Abstract
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.
Highlights
N [13C10]-carotene subjects over a 2 week period
atmospheric pressure chemical ionization (APCI) in positive mode offered greater linear dynamic range for both -carotene and retinoids compared with electrospray ionization (ESI)
Even if other MS methods such as gas chromatography/combustion/isotope-ratio MS and electron capture negative chemical ionization MS allow effective use of physiological doses of retinol [24, 25] and -carotene [26] tracers, these methods have the disadvantage of requiring extensive sample preparation, Fig. 4
Summary
N [13C10]-carotene subjects over a 2 week period. A reverse phase C18 column and binary mobile phase solvent system separated -carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Coadministration [13C10]-carotene acts of as [13C10] a reference dose for inter-individual variations in absorption and chylomicron clearance rates, and allows for simultaneous determination of an individual’s vitamin A status.—. Increasing the intake of provitamin A carotenoids, primarily through -carotene, is seen as a safe way of restoring the vitamin A reserves of an individual because high doses of preformed vitamin A have adverse health effects [3]. The current vitamin A equivalency ratio for -carotene is estimated at 12:1 (by weight) [4], large inter-individual variations in both absorption and conversion have been observed [5,6,7,8]. Retinyl esters are either stored in hepatic stellate cells or hydrolyzed back to retinol by the liver for repartition to other tissue compartments bound to retinol binding protein (RBP)
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