ARS | Publication request: An HPLC-MS method for d8-ß-carotene and d4-retinyl esters useful in studies of ß-carotene absorption and its conversion to vitamin A in humans

Submitted to: Journal of Lipid Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 3, 2012
Publication Date: February 3, 2012
Citation: Fleshman, M.K., Riedl, K., Novotny Dura, J., Schwartz, S.J., Harrison, E.H. 2012. An HPLC-MS method for d8-ß-carotene and d4-retinyl esters useful in studies of ß-carotene absorption and its conversion to vitamin A in humans. Journal of Lipid Research. 53:820-827.

Interpretive Summary: Intestinal absorption and metabolism of ß-carotene is of vital importance in humans, especially in populations that obtain the majority of their vitamin A from pro-vitamin A carotenoids. Five million children under the age of 5 years suffer from clinical eye disorders related to vitamin A deficiency. More than two hundred thirty million children have increased rates of infection related to inadequate vitamin A intake. Beta-carotene is a plant-based compound that forms vitamin A in the body and is an important target for dealing with the worldwide vitamin A problem, but the conversion of beta-carotene to vitamin A is complex and requires continuing developments in lab methodology to give increasingly accurate values for the vitamin A value of beta-carotene. Here we have developed a method for more completely analyzing the conversion of beta-carotene to vitamin A. During absorption, beta-carotene is converted to several forms of vitamin A, but previous methods could not distinguish between those different forms. We have elaborated the method to now distinguish among the different forms of vitamin A that appear in the blood during absorption. This new method will provide an opportunity for a more complete understanding of the conversion of beta-carotene to vitamin A. These results will be used by scientists studying the conversion of beta-carotene to vitamin A.

Technical Abstract: The intestinal absorption and metabolism of beta-carotene is of vital importance in humans, especially in populations that obtain the majority of their vitamin A from pro-vitamin A carotenoids. Mass spectrometry has provided a better understanding of the absorption of beta-carotene, the most potent pro-vitamin A carotenoid, through the use of stable isotopes of beta-carotene. We report here an HPLC/MS method that eliminates the need for complicated sample preparation and allows us to detect and quantify newly absorbed d8-beta-carotene as well as its d4-retinyl ester metabolites in human plasma and chylomicron fractions. Both retinoids and beta-carotene were recovered in a single, simple, extraction that did not involve saponification, thus allowing subsequent quantitation of individual fatty acyl esters of retinol. Separation of d8-beta-carotene and its d4-retinyl ester metabolites were achieved using the same C30 reversed phase liquid chromatography followed by mass spectrometry in selected ion reaction (SIR) and negative atmospheric pressure chemical ionization modes, respectively. Total time for the two successive runs was 30 minutes. This HPLC/MS method allowed us to quantify the absorption of intact d8-beta-carotene as well as its extent of conversion to d4-retinyl esters in humans after consumption of a single 5mg dose of d8-beta-carotene.