Determination of extracellular and intracellular enrichments of [1-C-13]-alpha-ketoisovalerate using enzymatically converted [1-C-13]-valine standardization curves and gas chromatography-mass spectrometry

We report a validated method for the determination of extra- and intracellu lar [1-C-13]-alpha-ketoisovalerate ([1-C-13]-KIV) enrichments by gas chroma tography-mass spectrometry. Standardization curves were prepared by enzymat ic oxidation of [1-C-13]-valine enriched standards of known composition. Sl opes of [1-C-13]-valine standardization curves (mean+/-SD: 0.99 +/- 0.02, n = 5) and [1-C-13]-KIV standardization curves (mean+/-SD: 0.98 +/- 0.01, n = 7) were not significantly different. The method was applied for thr deter mination of [1-C-13]-KIV enrichments in plasma and tissues during [1-C-13]- valine infusion in a piglet. [1-C-13]-KIV enrichment could be determined +/ - 0.1 MPE (C.V. 1%), and extracellular [1-C-13]-KIV enrichment was a reliab le estimate of intracellular (skeletal muscle, bone growth plate) [1-C-13]- KIV enrichment. (C) 1999 Elsevier Science B.V. All rights reserved.