Relative contribution of α-carotene to postprandial vitamin A concentrations in healthy humans after carrot consumption

Abstract

Background: Asymmetric α-carotene, a provitamin A carotenoid, is cleaved to produce retinol (vitamin A) and α-retinol (with negligible vitamin A activity). The vitamin A activity of α-carotene–containing foods is likely overestimated because traditional analytic methods do not separate α-retinol derivatives from active retinol.

Objective: This study aimed to accurately characterize intestinal α-carotene cleavage and its relative contribution to postprandial vitamin A in humans after consumption of raw carrots.

Design: Healthy adults (n = 12) consumed a meal containing 300 g raw carrot (providing 27.3 mg β-carotene and 18.7 mg α-carotene). Triglyceride-rich lipoprotein fractions of plasma were isolated and extracted, and α-retinyl palmitate (αRP) and retinyl palmitate were measured over 12 h postprandially via high-performance liquid chromatography–tandem mass spectrometry. The complete profile of all α-retinyl esters and retinyl esters was measured at 6 h, and total absorption of α- and β-carotene was calculated.

Results: αRP was identified and quantified in every subject. No difference in preference for absorption of β- over α-carotene was observed (adjusting for dose, 28% higher, P = 0.103). After absorption, β-carotene trended toward preferential cleavage compared with α-carotene (22% higher, P = 0.084). A large range of provitamin A carotenoid conversion efficiencies was observed, with α-carotene contributing 12–35% of newly converted vitamin A (predicted contribution = 25.5%). In all subjects, a majority of α-retinol was esterified to palmitic acid (as compared with other fatty acids).

Conclusions: α-Retinol is esterified in the enterocyte and transported in the blood analogous to retinol. The percentage of absorption of α-carotene from raw carrots was not significantly different from β-carotene when adjusting for dose, although a trend toward higher cleavage of β-carotene was observed. The results demonstrate large interindividual variability in α-carotene conversion. The contribution of newly absorbed α-carotene to postprandial vitamin A should not be estimated but should be measured directly to accurately assess the vitamin A capacity of α-carotene–containing foods. This trial was registered at clinicaltrials.gov as NCT01432210.

INTRODUCTION

Provitamin A carotenoids account for ∼70% of the vitamin A intake in the developing world (1, 2), where vitamin A deficiency remains a serious problem (3). A portion of newly absorbed provitamin A carotenoids is enzymatically cleaved at the central double bond in the small intestine by β-carotene dioxygenase 1 (BCO1) (Figure 1) (4–6). Cleavage of symmetrical β-carotene (containing 2 unsubstituted β-ionone rings) yields 2 molecules of retinal (i.e., vitamin A) (4), whereas cleavage of nonsymmetrical provitamin A carotenoids (e.g., α-carotene, β-cryptoxanthin, and γ-carotene, each containing only 1 β-ionone ring) results in 1 vitamin A and 1 non–vitamin A cleavage product. For α-carotene, this non–vitamin A cleavage product is α-retinal. Retinal is subsequently reduced to retinol and esterified to retinyl esters before they are packaged into chylomicrons, which are released into the bloodstream and sequestered by the liver (5).

FIGURE 1.

A fraction of the β- and α-carotene absorbed by the enterocyte is enzymatically cleaved to retinal and α-retinal (in the case of α-carotene). These products are then enzymatically reduced and esterified to fatty acids before being packaged into chylomicrons. BCO1, β-carotene oxygenase 1; LRAT, lecithin retinol acyltransferase.

α-Carotene is found in carrots, plantains, pumpkins, squash, red palm oil, and tangerines (7, 8). In animal models, α-retinyl acetate is absorbed (presumably as the cleaved form, α-retinol) and esterified to fatty acids in the enterocyte before transport to the liver, analogous to retinol (9–11). Although a 2014 report of a nonconventional approach to the rat growth assay suggested otherwise (12), α-retinol is reported to have ∼2% of the activity of vitamin A (13–15) and does not bind to retinol binding protein (16). The structural similarity of α-retinyl esters to retinyl esters causes co-elution with most HPLC methods (11, 14, 15). Although studies have assessed the absorption and conversion of α- and β-carotene to vitamin A in animals (9, 10, 17, 18) and humans (19, 20), vitamin A equivalence has likely been overestimated because of the contribution of α-retinyl esters to the newly converted retinyl esters. We developed a method to separate α-retinyl esters from retinyl esters for a more accurate quantitation of vitamin A derived from a meal containing α-carotene (21). The theoretical provitamin A potential of α-carotene should be exactly one-half that of β-carotene (Figure 1), as reflected in a 2001 Institute of Medicine report (22). However, studies show that the affinity (Michaelis constant) of various provitamin A carotenoids for BCO1 is not equal (6, 23, 24).

Here, we aimed to accurately assess the vitamin A derived from a carrot meal (containing both α- and β-carotene) postprandially in healthy humans. α-Retinyl palmitate (αRP) and retinyl palmitate (RP) were quantitated in the triglyceride-rich lipoprotein (TRL) fraction of plasma over 12 h after the meal. In addition, the full profile of all α-retinyl and retinyl esters was characterized at 6 h to observe esterification patterns of both α-retinol and retinol to various fatty acids. From these esterification patterns, total α-retinyl esters and retinyl esters formed were calculated, and total absorption of β- and α-carotene (including the cleaved proportion) was determined. The relative contribution of α-carotene to the newly converted vitamin A plasma pool was also assessed.

METHODS

Chemicals

RP was purchased from Sigma-Aldrich. Hexane, HPLC-grade methyl tert-butyl ether and methanol (MeOH), and Optima-grade water and MeOH were purchased from Fisher Scientific. Ammonium acetate was purchased from JT Baker. Retinyl myristate, linoleate, oleate, and stearate were synthesized as described previously (25). αRP was also synthesized as previously described (21).

Clinical subjects and experimental design

Twelve healthy subjects (6 men and 6 women) were recruited for a previously conducted study, as described (26). Subjects were aged 27.8 ± 4.3 y and had a BMI (in kg/m²) of 24.2 ± 2.7, total cholesterol of 168.9 ± 23.5 mg/dL, and triglycerides of 73.8 ± 64.2 mg/dL. Subjects abstained from consuming provitamin A carotenoids and preformed vitamin A for 2 wk before their day-long clinic visit. Subjects arrived at the clinic after an overnight fast, and a 0-h blood sample was drawn. Subjects then consumed a carrot-containing meal providing 27.3 mg β-carotene, 18.7 mg α-carotene, and 665 calories in total (18 g protein, 92 g carbohydrate, and 23 g lipid). Subjects consumed the meal within 30 min, ate a carotenoid-free lunch at 4.5 h, and were allowed to consume water ad libitum throughout the day. Postprandial blood samples were taken at 2, 3, 4, 5, 6, 8, 10, and 12 h. TRL fractions were isolated immediately from fresh blood plasma and samples were frozen at −80°C until extraction (27). The study was approved by the Institutional Review Board of The Ohio State University (protocol 2011H0159).

TRL extraction

Frozen samples were thawed in cold water and extracted as described previously (27). The dried extract was stored at −80°C for ≤5 d before analysis. Extracts were redissolved in 100 μL methyl tert-butyl ether and sonicated for 10 s, and 100 μL MeOH was added. Extracts were centrifuged at 21,130 × g for 2 min (model 5424; Eppendorf) and the supernatant was removed and subjected to HPLC–photodiode array–tandem mass spectrometry analysis.

HPLC–tandem mass spectrometry analysis of TRL extracts

The conditions developed to separate, identify, and quantify α-retinyl esters and retinyl esters were published previously (21). αRP and RP were quantified in all samples at all time points through the use of external standard curves. The complete profile of all α-retinyl esters and retinyl esters was also measured at 6 h.

Total α-retinyl esters and retinyl esters

The baseline-corrected trapezoidal approximation method was used to calculate the area under the time versus concentration curve (AUC) for αRP and RP. The AUC_RP and AUC_αRP values were then converted to represent total esters (sum of RP and αRP, myristate, linoleate, oleate, and stearate, respectively) based on the relative proportion of palmitate to other esters measured within each subject at 6 h. The esterification profile of 2 subjects at each time point was investigated and did not change over this period; thus, we assumed that the esterification profile measured for any given subject at 6 h is representative for each subject. These values are termed AUC_{retinyl esters} and AUC_{α-retinyl esters}.

Total β-carotene absorbed

Previously determined AUC values of α- and β-carotene in the same subjects from the same meal (26) were used to determine total absorption and total conversion efficiencies of α- and β-carotene. For these calculations, 100% stoichiometric conversion and esterification of the α- and β-carotene metabolites was assumed.

Total β-carotene absorbed encompasses both intact and newly cleaved β-carotene. To calculate this value, we first removed the contribution of α-carotene to retinyl esters. Because the cleavage of 1 molecule of α-carotene produces 1 α-retinyl ester and 1 retinyl ester, we calculated this contribution by subtracting the concentration of α-retinyl esters from total retinyl esters as follows:

X represents the retinyl esters derived solely from the cleavage of newly absorbed of β-carotene. Because stoichiometric cleavage of β-carotene produces 2 retinyl ester equivalents, we divided the remaining retinyl esters by a factor of 2 in our final absorption calculation:

Total α-carotene absorbed

The calculation for α-carotene is straightforward; we added the quantity of cleaved α-carotene to the previously measured AUC_α-carotene as follows:

β- and α-Carotene cleavage efficiency

β-Carotene cleavage efficiency was calculated as follows:

α-Carotene cleavage efficiency was calculated as follows:

The percentage of α-carotene’s contribution to the newly converted vitamin A pool was then calculated as follows:

Statistical analysis

R software (version 3.1.0) was used to perform all statistical analysis. Raw data for the maximum concentration, AUC_αRP, AUC_RP, AUC_{retinyl esters}, and AUC_{α-retinyl esters} were right skewed; thus, medians and 25% and 75% percentiles are also provided in Table 1. To determine whether there were differences in the esterification pattern of α-retinyl and retinyl esters, the profile of α-retinyl and retinyl linoleate, oleate, palmitate, and stearate within each subject at 6 h was evaluated through the use of a paired Wilcoxon’s Rank Sum test. The ratio of total β-carotene absorbed relative to total α-carotene absorbed was calculated for each subject, and this data set met the assumptions of equal variance and normality. A 2-tailed Student’s t test was performed to determine whether the ratio matched the proportion of β-carotene to α-carotene in the test meal (i.e., ratio = 1.46). Likewise, the ratio of the percentage of β-carotene conversion relative to the percentage of α-carotene conversion was calculated and met the assumptions of equal variance and normality. A 2-tailed Student’s t test was performed to determine whether the ratio matched the predicted value of 1 (i.e., equal percentages of cleaved β- and α-carotene relative to absorbed dose). P < 0.05 was considered statistically significant.

TABLE 1.

Maximum concentration, time of maximal concentration, AUC of αRP, RP, total α-retinyl esters, total retinyl esters, and ratio of retinyl esters to α-retinyl esters 12 h postprandially after consumption of a carrot-containing meal in 12 subjects¹

	Cmax, nmol/L plasma		Tmax, h		AUC, nmol · h/L
Subject	αRP	RP	αRP	RP	αRP	RP	α-Retinyl esters	Retinyl esters	Retinyl esters:α-retinyl esters ratio
1	52.6	176.4	6	6	292.8	879.6	485.4	1373.9	2.83
2	83.2	316.3	4	4	381.7	1331.9	656.0	1981.6	3.02
3	8.2	58.6	5	5	42.2	308.9	64.7	457.6	7.07
4	11.8	91.2	5	5	57.2	360.8	91.9	532.2	5.79
5	2.3	14.8	5	5	9.1	82.4	14.6	129.4	8.87
6	7.6	43.9	5	5	38.2	252.0	59.8	357.9	5.98
7	4.4	26.8	3	3	24.5	103.6	38.7	157.2	4.06
8	3.8	15.5	6	6	21.7	90.7	38.8	152.3	3.93
9	3.6	31.7	5	5	18.7	176.6	28.4	242.5	8.54
10	3.1	18.8	5	5	16.4	129.1	28.4	213.9	7.54
11	6.0	27.8	6	6	33.0	150.6	58.0	240.4	4.15
12	4.9	31.9	6	6	20.1	136.8	33.7	208.8	6.19
Mean ± SEM	16.0 ± 7.3	71.2 ± 25.9	5.1 ± 0.3	5.1 ± 0.3	79.6 ± 35.4	333.6 ± 110.7	133.2 ± 60.2	504.0 ± 166.5	5.7 ± 0.6
Median (25th, 75th percentiles)	5.4(3.8, 9.1)	31.8(24.8, 66.7)2	5.0(5.0, 6.0)	5.0(5.0, 6.0)	28.7(19.8, 45.9)	163.6(122.8, 321.9)	48.4(32.4, 71.5)	241.5(195.9, 476.2)	5.9(4.0, 7.2)

¹Because of right skewness, medians and 25th and 75th percentiles are also provided. C_max, maximum concentration; RP, retinyl palmitate; T_max, time of maximal concentration; αRP, α-retinyl palmitate.

²Median C_max was significantly different for RP than for αRP (Wilcoxon’s Signed Rank test, P = 0.007), whereas no significant difference in T_max was observed.

RESULTS

Concentrations of αRP and RP in plasma

The mean ± SEM baseline-corrected plasma TRL concentrations of αRP and RP in 12 subjects are presented in Figure 2. With the exception of αRP at hour 0, both analytes were identified in every subject at every time point, and large interindividual variability was observed. For 10 of 12 subjects, the shape of the AUC curve of RP qualitatively matched that of αRP, and the 2 curves could essentially be superimposed, varying only in absolute concentration. Absolute concentrations of RP were higher than those of αRP in plasma (4.19-fold higher, P = 0.007), which was anticipated based on the difference in the dose of α-carotene relative to β-carotene. Because of the right-skewedness of the data, the median maximum concentration, the time of maximum concentration, and total AUCs of αRP and RP are presented in Table 1. Although RP had an ∼6-fold higher median maximal plasma TRL concentration than αRP, both analytes reached the maxima at the same time.

FIGURE 2.

Mean baseline-corrected plasma TRL concentrations of αRP and RP over the course of 12 h after consumption of a carrot-containing meal. Concentrations of analytes are represented as means ± SEMs, n = 12. TRL, triglyceride-rich lipoprotein.

Total carotenoid absorption

Figure 3A depicts the total β- and α-carotene absorbed from the mixed carrot meal for each subject individually, taking into account the fraction of carotenes that were enzymatically cleaved by BCO1. More β-carotene was fed than α-carotene; thus, it was predicted that more β-carotene would be absorbed. After the differences in carotene dose administration were accounted for, there was not a significant difference in the absorption of total β-carotene compared with α-carotene (P = 0.103).

FIGURE 3.

The total carotenoid absorbed in each subject, including that cleaved to form retinyl esters/α-retinyl esters (A). Percentage of total carotenoid converted to retinoids relative to carotenoid absorbed (B). The percentage of α-carotene contribution to the newly formed retinyl ester in the same subject, respectively (C). Black bars represent β-carotene and white bars represent α-carotene. The line in panel C denotes the expected contribution of α-carotene (25.5%) to retinyl esters, assuming 100% absorption and conversion according to the provided dose. Note that the subject numbers in each panel represent the same individual (i.e., subject 1 in panel A is subject 1 in panels B and C).

Percentage of carotenoid cleavage

Figure 3B depicts the percentage of cleavage of β-carotene (to vitamin A) compared with α-carotene (to vitamin A and α-retinol) in the enterocyte for all subjects, relative to the dose absorbed within the same subject. A trend was observed for preferential cleavage of β-carotene compared with cleavage of α-carotene, relative to the absorbed dose (P = 0.084). A large range of cleavage was observed for both carotenoids (9–68% for β-carotene, and 6–63% for α-carotene); intraindividual variability was also high, with 8 subjects showing a preference for β-carotene as opposed to α-carotene, 3 subjects showing roughly equivalent cleavage of the 2 carotenoids, and 1 subject showing preferential cleavage of α-carotene. Furthermore, conversion preference (i.e., a preference for β-carotene or α-carotene cleavage) within a subject was not correlated with carotenoid absorption preference in the same subject (R² = 0.02 for β-carotene and R² = 0.33 for α-carotene; data not shown). A strong relationship exists between the percentage of conversion of β- and α-carotene, with an R² = 0.82 (i.e., individuals who convert more β-carotene also convert more α-carotene).

Figure 3C shows the percentage of the contribution of α-carotene–derived retinyl esters to the newly absorbed vitamin A. The predicted contribution assumed the following: 1) β- and α-carotene were equally absorbed from the carrot meal relative to the dose and 2) α-carotene provided one-half the vitamin A equivalence of β-carotene, which would be 25.5% (denoted by the black line in Figure 3C). That is, because the test meal contained 1.46 times more β-carotene than α-carotene, one would expect 2.92 molecules of vitamin A produced from β-carotene for each 1 IU of vitamin A from α-carotene. Two subjects had a higher percentage of contribution of α-carotene than expected, 3 subjects had the approximate percentage predicted, and the remaining 7 subjects had less α-carotene contribution than would be predicted solely by stoichiometry. AUCs for α- and β-carotene (as observed in the blood) and AUCs for α- and β-carotene adjusted to include the portion cleaved to produce α- and retinyl esters are provided in Supplemental Table 1.

Ester profiles of α-retinol and retinol

The proportions of various α-retinyl ester and retinyl ester species are presented in Figure 4. Palmitate was by far the most abundant ester in each subject. However, within a subject, the percentage of α-retinol esterified to palmitate was slightly but significantly lower than that for retinol (P < 0.001). The percentage of α-retinol esterified to linoleate (P = 0.012), oleate (P = 0.008), and stearate (P = 0.009) was higher than that of retinol. β-Retinyl myristate was detected in all subjects but one, whereas α-retinyl myristate was detected in only a few subjects and was below the limit of quantitation.

FIGURE 4.

Esterification profile of various retinyl (gray bars) and α-retinyl (white bars) species, expressed as a percentage of total retinyl and α-retinyl esters, respectively, at 6 h after the consumption of the carrot test meal. Values are expressed as means ± SEMs, n = 12. Asterisks indicate a significant difference between the percentage of α-retinyl and retinyl species of the same type (Wilcoxon’s Signed Rank test, P < 0.05).

DISCUSSION

Carrots have been well studied for provitamin A conversion, with most studies focusing solely on determining the total carotene contribution to vitamin A (17–19). Although other groups have demonstrated that α-retinol is esterified and cleared from the enterocyte into plasma in animal models (9, 11, 28), this is the first postprandial study, to our knowledge, to follow αRP in human plasma. The maximum concentration times for αRP and RP were identical for each subject, with striking similarity in the shape of individual AUCs of αRP and RP, suggesting little difference in incorporation into chylomicrons.

Measurement of α-retinyl products provides a unique tool to estimate the relative absorption of β- and α-carotene and the conversion of each into vitamin A. We did not observe a statistical difference in preference between absorption of β- compared with α-carotene when normalized to the difference in dose. Data in the literature are mixed on whether differences exist in absorption between α- and β-carotene and the directionality of this relation. Our observations are in greater alignment with previous acute and chronic human feeding studies of carrot puree (20) and carrot juice (29), respectively, although the nonsignificant differences noted herein are considerably smaller than those published previously. Other carrot (19, 30) and red palm oil (8) feeding studies report the opposite phenomenon, with more α-carotene absorbed than β-carotene relative to dose. However, these studies did not include the portion of carotenoid cleaved to form vitamin A and therefore only compared the intact α- and β-carotene circulating in the TRL fraction (8, 19) or plasma (30), anecdotally attributing the lower proportion of β-carotene to a higher amount of vitamin A conversion. In all instances, study populations were small (3–14 subjects), and total carotene dose varied widely from 7.3 mg (29) to 46 mg total carotene delivered herein.

Cell culture studies have demonstrated facilitated transport of α- and β-carotene across the apical membrane of the enterocyte via scavenger receptor class B member 1 (SR-B1) (31) and suggested that CD-36 may be involved as well (32). Sharing of a transporter provides the opportunity for competition for uptake. Indeed, Caco-2 studies have shown that cellular α-carotene uptake is significantly reduced with increasing initial concentrations of β-carotene, whereas the inverse phenomenon was not observed [i.e., initial concentrations of α-carotene had no effect on β-carotene uptake (33)].

The large interindividual differences observed in total carotene uptake may be partially attributed to single nucleotide polymorphisms (SNPs) correlated with provitamin A carotenoid uptake and circulating concentrations. Although SNPs were not measured in this study, previous work demonstrated that differences between circulating concentrations of α- and β-carotene were correlated to the same SNP in intron 5 of SR-B1 (34). Similarly, SNPs affiliated with the gene encoding for intestine specific homeobox, a transcriptional repressor of the SR-B1 transporter, as well as 4 genes involved in chylomicron response, were significantly correlated with postprandial β-carotene absorption (35). No study to our knowledge has yet assessed the effects of these SNPs on the differential uptake of α-carotene compared with β-carotene.

Herein, we observed a trend toward greater cleavage of β-carotene compared with α-carotene (mean 22%, P = 0.084). We hypothesized that we would observe less conversion of total absorbed α-carotene relative to β-carotene, owing to previous reports of the relative extent of conversion (36, 37) and to earlier work demonstrating that the catalytic efficiency of purified human BCO1 is 3 times higher with β-carotene compared with α-carotene (6). The reduced binding affinity could be attributable to the shift of a single double bond out of conjugation to form an ε-ring, allowing free rotation around the 6–7 bond in α-carotene (6). Studies revealed that SNPs in human BCO1 result in variable conversion of provitamin A carotenoids (35, 38–40). It is plausible that some of these SNPs result in enzymes with altered conformation, which could also affect provitamin A binding affinity. Other enzymes are also involved between cellular uptake of the parent carotenoid and the appearance of the resulting retinyl ester in the TRL fraction; these enzymes include retinal dehydrogenase, cellular retinol binding protein, lecithin retinol acyltransferase (LRAT), as well as the incorporation into the chylomicron. Thus, it is possible that differing interactions of α- and β-products with other enzymes and proteins in the enterocyte may influence the resulting concentrations measured.

A significantly higher percentage of α-retinol was esterified to linoleic, oleic, and stearic acids, and a lower percentage was esterified to palmitic acid, compared with retinol, although the magnitude of these differences is small (Figure 4). Palmitate, the dominant fatty acid ester for both species, comprised 65.6% ± 3.9% (mean ± SD) of the retinyl esters, consistent with previous reports (41), and 61.0% ± 3.3% of the α-retinyl esters. This observation suggests that minor differences in esterification patterns exist. LRAT is thought to be the primary enzyme responsible for catalyzing retinol esterification in the intestine (33) and is likely the primary enzyme responsible for esterifying α-retinol. Previous reports demonstrate that LRAT has higher affinity for all-trans retinol compared with 13-cis- or 3-dehydroretinol (42) or β-apo-carotenols of reduced chain lengths (43). Alteration of the retinoid substrate may also modify the fatty acid preferences of this enzyme for esterification.

Multiple studies have investigated the capacity of α-retinol derivatives to elicit a vitamin A response (10, 12–15). Although some vitamin A–like activity was observed with early in vitro cellular assays, the same activities later failed to translate in animal studies, likely because of the inability of α-retinol to bind to retinol binding protein for tissue distribution (44). One commonly used study is the curative rat growth assay (45). The ability of a vitamin A analog to rescue growth in deficient rats is tested with ≥2 doses, in addition to vitamin A tested with ≥2 doses, and the growth curves are plotted against the log dose to calculate relative biopotency (45). Multiple groups employing the curative rat growth assay reported no activity of α-retinal (13) and ∼2.1–2.6% activity of α-retinyl acetate (i.e., an ester of α-retinol) (14, 15). A recent report suggests that α-retinyl acetate may provide 40–50% of the bioactivity of retinol; however, only a single dose of both α-retinyl acetate and retinol was tested in this study, and nontraditional growth calculations were used, making it difficult to compare these findings with previous results (12). Taken together, the full body of literature suggests it is unlikely that any substantial bioactivity is derived from α-retinyl products in humans. However, α-retinyl esters resulting from an α-retinyl acetate dose fed to piglets was a useful chylomicron tag, because it tracked similarly to retinyl esters postprandially (0–24 h) (46). Our data further support this conclusion.

Together, between differences in absorption and conversion, our data reveal that the majority of our subjects absorbed similar quantities of β-carotene and α-carotene when adjusting for dose, and they cleaved β-carotene slightly more efficiently than α-carotene. Thus, α-carotene likely contributes to retinyl esters to a lesser degree than is assumed in calculations of retinol activity equivalents (Figure 3). Our study was performed in healthy humans, presumed to be vitamin A sufficient. Cell and animal experiments suggest that increased provitamin A absorption (47) and cleavage (36, 47, 48) would occur in vitamin A–deficient populations. However, studies investigating provitamin A cleavage in rats and mice should be interpreted cautiously, given differences in carotenoid absorption and metabolism between these species and humans (49, 50). BCO1 activity has also been demonstrated to be affected by other dietary factors, including fat, protein, antioxidants, and the presence of other carotenoids (51).

In conclusion, our data demonstrate that α-retinol is esterified to fatty acids and transported in a similar manner to retinol, although differences are observed in ester preference between α-retinol and retinol. From a mixed carotene meal, there was no preference for absorption of β-carotene when adjusting for dose (P = 0.103). A trend toward significance was noted, with higher cleavage efficiency of β-carotene relative to α-carotene (P = 0.084). Overall, our data confirm that carrot α-carotene substantially contributes to vitamin A, but that more consideration should be given to interindividual differences when estimating the provitamin A potential of this carotenoid. Larger studies are needed to better understand the true provitamin A equivalence of foods containing α-carotene.