Showing papers by "Albert H. van Gennip published in 2000"

Rapid Screening of High-Risk Patients for Disorders of Purine and Pyrimidine Metabolism Using HPLC-Electrospray Tandem Mass Spectrometry of Liquid Urine or Urine-soaked Filter Paper Strips

Abstract: Background: A rapid and specific screening method for patients at risk of inherited disorders of purine and pyrimidine metabolism is desirable because symptoms are varied and nonspecific. The aim of this study was to develop a rapid and specific method for screening with use of liquid urine samples or urine-soaked filter paper strips. Methods: Reverse-phase HPLC was combined with electrospray ionization (ESI), tandem mass spectrometry (MS/MS), and detection performed by multiple reaction monitoring. Transitions and instrument settings were established for 17 purines or pyrimidines. Stable-isotope-labeled reference compounds were used as internal standards when available. Results: Total analysis time of this method was 15 min, approximately one-third that of conventional HPLC with ultraviolet detection. Recoveries were 96–107% in urine with added analyte, with two exceptions (hypoxanthine, 64%; xanthine, 79%), and 89–110% in urine-soaked filter paper strips, with three exceptions (hypoxanthine, 65%; xanthine, 77%; 5-hydroxymethyluracil, 80%). The expected abnormalities were easily found in samples from patients with purine nucleoside phosphorylase deficiency, ornithine transcarbamylase deficiency, molybdenum cofactor deficiency, adenylosuccinase deficiency, or dihydropyrimidine dehydrogenase deficiency. Conclusions: HPLC-ESI MS/MS of urine allows rapid screening for disorders of purine and pyrimidine metabolism. The filter paper strips offer the advantage of easy collection, transport, and storage of the urine samples.

Pitfalls in the Diagnosis of Patients with a Partial Dihydropyrimidine Dehydrogenase Deficiency

Abstract: Background: Dihydropyrimidine dehydrogenase (DPD) catalyzes the degradation of thymine, uracil, and the chemotherapeutic drug 5-fluorouracil. To identify patients suffering from complete or partial DPD deficiency and to identify pitfalls that can preclude the proper diagnosis of patients with partial DPD deficiency, a sensitive and accurate assay is necessary. Methods: The activity of DPD was measured using [4-14C]thymine followed by separation of substrate and products with reversed-phase HPLC with on-line detection of the radioactivity. Results: Complete baseline separation of radiolabeled thymine and all degradation products was achieved within 15 min. The detection limit for dihydrothymine was 0.4 pmol. In lymphocytes, the DPD activity deviated from linearity at low protein concentrations (<0.2 g/L). Profoundly decreased activity of DPD was detected in the peripheral blood mononuclear cells (PBM cells) of two tumor patients when measured at low protein concentrations. Low DPD activity comparable to that observed in obligate heterozygotes was initially detected in PBM cells, containing substantial amounts of myeloid cells, from a patient suffering from 5-fluorouracil toxicity. However, after the patient experienced full clinical recovery, normal DPD activity was observed in the PBM cells. No significant differences in DPD activity were observed between exponentially growing fibroblasts and those at confluence. The range of DPD activities of obligate heterozygotes overlaps the range of DPD activities of controls. Conclusions: The low activity of DPD measured in PBM cells containing myeloid cells or that measured at a low protein concentration in the assay mixture is not indicative of heterozygosity for a mutant DPD allele. Although fibroblasts are suitable to establish a complete deficiency of DPD, unambiguous detection of heterozygotes is not possible.

Defects in Pyrimidine Degradation Identified by HPLC-Electrospray Tandem Mass Spectrometry of Urine Specimens or Urine-soaked Filter Paper Strips

Abstract: Background: Urinary concentrations of thymine, uracil, and their degradation products are useful indicators of deficiencies of enzymes of the pyrimidine degradation pathway. We describe a rapid, specific method to measure these concentrations to detect inborn errors of pyrimidine metabolism. Methods: We used urine or urine-soaked filter-paper strips as samples and measured thymine, uracil, and their degradation products dihydrothymine, dihydrouracil, N -carbamyl-β-aminoisobutyric acid, and N -carbamyl-β-alanine. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards. Results: All pyrimidine degradation products could be measured in one analytical run of 15 min. Detection limits were 0.4–4 μmol/L. The intraassay imprecision (CV) of urine samples with added compounds was 1.3–12% for liquid urines and 1.0–10% for filter-paper extracts of the urines. The interassay imprecision (CV) was 3–11% (100–200 μmol/L). Recoveries were 89–99% at 100–200 μmol/L and 95–106% at 1 mmol/L in liquid urines, and 93–103% at 100–200 μmol/L and 100–106% at 1 mmol/L in filter-paper samples. Correct identifications of deficiencies of the pyrimidine-degrading enzymes were readily made with urine samples from patients with known defects. Conclusions: HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders of the pyrimidine degradation pathway, and filter-paper samples allow easy collection, transport, and storage of urine samples.