Lipid metabolism I Biochemistry I Lecture 8 2013

Lipid metabolism I Biochemistry I Lecture 8 2013 (E. T. )

Major classes of lipids triacylglycerols prostanoids Energy nutrients steroids Derived lipids phospholipids glycerophospholipids leukotriens sphingofosfolipids Mainly structural components of membranes 2

Metabolisms of lipids metabolism of TG a FA 100 g/day Source of energy metabolism of structural lipids 2 g/day Triacylaglycerols are the most effective form of energy deposition. compound Glykogen TG Heat of combustion (k. J/g) 17 38 3

Triacylglycerols, fatty acids and esterified cholesterol are very hydrophobic they are not soluble in water unless they are emulsified or included in micelles in the presence of tensides. 4

lipids make the upper phase water 5

Four natural tensides work in fat digestion Tenside Type Origin Bile acids anionic from cholesterol in liver 2 -Acylglycerol non-ionic TAG hydrolysis in gut FA anions anionic TAG hydrolysis in gut Phospholipids amphoteric food 6

Emulsification of lipids in the intestine – formation of micelles colipase The main tensides are fatty bile acids 7

Lipid adsorption in the intestine Formation of mixed micelles from products of digestion Mixed micelles are composed of fatty acids, mono/diacylglycerols, bile acids, phospholipids and fat-soluble vitamins Bile acids, phospholipids and fatty acids function as tensides Intestinal lumen Mucosal cell (enterocyte) 8

In the extracellular fluids hydrophobic lipids are transported in the form of lipoprotein particles 9

Lipoprotein particles transport triacylglycerols and cholesterol in body fluids Hydrophobic core Superficial layer (hydrophilic surface) monolayer 10

Types of lipoproteins TG Chylomikron CM VLDL (very low density) PL CH Proteiny LDL (low density HDL (high density) 11

Metabolism of triacylglycerols 1. Hydrolytic cleavage of ester bonds 2. Metabolism of fatty acids and glycerol 12

Lipases are enzymes that catalyse hydrolysis of ester bonds of triacylglycerols releasing free fatty acids. O CH 2–O–C– O –C–O–CH O CH 2–O–C– Extracellular lipases Pancreatic lipase secreted into the duodenum, Lipoprotein lipase on the surface of the endothelium lining the capillaries Intracellular lipases Hormone-sensitive lipase of adipocytes mobilizing fat stores 13 Lysosomal lipase

Transport of fatty acids in ECT FA are released mainly from TG in adipocytes by the action of hormonsensitive lipase (hormonal regulation) or from lipoprotein particles FA in blood – carried by albumin (1 mmol/l, half-life 2 min) Transport of FA in cells • special membrane proteins facilitate the transport of FA across cytoplasmatic membrane • fatty acid binding proteins facilitate the intracellular transport • carnitine facilitates the transport across mitochondrial membrane 14

Degradation of lipids in the body adipocytes TG HSlipase Chylomicron, VLDL LPlipase FA Binding to albumin liver, muscle mitochondrie ER FA Binding to FABP -oxidation Binding to carnitin acetyl. Co. A Hormone-sensitive lipase in adipocytes is an intracellular lipase that through hydrolysis of triacylglycerols mobilizes the fat energy reserves. The activity of this lipase is controlled by hormones: Glucagon (at low blood glucose) 15 and adrenaline/noradrenaline (in stress)

Degradation of fatty acids: β-oxidation Fatty acids serve as an energy source for most of the cells (not for the nervous system and for red blood cells). The tissues gain fatty acids - either from lipoprotein particles after the triacylglycerols have been hydrolysed by lipoprotein lipase, - or as fatty acids mobilized by the action of hormones on the fat stores in adipose tissue and supplied bound onto albumin. Location: matrix of mitochondria 16

The utilization of fatty acids in the cells requires three stages of processing 1. Activation of FA by linking to coenzyme A 2. Transport of acyl Co. A into the mitochondrial matrix 3. β-Oxidation of acyl Co. A in the mitochondrial matrix to acetyl Co. A that enters the citrate cycle. 17

1. Activation of a fatty acid – synthesis of acyl coenzyme A NH 2 Coenzyme A CH 3 O O HS CH 2 HN Cysteamine N N C CH 2 HN β-Alanine O N N O C CH 2 O P~O P O CH 2 HO CH 3 O O O Pantoic acid Pantothenic acid O OH O P O O 3´–phospho ADP Acyls can be attached to the sulfanyl group by means of a thioester bond. 18

Synthesis of acyl-Co. A The synthesis of the high-energy acyl-Co. A thioester is catalysed by acyl-Co. A synthetases R–COO– + Co. A–SH R–CO–S-Co. A ATP AMP + 2 Pi Acyl-Co. A synthetases are located on the outer mitochondrial membrane. There is a loss of energy equivalent to 2 molecules of ATP, because the reaction is made irreversible by the hydrolysis of inorganic diphosphate (AMP + ATP 2 ADP). 19

2 Carnitine carries long-chain activated fatty acids into the mitochondrial matrix Acyl-Co. A itself cannot cross the inner mitochondrial membrane; instead, acyl groups are transferred to carnitine, transported across the membrane as acylcarnitine, and transferred back to Co. A within the mitochondrial matrix. Short-chain fatty acids (4 – 10 carbon atoms) do not require the carnitine shuttle, they can cross the inner mitochondrial membrane. CH 3 Carnitine H 3 C N CH 2–CH–CH 2–COO CH 3 OH – Trimethyl(2 -hydroxy-3 -carboxypropyl)ammonium 20

The transfers of acyls from acyl-Co. A to carnitine and from acylcarnitines to Co. A are catalysed by carnitine acyltransferases I and II. (also named carnitinpalmitoyltransferase CPT 1 and 2) CH 3 H 3 C N CH 2 CH 3 CH O CH 2 COOH C O Ester bond 21

Formation of acylcarnitine Intermembrane space + (CH 3)3 N CH 2 CH CH 2 COOH + O H 3 C C S Co. A OH Carnitinacyltransferase I or II Inhibition by malonyl-Co. A + (CH 3)3 N CH 2 CH O C CH 2 COOH + Co. ASH O CH 3 22

Transport of fatty acid into mitochondria – carnitine shuttle intermembrane space inner mitoch. membrane matrix acyl. Co. A RCO-S-Co. A Cn-OH Carnitin/acylkarnitin translocase Co. A-SH RCO-OCn acylcarnitine CPT 1 RCO-OCn Co. A-SH acylcarnitine CPT 2 Two forms of carnitinacyltransferase (also named carnitinpalmitoyltransferase CPT) 23

Sources and need of carnitine Synthesis in organism (10 -20 mg/day) Carnitine pool 20 g SAM + + Protein-CH 2 CH 2 NH 3 protein-CH 2 CH 2 N(CH 3)3 Side chain of lysine proteolysis Ascorbate is needed carnitine trimethyllysine Liver, kidney Transport in blood Intake in food: cca 100 mg/day ( meat, milk, also plant sources). Bioavailability - 75% Resorption in kidneys – 98 -99% is resorbed in tubuli 24

Carnitine deficiences • Liver diseases decreased synthesis • Malnutrition, vegetarian diet • Increased requirements for carnitine (pregnancy, burns, trauma) • Enzyme deficiency (transferases, translocase) Decreased ability of tissues to use long chain fatty acids as a metabolic fuel. Carnitine supplementation is necessary 25

Consequences of carnitine deficiency The ability to use fatty acids as a source of energy is reduced • Deficiency in liver – nonketotic hypoykemia during fasting -oxidation is necessary for provision of acetyl. Co. A for ketogenesis and ATP production in citric acid cycle • Deficiency in liver – muscle weakness, cramps during work 26

Inborn deficiency in carnitine transport Autosomal recesive deficiency of Na+-dependent carnitine transporter in muscle and kidney • Carnitine deficiency in muscle and heart • typically appear during infancy or early childhood and can include severe brain dysfunction (encephalopathy), a weakened and enlarged heart (cardiomyopathy), confusion, vomiting, muscle weakness, and low blood sugar (hypoglycemia). All individuals with this disorder are at risk for heart failure, liver problems, coma, and sudden death. Can be detected by expanded newborn screening by tandem mass spectrometry. Therapy: lifelong use of L-carnitine 27

FA-transport enzyme deficiency Inborn errors in fatty acids metabolism are components of newborn screening • CPT-I deficiency — affects the liver; severe hypoglycemia, total carnitine 150– 200 % of normal value. • CPT-II deficiency— cardiac and skeletal muscle, carnitine cca 25– 50 % mild (adult form) — rhabdomyolysis after prolonged exercise, starvation or at exposure to cold; severe (neonatal form) — cardiomyopathy, muscle weakness, congenital malformation. • Carnitin acylkarnitin translocase deficiency — hypoketotic hypoglycemia at fasting, arythmia, apnoe. Often death in infancy. 28

Carnitine as dietary supplement ? • The available research on L-carnitine supplementation does not appear to support claims of enhanced aerobic or anaerobic exercise performance. • Carnitine supplementation with supraphysiological doses above and beyond that which the body requires, does not result in increased fat oxidation at rest or during exercise in well-nourished individuals; • thus, it appears that we can synthesize the necessary amounts from a diet adequate in its precursors. • Athletes wishing to explore carnitine's purported benefits must be aware that the dietary supplement industry is not regulated and, therefore, product safety is not guaranteed. The bioavailability is 5 -10% • High doses (5 or more grams per day) may cause diarrhea. Other rare side effects include increased appetite, body odor, and rash. 29

Transport of fatty acids with the short chain Fatty acids with the chains shorter than 12 carbons do not require carnitine for their transport into the mitochondria. They freely cross the mitochondrial membrane. 30

3. -Oxidation of fatty acids • Main way of FA degradation • Fatty acid is enters the process in form of acyl-Co. A • -carbon is oxidized (C-3) • repetition of four reactions : dehydrogenation hydration dehydrogenation thiolysis by Co. A (fatty acid is shortened by two carbons and acetyl-Co. A is released) 31

(1) First dehydrogenation Saturated acyl Co. A α, β-Unsaturated acyl Co. A (2, 3 -unsaturated) configuration trans 32

(2) Hydration of double bond α, β-Unsaturated acyl Co. A β-Hydroxyacyl Co. A (L-3 -Hydroxy) Hydration is not a redox reaction, by addition of water to a double bond the sum of the oxidation numbers of both carbon atoms remain the same. 33

(3) Dehydrogenation of hydroxyacyl 34

(4) The final step: the thiolysis of 3 -oxoacyl-Co. A by Co. A-SH thiolase CC ACYL Co. A Shortened by two carbons Acetyl Co. A Substrate for the citrate cycle 35

! Distinguish: three types of lysis cleavage of substrate with water: Hydrolysis sucrose + H 2 O glucose + fructose (starch)n + H 2 O maltose + (starch)n-2 Phosphorolysis cleavage of O-glycosidic bond by phosphate: (glycogen)n + Pi (glycogen)n-1 + glucose-1 -P cleavage of C-C bond by sulfur of Co. A–SH Thiolysis β-oxidation of FA or utilization of KB, RCH 2 CO-SCo. A + Co. A-SH RCH 2 CO-SCo. A + CH 3 CO-SCo. A 36

-oxidation • 1. dehydrogenation FA oxidase acyl-Co. A dehydrogenase (FAD) • 2. hydration • 3 dehydrogenation (NAD+) • 4 thiolytic cleavage and transfer of acyl to Co. ASH 2 -enoyl-Co. A hydratase 3 -hydroxyacyl-Co. A dehydrogenase thiolase 37

Acyl-Co. A dehydrogenases (first reaction in β-o. ) 4 main types for FA with short chain (SCAD) mediate chain (MCAD) long chain (LCAD) very long chain (VLCAD) Examination of MCAD, LCAD and VLCAD deficiency is a component of newborn screening. MCAD deficiency One of the most common inborn errors of fatty acid metabolism. Under conditions of health this may not cause significant problems. However, when such individuals do not eat for prolonged periods or have increased energy requirements, the impairment of fatty acid oxidation may lead to fatty acid buildup, hypoglycemia, hyperammonemia and, possibly, sudden death. 38

The energetic yield of β-oxidation of palmitate – to 8 acetyl coenzymes A Palmitoyl Co. A + 7 FAD + 7 NAD+ + 7 H 2 O + 7 Co. A 8 acetyl Co. A + 7 FADH 2 + 7 NADH + 7 H+ 14 ATP + 21 ATP – 2 ATP (activation of palmitate) – and 8 acetyl Co. A in the citrate cycle 8 12 ATP = 96 ATP Net yield of complete palmitate oxidation to CO 2 14 ATP + 21 ATP – 2 ATP + 96 ATP = 129 ATP/palmitate 39

Net yield of the aerobic breakdown of glucose is 38 mol ATP / mol glucose (M = 180 g / mol; 6 mol C), i. e. 0. 21 mol ATP / g glucose, or 6. 3 mol ATP / mol C. Net yield of complete oxidation of palmitate is 129 mol ATP / mol palmitate (M = 256 g / mol; 16 mol C), i. e. 0. 50 mol ATP / g palmitate, or 8. 1 mol ATP / mol C. 40

Oxidation of unsaturated FA Oleic acid: cis 9 -C 18 cis 7 -C 16 cis 5 -C 14 cis 3 -C 12 isomerase trans 2 -C 12 Loss of FADH 2 Analogous process with -oxidation 41

FA with odd number of C provide propionyl-Co. A CO 2 + H 2 O D-methylmalonyl-Co. A COO - propionyl-Co. A H C CH 3 CH 2 CO -S-Co. A biotin ATP CH 3 CO-S-Co. A ADP racemase It is formed also by metabolism of some AA COO CH 2 -CH 2 CO-S-Co. A succinyl-Co. A B 12 COO CH 3 C H CO-S-Co. A L-methylmalonyl-Co. A 42

-oxidation in the peroxisome Very-long-chain fatty acids (20 C or longer) • preliminary -oxidation in peroxisomes • shortening of the chain • shortened FA is transferred to a mitochondrion FAD is the cofactor of -oxidation in peroxisome It is oxidized by molecular oxygen FADH 2 + O 2 → FAD + H 2 O 2 Energy is not obtained 43

-oxidation of FA is powerfull source of energy When does it take place? When the cells requires energy and availability of glucose is limited -oxidation is initiated by hormones in postabsorptive state or starvation, particularly in liver, muscle and myocardium 44

Lipids in postresorption phase (glucagon) liver Muscle, myocard FA Acetyl-Co. A FA FA-albumin Acetyl-Co. A FA + glycerol HSL TAG Adipose tissue Effect of glucagon 45

Mobilization of fat stores in postabsorptive phase (glucagon) Glucagon (or adrenaline) activate hormone sensitive lipase in adipose tissue • HSL cleaves triacylglycerols to fatty acids and glycerol • Fatty acids are released into the blood • the plasma level of free fatty acids increases • FA are taken up by the liver and other peripheral tissues (esp. muscle, myocard and kidney) at the rates proportional to the plasma concentration. 46

Formation of ketone bodies - ketogenesis OH CH 3–CH–CH 2–C O OH -Hydroxybutyric acid -2 H +2 H O O CH 3–C–CH 2–C O–H Acetoacetic acid – CO 2 O CH 3–C–CH 3 Acetone Ketone bodies are formed in the liver mitochondria and released into blood plasma. The two acids are detectable in plasma at any time, the usual ratio βhydroxybutyrate to acetoacetate is 3 – 6 (it reflects the intramitochondrial NADH/NAD+ ratio). There always traces of ketone bodies in urine, since there is no renal threshold for the two acids. Ketone bodies are readily metabolised in non-hepatic tissues. 47

Ketogenesis in liver mitochondria 4 C 2 C 2 C Acetoacetyl-Co. A 2 C Acetyl-Co. A H 2 O 6 C 3 -Hydroxy-3 -methylglutaryl-Co. A (HMG-Co. A) 4 C 2 C Acetoacetate (free) 3 C Acetyl-Co. A Acetone β-Hydroxybutyrate 4 C 48

The causes of increased keton bodies formation During fasting fatty acids are mobilized from adipose tissue and part of them is transported into the liver increased production of acetyl-Co. A by -oxidation capacity of citric cycle is overloaded (lack of oxalacetate) synthesis of keton bodies 49

Utilization of ketone bodies in non-hepatic tissues β-Hydroxybutyrate and acetoacetate are important in Acetoacetate is reactivated to acetoacetyl-Co. A through the transfer of Co. A from succinyl. Co. A. providing energy for peripheral tissues. β-Hydroxybutyrate Acetone is a waste product, eliminated by the kidney or expired, it can be smelt on the breath. (thioforase) are broken down in the citrate cycle 50

Formation and utilization of keton bodies CO 2 liver Keton bodies in blood Lack of oxaloacetate muscle Acetyl-Co. A FA CNS FA-albumin FA + glycerol-P TAG Adipose tissue Synthesis of thioforase is induced in brain after several days of starvation 51

The production of ketone bodies increases at high ratio glucagon/insulin, when fat stores are mobilized (prolonged fasting, starvation, uncontrolled diabetes mellitus type I). An extreme production of ketone bodies (ketosis) is very dangerous, because ketogenesis is a proton-producing process that disturbs acidbase balance (evoking ketoacidosis) and, through excretion of the two acids into urine, is a cause of serious loss of cations. Acetoacetic acid p. Ka = 3. 52 β-Hydroxybutyric acid p. Ka = 4. 70 52

Can be triacylglycerols formed de novo in the body? In human: fatty acids (except the essential) triacylglycerols can be synthesized 53

Fatty acids synthesis Location: Mainly liver, lactating mammary gland, in lesser extent adipocytes, brain When? sufficient amounts of acetyl. Co. A, that need not be utilized for production of energy ? After the meal, when sufficient amounts of glucose are available for production of acetyl Co. A, 54

Steps in fatty acid synthesis (cytoplasma) 1. Transport of acetyl-Co. A from matrix to cytoplasma 2. Malonyl-Co. A formation 3. Serie of reactions on fatty acid synthase enzyme complex 55

Transfer of acetyl Co. A to the cytosol acetyl-Co. A is formed in matrix of mitochondria mainly by oxidative decarboxylation of pyruvate (from glucose, amino acids) • acetyl-Co. A cannot freely penetrate the mitochondrial membrane • it is transported in form of citrate When it does occur? In case that citrate is not necessary for citric acid cycle 56

When the citrate is not necessary for citric acid cycle? -the well fed state – sufficient amounts of glucose are available producing acetyl Co. A, – low energy expenditure – high ATP concentrations within the cells inhibit degradation of acetyl Co. A in the citrate cycle, – absence of stress that activates mobilization of fat stores, free fatty acids released through the action of catecholamines inhibit fatty acid synthesis. 57

Transfer of acetyl Co. A to the cytosol pyruvate, AK MATRIX CYTOPLAZMA acetyl-Co. A + oxalacetate ADP + Pi oxalacetate acetyl-Co. A NADH NAD+ + H+ ATP Co. A malate citrate malat e citrate Co. A Blocked by ATP isocitrate 58

2. Synthesis of malonyl Co. A Acetyl. Co. A does not have energy enough to enter the synthesis of fatty acids Principle of carboxylation and decarboxylation Formation of malonyl. Co. A by carboxylation and its decarboxylation in the next step 59

Synthesis of malonyl Co. A is the rate-limiting step in fatty acid synthesis, catalysed by acetyl-Co. A carboxylase: ATP ADP + Pi O HN O NH + HCO 3– C O –Enzyme S Biotinyl–E CH 2–CO–S–Co. A COO– Malonyl Co. A N –COOH HN S Carboxybiotinyl–E CH 3–CO–S–Co. A Acetyl Co. A C O –Enzyme

Regulation of acetyl-Co. A carboxylase Activation by citrate Inhibition by acyl-Co. A with long chains (palmitate) Hormonal regulation: insulin glucagon, adrenalin 61

The fatty acyl synthase complex In mammals, the complex is a homodimer Each monomer is arranged in three domains carrying the seven catalytic activities. One of the two functional units One domain in both monomers includes the acyl carrier protein (ACP) area to which the phosphopantethein "arm" is attached Seven enzyme activities: AT Acetyl/acyl-Co. A transacylase MT Malonyl transacylase CE Condensing enzyme (Oxoacyl-PPt synthase) KR Oxoacyl reductase DH Hydroxyacyl dehydratase ER Enoyl reductase TE Palmitoyl thioesterase ACP domaine with phosphopantethein arm Two proteins with –SH group bind intermediates of the synthesis Both monomers cooperate so that each of them takes part on the 62 synthesis of two fatty acids processed simultaneously,

The flexible phosphopantethein "arm" of the synthase linked to a serine residue of acyl carrier protein ACP is found also in coenzyme A (as just one half of the coenzyme A molecule): CH 3 O C CH 2 O HS CH 2 HN Cysteamine C CH 2 HN HO CH 3 O NH O P~O CH 2–CH CO O β-Alanine Pantoic acid Pantothenic acid ACP The processed acyls attached to the sulfanyl group are carried from one active site of the synthase complex to another. 63

Reactions of fatty acid synthesis 1 Transfer of the acetyl group of acetyl Co. A to the sulfur of a cystein residue of the condensing enzyme. The reaction is catalysed by acetyl transacylase. SH ACP CO–CH 3 S Cys 64

2 The malonyl group is transferred to the sulphur atom of the phosphopantetheine attached to ACP. The reaction is catalysed by malonyl transacylase. COOH CH 2 CO S ACP CO–CH 3 S Cys 65

3 Condensation joining acetyl unit to a two-carbon part of the malonyl unit on phosphopantetheine. CO 2 is released. An acetoacetyl unit is formed. The reaction is catalysed by condensing enzyme (3 -oxoacyl synthase). CH 3 COOH CH 2 CO S ACP CO–CH 3 S Cys C=O CH 2 CO S ACP + CO 2 SH Cys 66

4 The first reduction catalysed by β-ketoacyl reductase with NADPH. The product is 3 -hydroxyacyl unit. CH 3 C=O + NADPH+H+ CH 2 CO CH 3 CH–OH + NADP+ CH 2 CO S S ACP SH Cys 67

5 Dehydration catalysed by 3 -hydroxyacyl dehydratase. The product is trans– 2–enoyl (named crotonyl) unit. CH 3 CH–OH CH 2 CO S ACP CH 3 CH CH CO SH S Cys ACP + H 2 O SH Cys 68

6 The second reduction catalysed by enoyl reductase with NADPH. The product is saturated acyl (now butyryl) unit. Initial acetyl was elongated by two carbon atoms. CH 3 CH CH CO S ACP + NADPH+H+ CH 3 CH 2 CO S + NADP+ SH SH Cys ACP 69

7 The saturated acyl is transferred to the cysteine sulfur atom on the condensing enzyme. The synthase is now ready for another round of elongation CH 3 CH 2 CO S ACP SH SH Cys ACP CH 3 CH 2 CO S Cys 70

After the completion of the first elongating cycle, new malonyl is "loaded“ on the sulfanyl group of PPt. In the second round of fatty acid synthesis, butyryl unit condenses with malonyl to form a C 6 -acyl, …… The elongation cycles continue until C 16 -acyl unit (palmitoyl) is formed. Palmitoyl unit is a good substrate for thioesterase that hydrolyses palmitoyl-PPt to yield palmitate (16: 0). 71

In mammals, palmitate is the major product of FA synthesis. A minor saturated product is stearate (18: 0). Further elongation of fatty acids is provided by similar mechanisms, but the elongating system is located on the membranes of endoplasmic reticulum 72

NADPH is required in the reductive steps of FA synthesis The main source of NADPH is the pentose phosphate pathway. A certain part of NADPH is supplied by the reaction catalysed by NADP+–linked malate enzyme ("malic enzyme“): Malate + NADP+ pyruvate + CO 2 + NADPH The reaction takes part on the transport of acetyl-Co. A (in the form of citrate) across the inner mitochondrial membrane. 73

The stoichiometry of fatty acid synthesis The synthesis of palmitate (C 16): The synthesis of malonyl Co. A 7 Acetyl Co. A + 7 CO 2 + 7 ATP 7 malonyl Co. A + 7 ADP + 7 Pi + 14 H+ The synthesis catalysed by the fatty acid synthase complex Acetyl Co. A + 7 malonyl Co. A + 14 NADPH + 20 H+ palmitate + 7 CO 2 + 14 NADP+ + 8 Co. A + 6 H 2 O The overall stoichiometry for the synthesis of palmitate is 8 Acetyl Co. A + 7 ATP + 14 NADPH + 6 H+ palmitate + 14 NADP+ + 8 Co. A + 6 H 2 O + 7 ADP + 7 Pi 74

Compare Feature FA -oxidation FA synthesis Localization mitochondria cytoplasm Acyl attached to Co. A-SH ACP Basic unit acetyl (C 2) Redox cofactors NAD+, FAD NADPH Enzymes separated dimer / complex Stimulated by glucagon insulin 75

Elongation of fatty acids Although palmitate (C 16) is the major product of the fatty acid synthase complex, and is the chief saturated fatty acid in human fat, stearate and oleate (C 18) are common and longer-chain fatty acids, arachidate (C 20), behenate (C 22) and lignocerate (C 24) occur in phospholipids. Elongation by enzymes bound to the endoplasmic reticulum: – Activation of palmitate by conversion to palmitoyl Co. A, – activation of acetyl Co. A by its carboxylation to malonyl Co. A, – elongation similar to synthesis catalysed by FA synthase complex, but the intermediates are Co. A-thioesters, not enzyme-bound acyls. The reductant is also NADPH. Elongation process in mitochondria (for the synthesis of fatty acids incorporated into mitochondrial lipids): – Reversal of the β-oxidation. 76

Desaturation of fatty acids In higher animals, only the desaturases are known which generate double bonds at carbons 9, 6, 5, and 4. 9 12 15 Mammals lack the enzymes to introduce double bonds at carbon atoms beyond C-9. Fatty acids containing double bonds beyond C-9 are synthesized by plants, they contain also 12 - and 15 -desaturase. Unsaturated fatty acids of the series n-6 are comprised in all plant oils (olive oil, sunflower oil etc. ). 15 -Desaturase is present predominantly in plants growing in cold water (algae, phytoplankton), then a high concentration of polyunsaturated fatty acyls of the series n-3 is in fish oils (fish 77 feeds phytoplankton).

Polyunsaturated fatty acids n-3 and n-6 are essential for animals They serve as precursors of eicosanoids (prostanoids and leukotrienes). If food intake is sufficient (vegetable oils, fish), linoleate (linoleic acid) and α-linolenate (linolenic ac. ) are precursors of other PUFA as arachidonate (n-6) and eicosapentaenoate (n-3), from which eicosanoids are formed. Linoleate 18: 2 (9, 12) 6 -desaturation γ-Linolenate 18: 3 (6, 9, 12) elongation Eicosatrienoate 20: 3 (8, 11, 14) 5 -desaturation Arachidonate 20: 4 (5, 8, 11, 14) α-Linolenate 18: 3 (9, 12, 15) 6 -desaturation Octadecatetraenoate 18: 4 (6, 9, 12, 15) elongation Eicosatetraenoate 18: 4 (8, 11, 14, 17) 5 -desaturation Eicosapentaenoate 18: 5 (5, 8, 11, 14, 17) 78

Elongation and desaturation of FA n-9 series 18: 0 18: 1 (9) n-6 series plants 18: 2 (9, 12) n-3 series phytoplankton 6 -desaturase 18: 2 (6, 9) 6 -desaturase 18: 3 (6, 9, 12) elongation 20: 2 (8. 11) elongation 22: 3 (7, 10, 13) 18: 4 (6, 9, 12, 15) elongation 20: 3 (8, 11, 14) 5 -desaturase 20: 3 (5, 8, 11) 18: 3 (9, 12, 15) 20: 4 (8, 11, 14, 17) 5 -desaturase 20: 5(5, 8, 11, 14, 17) 20: 4(5, 8, 11, 14) elongation 22: 4 (7, 10, 13, 16) 22: 5 (7, 10, 13, 16, 19) 79

Mechanism of long-chain fatty acyl-Co. As desaturation Location: smooth endoplasmic reticulum of liver cells. Desaturases are hydroxylating monooxygenases. The substrate is hydroxylated and after it water is eliminated from the hydroxylated product with the formation of the double bond. The reductant is NADH+H+, from which the electrons are carried by the flavine enzyme and the cytochrome b 5 to a desaturase. 80

Mechanism of long-chain fatty acyl-Co. A desaturation Example: Stearoyl–Co. A O 9 1 10 S Co. A O=O + NADH+H+ HO O H 1 H H Oleoyl–Co. A H S Co. A + H 2 O + NAD+ Co. A + H 2 O H O 1 S 81

Synthesis of triacylglycerols ER –liver, adipocytes, enterocytes 1. Synthesis of lysophosphatidate CH 2 OH CO RCOSCo. A HSCo. A NAD+ CH 2 OH ADP CHOH CH 2 OH glycerol * ATP CH 2 OH CO CH 2 O P NADH + H+ CH 2 OCOR NADPH + H+ NADP HSCo. A CH 2 OCOR CHOH CH 2 O P glycerol-3 P lysophosphatidate 82

2. Synthesis of phosphatidate Usually unsaturated lysophosphatidate 83

3. Synthesis of triacylglycerols Pi CH 2 OCOR CHOCOR CH 2 O P RCOSCo. A HSCo. A CH 2 OCOR hydrolase CH 2 OH CHOCOR CH 2 OCOR triacylglycerol PI, kardiolipin PC, PE, PS Intestine CM ER Lier VLDL Adipocytes deposition 84