Ronald A. Chalmers

Studies on the urinary acidic metabolites excreted by patients with β-methylcrotonylglycinuria, propionic acidaemia and methylmalonic acidaemia, using gas-liquid chromatography and mass spectrometry

Abstract The use of quantitative extraction, derivative preparation and gas—liquid chromatography of urinary acidic metabolites with component identification by mass spectrometry in the study of human metabolic disease is described. The results obtained with urine specimens from patients with β-methylcrotonylglycinuria, propionic acidaemia and methylmalonic aciduria are reported. These show associated defects in the metabolism of leucine and isoleucine in both of the former two disorders and illustrate the importance of the metabolism of propionyl-CoA in the biochemical features of all three diseases.

Quantitative studies on the urinary excretion of unconjugated aromatic acids in phenylketonuria

Abstract The urinary excretion of unconjugated aromatic acids (phenylglycollic (mandelic), 2-hydroxyphenylacetic, phenyllactic, phenylpyruvic, 4-hydroxy-phenyllactic and 4-hydroxyphenylpyruvic) by 41 cases of phenylketonuria, by 2 phenylketonuric children with apparently normal intelligence and by a phenylketonuric woman who was identified by the routine screening of antenatal patients for phenylketonuria has been studied. None of these patients was receiving a low phenylalanine diet when the urine collections were made. Possible correlations between the degree of organic aciduria and the patients degree of disability have been examined. The excretion of tyrosine metabolites (4-hydroxyphenyllactic and 4-hydroxyphenylpyruvic acids, without homogentisic aciduria) was a striking feature in all except one of the patients studied. These metabolites appear to be derived partly from dietary phenylalanine, and partly from endogenous tyrosine. The presence of some residual phenylalanine 4-hydroxylase (EC 1.14.3.1) activity is suggested, even in the most retarded patients. The possible location of this residual enzyme activity cannot be specified. 4-Hydroxyphenylpyruvate oxidase (EC 1.14.2.2) also appears to be inhibited in phenylketonuria. The study of a wide range of biochemical parameters may improve our ability to differentiate the atypical forms of phenylketonuria.

Betaine metabolism in human neonates and developing rats

Proton nuclear magnetic resonance spectroscopy has been used to demonstrate the presence of high concentrations of betaine (up to 0.75 mol/mol creatinine) in the urine of normal healthy human neonates. Betaine is not normally excreted in adults. Excretion of betaine from birth to 7 days old was monitored. The excretion of betaine in rats from 21 days after birth to 40-45 days old was also monitored. A peak in excretion in the rats of 1.5-3 mol/mol creatinine occurred between days 30-35. The presence of a high concentration of betaine in the urine is unlikely to be caused by a relative lack of betaine homocysteine methyl transferase activity compared with adults but may relate to the disposal of dietary choline during development.

Methylmalonic aciduria and propionic acidaemia studied by proton nuclear magnetic resonance spectroscopy

Proton nuclear magnetic resonance spectroscopy has been used to monitor changes in urinary metabolites in a patient with propionic acidaemia over a period of 10 months and in a patient with methylmalonic aciduria over a period of 11 days. Results could be obtained within 5-10 min of sample receipt. In the spectra on the patient with propionic acidaemia not only could fluctuations in 3-hydroxypropionate and propionylglycine excretion be followed, but also variations in creatine, glycine and betaine, which were often present at millimolar concentrations. The patient with methylmalonic aciduria had an acute episode of severe ketoacidosis during which the glycine excretion fell but creatine excretion rose and then fell on recovery from the episode. The changes in the creatine excretion may reflect disorders in intracellular energy supply. Nuclear magnetic resonance is a powerful technique for monitoring metabolic perturbations in the organic acidurias in real-time, allowing the planning and evaluation of therapy.

A method for the determination of volatile organic acids in aqueous solutions and urine, and the results obtained in propionic acidaemia, β-methylcrotonylglycinuria and methylmalonic aciduria

Abstract A rapid and simple method for the determination of the volatile acidic and neutral substances in aqueous solutions and urine is described and evaluated. The results obtained in the analysis of urine from normal humans and patients with propionic acidaemia, β-methylcrotonylglycinuria and methylmalonic aciduria are presented and discussed.

Gas chromatographic and mass spectrometric studies on urinary organic acids in a patient with congenital lactic acidosis due to pyruvate decarboxylase deficiency

Detailed studies, using gas chromatography and mass spectrometric methods, of the urinary organic acids excreted by a patient with proven pyruvate decarboxylase deficiency are reported. In addition to the greatly-increased levels of lactate and pyruvate, marked elevation in the levels of 2-oxoglutaric, malic, and isocitric acids were observed, with associated increases 2-hydroxyglutaric, fumaric, succinic, and glyceric acids, and reduced citric acid excretion. The levels of excretion during clinically static and acute periods are compared to those in a normal neonate and normal infants. The metabolites observed indicate a probable defect in the oxidation of pyruvate by pyruvate dehydrogenase and suggest the presence of secondary defects in the tricarboxylic acid cycle. Studies of this type may enable the relatively rapid identification of the probable underlying enzyme deficiency in cases of congenital lactic acidosis, prior to confirmatory enzyme studies.

ABNORMAL ORGANIC ACIDURIAS IN MENTALLY RETARDED PATIENTS

Urine specimens from 1778 mentally retarded patients and 420 age and sex matched non-retarded controls selected from a general practice have been analysed for non-amino organic acids by a quantitative extraction and gas chromatographic method. The compounds were identified by combined gas chromatography and mass spectrometry. Approximately 5% of the patients had an abnormal organic aciduria. The frequency of abnormalities was slightly higher (about 7%) in a group of 248 severely subnormal children, but not in cases with a family history of mental retardation, retarded sibs, or whose parents were consanguineous. The most frequently observed abnormalities were phenylalanine metabolites in cases of phenylketonuria (about 1%), increased excretion of benzoic acid (about 1%), and increased excretion of 2-oxoglutaric acid with or without raised urinary citric-acid levels (about 1%). The biochemical and clinical significance of these findings is being further investigated.

Effect of cotrimoxazole on the response to phenylalanine loading in man

Cotrimoxazole impairs the L-phenylalanine tolerance of control subjects as judged by the time course of the plasma phenylalanine concentration and the plasma ratios [phenylalanine]/[tyrosine] after oral and intravenous loading with phenylalanine. These subjects are presumed not to be heterozygous for any of the abnormal recessive genes which cause hyperphenylalaninaemia when they are present in the homozygous state. Patients with classical phenylketonuria did not show this effect. Cotrimoxazole did not affect the renal clearance of phenylalanine. The effect appears to be virtually entirely due to the trimethoprim component as opposed to the sulphamethoxazole component of cotrimoxazole.