Biochemical Genetics Unit

Analysis of the acylcarnitine profile is a powerful tool in the investigation of fatty acid oxidation defects (FAOD) and classical organic acidurias. FAOD include medium chain acyl-CoA dehydrogenase deficiency, very long chain acyl-CoA dehydrogenase deficiency and long chain hydroxyacyl-CoA dehydrogenase deficiency. β-oxidation of long chain fatty acids has an important role in energy production, a process that becomes critical during prolonged fasting. The clinical presentation of FAOD is variable but they typically present in early childhood with hypoketotic hypoglycaemia. Late-onset forms are also described which present in adulthood with muscle disease.

NB: Ketonuria does not exclude a FAOD

Plasma amino acids may be abnormal in a variety of amino acid disorders, including urea cycle defects and some organic acidurias. Investigations should be carried out, as far as possible, on samples taken when the patient is symptomatic. Dietary restrictions may cause characteristic patterns to disappear and result in false negative results. Plasma amino acids fluctuate depending on the protein intake and whether the patient is in a fed or fasted state. Patients receiving an intravenous amino acid mixture may have an abnormal amino acid pattern. Information on the type of diet and the timing of the sample in relation to meals may aid interpretation.

For the investigation of epileptic encephalopathy a paired plasma sample must accompany any CSF (see section CSF Amino Acids below). For information about sulfocysteine see sulfocysteine.

Analysis of amino acids in urine shows poor sensitivity and is subject to interferences from drug metabolites and other compounds. These problems are rarely observed in plasma samples. For this reason, amino acid analysis in plasma is the recommended test for the investigation of disorders of amino acid metabolism. This protocol follows the UK Metabolic Biochemistry Network Amino Acid guidelines and is consistent with practice in most UK metabolic laboratories. However urine amino acids analysis remains a useful test for the investigation of renal transport disorders (e.g. cystinuria), the assessment of renal tubular dysfunction (e.g. Fanconi syndrome, mitochondrial disease) and for some other specific disorders (e.g. hypophosphatasia). Please indicate on the request form that this is required as phosphoethanolamine is not normally reported.

In patients with cystinuria only cystine, ornithine, arginine and lysine will be reported. The solubility limit of cystine at pH 7 is approximately 1100 µmol/L; however this varies in urine samples. At concentrations above this, the patient is at high risk of stone formation.

Biotin is a cofactor for multiple carboxylases and the recycling of biotin requires the activity of the enzyme biotinidase. Typically biotinidase deficiency presents between 3-6 months of life with seizures. Treatment is with biotin replacement, which should be initiated prior to the result being available. Biotinidase is a relatively unstable enzyme; low results should be checked on a fresh sample if clinically indicated.

Gaucher disease is a lysosomal storage disorder resulting from an inherited deficiency of the enzyme b-glucosidase. This deficiency results in impaired breakdown of the lipid glucocerebroside and its subsequent accumulation in cells. Gaucher disease is characterised by markedly elevated chitotriosidase activity; symptomatic Gaucher patients typically exhibit concentrations 100 times greater than the reference range. However, chitotriosidase may be mildly increased in a number of other lysosomal storage disorders and other illnesses, such as sarcoidosis. Benign deficiency of chitotriosidase occurs in approximately 6% of Caucasians

This group of disorders is characterised by cerebral creatine deficiency, the main symptoms of which are learning disability and speech delay, and, in some patients, intractable seizures. Of the three disorders described; arginine:glycine amidinotransferase (AGAT) deficiency and guanidinoacetate methyltransferase (GAMT) deficiency show decreased creatine in the urine and plasma. Guanidinoacetate in urine and plasma is increased in GAMT deficiency and undetectable in AGAT deficiency. Defects in the creatine transporter (an X-linked disorder) result in an increase in the urine creatine/creatinine ratio in boys, girls with this condition may have normal creatine excretion.

Guanidinoacetate is stable in urine and plasma. Whilst creatine is stable in plasma, it is unstable in urine and concentrations increase within 1-2 hours of collection, leading to potentially false positive results for the creatine transporter defect or spuriously normal results in AGAT and GAMT.

Classical galactosaemia is caused by a deficiency of the enzyme galactose-1-phosphate uridyltransferase. Galactose is produced in the small intestine from the breakdown of dietary lactose into galactose and glucose. The presence of reducing substances in urine may be an important clue to diagnosis but equally can be misleading. False positives can occur in severe liver disease and false negatives can occur if lactose is not present in the diet, and therefore blood spot galactose-1-phosphate uridyltransferase is the recommended screening test. Carriers of galactosaemia cannot be detected by this screening method.

Please note: results are invalid if the sample has been collected within 6 weeks of a blood transfusion. If galactosaemia is suspected in a child who has had a blood transfusion please discuss alternative testing with one of the BGU clinical scientists.

The mucopolysaccharidoses are a group of inherited disorders characterised by the accumulation of glycosaminoglycans in the lysosomes. Children may appear normal at birth but later develop progressive skeletal abnormalities, coarse facies and hepatomegaly. Normal urine glycosaminoglycans consist mainly of chondroitin sulphate with traces of heparan and dermatan sulphates. Mucopolysaccharidoses are characterised by abnormal patterns of glycosaminoglycans in urine. The urine quantitative method has low sensitivity and in addition false positive results are common, particularly in young infants. False negative quantitative results may also be encountered, therefore if there is a strong clinical suspicion of a mucopolysaccharidosis, please specifically request GAG typing. If urine glycosaminoglycan typing and white cell enzymes are normal and a storage disorder is still suspected clinically, urinary oligosaccharide and sialic acid analysis should be considered.

Measurement of total homocysteine is offered for the diagnosis and monitoring of inherited defects in homocystine metabolism, such as classical homocystinuria and methionine synthase deficiency. Free homocystine is not recommended as it is only detectable in plasma and urine when the binding capacity of plasma proteins has been exceeded. The binding of homocystine to plasma protein, mainly albumin, seems to be saturable with a maximal capacity of about 140 mmol/L total homocysteine. Methionine is included in the analysis, to aid interpretation of any abnormal homocysteine results.

Methylmalonate can be measured as a marker of functional vitamin B12 deficiency or to diagnose and monitor patients with inherited disorders of methylmalonate or vitamin B12 metabolism.

Analysis of organic acids in urine can assist in the diagnosis of a number of disorders including those of amino acid metabolism (e.g. MSUD, urea cycle defects). In addition specific organic acids may be requested for specific disease conditions.

Orotate is an intermediate in the synthesis of pyrimidine nucleotides. In most defects of the urea cycle carbamoyl phosphate accumulates. This feeds into the pyrimidine biosynthetic pathway resulting in an excess of orotate.

Phenylketonuria is an autosomal recessive condition with an incidence of about 1 in 12,000. It is caused by a deficiency of phenylalanine hydroxylase which results in a marked increase in blood phenylalanine. Untreated, severe learning disability and spasticity ensues. Treatment is effective and consists of dietary phenylalanine restriction and supplementation of essential amino acids. Dried blood spot samples are used for monitoring and adjustment of the diet. The frequency of testing and the target concentration depends on the age of the patient

Sulfocysteine is formed in vivo by the attack of sulphite on free cysteine or on protein disulphide bonds and is increased where there is increased sulphite due to sulfite oxidase deficiency. This may be a primary deficiency or secondary to molybdenum cofactor deficiency. In contrast to urinary sulfite which is unstable and yields false positive and negative results, sulfocysteine is said to be a stable metabolite and an excellent marker of sulphite oxidase deficiency.

The determination of sweat chloride concentration is useful in the diagnosis of cystic fibrosis. Sweat testing can be performed after 2 weeks of age on infants greater than 3 kg that are normally hydrated and without significant systemic illness. If clinically important, sweat testing can be attempted after one week of age but will need repeating if insufficient sweat is collected. A repeat test is recommended when the result is abnormal or borderline and the genotype is not confirmatory.