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Alkaptonuria

Alkaptonuria (AKU) is a rare inherited genetic disorder of tyrosine metabolism characterized by the triad of Homogentisic aciduria, ochronosis and arthritis. It is one of the conditions in which Mendelian recessive inheritance was proposed.  It was also one of the four inborn errors of metabolism described by Garrod.

The most obvious sign in adults is a thickening and blue-black discoloration of the ear cartilage. This blue-black discoloration of connective tissue (including bone, cartilage, and skin) is caused by deposits of yellow or ochre-colored pigment, and is called Ochronosis.

Frequency

The condition is rare, affecting one in 250,000 to one million people worldwide. In US, the incidence is 1 case per 4 million populations.

Biochemical Defect

AKU is an autosomal recessive disorder, caused due to deficiency of Homogentisic acid oxidase (HGAO) which catalyzes the conversion of HGA (also called alkaptone) to maleyl acetoacetate (Figure-1). Inability to convert homogentisic acid to maleylacetoacetic acid results in accumulation of the former. Homogentisic acid is subsequently converted to benzoquinone acetic acid and spontaneously polymerized (Figure-1).

Pathophysiology

In the absence of the enzyme HGAO, Homogentisic acid and benzoquinone acetic acid (BQA) build up in the body (Figure-1).  Homogentisic acid is rapidly cleared in the kidney and excreted.

Although homogentisic acid blood levels are kept very low through rapid kidney clearance, over time homogentisic acid is deposited in cartilage throughout the body and is converted to the pigment like polymer through an enzyme-mediated reaction that occurs chiefly in collagenous tissues. As the polymer accumulates within cartilage, a process that takes many years, the normally transparent tissues become slate blue, an effect ordinarily not seen until adulthood.

The earliest sign of the disorder is the tendency for diapers to stain black. Throughout childhood and most of early adulthood, an asymptomatic, slowly progressive deposition of pigment like polymer material into collagenous tissues occurs.

In the fourth decade of life, external signs of pigment deposition, called ochronosis, begin to appear.

The slate blue, gray, or black discoloration of sclerae and ear cartilage is indicative of widespread staining of the body tissues, particularly cartilage. The hips, knees, and intervertebral joints are affected most commonly and show clinical symptoms resembling rheumatoid arthritis. Although unproven, the deposition of polymer is assumed to also cause an inflammatory response that results in calcium deposition in affected joints.

 

Clinical Manifestations

Most patients don’t have any symptoms throughout childhood or early adult life and it is not until they reach their 40′s that other signs of the disease start appearing.

  • One of the earliest signs is thickening of the ear cartilage (the pinna feels noticeably thickened and flexible). In addition the skin turns a blue-black color (Figure-2)

a2

 

 

Figure-2- Showing blackening of the ear cartilage

  • Earwax is often reddish-brown or jet-black.
  • Bones and cartilage of the lower back, knees, shoulders and hips are most affected. Firstly patients suffer low back pain with stiffness, followed by knee, shoulder and hip pain over the next 10 years. Cartilage becomes brittle and can break apart easily. In some cases this leads to spinal injuries such as prolapsed intervertebral discs.
  • Deposits around the trachea, larynx and bronchi may cause shortness of breath and difficulty breathing.
  • Deposits around the heart and blood vessels can calcify and lead to atherosclerotic plaques.
  • Pigmentation of the sclera of the eye usually occurs early on. This does not affect vision but appears as brown or grey deposits on the surface of the eye (Figure-3)

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Figure-3- Showing black spots on the sclera

  • Skin color changes are most apparent on areas exposed to the sun and where sweat glands are found (Figure-4)

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Figure-4- Showing blackening of the skin

  • Urine exposed to air can become dark; this is useful for diagnosing young children using diapers. The urine is malodorous.

 

 

 a5

 

 

 

 

 

 

 

 

 

 

Figure-5- Showing the darkening of urine on standing.

Diagnosis

Presumptive diagnosis can be made by adding sodium or potassium hydroxide to urine and observing the formation of a dark brown to black pigment on the surface layer of urine within 30 minutes to 1 hour.

The fresh urine of an alkaptonuric appears normal but starts darkening on exposure to the air. This is caused by oxidation and polymerization of the HGA that speeds up on alkalization. Hence, (strongly) acidic urine may not darken for many hours on standing. This may be one of the reasons why darkening of the urine may not be noted in an affected child and the diagnosis is delayed until adulthood when arthritis or ochronosis appears.

HGA is a strong reducing substance that produces a positive reaction with Benedict’s and Fehling’s reagent. With Fehling’s (FeCl3) reagent, it gives transient blue-green Color. 

The diagnosis of alkaptonuria is confirmed by measurement of HGA concentration in the urine by paper and thin layer chromatography and photometry.

HGA is not elevated in the blood but excreted in the urine in heavy amounts – as much as 4-8gm / day.

Treatment

Alkaptonuria is a life long disease. There is no cure for the condition.Prevention is not possible and the treatment is aimed at ameliorating symptoms. Reducing intake of the amino acids phenylalanine and tyrosine to the minimum required to sustain health (phenylalanine is an essential amino acid) can help slow the progression of the disease. Vitamin C has been found to slow down the conversion of homogentisic acid to the polymeric deposits in cartilage and bone. A dose of up to 1g/day is recommended for older children and adults.

Medical therapy is used to ameliorate the rate of pigment deposition. This minimizes articular and cardiovascular complications in later life.

Reduction of phenylalanine and tyrosine has reportedly reduced homogentisic acid excretion. Whether a mild dietary restriction from early in life would avoid or minimize later complications is not known, but such an approach is reasonable.

Prognosis

Life expectancy is normal although patients may be at increased risk of heart conditions and may require surgical treatments for spine, hip, knee and shoulder joint problems. Exogenous cutaneous Ochronosis has been successfully treated by laser.

 

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Q.5- a) Phase I reactions of detoxification

Refer power point presentation

http://www.namrata.co/metabolism-of-xenobiotics-detoxification-reactions-power-point-presentation/

b) Steps and regulation of cholesterol biosynthesis-

1) Synthesis

Refer- cholesterol metabolism- Subjective questions-set-1

http://www.namrata.co/cholesterol-metabolism-subjective-questions-set-1/

2) Regulation of cholesterol biosynthesis

Refer- cholesterol metabolism- Subjective questions-set-2

http://www.namrata.co/cholesterol-metabolism-subjective-questions-set-2/

c) Glucose Alanine cycle

Refer-Transamination and  Transaminases

http://www.namrata.co/transamination-and-transaminases/

and refer case study starvation

http://www.namrata.co/category/diet-and-nutrition/case-studies-diet-and-nutrition/

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

a) Alkaptonuria- Refer

http://www.namrata.co/alkaptonuria/

b) Homocystinuria   – Refer the link below

http://www.namrata.co/category/metabolism-proteins/case-studies-metabolism-of-proteins/

c) Diabetic ketoacidosis-    Refer the link below-

http://www.namrata.co/diabetes-mellitus-acute-complications/

 

 

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Q.4 b) Give a detailed description
of the factors that affect the enzyme activity in vivo. Support your answer by suitable graphs or diagrams.

Factors affecting enzyme activity

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,

 

 

 

 

invert

 

 

 

 

factor


 

 

 

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-Menton equation are useful in the analysis of enzyme inhibition.

 

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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.

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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

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

 

 

 

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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           

 

 

 

                                                                                                              

                                                                                                                                                                                                                               

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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.6-A 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.14-The 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.19-Which of the following glycolytic enzymes is used in gluconeogenesis?

a) Phosphofructokinase-1                           

b) Aldolase B

c) Phosphoglycerate kinase                        

d) Pyruvate kinase.                                                     

Q.20- Which 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,

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Iron absorption takes place largely in the proximal small intestine and is a carefully regulated process. In general, there is no regulation of the amounts of nutrients absorbed from the gastro intestinal tract. A notable exception is iron, the reason that absorption must be carefully regulated is that the body does not possess a physiological mechanism to eliminate much iron from the body. The small amount of iron that is lost each day (about 1-2 mg) is matched by dietary absorption of iron.

 Mechanism of iron absorption
 Iron is found in the diet  as ionic (non-haem) iron and haem iron. Absorption of these two forms of iron occurs by different mechanisms. Absorption is a multistep process involving the uptake of iron from the intestinal lumen across the apical cell surface of the villus enterocytes and the transfer out of the enterocyte across the basolateral membrane to the plasma. Ionic iron is present in the reduced (ferrous) or oxidised (ferric) state in the diet and the first step in the uptake of ionic iron involves the reduction of iron. Recently, a reductase that is capable of reducing iron from its ferric to ferrous state has been identified. It is a membrane bound haem protein called Dcytb that is expressed in the brush border of the duodenum. Next, ferrous ion is transported across the lumen cell surface by a transporter called divalent metal transporter 1 (DMT1) that can transport a number of other metal ions including copper, cobalt, zinc, and lead.

 

 

 

 

 

 

 

 

 

 

 

 

Figure- Showing the mechanism of iron absorption

 Once inside the gut cell, iron may be stored as ferritin or transported through the cell to be released at the basolateral surface to plasma transferrin through the membrane-embedded iron exporter, ferroportin. The function of ferroportinis negatively regulated by hepcidin,the principal iron regulatory hormone. More the Hepcidin levels lesser is the iron absorption and vice versa. In the process of release, iron interacts with another ferroxidase, hephaestin, which oxidizes the iron to the ferric form for transferrin binding. Hephaestin is similar to ceruloplasmin, the copper-carrying protein.

 The mechanism of absorption of haem iron has yet to be elucidated. Transfer across the brush border membrane is probably mediated by an unidentified haem receptor. Once inside, enterocyte iron is released from haem by haem oxygenase and either stored or transferred out of the enterocyte by a mechanism that is likely to be similar to that for ionic iron

 Factors affecting iron absorption

 Iron absorption is influenced by a number of physiologic states.

 1) Erythroid hyperplasia stimulates iron absorption, even in the face of normal or increased iron stores, and in this state hepcidin levels are inappropriately low. The molecular mechanism underlying this relationship is not known. Thus, patients with anemias associated with high levels of ineffective erythropoiesis absorb excess amounts of dietary iron. Over time, this may lead to iron overload and tissue damage. In iron deficiency, hepcidin levels are low and iron is much more efficiently absorbed from a given diet; the contrary is true in states of secondary iron overload.

 2)  Hypoxia-Both the rate of erythropoiesis and hypoxia regulate iron absorption. Expression of ferroportin and Dcytb are increased in hypoxia, resulting in more iron absorption.

 3)  Body Stores- Iron absorption is stimulated if the levels of  body stores are low. On the contrary, Hepcidin is produced excessively by hepatocytes when iron stores are full, hepcidin makes a complex with ferroportin promoting its degradation and thus iron is not  transported out of the enterocyte in to the blood. Iron  remains inside the cell in the form of ferritin till the life span of the cell

 

 

 

 

 

 

 

 

 

 

  Figure- showing influence of body iron stores on iron absorption

 4)  Inflammation can also stimulate hepcidin production resulting in lowered iron absorption.

 

 

 

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A teenage girl was brought to the medical centre because of her complaints that she used to get too tired when asked to participate in gym classes. A consulting neurologist found muscle weakness in girl’s arms and legs. When no obvious diagnosis could be made, biopsies of her muscles were taken for test.

Biochemistry revealed greatly elevated amounts of triglycerides  esterified with primary long chain fatty acids. Pathology reported the presence of significant numbers of lipid vacuoles in the muscle biopsy

What is the probable diagnosis?

What is the cause for these symptoms?

Case details

The most likely cause of these symptoms is carnitine deficiency.

Carnitine deficiency

 The amino acid carnitine is required for the transport of long-chain fatty acyl coenzyme  esters into myocyte mitochondria, where they are oxidised for energy. Carnitine is obtained from foods, particularly animal-based foods, and via endogenous synthesis.

Carnitine deficiency results from inadequate intake of or inability to metabolize the amino acid carnitine. It can cause a heterogeneous group of disorders. Muscle metabolism is impaired, causing myopathy, hypoglycemia, or cardiomyopathy.Infants typically present with hypoglycemic, hypoketotic encephalopathy. Most often, treatment consists of dietary l-carnitine.

 

                      

Carnitine

 

Basic concept

Fatty acids are activated on the outer mitochondrial membrane, whereas they are oxidised in the mitochondrial matrix. The activation is brought by converting the fatty acid into Acyl co A ester under the activity of Acyl co A synthetase (1). 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. The acyl group is transferred from the sulphur atom of CoA to the hydroxyl group of carnitine to form acyl carnitine. This reaction is catalysed by carnitine acyl transferase I (also called carnitine palmitoyl transferase I), which is bound to the outer mitochondrial membrane.(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) (4),  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 (See figure).

 

Figure showing the role of carnitine in transporting the activated fatty acids in to the mitochondria.

Pathogenesis

A number of diseases have been traced to a deficiency of carnitine, the transferase or the translocase. The symptoms of carnitine deficiency range from mild muscle cramping to severe weakness and even death.The muscle, kidney, and heart are the tissues primarily affected. Muscle weakness during prolonged exercise is an important characteristic of a deficiency of carnitine acyl transferase because muscle relies on fatty acids as a long-term source of energy. Medium-chain (C8-C10) fatty acids, which do not require carnitine to enter the mitochondria, are oxidised normally in these patients. These diseases illustrate that the impaired flow of a metabolite from one compartment of a cell to another can lead to a pathological condition.

Causes of 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.

·        Decreased endogenous synthesis of carnitine due to a severe liver disorder

·        Excess loss of carnitine due to diarrhoea,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

Primary Carnitine deficiency The underlying defect involves the plasma membrane sodium gradient–dependent carnitine transporter that is present in heart, muscle, and kidney. 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 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 of Carnitine deficiency

 Symptoms and the age at which symptoms appear depend on the cause. Carnitine deficiency may cause muscle necrosis, myoglobinuria, hypoglycemia, fatty liver, muscle aches, fatigue, and cardiomyopathy.

 1) The most common presentation is progressive cardiomyopathy with or without skeletal muscle weakness beginning at 2–4 yr of age.Energy deprived muscle cells are damaged.

 2) A smaller number of patients may present with fasting hypoketotic hypoglycemia during the 1st yr of life before the cardiomyopathy becomes symptomatic.Blockage of the transport of long chain fatty acids into mitochondria deprives the patient of energy production, as the fatty acid oxidation is impaired; all the energy needs are fulfilled by glucose oxidation. The resultant imbalance between demand and supply causes hypoglycemia. The compensatory ketosis in carnitine  induced hypoglycemia is not observed as the precursor, Acetyl co A is not available for ketone body production. The main source of Acetyl co is fatty acid oxidation  and that is impaired in carnitine deficiency.

3) Serious complications such as heart failure, liver problems, coma, and sudden unexpected death are also a risk.

Deficiencies in the Carnitine Acyl Transferase enzymes I and II can cause similar symptoms.

Diagnosis 

1)  Diagnosis of the carnitine transporter defect is aided by the fact that patients have extremely reduced carnitine levels in plasma and muscle (1–2% of normal).

2)  Fasting ketogenesis may be normal if liver carnitine transport is normal, but it may be impaired if dietary carnitine intake is interrupted.

3)  Hypoglycemia is a common finding. It is precipitated by fasting and strenuous exercise.

4) Muscle biopsy reveals significant lipid vacuoles.

Treatment

Treatment of this disorder with pharmacological doses of oral carnitine is highly effective in correcting the cardiomyopathy and muscle weakness as well as any impairment in fasting ketogenesis. 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.

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Case details

A 12- year-old girl who had a grossly enlarged abdomen reported to OPD . She had a history of frequent episodes of weakness, sweating and pallor that were eliminated by eating. Her development had been slow; she sat at the age of 1 year, walked unassisted at the age of 2 years, and was doing poorly in the school.

Physical examination revealed normal blood pressure,temperature and a normal pulse rate but a sub normal weight (23 Kg).The liver was enlarged, firm and was descended in to pelvis. The spleen was not palpable,nor were the kidneys. The remainder of the physical examination was within the normal limits.

Laboratory investigation report revealed, low blood glucose, low p H, high lactate, triglycerides, ketones and high free fatty acids. The liver biopsy revealed high glycogen content. Hepatic glycogen structure was normal. The enzyme assay performed on the biopsy tissue revealed very low glucose-6- phosphatase levels.

What is the probable diagnosis?

What is the possible treatment for this patient?

Case details

The girl is suffering from Von –Gierke’s disease. The clinical picture, biochemical findings, hypoglycemia and increased Hepatic Glycogen stores are all characteristic of Von –Gierke’s disease.

Von –Gierke’s disease

Glycogen storage disease (GSD) type I, is also known as Von Gierke’s disease or hepatorenal Glycogenesis. Von Gierke  described the first patient with GSD type I in 1929.

Basic concept- Glycogen is a readily mobilised storage form of glucose. It is a very large, branched polymer of glucose residues that can be broken down to yield glucose molecules when energy is needed. Most of the glucose residues in glycogen are linked by α-1,4-glycosidic bonds. Branches at about every tenth residue are created by α-1, 6-glycosidic bonds. 

Figure-1- showing structure of Glycogen
Glycogen is not as reduced as fatty acids are and consequently not as energy rich.

Why do animals store any energy as glycogen? Why not convert all excess fuel into fatty acids? 

Glycogen is an important fuel reserve for several reasons-

1)  The controlled break down of glycogen and release of glucose increase the amount of glucose that is available between meals. Hence, glycogen serves as a buffer to maintain blood-glucose levels.

2)   Glycogen’s role in maintaining blood-glucos elevels is especially important because glucose is virtually the only fuel used by the brain, except during prolonged starvation.

3)  Moreover, the glucose from glycogen i sreadily mobilised and is therefore a good source of energy for sudden,strenuous activity.

4)  Unlike fatty acids, the released glucose can provide energy in the absence of oxygen and can thus supply energy for anaerobic activity.

The two major sites of glycogen storage are the liver and skeletal muscle. The concentration of glycogen is higher in the liver than in muscle (10% versus 2% by weight), but more glycogen is stored in skeletal muscle overall because of its much greater mass. Glycogen is present in the cytosol in the form of granules ranging in-

 Figure-2- showing glycogen granules

-diameter from 10 to 40 nm .In the liver,glycogen synthesis and degradation are regulated to maintain blood-glucose levels as required to meet the needs of the organism as a whole. In contrast,in muscle, these processes are regulated to meet the energy needs of the muscle itself.

An Overview of Glycogen Metabolism

Glycogen degradation and synthesis are relatively simple biochemical processes.

Glycogen degradation consists of three steps:

 (1) The release of glucose 1-phosphate from glycogen,

(2) The remodelling of the glycogen substrate to permit further degradation, and

 (3) The conversion of glucose 1-phosphate into glucose 6-phosphate for further metabolism. (See Figure-2)

The glucose 6-phosphate derived from the breakdown of glycogen has three fates –

(a) It is the initial substrate for Glycolysis,

(b) It can be processed by the pentose phosphate pathway to yield NADPH and ribose derivatives; and

(c) It can be converted into free glucose for release into the bloodstream.

 This conversion takes place mainly in the liver and to a lesser extent in the intestines and kidneys.

 

 Figure -3- Showing glycogen degradation and the fate of glucose-6- phosphate.

Glycogen synthesis -requires an activated form of glucose, uridine diphosphate glucose (UDP-glucose), which is formed by the reaction of UTP and glucose 1-phosphate. UDP-glucose is added to the nonreducing end of glycogen molecules. Branching takes place after the addition of at least 12 glucose residues. As is the case for glycogen degradation, the glycogen molecule must be remodeled for continued synthesis.

The regulation of these processes is quite complex. Several enzymes taking part in glycogen metabolism allosterically respond to metabolites that signal the energy needs of the cell. These allosteric responses allow the adjustment of enzyme activity to meet the needs of the cell in which the enzymes are expressed. Glycogen metabolism is also regulated by hormonally stimulated cascades that lead to the reversible phosphorylation of enzymes, which alters their kinetic properties. Regulation by hormones allows glycogen metabolism to adjust to the needs of the entire organism. By both these mechanisms,glycogen degradation is integrated with glycogen synthesis. 

Figure-4 – showing an overview of glycogen metabolism

 Pathophysiology of Von –Gierke’s disease

Because of insufficient G6Pase activity,G6P cannot be converted into free glucose, but G6P is metabolised to lactic acid or incorporated into glycogen. In this way, large quantities of glycogen are formed and stored as molecules with normal structure in the cytoplasm of hepatocytes and renal and intestinal mucosa cells; therefore, enlarged liver and kidneys dominate the clinical presentation of the disease.

The chief biochemical alteration is hypoglycemia, while secondary abnormalities are hyperlactatemia, metabolic acidosis, hyperlipidemia, and hyperuricemia.

Hypoglycemia- The deficiency of G6Pase blocks the process of glycogen degradation and gluconeogenesis in the liver, preventing the production of free glucose molecules. As a consequence, patients with GSDtype I have fasting hypoglycemia. Hypoglycemia inhibits insulin secretion and stimulates glucagon and cortisol release.

Hyperlactatemia and acidosis- Undegraded G6P is metabolised to lactate, which is used in the brain as an alternative source of energy. The elevated blood lactate levels cause metabolic acidosis.

Hyperuricemia- Blood uric acid levels are raised because of the increased endogenous production and reduced urinary elimination caused by competition with the elevated concentrations of lactate, which should be excreted.

Hyperlipidemia- Elevated endogenous triglyceride synthesis and diminished lipolysis causes hyperlipidemia. Triglycerides increase the risk of fatty liver infiltration, which contributes to the enormous amount of liver enlargement. Despite significantly elevated serum triglyceride levels in patients, vascular lesions and atherosclerosis are rare complications.

Incidence

Patients with GSD type I account for 24.6%of all patients with GSD.

Inheritance

Type I glycogen storage disease is an autosomal recessive disorder .As with other genetically determined diseases,GSD type 1 develops during conception, yet the first signs of the disease may appear at birth or later.

 Clinical Manifestations

o  The earliest signs of the disease may develop shortly after birth and are caused by hypoglycemia and lactic acidosis.

o   Convulsions are a leading sign of disease.

o    Frequently,symptoms of moderate hypoglycemia, such as irritability,pallor, cyanosis, hypotonia, tremors, loss of consciousness, and apnoea, are present.

o   A leading sign of GSD type I is enlargement of the liver and kidneys. During the first weeks of life, the liver is normal size. It enlarges gradually thereafter, and in some patients, it even reaches the pubic symphysis. Enlargement of the abdomen due to hepatomegaly can be the first sign noted by the patient’s mother.

o   The patient’s face is characteristically reminiscent of a doll’s face (rounded cheeks due to fat deposition).

o   Mental development proceeds normally.

o   Growth is retarded and children affected with GSD type I never gain the height otherwise expected from the genetically determined potential of their families. The patient’s height is usually below the third percentile for their age. The onset of puberty is delayed.

o   Late complications of disease are renal function disturbance , renal stones, tubular defects, and hypertension, mainly in patients older than 20 years. Renal function deterioration progresses to terminal insufficiency,requiring dialysis and transplantation.

o   Skin and mucous membrane changes include the following:

  • Eruptive xanthomas develop on the extensor surfaces of the extremities.
  • Tophi or gouty arthritis may occur. Uric tophi often have the same distribution as xanthomas.
  • Many patients bleed easily, particularly from the nose. This tendency is a result of altered platelet function due to the platelets’ lower adhesiveness. Frequent and, occasionally, prolonged epistaxis may cause anaemia. At times, the bleeding may be so severe that blood transfusions are required.

 

Laboratory Investigations-

GSD type I: Serum glucose and blood pH levels are frequently decreased, while the serum lactate, uric acid,triglyceride, and cholesterol levels are elevated. Urea and creatinine levels might be elevated when renal function is impaired. The following laboratory values should be obtained:

  • Serum glucose and electrolyte levels (Higher anion gap may suggest lactic acidosis.)
  • Serum lactate level
  • Blood pH
  • Serum uric acid level
  • Serum triglyceride and cholesterol levels
  • Gamma glutamyl transferase level (Liver dysfunction)
  • CBC and differential (eg, anaemia, leucopenia, neutropenia)
  • Coagulation- Bleeding and clotting time
  • Urinalysis for aminoaciduria, proteinuria, and microalbuminuria in older patients
  • Urinary excretion levels of uric acid and calcium
  • Serum alkaline phosphatase, calcium, phosphorus, urea, and creatinine levels.

Imaging Studies

  • In GSD type I, liver and kidney ultrasonography should be performed for follow-up of organomegaly.
  •  Abdominal CT scanning or MRI is advised whenever the lesions are large, poorly defined, or are growing rapidly.

Other Tests

  • Glucagon and epinephrine tests do not cause a rise in glucose levels, but plasma levels of lactic acid are raised.
  • Orally administered galactose and fructose (1.75 g/kg) do not increase glucose levels, but plasma lactic acid levels do increase.
  • Glucose tolerance test (1.75 g/kg PO) progressively lowers lactic acid levels over several hours after the administration of glucose.

Treatment

Most children with GSD type I are admitted to the hospital to make a final diagnosis, to manage hepatomegaly or hypoglycemia.

 Because no specific treatment is available,symptomatic therapy is very important.

 Diet 

 The primary goal of treatment is to correct hypoglycemia and maintain a normoglycemic state. The normoglycemic state can be achieved with overnight nasogastric infusion of glucose, parental nutrition, or per oral administration of raw corn starch. Glucose molecules are continuously released by hydrolysis of corn starch in the digestive tract over 4 hours following its intake. The intake of fructose and galactose should be restricted because it has been shown that they can not be converted to glucose but they do increase lactic acid production. Limited intake of lipids is advisable for the existing hyperlipidemia.

 Medication

·     No specific drug treatment is recommended for GSD type I. Appropriately treat concurrent infections with antibiotics.

·     Allopurinol (Zyloprim),a xanthine oxidase inhibitor, therapy can reduce uric acid levels in the bloodand prevent occurrence of gout and kidney stones in adult life.

·    Hyperlipidemia can be reduced by lipid-lowering drugs (eg, 3-hydroxy-3-methylglutaryl coenzyme A [HMG-CoA]reductase inhibitors, fibric acid derivatives).

·     In patients with renal lesions,microalbuminuria can be reduced with Angiotensin-converting enzyme (ACE)inhibitor therapy. In addition to their antihypertensive effects, ACE inhibitors are renoprotective and reduce albuminuria. Nephrocalcinosis and renal calculi can be prevented with citrate therapy.

·  Additionally, for patients with GSD type I,the future may bring Adeno-associated virus vector – mediated gene therapy,which may result in curative therapy,

Complications

  • Bacterial infections and cerebral oedema are caused by prolonged hypoglycemia and metabolic acidosis.
  • Long-term complications encompass growth retardation, hepatic adenomas with a high rate of malignant change, xanthomas, gout, and renal dysfunction. Long-term complications result from metabolic disturbances, mostly hypoglycemia.
  • Acute hypoglycemia may be fatal, and long-term complications include irreversible damage to the CNS.
  • Early death usually caused by acute metabolic complications (eg, hypoglycemia, acidosis) or  bleeding in the course of various surgical procedures

Prognosis

 The prognosis is better than in the past provided that all the available dietary and medical measures are implemented.

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Under aerobic conditions regeneration of cytosolic NAD+ from cytosolic NADH is accomplished by transferring electrons across the mitochondrial membrane barrier to the electron transport chain where the electrons are transferred to oxygen.

There are two different shuttle mechanisms whereby this transfer of electrons across the membrane to regenerate cytosolic NAD+ can be accomplished, the glycerol 3-phosphate shuttle and the malate-aspartate shuttle.

1) The glycerol 3-phosphate shuttle (Figure-1) functions primarily in skeletal muscle and brain. The shuttle takes advantage of the fact that the enzyme glycerol-3-phosphate dehydrogenase exists in two forms, a cytosolic form that uses NAD+ as cofactor and a mitochondrial FAD-linked form.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-1- showing glycerol-3-Phosphate shuttle. G3P- glycerol-3-P, DHAP- Dihydroxy acetone-Phosphate

Cytosolic glycerol-3-phosphate dehydrogenase uses electrons from cytosolic NADH to reduce the glycolytic intermediate dihydroxyacetone phosphate to glycerol 3-phosphate, thereby regenerating cytosolic NAD+. The newly formed glycerol 3-phosphate is released from the cytosolic form of the enzyme and crosses to and is bound to the mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase, which is bound to the cytosolic side of the mitochondrial inner membrane. There the mitochondrial glycerol-3-phosphate dehydrogenase reoxidizes glycerol 3-phosphate to dihydroxyacetone phosphate (preserving mass balance) reducing its FAD cofactor to FADH2. Electrons are then passed  through complex II to coenzyme Q of the electron transport chain and on to oxygen generating two ATP molecules per electron pair and therefore per glycerol -3-phosphate.

2) The malate-aspartate shuttle, however, functions primarily in the heart, liver, and kidney (Figure 2). This shuttle requires cytosolic and mitochondrial forms of malate dehydrogenase and glutamate-oxaloacetate transaminase and two antiporters, the malate-α-ketoglutarate antiporter and the glutamate aspartate antiporter, which are both localized in the mitochondrial inner membrane. In this shuttle cytosolic NADH is oxidized to regenerate cytosolic NADby reducing oxaloacetate to malate by cytosolic malate dehydrogenase.(1)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-2-Showing Malate Aspartate shuttle

Malate is transported into the mitochondrial matrix while α-ketoglutarate is transported out by the malate-α-ketoglutarate antiporter, a seeming mass unbalance(2).

Next malate is oxidized back to oxaloacetate producing NADH from NAD+ in the mitochondrial matrix by mitochondrial malate dehydrogenase (3).

Oxaloacetate cannot be transported per se across the mitochondrial membrane. It is, instead transaminated to aspartate from the NH3 donor glutamate by mitochondrial glutamate-oxaloacetate transaminase (4). Aspartate is transported out of the matrix whereas glutamate is transported in by the glutamate-aspartate antiporter (5)in the mitochondrial membrane, obviating the apparent mass unbalance noted above.

The last step of the shuttle is catalyzed by cytosolic glutamate-oxaloacetate transaminase regenerating cytosolic oxaloacetate from aspartate and cytosolic glutamate from α-ketoglutarate(6) both of which were earlier transported in opposing directions by the malate- α- ketoglutarate antiporter. 

The net effect of this shuttle is to transport electrons from cytosolic NADH to mitochondrial NAD+. Therefore, those electrons can be presented by the newly formed NADH to electron transport system complex I thereby producing three ATPs by oxidative phosphorylation.

Note that depending on which shuttle is used (i.e., which tissue is catalyzing glycolysis) either two or three ATPs are produced by oxidative phosphorylation per triose phosphate going through the latter steps of glycolysis.


 

                                             ATP Formation in the Catabolism of Glucose 

 

Pathway

Reaction Catalyzed by

Method of ATP Formation

ATP per Mol of Glucose

Glycolysis

Glyceraldehyde 3-phosphate dehydrogenase

Respiratory chain oxidation of 2 NADH

6*

Phosphoglycerate kinase

Substrate level phosphorylation

2

Pyruvate kinase

Substrate level phosphorylation

2

 

 Total yield

10

Consumption of ATP for reactions of hexokinase and phosphofructokinase

–2

 

 

Net 8

Citric acid cycle

Pyruvate dehydrogenase

Respiratory chain oxidation of 2 NADH

6

Isocitrate dehydrogenase

Respiratory chain oxidation of 2 NADH

6

α-Ketoglutarate dehydrogenase

Respiratory chain oxidation of 2 NADH

6

Succinate thiokinase

Substrate level phosphorylation

2

Succinate dehydrogenase

Respiratory chain oxidation of 2 FADH2

4

Malate dehydrogenase

Respiratory chain oxidation of 2 NADH

6

 

 

Net 30

 

Total per mol of glucose under aerobic conditions

38

 

Total per mol of glucose under anaerobic conditions

2

 

This assumes that NADH formed in glycolysis is transported into mitochondria by the malate shuttle.

If the Glycerol-phosphate shuttle is used, then only 2 ATP will be formed per mol of NADH. At the step of glyceraldehyde-3-P dehydrogenase-4 ATP will be produced. Hence the total will be-6+30= 36 ATP. ( 6 from Glycolysis and 30 from PDH complex and TCA cycle. Since this shuttle operates. in skeletal muscle and brain, hence the total yield per glucose mol will be 2 ATP less as compared to other tissues.

Clinical Significance

In nonaerobic glycolysis, as in the case when a tissue is subjected to an ischemic episode (i.e., myocardial infarction), neither the ATP produced by the shuttle nor the ATPs produced by normal passage of electrons through the electron transport chain are produced because of oxygen insufficiency.

Therefore glycolysis must increase in rate to meet the energy demand. In damaged tissue this increased rate is compromised. Moreover the shuttle mechanisms to regenerate NADfrom NADH formed by glycolysis are unavailable.

Glycolysis under ischemic conditions satisfies the requirement for NAD+ by reducing pyruvate, the glycolytic end product under normal conditions, to lactate with the reducing equivalents of NADH.

The new end product lactate accumulates in muscle cells under ischemic conditions and damages cell walls with its low pH causing rupture and loss of cell contents such as myoglobin and troponin I. These compounds as well as other end products combine to cause increased cell rupture and pain.

 

 

 

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Vitamin B12 and folic deficiencies always coexist. Deficiency of one vitamin can precipitate the deficiency of the other vitamin also.

Folic acid participates in the transfer of single carbon units. Folate-dependent single-carbon transfer reactions are important in amino acid metabolism and in pathways leading to biosynthesis of DNA, RNA, membrane lipids, and neurotransmitters.

Structure of folic acid

Folic acid is a composite molecule, being made up of three parts: a pteridine ring system (6-methylpterin), para-aminobenzoic acid,and Glutamic acid.

Figure- 1-showing the structure and reduction of folic acid.

The Glutamic acid doesn’t participate in the coenzyme functions of folic acid. Instead, folic acid in the interior of the cell may contain a “chain” of three to eight (3–8) Glutamic acid residues,which serves as a negatively charged “handle” to keep the coenzyme inside cells and/or bound to the appropriate enzymes. The pteridine portion of the coenzyme and the p-aminobenzoic acid portion participate directly in the metabolic reactions of folate.

Reduction of folic acid

To carry out the transfer of 1-carbon units, NADPH must reduce folic acid twice in the cell. The 6-methyl pterin ring is reduced at each of the two N-C double bonds by folate reductase enzyme.

The resulting 5, 6, 7, 8-tetrahydrofolate is the acceptor of 1-carbon groups.(See figure-1)

Forms of folic acid as one carbon carrier

Tetrahydrofolate can carry one-carbon fragments attached to N-5 (Formyl, formimino, or methyl groups), N-10 (formyl) or bridging N-5–N-10(methylene or methenyl groups).

Sources of one carbon fragments

The major point of entry for one-carbon fragments into substituted folates is methylene-Tetrahydrofolate, which is formed by the reaction of glycine, serine,and choline with tetrahydrofolate. Serine is the most important source of substituted folates for biosynthetic reactions. One carbon fragments are also produced from the metabolism of Tryptophan and Histidine.

Utilization of one carbon fragments

Methylene-, methenyl-, and 10-formyl-tetrahydrofolates are interconvertible.

The N5,N10-methylene-tetrahydrofolate can either donate its single-carbon group directly, be oxidized by NADP to the methenyl form, or be reduced by NADH to the methyl form (See figure-2). Depending on the biosynthetic pathway involved, any of these species can donate the 1-carbon group to an acceptor. The methylene form donates its methyl group during the biosynthesis of thymidine nucleotides for DNA synthesis, the methenyl form donates its group as a Formyl group during purine biosynthesis, and the methyl form is the donor of the methyl group to sulfur during methionine formation. When one-carbon folates are not required, the oxidation of  formyl-tetrahydrofolate to yield carbon dioxide provides a means of maintaining a pool of free folate.

                         Figure-2- Showing the interconversion of various one carbon compounds

The conversion of 5,10-methylene-THF into 5-methyl-THF, which is catalyzed by MTHFR (5,10-methylene tetrahydrofolate   reductase), is irreversible. The only way to make further use of 5-methyl-THF and to maintain the folate cycle consists in the vitamin-B12-dependent remethylation of homocysteine to methionine (regenerating THF). The reaction is catalyzed by Methionine synthase. The methyl group transfer is therefore greatly dependent on 5-methyl-THF and the availability of vitamin-B12. In humans, this is the only known direct link of the metabolism of two vitamins; folic acid and vitamin-B12 both need each other ( See figure-3 below).

\

Th

 

 

Figure- 3- showing the interdependence of folic acid and vitamin B12 and the methylation cycle.

Methionine is activated to S-Adenosyl Methionine which acts as a methyl group donor, for a variety of reactions under the activity of Methyl Transferase enzyme. The methylated products serve important functions in the body.

In cases of vitamin-B12 deficiency,it is possible that, in spite of sufficient availability of folates (and5-methyl-THF), an intracellular deficiency of biologically active THF arises. This situation is called a ‘folate trap’ (or methyl group trap) because,on one hand, the concentration of 5-methyl-THF continues to rise and on the other hand, due to it being prevented from releasing methyl groups, a ‘metabolic dead-end situation’ develops, which leads to the inevitable blockage of the methylation cycle. The co-factors for the C1-transfers decrease and replication as well as the cell division rate are reduced.

Hence, the principal problem is the decreasing activity of methionine synthase under vitamin-B12 deficiency with secondary disorders affecting the folate metabolism and insufficient de-novo synthesis of purines and pyrimidines. There is therefore functional deficiency of folate, secondary to the deficiency of vitamin B12.

The deficiency in active folic acids first affects the quickly dividing and highly proliferating hematopoiesis cells in the bone marrow and can even lead to pancytopenia.

Clinically, there is no difference between vitamin-B12 deficiency anemia and folic acid deficiency anemia. If such anemia is treated with vitamin-B12, the blockage is immediately stopped and the blood count quickly normalizes. However, if the anemia is exclusively treated with folic acid, it is simply converted to dihydrofolate and THF.

Long-term therapy using high doses of folic acid could therefore conceal the real cause i.e. pernicious (vitaminB12-deficiency) anaemia for a long time. The serum folate continues to rise(congestion of non-regenerated 5-methyl-THF) while the intracellular folate concentration (erythrocytes) drops. This situation interrupts the methylation cycle with numerous cell processes, among them the synthesis of myelin, the nerve fiber lining, being blocked due to a deficiency of methyl groups.

A long undetected (causal) vitamin-B12-deficiency can therefore result in serious neurological damage.
Exclusive folic acid therapy can thus lead to neurological damage or even cause serious damage progression.

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Case Details

A  4 -year-old boy was brought for consultation for hematuria, edema of lower extremities as well as swollen right leg. He was the 12the born in a poor family, where one previous child died from malnutrition and dehydration in the period of infancy.  The child was fed only with cow’s milk and biscuits.

From the period of five months,the boy manifested irritability, sweating, poor appetite and cried when somebody touched him

At admission the baby was afebrile, pale, and malnourished; his hair was dry and cracked. Clinical evaluation showed no organomegaly, no neurological signs, gingival bleeding was there and only one tooth was present.

Laboratory findings were as follows

Red Blood Cell Count 3.5 million/mm3

Hemoglobin (Hb) 7 g/dl

Haemtocrit (Hct) 30%

Serum Iron low

Liver functional tests were in the normal range.

Ultrasound of kidney was normal.

Doppler of blood vessels of both legs was normal which excluded thrombophlebitis. Swelling of the right leg indicated radiological investigation. Massive subperiosteal hematoma on the right femur, dilatated metaphyses and general osteoporosis had been present on the radiogram.

What is the probable diagnosis for this child ?

Case details- The child is most probably suffering from Scurvy (Vitamin C deficiency). As the history suggests the child had been an ignored child, fed a diet deficient in fruits and vegetables and the signs and symptoms are also typical of scurvy. Bleeding gums, tooth loss, Sub periosteal hematoma and bonychanges are characteristic of scurvy. Iron deficiency anemia  is  also  there which is very common in scurvy due to various reasons.

Thus considering the osteoskeletal manifestations, malnutrition, anemia, irritability and bleeding tendencies as well as the radiological findings, deficiency of vitamin C is concluded.

Scurvy

Scurvy is a state of dietary deficiency of vitamin C (ascorbic acid). The human body lacks the ability to synthesize vitaminC and therefore depends on exogenous dietary sources to meet vitamin C needs. The enzyme, L-gluconolactone oxidase, which would usually catalyze the conversionof L-gluconolactone to L-ascorbic acid, is defective due to a mutation.

Vitamin C – An overview

Vitamin C (ascorbic acid) plays a role in collagen, carnitine,hormone, and amino acid formation. It is essential for wound healing and facilitates recovery from burns. Vitamin C is also an antioxidant, supports immune function, and facilitates the absorption of iron.

Causes of Vitamin C (Ascorbic Acid) Deficiency

Scurvy is caused by a dietary deficiency of vitamin C. The body’s pool of vitamin C can be depleted in 1-3 months.

  • Risk factors include the following:
    • Babies who are fed only cow’s milk during the first year of life are at risk.
    • Alcoholism  and conforming to food fads are risk factors.
    • Elderly individuals who eat a tea-and-toast diet are at risk. Retired people who live alone and those who eat primarily fast food face increased risk of deficiency.
    • Economically disadvantaged persons tend to not purchase foods high in vitamin C (eg, green vegetables, citrus fruits), which results in them being at high risk.
    • More recently, vitamin C deficiency has been noted in refugees who are dependent on external suppliers for their food and have limited access to fresh fruits and vegetables.
    • Cigarette smokers require increased intake of vitamin C because of lower vitamin C absorption and increased catabolism.
    • Pregnant and lactating women and those with thyrotoxicosis require increased intake of vitamin C because of increased utilization.
    • People with anorexia nervosa or anorexia from other diseases such as AIDS or cancer are at increased risk of vitamin C deficiency.
    • People with type 1 diabetes have increased vitamin C requirements, as do those on hemodialysis and peritoneal dialysis.
    • Because vitamin C is absorbed in the small intestine, people with disease of the small intestine such as Crohn, Whipple, and celiac disease are at risk.
    • Iron overload disorders may lead to renal vitamin C wasting.

Pathophysiology

Vitamin C is functionally most relevant for the triple-helix formation of collagen; a vitamin C deficiency results in impaired collagen synthesis. Proline and lysine hydroxylases are required for the post synthetic modification of procollagen to collagen. Vitamin C is necessary as a coenzyme for these hydroxylases. 

Formation of intercellular cement substances in connective tissues, bones, and dentin is defective, resulting in weakened capillaries with subsequent hemorrhage and defects in bone and related structures. Hemorrhaging is a hallmark feature of scurvy and can occur in any organ. Hair follicles are one of the common sites of cutaneous bleeding. 

Bone tissue formation becomes impaired, which, in children, causes bone lesions and poor bone growth. Fibrous tissue forms between the diaphysis and the epiphysis,and costochondral junctions enlarge. Densely calcified fragments of cartilage are embedded in the fibrous tissue. Subperiosteal hemorrhages, sometimes due to small fractures, may occur in children or adults.

Clinical Manifestations

  • Early symptoms are malaise and lethargy.
  • After 1-3 months, patients develop shortness of breath and bone pain.
  • Myalgias may occur because of reduced carnitine production.
  • Other symptoms include skin changes with roughness, easy bruising and petechiae, gum disease, loosening of teeth, poor wound healing, and emotional changes.

Figure-1- showing bleeding gums

  • Dry mouth and dry eyes similar to Sjögren syndrome may occur.
  • In the late stages, jaundice, generalized edema, oliguria, neuropathy, fever, and convulsions can be seen.
  • Vital signs: Hypotension may be observed late in the disease. This may be due to an inability of the resistance vessels to constrict in response to adrenergic stimuli.
  • Skin: Perifollicular hemorrhages (See figure),purpura, and ecchymoses are seen most commonly on the legs and buttocks where hydrostatic pressure is the greatest. Poor wound healing and breakdown of old scars may be seen.
  • Nails: Splinter hemorrhages may occur.

Figure- 2- showing hemorrhages in the nail bed

  • Head and neck: Gum swelling, friability, bleeding, and infection with loose teeth; mucosal petechiae; scleral icterus (late, probably secondary to hemolysis); and pale conjunctiva are seen. Conjunctival hemorrhage, flame-shaped hemorrhages, and cotton-wool spots may be seen. Bleeding into the periorbital area, eyelids, and retrobulbar space also can be seen. Alopecia may occur secondary to reduced disulfide bonding.
  • Chest and cardiovascular: Scorbutic rosary (ie, sternum sinks inward) may occur in children. High-output heart failure due to anemia can be observed. Bleeding into the myocardium and pericardial space has been reported.

Figure-3- showing scorbutic rosary

  • Extremities: Fractures, dislocations, and tenderness of bones are common in children. Bleeding into muscles and joints may be seen. Edema may occur late in the disease.
  • Gastrointestinal: Loss of weight secondary to anorexia is common. 

Figure-4- showing  perifollicular hemorrhages

Diagnosis

Diagnosis is usually made clinically in a patient who has skin or gingival signs and is at risk of vitamin C deficiency 

Laboratory Investigations

A plasma or leukocyte vitamin C level can confirm clinical diagnosis.

    • Scurvy occurs at levels generally less than 0.1 mg/dL.
    • Symptoms occur at levels below 2.5 mg/L, which is considered deficiency.
    • Levels of 2.5-5 mg/L indicate depletion.
    • Levels can be low in patients who have tuberculosis, rheumatic fever, or other chronic illnesses; those who smoke cigarettes; and patients on oral contraceptive drugs.
  • Capillary fragility can be checked by inflating a blood pressure cuff and looking for petechiae on the forearm.
  • Bleeding time, clotting time and Prothrombin time are normal.
  • An Fe deficiency anemia is generally observed.

Imaging Studies

Skeletal x-rays can help diagnose childhood (but not adult) scurvy. Changes are most evident at the ends of long bones, particularly at the knee.

·        Early changes resemble atrophy.

·        Loss of trabeculae results in a ground-glass appearance.

·        The cortex thins.

·        A line of calcified, irregular cartilage(white line of Fraenkel) may be visible at the metaphysis.

·        The epiphysis may be compressed.

·        Healing subperiosteal hemorrhages may elevate and calcify the periosteum.

 Differential Diagnosis

 In adults, scurvy must be differentiated from arthritis, hemorrhagic disorders, gingivitis, and protein-energy malnutrition.

Treatment

Patients should take ascorbic acid at 100mg 3-5 times a day until total of 4 g is reached, and then they should decrease intake to 100 mg daily.
Alternately, ascorbic acid may be taken at 1 g/d for the first 3-5 days followed by 300-500 mg/d for a week. Then the recommended daily allowance is resumed.

  • Divided doses are given because intestinal absorption is limited to 100 mg at one time.
  • Parenteral doses are necessary in those with gastro intestinal malabsorption.

    Diet

    Foods high in vitamin C include the following.

    • Citrus fruits, especially grapefruits and lemons
    • Vegetables, including broccoli, green peppers, tomatoes, potatoes, and cabbage
  • The recommended daily allowance for vitamin C varies. The current recommendation for adults is 120 mg daily, although a dose of 60 mg daily is all that is required to prevent scurvy.
  • Diets high in vitamin C have been claimed to lower the incidence of certain cancers, particularly esophageal and gastric cancers.    


    Toxicity

Taking>2 g of vitamin C in a single dose may result in abdominal pain, diarrhea,and nausea. Since vitamin C may be metabolized to oxalate, it is feared that chronic, high-dose vitamin C supplementation could result in an increased prevalence of kidney stones.. Thus, it is reasonable to advise patients with a past history of kidney stones to not take large doses of vitamin C. There is also an unproven but possible risk that chronic high doses of vitamin C could promote iron overload in patients taking supplemental iron.

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Case Details

A 6-year- old child with progressive hearing loss wasbrought for consultation. History revealed that the child was born normal butprogressively developed loss of hearing and loss of smell.  From the past few months the child wasfinding it difficult to locate the things at night time.

The child had dysmorphic features, a flat bridge of nose,and low-set ears.

On examination, pulse was irregular and the liver was enlarged.Laboratory investigations revealed low levels of plasma cholesterol, HDL andLDL. A diagnosis of Refsum disease was made.

 What is the defectin this disease?

Refsum disease

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 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-tetramethyl hexadecanoicacid) 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.

Reactions-

 

                                                  Phytanic acid

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).

 

 

Figure- showing steps of oxidation of phytanic acid. Pristanic acid is formed by alpha oxidation which subsequently undergoes beta oxidation to yield the final products.

Clinical manifestations

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

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 visualfields

·        Cataract

·        Signs resulting from cerebellar ataxia –

o   Progressive weakness

o   Foot drop

o   Loss of balance

·        Cardiac arrhythmias

·        Some individuals will have shortened bones in their fingers or toes.

·        The children usually have moderately dysmorphic features that may include epicanthal folds, a flat bridge of the nose,and low-set ears.

Laboratory Diagnosis

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

Imaging

Skeletal radiography is required to  estimate bone changes. 

Treatment

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

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.

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Case Details

A 22- year-old diabetic comes to the Accident and Emergency department. She gives a 2-day history of vomiting and abdominal pain. She is drowsy and her breathing is deep and rapid. There is distinctive smell from her breath

What is the most likely diagnosis?

What is the biochemical basis for all the presenting symptoms?
Which laboratory test would you request?
Case discussion The patient is most probably suffering from diabetic ketoacidosis. She is a known diabetic and the presenting symptoms like abdominal pain, vomiting, rapid breathing and distinctive smell of breath, all indicate associated ketoacidosis.
Basic concept Diabetic Ketoacidosis (DKA) is a state of inadequate insulin levels resulting in high blood sugar and accumulation of organic acids and ketones in the blood.  It is a potentially life-threatening complication in patients with diabetes mellitus. It happens predominantly in type 1 diabetes mellitus, but it can also occur in type 2 diabetes mellitus under certain circumstances.

Causes- DKA occurs most frequently in knownDiabetics. It may also be the first presentation in patients who had not been previously diagnosed as diabetics. There is often a particular underlying problem that has led to DKA episode. This may be-

1) Inter current illness such as Pneumonia,Influenza, Gastroenteritis, Urinary tract infection or pregnancy.

2) Inadequate Insulin administration may be due to defective insulin pen device or in young patient intentional missing of dose due to fear of weight gain.

3) Associated myocardial infarction, stroke or use of cocaine

4) Inadequate food intake- may be due to anorexia associated with infective process or due to eating disorder in children. 

Diabetic keto acidosis may occur in those previously known to have diabetes mellitus type 2 or in those who on further investigations turn out to have features of type 2 diabetes (e.g. obesity,strong family history); this is more common in African, African-American and Hispanic people. Their condition is then labelled ”ketosis-prone type 2 diabetes”.

Pathophysiology

DKA results from relative or absolute insulin deficiency combined with counter regulatory hormone excess (Glucagon, Catecholamines, cortisol, and growth hormone). The decreased ratio of insulin to Glucagon promotes Gluconeogenesis,glycogenolysis, and Ketone body formation in the liver, as well as increases in substrate delivery from fat and muscle (free fatty acids, amino acids) to the liver. 

a) Cause of hyperglycemia Uncontrolled IDDM leads to increased hepatic glucose output.First, liver glycogen stores are mobilized then hepatic gluconeogenesis is used to produce glucose. Insulin deficiency also impairs non-hepatic tissue utilization of glucose. In particular in adipose tissue and skeletal muscle,insulin stimulates glucose uptake. This is accomplished by insulin-mediated movement of glucose transporter proteins to the plasma membrane of these tissues.

Reduced glucose uptake by peripheral tissues in turn leads to a reduced rate of glucose metabolism. In addition, the level of hepatic Glucokinase is regulated by insulin. Therefore, a reduced rate of glucose phosphorylation in hepatocytes leads to increased delivery to the blood. Other enzymes involved in anabolic metabolism of glucose are affected by insulin(primarily through covalent modifications). The combination of increased hepatic glucose production and reduced peripheral tissues metabolism leads to elevated plasma glucose levels.

b) Cause of kenosis One major role of insulin is to stimulate the storage of food energy following the consumption of a meal. This energy storage is in the form of glycogen in hepatocytes and skeletal muscle. Additionally, insulin stimulates hepatocytes to synthesize triglycerides and storage of triglycerides in adipose tissue. In opposition to increased adipose storage of triglycerides is insulin-mediated inhibition of lipolysis. In uncontrolled IDDM there is a rapid mobilization of triglycerides leading to increased levels of plasma free fatty acids. 

The free fatty acids are taken up by numerous tissues (however, not the brain) and metabolized to provide energy.Free fatty acids are also taken up by the liver. Normally, the levels of malonyl-CoA are high in the presence of insulin. These high levels of malonyl-CoA inhibit carnitine palmitoyl Transferase I, the enzyme required for the transport of fatty acyl-CoA’s into the mitochondria where they are subject to oxidation for energy production.

Thus, in the absence of insulin,malonyl-CoA levels fall and transport of fatty acyl-CoA’s into the mitochondria increases. Mitochondrial oxidation of fatty acids generates acetyl-CoA which can be further oxidized in the TCA cycle. However, in hepatocytes the majorityof the acetyl-CoA is not oxidized by the TCA cycle but is metabolized into the ketone bodies, Acetoacetate and β-hydroxybutyrate.  TCA cycle is in a state of suppression due to non availability of oxaloacetate which is channeled towards pathway of gluconeogenesis in the absence of Insulin. 

These ketone bodies leave the liver and are used for energy production by the brain, heart and skeletal muscle. In IDDM, the increased availability of free fatty acids and ketone bodies exacerbates the reduced utilization of glucose furthering the ensuing hyperglycemia. Production of ketone bodies, in excess of the body’s ability to utilize them leads to ketoacidosis. In diabetics, this can be easily diagnosed by smelling the breath. A spontaneous breakdown product of Acetoacetate is acetone which is volatilized by the lungs producing a distinctive odor.

c) Causes of Acidosis and hyperventilationThe ketone bodies, however, have a low pH and therefore turn the blood acidic(metabolic acidosis). The body initially buffers this with the bicarbonate buffering system, but this is quickly overwhelmed and other mechanisms to compensate for the acidosis, such as hyperventilation to lower the blood carbon dioxide levels. This hyperventilation, in its extreme form, may be observed as Kussmaul respiration. Ketones, too,participate in osmotic diuresis and lead to further electrolyte losses. As a result of the above mechanisms, the average adult DKA patient has a total body water shortage of about 6 liters (or 100 ml/kg), in addition to substantial shortages in sodium, potassium, chloride, phosphate, magnesium and calcium. Glucose levels usually exceed 13.8 mmol/l or 250 mg/dl. 

Increased lactic acid production also contributes to the acidosis. The increased free fatty acids increase triglyceride and VLDL production. VLDL clearance is also reduced because the activity of insulin-sensitive lipoprotein lipase in muscle and fat is decreased. Most commonly, DKA is precipitated by increased insulin requirements, as might occur during a concurrent illness. Occasionally, complete omission of insulin by the patient with type 1 DM precipitates DKA.

Clinical manifestations- The symptoms of an episode of diabetic ketoacidosis usually evolve over the period of about 24 hours. Predominant symptoms are nausea and vomiting, pronounced thirst,excessive urine production and abdominal pain that may be severe.

Hyperglycemia is always present .In severe DKA, breathing becomes labored and of a deep, gasping character (a state referred to as “Kussmaul respiration”). The abdomen may be tender to the point that an acute abdomen may be suspected, such as acute pancreatitis, appendicitis or gastrointestinal perforation.  

Coffee ground vomiting(vomiting of altered blood) occurs in a minority of patients; this tends to originate from erosions of the esophagus. In severe DKA, there may be confusion, lethargy, stupor or even coma(a marked decrease in the level of consciousness).

On physical examination -there is usually clinical evidence of dehydration, such as a dry mouth and decreased skin turgor. If the dehydration is profound enough to cause a decrease in the circulating blood volume, tachycardia (a fast heart rate) and low blood pressure may be observed. Often, a ”ketotic”odor is present, which is often described as “fruity”. If Kussmaul respiration is present, this is reflected in an increased respiratory rate.

Small children with DKA are relatively prone to cerebral edema (swelling of the brain tissue), which may cause headache, coma, loss of the pupillary light reflex, and progress to death. It occurs in 0.7–1.0% of children with DKA, and has been described in young adults, but is  very rare in adults. It carries 20–50% mortality. 

 

Figure- showing causes and consequences of DKA

Diagnosis

Investigations-  Diabetic Ketoacidosis may be diagnosed when the combination of hyperglycemia (high blood sugars), ketones on urinalysis and acidosis are demonstrated.

Arterial blood gas measurement is usually performed to demonstrate the acidosis; this requires taking a blood sample from an artery.

In addition to the above, blood samples are usually taken to measure urea and creatinine (measures of kidney function, which may be impaired in DKA as a result of dehydration) and electrolytes.

Furthermore, markers of infection (complete blood count, C-reactive protein) and acute pancreatitis (amylase and lipase) may be measured.

Given the need to exclude infection, chest radiography and urinalysis are usually performed.If cerebral edema is suspected because of confusion, recurrent vomiting or other symptoms, computed tomography may be performed to assess its severity and to exclude other causes such as stroke.

Management

 The main aims in the treatment of diabetic ketoacidosis are replacing the lost fluids and electrolytes while suppressing the high blood sugars and ketone production with insulin.

a) Fluid replacement The amount of fluid depends on the estimated degree of dehydration. If dehydration is sosevere, rapid infusion of saline is recommended to restore circulating volume.

 b) Insulin is usually given continuously.

c) Potassium levels can fluctuate severely during the treatment of DKA, because insulin decreases potassium levels in the blood by redistributing it into cells. Serum potassium levels are initially often mildly raised even though total body potassium is depleted. Hypokalemia often follows treatment. This increases the risk of irregularities in the heart rate. Therefore, continuous observation of the heart rate is recommended, as well as repeated measurement of the potassium levels and addition of potassium to the intravenous fluids once levels fall below 5.3 mmol/l. If potassium levels fall below 3.3 mmol/l, insulin administration may need to be interrupted to allow correction of the hypokalemia.

d) Bicarbonate- Sodium bicarbonate solution is administered to rapidly improve the acid levels in the blood.

Cerebral edema- administration of fluids is slowed; intravenous Mannitol and hypertonic saline (3%) are used.

Prognosis

With appropriate therapy, the mortality of DKA is low (<5%) and is related more to the underlying or precipitating event, such as infection or myocardial infarction. The major non metabolic complication of DKA therapy is cerebral edema,which most often develops in children as DKA is resolving.

The etiology of and optimal therapy for cerebral edema are not well established, but over replacement of free water should be avoided. The other known complications of DKA therapy are, Hypoglycemia, hypokalemia and hypophosphatemia. Venous thrombosis, upper gastrointestinal bleeding, and acute respiratory distress syndrome occasionally complicate DKA.

Prevention of DKA

Following treatment, the physician and patient should review the sequence of events that led to DKA to prevent future recurrences. Foremost is patient education about the symptoms of DKA, its precipitating factors, and the management of diabetes during a concurrent illness.

During illness or when oral intake is compromised, patients should:

(1) frequently measure the capillary blood glucose;

(2) measure urinary ketones when the serum glucose > 16.5 mmol/L (300 mg/dL);

(3) drink fluids to maintain hydration;

(4) continue or increase insulin; and

(5) seek medical attention if dehydration, persistent vomiting, or uncontrolled hyperglycemia develop. Using these strategies, early DKA can be prevented or detected and treated appropriately on an outpatient basis.

DKA IN PREGNANCY-   DKA in pregnancy is of special concern. It tends to occur at lower plasma glucose levels and more rapidly than in non-pregnant patients and usually occurs in the second and third trimesters because of increasing insulin resistance. Fetal mortality rates have previously been reported as high as 30% rising to over 60% in DKA with coma. However with improvements in diabetic care the figure for fetal loss has been reported as low as 9% in some countries. Prevention, early recognition and aggressive management are vitally important to minimize fetal mortality.

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This is an enzyme of Uronic acid pathway. Uronic acid pathway is an alternative pathway for the oxidation of glucose that does not provide a means of producing ATP, but is utilized for the generation of the activated form of glucuronate, UDP-glucuronate which is mainly used for detoxification of foreign chemicals, some endogenous compounds like bilirubin,certain hormones, metabolites and for the synthesis of mucopolysaccharides.This pathway also produces Ascorbic acid in certain animals.

The unutilized Glucuronate produced in this pathway is converted to Xylulose-5 P which is further metabolized through HMP pathway.(Figure-1)

 

 

Figure1-showing the overview of Uronic acid pathway

 

Biological significance of UDP –Glucuronyl Transferase

UDP glucuronate the active form of Glucuronic acid, can readily donate the Glucuronic acid component under the catalytic activity of UDP –Glucuronyl Transferase for the following functions-

1)   Detoxification of foreign compounds and drugs- During detoxification, the glucuronate residues are covalently attached to lipid soluble substances. Since glucuronate residues are strongly polar, their attachment imparts polar character to these substances, making them water-soluble and readily excretable. Bilirubin, certain hormones and drugs are made more polar for renal excretion in this manner. UDP-Glucuronic acid is the Glucuronyl donor, and a variety of glucuronosyl transferases, present in both the endoplasmic reticulum and cytosol, are the catalysts. Molecules such as 2-acetylaminofluorene (a carcinogen), aniline,benzoic acid, meprobamate (a tranquilizer), phenol, and many steroids are excreted as glucuronides. The glucuronide may be attached to oxygen, nitrogen,or sulfur groups of the substrates. Glucuronidation is probably the most frequent conjugation reaction.

2)   Synthesis of Mucopolysaccharides-UDP Glucuronic acid is an essential component of Hyaluronic acid and heparin.

3)   Conjugation of Bilirubin-Bilirubin is nonpolar and would persist in cells (eg, bound to lipids) if not rendered water-soluble. Hepatocytes convert bilirubin to a polar form, which is readily excreted in the bile, by adding Glucuronic acid molecules to it. This process is called conjugation.

The conjugation of bilirubin is catalyzed by a specific Glucuronyl transferase.The enzyme is mainly located in the endoplasmic reticulum, uses UDP-Glucuronic acid as the glucuronosyl donor, and is referred to as bilirubin-UGT. BilirubinMonoglucuronide is an intermediate and is subsequently converted to the diglucuronide. Most of the bilirubin excreted in the bile of mammals is in the form of bilirubin diglucuronide.(Figure-2)

 

 

Figure- 2-showing the conjugation of bilirubin

Clinical significance Diminished activity of Bilirubin UDP Glucuronyl Transferase (UGT)

1) Neonatal “Physiologic Jaundice”

This transient condition is the most common cause of Unconjugated hyper-bilirubinemia. It results from an accelerated hemolysis around the time of birth and an immature hepatic system for the uptake, conjugation, and secretion of bilirubin. Not only is the bilirubin-UGT activity reduced, but there probably is reduced synthesis of the substrate for that enzyme,UDP-glucuronic acid. Since the increased amount of bilirubin is unconjugated,it is capable of penetrating the blood-brain barrier when its concentration in plasma exceeds that which can be tightly bound by albumin (20–25 mg/dL). This can result in a hyperbilirubinemic toxic encephalopathy, or kernicterus,which can cause mental retardation. Because of the recognized inducibility of this bilirubin UGT enzyme system, phenobarbital has been administered to jaundiced neonates and is effective in this disorder. In addition, exposure to blue light (phototherapy) promotes the hepatic excretion of unconjugated bilirubin by converting some of the bilirubin to other derivatives such as maleimide fragments and geometric isomers that are excreted in the bile.

2) Crigler-Najjar Syndrome, Type I; Congenital Nonhemolytic Jaundice

a) TypeI Crigler-Najjar syndrome -is a rare autosomal recessive disorder. It is characterized by severe congenital jaundice(serum bilirubin usually exceeds 20 mg/dL) due to mutations in the gene encoding bilirubin-UGT activity in hepatic tissues. The disease is often fatal within the first 15 months of life. Children with this condition have been treated with phototherapy, resulting in some reduction in plasma bilirubinlevels. Phenobarbital has no effect on the formation of bilirubin glucuronides in patients with type I Crigler-Najjar syndrome. A liver transplant may be curative. It should be noted that the gene encoding human bilirubin-UGT is part of a large UGT gene complex situated on chromosome 2. Many different substrates are subjected to glucuronosylation, so many glucuronosyl transferases are required.

b) Crigler-Najjar Syndrome Type II-This rare inherited disorder also results from mutations in the gene encoding bilirubin-UGT, but some activity of the enzyme is retained and the condition has a more benign course than type I. Serum bilirubin concentrations usually do not exceed 20 mg/dL. Patients with this condition can respond to treatment with large doses of phenobarbital.

3) Gilbert Syndrome

Again,this relatively prevalent condition is caused by mutations in the gene encoding bilirubin-UGT. It is more common among males. Approximately 30% of the enzyme’s activity is preserved and the condition is entirely harmless.

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Transketolase- is an enzyme of Pentose phosphate pathway.

The pentose phosphate pathway (also called Phospho Gluconate pathway or hexose mono phosphate shunt [HMP shunt]) is an alternative route for the metabolism of glucose. It does not lead to formation of ATP but has two major functions: (1) The formation of NADPH for synthesis of fatty acids and steroids, and (2) the synthesis of ribose for nucleotide and nucleic acid formation.

This pathway consists of two phases: the oxidative generation of NADPH and the non oxidative interconversion of sugars (Figure 1). In the oxidative phase, NADPH is generated when glucose 6-phosphate is oxidized to ribose 5-phosphate. This five-carbon sugar and its derivatives are components of RNA and DNA, as well as ATP, NADH, FAD, and coenzyme A.

 

In the nonoxidative phase, the pathway catalyzes the interconversion of three-, four-, five-, six-, and seven-carbon sugars in a series of nonoxidative reactions that can result in the synthesis of five-carbon sugars for nucleotide biosynthesis or the conversion of excess five-carbon sugars into intermediates of the glycolytic pathway. All these reactions take place in the cytosol.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1- showing an overview of HMP pathway.(GAP-Glyceraldehyde-3-Phosphate)

Transketolase transfers the two-carbon unit comprising carbons 1 and 2 of a ketose on to the aldehyde carbon of an aldosesugar. It therefore effects the conversion of a ketose sugar into an aldose with two carbons less and an aldose sugar into a ketose with two carbons more.The reaction requires Mg2+ and thiamine diphosphate (vitamin B1)as coenzyme. The two-carbon moiety transferred is probably glycoaldehyde boundto thiamine diphosphate. Thus, Transketolase catalyzes the transfer of the two-carbon unit from Xylulose 5-phosphate to ribose 5-phosphate, producing the seven-carbon ketose sedoheptulose 7-phosphate and the aldose glyceraldehyde 3-phosphate. These two products then undergo transaldolation.(Figure- 2)

 

 

 

 

 

 

 

 

Figure 2- showing the reaction catalyzedby Transketolase

Transaldolase catalyzes the transfer of a three-carbon Dihydroxyacetone moiety (carbons 1–3) from the ketose sedoheptulose 7-phosphate onto the aldose glyceraldehyde 3-phosphate to form the ketose fructose 6-phosphate and the four-carbon aldose Erythrose4-phosphate. (Figure-3)

 

 

 

 

 

 

 

 

Figure 3-showing the reaction catalyzed by Transaldolase

In a further reaction catalyzed by Transketolase, Xylulose5-phosphate serves as a donor of glycoaldehyde. In this case Erythrose4-phosphate is the acceptor, and the products of the reaction are fructose6-phosphate and glyceraldehyde 3-phosphate. (Figure 4)

 

 

 

 

 

 

 

 

Figure 4- showing the rearrangement of sugars to form glycolytic intermediates catalyzed by Transketolase

Clinical Significance- Since Transketolase requires the presence of Thiamine pyro phosphate(TPP) as a coenzyme, in the deficiency of TPP, Transketolase activity is grossly affected. Measurement of red blood cell Transketolase activity is an index for the determination of underlying Thiamine deficiency.


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Phospho enol pyruvate carboxy kinase- This is an enzyme of pathway of gluconeogenesis. Gluconeogenesis involves the formation of Glucose or Glycogen from non carbohydrate precursors.The major substrates are the glucogenic amino acids, lactate, glycerol, and propionate. These noncarbohydrate precursors of glucose are first converted into pyruvate or enter the pathway at later intermediates such as oxaloacetate and Dihydroxyacetone phosphate. In glycolysis, glucose is converted into pyruvate; in gluconeogenesis, pyruvate is converted into glucose. However,gluconeogenesis is not a reversal of glycolysis.

Phosphoenol pyruvate is formed from pyruvate by way of oxaloacetate through the action of pyruvate carboxylase and phosphoenolpyruvate carboxykinase.

Mitochondrial pyruvate carboxylase catalyses the carboxylation of pyruvate to oxaloacetate, an ATP-requiring reaction in which the vitamin biotin is the coenzyme. Biotin binds CO2 from bicarbonate as carboxybiotin prior to the addition of the CO2 to pyruvate. 

 Pyruvate carboxylase is a mitochondrial enzyme, whereas the other enzymes of gluconeogenesis are cytoplasmic.

Oxaloacetate, the product of the pyruvate carboxylase reaction, is reduced to malate inside the mitochondrion for transport to the cytosol. The reduction is accomplished by an NADH-linked malate dehydrogenase. When malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD+-linked malate dehydrogenase in the cytosol.

 

Figure- showing the transportation of oxaloacetate outside the mitochondrion in the form of Malate

Phosphoenol pyruvate carboxykinase, catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenol pyruvate 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.

Glucagon, which rises during starvation stimulates the production of phosphoenolpyruvate carboxykinase to increase the rate of gluconeogenesis.

ADP inhibits phosphoenolpyruvate carboxykinase. Hence, gluconeogenesis is favored when the cell is rich in biosynthetic precursors and ATP.
Figure- showing steps of Gluconeogenesis.Gluconeogenesis is not simply a reversal of glycolysis, Alternative reactions are there for the formation of Phosphoenolpyruvate from pyruvate which is finally converted to glucose by a series of reactions.

 

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HMG Co A reductase- 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), is an important control site in cholesterol biosynthesis, this enzyme catalyzes the formation of Mevalonate, the committed step in cholesterol biosynthesis. HMG-CoA reductase is an integral membrane protein in the endoplasmic reticulum and spans the membrane. The active site for this enzyme is found on the cytosolic side of the membrane.

The enzyme catalyzes the irreversible step,

 

Regulation of HMG co A reductase/ cholesterol Biosynthesis- HMG CoA reductase is controlled in multiple ways:

This regulation is mediated primarily by changes in the amount and activity of 3-hydroxy-3-methylglutaryl CoA reductase.

A) Regulation of enzyme activity-

1) Feed back inhibition -HMG-CoA reductase in liver is inhibited by Mevalonate, the immediate product of the pathway, and by cholesterol, the main product. The rate of cholesterol formation is highly responsive to the cellular level of cholesterol.

2) Covalent modification Insulin or thyroid hormone increases HMG-CoA reductase activity, whereas glucagon or glucocorticoids decrease it. Activity is reversibly modified by phosphorylation-dephosphorylation mechanisms, some of which may be cAMP-dependent and therefore immediately responsive to glucagon. Phosphorylation decreases the activity of the reductase.  This enzyme, like acetyl CoA carboxylase(which catalyzes the committed step in fatty acid synthesis, is switched off by an AMP-activated protein kinase. Thus, cholesterol synthesis ceases when the ATP level is low. (Insulin causes dephosphorylation, while glucagon causes phosphorylation).

3) Effect of statins-Becausethe enzyme HMG-CoA reductase is the rate-limiting step of cholesterol biosynthesis,this enzyme is the target for many cholesterol lowering drugs. Statins act by inhibiting HMG-CoA reductase and up-regulating LDL receptor activity. Examples currently in use include atorvastatin, simvastatin, fluvastatin, and pravastatin.

B) Regulation of concentration of HMG Co A reductase- The concentration of HMG Co A Reductase is regulated by three main mechanisms-

i)The rate of synthesis of reductase mRNA(Transcription ) – Transcription of  HMG Co A reductase gene is controlled by the sterol regulatory element binding protein (SREBP).

SREBPs are a family of proteins that regulate the transcription of a range of genes involved in the cellular uptake and metabolism of cholesterol and other lipids. 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 protein 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.Low concentrations of cholesterol increase the level of mRNA for HMG-CoA reductase, whereas high concentrations of cholesterol decrease the mRNA level.

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. It is feed back regulation.Dietary cholesterol also decreases the endogenous cholesterol synthesis. However, it is only hepatic synthesis that is inhibited by dietary cholesterol. .

ii)The rate of translation of reductase mRNA -is inhibited by non sterol metabolites derived from Mevalonate as well as by dietary cholesterol. Reverse occurs when Mevalonate concentration is low, hence translation is enhanced and amount ofHMG Co A reductase is increased.

iii) The degradation of the reductase is stringently controlled. In response to increasing concentrations of sterols such as cholesterol, the enzyme becomes more susceptible to proteolysis. A combination of these three regulatory devices can regulate the amount of enzyme over a 200-fold range.

 

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 a)  Acetyl co A carboxylase-Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonylCo A catalyzed by Acetyl co A carboxylase . This irreversible reaction is the committed step in fatty acidsynthesis.

 Acetyl CoA carboxylase, contains a biotin prosthetic group. The carboxyl group of biotin is covalently attached to the  epsilon amino group of a lysine residue, as in pyruvate carboxylase and propionyl CoA carboxylase .The enzyme is a multienzyme protein containing a variable number of identical subunits, each containing biotin, biotin carboxylase, biotin carboxyl carrier protein, and transcarboxylase, as well as a regulatory allosteric site.

 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.

 Regulation of Acetyl co A carboxylase- This enzyme is also the essential regulatory enzyme for fatty acid metabolism.

 a) Hormonal control- The carboxylase is controlled by three global signals glucagon, epinephrine, and insulin that correspond to the overall energy status of the organism. Insulin stimulates fatty acid synthesis by activating the carboxylase, whereas glucagon and epinephrine have the reverse effect.

 b) Allosteric modification-The levels of citrate, palmitoyl Co A, and AMP within a cell also exert control. Citrate, a signal that building blocks and energy are abundant,activates the carboxylase. Palmitoyl CoA and AMP, in contrast, lead to the inhibition of the carboxylase. Citrate facilitates the polymerization of the inactive octamers into active filaments. The level of citrate is high when both acetyl CoA and ATP are abundant.

The stimulatory effect of citrate on the carboxylase is antagonized by palmitoyl CoA, which is abundant when there is an excess of fatty acids. Palmitoyl CoA causes the filaments to disassemble into the inactive octamers.

Palmitoyl CoA also inhibits the translocase that transports citrate from mitochondria to the cytosol, as well as glucose 6-phosphate dehydrogenase, which generates NADPH in the pentose phosphate pathway

 c) Covalent Modification- is carried out by means of reversible phosphorylation and dephosphorylation mediated by hormonal action. AcetylCoA carboxylase is switched off by phosphorylation and activated by dephosphorylation. Epinephrine and glucagon activate protein kinase A to bring about phosphorylation. Hence, these catabolic hormones switch off fattyacid synthesis by keeping the carboxylase in the inactive phosphorylated state.

Insulin stimulates the carboxylase by causing its dephosphorylation by stimulating phosphatase enzyme.

 d)Response to Diet

Fatty acid synthesis and degradation are reciprocally regulated so that both are not simultaneouslyactive. In starvation, the level of free fatty acids rises because hormones such as epinephrine and glucagon stimulate adipose-cell lipase.  In well fed state, Insulin, in contrast, inhibits lipolysis. Acetyl CoA carboxylase also plays a role in the regulation of fatty acid degradation. Malonyl CoA, the product of the carboxylase reaction, is present at a high level when fuel molecules are abundant. Malonyl CoA inhibits carnitine acyl transferaseI, preventing access of fatty acyl CoA s to the mitochondrial matrix in times of plenty.

 e) Long-term control is mediated by changes in the rates of synthesis and degradation of the enzymes participating in fatty acid synthesis.Animals that have fasted and are then fed high-carbohydrate, low-fat diets show marked increases in their amounts of acetyl CoA carboxylase and fatty acid synthase (Another multienzyme complex of fatty acid bio synthetic pathway) within a few days. This type of regulation is known as adaptive control.

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Isoenzymes are enzymes that catalyze identical chemical reactions but are composed of different amino acid sequences. They are sometimes referred to as isozymes. Isoenzymes are produced by different genes and are not redundant despite their similar functions. They occur in many tissues throughout the body and are important for different developmental and metabolic processes.

Isoenzymes are useful biochemical markers and can be measured in the bloodstream to diagnose medical conditions. Isoenzymes can be differentiated from one another using gel electrophoresis. In gel electrophoresis, isoenzyme fragments are drawn through a thick gel by an electric charge. Each isoenzyme has a distinct charge of its own because of its unique amino acid sequence. This enables gel electrophoresis to separate the fragments into bands for identification. Some clinically important isoenzymes are as follows

1) Creatine Kinase(CK, CPK) is an enzyme found primarily in the heart and skeletal muscles, and to a lesser extent in the brain but not found at all in liver and kidney. Small amounts are also found in lungs, thyroid and adrenal glands. Significant injuryto any of these structures will lead to a measurable increase in CK levels. It is not found in red blood cells and its level is not affected by hemolysis.

Normal Value- serum activity varies from 10-50 IU/L at 30°C.

Elevations are found in:

  • Myocardial infarction
  • Crushing muscular trauma
  • Any cardiac or muscle disease, but not myasthenia gravis or multiple sclerosis
  • Brain injury
  • Hypothyroidism
  • Hypokalemia

After myocardial infarction- serum value is found to increase within hours, reaches a peak level in 24- 30 hours and returns to normal level in 2-4 days (usually in 72 hours). CK is a sensitive indicator in the early stages of myocardial ischemia. No increase in activity is found in heart failure and coronary insufficiency. In acute MI, CPK usually rises faster than SGOT and returns to normal faster than the SGOT. 

CK/CPK Isoenzymes
There are three Isoenzymes. Measuring them is of value in the presence of elevated levels of CK or CPK to determine the source of the elevation. Each iso enzyme is a dimer composed of two protomers‘M’ (for muscles) and ‘B’( for Brain). These isoenzymes can be separated by, Electrophoresis or by Ion exchange Chromatography. The three possible iso enzymes are;

Isoenzyme Electrophoretic mobility Tissue of origin  Mean percentage in blood
MM(CK3) Least Skeletal muscle

Heart muscle97-100%MB(CK2)IntermediateHeart muscle0-3% BB(CK1)MaximumBrain0%

  • Normal levels of CK/CPK are almost entirely MM, from skeletal muscle.
  • Elevated levels of CK/CPK resulting from acute myocardial infarction are about half MM and half MB. Myocardial muscle is the only tissue that contains more than five percent of the total CK activity as the CK2 (MB) isoenzyme.
  • Following an attack of acute myocardial infarction, this isoenzyme appears within 4 hours following onset of chest pain, reaches a peak of activity at approximately 24 hours and falls rapidly. MB accounts for 4.5- 20 % of the total CK activity in the plasma of the patients with recent myocardial infarction and the total isoenzyme is elevated up to 20-folds above the normal.
  • Atypical Isoenzymes- In addition to the above three isoenzymes two atypical iso enzymes of CPK have been reported. They are; Macro CK(CK-Macro) and Mitochondrial CK(CK-Mi).

o  Macro CK(CK-Macro)- It is formed by the aggregation of CK-BB with immunoglobulins usually with IgG but sometimes Ig A.It may also be formed by complexing CK-MM with lipoproteins. No specific disease has been found to be associated with this isoenzyme.

o  Mitochondrial CK(CK-Mi)- It is presentbound to the exterior surface of the inner mitochondrial membrane of muscle,liver and brain. It can exist in dimeric form or as oligomeric aggregates having molecular weight of approximately 35,000. It is only present in serum when there is extensive tissue damage causing breakdown of mitochondrial membrane and cell membrane. Thus its presence in serum indicates severe illness and cellular damage. It is not related with any specific disease states but it has been detected in certain cases of malignant tumors.

2) Aspartate amino Transferase (AST)

It is also called as Serum Glutamate Oxalo acetate Transaminase (SGOT). The level is significantly elevated in Acute MI.

Normal Value- 0-41 IU/L at 37°C

In acute MI- Serum activity rises sharply within the first 12 hours, with a peak level at 24 hours or over and returns to normal within 3-5 days. The rise depends on the extent of infarction. Re- infarction results in secondary rise of SGOT.

Prognostic significance- Levels> 350 IU/L are due to massive Infarction(Fatal), 

> 150 IU/L are associated with high mortality and levels < 50IU/L are associated with low mortality.

Other diseases- The rise in activity is also observed in muscle and hepatic diseases. These can be well differentiated from simultaneous estimations of other enzyme activities like SGPT etc, which do not show and rise in activity in Acute MI.

3) Alanine amino transferase (ALT)- Also called serum Glutamate pyruvate transaminase.

Normal serum level ranges between 0-45 IU/L at37oC.

Very high values are seen in Acute hepatitis,toxic or viral in origin. Both ALT and AST rise but ALT> AST. Moderate increase may be seen in chronic liver diseases such as Cirrhosis and Malignancy in liver. A sudden fall in AST level in hepatitis signifies bad prognosis.

4) Lactate dehydrogenase (LDH)

Lactate dehydrogenase catalyzes the reversible conversion of pyruvate and lactate. LDH is essential for anaerobic respiration. When oxygen levels are low,LDH converts pyruvate to lactate, providing a source of muscular energy.

Normal level-  55-140IU/L at 30°C. The levels in the upper range are generally seen in children. LDH level is 100 times more inside the RBCs than in plasma, and therefore minor amount of hemolysis results in false positive result.

In Acute MI-The serum activity rises within 12 to 24 hours, attains a peak at 48 hours (2 to 4 days) reaching about 1000 IU/L and then returns gradually to normal from 8 th to 14 th day. The magnitude of rise is proportional to the extent of myocardial infarction. Serum LDH elevation may persist for more than a week after CPK and SGOT levels have returned to normal levels.

Other diseases-The increase in serum activity of LDH is also seen in hemolytic anemia, hepatocellular damage, muscular dystrophies,carcinoma, leukemias, and any condition which causes necrosis of the body cells. Since the total LDH is increased in many diseases, so the study of Isoenzymes of LDH is of more significance.

Iso enzymes of LDH

LDH enzyme is tetramer with 4 subunits. The subunit may be either H(Heart) or M(Muscle) polypeptide chains. These two chains are the product of 2 different genes. Although both of them have the same molecular weight, there are minor amino acid variations.There can be 5 possible combinations; H4, H3M1, H2M2, H1M3. M4, these are 5different types of isoenzymes seen in all individuals.

No. of Isoenzyme Subunit make up of isoenzyme Electrophoretic mobility at pH8.6 Activity at 60°for 30 minutes Tissue origin Percentage in human serum(Mean)
LDH-1 H4 Fastest Not destroyed Heart muscle 30%
LDH-2 H3M1 Faster Not destroyed. RBC 35%
LDH-3 H2M2 Fast Partly destroyed Brain 20%
LDH-4 H1M3 Slow Destroyed Liver 10%
LDH-5 M4 Slowest Destroyed Skeletal Muscles 5%

Normally LDH- 2(H3M1) level in blood is greater than LDH-1, but this pattern is reversed in myocardial infarction, this is called ‘flipped pattern’. These iso enzymes are separated by cellulose acetate electrophoresis at pH 8.6.

5) Alkalinephosphatase (ALP )-is an enzyme that removes phosphate groups  from organic or inorganic compounds in the body. It is present in a number of tissues including liver, bone, intestine,and placenta. The activity of ALP found in serum is a composite of isoenzymes from those sites and, in some circumstances, placental or Regan isoenzymes. The optimum pH for enzyme action varies between 9-10. It is a zinc containing metalloenzyme and is localized in the cell membranes (ectoenzyme). It is associated with transport mechanism in the liver, kidney and intestinal mucosa.

 

Normal serum Level- of ALP ranges between 40-125 IU/L. In children the upper level of normal value may be more, because of increased osteoblastic activity.

Total Alkaline Phosphatase (ALP)

Serum ALP is of interest in the diagnosis of 2 main groups of conditions-hepatobiliary diseases and bone diseases associated with increased osteoblastic activity.  

Mild increase is observed in pregnancy, due to production of placental enzyme.

A moderate rise in ALP activity occurs inhepatic diseases such as infective hepatitis, alcoholic hepatitis or hepatocellular carcinoma. Moderate elevation of ALP may also be seen in other disorders such as Hodgkin’s disease, congestive heart failure, ulcerative colitis, regional enteritis, and intra-abdominal bacterial infections.

ALP elevations tend to be more marked (more than 3-fold) in extra hepatic biliary obstructions (eg, by stone or cancer of the head of the pancreas) than in intrahepatic obstructions, and the more complete the obstruction, the greater the elevation. With obstruction, serumALP activities may reach 10 to 12 times the upper limit of normal, returning to normal upon surgical removal of the obstruction. The ALP response to cholestatic liver disease is similar to the response o fgamma-glutamyltransferase (GGT), but more blunted. If both GGT and ALP are elevated, a liver source of the ALP is likely.  
The response of the liver to any form of biliary tree obstruction is to synthesize more ALP. The main site of new enzyme synthesis is the hepatocytes adjacent to the biliary canaliculi.

ALP also is elevated in disorders of the skeletal system that involve osteoblast hyperactivity and bone remodeling, suchas Paget’s disease rickets and osteomalacia, fractures, and malignant tumors.

Among bone diseases, the highest level o f ALP activity is encountered in Paget’s disease, as a result of the action of the osteoblastic cells as they try to rebuild bone that is being resorbed by the uncontrolled activity of osteoclasts. Values from 10 to 25 times the upper limit of normal are not unusual. 

Only moderate rises are observed in osteomalacia, while levels are generally normal in osteoporosis. In rickets,levels 2 to 4 times normal may be observed. Primary and secondary hyperparathyroidism are associated with slight to moderate elevations of ALP;the existence and degree of elevation reflects the presence and extent of skeletal involvement. 
Very high enzyme levels are present in patients with osteogenic bone cancer. 

A considerable rise in ALP is seen in children following accelerated bone growth.

Patients over age 60 can have a mildly elevated alkaline phosphatase (1–1½ times normal), while individuals with blood types O and B can have an elevation of the serum alkaline phosphatase after eating a fatty meal due to the influx of intestinal alkaline phosphatase into the blood.

ALP Isoenzymes

Electrophoresis is considered the most useful single technique for ALP isoenzyme analysis. By starch gel electrophoresis at pH 8.6 , at least 6 isoenzyme bands can be visualized.

1) Hepatic ALP isoenzyme- moves fastest towards the anode and occupies the same position as  Alpha 2 globulins. It is associated with biliary epithelium and is elevated in cholestatic processes. Various liver diseases (primary or secondary cancer, biliary obstruction) increase the liver isoenzyme.

2) Bone isoenzyme- It closely follows the hepatic enzyme and occupies the beta region. Osteoblastic bone tumors and hyperactivity of osteoblasts involved in bone remodeling (eg, Paget’s disease)increase the bone isoenzyme. Paget’s disease leads to a striking, solitary elevation of bone ALP.

3) Placental isoenzyme follows bone isoenzyme. It is heat stable isoenzyme and increases during last six weeks of pregnancy.

4) The intestinal isoenzyme-is the slowly moving band and follows the placental isoenzyme. It may be increased in patients with cirrhosis and in individuals who are blood group O or B secretors. Increased levels are also sen in patients undergoing hemodialysis.
  
Atypical ALP isoenzymes (Oncogenic markers)-In addition to 4 major isoenzymes, 2 more abnormal fractions are seen associated with tumors. These are Regan and Nagao isoenzymes. They are also called “Carcino placental ALP”, as they resemble placental isoenzymes.
Regan isoenzyme is elevated in various carcinomas of breast, lungs, colon and ovaries. Highest positivity is observed in carcinoma of ovary and uterus.

Rise in Nagao isoenzyme is observed in metastatic carcinoma of pleural surfaces and adenocarcinoma of pancreas and bile duct.

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Lipids absorbed from the diet and synthesized by the liver and adipose tissue must be transported between various cells and organs for utilization and storage. Since lipids are insoluble in water, the problem of transportation in the aqueous plasma is solved by associating nonpolar lipids (triacylglycerols and cholesteryl esters) with amphipathic lipids(phospholipids and cholesterol) and proteins to make water-miscible Lipoproteins.

General Structure of Lipo protein

 Lipoproteins Consist of a Nonpolar Core & a Single Surface Layer of Amphipathic Lipids

The nonpolar lipid core consists of mainly triacylglycerol and cholesteryl ester and is surrounded by a single surface layer of amphipathic phospholipid and cholesterol molecules (Figure-1). These are oriented so that their polar groups face outward to the aqueous medium. The protein moiety of a lipoprotein is known as an apolipoprotein or apoprotein,constituting nearly 70% of some HDL and as little as 1% of Chylomicons. Some apolipoproteins are integral and cannot be removed, whereas others can be freely transferred to other lipoproteins.

 

Figure-1- showing general structure of lipoprotein

 Classification of Lipoproteins

 Lipoproteins can be classified in three ways-

 1) Based on density

Because fat is less dense than water, the density of a lipoprotein decreases as the proportion of lipid to protein increases.  Lipoproteins with high lipid content will have low density and so float on centrifugation. Those with high protein content sediment easily and have a high density. They are separated by Ultracentrifugation. Depending upon the floatation constant (Sf), Five major groups of lipoproteins have been identified that are important physiologically and in clinical diagnosis. These are

 (i) Chylomicons, derived from intestinal absorption of triacylglycerol and other lipids; Density is generally less than0.95 while the mean diameter lies between 100- 500 nm

 (ii) Very low density lipoproteins(VLDL), derived from the liver for the export of triacylglycerol; density lies between 0.95- 1.006 and the mean diameter lies between 30-80 nm.

 (iii) Intermediate density lipoproteins (IDL) are  derived from the catabolism of VLDL,with a density  ranging intermediate between Very low densityand Low density lipoproteins i.e. ranging between 1.006-1.019 and the meandiameter ranges between 25-50nm.

 (iv)Low-density lipoproteins (LDL), representing a final stage in thecatabolism of VLDL; density lies between 1.019-1.063 and mean diameter lies between 18-28 nm

 (iv) High-density lipoproteins (HDL),involved in cholesterol transport and also in VLDL and chylomicron metabolism. Density ranges between 1.063-1.121 and the mean diameter varies between 5-15 nm. (Table)

 

Figure- 2-showing the relationship of density and mean diameter of lipoproteins

 

Triacylglycerol is the predominant lipid in chylomicron and VLDL, whereas cholesterol and phospholipid are the predominant lipids in LDL and HDL, respectively. (Table)

 2) Based on electrophoretic mobilities

Lipoproteins may be separated according to their electrophoretic properties into alpha , beta, pre-beta,and broad beta lipoproteins. The mobility of a  lipoprotein is mainly dependent upon protein content. Those with higher protein content will move faster towards the anode and those with minimum protein content will have minimum mobility.

 HDL are alpha , LDLbeta, VLDL pre-beta, and IDL are broad beta lipoproteins. Free fattyacids and albumin complex although not a lipoprotein is an important lipid fraction in serum and is the fastest moving fraction. Chylomicons remain at the origin since they have more lipid content. VLDL with less protein content than LDL move faster than LDL, this is due to nature of apoprotein present.

Table- showing the composition of lipoproteins. As the lipid content increases, density decreases and size increases, that is why Chylomicons are least dense but biggest in size, while HDL are rich in proteins , hence most dense but smallest in size.

 3)Based on nature of Apo- protein content

 One or more apolipoproteins (proteins or polypeptides) are present in each lipoprotein. The major apolipoproteins of HDL Alpha Lipoproteins) are designated A.The main apolipoprotein of LDL (beta -lipoprotein) is apolipoprotein B(B-100), which is found also in VLDL. Chylomicons contain a truncated form of apo B (B-48) that is synthesized in the intestine, while B-100 is synthesized in the liver. Apo B-100 is one of the longest single polypeptide chains known,having 4536 amino acids and a molecular mass of 550,000 Da. Apo B-48 (48% ofB-100) is formed from the same mRNA as apo B-100 after the introduction of a stop signal by an RNA editing enzyme. Apo C-I, C-II, and C-III are smaller polypeptides (molecular mass 7000–9000 Da) freely transferable between several different lipoproteins. Apo E is found in VLDL, HDL, Chylomicons, andchylomicron remnants; it accounts for 5–10% of total VLDL apolipoproteins in normal subjects.

 Functions of Apoproteins- Apolipoproteins carry out several roles: 

 (1) They can form part of the structure of the lipoprotein, eg, apo B is a structural component of VLDL and Chylomicons

 (2)They are enzyme cofactors, e.g. C-II for lipoprotein lipase, A-I for lecithin: cholesterolacyl transferase (LCAT), 

 (3) They act as enzyme inhibitors, eg, apo A-II and apo C-III for lipoprotein lipase, apo C-I for cholesteryl ester transfer protein;

 (4)They act as ligands for interaction with lipoprotein receptors in tissues, eg,apo B-100 and apo E for the LDL receptor, apo A-I for the HDL receptor. The functions of apo A-IV and apo D, however, are not yet clearly defined, although apo D is believed to be an important factor in human neuro degenerative disorders and acts as cholesteryl ester transfer protein required for the exchange of triglycerides and cholesteryl esters between VLDL,chylomicron remnants and HDL.

 All apoproteins are synthesized mainly in liver but small amounts can be synthesized in almost all organs.

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“Although chemical processes involved are the same but Fatty acid synthesis is not simply a reversal of fatty acid oxidation”.             

Fatty acid synthesis seems simply a reversal of the degradative pathway, but it consists of a new set of reactions, 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 malonylACP. The elongation reaction is driven by the release of CO2.
5. The reductant in fatty acid synthesis is NADPH, whereas the oxidants in fattyacid 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.
Over view of fatty acid oxidation- 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. 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. (Figure)

Overview of fatty acid synthesis- 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 (see Figure). 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. (See figure)

 
Figure-Showing the chemical processes involved in fatty acid synthesis and oxidation
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Q.1- 20 Multiple Choice questions                                              (10)

Q.2- Justify the following statements giving complete details-

a) “Presence of Oxygen is an absolute requirement for the functioning of electron transport chain”

 b)“TCA cycle is vital to life and is amphibolic in nature”    

c) “Although chemical processes involved are the same but Fatty acid oxidation is not simply a reversal of fatty acid synthesis. (4+4+2)

Q.3-    Discuss the followings-

a) General structure and classification of lipoproteins         

b) Isoenzymes and their clinical significance

 c)Iron absorption                                                                                 (4+4+2)

 

Q.4-    Describe the significance and details of the reaction catalyzed by each of the followings-         

a) Acetyl co A carboxylase

b) HMG Co A reductase

c) Phospho enol pyruvate carboxy kinase    

 d) Transketolase

e)  UDP Glucuronyl Transferase                                                        (2×5)

 

Q.5-    Discuss the biochemical defect, clinical manifestations and laboratory diagnosis of-

 a)Diabetic ketoacidosis   

 b) Refsum disease

 c) Scurvy                                                                                                       (4+3+3)

Q.6-    Explain the biochemical basis of the followings-           

 a) Excessive ingestion of fruit or sucrose containing diet can cause hyperlipidemia and Hyperuricemia       

  b) Vitamin B12 and folic deficiencies always coexist.

  c) Carnitine deficiency can cause non ketotic hypoglycemia 

  d) Complete oxidation of glucose in skeletal muscles yields 36 ATP molecules while in other tissues the yield is 38 ATPs.      

   e)  Hypoglycemia, Hyperuricemia and ketosis are commonly observed in Von Gierke’s disease.        

                                                                                                                             (2×5)

 

              

                                                                                                             

 


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