Proteomics and Metabolomics Shared Resource has developed a platform to quantify a number of known hydroxycholesterol isomers. Hydroxycholesterols are metabolites of cholesterol that exist at approximately three orders of magnitude lower concentration than the precursor, and exhibit signaling properties as diverse as immune response regulation (25-hydroxycholesterol) and estrogen receptor signaling (27-hydroxycholesterol).

level of intensity of hydroxycholesterols over time.

The method uses base hydrolysis to measure the total of free and esterified hydroxycholesterol of each isoform and has been specifically designed to perform chromatographic resolution of individual positional isomers, as demonstrated in the panel above. The quantification range used for plasma samples is 0.1 uM to 10 uM. Analysis is performed using Acquity UPLC and a Xevo TQ-S tandem mass spectrometer. The quantitative method utilizes LipidMaps stable-isotope internal standards for 22R, 22S, 24R/S, 27, 4b, 7a/b-hydroxycholesterols and cholesterol, and quantification is performed using quantitative standards in a bovine serum albumin matrix. The Skyline quantitative metabolomics package performs data analysis (example shown below).

Hydroxycholesterols analyzed in Skyline showing retention times, peak areas, calibration curves

The hydroxycholesterol panel has been specifically designed to measure biofluids, but different sample matrices can likely be accommodated based on discussions with Proteomics and Metabolomics Shared Resource. If you have questions about hydroxycholesterol quantification in our laboratory, please contact Will Thompson or Laura Dubois.