Warnecke Athanasia, Luessen Piet, Sandmann Jörg, Ikic Milos, Rossa Stefan, Gutzki Frank-Mathias, Stichtenoth Dirk O, Tsikas Dimitrios
Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 May 1;877(13):1375-87. doi: 10.1016/j.jchromb.2008.11.035. Epub 2008 Nov 30.
In the year 1992, S-nitrosoalbumin (SNALB) has been proposed to be the most abundant physiological carrier and pool of nitric oxide (NO) activity in human circulation, by which NO-dependent biological functions are regulated. The concentration, the metabolism and the mechanisms of the biological actions of SNALB are controversial and still incompletely understood. Moreover, the suitability of SNALB as a biomarker of diseases associated with altered NO bioactivity in human circulation has not been demonstrated convincingly so far. In the present study, we report on the development and application of a stable-isotope technique to study the pharmacokinetics of (15)N-labelled SNALB (S(15)NALB) in anesthetized rats. S(15)NALB was synthesized from albumin isolated by affinity chromatography from freshly prepared human plasma. This technique was also applied to study and quantify the formation of S(15)NALB from endogenous rat plasma albumin and intravenously applied S-[(15)N]nitrosoglutathione (GS(15)NO) or S-[(15)N]nitrosocysteine (S(15)NC) in anesthetized rats. In these investigations the mean arterial pressure (MAP) was monitored continuously. The elimination half-life (t(1/2)) of S(15)NALB from rat plasma was determined to be 4.1 min (t(1/2)alpha) and 9.4 min (t(1/2)beta). S(15)NALB (125 nmol) produced long-lasting decreases in MAP (by 49% for 18 min). Thirty minutes after intravenous (i.v.) injection of S(15)NALB (125 nmol), repeated i.v. injection of L-cysteine or D-cysteine (10 micromol each) produced repeatedly potent (by 44-55%) but short-lasting (about 4 min) MAP falls. Intravenously administered GS(15)NO and S(15)NC (each 500 nmol) could not be isolated from rat blood. (15)N-Labelled nitrite and nitrate were identified as the major metabolites of all investigated S-nitrosothiols in rat plasma. The results of this study suggest that in the rat S(15)NALB is a potent S-transnitrosylating agent and that the blood pressure-lowering effect of S(15)NALB and other S-nitrosothiols are mediated largely by L-cysteine via S-transnitrosylation to form S(15)NC that subsequently releases (15)NO. Our results also suggest that S-transnitrosylation of the single reduced cysteine moiety of albumin by endogenous GSNO or SNC in blood is possible but does not represent an effective mechanism to produce SNALB in vivo. This stable-isotope dilution GC-MS technique is suitable to perform in vivo studies on SNALB using physiologically and pharmacologically relevant doses.
1992年，有人提出S-亚硝基白蛋白（SNALB）是人体循环中最丰富的生理一氧化氮（NO）活性载体和储存库，通过它来调节依赖NO的生物学功能。SNALB的浓度、代谢及其生物学作用机制存在争议，目前仍未完全了解。此外，迄今为止，尚未令人信服地证明SNALB作为与人体循环中NO生物活性改变相关疾病的生物标志物的适用性。在本研究中，我们报告了一种稳定同位素技术的开发和应用，用于研究麻醉大鼠体内（15）N标记的SNALB（S（15）NALB）的药代动力学。S（15）NALB由通过亲和色谱从新鲜制备的人血浆中分离的白蛋白合成。该技术还用于研究和定量麻醉大鼠体内内源性大鼠血浆白蛋白与静脉注射的S-[(15)N]亚硝基谷胱甘肽（GS(15)NO）或S-[(15)N]亚硝基半胱氨酸（S(15)NC）形成S（15）NALB的情况。在这些研究中，连续监测平均动脉压（MAP）。大鼠血浆中S（15）NALB的消除半衰期（t(1/2)）经测定为4.1分钟（t(1/2)α）和9.4分钟（t(1/2)β）。S（15）NALB（125 nmol）可使MAP产生持久下降（18分钟内下降49%）。静脉注射（i.v.）S（15）NALB（125 nmol）30分钟后，重复静脉注射L-半胱氨酸或D-半胱氨酸（各10 μmol）可反复产生强效（下降44 - 55%）但持续时间短（约4分钟）的MAP下降。静脉注射的GS(15)NO和S(15)NC（各500 nmol）无法从大鼠血液中分离出来。（15）N标记的亚硝酸盐和硝酸盐被鉴定为大鼠血浆中所有研究的S-亚硝基硫醇的主要代谢产物。本研究结果表明，在大鼠中S（15）NALB是一种强效的S-转亚硝基化剂，S（15）NALB和其他S-亚硝基硫醇的降压作用主要由L-半胱氨酸通过S-转亚硝基化形成S（15）NC介导，随后释放（15）NO。我们的结果还表明，血液中内源性GSNO或SNC对白蛋白单个还原半胱氨酸部分的S-转亚硝基化是可能的，但并非体内产生SNALB的有效机制。这种稳定同位素稀释气相色谱 - 质谱技术适用于使用生理和药理相关剂量对SNALB进行体内研究。
