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-）是一种无处不在的环境有毒物质。尽管有很多研究，但鲜为人知的可能引起不利的健康影响。通常通过GC-MS顶空测定法确定CN暴露。但是，从该室中迅速清除了CN（血液中的T1/2 <1小时），并且在抽取血液样本以进行分析之前，几个半衰期经过是常见的。因此，评估高危人群中急性人类CN暴露是高度问题的。我们先前的研究表明，CN与免疫球蛋白（IgG）和人血清白蛋白（HSA）中的几种易感二硫键反应，在其中一种CYS组件上形成SCN加合物。现在，我们发现了一种新型的内源性HSA Cys567-SCN加合物，该加合物是通过与Cys558-Cys567，C末端二硫化物形成的。通过液相色谱法/质谱法/质谱法（LC-MS/MS），可以很容易地检测那些含有加合物的HSA分子的独特的19分钟C末端肽。在血浆中，CN与该HSA二硫化物的反应很快，并且与生理相关水平下的CN浓度线性相关。该加合物对化学处理和血浆酶降解非常稳定。通过使用LC-MS选择反应剂监测（LC-MS-SRM），通过与内源性（D6）肽标准同源物（M/Z 1278）进行内源性加合物的M/z 1272子离子的整合峰面积比较来实现定量。内源性加合物存在于市售的血浆中，以及来自13名志愿者的等离子体中，最高和最低之间的浓度差异约为3倍。总的来说，这些结果强烈表明内源性HSA CYS567-SCN加合物可以用作CN暴露的回顾性生物标志物。在此应用程序中，我们建议简化我们的测定，以减少样品制备时间，并确定是否可以从我们当前使用的LCQ平台转移检测，从而在定量上更精确，更强大的LC-Triple四倍体MS平台。使用优化的测定，我们将检验以下假设：HSA CYS567-SCN加合物的数量与全血中存在的游离CN浓度无关。作为研究的一部分，我们还将比较来自一群志愿者的对照匹配样本，预计CN正常暴露于一系列消防员。由于消防员靠近工业和住宅火烟雾，因此CN暴露过多的风险很长。公共卫生相关性：急性，中级和慢性氰化物暴露的长期健康影响知之甚少。这样做的主要原因是氰化物的快速代谢和清除率以及氰化物或其代谢物的当前测定法的不足，以准确检测暴露。我们提出的工作是测试一种新颖，内源性和容易检测到的人血清白蛋白氰化物加合物（HSA CYS567-SCN）是否可以用作氰化物暴露的回顾性生物标志物。使用美国开发的基于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可以比目前使用的测定法更准确地检测到控制和高风险种群（例如消防人员）之间的氰化物暴露差异。