Retrospective Cyanide Assay from a Unique HSA-CN Adduct

说明(申请人提供)：氰化物(CN=HCN+CN-)是一种普遍存在的环境毒物。尽管有过多的研究，但人们对其潜在的不良健康影响知之甚少。CN暴露通常是通过GC-MS顶空分析从血液中确定的。然而，CN从这个隔室中被迅速清除(血液中的T1/2为1小时)，在抽取血液样本进行分析之前，通常已经过了几个半衰期。因此，在高危人群中评估急性人类CN暴露是非常有问题的。我们以前的研究表明，CN与免疫球蛋白(Ig G)和人血清白蛋白(HSA)中的几个敏感的二硫键反应，在其中一个半胱氨酸成分上形成SCN加合物。我们现在发现了一种新型的内源性HSA Cys567-SCN加合物，它是通过与Cys558-Cys567，C-末端二硫键的CN反应而形成的。含有加合物的人血清白蛋白分子通过碱水解释放出一种独特的19聚体C-末端多肽，可通过LC-MS/MS(LC-MS/MS)轻松实现。在血浆中，CN与这种HSA二硫化物的反应是快速的，并且在生理水平上与CN浓度呈线性关系。加合物对化学处理和血浆酶降解都非常稳定。加合物的定量是通过使用LC-MS-SRM(LC-MS-SRM)将内源加合物的m/z 1272子离子与氚(D6)肽标准同系物(m/z 1278)的积分峰面积进行比较来实现的。内源性加合物存在于商业上可获得的血浆和13名志愿者的血浆中，最高和最低之间的浓度差约为3倍。总之，这些结果强烈表明，内源性HSA Cys567-SCN加合物可以作为CN暴露的追溯生物标志物。在这项应用中，我们建议简化我们的分析，以减少样品准备时间，并确定检测是否可以从我们目前使用的LC-三重四极杆MS平台转移到定量更精确和更坚固的LC-三重四极杆MS平台。使用优化的检测方法，我们将检验假设，即HSA Cys567-SCN加合物的数量与全血中存在的游离CN浓度无关。作为我们研究的一部分，我们还将比较一组志愿者与预期正常接触CN的志愿者与一组消防员的对照匹配样本。消防员由于长期接触工业和住宅火灾烟雾，因此暴露在过量的CN中的风险很高。公共卫生相关性：急性、中度和慢性氰化物暴露对健康的长期影响知之甚少。造成这种情况的主要原因是氰化物的快速新陈代谢和清除，以及目前对氰化物或其代谢物的检测方法不足以准确地检测暴露。我们建议的工作是测试一种新的、内源性的、容易检测到的人血清白蛋白氰化物加合物(HSA Cys567-SCN)是否可以作为氰化物暴露的追溯生物标志物。使用我们开发的基于MS的分析来量化HSA Cys567-SCN加合物，我们将测试加合物分析是否可以比目前使用的分析更准确地检测控制人群和高危人群(如消防员)之间氰化物暴露的假定差异。

DESCRIPTION (provided by applicant): Cyanide (CN = HCN + CN-) is a ubiquitous environmental toxicant. Despite a plethora of studies, little is known of its potential to cause adverse health effects. CN exposure is normally determined from blood by a GC-MS headspace assay. However, CN is rapidly cleared from this compartment (t1/2 in blood is < 1 hr), and it is common for several half-lives to have passed before blood samples are drawn for analysis. Consequently, assessment of acute human CN exposure in high-risk populations is highly problematic. Prior studies by us showed that CN reacts with several susceptible disulfide bonds in immunoglobulin (IgG) and human serum albumin (HSA) to form SCN adducts on one of the Cys components. We have now discovered a novel, endogenous, HSA Cys567-SCN adduct that is formed via CN reaction with the Cys558-- Cys567, C-terminal disulfide. Detection of a unique, 19-mer C-terminal peptide released by base hydrolysis of those HSA molecules containing the adduct is readily accomplished by liquid chromatography - mass spectrometry/mass spectrometry (LC-MS/MS). In plasma, reaction of CN with this HSA disulfide is rapid, and linearly related to the CN concentration at physiologically relevant levels. The adduct is very stable both to chemical treatment and to degradation by plasma enzymes. Quantitation of the adduct is achieved by integrated peak area comparison of a m/z 1272 daughter ion of the endogenous adduct with a deuterated (D6) peptide standard homologue (m/z 1278) using LC--MS-Selected Reactant Monitoring (LC-MS- SRM). The endogenous adduct is present in commercially available plasma and in the plasmas from 13 volunteers with an approximate 3-fold concentration difference between the highest and the lowest. Collectively, these results strongly suggest that the endogenous HSA Cys567-SCN adduct can serve as a retrospective biomarker of CN exposure. In this application, we propose to streamline our assay to reduce the time of sample preparation and to determine whether detection can be transferred from the LCQ platform, which we currently employ, to a quantitatively more precise and robust LC-triple quadrupole MS platform. Using the optimized assay, we will test the hypothesis that the quantity of the HSA Cys567-SCN adduct does not correlate with the free CN concentration that is present in whole blood. As part of our study, we will also compare control-matched samples from a cohort of volunteers with expected normal CN exposure to a cohort of firefighters. Firefighters are high risk for excessive CN exposure due to their proximity to industrial and residential fire smoke for prolonged periods. PUBLIC HEALTH RELEVANCE: The long-term health effects of acute, intermediate, and chronic cyanide exposure are poorly understood. Primary reasons for this are the rapid metabolism and clearance of cyanide and the inadequacy of current assays for cyanide, or its metabolites, to detect exposure accurately. Our proposed work is to test whether a novel, endogenous and readily detectable human serum albumin cyanide adduct (HSA Cys567-SCN) can serve as a retrospective biomarker of cyanide exposure. Using a MS-based assay developed by us to quantify the HSA Cys567-SCN adduct we will test whether adduct analysis can more accurately detect putative differences in cyanide exposure between control and high risk populations, such as firefighters, than currently used assays.