INTRACELLULAR HOMOCYSTEINE & METHIONINE KINETICS IN HUMANS

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Homocysteine is important because of its association with elevated levels of plasma homocysteine as an independent risk factor of heart disease. Because existing gas chromatography-mass spectrometry (GCMS) and HPLC methods were difficult to apply and assure complete and continued reduction of thiol-species in plasma and homocysteine concentration was difficult to measure precisely and sensitively enough to be able to obtain measurements of stable isotopic enrichments at tracer amounts, we have spent effort defining a new and novel method for measuring homocysteine by GCMS. The ultimate application is measurement of tracer enrichments in homocysteine with sufficient precision and sensitivity to be able to determine its rates of synthesis and disposal. The internal standard, [3,3,3',3',4,4,4',4',-2H8]homocystine is added to plasma samples to account for losses associated with the isolation, derivatization, and measurement of the natural homocysteine as previously used by other. We have modified this method in two ways: First, we use a novel reducing agent N,N'-dimethyl-N,N'-bis(mercaptoacetyl)hydrazine (DMH) for the reduction of disulfide bonds to reduced thiols. Secondly, the free thiols are then immediately alkylated with 4-vinyl pyridine to prevent permanently any reformation of the disulfide bridges. The plasma amino acids are separated and derivatized to form the t-butyldimethylsilyl derivatives as per our usual protocol. Using this new preparation scheme, we can measure amounts of homocysteine to <5 pmol and can determine plasma total homocysteine concentrations with an inter-day precision of 3.4% using 0.5-ml plasma samples. This method is also sensitive enough to determine enrichments of stable isotopic tracers (e.g. [1-13C]) with precisions typical of other amino acids (10.1 mole % excess tracer). This method will be used in our studies of sulfur amino acid metabolism in humans this year.