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Case Studies

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.

 

 

 

 

 

 

 

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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.

 

 

 

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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.

 

 

 

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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.

 

 

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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.

 

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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.

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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.

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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.

 

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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.

 

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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?

 

 


 

 

 

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A 2 –year-old child was brought to pediatrics  department for consultation. Mother of child reported that the child was born normally; there was no complication during delivery, and her other two children were also normal. The child was not taking feeds properly and was also not growing well.

There was history of increased frequency of urination from the previous few months for which the mother was very much apprehensive. The child avoided going to sun and there was apparent visual impairment.

On general physical examination, the child was found pale, thin and small for his age. There was slight hepatomegaly and he looked slightly dehydrated.

A urine sample was sent for complete analysis. The urine examination revealed presence of excessive amount of phosphates and amino acids. A thorough examination of eye was also done to know the cause of visual impairment. On slit lamp microscopic examination,characteristic crystals were found deposited in the cornea.

What is the possible defect?

What is the biochemical basis for phosphate and aminoacids in urine?

Case discussion-

The child is suffering from Cystinosis. Cystinosis is a Lysosomal storage disease characterized by the abnormal accumulation of the amino acid Cystine. Excess cystine forms crystals that can build up and damage cells. These crystals  negatively affect many systems in the body, especially the kidneys and eyes. Excessive urination, phosphaturia and aminoaciduria in the given patient are due to renal tubular damage while the visual impairment is due to deposition of cystine crystals in cornea.

Cystinosis

Inheritance- Cystinosis is an autosomal recessive genetic disease.

Biochemical defect-

Endogenous protein enters the lysosome, where acid hydrolases degrade it to its component amino acids, including cysteine. Within the lysosome, cysteine is readily oxidized to cystine (a disulfide of the aminoacid cysteine). In healthy individuals, both cystine and cysteine can normally enter the cytoplasm, where cystine is rapidly converted to cysteine by the reducing agent glutathione under the activity of oxidoreductase enzyme.Cytoplasmic cysteine is incorporated into protein or degraded to inorganic sulfate for excretion.

Cystinosis is caused by one of several mutations in the gene that encodes cystinosin, the cystine-lysosomal exporter. Because of the defect in cystinosin, cystine cannot leave the lysosomes and is accumulated there as birefringent, hexagonal, or rectangular crystals within cells of various organ systems.

Clinical Manifestations- There are three distinct types of cystinosis. In order of decreasing severity, they are –

·        Nephropathic Cystinosis (Infantile)

·        Intermediate Cystinosis (Juvenile)

·        Non-Nephropathicor ocular Cystinosis (Adult)

1) Nephropathic Cystinosis-In the infantile nephropathic form of cystinosis, the kidney is affected early in life by cystine crystals deposited in proximal tubule cells. This leads eventually to a Fanconi syndrome,characterized by wasting of substances reabsorbed in this nephron segment,including sodium, potassium, phosphate, calcium, magnesium, bicarbonate, and others. Metabolic acidosis and electrolyte disturbances ensue and contribute to the stunting of growth in children with cystinosis. Cystinosis is the most common inherited cause of Fanconi syndrome.

Patients usually present during the first year of life with polyuria, polydipsia, dehydration, metabolic acidosis (normal anion gap hyperchloremic acidosis), hypophosphatemic rickets, failure to thrive, and laboratory findings consistent with Fanconi’s syndrome. If untreated, renal failure develops by age 7-10 years.

Cystine continues to accumulate in other tissues,resulting in such complications as eye disease (eg, severe photophobia, corneal ulcerations, and retinal blindness), delayed puberty, hypothyroidism,pancreatic disease (eg, exocrine insufficiency, insulin-dependent diabetes mellitus), liver disease, swallowing difficulties, and CNS involvement

2)Intermediate Cystinosis-The signs and symptoms of intermediate cystinosis are the same as Nephropathic Cystinosis, but they occur at a later age.Intermediate cystinosis typically becomes apparent in affected individuals in adolescence. Malfunctioning kidneys and corneal crystals are the main initial features of this disorder. If intermediate Cystinosis is left untreated,complete kidney failure will occur, but usually not until the late teens to mid-twenties.

3) Nonnephropathic Cystinosis is considered a benign variant and is usually diagnosed by an ophthalmologist treating patients for photophobia. Photophobia may not begin until middle age and is not usually as debilitating as in the nephropathic form of the disease. Slit-lamp examination reveals corneal crystal deposits. In addition to the eye, cystine crystals are present in the bone marrow and leukocytes but are absent in the kidney and the retina.

Diagnosis- 

1) Serum electrolytes measurements are used to detect the presence of acidosis (hyperchloremic, normal anion gap) and severity of hypokalemia,hyponatremia, hypophosphatemia, and low bicarbonate concentration in patients with cystinosis. 

2) Blood gases may be used to detect metabolic acidosis and the degree of respiratory compensation.

3) Urine testing reveals low osmolality, glucosuria, and tubular proteinuria(including generalized amino aciduria). 

4) Measurements of urine electrolytes serve to detect the loss of bicarbonate and phosphaturia.

Definitive diagnosis and treatment monitoring are most often performed through measurement of white blood cell cystine level.

Other Investigations

  • Renal ultrasonography, Radiography for kidneys, ureters, and bladder (KUB) may be needed to evaluate possible urinary tract calcifications.
  • CT scanning and MRI are used to evaluate adult patients with infantile nephropathic cystinosis who have CNS symptoms.
  • Slit-lamp examination of the eyes reveals corneal and conjunctival cystine crystals (pathognomonic for cystinos is) as early as age 1 year, although photophobia does not usually become apparent until age 3-6 years.
  • Examination of the eye fundi may reveal the presence of peripheral retinopathy. In some patients, retinopathy may lead to blindness.

Treatment- Cystinosis is a common cause of the Fanconi Syndrome, a renal tubular disease.  By about one year of age,patients eliminate large volumes of urine and lose large amounts of salt and other minerals in their urine.

Replacement of urinary losses: The child must be well-hydrated and administered supplements of potassium and bicarbonate, as needed. Rickets should be treated with vitamin D and phosphate supplementation.Without specific treatment, these children progress to end-stage renal failure by an average age of nine years.  These patients can receive renal dialysis or transplantation, but even with successful transplantations, they develop abnormalities in other organs.The drug cysteamine slows the progression of cystinos is by removing the cystine from cells, but for the drug treatment to be effective, it must be taken every six hours. When administered regularly, cysteamine decreases the amount of cystine stored in lysosomes and correlates with conservation of renal function and improved growth. Cysteamine eye drops remove the cystine crystals in the cornea that can cause photophobia if left unchecked.


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Case Details

A 6 month- old Infant began to vomit occasionally and ceased to gain weight. At 9 months of age he was readmitted to the hospital.Routine examination and laboratory tests were normal but after one week he became drowsy, his temperature rose to 39.4 ° C, his pulse was elevated, and his liver was enlarged. The Electro Encephalogram (EEG) was grossly abnormal. Since the infant could not retain milk by tube feeding, Intravenous glucose was administered. Urine analysis showed abnormally high amount of Glutamine and Uracil. This suggested high amount of Ammonia concentration, which was confirmed by laboratory test.

What is the cause of hyper ammonemia in this patient?

Why were the urine glutamine and Uracil levels elevated?

How can such a patient be treated?

Case Discussion-The child is most probably suffering from hyperammonemia due to impaired urea formation. Hyperammonemia in a new born or very young infant is the characteristic sign of inherited defect in a gene for urea cycle enzymes. The enzyme affected in this patient seems to be Ornithine Transcarbamoylase as apparent from the 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. This case is unusual in that the symptoms took longer to appear. Urine glutamine excretion has increased because it is excreted in compensation for the inoperative urea cycle.  Free ammonia is toxic to brain. It is detoxified by conversion of Glutamate to glutamine. High Glutamine level indicates hyperammonemia that may be due to any liver pathology or may be due to defective urea cycle enzymes.

Ornithine Transcarbamoylase deficiency

The disease is characterized as X linked dominant because most females are also somewhat affected. Females usually respond well to treatment. A significant number of carrier females have hyperammonemia and neurologic compromise. The risk for hyperammonemia is particularly high in pregnancy and the postpartum period. The disease is much more severe in males than in females. The enzyme activity can range from 0% to 30% of the normal. 

Urea formation

(Urea cycle)

The urea cycle is the sole source of endogenous production of arginine and it is the principal mechanism for the clearance of waste nitrogen resulting from protein turnover and dietary intake. This extra nitrogen is converted into ammonia (NH3) and transported to the liver where it is processed. The urea cycle disorders (UCD) result from inherited molecular defects which compromise this clearance.

Reactions of urea cycle

Synthesis of 1 mol of urea requires 3 mol of ATP plus 1 mol each of ammonium ion and of the α-amino nitrogen of aspartate. Five enzymes catalyze the reactions of urea cycle (See Figure). 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. The major metabolic role of Ornithine, Citrulline, and argininosuccinate in mammals is urea synthesis. Urea synthesis is a cyclic process. Since the Ornithine consumed in 2nd reaction and is regenerated in last reaction, so there is no net loss or gain of Ornithine, Citrulline, argininosuccinate, or arginine. Ammonium ion, CO2, ATP, and aspartate are, however, consumed. Some reactions of urea synthesis occur in the matrix of the mitochondrion, other reactions in the cytosol (See Figure).

Reaction 1- Carbamoyl Phosphate Synthase I Initiates Urea Biosynthesis

Condensation of CO2, ammonia, and ATP to form Carbamoyl phosphate is catalyzed by mitochondrial Carbamoyl phosphate synthase I (CPS-1), A cytosolic form of this enzyme, Carbamoyl phosphate synthase II, uses glutamine rather than ammonia as the nitrogen donor and functions in pyrimidine biosynthesis. 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. Formation of Carbamoyl phosphate requires 2 mol of ATP, one of which serves as a phosphoryl donor.

Reaction -2-Carbamoyl Phosphate Plus Ornithine Forms Citrulline

L-Ornithine Transcarbamoylase (OTC) catalyzes transfer of the Carbamoyl group of Carbamoyl phosphate to Ornithine, forming Citrulline and orthophosphate. While the reaction occurs in the mitochondrial matrix, both the formation of Ornithine and the subsequent metabolism of Citrulline take place in the cytosol. Entry of Ornithine into mitochondria and exodus of Citrulline from mitochondria therefore involve mitochondrial inner membrane transport systems.

Reaction -3 Citrulline plus Aspartate Forms Argininosuccinate

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 and involves an intermediate formation of citrullyl-AMP. Subsequent displacement of AMP by aspartate then forms Argininosuccinate.

Reaction -4-Cleavage of Argininosuccinate Forms Arginine & Fumarate

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. These two reactions are analogous to reactions of the citric acid cycle but are catalyzed by cytosolic Fumarase and malate dehydrogenase. Transamination of oxaloacetate by glutamate aminotransferase then re-forms aspartate. (See Figure 2)The carbon skeleton of aspartate-fumarate thus acts as a carrier of the nitrogen of glutamate into a precursor of urea.

Reaction -5-Cleavage of Arginine Releases Urea & Re-Forms Ornithine

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. Ornithine and lysine are potent inhibitors of arginase, competitive with arginine. 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

Carbamoyl Phosphate Synthase I Is the Pacemaker Enzyme of the Urea Cycle

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. 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.

 

Figure-1- showing reactions of urea cycle

 

 Figure -2 showing the relationship of Urea cycle to TCA

Fate of Urea- 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. (See figure -3

 

 Figure-3 Showing the fate of urea

 Urea cycle disorders

1) Carbamoyl Phosphate synthetase (CPS-1) deficiency

Along with OTC deficiency, deficiency of CPSI is the most severe of the urea cycle disorders. Individuals with complete CPS-I deficiency rapidly develop hyperammonemia in the newborn period. Children who are successfully rescued from crisis are chronically at risk for repeated bouts of hyperammonemia.

2) Ornithine Transcarbamoylase deficiency (OTC deficiency)

Absence of OTC activity in males is as severe as CPSI deficiency. Approximately 15% of carrier females develop hyperammonemia during their lifetime and many require chronic medical management

3) Citrullinemia type I (ASS deficiency)

The hyperammonemia in this disorder is quite severe. Affected individuals are able to incorporate some waste nitrogen into urea cycle intermediates, which makes treatment slightly easier.

4) Argininosuccinic aciduria (ASL deficiency)

This disorder also presents with rapid-onset hyperammonemia in the newborn period. This enzyme defect is past the point in the metabolic pathway at which all the waste nitrogen has been incorporated into the cycle. Treatment of affected individuals often requires only supplementation of arginine. ASL deficiency is marked by chronic hepatic enlargement and elevation of transaminases. Biopsy of the liver shows enlarged hepatocytes, which may over time progress to fibrosis, the etiology of which is unclear. Affected individuals can also develop trichorrhexis nodosa, a node-like appearance of fragile hair, which usually responds to arginine supplementation. Affected individuals who have never had prolonged coma but nevertheless have significant developmental disabilities have been reported.

5) Arginase deficiency (hyperargininemia; ARG deficiency)

This disorder is not typically characterized by rapid-onset hyperammonemia. Affected individuals develop progressive spasticity and can also develop tremor, ataxia, and choreoathetosis. Growth is affected

6) NAG Synthase deficiency. Deficiency of this enzyme has been described in a number of affected individuals. Symptoms mimic those of CPSI deficiency; since CPSI is rendered inactive in the absence of NAG

Incidence

The incidence of UCDs(Urea cycle disorders)is estimated to be at least 1:25,000 births; partial defects may make the number much higher.

Clinical manifestations

Infants with a urea cycle disorder often appear normal initially but rapidly develop cerebral edema and the related signs of lethargy, anorexia, hyperventilation or hypo ventilation, hypothermia, Slurring of the speech, Blurring of vision, seizures, neurological posturing, and coma. In milder (or partial) urea cycle enzyme deficiencies, ammonia accumulation may be triggered by illness or stress at almost any time of life, resulting in multiple mild elevations of plasma ammonia concentration; the hyperammonemia is less severe and the symptoms more subtle. In individuals with partial enzyme deficiencies, the first recognized clinical episode may be delayed for months or years.

Laboratory diagnosis

The diagnosis of a urea cycle disorder is based on evaluation of clinical, biochemical, and molecular genetic data.

·    A plasma ammonia concentration of 150 mmol/L or higher is a strong indication for the presence of a UCD.

·    Plasma quantitative amino acid analysis can be used to diagnose a specific urea cycle disorder: plasma concentration of arginine may be reduced in all urea cycle disorders, except ARG(Arginase) deficiency, in which it is elevated five- to sevenfold;

·    Plasma concentration of Citrulline helps discriminate between the proximal and distal urea cycle defects, as Citrulline is the product of the proximal enzymes (OTC and CPSI) and a substrate for the distal enzymes (ASS, ASL, ARG).

·    Urinary Orotic acid is measured to distinguish CPSI deficiency and NAGS (N-Acetyl Glutamate Synthase) deficiency from OTC deficiency.

·     A definitive diagnosis of CPS-I deficiency, OTC deficiency, or NAGS deficiency depends on determination of enzyme activity from a liver biopsy specimen.

·     However, the combination of family history, clinical presentation, amino acid and Orotic acid testing, and, in some cases, molecular genetic testing is often sufficient for diagnostic confirmation, eliminating the risks of liver biopsy.

Treatment

The mainstays of treatment for urea cycle disorders include-

·    Dialysis to reduce plasma ammonia concentration,

·    Intravenous administration of arginine chloride and nitrogen scavenger drugs to allow alternative pathway excretion of excess nitrogen, Excess nitrogen is removed by intravenous phenyl acetate and that conjugate with glutamine and glycine, respectively, to form phenylacetylglutamine and Hippuric acid, water-soluble molecules efficiently excreted in urine.

·    Arginine becomes an essential amino acid (except in arginase deficiency) and should be provided intravenously to resume protein synthesis. If these measures fail to reduce ammonia, hemodialysis should be initiated promptly.

·   Restriction of protein for 24-48 hours to reduce the amount of nitrogen in the diet, providing calories as carbohydrates (intravenously as glucose) and fat (intralipid or as protein-free formula) to reduce catabolism,

·   Physiologic stabilization with intravenous fluids

·    Chronic therapy consists of a protein-restricted diet, phenyl butyrate (a more palatable precursor of phenyl acetate), arginine, or Citrulline supplements, depending on the specific diagnosis.

·   Liver transplantation should be considered in patients with severe urea cycle defects that are difficult to control medically.

Genetic counselling

Deficiencies of CPSI, ASS, ASL, NAGS, and ARG are inherited in an autosomal recessive manner. OTC deficiency is inherited in an X-linked manner. Prenatal testing using molecular genetic testing is available for five of the six urea cycle disorders

Differential diagnosis

A number of other disorders that perturb the liver can result in hyperammonemia and mimic the effects of a urea cycle disorder. The most common/significant ones are viral infection of the liver and vascular bypass of the liver.

 

Figure-4 showing the mechanism of Nitrogen removal by Glycine, phenyl acetate and Arginine

 

Figure- 5 showing an overview of Urea cycle. Glutamate is the ultimate precursor for both nitrogen atoms of urea. One through ammonia by oxidative deamination of Glutamate by glutamate dehydrogenase and the second through the activity of transaminase whereby aspartate is formed from Glutamate, hence both nitrogen actually come from Glutamate only.

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