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Subjective Questions

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.


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


  • 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.


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.


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


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


  • 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


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.


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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).




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


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.


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.


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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

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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.

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

Q.2- What is the role played by Glutathione in the absorption of amino acids?

Q.3- Discuss the disorders associated with the absorption of amino acids.

Q.4- Justify the reasoning that glutamic acid plays a pivotal role in the metabolism of amino acids.

Q.5- Alpha Methyldopa is a drug used in the treatment of hypertension. Explain its possible mode of action.(Hint- It is an inhibitor of DOPA Decarboxylase enzyme)

Q.6- Discuss the mechanism by which Ammonia is detoxified in the body.

Q.7-Describe the glucose-alanine cycle and explain its role in amino acid metabolism.

Q.8-Decarboxylation of some amino acids can lead to synthesis of physiologically important compounds. Give evidences in support of this statement.

Q.9- What is the significance of urea cycle apart from urea formation?

Q.10- Give the reactions of the pathway of urea synthesis that involve the participation of ATP

Q.11-What is oxidative deamination of amino acids? Give examples in support of your answer.

Q.12-Name two neurotransmitters that are derived from the metabolism of amino acids Show by means of reactions the mechanism of synthesis of each of them.

Q.13- What is transdeamination? State its importance and illustrate the answer giving reactions in support of your answer.

 Q.14-Discuss the biochemical roles of glutamate and glutamine in cell metabolism

 Q.15-How will you define a non-essential amino acid? Under what condition can a non-essential amino acid become essential? Explain clearly and illustrate your answer giving suitable example.

Q.16-Discuss briefly about the metabolic role of Tyrosine,giving examples and suitable reactions

Q.17-Describe transmethylation reactions giving suitable examples

Q.18-Show, by means of a diagram, the relationship between the urea cycle and the citric acid cycle

Q.19-What is meant by (a) ketogenic amino acid and (b)glucogenic amino acid ? Illustrate your answer with a named example of each.

 Q.20-Explain why phenylketonurics are warned against eating products containing the artificial sweetener aspartame (Nutrasweet; chemical name L-Aspartyl-L-Phenylalanine methyl ester)?

 Q.21-Why are polyamines important in mammalian metabolism? Write the reactions of polyamine biosynthesis and catabolism

 Q.22-Describe the importance of glutamic acid in the synthesis and catabolism of other amino acids

 Q.23-Name the immediate precursor and the enzyme catalyzing the formation of: (a) GABA (gamma-amino butyric acid) (b) Histamine  and (c) DOPA(dihydroxyphenylalanine).

 Q.24-Explain briefly why ammonia is highly toxic to brain cells?

 Q.25-A diet containing very little phenylalanine is used in the treatment of Phenylketonuria, what is the reason? Explain why it is necessary to supplement tyrosine in this diet.

 Q.26-Certain amino acid are described as glucogenic. Explain briefly what is meant by the term “glucogenic”, illustrating your answer with the metabolic reactions of three named glucogenic amino acid.

 Q.27-Outline the metabolic processes by Tryptophan is converted into hormones and neurotransmitters. Describe briefly the clinical condition produced by deficiencies in these processes.

 Q.28-Discuss the significance of Xanthurenic acid excretion test.

 Q.29-Show by means of a diagram the point of entry of phenylalanine, glutamine and methionine into the citric acid cycle

 Q.30-Outline the steps of urea cycle and state its importance.

 Q.31-Give the reaction catalyzed by a named (a) amino acid Decarboxylase and (b) aminotransferase

 Q.32-What is the origin of the nitrogen atoms in urea formation ?  Discuss the reason that deficiency of urea cycle enzymes especially Ornithine Trans Carbamoylase leads to Orotic aciduria

 Q.33-What is the P:O ratio when glutamate is oxidized by the glutamate dehydrogenase reaction? Show the reaction insupport of your answer. 

 Q.34-Outline the metabolic role of glycine, justifying the fact that it is nutritionally non-essential but functionally very essential.

 Q.35-Trace the metabolic origin of the following urinary constituents: (a) creatinine (b) urea and (c) ammonia. Discuss the significance of their altered excretion with suitable examples.

Q.36-Account for the biochemical changes in the blood of a phenylketonuric subject.

Q.37- What is the biochemical basis for pellagra like rashes in Hart nup disease ?

Q.38- What is the defect in Carcinoid syndrome?  What is the biochemical basis of increased HIAA (Hydroxy Indole Acetic acid excretion) in Carcinoid syndrome ?

Q.39- Metabolism of which amino acid is associated with FIGLU excretion test for the detection of underlying folic acid deficiency?

Q.40- What is the defect in Maple syrup urine disease? Discuss in brief about the symptoms, laboratory diagnosis and its treatment.

Q.41- What is the cause of  increased risk for ischemic heart disease in patients of Homocystinuria  Discuss in brief about the classification, clinical manifestations and laboratory diagnosis of Homocystinuria.

Q.42- Discuss the functions and therapeutic uses of nitric oxide

Q.43–Discuss briefly about the biological and clinical significance of Transaminases.

 Q.44- What is the defect in Cystinuria? Why is it associated with renal stone formation?

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