functions of fatty acids

Q.1– What are the major sources and functions of fatty acids?

Answer- A fatty acid contains a long hydrocarbon chain and a terminal carboxylate group. The fatty acids can be obtained from diet, by adipolysis or can be synthesized de novo from precursor molecules.

Functions-Fatty acids have four major physiological roles.

1) Fatty acids are building blocks of phospholipids and glycolipids. These amphipathic molecules are important components of biological membranes,

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 triacylglycerols (also called neutral fats or triglycerides), which are uncharged esters of fatty acids with glycerol. 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.

Q.2- “The processes of fatty acid synthesis and fatty acid degradation are, in many ways, the reverse of each other”, justify the statement giving an overview of the two processes.

Answer- Although fatty acids are both oxidized to acetyl-CoA and synthesized from acetyl-CoA, fatty acid oxidation is not the simple reverse of fatty acid biosynthesis but an entirely different process taking place in a separate compartment of the cell. 

Fatty acid degradation - The separation of fatty acid oxidation in mitochondria from biosynthesis in the cytosol allows each process to be individually controlled and integrated with tissue requirements. Each step in fatty acid oxidation involves acyl-CoA derivatives catalyzed by separate enzyme, utilizes NAD⁺ and FAD as coenzymes, and generates ATP. It is an aerobic process, requiring the presence of oxygen.

The process of degradation converts an aliphatic compound into a set of activated acetyl units (acetyl CoA) that can be processed by the citric acid cycle (Figure-1). An activated fatty acid is oxidized to introduce a double bond; the double bond is hydrated to introduce oxygen; the alcohol is oxidized to a ketone; and, finally, the four carbon fragment is cleaved by coenzyme A to yield acetyl CoA and a fatty acid chain two carbons shorter. If the fatty acid has an even number of carbon atoms and is saturated, the process is simply repeated until the fatty acid is completely converted into acetyl CoA units.

Fatty acid synthesis – is essentially the reverse of this process. Because the result is a polymer, the process starts with monomers in this case with activated acyl group (most simply, an acetyl unit) and malonyl units (Figure1). The malonyl unit is condensed with the acetyl unit to form a four-carbon fragment. To produce the required hydrocarbon chain, the carbonyl must be reduced. The fragment is reduced, dehydrated, and reduced again, exactly the opposite of degradation, to bring the carbonyl group to the level of a methylene group with the formation of butyryl CoA. Another activated malonyl group condenses with the butyryl unit and the process is repeated until a C16 fatty acid is synthesized.

Figure-1- showing the differentiation between fatty acid synthesis and fatty acid degradation

Q.-3- What is likely the reason that triacylglycerols rather than glycogen were selected in evolution as the major energy reservoir?

Answer- Triacylglycerols are highly concentrated stores of metabolic energy because they are reduced and anhydrous.

1) The yield from the complete oxidation of fatty acids is about 9 kcal g-1 (38 kJ g-1), in contrast with about 4 kcal g-1 (17 kJ g-1) for carbohydrates and proteins. The basis of this large difference in caloric yield is that fatty acids are much more reduced.

2)  Furthermore, triacylglycerols are nonpolar, and so they are stored in a nearly anhydrous form, whereas much more polar proteins and carbohydrates are more highly hydrated. In fact, 1 g of dry glycogen binds about 2 g of water. Consequently, a gram of nearly anhydrous fat stores more than six times as much energy as a gram of hydrated glycogen, which is likely the reason that triacylglycerols rather than glycogen were selected in evolution as the major energy reservoir.

3) In a 70-kg man, who has fuel reserves of 100,000 kcal (420,000 kJ) in triacylglycerols, 25,000 kcal (100,000 kJ) in protein (mostly in muscle), 600 kcal (2500 kJ) in glycogen, and 40 kcal (170 kJ) in glucose.

Triacylglycerols constitute about 11 kg of his total body weight. If this amount of energy were stored in glycogen, his total body weight would be 55 kg greater. The glycogen and glucose stores provide enough energy to sustain biological function for about 24 hours, whereas the Triacylglycerol stores allow survival for several weeks.

Q.4- Give an overview of provision of fatty acids from different sources for utilization as fuel molecules.

Answer-

Dietary lipids-Most lipids are ingested in the form of triacylglycerols but must be degraded to fatty acids for absorption across the intestinal epithelium.These are hydrophobic molecules, and have to be hydrolyzed and emulsified to very small droplets (micelles) before they can be absorbed.

Triacylglycerols in the intestinal lumen are incorporated into micelles formed with the aid of bile salts, amphipathic molecules synthesized from cholesterol in the liver and secreted from the gall bladder.

Incorporation of lipids into micelles (Figure-2) orients the ester bonds of the lipid toward the surface of the micelle, rendering the bonds more susceptible to digestion by pancreatic lipases that are in aqueous solution.

Figure-2 -showing micelle formation.

The lipases digest the triacylglycerols into free fatty acids and monoacylglycerol (Figure-3). These digestion products are carried in micelles to the intestinal epithelium where they are absorbed across the plasma membrane.

Figure-3- showing the action of Pancreatic Lipases. Lipases secreted by the pancreas convert triacylglycerols into fatty acids and monoacylglycerol for absorption into the intestine.

In the intestinal mucosal cells, the triacylglycerols are resynthesized from fatty acids and monoacylglycerols and then packaged into lipoprotein transport particles called chylomicrons, stable particles ranging from approximately 180 to 500 nm in diameter (Figure-4)

Figure-4- showing the structure of chylomicron

These particles are composed mainly of triacylglycerols, with apoprotein B-48 as the main protein component. Protein constituents of lipoprotein particles are called apolipoproteins. Chylomicrons also function in the transport of fat-soluble vitamins and cholesterol. The chylomicrons are released into the lymph system and then into the blood (Figure-5).  These particles bind to membrane-bound lipoprotein lipases, primarily at adipose tissue and muscle, where the triacylglycerols are once again degraded into free fatty acids and monoacylglycerol for transport into the tissue. The triacylglycerols are then resynthesized inside the cell and stored.

Figure-5- showing the Chylomicron Formation. Free fatty acids and monoacylglycerols are absorbed by intestinal epithelial cells. Triacylglycerols are resynthesized and packaged with other lipids and apoprotein B-48 to form chylomicrons, which are then released into the lymph system.

Endogenous lipids

Peripheral tissues gain access to the lipid energy reserves stored in adipose tissue through three stages of processing.

1) First, the lipids must be mobilized. In this process, triacylglycerols are degraded to fatty acids and glycerol,  (Figure-6) which are released from the adipose tissue and transported to the energy-requiring tissues.

Figure-6- showing the degradation of triglyceride by hormone sensitive lipase in the adipose tissue

The lipase of adipose tissue are activated on treatment of these cells with the hormones epinephrine, nor epinephrine, glucagon and adrenocorticotropic hormone. Thus, these hormones induce lipolysis.

2) Transportation of free fatty acids

Free fatty acids—also called unesterified (UFA) or nonesterified (NEFA) fatty acids—are fatty acids that are in the unesterified state. In plasma, longer-chain FFA are combined with albumin, and in the cell they are attached to a fatty acid-binding protein, so that in fact they are never really “free.” Shorter-chain fatty acids are more water-soluble and exist as the un-ionized acid or as a fatty acid anion. By these means, free fatty acids are made accessible as a fuel in other tissues.

Glycerol formed by lipolysis is absorbed by the liver and phosphorylated, oxidized to dihydroxyacetone phosphate, and then isomerized to glyceraldehyde 3-phosphate (Figure-7).This molecule is an intermediate in both the glycolytic and the gluconeogenic pathways.

Figure-7- showing the phosphorylation and subsequent utilization of glycerol as intermediates of glycolysis

3) In tissues, the fatty acids must be activated and transported into mitochondria for degradation. The fatty acids are then broken down in a step-by step fashion into acetyl CoA, which is then processed in the citric acid cycle.

Q.4- Enlist the other different ways by which fatty acids can be oxidised in different cells? Explain the mechanism of activation of fatty acids prior to catabolism,

Answer- Fatty acids can be oxidized by-

1) Beta oxidation- Major mechanism ,occurs in the mitochondria  matrix . It is the process by which fatty acids are  degraded by removal of 2-C units . The process begins with oxidation of the carbon that is “beta” to the carboxyl  carbon, so the process is called “beta oxidation” The 2-C units are released as acetyl CoA, not free acetate .

2) Alpha oxidation- Predominantly takes place in brain and liver, one carbon is lost in the form of CO2 per cycle.

3) Omega oxidation- Minor mechanism, but becomes important in conditions of impaired beta oxidation

4) Peroxisomal oxidation- Mainly for the trimming of the very long chain fatty acids.

Activation of fatty acids-Fatty acids must first be converted to an active intermediate before they can be catabolized. This is the only step in the complete degradation of a fatty acid that requires energy from ATP. In the presence of ATP and coenzyme A, the enzyme acyl-CoA synthetase (thiokinase) catalyzes the conversion of a fatty acid (or free fatty acid) to an “active fatty acid” or acyl-CoA, which uses one high-energy phosphate with the formation of AMP and PP_i (Figure-8 ). The PP_i is hydrolyzed by inorganic pyrophosphatase with the loss of a further high-energy phosphate, ensuring that the overall reaction goes to completion. Acyl-CoA synthetases are found in the endoplasmic reticulum, peroxisomes, and inside and on the outer membrane of mitochondria.

Figure-8- Showing the activation of fatty acids to form Acyl co A and AMP

The activation of a fatty acid is accomplished in two steps-

1) First, the fatty acid reacts with ATP to form an acyl adenylate. In this mixed anhydride, the carboxyl group of a fatty acid is bonded to the phosphoryl group of AMP. The other two phosphoryl groups of the ATP substrate are released as pyrophosphate. The sulfhydryl group of CoA then attacks the acyl adenylate, which is tightly bound to the enzyme, to form acyl CoA and AMP (Figure-9)

Figure-9-showing the 2 steps of Acyl co A formation

These partial reactions are freely reversible. Pyrophosphate is rapidly hydrolyzed by a pyrophosphatase, hence the overall reaction is driven forward.This reaction is quite favorable because the equivalent of two molecules of ATP is hydrolyzed, whereas only one high transfer-potential compound is formed.

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fatty acid oxidation

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:

• Inadequate intake (e.g., due to fad diets, lack of access, or long-term TPN)
• Inability to metabolize carnitine due to enzyme deficiencies (e.g., carnitine palmitoyl Transferase deficiency, methylmalonic aciduria, propionic acidemia, Isovaleric acidemia)
• Decreased endogenous synthesis of carnitine due to a severe liver disorder
• Excess loss of carnitine due to diarrhea, diuresis, or hemodialysis
• A hereditary disorder in which carnitine leaks from renal tubules (Primary carnitine deficiency)
• Increased requirements for carnitine when ketosis is present or demand for fat oxidation is high (eg, during a critical illness such as sepsis or major burns; after major surgery of the GI tract)
• Decreased muscle carnitine levels due to mitochondrial impairment (eg, due to use of zidovudine)
• Use of valproate.

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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Fatty acid oxidation

Q. – Discuss the minor pathways of oxidation of fatty acids.

Answer- The β oxidation accounts for the bulk of the energy production from fatty acids in human. These reactions must be supplemented by other mechanisms, so that all types of ingested fatty acids can be oxidised.

Over view of minor pathways of biological importance of fatty acid oxidation

1) α- Oxidation- Oxidation occurs at C-2 instead of C-3 , as in β oxidation

2) ω- Oxidation – Oxidation occurs at the methyl end of the fatty acid molecule.

3) Peroxisomal fatty acid oxidation- Occurs for the chain shortening of very long chain fatty acids.

Details

1) α- Oxidation

α- Oxidation- Takes place in the microsomes of brain and liver, involves decarboxylation process for the removal of single carbon atom at one time with the resultant production of an odd chain fatty acid that can be subsequently oxidized by beta oxidation for energy production. It is strictly an aerobic process. No prior activation of the fatty acid is required. The process involves hydroxylation of the alpha carbon with a specific α-hydroxylase enzyme that requires Fe⁺⁺ and vitamin C/FH4 as cofactors.

There are systems in many tissues for the hydroxylation of α –carbon of shorter chain fatty acids in order to start their oxidation.

Biological significance of alpha oxidation

Although the use of the α- Oxidation scheme is relatively less in terms of total energy production, but it is significant in the metabolism of dietary fatty acids that are methylated. A principal example of these is Phytanic acid.

1)  α- Oxidation  is most suited for the oxidation of phytanic acid, produced from dietary phytol, a constituent of chlorophyll of plants. Phytanic acid is a significant constituent of milk lipids and animal fats and normally it is metabolized by an initial α- hydroxylation followed by dehydrogenation and decarboxylation. Beta oxidation can not occur initially because of the presence of 3- methyl groups, but it can proceed after decarboxylation. The whole reaction produces three molecules of propionyl co A, three molecules of Acetyl co A, and one molecule of iso butyryl co A (Figure-1)

Phytanic acid

Figure-1- Phytanic acid is oxidised by Phytanic acid α oxidase (α- hydroxylase enzyme) to yield CO2 and odd chain fatty acid Pristanic acid that can be subsequently oxidised by beta oxidation.This process involves hydroxylation of the alpha carbon, removal of the terminal carboxyl group and concomitant conversion of the alpha hydroxyl group to a terminal carboxyl group, and linkage of CoA to the terminal carboxyl group. This branched substrate will function in the beta-oxidation process, ultimately yielding propionyl-CoA, acetyl Co As and, in the case of phytanic acid, 2-methyl propionyl CoA (Iso butyryl Co A)

2)The hydroxy fatty acids produced as intermediates of this pathway like Cerebronic acid can be used for the synthesis of cerebrosides and sulfatides

3) Odd chain fatty acid 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 co A .The number of acetyl co A depend upon the chain length. Propionyl co A is converted to Succinyl co A to gain entry in to TCA cycle for further oxidation.

Clinical significance of alpha oxidation of fatty acids

Refsum disease (RD)

Refsum disease (RD) is a neurocutaneous syndrome that is characterized biochemically by the accumulation of phytanic acid in plasma and tissues. Refsum first described this disease. Patients with Refsum disease are unable to degrade phytanic acid because of a deficient activity of Phytanic acid oxidase enzyme catalyzing the first step of phytanic acid alpha-oxidation.

Peripheral polyneuropathy, cerebellar ataxia, retinitis pigmentosa, and  Ichthyosis (rough, dry and scaly skin) are the major clinical components. The symptoms evolve slowly and insidiously from childhood through adolescence and early adulthood.

Biochemical defect

Refsum disease is an Autosomal recessive disorder characterized by defective alpha-oxidation of phytanic acid.Consequently, this unusual, exogenous C20-branched-chain (3, 7, 11, 15-tetramethylhexadecanoic acid) fatty acid accumulates in brain, blood and other tissues. It is almost exclusively of exogenous origin and is delivered mainly from dietary plant chlorophyll and, to a lesser extent, from animal sources. Blood levels of phytanic acid are increased in patients with Refsum disease. These levels are 10-50 mg/dL, whereas normal values are less than or equal to 0.2 mg/dL, and account for 5-30% of serum lipids. Phytanic acid replaces other fatty acids, including such essential ones as Linoleic and Arachidonic acids, in lipid moieties of various tissues. This situation leads to an essential fatty acid deficiency, which is associated with the development of ichthyosis.

Refsum disease is rare, with just 60 cases observed so far.

Clinical manifestations

Classic Refsum disease manifests in children aged 2-7 years; however, diagnosis usually is delayed until early adulthood. Infantile Refsum disease makes its appearance in early infancy. Symptoms develop progressively and slowly with neurologic and ophthalmic manifestations. The disease is characterized by

• Night blindness due to degeneration of the retina (retinitis pigmentosum)
• Loss of the sense of smell (anosmia)
• Deafness
• Concentric constriction of the visual fields
• Cataract
• Signs resulting from cerebellar ataxia –Cardiac arrhythmias
  • Progressive weakness
  • Foot drop
  • Loss of balance
• Some individuals will have shortened bones in their fingers or toes.
• The children usually have moderately dysmorphic features that may include epicanthal folds, a flat bridge of the nose, and low-set ears.

Laboratory Diagnosis

• Levels of plasma cholesterol and high- and low-density lipoprotein are often moderately reduced.
•  Blood phytanic acid levels are elevated.
• Cerebrospinal fluid (CSF) shows a protein level of 100-600 mg/dL.
• Routine laboratory investigations of blood and urine do not reveal any consistent diagnostic abnormalities.
• Phytanic oxidase activity estimation in skin fibroblast cultures is important

Imaging

Skeletal radiography is required to estimate bone changes.

Treatment

• Eliminate all sources of chlorophyll from the diet.
• The major dietary exclusions are green vegetables (source of phytanic acid) and animal fat (phytol).
• The aim of such dietary treatment is to reduce daily intake of phytanic acid from the usual level of 50 mg/d to less than 5 mg/d.
• Plasmapheresis – Patients may also require plasma exchange (Plasmapheresis) in which blood is drawn, filtered, and reinfused back into the body, to control the buildup of phytanic acid.

• The main indication for Plasmapheresis in patients with Refsum disease is a severe or rapidly worsening clinical condition.
• A minor indication is failure of dietary management to reduce a high plasma phytanic acid level.

Prognosis -in untreated patients generally is poor. Dysfunction of myelinated nerve fibers and the cardiac conduction system leads to central and peripheral neuropathic symptoms, impaired vision, and cardiac arrhythmias. The latter frequently are the cause of death.

In early diagnosed and treated cases, phytanic acid decreases slowly, followed by improvement of the skin scaling and, to a variable degree, reversal of recent neurological signs. Retention of vision and hearing are reported.

 Pharmacological up regulation of the omega-oxidation of phytanic acid may form the basis of the new treatment strategy for adult Refsum disease in the near future.

2) Omega oxidation of fatty acids

Another minor pathway for the fatty acid oxidation also involves hydroxylation and occurs in the endoplasmic reticulum of many tissues. In this case hydroxylation takes place 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, O2 and NADPH, as well as the necessary enzymes.

Hydroxy fatty acids can be further oxidised to a dicarboxylic acid via sequential reactions of Alcohol dehydrogenase and aldehyde dehydrogenases. The process occurs primarily with medium chain fatty acids.

The dicarboxylic acids so formed can be activated at either end of molecule to form a Co A ester, which can undergo beta oxidation to produce shorter chain dicarboxylic acids such as Adipic acids(C6) and succinic acid (C4).

The microsomal (endoplasmic reticulum, ER) pathway of fatty acid ω-oxidation represents a minor pathway of overall fatty acid oxidation.

However, in certain pathophysiological states, such as diabetes, chronic alcohol consumption, and starvation, the ω-oxidation pathway may provide an effective means for the elimination of toxic levels of free fatty acids. 

3) Peroxisomal oxidation of very long chain fatty acids-

Although most fatty acid oxidation takes place in mitochondria, some oxidation takes place in cellular organelles called peroxisomes(Figure-2).

Peroxisomes are a class of sub cellular organelles with distinctive morphological and chemical characteristics.

These organelles are characterized by high concentrations of the enzyme catalase, which catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. It has been suggested that peroxisomes may function in a protective role against oxygen toxicity. Several lines of evidence suggest that they are also involved in the lipid catabolism. A number of drugs used clinically to decrease triglyceride levels in patients cause a marked increase in peroxisomes.

Fatty acid oxidation in these organelles, which halts at octanyl CoA, may serve to shorten long chains to make them better substrates of b-oxidation in mitochondria. Peroxisomal oxidation differs from beta oxidation in the initial dehydrogenation reaction (Figure–2). In peroxisomes, a flavoprotein dehydrogenase transfers electrons to O2 to yield H2O2 instead of capturing the high-energy electrons as FADH2, 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 with their mitochondrial counterparts, although they are carried out by different isoforms of the enzymes.

Figure-2- Initiation of Peroxisomal Fatty Acid Degradation, The first dehydration in the degradation of fatty acids in peroxisomes requires a flavoprotein dehydrogenase that transfers electrons to O2 to yield H2O2.

The specificity of the peroxisomal enzymes is for somewhat longer chain fatty acids. Thus peroxisomal enzymes function to shorten the chain length of relatively long chain fatty acids to a point at which beta oxidation can be completed in mitochondria. Other peroxisomal reactions include chain shortening of dicarboxylic acids, conversion of cholesterol to bile acids and formation of ether lipids. Given these diverse metabolic roles it is not surprising that the congenital absence of functional peroxisomes, an inherited defect , known as Zellwegar syndrome, has such devastating effects.

Zellweger syndrome

Zellweger syndrome, also called cerebrohepatorenal syndrome is a rare, congenital disorder (present at birth), characterized by thereduction or absence of Peroxisomes in the cells of the liver, kidneys, and brain.

Biochemical defect

Zellweger syndrome is one of a group of four related diseases called peroxisome biogenesis disorders (PBD), which are part of a larger group of diseases known as the leukodystrophies.  These are inherited conditions that damage the white matter of the brain and also affect how the body metabolizes particular substances in the blood and organ tissues. It is characterized by an individual’s inability to beta-oxidize very-long chain fatty acids in the Peroxisomes of the cell, due to a genetic disorder in one of the several genes involved with peroxisome biogenesis. Zellweger syndrome is the most severe of the PBDs.  Infantile Refsum disease (IRD) is the mildest and neonatal adrenoleukodystrophy and rhizomelic chondrodysplasia have similar but less severe symptoms. 

Clinical Manifestations

The most common features of Zellweger syndrome include enlarged liver, high levels of iron and copper in the blood stream, and vision disturbances. Some affected infants may show prenatal growth failure. Symptoms at birth may include a lack of muscle tone, an inability to move and glaucoma. Other symptoms may include unusual facial characteristics, mental retardation, seizures, and an inability to suck and/or swallow. Jaundice and gastrointestinal bleeding may also occur. Of central diagnostic importance are the typical facial appearance (high forehead, unslanting palpebral fissures, hypo plastic supraorbital ridges, and epicanthal folds. More than 90% show postnatal growth failure.

Laboratory diagnosis

There are several noninvasive laboratory tests that permit precise and early diagnosis of peroxisomal disorders.  The  abnormally high levels of VLCFA( Very long chain fatty acids ), are most diagnostic.

Treatment

 There is no cure for Zellweger syndrome, nor is there a standard course of treatment.  Since the metabolic and neurological abnormalities that cause the symptoms of Zellweger syndrome are caused during fetal development, treatments to correct these abnormalities after birth are limited.  Most treatments are symptomatic and supportive.

Prognosis

The prognosis for infants with Zellweger syndrome is poor.  Most infants do not survive past the first 6 months, and usually succumb to respiratory distress, gastrointestinal bleeding, or liver failure.

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