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Q.- What is Reverse cholesterol transport ? Explain the biological/clinical significance of this process.


Discuss the concept of “reverse cholesterol transport” and indicate how its operation might contribute to a lowering of serum cholesterol.


Discuss the role of LCAT (Lecithin Cholesterol Acyl Transferase) in the cholesterol transport.

Answer-  The selective transfer of cholesterol from peripheral cells to HDLs, and from HDLs to the liver for bile acid synthesis, and to steroidogenic tissues for hormone synthesis, is a key component of cholesterol homeostasis. This is in part the basis for the inverse relationship seen between plasma HDL concentration and atherosclerosis, and because of this only HDL is designated as “Good cholesterol carrier”.

HDL– HDL particles serve as  circulating reservoir of apo CII (apo protein that is transferred to VLDL and Chylomicrons, and is an activator of lipoprotein lipase), and apo E (the apoprotein required for the receptor-mediated endocytosis of IDls and chylomicron remnants (Figure-1)


Figure-1 Showing the structure of HDL. The outer shell is made by apoproteins, phospholipids and free cholesterol and the inner core is made by Cholesteryl esters and triglycerides.


HDL is synthesized and secreted from both liver and intestine (Figure -2). However, apo C and apo E are synthesized in the liver and transferred from liver HDL to intestinal HDL when the latter enters the plasma. Nascent HDL consists of discoid phospholipid  bilayer containing apo A and free cholesterol.

Figure-2- Showing the role of HDL in reverse cholesterol transport. HDL has four forms, Discoidal HDL, HDL3 , HDL2 and Pre β  HDL.( C- Cholesterol, CE- Cholesteryl Ester, LCAT- Lecithin choleterol Acyl Transferase, PL- Phospholipid.

Reverse cholesterol transport involves-

1) Efflux of cholesterol from peripheral cells and esterification to form cholesteryl ester by LCAT

LCAT ( Lecithin Cholesterol Acyl Transferase) enzyme catalyzes the esterification of cholesterol to form Cholesteryl ester.

The reaction can be represented as follows-

Lecithin + Cholesterol ———->   Lysolecithin + Cholesteryl Ester

LCAT and the LCAT activator apo A-I—bind to the discoidal particles (Figure-1) and the surface phospholipid , free cholesterol  (extracted from peripheral cells) is converted into cholesteryl esters and lysolecithin .

The nonpolar cholesteryl esters move into the hydrophobic interior of the bilayer, whereas lysolecithin is transferred to plasma albumin. Thus, a nonpolar core is generated, forming a spherical, pseudomicellar HDL covered by a surface film of polar lipids and apolipoproteins. This aids the removal of excess unesterified cholesterol from lipoproteins and tissues .

2) Binding of the cholesteyl ester-rich HDL(HDL2) to liver and steroidogenic cells, and the selective transfer of cholesteryl esters in to these cells)- Figure-2

HDL Receptor

The class B scavenger receptor B1 (SR-B1) has been identified as an HDL receptor with a dual role in HDL metabolism.

a) In the liver and in steroidogenic tissues, it binds HDL via apo A-I, and cholesteryl ester is selectively delivered to the cells, although the particle itself, including apo A-I, is not taken up.

b) In the tissues, on the other hand, SR-B1 mediates the acceptance of cholesterol from the cells by HDL, which then transports it to the liver for excretion via the bile (either as cholesterol or after conversion to bile acids) in the process known as reverse cholesterol transport (Figure 2).

HDL cycle

HDL3, generated from discoidal HDL by the action of LCAT (Figure-2) accepts cholesterol from the tissues via the SR-B1 and the cholesterol is then esterified by LCAT, increasing the size of the particles to form the less dense HDL2.

HDL3 is then reformed, either after selective delivery of cholesteryl ester to the liver via the SR-B1 or by hydrolysis of HDL2 Phospholipid and triacylglycerol by hepatic lipase (Figure-2).This interchange of HDL2 and HDL3 is called the HDL cycle (Figure -2).

Free apo A-I is released by these processes and forms pre-HDL after associating with a minimum amount of Phospholipid and cholesterol. Surplus apo A-I is destroyed in the kidney.

HDL Transporter

A second important mechanism for reverse cholesterol transport involves the ATP-binding cassette transporter A1 (ABCA1) (Figure-2). ABCA1 is a member of a family of transporter proteins that couple the hydrolysis of ATP to the binding of a substrate, enabling it to be transported across the membrane. ABCA1 preferentially transfer cholesterol from cells to poorly lipidated particles such as pre-HDL or apo A-1, which are then converted to HDL3 via discoidal HDL (Figure -2). Pre-β HDL is the most potent form of HDL inducing cholesterol efflux from the tissues.

Clinical Significance

LCAT deficiency– Complete absence (Familial LCAT deficiency) or partial (Fish eye disease) deficiency results in a marked decrease in HDL primarily as a result of the hyper catabolism of lipid poor HDLs.

Atherosclerosis- There is an inverse relationship between HDL (HDL2) concentrations and coronary heart disease. This is consistent with the function of HDL in reverse cholesterol transport. Atherosclerosis is characterized by the deposition of cholesterol and cholesteryl ester from the plasma lipoproteins into the artery wall. Diseases in which prolonged elevated levels of VLDL, IDL, chylomicron remnants, or LDL occur in the blood (eg, diabetes mellitus, lipid nephrosis, hypothyroidism, and other conditions of hyperlipidemia) are often accompanied by premature or more severe atherosclerosis. LDL:HDL cholesterol ratio a good predictive parameter.

Low HDL levels

Causes of low HDL levels

  • Severely reduced plasma levels of HDL-C (<20 mg/dL) accompanied by triglycerides <400 mg/dL usually indicate the presence of a genetic disorder, such as a mutation in apoA-I, LCAT deficiency, or Tangier disease.
  • HDL-C levels <20 mg/dL are common in the setting of severe hypertriglyceridemia,
  • HDL-C levels <20 mg/dL also occur in individuals using anabolic steroids.
  • Secondary causes of more moderate reductions in plasma HDL (20–40 mg/dL) should be considered like smoking, diabetes mellitus Type 2, Gaucher’s disease and malnutrition.

Management of low HDL levels

  •  Smoking should be discontinued,
  • Obese persons should be encouraged to lose weight,
  • Sedentary persons should be encouraged to exercise, and diabetes should be optimally controlled.
  •  When possible, medications associated with reduced plasma levels of HDL-C should be discontinued.
  • The presence of an isolated low plasma level of HDL-C in a patient with a borderline plasma level of LDL-C should prompt consideration of LDL lowering drug therapy in high-risk individuals.
  • Statins increase plasma levels of HDL-C only modestly (~5–10%). Fibrates also have only a modest effect on plasma HDL-C levels (increasing levels ~5–15%), except in patients with coexisting hypertriglyceridemia, where they can be more effective.
  • Niacin is the most effective available HDL-C–raising therapeutic agent and can be associated with increases in plasma HDL-C by up to ~30%, although some patients do not respond to niacin therapy.


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Q.- Give a brief account of the steps of synthesis of Very Low density lipoprotein (VLDL). High light the clinical significance of impaired VLDL synthesis.

Answer- There are striking similarities in the mechanisms of formation of chylomicrons by intestinal cells and of VLDL by hepatic parenchymal cells (Figure -1), perhaps because—apart from the mammary gland—the intestine and liver are the only tissues from which particulate lipid is secreted.

Steps of synthesis-

1) Protein synthesis- The major apoprotein of VLDL, Apo B100 is synthesized in the rough endoplasmic reticulum. VLDL particles are stabilized by two lipoproteins apo B-100 and apo E (34 kd). Apo B-100, one of the largest proteins known (513 kd), is a longer version of apo B-48. Both apo B proteins are encoded by the same gene and produced from the same initial RNA transcript. In the intestine, RNA editing modifies the transcript to generate the mRNA for apo B-48, the truncated form.

Newly secreted VLDL contains only a small amount of apolipoproteins C and E, and the full complement is acquired from HDL in the circulation.

2) Lipid synthesis and formation of lipoprotein –In the fully fed state, apo B-100 is synthesized in excess of requirements for VLDL secretion and the surplus is destroyed in the liver (Figure-2). During translation of apo B-100, microsomal transfer protein-mediated lipid transport enables lipid to become associated with the nascent polypeptide chain.

The liver is a major site of triacylglycerol and cholesterol synthesis. Triacylglycerols and cholesterol in excess of the liver’s own needs are exported into the blood in the form of very low density lipoproteins (d<1.006 g cm-3).

After release from the ribosomes, these particles fuse with more lipids from the smooth endoplasmic reticulum, producing nascent VLDL.

3) Glycosylation and release of VLDL- After addition of carbohydrate residues in golgi apparatus, VLDL particles are released from the cell by reverse pinocytosis. VLDL are secreted into the space of Disse and then into the hepatic sinusoids through fenestrae in the endothelial lining.

Figure-1- Showing the steps of synthesis of VLDL in the liver cell, (RER- Rough endoplasmic reticulum, SER- Smooth endoplasmic reticulum, G- Golgi apparatus, N- Nucleus, S- Space of Disse and VLDL- very low density lipoprotein)

Clinical Significance

Fatty liver (steatosis)

 It is an abnormal accumulation of certain fats (triglycerides) inside liver cells.

Hepatic triacylglycerol synthesis provides the immediate stimulus for the formation and secretion of VLDL. Impaired VLDL formation or secretion leads to nonmobilization of lipid components from the liver, resulting in fatty liver.

Causes of fatty liver- Imbalance in the rate of triacylglycerol formation and export causes fatty liver. For a variety of reasons, lipid—mainly as triacylglycerol—can accumulate in the liver. Extensive accumulation is regarded as a pathologic condition. When accumulation of lipid in the liver becomes chronic, fibrotic changes occur in the cells that progress to cirrhosis and impaired liver function.

Fatty livers fall into two main categories-

A) More synthesis of Triglycerides or

B) Defective VLDL synthesis (Metabolic block)

A) More synthesis of Triglycerides-Triglycerides are synthesized in excess due to more availability of Fatty acid and glycerol. The fatty acids used are derived from two possible sources: (1) synthesis within the liver from acetyl-CoA derived mainly from carbohydrate (perhaps not so important in humans) and (2) uptake of free fatty acids from the circulation. The first source is predominant in the well-fed condition, when fatty acid synthesis is high and the level of circulating free fatty acids is low. As triacylglycerol does not normally accumulate in the liver under this condition, it must be inferred that it is transported from the liver in VLDL as rapidly as it is synthesized and that the synthesis of apo B-100 is not rate-limiting. Free fatty acids from the circulation are the main source during starvation, the feeding of high-fat diets, or in diabetes mellitus, when hepatic lipogenesis is inhibited.

Thus high carbohydrate diet stimulates de novo fatty acid synthesis by providing excess of Acetyl CoA and high fat feeding provides more flux of fatty acids from the diet that can be esterified to provide excess triglycerides.

B) Defective VLDL synthesis The second type of fatty liver is usually due to a metabolic block in the production of plasma lipoproteins, thus allowing triacylglycerol to accumulate.

Theoretically, the lesion may be due to-

 (1) A block in apolipoproteins synthesis

Causes- can be-

a) Protein energy Malnutrition

b) Impaired absorption

c) Presence of inhibitors of endogenous  protein synthesis e.g.- Carbon tetra chloride, Puromycin, Ethionine etc. The antibiotic puromycin, ethionine (α-amino-γ-mercaptobutyric acid), carbon tetrachloride, chloroform, phosphorus, lead, and arsenic all cause fatty liver and a marked reduction in concentration of VLDL (Figure-2). The action of ethionine is thought to be caused by a reduction in availability of ATP due to its replacing methionine in S-adenosylmethionine, trapping available adenine and preventing synthesis of ATP.

d) Hypobetalipoproteinemia- Defective apo B gene can cause impaired synthesis of apo B protein.

(2) A failure in provision of phospholipids that are found in lipoproteins-

a) A deficiency of choline, which has therefore been called a lipotropic factor can cause impaired formation of phosphatidyl choline (Lecithin),a glycerophospholipid (Figure-2)

b) Choline is formed by methylation from ethanolamine, with S-Adenosyl Methionine acting as a methyl group donor. Methionine deficiency can cause impaired choline synthesis and thus fatty liver besides other clinical defects.

c) Deficiency of essential fatty acids- can also lead to impaired Phospholipid synthesis

(3) Impaired Glycosylation- Orotic acid causes fatty liver; it is believed to interfere with glycosylation of the lipoprotein, thus inhibiting release, and may also impair the recruitment of triacylglycerol to the particles. In conditions of orotic aciduria (disorder of  pyrimidine nucleotide biosynthesis), fatty liver can be observed (Figure-2)

4) Impaired secretion of VLDL- oxidative stress is a common cause for membrane disruption of lipoprotein. The action of carbon tetrachloride probably involves formation of free radicals causing lipid peroxidation (Figure-2). Some protection against this is provided by the antioxidant action of vitamin E-C, beta carotene and selenium in the supplemented diets.


Figure-2- Showing the biochemical basis of fatty liver disease. Imbalance in the rate of triacylglycerol formation and export causes fatty liver.

Clinical conditions causing fatty liver-Clinically fatty liver is of two types-

1) Non alcoholic fatty liver- Fatty liver (with or without fibrosis) due to any condition except alcoholism is called nonalcoholic steatohepatitis (Macro vesicular steatosis).

Causes of nonalcoholic steatosis or NAFLD are

  • Obesity
  • Diabetes mellitus
  • Hypertriglyceridemia
  • Drugs- corticosteroids, amiodarone, diltiazem, tamoxifen, highly active antiretroviral therapy
  • Poisons (carbon tetrachloride and yellow phosphorus)
  • Endocrinopathies such as Cushing’s syndrome and hypopituitarism, hypobetalipoproteinemia and other metabolic disorders,
  • Obstructive sleep apnea,
  • Starvation

(The biochemical basis for each condition has been explained above)

2) Alcoholic fatty liver- Alcoholism leads to fat accumulation in the liver, hyperlipidemia, and ultimately cirrhosis. The fatty liver is caused by a combination of impaired fatty acid oxidation and increased lipogenesis, which is thought to be due to changes in the [NADH]/[NAD+] redox potential in the liver, and also to interference with the action of transcription factors regulating the expression of the enzymes involved in the pathways.

Oxidation of ethanol by alcohol and aldehyde dehydrogenase leads to excess production of NADH (Figure-3)


Figure-3- Showing steps of metabolism of alcohol

A) Effect of excess NADH – More triglyceride synthesis

1) The NADH generated competes with reducing equivalents from other substrates, including fatty acids, for the respiratory chain, inhibiting their oxidation and causing increased esterification of fatty acids to form triacylglycerol, resulting in the fatty liver.

2)  Oxidation of ethanol, leads to the formation of acetaldehyde, which is oxidized by aldehyde dehydrogenase, producing acetate. Acetate is converted to Acetyl coA and there is more fatty acid synthesis.

3) Accumulation of NADH causes more formation of Glycerol-3-P (shift of equilibrium of reaction), that can be used for the synthesis of triglycerides.

B) Improper apo- protein synthesis – Malnutrition is a common finding in chronic alcoholism There is less availability of essential amino acids.

C) Impaired Phospholipid synthesis-Due to malnutrition there is less availability of essential fatty acids and choline leading to defective Phospholipid synthesis.

D) Impaired secretion of VLDL- chronic alcohol consumption is associated with oxidative stress that can cause impaired VLDL secretion.

Thus multiple factors are responsible for alcoholic fatty liver disease

Lipotropic agents- Agents such as choline, Inositol, Methionine and other essential amino acids, essential fatty acids, anti oxidant vitamins, vitamin B12, folic acid and synthetic antioxidants which have the apparent effect of removal of fats from the liver cells, and thus prevent the formation of fatty liver are called lipotropic agents.


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Q,- Give a brief account of the role of lipoprotein lipase in the catabolism of lipoproteins. Discuss the clinical significance of its diminished activity.


What is the action of lipoprotein lipase and how is it activated or inhibited?

Answer- Triacylglycerols of Chylomicrons & VLDL are hydrolyzed by Lipoprotein Lipase.

1) Location-Lipoprotein lipase is located on the walls of blood capillaries, anchored to the endothelium by negatively charged proteoglycan chains of heparan sulfate (Figure-1). It has been found in heart, adipose tissue, spleen, lung, renal medulla, aorta, diaphragm, and lactating mammary gland, although it is not active in adult liver. It is not normally found in blood; however, following injection of heparin, lipoprotein lipase is released from its heparan sulfate binding into the circulation. Due to this reason heparin is also called ‘Clearing factor’. Reduced serum LPL activity, after an injection of intravenous heparin, confirms the diagnosis of either LPL or apoC-II deficiency.


Figure-1- Showing the attachment of lipoprotein lipase to the endothelial cells by proteoglycan chains of Heparan sulfate

2) Activators and Inhibitors-Both phospholipids and apo C-II are required as cofactors for lipoprotein lipase activity, while apo A-II and apo C-III act as inhibitors. In adipose tissue, insulin enhances lipoprotein lipase synthesis in adipocytes and its translocation to the luminal surface of the capillary endothelium.

 3) Catalytic action-Hydrolysis takes place while the lipoproteins are attached to the enzyme on the endothelium. Triacylglycerol is hydrolyzed progressively through a diacylglycerol to a monoacylglycerol and finally to free fatty acids plus glycerol.

 4) Fate of products of hydrolysis-Some of the released free fatty acids return to the circulation, attached to albumin, but the bulk is transported into the tissue . Glycerol is transported to liver for further utilization as a source of energy, as a substrate for gluconeogenesis or for reesterification to form triglycerides. In muscle, the fatty acids are oxidized to produce ATP and in adipose  cells they are reformed into TG for storage (Figure-2)

The action of lipoprotein lipase forms remnant lipoproteins

Figure-2- showing an overview of action of lipoprotein lipase and the fate of products

Reaction with lipoprotein lipase results in the loss of approximately 90% of the triacylglycerol of chylomicrons and VLDL, with the formation of chylomicron and  VLDL remnants or IDL (intermediate-density lipoprotein.

5) Organ specific variations in catalytic action-Heart lipoprotein lipase has a low Km( for triacylglycerol about one-tenth of that for the enzyme in adipose tissue. This enables the delivery of fatty acids from triacylglycerol to be redirected from adipose tissue to the heart in the starved state when the plasma triacylglycerol decreases. A similar redirection to the mammary gland occurs during lactation, allowing uptake of lipoprotein triacylglycerol fatty acid for milk fat synthesis. The VLDL receptor plays an important part in the delivery of fatty acids from VLDL triacylglycerol to adipocytes by binding VLDL and bringing it into close contact with lipoprotein lipase.

Clinical significance of diminished activity – The most common genetic defect leading to hypertriglyceridemia is a deficiency in LPL, which results in increased levels of both chylomicrons and VLDL. (TG levels >2000 mg/dL)- Type I- Hyperlipidemia (Chylomicronemia)

The cholesterol level may be normal or slightly elevated. LPL deficiency is inherited in an autosomal recessive pattern. Patients with LPL deficiency often present with recurrent episodes of pancreatitis in their childhood and may have other clinical signs of hypertriglyceridemia such as: xanthomas, hepatosplenomegaly, and lipemia retinalis.

Clinical diagnosis-

1)  LPL deficiency requires measurement of LPL activity in plasma following intravenous injection of heparin, which displaces the LPL from its heparan sulfate tether. However, heparin also releases hepatic lipase ( HL) into the plasma and the post-heparin plasma must be treated with antibodies specific to HL to remove it.

2) Alternatively, LPL can also be measured in adipose tissue, which has no HL activity.

3)  A deficiency in apoC-II will also show evidence of decreased postheparin  plasma LPL activity. An increase in LPL activity when normal apo C-II is added to the assay indicates that a defect in apoC-II is the culprit.


 Q- Give a brief account of the synthesis and catabolism of chylomicrons.

Answer- By definition, chylomicrons are found in chyle formed only by the lymphatic system draining the intestine. They are responsible for the transport of all dietary lipids into the circulation. Small quantities of VLDL are also to be found in chyle; however, most of the plasma VLDL are of hepatic origin. They are the vehicles of transport of triacylglycerol from the liver to the extrahepatic tissues.

Synthesis of Chylomicrons – (Figure-4)

1) Synthesis of Apo B48

Chylomicrons contain Apo B48, synthesized in the rough endoplasmic reticulum (RER). The synthesis of apo B48 is the result of RNA editing process. Coding information can be changed at the mRNA level by RNA editing. In such cases, the coding sequence of the mRNA differs from that in the cognate DNA. In liver, the single apoB gene is transcribed into an mRNA that directs the synthesis of a 100-kDa protein, apoB100. In the intestine, the same gene directs the synthesis of the primary transcript; however, a cytidine deaminase converts a CAA codon in the mRNA to UAA at a single specific site. Rather than encoding glutamine, this codon becomes a termination signal, and a 48-kDa protein (apoB48) is the result. ApoB100 and apoB48 have different functions in the two organs (Figure-3)


Figure-3- showing RNA editing

Clinical Significance- In abetalipoproteinemia (a rare disease), lipoproteins containing apo B are not formed and lipid droplets accumulate in the intestine and liver(Due to non formation of VLDL)

2) Synthesis of lipids and formation of lipoprotein- Long-chain fatty acids are esterified to yield to triacylglycerol in the mucosal cells and together with the other products of lipid digestion, are incorporated into lipoproteins in the SER, the main site of synthesis of triacylglycerol.


Figure-4 – Showing the Synthesis of Chylomicrons (RER- Rough endoplasmic reticulum, SER- smooth endoplasmic reticulum, G- Golgi apparatus, N- Nucleus, C-chylomicron.

3) Addition of Carbohydrate-Carbohydrate residues are added in the golgi apparatus.

4) Release and Transportation of Chylomicrons- After addition of carbohydrate residues in golgi apparatus, they are released from the cell by reverse pinocytosis. Chylomicrons pass into the lymphatic system and eventually enter the systemic circulation.

Catabolism of Chylomicrons

The clearance of chylomicrons from the blood is rapid, the half-time of disappearance being under 1 hour in humans. Larger particles are catabolized more quickly than smaller ones. Chylomicrons are acted upon by the enzyme lipoprotein lipase (Details given  under Lipoprotein lipase- see figure-5). Reaction with lipoprotein lipase results in the loss of approximately 90% of the triacylglycerol of chylomicrons and in the loss of apo C (which returns to HDL) but not apo E, which is retained. The resulting chylomicron remnant is about half the diameter of the parent chylomicron and is relatively enriched in cholesterol and cholesteryl esters because of the loss of triacylglycerol.


Figure-5- Showing the metabolic fate of Chylomicrons  The nascent chylomicrons acquire apo CII and E from HDL. After the action of lipoprotein lipase, apo CII is returned back to HDL while apo E is retained by the chylomicron remnants to get internalized through Apo B100,E receptors in the liver. Apo CII is an activator of lipoprotein lipase.

Chylomicron remnants are taken up by the liver by receptor-mediated endocytosis, and the cholesteryl esters and triacylglycerols are hydrolyzed and metabolized. Uptake is mediated by apo E (Figure –4), via two apo E-dependent receptors, the LDL (apo B-100, E) receptor and the LRP (LDL receptor-related protein). Hepatic lipase has a dual role: (1) it acts as a ligand to facilitate remnant uptake and (2) it hydrolyzes remnant triacylglycerol and phospholipid.

The products released are re utilized for the synthesis of VLDL.


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Q- Comment upon the degradation of cholesterol, Is it a source of energy ?

Answer- Cholesterol is not a source of energy , the ring structure of cholesterol cannot be metabolized to CO2 and H2O. Rather the intact sterol is eliminated from the body by conversion to bile acids and bile salts, and by secretion of cholesterol in to the bile, which transports it to the intestine for elimination.

Figure-1-Showing the structure of Coprostanol


Figure-2- Showing the structure of Cholestanol

Some of the cholesterol in the intestine is acted upon by bacteria before excretion. The primary compounds made are the isomers of Coprostanol (Figure-1)and Cholestanol (Figure-2) which are reduced derivatives of cholesterol. Together with cholesterol , these compounds make the bulk of the feces.

Q.-How does cholesterol liberated from LDL ( beta-lipoprotein) within the cell control the cell’s cholesterol metabolism? Discuss the clinical state associated with absent or reduced LDL uptake.

Answer- Cholesterol is transported in plasma in lipoproteins, and in humans the highest proportion is found in LDL.

Dietary cholesterol- Cholesteryl ester in the diet is hydrolyzed to cholesterol, which is then absorbed by the intestine together with dietary unesterified cholesterol and other lipids. With cholesterol synthesized in the intestines, it is then incorporated into chylomicrons. Of the cholesterol absorbed, 80–90% is esterified with long-chain fatty acids in the intestinal mucosa. Ninety-five percent of the chylomicron cholesterol is delivered to the liver in chylomicron remnants, and most of the cholesterol secreted by the liver in VLDL is retained during the formation of IDL and ultimately LDL (Figure-3)which is taken up by the LDL receptor in liver and extrahepatic tissues.

LDL Receptor- The LDLs (containing cholesteryl esters) are taken up by cells by a process known as receptor-mediated endocytosis. The LDL receptor mediates this endocytosis and is important to cholesterol metabolism.

LDL (apo B-100, E) receptors occur on the cell surface in pits that are coated on the cytosolic side of the cell membrane with a protein called clathrin. The glycoprotein receptor spans the membrane, the B-100 binding region being at the exposed amino terminal end.

Figure-3- Showing the structure of LDL. The outer hydrophilic shell is made up of Apo B100 , phospholipids and free cholesterol while the inner hydrophobic core is made up of Triglycerides and Cholesteryl esters.

 The LDL receptor gene is located on the short arm of chromosome 19, and the protein is composed of 860 amino acids. It is the primary determinant of hepatic LDL uptake, which normally processes approximately 70% of circulating LDL. Two ligands on LDL bind to the receptor, apolipoprotein B-100 (apoB-100) and apoE. and is, therefore, more accurately termed the B,E receptor. ApoE is found on most lipoproteins other than LDL, including very low-density lipoprotein (VLDL) and chylomicrons and their remnants, intermediate-density lipoprotein (IDL), and a subclass of high-density lipoprotein (HDL). The LDL receptor binds apoE with higher affinity than apoB-100, and some mutations in the receptor may spare uptake of LDL by allowing binding to apoE.


 Figure- 4- Showing uptake of LDL. The enzyme ACAT, uses mono unsaturated fatty acid for esterification of cholesterol (Oleic acid)

After LDL binding to the LDL receptor, the ligand-receptor complexes cluster on the plasma membrane in coated pits, which then invaginate forming coated vesicles. These coated vesicles are internalized and clathrin, the protein composing the lattice in membrane coated pits, is removed. These vesicles are now called endosomes and these endosomes fuse with the lysosome. The LDL receptor–containing membrane buds off and is recycled to the plasma membrane. Fusion of the lysosome and endosome releases lysosomal proteases that degrade the apoproteins into amino acids. Lysosomal enzymes also hydrolyze the cholesteryl esters to free cholesterol and fatty acids.

Fate of released free cholesterol(Regulation of cholesterol metabolism by internalized cholesterol)

1)The free cholesterol is released into the cell’s cytoplasm, and this free cholesterol is then available to be used by the cell. It may be used for the formation of bile acids, steroid  hormones, vitamin D or may be used as component of biological membranes depending upon the cell type.

2) Excess cholesterol is reesterified by acyl-CoA:cholesterol acyltransferase (ACAT), which uses fatty acyl-CoA as the source of activated fatty acid.

3) Free cholesterol affects cholesterol metabolism by inhibiting cholesterol biosynthesis. Cholesterol inhibits the enzyme hydroxy-methylglutaryl-CoA reductase (HMG-CoA reductase), which catalyzes an early rate-limiting step in cholesterol biosynthesis. HMG-CoA reductase is the target of the statin drugs in wide use for treating patients with elevated cholesterol levels.

4)  In addition, free cholesterol inhibits the synthesis of the LDL receptor, thus limiting the amount of LDLs that are taken up by the cell. This influx of cholesterol inhibits the transcription of the genes encoding HMG-CoA synthase—HMG-CoA reductase and other enzymes involved in cholesterol synthesis as well as the LDL receptor itself via the SREBP pathway, and thus coordinately suppresses cholesterol synthesis and uptake.

 cholesterol metabolism

 Figure-5- Showing the LDL uptake and the fate of free cholesterol

Clinical Significance of reduced LDL uptake

Familial hypercholesterolemia (FH)- FH is a disorder of absent or grossly malfunctioning low-density lipoprotein (LDL) receptors. LDL receptor function ranges from completely absent to approximately 25% of normal receptor activity.

In the absence of a functioning LDL receptor, LDL cholesterol levels are greatly elevated in individuals with this disease.

Also, when LDL is not internalized by hepatocytes, hepatic synthesis of cholesterol is not suppressed. This leads to further cholesterol production despite high levels of circulating cholesterol. Therefore, circulating cholesterol levels are increased dramatically. The total and LDLc levels of infants and children with homozygous FH are higher than 600 mg/dL. In patients with heterozygous FH, half the LDL receptors are normal and half are rendered ineffective by the mutation. These patients’ total cholesterol and LDLc levels are twice as high as the population average. LDLc levels of 200-400 mg/dL are common.

High levels of LDLc increase cholesterol uptake in nonhepatic cells that is independent of LDL receptors. These scavenger pathways allow cholesterol uptake by monocytes and macrophages, leading to foam cell formation, plaque deposition in the endothelium of coronary arteries, and premature CAD. Cholesterol also accumulates in other areas, particularly the skin, causing Xanthelasmas and a variety of xanthomas.

Homozygous children may have symptoms consistent with ischemic heart disease, peripheral vascular disease, cerebrovascular disease, or aortic stenosis. 

Healthy diet, regular exercise, and maintenance of desirable weight  along with pharmacological intervention is required for treating hypercholesterolemia.

Prognosis depends heavily on the extent to which LDLc levels can be reduced. Patients with homozygous FH have and extremely limited life expectancy without major medical intervention.



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Q.- What are gall stones? How is cholesterol metabolism related to gall stone formation ? Discuss in  detail about the biochemical basis, clinical manifestations, diagnosis and treatment of this problem.

Answer- Stones in the gall bladder (Gall stones)- (Figure-1)

A gallstone is a crystalline concretion formed within the gallbladder by accretion of bile components.


















Figure-1- Showing stones in the gall bladder.


The two main types of stones are cholesterol stones and pigmented stones.

Mixed type are also there which typically contain cholesterol, bile pigments, calcium ions and organic materials  (Calcium carbonate, palmitate phosphate), (Figure-4)

a) Cholesterol stones

Eighty percent of gallstones in the Western world are a result of cholesterol precipitation from the bile, a condition known as Cholelithiasis (Figure-2)











Figure-2-showing cholesterol stones.

Bile is a controlled mixture of cholesterol, bile acids, and phospholipids (with small amounts of bile pigments), and if cholesterol levels are elevated or bile acids/salts lowered, the ratio of the three major components changes leaving cholesterol less protected against the aqueous environment and more likely to precipitate. Bile salt (ionized, deprotonated form) and bile acid (neutral, protonated form) are to solubilize cholesterol thus preventing precipitation of cholesterol crystals and facilitating cholesterol excretion.

Causes of cholesterol stone formation

  • Excess HMG-CoA reductase activity, the rate-limiting enzyme in cholesterol biosynthesis; this condition is typically seen in the obese.
  • Alternatively, reduced levels of acyl- CoA: cholesterol acyl Transferase [ACAT],  the enzyme that esterifies cholesterol within cells, or
  •  Reduced levels of cholesterol 7α-hydroxylase can cause elevation of cholesterol. The initial and rate-limiting step of bile acid synthesis is oxidation of cholesterol to 7a-hydroxycholesterol by a mixed function oxidase from the cytochrome P450 super family, cholesterol 7a-hydroxylase (CYP7A1).
  • Deoxycholate, a secondary bile acid synthesized by intestinal bacteria, inhibits CYP7A1. Therefore, high levels of deoxycholate resulting from prolonged exposure of bile acids to intestinal bacteria may result in high levels of cholesterol in bile.
  • Due to impaired entero-hepatic circulation (ileal resection)
  • Infections (cause conversion of lecithin to lysolecithin), phospholipids  are less available to keep cholesterol soluble

When the bile becomes over saturated with cholesterol; the excess forms solid particles (cholesterol crystals). These microscopic crystals accumulate in the gallbladder, where they clump and grow into gallstones.

b) Pigmented stones are usually made of bilirubin and appear dark in color (Figure-3). These microscopic crystals accumulate in the gallbladder, where they clump and grow into gall stones. 











Figure-3-Showing Pigment stones

The stones may stay in the gallbladder or pass into bile ducts. Stones can block the cystic duct, common bile duct, or ampulla of Vater. Any stricture of the bile ducts can lead to a blockage or slow bile flow. Bacterial infections can develop when bile flow is slowed or blocked.











Figure-4- Showing mixed stones

Sometimes microscopic particles of cholesterol, calcium compounds, bilirubin, and other materials accumulate but do not form stones. This material is called biliary sludge. Sludge develops when bile remains in the gallbladder too long, for example, as it does during pregnancy. Gallbladder sludge usually disappears when its cause resolves, for example, when pregnancy ends. Sludge, however, can evolve into gallstones or pass into the biliary tract and block the ducts.

Incidence of Gall stones

Gallstones are more common in women than in men and increase in incidence in both sexes and all races with aging. In the United States, over 10% of men and 20% of women have gallstones by age 65 years; the total exceeds 20 million people. Although cholesterol gallstones are less common in black people, cholelithiasis attributable to hemolysis occurs in over a third of individuals with sickle cell anemia.

Risk factors

1) Genetic mutations that predispose persons to gallstones have been identified.

2) Obesity is a risk factor for gallstones, especially in women.

3) Rapid weight loss also increases the risk of symptomatic gallstone formation.

4) There is evidence that glucose intolerance and elevated serum insulin levels (insulin resistance syndrome) are risk factors for gallstones, and a high-intake of carbohydrate and high dietary glycemic load increase the risk of cholecystectomy in women. A low-carbohydrate diet and physical activity may help prevent gallstones.

5) Consumption of caffeinated coffee appears to protect against gallstones in women, and a high intake of polyunsaturated and monounsaturated fats reduces the risk of gallstones in men on an energy-balanced diet.

6) A high-fiber diet reduces the risk of cholecystectomy in women.

7) Hypertriglyceridemia may promote gallstone formation by impairing gallbladder motility.

8) The incidence of gallstones is high in individuals with Crohn’s disease; approximately one-third of those with inflammatory involvement of the terminal ileum have gallstones due to disruption of bile salt resorption that results in decreased solubility of the bile. T

8)The incidence of cholelithiasis is also increased in patients with diabetes mellitus  

9) The prevalence of gallbladder disease is increased in men (but not women) with cirrhosis and hepatitis C virus infection.

Clinical Manifestations

a) About 80% of people with gallstones do not have any symptoms for many years, if ever, particularly if the gallstones remain in the gallbladder.

b) Pain-Gallstones may cause pain. Pain develops when the stones pass from the gallbladder into the cystic duct, common bile duct, or ampulla of Vater and block the duct. Then the gallbladder dilates, causing pain called biliary colic. The pain is felt in the upper abdomen, usually on the right side. Eating a heavy meal can trigger biliary colic, but simply eating fatty foods does not. Gallstones do not cause belching or bloating. Nausea occurs only when biliary colic occurs.

Although most episodes of biliary colic resolve spontaneously, pain returns in 20 to 40% of people each year, and complications may develop. Between episodes, people feel well.

c) Inflammation-If the blockage persists, the gallbladder becomes inflamed .When the gallbladder is inflamed  infection may develop. The inflammation usually causes fever.

d) Blockage– Blockage of a bile duct can cause the ducts to dilate (Figure-5). It can also cause fever, chills, and jaundice. This combination of symptoms indicates that a serious infection called acute cholangitis has developed.












Figure -5- Showing Obstruction of Common bile duct by Gall stones

e) Septicemia- Bacteria can spread to the bloodstream and cause serious infections elsewhere in the body. Also, abscess can develop in the liver.

f) Pancreatitis-Stones that block the ampulla of Vater also can block the pancreatic duct, causing pancreatitis.

g) Perforation and Peritonitis-Inflammation of the gallbladder caused by gallstones can erode the gallbladder wall, sometimes resulting in perforation. Perforation results in leakage of the gallbladder contents throughout the abdominal cavity, causing severe peritonitis.

h) Gall stone ileus– A large gallstone that enters the small intestine can cause intestinal blockage, called a gallstone ileus. Though rare, this complication is more likely to occur in older people.


Diagnosis is   made by the characteristic pain in the upper abdomen. Sometimes gallstones are detected when an imaging test such as ultrasonography is done for other reasons.

Ultrasonography is essential. It is 95% accurate in detecting gallstones in the gallbladder. It is less accurate in detecting stones in the bile ducts, but it may show that the blockage has caused the ducts to dilate. Other diagnostic tests may be necessary.

They include magnetic resonance imaging (MRI) of the bile and pancreatic ducts, computed tomography (CT), and endoscopic retrograde cholangiopancreatography .

Blood test results are usually normal unless stones block the bile ducts. Then, the liver tests are abnormal, suggesting cholestasis. Results often include an increase in bilirubin and certain liver enzymes.


Gallstones that do not cause symptoms (silent gallstones) do not require treatment. If gallstones do cause pain, changing the diet (for example, to a low-fat diet) does not help.

Gallstones in the Gallbladder

If gallstones cause disruptive, recurring episodes of pain, surgical removal of the gallbladder cholecystectomy is recommended. Laparoscopic cholecystectomy is the treatment of choice for symptomatic gallbladder disease.  Removal of the gallbladder prevents episodes of biliary colic yet does not affect digestion. No special dietary restrictions are required after surgery.

Cheno- and ursodeoxycholic acids are bile salts that when given orally for up to 2 years dissolve some cholesterol stones and may be considered in occasional, selected patients who refuse cholecystectomy.

Gallstones in the Bile Ducts

Stones in the bile ducts are removed during endoscopic retrograde cholangiopancreatography (ERCP).



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Q- Give a brief account of the regulation of de novo synthesis of cholesterol.


Give the reaction catalyzed by HMG Co A reductase and elaborate on the significance of this reaction.

Answer-  Cholesterol can be obtained from the diet or it can be synthesized de novo. An adult on a low-cholesterol diet typically synthesizes about 800 mg of cholesterol per day. The liver is the major site of cholesterol synthesis in mammals, although the intestine also forms significant amounts. Regulation of cholesterol synthesis is exerted near the beginning of the pathway, at the HMG-CoA reductase step. this enzyme catalyzes the formation of mevalonate, the committed step in cholesterol biosynthesis.





HMG CoA reductase is controlled in multiple ways:

A) Fine control (Effect on catalytic activity)-

a) Feedback  inhibition– HMG-CoA reductase in liver is inhibited by Mevalonate, the immediate product of the pathway, and by cholesterol, the main product.

b) Covalent Modification Phosphorylation decreases the activity of the reductase. The enzyme exists in 2 forms- phosphorylated (Inactive)  and dephosphorylated (active) form. The phosphorylation is brought about by reductase kinase which itself is phosphorylated by  c-AMP dependent protein kinase ,through phosphorylation of another intermediary enzyme reductase kinase kinase (Figure-1)


Figure-1-Mechanism of regulation of HMG Co A reductase by covalent modification

The enzyme is converted back to its active form by dephosphorylation  mediated by  the action of protein phosphatase . Dephosphorylation is promoted by Insulin through stimulation of phosphatase , while phosphorylation is promoted by Glucagon through stimulation of c-AMP dependent phosphorylation cascade.

Insulin or thyroid hormone increases HMG-CoA reductase activity, whereas glucagon or glucocorticoids decrease it. Activity is reversibly modified by phosphorylation-dephosphorylation mechanisms, and therefore  cholesterol synthesis ceases when the ATP level is low. The reduced synthesis of cholesterol in starving animals is accompanied by a decrease in the activity of the enzyme.

c) Competitive inhibition– A family of drugs known as statins, have proved highly efficacious in lowering plasma cholesterol and preventing heart disease.The statins act as competitive inhibitors of the enzyme HMG-CoA reductase. Lovastatin (Figure-2) is a member of a class of drugs (Atorvastatin, fluvastatin, pravastatin and Simvastatin are others in this class) called statins that are used to treat hypercholesterolemia. These molecules mimic the structure of the normal substrate of the enzyme (HMG-CoA) and act as transition state analogues. While the statins are bound to the enzyme, HMG-CoA cannot be converted to mevalonic acid, thus inhibiting the whole cholesterol biosynthetic process.


Figure-2- Lovastatin, a Competitive Inhibitor of HMG-CoA Reductase. The part of the structure that resembles the 3-hydroxy-3-methylglutaryl moiety is shown in red.

d) A diurnal variation occurs in both cholesterol synthesis and reductase activity.

B) Coarse control- changes in the concentration of enzyme

a) Feedback regulation- The rate of cholesterol formation is highly responsive to the cellular level of cholesterol. This feedback regulation is mediated primarily by changes in the amount of 3-hydroxy-3-methylglutaryl CoA reductase. As  the intracellular cholesterol  concentration rises(dietary cholesterol brought by LDL) the gene for  HMG Co A reductase is repressed, However, it is only hepatic synthesis that is inhibited by dietary cholesterol. Reverse occurs when the intracellular cholesterol concentration goes down, the gene for HMG Co A reductase is induced. The rate of translation of reductase mRNA is also inhibited by nonsterol metabolites derived from mevalonate as well as by dietary cholesterol.

b) Sterol regulatory element binding protein (SREBP) dependent regulation- The rate of synthesis of reductase mRNA is controlled by the sterol regulatory element binding protein (SREBP). This transcription factor binds to a short DNA sequence called the sterol regulatory element (SRE) on the 5 side of the reductase gene. In its inactive state, the SREBP is anchored to the endoplasmic reticulum or nuclear membrane. When cholesterol levels fall, the amino-terminal domain is released from its association with the membrane by two specific proteolytic cleavages. The released protein migrates to the nucleus and binds the SRE of the HMG-CoA reductase gene, as well as several other genes in the cholesterol biosynthetic pathway, to enhance transcription. When cholesterol levels rise, the proteolytic release of the SREBP is blocked, and the SREBP in the nucleus is rapidly degraded. These two events halt the transcription of the genes of the cholesterol biosynthetic pathways.


Q.- Discuss the steps of formation of bile salts from cholesterol. How is this process regulated? What are the functions of bile salts?

Answer- Cholesterol is a precursor for other important steroid molecules: the bile salts, steroid hormones, and vitamin D. About 1 g of cholesterol is eliminated from the body per day. Approximately half is excreted in the feces after conversion to bile acids. The remainder is excreted as cholesterol.

Bile Salts

As polar derivatives of cholesterol, bile salts are highly effective detergents because they contain both polar and nonpolar regions. Bile salts are synthesized in the liver, stored and concentrated in the gall bladder, and then released into the small intestine. Bile salts, the major constituent of bile, solubilize dietary lipid . Solubilization increases in the effective surface area of lipids with two consequences: more surface area is exposed to the digestive action of lipases and lipids are more readily absorbed by the intestine. Bile salts are also the major breakdown products of cholesterol.

Steps of synthesis-

A) Synthesis of Primary bile acids

The primary bile acids are synthesized in the liver from cholesterol (Figure-3 ). These are  Cholic acid (found in the largest amount) and  Chenodeoxycholic acid.

Step -1- The 7-αhydroxylation of cholesterol is the first and principal regulatory step in the biosynthesis of bile acids and is catalyzed by cholesterol 7α-hydroxylase, a microsomal enzyme. A typical monooxygenase, it requires oxygen, NADPH, and cytochrome P450. (Figure-3)

 Step -2-Subsequent hydroxylation steps are also catalyzed by monooxygenases. The pathway of bile acid biosynthesis divides early into one subpathway leading to cholyl-CoA, characterized by an extra -OH group on position 12, and another pathway leading to chenodeoxycholyl-CoA (Figure-3).

A second pathway in mitochondria involving the 27-hydroxylation of cholesterol by sterol 27-hydroxylase as the first step is responsible for a significant proportion of the primary bile acids synthesized.





























Figure-3- Showing the formation of primary bile acids. Secondary bile acids are formed from primary bile acids by deconjugation and dehydroxylation of primary bile acids.

Step-3-The primary bile acids (Figure 26–7) enter the bile as glycine or taurine conjugates. Conjugation takes place in peroxisomes. In humans, the ratio of the glycine to the taurine conjugates is normally 3:1. In the alkaline bile, the bile acids and their conjugates are assumed to be in a salt form—hence the term “bile salts.”

B) Secondary Bile acids-A portion of the primary bile acids in the intestine is subjected to further changes by the activity of the intestinal bacteria. These include deconjugation and 7-dehydroxylation, which produce the secondary bile acids, deoxycholic acid and lithocholic acid (Figure-3)

Enterohepatic Circulation of Bile salts-The primary and secondary bile acids are absorbed almost exclusively in the ileum, and 98–99% are returned to the liver via the portal circulation. This is known as the enterohepatic circulation (Figure-4). However, lithocholic acid, because of its insolubility, is not reabsorbed to any significant extent. Only a small fraction of the bile salts escapes absorption and is therefore eliminated in the feces. Nonetheless, this represents a major pathway for the elimination of cholesterol. Each day the small pool of bile acids (about 3–5 g) is cycled through the intestine six to ten times and an amount of bile acid equivalent to that lost in the feces is synthesized from cholesterol, so that a pool of bile acids of constant size is maintained. This is accomplished by a system of feedback controls.




















Figure-4- showing the formation of primary and secondary bile acids; Enterohepatic circulation of bile salts.

Regulation of bile acid synthesis-The principal rate-limiting step in the biosynthesis of bile acids is at the cholesterol 7-α-hydroxylase reaction (Figure-3).

1) The activity of the enzyme is feedback-regulated .When the size of the bile acid pool in the enterohepatic circulation increases, transcription of the cholesterol 7-αhydroxylase gene is suppressed. Chenodeoxycholic acid is particularly important in causing repression.

2) 7-αhydroxylase activity is also enhanced by cholesterol of endogenous and dietary origin and regulated by insulin, glucagon, glucocorticoids, and thyroid hormone.




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Q – Discuss the structure and functions of cholesterol.

Answer- Cholesterol is the major sterol in the animal tissues.The structure of cholesterol consists of four fused rings (The rings in steroids are denoted by the letters A, B, C, and D.), with the carbons numbered in the sequence, and an eight numbered, and branched hydrocarbon chain attached to the D ring. Cholesterol contains two angular methyl groups: the C-19 methyl group is attached to C-10, and the C-18 methyl group is attached to C-13. The C-18 and C-19 methyl groups of cholesterol lie above the plane containing the four rings. A double is there between C5 and C6 (Figure-1-a and b)



















Figure-1-a) showing the structure of cholesterol, b)- Showing the numbering in the 4 fused rings.

Much of the plasma cholesterol is in the esterified form (with a fatty acid attached at carbon 3), which makes the structure even more hydrophobic and because of its hydrophobicity, cholesterol must be transported either in association with protein as a component of lipoprotein particle or solubilized by phospholipids and bile salts in the bile.)

Functions of cholesterol- Cholesterol is the most abundant sterol in humans and performs a number of essential functions in the body. For example-

1) It is a major constituent of the plasma membrane and of plasma lipoproteins.

2) It is a precursor of bile salts,

3)  It is a precursor of steroid hormones that include adrenocortical hormones, sex hormones, placental hormones etc

4) Also a precursor of vitamin D, cardiac glycosides, Sitosterol of the plant kingdom, and some alkaloids.

3) It is required for the nerve transmission. Cholesterol is widely distributed in all cells of the body but particularly abundant in nervous tissue.

As a typical product of animal metabolism, cholesterol occurs in foods of animal origin such as egg yolk, meat, liver, and brain. Plasma low-density lipoprotein (LDL) is the vehicle of uptake of cholesterol and cholesteryl ester into many tissues. Free cholesterol is removed from tissues by plasma high-density lipoprotein (HDL) and transported to the liver, where it is eliminated from the body either unchanged or after conversion to bile acids in the process known as reverse cholesterol transport .Cholesterol is a major constituent of gallstones. However, its chief role in pathologic processes is as a factor in the genesis of atherosclerosis of vital arteries, causing cerebrovascular, coronary, and peripheral vascular disease.

Q.-Discuss the steps of de novo synthesis of cholesterol

Answer- Cholesterol is derived from diet, de novo synthesis and from the hydrolysis of cholesteryl esters. A little more than half the cholesterol of the body arises by synthesis (about 700 mg/d), and the remainder is provided by the average diet. The liver and intestine account for approximately 10% each of total synthesis in humans. Virtually all tissues containing nucleated cells are capable of cholesterol synthesis, which occurs in the endoplasmic reticulum and the cytosol.

Acetyl co A acts as a precursor of cholesterol. All the 27 carbon atoms of cholesterol are derived from Acetyl co A.

Steps of synthesis of cholesterol

The biosynthesis of cholesterol may be divided into five steps:

(1) Synthesis of mevalonate from acetyl-CoA

(2) Formation of isoprenoid units from mevalonate by loss of CO2

(3) Condensation of six isoprenoid units to form squalene.

(4) Cyclization of squalene to give rise to the parent steroid, lanosterol.

(5) Formation of cholesterol from lanosterol

Details of reactions

(1) Synthesis of mevalonate from acetyl-CoA – HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) is formed by the reactions used in mitochondria to synthesize ketone bodies (Figure-2). However, since cholesterol synthesis is extramitochondrial, the two pathways are distinct. Initially, two molecules of acetyl-CoA condense to form acetoacetyl-CoA catalyzed by cytosolic thiolase. Acetoacetyl-CoA condenses with a further molecule of acetyl-CoA catalyzed by HMG-CoA synthase to form HMG-CoA, which is reduced to mevalonate by NADPH catalyzed by HMG-CoA reductase.This is the principal regulatory step in the pathway of cholesterol synthesis and is the site of action of the most effective class of cholesterol-lowering drugs, the HMG-CoA reductase inhibitors (statins).

























Figure-2- showing the formation of Mevalonate (Stage-1 of cholesterol biosynthesis) .The synthesis of mevalonate is the committed step in cholesterol formation. The enzyme catalyzing this irreversible step,3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), is an important control site in cholesterol biosynthesis.

Step 2—Formation of Isoprenoid Units: Mevalonate is phosphorylated sequentially by ATP by three kinases, and after decarboxylation (Figure-3) the active isoprenoid unit, isopentenyl diphosphate, is formed.





































Figure-3- Showing the Biosynthesis of squalene, ubiquinone, dolichol, and other polyisoprene derivatives.  A farnesyl residue is present in heme a of cytochrome oxidase. 

Step 3—Six Isoprenoid Units Form Squalene:

Squalene is synthesized from isopentenyl pyrophosphate by the reaction sequence-




Isopentenyl diphosphate is isomerized by a shift of the double bond to form dimethylallyl diphosphate, then condensed with another molecule of isopentenyl diphosphate to form the ten-carbon intermediate geranyl diphosphate (Figure-3 and 4).







Figure-4- Showing the conversion of IPP to Dimethyl Allyl prophosphate


A further condensation with isopentenyl diphosphate forms farnesyl diphosphate. Two molecules of farnesyl diphosphate condense at the diphosphate end to form squalene. Initially, inorganic pyrophosphate is eliminated, forming presqualene diphosphate, which is then reduced by NADPH with elimination of a further inorganic pyrophosphate molecule.

Step 4—Formation of Lanosterol: Squalene can fold into a structure that closely resembles the steroid nucleus (Figure-5). Before ring closure occurs, squalene is converted to squalene 2,3-epoxide by a mixed-function oxidase in the endoplasmic reticulum, squalene epoxidase. The methyl group on C14 is transferred to C13 and that on C8 to C14 as cyclization occurs, catalyzed by oxidosqualene:lanosterol cyclase.



















Figure-5- Showing the formation of Lanosterol from Squalene.

Step 5—Formation of Cholesterol: The formation of cholesterol from lanosterol takes place in the membranes of the endoplasmic reticulum and involves changes in the steroid nucleus and side chain (Figure-6). The methyl groups on C14 and C4 are removed to form 14-desmethyl lanosterol and then zymosterol. The double bond at C8–C9 is subsequently moved to C5–C6 in two steps, forming desmosterol. Finally, the double bond of the side chain is reduced, producing cholesterol. The exact order in which the steps described actually take place is not known with certainty.

Significance of Farnesyl pyrophosphate-

1) The polyisoprenoids  dolichol is formed from farnesyl diphosphate by the further addition of up to 16 isopentenyl diphosphate residues

2) Ubiquinone is formed from farnesyl diphosphate by the addition of or 3–7 isopentenyl diphosphate residues, respectively.

2) Some GTP-binding proteins in the cell membrane are prenylated with farnesyl or geranyl  geranyl (20 carbon) residues. Protein prenylation is believed to facilitate the anchoring of proteins into lipoid membranes and may also be involved in protein-protein interactions .



















Figure-6- Showing the formation of cholesterol from Lanosterol

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Q.-Enlist the substances required for de novo synthesis of fatty acids.

Answer-Fatty acids are synthesized by an extramitochondrial system, which is responsible for the complete synthesis of palmitate from acetyl-CoA in the cytosol. Following substances are required for the synthesis of fatty acids-

1)      Acetyl co A- As the initial substrate

2)      NADPH- As a donor of reducing equivalents

3)      Enzymes- Acetyl co A carboxylase and Fatty acid synthase, both are multienzyme complexes.

4)      Coenzymes and cofactors- Biotin, Mg++, Mn ++ and NADPH

5)      Energy in the form of ATP


Q- Give a  brief account of the structural characteristics of fatty acid synthase complex. Highlight the biological advantage of having a multienzyme complex.

Answer- In mammals, the fatty acid synthase complex is a dimer comprising two identical monomers, each containing all seven enzyme activities of fatty acid synthase on one polypeptide chain (Figure-1 and 2)

Each chain is folded into three domains joined by flexible regions (Figure-1).

1)      Domain 1, the substrate entry and condensation unit, contains acetyl transferase, malonyl transferase, and β-ketoacyl synthase (condensing enzyme).

2)      Domain 2, the reduction unit, contains the acyl carrier protein, β-ketoacyl reductase, dehydratase, and enoyl reductase.

3)      Domain 3, the palmitate release unit, contains the thioesterase.

Thus, seven different catalytic sites are present on a single polypeptide chain.

In bacteria and plants, the individual enzymes of the fatty acid synthase system are separate, and the acyl radicals are found in combination with a protein called the acyl carrier protein (ACP). However, in yeast, mammals, and birds, the synthase system is a multienzyme polypeptide complex that incorporates ACP, which takes over the role of CoA. It contains the vitamin pantothenic acid in the form of 4′-phosphopantetheine (Figure-1).

Figure-1 Schematic Representation of Fatty Acid Synthase.-Each of the identical chains in the dimer contains three domains. Domain 1 (blue) contains acetyl transferase (AT), malonyl transferase (MT), and condensing enzyme (CE). Domain 2 (yellow) contains acyl carrier protein (ACP), beta-ketoacyl reductase (KR), dehydratase (DH), and enoyl reductase (ER). Domain 3 (red) contains thioesterase (TE). The flexible phosphopantetheinyl group (green) carries the fatty acyl chain from one catalytic site on a chain to another, as well as between chains in the dimer.


Domain 1 of each chain of this dimer interacts with domains 2 and 3 of the other chain. Thus, each of the two functional units of the synthase consists of domains formed by different chains. Indeed, the arenas of catalytic action are the interfaces between domains on opposite chains.

Biological Advantage of having Multienzyme complex-

1)    An advantage of this arrangement is that the synthetic activity of different enzymes is coordinated since it is encoded by a single gene.

2)    A multienzyme complex consisting of covalently joined enzymes is more stable than one formed by noncovalent attractions.

3)     Furthermore, intermediates can be efficiently handed from one active site to another without leaving the assembly.


Figure-2- Fatty acid synthase multienzyme complex- The —SH of the 4′-phosphopantetheine of one monomer is in close proximity to the —SH of the cysteine residue of the ketoacyl synthase of the other monomer, suggesting a “head-to-tail” arrangement of the two monomers. Though each monomer contains all the partial activities of the reaction sequence, the actual functional unit consists of one-half of one monomer interacting with the complementary half of the other. Thus, two acyl chains are produced simultaneously.

Q- Discuss the steps of de novo synthesis of fatty acids, highlighting the roles of enzymes, coenzymes and the energy consumed during the process of synthesis.

 Answer- Fatty acid synthesis  takes places in three stages- Initiation, Elongation and Termination,

1) Initiation

Formation of Malonyl co A

Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA. This irreversible reaction is the committed step in fatty acid synthesis.

The synthesis of malonyl CoA is catalyzed by acetyl CoA carboxylase, which contains a biotin prosthetic group. The carboxyl group of biotin is covalently attached to the € amino group of a lysine residue, as in pyruvate carboxylase and propionyl CoA carboxylase.

As with these other enzymes, a carboxy biotin intermediate is formed at the expense of the hydrolysis of a molecule of ATP. The activated CO2 group in this intermediate is then transferred to acetyl CoA to form malonyl CoA.



Acyl carrier Protein-The intermediates in fatty acid synthesis are linked to an acyl carrier protein. Specifically, they are linked to the sulfhydryl  terminus of a phosphopantetheine group, which is, in turn, attached to a serine residue of the acyl carrier protein.

2) Elongation cycle in fatty acid synthesis- Elongation takes place in a cyclic manner, where  four processes  (Condensation, reduction, dehydration  and  reduction) are repeated till a fatty acid with a required chain length is synthesized.

Initially, a priming molecule of acetyl-CoA combines with a cysteine —SH group catalyzed by acetyl transacylase (Figure-3). Malonyl-CoA combines with the adjacent —SH on the 4′-phosphopantetheine of ACP of the other monomer, catalyzed by malonyl transacylase (reaction-1), to form acetyl (acyl)-malonyl enzyme.

Malonyl transacylase is highly specific, whereas acetyl transacylase can transfer acyl groups other than the acetyl unit, though at a much slower rate. Fatty acids with an odd number of carbon atoms are synthesized starting with propionyl ACP, which is formed from propionyl CoA by acetyl transacylase.

a) Condensation- The acetyl group attacks the methylene group of the malonyl residue, catalyzed by 3-ketoacyl synthase, forming 3-ketoacyl enzyme (acetoacetyl enzyme) (reaction 2), freeing the cysteine —SH group.

In the condensation reaction, a four-carbon unit is formed from a two carbon unit and a three-carbon unit, and CO2 is released.


Why is the four-carbon unit not formed from 2 two-carbon units? In other words, why are the reactants acetyl ACP and malonyl ACP rather than two molecules of acetyl ACP?

The answer is that the equilibrium for the synthesis of acetoacetyl ACP from two molecules of acetyl ACP is highly unfavorable. In contrast, the equilibrium is favorable if malonyl ACP is a reactant because its decarboxylation contributes a substantial decrease in free energy. In effect, ATP drives the condensation reaction, though ATP does not directly participate in the condensation reaction. Rather, ATP is used to carboxylate acetyl CoA to malonyl CoA. The free energy thus stored in malonyl CoA is released in the decarboxylation accompanying the formation of acetoacetyl ACP.

Although HCO3 – is required for fatty acid synthesis, its carbon atom does not appear in the product. Rather, all the carbon atoms of fatty acids containing an even number of carbon atoms are derived from acetyl CoA.

The acetoacetyl group is then delivered to three active sites in domain 2 of the opposite chain to reduce it to a butyryl unit.

b) Reduction-The next three steps in fatty acid synthesis reduce the keto group at C-3 to a methylene group . First, acetoacetyl ACP is reduced to d-3-hydroxybutyryl ACP. This reaction differs from the corresponding one in fatty acid degradation in two respects: (1) the d rather than the l isomer is formed; and (2) NADPH is the reducing agent, whereas NAD+ is the oxidizing agent in β oxidation. This difference exemplifies the general principle that NADPH is consumed in biosynthetic reactions, whereas NADH is generated in energy-yielding reactions.

c) Dehydration-d-3-hydroxybutyryl ACP is dehydrated to form trans-Δ 2-enoyl ACP (α-β unsaturated acyl ACP)

 d) Reduction-The final step in the cycle reduces α-β unsaturated acyl ACP to butyryl ACP.

NADPH is again the reductant, whereas FAD is the oxidant in the corresponding reaction in β-oxidation.

These last three reactions a reduction, a dehydration, and a second reduction convert acetoacetyl ACP into butyryl ACP, which completes the first elongation cycle. This saturated C4 unit then migrates from the phosphopantetheinyl sulfur atom on ACP to the cysteine sulfur atom on the condensing enzyme. The synthase is now ready for another round of elongation.

Second round of Elongation

In the second round of fatty acid synthesis, butyryl ACP condenses with malonyl ACP to form a C6-β-ketoacyl ACP. This reaction is like the one in the first round, in which acetyl ACP condenses with malonyl ACP to form a C4-β- ketoacyl ACP. Reduction, dehydration, and a second reduction convert the C6-β-ketoacyl ACP into a C6-acyl ACP, which is ready for a third round of elongation.


Figure-3 – Biosynthesis of long-chain fatty acids. Details of how addition of a malonyl residue causes the acyl chain to grow by two carbon atoms. (Cys, cysteine residue; Pan, 4′-phosphopantetheine.) 

3) Termination of fatty acid synthesis

The elongation cycles continue until C16-acyl ACP is formed.

Five more rounds of condensation and reduction produce a palmitoyl (C16) chain on the condensing enzyme, which is hydrolyzed to palmitate by the thioesterase on domain 3 of the opposite chain. This intermediate is a good substrate for a thioesterase that hydrolyzes C16-acyl ACP to yield palmitate and ACP. The thioesterase acts as a ruler to determine fatty acid chain length. In mammary gland, there is a separate thioesterase specific for acyl residues of C8, C10, or C12, which are subsequently found in milk lipids.The free palmitate must be activated to acyl-Co A before it can proceed via any other metabolic pathway. Its usual fate is esterification into acylglycerols, chain elongation or desaturation, or esterification to cholesteryl ester.

The equation  for the overall synthesis of palmitate from acetyl-Co A and malonyl-Co A is

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Q.- Discuss the important differences between fatty acid biosynthesis and fatty acid oxidation.

Answer-Fatty acid synthesis is not simply a reversal of the degradative pathway. Rather, it consists of a new set of reactions, again exemplifying the principle that synthetic and degradative pathways are almost always distinct. Some important differences between the pathways are:

1. Synthesis takes place in the cytosol, in contrast with degradation, which takes place primarily in the mitochondrial matrix.

2. Intermediates in fatty acid synthesis are covalently linked to the sulfhydryl groups of an acyl carrier protein (ACP), whereas intermediates in fatty acid breakdown are covalently attached to the sulfhydryl group of coenzyme A.

3. The enzymes of fatty acid synthesis in higher organisms are joined in a single polypeptide chain called fatty acid synthase. In contrast, the degradative enzymes do not seem to be associated.

4. The growing fatty acid chain is elongated by the sequential  addition of two-carbon units derived from acetyl CoA. The activated donor of two carbon units in the elongation step is malonyl ACP. The elongation reaction is driven by the release of CO2.

5. The reductant in fatty acid synthesis is NADPH, whereas the oxidants in fatty acid degradation are NAD + and FAD.

6. Elongation by the fatty acid synthase complex stops on formation of palmitate (C16). Further elongation and the insertion of double bonds are carried out by other enzyme systems.

Q. Acetyl co A, the precursor of fatty acids is produced in the mitochondrial matrix, but the enzymes of de novo fatty acid synthesis are extra mitochondrial, How is Acetyl co A transported out of the selectively permeable mitochondrial membrane.

Answer- Fatty acids are synthesized in the cytosol, whereas acetyl CoA is formed from pyruvate in mitochondria. Hence, acetyl CoA must be transferred from mitochondria to the cytosol. Mitochondria, however, are not readily permeable to acetyl CoA. Carnitine carries only long-chain fatty acids. The barrier to acetyl CoA is bypassed by citrate, which carries acetyl groups across the inner mitochondrial membrane. Citrate is formed in the mitochondrial matrix by the condensation of acetyl CoA with oxaloacetate (Figure-1 ).

















Figure-1- showing the transport of Acetyl co A in the form of citrate from mitochondrial matrix to cytosol

When present at high levels, citrate is transported to the cytosol, where it is cleaved by ATP-citrate Lyase, which increases in activity in the well-fed state. The acetyl-CoA is then available for malonyl-CoA formation and synthesis to palmitate (Figure-1).

Oxaloacetate formed in the transfer of acetyl groups to the cytosol must now be returned to the mitochondria.The inner mitochondrial membrane is impermeable to oxaloacetate. Hence, a series of bypass reactions are needed. Most importantly, these reactions generate much of the NADPH needed for fatty acid synthesis.

 First, oxaloacetate is reduced to malate by NADH. This reaction is catalyzed by a malate dehydrogenase in the cytosol.





Second, malate is oxidatively decarboxylated by an NADP + -linked malate enzyme (also called malic enzyme).




The pyruvate formed in this reaction readily enters mitochondria, where it is carboxylated to oxaloacetate by pyruvate carboxylase.





The sum of these three reactions is-




Thus, one molecule of NADPH is generated for each molecule of acetyl CoA that is transferred from mitochondria to the cytosol. Hence, eight molecules of NADPH are formed when eight molecules of acetyl CoA are transferred to the cytosol for the synthesis of palmitate. The additional six molecules of NADPH required for this process come from the pentose phosphate pathway.

Alternatively, malate itself can be transported into the mitochondrion, where it is able to re-form oxaloacetate. Note that the citrate (tricarboxylate) transporter in the mitochondrial membrane requires malate to exchange with citrate (Figure -1).

 Q- Discuss the central role of Acetyl co A in the metabolic pathways.

Answer- Acetyl CoA acts either as a metabolic substrate or product for various classes of biomolecules and as a major source of useful metabolic energy.

Sources of Acetyl co A- Acetyl co A, is produced from pyruvate, ketogenic amino acids, fatty acid oxidation, ketolysis and by alcohol metabolism (Figure-2).

Fate of Acetyl co A

 It is a substrate for TCA cycle and a precursor for fatty acids, cholesterol, ketone bodies and steroids. It is also required for detoxification reactions and for the synthesis of acetyl choline. Acetyl CoA can react “reversibly” in the degradation or synthesis of lipids and amino acids. This is not the case with carbohydrate metabolism. In mammals, it is impossible to use acetyl CoA to make carbohydrates. Acetyl co A forms the basis for the synthesis of steroids. Some steroids of importance include cholesterol, bile salts, sex hormones, aldosterone, and cortisol.










Figure- 2-Showing the central role of Acetyl co A


Q- Give a brief account of the sources of NADPH

Answer- Sources of NADPH- NADPH is involved as donor of reducing equivalents.

The oxidative reactions of the pentose phosphate pathway (Figure-3) are the chief source of the hydrogen required for the reductive synthesis of fatty acids. Significantly, tissues specializing in active lipogenesis—ie, liver, adipose tissue, and the lactating mammary gland—also possess an active pentose phosphate pathway. Moreover, both metabolic pathways are found in the cytosol of the cell, so there are no membranes or permeability barriers against the transfer of NADPH.








Figure-3- Showing the formation of NADPH in the Pentose phosphate pathway. Per Glucose-6-P two molecules of NADPH are produced.

 Other sources of NADPH include-

1) From Malate-The reaction that converts malate to pyruvate catalyzed by the “malic enzyme” (NADP malate dehydrogenase) is also an alternative source of NADPH (Figure-4)









Figure-4 – Showing the formation of NADPH from Malate by the activity of Malic enzyme

 2) From Isocitrate- The extra mitochondrial isocitrate dehydrogenase reaction though not a common source but does contribute to the formation of NADPH (Figure-5). There are three isoenzymes of isocitrate dehydrogenase. One, which uses NAD+, is found only in mitochondria. The other two use NADP+ and are found in mitochondria and the cytosol. Respiratory chain-linked oxidation of isocitrate proceeds almost completely through the NAD+-dependent enzyme.









Figure- 5- Showing the formation of NADPH from the activity of cytosolic isocitrate dehydrogenase


Q- Explain briefly about the reaction catalyzed by Acetyl co A carboxylase, What is the significance of this reaction?

Answer-The synthesis of malonyl CoA is catalyzed by acetyl CoA carboxylase. Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA. This irreversible reaction is the committed step in fatty acid synthesis.







Figure- 6- Showing the formation of Malonyl co A from Acetyl co A

Acetyl-CoA carboxylase has a requirement for the vitamin biotin (Figure-7)

Figure-7- Showing the attachment of Biotin to the enzyme and the formation of carboxy biotin. Biotin is the first acceptor of the carboxyl group

The enzyme is a multienzyme protein containing a variable number of identical subunits, each containing-

1) Biotin

2) Biotin carboxylase,

3) Biotin carboxyl carrier protein

4) Transcarboxylase,

5) A regulatory allosteric site.

Bicarbonate as a source of CO2 is required in the initial reaction for the carboxylation of acetyl-CoA to malonyl-CoA in the presence of ATP and acetyl-CoA carboxylase.

The reaction takes place in two steps: (1) carboxylation of biotin involving ATP and (2) transfer of the carboxyl to acetyl-CoA to form malonyl-CoA (Figure-8)








Figure-8 – Showing the role of biotin in the carboxylation of Acetyl co A

The first reaction which includes the carboxylation of biotin to form carboxy biotin is catalyzed with the biotin subunit of acetyl-CoA carboxylase. This portion of the mechanism is ATP dependent; also the bicarbonate provides the CO2. The second step of this mechanism requires that the carboxyl group be transferred from the biotin to the acetyl-CoA to form malonyl-CoA.  This reaction thermodynamically should not be spontaneous, but because it is coupled with the hydrolysis of ATP to ADP this reaction goes. This hydrolysis of ATP also is one main reason this reaction is the committed step of this metabolic cycle. This mechanism is very much like the pyruvate kinase that is a major committed step in glycolysis that converts phosphoenolpyruvate to pyruvate.

Biological significance

Acetyl co A carboxylase is the rate controlling enzyme in the pathway of lipogenesis. It is regulated by-

1)      Allosteric modification- Acetyl-CoA carboxylase is an allosteric enzyme and is activated by citrate, which increases in concentration in the well-fed state and is an indicator of a plentiful supply of acetyl-CoA. Citrate converts the enzyme from an inactive dimer to an active polymeric form, with a molecular mass of several million. Inactivation is promoted by long-chain acyl-CoA molecules.

2)      Feedback Inhibition- The enzyme is inhibited by malonyl co A and Palmitoyl co A, an example of negative feedback inhibition by a product of a reaction.Thus, if acyl-CoA accumulates because it is not esterified quickly enough or because of increased lipolysis or an influx of free fatty acids into the tissue, it will automatically reduce the synthesis of new fatty acid. Acyl-CoA also inhibits the mitochondrial tricarboxylate transporter, thus preventing activation of the enzyme by egress of citrate from the mitochondria into the cytosol.

3)      Covalent Modification-Acetyl-CoA carboxylase is also regulated by hormones such as glucagon, epinephrine, and insulin via changes in its phosphorylation state (Figure-8)










Figure-8- Regulation of acetyl-CoA carboxylase by phosphorylation/dephosphorylation, the enzyme is inactivated by phosphorylation by AMP-activated protein kinase (AMPK) . Glucagon (and epinephrine) increase cAMP, and thus activate this latter enzyme via cAMP-dependent protein kinase. Insulin activates acetyl-CoA carboxylase by causing dephosphorylation mediated by protein phosphatase.

4)      Induction and Repression- Insulin is an important hormone causing gene expression and induction of enzyme biosynthesis, and glucagon (via cAMP) antagonizes this effect. Feeding fats containing polyunsaturated fatty acids coordinately regulates the inhibition of expression of key enzymes of glycolysis and lipogenesis. These mechanisms for longer-term regulation of lipogenesis take several days to become fully manifested and augment the direct and immediate effect of free fatty acids and hormones such as insulin and glucagon.

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Q. Give the details of the energy yield from the complete oxidation of ketone bodies.

Answer-The energy provided to the peripheral tissues from acetoacetate and for beta-hydroxybutyrate are shown below:

Reaction Energy product Multiplier ATP equivalents
Co A transferase – GTP (No GTP formation  from Succinyl coA to Succinate in TCA cycle 1 -1
Acetyl co A OxidationIn TCA cycle 12 ATP 2 24
Acetoacetate oxidationTotal 23
β-OH ButyrateDehydrogenase 1NADH-3ATP 1 3
Total β-OH Butyrate oxidation  26


This may be appreciated when it is realized that complete oxidation of 1 mol of palmitate involves a net production of 129 mol of ATP via beta oxidation and CO2 production in the citric acid cycle, whereas only 23 mol of ATP are produced when acetoacetate is the end product and only 26 mol when 3-hydroxybutyrate is the end product. Thus, ketogenesis may be regarded as a mechanism that allows the liver to oxidize increasing quantities of fatty acids within the constraints of a tightly coupled system of oxidative phosphorylation.

Q.- Discuss in brief about  the regulation of ketosis.


What is the effect of insulin, glucagon, or epinephrine upon lipolysis in adipose tissue? How can a decrease in the insulin/glucagon ratio explain the increased production of ketone bodies during a fast?

Answer- Ketogenesis is regulated at three steps-

(1) Lipolysis in Adipose tissues- Ketosis does not occur unless there is an increase in the level of circulating free fatty acids that arise from lipolysis of triacylglycerol in adipose tissue. Fatty acid release from adipose tissue is controlled via the activity of hormone-sensitive lipase (HSL). When glucose levels fall, pancreatic glucagon secretion increases resulting in phosphorylation of adipose tissue HS (Figure-1), thus resulting in increased hepatic ketogenesis due to increased substrate (free fatty acids) delivery from adipose tissue. Conversely, insulin, released in the well-fed state, inhibits ketogenesis via the triggering dephosphorylation and inactivation of adipose tissue HSL.


















Figure-1- Showing the degradation of Triglycerides in adipose cell by hormone sensitive lipase. Hormone sensitive lipase exists in two forms inactive- dephosphorylated (brought by Insulin) and active phosphorylated form (brought by glucagon, ACTH and catecholamines). Insulin promotes lipogenesis while the other hormones  promote lipolysis.

Free fatty acids are the precursors of ketone bodies in the liver. The liver, both in fed and in fasting conditions, extracts about 30% of the free fatty acids passing through it, so that at high concentrations (After high fat diet or in conditions of excessive lipolysis) the flux passing into the liver is substantial. Therefore, the factors regulating mobilization of free fatty acids from adipose tissue are important in controlling ketogenesis.

2) Fate of  Fatty acid- After uptake by the liver, free fatty acids are either oxidized to CO2 or ketone bodies or esterified to triacylglycerol and phospholipid.  If the liver has sufficient supplies of glycerol-3-phosphate, most of the fats will be turned to the production of triacylglycerols.

There is regulation of entry of fatty acids into the oxidative pathway by carnitine Acyl transferase-I (CAT-I), and the remainder of the fatty acid taken up is esterified. CAT-I activity is low in the fed state, leading to depression of fatty acid oxidation. Malonyl-CoA, the initial intermediate in fatty acid biosynthesis (Figure-2), formed by acetyl-CoA carboxylase in the fed state, is a potent inhibitor of CAT-I . Under these conditions, free fatty acids enter the liver cell in low concentrations and are nearly all esterified to acylglycerols and transported out of the liver in very low density lipoproteins (VLDL).

















Figure-2- Showing the inhibition of CAT-1 by Malonyl Co A

However, CAT-1 activity is higher in starvation, allowing fatty acid oxidation to increase. Since the concentration of free fatty acids increases with the onset of starvation, acetyl-CoA carboxylase is inhibited directly by acyl-CoA, and malonyl-CoA level decreases, releasing the inhibition of CAT-I and allowing more acyl-CoA to be oxidized. These events are reinforced in starvation by decrease in the [insulin]/[glucagon] ratio.  
















Figure-3-Showing the regulation of Acetyl co A carboxylase by covalent modification. During starvation glucagon causes inhibition of Acetyl co A carboxylase by c AMP dependent phosphorylation. Reverse occurs in the presence of Insulin.


Glucagon In addition, results in phosphorylation and inhibition of acetyl-CoA carboxylase (ACC), the rate limiting enzyme of de novo fatty acid synthesis (Since the enzymes gets inactivated upon phosphorylation). Conversely, under conditions of insulin release, in fed state, hepatic ACC is activated, by dephosphorylation and the excess acetyl-CoA  is converted into malonyl-CoA and then to fatty acids, CAT-1 is inhibited and fatty acid oxidation is also inhibited. Thus, -oxidation from free fatty acids is controlled by the CAT-I gateway into the mitochondria.

(3) Fate of Acetyl co A- In turn, the acetyl-CoA formed in  beta-oxidation is oxidized in the citric acid cycle, or it enters the pathway of ketogenesis to form ketone bodies. If the hepatic demand for ATP is high the fate of acetyl-CoA is likely to be further oxidation to CO2. This is especially true under conditions of hepatic stimulation by glucagon which results in increased gluconeogenesis and the energy for this process is derived primarily from the oxidation of fatty acids supplied from adipose tissue.

As the level of serum free fatty acids is raised, proportionately more free fatty acids are  converted to ketone bodies and less are oxidized via the citric acid cycle to CO2. The partition of acetyl-CoA between the ketogenic pathway and the pathway of oxidation to CO2 is so regulated that the total free energy captured in ATP which results from the oxidation of free fatty acids remains constant as their concentration in the serum changes.

Q.- Enlist the conditions causing ketosis,  Discuss the underlying defect in each of them responsible for causing ketosis.

Answer- The production of ketone bodies occurs at a relatively low rate during normal feeding and under conditions of normal physiological status. Ketosis is basically observed in conditions of glucose deprivation and excess lipolysis.

A) Conditions causing glucose deprivation- Normal physiological responses to carbohydrate shortages cause the liver to increase the production of ketone bodies from the acetyl-CoA generated from fatty acid oxidation. This allows the heart and skeletal muscles primarily to use ketone bodies for energy, thereby preserving the limited glucose for use by the brain. The common causes of glucose deprivation are as follows-

a) Starvation

b) Chronic alcoholism

c) Von- Gierke’s disease

d) Heavy exercise

e) Low carbohydrate diet- For weight loss

f) Glycogen storage disease type 6(Due to phosphorylase kinase deficiency)

g) Pyruvate carboxylase deficiency

B) Conditions causing excessive Lipolysis- All conditions causing hypoglycemia cause lipolysis to compensate for the energy needs, but in uncontrolled diabetes mellitus (Type 1 especially) glucose is available yet cannot be utilized due to insulin deficiency. There is an imbalance between Insulin to Glucagon ratio. Excess glucagon in such conditions induces a state of catabolism , causing lipolysis and thus enhanced ketogenesis. Similarly extreme stress and glucagon producing tumors can cause ketosis.

C) Prolonged ether anesthesia, toxaemia of pregnancy and certain conditions of alkalosis associated with excessive vomiting can also cause ketosis.

D) Nonpathologic forms of ketosis are found under conditions of high-fat feeding and after severe exercise in the post absorptive state.


Q. What is the biochemical basis of ketosis in prolonged fasting or starvation

Answer-Prolonged fasting may result from an inability to obtain food, from the desire to lose weight rapidly, or in clinical situations in which an individual cannot eat because of trauma, surgery, neoplasms, burns etc. In the absence of food the plasma levels of glucose, amino acids and triacylglycerols fall, triggering a decline in insulin secretion and an increase in glucagon release. The decreased insulin to glucagon ratio, and the decreased availability of circulating substrates, make this period of nutritional deprivation a catabolic state, characterized by degradation of glycogen, triacylglycerol and protein. This sets in to motion an exchange of substrates between liver, adipose tissue, muscle and brain that is guided by two priorities (i) the need to maintain glucose level to sustain the energy metabolism of brain ,red blood cells and other glucose requiring cells and (ii) to supply energy to other tissues by mobilizing fatty acids from adipose tissues and converting them to ketone bodies to supply energy to other cells of the body (Figure-4)


















Figure-4 Showing the distribution of fuels in different tissues during starvation

After about 3 days of starvation, the liver forms large amounts of acetoacetate and d3- hydroxybutyrate. Their synthesis from acetyl CoA increases markedly because the citric acid cycle is unable to oxidize all the acetyl units generated by the degradation of fatty acids. Gluconeogenesis depletes the supply of oxaloacetate, which is essential for the entry of acetyl CoA into the citric acid cycle. Consequently, the liver produces large quantities of ketone bodies, which are released into the blood. At this time, the brain begins to consume appreciable amounts of acetoacetate in place of glucose. After 3 days of starvation, about a third of the energy needs of the brain are met by ketone bodies. The heart also uses ketone bodies as fuel. After several weeks of starvation, ketone bodies become the major fuel of the brain. Acetoacetate is activated by the transfer of CoA from succinyl CoA to give acetoacetyl CoA .Cleavage by Thiolase then yields two molecules of acetyl CoA, which enter the citric acid cycle.

In essence, ketone bodies are equivalents of fatty acids that can pass through the blood-brain barrier. Only 40 g of glucose is then needed per day for the brain, compared with about 120 g in the first day of starvation. The effective conversion of fatty acids into ketone bodies by the liver and their use by the brain markedly diminishes the need for glucose. Hence, less muscle is degraded than in the first days of starvation. The breakdown of 20 g of muscle daily compared with 75 g early in starvation is most important for survival.

A person’s survival time is mainly determined by the size of the triacylglycerol depot.

Q. Discuss in detail about the causes, clinical manifestations, laboratory diagnosis and treatment of diabetic ketoacidosis.

Answer- Explained in Complications of diabetes Mellitus.


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Q- What are ketone bodies ? Discuss their biological significance ?

Answer- Ketone bodies can be regarded as  water-soluble, transportable form of acetyl units. Fatty acids are released by adipose tissue and converted into acetyl units by the liver, which then exports them as ketone bodies.

Acetoacetate, D(-3) -hydroxybutyrate (Beta hydroxy butyrate), and acetone are often referred to as ketone bodies (Figure-1).






Figure-1- showing the structure of ketone bodies.

The term “ketones”  is a misnomer because 3-hydroxybutyrate is not a ketone and there are ketones in blood that are not ketone bodies, eg, pyruvate, fructose.

Biological Significance

Ketone bodies serve as a fuel for extra hepatic tissues

The  brain  is an  important  organ.  It  is  metabolically  active  and  metabolically privileged. The brain generally uses 60-70% of total body glucose requirements, and always  requires  some  glucose for  normal  functioning.  Under  most  conditions, glucose is essentially the sole energy source of the brain. The brain cannot use fatty acids, which  cannot cross  the  blood-brain  barrier. Because animals  cannot synthesize significant amounts of glucose from fatty acids, as glucose availability decreases, the brain is forced to use either amino acids or ketone bodies for fuel.

Individuals eating diets extremely high in fat and low in carbohydrates,or starving, or suffering  from  a severe lack of insulin  (Type I diabetes  mellitus) therefore increase the synthesis and utilization of ketone bodies

During high rates of fatty acid oxidation, primarily in the liver, large amounts of acetyl-Co A are generated. These exceed the capacity of the TCA cycle, and one result is the synthesis of ketone bodies. The synthesis of the ketone bodies (ketogenesis) occurs in the liver mitochondria allowing this process to be intimately coupled to rate of hepatic fatty acid oxidation. Conversely, the utilization of the ketones (ketolysis) occurs in the peripheral cells, in the cytosol.

The acetyl CoA formed in fatty acid oxidation enters the citric acid cycle only if fat and carbohydrate degradation are appropriately balanced. The reason is that the entry of acetyl CoA into the citric acid cycle depends on the availability of oxaloacetate for the formation of citrate, but the concentration of Oxaloacetate is lowered if carbohydrate is unavailable or improperly utilized. Oxaloacetate is normally formed from pyruvate, the product of glycolysis, by pyruvate carboxylase (Figure-2).This is the molecular basis of the adage that fats burn in the flame of carbohydrates.

















Figure-2-showing the pathway of ketogenesis in conditions of non availability of Oxaloacetate

In fasting or diabetes, oxaloacetate is consumed to form glucose by the gluconeogenic pathway (figure-2) and hence is unavailable for condensation with acetyl CoA. Under these conditions, acetyl CoA is diverted to the formation of acetoacetate and β-hydroxybutyrate.

These substances diffuse from the liver mitochondria into the blood and are transported to peripheral tissues. These ketone bodies were initially regarded as  degradation products of little physiological value. However, the results of studies revealed that these derivatives of acetyl CoA are important molecules in energy metabolism. Acetoacetate and β-hydroxybutyrateare normal fuels of respiration and are quantitatively important as sources of energy. Indeed, heart muscle and the renal cortex use acetoacetate in preference to glucose. In contrast, the brain adapts to the utilization of acetoacetate during starvation and diabetes. In prolonged starvation,75% of the fuel needs of the brain are met by ketone bodies.

Q.- Describe the pathway for the synthesis of ketone bodies by naming substrates, the first ketone body made in the pathway, the next two ketone bodies made in the pathway, the intermediates in the pathway that can be used either for ketone body synthesis or cholesterol synthesis, and the enzyme that actually produces the first ketone body as a product.

Answer-Ketogenesis takes place in liver using Acetyl co A as a substrate or a precursor molecule. Enzymes responsible for ketone body formation are associated mainly with the mitochondria.

Steps of synthesis-Acetoacetate (First ketone body) is formed from acetyl CoA in three steps (Figure-3 ).

1)Two molecules of acetyl CoA condense to form acetoacetyl CoA. This reaction, which is catalyzed by thiolase, is the reverse of the thiolysis step in the oxidation of fatty acids.

2) Acetoacetyl CoA then reacts with acetyl CoA and water to give 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) and CoA. The reaction is catalyzed by HMG co A synthase. This enzyme is exclusively present in liver mitochondria. There are two isoforms of this enzyme-cytosolic and mitochondrial. The mitochondrial enzyme is needed for ketogenesis while the cytosolic form is associated with cholesterol biosynthesis.

This condensation resembles the one catalyzed by citrate synthase. This reaction, which has a  favorable equilibrium owing to the hydrolysis of a thioester linkage, compensates for the unfavorable equilibrium in the formation of acetoacetyl CoA.

3) 3-Hydroxy-3-methylglutaryl CoA is then cleaved to acetyl CoA and acetoacetate in the presence of HMG Co A  lyase (Figure-3)

The carbon atoms split off in the acetyl-CoA molecule are derived from the original acetoacetyl-CoA molecule. Both enzymes must be present in mitochondria for ketogenesis to take place. This occurs solely in liver and rumen epithelium,

The sum of these reactions is




The other two ketone bodies-Acetone and D(-)- 3-Hydroxybutyrate are formed from Acetoacetate, the primary ketone body.

4) Acetone is formed by decarboxylation in the presence of decarboxylase enzyme and, because it is a beta-keto acid, acetoacetate also undergoes a slow, spontaneous decarboxylation to acetone. The odor of acetone may be detected in the breath of a person who has a high level of acetoacetate in the blood.  “Acetone-breath” has been used as  a  crude  method  of  diagnosing  individuals  with  untreated Type I diabetes mellitus.

 5) D (-)-3-Hydroxybutyrate is formed by the reduction of acetoacetate in the mitochondrial matrix by D(-)3-hydroxybutyrate dehydrogenase. D(-)-3-Hydroxybutyrate is quantitatively the predominant ketone body present in the blood and urine in ketosis.

The β-hydroxybutyrate dehydrogenase reaction has two functions: 1) it stores energy equivalent to an NADH in the ketone body for export to the tissues, and 

2) it  produces  a  more  stable molecule.

Acetoacetate and β-hydroxybutyrate, in particular, also serve as major substrates for the biosynthesis of neonatal cerebral lipids.

The ratio of β hydroxybutyrate to acetoacetate depends on the NADH/NAD+ ratio inside mitochondria. if NADH concentration is high, the liver releases a higher proportion of β-hydroxybutyrate.

In vivo, the liver appears to be the only organ in nonruminants to add significant quantities of ketone bodies to the blood. Extrahepatic tissues utilize them as respiratory substrates. The net flow of ketone bodies from the liver to the extrahepatic tissues results from active hepatic synthesis coupled with very low utilization. The reverse situation occurs in extra hepatic tissues.

While an active enzymatic mechanism produces acetoacetate from acetoacetyl-CoA in the liver, acetoacetate once formed cannot be reactivated directly except in the cytosol, where it is used in a much less active pathway as a precursor in cholesterol synthesis. This accounts for the net production of ketone bodies by the liver.

Why are three enzymes required to synthesize acetoacetate?

An enzyme that cleaves the thioester  bond of the thiolase  product  acetoacetyl-CoA  would  also produce acetoacetate, but such a thioesterase does not seem to exist. The reason for the multienzyme pathway is not really understood. However, the pathway that does exist is not especially wasteful; the third acetyl-CoA used merely acts catalytically.

Because the cell needs to have HMG-CoA synthase for other purposes, the choice is in having HMG-CoA lyase. It is possible that having two mitochondrial enzymes (HMG-CoA synthase and HMG-CoA lyase) reuired for ketone body synthesis assists in controlling the pathway.



































Figure-3- Showing the steps of ketogenesis

Q.- Name the tissues that oxidize ketone bodies. Why not the liver? What happens to blood ketone bodies? Name the intermediates in the pathway from β-Hydroxybutyrate to acetyl CoA.

Answer- The ketone bodies are water soluble and are transported across the inner mitochondrial membrane as well as across the blood-brain barrier and cell membranes. Thus they can be used as a fuel source by a variety of tissues including the CNS. They are preferred substrates for aerobic muscle and heart, thus sparing glucose when they are available.

Tissues that can use fatty acids can generally use ketone bodies in addition to other energy sources. The exceptions are the liver and the brain. The liver synthesizes ketone bodies, but has little β-ketoacyl-CoA transferase, and therefore little ability to convert acetoacetate into acetyl-CoA. The brain does not normally use fatty acids, which do not cross the blood-brain barrier; under ordinary circumstances, the brain uses glucose as its sole energy source.

The metabolic rate of the brain is essentially constant. While other tissues reduce their metabolic requirements during starvation, the brain is unable to do so. After a few  days  of  fasting,  the  brain  undergoes  metabolic  changes  to  adapt  to  the decreased availability of glucose. One major change is increased amounts  of the enzymes necessary to metabolize ketone bodies.

Ketone bodies are utilized by extrahepatic tissues via a series of cytosolic reactions (Figure-4) that are essentially a reversal of ketone body synthesis, the ketones must be reconverted to acetyl CoA in the mitochondria:













Figure-4- Showing the steps of utilization of ketone bodies.

Steps- (Figure-4)

1) Beta-hydroxybutyrate, is first oxidized to acetoacetate with the production of one NADH (1). It is important to appreciate that under conditions where tissues are utilizing ketones for energy production their NAD+/NADH ratios are going to be relatively high, thus driving the β-hydroxybutyrate dehydrogeanse catalyzed reaction in the direction of acetoacetate synthesis. 

2) Coenzyme A must be added to the acetoacetate. The thioester bond is a high energy bond, so ATP equivalents must be used. In this case the energy comes from a trans esterification of the CoAS from succinyl CoA to acetoacetate by Coenzyme A transferase (2), also called Succinyl co A : Acetoacetate co A transferase, also known as Thiophorase.

The succinyl CoA comes from the TCA cycle. This reaction bypasses the succinyl-CoA synthetase step of the TCA cycle, hence there is no GTP formation at this steps although it does not alter the amount of carbon in the cycle.

The liver has acetoacetate available to supply to other organs because it lacks this particular CoA transferase and that is the reason that “Ketone bodies are synthesized in the liver but utilized in the peripheral tissues”. The latter enzyme is present at high levels in most tissues except the liver. Importantly, very low level of enzyme expression in the liver allows the liver to produce ketone bodies but not to utilize them. This ensures that extrahepatic tissues have access to ketone bodies as a fuel source during prolonged fasting and starvation, and also,lack of this enzyme in the liver prevents the futile cycle of synthesis and breakdown of acetoacetate.

3) The acetoacetyl CoA is now cleaved to two acetyl CoA’s with Thiolase (3).

This implies that the TCA cycle must be running to allow ketone body utilization; a fact  which is necessarily true,  because  the TCA cycle is necessary to allow generation of energy from acetyl-CoA.

D(-)-3-Hydroxybutyrate is oxidized to produce acetoacetate as well as NADH for use in oxidative phosphorylation.

If the blood level is raised, oxidation of ketone bodies increases until, at a concentration of approximately 12 mmol/L, they saturate the oxidative machinery. When this occurs, a large proportion of the oxygen consumption may be accounted for by the oxidation of ketone bodies.

In most cases, ketonemia is due to increased production of ketone bodies by the liver rather than to a deficiency in their utilization by extrahepatic tissues. While acetoacetate and D(-)-3-hydroxybutyrate are readily oxidized by extrahepatic tissues, acetone is difficult to oxidize in vivo and to a large extent is volatilized in the lungs.

Both β-hydroxybutyrate and acetoacetate are organic acids. These compounds are released in the protonated form, which means that their release tends to lower the pH of the blood. In  normal  individuals, other mechanisms compensate  for  the increased proton release. Individuals with untreated Type I diabetes mellitus often release  ketone  bodies  in  such  large  quantities  that the normal  pH-buffering mechanisms are overloaded; the reduced pH, in combination with a number of other metabolic  abnormalities  associated  with lack  of  insulin results  in  diabetic ketoacidosis, a life-threatening acute disorder of Type I diabetes. In most cases, the increase in ketone body concentration in blood is due to increased synthesis in liver; in severe ketoacidosis, cells begin to lose ability to use ketone bodies also.


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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.


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


Skeletal radiography is required to estimate bone changes.


  • 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 the reduction 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.


 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.


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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Q.- What  are  the causes  of hypoglycemia and hypo ketosis in carnitine deficiency ?



The sources of glucose in normal health are-

1) Diet

2) Glycogen degradation

3) Gluconeogenesis

Dietary supply is sporadic and glycogen stores are sufficient only for 12-16 hours. The main supply of glucose is through gluconeogenesis in conditions of increased demand .

Carnitine deficiency causes Impaired gluconeogenesis  due to impaired beta oxidation of fatty acids since there is-

a) Insufficient  energy – Low ATP

b) Impaired activity of Pyruvate carboxylase- Acetyl co A is a positive allosteric modifier of Pyruvate carboxylase. In conditions of impaired fatty acid oxidation, Acetyl co A pool is small enough to sufficiently  activate pyruvate carboxylase. as a result there is less formation of Oxaloacetate and decrease rate of gluconeogenesis.

Thus hypoglycemia in carnitine deficiency is basically due to an imbalance between demand and supply of glucose. The demand is more, since the energy needs are fulfilled by glucose oxidation only.

Hypo ketosis- Impaired beta oxidation in carnitine deficiency results in less of Acetyl co A to be used for ketogenesis.



















Figure-1- showing the role of Acetyl co A as a positive allosteric modifier for Pyruvate carboxylase enzyme.


Q.2- Discuss in brief about the disorders associated with impaired  beta oxidation of fatty acids.

Answer- 1) Carnitine deficiency and deficiencies of CAT-1 and CAT-2 enzymes (Separately explained)

2) Jamaican Sickness- Jamaican vomiting sickness is caused by eating the unripe fruit of the akee tree, which contains the toxin hypoglycin, which inactivates medium- and short-chain acyl-CoA dehydrogenase, inhibiting β oxidation and causing hypoglycemia.

3) Dicarboxylic aciduria is characterized by the excretion of C6–C10 -dicarboxylic acids and by nonketotic hypoglycemia, and is caused by a lack of mitochondrial medium-chain acyl-CoA dehydrogenase.

4) Acute fatty liver of pregnancy- Acute fatty liver of pregnancy usually manifests in the second half of pregnancy, usually close to term, but may also develop in the postpartum period. The patient developed symptoms of hepatic dysfunction at 36 weeks of gestation. Short history of illness, hypoglycemia, liver failure, renal failure, and coagulopathy , all are observed  in Acute fatty liver of pregnancy, Typically, diagnosis is made based on an incidental finding of abnormal liver enzyme levels. Affected patients may become jaundiced or develop  encephalopathy  from liver failure, usually reflected by an elevated ammonia level. Profound hypoglycemia is common.

Q.- Discuss the oxidation of odd chain and unsaturated fatty acids.


1) oxidation of odd chain fatty acids- Fatty acids with an odd number of carbon atoms are oxidized by the pathway of β-oxidation, producing acetyl-CoA, until a three-carbon (propionyl-CoA) residue remains (Figure-2). This compound is converted to succinyl-CoA, a constituent of the citric acid cycle (Figure 3).











Steps of conversion of propionyl co A to Succinyl co A-

Propionyl-CoA is carboxylated at the expense of the hydrolysis of an ATP to yield the d isomer of methylmalonyl CoA (Figure 3). This carboxylation reaction is catalyzed by propionyl CoA carboxylase, a biotin dependent  enzyme that is homologous to and has a catalytic mechanism like that of pyruvate carboxylase. The d isomer of methylmalonyl CoA is racemized to the l isomer, the substrate for a mutase that converts it into succinyl CoA by an intramolecular rearrangement. This isomerization is catalyzed by methylmalonyl CoA mutase, which contains a derivative of vitamin B12, cobalamin, as its coenzyme.










Figure-3-Showing the conversion of propionyl co A to Succinyl co A. Propionyl CoA, generated from fatty acids with an odd number of carbons as well as from some amino acids, is converted into the citric acid cycle intermediate succinyl CoA.

 Deficiency of Vitamin B12 causes Methyl malonic aciduria.

Hence, the propionyl residue from an odd-chain fatty acid is the only part of a fatty acid that is glucogenic. Acetyl CoA  cannot be converted into pyruvate or oxaloacetate in animals. The two carbon atoms of the acetyl group of acetyl Co A enter the citric acid cycle, but two carbon atoms leave the cycle in the decarboxylations catalyzed by isocitrate dehydrogenase and a-ketoglutarate dehydrogenase. Consequently, oxaloacetate is regenerated, but it is not formed de novo when the acetyl unit of acetyl CoA is oxidized by the citric acid cycle.

Acetyl co A cannot be converted to Pyruvate since the reaction  catalyzed by Pyruvate dehydrogenase complex for the conversion of pyruvate to Acetyl co A is irreversible.

Oxidation of unsaturated fatty acids

 In the oxidation of unsaturated fatty acids , most of the reactions are the same as those for saturated fatty acids, only two additional enzymes an isomerase and a reductase are needed to degrade a wide range of unsaturated fatty acids.

 For example In the oxidation of palmitoleate,  C16 unsaturated fatty acid, which has one double bond between C-9 and C- 10, is activated and transported across the inner mitochondrial membrane in the same way as saturated fatty acids.

Palmitoleoyl  Co A then undergoes three cycles of degradation, which are carried out by the same enzymes as in the oxidation of saturated fatty acids. However, the cis-D 3-enoyl CoA formed in the third round is not a substrate for acyl CoA dehydrogenase. The presence of a double bond between C-3 and C-4 prevents the formation of another double bond between C-2 and C-3. This impasse is resolved by a new reaction that shifts the position and configuration of the cis-D 3 double bond. An isomerase converts this double bond into a trans- D 2 double bond. The subsequent reactions are those of the saturated fatty acid oxidation pathway, in which the trans- D 2-enoyl CoA is a regular substrate (Figure-4)



















Figure-4-Showing the oxidation of Palmitoleoyl co A, mono unsaturated fatty acid.

 A different set of enzymes  is required for the oxidation of Linoleic acid. a C18 polyunsaturated fatty acid with cis-Δ 9 and cis-Δ 12 double bonds (Figure 5). The cis- Δ 3 double bond formed after three rounds of  β oxidation is converted into a trans- Δ 2 double bond by the a aforementioned isomerase. The acyl CoA produced by another round of β oxidation contains a cis- Δ 4 double bond. Dehydrogenation of this species by acyl CoA dehydrogenase yields a 2,4-dienoyl intermediate, which is not a substrate for the next enzyme in the β -oxidation pathway. This impasse is circumvented by 2,4-dienoyl CoA reductase, an enzyme that uses NADPH to reduce the 2,4-dienoyl intermediate to trans-D 3-enoyl CoA. cis-Δ 3-Enoyl CoA isomerase then converts trans– Δ 3-enoyl CoA into the trans- Δ 2 form, a customary intermediate in the beta-oxidation pathway.. Only two extra enzymes are needed for the oxidation of any polyunsaturated fatty acid. Odd-numbered double bonds are handled by the isomerase, and even-numbered ones by the reductase and the isomerase.




















Figure-5- Showing the Oxidation of Linoleoyl CoA. The complete oxidation of the diunsaturated fatty acid linoleate is facilitated by the activity of enoyl CoA isomerase and 2,4-dienoyl CoA reductase.

Energy yield is less by the oxidation of unsaturated fatty acids since they are less reduced. Per double bonds 2 ATP are less formed, since the first step of dehydrogenation to introduce double bond is not required, as the double is already existing, FADH2 is not formed, and hence loss of 2 ATP per pre -existing double bond.


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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 transferaseI), 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 carnitine acyltransferase 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.


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 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.


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 Δ2trans-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 G for 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 to ETF: 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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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.


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 PPi (Figure-8 ). The PPi 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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1.  Describe the role of bile salts in the digestion and absorption of lipids.

2.  What is steatorrhea? What are the conditions that can cause it and what is the outcome?

3.  Discuss the steps of beta oxidation of a fatty acid with 16 carbon atoms. What will be the net energy yield per mole upon its complete oxidation?

4.  How is fatty acid oxidation switched off when the active synthesis of fatty acid is in process?


5.  Discuss the regulation of fatty acid oxidation.

6.  Which process out of fatty acid synthesis or fatty acid oxidation would predominate during starvation? Discuss the steps,energetics and regulation of that pathway?

7.  Give a brief account of Alpha oxidation of fatty acids. What is its biological or clinical significance?

8. If a person fails to metabolize Phytanic acid which is a normal component of dairy products and green vegetables, what could be the possible defect? What would be the clinical manifestations and how can this defect be treated?


9.  Discuss the causes, consequences,laboratory diagnosis and treatment of Refsum disease.

10. What is the role played by Carnitine in mitochondrial oxidation of long chain fatty acids?

11. What is the cause of non ketotic hypoglycemia in Carnitine deficiency?

12. Discuss the causes, consequences and laboratory diagnosis of-

 i) Jamaican sickness ii) Sudden cot death syndrome iii) Zellweger syndrome

13.-What is ketosis ? Outline the steps of formation and utilization of ketone bodies.

14.- Why it is said that ketone bodies are synthesized in liver and utilized in the peripheral tissues?

15- Give a flow chart representation of biochemical basis of ketosis in starvation and Diabetes mellitus.

16. What are the clinical features,laboratory findings and possible treatment of Ketosis?

17-Discuss the steps of de novo biosynthesis of fatty acids. How is this pathway regulated?

18- Give a diagrammatic representation of fatty acid synthase complex.

19- Discuss the regulation of fatty acid synthesis in the presence of i) Excess of citrate ii) Palmitic acid and describe the  long term control of  fatty acid synthesis if a person has  developed a compulsive urge to eat carbohydrate rich diet.

20- De novo Fatty acid synthesis is a cytoplasmic process while its precursor Acetyl co A is present in  the mitochondrion,how is that transported out into the cytoplasm ?

21- “Fatty acid oxidation is not simply a reversal of fatty acid synthesis”, justify the statement giving the details of differences.

22- Discuss the steps and regulation of de novo synthesis of cholesterol.

23- Discus the significance of reaction catalyzed by HMG Co A reductase.

24- What is the normal level of serum total cholesterol? What are the different conditions in which variations of serum total cholesterol level are observed?

25 Differentiate between LCAT (Lecithin Cholesterol Acyl  Transferase) and ACAT (Acyl cholesterol acyl transferase) by giving at least three points of differences.

26- Discuss the steps of formation of bile acids. How is this pathway regulated?

27- How do bile acid sequestrants help in lowering serum total cholesterol levels?

28- Why it is said that equimolar amount of bile salts and phospholipids are needed during excretion of cholesterol in bile? What will happen if the bile salts or phospholipids are deficient?

29- What do you understand by cholelithiasis? What is the biochemical defect and what are the clinical manifestations in the affected patients?

30- What are lipoproteins? Discuss the general structure and classification of lipoproteins.

31- Discuss the functions of different apo-proteins giving examples.

32- What is meant by down regulation of LDL receptors? What is the clinical significance associated with it?

33- Discuss the role of lipoprotein lipase in the metabolism of lipoproteins. What is clearing factor? What will be the clinical outcome if lipoprotein lipase is deficient?

34- Give a detailed account of hyperlipidemia. What is the possible line of treatment for this group of disorders?

35.- Discuss the central role of Acetyl coA  giving the details of its sources and channels of utilization.

36- Give a brief account of hypolipidemic drugs.

37- How do polyunsaturated fatty acids help in lowering serum total cholesterol levels?

38- Give a brief account of metabolism of HDL highlighting the process of reverse cholesterol transport.

39- Why is LDL called a bad cholesterol while HDL a good cholesterol?

40- What is Lp (a) ? Discuss its clinical significance?

41- Outline the reaction catalyzed by each of the following enzymes-

i) Acetyl coA carboxylase

ii) HMG Co A reductase

iii) Thiokinase

iv) Thiophorase

v) Thiolase

vi) 7-Alpha hydroxylase

vii) Acyl coA dehydrogenase

42-What is fatty liver? Enlist the causes and discuss the role of lipotropic agents.

43- What is the product of oxidation of a fatty acid with odd number of carbon atoms?

44- Why it is partly correct to say that fats can not be converted to glucose?

45- What is Atherosclerosis? Discuss in detail the causes, pathogenesis, laboratory findings and its possible treatment. 

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