Stable isotope dilution assay for branched chain alpha-hydroxy-and alpha-ketoacids: serum concentrations for normal children.

A stable isotope dilution assay is described for the six branched chain alpha-hydroxyacids and alpha-ketoacids that accumulate in serum in maple syrup urine disease. The method employs sodium borodeuteride reduction of the alpha-ketoacids in situ to once-labeled hydroxyacids, addition of a more extensively labeled analog as internal standard, isolation of the organic acids by ether extraction, and quantitation by gas chromatography/mass spectrometry in selected ion monitoring mode. A set of three areas are obtained for each of the three branched chain hydroxyacid peaks, and these are corrected for natural abundance heavy isotope content and for isotopic impurities in the labeled standards and precursors. The corrected areas are then converted to concentrations of the endogenous three hydroxyacids and the original three ketoacids in serum.