Metabolite Measurements

Amino acids and acylcarnitines in dried blood spots (DBS) were analyzed by electrospray tandem mass spectrometry (LC-MS/MS) for C4-OH (composed of 3-hydroxyisobutyryl-carnitine, 3-hydroxy-butyryl-carnitine, and malonylcarnitine [C3-DC]). Samples were prepared as follows: a 3-mm diameter disc was punched in a microtiter plate, 100 μl internal standard solution was added, and the plate was covered. After gentle shaking at 45°C for 45 min, the solvent after elution was transferred into a new microtiter plate and analyzed immediately. An LCMS-8040 (Shimadzu, Japan) in positive mode was utilized for acylcarnitines and amino acids analysis. For MS/MS data acquisition, the peak including C4-OH (248.2>85) was observed with multiple reaction monitoring.

Seven patients’ urine metabolites, including 2,3-dihydroxy-2-methylbutyrate (23DH2MB), were analyzed by standard gas chromatography-mass spectrometry (GC-MS). Briefly, 100 μl urine was incubated with 40 units urease at 37°C for 15 min. After adding an internal standard (20 μg of n-heptadecanoic acid), the sample was centrifuged to deproteinize by adding 1,000 μl of ethanol, then the supernatant was evaporated. The residue was completely dried under a nitrogen stream for 3 min and derivatized with 100 μl of N, O-bis(trimetnylsilily) trifluorcetamide and 10 μl of trimethylchlorosilane at 90°C for 40 min (Zhang et al., 2000). A GCMS-QP2010 Ultra GC-MS system (Shimadzu, Kyoto, Japan) was used with an ultra Alloy capillary column (30 × 0.25 mm internal diameter with 0.25-μm film thickness, Frontier Lab, Fukushima, Japan). The temperature was programmed to increase from 60 to 350°C at 17°C/min. Next, 2 μl of derivated sample was injected in split mode. The mass chromatographic quantitation of urine metabolites including 23DH2MB was based on the fragment ion peaks area compared with the corresponding urine creatinine fragment ion area ratio.

Through retrospective re-analysis, quantitative urine screening for S-(2-caboxypropyl) cysteamine (SCPCM) was measured by LCMS/MS in urine of cases. This project was not routinely performed in the metabolic laboratory. The process included sample and label pretreatment. The SCPCM concentration in urine of 140 control children was determined by the detection and verification of an SCPCM standard, and the reference value was obtained using the SCPCM standard concentration curve.