Abstract
Retinol (vitamin A) circulates at 1-4 μM concentration and is easily measured in serum. However, retinol is biologically inactive. Its metabolite, retinoic acid (RA), is believed to be responsible for biological effects of vitamin A, and hence the measurement of retinol concentrations is of limited value. A UHPLC-MS/MS method using isotope-labeled internal standards was developed and validated for quantitative analysis of endogenous RA isomers and metabolites. The method was used to measure retinoids in serum samples from 20 healthy men. In the fed state, the measured concentrations were 3.1 ± 0.2 nM for atRA, 0.1 ± 0.02 nM for 9-cisRA, 5.3 ± 1.3 nM for 13-cisRA, 0.4 ± 0.4 nM for 9,13-dicisRA, and 17.2 ± 6.8 nM for 4oxo-13-cisRA. The concentrations of the retinoids were not significantly different when measured after an overnight fast (3.0 ± 0.1 nM for atRA, 0.09 ± 0.01 nM for 9-cisRA, 3.9 ± 0.2 nM for 13-cisRA, 0.3 ± 0.1 nM for 9,13-dicisRA, and 11.9 ± 1.6 nM for 4oxo-13-cisRA). 11-cisRA and 4OH-RA were not detected in human serum. The high sensitivity of the MS/MS method combined with the UHPLC separation power allowed detection of endogenous 9-cisRA and 4oxo-atRA for the first time in human serum.
Highlights
Retinol circulates at 1–4 M concentration and is measured in serum
Acetonitrile, methanol, water, and formic acid used in the Ultrahigh-performance liquid chromatography (UHPLC)-MS/MS method were from Fischer Scientific (Pittsburg, PA), and all were Optima LC/MS grade
The fragmentation and specific MS/MS ion transitions (SRM transition) for each retinoic acid (RA) isomer, for the isotope-labeled internal standards, and for the metabolites were optimized for maximum sensitivity
Summary
Retinol (vitamin A) circulates at 1–4 M concentration and is measured in serum. Retinoic acid (RA), is believed to be responsible for biological effects of vitamin A, and the measurement of retinol concentrations is of limited value. The concentrations of the retinoids were not significantly different when measured after an overnight fast (3.0 ± 0.1 nM for atRA, 0.09 ± 0.01nM for 9-cisRA, 3.9 ± 0.2 nM for 13-cisRA, 0.3 ± 0.1 nM for 9,13-dicisRA, and 11.9 ± 1.6 nM for 4oxo-13cisRA). The high sensitivity of the MS/MS method combined with the UHPLC separation power allowed detection of endogenous 9-cisRA and 4oxo-atRA for the first time in human serum.—Arnold, S. A sensitive and specific method for measurement of multiple retinoids in human serum with UHPLC-MS/MS. Retinol (vitamin A) is the main circulating retinoid, but it is biologically inactive and is metabolized in the body to active retinoic acid (RA). 9-cisRA binds to RXR with a significantly higher affinity than atRA, and 4oxo-atRA and 4OH-atRA have been shown to bind to RAR with similar affinity as atRA [10, 12, 15]
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