Q- Discuss the role of carnitine in fatty acid oxidation.
Answer- Fatty acids are activated on the outer mitochondrial membrane, whereas they are oxidized in the mitochondrial matrix. A special transport mechanism is needed to carry long-chain acyl CoA molecules across the inner mitochondrial membrane. Activated long-chain fatty acids are transported across the membrane by conjugating them to carnitine, a zwitterionic alcohol.
Carnitine (ß-hydroxy-Υ-trimethyl ammonium butyrate), (CH3)3N+—CH2—CH(OH)—CH2—COO–, is widely distributed and is particularly abundant in muscle. Carnitine is obtained from foods, particularly animal-based foods, and via endogenous synthesis.
The transportation across the inner mitochondrial membrane through carnitine shuttle involves three steps-
1) The acyl group is transferred from the sulfur atom of CoA to the hydroxyl group of carnitine to form acyl carnitine.(Figure-1).This reaction is catalyzed by carnitine acyl transferase I (also called carnitine palmitoyl transferase-I), which is bound to the outer mitochondrial membrane.
Figure- 1-Showing the formation of acyl carnitine
2) Acyl carnitine is then shuttled across the inner mitochondrial membrane by a translocase.
3) The acyl group is transferred back to CoA on the matrix side of the membrane. This reaction, which is catalyzed by carnitineacyltransferase II (carnitine palmitoyl transferase II), is simply the reverse of the reaction that takes place in the cytosol.
Finally, the translocase returns carnitine to the cytosolic side in exchange for an incoming acyl carnitine (Figure-2)
Figure-2- showing the transportation of acyl co A in to to the mitochondrial matrix through carnitine shuttle
This counter-transport system provides regulation of the uptake of fatty acids into the mitochondrion for oxidation. As long as there is free CoA available in the mitochondrial matrix, fatty acids can be taken up and the carnitine returned to the outer membrane for uptake of more fatty acids. However, if most of the CoA in the mitochondrion is acylated, then the fatty acid uptake is inhibited.
This carnitine shuttle also serves to prevent uptake into the mitochondrion (and hence oxidation) of fatty acids synthesized in the cytosol in the fed state; malonyl CoA (the precursor for fatty acid synthesis) is a potent inhibitor of carnitine palmitoyl transferase I in the outer mitochondrial membrane.
Short and medium chain fatty acids do not require carnitine for their transportation across the inner mitochondrial membrane.
Carnitine deficiency-
Causes of carnitine deficiency include the following:
Primary Carnitine deficiency- The underlying defect involves the plasma membrane sodium gradient–dependent carnitine transporter that is present in heart, muscle, and kidney(Figure-2).This transporter is responsible both for maintaining intracellular carnitine concentrations 20- to 50-fold higher than plasma concentrations and for renal conservation of carnitine. Primary carnitine deficiency has an Autosomal recessive pattern of inheritance. Mutations in the SLC22A5 gene lead to the production of defective carnitine transporters. As a result of reduced transport function, carnitine is lost from the body and cells are not supplied with an adequate amount of carnitine.
Clinical manifestations
Symptoms and the age at which symptoms appear depend on the cause.
1) Carnitine deficiency may cause muscle necrosis, myoglobinuria, lipid-storage myopathy, hypoglycemia, fatty liver, and hyperammonemia with muscle aches, fatigue, confusion, and cardiomyopathy.
2) A smaller number of patients may present with fasting hypoketotic hypoglycemia during the 1st yr of life before the cardiomyopathy becomes symptomatic.
3) Blockage of the transport of long chain fatty acids into mitochondria not only deprives the patient of energy production, but also disrupts the structure of the muscle cells with the accumulation of lipid droplets. ).
4) Serious complications such as heart failure, liver problems, coma, and sudden unexpected death are also a risk.
5) Acute illness due to primary carnitine deficiency can be triggered by periods of fasting or illnesses such as viral infections, particularly when eating is reduced.
Hypoglycemia in carnitine deficiency, is a consequence of impaired fatty acid oxidation with the resultant imbalance between demand and supply of glucose which is the sole of source of energy in such individuals.
Deficiencies in the carnitine acyl Transferase enzymes I and II can cause similar symptoms. Inherited CAT-I deficiency affects only the liver, resulting in reduced fatty acid oxidation and ketogenesis, with hypoglycemia. CAT-II deficiency affects primarily skeletal muscle and, when severe, the liver.
Diagnosis
1) Diagnosis of the carnitine transporter defect is aided by the fact that patients have extremely reduced carnitine levels in plasma and muscle (1–2% of normal). Heterozygote parents have plasma carnitine levels approximately 50% of normal.
2) Fasting ketogenesis may be normal if liver carnitine transport is normal, but it may be impaired if dietary carnitine intake is interrupted and there is associated liver disorder.
3)The fasting urinary organic acid profile may show a hypoketotic dicarboxylicaciduria pattern if hepatic fatty acid oxidation is impaired, but it is otherwise unremarkable.
4) The defect in carnitine transport can be demonstrated clinically by severe reduction in renal carnitine threshold (In primary carnitine deficiency)
Treatment
Treatment of this disorder with pharmacologic doses of oral carnitine is highly effective in correcting the cardiomyopathy and muscle weakness as well as any impairment in fasting ketogenesis. Muscle total carnitine concentrations remain less than 5% of normal on treatment. All patients must avoid fasting and strenuous exercise. Some patients require supplementation with medium-chain triglycerides and essential fatty acids (eg, Linoleic acid, Linolenic acid). Patients with a fatty acid oxidation disorder require a high-carbohydrate, low-fat diet.
Q.- Discuss the steps of beta oxidation of fatty acids, highlighting the enzymes and coenzymes involved.
Answer-
Overview of beta oxidation
A saturated acyl Co A is degraded by a recurring sequence of four reactions:
1) Oxidation by flavin adenine dinucleotide (FAD)
2) Hydration,
3) Oxidation by NAD+, and
4) Thiolysis by Co A
The fatty acyl chain is shortened by two carbon atoms as a result of these reactions, and FADH2, NADH, and acetyl Co A are generated. Because oxidation is on the β carbon and the chain is broken between the α (2)- and β (3)-carbon atoms—hence the name – β oxidation .
Actually, the reactions in the fatty acid cycle closely resemble the last three steps of the citric acid cycle.
Step-1- Dehydrogenation-The first step is the removal of two hydrogen atoms from the 2(α)- and 3(β)-carbon atoms, catalyzed by acyl-CoA dehydrogenase and requiring FAD. This results in the formation of Δ2-trans-enoyl-CoA and FADH2.
As in the dehydrogenation of succinate in the citric acid cycle, FAD rather than NAD+ is the electron acceptor because the value of Δ Gfor this reaction is insufficient to drive the reduction of NAD+. Electrons from the FADH2 prosthetic group of the reduced acyl CoA dehydrogenase are transferred to a second flavoprotein called electron-transferring flavoprotein (ETF). In turn, ETF donates electrons toETF: ubiquinone reductase, an iron-sulfur protein. Ubiquinone is thereby reduced to ubiquinol, which delivers its high-potential electrons to the second proton-pumping site of the respiratory chain. Consequently, 2 (1.5) molecules of ATP are generated per molecule of FADH2 formed in this dehydrogenation step, as in the oxidation of succinate to fumarate.
Step-2- Hydration- Water is added to saturate the double bond and form 3-hydroxyacyl-CoA, catalyzed byΔ 2-enoyl-CoA hydratase.
Step-3- dehydrogenation-The 3-hydroxy derivative undergoes further dehydrogenation on the 3-carbon catalyzed by L(+)-3-hydroxyacyl-CoA dehydrogenase to form the corresponding 3-ketoacyl-CoA compound. In this case, NAD+ is the coenzyme involved.
Step-4- Thiolysis- Finally, 3-ketoacyl-CoA is split at the 2,3- position by thiolase (3-ketoacyl-CoA-thiolase), forming acetyl-CoA and a new acyl-CoA two carbons shorter than the original acyl-CoA molecule.
The acyl-CoA formed in the cleavage reaction reenters the oxidative pathway at reaction 2 (Figure 3). In this way, a long-chain fatty acid may be degraded completely to acetyl-CoA (C2 units). Since acetyl-CoA can be oxidized to CO2 and water via the citric acid cycle (which is also found within the mitochondria), the complete oxidation of fatty acids is achieved (Figure-3)
Fatty acyl chains containing from 12 to 18 carbon atoms are oxidized by the long-chain acyl CoA dehydrogenase. The medium-chain acyl CoA dehydrogenase oxidizes fatty acyl chains having from 14 to 4 carbons, whereas the short-chain acyl CoA dehydrogenase acts only on 4- and 6- carbon acyl chains. In contrast, β keto thiolase, hydroxy acyl dehydrogenase, and enoyl CoA hydratase have broad specificity with respect to the length of the acyl group.
Figure-3 showing the steps of beta oxidation
The overall reaction can be represented as follows-
Energy yield by the complete oxidation of one mol of Palmitic acid
The degradation of palmitoyl CoA (C16-acyl Co A) requires seven reaction cycles. In the seventh cycle, the C4-ketoacyl CoA is thiolyzed to two molecules of acetyl CoA.
Approximately 3 ( 2.5) molecules of ATP are generated when the respiratory chain oxidizes each of the 7 molecules of NADH, whereas 2( 1.5) molecules of ATP are formed for each of the 7 molecules of FADH2 because their electrons enter the chain at the level of ubiquinol.
The oxidation of acetyl CoA by the citric acid cycle yields 12(10) molecules of ATP. Hence, the number of ATP molecules formed in the oxidation of palmitoyl CoA is 14( 10.5) from the 7 molecules of FADH2, 21(17.5) from the 7 molecules of NADH, and 96 ( 80) from the 8 molecules of acetyl CoA, which gives a total of 131(108).
The equivalent of 2 molecules of ATP is consumed in the activation of palmitate, in which ATP is split into AMP and 2 molecules of Pi. Thus, the complete oxidation of a molecule of palmitate yields 129 ( 106) molecules of ATP.
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