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

Elevated levels of plasma homocysteine have been established as an independ ent risk factor for cardiovascular disease. Homocysteine is in low concentr ation 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 homoc ysteine have difficulty in reducing and maintaining the reduction of homocy steine for measurement. We describe a GC/MS method that first reduces the d isulfides in the physiological sample matrix:and then immediately alkylates the free thiols with 4-vinylpyridine to prevent the reformation of the dis ulfide 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, deri vatization, and measurement of the natural homocysteine, The amino acids ar e separated and derivatized to form the tert-butyldimethylsilyl derivatives . This method requires only 50 mu L of plasma to measure homocysteine conce ntrations to 5 mu M. Total homocysteine concentrations in plasma can be mea sured routinely from 0.5-mL samples with relative intra- and interday preci sions of 1.3 and 4.0%, respectively. This method is sensitive enough to det ermine tracer enrichments of [1-C-13]homocysteine with a detection limit of <0.3 mol % excess and an average tracer precision of 0.6%.