Biochemistry Second Year, Coll

Biochemistry –Second year, Coll. of Medicine-Baghdad Univ. .2011-2012 .Dr.Basil Oied Mohammed Saleh Subjec:Lipid

Lecture 5,6,7

Objective: To illustrate the steps involved in production of energy ATP from . oxidation of fatty acids

The fate of the saturated Fatty Acids The synthesized saturated fatty acids, mainly in the liver and those derived from the diet as chylomicrone as mentioned ,Lecture 2 are stored as triglyceride(triacylglycerol) in adipose tissues and represents the principal energy reserve in human body. TG which referred to as fat when its solid and as oil when it is liquid at room temperature is not normally stored in the liver as fatty liver is the early stage in different forms of chronic liver diseases; alcoholic liver cirrhosis. The synthesis of TG occurred in the liver and adipose tissues. In the steps of TG synthesis pathway, the synthetic units are; glycerol and fatty acids which firstly must be activated into active forms to be ready for their reaction. Glycerol is converted into glycerol phosphate(active form) which in the liver is achieved by Glycerol kinase enzyme, the enzyme present only in the liver

Glycerol kinase Gylcerol …………………………………….» Glycerol phosphate ATP……ADP+Pi

The formation of activated glycerol; glycerolphosphate is also activated by another enzyme; the Glycerol phosphate dehydrogenase(GlyPDH) but in :different reaction

Glycolysis pathway GlyPDH Glucose………………………..» DHAP………..……….» Glycerol phosphate + NADH….NAD This latter reaction occurred in liver and adipose tissues because the .activated enzyme is found in both these two organs Note: The adipocytes can take glucose from the circulation only in the presence of Insulin, so in the case of fasting state or pathological cases as in DM when the blood glucose and so the Insulin levels are normally low, the adipocytes uptake of glucose and the synthesis of glycerol will be limited with consequent inhibition of TG pathway synthesis. So, the pathway of fatty acids and TG synthesis is stimulated in the presence of Insulin Hormone and inhibited in its .absence The second substance in the synthesis of fatty acid, which activated by its conversion into acyl-CoA , the active form of fatty acid the reaction is .(stimulated by the enzyme Thiokinases(or Fatty acyl CoA synthetases

Thiokinases RCOOH………………………………↑…….». RCO CoA ATP….»AMP+PPi CoA The activated two substances; Glycerol phosphate and Acyl CoA reacted in :four steps to form TG

(1) Acyltransferase Glycerol phosphate+ Acyl CoA…↓….» Monoacylglycerol(Lysophosphatidic (acid) CoA ↓ (2 Acyltransferase ↓← AcylCoA ↓ ( Diacylglycerol(phosphatidic acid ↓ (H2O→ ↓→ Pi (3 ↓ (Diacylgylcerol(Diglyceride

(4) Acyltransferase

Diglyceride………………………………»TG ↑ AcylCoA The TG is stored in the adipose tissues as anhydrous form(insoluble in water) and occupied the major space of adipocytes cytoplasm and is ready for .mobilization to be used for production of energy when there is need

Mobilization and Oxidation of Fat

The stored TG in adipose tissues is released in the form of saturated fatty acids and glycerol when there is need for the stored energy;prolonged fastin g, starvation and DM. This process is referred to as LIPOLYSIS, which is the opposite to lipogenesis. The Lipolysis is stimulated in the presence of the Insulin antagonist, principally the Glucagon, adrenaline, ACTH, cortisol,TSH and noradrenaline. Insulin is the potent inhibitor of lipolysis pathway.The key enzyme in the lipolysis is: Hormone sensitive -lipoprotein Lipase(HS-LPL), the enzyme which act only under stimulation of the insulin anagonist hormones. These hormones by their binding to their CM receptors increased the synthesis of the intracellular concentration of the second messanger c AMP, which :inturn converts the HS-LPL from its inactive form into active form

cAMP+PPi ↑ . Hormones………………»CM(Receptors). ATP this Hs+Receptors binding Reaction leads to increase Adrenaline,glucagon…. the cAMP intracellular levels

The formed intracellular cAMP stimulates the formation of active protein :kinase which is important in conversion of HS-LPL into its active form

Active Protein Kinase (HS-LPL( dephosphorylste form)………↑…………» HS-LPL (phosphorlyated (Inactive form) cAMP (Active form ) ↑ Hs .

:Now HS-LPL «..…TG…………………↓………………..» Diacylglycerol in adipocytes Fatty acids ↓→fatty These fatty acids are HIS-LPL ↓ acids Able to penetrate the CM Monoacylglycerol Of adipocytes and released ↓ fatty Into the circulation. . ↓ acids Glycerol

The formed saturated Fatty acids in these three steps and the glycerol in the last step are released into the circulation. The last two enzymes HIS-LPL hormone insensitive LPL are different from the HS-LPL one because these enzymes act in the absence of hormonal action, and so the regulatory step in the lipolysis is that activated by HS-LPL. The TG, and definitly the contained fatty acids represset the concentrated stores of metabolic energy in comparison with that yeilded from carbohydrate and protein, the two other major nutrients because fatty acids have the highly reduced hydrogen and are largly .anhydrous

The next note is who the human body deals with the products of the metabolized TG; the fatty acids and glycerol. The fatty acids formed in adipocytes are either reuesd again in adipocytes(intraadipocytes) for the synthesis of new molecules of TG(lipogenesis) as the two pathways of liogenesis and lipolysis are in dynamic state. The other fate of fatty acids is their release of the formed fatty acids in the circulation, where they are combined with blood albumin to be able to transport in the blood??. The transporting of fatty acids in the blood is restricteted to those of long chain, while of the short chain not need for albumin carrier. Fatty acids are transported to different tissues, predemonantly the liver and skeletal and cardiac muscles.The Red Blood Cells and the Brain are the only organs that not uesd the fatty acids as fuel irrespective of their blood levels because of lack of RBCs of mitochondria and inability of fatty acids to pass the blood-brain carrier. In the liver the entered fatty acids are either used for resynthesis of TG in the cytoplasm and :transported again to adipose tissues as VLDL

Fatty acids+ glycerol……..» TG………….» VLDL…….» adipose tissues(stored .(metabolic enery

The second pathway of the entered saturated fatty acids is their utilization in the production of chemical energy ATP. The latter pathway occurred in the liver and other tissues, mainly Muscles; cardiac and skeletal. Muscles used fatty acids even in the presence of glucose as a source of energy which spare the glucose for other tissues dependent on glucose. The pathway of utilization of saturated (even C2, C4,…C16,….C20….) fatty acids is the oxidation pathway which includes; alpha(α), beta(β), and gamma(γ). The β-oxidation pathway is the major pathway of production of energy ATP which occurred in the mitochondria(definitly matrix of mitochondria). This means that the entered fatty acids must be transported from the cytoplasm in the mitochondria across its membrane, the shorter and medium chain fatty acids can pass into mitochondria directly without need for any transporter system. The long chain fatty acids cannot pass cross the mitochondrial memebrane MM by themselves, but only in the presence of transporter system referred to as Carnitine system. So, the long chain fatty acids,more than 12 C cannot be oxidized to produce energy only in the presence of carnitine system. Carnitine system consisted of three enzymes and carnitine substance; carnitineacyle transferase I CAT-I, .carnitine acylcarnitine translocase, and carnitine acyl transferase II ACT-II

The first step in oxidation of saturated (Even)fatty acids is the formation of :active form Fatty acyl-CoA Thiokinase (Fatty acids RCCOH………………………..» Fatty acyl CoA (RCOCoA ATP……AMP+PPi

This reaction is activated by the Thiokinase(Fatty acyl CoAsynthetase enzyme), an outer M M enzyme location, this reaction occurred in the cytoplasm and : then transported across the MM

: Carnitine Transporter System

CAT-I (Fatty acylCoA (cytoplasm)…………………» Fattyacyl carnitine(interMM space . ↑ . (Carnitine molecule(inter MM space carnitine→ ((from Inner Mitoch. Matrix . . . BY Carnitineacylacarnitine Translocase enzyme . . ↓ (Fattyacylcarnitine(inner MM;Mitoch. Matrix . . . ← ← carnitine . To inter MM space . . . ↓ (Fatty acyl CoA(Inner MM; Mitoch. matrix Ready for β-oxidation pathway

The Mitoch. Matrix fatty acyl CoA is now ready for reactions of β-oxidation :pathway

RCO CoA(e.g; CH3(CH2)12CH2-CH2COCoA Palmitoylacyl CoA . . AcylCoA Dehydrogenase. NAD…»NADH H» Respiratory . chain . system ↓ CH3(CH2)12CH=CHCOCoA unsaturated fatty acyl (Enoyl CoA) . . Enoyl CoA Hydratase . →H2O ↓

CH3(CH2)12CHOHCH2COCoA β-hydroxyfattyacyl CoA . Β-hydroxyfattyacylCoA Dehydrogenase FAD…»FADH2»Respiratory . . Chain system ↓ CH3(CH2)12COCH2COCoA β-Ketoacyl CoA . → CoA.SH . Thiolase CH3CoA acetyl CoA…» CAC →→↓ ↓ pathway CH3(CH2)12CoA (Fattyacyl CoA(less-C2

So with each run or turn of these reaction of β-oxidation of fattyacyl, the product is the fattyacyl of less two carbon unit C2 of the original one(C16….»C14) Plus the Acetyl-CoA which is the substrate of Citric Acid Cycle CAC pathway with end products are CO2+H2O. These above steps or reactions continue with removal of C2 until the remainder fattyacyl is CH3COCH2CoA C4 which splitted by thiolase enzyme into two C2 2Aetyl CoA. So, for fattyacyl C16 Palmitoyl acyl CoA there is need for 7 turn of β- :oxidation steps or reaction C16…1.C14…2…C12…3….C10…4.C8…5.C6…6….C4→ 2 Acetyl CoA ↓ ↓ ↓ ↓ ↓ ↓ Acetyl CoA AcetylCoA Acetyl CoA Acetyl CoA Acetyl CoA Acetyl CoA

So, the overall pathway of β-oxidation of C16 palmitic saturated acid leads to formation of 8 aceyl CoA by 7 turn or cycles(consisted of 4 steps or reactions), these aceyl CoA molecules then enter CAC with formation of 3NADH H, 1 FADH2, and 1 GTP(GTP….ATP). The net amounts of ATPs that yeilded from :complete oxidation of palmitic acid C16; β-oxidation+CAC is turn of β-oxidation…….» 7 NADH H……..» 7×3=21 ATP 7 FADH2 ………» 7× 2= 14 ATP 7

Acetyl CoA 8(3NADH H+ FADH2+GTP=12 ATP)» 96 ATP 8 CAC The total ATP s= 131 ATPs 2ATPs- The net amounts of ATPs: 129 ATPs

Calculate the amounts of NADH H, FADH2 and ATPs from 1. Β-oxidation 2. .??Complete oxidation to CO2+H2O

The regulatory step in β-oxidation pathway is at the CAT-I step.Malonyl CoA, the intermediate of fatty acids synthesis, is the inhibitor of by β-oxidation by inhibiting of CPT-I enzyme of carnitine system. The inhibiting of CPT-I results in preventing the fatty (long chain)acyl CoA from entry into mitochondrial matrix and so from oxidation in β-pathway. So, in case of CHO intake(post CHO meal, or high caloric intake) the levels of glucose and insulin are high, the amounts of citrate and so of Malonyl CoA are also high, the synthesized malonyl CoA will inhibits the CPT-I and therefore the newly synthesized fatty acids cannot not pass into the mitochondrial matrix and so will be reacted with glycerol to form TG, → VLDL which secreted into circulation and transported to the adipose tissues(energy storage form) and muscles(energy source). The vice versa, in prolonged fasting state the glucose and insulin are low, the malonyl CoA low, the inhibition of CPT-I will be removed and the entered fatty .acids into the cytoplasm will be proceed into the β-oxidation pathway

Carnitine substance are found in meat(exogenous sources). In human body carnitine is synthesizes endogenously fro Lysine and Methionine amion acids in the liver and kidney but not the muscles. However, the synthesized carnitine will be transported to the muscles; cardiac and skeletal where they are used foe fatty acids oxidation to produce the ATP. About 97 % of the body ΄s carnitine .is predominant in the muscles

:Disorders of Carnitine Deficiences and β-Oxidation Impairment

Carnitine deficiency which may be primary and secondary leads to decrease utilization of long chain fatty acid LCFA as source of energy, pariculary in the muscles, with dependent mainly on the glucose and so the occurrence of Hypoglycemia.The primary causes are:1. genetic defect or deficiency of CAT-I which affects mainly the Liver and prevent it from utilization of LCFA to produce of energy and so decrease its ability for formation of glucose and spare it for those glucose dependent tissues(RBCs, brain, kidney…). The outcome of this defect is hypoglycemia, coma and death. 2. Genetic defect or deficiency of CAT-II which affects mainly the muscles(cardiac and skeletal) and leads to defect in utilization of LCFA with cardiomyopathy, muscle weakness and myoglobinemia after prolonged exercise??. Tratment, by low lipid or fat diet and rich CHO , but high amounts of short and medium fatty acids is recommended ??. supplementation with carnitine is also recommended in case .of carnitine deficiency

Impairment of β-oxidation including the genetic deficiency of Medium Chain Fatty Acyl CoA Dehydrogenaes enzyme MCAD leads to defect in β-oxidation of MCFA which is predominant in milk and so affects mainly the infants who are dependent on milk in their nourishment or feeding. MCAD disease is autosomal recessive disease and is the most common inborn error of metabolism and the most common inborn error of fatty acid oxidation. MCAD disease is considered as one of the sudden infant death syndrome SIDS or Reye .syndrome

The oxidation of Odd number(C1,…C3,….C15,…..) saturated fatty acids also proceeded as the even number in β-oxidation, but the pathway terminates with C5which splitted into the acetyl CoA + C3 Propionyl CoA. The acetyl CoA also oxidized in CAC, but the propionyl CoA is metabolized by different pathway as :follows Carboxylase CO2 -Propinoyl CoA………………………↓…..» D-Methyl malonyl CoA COO | . ATP……..ADP+Pi Biotin Co E . H- C-CH3 | Racemase . CO CoA . ↓ -L-Methylmalonyl CoA COO | CoE Vit. B12 . H3C -C-H | . Mutase . CO CoA ↓ Gluconeogenesis ← Succinyl CoA ↓ (Formation of glucose from fatty acids) CAC

So the only saturated fatty acids that can be used in production of glucose(gluconeogenesis pathway) is the odd nembeer fatty acids, while the naturally endogenous synthesized fatty acids(the even number) cannot be used .??as sourcer of gluconeogenesis

Oxidation of very long saturated fatty acids VLCFA which predominant in nervous tissues is also by β-oxidation but in Peroxisome organelle instead of mitochondria, this pathway terminates in shorter fatty acids and acetyl CoA which transferred to the mitochondria for further complete oxidation by also β- oxidation and subsequent CAC. Zellweger syndrome is characterized by accumulation of VLCFAs in tissues and blood due to defect in biogenesis of peroxisome organelle(congenital defect) or genetic defect in transporting of VLCFA across the peroxisome membrane resulting in X-linked .Adrenoleukodystrophy

Phytanic acid is branched fatty acid with methyl group at C3(β position). This fatty acid cannot be oxidized by Fatty acyl CoA DH of β-oxidaton, instead is firstly oxidized by α-hydoxylase enzyme to produce the acyl-CoA derivatives which then

proceeded in β-oxidation pathway. Refsum disease is characterized by primarily the neurologic symptoms due to accumulation of phytanic acid in blood and tissues because of genetic deficiency of α-hydroxylase enzyme and its .treatment is by dietary restriction of this fatty acid