Liver Research Unit, Medica Sur Clinic & Foundation, Mexico City, Mexico.
Medical, Dental and Health Sciences Master and Doctorate Program, National Autonomous University of Mexico, Mexico City, Mexico.
PLoS One. 2024 Nov 19;19(11):e0313044. doi: 10.1371/journal.pone.0313044. eCollection 2024.
Unconjugated bilirubin (UCB) is a byproduct of the heme group that indicates irregularities in the metabolism of several important biological molecules, such as hemoglobin. UCB is processed by hepatic UGT1A1, which catalyzes its conjugation to the metabolites bilirubin diglucuronide (BDG) and bilirubin monoglucuronide (BMG). The serum concentrations of BDG and BMG may indicate liver injury or dysfunction. The aim of this study was to standardize and validate a method for the identification and simultaneous quantification of BMG, BDG and UCB by LC‒MS/MS.
Liquid‒liquid extraction allows the separation of UCB, BMG and BDG from the serum of healthy subjects or patients with liver injury. Detection and quantification were performed using an LC‒MS/MS method. Compound separation was achieved with a BEH-C18 column at 40°C. The mobile phase was prepared with 5 mM ammonium acetate (pH 6) and acetonitrile, and a flow gradient was applied.
This is the first study to directly quantify BMG and UCB levels in human serum; no postcalculations or correction factors are needed. However, BDG quantification requires calculations and a correction factor. We identified the molecular species with ionic transitions m/z1+ 585.4 > 299.2 for UCB, 761.3 > 475.3 for BMG, 937.3 > 299.5 for BDG and mesobilirubin 589.4 > 301.3 (IS).
The procedures used in this study allowed the simultaneous identification and quantification of the molecular species of bilirubin, BDG, BMG and UCB. Analysis of the serum levels in patients with hyperbilirubinemia revealed that patients with acute-on-chronic liver failure had elevated levels of these species.
未结合胆红素（UCB）是血红素代谢产物的一种副产物，提示血红蛋白等多种重要生物分子的代谢异常。UCB 由肝 UGT1A1 处理，该酶催化其与胆红素二葡萄糖醛酸（BDG）和胆红素单葡萄糖醛酸（BMG）代谢物结合。BDG 和 BMG 的血清浓度可能表明肝脏损伤或功能障碍。本研究旨在建立并验证一种通过 LC-MS/MS 同时鉴定和定量检测 BMG、BDG 和 UCB 的方法。
液液萃取法可将 UCB、BMG 和 BDG 从健康受试者或肝损伤患者的血清中分离出来。采用 LC-MS/MS 方法进行检测和定量。化合物分离在 40°C 下使用 BEH-C18 柱进行。流动相由 5 mM 乙酸铵（pH 6）和乙腈组成，并应用梯度洗脱。
这是首次直接定量检测人血清中 BMG 和 UCB 水平的研究，无需进行后计算或校正因子。但 BDG 的定量需要计算和校正因子。我们鉴定出 UCB 的分子离子转移 m/z1+585.4 > 299.2，BMG 为 761.3 > 475.3，BDG 为 937.3 > 299.5，间胆素为 589.4 > 301.3（IS）。
本研究中采用的程序允许同时鉴定和定量检测胆红素、BDG、BMG 和 UCB 的分子种类。对高胆红素血症患者的血清水平分析显示，急性肝衰竭伴慢性肝病患者这些物质的水平升高。
