DISORDERS OF AMINO ACID METABOLISM

Proteins are complex organic nitrogenous compounds made up of smaller units-amino acids, which are part of almost all enzymes required for human metabolism. Common IEMs of amino acid metabolism are as follows: Phenylketonuria: Phenylalanine is an essential amino acid, which is degraded into tyrosine, if not utilized.

Clinically, children with classic PKU, which accounts for gt;98% cases, are normal at birth and usually manifest at 2-3 months with—(a) progressive mental retardation,

(b) neurological features, e.g. irritability, seizures and hypertonia (c) fair complexion with blond hair, blue eyes and occasionally eczema due to tyrosine deficiency, which is essential for melanin production, and (d) mousy odor in urine due to excretion of alternate phenylalanine products.

Diagnosis depends on—(a) plasma phenylalanine levels gt;2 mg/dl, with (b) altered plasma phenylalanine: tyrosine ratio (gt;1:3). Urinary excretion of alternate metabolites may be used for screening as Guthrie test or Ferric chloride test (Add few drops of 1% ferric chloride to fresh urine, which turns deep green due to presence of phenylpyruvic acid). Molecular diagnosis is confirmatory.

Neonatal screening for PKU is widely practiced in many countries, involves whole-blood phenylalanine assay after 48-72 hours of birth by a semi-quantitative bacterial inhibition assay on dried blood spot.

Treatment aims to reduce phenylalanine/metabolites levels to minimize brain damage. Controlled dietary restriction of phenylalanine using commercial formula, is the key of successful management, which should begin within 1-2 weeks of life and continued till at least 8-10 years.

During dietary restriction, plasma phenylalanine levels should be closely monitored to maintain between 3-15 mg/dl, as over-restriction may lead to deficiency. Adequate dietary intake of tyrosine is essential as its endogenous synthesis is impaired in PKU.

Pregnant mothers with untreated PKU are at risk for (a) spontaneous abortions, (b) babies with mental retardation, microcephaly, agenesis of corpus callosum and heart defects. Prospective mothers should begin low phenylalanine diet before conception to maintain plasma phenylalanine levels lt;10 mg/dl.

Malignant PKU, carries poor prognosis with rapid neurological deterioration within few months of life. Apart from dietary phenylalanine restriction, some cases may benefit with co-factor BH4 supplementation and/or therapy with neurotransmitter precursors, e.g. L-dopa or 5-HT, to prevent neurological damage.

Tyrosinemia: Tyrosine is a precursor of many important human proteins, e.g. dopamine, norepinephrine, epinephrine, melanin and thyroxine. Hypertyrosinemia is seen in many inherited enzyme defects or acquired disorders, e.g. liver failure, scurvy and hypothyroidism.

Clinically, Hereditary tyrosinemia (Tyrosinemia type I) is an autosomal recessive deficiency of Fumarylacetoacetate hydrolase enzyme, which presents with:

• Failure to thrive and developmental retardation,

• Acute hepatic crisis with fever, vomiting, jaundice, hypoglycemia, hemorrhagic tendencies and a cabbage like odor due to accumulated methionine metabolites. Crises are often precipitated by intercurrent infections and resolve spontaneously or progress to cirrhosis or hepatic carcinoma in survivors.

• Episodic acute peripheral neuropathy, also triggered by intercurrent infections, presents with severe leg pain, hypertonia and motor weakness.

• Fanconi-like syndrome due to renal tubular dysfunction with metabolic acidosis, hyperphosphaturia with nephrocalcinosis and hypophosphatemia with vitamin D resistant rickets.

Early onset cases usually die by 2 years, while others may survive till 8-10 years before death due to cirrhosis or hepatic tumors.

Diagnosis must be suspected on presence of succiny­lacetoacetate and succinylacetone in serum and urine, confirmed by molecular test by sanger or next generation sequencing. Prenatal diagnosis by NGS or enzyme assay in chorionic villous biopsy or succinylacetone levels in amniotic fluid is possible.

Treatment includes dietary restriction of tyrosine and phenylalanine to retard progression of disease, though liver transplant as early as possible is the only effective therapy. An alternate enzyme inhibitor Nitisinone or NTBC (PO 1 mg/day), an inhibitor of the upstream enzyme 4-HPP,) is the mainstay of treatment to prevents toxic metabolite formation. However, as it does not restrict tyrosine formation, diet restriction is mandatory. Liver transplant is recommended only in case of acute liver failure and hepatocellular carcinoma.

Tyrosinemia type II (oculocutaneous tyrosinemia) presents with mental retardation, self-mutilating behavior and herpetiform corneal ulcers in infancy due to tyrosine deposition; and palmar/planter punctuate hyperkeratosis in older children, which responds well to dietary restriction.

Transient tyrosinemia of newborn is usually seen in preterms on high-protein diet, due to delayed matu­ration of enzyme 4-hydroxy phenylpyruvate dioxygenase. Most cases are asymptomatic but some may develop lethargy, poor feeding, decreased motor activity due to high phenylalanine levels. While most cases recver spontaneously in 3-4 weeks, symptomatic cases need dietary protein restriction and vitamin C supple­mentation (200-400 mg/d).

Albinism denotes a group of genetic disorders with defective biosynthesis and distribution of melanin due to enzyme tyrosinase deficiency, despite normal number of melanocytes.

Clinically, Albinism may be generalized or localized, though all variants have some degree of hypopig­mentation of hair and skin, with/without eye involve­ment. Hypopigmented skin tends to burn on exposure to sunlight, hair are white and silky and those with ocular involvement have translucent/pinkish or bluish sclera with photophobia, diminished visual acuity and strabismus. Blindness and skin cancers are common in severe albinism.

Management depends on severity and includes: (a) avoidance of sun exposure and use of sun-protection creams, (b) ophthalmic support with anti-glare lenses,

(c) vocational support, and (d) genetic counseling.

Important variants of albinism include:

• Oculocutaneous albinism type I (OCA-1), is the most severe form with complete lack of pigment in skin/ hair and eyes since birth, though some pigmentation may occur with advancing age.

• Oculocutaneous albinism type II (OCA-2), the commonest type with partial tyrosinase activity, presents at birth with some pigmentation of skin/eyes and yellowish hair and continue to collect pigment throughout the life.

• Ocular albinism is limited to eyes with near-normal skin/hair pigmentation and hair bulb tyrosinase activity. Most cases are X-linked.

• Partial albinism may also be associated with immuno­deficiency states, e.g. Chediak-Higashi syndrome (with neutropenia and increased susceptibility to infection), and Hermansky-Pudlak syndrome (with platelet dysfunction and bleeding tendencies).

• Localized albinism, limited to a small part of skin/ hair, is usually auotosomal dominant and may be present at birth or develop over time. Two important syndromes associated with localized albinism are - Piebaldism with white forelock at birth with/out white macules over face, trunk and limbs; and Waardenburg syndrome with white forelock, heterochromia iridis and sensorineural deafness.

Homocystinuria, an autosomal recessive disorder of methionine metabolism, is the second commonest error of amino acid metabolism. Classic homocystinuria, the commonest variant, denotes deficiency of enzyme cystathionine ^-synthetase, which leads to accumulation of homocystine (an intermediate metabolite) and methionine (dietary amino acid) in various body fluids and tissues.

Clinically, these cases are asymptomatic in infancy and usually manifest at 3-4 years of age with:

• Ocular complications, e.g. ectopia lentis with consequent myopia, iridodonesis, glaucoma, cataracts and retinal detachment/optic atrophy;

• Skeletal manifestations with generalized osteoporosis and marfanoid features, e.g. tall and thin stature, arachnodactyly, scoliosis, pectus excavatum, genu valgum, etc.;

• Neurological manifestations, e.g. mental retardation and psychiatric/behavioral disorders; and

• Susceptibility to thromboembolic episodes, e.g. cere­brovascular strokes, renovascular hypertension, pulmonary embolism, etc.

Diagnosis rests on elevated levels of homocystine (gt;100 #956;mol#8725;L) and methionine (gt;50 #956;mol#8725;L) in plasma as well as in urine. It is confirmed by enzyme assay or molecular testing in liver biopsy specimen. Cyanide nitroprusside test is a simple screening test in these cases to detect presence of homocystine in urine.

Prenatal diagnosis is possible by enzyme assay in cultured amniotic cells or chorionic villi. Whole blood methionine estimation is used for neonatal screening. Treatment with high doses of Pyridoxine (200-1000 mg#8725;d) is effective in ~40% cases and should be continued throughout the life, along with dietary methionine restriction and cysteine, folate and B₁₂ supplementation

Non-responsive cases may benefit with betaine therapy (trimethylglycine 100 mg/kg/day q12hr), which stimulates reconversion of homocystine into methionine. Hartnup disease, an autosomal recessive disorder with mutation in SLC6A19 gene causing defective transport of various neutral amino acids, specially tryptophan, across the gut mucosa and renal tubules is not uncommon but usually asymptomatic.

Clinically, these cases present with:

• Cutaneous photosensitivity with pellagra-like rash or chronic eczema, and

• Intermittent episodes of cerebellar ataxia and psychiatric disturbances.

Symptoms are usually exacerbated after diarrhea or low-protein diet. Long, spontaneous remissions are common.

Diagnosis rests on: (a) selective aminoaciduria with normal excretion of non-neutral amino acids, e.g. arginine, proline or hydroxyproline, and (b) heavy urinary excretion of indole derivatives, e.g. indican. Indicanuria, on oxidation into indigo-blue after air exposure, leads to blue staining of diapers*. Plasma levels of tryptophan and other neutral amino acids are usually normal due to alternative modes of absorption.

*Blue-diaper syndrome is a familial disorder with indicanuria, hypercalcemia and nephrocalcinosis.

Treatment includes high protein diet to replenish urinary loss of amino acids along with nicotinamide supplements (50-300 mg#8725;day) in symptomatic cases and avoidance of sun-exposure.

Alkaptonuria, the first identified IEM, is a rare autosomal recessive disorder with deficiency of homogentisic acid oxidase enzyme, essential for degradation of homogentisic acid-an intermediate metabolite of tyrosine metabolism. Consequent accumulation of homogentisic acid in tissues leads to black pigmentation and destruction of connective tissues (ochronosis) and increased excretion in urine, which turns black on oxidation.

Clinically, these cases are essentially asymptomatic in childhood except black discoloration of urine on standing, specially if it is alkaline.

Most cases manifest in adults with: (a) ochronosis, i.e. black pigmentation of sclera and ear/nose cartilage, (b) progressively degenerative arthritis, specially involving spine and large joints. Nephrolithiasis due to renal pigment deposits may develop in later stages.

Diagnosis is based on elevated levels of homogentisic acid in urine and bi-allelic pathogenic variant in HGD gene. Homogentisic acid is a strong antioxidant and its presence in urine is indicated by black reaction with Benedict or Fehling agent. (d/d black urine include phenol poisoning and melanotic tumors)

Treatment is non-specific, though vitamin C therapy (PO 1 gm/d) and Nitisinone (PO 1 mg/day) may delay ochronosis and degenerative arthritic changes. NTBC has shown to slow progression of disease and is approved for treatment in some European countries. Physiotherapy is recommended to promote strength and flexibility.

Organic acidemia or aciduria, is a group of amino- acidopathies due to enzymatic defects in catabolism of three essential branched-chain amino acids, i.e. valine, leucine and isoleucine. Most of these disorders present in neonatal period with persistent metabolic acidosis and neurological dysfunction, with/without hyperammonemia.

Organic acidosis with wide anion gap (gt;15 mEq/L) is the hallmark of these disorders, though many other inborn errors with metabolic acidosis (Table 11.16) must be excluded in differential diagnosis.

Maple syrup urine disease (MSUD) is a rare autosomal recessive defect in decarboxylation of these branched chain amino acids due to deficiency of a complex enzyme system—branched-chain-ketoacid dehydrogenase. Consequent accumulation of intermediate metabolites in body fluids and their excretion in urine leads to metabolic acidosis, neurological manifestations and a typical maple syrup-like odor in urine.

TABLE 11.16: IEMs with metabolic acidosis

a. Carbohydrate defects (Hyperketotic acidosis)

- Disorders of gluconeogenesis, e.g. GSD

- Disorders of pyruvate metabolism

- Ketotic hypoglycemia

b. Fatty-acid oxidation defects (Hypoketotic acidosis)

- Medium chain aceyl CoA dehyrogenase deficiency

- Carnitine deficiency

- Biotinidase deficiency

c. Amino acid defects (Organic acidemia)

- Branched-chain organic acidurias

- Others: Hyperlysinemia

Clinically, classic MSUD manifests within first week of life with poor feeding, vomiting and tachypnea; and rapidly progress to develop neurological features, i.e. coma, seizures and alternate periods of hypotonia and hypertonia (extensor spasms). Most cases die in infancy due to severe ketoacidosis and cerebral edema, while mental retardation and neurological deficit is common among survivors.

Other variants present with intermittent ketoacidosis and neurological manifestations during stress, e.g. infections; or a mild late-onset disease beyond infancy with mental retardation, seizures and typical urinary odor. Diagnosis must be suspected in a case with typical urinary odor, metabolic acidosis, hypoglycemia and ketonuria with navy blue color on ferric chloride test. A useful screening test is the development of yellowish precipitate on addition of dinitrophenylhydrazine in urine. Confirmation requires NGS and aminoacidogram to show marked elevation of branched amino acids.

Treatment includes: (a) life-long dietary restriction of branched-chain amino acids, especially leucine by using BCAA free formula. Supplementation with isoleucine and valine may be required; (b) exchange transfusion or hemodialysis to eliminate accumulated organic acids in acute phase, and (c) prevention of catabolic states, i.e. infections, which may precipitate the crisis. Thiamine supplementation may be beneficial in some mild cases. Liver transplantation is the definitive treatment.

Disorders of branched chain amino acids, other than MSUD, e.g. Methylmalonic aciduria (MMA), Isovaleric academia (IVA), and Propionic aciduria (PA) present with similar manifestations, i.e. metabolic ketoacidosis and neurological manifestations in early infancy and need to be differentiated on plasma aminoacidgram and Gas Chromatography Mass Spectrometry (GCMS) urine in isovaleric acidemia has typical 'sweaty feet' odor.

Management includes supplemental vitamin B1₂ to those MMA cases who are known to be vitamin B₁₂ responsive; restricting natural protein, particularly of propiogenic amino acid precursors, while maintaining a high-calorie diet. Carnitine supplementation is required to improve the excretion of propionic acid in PA. Metronidazole is given to reduce the propionate producing gut bacteria. Liver transplant is the definitive management.

Urea cycle defects (hyperammonemia): Ammonia, a highly neurotoxic substance, is regularly produced on catabolism of various amino acids and rapidly degraded in liver by Krebs-Henseleit cycle to urea for excretion.

Hyperammonemia is seen in many inherited defects, e.g. (a) urea cycle defects and (b) organic acidemias, etc.; or acquired hepato-cellular disorders, e.g. (c) acute hepatic failure, (d) Reye syndrome, and (e) portal vein obstruction.

Urea cycle defects include inherited deficiency in one of the five major Krebs-cycle enzymes, namely carbamyl phosphate synthetase (CPS), ornithine trans carbamylase (OTC), argininosuccinate synthetase (AS), argininosuccinate lyase (AL) and arginase; leading to hyperammonemia and neurological features. All of them are autosomal recessive except OTC deficiency, which is commonest among all urea-cycle defects and inherited as X-linked dominant. Clinical manifestations of urea cycle defects vary according to the type and severity of enzyme defects, broadly divided into:

a. Severe neonatal hyperammonemia (gt;1000 #956;mol#8725;L) due to complete urea-cycle enzyme defects, presents within few hours / days of birth with poor feeding, vomiting, hypotonia and tachypnea; rapidly progressing to coma, intractable seizures and death, if untreated.

b. Moderate neonatal hyperammonemia (200-400 #956;mol#8725;L) due to partial urea-cycle enzyme defects or organic acidemias, presents in newborns with mild features, e.g. unexplained lethargy and poor feeding.

c. Late-onset hyperammonemia is asymptomatic till the baby is on low-protein breast milk, only to manifest after introduction of high-protein weaning foods or infections. These cases present with intermittent episodes of vomiting, lethargy, seizures and coma, often misdiagnosed as recurrent Reye syndrome. Plasma ammonia levels are high (200-500 #956;mol#8725;L), but only during episodes.

d. Transient neonatal hyperammonemia, an unexplained hyperammonemic state is usually seen in preterms, which is either asymptomatic or manifests soon after birth with features mentioned in 'a' above. Most cases recover spontaneously after 6-8 weeks, unless die during acute crisis.

Diagnosis depends on high index of suspicion (often misdiagnosed as neonatal septicemia) and presence of hyperammonemia gt;200 #956;mol#8725;L. Identification of specific urea-cycle enzyme defects depends on amino acid profile in plasma or urine.

Treatment of acute hyperammonemic states include:

• Complete elimination of proteins in diet, except an IV mixture of essential amino acids as 0.25 gm/kg/day;

• Adequate provision of calories as IV dextrose and lipids, to prevent endogenous protein breakdown;

• Gut sterilization with lactulose therapy to reduce intestinal ammonia production by bacterial activity;

• Urinary elimination of ammonia in excretable conjugate forms with sodium benzoate and arginine (PO 250-500 mg/kg/day). Arginine is not used in arginase deficiency.

• Hemodialysis or hemofiltration in severe cases.

On recovery, these cases should continue on low protein diet (lt;1 gm/kg/d) and chemical conjugates,

e. g. sodium benzoate, sodium phenylacetate etc (orally, in abovementioned doses), along with avoidance of catabolic states, e.g. infections and heavy meals.

11.6.2