Gas chromatographic determination of urinary vanilglycolic acid, vanilglycol, vanilacetic acid and vanillactic acid-chemical parameters for the diagnosis of neurogenic tumours and the evaluation of their treatment.

A gas chromatographic method is described for the determination of urinary vanilglycolic acid, vanilglycol, vanilacetic acid and vanillactic acid as their trimethylsilyl derivatives. These metabolites are chemical parameters for the dignosis of neurogenic tumours. Their determination is especially recommended in order to evaluate the effect of the therapy. Results in normals and controls are given. Data in a number of selected patients with neuroblastoma, ganglioneuroma and phaeochromocytoma are presented and discussed.

The occurrence of lactyl lactate and succinyl lactate in the urine of patients screened for inherited metabolic disease.

The gas chromatographic and mass spectrometric identification of lactyl lactate and succinyl lactate, both present in human urine, is described. In the gas chromatogram lactyl lactate (as TMS derivative) presented as two peaks: the L,L- and/or D,D-form as well as the D,L- and/or the L,D-enantiomer. Both L- and D-lactyl succinate were excreted simultaneously. Lactyl lactate was observed in many patients; succinyl lactate only a few times and only together with lactyl lactate. No correlation with (endogenous) urinary lactate could be established. Presumably these compounds are products of the intestinal bacteria.

1,6-Anhydro-beta-D-glucopyranose (beta-glucosan), a constituent of human urine.

When screening for abnormal urinary saccharides with one-dimensional thin-layer chromatography, an unknown component was observed in a position just above that of xylose. This compound was studied by gas chromatography-mass spectrometry and identified as the anhydro sugar beta-glucosan. It was observed in approximately 20% of all urine samples investigated by thin-layer chromatography. Excretory levels varied widely from zero up to 5.3 mmol/l. No correlation with age or disease could be established. The compound was thought to be of exogenous origin.

The variability of metabolite excretion in propionicacidaemia.

Random urine samples from eight patients with propionicacidaemia were analyzed by gas chromatography and mass spectrometry in order to see if a consistent metabolite pattern with a high diagnostic value could be found. However, wide variations were observed. The presence of 3-hydroxypropionate and/or methylcitrate were considered to be diagnostic of propionyl-CoA carboxylase deficiency. In addition, samples from ketotic periods frequently contained 3-hydroxy-n-valerate and 3-oxo-n-valerate.

The absolute configuration of urinary 2-hydroxybutyric acid in patients with ketosis and lactic acidosis.

The absolute configurations of urinary 2-hydroxybutyrate and 3-hydroxybutyrate were determined in patients with lactic acidemia and ketosis by capillary gas-liquid chromatography of their O-acetylated (--)-menthyl ester derivatives. 2-Hydroxybutyrate had the L-configuration, whereas 3-hydroxybutyrate was in the D-configuration.

Urinary excretion of 2-methyl-2,3-butanediol and 2,3-pentanediol in patients with disorders of propionate and methylmalonate metabolism.

Urine samples from patients with propionic acidemia and from a patient with methylmalonic acidemia contained unknown non-acidic metabolites by gas chromatography/mass spectrometry after ethyl acetate extraction. It could be demonstrated by mass spectrometric studies and by synthesis of reference compounds that the major metabolite was 2-methyl-2,3-butanediol, while smaller amounts of 2,3-pentanediol were also present. These diols were present in abnormal amounts in these patients during attacks of metabolic decompensation.

Gas chromatography method for the separation of amino acids enantiomers in plasma and urine. Application in a case of short bowel syndrome.

Urinary amino acids were isolated from the urine of healthy controls and a patient with a short bowel syndrome. Following derivatization with isopropyl alcohol/HCl and trifluoroacetic anhydride the amino acid enantiomers were separated by gas chromatography on a Chirasil-L-Val column. All subjects excreted D-alanine (10-30% of total Ala). The percentage D-alanine was higher in the patient with the short bowel syndrome. The excretion of D-alanine did not correlate with the D-lactate excretion. An intestinal origin for the D-amino acids is the most probable explanation.

Glutaric aciduria type II: report on a previously undescribed metabolic disorder.

A report is given on a hitherto undescribed metabolic disorder, characterized clinically by fatal neonatal acidosis, hypoglycemia and a strong 'sweaty-feet' odour. Biochemical features were a massive urinary excretion of glutaric and lactic acids. Isobutyric, isovaleric and alpha-methylbutyric acids were also greatly increased, followed by adipic, ethylmalonic, alpha-hydroxybutyric, n-butyric, beta-hydroxybutyric, sebacic, suberic, propionic, alpha-hydroxyisovaleric and hexanoic acids. The serum level of glutaric acid was highly elevated. In the serum there were also abnormal levels of lactic, alpha-hydroxybutyric, adipic, suberic, p-hydroxyphenyllactic, myristic, hexadecenoic, palmitic, oleic and stearic acids. Plasma lysine and valine were also elevated. Degradation of 14C-labelled glutaric acid and 14C-labelled branched-chain amino acids, alpha-ketoisovaleric and alpha-ketoisocaproic acids in intact fibroblasts was decreased, whereas that of pyruvic acid was normal. The defect was tentatively supposed to be localized at the level of the metabolism of a range of acyl-CoA compounds. The name glutaric aciduria 'type II' is proposed for the patient's disease.

Isovalerylglucuronide, a new urinary metabolite in isovaleric acidemia. Identification problems due to rearrangement reactions.

Isovaleryl-beta-D-glucuronide, a new metabolite in the urine of patients with isovaleric acidemia, is described. Its gas chromatographic and mass spectrometric parameters are presented. In alkaline solution this glucuronide exhibited intramolecular rearrangements, resulting in isomers bearing the acyl moiety on C-2, C-3 and C-4. The isomers showed similar mass spectra but different positions on the gas chromatogram. In the index patient isovalerylglucuronide was a main metabolite, but the excretion was a transient phenomenon. Only traces of isovalerylglucuronide could also be detected in the urine of three other patients with isovaleric acidemia. The significance of this metabolite for the detoxication of isovalerate in isovaleric acidemia is discussed.

Organic acid excretion in a patient with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency: facts and artefacts.

The organic acid excretion in a patient with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency is described. In addition to 3-hydroxy-3-methylglutaric, 3-methylglutaconic, 3-methylglutaric and 3-hydroxyisovaleric acids, substantial amounts of glutaric and adipic acids were repeatedly observed. 3-Methylcrotonic acid and possibly one of its isomers was found to be formed artificially from 3-methylglutaconic acid.

Octanoylglucuronide excretion in patients with a defective oxidation of medium-chain fatty acids.

Octanoyl-beta-D-glucuronide was identified in the urine of five patients with hypoketotic hypoglycemia and dicarboxylic aciduria due to a defective beta-oxidation of medium-chain fatty acids. Two subjects who ingested large amounts of medium-chain triglycerides also excreted large amounts of the glucuronide. The substance was extracted from the urine with ethyl acetate and analyzed by: (1) gas chromatography/mass spectrometry (GC-MS) of the trimethylsilyl derivative and (2) preparative one-dimensional thin-layer chromatography followed by enzymatic hydrolysis with beta-glucuronidase and again GC-MS. A quantitative analysis was performed indirectly by measuring the urinary bound octanoate after the removal of octanoylcarnitine. Octanoylglucuronide represents an additional mechanism for the detoxification of octanoate; its formation may be of help for the maintenance of carnitine homeostasis in patients with medium-chain acyl-CoA dehydrogenase deficiency.

(2-Ethoxyethoxy)acetic acid: an unusual compound found in the gas chromatographic analysis of urinary organic acids.

An unknown acidic compound was detected in a number of urine samples from patients with a suspected metabolic disorder and some patients treated with chemotherapy. The structure of this compound has been characterized as (2-ethoxyethoxy)acetic acid, using a gas chromatography/mass spectrometry/computer system. The authentic compound was synthesized and compared with the unknown. Urinary (2-ethoxyethoxy)acetic acid is assumed to be formed endogenously from an exogenous precursor, probably 2-(2-ethoxyethoxy)ethanol.

Permanent chemical phenylketonuria and a normal phenylalanine tolerance in two sisters with a normal mental development.

A positive ferric chloride reaction was found at routine examination of the urine of a 14 year old mentally normal girl, admitted for complaints of headache and other meningitis-like symptoms. It turned out that she excreted permanently increased amounts of phenylpyruvic, phenyllactic and o-hydroxyphenylacetic acids, but phenylacetic acid (free plus conjugated) was normal. Fasting serum phenylalanine was not increased nor was urinary phenylalanine. On loading with L-phenylalanine (100 mg/kg) a normal serum phenylalanine response followed, but urinary phenylpyruvic, phenyllactic and o-hydroxyphenylacetic acids increased further. Phenylacetic acid responded too, but remained in the normal range. In addition to the above-mentioned abnormalities the urine contained a still unidentified abnormal acid, which also increased after loading with phenylalanine. Her 12 year old healthy sister showed the same chemical abnormality. Two older brothers and the parents had normal excretions. The enzyme defect has not been identified. As a possibility the defective decarboxylation of phenylpyruvic acid is proposed.

Tyrosinemia and tyrosyluria in healthy prematures: time courses not vitamin C-dependent.

Tyrosyluria and for a part also tyrosinemia were studied in 60 healthy prematures of various birth weights and gestational ages. The first analyses were performed between the 6th and the 14th day after birth. A normal milk diet was given and the protein-intake was between 3 and 4 g/kg. After the first collection of urine half the patients received extra ascorbic acid, 100 mg/kg daily. Urinary analyses of tyrosine and p-hydroxyphenyl metabolites were performed once a week, until the excretion of p-hydroxyphenylpyruvic plus p-hydroxyphenyllactic acids was lower than 5 mmoles per gram creatinine. In 22 out of the 60 prematures (or 37%) a tyrosyluria of more than 5 mmoles/g creatinine and in 19 out of 44 (43%) patients analysed serum tyrosine was higher than 5 mg/100 ml at first analysis. No inverse correlation between tyrosyluria and tyrosinemia on the one hand and birth weight and gestational age on the other hand existed. But in children with a delayed intra-uterine development the incidence of tyrosyluria was higher as prematurity was more pronounced. Ascorbic acid had no effect on the rate of disappearance of tyrosyluria. It was concluded that the addition of extra vitamin C to the diet of prematures is not useful for the normalization of tyrosine metabolism.

Deficiency of fumarylacetoacetase in a patient with hereditary tyrosinemia.

A patient is described with type I tyrosinemia characterized by urinary excretion of succinylacetone together with increased excretion of tyrosine, p-hydroxyphenyllactic, p-hydroxyphenylpyruvic and p-hydroxyphenylacetic acids. Fumarylacetoacetase was measured in a liver biopsy and found to be very low compared to control liver. Furthermore the mass spectra of succinylacetone and fumarylacetoacetate (methoxime-TMS derivatives) are reported. Control jejunal mucosa, leucocytes and fibroblasts showed no enzyme activity; hence the prenatal diagnosis of this disease by measuring the fumarylacetoacetase activity in cultured amniotic fluid cells is not possible at present.

D-Glyceric acidemia in a patient with chronic metabolic acidosis.

A patient is described with glyceric acidemia and glyceric aciduria. The main clinical problems in infancy were severe metabolic acidosis and failure to thrive. The patient needs permanent treatment with bicarbonate. Hyperglycinemia, as described in the first case discovered elsewhere, was not present. The glyceric acid was found to have the D-configuration, as analyzed by capillary gas chromatography of its di-O-acetyl-l-menthyl ester. The abnormality may result from a defect in serine metabolism.

Urinary excretion of deuterated metabolites in patients with tyrosinemia type I after oral loading with deuterated L-tyrosine.

1. The metabolic fate of orally given deuterated L-tyrosine, 50 mg/kg body weight, was investigated in seven patients with tyrosinemia type I in order to obtain evidence that the primary defect is at the level of fumarylacetoacetase. 2. The absence of fumarylacetoacetase could be proved in liver biopsy specimens obtained from four patients. 3. All patients excreted deuterated succinylacetoacetate and deuterated succinylacetone was detected in six out of seven. The total amount of these compounds was rather low; maximal 8.3% of the dose. The peak of the excretion occurred 3-6 h after loading, indicating an endogenous formation of the metabolites. 4. All patients excreted deuterated 4-hydroxyphenyl acids, probably reflecting secondary 4-hydroxyphenylpyruvate dioxygenase deficiency connected with liver damage. 5. No evidence for other secondary routes of tyrosine metabolism was found.