5-Oxoprolinase deficiency: report of the first human OPLAH mutation.

Gamma-glutamyl cycle is a six-enzyme cycle that represents the primary pathway for glutathione synthesis and degradation. 5-Oxoprolinase deficiency is an extremely rare disorder of the gamma-glutamyl cycle with only eight patients reported to date. Debate continues as to whether this is a benign biochemical defect because of the heterogeneity of the clinical presentation which ranges from normal to significant neurological involvement. Here, we report the first molecularly characterized patients with 5-oxoprolinase deficiency due to a mutation in OPLAH (which encodes 5-oxoprolinase). The largely benign clinical course of the patients described herein despite persistent 5-oxoprolinuria highlights the importance of establishing a molecular diagnosis in the few cases with abnormal neurological outcome to exclude potentially overlapping biochemical defects and to explore potential genotype/phenotype correlation.

Determination of L-pipecolic acid in plasma using chiral liquid chromatography-electrospray tandem mass spectrometry.

BACKGROUND: L-pipecolic acid (L-PA), an important biochemical marker for the diagnosis of peroxisomal disorders, is usually determined as the racemate. We developed a chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of L-PA in plasma. METHODS: We used a narrow bore chiral macrocyclic glycopeptide teicoplanin column for the enantioseparation of D-pipecolic acid (D-PA) and L-PA and interfaced the column directly to the electrospray source of a tandem mass spectrometer. We used phenylalanine-d5 as internal standard added to 50 microL of plasma followed by deproteinization, evaporation, and injection. The analysis was performed in the selected-reaction monitoring mode using two transitions: m/z 130-->m/z 84 for PA, and m/z 171-->m/z 125 for phenylalanine-d5. L-PA eluted at 7 min, and D-PA eluted at 11.7 min, whereas phenylalanine-d5 eluted at 6 min. The turnaround time for the assay was 20 min. RESULTS: Linear calibration curves were obtained in the range of 0.5-80 micromol/L. At a plasma concentration of 1.0 micromol/L, the signal-to-noise ratio was 50:1. The intra- and interassay variations were 3.1-7.9% and 5.7-13%, respectively, at concentrations of 1-50 micromol/L. Mean recoveries of L-PA added to plasma were 95% (5 micromol/L) and 102% (50 micromol/L). The method clearly distinguished between healthy individuals and peroxisomal disease patients. CONCLUSIONS: The novel LC-MS/MS method is simple, rapid, and stereoselective, and uses only 50 microL of plasma, no derivatizing reagents, and a commercially available internal standard. Sample preparation is not complex and is faster than for all other methods.