Main Menu

Advertisement

Metabolism – Proteins

In a 39-year-old woman who just gave birth, chorionic villus sampling was performed, and a battery of genetic panels was assessed on the new born. One marker indicated a defective Cystathionine -β- Synthase. Which of the following compounds you most likely expect to be elevated in the blood of the infant at birth if the mother was not treated properly?

A) Valine

B) Methionine

C) Threonine

D) Glutamine

E) Cysteine

The correct answer is- B) – Methionine.

This seems to be a case of” Classical homocystinuria”, that occurs due to defective Cystathionine-β- Synthase activity.

Homocystinuria is a disorder of methionine metabolism, leading to an abnormal accumulation of homocysteine and its metabolites (homocystine, homocysteine-cysteine complex, and others) in blood and urine. Normally, these metabolites are not found in appreciable quantities in blood or urine.

Basic Concept-

Most homocysteine, an intermediate compound of Methionine degradation, is normally remethylated to Methionine. This Methionine-sparing reaction is catalyzed by the enzyme Methionine Synthase, which requires a metabolite of folic acid (5-methyltetrahydrofolate) as a methyl donor and a metabolite of vitamin B12 (Methylcobalamin) as a cofactor .Only 20–30% of total homocysteine (and its dimer homocystine) is in free form in the plasma of normal individuals. The rest is bound to protein.

The accumulation of homocysteine and its metabolites is caused by disruption of any of the 3 interrelated pathways of methionine metabolism—

1) Deficiency in the Cystathionine β-synthase (CBS) enzyme (Type I/Classical Homocystinuria)

2) Defective methyl cobalamin synthesis -Type II

3) Abnormality in Methylene tetrahydrofolate reductase (MTHFR) – Type III

Three different cofactors/vitamins—pyridoxal 5-phosphate, methylcobalamin, and folate—are necessary for the 3 different metabolic paths.

The pathway, starting at methionine, progressing through homocysteine, and onward to cysteine, is termed the trans- sulfuration pathway. Conversion of homocysteine back to methionine, catalyzed by MTHFR and methylcobalamin, is termed the remethylation pathway. (Figure-1).

 methionine metab

 

Figure-1- Remethylation and Transsulfuration pathway of methionine metabolism

Defective Cystathionine beta Synthase

 (Classical Homocystinuria)

Inheritance

Homocystinuria is inherited in families as an autosomal recessive trait.

Clinical Manifestations

Infants with this disorder are normal at birth.

  • Clinical manifestations during infancy are nonspecific and may include failure to thrive and developmental delay.
  • The diagnosis is usually made after 3 yr of age, when subluxation of the ocular lens (ectopia lentis) occurs (figure-2). This causes severe myopia and iridodonesis (quivering of the iris), astigmatism, glaucoma, cataract, retinal detachment, and optic atrophy may develop later in life.

Dislocated lens

Figure-2- Dislocated lens

  • Progressive mental retardation is common. Normal intelligence, however, has been reported.
  • Affected individuals with homocystinuria manifest skeletal abnormalities resembling those of Marfan syndrome; they are usually tall and thin with elongated limbs and arachnodactyly (figure-3), scoliosis (figure-4), pectus excavatum, genu valgum, pes cavus, high arched palate, and crowding of the teeth are common.

Arachynodactyly

Figure-3- Arachnodactyly

scoliosis

Figure-4- Scoliosis

  • Patients usually have fair complexions, blue eyes, and a peculiar malar flush.
  • Thromboembolic episodes involving both large and small vessels, especially those of the brain, are common and may occur at any age.

Laboratory findings

  • Amino acid screen of blood and urine – Elevations of both Methionine and homocystine in body fluids are the diagnostic laboratory findings, as in the given case.
  • Total plasma homocysteine is extremely elevated (usually >100μ M).
  • Cystine is low or absent in plasma.
  • The urine screening test for sulfur-containing amino acids, called the cyanide nitroprusside test, can be undertaken;
  • Liver biopsy and enzyme assay are diagnostic
  • Skeletal x-ray reveals generalized osteoporotic changes
  • Skin biopsy with a fibroblast culture (The diagnosis may be established by assay of the enzyme in liver biopsy specimens, cultured fibroblasts)
  • Standard ophthalmic examination is diagnostic for various eye changes
  • Genetic testing can be helpful.

Treatment

  • A high dose of vitamin B6 causes dramatic improvement in patients who are responsive to this therapy.
  • The cysteine deficiency must be made up from dietary sources.
  • Supplementation with pyridoxine, folic acid, B12 or trimethyl glycine (Betaine) reduces the concentration of homocysteine considerably in the bloodstream.
  • A low Methionine diet is also recommended.
  • Existing mental retardation can be improved by symptomatic treatment

As regards other options,

The amino acids such as

Valine, Threonine, Glutamine, or Cysteine are not found in high concentration in blood in conditions of defective Cystathionine-β-synthase activity. Cysteine concentration is rather decreased in such defect.

 

 

 

 

 

 

 

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

A 10-month-old infant was admitted to the hospital in a state of coma. Examination revealed a high body temperature, rapid pulse rate, abnormal electroencephalogram and hepatomegaly. Analysis of his urine showed abnormally high amounts of glutamine and uracil, which suggested a high blood ammonium ion concentration. Considering the data, which enzyme might be defective in this patient?

A. Arginase

B. Carbamoyl phosphate synthetase I

C. Glutamate dehydrogenase

D. Glutaminase

E. Ornithine transcarbamoylase

The correct answer is- E- Ornithine Transcarbamoylase.

High amounts of glutamine in blood, urine or CSF indicate underlying hyperammonemia.

Glutamine is produced during the course of detoxification of ammonia.

Hyperammonemia and Glutamine levels

Ammonia is produced as a result of various metabolic activities. It has to be detoxified immediately else can prove toxic to the brain cells and other tissues.

Mechanism of Ammonia detoxification

1) The first line of defense- Glutamate condenses with ammonia to produce Glutamine. The reaction catalyzed can be represented as follows (figure-1):

Glutamine synthetase

Figure-1- Glutamate to Glutamine conversion is catalyzed by Glutamine synthetase. It is an energy requiring process, ATP acts as a source of energy.

Glutamine is transported to liver.The nitrogen of glutamine can be converted to urea in the liver (Figure -2).

Glutaminase

Figure-2- Hydrolytic release of the amide nitrogen of glutamine as ammonia,is catalyzed by glutaminase in liver. Ammonia thus released is detoxified producing urea.

2) Second line of defense

In conditions of excess ammonia release the second line of defense involves the formation of Glutamate from Alpha keto glutarate (intermediate of TCA cycle) that can be subsequently used for Glutamine synthesis (as explained above and also shown below). The reactions can be represented as:

Detoxification of ammonia

Figure-3-Steps of detoxification of ammonia. In the first step the reaction is catalyzed by Glutamate dehydrogenase, a unique enzyme that can use any of NAD+ or NADP+ as a coenzyme. The reaction is although reversible, but in the liver the reaction is directed towards Alpha keto glutarate formation and the released ammonia is used for the urea formation. In conditions of hyperammonemia, the reaction is favored towards glutamate formation. Glutamate to Glutamine is an energy requiring irreversible reaction catalyzed by Glutamine synthetase.

 

Thus, Glutamine is the end product of detoxification of ammonia; therefore the glutamine levels are directly proportional to the ammonia levels.

Overview of causes of hyperammonemia

There can be congenital or acquired causes of hyperammonemia. Urea cycle disorders are responsible for congenital hyperammonemia, whereas the cirrhosis of liver or other conditions causing liver failure are responsible for Acquired hyperammonemia.

The symptoms of ammonia intoxication include- Slurring of speech, blurring of vision, tremors, convulsions, coma and death. The symptoms are due to energy depletion (TCA cycle suppression due to depletion of alpha ketoglutarate) and hyperexcitation caused as a result of excess serotonin (excitatory)  but decreased GABA (gamma amino butyric acid- inhibitory neurotransmitter) formation.

Hyperammonemia and urea cycle disorders

The deficiencies of urea cycle enzymes cause hyperammonemia and corresponding increase in glutamine levels.

In the given case the clinical manifestations are indicative of hyperammonemia due to a defect in the urea cycle, and the simultaneous rise of uracil in urine indicates excess pyrimidine biosynthesis, that might be due to divergence of unutilized Carbamoyl-P towards the pathway of pyrimidine biosynthesis. The Carbamoyl-phosphate accumulates only if there is deficiency of Ornithine transcarbamoylase. The mitochondrial Carbamoyl-P leaks into the cytoplasm so as to be channeled towards pathway of pyrimidine biosynthesis (figure-4).

OTC-deficiency

Figure-4- Urea cycle disorder causing orotic aciduria. Ornithine transcarbamoylase deficiency leaves excess of Carbamoyl -P that acts as a substrate for pyrimidine biosynthesis.

As regards other options

A. Arginase- Catalyzes the conversion of Arginine to urea and ornithine (figure-4)

B. Carbamoyl phosphate synthetase I- catalyzes the first step of urea cycle. It is a rate limiting enzyme. There is a cytoplasmic Carbamoyl-P synthetase-II, which is the first enzyme of pathway of pyrimidine pathway. Thus Carbamoyl P is produced both in the urea cycle as well in the pyrimidine biosynthetic pathway by different enzymes. The mitochondrial Carbamoyl P, as in this case, can leak to cytoplasm to be subsequently utilized for pyrimidine biosynthesis if there is a block at the level of its utilization forming Citrulline in urea cycle.

C. Glutamate dehydrogenase- The reaction catalyzed by Glutamate dehydrogenase has been shown above (figure-3)

D. Glutaminase- Catalyzes the hydrolysis of Glutamine to glutamate (figure-2).

Thus ornithine transcarbamoylase deficiency is the most appropriate answer. Hyperammonemia and rise in glutamine levels can be observed in other urea cycle disorders as well but the simultaneous rise of orotic acid or uracil nucleotide in urine occurs only in ornithine transcarbamoylase deficiency.

 

 

 

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

Case details

A 55-year-old man suffers from cirrhosis of liver. Toxins such as ammonia are not properly metabolized by the liver and can damage brain. Which of the following compounds should be in highest concentration in brain as a result of detoxification of ammonia?

A. Alpha Ketoglutarate

B.Glutamate

C.Glutamine

D.GABA (Gamma amino butyric acid)

E. Asparagine 

The correct answer is C-Glutamine

Basic concept

Ammonia is produced as a result of various metabolic activities. It has to be detoxified immediately else can prove toxic to the brain cells and other tissues.

Mechanism of Ammonia detoxification

1) The first line of defense- Glutamate condenses with ammonia to produce Glutamine. The reaction catalyzed can be represented as follows (figure-1)

 

 Reaction catalyzed by Glutamine synthetase

Figure-1- Glutamate to Glutamine conversion is catalyzed by Glutamine synthetase. It is an energy requiring process, ATP acts as a source of energy.

Glutamine is transported to liver.The nitrogen of glutamine can be converted to urea in the liver (Figure -2).

 Reaction by Glutaminase

Figure-2- Hydrolytic release of the amide nitrogen of glutamine as ammonia,is catalyzed by glutaminase in liver. Ammonia thus released is detoxified producing urea.

2) Second line of defense

In conditions of excess ammonia release the second line of defense involves the formation of Glutamate from Alpha ketoglutarate (intermediate of TCA cycle) that can be subsequently used for Glutamine synthesis as explained above . The reaction can be represented as follows :

 

 role of glutamate

Figure-3- Ammonia detoxification. In the first step the reaction is catalyzed by Glutamate dehydrogenase, a unique enzyme that can use either of NAD+ or NADP+ as a coenzyme. The reaction is reversible, but in the liver the reaction is directed towards  Alpha ketoglutarate formation and the released ammonia is used for urea synthesis. Glutamate to Glutamine is an energy requiring irreversible reaction catalyzed by Glutamine synthetase.

Implication of Ammonia Intoxication

In the process of detoxification of Ammonia, some of the biologically important compounds are depleted whereas some are produced in highly excess amounts to cause toxicity.

Glutamine is the final product of detoxification which is transported out of the brain cells in exchange with tryptophan. Tryptophan is a precursor of Serotonin, the excess of which causes a state of hyper excitation.

Glutamate and Alpha ketoglutarate are depleted in this process of ammonia detoxification.

Decreased Glutamate, produces less GABA(figure-4), which is an inhibitory transmitter, thus again the result is a state of hyper excitation.

 GABA synthesis

Figure-4- Glutamate is decarboxylated to produce Gamma AminoButyric acid (GABA). The reaction is catalyzed by Glutamate decarboxylase, that requires the presence of Vitamin B6.

The second compound that is decreased is Alpha ketoglutarate, an intermediate of TCA cycle and the depletion of which causes overall suppression of TCA cycle resulting in a state of energy depletion.

The symptoms of ammonia intoxication include- Slurring of speech, blurring of vision, tremors, convulsions,coma and death. The biochemical basis of such symptoms is energy depletion and hyperexcitation due to excess serotonin formation and decreased GABA synthesis. In Cirrhosis of liver, the conversion of ammonia to urea is impaired, the resulting hyperammonemia), proves toxic to brain. In severe liver disorders blood and CSF Glutamine levels are increased (a diagnostic feature of hepatic encephalopathy).

As regards other options

A. Alpha Ketoglutarate- its concentration is decreased, as explained above.

B. Glutamate- Its concentration is also decreased

C. Glutamine concentration is increased.

D.GABA (Gamma amino butyric acid)- levels are decreased.

E. Asparagine- levels are not much affected in ammonia intoxication.

 

 

 

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

Introduction

Transamination interconverts pairs of α-amino acids and α-keto acids. During transamination, the alpha amino group of an amino acid (amino acid R1) is transferred to a α keto acid (keto acid R2), this produces a new keto acid while from the original keto acid, a new amino acid is formed (Figure-1)

Transamination

Figure-1- The transfer of amino group from a donor amino acid to a keto acid for the formation of a new amino acid and a new keto acid

Characteristic features

  • The general process of transamination is reversible and is catalyzed by transaminases, also called amino transferases that require B6-Phosphate as coenzyme.
  • Most of the amino acids act as substrate for the transaminases but the amino acids like lysine, threonine, proline, and hydroxy proline do not participate in transamination reactions.
  • Transamination is not restricted to α -amino groups. The δ-amino group of ornithine and the ε -amino group of lysine—readily undergoes transamination.
  • The most common compounds involved as a donor/acceptor pair in transamination reactions are glutamate and α-ketoglutarate (α-KG), which participate in reactions with many different aminotransferases.

Role of B6 Phosphate as a coenzyme

The coenzyme pyridoxal phosphate (PLP) is present at the catalytic site of aminotransferases, PLP, is a derivative of vitamin B6.

During transamination, bound PLP serves as a carrier of amino groups. Rearrangement forms an α-keto acid and enzyme-bound Pyridoxamine phosphate, which forms a Schiff base with a second keto acid (Figure-2).

Role of B6-P

Figure-2 – The transfer of α-amino group from donor amino acid to Pyridoxal phosphate forms Pyridoxamine phosphate, and a keto acid. The α-amino group is finally passed on to acceptor α-keto acid to form a new amino acid.

Significance of Transamination

Biological significance

  • Transamination is used both for the catabolic as well as anabolic processes.
  • The resultant α-Keto acid can be completely oxidized to provide energy, glucose, fats or ketone bodies depending upon the cellular requirement.
  • Since it is a reversible process, it is also used for the synthesis of non-essential amino acids.

Clinical significance of transaminases.

Serum aminotransferases such as serum glutamate-oxaloacetate-aminotransferase (SGOT) (also called aspartate aminotransferase, AST) and serum glutamate-pyruvate aminotransferase (SGPT) (also called alanine transaminase, ALT) have been used as clinical markers of tissue damage, with increasing serum levels indicating an increased extent of damage.

1) AST is found in the liver, cardiac muscle, skeletal muscle, kidneys, brain, pancreas, lungs, leukocytes, and erythrocytes in decreasing order of concentration.

Reaction catalyzed (figure-3) can be represented as follows-

AST

Figure-3- Reaction catalyzed by AST

Normal serum activity is 0-41 IU/L. The concentration of the enzyme is very high in myocardium. The enzyme is both cytoplasmic as well as mitochondrial in nature.

Diagnostic significance of Aspartate amino transferase

Marker of Acute myocardial infarction- In acute MI the serum activity of AST rises sharply within the first 12 hours, with a peak level of 24 hours or over and returns to normal within 3 to 5 days.

Extra cardiac conditions

  • Elevation of AST can also be seen in Muscle disorders like muscular dystrophies- myositis etc.
  • Increase activity  of AST is also observed in acute pancreatitis, leukemias and acute hemolytic anemias
  • In normal health slight rise of AST level can be observed after  prolonged exercise
  • Alcohol consumption increases AST levels (Alcohol induces AST synthesis)

2) ALT (Alanine amino transferase) is found primarily in the liver.

Reaction catalyzed (figure-4) can be represented as follows-

ALT

Figure-4- Reaction catalyzed by ALT

The normal serum activity ranges between 0-45 IU/L

Diagnostic significance of Alanine amino transferase

1) Liver Diseases- The aminotransferases are normally present in the serum in low concentrations. These enzymes are released into the blood in greater amounts when there is damage to the liver cell membrane resulting in increased permeability. These are sensitive indicators of liver cell injury and are most helpful in recognizing acute hepatocellular diseases such as hepatitis. Any type of liver cell injury can cause modest elevations in the serum aminotransferases. ALT is a better indicator of liver cell injury as compared to AST.

2) Alcoholic liver cell injury- Both AST and  ALT levels are increased.

3) Glucose Alanine cycle- Alanine transaminase has an important function in the delivery of skeletal muscle carbon and nitrogen (in the form of alanine) to the liver (Figure-5).

Glucose Alanine cycle

Figure-5- In skeletal muscle, pyruvate is transaminated to alanine, thus affording an additional route of nitrogen transport from muscle to liver. In the liver alanine transaminase transfers the ammonia to a-KG, forming Glutamate and pyruvate. Glutamate is oxidatively deaminated forming ammonia that is detoxified to form urea. The pyruvate can then be diverted into the pathway of gluconeogenesis. This process is referred to as the glucose-alanine cycle.

 

 

 

 

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

Urea is the end product of nitrogen metabolism

Introduction

  • The continuous degradation and synthesis of cellular proteins occur in all forms of life.
  • Each day, humans turn over 1–2% of their total body protein, principally muscle protein.
  • Of the liberated amino acids, approximately 75% are reutilized.
  • Since excess amino acids are not stored, those not immediately incorporated into new protein are rapidly degraded to amphibolic intermediates.
  • The excess nitrogen forms urea (figure-1).

 Fate of amino acids

Figure-1- Fate of Amino acids, the ammonia released from the amino group of the amino acids is detoxified through urea formation and the carbon skeleton is used either for the synthesis of glucose, ketone bodies or is completely oxidized in TCA cycle to provide energy.

Urea formation (Urea cycle)

Characteristics of urea cycle

  • Urea is the major disposal form of amino groups
  • It accounts for 90% of the nitrogen containing components of urine
  • The urea cycle is the sole source of endogenous production of arginine
  • Urea formation takes place in liver,
  • Urea excretion occurs through kidney

Substances required for urea formation

1) Amino acids- 6 amino acids participate in urea formation, which are-

  • Ornithine
  • Citrulline
  • Aspartic acid
  • Argininosuccinic acid
  • Arginine and
  • N-Acetyl Glutamate

Of the six participating amino acids, N-acetyl glutamate functions solely as an enzyme activator. The others serve as carriers of the atoms that ultimately become urea.

2) Energy

  • Synthesis of 1 mol of urea requires 3 mol of ATP

3) Amino group

  • 1 mol each of ammonium ion and of the α-amino nitrogen of aspartate.

4) Enzymes

  • Five enzymes catalyze the reactions of urea cycle

5) Carbon dioxide

  • CO2 and ammonia both are waste products and are eliminated as urea from the body
  • CO2 is added in the form of bicarbonate ion

 Site of urea formation

  • Urea synthesis is a cyclic process.
  • The first two reactions of urea synthesis occur in the matrix of the mitochondrion, the remaining reactions occur in the cytosol

Steps of urea formation

Step-1- Formation of Carbamoyl-Phosphate (figure-2)

  • Condensation of CO2, ammonia, and ATP to form Carbamoyl phosphate is catalyzed by mitochondrial Carbamoyl phosphate synthase I (CPS-1)
  • Formation of Carbamoyl phosphate requires 2 mol of ATP, one of which serves as a phosphoryl donor.
  • Carbamoyl phosphate synthase I, the rate-limiting enzyme of the urea cycle, is active only in the presence of its allosteric activator N-acetyl glutamate, which enhances the affinity of the synthase for ATP.

Step-2- Formation of Citrulline (figure-2)

  • The Carbamoyl group of Carbamoyl phosphate  is transferred to ornithine, forming Citrulline and Ortho Phosphate
  • The reaction is catalyzed by Ornithine trans Carbamoylase
  • Subsequent metabolism of Citrulline take place in the cytosol.
  • Entry of ornithine into mitochondria and exit of citrulline from mitochondria involves mitochondrial inner membrane transport systems

Clinical Significance

  • Ornithine Transcarbamoylase deficiency causes enhanced excretion of Uracil.
  • Excessive excretion of Uracil or its precursor Orotic acid, results from an accumulation of Carbamoyl phosphate in the mitochondria.
  • In the absence of Ornithine Transcarbamoylase, Carbamoyl phosphate accumulates and leaks in to the cytoplasm, where it can be used to make Carbamoyl Aspartate, the first intermediate in the pathway of pyrimidine nucleotide biosynthesis.

Step-3- Formation of Argininosuccinate (figure-2)

  • Argininosuccinate synthase (ASS) links L- Aspartate and Citrulline via the amino group of aspartate and provides the second nitrogen of urea.
  • The reaction requires ATP, production of argino-succinate is an energetically expensive process, since the ATP is split to AMP and pyrophosphate.
  • The pyrophosphate is then cleaved to inorganic phosphate using pyrophosphatase, so the overall reaction costs two equivalents of high energy phosphate per mole.

Step-4- Cleavage of Argininosuccinate (figure-2)

  • Cleavage of argininosuccinate catalyzed by argininosuccinate lyase (ASL), proceeds with retention of nitrogen in arginine and release of the aspartate skeleton as fumarate.
  • Addition of water to fumarate forms L-malate, and subsequent NAD+-dependent oxidation of malate forms oxaloacetate (figure-3).
  • Transamination of oxaloacetate by glutamate aminotransferase then re-forms aspartate. carbon skeleton of aspartate-fumarate thus acts as a carrier of the nitrogen of glutamate into a precursor of urea

 

 Urea cycle

Figure-2-Steps of urea formation, the first two reactions are mitochondrial and the remaining reactions take place in the cytoplasm.

Relationship of Urea cycle with TCA cycle

 Fate of fumarate

Figure-3- Urea cycle and TCA cycle are linked together through fumarate.

Step-5- Cleavage of Arginine(figure-2)

  • Hydrolytic cleavage of the guanidino group of arginine, catalyzed by liver Arginase (ARG1) releases urea, the other product, Ornithine, reenters liver mitochondria for additional rounds of urea synthesis.
  • Arginine also serves as the precursor of the potent muscle relaxant nitric oxide (NO) in a Ca2+-dependent reaction catalyzed by NO synthase

Regulation of Urea formation

  • The activity of Carbamoyl phosphate synthase I is determined by N-acetyl glutamate, whose steady-state level is dictated by its rate of synthesis from acetyl-CoA and glutamate and its rate of hydrolysis to acetate and glutamate.
  • These reactions are catalyzed by N-acetyl glutamate synthase and N-acetyl glutamate hydrolase, respectively (figure-4).

Formation and degradation of N-Acetyl glutamate 

Figure-4-Formation and hydrolysis of N-Acetyl glutamate

  • Major changes in diet can increase the concentrations of individual urea cycle enzymes 10- to 20-fold.
  • Starvation, for example, elevates enzyme levels, presumably to cope with the increased production of ammonia that accompanies enhanced protein degradation
  • Regulation is also achieved by linkage of mitochondrial glutamate dehydrogenase with CPS-1

Fate of Urea (figure-5)

  • Urea formed in the liver is transported through circulation to kidneys for excretion through urine.
  • It is also transported to intestine where it is decomposed by Urease produced by microbial action.
  • Ammonia liberated by this activity is transported by portal circulation to liver where it is detoxified back to urea.
  • A fraction of ammonia goes to systemic circulation.

 Fate of urea

Figure-5- Urea formed in the liver is mainly excreted through kidney in urine. A fraction of urea is transported to intestine where it is acted upon by bacterial urease. The ammonia thus released either goes through portal circulation to liver for reconversion to urea or enters systemic circulation.

 

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

http://www.slideshare.net/namarta28/amino-acid-catabolism-ii

 

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

Serine

Serine is a hydroxyl group containing, nutritionally non-essential, one carbon donor and glucogenic amino acid.

Structure– Chemically it is α- Amino –β- Hydroxy Propionic acid (figure-1)

 serine

Figure-1- Chemical structure of serine

Synthesis

1)  From Glycine

Glycine and serine can be inter- converted by the action of serine hydroxy methyl transferase (figure-2).

serine to glycine conversion 

Figure-2– Serine to glycine interconversion

2) From 3- Phospho Glycerate (figure-3)

This is the major source of serine in the body. The reactions involved are as follows

Step-1- Dehydrogenation

The reaction is catalyzed by dehydrogenase enzyme

3- Phosphoglycerate——–> 3-Phosphohyroxy pyruvate

Step-2- Transamination

The reaction is catalyzed by phospho serine transaminase enzyme; the alpha amino group is donated by glutamate

3-Phosphohyroxy pyruvate <———–> Phosphoserine

Step-3 Dephosphorylation

The reaction is catalyzed by phosphatase enzyme

Phosphoserine——–> Serine + H3PO4

Synthesis of serine from 3-phosphoglycerate

Figure-3- Synthesis of serine from 3-Phosphoglycerate

3) From Hydroxy pyruvate

Hydroxy pyruvate can be transaminated to form serine

Alanine + Hydroxy pyruvate——–> Pyruvate +Serine

Catabolism of Serine

1)  Non oxidative deamination- Serine can be non oxidatively deaminated to form pyruvate, hence it is glucogenic amino acid (figure-4)

 Non oxidative deamination of serine

Figure-4- The first step of the reaction sequence is catalyzed by dehydratase enzyme that requires B6-P as a coenzyme. The second step is same as oxidative deamination i.e. hydration followed by deamination.

2)  Conversion to Glycine- It can also be converted to glycine depending upon the cellular requirement.

3) Transamination– Serine can be transaminated to form hydroxy pyruvate

Metabolic role of serine

1) One carbon donor- During the conversion of serine to glycine one carbon fragment is transferred to THF forming N5N10 Methylene THF (figure-2).

2) Synthesis of Cysteine- Serine contributes its carbon skeleton for the synthesis of cysteine, the- SH group is donated by Methionine (figure-5). It is a two-step process and the reactions are as follows-

Synthesis of cysteine form serine and methionine 

Figure-5- Synthesis of Cysteine from Serine and Methionine. Homocysteine is a metabolic product of Methionine

3) Synthesis of phosphoproteins- Serine acts as a carrier of phosphate group in phosphoproteins- like casein, vitellin etc.

4) Synthesis of phospholipids- Phosphatidyl serine is biologically an important phospholipid (figure-6).

 phosphatidylserine

Figure-6-Chemical structure of Phosphatidyl serine

5) Synthesis of Sphingosine- Sphingosine the alcohol present in sphingolipids is synthesized by the condensation of Palmitic acid and serine.

6) Synthesis of Ethanolamine- Serine is decarboxylated to form ethanolamine. Ethanolamine can be used either for the synthesis of choline by subsequent methylation reactions or it is as such used for the synthesis of Phosphatidyl ethanolamine, an important phospholipid and a lipotropic agent.

7) Regulation of enzyme activity- The hydroxyl group of serine can be reversibly phosphorylated or dephosphorylated to regulate the enzyme activity (Figure-7).This is covalent modification and is an important mechanism to regulate the activity of many enzymes. For example- Glycogen synthase a key regulatory enzyme for glycogen synthesis gets activated upon dephosphorylation, whereas phosphorylase, an enzyme of glycogen degradation becomes active upon phosphorylation. All the enzymes under the influence of Insulin are active in the dephosphorylated form while the enzymes under the influence of glucagon are active in the phosphorylated form.

 covalent modification

Figure-7- Reversible phosphorylation and dephosphorylation of serine residues for regulation of enzyme activity (covalent modification)

Apart from that serine is also found at the active site of many enzymes- Serine proteases- Coagulation factors and trypsin.

8) Formation of O- glycosidic linkages- In glycoproteins the carbohydrate groups are generally linked either by O-Glycosidic linkages or by N- Glycosidic linkages. The O-Glycosidic linkages are provided by –OH group of either serine or threonine, whereas the N-glycosidic linkages are provided by NH2 group of Asparagine (figure-8).

 Glycosidic linkages

Figure-8- O and N-Glycosidic linkages

9) Incorporation in to tissue proteins- Like other amino acids serine is also incorporated in to  tissue proteins.

10) Glucogenic- Serine undergoes non oxidative deamination to form Pyruvic acid that can be channeled towards pathway of gluconeogenesis.

Serine analogues- Azaserine and Cycloserine are serine analogues. They are used as drugs to inhibit nucleotide biosynthesis. Azaserine is an anticancer drug, whereas Cycloserine is used as an antitubercular drug.

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

Metabolic role of glycine

Although nutritionally glycine is a non-essential amino acid but functionally it is very essential. The important functions of Glycine can be summarized as follows

i) As a constituent of protein

Glycine being a simple amino acid is found where the polypeptide bends as in beta bends or loops.

A striking characteristic of collagen is the occurrence of glycine residues at every third position of the triple helical portion of the alpha chain. This is necessary because glycine is the only amino acid small enough to be accommodated in the limited space available down the central core of the triple helix. This repeating structure, represented as (Gly-X-Y)n (figure-1), is an absolute requirement for the formation of the triple helix. While X and Y can be any other amino acids, about 100 of the X positions are proline and about 100 of the Y positions are hydroxy proline. Proline and hydroxy proline confer rigidity on the collagen molecule.

Collagen structure

Figure-1- Glycine is the most abundantly found amino acid in the structure of collagen. Every third amino acid in the structure of alpha helix is glycine.

ii) One carbon donor

The major pathway of glycine catabolism involves the cleavage of glycine to form CO2, NH4+ and N5N10 Methylene tetra hydro folate (Figure-2). Hence it acts as a donor of one carbon fragment.

Glycine cleavage system

Figure-2- The reaction is catalyzed by glycine cleavage system, N5,N10 Methylene tetra hydro folate acts as a carrier of one carbon fragment

iii) Synthesis of Glutathione

Glutathione is a tripeptide containing three amino acids- Glutamic acid, cysteine and glycine (gamma glutamyl cysteinyl glycine) – Figure-3. It is an important reducing agent, helps in maintaining the integrity of the red blood cells; also acts as a coenzyme in many reduction reactions.

Structure of glutathione

Figure-3- Structure of glutathione, GSH represents the reduced form of glutathione, the –SH group is contributed by cysteine

iv) Synthesis of creatine

Creatine (methyl guanido acetic acid ) is synthesized from three amino acids-Methionine, arginine and glycine. Methyl group is donated by Methionine; guanido group is contributed by Arginine and Acetic acid group comes from glycine. Creatine is stored in the muscle in the phosphorylated from- Creatine-P, a high energy compound. Creatinine is the anhydrous form of creatine (figure-4)

Synthesis of creatine

Figure-4- Role of glycine in the synthesis of creatine. Creatine –P also called phosphocreatine,  is a high energy compound that can be non enzymatically converted to creatinine, the excretable form of creatine.

v) Synthesis of purine nucleotide

Glycine contributes its entire structure for the formation of C4, C5 and N7 of purine nucleus (figure-5)

Components of purine ring

Figure-5- C4,C5 and N7 are derived form Glycine. Three amino acids – Glycine, aspartic acid and glutamine, contribute towards formation of  purine ring.

vi)  Synthesis of bile salts

Cholyl co A derived from cholesterol conjugates with glycine to form Glycocholic acid, a bile acid which is secreted in the bile in the form of sodium salt- Sodium glycocholate(figure-6).

Bile salts are required for the digestion and absorption of fats.
Role of glycine in bile salt formation

Figure- 6-Taurene is derived from cystiene. Glycocholic acid, tauro and glycochenodeoxy cholic acid are primary bile acids. The primary bile acids are converted to secondary bile acids by 7-Alpha dehydroxylation and deconjugation. Deoxy cholic acid and lithocholic acid are secondary bile acids.

vii) Detoxification

Aromatic compounds like benzoic acid obtained from diet are detoxified by conjugation with glycine to form hippuric acid (figure-7) which is excreted in urine.  This reaction takes place exclusively in liver. Hippuric acid excretion test is carried out to determine the functional status of liver.

Many drugs, drug metabolites, and other compounds with carboxyl groups are excreted in the urine as glycine conjugates.

Hippuric acid synthesis

Figure-7- Sodium benzoate is given as a loading dose and the amount of hippuric acid excreted in urine is estimated to determine the functional status of liver.

viii) Synthesis of heme

The two starting materials for heme synthesis are succinyl-CoA, derived from the citric acid cycle in mitochondria, and the amino acid glycine. By a series of reactions heme is synthesized (figure-8) that can be used for the synthesis of hemoglobin and other hemo proteins.

Role of glycine in heme synthesis

 

Figure-8- By a series of steps porphoblinogen is converted to heme.

ix) Synthesis of Glucose

Glycine is glucogenic in nature. During the course of its metabolism it is converted to serine (figure-9) which is non oxidatively deaminated to from pyruvate. Pyruvate is further channeled towards pathway of gluconeogenesis.

Serine and glycine interconversion

Figure-9- Glycine and serine are inter convertible. Serine forms pyruvate upon non oxidative deamination  which is a substrate for gluconeogenesis.

x) Glycine as a neurotransmitter

Glycine itself acts as neurotransmitter to regulate brain activities.

 

Clinical significance

Non ketotic hyperglycinemia- It is due to defect in the glycine cleavage system. Glycine level is found to be higher in blood, C.S.F and urine. Severe mental retardation and convulsions are observed. There is no permanent cure for this disorder only symptomatic treatment can be given.

Glycinuria

The disease is characterized by excessive excretion of glycine in urine. Urinary excretion of glycine ranges from 600-1000 mg/dl. Plasma level of glycine remains normal.

Biochemically there is no enzyme deficiency. The defect is attributed to renal tubular reabsorption of glycine. The tendency to oxalate stone formation is increased.

Hyperoxaluria

The disorder is characterized by continuous high excretion of oxalates. Biochemically it is a protein targeting effect. There is excessive oxalate formation from Glycine. The patients present with progressive bilateral calcium oxalate urolithiasis, recurrent urinary infections and renal damage.

Death occurs in childhood or early adult life from renal failure or hypertension.

 

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

Metabolism of Glycine

Characteristics

  • Simple amino acid
  • Optically inactive
  • Nutritionally non-essential
  • Glucogenic in nature

Chemistry

Chemically glycine is amino acetic acid.

Glycine structure

Figure-1- Structure of glycine, since it lacks an asymmetric carbon atom, hence it is optically inactive.

Synthesis of Glycine

Glycine can be synthesized in three different ways-

1) From Serine

The alpha carbon of serine becomes Alpha carbon of glycine, whereas the beta carbon is channeled to one carbon pool. The reaction can be represented as –

Serine to glycine conversion

Figure-2- The reaction is catalyzed by Serine hydroxy methyl transferase, tetra hydrofolate is converted to N5, N10 Methylene tetra hydro folate

2) From Threonine

Glycine can also be synthesized from threonine by the action of threonine aldolase

Threonine to glycine conversion

3) De novo synthesis

Glycine can also be synthesized from its precursor molecules i.e. from CO2, NH4+ and one carbon unit. The reaction is catalyzed by Glycine synthase system. (Figure-3).

Glycine synthase system

Figure-3- The reaction is reversible; the same enzyme catalyzes the degradation of glycine also.

Catabolism of Glycine

1) Oxidative deamination

Glycine undergoes oxidative deamination. The reaction is catalyzed by Glycine oxidase, an enzyme that requires FAD as a coenzyme (figure-4). The reduced form of FAD (FADH2) is not oxidized through electron transport chain, it is oxidized at the expense of molecular oxygen forming H2O2.. The decomposition of H2O2.takes place by Catalase forming water and molecular oxygen that can be reutilized. That is the reason that amino acid oxidases and catalases are found together so as to decompose H2O2 quickly as soon as it is generated.

Oxidative deamination of glycine

Figure-4- The reaction proceeds through two steps, initially an imino acid is formed that undergoes hydration and deamination to produce glyoxalate.

2) Transamination- Like other amino acids, Glycine can undergo transamination to form Alpha keto acid (Glyoxalate)- Figure-5. The reaction is catalyzed by Alanine glyoxalate transaminase that requires B6-P as a coenzyme.

Fate of glyoxalate- Glyoxalate can undergo decarboxylation to produce formate that enters one carbon pool, hence this way glycine is a one carbon donor .

Alternatively glyoxalate can also be converted to oxalate by oxidation

Clinical Significance

Genetic defects in alanine-glyoxalate transaminase (either low activity or, rarely, a mutation that leads to the enzyme being in mitochondria rather than peroxisomes) results in hyperoxaluria. The glyoxalate formed by glycine oxidase cannot be recycled to glycine by transamination, but accumulates, and is a substrate for oxidation catalyzed by lactate dehydrogenase, forming oxalate. Oxalate crystallizes in the liver and kidneys, leading, to stone formation in urinary tract or  in severe cases, to early death.

Transamination of glycine

Figure-5- Glyoxalate is the end product of both transamination as well as oxidative deamination reactions

3) Formation of serine- Glycine can be converted to serine, which by non oxidative deamination can produce pyruvate, thus glycine can be considered glucogenic (figure-6).

Non oxidative deamination of serine

Figure- 6- The first step of the reaction sequence is catalyzed by dehydratase enzyme that requires B6-P as a coenzyme. The second step is same as oxidative deamination i.e. hydration followed by deamination.

4) Glycine cleavage- Major pathway involves the cleavage of glycine to form CO2, NH4+and N5N10 Methylene tetra hydro folate. The reaction is reversible and the glycine cleavage system is a multienzyme complex comprising of-

i) Glycine decarboxylase- P protein

ii) Amino methyl transferase- T protein

iii) Hydrogen carrier protein- H protein

iv) Dihydrolipoyl dehydrogenase- L -Protein

The net reaction can be represented as follows-

Glycine + H4folate + NAD+ ↔ 5,10-methylene-H4folate + CO2 + NH3 + NADH + H+

To be continued….

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

http://www.slideshare.net/namarta28/amino-acid-catabolism-part1

 

 

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

Case details

A 12-year-old boy was admitted to the hospital with a red scaly rash and mild cerebellar ataxia. His mother thought that the boy is suffering from Pellagra because the same symptoms in her older daughter had been so diagnosed earlier. The boy did not have the usual dietary deficiency form of Pellagra, but large amounts of free amino acids were found in his urine. When the older daughter had a recurrent attack of ataxia, it was found that her urine also contained excessive amount of amino acids. Two other siblings had the sane aminoaciduria; however four others were normal. The parents were asymptomatic, but a family history revealed that they were first cousins.

Assuming that this defect is inherited, what abnormality could account for the unusual amount of amino acids in urine and what is its relationship with pellagra like rashes?

How can this defect be treated?

Case Discussion

The boy is suffering from Hart Nup Disease.

Hartnup disease is an autosomal recessive disorder caused by impaired neutral (i.e., mono amino mono carboxylic) amino acid transport in the apical brush border membrane of the small intestine and the proximal tubule of the kidney. Patients present with pellagra like skin eruptions, cerebellar ataxia, and gross aminoaciduria.

The disorder was first observed in the Hartnup family of London; 4 of the 8 family members presented with aminoaciduria, a rash resembling pellagra, and cerebellar ataxia.

Hart Nup Disease

Inheritance

 Hartnup disease is inherited as an autosomal recessive trait. Heterozygote are normal. Consanguinity is common. The causative gene, SLC6A19 is defective.Mutations in the SLC6A19 gene, which encodes the B0 AT1, sodium-dependent, chloride-independent, neutral amino acid transporter, cause a failure of the transport of neutral amino acids in the small intestine and the renal tubules (Figure-1)

Pathogenesis

A person with Hartnup disease cannot absorb amino acids properly from the intestine (Figure-1) and from tubules in the kidneys. Excessive amounts of amino acids, such as Tryptophan, are excreted in the urine. The body is thus left with inadequate amounts of amino acids, which are the building blocks of proteins.

 

Tryptophan Absorption

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-1- Showing the mechanism of absorption of Tryptophan through a specific transporter.

With too little Tryptophan in the blood, the body is unable to make a sufficient amount of the B-complex vitamin niacinamide, (Figure-2 and 3) particularly under stress when more vitamins are needed. As a result, tryptophan and niacin deficiencies generate similar symptoms.

 

 Tryptophan metabolism

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-2- Showing tryptophan metabolism. Tryptophan is mainly metabolized through Kynurenine Anthranilate pathway(Major pathway), A fraction of tryptophan is  also metabolized alternatively to produce niacin . 60 mg of tryptophan produces 1 mg of niacin.

 

Amino acids are retained within the intestinal lumen, where they are converted by bacteria to indolic compounds that can be toxic to the CNS. Tryptophan is converted to Indole in the intestine. Following absorption, indole is converted to 3-hydroxyindole (ie, indoxyl, indican) in the liver, where it is conjugated with potassium sulfate or glucuronic acid. Subsequently, it is transported to the kidneys for excretion (i.e., Indican uria).

Other Tryptophan degradation products, including kynurenine and serotonin,(Figure-3) are also excreted in the urine. Tubular renal transport is also defective, contributing to gross aminoaciduria. Neutral amino acids are also found in the feces.

 Niacin and serotonin pathway of tryptophan metabolism

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-3- showing the alternative pathways of tryptophan metabolism. Serotonin and niacin deficiencies are mainly responsible for symptoms of hartnup disease

 Frequency

With an overall prevalence of 1 case per 24,000 population, (range, 1 case per 18,000-42,000 population), Hartnup disease ranks among the most common amino acid disorders in humans.

Clinical Manifestations

Hartnup disease manifest during infancy with variable clinical presentation: failure to thrive, photosensitivity, intermittent ataxia, nystagmus and tremors. Symptoms may be triggered by sunlight, fever, drugs, or emotional or physical stress. A period of poor nutrition nearly always precedes an attack. Most symptoms occur sporadically and are caused by a deficiency of niacinamide.

 A rash develops on parts of the body exposed to the sun (Figure-4). Mental retardation, short stature, headaches, unsteady gait, and collapsing or fainting are common. Psychiatric problems (such as anxiety, rapid mood changes, delusions, and hallucinations) may also result. Cutaneous signs usually precede the neurological manifestations.

 

 Pellagra

 

 

 

 

 

 

 

 

 

 

Figure-4- Pellagra like rash due to niacin deficiency

Diagnosis

Urine Analysis –   Neutral amino acids, Tryptophan, Indole derivatives and Tryptophan degradation products are present in urine.

Treatment

A high-protein diet can overcome the deficient transport of neutral amino acids in most patients. Poor nutrition leads to more frequent and more severe attacks of the disease, which is otherwise asymptomatic. Avoiding excessive exposure to sunlight, wearing protective clothing, and using physical and chemical sunscreens are mandatory. Advise patients to avoid other aggravating factors, such as photosensitizing drugs, as much as possible. In patients with niacin deficiency and symptomatic disease, daily supplementation with nicotinic acid or nicotinamide reduces both number and severity of attacks. Neurological and psychiatric treatment is needed in patients with severe CNS involvement.

 Prognosis

 Hartnup disease is manifested by a wide clinical spectrum. Most patients remain asymptomatic, but, in a minority of patients, skin photosensitivity and neurological and psychiatric symptoms may have a considerable influence on quality of life. Rarely, severe CNS involvement may lead to death.

 

 

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

Explain why phenylketonurics are warned against eating products containing the artificial sweetener aspartame (Nutrasweet; chemical name L-Aspartyl-L-Phenylalanine methyl ester)?

Discuss the biochemical defect, clinical manifestations, laboratory diagnosis and treatment of Phenylketonuria.

Aspartame contains Aspartic acid and phenyl alanine. The patients suffering from Phenylketonuria have high levels of phenyl alanine, any further increase in phenylalanine can prove harmful to the patient.

(See the details below )

Phenylketonuria (PKU)

Phenylketonuria (PKU) is an inherited error of metabolism caused by deficiency of the enzyme phenylalanine hydroxylase. Loss of this enzyme results in mental retardation, organ damage, and unusual posture and can, in cases of maternal PKU, result in severely compromised pregnancy.

Incidence

Classic PKU and the other causes of hyperphenylalaninemia affect about one of every 10,000 to 20,000 Caucasian or Oriental births. The incidence in African Americans is far less. These disorders are equally frequent in males and females.

Biochemical defect

Deficiency of the enzyme phenylalanine hydroxylase or of its cofactor (Figure-1)  causes accumulation of phenylalanine in body fluids and the central nervous system (CNS).

 Phenyl alanine hydroxylase

 

Figure-1- showing the conversion of phenyl alanine to Tyrosine. The reaction is catalyzed by phenyl alanine hydroxylase . The enzyme requires tetrahydrobiopterine as a cofactor.

Overview of Phenyl alanine metabolism –Phenyl alanine is metabolized through formation of Tyrosine. The first enzyme in the catabolic pathway for phenylalanine (Figure-1 and 2), phenylalanine hydroxylase, catalyzes the hydroxylation of phenylalanine to tyrosine. Phenylalanine hydroxylase inserts one of the two oxygen atoms of O2 into phenylalanine to form the hydroxyl group of tyrosine; the other oxygen atom is reduced to H2O by the NADH/NADPH also required in the reaction. This is one of a general class of reactions catalyzed by enzymes called mixed-function oxidases , all of which catalyze simultaneous hydroxylation of a substrate by O2 and reduction of the other oxygen atom of O2 to H2O. Phenylalanine hydroxylase requires a cofactor,tetrahydrobiopterin, which carries electrons from NADH/NADPH to O2 in the hydroxylation of phenylalanine. During the hydroxylation reaction the coenzyme is oxidized to dihydrobiopterin (Figure-2). It is subsequently reduced again by the enzyme dihydrobiopterin reductase in a reaction that requires NADH/NADPH.

 Metabolism of phenylalanine

Figure-2- Showing the metabolism of phenylalanine . The different enzyme deficiencies cause different disorders  with different clinical manifestations. Under normal conditions phenylalanine is catabolized to produce fumarate and acetoacetate, thus it is both glucogenic as well as ketogenic.

 

The severity of hyperphenylalaninemia depends on the degree of enzyme deficiency and may vary from very high plasma concentrations (>20mg/dL, or >1200µM, “classic PKU”) to mildly elevated levels (2–6mg/dL or 120–360µM). In affected infants with plasma concentrations over 20mg/dL, excess phenylalanine is metabolized to phenylketones (phenylpyruvate and phenyl acetate  through a secondary pathway of phenylalanine metabolism. In this minor pathway phenylalanine undergoes transamination with pyruvate to yield phenylpyruvate (Figure 3) Phenylalanine and phenylpyruvate accumulate in the blood and tissues and are excreted in the urine: hence the name of the condition, phenylketonuria. Much of the phenylpyruvate is either decarboxylated to produce phenylacetate or reduced to form phenyllactate. Phenylacetate is excreted in  conjugation form with Glutamine that imparts a characteristic odor (Mousy odor) to the urine and that has been used to detect PKU in infants. The accumulation of phenylalanine or its metabolites in early life impairs the normal development of the brain, causing severe mental retardation. Excess phenylalanine may compete with other amino acids for transport across the blood-brain barrier, resulting in a depletion of some required metabolites.

 Alternative pathways of phenylalanine metabolism

Figure-3- Alternative pathways for catabolism of phenylalanine in phenylketonurics. Phenylpyruvate accumulates in the tissues, blood, and urine. Phenylacetate and phenyllactate can also be found in the urine.

Clinical manifestations

CLASSICAL  PKU

  • The affected infant is normal at birth.
  • Mental retardation may develop gradually and may not be evident for the first few months. It is usually severe, and most patients require institutional care if the condition remains untreated. Mental retardation is due to direst toxic effect of phenyl alanine as well as due to impaired formation of catecholamines .
  •  Vomiting, sometimes severe enough to be misdiagnosed as pyloric stenosis, may be an early symptom.
  • Older untreated children become hyperactive with purposeless movements, rhythmic rocking, and athetosis.
  • On physical examination these infants are fairer in their complexion (Figure-5) than unaffected siblings. There is impaired formation of melanin, since tyrosine is a precursor of melanin (Figure-4).
  • Some may have a seborrheic or eczematous rash, which is usually mild and disappears as the child grows older.
  • These children have an unpleasant odor of phenyl acetic acid, which has been described as musty or mousy.
  • There are no consistent findings on neurologic examination. However, most infants are hypertonic with hyperactive deep tendon reflexes.
  •  About 25% of children have seizures, and more than 50% have electroencephalographic abnormalities.
  •  Microcephaly, prominent maxilla with widely spaced teeth, enamel hypoplasia, and growth retardation are other common findings in untreated children.

 Role of tyrosine

 

 

 

 

 

 

 

 

 

Figure-4- showing the metabolic  role of tyrosine. There is impaired formation of melanin and catecholamines , manifested by blond hair, lighter skin and mental retardation.

MILDER FORMS OF PKU

1.Non-PKU Hyperphenylalaninemia.

In any screening program for PKU, a group of infants are identified in whom initial plasma concentrations of phenylalanine are above normal (2mg/dL, 120µM) but less than 20mg/dL (1200µM). These infants do not excrete phenylketones. Clinically, these infants may remain asymptomatic but progressive brain damage may occur gradually with age. These patients have milder deficiencies of phenylalanine hydroxylase or its cofactor tetrahydrobiopterin (BH4) than those with classic PKU.

2.Hyperphenylalaninemia from Deficiency of the Cofactor Tetrahydrobiopterin (BH4)-

In 1–2% of infants with hyperphenylalaninemia, the defect resides in one of the enzymes necessary for production or recycling of the cofactor BH4 .These infants are diagnosed as having PKU, but they deteriorate neurologically despite adequate control of plasma phenylalanine. BH4 is the cofactor for phenylalanine, tyrosine, and Tryptophan hydroxylase. The latter two hydroxylase are essential for biosynthesis of the neurotransmitters dopamine and serotonin.

Plasma phenylalanine levels may be as high as those in classic PKU or in the range of milder forms of hyperphenylalaninemia. Neurologic manifestations, such as loss of head control, truncal hypotonia (floppy baby), drooling, swallowing difficulties, and myoclonic seizures, develop after 3 months of age despite adequate dietary therapy.

3. Maternal Phenylketonuria

A number of women with Phenylketonuria who have been treated since infancy will reach adulthood and become pregnant. If maternal phenylalanine levels are not strictly controlled before and during pregnancy, their offspring are at increased risk for congenital defects and Microcephaly. After birth, these children have severe mental and growth retardation. Pregnancy risks can be minimized by continuing lifelong phenylalanine-restricted diets and assuring strict phenylalanine restriction 2 months prior to conception and throughout gestation.

 PKU

Figure-5- showing  blond hair and eczematous rashes in a child suffering from PKU

DIAGNOSIS

Because of gradual development of clinical manifestations of hyperphenylalaninemia, early diagnosis can only be achieved by mass screening of all newborn infants.

NEONATAL SCREENING FOR HYPERPHENYLALANINEMIA

  • The bacterial inhibition assay of Guthrie,(Figure-6) which was the first and still the most widely used method for the purpose, is being replaced by more precise and quantitative methods (fluorometric and tandem mass spectrometry).
  • Blood phenylalanine in affected infants with PKU may rise to diagnostic levels as early as 4hr after birth even in the absence of protein feeding. It is recommended, however, that the blood for screening be obtained in the first 24–48hr of life after feeding protein to reduce the possibility of false-negative results, especially in the milder forms of the condition
  • Plasma Phenyl Alanine levels In infants with positive results from the screen for hyperphenylalaninemia, diagnosis should be confirmed by quantitative measurement of plasma phenylalanine . A normal blood phenylalanine level is about 1 mg/dl. In classic PKU, levels may range from 6 to 80mg/dl, but are usually greater than 30mg/dl. Levels are somewhat less in the other disorders of hyperphenylalaninemia.  .
  • Identification and measurement of phenylketones in the urine has no place in any screening program. However, in countries and places where such programs are not in effect, identification of phenylketones in the urine by ferric chloride may offer a simple test for diagnosis of infants with developmental and neurologic abnormalities.
  • Once the diagnosis of hyperphenylalaninemia is established, deficiency of cofactor (BH4) should be ruled out in all affected infants.
  • BH4 loading test. An oral dose of BH4 (20mg/kg) normalizes plasma phenylalanine in patients with BH4 deficiency within 4–8hr.
  • Enzyme assay-The activity of Dihydropteridine Reductase can be measured in the dry blood spots on the filter paper used for screening purposes. The other enzymes required for the synthesis of BH4 can also be similarly estimated.

 guthrie test

 Figure-6- showing Guthrie card

Treatment

The goal of PKU treatment is to maintain the blood level of phenylalanine between 2 and 10 mg/dl. Some phenylalanine is needed for normal growth. This requires a diet that has some phenylalanine but in much lower amounts than normal.

High protein foods, such as: meat, fish, poultry, eggs, cheese, milk, dried beans, and peas are avoided. Instead, measured amounts of cereals, starches, fruits, and vegetables, along with a milk substitute are usually recommended.

In some clinics, a phenylalanine ‘challenge’ may be suggested to evaluate whether or not the child continues to require a low phenylalanine diet. This test identifies those few persons with a transient or ‘variant’ form of the disorder.

 No dietary restriction is currently recommended for infants whose phenylalanine levels are between 2–6mg/dL. Plasma concentrations of phenylalanine in treated patients should be maintained as close to normal as possible.

Because phenylalanine is not synthesized by the body, “over treatment” may lead to phenylalanine deficiency manifested by lethargy, failure to thrive, anorexia, anemia, rashes, diarrhea, and even death; moreover, tyrosine becomes an essential amino acid in this disorder and its adequate intake must be ensured.

The current recommendation is that all patients be kept on a phenylalanine-restricted diet for life, in order to promote maximal development and cognitive abilities.

Oral administration of the cofactor (BH4 ) to patients with milder forms of hyperphenylalaninemia from phenylalanine hydroxylase deficiency may produce significant reductions.

 

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

Polyamines

Polyamines are low molecular weight aliphatic polycations, highly charged and ubiquitously present in all living cells. The naturally abundant polyamines are –

1)  Putrescine – H2N–(CH2)4–NH2 (diamine)

 2) Spermidine- H2N–(CH2)4–NH–(CH2)3–NH2 (triamine)

 3) Spermine H2N–(CH2)3–NH–(CH2)4–NH–(CH2)3–NH2 (tetramine)

Significance of Polyamines

The polyamines are involved in a large number of cellular processes. They  exert their role through ionic interactions, owing to their unique structural feature of regularly spaced positive charges. Some of the important functions are as follows-

1) Modulation of chromatin structure

2) Gene transcription and translation

3) DNA stabilization

4) Signal transduction

5) Cell growth and proliferation

6) Membrane stability

7) Functioning of ion channels 

8) Receptor-ligand interactions

9) Pharmacologic doses of polyamines are hypothermic and hypotensive

Since their primary and secondary amino groups are all protonated at physiological pH, putrescine is divalent, spermidine trivalent and spermine tetravalent organic cation. In the cells polyamines interact electrostatically with negatively charged moieties such as DNA, RNA, proteins and phospholipids.The unique feature of polyamine structure compared to inorganic cations like Mg2+ or Ca2+ is that they have positive charges at defined distances and between them methylene groups that can participate in hydrophobic interactions. Thus polyamines form stronger and more specific interactions than inorganic cations.

There is equilibrium between polyamines that are bound to different polyanionic molecules (mainly DNA and RNA) and free polyamines. The free polyamine pool represents 7-10% of the total cellular polyamine content. Only the free intracellular polyamines are available for immediate cellular needs and therefore are subject to strict regulation. Polyamines are maintained within very narrow range because decrease in their concentrations inhibits cell proliferation while excess appears to be toxic.Therefore, the free polyamine pools are regulated in a very fast, sensitive and precise manner.

Synthesis of Polyamines

Polyamine synthesis occurs in the cytoplasm of cells .Polyamines are synthesized from two amino acids: L-Methionine and L-Ornithine (an amino acid that is not incorporated  into tissue proteins, but is an intermediate  of urea cycle).

In mammalian cells, putrescine is formed by decarboxylation of ornithine, a reaction catalyzed by the enzyme ornithine decarboxylase (ODC). Ornithine is available from the plasma and can also be formed within the cell from arginine by the action of arginase. It is possible that arginase, which is much more widely distributed than other enzymes of the urea cycle, is present in extrahepatic tissues to ensure the availability of ornithine for polyamine production. Arginase can, therefore, be thought of as an initial step in polyamine biosynthesis.

For the synthesis of Putrescine , the amino propyl group must be added. This amino propyl moiety is derived from methionine, which is first converted into S-adenosylmethionine and is then decarboxylated. The resulting decarboxylated S-adenosylmethionine is used as an aminopropyl donor in an analogous manner to the use of S-adenosylmethionine itself as a methyl donor. Once it has been decarboxylated, S-adenosylmethionine is committed to Polyamine synthesis. Therefore the concentration of decarboxylated S- adenosylmethionine is kept low and constitutes the rate-limiting factor in spermidine formation. 

Synthesis of spermidine and spermine require the action of two enzymes: first, the S-adenosyl-methionine decarboxylase (AdoMetDC) for the synthesis of decarboxylated S-Adenosyl Methionine, the aminopropyl donor; and second, a transferase enzyme (spermidine synthase or spermine synthase) which catalyze the transfer of the aminopropyl group to the primary amine groups of putrescine or spermidine, respectively (decarboxylated S-Adenosyl Methionine reacts with Putrescine in the presence of Spermidine synthase forming Spermidine and that reacts with another molecule of decarboxylated S-Adenosyl Methionine in the presence of Spemine synthase forming Spermine) (Figure-1)

Ornithine decarboxylase is a B6-P dependent enzyme. It is present in very small amounts in quiescent cells, and its activity can be increased many fold within a few hours of exposure to  hormones, drugs, tissue regeneration, and growth factors. Spermidine  synthase and spermine synthase are discrete enzymes each specific for its own particular substrate.

The other product of the aminopropyl transferase reactions is 5’-methylthioadenosine. Although this nucleoside is produced in stoichiometric amounts with the polyamines, its concentration in the cell is kept low and is rapidly degraded.

Regulation of polyamine biosynthesis

Ornithine decarboxylase and S-Adenosyl decarboxylase are inducible enzymes with short half lives. Hormones like Growth hormone, corticosteroids, testosterone and growth factors increase the activity of Ornithine decarboxylase. Spermidine synthase and spermine synthase are non inducible enzymes.

The activity of S-Adenosyl methionine decarboxylase is inhibited by decarboxylated S-adenosyl Methionine and activated by Putrescine.

Interconversion

An interconversion or recycling pathway converts spermidine and spermine back to putrescine. Spermidine synthase and spermine synthase reactions are effectively irreversible, but it has been known for many years that conversion of spermine into spermidine and spermidine into putrescine can occur in vivo. This interconversion takes place by the action of two enzymes, spermidine-N1-acetyltransferase and polyamine oxidase. The former enzyme uses acetyl CoA to convert spermidine to N1-acetylspermidine and Spermine to N1-acetylspermine. The N1-acetylspermidine or N1-acetylspermine is then oxidized by polyamine oxidase , which cleaves the polyamine at a secondary amino nitrogen to release 3- acetamidopropion aldehyde  and putrescine or spermidine,  depending upon the substrate (Figure-1)

Degradation of Polyamines              

The enzyme polyamine oxidase present in liver peroxisomes oxidizes Spermine to Spermidine, that undergoes oxidation by the same enzyme to form Putrescine. Diamino propane is released, both these are converted to β-amino propane aldehyde. Putrescine is finally oxidized to NH+ and CO2. Major portions of polyamines are excreted in urine as acetylated derivatives (Figure-2)

Clinical Significance

The drug DFMO (Difluoromethyl ornithine) is a powerful inhibitor of polyamine biosynthesis. It inhibits ornithine decarboxylase enzyme and is used for the treatment of African sleeping sickness.

polyamine1

Figure 1. Biosynthesis and interconversion of polyamines. The enzymes catalyzing reactions are: 1.L-methionine S-adenosyltransferase, 2. S-adenosylmethionine decarboxylase , 3. Ornithine decarboxylase , 4. Spermidine synthase , 5. Spermine synthase, 6. Spermidine/spermine N1-acetyltransferase , and 7. Polyamine oxidase.

poly2

 

Figure-2 Showing the degradation of polyamines

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

Case details

An obese, middle-aged man was brought to the emergency room after an automobile accident. The patient stated that he had been short of breath and dizzy just before the accident. There was chest discomfort also.

The patient was admitted for observation and blood samples for enzyme assays were collected periodically. A chest X- ray was taken. And a12-lead ECG was recorded.

On general examination, his blood pressure was 150/90 mm Hg, pulse was 60/min. and he was sweating profusely. There was no evidence of cardiac failure. His initial ECG showed changes in certain leads, indicative of myocardial ischemia. Enzyme profile was normal. The patient was diagnosed to be having Angina. He was immediately shifted to the cardiac care unit. Treatment with nitroglycerine was started to dilate his coronaries.

 What is the rationale behind this treatment with nitroglycerine?

 Case Discussion

 Nitroglycerin, amyl nitrite, (isobutyl nitrite or similar) and other nitrite derivatives are used in the treatment of heart disease: The compounds are converted to nitric oxide, which in turn dilates the coronary artery, thereby increasing its blood supply. These drugs, however, are predominantly vasodilators; dilating peripheral veins and hence reducing venous return and preload to the heart. This reduces the oxygen requirement of the myocardium and subsequently lessens the anginal pain felt with myocardial ischemia.

Nitric oxide- Nitric oxide is a highly reactive gas. It participates in many chemical reactions. Endothelium-derived relaxing factor was originally the name given to several proposed factors causing vasodilatation. The major endothelial derived relaxing factor was later discovered to be nitric oxide (NO).

Nitric oxide synthesis

Nitric oxide is synthesized by nitric oxide synthase (NOS). There are three isoforms of the NOS enzyme:

1) Endothelial (eNOS),

2) Neuronal (nNOS), and

3) Inducible (iNOS) – each with separate functions.

The neuronal enzyme (NOS-1) and the endothelial isoform (NOS-3) are calcium-dependent and produce low levels of gas as a cell signaling molecule. The inducible isoform (NOS-2) is calcium independent and produces large amounts of gas which can be cytotoxic.

NOS oxidizes the guanidine group of L-arginine in a process that consumes five electrons and results in the formation of NO with stoichiometric formation of L-Citrulline. The process involves the oxidation of NADPH and the reduction of molecular oxygen (figure-1).The transformation occurs at a catalytic site adjacent to a specific binding site of L-arginine.

 n1

 

 

 

 

 

 

 

 

Figure-1- showing the formation of Nitric oxide from Arginine, the enzyme is Nitric oxide synthase

Functions of NO

NO is an important regulator and mediator of numerous processes in the nervous, immune and cardiovascular systems, including smooth muscle relaxation. The various functions are as follows-

1) Vasodilatation- Nitric Oxide (NO) is of critical importance as a mediator of vasodilatation in blood vessels. It is induced by several factors, and once synthesized by eNOS it results in activation of guanylate cyclase to form cGMP, which in turn causes phosphorylation of several proteins through activation of c GMP dependent protein kinases that cause smooth muscle relaxation by decreasing the intracellular Ca++ concentration (Figure-2).The vasodilator action of nitric oxide plays a key role in renal control of extra cellular fluid homeostasis and is essential for the regulation of blood flow and blood pressure.

NO also inhibits the aggregation of platelets and thus keeps inappropriate clotting from interfering with blood flow.

Nitric oxide also acts on cardiac muscle to decrease contractility and heart rate. NO contributes to the regulation of cardiac contractility. Emerging evidence suggests that coronary artery disease (CAD) is related to defects in generation or action of NO. Adequate availability of NO prevents the coronary artery disease.

 n2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-2- showing the mechanism of vasodilatation by NO

 2) Erection

The vasodilatatory effect of NO, in turn, also plays a role in development and maintenance of erection. Vasodilatation of blood vessels supplying the corpus cavernosum results in more blood flowing in and hence erection. This is the biological basis of sildenafil (Viagra), which works to inhibit the enzyme phosphodiesterase PDE5 that lowers the cGMP concentration by converting it back to GMP.

3) Other Actions on Smooth Muscles –

The peristaltic movements are aided by the relaxing effect of NO on the smooth muscle in its walls.

NO also inhibits the contractility of the smooth muscle wall of the uterus.  Near term the production of NO decreases.

Release of NO around the glomeruli of the kidneys increases blood flow through them thus increasing the rate of filtration and urine formation.

4) Immune system- Macrophages, certain cells of the immune system, produce nitric oxide in order to kill invading bacteria. In this case, the nitric oxide Synthase is inducible NOS.

Under certain conditions, there is excess production of nitric oxide by macrophages (e.g. during fulminant infections), leading to vasodilatation, probably one of the main causes of hypotension in sepsis. The inducible isoform of nitric oxide synthase is expressed and produces cytotoxic levels of nitric oxide.

5) Neurotransmitter- Nitric oxide also serves as a neurotransmitter between nerve cells. Unlike most other neurotransmitters that only transmit information from a presynaptic to a postsynaptic neuron, the small nitric oxide molecule can diffuse all over and can thereby act on several nearby neurons, even on those not connected by a synapse. It is conjectured that this process may be involved in memory through the maintenance of long-term potentiation. Nitric oxide is an important non-adrenergic, non-cholinergic (NANC) neurotransmitter in various parts of the gastrointestinal tract. It causes relaxation of the gastrointestinal smooth muscle. In the stomach it increases the capacity of the fundus to store food/fluids.

6) Deficiency of Nitric oxide- People with diabetes usually have lower levels of Nitric Oxide than patients without diabetes. Diminished supply of Nitric Oxide can lead to vascular damage, such as endothelial dysfunction and vascular inflammation. Vascular damage can lead to decreased blood flow to the extremities, causing the diabetic patient to be more likely to develop Neuropathy, non-healing ulcers, and be at a greater risk for lower limb amputation.

Thus nitric oxide, is cardio protective, maintains kidney functions, acts as a potent neurotransmitter and has an important role in immune system.

 

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

Q.1- Justify the reasoning that glutamic acid plays a pivotal role in the metabolism of amino acids

Answer- Glutamate occupies a central place in amino acid metabolism. Basically it acts as a collector of amino group of the amino acids. Free ammonia is toxic to the body especially to brain cells, it is transported in the bound form to liver where it is finally detoxified forming urea.

Amino acids not needed as building blocks are degraded to specific compounds. The major site of amino acid degradation in mammals is the liver. The amino group must be removed, in as much as there are no nitrogenous compounds in energy-transduction pathways. Amino group can be transferred (Transamination) or it can be removed in the form of ammonia (Deamination). The α-keto acids that result from amino acids are metabolized so that the carbon skeletons can enter the metabolic main stream as precursors to glucose or citric acid cycle intermediates.

The formation and fate of glutamate and significance of these processes related to metabolism  of amino acids can be explained as follows-

Sources of Glutamate include-

1) Transamination of amino acids

2) Hydrolysis of Glutamine

3) Metabolic product of amino acids

Fate of Glutamate

1) Oxidative deamination to from Alpha keto glutarate

2) Amination  to form Glutamine

3) Decarboxylation to form GABA (Gamma amino butyric acid)

4) Formation of N-Acetyl Glutamate

All processes except GABA formation are involved in the catabolism  of amino acids and transport of amino group or ammonia.

1) Transamination and role of Glutamate

a) General reactions

Aminotransferases catalyze the transfer of an α-amino group from an α-amino acid to an α-keto acid. These enzymes, also called transaminases, generally funnel α-amino groups from a variety of amino acids to α-keto-glutarate for conversion into NH4 +(Figure-1)

Figure-1- showing the transfer of alpha amino  group to an-α keto acid catalyzed by amino transferase

The α -amino group of many amino acids is transferred to α -ketoglutarate to form glutamate, which is then oxidatively deaminated to yield ammonium ion (NH4) (Figure-2)

Figure-2- showing the  general role of glutamate in the transfer of amino group of amino acid that can be subsequently  removed as ammonium ion.

All the amino acids except lysine, threonine, proline, and hydroxyproline participate in transamination. Transamination is readily reversible, and aminotransferases also function in amino acid biosynthesis.

b) Special reactions

1) Aspartate aminotransferase, one of the most important of these enzymes, catalyzes the transfer of the amino group of aspartate to α-ketoglutarate.

2) Alanine aminotransferase catalyzes the transfer of the amino group of alanine to α -ketoglutarate.

2) Deamination of glutamate- The nitrogen atom that is transferred to α-ketoglutarate in the transamination reaction (forming Glutamate) is converted into free ammonium ion by oxidative deamination. This reaction is catalyzed by glutamate dehydrogenase. This enzyme is unusual in being able to utilize either NAD+ or NADP+, at least in some species. The reaction proceeds by dehydrogenation of the C-N bond, followed by hydrolysis of the resulting Schiff base.

Figure-3- Showing the oxidative deamination of Glutamate to from α- Keto glutarate. Glutamate carries the amino group of amino acids from peripheral tissue to liver to be released as ammonium ions .

The equilibrium for this reaction favors glutamate; the reaction is driven by the consumption of ammonia. Glutamate dehydrogenase is located in mitochondria, as are some of the other enzymes required for the production of urea. This compartmentalization sequesters free ammonia, which is toxic.

The sum of the reactions catalyzed by aminotransferases and glutamate dehydrogenase is-

In most terrestrial vertebrates, NH4 + is converted into urea, which is excreted (Figure-4)

 

 

Figure-4-Showing the  process of transdeamination and the role of glutamate

Role of Glutamate and Glutamate dehydrogenase- In majority of the transamination reactions alpha keto glutarate is the acceptor keto acid forming Glutamate, that is oxidatively deaminated in the liver by Glutamate dehydrogenase to form alpha keto glutarate and ammonia. Conversion of α-amino nitrogen to ammonia by the concerted action of glutamate aminotransferase and GDH is often termed “transdeamination.” Thus Transamination and deamination are coupled processes though they occur at distant places and in these two processes Glutamate occupies the central place.

Regulation of Glutamate dehydrogenase– The activity of glutamate dehydrogenase is allosterically regulated. The enzyme consists of six identical subunits. Guanosine triphosphate (GTP) and adenosine triphosphate (ATP) are allosteric inhibitors, whereas guanosine diphosphate (GDP) and adenosine diphosphate (ADP) are allosteric activators. Hence, a lowering of the energy charge (more of ADP and GDP) accelerates the oxidation of amino acids favoring formation of alpha keto glutarate that can be channeled towards TCA cycle for complete oxidation to provide energy.

3) Glucose alanine cycle and the role of Glutamate- The transport of amino group of amino acids also takes place in the form of Alanine.

Nitrogen is transported from muscle to the liver in two principal transport forms. Glutamate is formed by transamination reactions, but the nitrogen is then transferred to pyruvate to form alanine, which is released into the blood. The liver takes up the alanine and converts it back into pyruvate by transamination. The pyruvate can be used for gluconeogenesis and the amino group eventually appears as urea. This transport is referred to as the alanine cycle. It is reminiscent of the Cori cycle and again illustrates the ability of the muscle to shift some of its metabolic burden to the liver (Figure-5)


 Figure-5- The Glucose- Alanine Cycle- Glutamate in muscle is transaminated to alanine, which is released into the blood stream. In the liver, alanine is taken up and converted into pyruvate for subsequent metabolism.

) Glutamate and Glutamine relationship

Ammonia Nitrogen can also be transported as glutamine. This is the first line of defense in brain cells. Glutamine synthetase catalyzes the synthesis of glutamine from glutamate and NH4 + in an ATP-dependent reaction (Figure-6)

Figure-6- Showing the synthesis of glutamine form glutamate

The nitrogen of glutamine can be converted into urea in the liver.

Hydrolytic release of the amide nitrogen of glutamine as ammonia, catalyzed by glutaminase (Figure -7 )strongly favors glutamate formation. The concerted action of glutamine synthase and glutaminase thus catalyzes the interconversion of free ammonium ion and glutamine.

Figure-7- showing the hydrolysis of glutamine by glutaminase.

Renal glutaminase activity is associated with maintenance of acid base metabolism.

5) Glutamate as a metabolic product– Glutamate is produced  directly from the metabolism of Proline, Arginine and Histidine, that can be oxidatively deaminated to form Alpha keto glutarate and ammonia. (Figure-8)

 

Figure-8- Showing the  formation  of glutamate from amino acids  like Arginine, Histidine and Proline

 6) Glutamate as an activator for urea formation– Glutamate in the form of N-Acetyl Glutamate  acts as a positive allosteric modifier for Carbamoyl phosphate synthetase-1 , the first  and the rate limiting enzyme of urea cycle. Carbamoyl phosphate synthase I, is active only in the presence of its allosteric activator N-acetylglutamate, which enhances the affinity of the synthase for ATP (Figure-9)

 

 

Figure-9- Showing the role of N-Acetyl Glutamate as a positive modifier for CPS-1

6) Formation of GABA- GABA, an inhibitory neurotransmitter is produced from the decarboxylation of glutamic acid by glutamate decarboxylase enzyme in the presence of B6-P (Figure-10)

 

Figure-10– showing the synthesis of GABA from glutamate.

Ammonia intoxication  and role of glutamate- Excess of ammonia depletes glutamate and hence GABA level in brain, To compensate for glutamate, alpha keto glutarate is used , the decrease concentration of which subsequently depresses TCA and thus deprives brain cells of energy.  Excess Glutamine is exchanged with Tryptophan , a precursor of Serotonin , resulting in hyper excitation. The symptoms of ammonia intoxication are all due to energy depletion and a state of hyperexcitation.

Thus to conclude, Glutamate  represents the major transporter of amino group of amino acids and has a central role in both  the catabolism of amino acids as well in the synthesis of non- essential amino acids( through Transamination reactions).

 

 

 

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

Q.1- Alpha Methyl dopa is a drug used in the treatment of hypertension. Explain its possible mode of action.

0r

Describe the steps of catecholamines synthesis  and degradation and highlight the clinical significance of these reactions.

Answer- Catecholamines are synthesized from Tyrosine.

Cells in the adrenal medulla synthesize and secrete epinephrine and nor epinephrine. In humans  80% of the catecholamine output is epinephrine.

Synthesis and Secretion of Catecholamines

Synthesis of catecholamines begins with the amino acid tyrosine, which is taken up by chromaffin  cells in the medulla and converted to norepinephrine and epinephrine through the following steps :

1) Tyrosine is hydroxylated to DOPA (Dihydroxy Phenyl Alanine) by Tyrosinase (Figure-1), that requires BH4 (Tetra hydro biopterine) and NADPH. The reaction is similar to hydroxylation of phenyl alanine to form Tyrosine. Tyrosinase  meant for catecholamine synthesis is different for the one required for Melanin synthesis.

2) Dopa decarboxylase, a Pyridoxal phosphate (B6-P)-dependent enzyme, forms dopamine by decarboxylation of DOPA (Figure-1).

3) Subsequent hydroxylation of Dopamine  by dopamine -β-oxidase then forms norepinephrine (Figure-1). The enzyme requires molecular oxygen, vitamin C and  Copper ion for its activity.

4) In the adrenal medulla, phenyl ethanolamine-N-methyltransferase utilizes S-adenosylmethionine to methylate the primary amine of norepinephrine, forming epinephrine (Figure-1).

Figure-1- showing the steps of synthesis of Catecholamines

Norepinephrine and epinephrine are stored in electron-dense granules which also contain ATP and several neuropeptides. Secretion of these hormones is stimulated by acetylcholine release from preganglionic sympathetic fibers innervating the medulla. Many types of “stresses” stimulate such secretion, including exercise, hypoglycemia and trauma. Following secretion into blood, the catecholamines bind loosely to and are carried in the circulation by albumin and perhaps other serum proteins.

Adrenergic Receptors and Mechanism of Action

These hormones bind adrenergic receptors on target cells, where they induce essentially the same effects as direct sympathetic nervous stimulation.  There are multiple receptor types which are differentially expressed in different tissues and cells. The alpha and beta adrenergic receptors and their subtypes were originally defined by differential binding of various agonists and antagonists and, more recently, by analysis of molecular clones.

Receptor

Effectively Binds

Effect of Ligand Binding

Alpha1 Epinephrine, Norepinphrine Increased free calcium
Alpha2 Epinephrine, Norepinphrine Decreased cyclic AMP
Beta1 Epinephrine, Norepinphrine Increased cyclic AMP
Beta2 Epinephrine Increased cyclic AMP

 

Physiologic Effects of Medullary Hormones

In general, circulating epinephrine and norepinephrine released from the adrenal medulla have the same effects on target organs as direct stimulation by sympathetic nerves, although their effect is longer lasting. Additionally, of course, circulating hormones can cause effects in cells and tissues that are not directly innervated. The physiologic consequences of medullary catecholamine release are justifiably framed as responses which aid in dealing with stress. A listing of some major effects mediated by epinephrine and norepinephrine are:

  • Increased rate and force of contraction of the heart muscle: this is predominantly an effect of epinephrine acting through beta receptors.
  • Constriction of blood vessels: norepinephrine, in particular, causes widespread vasoconstriction, resulting in increased resistance and hence arterial blood pressure.
  • Dilation of bronchioles: assists in pulmonary ventilation.
  • Stimulation of lipolysis in fat cells: this provides fatty acids for energy production in many tissues and aids in conservation of dwindling reserves of blood glucose.
  • Increased metabolic rate: oxygen consumption and heat production increase throughout the body in response to epinephrine. Medullary hormones also promote breakdown of glycogen in skeletal muscle to provide glucose for energy production.
  • Dilation of the pupils: particularly important under conditions of low ambient light.
  • Inhibition of certain “non-essential” processes: an example is inhibition of gastrointestinal secretion and motor activity.

Common stimuli for secretion of adrenomedullary hormones include exercise, hypoglycemia, hemorrhage and emotional distress. The alpha and Beta blockers are used as drugs to inhibit the action of catecholamines.

Catecholamine degradation

Catecholamines are degraded in the liver by two enzymes, COMT( Catechol-O-Methyl-Transferase) and MAO(Mono amine Oxidase). By the action of COMT, epinephrine and Nor epinephrine are converted to metanephrine and nor metanephrine respectively. Both these products are further acted upon by MAO to form VMA (Vanillyl Mandelic acid) and MOPG (3-Methoxy 4- hydroxyphenylglycol). These products are further excreted in urine. Epinephrine and nor epinephrine can be acted upon directly also by MAO to form DOPG and DOMA (Figure-2).The Excretory products are increased in Pheochromocytoma and that forms the basis for the diagnostic test.

Figure-2- showing the steps of degradation of Catecholamines

Clinical Significance-

Methyldopa (L-α-Methyl-3,4-dihydroxyphenylalanine; AldometAldorilDopametDopegyt, etc.) is a drug used as a sympatholytic or antihypertensive agent . It is less commonly used now following  the introduction of alternative safer classes of agents. However, it continues to have a role in otherwise difficult to treat hypertension and gestational hypertension (also known as pregnancy-induced hypertension (PIH)) and pre eclampsia.

Mechanism of action- Methyldopa has a dual mechanism of action:

  • It is a competitive inhibitor of the enzyme DOPA decarboxylase, also known as aromatic L-amino acid decarboxylase, which converts L-DOPA into dopamine. This inhibition results in reduced dopaminergic and adrenergic neurotransmission in the peripheral nervous system. This effect may lower blood pressure and cause central nervous system effects such as depression, anxiety, apathy, and parkinsonism.
  • It is converted to α-methylnorepinephrine by dopamine beta-hydroxylase (DBH). α-methylnorepinephrine is an agonist of presynaptic central nervous system α2-adrenergic receptors. Activation of these receptors in the brainstem appears to inhibit sympathetic nervous system output and lower blood pressure.

Figure-3  showing the  mechanism of action of alpha methyl DOPA. 

2) L-DOPA-L-DOPA crosses the protective blood–brain barrier, whereas dopamine itself cannot. Thus, L-DOPA is used to increase dopamine concentrations in the treatment of Parkinson’s disease and dopamine-responsive dystonia.

Once L-DOPA has entered the central nervous system, it is converted into dopamine by the enzyme  DOPA decarboxylase (DDC). Pyridoxal phosphate (vitamin B6) is a required cofactor in this reaction, and may occasionally be administered along with L-DOPA, usually in the form of pyridoxine.

3) Dopamine drips are intravenous deliveries of dopamine, that can be necessary for a hemodynamically unstable patient, at risk of shock caused by low blood pressure. These can include patients with a recent history of open heart surgery, heart attacks, or renal failure.

4) Pheochromocytoma- Pheochromocytomas and paragangliomas are catecholamine-producing tumors derived from the sympathetic or parasympathetic nervous system. Elevated plasma and urinary levels of catecholamines and the methylated metabolites, metanephrines, are the cornerstone for the diagnosis.

 

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

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)

a3

 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)

a4

 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.

 

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

Q.- Give a brief description of the process of Transamination, highlight the role of B6 phosphate in this process.

Answer- Transamination interconverts pairs of α -amino acids and α -keto acids. During Transamination, the amino group of an amino acid (amino acid R 1) is transferred to a keto acid (keto acid R 2), this produces a  new keto acid while from the original keto acid, a new amino acid is formed (Figure-1)

Figure-1- showing the transfer of amino group from a donor amino acid to a keto acid for the formation of a new amino acid and a new keto acid

The general process of transamination is reversible and is catalyzed by a transaminase, also called amino transferase that require B6-Phosphate as  a coenzyme.

Most of the amino acids act as substrate for the transaminases but the amino acids like lysine, threonine, proline, and hydroxyproline do not participate in transamination reactions.

Transamination is not restricted to α -amino groups. The δ-amino group of ornithine and the  ε-amino group of lysine—readily undergoes transamination.

Role of B6 Phosphate as a coenzyme

The coenzyme pyridoxal phosphate (PLP) is present at the catalytic site of aminotransferases and of many other enzymes that act on amino acids. PLP, is  a derivative of vitamin B6 (Figure-2).

Figure-2- Showing the structure of B6-Phosphate

1) B6-P forms an enzyme-bound Schiff base intermediate that can rearrange in various ways-

B6 bound to enzyme

Figure-3-In the “resting” state, the aldehyde group of pyridoxal phosphate is in a Schiff base linkage to the ε-amino group of an enzyme lysine side-chain.

2) During transamination, bound PLP serves as a carrier of amino groups (Figure-5 and 6)

3) Rearrangement forms an α -keto acid and enzyme-bound Pyridoxamine phosphate(Figure-4, 5 and 6), which forms a Schiff base with a second keto acid (Figure-5).

Figure-4-The α-amino group of a substrate amino acid displaces the enzyme lysine, to form a Schiff base linkage to PLP. 

Figure-5- A different a-keto acid reacts with PMP (Pyridoxamine phosphate) and the process reverses, to complete the reaction.

 

Figure-6 -Overall reaction showing the role of B6-Phosphate,  the transfer of  α-amino group from  donor amino acid to Pyridoxal phosphate forms Pyridoxamine phosphate, and a keto acid. The α-amino group is finally passed on to an acceptor  an α-keto acid to form a new amino acid.

Significance of Transamination – Transamination is used both for the catabolic as well as anabolic processes. The resultant α-Keto acid can be completely oxidized to provide energy, glucose, fats or ketone bodies depending upon the cellular requirement. Since it is a reversible process, it  is also used or the synthesis of non-essential amino acids. Some points of significance are as follows-

  • Once the keto acids have been formed from the appropriate amino acids by transamination, they may be used for several purposes. The most obvious is the complete metabolism into carbon dioxide and water by the citric acid cycle.
  • However, if there are excess proteins in the diet those amino acids  that are converted into pyruvic acid and acetyl CoA can be converted into lipids by the lipogenesis process. If carbohydrates are lacking in the diet or if glucose cannot get into the cells (as in diabetes), then those amino acids converted into pyruvic acid and oxaloacetic acids can be converted into glucose or glycogen.
  • The most usual and major keto acid involved with transamination reactions is alpha-ketoglutaric acid, an intermediate in the citric acid cycle.
  • All of the amino acids can be converted through a variety of reactions and transamination into a keto acid which is a part of or feeds into the citric acid cycle. 
  • In addition to the catabolic function of transamination reactions, these reactions can also be used to synthesize amino acids needed or not present in the diet. An amino acid may be synthesized if there is an available “root” keto acid with a synthetic connection to the final amino acid. Since an appropriate “root” keto acid does not exist for eight amino acids, (lys, leu, ile, met, thr, try, val, phe), they are essential and must be included in the diet because they cannot be synthesized. Transaminases equilibrate amino groups among available  α-keto acids. This permits synthesis of non-essential amino acids, using amino groups derived from other amino acids and carbon skeletons synthesized in the cell. Thus a balance of different amino acids is maintained, as proteins of varied amino acid contents are synthesized. 
  • Glutamic acid usually serves as the source of the amino group in the transamination synthesis of new amino acids. The reverse of the reactions are the most obvious methods for producing the amino acids alanine and aspartic acid.
  • In addition to equilibrating amino groups among available  α-keto acids, transaminases funnel amino groups from excess dietary amino acids to those amino acids (e.g., glutamate) that can be deaminated. Carbon skeletons of deaminated amino acids can be catabolized for energy or used to synthesize glucose or fatty acids for energy storage.

Figure-7 – Glutamate is the ultimate collector of amino groups of amino acids, In the liver it is rapidly deaminated, ammonia thus released is detoxified by forming urea

Q.- Discuss the clinical significance of transaminases.

Answer The enzymes catalyzing transamination process exist for all amino acids except threonine and lysine. The most common compounds involved as a donor/acceptor pair in transamination reactions are glutamate and  α-ketoglutarate ( α-KG), which participate in reactions with many different aminotransferases. Serum aminotransferases such as serum glutamate-oxaloacetate-aminotransferase (SGOT) (also called aspartate aminotransferase, AST) and serum glutamate-pyruvate aminotransferase (SGPT) (also called alanine transaminase, ALT) have been used as clinical markers of tissue damage, with increasing serum levels indicating an increased extent of damage. 

1) AST is found in the liver, cardiac muscle, skeletal muscle, kidneys, brain, pancreas, lungs, leukocytes, and erythrocytes in decreasing order of concentration.

Reaction catalyzed can be represented as follows-

 

Figure-8- Showing the reaction catalyzed by AST (Aspartate amino transferase)

Normal serum activity is  0-41 IU/L. The concentration of the enzyme is very high in myocardium. The enzyme is both cytoplasmic as well as mitochondrial in nature.

2) ALT is found primarily in the liver.

Reaction catalyzed can be represented as follows-

Figure-9 – Showing the reaction catalyzed by ALT(Alanine amino transferase)

The normal serum activity ranges between 0-45 IU/L.

Diagnostic significance of amino transferases-

I) Liver Diseases- The aminotransferases are normally present in the serum in low concentrations. These enzymes are released into the blood in greater amounts when there is damage to the liver cell membrane resulting in increased permeability.These are sensitive indicators of liver cell injury and are most helpful in recognizing acute hepatocellular diseases such as hepatitis. Any type of liver cell injury can cause modest elevations in the serum aminotransferases.

  • Levels of up to 300 U/L are nonspecific and may be found in any type of liver disorder.
  • Striking elevations—i.e., aminotransferases > 1000 U/L—occur almost exclusively in disorders associated with extensive hepatocellular injury such as (1) viral hepatitis, (2) ischemic liver injury (prolonged hypotension or acute heart failure), or (3) toxin- or drug-induced liver injury.
  • In most acute hepatocellular disorders, the ALT is higher than or equal to the AST.
  • An AST: ALT ratio > 2:1 is suggestive while a ratio > 3:1 is highly suggestive of alcoholic liver disease.
  • The AST in alcoholic liver disease is rarely >300 U/L and the ALT is often normal. A low level of ALT in the serum is due to an alcohol-induced deficiency of Pyridoxal phosphate.
  • In obstructive jaundice the aminotransferases are usually not greatly elevated. One notable exception occurs during the acute phase of biliary obstruction caused by the passage of a gallstone into the common bile duct. In this setting, the aminotransferases can briefly be in the 1000–2000 U/L range. However, aminotransferase levels decrease quickly, and the liver function tests rapidly evolve into one typical of cholestasis.

2) Acute myocardial infarction- In acute MI the serum activity rises sharply within the first 12 hours, with a peak level of 24 hours or over and returns to normal within 3 to 5 days.

  • Levels > 350 IU/L are usually fatal and signify massive infarction
  • Levels < 50 IU/L are associated with low mortality
  • The rise depends upon the size of infarction
  • There is no rise of ALT in acute MI
  • Reinfarction results in secondary rise of AST

3) Extra cardiac and extra hepatic conditions-

  • Elevation of AST can also be seen in Muscle disorders like muscular dystrophies- myositis etc.
  • Increase activity  of AST is also observed in acute pancreatitis, leukemias and acute hemolytic anemias
  • In normal health slight rise of AST level can be observed after prolonged exercise

4) Glucose Alanine cycle- Alanine transaminase has an important function in the delivery of skeletal muscle carbon and nitrogen (in the form of alanine) to the liver. In skeletal muscle, pyruvate is transaminated to alanine, thus affording an additional route of nitrogen transport from muscle to liver. In the liver alanine transaminase transfers the ammonia to  α-KG and regenerates pyruvate. The pyruvate can then be diverted into gluconeogenesis. This process is referred to as the glucose-alanine cycle.

Figure-10- Glucose Alanine cycle functions to transport amino group of amino acids in the form of alanine from skeletal muscle to liver

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

Q.1- What is the biological advantage of secretion of proteolytic enzymes  in the zymogen form ?

Answer- Certain proteins are synthesized and secreted as inactive precursor proteins known as proproteins. The proproteins of enzymes are termed pro enzymes or zymogens. Selective proteolysis convert a proprotein by one or more successive proteolytic “clips” to a form that exhibits the characteristic activity of the mature protein, eg, its enzymatic activity. (Figure-1)

Figure-1- showing the selective cleavage of a zymogen to form an active enzyme

Examples of other Proproteins or Zymogens

Proteins synthesized as proproteins include the hormone insulin (proprotein = Proinsulin), the digestive enzymes pepsin, trypsin, and chymotrypsin (proproteins = pepsinogen, trypsinogen, and chymotrypsinogen, respectively), several factors of the blood clotting and blood clot dissolution cascades and the connective tissue protein collagen (proprotein = procollagen).

Biological advantage of having  proteolytic zymogens

The synthesis and secretion of proteases as catalytically inactive proenzymes protects the tissue of origin (e.g., the pancreas) from auto digestion, such as can occur in  Acute pancreatitis.

Physiologic processes such as digestion are intermittent but fairly regular and predictable. Enzymes needed intermittently but rapidly often are secreted in an initially inactive form since the secretion process or new synthesis of the required proteins might be insufficiently rapid for response to a pressing pathophysiologic demand . Proenzymes facilitate rapid mobilization of an activity in response to physiologic demand.

Q.2- Give a brief account of the mechanism of absorption of products of digestion of protein, highlighting the role of Glutathione in this process.

Answer- Digestive products of protein can be absorbed as amino acids, dipeptides, and tripeptides (in contrast to carbohydrates, which can only be absorbed as monosaccharides). The absorption of amino acids takes place mainly in the small intestine. There are two mechanisms for amino acid absorption-

A) Carrier protein transport system (Figure-2)

  • It is the main mechanism for amino acid absorption
  • It is an active and energy requiring process.
  • The needed energy is provided by ATP
  • There are approximately 7 carrier proteins, each specific for a group of amino acids
  • These carrier proteins are Sodium dependent symport systems
  • Each transporter has two binding sites, one for sodium and the other for an amino acid.
  • Absorption of dipeptides and tripeptides is faster than absorption of free amino acids.
  • Na+dependent cotransport of dipeptides and tripeptides also occurs in the luminal membrane.
  • After the dipeptides and tripeptides are transported into the intestinal
    cells, cytoplasmic peptidases hydrolyze them to amino acids.
  • After absorption the amino acids are transported to the portal circulation by facilitated diffusion. Na + is expelled out of the cell in exchange for K+ through the Na+ -K+ ATPase pump (Figure-2).

 Figure-2-Mechanism of absorption of amino acids, dipeptides, and tripeptides by intestinal epithelial cells. Each is absorbed by Na+-dependent co transport.

Clinical significance

1) Cystinuria- Common transporter for cystine, ornithine, arginine and lysine(COAL) is present in gut and renal tubules. Deficiency of transporter results in loss of these  amino acids in the feces and urine.

2) Hart- Nup Disease-The  transporter for tryptophan  and neutral amino acid is deficient. There is  reduced  absorption of tryptophan , tryptophan deficiency produce neurological and skin manifestation (pellagra-like rashes).Neurological symptoms are due to the fact that tryptophan is a precursor for serotonin and melatonin, while skin rashes are due to deficiency of niacin,  since niacin can be synthesized from tryptophan.

3) Food allergies– Relatively large peptides may be absorbed intact, either by uptake into mucosal epithelial cells (transcellular) or by passing between epithelial cells (paracellular). Many such peptides are large enough to stimulate antibody formation—this is the basis of allergic reactions to foods.

B) Glutathione transport system (Υ- Glutamyl cycle)- Glutathione is used to transport  neutral amino acids in intestine, brain and kidney tubules.

Glutathione. This tripeptide consists of a cysteine residue flanked by a glycine residue and a glutamate residue that is linked to cysteine by an isopeptide bond between glutamate’s side-chain carboxylate group and cysteine’s amino group.(Figure-3)

 

Figure-3- showing the structure of glutathione (Gamma glutamyl cysteinyl glycine)

Role of glutathione in the absorption of amino acids (Figure-4)

  • Glutathione reacts with amino acid to form gamma glutamyl amino acid.This is catalyzed by Gamma glutamyl Transferase (GGT) in the presence of Na + (figure-2) to form Υ- Glutamyl amino acid and cysteinyl glycine.
  • The Υ-Glutamyl amino acid is then cleaved to give free amino acid and 5-oxo proline.
  •  Amino acid during this process is transported inside the cell.
  • It is an energy requiring process, which is supplied by the hydrolysis of peptide bond of Glutathione.
  • 5-oxo proline in the presence of the enzyme 5-oxo prolinase and ATP forms Glutamic acid
  • Cysteinyl glycine formed in the first step is cleaved to form cysteine and glycine.
  • Glutamic acid combines with cysteine first to form glutamyl cysteine and then combines with glycine to form glutathione.
  • Glutathione is regenerated again  and that completes the Υ- Glutamyl cycle.
  • The transport of one amino acid and regeneration of Glutathione requires 3  molecules of ATP.

 

Figure-4- showing the role of glutathione in the absorption of amino acids

Clinical  Significance- The deficiency of 5 oxoprolinase causes oxoprolinuria

Q.3- What is nitrogen balance ? Explain its significance and enlist the conditions causing deviations in the nitrogen balance.

Answer- The state of protein nutrition can be determined by measuring the dietary intake and output of nitrogenous compounds from the body. Although nucleic acids also contain nitrogen, protein is the major dietary source of nitrogen and measurement of total nitrogen intake gives a good estimate of protein intake (mg N x 6.25 = mg protein, as N is 16% of most proteins). The output of N from the body is mainly in urea and smaller quantities of other compounds in urine, undigested protein in feces; significant amounts may also be lost in sweat and shed skin. The difference between intake and output of nitrogenous compounds is known as nitrogen balance (Figure-5)

Figure-5- Showing nitrogen balance. The intake of nitrogen is in the form of dietary proteins while the output is through urine and feces in the form of undigested proteins, urea, uric acid, creatinine, ammonia and amino acids.

States of nitrogen balance

Three states can be defined-

1)  Nitrogen equilibrium– In a healthy adult, nitrogen balance is in equilibrium, when intake equals output, and there is no change in the total body content of protein.

Intake = output : N equilibrium

2) Positive nitrogen balance– when the excretion of nitrogenous compounds is less than the dietary intake and there is net retention of nitrogen in the body as protein.

Intake > output: positive N balance

Examples- In a growing child, a pregnant woman, or a person in recovery from illness there is positive nitrogen balance.

3) Negative nitrogen balance– There is net loss of protein nitrogen from the body

In response to trauma or infection, or if the intake of protein is inadequate to meet requirements there is negative nitrogen balance.

Intake < output: negative N balance

Significance of nitrogen balance

1) Growth-The continual catabolism of tissue proteins creates the requirement for dietary protein, even in an adult who is not growing; although some of the amino acids released can be reutilized, much are used for gluconeogenesis in the fasting state.The average daily requirement is 0.6 g of protein/kg body weight (0.75 allowing for individual variation), or approximately 50 g/day. Average intakes of protein in developed countries are of the order of 80–100 g/day, ie, 14–15% of energy intake. Because growing children are increasing the protein in the body, they have a proportionally greater requirement than adults and should be in positive nitrogen balance.. In some countries, protein intake may be inadequate to meet these requirements, resulting in stunting of growth.

2) Illness and convalescence– Negative nitrogen balance is seen immediately after acute illnesses like surgery, trauma and burns.

One of the metabolic reactions to a major trauma, such as a burn, a broken limb, or surgery, is an increase in the net catabolism of tissue proteins. As much as 6–7% of the total body protein may be lost over 10 days.

Chronic illnesses like malignancy, uncontrolled diabetes mellitus and other debilitating diseases also show negative nitrogen balance

Prolonged bed rest results in considerable loss of protein because of atrophy of muscles. Protein is catabolized as normal, but without the stimulus of exercise, it is not completely replaced.

Lost protein is replaced during convalescence, when there is positive nitrogen balance. A normal diet is adequate to permit this replacement.

3) Hormones- Insulin , growth hormone and androgens promote positive nitrogen balance while corticosteroids induce negative nitrogen balance.

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

Q.1- A 10 -year-old boy develops convulsions. After running an Electroencephalogram(EEG), a neurologist determines that the child has epilepsy. He is started on benzodiazepine which promotes the activity of GABA. GABA is derived from Glutamate by which of the following reactions ?

a) Transamination

b) Decarboxylation

c) Deamination

d) Hydroxylation

e) Dehydrogenation

Q.2- A couple of African- American descent gives birth to a boy  after an otherwise uneventful pregnancy. The child is exceptionally fair skinned and has almost white hair. Further examination reveals red pupils.  A postnatal screen is likely to confirm the deficiency of which of the following enzymes ?

a) Glutathione reductase

b) Glutathione peroxidase

c) Tyrosinase

d) Methionine synthase

e) Cystathionine beta synthase

Q.3- A 16- year -old girl is found by her parents unconscious on the bathroom floor with an empty bottle of acetaminophen in the toilet. She is rushed to the hospital where she is given several doses of N-acetyl Cysteine. Acetaminophen overdose is potentially life threatening because it depletes the cellular stores of Glutathione. Which of the following amino acid is another component of Glutathione besides Cysteine and Glutamic acid ?

a) Methionine

b) Arginine

c) Lysine

d) Ornithine

e) Glycine

Q.4-  A 56- year-old man with  long-standing poorly controlled diabetes mellitus visits his primary care physician for a follow-up after a recent hospitalization. The patient experienced an episode of acute renal failure while in hospital and his serum creatinine rose to 2.4 mg/dl ( Normal, 0.7- 1.5 mg/dl). Creatinine, a marker of kidney function, is derived from which of the following precursors ?

a) Arginine, Lysine and Glutamine

b) Glutamic acid, Cysteine and Glycine

c) Methionine, Serine and Glycine

d) Methionine, Arginine and Glycine

e) Cysteine, Glycine and Arginine.

Q.5- S- Adenosyl  Methionine (Active Methionine) is required for the synthesis  of which of the following compounds ?

a) Thyroid hormone

b) Melanin

c) Epinephrine

d) Serotonin

e) Bile salts.

Q.6- Which of the following compounds is formed from  hydroxylation requiring vitamin C and subsequent methylation ?

a) Histamine

b) Dopamine

c) Epinephrine

d) Creatine

e) Melanin

Q.7- A 40-year old woman complains of deceased energy, significant weight gain and cold intolerance. She is seen by her family physician, who has diagnosed her to be having hypothyroidism (Low  level of thyroid hormone). Which of the following is a precursor for thyroid hormone ?

a)  DOPA

b) Glutamine

c)  Tyrosine

d) Tryptophan

e) Threonine.

Q.8- A 63 -year old woman reports a long history of joint pains. Her fingers are severely deformed secondary to rheumatoid arthritis. Upon visiting a rheumatologist, she is started on methotrexate. This drug inhibits which of the following conversions ?

a) Dopamine to norepinephrine conversion

b) Tyrosine to Dopa

c) Dihydrofolate to Tetra hydro folate

d) Phenyl Alanine to Tyrosine

e) N-Acetyl serotonin to melatonin

Q.9-  A 59-year-old woman develops a shuffling gait and a pin- rolling tremor. She is referred to a neurologist for evaluation. After a thorough workup, a diagnosis of Parkinson disease is made and the patient is placed on Mono amine oxidase inhibitor. The drug in this case, is given to decrease the degradation of which of the followings ?

a) Serotonin

b) Dopamine

c) Nicotinamide

d) Melatonin

e) Nitric oxide

Q.10- During a medical rotation, a medical student volunteered for a respiratory physiology examination that determines basal metabolic rate and the respiratory quotient. She followed the protocol for a resting individual in the post absorptive state. Which of the following amino acids would be found in the highest concentration in the serum ?

a) Alanine and Glutamine

b) Arginine and Ornithine

c) Glutamate and Aspartate

d)  Branched chain amino acids

e) Hydrophobic amino acids

Q.11- A 27- year-old semiprofessional  tennis player seeks advice from a hospital -based nutritionist concerning his diet supplements. His coach had given him amino acid supplements consisting of phenyl alanine and tyrosine. The rationale was that these precursors to several neurotransmitters will “help his brain focus” on his game. In reality, the excess amino acids are used for energy, with a poor and eclectic diet. Phenyl Alanine upon metabolism, enter TCA cycle as which of the following ?

a) Oxalo acetate

b) Citrate

c) Succinyl co A

d) Fumarate

e) α- Keto glutarate

Q.12- In a 39-year-old woman who just gave birth, chorionic villus sampling was performed, and a battery of genetic panels was assessed on the new-born. One marker indicated a defective Cystathionine -β- Synthase. Which of the following compounds you most likely expect to be elevated  in the blood of the infant at birth if the mother was not treated properly ?

a) Valine

b) Homocysteine

c) Threonine

d) Glutamine

e) Cysteine

Q.13- A 55-year-old man suffers from cirrhosis of liver. Toxins such as ammonia are not properly metabolized by the liver and can damage brain. Which of the following compounds is expected to be in highest concentration in brain  as a result of detoxification of ammonia?

a) Alpha keto glutarate

b) Glutamate

c)  Glutamine

d) GABA

e) Asparagine

Q.14- Which of the following enzymes requires adenosine triphosphate (ATP) to mediate its reactions ?

a) Argino Succinate lyase

b) Argino Succinate synthetase

c) Arginase

d) Glutaminase

e) Ornithine transcarbamoylase

Q.15- Which of the following amino acids is not converted to Acetyl co A upon metabolism ?

a) Tyrosine

b) Leucine

c) Tryptophan

d) Lysine

e) Valine

 

 

Key to Answers-

1)-b,      2)-c,      3)-e,      4)- d,     5)- c,      6)-c,       7)-c,       8)-c,       9)-b,      10)-a,    11)- d,   12-b,     13)-C

14)-b,    15)-e

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

Case Details- An adolescent girl develops hemiballismus (repetitive throwing motions of the arm) after anesthesia for a routine operation. She is tall and lanky, and it is noted that she and her sister both had previous operations for dislocated lenses of the eyes.

The symptoms are suspicious for the disease homocystinuria.

What is the nature of disease, how can this be treated?

Case discussion

Homocystinuria (Homocysteinemia) Homocystinuria is a disorder of methionine metabolism, leading to an abnormal accumulation of homocysteine and its metabolites (homocystine, homocysteine-cysteine complex, and others) in blood and urine. Normally, these metabolites are not found in appreciable quantities in blood or urine.

Homocystinuria is an autosomal recessively inherited defect in the transsulfuration pathway (homocystinuria I) or methylation pathway (homocystinuria II and III).

Homocysteinemia

Homocysteinemia, a separate but related entity, is defined as elevation of the homocysteine level in blood. This condition has also been referred to as homocyst(e)inemia to reflect metabolites that may accumulate. A mild elevation of plasma homocysteine may exist without homocystinuria.

Homocysteinemia may be due to a genetic predisposition to abnormal activity in the same pathways as homocystinuria. Nutritional and environmental factors, as well as specific medications, may worsen this abnormality and provoke symptoms.

Basic Concept-

The pathway, starting at methionine, progressing through homocysteine, and onward to cysteine, is termed the transsulfuration pathway. Conversion of homocysteine back to methionine, catalyzed by MTHFR and methylcobalamin, is termed the remethylation pathway. A minor amount of remethylation takes place via an alternative route using betaine as the methyl donor (Figure-1)

Most homocysteine, an intermediate compound of Methionine degradation, is normally remethylated to Methionine. This Methionine-sparing reaction is catalyzed by the enzyme Methionine Synthase, which requires a metabolite of folic acid (5-methyltetrahydrofolate) as a methyl donor and a metabolite of vitamin B12 (Methylcobalamin) as a cofactor .Only 20–30% of total homocysteine (and its dimer homocystine) is in free form in the plasma of normal individuals. The rest is bound to protein.

Pathophysiology

The accumulation of homocysteine and its metabolites is caused by disruption of any of the 3 interrelated pathways of methionine metabolism—deficiency in the cystathionine B-synthase (CBS) enzyme, defective methylcobalamin synthesis, or abnormality in methylene tetrahydrofolate reductase (MTHFR).

Clinical syndromes resulting from each of these metabolic abnormalities have been termed homocystinuria I, II, and III. Three different cofactors/vitamins—pyridoxal 5-phosphate, methylcobalamin, and folate—are necessary for the 3 different metabolic paths.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-1- showing metabolism of Methionine. Homoserine and  cysteine are produced by cleavage of cystothionine, homoserine is further metabolized to form propionyl co A , that gains entry in TCA cycle as succinyl co A

Classification

Three major forms of homocysteinemia and homocystinuria have been identified

1) Homocystinuria due to Cystathionine beta Synthetase deficiency

(Classical Homocystinuria)

Inheritance

Homocystinuria is inherited in families as an autosomal recessive trait.

Clinical Manifestations

Infants with this disorder are normal at birth.

  • Clinical manifestations during infancy are nonspecific and may include failure to thrive and developmental delay.
  •  The diagnosis is usually made after 3 yr of age, when subluxation of the ocular lens (ectopia lentis) occurs. This causes severe myopia and iridodonesis (quivering of the iris), astigmatism, glaucoma, cataract, retinal detachment, and optic atrophy may develop later in life.
  • Progressive mental retardation is common. Normal intelligence, however, has been reported.
  •  Affected individuals with homocystinuria manifest skeletal abnormalities resembling those of Marfan syndrome; they are usually tall and thin with elongated limbs and arachnodactyly. Scoliosis, pectus excavatum genu valgum, pes cavus, high arched palate, and crowding of the teeth are common.
  •  Patients usually have fair complexions, blue eyes, and a peculiar malar flush. Generalized osteoporosis, especially of the spine, is the main radiographic finding.
  • Thromboembolic episodes involving both large and small vessels, especially those of the brain, are common and may occur at any age.

Laboratory findings

Amino acid screen of blood and urine – Elevations of both Methionine and homocystine in body fluids are the   diagnostic laboratory findings.

  • Total plasma homocysteine is extremely elevated (usually >100μ M).
  • Cystine is low or absent in plasma.
  • The urine screening test for sulfur-containing amino acids, called the cyanide nitroprusside test, can be undertaken;
  • Liver biopsy and enzyme assay are diagnostic
  • Skeletal x-ray reveals generalized osteoporotic changes
  • Skin biopsy with a fibroblast culture (The diagnosis may be established by assay of the enzyme in liver biopsy specimens, cultured fibroblasts)
  • Standard ophthalmic examination is diagnostic for various eye changes
  • Genetic testing can be helpful

Treatment

  • High doses of vitamin B6 causes dramatic improvement in patients who are responsive to this therapy.
  • The cysteine deficiency must be made up from dietary sources.
  • Supplementation with pyridoxine, folic acid, B12 or trimethyl glycine (betaine) reduces the concentration of homocysteine considerably in the bloodstream.
  • A low Methionine diet is also recommended.
  • Existing mental retardation can not be improved by symptomatic treatment

 2) Other form of homocystinuria

They are the result of impaired remethylation of homocysteine to Methionine.

This can be caused by defective Methionine synthase or reduced availability of two essential cofactors, 5-methyltetrahydrofolate and methylcobalamin (methyl-vitamin B12).

Affected children present with vomiting, poor feeding, lethargy, hypotonia and developmental delay.

Laboratory diagnosis reveals megaloblastic anemia, Hyperhomocysteinemia and low Methionine levels in blood.

Diagnosis is confirmed by enzyme assay in cultured fibroblasts.

Treatment – supplementation  with vitamin B12.

3) MTHFR (Methylene tetra hydro folate reductase) DNA Gene mutation has been associated with an increased risk for Hyperhomocysteinemia. MTHFR is involved in the methylation of homocysteine to Methionine. The enzyme causes reduction of N5 N10 Methylene tetrahydrofolate to N5 Methyl tetra hydro folate. Individuals with MTHFR gene mutations that reduce enzyme activity may develop hyperhomocysteinemia and thus be at risk for vascular disease.

Complete absence of enzyme causes convulsions, coma and death in untreated cases.Partial deficiency causes mental retardation, microcephaly and convulsions.

HYPERHOMOCYSTEINEMIA

In the absence of significant homocystinuria, it is found in some heterozygotes for the genetic defects noted above or in homozygotes for milder variants. Changes of homocysteine levels are also observed with increasing age; with smoking; in postmenopausal women; in patients with renal failure, hypothyroidism, leukemias, inflammatory bowel disease, or psoriasis; and during therapy with drugs such as methotrexate, nitrous oxide, isoniazid, and some antiepileptic agents. 

Consequences of Hyperhomocysteinemia:  Homocysteine acts as an atherogenic agent. An increase in total plasma homocysteine represents an independent risk factor for coronary, cerebrovascular, and peripheral arterial disease as well as for deep-vein thrombosis. Homocysteine is synergistic with hypertension and smoking, and it is additive with other risk factors that predispose to peripheral arterial disease. In addition, hyperhomocysteinemia and folate and vitamin B12 deficiency have been associated with an increased risk of neural tube defects in pregnant women. Vitamin supplements are effective in reducing plasma homocysteine levels in these cases.

Prognosis

Although no cure exists for homocystinuria, vitamin B6 therapy can help about half of people affected by the condition.

If the diagnosis is made while a patient is young, starting a low Methionine diet quickly can prevent some mental retardation and other complications of the disease. For this reason, some states screen for homocystinuria in all newborns.

Patients with persistent rises in blood homocysteine levels are at increased risk for Thromboembolic episodes which can cause significant medical problems and shorten lifespan.

 

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

http://www.slideshare.net/namarta28/biochemistry-for-medics

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


Disease Biochemical defect and pathogenesis Laboratory findings Clinical Manifestations Treatment
Alkaptonuria It is caused by a defect in the enzyme Homogentisic acid oxidase.Due to deficiency Homogentisic acid remains in the body and slowly and progressively get deposited in bones and cartilages where it turns into a pigmented polymeric material. 

These polymerised Homogentisic acid products are excreted in large amounts in urine and impart the black color to urine.

 

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.

 

 

Diagnosis can be confirmed by demonstrating the presence of Homogentisic acid in the urine.This may be done by paper chromatography  or Thin-layer chromatography.  The earliest signs is thickening of the ear cartilage, skin turns a blue-black colour,Bones and cartilage of the lower back, knees, shoulders and hips are most affected, Deposits around the trachea, larynx and bronchi may cause shortness of breath and difficulty in breathing deposits around the heart and blood vessels can calcify and lead to atherosclerotic plaques,  

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

 

Sclera becomes pigmented but this does not affect the vision.

 

Reducing intake of the amino acids phenylalanine and tyrosine to the minimum .Vitamin C has been found to slow down the conversion of Homogentisic acid to the polymeric deposits in cartilage and bone.
Albinism Albinism is a defect of melanin production that results in little or no color (pigment) in the skin, hair, and eyes.There are three  main categories of albinism in humans:In oculo cutaneous albinism pigment is lacking in the eyes, skin and hair.In ocular albinism, only the eyes lack pigment.

 People who have ocular albinism have generally normal skin and hair color.

In patchy albinism– patches of depigmentation  are there on skin and hair

 

The diagnosis of the condition is based on the appearance of skin, hair, and eyes.Genetic testing offers the most accurate way to diagnose albinism and its type. Absence of coloring from the hair, skin, or iris of the eye, Long-term sun exposure greatly increases the risk of skin damage and skin cancers, including an aggressive form of skin cancer called melanoma, in people with this condition. Symptomatic treatment. Sun screen lotions and sun glasses are very helpful.Genetic counseling is  recommended  if the disease runs in families.
Phenylketonuria (PKU) Deficiency of the enzyme phenylalanine hydroxylase or of its cofactor tetrahydrobiopterin causes accumulation of phenylalanine in body fluids and the central nervous system (CNS).

 

In classic PKU, levels may range from 6 to 80mg/dl, but are usually greater than 30mg/dl. Identification and measurement of phenylketones in the urine has no place in any screening program.

 

Identification of phenylketones in the urine by ferric chloride may offer a simple test for diagnosis of infants with developmental and neurologic abnormalities.

CLASSICAL    PKU- The infants are normal at birth, Mental retardation may develop gradually, Vomiting, sometimes severe enough to be misdiagnosed as pyloric stenosis, may be an early symptom. Older untreated children become hyperactive with purposeless movements. On physical examination these infants are fairer in their complexion than unaffected siblings. About 25% of children have seizures, and more than 50% have electroencephalographic abnormalities.

Microcephaly, prominent maxilla with widely spaced teeth, enamel hypoplasia, and growth retardation are other common findings in untreated children.

 

 

The goal of PKU treatment is to maintain the blood level of phenylalanine between 2 and 10 mg/dl.Some phenylalanine is needed for normal growth.This requires a diet that has some phenylalanine but in much lower amounts than normal.Because phenylalanine is not synthesized by the body, “over treatment” may lead to phenylalanine deficiency manifested by lethargy, failure to thrive, anorexia, anemia, rashes, diarrhea, and even death; moreover, tyrosine becomes an essential amino acid in this disorder and its adequate intake must be ensured.

Oral administration of the cofactor (BH4 ) to patients with milder forms of hyperphenylalaninemia from phenylalanine hydroxylase deficiency may produce significant reductions.

 

 

 

Hart Nup Disease It is caused by impaired neutral (i.e., monoaminomonocarboxylic) amino acid transport in the apical brush border membrane of the small intestine and the proximal tubules of the kidney. Excessive amounts of amino acids, such as Tryptophan, are excreted in the urine.The body is thus left with inadequate amounts of amino acids. Urine Analysis –   Neutral amino acids, Tryptophan, Indole derivatives and Tryptophan degradation products are present in urine. Patients present with pellagra like skin eruptions, cerebellar ataxia,, nystagmus , tremor, failure to thrive, photosensitivity,  and gross aminoaciduria.Symptoms may be triggered by sunlight, fever, drugs, emotional or physical stress. A high-protein diet can overcome the deficient transport of neutral amino acids in most patients. Avoiding excessive exposure to sunlight, wearing protective clothing, and using physical and chemical sunscreens are mandatory, daily supplementation with nicotinic acid or nicotinamide reduces both number and severity of attacks.
Maple syrup urine disease MSUD is caused by a deficiency of the branched-chain alpha-Keto acid dehydrogenase complex (BCKDH), leading to a buildup of the branched-chain amino acids (leucine, Isoleucine, and Valine) and their toxic by-products in the blood and urine. Blood and urine in this disease have elevated levels of branched amino acids and their keto derivatives. The keto derivatives cause acidosis. Poor feeding, vomiting, dehydration, lethargy, hypotonia, seizures, Ketoacidosis, opisthotonus, pancreatitis, coma and neurological decline. The disease is characterized in an infant by the presence of sweet-smelling urine, with an odor similar to that of maple syrup. Infants with this disease seem healthy at birth but if left untreated suffer severe brain damage, and eventually die.  A diet with minimal levels of the amino acids leucine, Isoleucine, and Valine must be maintained in order to prevent neurological damage.As these three amino acids are required for proper metabolic functions, specialized protein preparations containing substitutes and adjusted levels of the amino acids have been synthesized and tested, allowing MSUD patients to meet normal nutritional requirements without causing harm.
Cystinuria The amino acid transporter for cystine and other amino acids like Arginine, ornithine and Lysine, is defective both in the intestine and in PCT of nephron. This leads to retention of cystine and other amino acids in urine. Cystine gets precipitated in acidic pH 1)Sodium cyanide nitroprusside test. in the presence of cystine a purple-red colour is observed.2) Flat x-ray of the kidney, ureters and bladder (KUB), is undertaken to visualize the stones.3) Microscopic examination  of urine  reveals flat hexagonal crystals of cystine.  Recurrent nephrolithiasis. Possible complications include obstruction of the urinary tract, which can predispose to infection of the urine ,fever may be apparent, and white blood cells are noted in the urine. Unrelieved obstruction leads to renal dysfunction, renal failure and the need for dialysis are quite rare. Medical therapy is directed toward dissolution of existing calculi and prevention of new stone formation.Increasing urine volume by generous oral fluid intake is beneficial.Cystine solubility can be improved by urinary alkalinization and where necessary by the administration of thiol chelators, particularly D-penicillamine or mercaptopropionyl glycine.

 

Carcinoid syndrome Carcinoid syndrome refers to the array of symptoms that occur secondary to Carcinoid tumors. Many of the symptoms of carcinoid syndrome are produced by serotonin or its metabolites. 24 hour urine levels of 5-HIAA (5-hydroxyindoleacetic acid), a breakdown product of serotonin. Patients with carcinoid syndrome usually excrete >25 mg of 5-HIAA per day. For localization of both primary lesions and metastasis, the initial imaging method is Octreoscan,  Flushing ,pellagra like rash, Secretory diarrhea and abdominal cramps are also characteristic features of the syndrome. When the diarrhea is intensive it may lead to electrolyte disturbance and dehydration. Other associated symptoms are nausea, and vomiting. About 50% of patients have cardiac abnormalities, caused by serotonin-induced fibrosis of the tricuspid and pulmonary valves. Symptomatic treatment,.octreotide (a somatostatin analogue that neutralizes serotonin and decreases urinary 5-HIAA),methysergide maleate (antiserotonin agent but not used because of serious side effect of retroperitoneal fibrosis),cyproheptadine (an antihistamine drug).Surgical resection of tumor and chemotherapy (5-FU and doxorubicin) are the alternative treatments. 
Homocystinuria (Homocysteinemia).

 

 

Three major forms of homocysteinemia and Homocystinuria have been identified :1) Homocystinuria due to Cystathionine beta Synthetase deficiency,(Classical Homocystinuria),2)  The other forms can be caused by defective Methionine synthase or reduced availability of two essential cofactors, 5-methyltetrahydrofolate and Methylcobalamine (methyl-vitamin B12).

 

Elevations of both Methionine and homocystine in body fluids are the diagnostic laboratory findings.Total plasma homocysteine is extremely elevated (usually >100 M). 

Cystine is low or absent in plasma.

 

The diagnosis may be established by assay of the enzyme in liver biopsy specimens, cultured fibroblasts.

 

Failure to thrive and developmental delay. Subluxation of the ocular lens (ectopia lentis) occurs. This causes severe myopia and iridodonesis (quivering of the iris), Astigmatism, glaucoma, cataracts, retinal detachment, and optic atrophy may develop later in life.  

Progressive mental retardation is common, skeletal abnormalities resemble those of Marfan syndrome;

Patients usually have fair complexions, blue eyes, and a peculiar malar flush.

Generalized osteoporosis, especially of the spine, is the main radiographic finding.

 

Thromboembolic episodes involving both large and small vessels, especially those of the brain, are common and may occur at any age.

 

 

 

Supplementation with pyridoxine, folic acid, B12 or trimethylglycine (betaine) reduces the concentration of homocysteine considerably in the blood stream. Those who do not respond require a low Methionine diet.
Cystinosis Cystinosis occurs due to a mutation in the gene CTNS, located on chromosome 17, which codes for cystinosin, the lysosomal cystine transporter. The accumulation is caused by abnormal transport of Cystine from lysosomes, resulting in a massive intra-lysosomal cystine accumulation in tissues. Via an as yet unknown mechanism, lysosomal cystine appears to amplify and alter apoptosis in such a way that cells die inappropriately, leading to loss of renal epithelial cells. This results in renal Fanconi syndrome and similar loss in other tissues can account for the short stature, retinopathy, and other features of the disease.  Definitive diagnosis and treatment monitoring are most often performed through measurement of white blood cell cystine level using tandem mass spectrometry.

 

 

Symptoms are first seen at about 3 to 18 months of age with profound polyuria (excessive urination), followed by poor growth, photophobia, and ultimately kidney failure by age 6 years in the nephropathic form. The other forms are Intermediate cystinosis (Juvenile) and Non-nephropathic or ocular cystinosis (Adult) forms. The drug cysteamine slows the progression of cystinosis by removing the cystine from cells, but for the drug treatment to be effective, it must be taken every six hours. Without specific treatment, these children progress to end-stage renal failure by an average age of nine years. Cystinosis is a common cause of the Fanconi Syndrome, a renal tubular disease.  

 

 

 

 

 

 

                                                    

 

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

Q.1- Which of the following is a common compound shared by the TCA cycle and the Urea cycle?

a) α- Keto glutarate

b) Succinyl co A

c) Oxalo acetate

d) Fumarate

Q.2-Which of the followings is a common nitrogen acceptor for all reactions involving transaminases?

a) α- Keto glutarate

b) Pyruvate

c) Oxaloacetate

d) Acetoacetate

Q.3- In a 55- year-old man, who has been diagnosed with cirrhosis of liver, Ammonia is not getting detoxified and can damage brain. Which of the following amino acids can covalently bind ammonia, transport and store in a non toxic form?

a) Aspartate

b) Glutamate

c) Serine

d) Cysteine

Q.4- In a new born presenting with refusal to feeds and irritability, a deficiency of Cystathionine –β– synthase has been diagnosed, which of the following compounds is expected to be elevated in blood?

a) Serine

b) Glutamate

c) Homocysteine

d) Valine

Q.5 -A 3- month-old child is being evaluated for vomiting and an episode of convulsions, Laboratory results show hyperammonemia and Orotic aciduria. Which of the following enzyme defect is likely to be there?

a) Glutaminase

b) Arginase

c) Argino succinic acid synthase

d) Ornithine Transcarbamoylase

Q.6- Which out of the following amino acids is not converted to Succinyl co A?

a) Methionine

b) Valine

c) Isoleucine

d) Histidine

Q.7-All of the following compounds are synthesized by transmethylation reactions, except-

a) Choline

b) Epinephrine

c) Creatine

d) Ethanolamine

Q.8- A patient diagnosed with Hart Nup disease, (due to deficiency of transporter required for the absorption of amino acid tryptophan), has been brought with skin rashes and suicidal tendencies. Tryptophan is a precursor for many compounds, the deficiencies of which can cause the said symptoms. Which out of the following compounds is not synthesized by tryptophan?

a) Serotonin

b) Epinephrine

c) Melatonin

d) Niacin

Q.9- Histamine, a chemical mediator of allergies and anaphylaxis, is synthesized from amino acid Histidine by which of the following processes?

a) Deamination

b) Decarboxylation

c) Transamination

d) Dehydrogenation

Q.10- The synthesis of all of the following compounds except one is deficient in a patient suffering from Phenylketonuria-

a) Melanin

b) Melatonin

c) Catecholamines

d) Thyroid hormone

Q.11- The diet of a child suffering from Maple syrup urine disease (an amino acid disorder), should be low, in which out of the following amino acids content?

a) Branched chain amino acids

b) Phenylalanine Alanine

c) Methionine

d) Tryptophan

Q.12- Which out of the following amino acids in not required for creatine synthesis?

a) Methionine

b) Serine

c) Glycine

d) Arginine

Q.13- All of the following substances are synthesized from Cysteine, except-

a) Taurine

b) Mercaptoethanolamine

c) Melanin

d) Pyruvate

Q.14- Urea is synthesized in –

a) Cytoplasm

b) Mitochondria

c) Both cytoplasm and mitochondria

d) In lysosomes

Q.15-Blood urea decreases in all of the following conditions, except-

a) Liver cirrhosis

b) Pregnancy

c) Renal failure

d) Urea cycle disorders

Q.16- All of the following amino acids are donors of one carbon compounds except-

a) Histidine

b) Tyrosine

c) Tryptophan

d) Serine

Q.17- The two nitrogen of urea are derived from-

a) Aspartate and Ammonia

b) Glutamate and ammonia

c) Argino succinate and ammonia

d) Alanine and ammonia

Q.18- Which out of the following amino acids is not required for the synthesis of Glutathione?

a) Serine

b) Cysteine

c) Glutamic acid

d) Glycine

Q.19- The first line of defence in brain in conditions of hyperammonemia is-

a) Urea formation

b) Glutamine synthesis

c) Glutamate synthesis

d) Asparagine formation

Q.20- Which coenzyme out of the followings is required for the oxidative deamination of most of amino acids?

a) Folic acid

b) Pyridoxal- P

c) FMN

d) FAD

Q.21-Chose the incorrect statement about amino acid Glycine-

a) One carbon donor

b) Required for the synthesis of haem

c) Forms oxalates upon catabolism

d) Both glucogenic as well as ketogenic

Q.22- Which out of the followings is required as a coenzyme for the transamination reactions?

a) Coenzyme A

b) Pyridoxal-P

c) Folic acid

d) Cobalamine

Q.23- A patient diagnosed with Homocystinuria should be supplemented with all of the following vitamins except-

a) Vitamin C

b) Folic acid

c) Vitamin B12

d) Pyridoxal- P

Q.24- In a patient suffering from Cystinuria, which out of the following amino acids is not seen in urine of affected patients?

a) Arginine

b) Methionine

c) Lysine

d) Ornithine

Q.25- Positive nitrogen balance is seen in all of the following conditions except-

a) Pregnancy

b) Growth

c) Fever

d) Convalescence

Q.26- The L-amino acids are absorbed from intestine by-

a) Active transport

b) Passive diffusion

c) Pinocytosis

d) Facilitated diffusion

Q.27- A child presented with increased frequency of urination, photophobia and impairment of vision. Which out of the following defects could be responsible for the said symptoms?

a) Tyrosinosis

b) Cystinosis

c) Alkaptonuria

d) Albinism

Q.28- Which out of the following statements about Glutamate dehydrogenase is correct?

a) Required for transamination reactions

b) Universally present in all the cells of the body

c) Can utilize either of NAD+ /NADP+

d) Catalyzes conversion of glutamate to glutamine

Q.-29-A child was brought to paediatric OPD with complaint of passage of black colored urine. A disorder of Phenylalanine metabolism was diagnosed. A low phenylalanine diet and a supplementation of vitamin C were recommended. Which enzyme defect is expected in this child?

a) Phenyl alanine hydroxylase

b) Tyrosine transaminase

c) Homogentisic acid oxidase

d) Hydrolase

Q.30- Dopamine is synthesized from which of the following amino acids?

a) Tyrosine

b) Tryptophan

c) Histidine

d) Methionine

Q.31- In mammalian tissue serine can be a biosynthetic precursor for which amino acid?

a) Methionine

b) Glycine

c) Arginine

d) Lysine

Q.32- Hydroxylation of Phenyl Alanine to Tyrosine requires all except

a) Glutathione

b) Tetra hydrobiopterin

c) Molecular oxygen

d) NADPH

Q.33- The amino acid that undergoes oxidative deamination at a highest rate is-

a) Glutamine

b) Glutamate

c) Aspartate

d) Alanine

Q.34- All of the following statements regarding serotonin are true except-

a) Causes vasodilatation

b) Causes broncho constriction

c) Metabolized to 5-hydroxy Indole acetic acid

d) Causes diarrhoea

Q.35- Choose the incorrect statement about cysteine-

a) Carbon skeleton is provided by serine

b) Sulfur group is provided by Methionine

c) Forms Hippuric acid for detoxification of xenobiotics

d) Required for Bile salt formation

 

 Answers-

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

19) – b, 20) – c, 21) – d, 22) – b,23) -a, 24)- b, 25) – c, 26) – a, 27) – b, 28) – c, 29) – c, 30) – a, 31) – b,32) – a, 33) – b, 34) – a,35) – c.

 


 

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

Case study-1

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. They had delayed disposing one of the child’s diapers and noted black discoloration where the urine had collected. Later, they realized that all of the child’s diapers would turn black if kept unwashed for a longer time.

The attending pediatrician examined the child and explained them that it was due to an amino acid disorder.

Which amino acid pathway is implicated in this phenomenon?

What is the cause of blackening of the diapers?

How can this defect be treated?

 

Case Study-2

A 12-year-old boy was admitted to the hospital with a red scaly rash and mild cerebellar ataxia. His mother thought that the boy is suffering from Pellagra because the same symptoms in her older daughter had been so diagnosed earlier. The boy did not have the usual dietary deficiency form of Pellagra, but large amounts of free amino acids were found in his urine. When the older daughter had a recurrent attack of ataxia, it was found that her urine also contained excessive amount of amino acids. Two other siblings had the sane aminoaciduria; however four others were normal. The parents were asymptomatic, but a family history revealed that they were first cousins.

Assuming that this defect is inherited, what abnormality could account for the unusual amount of amino acids in urine and what is its relationship with pellagra like rashes?

How can this defect be treated?

 

Case study-3

A 2-week –old infant with refusal to feed lethargy, excessive cry and irritability, responded positively to a test for Phenylketonuria, A diagnosis of Classical  Phenylketonuria (PKU) was made and the child was maintained on a low Phenyl Alanine diet.Serum Phenyl Alanine was 30 mg/dl and Tyrosine was 2mg/dl. Ferric chloride test was positive for urine. A diagnosis of Classical Phenylketonuria (PKU) was made and the child was maintained on a low Phenyl Alanine diet.

What enzymatic reactions are defective in the patient with PKU?

What are the physiological consequences of PKU and why it should be detected as early as possible?

What is the treatment for this disease?

 

 Case study-4

A child presented with severe vomiting, dehydration and fever. History revealed that the child was born normal but was not growing well from the last few months. There was progressive mental retardation. Urine analysis revealed the presence of branched amino acids and their Keto acids in high amount. Preliminary results from the blood amino acid screen showed two elevated amino acids with non polar side chains.

Blood studies showed acidosis with a low bicarbonate concentration. The urine of the patient had a smell of burnt sugar. Urine analysis revealed the presence of branched amino acids and their Keto acids in high amount. Preliminary results from the blood amino acid screen showed two elevated amino acids with non polar side chains.

What is the probable defect?

What is the basis for these symptoms?

 

Case study-5

A 38- year- old female reported with a dull pain in the left flank. The pain was radiating towards left leg. She was in real agony. She reported that there was fever and inability to pass urine from the last few days. History revealed that she was admitted twice with the similar symptoms in the previous six months. .

On general physical examination, she was found to be anemic, pulse was 80/minute, B.P. was 130/90 mm Hg and the abdomen was tender to touch. The patient was admitted for observation and treatment. There was Costovertrebral angle tenderness on Murphy’s punch.

Routine urinalysis revealed the presence of RBCs, pus cells, WBC casts, characteristic hexagonal crystals and amino acids. A diagnosis of aminoaciduria was made.

What is the probable defect? Which amino acid is expected to be there in urine?

What is the possible treatment?

 

Case study-6

A 54-year-old post menopausal woman, presented to internist for care of hot flashes that have returned 2 years after menopause. The symptoms occurred mostly after meals, when she drank wine, or when she went for running. There was history of hypertension and frequent diarrhea also. The patient was referred to gynecologist. The general physical examination was entirely normal. Hormone-replacement therapy (HRT) was prescribed to the patient and she was referred to gastroenterologist (GI) for Irritable Bowel Syndrome (IBS) treatment.  She experienced 2 hot flashes with no sweating during the examination.

Patient returned 3 months later with no improvement in hot flashes. A different formulation of  HRT was prescribed. Patient returned to GI doctor 1 year later with worsening of her IBS. Patient reported cramping, abdominal pain, and increased episodes of diarrhea. She had to wake up at night with diarrhea. Patient discontinued her HRT   although they were ineffective. After 1 year, patient was in need of emergency care. She presented with acute bowel obstruction and was taken to the operating room.

There was marked fibrosis of the terminal ileum with multiple hairpin turns of the bowel and a small tumor in the terminal ileum. Pathological report concluded that the patient had Carcinoid syndrome.

What is the probable defect in Carcinoid syndrome?

How can this be diagnosed at the earliest possible? What is the biochemical basis for all the clinical manifestations?

What is the prognosis of this disease?

 

 


 

 

 

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

Advertisement