Method for the Simultaneous Determination of Retinol and β-Carotene Concentrations in Human Tissues and Plasma

Abstract

To understand differential tissue distribution of retinoids and carotenoids, as it might influence biological processes in humans, we developed and demonstrated a method for measuring them in selected human tissues. The method includes internal standards and a secondary reference standard to eliminate the need for external standard calibration and to minimize sample-handling errors. Tissues were digested (saponified) in ethanolic KOH. Retinol and β-carotene were extracted with organic solvent containing internal standards. Analytes were separated using isocratic liquid chromatography and quantified at 325 nm for retinol and 450 nm for β-carotene. Plasma was analyzed in a similar way but without saponification. Retinal-O-ethyloxime and β-apo-12′-carotenal-O-t-butyloxime served as internal standards. Plasma, breast, and fat from breast surgery patients and colon, liver, muscle, and fat from colon surgery patients were analyzed. Within-day relative standard deviations (RSDs) for plasma were <0.04 for β-carotene and <0.03 for retinol, between-day RSDs were <0.05 for β-carotene and <0.04 for retinol. Saponification ensured complete extraction of retinol and β-carotene and removal of triglycerides that “foul” chromatographic columns. It seems retinol and β-carotene concentrations in tissues and blood of cancer patients are the same or higher than those in corresponding tissues of patients without these cancers.