Metabolic Screen (Plasma Amino Acids and Urine Organic Acids)

The metabolic screen is a group of tests to detect inborn errors of metabolism, a large set of over 1000 inherited disorders of amino acid, organic acid, fatty acid, carbohydrate and urea cycle metabolism.

Plasma amino acids (quantitative, by ion exchange chromatography or liquid chromatography with tandem mass spectrometry) and urine organic acids (qualitative and semi-quantitative, by gas chromatography with mass spectrometry) are the first line pair.

They are usually accompanied by plasma ammonia, lactate, glucose, ketones, blood gas, acylcarnitine profile and urine orotic acid, and are diagnostic in most amino acidopathies, organic acidurias and urea cycle disorders.

Individually rare, inborn errors of metabolism affect about 1 in 800 to 1 in 2500 births as a group. Presentation in the neonate with acute encephalopathy, vomiting, seizures, hypoglycaemia, acidosis or hyperammonaemia after a symptom free interval is typical, and later presentation with developmental delay, episodic decompensation, cardiomyopathy, liver failure or neurological regression occurs in older children and adults. Early diagnosis changes outcomes, and samples must be taken during acute illness, because results can normalise between episodes.

Indications. Unexplained encephalopathy, coma, seizures, vomiting, lethargy or poor feeding in a neonate or infant.

• Metabolic acidosis with a raised anion gap
• hyperammonaemia
• hypoglycaemia (especially with ketosis or without ketones)
• raised lactate
• unusual odour of urine or sweat
• developmental regression
• unexplained liver failure
• cardiomyopathy
• rhabdomyolysis
• Reye like illness
• recurrent episodes triggered by fasting, infection or protein intake
• family history of sudden infant death or consanguinity

Positive newborn screening result for confirmation.

Specimen. Plasma: heparin or EDTA, 1 to 2 mL, separated within 30 minutes and frozen, taken ideally during illness and before transfusion or dextrose infusion. Urine: random first sample 5 to 10 mL, frozen at minus 20 °C, taken during the acute episode, without preservative. Dried blood spot on filter paper for acylcarnitines. CSF (amino acids, glycine, lactate) and simultaneous plasma glycine for non-ketotic hyperglycinaemia. Critical samples are taken before treatment is given where this is safe.

Plasma amino acids. Quantitative profile of about 30 amino acids, with typical patterns.

• Phenylketonuria: phenylalanine over 120 micromol/L (usually over 1200), tyrosine low or normal.
• Tyrosinaemia type 1: raised tyrosine, methionine and succinylacetone (urine).
• Maple syrup urine disease: raised leucine, isoleucine, valine and alloisoleucine.
• Homocystinuria: raised methionine (in cystathionine beta synthase deficiency) and homocystine, with low cystine.
• Urea cycle disorders: raised glutamine and alanine, low or absent citrulline in proximal defects (ornithine transcarbamylase and carbamoyl phosphate synthetase deficiency), high citrulline in citrullinaemia type 1, argininosuccinate in argininosuccinic aciduria, and raised arginine in arginase deficiency.
• Non-ketotic hyperglycinaemia: high glycine in plasma and CSF with a CSF to plasma glycine ratio over 0.08.
• Others: nonspecific changes occur with liver disease, renal failure, parenteral nutrition, haemolysis and poor sample handling.

Urine organic acids. Gas chromatography with mass spectrometry identifies abnormal acids.

• Organic acidurias (methylmalonic, propionic, isovaleric aciduria, glutaric aciduria type 1, 3-methylcrotonyl-CoA carboxylase deficiency): characteristic metabolites (methylmalonic acid, 3-hydroxypropionate, methylcitrate, isovalerylglycine, glutaric and 3-hydroxyglutaric acids). Acute illness with ketosis, high anion gap acidosis and hyperammonaemia in neonates.
• Fatty acid oxidation disorders (medium chain acyl-CoA dehydrogenase deficiency): dicarboxylic aciduria with hypoketotic hypoglycaemia during fasting.
• Ketone body utilisation defects, mitochondrial disorders (lactic aciduria, Krebs cycle intermediates), glycogen storage disease type I (lactate and ketones), and tyrosinaemia type 1 (succinylacetone).
• Orotic aciduria: raised in ornithine transcarbamylase deficiency and distinguishes it from carbamoyl phosphate synthetase deficiency (low orotic acid).
• Normal in many amino acid, lysosomal and peroxisomal disorders, so a normal result does not exclude a metabolic disease.

Associated first line tests. Ammonia (neonatal normal under 110 micromol/L, hyperammonaemia over 150 micromol/L needing urgent action), lactate (normal under 2 mmol/L), glucose, plasma ketones, blood gas and anion gap, liver function, creatine kinase, uric acid, urinary reducing substances (galactosaemia), and acylcarnitine profile.

Critical values and findings needing immediate action.

• Ammonia over 150 micromol/L in a neonate or infant with encephalopathy, or over 100 micromol/L in children and adults with unexplained encephalopathy: metabolic emergency with protein free nutrition, intravenous glucose and insulin to reverse catabolism, nitrogen scavengers (sodium benzoate, sodium phenylbutyrate), arginine, and haemodialysis if over 400 to 500 micromol/L or if coma is present.
• Severe metabolic acidosis with raised anion gap and ketosis in a neonate: suspected organic acidaemia, stop protein, provide glucose, carnitine and hydroxocobalamin as indicated, and urgent specialist discussion.
• Hypoglycaemia with low ketones: fatty acid oxidation disorder or hyperinsulinism, critical sample (glucose, ketones, insulin, free fatty acids, acylcarnitines) is taken at the time of hypoglycaemia.
• Positive urine reducing substances with vomiting and jaundice in a neonate: galactosaemia, galactose free feeding urgently, and risk of Escherichia coli sepsis.
• Intractable seizures unresponsive to anticonvulsants in a neonate: pyridoxine, pyridoxal phosphate and folinic acid, and non-ketotic hyperglycinaemia testing.

Interferences and pitfalls.

• Samples taken between episodes can be normal, particularly for organic acidurias and urea cycle disorders. The test is repeated during illness.
• Delayed separation of plasma raises many amino acids (glutamate, aspartate, ornithine) and lowers glutamine and cystine. Haemolysis releases intracellular amino acids.
• Parenteral nutrition, intravenous amino acids, drugs (valproate, antibiotics, benzoate) and ketoacidosis from other causes alter patterns.
• Bacterial contamination of urine, drugs (aspirin, paracetamol metabolites, valproate) and dietary compounds produce spurious organic acid peaks.
• Normal organic acids occur in some disorders (urea cycle defects except orotic acid), so tests are combined.
• Results require interpretation by metabolic specialists, and confirmation by enzyme assay or gene sequencing.
• Treatment in an emergency begins before results, and anabolic support (glucose) is continued.
• Newborn screening by tandem mass spectrometry misses some late onset or milder cases, and a normal screen does not exclude disease.
• Sodium valproate, which causes secondary carnitine deficiency and hyperammonaemia, is stopped in suspected urea cycle disorders.

Special groups.

• Neonates: sudden deterioration after feeding is typical, and urine odours (maple syrup, sweaty feet, mousy, cabbage) provide clues.
• Older children and adults: late onset ornithine transcarbamylase deficiency, homocystinuria, and mitochondrial disease present with episodic encephalopathy, psychiatric symptoms, stroke or liver failure.
• Pregnancy: women with phenylketonuria need strict dietary control (phenylalanine under 360 micromol/L) before conception to prevent maternal phenylketonuria syndrome in the infant.
• Regions: consanguinity increases frequency, and newborn screening coverage is limited in many low resource regions.

Follow up tests. Acylcarnitine profile, plasma and urine orotic acid, urine succinylacetone, very-long-chain fatty acids, transferrin isoforms for congenital disorders of glycosylation, lysosomal enzymes, CSF lactate, glycine and neurotransmitters, enzyme assays on leucocytes or fibroblasts, targeted gene panels or exome sequencing, skin biopsy, brain MRI and MR spectroscopy, echocardiography, ophthalmological assessment and dietetic assessment.