Reference intervals for orotic acid in urine, plasma and dried blood spot using hydrophilic interaction liquidchromatography–tandem mass spectrometry

摘要

Orotic acid (OA), a marker of hereditary orotic aciduria, is usually used for the differential diagnosis of\udsome hyperammonemic inherited defects of urea cycle and of basic amino acid transporters. This study\udwas aimed to establish age related reference intervals of OA in urine, and for the first time in plasma,\udand dried blood spot (DBS) from 229 apparently healthy subjects aged from three days to 40 years.\udThe quantification of OA was performed by a previously implemented method, using a stable isotope\uddilution with 1,3-[15N2]-orotic acid and hydrophilic interaction liquid chromatography–tandem mass\udspectrometry (HILIC–MS/MS). The method has proved to be sensitive and accurate for a quantitative\udanalysis of OA also in DBS and plasma. According to previous studies, urinary OA levels (mmol/mol of\udcreatinine) decrease significantly with age. The upper limits (as 99th %ile) were of 3.44 and 1.30 in groups\udaged from three days to 1 year (group 1) and from 1 year to 12 years (group 2), respectively; in teenagers\ud(from 13 to 19 years; group 3) and adults (from 20 to 40 years; group 4) urinary levels became more\udstable and the upper limits were of 0.64 and 1.21, respectively. Furthermore, OA levels in DBS (M) also\udresulted significantly higher in subjects of group 1 (upper limit of 0.89) than in subjects of groups 2, 3 and\ud4 (upper limits of 0.24, 0.21, and 0.29, respectively). OA levels in plasma (M) were significantly lower\udin subjects of group 3 (upper limit of 0.30) than in subjects of groups 1, 2, and 4 (upper limits of 0.59,\ud0.48, and 0.77, respectively). This method was also employed for OA quantification in plasma and DBS\udof 17 newborns affected by urea cycle defects, resulting sensitive and specific enough to screen these\uddisorders.