Main Menu


November 2012

Please help "Biochemistry for Medics" by CLICKING ON THE ADVERTISEMENTS above!

Q.5- a) Phase I reactions of detoxification

Refer power point presentation

b) Steps and regulation of cholesterol biosynthesis-

1) Synthesis

Refer- cholesterol metabolism- Subjective questions-set-1

2) Regulation of cholesterol biosynthesis

Refer- cholesterol metabolism- Subjective questions-set-2

c) Glucose Alanine cycle

Refer-Transamination and  Transaminases

and refer case study starvation

Q.6- Giving the biochemical defect, pathogenesis, clinical manifestations, laboratory diagnosis and possible treatment, describe the following diseases-

a) Alkaptonuria- Refer

b) Homocystinuria   – Refer the link below

c) Diabetic ketoacidosis-    Refer the link below-



Please help "Biochemistry for Medics" by CLICKING ON THE ADVERTISEMENTS above!

A complete, balanced set of enzyme activities is of fundamental importance for maintaining homeostasis.

Numerous factors affect the reaction rate

The kinetic theory—also called the collision theory—of chemical kinetics states that for two molecules to react they must –

(1) approach within bond-forming distance of one another, or “collide”; and

(2) must possess sufficient kinetic energy to overcome the energy barrier for reaching the transition state.

It therefore follows that anything that increases the frequency or energy of collision between substrates will increase the rate of the reaction in which they participate.

1) Temperature

Raising the temperature increases the kinetic energy of molecules. Increasing the kinetic energy of molecules also increases their motion and therefore the frequency with which they collide. This combination of more frequent and more highly energetic and productive collisions increases the reaction rate. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. The Q10, or temperature coefficient, is the factor by which the rate of a biologic process increases for a 10 °C increase in temperature. For the temperatures over which enzymes are stable, the rates of most biologic processes typically double for a 10 °C rise in temperature (Q10 = 2).

Effect of temperature

 Figure-1- showing the effect of temperature on enzyme catalyzed reaction.

The reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature (Figure-1). Because most animal enzymes rapidly become denatured at temperatures above 40oC, most enzyme determinations are carried out somewhat below that temperature. Some enzymes lose their activity when frozen.The optimal temperatures of the enzymes in higher organisms rarely exceed 50 °C, while enzymes from thermophilic bacteria found in hot springs, for instance, may still be active at 100 °C.

Changes in the rates of enzyme-catalyzed reactions that accompany a rise or fall in body temperature constitute a prominent survival feature for “cold-blooded” life forms such as lizards or fish, whose body temperatures are dictated by the external environment. However, for mammals and other homoeothermic organisms, changes in enzyme reaction rates with temperature assume physiologic importance only in circumstances such as fever or hypothermia.

2) Hydrogen Ion Concentration

The rate of almost all enzyme-catalyzed reactions exhibits a significant dependence on hydrogen ion concentration. Most intracellular enzymes exhibit optimal activity at pH values between 5 and 9 (Figure-2).The pH optimum—i. e., the pH value at which enzyme activity is at its maximum—is often close to the pH value of the cells (i. e., pH 7). However, there are also exceptions to this. For example, the proteinase pepsin , which is active in the acidic gastric lumen, has a pH optimum of 2, while other enzymes (at least in the test tube) are at their most active at pH values higher than 9 (Figure-3)

When the activity is plotted against pH, a bell-shaped curve is usually obtained (Figure-2)


 Figure-2- Showing the effect of pH on enzyme catalyzed reaction


 Figure-3- Except for Pepsin, acid phosphatase and alkaline phosphatase, most enzyme  have optimum pH between 5 to 9

The relationship of activity to hydrogen ion concentration reflects the balance between enzyme denaturation at high or low pH and effects on the charged state of the enzyme, the substrates, or both. For enzymes whose mechanism involves acid-base catalysis, the residues involved must be in the appropriate state of protonation for the reaction to proceed. The binding and recognition of substrate molecules with dissociable groups also typically involves the formation of salt bridges with the enzyme. The most common charged groups are the negative carboxylate groups and the positively charged groups of protonated amines. Gain or loss of critical charged groups thus will adversely affect substrate binding and thus will retard or abolish catalysis.

 3) Substrate concentration

The frequency with which molecules collide is directly proportionate to their concentrations. For two different molecules A and B, the frequency with which they collide will double if the concentration of either A or B is doubled. If the concentrations of both A and B are doubled, the probability of collision will increase fourfold.

For a typical enzyme, as substrate concentration is increased; vi increases until it reaches a maximum value Vmax (Figure -4)When further increases in substrate concentration do not further increase Vmax the enzyme is said to be “saturated” with substrate. If a curve is plotted,the shape of the curve that relates activity to substrate concentration (Figure-4) is hyperbolic.

At any given instant, only substrate molecules that are combined with the enzyme as an ES complex can be transformed into product. Second, the equilibrium constant for the formation of the enzyme-substrate complex is not infinitely large. Therefore, even when the substrate is present in excess, (points A and B of Figure), only a fraction of the enzyme may be present as an ES complex. At points A or B, increasing or decreasing [S] therefore will increase or decrease the number of ES complexes with a corresponding change in vi. The rate of reaction is substrate dependent  (First order reaction)- Figure-4

At point C (Figure), essentially all the enzyme is present as the ES complex. Since no free enzyme remains available for forming ES, further increases in [S] cannot increase the rate of the reaction . Reaction rate therefore becomes independent of substrate concentration (Zero order reaction).

Figure-4- Showing the relationship of reaction rate with substrate concentration

4) Effect of Enzyme concentration

In order to study the effect of increasing the enzyme concentration upon the reaction rate, the substrate must be present in an excess amount; i.e., the reaction must be independent of the substrate concentration. Any change in the amount of product formed over a specified period of time will be dependent upon the level of enzyme present. Graphically this can be represented as:

Figure-5- Showing the effect of increasing or decreasing enzyme  concentration on reaction rate

These reactions are said to be “zero order” because the rates are independent of substrate concentration, and are equal to some constant k. The formation of product proceeds at a rate which is linear with time. The addition of more substrate does not serve to increase the rate. In zero order kinetics, allowing the assay to run for double time results in double the amount of product (Figure-5)

The amount of enzyme present in a reaction is measured by the activity it catalyzes. The relationship between activity and concentration is affected by many factors such as temperature, pH, etc. An enzyme assay must be designed so that the observed activity is proportional to the amount of enzyme present in order that the enzyme concentration is the only limiting factor. It is satisfied only when the reaction is zero order.

Enzyme activity is generally greatest when substrate concentration is unlimiting.

5) Effect of product concentration

The enzyme activity declines when the products start accumulating. This is called product or feed back inhibition. Under certain conditions reverse reaction may be favored forming back the substrate.

6)  Effect of activators and Coenzymes

The activity of certain enzymes is greatly dependent on metal ion activators and coenzymes. Vitamins act as coenzymes in a variety of reactions.

7) Effect of modulators and Inhibitors

The enzyme activity is reduced in the presence of an inhibitor and on the other hand the enzyme activity may be increased in the presence of a positive modifier.

Michaelis-Menten Kinetics

In typical enzyme-catalyzed reactions, reactant and product concentrations are usually hundreds or thousands of times greater than the enzyme concentration. Consequently, each enzyme molecule catalyzes the conversion to product of many reactant molecules. In biochemical reactions, reactants are commonly known as substrates. The catalytic event that converts substrate to product involves the formation of a transition state, and it occurs most easily at a specific binding site on the enzyme. This site, called the catalytic site of the enzyme, has been evolutionarily structured to provide specific, high-affinity binding of substrate(s) and to provide an environment that favors the catalytic events. The complex that forms, when substrate(s) and enzyme combine, is called the enzyme substrate (ES) complex. Reaction products arise when the ES complex breaks down releasing free enzyme.

Between the binding of substrate to enzyme, and the reappearance of free enzyme and product, a series of complex events must take place. At a minimum an ES complex must be formed; this complex must pass to the transition state (ES*); and the transition state complex must advance to an enzyme product complex (EP). The latter is finally competent to dissociate to product and free enzyme. The series of events can be shown thus:

E + S <——> ES <——> ES* <——> EP <——> E + P

The kinetics of simple reactions like that above were first characterized by biochemists Michaelis and Menten. The concepts underlying their analysis of enzyme kinetics continue to provide the cornerstone for understanding metabolism today, and for the development and clinical use of drugs aimed at selectively altering rate constants and interfering with the progress of disease states.

The Michaelis-Menten equation is a quantitative description of the relationship among the rate of an enzyme- catalyzed reaction [v1], the concentration of substrate [S] and two constants, Vmax and km (which are set by the particular equation). The symbols used in the Michaelis-Menten equation refer to the reaction rate [v1], maximum reaction rate (V max), substrate concentration [S] and the Michaelis-Menten constant (km).

The Michaelis-Menten equation can be used to demonstrate that at the substrate concentration that produces exactly half of the maximum reaction rate, i.e. ½ V max the substrate concentration is numerically equal to Km. This fact provides a simple yet powerful bioanalytical tool that has been used to characterize both normal and altered enzymes, such as those that produce the symptoms of genetic diseases.

 Thus, the Michaelis constant, km is the substrate concentration at which V1 is half the maximal velocity (Vmax /2) attainable at a particular concentration of enzyme. km thus has the dimensions of substrate concentration.

The dependence of initial reaction velocity on [S] and Km may be illustrated by evaluating the Michaelis-Menten equation under three conditions.

(1) When [S] is much less than km, the term km + [S] is essentially equal tokm. Replacing Km + [S] with Km reduces equation to

Since V max and km are both constants, their ratio is a constant (k). In other words, when [S] is considerably below km, V max is proportionate to k[S]. The initial reaction velocity therefore is directly proportionate to [S].

(2) When [S] is much greater than km, the term km + [S] is essentially equal to [S]. Replacing km + [S] with [S] reduces equation to

Thus, when [S] greatly exceeds km, the reaction velocity is maximal (V max) and unaffected by further increases in substrate concentration.

(3) When [S] = km

Equation states that when [S] equals km, the initial velocity is half-maximal. Equation also reveals that km  is—and may be determined experimentally from—the substrate concentration at which the initial velocity is half-maximal.


Figure-6-Plot of substrate concentration versus reaction velocity

The key features of the plot are marked by points A, B and C. At high substrate concentrations the rate represented by point C the rate of the reaction is almost equal to V max and the difference in rate at nearby concentrations of substrate is almost negligible. If the Michaelis-Menten plot is extrapolated to infinitely high substrate concentrations, the extrapolated rate is equal to V max When the reaction rate becomes independent of substrate concentration, or nearly so, the rate is said to be zero order. (Note that the reaction is zero order only with respect to this substrate. If the reaction has two substrates, it may or may not be zero order with respect to the second substrate). The very small differences in reaction velocity at substrate concentrations around point C (near V max) reflect the fact that at these concentrations almost all of the enzyme molecules are bound to substrate and the rate is virtually independent of substrate, hence zero order. At lower substrate concentrations, such as at points A and B, the lower reaction velocities indicate that at any moment only a portion of the enzyme molecules are bound to the substrate. In fact, at the substrate concentration denoted by point B, exactly half the enzyme molecules are in an ES complex at any instant and the rate is exactly one half of V max At substrate concentrations near point A the rate appears to be directly proportional to substrate concentration, and the reaction rate is said to be first order.

A Linear Form of the Michaelis-Menten Equation Is Used to determine km & V max

The direct measurement of the numeric value of V max and therefore the calculation of km often requires impractically high concentrations of substrate to achieve saturating conditions. A linear form of the Michaelis-Menten equation circumvents this difficulty and permits V max and km to be extrapolated from initial velocity data obtained at less than saturating concentrations of substrate. Starting with equation,



 and simplify

Equation is the equation for a straight line, y = ax + b, where y = 1/vi and x = 1/[S]. A plot of 1/vi as y as a function of 1/[S] as x therefore gives a straight line whose y intercept is 1/ V max and whose slope is km / V max. Such a plot is called a double reciprocal or Lineweaver-Burk plot (Figure-7). Setting the y term of equation  equal to zero and solving for x reveals that the x intercept is -1/Km.


Figure- 7-showing A Lineweaver-Burk Plot Plots of 1/v versus 1/[S] yield straight lines having a slope of Km/Vmax and an intercept on the ordinate at 1/Vmax.

An alternative linear transformation of the Michaelis-Menten equation is the Eadie-Hofstee transformation: and when v/[S] is plotted on the y-axis versus v on the x-axis, the result is a linear plot with a slope of –1/Km and the value Vmax/Km the intercept on the y-axis and as the Vmax intercept on the x-axis.

Both the Lineweaver-Burk and Eadie-Hofstee transformation of the Michaelis-Menten equation are useful in the analysis of enzyme inhibition.




Please help "Biochemistry for Medics" by CLICKING ON THE ADVERTISEMENTS above!

Q.4-a) Enlist the important non carbohydrate precursors of Glucose and show the entry of any two of them in the main pathway of gluconeogenesis.                              

Answer- Gluconeogenesis is the process of converting noncarbohydrate precursors to glucose or glycogen. The major substrates (non carbohydrate precursors ) are the glucogenic amino acids, lactate, glycerol, propionate and the intermediates of TCA cycle.

Liver and kidney are the major gluconeogenic tissues. Gluconeogenesis meets the needs of the body for glucose when sufficient carbohydrate is not available from the diet or glycogen reserves. A supply of glucose is necessary especially for the nervous system and erythrocytes. Failure of gluconeogenesis is usually fatal.

Gluconeogenesis involves Glycolysis,  Citric Acid Cycle, Plus Some Special Reactions –

Three nonequilibrium reactions in glycolysis , catalyzed by hexokinase, phosphofructokinase and pyruvate kinase, prevent simple reversal of glycolysis for glucose synthesis.  These are considered thermodynamic barriers of gluconeogenesis and are circumvented by alternative reactions-

1) Pyruvate to  Phosphoenolpyruvate

Reversal of the reaction catalyzed by pyruvate kinase in glycolysis involves two endothermic reactions. Mitochondrial pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate, an ATP-requiring reaction in which the vitamin biotin is the coenzyme. (Figure-1). A second enzyme, phosphoenolpyruvate carboxykinase, catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate using GTP as the phosphate donor. In liver and kidney, the reaction of succinate thiokinase in the citric acid cycle produces GTP (rather than ATP as in other tissues), and this GTP is used for the reaction of phosphoenolpyruvate carboxykinase, thus providing a link between citric acid cycle activity and gluconeogenesis, to prevent excessive removal of oxaloacetate for gluconeogenesis, which would impair citric acid cycle activity.

Fructose 1,6-Bisphosphate  to  Fructose 6-Phosphate

The conversion of fructose 1,6-bisphosphate to fructose 6-phosphate, for the reversal of glycolysis, is catalyzed by fructose 1,6-bisphosphatase. Its presence determines whether a tissue is capable of synthesizing glucose (or glycogen) not only from pyruvate, but also from triose phosphates.

Glucose 6-Phosphate & Glucose

The conversion of glucose 6-phosphate to glucose is catalyzed by glucose 6-phosphatase. It is present in liver and kidney, but absent from muscle and adipose tissue, which therefore, cannot export glucose into the bloodstream.


Figure-1-Showing an overview of gluconeogenesis, the thermodynamic barriers and the alternative reactions, the entry of major substrates in to the main pathway of gluconeogenesis

Entry of substrates of gluconeogenesis in to the main pathway-

A) Glucogenic amino acids- Amino acids are derived from the dietary proteins, tissue proteins or from the breakdown of skeletal muscle proteins during starvation. After transamination or deamination, glucogenic amino acids yield either pyruvate or intermediates of the citric acid cycle. Amino acids that are degraded to acetyl CoA or Acetoacetyl CoA are termed ketogenic amino acids because they can give rise to ketone bodies or fatty acids. Amino acids that are degraded to pyruvate, α-ketoglutarate, succinyl CoA, fumarate, or oxaloacetate are termed glucogenic amino acids (Figure-2).The net synthesis of glucose from these amino acids is feasible because these citric acid cycle intermediates and pyruvate can be converted into phosphoenolpyruvate. The entry of each of the  glucogenic amino acids in to the pathway of gluconeogenesis is as follows-

1) Pyruvate is the point of entry for alanine, serine, cysteine, glycine, threonine, and tryptophan (Figure-2). The transamination of alanine directly yields pyruvate.

 2) Oxalo acetate- Aspartate and asparagine are converted into oxaloacetate, a citric acid cycle intermediate. Aspartate, a four-carbon amino acid, is directly transaminated to oxaloacetate.

 3) α-Ketoglutarate is the point of entry of several five-carbon amino acids that are first converted into glutamate.(Figure-2)

4) Succinyl CoA is a point of entry for some of the carbon atoms of methionine, isoleucine, and valine. Propionyl CoA and then Methylmalonyl CoA are intermediates in the breakdown of these three nonpolar amino acids.

5) Fumarate is the point of entry for Aspartate, Phenyl alanine and Tyrosine.


Figure- 2- Amino acids forming  Acetyl co A or Acetoacetyl co A are not considered glucogenic, they are called ketogenic amino acids since acetyl co A is a precursor for ketone bodies. All other amino acids which form pyruvate or intermediates of TCA cycle are considered glucogenic.

 B) Lactate- Lactate is formed by active skeletal muscle when the rate of glycolysis exceeds the rate of oxidative metabolism. Lactate is readily converted into pyruvate by the action of lactate dehydrogenase (Figure-3)


 Figure-3- Reaction showing the inter conversion of lactate and pyruvate


Lactate is a major source of carbon atoms for glucose synthesis by gluconeogenesis. During anaerobic glycolysis in skeletal muscle, pyruvate is reduced to lactate by lactate dehydrogenase (LDH). This reaction serves two critical functions during anaerobic glycolysis. First, in the direction of lactate formation the LDH reaction requires NADH and yields NAD+ which is then available for use by the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis. These two reactions are, therefore, intimately coupled during anaerobic glycolysis. Secondly, the lactate produced by the LDH reaction is released to the blood stream and transported to the liver where it is converted to glucose. The glucose is then returned to the blood for use by muscle as an energy source and to replenish glycogen stores. This cycle is termed the Cori cycle (Figure-4)















Figure-4- Cori cycle, the interchange of lactate and glucose between skeletal muscle and Glucose

C) Propionate- Propionate is a major precursor of glucose in ruminants; it enters gluconeogenesis via the citric acid cycle. After esterificaton with CoA, Propionyl-CoA is carboxylated to D-Methylmalonyl-CoA, catalyzed by Propionyl-CoA carboxylase, a biotin-dependent enzyme (Figure-5). Methylmalonyl-CoA racemase catalyzes the conversion of D-Methylmalonyl-CoA to L-Methylmalonyl-CoA, which then undergoes isomerization to succinyl-CoA catalyzed by Methylmalonyl-CoA mutase. In non-ruminants, including humans, propionate arises from the Beta -oxidation of odd-chain fatty acids that occur in ruminant lipids, as well as the oxidation of isoleucine and the side-chain of cholesterol, and is a (relatively minor) substrate for gluconeogenesis. Methylmalonyl CoA Isomerase/ mutase is a vitamin B12 dependent enzyme, and in deficiency methylmalonic acid is excreted in the urine (methylmalonic aciduria). (Figure-5)


Figure-5- showing the fate of Propionyl co A 

D) Glycerol-The hydrolysis of triacylglycerols in fat cells yields glycerol and fatty acids. Glycerol may enter either the gluconeogenic or the glycolytic pathway at Dihydroxyacetone phosphate; however, the carbons of the fatty acids cannot be utilized for net synthesis of glucose. In the fasting state glycerol released from lipolysis of adipose tissue triacylglycerol is used solely as a substrate for gluconeogenesis in the liver and kidneys.This requires phosphorylation to glycerol-3-phosphate by glycerol kinase and dehydrogenation to Dihydroxyacetone phosphate (DHAP) by glyceraldehyde-3-phosphate dehydrogenase (G3PDH). The G3PDH reaction is the same as that used in the transport of cytosolic reducing equivalents into the mitochondrion for use in oxidative phosphorylation. This transport pathway is called the glycerol-phosphate shuttle.




 Figure-6- showing the conversion of glycerol to dihydroxy acetone phosphate

Glycerol kinase is absent in adipose tissue, so glycerol released by hydrolysis of triglycerides  can not be utilized for re esterificaton, it is a waste product, It is carried through circulation to the liver and is used for gluconeogenesis or glycolysis as the need may be. In fact adipocytes require a basal level of glycolysis in order to provide them with DHAP as an intermediate in the synthesis of triacylglycerols.

Please help "Biochemistry for Medics" by CLICKING ON THE ADVERTISEMENTS above!

Q.1- a) Diagrammatic representation of ATP synthase complex

Figure-1- showing ATP Synthase complex.

ATP synthase  complex– is embedded in the inner membrane, together with the respiratory chain complexes .The enzyme complex consists of an F0 sub complex which is a disk of “C” protein subunits. Attached is a Υ subunit in the form of a “bent axle.” Protons passing through the disk of “C” units cause it and the attached Υ subunit to rotate. The Υ subunit fits inside the F1 sub complex of three α and three β subunits, which are fixed to the membrane and do not rotate. The flow of protons through F0 causes it to rotate, driving the production of ATP in the F1 complex.

 ii) Visual Cycle

In the retina, retinaldehyde functions as the prosthetic group of the light-sensitive opsin proteins, forming rhodopsin (in rods) and iodopsin (in cones). Any one cone cell contains only one type of opsin, and is sensitive to only one color.

In the pigment epithelium of the retina, all-trans-retinol is isomerized to 11-cis-retinol and oxidized to 11-cis-retinaldehyde. This reacts with a lysine residue in opsin, forming the holoprotein rhodopsin .

The absorption of light by rhodopsin causes isomerization of the retinaldehyde from 11-cis to all-trans, and a conformational change in opsin. This results in the release of retinaldehyde from the protein, and the initiation of a nerve impulse.

The formation of the initial excited form of rhodopsin, bathorhodopsin, occurs within picoseconds of illumination. There is then a series of conformational changes leading to the formation of metarhodopsin II, which initiates a guanine nucleotide amplification cascade and then a nerve impulse. The final step is hydrolysis to release all-trans-retinaldehyde and opsin. The key to initiation of the visual cycle is the availability of 11-cis-retinaldehyde, and hence vitamin A. In deficiency, both the time taken to adapt to darkness and the ability to see in poor light are impaired.



Figure-2- Showing visual cycle

iii) Carnitine shuttle

Figure-3- Showing carnitine shuttle.

Carnitine – (CH3)3N+—CH2—CH(OH)—CH2—COO,  is widely distributed and is particularly abundant in muscle.

Long-chain acyl-CoA (or FFA) will not penetrate the inner membrane of mitochondria. However, carnitine palmitoyl transferase-I, present in the outer mitochondrial membrane, converts long-chain acyl-CoA to acylcarnitine, which is able to penetrate the inner membrane and gain access to the Β-oxidation system of enzymes (Figure -3).

Carnitine-acyl carnitine translocase acts as an inner membrane exchange transporter. Acyl carnitine is transported in, coupled with the transport out of one molecule of carnitine. The acyl carnitine then reacts with CoA, catalyzed by carnitine palmitoyl transferase-II, located on the inside of the inner membrane. Acyl-CoA is re-formed in the mitochondrial matrix, and carnitine is liberated.  

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 may cause muscle necrosis, myoglobinuria, lipid-storage myopathy, hypoglycemia, fatty liver, and Hyperammonemia with muscle aches, fatigue, confusion, and cardiomyopathy.


Q.3-  a) Give a brief account of the role of niacin as a coenzyme, highlight the important reactions and  mention the names of the enzymes requiring niacin as a coenzyme. What would be the clinical implications of its deficiency?

Answer- The term niacin refers to both nicotinic acid and its amide derivative, nicotinamide (niacinamide) Both are used to form the coenzymes nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+).

Niacin is a member of the water-soluble B- vitamin complex. The amino acid tryptophan can be converted to nicotinic acid in humans; therefore niacin is not really a vitamin provided that an adequate dietary supply of tryptophan is available. Some 60 mg of tryptophan is equivalent to 1 mg of dietary niacin.

Redox reactions – As many as 200 enzymes require the niacin coenzymes, NAD+ and NADP+, mainly to accept or donate electrons for redox reactions.

NAD+ functions most often in energy producing reactions involving the degradation (catabolism) of carbohydrates, fats, proteins, and alcohol.

NADP+ functions more often in biosynthetic (anabolic) reactions, such as in the synthesis of all macromolecules, including fatty acids and cholesterol.

Some of the important enzymes requiring NAD+ and NADP+ are as follows-

NAD+ dependent enzymes –

1)     Glyceraldehyde-3-phosphae dehydrogenase

2)     Pyruvate dehydrogenase complex

3)     Mitochondrial Isocitrate dehydrogenase

4)     Alpha keto glutarate dehydrogenase complex

5)     Malate dehydrogenase

6)     Lactate dehydrogenase

7)     Beta hydroxy acyl co A dehydrogenase

8)    Cytosolic glycerol-3-phosphate dehydrogenase

NADPH dependent enzymes

1)      HMG co A reductase

2)     Enoyl reductase

3)     Keto acyl reductase

4)     Dihydrofolate reductase

5)     Met hemoglobin reductase

6)     Ribonucleotide reductase

Non-redox reactions –

The niacin coenzyme, NAD+, is the substrate (reactant) for two classes of enzymes-

(mono-ADP-ribosyltransferases and poly-ADP-ribose polymerase) that separate the niacin moiety from NAD+ and transfer ADP-ribose to proteins.

1) Mono-ADP-ribosyltransferases –These enzymes and their products, ADP-ribosylated proteins, are thought to play a role in cell signaling by affecting G-protein activity. G-proteins are proteins that bind guanosine-5′-triphosphate (GTP) and act as intermediaries in a number of cell-signaling pathways.

2) Poly-ADP-ribose polymerases (PARPs) are enzymes that catalyze the transfer of many ADP-ribose units from NAD+ to acceptor proteins. PARPs appear to function in DNA repair and stress responses, cell signaling, transcription, regulation or apoptosis, chromatin structure, and cell differentiation, suggesting a possible role for NAD+ in cancer prevention

3) A third class of enzymes (ADP-ribosyl cyclase) catalyzes the formation of cyclic ADP-ribose, a molecule that works within cells to provoke the release of calcium ions from internal storage sites and probably also plays a role in cell signaling.

Niacin deficiency causes -Pellagra


Pellagra is clinically manifested by the 4 D’ s: photosensitive dermatitis, diarrhea, dementia, and death.

The early symptoms of pellagra include loss of appetite, generalized weakness, irritability, abdominal pain and vomiting.

Later symptoms are bright red glossitis, chronic or recurrent diarrhea (watery, but occasionally bloody), which leads to a state of malnutrition and cachexia.

The characteristic skin rash is characterized by pigmentation and scaling, particularly involving the sun exposed areas. Pellagra can affect any part of the body surface, but it more frequently appears in certain areas. The usual sites are the dorsal surfaces of the hands, face, neck, arms, and feet.




Figure-4-  showing  rashes in the sun exposed region in Pellagra As pellagra advances, neuropsychiatric symptoms such as photophobia, asthenia, depression, hallucinations, memory loss, and psychosis begin. The patient become disoriented, confused, and delirious; stupor and death result if untreated.

Causes – Pellagra is the late stage of severe niacin deficiency.

  • Primary pellagra results from inadequate nicotinic acid (ie, niacin) and/or tryptophan intake in the diet.
  • Secondary pellagra occurs when adequate quantities of niacin are present in the diet, but other diseases or conditions interfere with its absorption and/or processing. Such conditions include the following:
    • Prolonged diarrhea
    • Long-term alcoholism
    • Chronic colitis
    • Ileitis terminalis
    • Cirrhosis of the liver
    • Tuberculosis of the GI tract
    • Malignant carcinoid tumor
    • Hartnup syndrome

Isoniazid induced niacin deficiency: Isoniazid causes a depletion of Pyridoxal phosphate, that is required for the production of niacin from tryptophan.

Treatment -Because multiple deficiencies are common, a balanced diet, including other B vitamins (particularly riboflavin and pyridoxine), is needed. Nicotinamide is usually used to treat deficiency, because nicotinamide, unlike nicotinic acid (the most common form of niacin), does not cause flushing, itching, burning, or tingling sensations. Nicotinamide is given in doses ranging from 40 to 250 mg/day in divided doses 3 to 4 times a day.

Niacin (nicotinic acid) in large amounts is sometimes used to lower LDL cholesterol and triglyceride levels and to increase HDL cholesterol levels.

b) Give a brief description of the abnormalities that can lead to fatty liver formation. Support your answer by a suitable flow chart or a diagram summarizing the mechanisms involved in fatty liver formation.

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-5). 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-5)

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-5)

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-5). Some protection against this is provided by the antioxidant action of vitamin E-C, beta carotene and selenium in the supplemented diets.


Figure-5- 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

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-6)

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) 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)  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.

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

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

E) 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.



Figure-6- Showing steps of metabolism of alcohol




Please help "Biochemistry for Medics" by CLICKING ON THE ADVERTISEMENTS above!

Q.1-  20 Multiple Choice questions on a separate sheet.                   

Q.2- Give a diagrammatic representation of each of the followings-

i) ATP synthase complex              

ii) Visual Cycle                   

iii) Carnitine shuttle

Mention the functional/clinical significance of each of them.    

 Q.3- a) Give a brief account of the role of niacin as a coenzyme, highlight the important reactions and  mention the names of the enzymes requiring niacin as a coenzyme. What would be the clinical implications of its deficiency?

b) Give a brief description of the abnormalities that can lead to fatty liver formation. Support your answer by a suitable flow chart or a diagram summarizing the mechanisms involved in fatty liver formation.  

Q.4-a) Enlist the important non carbohydrate precursors of Glucose and show the entry of any two of them in the main pathway of gluconeogenesis.                              

b) Give a detailed description of the factors that affect the enzyme activity in vivo.

Support your answer by suitable graphs or diagrams.                                     

Q.5- Write short note on-

a) Phase 1 reactions of detoxification

b) Steps and regulation of cholesterol synthesis

c) Glucose- Alanine cycle                                                                                                

Q.6- Giving the biochemical defect, pathogenesis, clinical manifestations, laboratory diagnosis and possible treatment, describe the following diseases-

a) Alkaptonuria                

b) Homocystinuria          

c) Diabetic ketoacidosis           






Please help "Biochemistry for Medics" by CLICKING ON THE ADVERTISEMENTS above!

Q.1-Glycogen phosphorylase, which mobilizes glycogen for energy, requires which of the followings as a cofactor?

a)Pyridoxal phosphate                                                                  

b)Tetra hydro folate

c) Adenosyl Cobalamine                                                             

d) Coenzyme A                                 

Q.2.Which statement out of the followings is incorrect about the effect of increasing temperature on enzyme activity-

a) Raising the temperature increases the kinetic energy of molecules

b) A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%.

c) Most animal enzymes rapidly become denatured at temperatures above 40oC

d) Storage of enzymes at 5°C or below is generally not suitable.                                          

Q. 3.-A 2 -week –old child was brought to the emergency. The parents were fearful that the child had been given some poison as they noted black discoloration on the diaper. A diagnosis of Alkaptonuria was made and the child was given Vitamin C as a supplement. Alkaptonuria occurs due to reduced activity of Homogentisic acid oxidase enzyme. What is the role played by vitamin C in this defect?

a) Acts as an oxidant                                                      

b) Acts as a coenzyme

c) Acts as an inducer                                                      

d) Acts as a positive allosteric modifier

Q.4 – Sub Acute Combined Degeneration of the Spinal Cord is found in the deficiency of-

a) B12                                                                                  

b) B6

c) Niacin                                                                              

d) B1                                                             

Q.5-The enzyme Ribonucleotide Reductase requires the presence of-

a) Inositol                                                                           

b) B1

c) B12                                                                                   

d) B2                                                   

Q.6A person on ingestion of Primaquine develops hemolytic anemia, what is the possible defect?

a) Deficiency of Iron                                                      

b) Vitamin K deficiency

c) Glucose-6-P dehydrogenase deficiency           

d) Vitamin C deficiency                 

Q.7-All of the following processes except one are mitochondrial-

a) Glycolysis                                                                      

b) TCA cycle

c) Beta oxidation of fatty acids                                  

d) Ketogenesis                                

Q.8-The primary enzyme for utilization of ketone bodies is-

a) Thiokinase                                                                    

b) Thioesterase

c) Thiophorase                                                                 

d) Thiolase                                                      

Q.9- Which of the following enzymes does not have an impaired activity in Vitamin B1 deficiency?

a) Succinate dehydrogenase                                      

b) Pyruvate dehydrogenase

c) Transketolase                                                              

d) Alpha keto glutarate dehydrogenase              

Q.10- Which of the following compounds is formed from hydroxylation requiring vitamin C and subsequent methylation?

a) Histamine                                                                      

b) Dopamine

c) Epinephrine                                                                  

d) Creatine                                                       

Q.11-Which of the following amino acids is not converted to Acetyl co A upon metabolism?

a) Tyrosine                                                                        

b) Leucine

c) Tryptophan                                                                   

d) Valine                                                              

Q.12-Which of the following lipid lowering drugs act by inhibiting the absorption of dietary cholesterol?

a) Statins                                                                            

b) Ezetimibe

c) Colestipol                                                                      

d) Clofibrate                                                      

Q.13-The synthesis of HMG Co A can take place-

a) Only in mitochondria                                                

b) Only in cytoplasm of all nucleated mammalian cells

c) In the endoplasmic reticulum of all mammalian cells

d) In both cytosol and mitochondria.                                                                                                    

Q.14The complete oxidation of odd chain fatty acid produces which of the followings?

a) Acetyl co A only                                          

b) Acetyl co A and Propionyl co A

c) Butyryl co A                                                  

d) Propionyl co A only                                                 

Q.15-All except one are incorrect about oxidases-

a) Oxidases catalyze reactions involving hydrogen peroxide.

b) Oxidases catalyze reaction using oxygen as a hydrogen acceptor

c) Oxidases catalyze reactions using Niacin as coenzyme

d) Oxidases catalyze reactions of direct incorporation of oxygen in to the substrate.       

Q.16-The enzymes of mitochondrial matrix include all except-

a) Enzymes of fatty acid oxidation                            

b) Creatine kinase

c) Enzymes of TCA cycle                                               

d) Pyruvate dehydrogenase complex         

Q.17- Which of the following intermediates of TCA cycle cannot be utilized for gluconeogenesis?

a) Succinate                                                       

b) Malate

c) α-Keto glutarate                                         

d) Acetyl co A.                                                                        

Q.18-Malonate is an inhibitor of-

a) Citrate synthase                                         

b) Aconitase

c)  Succinate dehydrogenase                     

d) Malate dehydrogenase.                                              

Q.19Which of the following glycolytic enzymes is used in gluconeogenesis?

a) Phosphofructokinase-1                           

b) Aldolase B

c) Phosphoglycerate kinase                        

d) Pyruvate kinase.                                                     

Q.20Which of the following is most important for maintenance of blood glucose during fasting?

a) Liver                                                               

b) Heart

c) Skeletal muscle                                           

d) Lysosome                                                                    


Key to Answers- 1)-a,  2)-d, 3)-b, 4)-a, 5)- c, 6)- c, 7)-a, 8)- c, 9)-a, 10)-c, 11)- d, 12- b, 13)-d, 14)-b, 15)- d, 16)-b, 17)-d, 18)-c, 19)-c, 20)-a,

Please help "Biochemistry for Medics" by CLICKING ON THE ADVERTISEMENTS above!