Human Nutrition Research Centre, Newcastle University, Newcastle Upon Tyne, UK.
J Lipid Res. 2014 Feb;55(2):319-28. doi: 10.1194/jlr.D040204. Epub 2013 Oct 24.
Isotope dilution is currently the most accurate technique in humans to determine vitamin A status and bioavailability/bioconversion of provitamin A carotenoids such as β-carotene. However, limits of MS detection, coupled with extensive isolation procedures, have hindered investigations of physiologically-relevant doses of stable isotopes in large intervention trials. Here, a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analytical method was developed to study the plasma response from coadministered oral doses of 2 mg [(13)C10]β-carotene and 1 mg [(13)C10]retinyl acetate in human subjects over a 2 week period. A reverse phase C18 column and binary mobile phase solvent system separated β-carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Selected reaction monitoring of analytes was performed under atmospheric pressure chemical ionization in positive mode at m/z 537→321 and m/z 269→93 for respective [(12)C]β-carotene and [(12)C] retinoids; m/z 547→330 and m/z 274→98 for [(13)C10]β-carotene and [(13)C5] cleavage products; and m/z 279→100 for metabolites of [(13)C10]retinyl acetate. A single one-phase solvent extraction, with no saponification or purification steps, left retinyl esters intact for determination of intestinally-derived retinol in chylomicrons versus retinol from the liver bound to retinol binding protein. Coadministration of [(13)C10]retinyl acetate with [(13)C10]β-carotene not only acts as a reference dose for inter-individual variations in absorption and chylomicron clearance rates, but also allows for simultaneous determination of an individual's vitamin A status.
同位素稀释法是目前测定人体维生素 A 状态和视黄醇前体类胡萝卜素（如β-胡萝卜素）生物利用度/生物转化率最准确的技术。然而，由于 MS 检测的局限性以及广泛的分离程序，限制了在大型干预试验中对稳定同位素生理相关剂量的研究。在此，开发了一种灵敏的液相色谱-串联质谱（LC/MS/MS）分析方法，用于研究人体在 2 周时间内同时口服 2mg[（13）C10]β-胡萝卜素和 1mg[（13）C10]视黄醇乙酸酯后血浆中的反应。反相 C18 柱和二元流动相溶剂系统在 7 分钟的运行时间内分离了β-胡萝卜素、视黄醇、视黄醇乙酸酯、视黄醇亚油酸酯、视黄醇棕榈酸酯/视黄醇油酸酯和视黄醇硬脂酸酯。分析物的选择反应监测在大气压化学电离正模式下进行，m/z537→321 和 m/z269→93 分别用于各自的[（12）C]β-胡萝卜素和[（12）C]视黄醇；m/z547→330 和 m/z274→98 用于[（13）C10]β-胡萝卜素和[（13）C5]裂解产物；m/z279→100 用于[（13）C10]视黄醇乙酸酯的代谢物。单相溶剂萃取，无需皂化或纯化步骤，使视黄醇酯保持完整，用于测定乳糜微粒中肠源视黄醇与与视黄醇结合蛋白结合的肝脏视黄醇的比值。[（13）C10]视黄醇乙酸酯与[（13）C10]β-胡萝卜素同时给药不仅可以作为吸收和乳糜微粒清除率个体间差异的参考剂量，还可以同时确定个体的维生素 A 状态。
