The authors are grateful for the comments of Dr. Achim
Treumann (Proteomics and Biological Mass Spectrometry
Facility, Newcastle University) during manuscript preparation.
esters or -carotene absorbed (37). Thus, the current
method can detect intestinally-derived retinyl esters with
more accuracy compared with methods employing TRL
separations (27, 37, 38).
The current method also allows -carotene bioefficacy
and vitamin A dilution to be studied concurrently due to
differential extrinsic [13C] labeling of administered com-
pounds. [13C] isotopes were selected because deuterated
compounds are subject to hydrogen-deuterium exchange
and possess different physicochemical characteristics re-
sulting in altered LC retention times and solvent extraction
efficiencies (2, 11, 28). Position of [13C10] labels around
the centric 15,15′ double bond on the -carotene mole-
cule allowed BCMO1 [13C5] cleavage products to be distin-
guished from [13C10] metabolites of [13C10]retinyl acetate.
Although both [13C10] and [13C5] metabolites displayed
similar plasma kinetic profiles, concentrations of [13C5]
retinol and retinyl esters were 3- to 4-fold lower even
though twice the dose of [13C10]-carotene was adminis-
tered. It is known that intestinal absorption of synthetic
-carotene is limited although bioavailability is distinctly
enhanced when dissolved in oil (39). Regarding retinyl es-
ters, both [13C10] and [13C5]retinol were preferentially es-
terified to palmitate and oleate. However, subsequent
specificities of [13C5]retinol for linoleate and [13C10]retinol
for stearate were observed, which suggests differences in
subcellular compartmentalization between preformed
retinol and retinol from provitamin A sources in the en-
terocyte before incorporation in chylomicrons.
Retinyl acetate was coadministered with -carotene as
a reference dose to correct for inter- and intra-individual
variations in intestinal absorption and chylomicron clear-
ance rates (37). The [13C10]retinyl acetate dose can also be
used to determine total body vitamin A reserves after a suf-
ficient period (circa 3 days) of isotope dilution with en-
dogenouspools(1).Insomepreviousstudies,thereference
dose was not administered concomitantly with -carotene
to avoid competition during intestinal absorption (12, 14).
Single doses of -carotene have ranged from 5 to 126 mg
due to analytical detection limits dictating the minimum
dose that can be administered to human subjects. How-
ever, -carotene bioefficacy is dose-dependent when
>4 mg is ingested (40), while doses >6 mg perturb the
steady-state equilibrium in the blood (41). The 2 mg utilized
in the current study represents a true physiological dose
according to the estimated daily intake of -carotene in
UK and US populations (39). Although lower doses have
been administered daily over a prolonged period to reach
a plateau of isotopic enrichment in the blood (15, 16),
multiple dosing cannot establish uptake kinetics.
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In summary, this new sensitive analytical method allows
for the simultaneous study of -carotene bioefficacy and
vitamin A status in human subjects at physiological doses
for at least 2 weeks. The simple extraction procedure and
single 7 min LC/MS run-time for all analytes makes the
method applicable to the high throughput of samples gen-
erated in large human intervention studies.
LC/MS/MS of [13C]-carotene and [13C]-vitamin A
327