使用極致高效液相層析串聯質譜儀與化學同位素標記法檢測已知與未知的血紅蛋白加成物

Abstract

談到法醫毒物學，一般大眾首先想到毒殺、中毒、酒精與毒品濫用造成的社會問題，但不可忽視的還有「環境汙染與公害」。隨經濟與科技發展，稱作異性生物質（xenobiotics）的環境毒物，亦無聲無息地影響人類健康，而慢性影響導致的病害與死亡，往往難以證明與鑑定，成為法醫毒物學的一大挑戰。 其中由生活上單純飲食攝入之異型性生物質，更可說是無人能倖免。這些分子改變了人體組織結構，也可能因此造成病變。好比存在高溫烹調澱粉食物中的丙烯醯胺（acrylamide），惡名昭彰，乃因丙烯醯胺為一α, β-unsaturated carbonyl 化合物，可和血紅蛋白形成加成物（acrylamide adduct）。又血紅蛋白有容易取得、半衰期長的特性，使acrylamide adduct在過往文獻中，已引起許多討論，亦被當作丙烯醯胺暴露指標（biomarker）。然而，加成物與疾病的關聯仍尚未明朗，學者們也正試圖發現除丙烯醯胺外，更多未知的血紅蛋白加成物。 因此，本研究一方面透過現今準確度最高之液相層析儀－極致高效液相層析（UPLC）串聯質譜儀，在萃取最佳化後，測試臨床不孕症病患（N=29）血中acrylamide adduct濃度，一方面使用UPLC串聯高解析度質譜儀，搭配化學同位素標記法（chemical isotope labeling）與統計，找出一般血中可能含有的未標定加成物，及比較受汙染之血液可能出現的加成物。結果為臨床檢體有4位測得acrylamide adduct，濃度由41.17pmol/g Hb至117.58pmol/g Hb；而未標定加成物方面，篩出79至105對可能為加成物（或其碎片）之分子，然受汙染血液和空白血液並未有加成物上之明顯差異。若須明確鑑定出是何種分子形成加成物，則需進一步分析，例如使用串聯質譜儀（MSMS）方法產生離子碎片。 研究應用則期待，未來能以成熟UPLC-MS方法檢驗acrylamide adduct，及其他已知結構之血紅蛋白加成物；也能以CIL-UPLC-HRMS方法，初步篩出不同族群血液中，所存在未知結構之血紅蛋白加成物。俾利法醫毒物學家對環境汙染物之健康影響，獲得更深入的了解，以及可靠的鑑定依據。 關鍵字：環境汙染物、丙烯醯胺、血紅蛋白加成物、化學同位素標記法、極致高效液相層析、高解析質譜儀

When it came to forensic toxicology, most of us brought “murder with poison”, “intoxication”, and “drugs and alcohol abuse” into mind. However, one more thing that couldn’t be ignored – people were always affected by environmental pollutants. With the development of science and technology, many poisonous substances called xenobiotics also came up. Unconsciously, these toxins affected our health, even causing irreversible disease as well as death. Although very challenging, forensic toxicology took the responsibility of identifying the controversial xenobiotics and their disadvantages to health. Xenobiotics from foods would be an issue. These small molecules somehow changed tissue structure in human bodies. The most notorious one, acrylamide, an α, β-unsaturated carbonyl compound common in carbohydrate-rich foods cooked at high temperatures (>120°C), would form covalent adducts with hemoglobin (known as “acrylamide adduct”). Owing to the accessibility and long lifetime of the red blood cells, acrylamide adducts had been discussed in much literature and regarded as a biomarker of acrylamide exposure. Even though the relationship between adduct level and human disease was still not fully understood, many scientists were dedicated to exploring unknown-structured (in other words, untargeted) hemoglobin adducts simultaneously. In our study, intending to study acrylamide adduct levels in infertility patients from NTUH, we applied Edman degradation and UPLC-MS system as analytical procedures. Different from traditional LC-MS, UPLC-MS was invented with higher sensitivity and accuracy. Furthermore, for detecting untargeted hemoglobin (Hb) adducts, we designed a chemical isotope labeling (CIL) method together with UPLC-HRMS (Q-TOF) analysis; meanwhile, we compared the background Hb adducts in polluted blood samples to those in unpolluted blood samples. Results showed acrylamide adduct was detected in 4 of 29 infertility patients, with levels ranging from 41.17 pmol/g Hb to 117.58 pmol/g Hb; concerning untargeted adducts analysis, 79 to 105 possible adducts (fragments) were screened out. However, there was no obvious background adduct difference between polluted samples and unpolluted samples. Besides, MSMS mode was suggested for adduct fragmentation to identify real Hb adducts. In conclusion, our idea emphasized the future work of detecting acrylamide adducts or other targeted adducts via the UPLC-MS approach; on the other hand, with an application of CIL-UPLC-HRMS, scientists could rapidly screen out untargeted Hb adducts in specific groups like smokers. Working with that reliable evidence, forensic toxicologists could come to conclusions without prejudice. Keywords: Environmental pollutants, acrylamide, Hb adduct, CIL, UPLC, HRMS