP). Although carbohydrates, fats, and proteins can all be processed and consumed as reactant, it is the preferred method of pyruvate breakdown in glycolysis and requires that pyruvate enter the mitochondrion in order to be fully oxidized by the Krebs cycle. The product of this process is energy in the form of ATP (Adenosine triphosphate), by substrate-level phosphorylation, NADH and FADH2 Simplified reaction:

C6H12O6 (aq) + 6 O2 (g) → 6 CO2 (g) + 6 H2O (l) ΔG = -2880 kJ per mole of C6H12O6

The negative ΔG indicates that the reaction can happen spontaneously The reducing potential of NADH and FADH2 is converted to more ATP through an electron transport chain with oxygen as the "terminal electron acceptor". Most of the ATP produced by aerobic cellular respiration is made by oxidative phosphorylation. This works by the energy released in the consumption of pyruvate being used to create a chemiosmotic potential by pumping protons across a membrane. This potential is then used to drive ATP synthase and produce ATP from ADP. Biology textbooks often state that 38 ATP molecules can be made per oxidised glucose molecule during cellular respiration (2 from glycolysis, 2 from the Krebs cycle, and about 34 from the electron transport system). However, this maximum yield is never quite reached due to losses (leaky membranes) as well as the cost of moving pyruvate and ADP into the mitochondrial matrix and current estimates range around 29 to 30 ATP per glucose. Aerobic metabolism is 19 times more efficient than anaerobic metabolism (which yields 2 mol ATP per 1 mol glucose). They share the initial pathway of glycolysis but aerobic metabolism continues with the Krebs cycle and oxidative phosphorylation. The post glycolytic reactions take place in the mitochondria in eukaryotic cells, and in the cytoplasm in prokaryotic cells.

Glycolysis Glycolysis is a metabolic pathway that is found in the cytosol of cells in all living organisms and is anaerobic (that is, oxygen is not required). The process converts one molecule of glucose into two molecules of pyruvate(pyruvic acid), it makes energy in the form of two net molecules of ATP. Four molecules of ATP per glucose are actually produced; however, two are consumed for the preparatory phase. The initial phosphorylation of glucose is required to destabilize the molecule for cleavage into two triose sugars. During the pay-off phase of glycolysis, four phosphate groups are transferred to ADP by substrate-level phosphorylation to make four ATP, and two NADH are produced when the triose sugars are oxidized. The overall reaction can be expressed this way: Glucose + 2 NAD+ + 2 Pi + 2 ADP → 2 pyruvate + 2 NADH + 2 ATP + 2 H+ + 2 H2O

Oxidative decarboxylation of pyruvate The pyruvate is oxidized to acetyl-CoA and CO2 by the Pyruvate dehydrogenase complex, a cluster of enzymes—multiple copies of each of three enzymes—located in the mitochondria of eukaryotic cells and in the cytosol of prokaryotes. In the process one molecule of NADH is formed per pyruvate oxidized, and 3 molecules of ATP are formed for each molecule of pyruvate(pyruvic acid.) This step is also known as the link reaction, as it links glycolysis and the Krebs cycle.

Citric acid cycle This is also called the Krebs cycle or the tricarboxylic acid cycle. When oxygen is present, acetyl-CoA is produced from the pyruvate molecules created from glycolysis. Once acetyl-CoA is formed, two processes can occur, aerobic or anaerobic respiration. When oxygen is present, the mitochondria will undergo aerobic respiration which leads to the Krebs cycle. However, if oxygen is not present, fermentation of the pyruvate molecule will occur. In the presence of oxygen, when acetyl-CoA is produced, the molecule then enters the citric acid cycle (Krebs cycle) inside the mitochondrial matrix, and gets oxidized to CO2 while at the same time reducing NAD to NADH. NADH can be used by the electron transport chain to create further ATP as part of oxidative phosphorylation. To fully oxidize the equivalent of one glucose molecule, two acetylCoA must be metabolized by the Krebs cycle. Two waste products, H2O and CO2, are created during this cycle. The citric acid cycle is an 8-step process involving 18 different enzymes. Throughout the entire cycle, acetyl-CoA changes into citrate, cis-aconitate, isocitrate, α-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, and finally, oxaloacetate. The net energy gain from one cycle is 3 NADH, 1 FADH2, and 1 GTP; the GTP may subsequently be used to produce ATP. Thus, the total energy yield from one whole glucose molecule (2 pyruvate molecules) is 6 NADH, 2 FADH2, and 2 ATP.

Oxidative phosphorylation In eukaryotes, oxidative phosphorylation occurs in the mitochondrial cristae. It comprises the electron transport chain that establishes a proton gradient (chemiosmotic potential) across the inner membrane by oxidizing the NADH produced from the Krebs cycle. ATP is synthesised by the ATP synthase enzyme when the chemiosmotic gradient is used to drive the phosphorylation of ADP. The electrons are finally transferred to exogenous oxygen and, with the addition of two protons, water is formed. the table below are for one glucose molecule being fully oxidized into carbon dioxide. It is assumed that all the reduced coenzymes are oxidized by the electron transport chain and used for oxidative phosphorylation.

Step

coenzyme yield

ATP yield

Source of ATP

Glycolysis preparatory phase

-2

Glycolysis pay-off phase

2 NADH

4 6

Phosphorylation of glucose and fructose 6phosphate uses two ATP from the cytoplasm. Substrate-level phosphorylation Oxidative phosphorylation

2 NADH

6

Oxidative phosphorylation

6 NADH 2 FADH2

2 18 4

Substrate-level phosphorylation Oxidative phosphorylation Oxidative phosphorylation

Oxidative decarboxylation of pyruvate Krebs cycle

Total yield

From the complete oxidation of one glucose 38 ATP molecule to carbon dioxide and oxidation of all the reduced coenzymes.

Although there is a theoretical yield of 38 ATP molecules per glucose during cellular respiration, such conditions are generally not realized due to losses such as the cost of moving pyruvate (from glycolysis), phosphate, and ADP (substrates for ATP synthesis) into the mitochondria. All are actively transported using carriers that utilise the stored energy in the proton electrochemical gradient. • • •

Pyruvate is taken up by a specific, low km transporter to bring it into the mitochondrial matrix for oxidation by the pyruvate dehydrogenase complex. The phosphate translocase is a symporter and the driving force for moving phosphate ions into the mitochondria is the proton motive force. The adenine nucleotide carrier is an antiporter and exchanges ADP and ATP across the inner membrane. The driving force is due to the ATP (-4) having a more negative charge than the ADP (-3) and thus it dissipates some of the electrical component of the proton electrochemical gradient.

The outcome of these transport processes using the proton electrochemical gradient is that more than 3 H+ are needed to make 1 ATP. Obviously this reduces the theoretical efficiency of the whole process and the likely maximum is closer to 28-30 ATP molecules. In practice the efficiency may be even lower due to the inner membrane of the mitochondria being slightly leaky to protons. Other factors may also dissipate the proton gradient creating an apparently leaky mitochondria. An uncoupling protein known as thermogenin is expressed in some cell types and is a channel that can transport protons. When this protein is active in the inner membrane it short circuits the coupling between the electron transport chain and ATP synthesis. The potential energy from the proton gradient is not used to make ATP but generates heat. This is particularly important in brown fat thermogenesis of newborn and hibernating mammals.

Fermentation Without oxygen, pyruvate (pyruvic acid) is not metabolized by cellular respiration but undergoes a process of fermentation. The pyruvate is not transported into the mitochondrion, but remains in the cytoplasm, where it is converted to waste products that may be removed from the cell. This serves the purpose of oxidizing the electron carriers so that they can perform glycolysis again and removing the excess pyruvate. This waste product varies depending on the organism. In skeletal muscles, the waste product is lactic acid. This type of fermentation is called lactic acid fermentation. In yeast, the waste products are ethanol and carbon dioxide. This type of fermentation is known as alcoholic or ethanol fermentation. The ATP generated in this process is made by substrate-level phosphorylation, which does not require oxygen. Fermentation is less efficient at using the energy from glucose since 2 ATP are produced per glucose, compared to the 38 ATP per glucose produced by aerobic respiration. This is because the waste products of fermentation still contain plenty of energy. Ethanol, for example, can be used in gasoline (petrol) solutions. Glycolytic ATP, however, is created more quickly. For prokaryotes to continue a rapid growth rate when they are shifted from an aerobic environment to an anaerobic environment, they must increase the rate of the glycolytic reactions. For multicellular organisms, during short bursts of strenuous activity, muscle cells use fermentation to supplement the ATP production from the slower aerobic respiration, so fermentation may be used by a cell even before the oxygen levels are depleted, as is the case in sports that do not require athletes to pace themselves, such as sprinting.

Anaerobic Respiration Anaerobic respiration is used by some microorganisms in which neither oxygen (aerobic respiration) nor pyruvate or pyruvate derivative (fermentation) is the final electron acceptor. Rather, an inorganic acceptor (for example, Sulfur) is used.

Chapter- 4

Cholesterol

Cholesterol

IUPAC name (3β)-cholest-5-en-3-ol Other names (10R,13R)-10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4, 7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta [a]phenanthren-3-ol Identifiers CAS number 57-88-5 PubChem 5997

ChemSpider UNII

5775 97C5T2UQ7J Properties Molecular formula C27H46O Molar mass 386.65 g/mol Appearance white crystalline powder Density 1.052 g/cm3 Melting point 148–150 °C Boiling point 360 °C (decomposes) Solubility in water 0.095 mg/L (30 °C) soluble in acetone, benzene, chloroform, Solubility ethanol, ether, hexane, isopropyl myristate, methanol

Microscopic appearance of cholesterol crystals in water. Photo taken under polarized light. Cholesterol is a waxy steroid metabolite found in the cell membranes and transported in the blood plasma of all animals. It is an essential structural component of mammalian cell membranes, where it is required to establish proper membrane permeability and fluidity.

In addition, cholesterol is an important component for the manufacture of bile acids, steroid hormones, and fat-soluble vitamins including Vitamin A, Vitamin D, Vitamin E, and Vitamin K. Cholesterol is the principal sterol synthesized by animals, but small quantities are synthesized in other eukaryotes, such as plants and fungi. It is almost completely absent among prokaryotes, which include bacteria. Although cholesterol is an important and necessary molecule for animals, a high level of serum cholesterol is an indicator for diseases such as heart disease. The name cholesterol originates from the Greek chole- (bile) and stereos (solid), and the chemical suffix -ol for an alcohol, as François Poulletier de la Salle first identified cholesterol in solid form in gallstones, in 1769. However, it was only in 1815 that chemist Eugène Chevreul named the compound "cholesterine".

Physiology Since cholesterol is essential for all animal life, it is primarily synthesized from simpler substances within the body. However, high levels in blood circulation, depending on how it is transported within lipoproteins, are strongly associated with progression of atherosclerosis. For a person of about 68 kg (150 pounds), typical total body cholesterol synthesis is about 1 g (1,000 mg) per day, and total body content is about 35 g. Typical daily additional dietary intake in the United States is 200–300 mg. The body compensates for cholesterol intake by reducing the amount synthesized. Cholesterol is recycled. It is excreted by the liver via the bile into the digestive tract. Typically about 50% of the excreted cholesterol is reabsorbed by the small bowel back into the bloodstream. Phytosterols can compete cholesterol reabsorption in intestinal tract back into the intestinal lumen for elimination.

Function Cholesterol is required to build and maintain membranes; it modulates membrane fluidity over the range of physiological temperatures. The hydroxyl group on cholesterol interacts with the polar head groups of the membrane phospholipids and sphingolipids, while the bulky steroid and the hydrocarbon chain are embedded in the membrane, alongside the nonpolar fatty acid chain of the other lipids. In this structural role, cholesterol reduces the permeability of the plasma membrane to protons (positive hydrogen ions) and sodium ions. Within the cell membrane, cholesterol also functions in intracellular transport, cell signaling and nerve conduction. Cholesterol is essential for the structure and function of invaginated caveolae and clathrin-coated pits, including caveola-dependent and clathrindependent endocytosis. The role of cholesterol in such endocytosis can be investigated by using methyl beta cyclodextrin (MβCD) to remove cholesterol from the plasma membrane. Recently, cholesterol has also been implicated in cell signaling processes, assisting in the formation of lipid rafts in the plasma membrane. In many neurons, a

myelin sheath, rich in cholesterol, since it is derived from compacted layers of Schwann cell membrane, provides insulation for more efficient conduction of impulses. Within cells, cholesterol is the precursor molecule in several biochemical pathways. In the liver, cholesterol is converted to bile, which is then stored in the gallbladder. Bile contains bile salts, which solubilize fats in the digestive tract and aid in the intestinal absorption of fat molecules as well as the fat-soluble vitamins, Vitamin A, Vitamin D, Vitamin E, and Vitamin K. Cholesterol is an important precursor molecule for the synthesis of Vitamin D and the steroid hormones, including the adrenal gland hormones cortisol and aldosterone as well as the sex hormones progesterone, estrogens, and testosterone, and their derivatives. Some research indicates that cholesterol may act as an antioxidant.

Dietary sources Animal fats are complex mixtures of triglycerides, with lesser amounts of phospholipids and cholesterol. As a consequence, all foods containing animal fat contain cholesterol to varying extents. Major dietary sources of cholesterol include cheese, egg yolks, beef, pork, poultry, and shrimp. Human breast milk also contains significant quantities of cholesterol. The amount of cholesterol present in plant-based food sources is generally much lower than animal based sources. In addition, plant products such as flax seeds and peanuts contain cholesterol-like compounds called phytosterols, which are suggested to help lower serum cholesterol levels. Total fat intake, especially saturated fat and trans fat, plays a larger role in blood cholesterol than intake of cholesterol itself. Saturated fat is present in full fat dairy products, animal fats, several types of oil and chocolate. Trans fats are typically derived from the partial hydrogenation of unsaturated fats, and do not occur in significant amounts in nature. Trans fat is most often encountered in margarine and hydrogenated vegetable fat, and consequently in many fast foods, snack foods, and fried or baked goods. A change in diet in addition to other lifestyle modifications may help reduce blood cholesterol. Avoiding animal products may decrease the cholesterol levels in the body not only by reducing the quantity of cholesterol consumed but also by reducing the quantity of cholesterol synthesized. Those wishing to reduce their cholesterol through a change in diet should aim to consume less than 7% of their daily calories from saturated fat and less than 200 mg of cholesterol per day. The view that a change in diet (to be specific, a reduction in dietary fat and cholesterol) can lower blood cholesterol levels, and thus reduce the likelihood of development of, among others, coronary artery disease (CAD) leading to coronary heart disease (CHD)

has been challenged. An alternative view is that any reductions to dietary cholesterol intake are counteracted by the organs such as the liver, which will increase or decrease production of cholesterol to keep blood cholesterol levels constant. Another view is that although saturated fat and dietary cholesterol also raise blood cholesterol, these nutrients are not as effective at doing this as is animal protein.

Synthesis All animal cells manufacture cholesterol with relative production rates varying by cell type and organ function. About 20–25% of total daily cholesterol production occurs in the liver; other sites of higher synthesis rates include the intestines, adrenal glands, and reproductive organs. Synthesis within the body starts with one molecule of acetyl CoA and one molecule of acetoacetyl-CoA, which are dehydrated to form 3-hydroxy-3methylglutaryl CoA (HMG-CoA). This molecule is then reduced to mevalonate by the enzyme HMG-CoA reductase. This step is the regulated, rate-limiting and irreversible step in cholesterol synthesis and is the site of action for the statin drugs (HMG-CoA reductase competitive inhibitors). Mevalonate is then converted to 3-isopentenyl pyrophosphate in three reactions that require ATP. This molecule is decarboxylated to isopentenyl pyrophosphate, which is a key metabolite for various biological reactions. Three molecules of isopentenyl pyrophosphate condense to form farnesyl pyrophosphate through the action of geranyl transferase. Two molecules of farnesyl pyrophosphate then condense to form squalene by the action of squalene synthase in the endoplasmic reticulum. Oxidosqualene cyclase then cyclizes squalene to form lanosterol. Finally, lanosterol is then converted to cholesterol. Konrad Bloch and Feodor Lynen shared the Nobel Prize in Physiology or Medicine in 1964 for their discoveries concerning the mechanism and regulation of cholesterol and fatty acid metabolism.

Regulation of cholesterol synthesis Biosynthesis of cholesterol is directly regulated by the cholesterol levels present, though the homeostatic mechanisms involved are only partly understood. A higher intake from food leads to a net decrease in endogenous production, whereas lower intake from food has the opposite effect. The main regulatory mechanism is the sensing of intracellular cholesterol in the endoplasmic reticulum by the protein SREBP (sterol regulatory element-binding protein 1 and 2). In the presence of cholesterol, SREBP is bound to two other proteins: SCAP (SREBP-cleavage-activating protein) and Insig1. When cholesterol levels fall, Insig-1 dissociates from the SREBP-SCAP complex, allowing the complex to migrate to the Golgi apparatus, where SREBP is cleaved by S1P and S2P (site-1 and -2 protease), two enzymes that are activated by SCAP when cholesterol levels are low. The cleaved SREBP then migrates to the nucleus and acts as a transcription factor to bind to the SRE (sterol regulatory element), which stimulates the transcription of many genes. Among these are the low-density lipoprotein (LDL) receptor and HMG-CoA reductase. The former scavenges circulating LDL from the bloodstream, whereas HMG-CoA

reductase leads to an increase of endogenous production of cholesterol. A large part of this signaling pathway was clarified by Dr. Michael S. Brown and Dr. Joseph L. Goldstein in the 1970s. In 1985, they received the Nobel Prize in Physiology or Medicine for their work. Their subsequent work shows how the SREBP pathway regulates expression of many genes that control lipid formation and metabolism and body fuel allocation. Cholesterol synthesis can be turned off when cholesterol levels are high, as well. HMG CoA reductase contains both a cytosolic domain (responsible for its catalytic function) and a membrane domain. The membrane domain functions to sense signals for its degradation. Increasing concentrations of cholesterol (and other sterols) cause a change in this domain's oligomerization state, which makes it more susceptible to destruction by the proteosome. This enzyme's activity can also be reduced by phosphorylation by an AMPactivated protein kinase. Because this kinase is activated by AMP, which is produced when ATP is hydrolyzed, it follows that cholesterol synthesis is halted when ATP levels are low.

Plasma transport and regulation of absorption Cholesterol is only slightly soluble in water; it can dissolve and travel in the water-based bloodstream at exceedingly small concentrations. Since cholesterol is insoluble in blood, it is transported in the circulatory system within lipoproteins, complex spherical particles which have an exterior composed of amphiphilic proteins and lipids whose outwardfacing surfaces are water-soluble and inward-facing surfaces are lipid-soluble; triglycerides and cholesterol esters are carried internally. Phospholipids and cholesterol, being amphipathic, are transported in the surface monolayer of the lipoprotein particle. In addition to providing a soluble means for transporting cholesterol through the blood, lipoproteins have cell-targeting signals that direct the lipids they carry to certain tissues. For this reason, there are several types of lipoproteins within blood called, in order of increasing density, chylomicrons, very-low-density lipoprotein (VLDL), intermediatedensity lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). The more cholesterol and less protein a lipoprotein has the less dense it is. The cholesterol within all the various lipoproteins is identical, although some cholesterol is carried as the "free" alcohol and some is carried as fatty acyl esters referred to as cholesterol esters. However, the different lipoproteins contain apolipoproteins, which serve as ligands for specific receptors on cell membranes. In this way, the lipoprotein particles are molecular addresses that determine the start- and endpoints for cholesterol transport. Chylomicrons, the least dense type of cholesterol transport molecules, contain apolipoprotein B-48, apolipoprotein C, and apolipoprotein E in their shells. Chylomicrons are the transporters that carry fats from the intestine to muscle and other tissues that need fatty acids for energy or fat production. Cholesterol which is not used by muscles remains in more cholesterol-rich chylomicron remnants, which are taken up from the bloodstream by the liver.

VLDL molecules are produced by the liver and contain excess triacylglycerol and cholesterol that is not required by the liver for synthesis of bile acids. These molecules contain apolipoprotein B100 and apolipoprotein E in their shell. During transport in the bloodstream, the blood vessel cleave and absorb more triacylglycerol from IDL molecules, which contain an even higher percentage of cholesterol. The IDL molecules have two possible fates: Half are into metabolism by HTGL, taken up by the LDL receptor on the liver cell surfaces and the other half continue to lose triacylglycerols in the bloodstream until form LDL molecules, which have the highest percentage of cholesterol within them. LDL molecules, therefore, are the major carriers of cholesterol in the blood, and each one contains approximately 1,500 molecules of cholesterol ester. The shell of the LDL molecule contains just one molecule of apolipoprotein B100, which is recognized by the LDL receptor in peripheral tissues. Upon binding of apolipoprotein B100, many LDL receptors become localized in clathrin-coated pits. Both the LDL and its receptor are internalized by endocytosis to form a vesicle within the cell. The vesicle then fuses with a lysosome, which has an enzyme called lysosomal acid lipase that hydrolyzes the cholesterol esters. Now within the cell, the cholesterol can be used for membrane biosynthesis or esterified and stored within the cell, so as to not interfere with cell membranes. Synthesis of the LDL receptor is regulated by SREBP, the same regulatory protein as was used to control synthesis of cholesterol de novo in response to cholesterol presence in the cell. When the cell has abundant cholesterol, LDL receptor synthesis is blocked so that new cholesterol in the form of LDL molecules cannot be taken up. On the converse, more LDL receptors are made when the cell is deficient in cholesterol. When this system is deregulated, many LDL molecules appear in the blood without receptors on the peripheral tissues. These LDL molecules are oxidized and taken up by macrophages, which become engorged and form foam cells. These cells often become trapped in the walls of blood vessels and contribute to artherosclerotic plaque formation. Differences in cholesterol homeostasis affect the development of early atherosclerosis (carotid intimamedia thickness). These plaques are the main causes of heart attacks, strokes, and other serious medical problems, leading to the association of so-called LDL cholesterol (actually a lipoprotein) with "bad" cholesterol. Also, HDL particles are thought to transport cholesterol back to the liver for excretion or to other tissues that use cholesterol to synthesize hormones in a process known as reverse cholesterol transport (RCT). Having large numbers of large HDL particles correlates with better health outcomes. In contrast, having small numbers of large HDL particles is independently associated with atheromatous disease progression within the arteries.

Metabolism, recycling and excretion Cholesterol is susceptible to oxidation and easily forms oxygenated derivatives known as oxysterols that can be formed by three different mechanisms, autoxidation, oxidation secondary to lipid peroxidation and by cholesterol metabolizing enzymes. A great interest

in oxysterols arose when it was shown that they exerted inhibitory actions on cholesterol biosynthesis, a finding that subsequently became known as the “oxysterol hypothesis”. Additional roles for oxysterols in human physiology include their participation in bile acid biosynthesis, their function as transport forms of cholesterol and as regulators of gene transcription. Cholesterol is oxidized by the liver into a variety of bile acids. These in turn are conjugated with glycine, taurine, glucuronic acid, or sulfate. A mixture of conjugated and non-conjugated bile acids along with cholesterol itself is excreted from the liver into the bile. Approximately 95% of the bile acids are reabsorbed from the intestines and the remainder lost in the feces. The excretion and reabsorption of bile acids forms the basis of the enterohepatic circulation which is essential for the digestion and absorption of dietary fats. Under certain circumstances, when more concentrated, as in the gallbladder, cholesterol crystallises and is the major constituent of most gallstones, although lecithin and bilirubin gallstones also occur less frequently.

Significance Hypercholesterolemia According to the lipid hypothesis, abnormal cholesterol levels (hypercholesterolemia)— that is, higher concentrations of LDL and lower concentrations of functional HDL—are strongly associated with cardiovascular disease because these promote atheroma development in arteries (atherosclerosis). This disease process leads to myocardial infarction (heart attack), stroke, and peripheral vascular disease. Since higher blood LDL, especially higher LDL particle concentrations and smaller LDL particle size, contribute to this process more than the cholesterol content of the LDL particles, LDL particles are often termed "bad cholesterol" because they have been linked to atheroma formation. On the other hand, high concentrations of functional HDL, which can remove cholesterol from cells and atheroma, offer protection and are sometimes referred to as "good cholesterol". These balances are mostly genetically determined but can be changed by body build, medications, food choices, and other factors. Conditions with elevated concentrations of oxidized LDL particles, especially "small dense LDL" (sdLDL) particles, are associated with atheroma formation in the walls of arteries, a condition known as atherosclerosis, which is the principal cause of coronary heart disease and other forms of cardiovascular disease. In contrast, HDL particles (especially large HDL) have been identified as a mechanism by which cholesterol and inflammatory mediators can be removed from atheroma. Increased concentrations of HDL correlate with lower rates of atheroma progressions and even regression. A 2007 study pooling data on almost 900,000 subjects in 61 cohorts demonstrated that blood total cholesterol levels have an exponential effect on cardiovascular and total mortality, with the association more pronounced in younger subjects. Still, because cardiovascular disease is relatively rare in the younger population, the impact of high cholesterol on health is still larger in older people. Elevated levels of the lipoprotein fractions, LDL, IDL and VLDL are regarded as atherogenic (prone to cause atherosclerosis). Levels of these fractions, rather than the

total cholesterol level, correlate with the extent and progress of atherosclerosis. On the converse, the total cholesterol can be within normal limits, yet be made up primarily of small LDL and small HDL particles, under which conditions atheroma growth rates would still be high. In contrast, however, if LDL particle number is low (mostly large particles) and a large percentage of the HDL particles are large, then atheroma growth rates are usually low, even negative, for any given total cholesterol concentration. Recently, a post-hoc analysis of the IDEAL and the EPIC prospective studies found an association between high levels of HDL cholesterol (adjusted for apolipoprotein A-I and apolipoprotein B) and increased risk of cardiovascular disease, casting doubt on the cardioprotective role of "good cholesterol" Multiple human trials utilizing HMG-CoA reductase inhibitors, known as statins, have repeatedly confirmed that changing lipoprotein transport patterns from unhealthy to healthier patterns significantly lowers cardiovascular disease event rates, even for people with cholesterol values currently considered low for adults. As a result, people with a history of cardiovascular disease may derive benefit from statins irrespective of their cholesterol levels, and in men without cardiovascular disease there is benefit from lowering abnormally high cholesterol levels ("primary prevention"). Primary prevention in women is practiced only by extension of the findings in studies on men, since in women, none of the large statin trials has shown a reduction in overall mortality or in cardiovascular end points. The 1987 report of National Cholesterol Education Program, Adult Treatment Panels suggest the total blood cholesterol level should be: < 200 mg/dL normal blood cholesterol, 200–239 mg/dL borderline-high, > 240 mg/dL high cholesterol. The American Heart Association provides a similar set of guidelines for total (fasting) blood cholesterol levels and risk for heart disease: Level mg/dL Level mmol/L Interpretation < 200

< 5.0

Desirable level corresponding to lower risk for heart disease

200–240

5.2–6.2

Borderline high risk

> 240

> 6.2

High risk

However, as today's testing methods determine LDL ("bad") and HDL ("good") cholesterol separately, this simplistic view has become somewhat outdated. The desirable LDL level is considered to be less than 100 mg/dL (2.6 mmol/L), although a newer upper limit of 70 mg/dL (1.8 mmol/L) can be considered in higher risk individuals based on some of the above-mentioned trials. A ratio of total cholesterol to HDL—another useful measure—of far less than 5:1 is thought to be healthier. Of note, typical LDL values for children before fatty streaks begin to develop is 35 mg/dL. Total cholesterol is defined as the sum of HDL, LDL, and VLDL. Usually, only the total, HDL, and triglycerides are measured. For cost reasons, the VLDL is usually estimated as one-fifth of the triglycerides and the LDL is estimated using the Friedewald formula (or a variant): estimated LDL = [total cholesterol] − [total HDL] − [estimated VLDL]. VLDL

can be calculated by dividing total triglycerides by 5. Direct LDL measures are used when triglycerides exceed 400 mg/dL. The estimated VLDL and LDL have more error when triglycerides are above 400 mg/dL. Given the well-recognized role of cholesterol in cardiovascular disease, it is surprising that some studies have shown an inverse correlation between cholesterol levels and mortality. A 2009 study of patients with acute coronary syndromes found an association of hypercholesterolemia with better mortality outcomes. In the Framingham Heart Study, in subjects over 50 years of age they found an 11% increase overall and 14% increase in CVD mortality per 1 mg/dL per year drop in total cholesterol levels. The researchers attributed this phenomenon to the fact that people with severe chronic diseases or cancer tend to have below-normal cholesterol levels. This explanation is not supported by the Vorarlberg Health Monitoring and Promotion Programme, in which men of all ages and women over 50 with very low cholesterol were increasingly likely to die of cancer, liver diseases, and mental diseases. This result indicates that the low-cholesterol effect occurs even among younger respondents, contradicting the previous assessment among cohorts of older people that this is a proxy or marker for frailty occurring with age. The vast majority of doctors and medical scientists consider that there is a link between cholesterol and atherosclerosis as discussed above; a small group of scientists, united in The International Network of Cholesterol Skeptics, questions the link.

Hypocholesterolemia Abnormally low levels of cholesterol are termed hypocholesterolemia. Research into the causes of this state is relatively limited, but some studies suggest a link with depression, cancer, and cerebral hemorrhage. In general, the low cholesterol levels seem to be a consequence of an underlying illness, rather than a cause.

Cholesterol testing It is recommended by the American Heart Association to test cholesterol every 5 years for people aged 20 years or older. A blood sample after 12-hour fasting is taken by a doctor or a home cholesterolmonitoring device to determine a lipoprotein profile. This measures total cholesterol, LDL (bad) cholesterol, HDL (good) cholesterol, and triglycerides. It is recommended to test cholesterol at least every 5 years if a person has total cholesterol of 200 mg/dL or more, or if a man over age 45 or a woman over age 50 has HDL (good) cholesterol less than 40 mg/dL, or there are other risk factors for heart disease and stroke. (In different countries measurements are given in mg/dL or mmol/L; 1 mmol/L is 38.665 mg/dL.)

Interactive pathway map

Statin Pathway

Cholesteric liquid crystals Some cholesterol derivatives, (among other simple cholesteric lipids) are known to generate the liquid crystalline cholesteric phase. The cholesteric phase is in fact a chiral nematic phase, and changes colour when its temperature changes. This makes cholesterol derivatives useful for indicating temperature in liquid crystal display thermometers and in temperature-sensitive paints.

Chapter- 5

Eicosanoid

Pathways in biosynthesis of eicosanoids from arachidonic acid: there are parallel paths from EPA & DGLA. In biochemistry, eicosanoids are signaling molecules made by oxidation of twentycarbon essential fatty acids, (EFAs). They exert complex control over many bodily systems, mainly in inflammation or immunity, and as messengers in the central nervous system. The networks of controls that depend upon eicosanoids are among the most complex in the human body. Eicosanoids derive from either omega-3 (ω-3) or omega-6 (ω-6) EFAs. The ω-6 eicosanoids are generally pro-inflammatory; ω-3's are much less so. The amounts and balance of these fats in a person's diet will affect the body's eicosanoid-controlled functions, with effects on cardiovascular disease, triglycerides, blood pressure, and arthritis. Anti-inflammatory drugs such as aspirin and other NSAIDs act by downregulating eicosanoid synthesis.

There are four families of eicosanoids—the prostaglandins, prostacyclins, the thromboxanes and the leukotrienes. For each, there are two or three separate series, derived either from an ω-3 or ω-6 EFA. These series' different activities largely explain the health effects of ω-3 and ω-6 fats.

Nomenclature "Eicosanoid" (eicosa-, Greek for "twenty") is the collective term for oxygenated derivatives of three different 20-carbon essential fatty acids: • • •

Eicosapentaenoic acid (EPA), an ω-3 fatty acid with 5 double bonds; Arachidonic acid (AA), an ω-6 fatty acid, with 4 double bonds; Dihomo-gamma-linolenic acid (DGLA), an ω-6, with 3 double bonds.

Current usage limits the term to the leukotrienes (LT) and three types of prostanoids— prostaglandins (PG) prostacyclins (PGI), and thromboxanes (TX). This is the definition used here. However, several other classes can technically be termed eicosanoid, including the hepoxilins, resolvins, isofurans, isoprostanes, lipoxins, epi-lipoxins, epoxyeicosatrienoic acids (EETs) and endocannabinoids. LTs and prostanoids are sometimes termed 'classic eicosanoids' in contrast to the 'novel', 'eicosanoid-like' or 'nonclassic eicosanoids'. A particular eicosanoid is denoted by a four-character abbreviation, composed of: • • •

Its two letter abbreviation (above), One A-B-C sequence-letter; and A subscript, indicating the number of double bonds.

Examples are: • •

The EPA-derived prostanoids have three double bonds, (e.g. PGG3, PGH3, PGI3, TXA3) while its leukotrienes have five, (LTB5). The AA-derived prostanoids have two double bonds, (e.g. PGG2, PGH2, PGI2, TXA2) while its leukotrienes have four, (LTB4).

Furthermore, stereochemistry may differ among the pathways, indicated by Greek letters, e.g. for (PGF2α).

Biosynthesis Two families of enzymes catalyze fatty acid oxygenation to produce the eicosanoids: • •

Cyclooxygenase, or COX, generates the prostanoids. Lipoxygenase, or LOX, in several forms. 5-lipoxygenase (5-LO) generates the leukotrienes.

Eicosanoids are not stored within cells, but are synthesized as required. They derive from the fatty acids that make up the cell membrane and nuclear membrane. Eicosanoid biosynthesis begins when cell is activated by mechanical trauma, cytokines, growth factors or other stimuli. (The stimulus may even be an eicosanoid from a neighboring cell; the pathways are complex.) This triggers the release of a phospholipase at the cell membrane. The phospholipase travels to the nuclear membrane. There, the phospholipase catalyzes ester hydrolysis of phospholipid (by A2) or diacylglycerol (by phospholipase C). This frees a 20-carbon essential fatty acid. This hydrolysis appears to be the rate-determining step for eicosanoid formation. The fatty acids may be released by any of several phospholipases. Of these, type IV cytosolic phospholipase A2 (cPLA2) is the key actor, as cells lacking cPLA2 are generally devoid of eicosanoid synthesis. The phospholipase cPLA2 is specific for phospholipids that contain AA, EPA or GPLA at the SN2 position. Interestingly, cPLA2 may also release the lysophospholipid that becomes platelet-activating factor.

Peroxidation and reactive oxygen species Next, the free fatty acid is oxygenated along any of several pathways. The eicosanoid pathways (via lipoxygenase or COX) add molecular oxygen (O2). Although the fatty acid is symmetric, the resulting eicosanoids are chiral; the oxidation proceeds with high stereospecificity. The oxidation of lipids is hazardous to cells, particularly when close to the nucleus. There are elaborate mechanisms to prevent unwanted oxidation. COX, the lipoxygenases and the phospholipases are tightly controlled—there are at least eight proteins activated to coordinate generation of leukotrienes. Several of these exist in multiple isoforms. Oxidation by either COX or lipoxygenase releases reactive oxygen species (ROS) and the initial products in eicosanoid generation are themselves highly reactive peroxides. LTA4 can form adducts with tissue DNA. Other reactions of lipoxygenases generate cellular damage; murine models implicate 15-lipoxygenase in the pathogenesis of atherosclerosis. The oxidation in eicosanoid generation is compartmentalized; this limits the peroxides' damage. The enzymes which are biosynthetic for eicosanoids (e.g. glutathione-Stransferases, epoxide hydrolases and carrier proteins) belong to families whose functions are largely involved with cellular detoxification. This suggests that eicosanoid signaling may have evolved from the detoxification of ROS. The cell must realize some benefit from generating lipid hydroperoxides close-by its nucleus. PGs and LTs may signal or regulate DNA-transcription there; LTB4 is ligand for PPARα.

Structures of Selected Eicosanoids

Prostaglandin E1. The 5-member ring is characteristic of the class

Thromboxane A2. Oxygens have moved into the ring

Leukotriene B4. Note the 3 conjugated double bonds

Prostacyclin I2. The second ring distinguishes it from the prostaglandins

Leukotriene E4, an example of a cysteinyl leukotriene

Prostanoid pathways Cyclooxygenase (COX) catalyzes the conversion of the free essential fatty acids to prostanoids by a two-step process. First, two molecules of O2 are added as two peroxide linkages, and a 5-member carbon ring is forged near the middle of the fatty acid chain. This forms the short-lived, unstable intermediate Prostaglandin G (PGG). Next, one of the peroxide linkages sheds a single oxygen, forming PGH. All three classes of prostanoids originate from PGH. All have distinctive rings in the center of the molecule. They differ in their structures. The PGH compounds (parents to all the rest) have a 5-carbon ring, bridged by two oxygens (a peroxide.) As the example in Structures of Selected Eicosanoids figure shows, the derived prostaglandins contain a

single, unsaturated 5-carbon ring. In prostacyclins, this ring is conjoined to another oxygen-containing ring. In thromboxanes the ring becomes a 6-member ring with one oxygen. The leukotrienes do not have rings.

Leukotriene pathways The enzyme 5-lipoxygenase (5-LO) uses 5-lipoxygenase activating protein (FLAP) to convert arachidonic acid into 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which spontaneously reduces to 5-hydroxyeicosatetraenoic acid (5-HETE). The enzyme LTA synthase acts on 5-HPETE to convert it into leukotriene A4 (LTA4), which may be converted into LTB4 by the enzyme leukotriene A4 epoxide hydrolase. Eosinophils, mast cells, and alveolar macrophages use the enzyme leukotriene C4 synthase to conjugate glutathione with LTA4 to make LTC4, which is transported outside the cell, where a glutamic acid moiety is removed from it to make LTD4. The leukotriene LTD4 is then cleaved by dipeptidases to make LTE4. The leukotrienes LTC4, LTD4 and LTE4 all contain cysteine and are collectively known as the cysteinyl leukotrienes.

Function and pharmacology Metabolic actions of selected prostanoids and leukotrienes† Stimulation of platelet PGD2 Promotion of sleep TXA2 aggregation; vasoconstriction Smooth muscle contraction; PGE2 inducing pain, heat, fever; 15d-PGJ2 Adipocyte differentiation bronchoconstriction PGF2α Uterine contraction LTB4 Leukocyte chemotaxis Inhibition of platelet aggregation; Anaphylaxis; bronchial smooth PGI2 Cysteinyl-LTs vasodilation; embryo implantation muscle contraction. †

Shown eicosanoids are AA-derived; EPA-derived generally have weaker activity

Eicosanoids exert complex control over many bodily systems, mainly in inflammation or immunity, and as messengers in the central nervous system. They are found in most living things. In humans, eicosanoids are local hormones that are released by most cells, act on that same cell or nearby cells (i.e., they are autocrine and paracrine mediators), and then are rapidly inactivated. Eicosanoids have a short half-life, ranging from seconds to minutes. Dietary antioxidants inhibit the generation of some inflammatory eicosanoids, e.g. trans-resveratrol against thromboxane and some leukotrienes. Most eicosanoid receptors are members of the G protein-coupled receptor superfamily.

Receptors: There are specific receptors for all eicosanoids

Leukotrienes: • • •

CysLT1 (Cysteinyl leukotriene receptor type 1) CysLT2 (Cysteinyl leukotriene receptor type 2) BLT1 (Leukotriene B4 receptor)

Prostanoids: • •

• • •

PGD2: DP-(PGD2) PGE2: o EP1-(PGE2) o EP2-(PGE2) o EP3-(PGE2) o EP4-(PGE2) PGF2α: FP-(PGF2α) PGI2 (prostacyclin): IP-(PGI2) TXA2 (thromboxane): TP-(TXA2)

The ω-3 and ω-6 series

“

The reduction in AA-derived eicosanoids and the diminished activity of the alternative products generated from ω-3 fatty acids serve as the foundation for explaining some of the beneficial effects of greater ω-3 intake.

”

—Kevin Fritsche, Fatty Acids as Modulators of the Immune Response

Arachidonic acid (AA; 20:4 ω-6) sits at the head of the 'arachidonic acid cascade'—more than twenty different eicosanoid-mediated signaling paths controlling a wide array of cellular functions, especially those regulating inflammation, immunity and the central nervous system. In the inflammatory response, two other groups of dietary essential fatty acids form cascades that parallel and compete with the arachidonic acid cascade. EPA (20:5 ω-3) provides the most important competing cascade. DGLA (20:3 ω-6) provides a third, less prominent cascade. These two parallel cascades soften the inflammatory effects of AA and its products. Low dietary intake of these less-inflammatory essential fatty acids, especially the ω-3s, has been linked to several inflammation-related diseases, and perhaps some mental illnesses. The U.S. National Institutes of Health and the National Library of Medicine state that there is 'A' level evidence that increased dietary ω-3 improves outcomes in hypertriglyceridemia, secondary cardiovascular disease prevention and hypertension.

There is 'B' level evidence ('good scientific evidence') for increased dietary ω-3 in primary prevention of cardiovascular disease, rheumatoid arthritis and protection from ciclosporin toxicity in organ transplant patients. They also note more preliminary evidence showing that dietary ω-3 can ease symptoms in several psychiatric disorders. Besides the influence on eicosanoids, dietary polyunsaturated fats modulate immune response through three other molecular mechanisms. They (a) alter membrane composition and function, including the composition of lipid rafts; (b) change cytokine biosynthesis and (c) directly activate gene transcription. Of these, the action on eicosanoids is the best explored. Mechanisms of ω-3 action

EFA sources: Essential fatty acid production and metabolism to form eicosanoids. At each step, the ω-3 and ω-6 cascades compete for the enzymes. The eicosanoids from AA generally promote inflammation. Those from EPA and from GLA (via DGLA) are generally less inflammatory, or inactive, or even antiinflammatory. The figure shows the ω-3 and -6 synthesis chains, along with the major eicosanoids from AA, EPA and DGLA.

Dietary ω-3 and GLA counter the inflammatory effects of AA's eicosanoids in three ways, along the eicosanoid pathways: • • •

Displacement—Dietary ω-3 decreases tissue concentrations of AA, so there is less to form ω-6 eicosanoids. Competitive inhibition—DGLA and EPA compete with AA for access to the cyclooxygenase and lipoxygenase enzymes. So the presence of DGLA and EPA in tissues lowers the output of AA's eicosanoids. Counteraction—Some DGLA and EPA derived eicosanoids counteract their AA derived counterparts.

Role in inflammation Since antiquity, the cardinal signs of inflammation have been known as: calor (warmth), dolor (pain), tumor (swelling) and rubor (redness). The eicosanoids are involved with each of these signs. Redness—An insect's sting will trigger the classic inflammatory response. Short acting vasoconstrictors — TXA2—are released quickly after the injury. The site may momentarily turn pale. Then TXA2 mediates the release of the vasodilators PGE2 and LTB4. The blood vessels engorge and the injury reddens. Swelling—LTB4 makes the blood vessels more permeable. Plasma leaks out into the connective tissues, and they swell. The process also loses pro-inflammatory cytokines. Pain—The cytokines increase COX-2 activity. This elevates levels of PGE2, sensitizing pain neurons. Heat—PGE2 is also a potent pyretic agent. Aspirin and NSAIDS—drugs that block the COX pathways and stop prostanoid synthesis—limit fever or the heat of localized inflammation.

Pharmacy: Eicosanoid, eicosanoid analogs and receptor agonists/antagonists used as medicines

Medicine

Type

Medical condition or use

Alprostadil

PGE1

Erectile dysfunction, maintaining a patent ductus arteriosus in the fetus

Beraprost

PGI1 analog

Pulmonary hypertension, avoiding reperfusion injury

Bimatoprost

PG analog

Glaucoma, ocular hypertension

Carboprost

PG analog

Labor induction, abortifacient in early pregnancy

Dinoprostone

PGE2

Labor induction

Iloprost

PGI2 analog

Pulmonary arterial hypertension

Latanoprost

PG analog

Glaucoma, ocular hypertension

Misoprostol

PGE1 analog

Stomach ulcers, labor induction, abortifacient

Montelukast

LT receptor antagonist

Asthma, seasonal allergies

Travoprost

PG analog

Glaucoma, ocular hypertension

Treprostinil

PGI analog

Pulmonary hypertension

U46619

Longer lived TX analog

Research only

Zafirlukast

LT receptor antagonist

Asthma

Action of prostanoids Prostanoids mediate local symptoms of inflammation: vasoconstriction or vasodilation, coagulation, pain and fever. Inhibition of cyclooxygenase, specifically the inducible COX-2 isoform, is the hallmark of NSAIDs (non-steroidal anti-inflammatory drugs), such as aspirin. COX-2 is responsible for pain and inflammation, while COX-1 is responsible for platelet clotting actions.

Prostanoids activate the PPARγ members of the steroid/thyroid family of nuclear hormone receptors, directly influencing gene transcription.

Action of leukotrienes Leukotrienes play an important role in inflammation. There is a neuroendocrine role for LTC4 in luteinizing hormone secretion. LTB4 causes adhesion and chemotaxis of leukocytes and stimulates aggregation, enzyme release, and generation of superoxide in neutrophils. Blocking leukotriene receptors can play a role in the management of inflammatory diseases such as asthma (by the drugs montelukast and zafirlukast), psoriasis, and rheumatoid arthritis. The slow reacting substance of anaphylaxis comprises the cysteinyl leukotrienes. These have a clear role in pathophysiological conditions such as asthma, allergic rhinitis and other nasal allergies, and have been implicated in atherosclerosis and inflammatory gastrointestinal diseases. They are potent bronchoconstrictors, increase vascular permeability in postcapillary venules, and stimulate mucus secretion. They are released from the lung tissue of asthmatic subjects exposed to specific allergens and play a pathophysiological role in immediate hypersensitivity reactions. Along with PGD, they function in effector cell trafficking, antigen presentation, immune cell activation, matrix deposition, and fibrosis.

History In 1930, gynecologist Raphael Kurzrok and pharmacologist Charles Leib characterized prostaglandin as a component of semen. Between 1929 and 1932, Burr and Burr showed that restricting fat from animal's diets led to a deficiency disease, and first described the essential fatty acids. In 1935, von Euler identified prostaglandin. In 1964, Bergström and Samuelsson linked these observations when they showed that the "classical" eicosanoids were derived from arachidonic acid, which had earlier been considered to be one of the essential fatty acids. In 1971, Vane showed that aspirin and similar drugs inhibit prostaglandin synthesis. Von Euler received the Nobel Prize in medicine in 1970, which Samuelsson, Vane, and Bergström also received in 1982. E. J. Corey received it in chemistry in 1990 largely for his synthesis of prostaglandins.

Chapter- 6

Fatty Acid

Butyric acid, a short-chain fatty acid A fatty acid is a carboxylic acid with a long unbranched aliphatic tail (chain), which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of four to 28 carbons. Fatty acids are usually derived from triglycerides or phospholipids. When they are not attached to other molecules, they are known as "free" fatty acids. Fatty acids are important sources of fuel because their metabolism yield large quantities of ATP. Many cell types can use either glucose or fatty acids for this purpose. In particular, heart and skeletal muscle prefer fatty acids. The brain cannot use fatty acids as a source of fuel; it relies on glucose or on ketone bodies.

Types of fatty acids

Three dimensional representations of several fatty acids Fatty acids can be saturated and unsaturated, depending on double bonds. They differ in length as well.

Long and short fatty acids In addition to saturation, fatty acids have different lengths, often categorized as short, medium, or long. • • • •

Short-chain fatty acids (SCFA) are fatty acids with aliphatic tails of fewer than six carbons. Medium-chain fatty acids (MCFA) are fatty acids with aliphatic tails of 6–12. carbons, which can form medium-chain triglycerides. Long-chain fatty acids (LCFA) are fatty acids with aliphatic tails longer than 12 carbons. Very-Long-chain fatty acids (VLCFA) are fatty acids with aliphatic tails longer than 22 carbons

Unsaturated fatty acids

Comparison of the trans isomer (top) Elaidic acid and the cis-isomer oleic acid Unsaturated fatty acids resemble saturated fatty acids, except that the chain has one or more double-bonds between carbon atoms. The two carbon atoms in the chain that are bound next to either side of the double bond can occur in a cis or trans configuration. cis A cis configuration means that adjacent hydrogen atoms are on the same side of the double bond. The rigidity of the double bond freezes its conformation and, in the case of the cis isomer, causes the chain to bend and restricts the conformational freedom of the fatty acid. The more double bonds the chain has in the cis configuration, the less flexibility it has. When a chain has many cis bonds, it becomes quite curved in its most accessible conformations. For example, oleic acid, with one double bond, has a "kink" in it, whereas linoleic acid, with two double bonds, has a more pronounced bend. Alphalinolenic acid, with three double bonds, favors a hooked shape. The effect of this is that, in restricted environments, such as when fatty acids are part of a phospholipid in a lipid bilayer, or triglycerides in lipid droplets, cis bonds limit the ability of fatty acids to be closely packed, and therefore could affect the melting temperature of the membrane or of the fat.

trans A trans configuration, by contrast, means that the next two hydrogen atoms are bound to opposite sides of the double bond. As a result, they do not cause the chain to bend much, and their shape is similar to straight saturated fatty acids. In most naturally occurring unsaturated fatty acids, each double bond has three n carbon atoms after it, for some n, and all are cis bonds. Most fatty acids in the trans configuration (trans fats) are not found in nature and are the result of human processing (e.g., hydrogenation). The differences in geometry between the various types of unsaturated fatty acids, as well as between saturated and unsaturated fatty acids, play an important role in biological processes, and in the construction of biological structures (such as cell membranes). Examples of Unsaturated Fatty Acids Common name Myristoleic acid Palmitoleic acid Sapienic acid Oleic acid

Δx

Chemical structure

C:D n−x

CH3(CH2)3CH=CH(CH2)7COOH

cis-Δ9 14:1 n−5

CH3(CH2)5CH=CH(CH2)7COOH

cis-Δ9 16:1 n−7

CH3(CH2)8CH=CH(CH2)4COOH

cis-Δ6 16:1 n−10

cis-Δ9 18:1 n−9 cis,cis Linoleic CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH 18:2 n−6 acid Δ9,Δ12 cis,cis, α-Linolenic CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COO cis18:3 n−3 acid H Δ9,Δ12 15 ,Δ cis,cis, cis,cis Arachidonic CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH= 20:4 n−6 acid CH(CH2)3COOHNIST Δ5Δ8, 11 1 Δ ,Δ CH3(CH2)7CH=CH(CH2)7COOH

4

cis,cis, cis,cis, Eicosapenta CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH cis20:5 n−3 enoic acid CH2CH=CH(CH2)3COOH Δ5,Δ8, Δ11,Δ1 4 17 ,Δ Erucic acid CH3(CH2)7CH=CH(CH2)11COOH cis22:1 n−9

Δ13 cis,cis, cis,cis, cis,cis Docosahexa CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH 22:6 n−3 enoic acid CH2CH=CHCH2CH=CH(CH2)2COOH Δ4,Δ7, Δ10,Δ1 3 16 ,Δ , Δ19 Essential fatty acids Fatty acids that are required by the body but cannot be made in sufficient quantity from other substrates, therefore must be obtained from food and are called essential fatty acids. Essential fatty acids are polyunsaturated fatty acids and are the parent compounds of the omega-6 and omega-3 fatty acid series, respectively. Humans lack the ability to introduce double bonds in fatty acids beyond carbons 9 and 10, Two fatty acids are essential in humans, linoleic acid (LA) and alpha-linolenic acid (ALA). They are widely distributed in plant oils. In addition, fish, flax, and hemp oils contain the longer-chain omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

Saturated fatty acids Saturated fatty acids are long-chain carboxylic acids that usually have between 12 and 24 carbon atoms and have no double bonds. Thus, saturated fatty acids are saturated with hydrogen (since double bonds reduce the number of hydrogens on each carbon). Because saturated fatty acids have only single bonds, each carbon atom within the chain has 2 hydrogen atoms (except for the omega carbon at the end that has 3 hydrogens). Examples of Saturated Fatty Acids Common name Chemical structure C:D Lauric acid CH3(CH2)10COOH 12:0 Myristic acid CH3(CH2)12COOH 14:0 Palmitic acid CH3(CH2)14COOH 16:0 Stearic acid CH3(CH2)16COOH 18:0 Arachidic acid CH3(CH2)18COOH 20:0 Behenic acid CH3(CH2)20COOH 22:0 Lignoceric acid CH3(CH2)22COOH 24:0 Cerotic acid CH3(CH2)24COOH 26:0

Nomenclature

Numbering of carbon atoms Several different systems of nomenclature are used for fatty acids. The following table describes the most common systems. System

Trivial nomenclature

Systematic nomenclature

Δx nomenclature

n−x nomenclature

Example

Explanation Trivial names (or common names) are nonsystematic historical names, which are the most frequent naming system used in literature. Most Palmitoleic acid common fatty acids have trivial names in addition to their systematic names (see below). These names frequently do not follow any pattern, but they are concise and often unambiguous. Systematic names (or IUPAC names) derive from the standard IUPAC Rules for the Nomenclature of Organic Chemistry, published in 1979, along with a recommendation published specifically for lipids in (9Z)-octadecenoic 1977. Counting begins from the carboxylic acid end. Double bonds are labelled with cis-/trans- notation or acid E-/Z- notation, where appropriate. This notation is generally more verbose than common nomenclature, but has the advantage of being more technically clear and descriptive. In Δx (or delta-x) nomenclature, each double bond is indicated by Δx, where the double bond is located on the xth carbon–carbon bond, counting from the carboxylic acid end. Each double bond is preceded by cis,cis-Δ9,Δ12 a cis- or trans- prefix, indicating the conformation of octadecadienoic the molecule around the bond. For example, linoleic acid acid is designated "cis-Δ9, cis-Δ12 octadecadienoic acid". This nomenclature has the advantage of being more verbose than systematic nomenclature, but is no more technically clear or descriptive. n−x (n minus x; also ω−x or omega-x) n−3 nomenclature both provides names for individual compounds and classifies them by their likely

18:3 18:3, n−6 Lipid numbers 18:3, cis,cis,cisΔ9,Δ12,Δ15

biosynthetic properties in animals. A double bond is located on the xth carbon–carbon bond, counting from the terminal methyl carbon (designated as n or ω) toward the carbonyl carbon. For example, α-Linolenic acid is classified as a n−3 or omega-3 fatty acid, and so it is likely to share a biosynthetic pathway with other compounds of this type. The ω−x, omega-x, or "omega" notation is common in popular nutritional literature, but IUPAC has deprecated it in favor of n−x notation in technical documents. The most commonly researched fatty acid biosynthetic pathways are n−3 and n−6, which are hypothesized to increase or decrease inflammation. Lipid numbers take the form C:D, where C is the number of carbon atoms in the fatty acid and D is the number of double bonds in the fatty acid. This notation can be ambiguous, as some different fatty acids can have the same numbers. Consequently, when ambiguity exists this notation is usually paired with either a Δx or n−x term.

Production Fatty acids are usually produced industrially by the hydrolysis of triglycerides), with the removal of glycerol. Phospholipids represent another source. Some fatty acids are produced synthetically by hydrocarboxylation of alkenes.

Free fatty acids The biosynthesis of fatty acids involves the condensation of acetyl-CoA. Since this coenzyme carries a two-carbon-atom group, almost all natural fatty acids have even numbers of carbon atoms. The "uncombined fatty acids" or "free fatty acids" found in organim come from the breakdown of a triglyceride. Because they are insoluble in water, thse fatty acids are transported (solubilized, circulated) while bound to plasma protein albumin. The levels of "free fatty acid" in the blood are limited by the availability of albumin binding sites.

Fatty acids in dietary fats The following table gives the fatty acid, vitamin E and cholesterol composition of some common dietary fats.

Saturated Monounsaturated Polyunsaturated Cholesterol g/100g Animal fats Lard Duck fat Butter Vegetable fats Coconut oil Palm oil Cottonseed oil Wheat germ oil Soya oil Olive oil Corn oil Sunflower oil Safflower oil Hemp oil Canola/Rapeseed oil

g/100g

g/100g

mg/100g

Vitamin E mg/100g

40.8 33.2 54.0

43.8 49.3 19.8

9.6 12.9 2.6

93 100 230

0.00 2.70 2.00

85.2 45.3 25.5 18.8 14.5 14.0 12.7 11.9 10.2 10

6.6 41.6 21.3 15.9 23.2 69.7 24.7 20.2 12.6 15

1.7 8.3 48.1 60.7 56.5 11.2 57.8 63.0 72.1 75

0 0 0 0 0 0 0 0 0 0

.66 33.12 42.77 136.65 16.29 5.10 17.24 49.0 40.68

5.3

64.3

24.8

0

22.21

Reactions of fatty acids Fatty acids exhibit reactions like other carboxylic acid, i.e. they undergo esterification and acid-base reactions.

Acidity Fatty acids do not show a great variation in their acidities, as indicated by their pKas. Nonanoic acid, for example, has a pKa of 4.96, being only slightly weaker than acetic acid (4.76). As the chain length increases the solubility of the fatty acids in water decreases very rapidly, so that the longer-chain fatty acids have minimal effect on the pH of an aqueous solution. Even those fatty acids that are insoluble in water will dissolve in warm ethanol, and can be titrated with sodium hydroxide solution using phenolphthalein as an indicator to a pale-pink endpoint. This analysis is used to determine the free fatty acid content of fats; i.e., the proportion of the triglycerides that have been hydrolyzed.

Hydrogenation and hardening Hydrogenation of unsaturated fatty acids is widely practiced to give saturated fatty acids, which are less prone toward rancidification. Since the saturated fatty acids are higher melting that the unsaturated relatives, the process is called hardening. This technology is

used to convert vegetable oils into margarine. During partial hydrogenation, unsaturated fatty acids can be isomerized from cis to trans configuration. More forcing hydrogenation, i.e. using higher pressures of H2 and higher temperatures, converts fatty acids fatty alcohols. Fatty alcohols are, however, more easily produced from fatty acid esters. In the Varrentrapp reaction certain unsaturated fatty acids are cleaved in molten alkali, a reaction at one time of relevance to structure elucidation.

Auto-oxidation and rancidity Unsaturated fatty acids undergo a chemical change known as auto-oxidation. The process requires oxygen (air) and is accelerated by the presence of trace metals. Vegetable oils resists this process because they contain antioxidants, such as tocopherol. Fats and oils often are treated with chelating agents such as citric acid to remove the metal catalysts.

Ozonolysis Unsaturated fatty acids are susceptible to degradation by ozone. This reaction is practiced in the production azelaic acid ((CH2)7(CO2H)2) from oleic acid.

Circulation Digestion and intake Short- and medium-chain fatty acids are absorbed directly into the blood via intestine capillaries and travel through the portal vein just as other absorbed nutrients do. However, long-chain fatty acids are too large to be directly released into the tiny intestine capillaries. Instead they are absorbed into the fatty walls of the intestine villi and reassembled again into triglycerides. The triglycerides are coated with cholesterol and protein (protein coat) into a compound called a chylomicron. Within the villi, the chylomicron enters a lymphatic capillary called a lacteal, which merges into larger lymphatic vessels. It is transported via the lymphatic system and the thoracic duct up to a location near the heart (where the arteries and veins are larger). The thoracic duct empties the chylomicrons into the bloodstream via the left subclavian vein. At this point the chylomicrons can transport the triglycerides to where they are needed.

Distribution Blood fatty acids are in different forms in different stages in the blood circulation. They are taken in through the intestine in chylomicrons, but also exist in very low density lipoproteins (VLDL) and low density lipoproteins (LDL) after processing in the liver. In addition, when released from adipocytes, fatty acids exist in the blood as free fatty acids.

It is proposed that the blend of fatty acids exuded by mammalian skin, together with lactic acid and pyruvic acid, is distinctive and enables animals with a keen sense of smell to differentiate individuals.

Chapter- 7

Phospholipid

Polar group of the molecule, highlighted in red The U indicates the uncharged hydrophobic portion of the molecule, highlighted in blue.

Phosphatidyl choline is the major component of lecithin. It is also a source for choline in the synthesis of acetylcholine in cholinergic neurons.

Cell membranes consist of phospholipid bilayers Phospholipids are a class of lipids and are a major component of all cell membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a phosphate group, and a simple organic molecule such as choline; one exception to this rule is sphingomyelin, which is derived from sphingosine instead of glycerol. The first phospholipid identified as such in biological tissues was lecithin, or phosphatidylcholine, in the egg yolk, by Theodore Nicolas Gobley, a French chemist and pharmacist, in 1847.

Amphipathic character The 'head' of a phospholipid is hydrophilic (attracted to water), while the hydrophobic 'tails' repel water. The hydrophillic head contains the negatively charged phosphate group, and may contain other polar groups. The hydrophobic tail usually consists of long fatty acid hydrocarbon chains. When placed in water, phospholipids form a variety of structures depending on the specific properties of the phospholipid. These specific properties allow phospholipids to play an important role in the phospholipid bilayer. In biological systems, the phospholipids often occur with other molecules (e.g., proteins, glycolipids, cholesterol) in a bilayer such as a cell membrane. Lipid bilayers occur when hydrophobic tails line up against one another, forming a membrane with hydrophilic heads on both sides facing the water. This type of membrane is partially permeable, capable of elastic movement, and has fluid properties, in which embedded proteins (integral or peripheral proteins) and phospholipid molecules are able to move laterally. Such movement can be described by the Fluid Mosaic Model, that describes the membrane as a mosaic of lipid molecules that act as a solvent for all the substances and proteins within it, so proteins and lipid molecules are then free to diffuse laterally through the lipid matrix and migrate over the membrane. Cholesterol contributes to membrane fluidity by hindering the packing together of phospholipids. However, this model has now been superseded, as through the study of lipid polymorphism it is now known that the behaviour of lipids under physiological (and other) conditions is not simple.

Types of phospholipid Diacylglyceride structures • • • • •

Phosphatidic acid (phosphatidate) (PA) Phosphatidylethanolamine (cephalin) (PE) Phosphatidylcholine (lecithin) (PC) Phosphatidylserine (PS) Phosphoinositides: o Phosphatidylinositol (PI) o Phosphatidylinositol phosphate (PIP) o Phosphatidylinositol bisphosphate (PIP2) and o Phosphatidylinositol triphosphate (PIP3).

Phosphosphingolipids • • •

Ceramide phosphorylcholine (Sphingomyelin) (SPH) Ceramide phosphorylethanolamine (Sphingomyelin) (Cer-PE) Ceramide phosphorylglycerol

Simulations Computational simulations of phospholipids are often performed using molecular dynamics with force fields such as GROMOS, CHARMM, or AMBER.

Phospholipid synthesis Phospholipid synthesis occurs in the cytosol adjacent to ER membrane that is studded with proteins that act in synthesis (GPAT and LPAAT acyl transferases, phosphatase and choline phosphotransferase) and allocation (flippase and floppase). Eventually a vesicle will bud off from the ER containing phospholipids destined for the cytoplasmic cellular membrane on its exterior leaflet and phospholipids destined for the exoplasmic cellular membrane on its inner leaflet.

In signal transduction Some types of phospholipid can be split to produce products that function as second messengers in signal transduction. Examples include phosphatidylinositol (4,5)bisphosphate (PIP2), that can be split by the enzyme Phospholipase C into inositol triphosphate (IP3) and diacylglycerol (DAG), which both carry out the functions of the Gq type of G protein in response to various stimuli and intervene in various processes from long term depression in neurons to leukocyte signal pathways started by chemokine receptors. Phospholipids also intervene in prostaglandin signal pathways as the raw material used by lipase enzymes to produce the prostaglandin precursors. In plants they serve as the raw material to produce Jasmonic acid, a plant hormone similar in structure to prostaglandins that mediates defensive responses against pathogens.

Food technology Phospholipids can also act as an emulsifier, enabling oils to dissolve in water. Phospholipids called lecithin are extracted out of cooking oil and then used as food additives in many things such as bread and can also be purchased separately in a health food store.

Phospholipid derivatives •

Natural phospholipid derivates: egg PC, egg PG, soy PC, hydrogenated soy PC, sphingomyelin as natural phospholipids.

•

Synthetic phospholipid derivates:

o o o o o o

Phosphatidic acid (DMPA, DPPA, DSPA) Phosphatidylcholine (DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC) Phosphatidylglycerol (DMPG, DPPG, DSPG, POPG) Phosphatidylethanolamine (DMPE, DPPE, DSPE DOPE) Phosphatidylserine (DOPS) PEG phospholipid (mPEG-phospholipid, polyglycerin-phospholipid, funcitionalized-phospholipid, terminal activated-phospholipid)

Abbreviations used and chemical information of glycerophospholipids Abbreviation DDPC DEPA-NA DEPC DEPE DEPG-NA DLOPC DLPA-NA DLPC DLPE DLPG-NA

DLPG-NH4 DLPS-NA DMPA-NA

CAS 343644-0 8072431-8 5664939-9 988-072

Name 1,2-Didecanoyl-sn-glycero-3phosphocholine 1,2-Dierucoyl-sn-glycero-3phosphate (Sodium Salt) 1,2-Dierucoyl-sn-glycero-3phosphocholine 1,2-Dierucoyl-sn-glycero-3phosphoethanolamine 1,2-Dierucoyl-sn-glycero3[Phospho-rac-(1-glycerol...) (Sodium Salt) 998-06- 1,2-Dilinoleoyl-sn-glycero-31 phosphocholine 1,2-Dilauroyl-sn-glycero-3phosphate (Sodium Salt) 18194- 1,2-Dilauroyl-sn-glycero-325-7 phosphocholine 1,2-Dilauroyl-sn-glycero-3phosphoethanolamine 1,2-Dilauroyl-sn-glycero3[Phospho-rac-(1-glycerol...) (Sodium Salt) 1,2-Dilauroyl-sn-glycero3[Phospho-rac-(1-glycerol...) (Ammonium Salt) 1,2-Dilauroyl-sn-glycero-3phosphoserine (Sodium Salt) 1,2-Dimyristoyl-sn-glycero-380724-3 phosphate (Sodium Salt)

Type Phosphatidylcholine Phosphatidic acid Phosphatidylcholine Phosphatidylethanolamine Phosphatidylglycerol Phosphatidylcholine Phosphatidic acid Phosphatidylcholine Phosphatidylethanolamine Phosphatidylglycerol

Phosphatidylglycerol Phosphatidylserine Phosphatidic acid

DMPC DMPE DMPG-NA

1819424-6 988-072 6723280-8

DMPG-NH4

DMPG-NH4/NA DMPS-NA DOPA-NA DOPC DOPE DOPG-NA DOPS-NA DPPA-NA

423595-4 4004-516270069-0 7061414-1 7106587-7

DPPC

63-89-8

DPPE

923-615

DPPG-NA

6723281-9

DPPG-NH4

7354870-6

DPPS-NA DSPA-NA

10832118-2

1,2-Dimyristoyl-sn-glycero-3phosphocholine 1,2-Dimyristoyl-sn-glycero-3phosphoethanolamine 1,2-Dimyristoyl-sn-glycero3[Phospho-rac-(1-glycerol...) (Sodium Salt) 1,2-Dimyristoyl-sn-glycero3[Phospho-rac-(1-glycerol...) (Ammonium Salt) 1,2-Dimyristoyl-sn-glycero3[Phospho-rac-(1-glycerol...) (Sodium/Ammonium Salt) 1,2-Dimyristoyl-sn-glycero-3phosphoserine (Sodium Salt) 1,2-Dioleoyl-sn-glycero-3phosphate (Sodium Salt) 1,2-Dioleoyl-sn-glycero-3phosphocholine 1,2-Dioleoyl-sn-glycero-3phosphoethanolamine 1,2-Dioleoyl-sn-glycero3[Phospho-rac-(1-glycerol...) (Sodium Salt) 1,2-Dioleoyl-sn-glycero-3phosphoserine (Sodium Salt) 1,2-Dipalmitoyl-sn-glycero-3phosphate (Sodium Salt) 1,2-Dipalmitoyl-sn-glycero-3phosphocholine 1,2-Dipalmitoyl-sn-glycero-3phosphoethanolamine 1,2-Dipalmitoyl-sn-glycero3[Phospho-rac-(1-glycerol...) (Sodium Salt) 1,2-Dipalmitoyl-sn-glycero3[Phospho-rac-(1-glycerol...) (Ammonium Salt) 1,2-Dipalmitoyl-sn-glycero-3phosphoserine (Sodium Salt) 1,2-Distearoyl-sn-glycero-3phosphate (Sodium Salt)

Phosphatidylcholine Phosphatidylethanolamine Phosphatidylglycerol

Phosphatidylglycerol

Phosphatidylglycerol Phosphatidylserine Phosphatidic acid Phosphatidylcholine Phosphatidylethanolamine Phosphatidylglycerol Phosphatidylserine Phosphatidic acid Phosphatidylcholine Phosphatidylethanolamine Phosphatidylglycerol

Phosphatidylglycerol Phosphatidylserine Phosphatidic acid

DSPC DSPE DSPG-NA

DSPG-NH4 DSPS-NA

816-944 106979-0

1,2-Distearoyl-sn-glycero-3phosphocholine 1,2-Distearoyl-sn-glycero-3phosphoethanolamine 1,2-Distearoyl-sn-glycero672323[Phospho-rac-(1-glycerol...) 82-0 (Sodium Salt) 1,2-Distearoyl-sn-glycero1083473[Phospho-rac-(1-glycerol...) 80-4 (Ammonium Salt) 1,2-Distearoyl-sn-glycero-3phosphoserine (Sodium Salt)

Egg Sphingomyelin empty Liposome EPC HEPC HSPC HSPC LYSOPC MYRISTIC LYSOPC PALMITIC LYSOPC STEARIC Milk Sphingomyelin MPPC

1819424-6 1736416-8 1942057-6

1-Myristoyl-2-palmitoyl-snglycero 3-phosphocholine

MSPC PMPC POPC

2685331-6

POPE POPG-NA PSPC

Egg-PC Hydrogenated Egg PC High purity Hydrogenated Soy PC Hydrogenated Soy PC 1-Myristoyl-sn-glycero-3phosphocholine 1-Palmitoyl-sn-glycero-3phosphocholine 1-Stearoyl-sn-glycero-3phosphocholine

8149005-3

Phosphatidylcholine Phosphatidylethanolamine Phosphatidylglycerol

Phosphatidylglycerol Phosphatidylserine

Phosphatidylcholine Phosphatidylcholine Phosphatidylcholine Phosphatidylcholine Lysophosphatidylcholine Lysophosphatidylcholine Lysophosphatidylcholine Phosphatidylcholine

1-Myristoyl-2-stearoyl-snPhosphatidylcholine glycero-3–phosphocholine 1-Palmitoyl-2-myristoyl-snPhosphatidylcholine glycero-3–phosphocholine 1-Palmitoyl-2-oleoyl-sn-glyceroPhosphatidylcholine 3-phosphocholine 1-Palmitoyl-2-oleoyl-sn-glyceroPhosphatidylethanolamine 3-phosphoethanolamine 1-Palmitoyl-2-oleoyl-sn-glycero3[Phospho-rac-(1-glycerol)...] Phosphatidylglycerol (Sodium Salt) 1-Palmitoyl-2-stearoyl-snPhosphatidylcholine glycero-3–phosphocholine

SMPC SOPC SPPC

1-Stearoyl-2-myristoyl-snPhosphatidylcholine glycero-3–phosphocholine 1-Stearoyl-2-oleoyl-sn-glyceroPhosphatidylcholine 3-phosphocholine 1-Stearoyl-2-palmitoyl-snPhosphatidylcholine glycero-3-phosphocholine

Chapter- 8

Sphingolipid and Steroid

Sphingolipid

General chemical structure of sphingolipids. Different substituents (R) give: H -> ceramide phosphocholine -> sphingomyelin sugar(s) -> glycosphingolipid(s). Sphingolipids are a class of lipids derived from the aliphatic amino alcohol sphingosine. These compounds play important roles in signal transmission and cell recognition. Sphingolipidoses, or disorders of sphingolipid metabolism, have particular impact on neural tissue.

Structure The long-chain bases, sometimes simply known as sphingoid bases, are the first nontransient products of de novo sphingolipid synthesis in both yeast and mammals. These compounds, specifically known as phytosphingosine and dihydrosphingosine (also known as sphinganine, although this term is less common), are mainly C18 compounds, with somewhat lower levels of C20 bases. Ceramides and glycosphingolipids are N-acyl derivatives of these compounds. The sphingosine backbone is O-linked to a (usually) charged head group such as ethanolamine, serine, or choline.

The backbone is also amide-linked to an acyl group, such as a fatty acid.

Types •

Ceramide is the fundamental structural unit common to all sphingolipids. They consist simply of a fatty acid chain attached through an amide linkage to sphingosine.

There are three main types of sphingolipids, differing in their head groups: • •

•

Sphingomyelins have a phosphocholine or phosphoethanolamine molecule with an ester linkage to the 1-hydroxy group of a ceramide. Glycosphingolipids, which differ in the substituents on their head group (see image). Glycosphingolipids are ceramides with one or more sugar residues joined in a β-glycosidic linkage at the 1-hydroxyl position. o Cerebrosides have a single glucose or galactose at the 1-hydroxy position.  Sulfatides are sulfated cerebrosides. Gangliosides have at least three sugars, one of which must be sialic acid.

Mammalian sphingolipid metabolism De novo sphingolipid synthesis begins with formation of 3-keto-dihydrosphingosine by serine palmitoyltransferase. The preferred substrates for this reaction are palmitoyl-CoA and serine. However, studies have demonstrated that serine palmitoyltransferase has some activity toward other species of fatty acyl-CoA and alternative amino acids, and the diversity of sphingoid bases has recently been reviewed. Next, 3-ketodihydrosphingosine is reduced to form dihydrosphingosine. Dihydrosphingosine is acylated by a (dihydro)-ceramide synthase, such as Lass1p or Lass2p (also termed as CerS), to form dihydroceramide. This is desaturated to form ceramide. Ceramide may subsequently have several fates. It may be phosphorylated by ceramide kinase to form ceramide-1-phosphate. Alternatively, it may be glycosylated by glucosylceramide synthase or galactosylceramide synthase. Additionally, it can be converted to sphingomyelin by the addition of a phosphorylcholine headgroup by sphingomyelin synthase. Diacylglycerol is generated by this process. Finally, ceramide may be broken down by a ceramidase to form sphingosine. Sphingosine may be phosphorylated to form sphingosine-1-phosphate. This may be dephosphorylated to reform sphingosine. Breakdown pathways allow the reversion of these metabolites to ceramide. The complex glycosphingolipids are hydrolyzed to glucosylceramide and galactosylceramide. These lipids are then hydrolyzed by beta-glucosidases and beta-galactosidases to regenerate ceramide. Similarly, sphingomyelin may be broken down by sphingomyelinase to form ceramide.

The only route by which sphingolipids are converted to non-sphingolipids is through sphingosine-1-phosphate lyase. This forms ethanolamine phosphate and hexadecenal.

Functions of mammalian sphingolipids Sphingolipids are commonly believed to protect the cell surface against harmful environmental factors by forming a mechanically stable and chemically resistant outer leaflet of the plasma membrane lipid bilayer. Certain complex glycosphingolipids were found to be involved in specific functions, such as cell recognition and signaling. Cell recognition depends mainly on the physical properties of the sphingolipids, whereas signaling involves specific interactions of the glycan structures of glycosphingolipids with similar lipids present on neighboring cells or with proteins. Recently, relatively simple sphingolipid metabolites, such as ceramide and sphingosine1-phosphate, have been shown to be important mediators in the signaling cascades involved in apoptosis, proliferation, and stress responses. Ceramide-based lipids selfaggregate in cell membranes and form separate phases less fluid than the bulk phospholipids. These sphingolipid-based microdomains, or "lipid rafts" were originally proposed to sort membrane proteins along the cellular pathways of membrane transport. At present, most research focuses on the organizing function during signal transduction. Sphingolipids are synthesized in a pathway that begins in the ER and is completed in the Golgi apparatus, but these lipids are enriched in the plasma membrane and in endosomes, where they perform many of their functions. Transport occurs via vesicles and monomeric transport in the cytosol. Sphingolipids are virtually absent from mitochondria and the ER, but constitute a 20-35 molar fraction of plasma membrane lipids.

Yeast sphingolipids Because of the incredible complexity of mammalian systems, yeast are sometimes used as a model organism for working out new pathways. These single-celled organisms are often more genetically tractable than mammalian cells, and strain libraries are available to supply strains harboring almost any non-lethal open reading frame single deletion. The two most commonly used yeasts are Saccharomyces cerevisiae and Schizosaccharomyces pombe, although research is also done in the pathological yeast Candida albicans. In addition to the important structural functions of complex sphingolipids (inositol phosphorylceramide and its mannosylated derivatives), the sphingoid bases phytosphingosine and dihydrosphingosine (sphinganine) play vital signaling roles in S. cerevisiae. These effects include regulation of endocytosis, ubiquitin-dependent proteolysis (and, thus, regulation of nutrient uptake), cytoskeletal dynamics, the cell cycle, translation, posttranslational protein modification, and the heat stress response. Additionally, modulation of sphingolipid metabolism by phosphatidylinositol (4,5)bisphosphate signaling via Slm1p and Slm2p and calcineurin has recently been described. Additionally, a substrate-level interaction has been shown between complex sphingolipid

synthesis and cycling of phosphatidylinositol 4-phosphate by the phosphatidylinositol kinase Stt4p and the lipid phosphatase Sac1p.

Plant sphingolipids Higher plants contain a wider variety of sphingolipids than animals and fungi.

Disorders There are several disorders of sphingolipid metabolism, known as sphingolipidoses. The most common is Gaucher's disease. Also of note is Fabry's disease, an X-linked recessive condition wherein a buildup of glycosphingolipids in lysosomes of various tissues is due to alpha-galactosidase deficiency. These patients tend to present with peripheral neuropathies and develop chronic renal conditions.

Additional image

Sphingosine

Steroid

IUPAC recommended ring lettering (left) and atom numbering (right) of cholestane, a prototypical steroid skeleton. The four rings A-D form the gonane nucleus of the steroid.

Stick model of the steroid lanosterol. The total number of carbons (30) reflects its triterpenoid origin. A steroid is a type of organic compound that contains a specific arrangement of four cycloalkane rings that are joined to each other. Examples of steroids include the dietary fat cholesterol, the sex hormones estradiol and testosterone, and the anti-inflammatory drug dexamethasone.

The core of steroids is composed of seventeen carbon atoms bonded together that take the form of four fused rings: three cyclohexane rings (designated as rings A, B, and C in the figure to the right) and one cyclopentane ring (the D ring). The steroids vary by the functional groups attached to this four ring core and by the oxidation state of the rings. Sterols are special forms of steroids, with a hydroxyl group at position-3 and a skeleton derived from cholestane. Hundreds of distinct steroids are found in plants, animals, and fungi. All steroids are made in cells either from the sterols lanosterol (animals and fungi) or from cycloartenol (plants). Both lanosterol and cycloartenol are derived from the cyclization of the triterpene squalene.

Structure Steroids are a class of organic compounds with a chemical structure that contains the core of gonane or a skeleton derived therefrom. Usually, methyl groups are present at the carbons C-10 and C-13. At carbon C-17 an alkyl side chain may also be present.

The basic skeleton of a steroid, with standard stereo orientations. R is a side-chain.

Numbering of carbon atoms in gonane

Cholestane, a typical steroid Gonane is the simplest possible steroid and is composed of seventeen carbon atoms, bonded together to form four fused rings. The three cyclohexane rings (designated as rings A, B, and C in the figure above right) form the skeleton of phenanthrene; ring D has a cyclopentane structure. Hence, together they are called cyclopentaphenanthrene. Commonly, steroids have a methyl group at the carbons C-10 and C-13 and an alkyl side chain at carbon C-17. Further, they very by the configuration of the side chain, the number of additional methyl groups and the functional groups attached to the rings. For example the hydroxyl group at position C-3 in sterols.

Cholesterol

Cholic acid

Medrogestone

Classification Taxonomical/Functional Some of the common categories of steroids: •

Animal steroids o Insect steroids  Ecdysteroids such as ecdysterone o Vertebrate steroids  Steroid hormones



 • •

Sex steroids are a subset of sex hormones that produce sex differences or support reproduction. They include androgens, estrogens, and progestagens.  Corticosteroids include glucocorticoids and mineralocorticoids. Glucocorticoids regulate many aspects of metabolism and immune function, whereas mineralocorticoids help maintain blood volume and control renal excretion of electrolytes. Most medical 'steroid' drugs are corticosteroids.  Anabolic steroids are a class of steroids that interact with androgen receptors to increase muscle and bone synthesis. There are natural and synthetic anabolic steroids. In popular language, the word "steroids" usually refers to anabolic steroids. Cholesterol, which modulates the fluidity of cell membranes and is the principal constituent of the plaques implicated in atherosclerosis.

Plant steroids o Phytosterols o Brassinosteroids Fungus steroids o Ergosterols

Structural It is also possible to classify steroids based upon their chemical composition. Examples from this classification include: Class Examples Number of carbon atoms Cholestanes cholesterol 27 Cholanes cholic acid 24 Pregnanes progesterone 21 Androstanes testosterone 19 Estranes estradiol 18 Gonane (or steroid nucleus) is the parent (17-carbon tetracyclic) hydrocarbon molecule without any alkyl sidechains.

Metabolism Steroids include estrogen, cortisol, progesterone, and testosterone. Estrogen and progesterone are made primarily in the ovary and in the placenta during pregnancy, and testosterone in the testes. Testosterone is also converted into estrogen to regulate the supply of each, in the bodies of both females and males. Certain neurons and glia in the

central nervous system (CNS) express the enzymes that are required for the local synthesis of pregnane neurosteroids, either de novo or from peripherally-derived sources. The rate-limiting step of steroid synthesis is the conversion of cholesterol to pregnenolone, which occurs inside the mitochondrion.

Simplified version of latter part of steroid synthesis pathway, where the intermediates isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) form geranyl pyrophosphate (GPP), squalene and, finally, lanosterol, the first steroid in the pathways. Some intermediates are omitted for clarity. Steroid metabolism is the complete set of chemical reactions in organisms that produce, modify, and consume steroids. These metabolic pathways include: • • •

steroid synthesis – the manufacture of steroids from simpler precursors steroidogenesis – the interconversion of different types of steroids steroid degradation.

A. Steroid biosynthesis Steroid biosynthesis is an anabolic metabolic pathway that produces steroids from simple precursors. This pathway is carried out in different ways in animals than in many other organisms, making the pathway a common target for antibiotics and other anti-

infective drugs. In addition, steroid metabolism in humans is the target of cholesterollowering drugs such as statins. It starts in the mevalonate pathway in humans, with Acetyl-CoA as building blocks, which form DMAPP and IPP. In following steps, DMAPP and IPP form lanosterol, the first steroid. Further modification belongs to the succeeding steroidogenesis. Mevalonate pathway

Mevalonate pathway The mevalonate pathway or HMG-CoA reductase pathway starts with and ends with dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). Regulation and feedback

Several key enzymes can be activated through DNA transcriptional regulation on activation of SREBP (Sterol Regulatory Element-Binding Protein-1 and -2). This intracellular sensor detects low cholesterol levels and stimulates endogenous production by the HMG-CoA reductase pathway, as well as increasing lipoprotein uptake by upregulating the LDL receptor. Regulation of this pathway is also achieved by controlling the rate of translation of the mRNA, degradation of reductase and phosphorylation. Pharmacology

A number of drugs target the mevalonate pathway: • •

Statins (used for elevated cholesterol levels) Bisphosphonates (used in treatment of various bone-degenerative diseases)

Plants and bacteria

In plants and bacteria, the non-mevalonate pathway uses pyruvate and glyceraldehyde 3phosphate as substrates. DMAPP to lanosterol Isopentenyl pyrophosphate and dimethylallyl pyrophosphate donate isoprene units, which are assembled and modified to form terpenes and isoprenoids, which are a large class of lipids that include the carotenoids, and form the largest class of plant natural products. Here, the isoprene units are joined together to make squalene and then folded up and formed into a set of rings to make lanosterol. Lanosterol can then be converted into other steroids such as cholesterol and ergosterol.

Human Steroidogenesis

B. Steroidogenesis Steroidogenesis is the biological process by which steroids are generated from cholesterol and transformed into other steroids. The pathways of steroidogenesis differ between different species, but the pathways of human steroidogenesis are shown in the figure. Products of steroidogenesis include: • • •

androgens o testosterone estrogens and progesterone corticoids o cortisol o aldosterone

C. Elimination Steroids are oxidized mainly by cytochrome P450 oxidase enzymes, such as CYP3A4. These reactions introduce oxygen into the steroid ring and allows the structure to be broken up by other enzymes, to form bile acids as final products. These bile acids can then be eliminated through secretion from the liver in the bile. The expression of this oxidase gene can be upregulated by the steroid sensor PXR when there is a high blood concentration of steroids.

Chapter- 9

Triglyceride

Example of an unsaturated fat triglyceride. Left part: glycerol, right part from top to bottom: palmitic acid, oleic acid, alpha-linolenic acid, chemical formula: C55H98O6 A triglyceride (triacylglycerol, TAG or triacylglyceride) is an ester derived from glycerol and three fatty acids. It is the main constituent of vegetable oil and animal fats.

Chemical structure Triglycerides are formed by combining glycerol with three molecules of fatty acid. The glycerol molecule has three hydroxyl (HO-) groups. Each fatty acid has a carboxyl group (COOH). In triglycerides, the hydroxyl groups of the glycerol join the carboxyl groups of the fatty acid to form ester bonds: HOCH2CH(OH)CH2OH + RCO2H + R'CO2H + R''CO2H → RCO2CH2CH(O2CR')CR'' + 2O The three fatty acids (RCO2H, R'CO2H, R''CO2H in the above equation) are usually different, but many kinds of triglycerides are known. The chain lengths of the fatty acids in naturally occurring triglycerides vary, but most contain 16, 18, or 20 carbon atoms. Natural fatty acids found in plants and animals are typically composed only of even numbers of carbon atoms, reflecting the pathway for their biosynthesis from the twocarbon building block acetyl CoA. Bacteria, however, possess the ability to synthesise odd- and branched-chain fatty acids. As a result, ruminant animal fat contains oddnumbered fatty acids, such as 15, due to the action of bacteria in the rumen. Many fatty acids are unsaturated, some are polyunsaturated, e.g., those derived from linoleic acid.

Most natural fats contain a complex mixture of individual triglycerides. Because of this, they melt over a broad range of temperatures. Cocoa butter is unusual in that it is composed of only a few triglycerides, derived from palmitic, oleic, and stearic acids.

Metabolism The enzyme pancreatic lipase acts at the ester bond, hydrolysing the bond and "releasing" the fatty acid. In triglyceride form, lipids cannot be absorbed by the duodenum. Fatty acids, monoglycerides (one glycerol, one fatty acid) and some diglycerides are absorbed by the duodenum, once the triglycerides have been broken down. Triglycerides, as major components of very-low-density lipoprotein (VLDL) and chylomicrons, play an important role in metabolism as energy sources and transporters of dietary fat. They contain more than twice as much energy (9 kcal/g or 38 kJ/g) as carbohydrates and proteins. In the intestine, triglycerides are split into monoacylglycerol and free fatty acids in a process called lipolysis, with the secretion of lipases and bile, which are subsequently moved to absorptive enterocytes, cells lining the intestines. The triglycerides are rebuilt in the enterocytes from their fragments and packaged together with cholesterol and proteins to form chylomicrons. These are excreted from the cells and collected by the lymph system and transported to the large vessels near the heart before being mixed into the blood. Various tissues can capture the chylomicrons, releasing the triglycerides to be used as a source of energy. Fat and liver cells can synthesize and store triglycerides. When the body requires fatty acids as an energy source, the hormone glucagon signals the breakdown of the triglycerides by hormone-sensitive lipase to release free fatty acids. As the brain cannot utilize fatty acids as an energy source (unless converted to a ketone), the glycerol component of triglycerides can be converted into glucose, via glycolysis by conversion into Dihydroxyacetone phosphate and consequently into Glyceraldehyde 3-phosphate, for brain fuel when it is broken down. Fat cells may also be broken down for that reason, if the brain's needs ever outweigh the body's. Triglycerides cannot pass through cell membranes freely. Special enzymes on the walls of blood vessels called lipoprotein lipases must break down triglycerides into free fatty acids and glycerol. Fatty acids can then be taken up by cells via the fatty acid transporter (FAT).

Role in disease In the human body, high levels of triglycerides in the bloodstream have been linked to atherosclerosis (hardening of the arteries), and, by extension, the risk of heart disease and stroke. However, the relative negative impact of raised levels of triglycerides compared to that of LDL:HDL ratios is as yet unknown. The risk can be partly accounted for by a strong inverse relationship between triglyceride level and HDL-cholesterol level.

Guidelines The American Heart Association has set guidelines for triglyceride levels: Level mg/dL Level mmol/L Interpretation 5.65

Very high: high risk

Please note that this information is relevant to triglyceride levels as tested after fasting 8 to 12 hours. Triglyceride levels remain temporarily higher for a period of time after eating.

Reducing triglyceride levels Diets high in carbohydrates, with carbohydrates accounting for more than 60% of the total caloric intake, can increase triglyceride levels. Of note is how the correlation is stronger for those with higher BMI (28+) and insulin resistance (more common among overweight and obese) is a primary suspect cause of this phenomenon of carbohydrateinduced hypertriglyceridemia. There is evidence that carbohydrate consumption causing a high glycemic index can cause insulin overproduction and increase triglyceride levels in women. Adverse changes associated with carbohydrate intake, including triglyceride levels, are stronger risk factors for heart disease in women than in men. Triglyceride levels are also reduced by exercise, omega-3 fatty acids from fish, flax seed oil, and other sources. Recommendation in the U.S. is that one ingest up to 3 grams a day of such oils. It has been found that residents in Western countries do not ingest sufficient quantity of food with omega-3. In Europe, the recommendation is for up to 2 grams. However, omega-3 consumption should be balanced with omega-6 fatty acids, in a ω6/ω-3 ratio between 1:1 and 4:1 (i.e., no more than four grams omega-6 for every one of omega-3). Carnitine has the ability to lower blood triglyceride levels. In some cases, fibrates have been used to bring down triglycerides substantially. Heavy use of alcohol can elevate triglycerides levels.

Industrial uses Linseed oil and related oils are important components of useful products used in oil paints and related coatings. Linseed oil is rich in di- and triunsaturated fatty acid components, which tend to harden in the presence of oxygen. The hardening process is peculiar to these so-called "drying oils". It is caused by a polymerization process that begins with oxygen attacking the carbon backbone. Triglycerides are also split into their components via transesterification during the manufacture of biodiesel. The resulting fatty acid esters can be used as fuel in diesel engines. The glycerin has many uses, such as in the manufacture of food and in the production of pharmaceuticals.

Staining Staining for fatty acids, triglycerides, lipoproteins, and other lipids is done through the use of lysochromes (fat-soluble dyes). These dyes can allow the qualification of a certain fat of interest by staining the material a specific color. Some examples: Sudan IV, Oil Red O, and Sudan Black B.

Interactive pathway map

Statin Pathway

Chapter- 10

Bile Acid

Bile acids are steroid acids found predominantly in the bile of mammals. Bile salts are bile acids compounded with a cation, usually sodium. In humans, the salts of taurocholic acid and glycocholic acid (derivatives of cholic acid) represent approximately eighty percent of all bile salts. The two major bile acids are cholic acid, and chenodeoxycholic acid. Bile acids, glycine and taurine conjugates, and 7-alpha-dehydroxylated derivatives (deoxycholic acid and lithocholic acid) are all found in human intestinal bile. An increase in bile flow is exhibited with an increased secretion of bile acids. The main function of bile acid is to facilitate the formation of micelles, which promotes processing of dietary fat.

Production and distribution Bile acids are made in the liver by the cytochrome P450-mediated oxidation of cholesterol. They are conjugated with taurine or the amino acid glycine, or with a sulfate or a glucuronide, and are then stored in the gallbladder, which concentrates the salts by removing the water. In humans, the rate limiting step is the addition of a hydroxyl group on position 7 of the steroid nucleus by the enzyme cholesterol 7 alpha-hydroxylase. Upon eating a meal, the contents of the gallbladder are secreted into the intestine, where bile acids serve the purpose of emulsifying dietary fats. Bile acids serve other functions, including eliminating cholesterol from the body, driving the flow of bile to eliminate catabolites from the liver, emulsifying lipids and fat soluble vitamins in the intestine to form micelles that can be transported via the lacteal system, and aiding in the reduction of the bacteria flora found in the small intestine and biliary tract. Bile acid refers to the protonated (-COOH) form. Bile salt refers to the deprotonated or ionized (-COO-) form. Conjugated bile acids are more efficient at emulsifying fats because at intestinal pH, they are more ionized than unconjugated bile acids. Synthesis of bile acids is a major route of cholesterol metabolism in most species other than humans. The body produces about 800 mg of cholesterol per day and about half of that is used for bile acid synthesis. In total about 20-30 grams of bile acids are secreted into the intestine daily. about 90% of excreted bile acids are reabsorbed by active transport in the ileum and recycled in what is referred to as the enterohepatic circulation which moves the bile salts from the intestinal system back to the liver and the

gallbladder. This allows a low rate of daily synthesis, but high secretion to the digestive system. Bile is also used to break down fat globules into tiny droplets. Bile from slaughtered animals can be used in the preparation of soap.

Types

IUPAC recommended ring lettering (left) and atom numbering (right) of the steroid skeleton.The four rings A-D form a sterane core.

Cholesterol with numbering Bile salts constitute a large family of molecules, composed of a steroid structure with four rings, a five or eight carbon side-chain terminating in a carboxylic acid, and the

presence and orientation of different numbers of hydroxyl groups. The four rings are labeled from left to right (as commonly drawn) A, B, C, and D, with the D-ring being smaller by one carbon than the other three. The hydroxyl groups have a choice of being in 2 positions, either up (or out) termed beta (often drawn by convention as a solid line), or down, termed alpha (seen as a dashed line in drawings). All bile acids have a hydroxyl group on position 3, which was derived from the parent molecule, cholesterol. In cholesterol, the 4 steroid rings are flat and the position of the 3-hydroxyl is beta. In many species, the initial step in the formation of a bile acid is the addition of a 7-alpha hydroxyl group. Subsequently, in the conversion from cholesterol to a bile acid, the junction between the first two steroid rings (A and B) is altered, making the molecule bent, and in this process, the 3-hydroxyl is converted to the alpha orientation. Thus, the default simplest bile acid (of 24 carbons) has two hydroxyl groups at positions 3-alpha and 7-alpha. The chemical name for this compound is 3-alpha,7-alpha-dihydroxy-5-betacholan-24-oic acid, or as it is commonly known, chenodeoxycholic acid. This bile acid was first isolated from the domestic goose, from which the "cheno" portion of the name was derived. Another bile acid, cholic acid (with 3 hydroxyl groups) had already been described, so the discovery of chenodeoxcholic acid (with 2 hydroxyl groups) made the new bile acid a "deoxycholic acid" in that it had one less hydroxyl group than cholic acid. The 5-beta portion of the name denotes the orientation of the junction between rings A and B of the steroid nucleus (in this case, they are bent). The term "cholan" denotes a particular steroid structure of 24 carbons, and the "24-oic acid" indicates that the carboxylic acid is found at position 24, which happens to be at the end of the side-chain. Chenodeoxycholic acid is made by many species, and is quite a functional bile acid. Its chief drawback lies in the ability of intestinal bacteria to remove the 7-alpha hydroxyl group, a process termed dehydroxylation. The resulting bile acid has only a 3-alpha hydroxyl group and is termed lithocholic acid (litho = stone). It is poorly water-soluble and rather toxic to cells. Bile acids formed by synthesis in the liver are termed "primary" bile acids, and those made by bacteria are termed "secondary" bile acids. As a result, chenodeoxycholic acid is a primary bile acid, and lithocholic acid is a secondary bile acid. To avoid the problems associated with the production of lithocholic acid, most species add a third hydroxyl group to chenodeoxycholic acid. In this manner, the subsequent removal of the 7-alpha hydroxyl group by intestinal bacteria will result in a less toxic, still functional dihydroxy bile acid. Over the course of vertebrate evolution, a number of positions have been chosen for placement of the third hydroxyl group. Initially, the 16alpha position was favored, particularly in birds. Later, this position was superseded by a large number of species selecting position 12-alpha. Primates (including humans) utilize 12-alpha for their third hydroxyl group position. The resulting primary bile acid in humans is 3-alpha,7-alpha,12-alpha-trihydroxy-5-beta-cholan-24-oic acid, or as it is commonly called, cholic acid. In the intestine, cholic acid is dehydroxylated to form the dihydroxy bile acid deoxycholic acid. In many vertebrate orders still subject to speciation, new species are

discarding 12-alpha hydroxylation in favor of a hydroxy group on position 23 of the sidechain. Vertebrate families and species exist that have experimented with and utilize just about every position imaginable on the steroid nucleus and side-chain. The principal bile acids are:

Cholic acid

Chenodeoxycholic acid

Glycocholic acid

Taurocholic acid

Deoxycholic acid

Lithocholic acid In humans, the most important bile acids are cholic acid, deoxycholic acid, and chenodeoxycholic acid. Prior to secretion by the liver, they are conjugated with either the amino acid glycine or taurine. Conjugation increases water solubility, preventing passive

re-absorption once secreted into the small intestine. As a result, the concentration of bile acids in the small intestine can stay high enough to form micelles and solubilize lipids. "Critical micellar concentration" refers to both an intrinsic property of the bile acid itself and amount of bile acid necessary to function in the spontaneous and dynamic formation of micelles. Bile acid are essentially steroid except that they are secreted from the liver and have direct metabolic actions in the body.

Regulation As surfactants or detergents, bile acids are potentially toxic to cells, and their concentrations are tightly regulated. They function as a signaling molecule in the liver and the intestines by activating a nuclear hormone receptor, FXR, also known by its gene name NR1H4. Such activation inhibits synthesis of bile acid in the liver when bile acid levels are too high. Emerging evidence associates FXR activation with alterations in triglyceride metabolism, glucose metabolism, and liver growth.

Clinical significance Since bile acids are made from endogenous cholesterol, the enterohepatic circulation of bile acids may be disrupted to lower cholesterol. Bile acid sequestrants bind bile acids in the gut, preventing reabsorption. In so doing, more endogenous cholesterol is shunted into the production of bile acids, thereby lowering cholesterol levels. The sequestered bile acids are then excreted in the feces. Tests for bile acids are useful in both human and veterinary medicine, as they help to diagnose a number of conditions, including cholestasis, portosystemic shunt, and hepatic microvascular dysplasia.