BIOCHEM Fatty acids and Eicosanoids.pdf

METABOLISM OF LIPIDS (FATTY ACIDS & EICOSANOIDS) JUDELYN T. TY-UY, M.D. MANILA CENTRAL UNIVERSITY COLLEGE OF MEDICINE Department of Biochemistry  OBJECTIVES: 1. Explain the processes involved in the synthesis of Fatty acids and Eicosanoids. 2. Correl...

METABOLISM OF LIPIDS (FATTY ACIDS & EICOSANOIDS) JUDELYN T. TY-UY, M.D. MANILA CENTRAL UNIVERSITY COLLEGE OF MEDICINE Department of Biochemistry  OBJECTIVES: 1. Explain the processes involved in the synthesis of Fatty acids and Eicosanoids. 2. Correlate the effects of diet and nutrition in the biosynthesis of fatty acids. 3. Detect the effect of pharmacologic agents with the enzymes involved in the metabolism of eicosanoids  BIOSYNTHESIS OF FATTY ACIDS Fatty acids are oxidized or degraded to acetyl CoA and synthesized from acetyl CoA Acetyl CoA FATTY ACIDS CH3-CO~S-CoA Their oxidation/degradation is not simply the reverse of their biosynthesis  BIOSYNTHESIS OF FATTY ACIDS Fatty acid synthesis occurs in the cytosol Fatty acid oxidation occurs in the mitochondria LIPOGENESIS OCCURS IN THE CYTOSOL It occurs in the liver, kidney, brain, lung, mammary gland & adipose tissue Major sites: Liver, adipose tissue and mammary gland Acetyl-CoA  immediate substrate LIPOGENESIS OCCURS IN THE CYTOSOL Who will provide the carbon for FA synthesis? End product: Free Palmitate  where all other FA are made by its modification  FATTY ACID ACTIVATION In the presence of ATP & coenzyme A, the enzyme acyl-CoA synthetase catalyzes the conversion of FFA to an “active FA” or acyl-CoA ENZYMES INVOLVED IN FATTY ACID SYNTHESIS Malonyl-CoA FASN ACC Palmitate Acetyl-CoA ACC - Acetyl CoA carboxylase FASN - Fatty acid synthase RATE LIMITING STEP IN FATTY ACID SYNTHESIS Formation of Malonyl-CoA through the carboxylation of acetyl-CoA by the enzyme acetyl-CoA carboxylase BIOTIN RATE LIMITING STEP IN FATTY ACID SYNTHESIS 2 step reaction in the synthesis of Malonyl-CoA: 1. Carboxylation of biotin by biotin carboxylase, ATP & - HCO3 2. Transfer of the carboxyl group to acetyl-CoA Acetyl CoA carboxylase Acetyl-CoA carboxylase - Enz-biotin-COO Enz-biotin biotin carboxylase - ATP + ADP + Pi - HCO3 Biotin carboxylase  The enzyme acetyl CoA carboxylase can also be activated or inactivated Dephosphorylation of the ACC enzyme will make it active  promoting formation of Malonyl CoA Phosphorylation of ACC will make it inactive RATE LIMITING STEP IN FATTY ACID SYNTHESIS BIOTIN Activated: 1. Well-fed state  Citrate 2. Insulin  dephosphorylation of the ACC enzyme Inactivated by: 1.Glucagon/Epinephrine  phosphorylation of the enzyme 2.Accumulation of long chain acyl-CoA  FATTY ACID SYNTHESIS: LIPOGENESIS Fatty acids are synthesized by the sequential addition of 2-carbon units from malonyl Co-A to the activated end of the chain by FATTY ACID SYNTHASE 2 prosthetic groups: 1. -SH group of cysteine 2. -SH group of phosphopantetheine – acyl carrier protein domain of FA synthase Pant-SH HS-Cys Cys-SH HS-Pant  FATTY ACID SYNTHESIS: LIPOGENESIS FA synthase is a multienzyme complex Each unit contains 7 enzymes & a protein (Vitamin B5 - Pantothenic acid) Enoyl Malonyl Pant-SH Hydratase HS-Cys reductase Ketoacyl transacyl Pant-SH HS-Cys reductase ase Acetyl ACP Thioestera transacyl se ase Cys-SH HS-Pant Cys- Ketoacyl SH synthase Cys-SH HS-Pant Pant-SH  FATTY ACID SYNTHESIS: LIPOGENESIS It is a dimer with two identical units Pant-SH Pant-SH Cys- SH  FATTY ACID SYNTHESIS: LIPOGENESIS 1. A priming molecule of acetyl-CoA (2-carbon unit) combines with a cysteine (-SH) group catalyzed by acetyl transacylase 2. Malonyl-CoA (3-carbon unit) combines to adjacent –SH on the phosphopantetheine of the ACP catalyzed by malonyl transacylase Acetyl Malonyl Ketoacyl transacylase transacylase synthase Acetyl-malonyl enzyme  FATTY ACID SYNTHESIS: LIPOGENESIS 3. The acetyl group attacks the methylene group of the malonyl residue, catalyzed by the ‘condensing enzyme’ : ketoacyl synthase forming 3-ketoacyl enzyme; freeing the cyteine –SH group & liberating CO2 4. The 3-ketoacyl-ACP is reduced to -hydroxyacyl-ACP by - ketoacyl reductase; using NADPH as an electron donor 5. -hydroxyacyl-ACP is dehydrated by hydratase to an unsaturated acyl enzyme 6. Then reduced by enoyl reductase to a saturated acyl CoA using a second NADPH as a reductant  FATTY ACID SYNTHESIS: LIPOGENESIS Ketoacyl Enoyl Hydratase reductase reductase 3-ketoacyl enz -hydroxyacyl Unsaturated acyl Saturated acyl enz enz enz  FATTY ACID SYNTHESIS: LIPOGENESIS 7. A new malonyl-CoA combines with the –SH of phosphopantetheine, displacing the saturated acyl to the free cysteine –SH group 8. Sequence is repeated six more times until a saturated 16- carbon acyl (palmityl) has been assembled Malonyl transacylase Saturated acyl enz  FATTY ACID SYNTHESIS: LIPOGENESIS 8. When the saturated acyl enzyme is 16 carbon atoms long, thioesterase catalyzes the hydrolysis of the thioester linking the FA to phosphopantetheine  16-C saturated palmitate is the final product of FATTY ACID SYNTHASE complex Thioesterase C=O CH2-(CH2)7-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3 Saturated acyl enz  FATTY ACID SYNTHESIS: LIPOGENESIS With acetyl CoA as the primer & malonyl CoA as the donor of subsequent carbon atoms, the overall reaction is: Acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H+ + Palmitate + 7 CO2 + 14 NADP + 8 CoASH + 7 H20  FATTY ACID SYNTHESIS: LIPOGENESIS In the mammary gland: Usually found in breast milk lipids – Remember lipogenesis occurs in the mammary gland, and that the breast milk would need to produced short and medium chain fatty acids only, in order for the lingual and gastric lipases in children to digest the chains. Separate thioesterase specific for acyl residues of C8, C10, C12  ACETYL-COA Principal building block of FA Act as a primer for the synthesis of even numbered fatty acids Propionyl-CoA for the synthesis of long chain FA having odd number of carbon atoms Propionyl-CoA SOURCE OF ACETYL-COA Formed from glucose via the oxidation of pyruvate w/in the mitochondria Doesn’t readily diffuse to the cytosol Need to condense with oxaloacetate to form citrate (citric acid/TCA cycle) Citrate will be translocated from the mitochondria to the cytosol via the tricarboxylate transporter Citrate undergoes cleavage to acetyl-CoA & oxaloacetate catalyzed by ATP-citrate lyase  SOURCES OF NADPH: 1. Pentose phosphate pathway – chief source of the hydrogen for the reductive synthesis of FA - PPP pathway also occurs in the cytosol  no membrane barrier against the transfer of NADPH 2. Conversion of malate to pyruvate by malic enzyme (NADP malate dehydrogenase) 3. Extramitochondrial isocitrate dehydrogenase reaction  FATTY ACID SYNTHESIS: LIPOGENESIS Free palmitate must be activated to acyl-CoA before it can proceed to any metabolic pathways Fate of palmitoyl-CoA 1. Esterification to acylglycerols (MAG,DAG,TAG,PL) 2. Esterification to cholesteryl esters 3. Chain elongation 4. Desaturation: monoenoic  PUFA  CHAIN ELONGATION Fatty acid elongation occurs in the endoplasmic reticulum Elongates saturated & unsaturated fatty acyl CoA from C10 upward by 2 carbons sequence using malonyl CoA In the brain, there are one or more elongation systems, which synthesize longer fatty acid chains Elongation of stearyl Co A increases during myelination in order to provide C22 & C24 fatty acids for sphingolipids CHAIN ELONGATION Fatty acyl elongase system: Malonyl-CoA as the acetyl donor NADPH provides the reducing power Palmitoyl CoA is converted almost exclusively to stearate (18:0)  FORMATION OF MONOENOIC ACIDS Fatty acid desaturation involves reactions & enzymes that introduce double bonds in the FA chain Considered to be responsible for the formation of nonessential monounsaturated FA from saturated FA Double bond introduced is nearly always in the 9 position  FORMATION OF MONOENOIC ACIDS Catalyzed by 9 desaturase in the endoplasmic reticulum O2+NADH+ H+ Δ9 Desaturase Cyt b5 Palmitoleoyl-CoA NAD++2H2 O  FORMATION OF PUFA In mammals, double bonds can be added only to the proximal half of the fatty acyl CoA  double bonds can not be added beyond C9 In humans, there are 4 distinct desaturases, each with a different specificity: 9, 6, 5, 4 As a rule, addition of double bonds are always separated from each other by a methylene group -CH2CH=CHCH2CH=CHCH2CH=CHCH2-  FORMATION OF PUFA Long-chain unsaturated FA of metabolic significance: 1.Palmitoleic & oleic are not essential in the diet 2.Linoleic (6) & -linolenic acids (3) – are the only FA known to be essential for complete nutrition  FORMATION OF PUFA From the nutritionally essential FA  other 3 & 6 families can be synthesized by a combination of chain elongation & desaturation  ARACHIDONIC ACID 6, 20:4, 5,8,11,14 present in membranes & accounts for 5–15% of the FA in phospholipids Usually attached in the C2 of the glycerol moiety of phospholipids  DOCOSAHEXAENOIC ACID 3, 22:6 (4,7,10,13,16,19) Synthesized from -linolenic acid or obtained directly from fish oils Present in high concentrations in retina, cerebral cortex, testis, and sperm  DOCOSAHEXAENOIC ACID Particularly needed for development of the brain & retina and is supplied via the placenta and milk Retinitis pigmentosa: have low blood levels of DHA  CLINICAL ASPECT Rats fed with purified nonlipid diet containing vitamins A and D  exhibit a reduced growth rate & reproductive deficiency Humans: Dry, scaly rah, hair loss/depigmentation, poor wound healing, growth restriction in children, and increase susceptibility to infection Tx: addition of linoleic, -linolenic to the diet  NUTRITIONAL STATE REGULATES LIPOGENESIS Rate is HIGH Rate is LOW High carbohydrate diet Restricted caloric diet Well-fed state High fat diet Insulin Deficiency of insulin Sucrose > Glucose (DM) Glucagon Epinephrine  NUTRITIONAL STATE REGULATES LIPOGENESIS Rate is HIGH Increase activity: High carbohydrate diet Acetyl CoA Well-fed state carboxylase Insulin Fatty acid synthase Sucrose > Glucose Desaturase Elongation system Fructose content of sucrose bypasses the phosphofructokinase control point in glycolysis thus flooding the lipogenic pathway  EICOSANOIDS Active compounds: Roles: – Prostaglandins (PG) Biological effects on – Thromboxanes (TX) inflammatory responses – Prostacyclins On the intensity & – Leukotrienes (LT) duration of pain & fever – Lipoxins (LX) On reproductive function Inhibit gastric acid Act mainly as local secretion hormones affecting the Regulates BP  cells that produced vasodilation or them or neighboring constriction cells Inhibits platelet aggregation & thrombosis  EICOSANOIDS Major source of arachidonic acid is through its release from cellular stores LIPID BILAYER Phosphatidylcholine Phosphatidylinositol Phospholipase C Phospholipase A2 Diacylglycerol Arachidonic acids MAG  EICOSANOIDS Arachidonate & some other C20 PUFA Usually from cleavage of the beta acyl chain of phospholipids Dietary source: Linoleic 6  immediate dietary precursor of arachidonate -linolenic acids (3)  eicosapentaenoate precursor  DHA PHOSPHOLIPASES Phospholipase A2 removes fatty acyl group on carbon 2 Phospholipase C cleaves the bond between C3 to phosphate  METABOLISM OF EICOSANOIDS Cyclooxygenase pathway  PG/TX Lipoxygenase Lipoxygenase pathway  LT/LX Cycloxygenase  BASIC STRUCTURE PROSTAGLANDINS THROMBOXANE 20 carbon atoms w/ an internal 20 carbon atoms saturated 5 C ring (cyclopentane Contains a 6 membered ring ring) (cyclopentane ring with an Hydroxyl group at C15 oxygen atom – oxane ring) Double bond bet C13-14 Hydroxyl group at C15 Various substituents on the ring: Double bond bet C13-14 usually hydroxyl or keto group at C9 or C11  CYCLOOXYGENASE PATHWAY Involves the consumption of two molecules of O2 catalyzed by cyclooxygenase (COX) (aka prostaglandin H synthase), an enzyme that has two activities: cyclooxygenase & peroxidase 2 isoenzymes of COX: COX-1 & COX-2. The product, an endoperoxide (PGH), is converted to PROSTAGLANDIN D/E/F, THROMBOXANES, PROSTACYCLINS  BASIC STRUCTURES LEUKOTRIENES LIPOXINS 20 carbon atoms 20 carbon atoms Epoxide or hydroxyl group at Contains 3 hydroxyl grps C5 Four double bonds Four double bonds Four of them are conjugated – Three of them are conjugated (tetraenoic) – (Trienoic) LTA4 LXA4 LXB4  LIPOXYGENASE PATHWAY Lipoxygenase catalyzes incorporation of O2 onto a carbon (C5,12,15) forming a hydroperoxy (-OOH) group, which is further converted to an epoxide or a hydroxyl group 3 of the double bonds are conjugated to form a triene Only 5-lipoxygenase forms LEUKOTRIENES LIPOXINS are formed by the action of 15- lipoxygenase followed by 5-lipoxygenase  SOME FUNCTIONS OF EICOSANOIDS PGI2, PGE2, PGD2 PGF2 TXA2 Increase: Increase: Increase: Vasodilation Vasoconstriction Vasoconstriction Bronchoconstriction Platelet aggregation Smooth muscle Lymphocyte proliferation contraction Bronchoconstriction Decrease: Platelet aggregation Leucocyte aggregation T cell proliferation Lymphocyte migration LTB4 LTC4, LTD4, LTE4 LX Increase: Increase: Increase: Vascular permeability Bronchoconstriction Chemotaxis T cell proliferation Vascular permeability Stimulate superoxide Leukocyte activation & Slow-reacting substance of anion production in migration anaphylaxis leukocytes  PHARMACOLOGY GLUCOCORTICOIDS Induce the synthesis of lipocortins or macrocortins that inhibits the activity of phospholipase A2 ASPIRIN Suppresses the production of PG/TX by irreversibly inhibiting cyclooxygenase Inhibits both COX1 & COX2 IBUPROFEN, NAPROXEN, Reversibly inhibits cyclooxygenase MEFENAMIC ACID both COX1 & COX2 PARACETAMOL Selectively inhibit COX2 in the CNS COXIBS Selective COX2 inhibitors ZILEUTON (ZYFLO) Inhibitor of 5-lipoxygenase LATANOPROST Prostaglandin analoque used to reduce intraocular pressure in glaucoma MONTELUKAST Blocks the activation of LTC4, LTD4, LTE4 By blocking the cysteinyl leukotriene receptor 1 (CysLTR-1)