INTRACELLULAR HOMOCYSTEINE & METHIONINE KINETICS IN HUMANS

同型半胱氨酸之所以重要，是因为它与血浆同型半胱氨酸水平升高有关，同型半胱氨酸是心脏病的一个独立危险因素。由于现有的气相色谱-质谱仪(GCMS)和高效液相色谱方法很难应用并保证血浆中硫醇物种的完全和持续减少，而同型半胱氨酸的浓度很难精确和灵敏地测量到足够高的示踪量的稳定同位素富集物，因此我们努力定义了一种新的GCMS测定同型半胱氨酸的新方法。最终的应用是测量同型半胱氨酸中的示踪剂富集量，具有足够的精密度和灵敏度，能够确定其合成和处置的速度。将内标[3，3，3‘，3’，4，4，4‘，4’，-2H8]同型半胱氨酸添加到血浆样品中，以解决与分离、衍生化和测量天然同型半胱氨酸有关的损失。我们从两个方面对该方法进行了改进：首先，我们使用了一种新型的还原剂N，N‘-二甲基-N，N’-双(硫代乙酰基)肼(DMH)来将二硫键还原为还原的硫醇。其次，游离的硫醇随后立即与4-乙烯基吡啶烷基化，以永久防止二硫键的任何重整。根据我们通常的方案，血浆氨基酸被分离和衍生化以形成叔丁基二甲基硅基衍生物。使用这种新的制备方案，我们可以测量同型半胱氨酸的量到5 pmol，并可以使用0.5毫升的血浆样品以3.4%的日间精密度测定血浆总同型半胱氨酸浓度。这种方法也足够灵敏，可以用其他氨基酸的典型精密度(10.1摩尔%过量示踪剂)来测定稳定同位素示踪剂(例如[1-13C])的富集度。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.