用于人血浆中 4β-和 4α-羟基胆固醇的灵敏 UHPLC-MS/MS 定量方法，用于准确的 CYP3A 表型分析。,Journal of Lipid Research

当前位置： X-MOL 学术 › J. Lipid Res. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

用于人血浆中 4β-和 4α-羟基胆固醇的灵敏 UHPLC-MS/MS 定量方法，用于准确的 CYP3A 表型分析。
Journal of Lipid Research ( IF 5.0 ) Pub Date : 2022-02-15 , DOI: 10.1016/j.jlr.2022.100184
Yosuke Suzuki ₁ , Ayako Oda ₁ , Jun Negami ₁ , Daiki Toyama ₁ , Ryota Tanaka ₂ , Hiroyuki Ono ₂ , Tadasuke Ando ₃ , Toshitaka Shin ₃ , Hiromitsu Mimata ₃ , Hiroki Itoh ₂ , Keiko Ohno ₁

Affiliation

4β-羟基胆固醇 (4β-OHC) 由 CYP3A4 和 CYP3A5 形成，作为 CYP3A 活性的内源性表型探针引起了人们的关注。然而，4β-OHC 也会因体外由于储存失调而发生的胆固醇自氧化而增加，而在体内则因氧化应激或炎症而增加，与 CYP3A 活性无关。4α-羟基胆固醇 (4α-OHC) 是 4β-OHC 的立体异构体，也是通过胆固醇的自氧化而非 CYP3A 形成的，因此可能具有反映胆固醇自氧化状态的临床潜力。在本研究中，我们建立了一种使用超高效液相色谱与串联质谱联用 (UHPLC-MS/MS) 同时定量人血浆中 4β-OHC 和 4α-OHC 的灵敏方法。血浆样品通过皂化、两步液液萃取、使用吡啶甲酸进行衍生化。使用电喷雾电离监测 4β-OHC 和 4α-OHC 二吡啶甲酸酯的强烈 [M+H]+ 信号。该测定满足美国食品和药物管理局对生物分析方法验证指南的要求，4β-OHC 和 4α-OHC 的定量下限均为 0.5 ng/mL。4β-OHC 的人血浆表观回收率为 88.2% 至 101.5%，4α-OHC 为 91.8% 至 114.9%。此外，4β-OHC 的基质效应在 86.2% 和 117.6% 之间变化，4α-OHC 的基质效应在 89.5% 和 116.9% 之间变化。经验证的测定法测量的健康志愿者、3-5 期慢性肾病 (CKD) 患者和 5D 期 CKD 患者的血浆 4β-OHC 和 4α-OHC 浓度在所有样品的校准范围内。

"点击查看英文标题和摘要"

Sensitive UHPLC-MS/MS quantification method for 4β- and 4α-hydroxycholesterol in human plasma for accurate CYP3A phenotyping.

4β-Hydroxycholesterol (4β-OHC) is formed by CYP3A4 and CYP3A5 and has drawn attention as an endogenous phenotyping probe for CYP3A activity. However, 4β-OHC is also increased by cholesterol autooxidation occurring in vitro due to dysregulated storage and in vivo by oxidative stress or inflammation, independent of CYP3A activity. 4α-hydroxycholesterol (4α-OHC), a stereoisomer of 4β-OHC, is also formed via autooxidation of cholesterol, not by CYP3A, and thus may have clinical potential in reflecting the state of cholesterol autooxidation. In this study, we establish a sensitive method for simultaneous quantification of 4β-OHC and 4α-OHC in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Plasma samples were prepared by saponification, two-step liquid-liquid extraction, and derivatization using picolinic acid. Intense [M+H]+ signals for 4β-OHC and 4α-OHC di-picolinyl esters were monitored using electrospray ionization. The assay fulfilled the requirements of the US Food and Drug Administration guidance for bioanalytical method validation, with a lower limit of quantification of 0.5 ng/mL for both 4β-OHC and 4α-OHC. Apparent recovery rates from human plasma ranged from 88.2% to 101.5% for 4β-OHC, and 91.8% to 114.9% for 4α-OHC. Additionally, matrix effects varied between 86.2% and 117.6% for 4β-OHC, and between 89.5% and 116.9% for 4α-OHC. Plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers, stage 3-5 chronic kidney disease (CKD) patients, and stage 5D CKD patients as measured by the validated assay were within the calibration ranges in all samples. We propose this novel quantification method may contribute to accurate evaluation of in vivo CYP3A activity.

更新日期：2022-02-15

点击分享查看原文

点击收藏

公开下载

阅读更多本刊新发论文本刊介绍/投稿指南