Luna Leah G, Green Brett J, Zhang Fagen, Arnold Scott M, Siegel Paul D, Bartels Michael J
Toxicology, Environmental Research and Consulting, The Dow Chemical Company, United States.
Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, United States.
Toxicol Rep. 2014;1:743-751. doi: 10.1016/j.toxrep.2014.09.009.
4,4'-Methylene diphenyl diisocyanate (herein 4,4'-MDI) is used in the production of polyurethane foams, elastomers, coatings, adhesives and the like for a wide range of commercial products. Occupational exposure to MDI levels above current airborne exposure limits can elicit immune mediated hypersensitivity reactions such as occupational asthma in sensitive individuals. To accurately determine exposure, there has been increasing interest in developing analytical methods to measure internal biomarkers of exposure to MDI. Previous investigators have reported methodologies for measuring MDI diamine metabolites and MDI-Lysine (4,4'-MDI-Lys) adducts. The purpose of this study was to develop and validate an ultra performance liquid chromatography isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) quantitation method a signature peptide approach to enable biomonitoring of 4,4'-MDI adducted to human serum albumin (HSA) in plasma. A murine, anti-4,4'-MDI monoclonal IgM antibody was bound to magnetic beads and utilized for enrichment of the MDI adducted HSA. Following enrichment, trypsin digestion was performed to generate the expected 414 site (primary site of adduction) 4,4'-MDI-adducted HSA signature peptide that was quantified by UPLC-ID/MS/MS. An Agilent 6530 UPLC/quadrupole time of flight MS (QTOF) system was utilized for intact adducted protein analysis and an Agilent 6490 UPLC/MS/MS system operated in multiple reaction monitoring (MRM) mode was utilized for quantification of the adducted signature peptide biomarker both for and worker serum samples. Worker serum samples were initially screened utilizing the previously developed 4,4'-MDI-Lys amino acid method and results showed that 12 samples were identified as quantifiable for 4,4'-MDI-Lys adducts. The signature peptide adduct approach was applied to the 12 worker samples identified as quantifiable for 4,4'-MDI-Lys adducts. Results indicated no positive results were obtained above the quantification limit by the signature peptide approach. If the 414 site of lysine adduction accounted for 100% of the 4,4'-MDI adductions in the signature peptide adduct approach, the three highest quantifiable samples by the 4,4'-MDI-Lys method should have at least been detectable by the signature peptide method. Results show that although the 4,4'-MDI signature peptide approach is more selective, it is 18 times less sensitive than the 4,4'-MDI-Lys method, thus limiting the ability to detect adduct levels relative to the 4,4'-MDI-Lys amino acid method.
4,4'-亚甲基二苯基二异氰酸酯（以下简称4,4'-MDI）用于生产聚氨酯泡沫、弹性体、涂料、粘合剂等多种商业产品。职业接触高于当前空气暴露限值的MDI水平，可能会在敏感个体中引发免疫介导的超敏反应，如职业性哮喘。为了准确测定暴露情况，人们对开发测量MDI暴露内部生物标志物的分析方法越来越感兴趣。此前的研究人员已经报道了测量MDI二胺代谢物和MDI-赖氨酸（4,4'-MDI-Lys）加合物的方法。本研究的目的是开发并验证一种超高效液相色谱同位素稀释串联质谱（UPLC-ID/MS/MS）定量方法——一种特征肽方法，用于对血浆中与人类血清白蛋白（HSA）结合的4,4'-MDI进行生物监测。一种鼠源抗4,4'-MDI单克隆IgM抗体与磁珠结合，用于富集与MDI结合的HSA。富集后，进行胰蛋白酶消化，以生成预期的414位点（加合的主要位点）4,4'-MDI加合的HSA特征肽，通过UPLC-ID/MS/MS进行定量。使用安捷伦6530 UPLC/四极杆飞行时间质谱（QTOF）系统进行完整加合蛋白分析，使用安捷伦6490 UPLC/MS/MS系统在多反应监测（MRM）模式下运行，对加合特征肽生物标志物进行定量，用于患者和工人血清样本。最初使用先前开发的4,4'-MDI-Lys氨基酸方法对工人血清样本进行筛查，结果显示有12个样本被鉴定为可定量检测4,4'-MDI-Lys加合物。将特征肽加合方法应用于12个被鉴定为可定量检测4,4'-MDI-Lys加合物的工人样本。结果表明，特征肽方法在定量限以上未获得阳性结果。如果赖氨酸加合的414位点在特征肽加合方法中占4,4'-MDI加合的100%，那么4,4'-MDI-Lys方法中三个最高可定量样本至少应该能用特征肽方法检测到。结果表明，尽管4,4'-MDI特征肽方法更具选择性，但它的灵敏度比4,4'-MDI-Lys方法低18倍，因此相对于4,4'-MDI-Lys氨基酸方法，其检测加合物水平的能力受到限制。
