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A gas chromatographic-mass spectrometric method is described for the assay of propionic acid and of isovaleric acid in physiological fluids by isotope dilution. The acids are derivatized to the pentafluorobenzyl esters to decrease volatility to render them suitable for GC-MS analysis. The following reference values were found. Propionic acid: plasma 0.54 +/- 0.38 mumol/l (n = 13, range 0.03-1.38 mumol/l), urine 1.7 +/- 1.6 mumol/mmol creatinine (n = 9, range 0.1-4.9 mumol/mmol creatinine). Isovaleric acid: plasma 0.89 +/- 0.93 mumol/l (n = 10, range 0.01-3.03 mumol/l), urine 0.38 +/- 0.51 mumol/mmol creatinine (n = 10, range 0.01-1.70 mumol/mmol creatinine).

A comprehensive program for proficiency testing of biochemical genetics laboratories is described. Inborn errors of metabolism of amino acids, organic acids, glycosaminoglycans and oligosaccharides are covered, as well as the assay of galactose-1-phosphate. Participants are not only required to report the analytical results but to give an interpretation of the results as well. Sixteen rounds of testing have been completed. A slow, but definite improvement in performance can be observed.

Previous studies on the effect of hyperphenylalaninaemia on the development of the muscarinic acetylcholine receptor in the cerebrum of the rat, using alpha-methylphenylalanine-induced hyperphenylalaninaemia, have shown a gradual and steady decrease in the number of binding sites for this neurotransmitter. The HPH-5 mouse, a phenylalanine hydroxylase mutant, can be hyperphenylalaninaemic without the use of a hydroxylase inhibitor. By employing quantitative autoradiography using [3H]quinuclinidylbenzilate to label muscarinic acetylcholine receptors, a refined analysis of this decrease in neurotransmitter binding sites can be made. The decrease was confirmed and is therefore due to the hyperphenylalaninaemia per se and not to the use of the inhibitor. Various areas of the brain reacted differently to hyperphenylalaninaemia, from no change (putamen) to a gradual decrease (external layer of the olfactory bulb, parietal, occipital and cingulate areas of the cerebral cortex, CA1 and CA3 layer of the hippocampus) to a decrease preceded by a transient increase (frontal area of the cerebral cortex, caudate nucleus). The extent of these changes depends on the duration of exposure to hyperphenylalaninaemia as well as on the degree of brain maturation, but can even be observed in the brain of the adult mouse on a hyperphenylalaninaemic regimen for 11 days. Since the hippocampus has been shown to be involved in the long-term storage of information, damage to this structure by hyperphenylalaninaemia may provide a clue to the global mental retardation observed in untreated PKU.

A female newborn probably with a variant form of galactosialidosis is described. The patient, in addition to the common findings seen in early infantile forms of classical galactosialidosis, displayed an unusual combination of congenital malformations including complex cyanotic congenital heart disease with dextrocardia and situs inversus.

Myelin turnover has been studied in the 25-day-old HPH-5 mouse, a phenylalanine hydroxylase-deficient mouse mutant. The half-life of the fast component of myelin decreased from 15 days in control mice to 4.5 days at blood phenylalanine levels of 2.5 mmol/L. The slow component of myelin seems also to be affected by the high phenylalanine level. These observations confirm similar observations obtained with chemically induced models of hyperphenylalaninaemia and are therefore due to the hyperphenylalaninaemia per se, independently of the inhibitors of phenylalanine hydroxylase. An intermediate blood level of phenylalanine (0.7 mmol/L) likewise seems to interfere with myelin metabolism, although to a lesser degree.

Urinary oligosaccharides can be separated by high-performance anion-exchange chromatography using a Dionex CarboPac PA1 column, elution with aqueous sodium hydroxide and sodium acetate solutions and detection by pulsed amperometry. Each of the urines of patients with glycoprotein degradation disorders yielded a pattern of oligosaccharide excretion unique for that disorder, facilitating an unambiguous diagnosis. The method is sensitive (10 microliters of urine required) and fast (40 min).

Two male siblings presented in the first 6 weeks of life with emesis, diarrhoea, metabolic acidosis and lethargy. A male sibling had previously died at 14 months of age from liver failure of unknown aetiology. Both of the current cases had mild hyperammonaemia with normal orotic acid, organic acid and argininosuccinic acid levels. Citrulline and arginine levels were normal or mildly decreased. One of the brothers was biopsied and had no detectable N-acetylglutamate synthetase activity and normal values for other enzymes of the urea cycle in liver. Treatment with a low-protein diet and sodium benzoate/sodium phenylacetate resulted in near normal blood ammonia levels, except during viral illness. Subsequent neurological development has been normal to mildly delayed. These patients differ from those previously described with N-acetylglutamate synthetase deficiency in that their presentation and subsequent course were relatively benign.

The effect of increasing blood levels of valine, isoleucine and leucine on the fluxes through tyrosine and tryptophan hydroxylase in brain has been calculated and analysed in terms of flux control coefficients. It is concluded that any beneficial effect of increasing the concentration of these amino acids in phenylketonuria patients is not due to a decrease in brain phenylalanine or an improved neurotransmitter synthesis through tyrosine or tryptophan hydroxylase.

The transport of the eight amino acids (phenylalanine, tyrosine, tryptophan, valine, leucine, isoleucine, histidine and methionine) using the large neutral amino acid transporter of the blood-brain barrier (BBB) has been calculated using published kinetic data. The fate of the amino acids has been followed from blood to interstitial space, to cell and through metabolism which included, for tyrosine and tryptophan, the hydroxylases. The system was analysed in terms of flux control coefficients. Since the summation theorem did not hold, the system clearly behaved as a non-homogeneous system. At physiological levels of these eight amino acids, the largest contribution to the control of the flux of tyrosine is given by the hydroxylase step, followed by the diffusional component of the transport across the BBB. For tryptophan it is the hydroxylase step, followed by the carrier-mediated transport across the BBB. For the other amino acids it is the metabolism, followed by the diffusional component of the BBB transport. These parameters for tyrosine and tryptophan were determined at increased levels of blood phenylalanine, tyrosine or histidine. The flux through tryptophan hydroxylase can be affected by high blood levels of tyrosine and histidine to values also observed in hyperphenylalaninaemia. Since hypertyrosinaemia (type II) and hyperhistidinaemia are not associated with mental retardation, it is concluded that interference with transport across the BBB of tyrosine and tryptophan, as well as the flux through tryptophan hydroxylase leading to the synthesis of 5-hydroxytryptamine, do not contribute to the cause of permanent brain dysfunction in hyperphenylalaninaemia. It can be calculated that addition of tyrosine to the diet to raise the blood tyrosine level in phenylketonuria patients may have a beneficial effect for the synthesis of neurotransmitters derived from tyrosine.

A patient presenting with a deep metabolic acidosis after birth is described. Gas chromatographic analysis of short chain fatty acid and non volatile organic acids revealed the presence of both propionic and methylmalonic acid. In plasma obtained immediately after death the propionic- and methylmalonic acid concentrations were measured after separation of both acids by thin layer chromatography. The propionic acid concentration was about 5 mM while the methylmalonic acid concentration was 2.6 mM. The methylmalonic acid concentration in urine was 6.8 mM. Propionyl-CoA carboxylase activity measured in leucocytes and liver-mitochondria revealed normal values (53 pmoles/min/mg protein and 6.5 nmoles/min/mg protein respectively). 2-14C-Methyl-malonate oxydation in intact fibroblasts was totally blocked in the patient's cells. The methylmalonyl-CoA carbonyl mutase activity was found to be absent in the patient's fibroblasts. Addition of vit. B12 coenzyme to the incubation mixture stimulated 14C-succinate formation in the control cells but not in the patient's cells.