R. W. E. Watts

The quantitative extraction and gas-liquid chromatographic determination of organic acids in urine

A method for the quantitative extraction and gas-liquid chromatographic determination of organic acids in aqueous solution and biological fluids has been developed. The acids are extracted by a DEAE-Sephadex anion-exchange column, neutral and basic compounds are removed by washing with water and the acids are eluted with a pyridinium acetate buffer. The ethoximes of oxo-acids are formed in the eluate to stabilise these compounds and the eluate is then freeze-dried under carefully controlled conditions. The residue of dry free acids and their pyridinium salts is trimethylsilylated and the trimethylsilyl derivatives are separated and quantitatively determined by gas-liquid chromatography with the use of internal standards. The method would not be suitable for the determination of formic, acetic, propionic or butyric acid because of their volatility.The accuracy and precision of the method, which can be applied to other protein-free biological fluids as well as urine, are discussed.

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.

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.

Measurement of DNA in isolated granulocytes by the ethidium technique.

Abstract A method is described for measuring DNA that is based on the quantitative effect of DNase on the fluorescence of DNA with ethidium bromide and is suitable for measuring the DNA content of 1 × 10 6 granulocytes. It is proposed that DNA measured by this method is a useful parameter to which the results of other analyses on separated granulocytes may be referred.

Suppression of glycine-15N incorporation into urinary uric acid by adenine-8-13C in normal and gouty subjects

Adenine inhibited the de novo synthesis of purines in both normal and gouty man as shown by inhibition of the incorporation of glycine-(15)N into urinary uric acid without altering the incorporation of glycine-(15)N into urinary creatinine. The diminished purine synthesis did not result in a diminution in the 24 hr excretion of uric acid. This observation was explainable in part by the prompt conversion of adenine to uric acid. In addition to this direct conversion, adenine-8-(13)C provided a slow and prolonged contribution to urinary uric acid.A feedback inhibition of purine synthesis by nucleotides derived from adenine provides the best interpretation of these results.

Derivatives for the identification and quantitative determination of some keto and aldo-carboxylic acids by gas-liquid chromatography

Studies on the derivatives of some physiologically and pathologically important keto and aldo-acids (pyruvic, glyoxylic, 2-oxoglutaric, oxaloacetic and 4-hydroxyphenylpyruvic) have shown that the O-ethoxime trimethylsilyl esters are the most suitable for quantitative gas-liquid chromatography. The O-trimethylsilyl oxime trimethylsilyl esters were thermally unstable under the conditions used. The O-methyl and O-benzyl oxime trimethylsilyl esters are also suitable for gas-liquid chromatography and would be useful in identification studies. The O-benzyl oxime trimethylsilyl derivatives are suggested as being especially suitable when only the keto and aldo-acids are to be studied, and it is not necessary to identify the lower boiling trimethylsilyl esters of other carboxylic acids on the same chromatogram.Two aldehyde-specific reagents, dimedone and NN′-diphenylethylenediamine, were also examined for use with glyoxylic acid. The trimethylsilyl ester of 1,3-diphenylimidazolidine-2-carboxylic acid (the derivative of glyoxylic acid with NN′-diphenylethylenediamine) has a high boiling-point and is suitable for quantitative gas-liquid chromatography. This derivative should be especially useful in studied that are particularly concerned with the metabolism of glyoxylic acid or possibly other aldehydes.The retention times of the derivatives relative to the retention times of n-tetracosane and n-hexacosane are compared with the corresponding values for the trimethylsilyl derivatives of some physiologically related carboxylic acids that do not contain keto or aldo groups.

Purine de novo synthesis in liver and developing rat brain, and the effect of some inhibitors of purine nucleotide interconversion.

The rate of purine de novo synthesis from sodium formate in developing rat brain falls in the late gestational stages to birth, rises again in the 1st week of life and then decreases rapidly to the 3rd week, and continues declining up to 8 weeks of life (adulthood). The changes in the overall purine biosynthetic rate with respect to time are similar to those in the activity of the rate-limiting enzyme [amidophosphoribosyltransferase (phosphoribosyl diphosphate amidotransferase; EC 2.4.2.14)]. Azaserine [O-diazoacetyl-L-serine], a known inhibitor of glutamine requiring metabolic steps, inhibits purine de novo synthesis by more than 90%. This confirms that the method used to assess purine de novo synthesis in fact does so. The effects of virazole [1-beta-ribofuranosyl-1-H,1,2,4-triazole-3-carboxamide], an inhibitor of IMP dehydrogenase (EC 1.2.1.14), and of alanosine [L-2-amino-3-(hydroxynitrosamino)propanoic acid] an inhibitor of adenylosuccinate synthetase (EC 6.3.4.4), on the rate of purine de novo synthesis were investigated in liver and brain tissue. The effect of the xanthine oxidase inhibitor allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] was also investigated in liver tissue. The biosynthesis of the purines which were extruded into the incubation medium as well as those which remained in the tissue was studied. Only inhibitory effects were observed, and these were confined to the purines remaining in the tissue. Allopurinol was completely inert from this viewpoint. The results are compared with those of other workers using lymphoid cells, and emphasize the differences in the control of de novo purine synthesis in different tissues and under different conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the quantitative freeze-drying of aqueous solutions of some metabolically important aliphatic acids prior to gas-liquid chromatographic analysis

These studies have shown that the over-all losses observed are due to volatilisation during the freeze-drying process. These losses are related to the variation of the vapour or sublimation pressures of the acids with temperature, and are also related to their latent heats of vaporisation or sublimation.Reliable data on the latent heats of vaporisation and sublimation of the acids of interest are seldom available and a method for the estimation of the latent heats, based on group contributions, has been developed. The reliability of the method, which is generally applicable to organic compounds, is discussed.Thermochemical data derived from the use of this method have been used in conjunction with reliable published experimental data to determine the optimum freeze-drying conditions for the complete quantitative recovery of all but the most volatile of the acids studied, with a minimum of preliminary experimental work.

Problems in the Behavioural Treatment of Self-injury in the Lesch-Nyhan Syndrome

The results are described of a behavioural programme designed to modify self‐injurious behaviour of a child with Lesch‐Nyhan syndrome. The treatment combined extinction of the injurious behaviour and reinforcement of alternative behaviour, and was successful in the controlled hospital environment. However, an attempt to teach the parents to continue the treatment at home failed. The results are discussed in terms of the possible relationship between organic and environmental factors in maintaining the injurious behaviour, and the importance of analysing both the behaviour itself and the factors (including familial) maintaining it. It is suggested that parents should be advised about management of behavioural problems at an early stage.