INTRACELLULAR HOMOCYSTEINE & METHIONINE KINETICS IN HUMANS

同型半胱氨酸很重要，因为它与血浆同型半胱氨酸水平升高有关，是心脏病的独立危险因素。由于现有的气相色谱-质谱（GCMS）和高效液相色谱（HPLC）方法难以应用，也难以保证血浆中硫醇类物质的完整和持续减少，而同型半胱氨酸浓度也难以精确和敏感地测量，无法获得示踪量下稳定同位素富集的测量结果，因此我们花了很多时间来定义一种新的用GCMS测量同型半胱氨酸的方法。最终的应用是测量同型半胱氨酸的示踪剂浓度，具有足够的精度和灵敏度，能够确定其合成和处理速率。血浆样品中加入内标[3,3,3',3',4,4,4',4'，-2H8]同型半胱氨酸，以弥补与分离，衍生化和测量天然同型半胱氨酸相关的损失。我们从两个方面对该方法进行了改进：首先，我们使用一种新型还原剂N，N'-二甲-N，N'-双（巯基乙酰）肼（DMH）将二硫键还原为还原性硫醇。其次，游离的硫醇立即与4-乙烯基吡啶烷基化，以永久防止二硫桥的任何重组。血浆氨基酸被分离和衍生，形成t-丁基二甲基硅基衍生物，按照我们通常的方案。使用这种新的制备方案，我们可以测量到<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%的日间精度测定血浆总同型半胱氨酸浓度。该方法还具有足够的灵敏度，可以测定稳定同位素示踪剂（例如[1-13C]）的富集程度，其精度与其他氨基酸相当（示踪剂过量10.1摩尔%）。这种方法将在今年用于我们对人类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.