Human lung type II cell derived A549 epithelial cancer cells and HepG2 hepatocytes constitutively express cytochrome P4504F2, a P450 we previously identified as a tocopherol-omega-hydroxylase. To determine if A549 cells would metabolize tocochromanols via the omega-hydroxylase pathway, we compared the metabolism of tocopherols (alpha-, gamma-, delta-TOH) and tocotrienols (alpha-, gamma-, delta-T3) in these 2 cell lines. Cultures were incubated with alpha-, gamma-, or delta-TOH, or the analogous T3s, and synthesis of their metabolites quantitated by GC-MS. A549 cells metabolized all tocochromanols 2-3 times more extensively than HepG2 cells (P < 0.001) except alpha-TOH, a difference not related to cell uptake of substrate but rather was reflective of greater microsomal TOH-omega-hydroxylase enzyme activity. Notably, 9'-carboxychromanols were the major metabolites of all gamma- and delta-TOHs and T3s in A549 cultures, whereas 3'- and 5'-carboxychromanols predominated in HepG2 cultures. Accumulation of 9'-carboxychromanols in A549 cultures was due to their inefficient conversion to 7'-carboxychromanols relative to HepG2 cells. Sesamin inhibited tocochromanol metabolism in both cells types, and neither cell type exhibited evidence of alternative (sesamin-insensitive) pathways of metabolism. TOH-omega-hydroxylase activity was undetectable in rat primary lung type II cells, suggesting that expression of activity was associated with transformation of normal type II cells to cancer cells. Long-chain carboxychromanol metabolites of gamma-TOH and other forms of vitamin E can be biosynthesized in A549 cultures for assessment of their biological activity, including their potential inhibition of synthesis of inflammatory mediators.

Tocopherols are known to undergo metabolism to phytyl chain-shortened metabolites excreted in urine. We sought to characterize the pathway, including associated enzymes, involved in this biotransformation. We previously found that human hepatoblastoma (HepG2) cultures metabolized tocopherols to their corresponding short-chain carboxychromanols. Putative metabolites of gamma-tocopherol that contained intact chromanol moieties were structurally identified using HepG2 cultures and electron impact gas chromatography-mass spectrometry. A microsomal assay for synthesis of the initial omega-oxidation metabolites was developed and used to screen several recombinant human liver cytochrome P450 isozymes for omega-hydroxylase activity. Seven metabolites of gamma-tocopherol were identified in HepG2 cultures, including 13'-hydroxy-gamma-TOH and all six carboxychromanols predicted by sequential omega-oxidation truncation. Rat and human liver microsomes catalyzed synthesis of 13'-OH- and 13'-COOH-gamma-TOH, but not other metabolites, in the presence of NADPH. Inclusion of NAD favored synthesis of the 13'-COOH metabolite. Recombinant CYP4F2, but not other major human liver CYP isoforms (including CYP3A4 and 3A7), exhibited tocopherol-omega-hydroxylase activity. Liver microsomes and recombinant CYP4F2 both exhibited substrate preference for gamma-TOH over alpha-TOH, and recent studies show that tocotrienols are catabolized more extensively than the corresponding tocopherols. Comparative rates of omega-oxidation of tocochromanols in hepatocytes are inversely related to biopotency and directly related to cytotoxicity of these substances in macrophages. The liver contains a cytochrome P450-mediated pathway that preferentially catabolizes "non-alpha" tocochromanols to excretable metabolites. This metabolic pathway appears central to the optimization of tissue tocochromanol status.

Alkylresorcinols (AR) are a class of amphiphilic phenolic lipids present in high amounts in wheat and rye bran. They have been reported to be both growth retarding and innocuous when fed to rats, and to have a broad range of bioactivities in vitro, suggested to be related to their ability to bind to proteins and modify membranes. This study was designed to test the effects of AR (purified from rye bran) on growth, tocopherol levels, and cholesterol levels in rats. Rats were fed 1 of 4 different levels of AR for 4 wk: 0 (control), 1 , 2, and 4 g/kg diet. AR did not affect final body, liver, or lung weights. The AR diets increased the levels of -tocopherol in liver and lungs (P 0.05). To investigate whether AR could have increased -tocopherol levels via inhibition of tocopherol-hydroxylase, HepG2 cells were incubated with AR and the metabolism of -tocopherol measured. AR significantly inhibited the conversion of -tocopherol to its water-soluble hydroxychroman metabolite in vitro, indicating that AR may increase -tocopherol levels via inhibition of tocopherol metabolism in vivo. The 4 g AR/kg diet decreased liver cholesterol (P 0.001), but did not affect plasma lipids. AR were detected in the perirenal adipose tissue samples of rats fed AR, indicating that they can accumulate in the fatty tissues of rats. High levels of dietary AR moderately affect -tocopherol, possibly via inhibition of tocopherol metabolism, and decrease liver cholesterol in rats. J. Nutr. 134: 506–510, 2004.

Alkylresorcinols (AR) are a class of amphiphilic phenolic lipids present in high amounts in wheat and rye bran. They have been reported to be both growth retarding and innocuous when fed to rats, and to have a broad range of bioactivities in vitro, suggested to be related to their ability to bind to proteins and modify membranes. This study was designed to test the effects of AR (purified from rye bran) on growth, tocopherol levels, and cholesterol levels in rats. Rats were fed 1 of 4 different levels of AR for 4 wk: 0 (control), 1, 2, and 4 g/kg diet. AR did not affect final body, liver, or lung weights. The AR diets increased the levels of gamma-tocopherol in liver and lungs (P < 0.05). To investigate whether AR could have increased gamma-tocopherol levels via inhibition of tocopherol-omega-hydroxylase, HepG2 cells were incubated with AR and the metabolism of gamma-tocopherol measured. AR significantly inhibited the conversion of gamma-tocopherol to its water-soluble hydroxychroman metabolite in vitro, indicating that AR may increase gamma-tocopherol levels via inhibition of tocopherol metabolism in vivo. The 4 g AR/kg diet decreased liver cholesterol (P < 0.001), but did not affect plasma lipids. AR were detected in the perirenal adipose tissue samples of rats fed AR, indicating that they can accumulate in the fatty tissues of rats. High levels of dietary AR moderately affect gamma-tocopherol, possibly via inhibition of tocopherol metabolism, and decrease liver cholesterol in rats.

Vitamin E in foodstuffs is a mixture of tocopherols. In mouse Mutatect tumors, a model designed to detect DNA mutations, the hypoxanthine phosphoribosyltransferase (Hprt) gene mutation frequency is associated with the number of tumor-infiltrating neutrophils and both are markedly decreased in mice fed high levels of alpha-tocopherol. Dietary alpha-tocopherol is also associated with a decrease in neutrophil-associated loss of an interleukin 8 (IL-8)-expressing transgene in this tumor model. We examined Hprt gene mutation frequency (expressed as the number of 6-thioguanine-resistant colonies per 10(5) clonable tumor cells), IL-8 transgene loss, and myeloperoxidase activity (an indirect measure of neutrophil number) in tumors from Mutatect mice fed diets supplemented with various concentrations of D-alpha-tocopherol acetate and/or D-gamma-tocopherol acetate or neither tocopherol for 4 weeks. Hprt gene mutation frequency and myeloperoxidase activity were statistically significantly lower in tumor cells from mice fed alpha-tocopherol at 50 or 100 mg/kg body weight per day than in tumor cells from mice fed 0 mg/kg body weight per day alpha-tocopherol (P<.001 for each comparison). IL-8 transgene loss occurred in 28 of 28 tumors (100%; 95% confidence interval [CI] = 86% to 100%) from mice fed alpha-tocopherol at 50 mg or less/kg body weight per day and seven of 18 tumors (39%; 95% CI = 24% to 54%) from mice fed 100 mg/kg body weight per day (P<.001, Fisher's exact test, referent groups [pooled] 0, 25, and 50 mg/kg). gamma-Tocopherol had no detectable effect on any of the three endpoints. Thus, dietary alpha-tocopherol decreases two forms of genetic instability in a dose-dependent manner in this experimental tumor model.

The effects of dietary (+)-catechin (CAT) and BHT on plasma and tissue concentrations of alpha-tocopherol (alpha-T), gamma-tocopherol (gamma-T) and cholesterol (C) were studied in male Sprague-Dawley rats. The rats were fed the compounds during a 4-wk period at concentrations of 2 g/kg in standardized diets, low but adequate in vitamin E, with 2 g/kg cholesterol. The CAT-regimen did not affect weight gain, feed intake or organ weights. BHT did not affect feed intake but lowered the body weight and the amount of liver lipids and increased the weights of livers and lungs relative to the body weight. Rats consuming CAT had 2.5-3.5-fold increased plasma, liver and lung alpha-T concentrations, but C concentrations remained unchanged. BHT-feeding resulted in 2.4- and 1.7-fold elevation in alpha-T but approximately 50% decrease in gamma-T concentrations in blood plasma and liver, respectively. BHT also lowered total C in the liver without affecting the concentration of C in the liver lipids. To investigate whether the alpha-T-sparing action of the studied compounds was due to the inhibition of tocopherol-omega-hydroxylase, HepG2 cells were incubated with CAT or BHT in the presence of delta-tocopherol (delta-T) and the 3'- and 5'-delta-carboxychromanol metabolites in the media were analyzed by GC/MS. Neither CAT nor BHT inhibited tocopherol-omega-hydroxylase activity in hepatocyte cultures; CAT was also inactive in a rat microsomal assay. In conclusion, both dietary CAT and BHT markedly increased alpha-T concentrations in plasma and organs of Sprague-Dawley rats by a mechanism that apparently does not involve inhibition of tocopherol-omega-hydroxylase, a key enzyme in tocopherol catabolism.

Red palm oil (RPO) contains high concentrations of beta- and alpha-carotene, and is presumed to possess a higher vitamin A value than other foods. The objective was to determine the metabolic vitamin A and carotene values of refined red palm oil in healthy adult subjects, using a stable isotope reference method. Twelve healthy subjects were administered a small standardised meal containing 10 g RPO (2.4 mg beta-carotene and 1.8 mg alpha-carotene) in a blended juice-based drink also containing 2 mg tetradeuterated retinyl acetate (d(4) -RA) as a metabolic reference. At baseline and at several times after the test meal, the concentrations of carotenes and of d(4) - and d(0) -(unlabelled) retinyl esters, in the plasma chylomicron-rich (d < 1.006) fraction were determined by high high-performance liquid chromatography and gas chromatography mass spectrometry, respectively. The masses of palm oil-derived vitamin A and carotenes absorbed ('yield') were calculated assuming 80% absorption of the d(4) -RA reference dose. The mean yield of retinol from the RPO was 0.41 mg, ranging from 0.17 mg to 0.86 mg. The mean yields of beta- and alpha-carotene were 0.29 mg and 0.25 mg, respectively, suggesting that beta-carotene was more extensively metabolised than alpha-carotene. Subjects assimilated an average of 23% of the dose of carotenes, as the sum of retinol and unmetabolised carotenes. The vitamin A values of red palm oil obtained under these conditions, a mean of 0.17 mg retinol absorbed per mg beta-carotene consumed (beta-carotene : retinol equivalency of 5.7:1) is higher than that of all other vegetable sources we have evaluated to date.

The extent to which processing affects the carotene or vitamin A value of foods is poorly understood. An extrinsic reference method was used to estimate the mass of carotenes and vitamin A derived from various preparations made from the same lot of carrots. Using a repeated-measures design, nine healthy adult subjects consumed test meals of either carrot puree (commercial baby food) or boiled-mashed carrots on separate days; six of the subjects also consumed a test meal of raw-grated carrot. Test meals supplied 34.7 micromol (18.6 mg) carrot beta-carotene (beta C), plus 6 micromol deuterium-labeled retinyl acetate (d(4)-RA) in oil solution. Baseline-adjusted carotene and retinyl ester (R-ester) area-under-curve (AUC) responses in the triacylglycerol-rich lipoprotein (TRL) fraction (0-8.5 h) were determined using HPLC and gas chromatography-mass spectrometry. The masses of absorbed beta C, alpha-carotene (alpha C) and R-ester were estimated by comparing their AUC values with that of deuterium-labeled retinyl ester (d(4)-R-ester), assuming the latter represented 80% of the d(4)-RA reference dose. Absorption of beta C and alpha C was approximately twofold greater from carrot puree than from boiled-mashed carrots, whereas the retinol yield was only marginally (P = 0.11) influenced by treatment. Carotene and R-ester absorption from raw-grated carrot was intermediate to, and did not differ significantly from the cooked preparations. The vitamin A yield (puree, 0.53 mg; boiled-mashed, 0.44 mg) of cooked carrot containing 18.6 mg beta C was substantially less than that predicted by current convention and limited primarily by intestinal carotene uptake. Processing can therefore significantly improve bioavailability of carrot carotenes, and in some cases influence the carotene value more than the intrinsic vitamin A value.

BACKGROUND:The amounts of vitamin A that are metabolically derived from specific carotene-containing foods are largely unknown.OBJECTIVE:We sought to develop an improved method for estimating the metabolic vitamin A potential of provitamin A carotenoids by using [2H4]retinyl acetate (d4-RA) as an extrinsic reference standard.DESIGN:Healthy subjects consumed a standardized test meal containing 6 mg beta-carotene as either raw carrot or spinach, either 20 or 1 g added fat, and 6.0 micromol d4-RA. Concentrations of unlabeled (d0) retinyl esters (RE), labeled (d4) RE, and carotenoids in the plasma triacylglycerol-rich lipoprotein fraction (d < 1.006 kg/L) were determined in serial blood samples with HPLC and gas chromatography-mass spectrometry. Baseline-corrected areas under the curve for d0-RE, d4-RE, and carotenoids were calculated, and the masses of absorbed d0-retinol and carotenes were estimated assuming 80% absorption of the d4-RA reference dose.RESULTS:In trials with ample (20 g) fat (n = 6), 7 +/- 4% of the 6 mg beta-carotene ingested was taken up as beta-carotene plus RE with 0.3 +/- 0.1 mg as retinol. Test meals without carotenes yielded no beta-carotene or d0-RE response and there was no effect of treatment (either fat amount or vegetable, n = 6) on the mean d4-RE area under the curve. The lower-than-expected vitamin A yields were attributed to poor intestinal uptake rather than to low conversion of beta-carotene to RE.CONCLUSION:The triacylglycerol-rich lipoprotein and d4-RA method, which controls for variation in chylomicron kinetics in vivo and RE recovery during analysis, is useful for obtaining quantitative estimates of the vitamin A potential of single meals.

Carboxychroman metabolites of the major dietary tocopherols are excreted in human urine, but the mechanism of their synthesis is unknown. We employed well-characterized inhibitors of specific cytochrome P-450 (CYP) enzymes to determine which form was likely involved in tocopherol side chain oxidation. Ketoconozole (1.0 microM), a potent and selective inhibitor of CYP3A, substantially inhibited metabolism of gamma- and alpha-tocopherol in rat primary hepatocytes, and metabolism of gamma- and delta-tocopherol in HepG2/C3A cells. Sulphaphenazole and cyclosporin, inhibitors of CYP2C and CYP27, respectively, were without effect. Sesamin, a sesame lignan that causes elevation of tissue tocopherol concentration in rats, strongly inhibited tocopherol metabolism by HepG2/C3A cells at 1.0 microM. These results support a CYP3A-dependent mechanism of side chain metabolism of tocopherols to water-soluble carboxychromans, and provide the first evidence of a specific enzyme involved in vitamin E metabolism. The data further suggest that sesamin increases tissue tocopherol concentration by inhibiting tocopherol catabolism.

HepG2 cells were incubated with a medium containing fetal bovine serum enriched with RRR-γ-tocopherol (γ-TOH). After 48 h the medium was extracted and analyzed for γ-TOH metabolites by gas chromatography-mass spectrometry. In addition to γ-CEHC, the 3′-carboxychroman metabolite of γ-TOH previously reported in human urine, these cells secreted a second substance whose extraction and mass spectral characteristics were consistent with those of the 5′-carboxychroman analog of γ-CEHC, 2,7,8-trimethyl-2-(δ-carboxymethylbutyl)-6-hydroxychroman. This is the first report of metabolism of γ-TOH to carboxychroman metabolites in cell culture. Analysis of human urine samples revealed the consistent presence of the novel 5′-carboxychroman metabolite, along with that of γ-CEHC. Oral supplementation with purified RRR-γ-TOH resulted in elevated urinary concentrations of both metabolites, although the concentration of the 5′-γ-carboxychroman metabolite was consistently and substantially less than that of γ-CEHC. The presence of both metabolites is consistent with the involvement of an ω-oxidation-like process in the phytyl tail shortening of γ-TOH to water soluble metabolites excreted in urine.

There is growing need for accurate information regarding the bioavailability of carotenoids, both with respect to carotenoidsper seand to the vitamin A value of provitamin A carotenoids in foods or supplement preparations. Little quantitative information is currently available, owing primarily to the lack of adequate methods to assess carotenoid bioavailability. Methods applied to xenobiotic drugs are in most cases not useful for carotenoids, many of which circulate in appreciable quantities in human plasma. Reported ranges of carotenoid bioavailability (% dose absorbed) range from 1–99, and variability is generally high both within and between treatments. With the current methods, relative bioavailability is more readily assessed than absolute bioavailability. The most commonly applied methods include measuring the increase in plasma carotenoid concentration following chronic intervention, and use of postprandial chylomicron (PPC) carotenoid or retinyl ester response following a single dose of carotenoid. The advantages and limitations of these approaches, together with examples of each, are discussed. A new PPC approach utilizing extrinsic-stable-isotope-labelled vitamin A (2H4-labelled retinyl acetate) is under development in our laboratory, and examples of its application are presented. The currently available data suggest that oil solutions of carotenoids are more bioavailable than those from food matrices, and heating can improve the bioavailability of carotenoids from some food products. Increased availability of labelled carotenoids and retinoids should aid the development of reliable methods of carotenoid bioavailability assessment. Such data are needed for dietary recommendations, supplement formulation, and design of intervention strategies involving carotenoids.

Little is known of the post-absorptive, metabolic fate of γ-tocopherol, the major form of vitamin E in North American diets. The objective of this study was to determine the extent of urinary excretion of 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman (γ-CEHC), a recently identified metabolite of γ-tocopherol. A method for measurement of urinary γ-CEHC was developed, using gas chromatography–mass spectrometry (GC–MS) with a deuterated internal standard, 2,7,8-trimethyl-2-(β-carboxyethyl)-(3,4-2H2)-6-hydroxychroman (d2-γ-CEHC). This standard was synthesized by dehydrogenation of 6-acetyl-γ-CEHC followed by deuteration of the resulting 3,4-double bond. The use of d2-γ-CEHC resulted in accurate determinations of the concentration of d0-γ-CEHC in human urine. Urine samples containing added d2-γ-CEHC were treated with β-glucuronidase, extracted with an organic solvent, and analyzed by GC–MS. Analysis of 24-h urine pools from healthy subjects revealed γ-CEHC concentrations, normalized against creatinine, ranging from 2.5 to 31.5 μmol/g creatinine, or a total of 4.6 to 29.8 μmol per day. These results correspond to 2–12 mg γ-tocopherol excreted daily as γ-CEHC in the urine. Given an estimated mean intake of γ-tocopherol of 20 mg/day, catabolism of γ-tocopherol to γ-CEHC, followed by glucuronide conjugation and urinary excretion, is a major pathway for elimination of γ-tocopherol in humans.—Swanson, J. E., R. N. Ben, G. W. Burton, and R. S. Parker. Urinary excretion of 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman is a major route of elimination of γ-tocopherol in humans. J. Lipid Res. 1999. 40: 665–671.

OBJECTIVES The aims of this study were to determine if ultraviolet light (UV) is immunosuppressive in healthy older males, if beta-carotene (betaC) supplementation could prevent any observed UV-induced immunosuppression, and to compare these effects with those observed previously in younger men. METHODS The study was a placebo-controlled, randomized trial that employed a 2 x 2 factorial design. Healthy older men (mean age 65.5 years) received 30 mg betaC or placebo daily throughout the 47-day trial, while on a low carotenoid diet. After 28 days, half of each group received 12 suberythemic exposures to UV over a 16-day period. Delayed-type hypersensitivity (DTH) tests and plasma carotenoid assays were performed at baseline, pre-UV and post-UV time points, with DTH testing performed on an area of skin protected from UV exposure. RESULTS UV exposure resulted in significantly suppressed DTH response in the placebo group but not in the betaC-UV group. While there was no significant interaction between betaC supplementation and UV on DTH response, there was a significant inverse relationship between final plasma betaC concentration and extent of UV-induced suppression of DTH response. A similar correlation existed among subjects not exposed to UV. CONCLUSIONS Suberythemic UV exposure was immunosuppressive, as measured by DTH response, in healthy older men as in younger men. Higher plasma betaC was significantly associated with maintenance of DTH response, although the extent of protective effect of betaC appeared less than previously observed in younger subjects. The attenuated effect of betaC in the older UV-exposed subjects may have resulted in part from muted plasma betaC responses to betaC supplementation and/or higher plasma vitamin E levels than those of younger men. The finding that stronger DTH responses were associated with higher plasma betaC concentrations in both UV and non-UV subjects further supports a role for this nutrient in immunomodulation.

We examined the relationship between body composition and changes in plasma carotenoid concentration in response to dietary carotenoid restriction or beta-carotene (beta C) supplementation in healthy older men. Subjects (mean age 65 y) were assigned randomly to supplement (30 mg beta C/d) or placebo groups, and all subjects consumed a standard low carotenoid basal diet plus 1.5 mg beta C/d as carrots. Body composition was measured at baseline by hydrodensitometry, and plasma carotenoids were measured at baseline and after 28 d of treatment by HPLC. Baseline plasma total carotenoid concentration was significantly and negatively correlated with body mass index (BMI) and fat-free mass (FFM) but not with fat mass, whereas baseline beta C concentration was negatively associated with all three variables. The increase in plasma beta C concentration in response to beta C supplementation was significantly and inversely correlated with BMI and FFM but not with fat mass. Likewise, the decline in plasma total carotenoid concentration in the placebo group was also significantly and inversely related to BMI and FFM but not to fat mass. Thus, FFM seems to be an important determinant of plasma carotenoid concentrations and to explain a substantial portion of the often-observed relationship between BMI and blood carotenoid levels. Fat-free mass seems to represent a dynamic reservoir that dampens short-term changes in plasma carotenoid concentrations during fluctuation in carotenoid intake.

Absorption and metabolism of [13C]9-cis-beta-carotene ([13C]9c beta C) was studied in three subjects after a single oral dose. Subjects given 1.0 mg [13C]beta-carotene (mean: 99.4% 9-cis-beta-carotene, 0.6% all-trans-beta-carotene; dose A) had substantial concentrations of [13C]all-trans-beta-carotene ([13C]tr beta C) and [13C]all-trans retinol ([13C]retinol) but very low concentrations of [13C]cis-beta-carotene ([13C]cis beta C) in saponified plasma 5 h after dosing, as determined by HPLC and isotope-ratio mass spectrometry. There was no evidence of appreciable absorption of [13C]9-cis retinol. To determine the proportion of [13C]tr beta C and [13C]retinol derived from [13C]9c beta C, a second set of studies in the same subjects was performed with the same isomeric composition except with 13C labeling only in all-trans-beta-carotene (dose B). The results indicated that > 95% of plasma [13C]tr beta C and [13C]retinol observed after dose A was derived from [13C]9c beta C. The concentrations of [13C]tr beta C observed, in excess of that derived from the trace amounts of [13C]tr beta C in the dose, indicated that a significant proportion of the [13C]9c beta C dose was isomerized to [13C]tr beta C before entering the bloodstream. Although precise quantitative estimates of the extent of isomerization of 9-cis-beta-carotene could not be made, it is apparent that cis-trans isomerization of 9-cis-beta-carotene to all-trans-beta-carotene contributed to the near absence of postprandial plasma 9-cis-beta-carotene after its oral administration in humans. The observation of different ratios of beta-carotene to retinol between the two dosing protocols suggests that isomerization did not occur exclusively before uptake by the intestinal mucosa. These results indicate that isomerization of ingested 9-cis-beta-carotene before its secretion into the bloodstream limits the potential supply of 9-cis retinoids to tissues, and increases the vitamin A value of 9-cis-beta-carotene.

This chapter reviews the biological relevance of β-carotene (βC) to human health. Previous approaches to model quantitative aspects of βC absorption and metabolism are also discussed. In addition, the chapter discusses the potential merits of the development of a 13 C-based stable tracer approach, and its uses for βC modeling in humans. The development and application of mathematical and compartmental kinetic models, using stable isotope tracer techniques, offers the advantages of (1) safely studying βC metabolism in humans, (2) providing predictive information regarding βC biokinetics, and (3) aiding in the evaluation of the role(s) of βC in various biological responses. Stable tracer approaches to the study of βC metabolism in humans offer several advantages over the use of unlabeled or radioactive βC. β-Carotene enriched with 13 C, coupled with the use of gas chromatography-combustion- high precision isotope ratio mass spectrometry, constitutes a safe and sensitive approach that requires only small doses of βC typical of daily dietary intake and that do not perturb endogenous pool sizes.