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Background: The diagnosis of inborn errors of purine and pyrimidine metabolism is often difficult. We examined the potential of 1H-NMR as a tool in evaluation of patients with these disorders., Methods: We performed 1H-NMR spectroscopy on 500 and 600 MHz instruments with a standardized sample volume of 500 microL. We studied body fluids from 25 patients with nine inborn errors of purine and pyrimidine metabolism., Results: Characteristic abnormalities could be demonstrated in the 1H-NMR spectra of urine samples of all patients with diseases in the pyrimidine metabolism. In most urine samples from patients with defects in the purine metabolism, the 1H-NMR spectrum pointed to the specific diagnosis in a straightforward manner. The only exception was a urine from a case of adenine phosphoribosyl transferase deficiency in which the accumulating metabolite, 2,8-dihydroxyadenine, was not seen under the operating conditions used. Similarly, uric acid was not measured. We provide the 1H-NMR spectral characteristics of many intermediates in purine and pyrimidine metabolism that may be relevant for future studies in this field., Conclusion: The overview of metabolism that is provided by 1H-NMR spectroscopy makes the technique a valuable screening tool in the detection of inborn errors of purine and pyrimidine metabolism., (Copyright 1999 American Association for Clinical Chemistry)

Treatment of MOLT F4 lymphoblasts with 6-mercaptopurine (6-MP) resulted in a decrease of ATP and a depletion of S-adenosylmethionine (AdoMet). To investigate whether this might affect the methylation of DNA, we treated MOLT F4 lymphoblasts with increasing concentrations of 6-MP, followed by labeling with [methyl-14C]methionine and [methyl-3H]thymidine. After DNA isolation, we measured the incorporated radioactivity and determined the 14C/3H ratio as a measure for the methylation of newly formed DNA. The 14C/3H ratio was decreased by 17% with 1 mumol/L 6-MP; treatment with increasing concentrations of 6-MP up to 10 mumol/L showed a further decrease to 70%, in comparison with untreated cells. To demonstrate that the methylation of deoxycytidine residues in DNA was reduced, we quantified hydrolyzed DNA by HPLC. The 14C/3H ratio showed a decrease with increasing 6-MP concentrations, indicating that treatment with 6-MP resulted in hypomethylation of DNA.

6-Mercaptopurine, a hypoxanthine antimetabolite, is used in the treatment of acute lymphoblastic leukemia (ALL) in children. Extensively metabolized before it exerts cytotoxic action, it is catabolized into 6-mercapto-2,8-dihydroxypurine (thiouric acid), which is excreted by the kidneys. We describe a metabolite of 6-mercaptopurine, 6-methylmercapto-8-hydroxypurine, whose presence has not been previously reported in plasma. This compound was found in high concentrations in plasma during high-dose 6-mercaptopurine infusions (1300 mg/m2 in 24 h). This previously unknown compound was identified by reversed-phase HPLC with absorbance detection and by gas chromatography-mass spectrometry. The pathways leading to 6-methylmercapto-8-hydroxypurine in vivo are not yet fully understood. In a group of 17 patients treated with four courses of high-dose 6-mercaptopurine infusions according to the ALL-8 treatment protocol of the Dutch Childhood Leukemia Study Group, the steady-state concentrations of 6-methylmercapto-8-hydroxypurine in plasma were one-fifth of the parent drug concentrations, with wide interindividual variation. The formation of high concentrations of 6-methylmercapto-8-hydroxypurine in plasma, especially during the infusion, probably indicates another catabolic pathway of high-dose 6-mercaptopurine, apart from its conversion into thiouric acid.

Disturbances in the metabolism of purines and pyrimidines in neurologically affected patients can be reflected by aberrant concentrations of nucleosides and nucleobases in cerebrospinal fluid (CSF). However, normal values, especially for children at different ages, are lacking. We collected 1000 specimens of CSF from subjects ranging in age from newborn to 18 years, who were undergoing a diagnostic lumbar puncture for several clinical indications. Of these, 78 samples could be used retrospectively as a reference according to our criteria. The analyses were performed with a modified HPLC procedure. None of the substances shows age-dependency except uridine and uric acid. Uridine increases with age, and uric acid increases with age in boys older than 12 years.

The toxicity of guanosine and deoxyguanosine in the presence or absence of the purine nucleoside phosphorylase inhibitor, 8-aminoguanosine, for malignant lymphoid cell lines and mitogen-stimulated peripheral blood lymphocytes has been studied. Deoxyguanosine inhibited the proliferation of lymphoid cells more strongly than guanosine. Addition of 100 microM 8-aminoguanosine neither enhanced nor diminished the toxicity of guanosine to the lymphoid cells. Only the toxicity of deoxyguanosine for the leukemic T cell line, MOLT 4, and the leukemic nonBnonT cell line, KM-3, was enhanced by the addition of 100 microM 8-aminoguanosine. These data suggest a possible role of purine nucleoside phosphorylase inhibitors in the treatment of lymphoproliferative disorders of the T-acute lymphoblastic leukemia (ALL) as well as the nonBnonT-ALL subclass.