Retrospective Cyanide Assay from a Unique HSA-CN Adduct

对独特的 HSA-CN 加合物进行回顾性氰化物测定

基本信息

批准号：
7894711
负责人：
Kenneth M. Aldous
金额：
$ 15.1万
依托单位：
WADSWORTH CENTER
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-16 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7894711
关键词：
Acute Age Anticoagulants Area Biological Assay Biological Markers Blood Blood specimen Burn injury C-terminal Cessation of life Chemicals Chronic Confounding Factors (Epidemiology)Cyanides Data Databases Daughter Dependence Detection Disulfides Enzymes Exposure to Fire - disasters Gases Goals Half-Life Health Homologous Gene Hour Human Hydrolysis Immunoglobulin G Immunoglobulins In Vitro Individual Ions Lead Length Mass Spectrum Analysis Measurement Metabolism Monitor Occupations Peptides Plasma Population Preparation Questionnaires Reaction Recovery Reproducibility Route Salts Sampling Serum Serum Albumin Smoke Smoking History Solid Testing Time Toxic Environmental Substances Validation Whole Blood Work adduct base cohort disulfide bond high risk human subject improved indexing insight liquid chromatography mass spectrometry mass spectrometer novel public health relevance sample collection sex suicidal volunteer weapons

项目摘要

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.

描述（由申请人提供）：氰化物（CN = HCN + CN-）是一种普遍存在的环境毒物。尽管进行了大量研究，但人们对其对健康造成不良影响的潜力知之甚少。 CN 暴露通常通过 GC-MS 顶空测定从血液中测定。然而，CN 会迅速从该隔间中清除（血液中的 t1/2 < 1 小时），并且在抽取血样进行分析之前通常会经过几个半衰期。因此，对高危人群中人类急性氯化萘暴露的评估存在很大问题。我们之前的研究表明，CN 与免疫球蛋白 (IgG) 和人血清白蛋白 (HSA) 中的几个易受影响的二硫键发生反应，在其中一种 Cys 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 CN 与 Cys558-Cys567、C 末端二硫键反应形成的。通过液相色谱-质谱/质谱 (LC-MS/MS) 可以轻松检测含有加合物的 HSA 分子的碱水解所释放的独特 19 聚体 C 端肽。在血浆中，CN 与 HSA 二硫化物的反应很快，并且与生理相关水平的 CN 浓度呈线性相关。该加合物对于化学处理和血浆酶降解都非常稳定。使用 LC-MS 选择反应物监测 (LC-MS)，通过内源加合物的 m/z 1272 子离子与氘代 (D6) 肽标准同源物 (m/z 1278) 的积分峰面积比较来实现加合物的定量- SRM）。内源性加合物存在于市售血浆和 13 名志愿者的血浆中，最高浓度与最低浓度之间存在大约 3 倍的差异。总的来说，这些结果强烈表明内源性 HSA Cys567-SCN 加合物可以作为 CN 暴露的回顾性生物标志物。在此应用中，我们建议简化我们的测定，以减少样品制备时间，并确定检测是否可以从我们目前使用的 LCQ 平台转移到定量更精确和稳健的 LC-三重四极杆 MS 平台。使用优化的测定，我们将测试 HSA Cys567-SCN 加合物的数量与全血中存在的游离 CN 浓度不相关的假设。作为我们研究的一部分，我们还将比较一组志愿者的对照样本与一组消防员的预期正常 CN 暴露量。由于消防员长时间接近工业和住宅火灾烟雾，因此他们面临着过量 CN 暴露的高风险。公众健康相关性：人们对急性、中度和慢性氰化物暴露的长期健康影响知之甚少。造成这种情况的主要原因是氰化物的快速代谢和清除，以及目前氰化物或其代谢物的检测方法不足以准确检测暴露情况。我们提出的工作是测试一种新型、内源性且易于检测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回顾性生物标志物。使用我们开发的基于 MS 的测定法来量化 HSA Cys567-SCN 加合物，我们将测试加合物分析是否能够比目前使用的测定法更准确地检测对照人群和高风险人群（例如消防员）之间氰化物暴露的推定差异。