Screening and diagnosis of β‐ureidopropionase deficiency by gas chromatographic/mass spectrometric analysis of urine

Abstract

Dihydropyrimidine dehydrogenase (DHPDase), dihydropyrimidinase (DHPase) and beta-ureidopropionase (betaUPase) are the enzymes that catalyze the first, second, and third steps of the degradation of pyrimidines, respectively. beta-Ureidopropionate (betaUP) and beta-ureidoisobutyrate (betaUIB) are increased in the urine of patients with betaUPase deficiency. The original case in which betaUPase deficiency was discovered by NMR spectroscopy was an 11-month-old patient who presented with hypotonia and dystonic movement. We detected a second but asymptomatic case during a pilot study of neonatal screening with filter-paper urine, urease pretreatment and gas chromatography/mass spectrometry (GC/MS). The urease pretreatment of urine without fractionation resulted in a high recovery of these polar ureide compounds and allowed the highly sensitive GC/MS detection and diagnosis of betaUPase deficiency. betaUP and betaUIB were identified using GC/MS techniques. In the urine of the neonate with betaUPase deficiency, betaUP and betaUIB were persistently increased. Thymine, 5,6-dihydrothymine and 5,6-dihydrouracil were increased only moderately but significantly. It is known that thymine and uracil increase markedly in DHPDase deficiency, and 5,6-dihydrothymine and 5,6-dihydrouracil increase in DHPase deficiency. Therefore, betaUPase deficiency can be differentially diagnosed from the first and second enzyme deficiencies. Application of this specific and sensitive diagnostic procedure will lead to an understanding of the clinical heterogeneity of betaUPase deficiency. Furthermore, the identification of patients with defects in pyrimidine metabolism will enable doctors to avoid cancer chemotherapy with pyrimidine analogues such as 5-fluorouracil, which could be dangerous for these patients.

Liquid chromatography–tandem mass spectrometric assay for the analysis of uracil, 5,6-dihydrouracil and β-ureidopropionic acid in urine for the measurement of the activities of the pyrimidine catabolic enzymes

“…Developing a chromatographic assay for urine was initially started at our laboratory using GC-MS according to the method of Kuhara et al [19,30,32] using our labelled ISs. Unfortunately, the validation of this assay was not successful due to batch-to-batch variation of the N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS) reagent, resulting in a poor yield of the derivatization for several batches.…”

“…The pooled "blank" urine, representing an average individual or patient, as well as three spiked samples of the same urine were also used for the validation. QC-A was spiked with 1824 M uracil, representing total DPD deficiency [19,21]; QC-B with 912 M uracil and 1774 M UH2, representing total DHP deficiency [19,21,22,33] and QC-C with 365 M uracil, 709 M UH2 and 1535 M bUP, representing total UP deficiency [13,30]. Precisions and accuracies were determined by sextuple analysis of each validation sample in three analytical runs on three separate days for all QCs (total: n = 18).…”

“…A 100% conversion of this reaction would result in a yield of 5.6 mM bUP . Remaining UH2 was not detected in the product using a GC-MS assay according to Kuhara et al [19,30,32]. One liter of artificial urine comprised 1.3 g sodium dihydrogen phosphate monohydrate, 0.51 g disodium hydrogen phosphate dihydrate, 0.58 g trisodium citrate dihydrate, 6.8 g sodium chloride, 12 g ureum, 0.90 g calcium chloride dehydrate, 2.5 g disodium sulfate, 0.75 g magnesium sulfate, 2.3 g ammonium chloride and 6.1 g potassium chloride.…”

“…60 M for each IS) in different matrices Stability data (±S.D. ; n = 4), of the three analytes under different storage conditions QC-C: pooled urine spiked with 365 M uracil, 710 M UH2 and 1535 M bUP.b Samples were measured using a GC-MS assay according to Kuhara et al[19,30,32]. c n = 3.…”

Noninvasive human metabolome analysis for differential diagnosis of inborn errors of metabolism

“…One can screen for and make a chemical diagnosis of more than 130 IEMs, including branched chain amino acids (22 etiologies), primary hyperammonemias (8 etiologies), aromatic amino acids (17 etiologies), pyrimidines and purines (9 etiologies) [36][37][38][39][40]. Galactose [2,41], glucose and fructose can also be targets as well as their oxidized and reduced metabolites.…”