Measurement of homocysteine concentrations and stable isotope tracer enrichments in human plasma

Elevated levels of plasma homocysteine have been established as an independent risk factor for cardiovascular disease. Homocysteine is in low concentration in plasma (5-15 mu M) and is bound to other thiols (e.g,, cysteine in plasma proteins) via disulfide bonds. Existing methods for measuring homocysteine have difficulty in reducing and maintaining the reduction of homocysteine for measurement. We describe a GC/MS method that first reduces the disulfides in the physiological sample matrix:and then immediately alkylates the free thiols with 4-vinylpyridine to prevent the reformation of the disulfide bonds. We use a deuterated internal standard, [3,3,3',3',4,4,4',4'-H-2(8)]homocystine to account for losses associated with the isolation, derivatization, and measurement of the natural homocysteine, The amino acids are separated and derivatized to form the tert-butyldimethylsilyl derivatives. This method requires only 50 mu L of plasma to measure homocysteine concentrations to 5 mu M. Total homocysteine concentrations in plasma can be measured routinely from 0.5-mL samples with relative intra- and interday precisions of 1.3 and 4.0%, respectively. This method is sensitive enough to determine tracer enrichments of [1-C-13]homocysteine with a detection limit of <0.3 mol % excess and an average tracer precision of 0.6%.