Determination of extracellular and intracellular enrichments of [1-(13)C]-alpha-ketoisovalerate using enzymatically converted [1-(13)C]-valine standardization curves and gas chromatography-mass spectrometry.
We report a validated method for the determination of extra- and intracellular [1-(13)C]-alpha-ketoisovalerate ([1-(13)C]-KIV) enrichments by gas chromatography-mass spectrometry. Standardization curves were prepared by enzymatic oxidation of [1-(13)C]-valine enriched standards of known composition. Slopes of [1-(13)C]-valine standardization curves (mean+/-SD: 0.99+/-0.02, n=5) and [1-(13)C]-KIV standardization curves (mean+/-SD: 0.98+/-0.01, n=7) were not significantly different. The method was applied for the determination of [1-(13)C]-KIV enrichments in plasma and tissues during [1-(13)C]-valine infusion in a piglet. [1-(13)C]-KIV enrichment could be determined+/-0.1 MPE (C.V. 1%), and extracellular [1-(13)C]-KIV enrichment was a reliable estimate of intracellular (skeletal muscle, bone growth plate) [1-(13)C]-KIV enrichment.
Department of Clinical Chemistry, University Hospital Vrije Universiteit, Amsterdam, The Netherlands. w.kulik@azvu.nl
W
W
Kulik
C
C
Jakobs
K
K
de Meer
D000818:Animals; D002247:Carbon Isotopes; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D045782:Hemiterpenes; D007651:Keto Acids / Q000032:analysis*; D012015:Reference Standards; D015203:Reproducibility of Results; D013552:Swine; D014633:Valine / Q000032:analysis*
D002247:Carbon Isotopes; D045782:Hemiterpenes; D007651:Keto Acids; C001505:alpha-ketoisovalerate; D014633:Valine