INTRACELLULAR HOMOCYSTEINE & METHIONINE KINETICS IN HUMANS

同型半胱氨酸是重要的，因为它与血浆同型半胱氨酸水平升高作为一个独立的危险因素的心脏病。由于现有的气相色谱-质谱法（GCMS）和HPLC方法难以应用并确保血浆中巯基物质的完全和持续减少，并且高半胱氨酸浓度难以足够精确和灵敏地测量以能够在示踪量下获得稳定同位素富集的测量结果，因此我们已经花费精力定义了通过GCMS测量高半胱氨酸的新的新颖方法。 最终的应用是以足够的精度和灵敏度测量高半胱氨酸中的示踪剂富集，以能够确定其合成和处置速率。 将内标物[3，3，3 '，3'，4，4，4 '，4'，-2 H8]同型半胱氨酸加入血浆样品中，以解释与先前使用的天然同型半胱氨酸的分离、衍生化和测量相关的损失。 我们在两个方面改进了该方法：首先，我们使用一种新的还原剂N，N '-二甲基-N，N'-双（巯基乙酰基）肼（DMH）将二硫键还原为还原硫醇。 其次，游离硫醇然后立即用4-乙烯基吡啶烷基化以永久防止二硫键的任何重新形成。 根据我们的常规方案，分离血浆氨基酸并衍生化以形成叔丁基二甲基甲硅烷基衍生物。 使用这种新的制备方案，我们可以测量<5 pmol的同型半胱氨酸的量，并且可以使用0.5 ml血浆样品以3.4%的日间精密度测定血浆总同型半胱氨酸浓度。 该方法也足够灵敏，以其他氨基酸的典型精度（10.1摩尔%过量示踪剂）测定稳定同位素示踪剂（例如[1- 13 C]）的富集。 这种方法将用于我们今年对人体含硫氨基酸代谢的研究。

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.