Biochemistry LC8 Fatty Acid & Tag Metabolism PDF

METABOLISM - linked series of chemical reactions that begins with a particular molecule and converts COURSE OUTLINE it into some other molecule or molecules in a...

METABOLISM - linked series of chemical reactions that begins with a particular molecule and converts COURSE OUTLINE it into some other molecule or molecules in a carefully defined fashion I. OVERVIEW OF LIPID METABOLISM A. Lipolysis B. Lipogenesis Types of metabolic pathways: II. FATTY ACIDS 1. Fuel (CH2O) catabolism > CH2 + H2O + A. TYPES OF FATTY ACID energy OXIDATION/DEGRADATION 2. Energy + small molecules anabolism– B. Biosynthesis of FATTY ACID complex molecules C. FATTY ACID SYNTHESIS VS 3. Some pathways can be either anabolic or DEGRADATION catabolic, depending on the energy D. ACTIVATION OF FATTY ACIDS conditions in the cell. They are referred to D.1. DISORDERS ASSOCIATED as amphibolic pathways. WITH IMPAIRED BETA - Needless to say lipids (fatty acids, OXIDATION triglycerides, phospholipids and E. KETOGENESIS cholesterol) And ketone bodies F. FATTY ACID BIOSYNTHESIS are metabolized by our cells G. SYNTHESIS OF ARACHIDONIC according to their physiological ACID FROM LINOLEIC ACID demands (energy, homeostasis) H. HYPERTRIGLYCERIDEMIA I. SHORT-TERM REGULATION A. LIPOLYSIS OF FATTY ACID SYNTHESIS J. MOBILIZATION OF FATTY Lipolysis = Triacylglycerol (TAG) ACIDS FROM ADIPOSE Hydrolysis = Catabolism and mobilization TISSUES of stored fats (in adipocyte) during K. TRIACYLGLYCEROLS starvation L. ROLE OF LIPIDS IN THE DIET initiated by hormone-sensitive lipase M. IDENTIFICATION OF FATS AND (HSL), which hydrolyzes the FAs at C1 OILS and/or C3 of TAG. Regulated by hormones through cAMP- dependent interconversion III. REFERENCES The amount of fatty acids (FAs) released depends on the activity of this enzyme. In the blood, FAs are transported in free form FATTY ACID AND TAG METABOLISM (non-esterified). Learning Objectives: Only short chain fatty acids are soluble in - Basic characteristics of lipids by defining blood. Longer, less water-soluble fatty simple and complex lipids and identify the acids are transported by being bound to lipid classes in each group serum albumin - Clinical implications of non-degradation of Glycerol diffuses into the plasma and is complex lipids taken upon by tissues like liver and kidney - Types of oxidation of fatty acids with active glycerol kinase (absent in - Clinical implications of impaired fatty acid adipocytes). oxidation TAG stores continually undergo lipolysis and reesterification. I. OVERVIEW OF LIPID METABOLISM B. LIPOGENESIS Lipogenesis = TAG synthesis The pathway is active when overall energy demand is low and carbohydrate input exceeds total body needs. The carbohydrate content of food is high, while storage of carbohydrates in the body is limited. TAGs come from lipoproteins: VLDLs and Chylomicrons Formed in the liver and then intestines and Figure 1 delivered by the blood to the liver and peripheral tissues. BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 Lipoprotein lipase, in the inner surface of acids and monoacylglycerides (MAG) blood capillaries cleaves TAGs into before the enterocytes uptake- the glycerol and fatty acids, which are taken enterocyte MAG serve as a substrate for up by adipocytes and converted back (re acylation in a two step process yielding esterified) into fats. triglyceride. WELL FED STATE Within adipose tissue, there is no expression of TAG synthesis glycerol kinase so the building block f9r triglycerides Lipogenesis in this tissue is the glycolytic intermediate, 3 (FA) + glycerol = TAG dihydroxyacetone phosphate (DHAP). Insulin (+) Dephosphorylation of enzymes The DHAP is reduced to Liver, adipose tissue glycerol-3-phosphate by cytosolic glycerol-3- phosphate dehydrogenase and the remaining reaction of triglyceride synthesis is the same as for all other tissues. Figure 2 FASTING/ STARVATION / DM TAG breakdown Lipolysis TAG= 3 ( FA) + glycerol Glucagon/ epinephrine/ NE Phosphorylation of enzymes Adipose tissue Figure 4 When low glucose of the blood trigger the release of glucagon 1. The hormone binds its receptor in the adipocyte membrane and thus 2. (+) adenylyl cyclase, via a G protein, to produce cAMP. This activates PKA, which phosphorylates Figure 3 3. the hormone- sensitive lipase and Triglycerides, TG, TAG, - the major tissues 4. perilipin molecules on the surface of the for synthesis are the small intestines, the lipid droplet. Phosphorylation of perilipin liver, and adipocytes. permits hormone- sensitive lipase access Except for the intestine and adipocytes, to the surface of the lipid droplet; where triglycerides begins with glycerol-3- 5. it hydrolyzes triacylglycerols to free fatty phosphate acids and monoacylglycerols (MAG) Glycerol is first phosphorylated by glycerol 6. Fatty acids leave the adipocyte and bind kinase and then activated fatty acids (fatty serum albumin in the blood, and are acyl-CoA) serve as the substrates for carried in the blood; they are released from addition generating phosphatidic acid. the albumin and The phosphate group is removed and the 7. enter a specific cell via a specific fatty acid last fatty acid is added. transporter where In the smallest intestine, dietary 8. The FAs oxidize to CO2, and the energy of triglycerides are hydrolyzed to free fatty oxidation is conserved in ATP. PREPARED BY: BATCH 2028 1D 2 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 2. Many proteins are modified by the covalent attachment of fatty acids, which target them to membrane locations 3. Fatty acids are fuel molecules. They are stored as TAG. Fatty acids mobilized from triacylglycerols are oxidized to meet the energy needs of a cell or organism. 4. Fatty acid derivatives serve as hormones and intracellular messengers e.g. steroids, sex hormones and Prostaglandins. A. TYPES OF FATTY ACID OXIDATION/DEGRADATION 1. Beta oxidation- Major mechanism, occurs in the mitochondrial matrix. 2-C units are released as acetyl CoA per cycle. 2. Alpha oxidation - Predominantly takes Figure 5 place in the brain and liver, one carbon is lost in the form of CO2 per cycle. 1. Glucagon - inhibits FA synthesis by 3. Omega oxidation - Minor mechanism, but inhibiting the key enzyme acetyl CoA becomes important in conditions of carboxylase. impaired beta oxidation 2. Insulin - increases FA synthesis in several 4. Peroxisomal oxidation - Mainly for the ways: trimming of very long chain fatty acids. by decreasing lipolysis Activation of protein phosphatase B. BIOSYNTHESIS OF FATTY ACID Stimulating synthesis and activation of citrate lyase. (Inner 1. Reverse of beta oxidation and outer mitochondria) 2. Condensation of 2 carbon units Enhancing the formation of acetyl 3. Three processes CoA by stimulating glycolysis Transport of acetyl CoA into cytosol Formation of malonyl CoA Assembly of fatty acids C. FATTY ACID SYNTHESIS VS DEGRADATION Figure 6 II. FATTY ACIDS Functions: - Fatty acids have four major physiological roles. 1. Fatty acids are building blocks of Figure 7. Fatty Acid Synthesis vs. Degradation phospholipids and glycolipids. PREPARED BY: BATCH 2028 1D 3 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 The conversion is catalyzed by fatty SYNTHESIS DEGRADATION acyl-CoA synthetase or thiokinase and cytosol Mitochondria inorganic pyrophosphatase. The thiol Requires NADH, NADPH FADH2 group of coenzyme A attacks the Acyl carrier CoA acyl-adenylate, displacing AMP and protein L-isomer forming the thioester fatty acyl-CoA. CO₂ No CO₂ activation No citrate STEP II: Transport of fatty acid from cytosol D-isomer Enzymes as into mitochondria via the acylcarnitine/ carnitine citrate lon independent transporter. Multi-enzyme proteins complex 2 carbon 2 carbon units split off units added, as as 3 acetyl CoA carbon malonyl CoA Table 1. SIZE NUMBER CATABOLISM MEMBRANE Figure 9 CLASS OF SITE TRANSPORT CARBONS SCFA (20 peroxisome (CPT-1) present on the outer membrane of long mitochondria. Table 2. Transport and Oxidation Sites of Different FFAS Fatty acylcarnitine then enters into the mitochondrial matrix with the help of the carnitine-acyl-carnitine transporter protein (carnitine shuttle). Finally, carnitine acyl transferase II (CAT-II) transfers the acyl group from fatty acylcarnitine to mitochondrial coenzyme A, freeing carnitine to return to the inter membrane space through the same transporter protein. The carnitine is released and returned to the cytosol for reuse. - Carnitine (made up of lysine and methionine): Transports the long-chain fatty acids from Figure 8. FA Synthesis vs. B-Oxidation Overview cytosol to mitochondria for ẞ-oxidation D. ACTIVATION OF FATTY ACIDS - It has three enzymes, CPT I, CPT STEP I: Activation of a fatty acid by conversion II, and translocase to a fatty acyl CoA After fatty acid enters into the cell, it is CPT1 (Carnitine Palmitoyltransferase 1) converted into fatty acid CoA. The Location: Found on the outer mitochondrial carboxylate ion is adenylated by ATP, to membrane. form a fatty acyl-adenylate and PPi. The Function: Catalyzes the acyl group transfer PPi is immediately hydrolyzed to two from long-chain fatty acyl-CoA to carnitine, molecules. forming acyl-carnitine, essential for PREPARED BY: BATCH 2028 1D 4 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 transporting fatty acids into the e- removed transferred to FAD mitochondria. Hydration of double bond Isoforms: There are three isoforms- CPTA Dehydration of ẞ-hydroxyl group (liver), CP1B (muscle, and CPT1G (brain). to a ketone Regulation: Inhibited by malonyl-CoA, a -e- removed transferred to NAD+ key regulator of fatty acid oxidation. Acylation- addition of CoA and CPT2 (Carnitine Palmitoyltransferase 2) production of acetyl-CoA Location: Found on the inner mitochondrial membrane. *These four steps make one complete Function: Converts acyl-carnitine back to cycle of beta-oxidation. In each cycle, one acyl-CoA and carnitine inside the acetyl CoA, one FADH2, and one NADH mitochondria, allowing the fatty acids to are produced undergo ẞ-oxidation. Deficiency: CPT deficiency can lead to muscle weakness, hypoglycemia, and other metabolic issues - There are three forms of CPT deficiency: myopathic (most common), severe infantile hepatocardiomuscular, and lethal neonatal Figure 11 Energy Yield from ẞ-oxidation of Palmitic Acid Palmitic acid yields 7 NADH + 7 FADH2 + 8 acetyl CoA in 7 cycles of mitochondrial beta oxidation. Every acetyl CoA yields 3 NADH + 1 FADH2 + 1 GTP (=ATP) during the Krebs cycle. Considering an average production of 2.5 ATP per NADH and 1.5 ATP per FADH2 using the respiratory chain, we have 108 ATP molecules. However, 2 ATP molecules are used for the initial activation of every fatty acid that Figure 10 is going to be oxidized in the mitochondria. Patients undergoing dialysis can lose ○ Hence, a net of 106 ATP carnitine, which may predispose to fatty molecules are produced. liver. Step III: Beta oxidation a. Dehydrogenation of the fatty acyl-CoA to make a trans double bond between α and ẞ carbon. Short, medium, and long chain acyl- CoAdehydrogenases PREPARED BY: BATCH 2028 1D 5 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 Oleoyl-CoA then undergoes three ẞ-oxidation cycles to yield three molecules of acetyl-CoA, 3 NADH and 3 FADH2, and the coenzyme A ester of a Δ3, 12-carbon unsaturated fatty acid cis-Δ3-dodecenoyl-CoA. This product cannot serve as a substrate for enoyl-CoA hydratase, which acts only on trans double bonds. The auxiliary enzyme, Δ3, Δ2-enoyl-CoA isomerase isomerizes the cis-Δ3-enoyl-CoA to the trans-Δ2-enoyl-CoA, which is converted by enoyl CoA hydratase into the corresponding ß- hydroxyacyl CoA (trans-Δ2-dodecenoyl CoA). This intermediate is now acted upon by the remaining enzymes of ß oxidation to yield acetyl CoA and the coenzyme A ester of a 10-carbon saturated fatty acid, decanoyl CoA. The latter undergoes four more passes through the ß- oxidation pathway to yield five more molecules of acetyl CoA Altogether, nine molecules of acetyl CoA are produced from one molecule of 18-carbon oleate Figure 12 2.5 ATPs per NADH = 17.5 1.5 ATPs per FADH2 = 10.5 10 ATPs per acetyl CoA = 80 Total = 108 ATPs 2 ATP equivalents (ATP -> AMP + PPi -> 2 Pi) consumed during activation of palmitate to Palmitoyl CoA Net Energy output = 108 - 2 = 106 ATP b. ẞ-oxidation of Unsaturated Fatty Acids: ○ b1. ẞ-oxidation of Mono-Unsaturated Fatty Acids Figure 13 (e.g. Oleic Acid) Two auxiliary enzymes, an isomerase and b2. ß-oxidation of Poly-Unsaturated Fatty Acids a reductase are needed for the ẞ-oxidation (E.g. Linoleic acid) of common unsaturated fatty acids. 18-carbon Linoleate (Linoleic acid), has a Oleate (Oleic Acid) - 18-carbon cis- Δ9, cis-Δ12 configuration and enters mono-unsaturated FA with a cis double the ß-oxidation cycle as linoleoyl CoA bond - C-9 and C-10 (denoted ∆9 ). (Δ9,12). In the first step of oxidation, oleate is converted to oleoyl CoA. Linoleoyl COA undergoes three ß-oxidation cycles to yield three molecules PREPARED BY: BATCH 2028 1D 6 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 of acetyl CoA and the coenzyme A ester of When this is oxidized and cleaved the a 12-carbon unsaturated fatty acid with a products are acetyl-CoA and propionyl cis-Δ3,cis- Δ6 CoA This intermediate cannot be used by the Complete oxidation of this Propionyl CoA enzyme of the ß-oxidation pathway requires three extra reactions. because its double bonds are in the wrong Propionyl CoA is first carboxylated to form position and have the wrong configuration the D-methyl malonyl CoA by propionyl (cis, not trans). CoA carboxylase Hence, oxidation requires a second The D-methyl malonyl CoA thus formed is auxiliary enzyme, NADPH - dependent enzymatically epimerized to L-methyl 2,4-dienoyl- CoA reductase, in addition to malonyl CoA by methylmalonyl CoA enoyl CoA isomerase epimerase. The L-methylmalonyl-CoA then undergoes The combined action of these two an intramolecular rearrangement to form enzymes converts a trans-Δ2, succinyl- CoA, which can enter the citric cis-Δ4-dienoyl CoA intermediate to the acid cycle. This rearrangement is catalyzed by methyl trans-Δ2-enoyl-CoA substrate that can malonyl CoA mutase, which requires as its enter the ß-oxidation pathway and be coenzyme 5'- deoxyadenosylcobalamin, or degraded to six molecules of acetyl CoA. coenzyme B12, which is derived from The overall result is the conversion of vitamin B (cobalamin). linoleate to nine molecules of acetyl CoA. Figure 15 Figure 14 Conversion of propionyl-CoA to succinyl-CoA. Succinyl-CoA, an intermediate of the TCA cycle, Reaction can form malate, which can be converted to glucose ATP Yield in the liver through the process of gluconeogenesis. Activation of palmitate to palmitoyl CoA-2 NADPH Certain AA also form glucose by this route -3 D.1 DISORDERS ASSOCIATED WITH IMPAIRED 8 rounds of ß-oxidation BETA OXIDATION Oxidation of 9 acetyl CoA ( 9x10= 90) Oxidation of 6 FADH (6x1.5=9) 1. Deficiencies of carnitine or carnitine Oxidation of 8 NADH. ( 8x2.5 =20) transferase or translocase Symptoms include muscle - Less two FADH since Linoleoyl-CoA has cramps during exercise, severe two double bonds; the dehydrogenation weakness, and death. step will be bypassed twice. Muscle weakness related to the importance of fatty acids as a c. ẞ-oxidation of Fatty Acids with Odd long-term energy source Number of Carbon Atoms Hypoglycemia and hypoketosis are common findings PREPARED BY: BATCH 2028 1D 7 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 A diet containing medium-chain to serve as fuel, blood glucose levels may fall to fatty acids is recommended since extremely low levels. Fatty acid levels rise because they do not require carnitine of decreased - oxidation - thus dicarboxylic acids shuttle to mitochondria. are excreted in the urine. Results in decreased levels of acetyl-CoA, the substrate for ketone body synthesis. 3. Dicarboxylic aciduria is characterized by i) Excretion of C 6-C 10 dicarboxylic acids ii) Nonketotic hypoglycemia which is caused by lack of mitochondrial medium chain acyl-CoA dehydrogenases 4. Acute Fatty Liver of Pregnancy - Manifests in the second half of pregnancy, usually close to term, but may also Figure 16 develop in the postpartum period. - The patient developed symptoms of (1) the transporter for carnitine uptake into muscle, hepatic dysfunction at 36 weeks of (2) CPTI gestation. Short history of illness, (3) carnitine acylcarnitine translocase hypoglycemia, liver failure, renal failure, (4) CPTII. and coagulopathy are observed - Diagnosis is made based on an incidental Classical CPTIl deficiency is most commonly finding of abnormal liver enzyme levels. characterized by adolescent-to-adult onset - Affected patients may become jaundiced recurrent episodes of acute myoglobinuria due to or develop encephalopathy from liver prolonged exercise or fasting. Becomes weak, failure, usually reflected by an elevated hypoglycemic, hypoketosis. Lipid deposits are found ammonia level. in skeletal muscles. Both creatine phosphokinase - Profound hypoglycemia is common. (CPK) and long chain acylcarnitines are elevated in the blood. Fatty Acid Oxidation - Minor route for FA oxidation in ER of liver 2. Jamaican Sickness and kidney for 10-12 C MCFAs. - Jamaican vomiting sickness is caused by - Introduces OH- to w-carbon Involving eating the unripe fruit of ackee tree, which molecular oxygen, cytochrome P450 and contains the toxin hypoglycin, which NADPH. inactivates medium and short-chain - Two subsequent oxidation reactions on acyl-CoA dehydrogenases, inhibiting w-carbon produces dicarboxylic acid. ẞ-oxidation and thereby causing - ER - Mitochondria for B-oxidation hypoglycemia. produces Succinate. - The pathway was upregulated in conditions that limit B- oxidation (MCADD). - It involves hydroxylation and occurs in the ER of many tissues. - Hydroxylation occurs on the methyl carbon at the other end of the molecule from the carboxyl group or on the carbon next to the methyl end. - It uses the "mixed function oxidase" type of reaction requiring Cytochrome P450, 02, and NADPH, as well as the necessary enzymes. - Hydroxy fatty acids can be further oxidized to a dicarboxylic acid via sequential Figure 17 reactions of Alcohol dehydrogenase and aldehyde dehydrogenases. Because more glucose must be oxidized to compensate for the decreased ability of fatty acids PREPARED BY: BATCH 2028 1D 8 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 Significance Of Omega Oxidation Defined as the oxidation of fatty acid - The microsomal (ER) pathway of fatty acid (methyl group at beta carbon) with the w-oxidation represents a minor pathway of removal of one carbon unit adjacent to the overall FA oxidation. a- carbon from the carboxyl end. - However, in certain pathophysiological The carbon unit is removed in the form of states, such as diabetes, chronic alcohol CO2 consumption, and starvation, the Alpha oxidation occurs in those fatty acids w-oxidation pathway may provide an that have a methyl group (-CH3) at the effective means for the elimination of toxic beta-carbon, which blocks beta-oxidation levels of free fatty acids. There is no production of ATP. Biological Significance Of Alpha Oxidation The hydroxy fatty acids produced as intermediates of this pathway like Cerebronic acid can be used for the synthesis of cerebrosides and sulfatides Odd chain fatty acids produced upon decarboxylation in this pathway can be used for the synthesis of sphingolipids and can also undergo beta- oxidation to form propionyl co A and Acetyl coA. Clinical Significance Of Alpha Oxidation Refsum's disease (RD)-is a Figure 18 neurocutaneous syndrome that is Alpha Oxidation characterized biochemically by the Takes place in the microsomes of the brain accumulation of phytanic acid in plasma and liver, and tissues. Involves the decarboxylation process for Patients with Refsum disease are unable the removal of a single carbon atom at one to degrade phytanic acid because of time with the resultant production of an deficient activity of the Phytanic acid odd-chain fatty acid that can be oxidase enzyme catalyzing the first step of subsequently oxidized by beta-oxidation phytanic acid alpha- oxidation for energy production It is strictly an aerobic process. Peripheral polyneuropathy, cerebellar ataxia retinitis No prior activation of the fatty acid is pigmentosa, and Ichthyosis (rough, dry, and scaly required. skin) are the major clinical components. The The process involves hydroxylation of the symptoms evolve slowly and insidiously from alpha carbon with a specific a-hydroxylase childhood through adolescence and early that requires Fe++ and vitamin C/FH4 as adulthood. cofactors. Peroxisomal Oxidation Of Very Long Chain Fatty Acids In peroxisomes, a flavoprotein dehydrogenase transfers electrons to O, to yield H2O, instead of capturing the high-energy electrons as FADH, as occurs in mitochondrial beta- oxidation. Catalase is needed to convert the hydrogen peroxide produced in the initial reaction into water and oxygen. Subsequent steps are identical to their mitochondrial counterparts, Figure 19 PREPARED BY: BATCH 2028 1D 9 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 They are carried out by different isoforms of the A and therefore phosphorylates. ACC, enzymes.The specificity of the peroxisomal inhibiting its activity enzymes is for longer-chain fatty acids. Thus So instead of FA synthesis, the peroxisomal enzymes function to shorten the chain predominant pathway is beta- oxidation, length of relatively long chain fatty acids to a point forming a lot of acetyl Coa at which beta oxidation can be completed in Acetyl CoA goes to Kreb's cycle. mitochondria. Figure 20 Significance Of Peroxisomal Oxidation Peroxisomal reactions include chain shortening of very long chain fatty acids, dicarboxylic acids, conversion of cholesterol to bile acids and formation of ether lipids. Figure 21 The congenital absence of functional peroxisomes, Regulation of B-oxidation an inherited defect, causes Zellweger syndrome. (1) Hormones control the supply of fatty acids in the blood. ZELLWEGER SYNDROME (2) Carnitine palmitoyltransferase I is inhibited by Most common malonyl-CoA, which is synthesized by acetyl-CoA Aka cerebrohepatorenal syndrome is a carboxylase (ACC). AMP-PK is the AMP-activated rare, congenital disorder (present at birth) protein kinase. characterized by the reduction or absence (3) The rate of ATP use controls the rate of the of Peroxisomes in the cells of the liver, electron-transport chain, which regulates the kidneys, and brain. oxidative enzymes of -oxidation and the TCA cycle. The most common features of Zellweger syndrome include vision disturbances, prenatal growth failure, lack of muscle tone, unusual facial characteristics, mental retardation, seizures, and an inability to suck and/or swallow. The abnormally high levels of VLCFA( Very long chain fatty acids ) are most diagnostic There is no cure for Zellweger syndrome, nor is there a standard course of treatment Most treatments are symptomatic and supportive Figure 22 Most infants do not survive past the first 6 months B-oxidation is strictly an aerobic pathway, anc usually succumb to respiratory distress, dependent on oxygen, a good blood gastrointestinal bleeding, or liver failure.Short- term Supply, and adequate levels of Regulation of Fatty Acid Oxidation mitochondria. Glucagon and Epinephrine phosphorylate Tissues that lack mitochondria, such as the acetyl CoA carboxylase which forms red blood cells, cannot oxidize fatty acids CAMP which then activates protein kinase by B-oxidation PREPARED BY: BATCH 2028 1D 10 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 Fatty acids also do not serve as a significant fuel for the brain They are not used by adipocytes, whose function is to store triacylglycerols to provide fuel for other tissues. Those tissues that do not use fatty acids as a fuel, or use them only to a limited extent, can use ketone bodies instead. E. KETOGENESIS Acetoacetate is the primary ketone body while beta-hydroxybutyrate and acetone are secondary ketone bodies. They are synthesized exclusively by the liver mitochondria. Figure 24 After oxidation, cells produce a significant amount of ketone bodies, especially when deprived of nutrients. In a state of starvation, acetyl-CoA becomes a primary energy source, leading to the formation of ketone bodies. The process begins with the condensation of two molecules of acetyl-CoA, catalyzed by thiolase, which releases coenzyme A and forms acetoacetyl-CoA. Next, synthetase facilitates the reaction with another molecule of acetyl-CoA to Figure 23 form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). With the subsequent release of acetyl-CoA, the first STEP 1: Condensation- two molecules of ketone body, acetoacetate, is produced. acetyl-CoA are condensed to form acetoacetyl-CoA. STEP 2 : Production of HMG-CoA- one more acetyl-CoA is added to acetoacetyl-CoA (beta-hydroxy beta-methyl glutaryl-CoA). The enzyme is HMG-CoA synthetase. Mitochondrial HMG-CoA is used for ketogenesis, while cytosolic fraction is used for cholesterol synthesis. STEP 3: Lysis- HMG-CoA is lysed to form acetoacetate. HMG-CoA lyase is present only in the liver. STEP 4: Reduction- beta-hydroxybutyrate is formed by the reduction of acetoacetate. Figure 25 STEP 5: Spontaneous decarboxylation- acetone is formed Note: Acetone is a volatile ketone body that is excreted primarily through respiration. Its excessive accumulation is often associated with type 1 PREPARED BY: BATCH 2028 1D 11 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 diabetes mellitus, where a lack of insulin production KETOGENESIS IN DIABETES MELLITUS AND can lead to the development of diabetic STARVATION ketoacidosis (DKA). In this condition, the absence of insulin causes uncontrolled fatty acid breakdown, leading to an overproduction of ketone bodies, including acetone, which can result in life-threatening acidosis if not managed promptly. The liver is the primary site of ketogenesis; however, it cannot use ketone bodies as an energy source due to the absence of the enzyme succinyl-CoA: acetoacetate CoA transferase (thiophorase), which is required for ketone body utilization. Once formed, ketone bodies are released into the bloodstream, where they are taken up by extrahepatic tissues such as the brain, heart, and muscles, and used as an alternative energy source, especially during periods of low glucose availability. Figure 27 F. FATTY ACID BIOSYNTHESIS Figure 28 Figure 26 Citrate Shuttle (at mitochondria and cytosol) In the QUESTION: can acetone be glucogenic? citrate shuttle, acetyl-CoA is first converted to citrate A propanediol pathway of acetone in the mitochondria. Citrate is then transported into metabolism has been proposed which the cytoplasm, where it is cleaved into acetyl-CoA is glycogen and oxaloacetate. The cytoplasmic acetyl-CoA is - Acetone is generally considered used for fatty acid synthesis, while oxaloacetate is non-glucogenic, but a proposed metabolic recycled back to the mitochondria, either as malate pathway, known as the propanediol or pyruvate. During this process, NADPH is pathway, suggests that acetone can be produced converted into glucose. In this pathway, acetone is first metabolized into 1,2-propanediol, which can then be further processed into intermediates that feed into gluconeogenesis, potentially contributing to glucose production. PREPARED BY: BATCH 2028 1D 12 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 1. Synthesis/ formation of malonyl CoA - the three carbon precursor for fatty acid synthesis is made from acetyl-CoA and CO2 - the reaction is catalyzed by acetyl CoA carboxylase(ACC) in the cytosol - ACC is a bifunctional enzyme- biotin carboxylase and transcarboxylase. - ACC contains biotin, natires carrier of CO2 - Biotin shuttles between two active sites Figure 29 Acetyl-CoA is carboxylated by acetyl-CoA carboxylase (ACC), using bicarbonate and ATP, to - in the cytosol, citrate is cleaved by citrate lyase to form malonyl-CoA. reform acetyl-CoA and oxaloacetate. It provides starting material acetyl CoA for de novo synthesis of ACETYL COA CARBOXYLASE fatty acids. → catalyzes the rate-limiting step (COMMITTED - circuitous route is required because pyruvate STEP) in FA synthesis, which is the formation of dehydrogenase, the enzyme that converts pyruvate malonyl CoA from the carboxylation of acetyl CoA. to acetyl-CoA, is found only in mitochondria and - Acetyl CoA carboxylase has biotin as a because acetyl-CoA cannot directly cross the prosthetic group,a common feature in Co2 mitochondrial membrane. Acetyl-CoA from the binding enzymes. mitochondrial oxidation of pyruvate is shuttled to the - It adds CO2 in the form of bicarbonate cytosol as citrate HCO3- with a concomitant use of ATP. - oxaloacetate is shuttled back to the mitochondrial - Just like other committed steps, this step is matrix as pyruvate via malic reaction. also irreversible. - the NADPH that is generated by malic enzyme, along with the pentose phosphate pathway, is used for the reduction reactions that occur on the fatty acid synthase complex. Figure 30 Acetyl CoA - immediate substrate and building block of FA; formed from (CH2O)n via oxidation of Figure 31 pyruvate in mitochondria; transported from NOTE: mitochondria to the cytosol via the CITRATE - ACC rate limiting enzyme of FAS: citrate TRANSPORT SYSTEM. allosterically activates ACC - phosphorylation by AMP-PK inhibits the Acetyl CoA from the mitochondrial oxidation of enzyme while activated by pyruvate is shuttled to the cytosol as citrate (upon dephosphorylation reacting with oxaloacetate). Oxaloacetate is shuttled back to the mitochondrial matrix as pyruvate via MALIC REACTION. PREPARED BY: BATCH 2028 1D 13 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 Figure 32 Figure 33 - The reaction below is the key regulatory step. - The enzyme is activated by Figure 34 dephosphorylation. - In animal cells, acetyl-CoA carboxylase - prokaryotes, this latter domain is a exists as an equilibrium between an separate protein called ACP or acyl carrier inactive dimer and an active polymer. protein. - citrate act as a precursor and an allosteric - large, dimeric, multidomain, multifunctional activator, shifting the equilibrium towards - localized to cytosol the active polymer state, while long chain - expressed in liver, adipose, brain, kidney, fatty acyl-CoA (palmitoyl-CoA is an lung, and mammary inhibitor) shifts the equilibrium towards - It is a dimer, they are arranged head to tail. inactive dimer. These subunits carry on the different 4 -the problem is that the true and product is reactions the cell utilizes every cycle of the triacylglycerol, and its synthesis keeps the pathway. fatty acyl-COA level down.thus, there is no regulatory limit on triglyceride production Transacylation reaction - The initial reaction attaches and activates intermediates for fatty acid synthesis to Enzyme Activator Inhibitor ACP (Acyl Carrier Protein). Acetyl-CoA Citrate, Acyl-CoA, Acetyl transacylase - Enzyme that carboxylase insulin glucagon catalyzes the binding of acetyl CoA to the Carnitine Malonyl-CoA ACP palmitoyl - Malony| transacylase- Enzyme that transferase I catalyzes the binding of malonyl Co to the Table 3 ACP - Coenzyme A Counterpart of Fatty Acid - Fatty acid synthase - a multienzyme - Synthase in beta oxidation complex consisting of a dimer, each monomer of which has seven different catalytic activities plus a domain that covalently binds a molecule of 4' - phosphopantetheine Figure 35 PREPARED BY: BATCH 2028 1D 14 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 Reactions of fatty acid synthase - The acyl carrier protein shuttles the growing chain from one active site to Each subunit of the enzyme binds acetyl residues another during the four step reaction. as thioesters at two different SH groups: at one peripheral cysteine residue (Cys-SH) and one Here, the acetyl and malonyl moieties first central 4-phosphopantetheine group (Pan-SH). Pan condense, with the release of the malonyl SH, which is very similar to coenzyme A, is carboxyl group as CO2. covalently bound to a protein segment of the Secondly, the malonyl-CoA provides the synthase known as the acyl-carrier protein (ACP). two carbon units that are added to the growing fatty acyl chain. Then, the elongation of fatty acid chain occur in the endoplasmic reticulum Lastly, the methyl group of acetyl-CoA becomes the carbon(the terminal methyl group) of palmitate. Each new two carbon unit is added to the carboxyl end of the growing fatty acid chain. Figure 36 This part functions like a long arm that passes the substrate from one reaction center to the next. The two subunits of fatty acid synthase cooperate in this process; the enzyme is therefore only capable of functioning as: 1. Palmitate synthesis Figure 38 Figure 37 Figure 39 - contains a covalently attached prosthetic group 4’-phospho-pantetheine - the acyl carrier protein delivers acetate(in the first step) or malonate (in all the next steps) to the fatty acid synthase PREPARED BY: BATCH 2028 1D 15 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 Entry of electrons from fatty acid oxidation in the Enzyme Product Reaction catalyzed mitochondrial respiratory chain 3-Ketoacyl Acetoacetyl Condensation ACP synthase ACP 3-Ketoacyl 3-OH Butyryl 1st Reduction ACP ACP reductase 3-OHACYL Alpha beta dehydrase transbutenoyl ACP Enoyl Butyryl ACP 2nd Reduction Figure 41 Reductase Table 4 Stoichiometry of palmitate synthesis: 8 acetylCoA+7 ATP +14 NADPH > palmitate + 14 NADP+ + 8 CoASH + water + 7 ADP + 7 Pi NADPH+H= 14 Acetyl CoA needed by the cell to synthesize palmitic acid= 8 citrate= 8 because from 8 citrates, 8 acetyl CoA will be formed by citrate lyase enzyme OAA= 8 converted to pyruvate > (8 NADPH+H is generated) Figure 42 As long as citrate is in the cytoplasm, acetyl CoA carboxylase is stimulated. Figure 43 - Transfer of electrons from acyl-CoA dehydrogenase to the electron-transport Figure 40 chain. - a FAD is tightly bound to each protein in these three electron-transfer reactions. PREPARED BY: BATCH 2028 1D 16 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 ETF, electron transfer flavoprotein; 1. Linoleic acid is activated to form ETF-QO, electron-transferring linoleyl-CoA which is dehydrogenated to flavoprotein-coenzyme Q(CoQ) form y-linolenyl-CoA oxidoreductase. 2. y-linolenyl-CoA is converted to dihomo-y-linolenyl-CoA by the addition of Chain elongation and saturation of fatty acids one molecule of acetyl CoA in the microsomal system of chain elongation 3. Dihomo-y-linolenyl-CoA forms arachidonic acid by another dehydrogenation Pyridoxal-P is required as a coenzyme in the second step. H. HYPERTRIGLYCERIDEMIA ○ Elevated levels of TAGs, termed hypertriglyceridemia, are linked to an increased Figure 44 susceptibility to atherosclerosis and cardiovascular disease (CVD). To introduce a double bond in a saturated fatty acid, ○ Excessive consumption of TGs, particularly we need a source of electrons and protons coming unhealthy saturated fats, can contribute to various from NADH+H with the help of cytochrome B5 health complications, including heart disease, reductase, in the form of FAD. arterial hardening, obesity, stroke, and even - NADH+H transfers electrons to FAD and mortality. becomes reduced to FADH2 which will 4. ○ Hypertriglyceridemia may arise from again oxidize. diverse factors such as genetic - FADH2 will transfer electrons and protons predisposition, such as familial to the ferric form that will be reduced to chylomicronemia syndrome, as well as ferrous forming a double bond with water conditions like obesity, metabolic molecule removal. syndrome, diabetes mellitus, excessive Processes require molecular O2 as a reactant in alcohol intake, hypothyroidism, kidney unsaturated fatty acid biosynthesis. disorders like nephrotic syndrome, paraproteinemia, systemic lupus erythematosus, anorexia nervosa, glycogen storage diseases, sepsis, pregnancy, and certain medications such as steroids, estrogens anabolic steroids, tamoxifen, thiazides, non- cardioselective beta blockers, cyclophosphamide, cyclosporine, and protease inhibitors. I. SHORT-TERM REGULATION OF FATTY ACID Figure 45 SYNTHESIS G. SYNTHESIS OF ARACHIDONIC ACID FROM LINOLEIC ACID Figure 47 1. In the cytoplasm, there will be an occurrence of glycolysis to form a lot of pyruvate 2. Pyruvate goes into the matrix through pyruvate translocase situated in the inner Figure 46 membrane. PREPARED BY: BATCH 2028 1D 17 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 3. Pyruvate in the mitochondrial matrix will 3. Insulin and glucagon are peptide then be oxidatively decarboxylated by hormones, so they cannot just traverse the pyruvate dehydrogenase to become acetyl plasma membrane due to the nature of the CoA. structure. 4. Acetyl CoA will condense with OAA to form 4. For them to be able to transmit their citrate with the help of citrate synthase. If message inside the cell, they must there is a lot of energy, excess citrate will possess their receptors expressed on the go out of the cytoplasm and will be membrane. converted back to acetyl-CoA and Epinephrine is derived from tyrosine oxaloacetate with the help of hormone. ATP-dependent citrate lyase enzyme. 5. The receptor in the plasma membrane, 5. Acetyl- CoA will be acted upon by epinephrine and glucagon have to bind to acetyl-CoA carboxylase to form the receptor forming a hormone-receptor malonyl-CoA and thereby will be taken complex to activate the G-protein over by fatty acid synthase to form fatty 6. Activated G-protein then activates adenylyl acids such as palmitate. cyclase (AC), which tries to cyclize ATP to 6. OAA will be subsequently reduced by form the 2nd messenger, which is the malate dehydrogenase cytosolic with the cyclic AMP (cAMP). help of NADH to form malate. 7. cAMP activates the inactive protein kinase 7. Malate can either go again inside the A. mitochondrial matrix or it can be 8. If there is an activated protein kinase A, oxidatively decarboxylated by malate proteins and enzymes will receive enzyme (or NADP- dependent malate phosphate groups. Therefore, these dehydrogenase enzyme) with the help of enzymes and proteins are inactivated due NADP, which after oxidizing malate back to to their phosphorylation. pyruvate, the cells generate NADPH+H (much needed reducing equivalent needed K. TRIACYLGLYCEROLS (TAGs) for fatty acid synthesis). - Triacylglycerols represent a vital class of lipids with substantial implications in J. MOBILIZATION OF FATTY ACIDS FROM biochemistry, notably concerning lipid ADIPOSE TISSUES metabolism and cardiovascular well-being. - Comprising one glycerol unit and three fatty acid chains of variable lengths and degrees of hydrogen saturation, TAGs exhibit water-insolubility. - To facilitate their absorption in the small intestine, bile acids are excreted to emulsify TAGs, rendering them water-soluble. - Additionally, the pancreas secretes lipase, an enzyme pivotal in the breakdown of TAGs, to aid in their absorption - They play several important roles in the human body. ENERGY STORAGE - TAGs serve as the principal mode of energy storage within the body - They accumulate surplus calories not promptly utilized, and hormonal cues Figure 48 trigger the release of TAGs into the bloodstream during intervals between 1. There is insulin predominance during FA meals when energy is needed. synthesis. 2. The confirmation of acetyl-CoA REGULATION OF BODY TEMPERATURE carboxylase is in the dephosphorylated - TAGs contribute to the regulation of body form (active form). temperature by furnishing an energy PREPARED BY: BATCH 2028 1D 18 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 source for thermogenesis, the mechanism Equipment responsible for heat production. 5 mL measuring pipette (1) Centrifuge (1) DIETARY SOURCE Cuvettes (3) - TAGs represent a prominent dietary lipid Micropipettes (2) form in fats and oils obtained from plant Pipette Aspirator (1) and animal sources. Spectrophotometer (1) - TAGs are included in processed foods to Test tube stand (1) serve as lubricants and improve eating Test tubes (5) characteristics. Venipuncture set (1) Water bath (1) HYPERTRIGLYCERIDEMIA - Elevated levels of TAGs are linked to an Reagents increased susceptibility to atherosclerosis Distilled water and cardiovascular disease Standard solution of triacylglycerol - Excessive consumption of TAGs, (triolein) (2.29 mmol/) particularly unhealthy saturated fats can Working reagent (lipase, glycerol kinase, contribute to various health complications, glycerol phosphate, peroxidase, including heart disease, obesity, stroke, chlorophenol, 4-aminoantipyrine in a buffer and even mortality solution) - Hyprertriglyceridemia may arise from diverse factors such as genetic Specimen for Examination --Serum predisposition, such as familial Procedures chylomicronemia syndrome, as well as Label test tubes as "blank," "serum," and conditions like obesity, metabolic "standard," syndrome, diabetes mellitus, excessive Place 0.03 mL of distilled water in the alcohol intake, hypothyroidism, kidney "blank" test tube. disorders and certain medications such as Place 0.03 mL of serum (sample) in the steroids , estrogens, anabolic steroids etc. "serum" test tube Place 0.03 mL of the standard solution of triacylglycerol in the "standard" test tube Add 3 mL of the working reagent to each test tube Incubate in the water bath for 20 minutes at a temperature ranging between 20-25°C Adjust the spectrophotometer to "zero" using the "blank” Measure the optical density of both experimental and standard samples relative to water using a wavelength of 540 nm Calculation TAG concentration (mmol/1) = Eex/Est X Cst *Ess - optical density of the serum (sample) Figure 49 *Est - optical density of the standard solution TRIGLYCERIDES TEST *Cst - concentration of the standard Objective To describe the mechanisms solution involved in lipogenesis, lipolysis and the oxidation of fatty acids. Why do I need a triglycerides test? To acquire hands-on proficiency in Your healthcare provider may order a lipid profile, quantitatively measuring triacylglycerol including triglycerides. levels in serum. **How often you need to have a lipid profile test depends on your age, sex, and your risk of PREPARED BY: BATCH 2028 1D 19 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 developing heart disease. The general age Dietary fat has a high "satiety value", i.e. recommendations for testing blood lipid level the ability to satisfy hunger. Supply of polyunsaturated FA: Food fats For ages 2 to 19: should contain adequate amounts of In general, start testing between ages nine polyunsaturated FA to supply at least 1 and 11. Repeat the test every five years, percent of total calories in adult men and 4 If there is a family history of high blood percent of the same in children. The cholesterol, heart attack, stroke, or other nutritional significance of the risks developing heart disease, test as polyunsaturated "EFA" has already been early as age two. discussed. For ages 20 and older, tests should be done: Quality of fat: Chain length and saturation Every five years for: Males between ages of FA and MP of TG influence the nutritive of 20 and 45 ; Females between the ages quality of food fats. Palmitic acid (C16) has 20 and 50. a digestibility of about 70 percent only. Every 1 to 2 years for: Males age 45 and Unsaturated TG are also more readily older ; Females age 55 and older absorbed than saturated ones. Oils of Every year for adults over age 65 vegetables and seeds (e.g. sunflower oil, You may need to be tested more often if you have a groundnut oil, soybean oil, mustard oil) higher risk for heart disease because you: contain mainly unsaturated FA and are Have a family health history of early heart preferable. disease (a parent or sibling with heart Medium -chain TG has been used in the disease before age 55 for males, and treatment of chyluria and chylothorax as before age 65 for females) they are absorbed directly in portal blood Smoke Excess of fats in diet: An excessive high Are overweight or have obesity fat intake inhibits gastric secretion and Have unhealthy eating habits motility, producing anorexia and gastric Don't get enough exercise discomfort. Have diabetes Have high blood pressure Dietary Role of Trans Fatty Acids (TFAs) or Are a male age 45 or older, or a female Trans Fats age 50 or older Ask your doctor how often you need to Trans fatty acids are principally artificial have your blood lipid levels tested. fats but a small amount of TFAs occur naturally in meat and dairy products L. ROLE OF LIPIDS IN THE DIET Trans fatty acids are formed artificially The main function of dietary lipids, like that during the hydrogenation of vegetable oils. of carbohydrates, is to provide energy, In this process, the vegetable oils which largely through oxidation of their are liquid turn solid. constituent free fatty acids. Hydrogenation improves the shelf-life and The dietary lipids serve another indirect palatability of oils and thus they are used function, serving as "carriers" of certain in the food industry. Examples of fat-soluble vitamins (vit A, D, E, and K) and hydrogenated fats include provitamins like carotenes, which because Naturally occurring TFAs: Some TFA is of their solubility in fats, occur in nature found naturally in small amounts in meat mainly in association with these and dairy products. Effects of TFAs in our substances. body: TFAs are found to be a greater risk Lipids may also exert a relatively minor for chronic degenerative diseases. TFAs "protein-sparing effect", apart from their are more atherogenic than saturated fatty calorie contribution. acids. Requirement: Neutral fats (TG), TFAs alter secretion and composition of comprising the largest fraction of food apo-100 containing lipoproteins LDL and lipids are quantitatively the most important VLDL of these substances. Under usual TFA is involved in altering metabolism, conditions, fats provide 20 to 35% of the Viz.: calories of the diet, i.e. 1 to 2 gm/kg of Increasing catabolism of apo-A1 body weight in the average moderately Increasing LDL levels (bad active adult cholesterol) PREPARED BY: BATCH 2028 1D 20 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 Decreasing HDL (good 5. Iodine Number cholesterol) level Definition: The iodine number is defined as the Increasing the abnormal clotting number of grams of iodine absorbed by 100 grams of blood of a given fat or oil. Studies have also reported a Significance and Use: The iodine number is a positive correlation of trans fatty measure of the degree of unsaturation of a fat or oil. acids with diabetes type-2 and The higher the iodine number, the greater the some cancers. unsaturation. The determination of the iodine number is useful to the chemist in determining the M. IDENTIFICATION OF FATS AND OILS quality of an oil or its freedom from adulteration. Sometimes it becomes necessary to Identify a pure fat 6. Acetyl Number Assess the degree of adulteration Some of the fatty acid residues in fats contain - OH Determine the proportions of different groups. In order to determine the proportion of types of fat in a mixture. these, they are acetylated by means of acetic anhydride. Thus an acetyl group is introduced 1.Saponification Number wherever a free-OH group is present. After washing Definition: The number of mgms of KOH required to out the excess acetic anhydride and acetic acid saponify the free and combined FA in one gram of a liberated, the acetylated fat can be dried and given fat is called its saponification number. weighed and the acetic acid in combination Basis: the amount of alkali needed to saponify a determined by titration with standard alkal after it given quantity of fat will depend upon the number of has been set free. The acetyl number is thus a -CO0H groups present. Thus fats containing measure of the number of -OH groups present. short-chain fatty acids will have more -COOH Definition: The number of mgms of KOH required to groups per gram than long-chain fatty acids and this neutralize the acetic acid obtained by saponification will take up more alkali and hence will have a higher of 1 gm of fat after it has been acetylated. saponification number. Examples: Castor oil because of its high content of ricinoleic acid has a high acetyl number. Examples: Butter containing a larger proportion of short-chain Fatty acid composition of three food fats fatty acids, such as butyric and caproic acids, has a Olive oil, butter, and beef fat consist of relatively high saponification number from 220 to mixtures of triacylglycerols, differing in 230. Oleomargarine, with more long-chain fatty their fatty acid composition acids, has a saponification number of 195 or less. The melting points of these fats-and hence their physical state at room temperature 2. Acid Number (25 °C)-are a direct function of their fatty Definition: Number of mgms of KOH required to acid composition neutralize the fatty acids in a gm of fat is known as Olive oil has a high proportion of the acid number. long-chain (C16 and (18) unsaturated fatty Significance: The acid number indicates the degree acids, which accounts for its liquid state at of rancidity of the given fat. 25 °C The higher proportion of long-chain (Ci6 3. Polenske Number and (8) saturated fatty acids in butter Definition: lt is the number of milliliters of 0.1 normal increases its melting point, so butter is a KOH required to neutralize the insoluble fatty acids soft solid at room temperature (those not volatile with steam distillation) from 5 Beef fat, with an even higher proportion of grams of fat. long-chain saturated fatty acids, is a hard solid. 4. Reichert-Meissl Number Definition: It is the number of milliliters of 0.1 (N) alkali required to neutralize the soluble volatile fatty acids distilled from 5 gm of fat. Significance: The Reichert-Meissl measures the amount of volatile soluble fatty acids. By saponification of fat, acidification and steam distillation, the volatile soluble acids may be separated and determined quantitatively. Figure 50 PREPARED BY: BATCH 2028 1D 21 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 Laboratory phosphorylation of glycerol to Reports/Discussions glycerophosphate NADH Show your calculation formation can be measured Explain the principle and the connected spectrophotometrically at 340 nm enzymatic reactions in the methods used ○ The resulting H202 is measured What is the normal value for serum TAG? Describe the diagnostic and clinical significance of TAG determination. Self-Study Questions What are triacylglycerols (TAGs), and what roles do they play in the human body? Describe the structure of a triacylglycerol molecule and explain how it contributes to its functions. What are the main dietary sources of triacylglycerols? Discuss the process of lipogenesis and its significance in lipid metabolism. Explain the process of lipolysis and its role in energy metabolism. How are triacylglycerol levels regulated in Figure 51 the body? What health conditions are associated with Triglyceride Test elevated levels of triacylglycerols? The triglyceride level test helps measure the Describe the laboratory methods used for amount of triglycerides in your blood. Triglycerides the quantitative determination of are a type of fat, or lipid, found in the blood. The triacylglycerol levels in serum. test results of this test help your doctor determine What are the implications of abnormal your risk of developing heart disease. Another triacylglycerol levels for cardiovascular name for this test is a triacylglycerol test health? How can lifestyle modifications and Normal Range pharmacological interventions help 40 to 160 mg/dL manage dyslipidemia-associated With elevated triacylglycerol levels? Causes of High Results Poor diet (high in sugars and fats) Triglycerides Obesity most commonly used for clinical or Excessive alcohol consumption epidemiologic purposes are based on the Uncontrolled diabetes hydrolysis of triglycerides and the Hypothyroidism measurement of glycerol overestimated if endogenous unesterified glycerol is not subtracted also because of the presence of monoglyceride and diglyceride molecules Enzymatic methods ○ universally used for TG analysis in the clinical laboratory. ○ relatively specific, rapid, and easy to use. ○ performed directly in plasma or serum and are not subject to interference by phospholipids or glucose. Figure 52 ○ Common to most enzymatic methods is the hydrolysis of triglycerides to free fatty acids and glycerol, followed by the PREPARED BY: BATCH 2028 1D 22 BIOCHEMISTRY LC8: FATTY ACID AND TAG METABOLISM DR. A. VIESTA DATE: 10/15/2024 Triglycerides Test Interpretation Result Interpretation or = 500 mg/dL Very high, significant risk Table 5 Reference(s): 1. Dr. A. Viesta (2024). Lecture and Powerpoint Presentation. PREPARED BY: BATCH 2028 1D 23