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The Journal of Nutrition Vol. 127 No. 10 October 1997, pp. 1993-1999
Copyright ©1997 by the American Society for Nutritional Sciences

beta -Carotene Isomers in Human Serum, Breast Milk and Buccal Mucosa Cells after Continuous Oral Doses of All-Trans and 9-Cis beta -Carotene1,2,3

Elizabeth J. Johnson4, Jian Qin, Norman I. Krinsky*, and Robert M. Russell

Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University and * Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED

ABSTRACT

The concentrations of all-trans beta -carotene (tBC) and 9-cis beta -carotene (9cBC) isomers in serum, breast milk and buccal mucosa cells were determined after continuous oral doses as a simple, non-invasive method to determine whether differences in tissue uptake are important determinants of serum responses. Twelve healthy lactating women were recruited for a nonresidential study. On d 1, blood samples were obtained from fasting subjects for baseline concentrations of beta -carotene isomers. Over a 1-wk period, subjects were given either seven doses of a placebo (n = 4) or seven doses of naturally occurring BC (n = 8) derived from Dunaliella bardawil (64 mg tBC, 69 mg 9cBC). Subjects were instructed to consume a single beta -carotene dose along with a meal containing adequate fat each day for 1 wk. On d 2, 3, 5 and 8, blood samples and breast milk were collected from fasting subjects. On d 1 and 8, buccal mucosa cells were collected. Samples were analyzed for carotenoids by HPLC. In the experimental group, the mean serum concentration of tBC significantly increased to seven times the baseline level by the end of the supplementation period (P < 0.0001). The serum concentration of 9cBC significantly increased to three times the baseline level by the end of the supplementation period (P < 0.0001). The changes in milk and buccal mucosa cells levels of tBC and 9cBC followed a pattern similar to that for serum, showing significant increases at the end of the supplementation period. In the control group, the serum, milk and buccal mucosa cell concentrations of BC isomers did not change. This study confirms the previously reported differences in the serum response curves of tBC and 9cBC and provides evidence that there is no difference in tissue uptake of tBC and 9cBC.

KEY WORDS: beta -carotene isomers · absorption · tissue uptake · humans

INTRODUCTION

beta -Carotene (BC) can exist in foods and human tissues as several different geometric isomers, including the all-trans, 9-cis, 13-cis, and 15-cis isomers (Chandler and Schwartz 1987). The major BC isomer in human serum is all-trans BC, with small or negligible amounts of 13-cis and 9-cis BC (Stahl et al. 1992 and 1993, Tamai et al. 1995). However, there are considerable amounts of 9-cis and 13-cis BC present in human tissue (Stahl et al. 1993), and it has been suggested that these compounds may possess isomer-specific functions (Stahl et al. 1993). There is particular interest in all-trans and 9-cis BC because their metabolites, all-trans and 9-cis retinoic acid, are active in the regulation of gene expression (Heyman et al. 1992, Levin et al. 1992).

Studies on the bioavailability of all-trans BC vs. 9-cis BC in humans are few. Both trans and cis isomers are present in foods. For example, cis BC isomers are more prevalent in apricots, whereas trans BC isomers are more prevalent in carrots. In rats and chickens, it seems that cis BC isomers are more likely to be absorbed intact, whereas trans BC isomers are more likely to be broken down to vitamin A. However, data examining the serum response to a single large oral dose of either all-trans BC or 9-cis BC in men indicate that the all-trans isomer attains a far greater postprandial concentration, but that the 9-cis isomer reaches peak levels sooner (Johnson et al. 1996). It is difficult to determine if these serum response differences are due to differences in intestinal absorption, conversion to vitamin A or rates of uptake of BC from the circulation by peripheral tissues. Given that tissue concentrations of all-trans BC and 9-cis BC differ, tissue uptake of these isomers may likewise differ. Examination of the concentration of BC isomers in breast milk and buccal mucosa cells after continuous oral doses of BC isomers is a simple, non-invasive method to determine whether differences in tissue uptake are important determinants of serum responses.

MATERIALS AND METHODS

Subjects. Healthy lactating women (1-8 mo postpartum, 18-40 y old) were recruited from La Leche League International. Blood samples were obtained and screened for normal hematological variables, serum albumin, liver function and kidney function. Subjects with a history of fat malabsorption, active small bowel disease or resection, atrophic gastritis, hyperlipidemia, insulin-requiring diabetes, alcoholism, pancreatic disease or bleeding disorders were excluded from the study. Subjects were excluded if they were taking drugs that interfere with fat absorption or metabolism. Subjects were not greater that 30% above their pre-pregnancy weight. Moreover, subjects were not allowed to take vitamin supplements containing BC or more than one times the RDA for vitamin A. No smoking or alcohol consumption was permitted during the course of the study.

Study design. Subjects (n = 12) were involved in a nonresidential study. Two weeks prior to the study, blood (10 mL) was collected for pre-study measurements of carotenoids. For 2 wk before the study and for the duration of the supplementation period of the study, subjects were given instructions to consume a low carotene diet while at home to lower blood carotenoid levels. Food diaries were kept for 3 d of the 2 wk before initiation of the study (d 1) and for 3 d midway through the study, as a check for carotenoid consumption.

On d 1 of the study, a 10-mL blood sample was obtained from fasting subjects and analyzed for baseline levels of all-trans and 9-cis BC. The subjects were then given a breakfast containing no carotenoids. Other meals were consumed from a self selected diet. The subjects were given either seven doses of a placebo (n = 4) or seven doses of a naturally occurring BC derived from Dunaliella bardawil (64 mg all-trans BC and 69 mg 9-cis BC; Henkel Corp., LaGrange, IL) (n = 8). This is ~40 times typical intakes for premenopausal women (Yong et al. 1994). The women were randomly assigned to the groups. The subjects were instructed to consume a single dose along with a meal containing fat each day for 1 wk beginning on d 1 of the study. On study d 2, 3, 5 and 8, blood samples (10 mL) were collected from fasting subjects. One month after the last dose, another 10-mL blood sample was obtained. On each of the blood sampling days, breast milk samples (10 mL) were collected by hand expression. Given the inter and intraindividual variability in carotenoids of breast milk (Guiliano et al. 1994), foremilk was collected for all breast milk samples and sampling was done at the same time of day throughout the study.

On study 1 and 8 and at 1 mo after the last dose, buccal mucosa cells were collected by the method of Peng et al. (1995). Briefly, after rinsing their mouths vigorously with drinking water, the subjects were asked to brush the inside of their cheeks with a soft toothbrush, ~1 minute each side. After the brushing, they were asked to rinse their mouths with 30 mL of distilled water and deposit the rinsing solution into a 50 mL vial. The toothbrush was then washed with 20 mL water, which was deposited into the vial. Buccal mucosa cells were centrifuged (800 × g for 10 min at 4°C), and the supernatant was discarded. To the pellet was added 10 mL of phosphate buffer solution, pH 7.4. After vortexing, the sample was centrifuged (800 × g for 5 min at 4°C, and the supernatant was removed. To the pellet was added 0.85 mL of cold phosphate buffer solution, pH 7.4.

Serum was prepared from blood samples (800 × g for 15 min at 4°C). Serum, buccal mucosa cells and breast milk samples were stored at -70°C until analysis for carotenoids. During collection and analyses, all samples were protected from light.

Chemicals. HPLC-grade methanol and water were purchased from J. T. Baker Chemical (Phillipsburg, NJ). Methyl-tert-butyl ether, carotenoids used for HPLC standard curves, and ammonium acetate were purchased from Sigma Chemical (St. Louis, MO). Solvents were passed through a 0.45-µm membrane filter and degassed prior to use. Echinenone was from Roche (Nutley, NJ). All carotenoid standards were stored at -70°C.

Serum extraction for carotenoids. Serum was prepared for extraction using a 150-µL sample and 1 mL of 9 g/L saline. Echinenone, in ethanol, was added as an internal standard. The mixture was extracted by using 2 mL of CHCl3-CH3OH (2:1,v/v). The mixture was vortexed and then centrifuged at 800 × g at 4°C for 15 min. The CHCl3 layer was removed and evaporated to dryness under nitrogen. A second extraction was performed on the mixture using 3 mL of hexane. The mixture was vortexed and centrifuged as above. The hexane layer was combined with the first extraction and evaporated to dryness under nitrogen. The residue from serum was redissolved in 150 µL of ethanol, vortexed and sonicated for 30 s. A 50-µL aliquot was used for HPLC analysis.

Buccal mucosa cell extraction for carotenoids. Buccal mucosa cells were extracted for carotenoids according to the method of Peng et al. (1995). Briefly, frozen cells were allowed to thaw at room temperature for a few minutes. After vortexing, a 10-µL aliquot was taken for protein determination using bicinchoninic acid (Smith et al. 1985). To 1.0 mL of cells, 1-2 mg of butylated hydroxytoluene crystals and 200 µL of 1% protease solution (pronase E from Streptomyces griseus, Sigma Chemical) were added, and the tubes were incubated at 37°C for 45 min. After the incubation, the samples were treated with 400 µL of 1% sodium dodecyl sulfate in ethanol containing 0.1% butylated hydroxytoluene (wt/vol/wt). Echinenone, in ethanol, was added as an internal standard. The mixture was extracted using 3 mL of ether-hexane (2:1, v/v). The mixture was vortexed and then centrifuged at 800 × g at 4°C for 15 min. The upper layer was removed. The ether-hexane extraction was repeated and combined with the first extraction and evaporated to dryness under nitrogen. The residue was redissolved in 150 µL of ethanol, vortexed and sonicated for 30 s. A 50-µL aliquot was used for HPLC analysis. Buccal mucosa cells concentration of carotenoids is expressed as picomoles per milligram of protein.

Breast milk extraction for carotenoids. Carotenoids were extracted from 2 mL of breast milk. Samples were lyophilized (20 h at -20°C, <0.03 kg/m2). To the sample was added 100 µL of 12% pyrogallol in ethanol, 200 µL of 30% KOH and 1 mL of ethanol. The mixture was vortexed and incubated at 37°C for 2 h. After incubation, the sample was cooled down to room temperature and 1 mL of H2O was added, and the mixture was vortexed. Echinenone in ethanol (100 µL) was added as an internal standard. The mixture was extracted by using 3 mL of ether-hexane (2:1, v/v). The mixture was vortexed and then centrifuged at 800 × g at 4°C for 5 min. The upper layer was removed. The extraction with ether-hexane was repeated and the upper layers combined. To the extract was added 1 mL of H2O. The mixture was vortexed and ethanol was added to make the solution clear. The mixture was centrifuged for 5 min at 800 × g. The H2O layer (lower layer) was removed and discarded. Another 1 mL of H2O was added and removed as above. The extract was evaporated to dryness under nitrogen. The residue was redissolved in 150 µL of ethanol, vortexed and sonicated for 30 s. A 50-µL aliquot was used for HPLC analysis.

HPLC analysis. All-trans and 9-cis BC were separated and quantified using a reverse-phase, gradient HPLC method. The identity of 9-cis BC was confirmed by coelution with a standard and the absorption spectra of BC isomers. Analysis of absorption spectra of the 9-cis BC peak from serum samples found that zeta -carotene coeluted with 9-cis BC. The absolute amount of zeta -carotene could not be determined because a standard was not available. Therefore, the relative amount of zeta -carotene was determined by measuring peak areas at the optimal absorption wavelength for zeta -carotene (400 nm). The system consisted of a Series 410 LC pump (Perkin-Elmer, Norwalk, CT), Waters 717 plus autosampler (Millipore, Milford, MA), a C30 carotenoid column (3 µm, 150 × 4.6 mm, YMC, Wilmington, NC), HPLC column temperature controllers (model 7950 column heater/chiller, Jones Chromatography, Lakewood, CO), Waters 994 programmable photodiode array detector (400 and 450 nm) and a Waters 840 digital 350 data station. The HPLC mobile phase was methanol-methyl-tert-butyl ether-water (83:15:2, v/v/v, with 1.5% ammonium acetate in H2O, solvent A) and methanol-methyl-tert-butyl ether-water (8:90:2, v/v/v, with 1 g/100 mL ammonium acetate in H2O, solvent B). The gradient procedure at a flow rate of 1 mL/min at 16°C was as follows: The procedure began at 100% solvent A and went to 93% solvent A and 7% solvent B over a 1-min linear gradient. This was followed by a 3-min hold at 93% solvent A, followed by a 17-min linear gradient to 45% solvent A and a 1-min hold at 45% solvent A, then an 11-min linear gradient to 95% solvent B, a 4-min hold at 95% solvent B, and finally a 2-min gradient back to 100% solvent A. The system was held at 100% solvent A for 10 min for equilibration back to initial conditions. Using this method, lutein, zeaxanthin, cryptoxanthin, alpha -carotene, 13-cis beta -carotene, all-trans beta -carotene, and 9-cis beta -carotene are adequately separated. Also, four geometrical isomers of lycopene (15-cis, 13-cis, 9-cis, and all-trans lycopenes) are separated. A typical HPLC chromatogram for breast milk is shown in Figure 1. Carotenoids were quantified by determining peak areas in the HPLC chromatograms calibrated against known amounts of standards. Levels were corrected for extraction and handling losses by monitoring the recovery of the internal standards. The lower limit of detection was 0.2 pmol for carotenoids.
Fig. 1. HPLC chromatogram of the major carotenoids in human breast milk. The numbered peaks are as follows: 1, lutein; 2, zeaxanthin; 3, cryptoxanthin; 4, echinenone (internal standard); 5, 13-cis beta -carotene, 6, alpha -carotene; 7, all-trans beta -carotene; 8, 9-cis/zeta -carotene; 9, 13-cis lycopene; 10, 9-cis lycopene; 11, trans lycopene.
[View Larger Version of this Image (13K GIF file)]

Statistics. Results are expressed as means ± SEM. Significant differences from baseline were measured using ANOVA at the 95% confidence level (StatView 4.51, 1995, Abacus Concepts, Berkeley, CA). The Bonferroni/Dunn test was used when the F test was significant (StatView). The area under the curve (AUC) was measured by trapezoidal approximation after subtracting the baseline concentration (Kaleidograph 3.0.S, 1994, Abelbeck Software, Reading, PA). For each isomer, significant differences between AUC for experimental and control groups were compared using the Mann-Whitney U test (StatView). Also, for each group, significant differences between AUC for each isomer were compared using ANOVA.

RESULTS

Serum. The mean serum all-trans BC response to a continuous oral dose of BC derived from Dunaliella bardawil and placebo is presented in Figure 2. The mean serum concentration of all 12 subjects on d 1 significantly decreased from pre-study values (P < 0.007), indicating adherence to a low carotenoid diet for the 2-wk pre-study period. This compliance was also indicated by the food diaries.
Fig. 2. Serum all-trans beta -carotene (panel A) and 9-cis beta -carotene (panel B) concentrations in lactating women in the control group (no beta -carotene, n = 4) and in the experimental group (64 mg all-trans beta -carotene and 69 mg 9-cis beta -carotene/d for 7 d, n = 8) while consuming a diet low in carotenoids. Solid points are significantly different from baseline (d 1) (P < 0.05). Note the difference in scale between panels A and B. Values are means ± SEM.
[View Larger Version of this Image (13K GIF file)]

In the experimental group, the mean serum concentration of all-trans BC significantly increased from baseline during the dosing period (Fig. 2). The increase was observed by d 2 (P < 0.001) and steadily increased to seven times the baseline (d 1) level by the end of the supplementation period (d 8, P < 0.0001). At the 1-mo post-study measure, the mean serum concentration of all-trans BC decreased to the pre-study level but remained significantly greater than the d 1 value (P < 0.007). In the placebo-fed control group, the serum concentration of all-trans BC did not change throughout the supplementation period and the post-study serum all-trans BC concentration was not different from baseline (Fig. 2).

The serum 9-cis BC response curve to a continuous oral dose of BC and placebo is presented in Figure 2. The results include serum zeta -carotene; however, the contribution of zeta -carotene is assumed to be small. According to the dietary diaries, the subjects were not consuming foods containing zeta -carotene, i.e., tomatoes and tomato products, during pre-study and supplemental periods. Additionally, the peak areas of zeta -carotene measured at 400 nm did not change throughout the study.

The pre-study and d 1 serum concentrations of all-trans BC of the 12 subjects were ~40 times greater than those for 9-cis BC. The serum 9-cis-BC concentration of all 12 subjects on d 1 significantly decreased from pre-study values (P < 0.019). In the experimental group, the serum concentration of 9-cis BC significantly increased from baseline during the dosing period, with a twofold increase over baseline by the end of the supplementation period (P < 0.0001, Fig. 2). At the 1-mo post-study measure, serum concentration of 9-cis BC decreased to the pre-study level. In the control group, the serum concentration of 9-cis BC did not change throughout the supplementation period and the post-study serum 9-cis BC concentration was not different from baseline.

The serum AUC response to the continuous oral dose of BC isomers for all-trans BC was more than 100-fold greater than that for 9-cis BC (P < 0.0001, Table 1). The AUC response was significantly greater in the experimental group than in the control group for both isomers of BC (P < 0.0001, Table 1). In the control group, there was no difference between all-trans and 9-cis BC in the AUC response (Table 1).

Table 1. Serum beta -carotene response [area under the curve (AUC)] in lactating women1,2

[View Table]

Breast milk. The pre-study concentration of all-trans BC in breast milk of the 12 subjects did not differ from that of d 1 (0.916 ± 0.255 and 0.706 ± 0.216 µmol/L, respectively). The changes in breast milk concentration of all-trans BC in response to a continuous oral dose of BC followed a pattern similar to that for serum. A significant increase in concentration was observed by d 3 (P < 0.009, Fig. 3) and steadily increased to six times the baseline level (d 1) by the end of the supplementation period (d 8, P < 0.0001, Fig. 3). At the 1-mo post-study measure, the breast milk concentration of all-trans BC decreased but was still significantly greater than the d 1 value (P < 0.022, Fig. 3). In the control group, the breast milk concentration of all-trans BC did not change throughout the supplementation period, and the post-study serum all-trans BC concentration was not different from baseline (Fig. 3).
Fig. 3. All-trans beta -carotene (panel A) and 9-cis beta -carotene (panel B) concentrations in breast milk in women in the control group (no beta -carotene, n = 4) and in the experimental group (64 mg all-trans beta -carotene and 69 mg 9-cis beta -carotene/d for 7 d, n = 8) while consuming a diet low in carotenoids. Solid points are significantly different from baseline (d 1) (P < 0.05). Note the difference in scale between panels A and B. Values are means ± SEM.
[View Larger Version of this Image (13K GIF file)]

The 9-cis BC response to a continuous oral dose of BC and placebo in breast milk is presented in Figure 3. The pre-study and d 1 concentrations of 9-cis BC of the 12 subjects was approximately 3% that of all-trans BC. The pre-study 9-cis-BC concentrations in breast milk of the 12 subjects were not different from d 1 values (0.024 ± 0.004 and 0.028 ± 0.006 µmol/L, respectively). In the experimental group, the serum concentration of 9-cis BC significantly increased from baseline during the dosing period, with a twofold increase over baseline by the end of the supplementation period (P < 0.007, Fig. 3). At the 1-mo post-study measure, breast milk concentration of 9-cis BC remained significantly greater than baseline (P < 0.021, Fig. 3). In the control group, the breast milk concentration of 9-cis BC did not change throughout the supplementation period, and the post-study serum 9-cis BC concentration was not different from baseline (Fig. 3).

The breast milk AUC response to the continuous oral dose of BC isomers for all-trans BC was approximately a 100-fold greater than that for 9-cis BC (P < 0.0001, Table 2). The AUC response was significantly greater in the experimental group than in the control group for each of the isomers of BC (P < 0.015, Table 2). In the control group, there was no difference in the AUC response between all-trans and 9-cis BC (Table 2).

Table 2. Breast milk beta -carotene response [area under the curve (AUC)] in lactating women1,2

[View Table]

Buccal mucosa cells. The baseline concentration of all-trans BC in buccal mucosa cells did not differ between the control and experimental groups (Fig. 4). After 1 wk of supplementation with the BC isomers, the mean concentration of all-trans BC significantly increased to six times the mean baseline value (P < 0.005, Fig. 4). The level remained significantly greater than baseline 1 mo after the study (P < 0.020, Fig. 4). In the control group, the buccal mucosa cells concentration of all-trans BC did not change throughout the supplementation period, and the post-study concentration was not different from baseline (Fig. 4).
Fig. 4. All-trans beta -carotene (panel A) and 9-cis beta -carotene (panel B) concentrations in buccal mucosa cells in lactating women in the control group (no beta -carotene, n = 4) and in the experimental group (64 mg all-trans beta -carotene and 69 mg 9-cis beta -carotene/d for 7 d, n = 8) while consuming a diet low in carotenoids. Day 1 is baseline, d 8 is at the end of the supplementation, and post-study is 1 mo after the supplementation. Note the difference in scale between panels A and B. Values are means ± SEM. *Significantly different (P < 0.05) from d 1 (experimental group).
[View Larger Version of this Image (17K GIF file)]

Only one of the eight subjects in the experimental group had measurable 9-cis BC in buccal mucosa cells at baseline (0.050 pmol/mg protein). After 1 wk of supplementation with the BC isomers, 9-cis BC was detected in all but one subject. The mean concentration of 9-cis BC on d 8 was significantly greater than the mean baseline value (P < 0.022, Fig. 4). One month after the study, 9-cis BC was detected in four of the subjects in the experimental group (Fig. 4). In the control group, 9-cis BC was not detected in buccal mucosa cells throughout the entire study.

DISCUSSION

Most studies examining all-trans and 9-cis BC bioavailabilities have measured serum responses to a single oral dose of all-trans and 9-cis BC. It generally has been found that the serum response to all-trans BC is much greater than that for 9-cis BC. However, there are several factors that may influence the serum response to an oral BC dose, e.g., efficiency of absorption, breakdown and tissue uptake. In this study, in addition to serum concentrations, we measured buccal mucosa cell and breast milk concentrations of BC isomers after continuous oral doses of all-trans and 9-cis BC to determine whether differences in tissue uptake are important determinants of serum responses.

In an earlier report, we observed that continuous administration of an approximately 50:50 mixture of all-trans and 9-cis BC resulted in an increase of both isomers in serum, although the increase in the 9-cis isomer was much less pronounced than that of all-trans BC (Gaziano et al. 1995). Other studies examining the serum response to a single oral dose of 9-cis BC have been consistent in demonstrating minimal increases in serum concentrations of this isomer (Ben-Amotz and Levy 1996, Stahl et al. 1993, Morinobu et al. 1994). Our study confirms these reports.

The smaller increases in serum concentrations of 9-cis BC compared with all-trans BC may indicate selective intestinal absorption of the all-trans isomer. Evidence supporting this comes from the work of Stahl et al. (1995), who studied the isomeric BC pattern in chylomicrons as an indicator of gut absorption after ingestion of a mixture of all-trans and 9-cis BC. Concentrations of all-trans BC in chylomicrons increased substantially after ingestion of the mixture. The rise of 9-cis BC was only 2-10% of that of the all-trans isomer and did not reflect the isomer pattern of the ingested mixture. The authors concluded that an isomer-selective mechanism, located at the level of the intestinal mucosa cell, was related to specific uptake or incorporation steps and excluded the 9-cis isomer from accumulation in chylomicrons. In our design, we did not evaluate gut absorption. Given the data of Stahl et al. (1995) and the extremely large difference in the increase in serum concentrations of these two isomers in our study with continuous dosing, it seems most likely that all-trans BC is better absorbed than 9-cis BC.

It is possible that there is a difference in the metabolism of the different isomers that could account for the difference seen in the serum response. Evidence for this comes from Ben-Amotz and Levy (1996), who reported that there was a preferential absorption of all-trans BC over 9-cis BC, in parallel with the appearance of a high serum concentration of oxidized dienic products with supplementation with all-trans BC compared with the low concentration of serum-oxidized dienic products with supplementation with a natural BC source. Their study suggested that 9-cis BC acts as an in vivo lipophilic antioxidant more efficiently than does all-trans BC.

Assuming similar absorption efficiencies for the two BC isomers, isomerization of 9-cis to all-trans BC may also explain the small serum response of 9-cis BC. The work of You et al. (1996) provides strong evidence for isomerization of 9-cis BC during absorption in humans. They demonstrated that substantial amounts of [13C]all-trans BC and [13C]retinol appeared in plasma after ingestion of [13C]9-cis BC. In the present study, we were not able to distinguish between increases in serum concentration of all-trans BC resulting from intake of all-trans BC and increases resulting from the simultaneous intake of 9-cis BC that had been isomerized. Future study could examine the changes in serum concentrations of all-trans BC after an oral dose of 9-cis BC. However, purified preparations of 9-cis BC for this purpose are not available.

A small serum response of 9-cis BC to an oral dose of 9-cis BC may also be due to a rapid tissue uptake of the isomer. In fact, it has been reported that 9-cis BC contributes up to 25% of the total BC in human liver and 10% in human adrenals (Stahl et al. 1992). In this present study, buccal mucosa cells and breast milk concentrations of all-trans and 9-cis BC in women supplemented with BC were measured as an indication of the tissue uptake of these isomers. In both buccal mucosa cells and breast milk, there was an increase in all-trans and, to a much lesser extent, 9-cis BC. This suggests that there is no difference in tissue uptake of all-trans BC and 9-cis BC. However, the possibility of isomerization of 9-cis to all-trans BC in tissues remains.

As has been observed with serum response to oral doses of BC, we also observed individual variation in the buccal mucosa cells and breast milk responses. This may indicate that the difference between low responders and high responders (as defined by serum response) is indeed due to variation in the absorption, rather than metabolism or tissue uptake, and that the serum response is reflective of the amount that gets into tissue. Another indication that the variation in the serum response to an oral dose of BC is due to differences among individuals in the absorption of BC comes from the AUC data. We determined the AUC of the serum and breast milk concentrations vs. time plot as a method of estimating the absorption and tissue uptake of BC. For each isomer, AUC were approximately the same in serum and breast milk. This indicates that the BC that gets absorbed is taken into tissues.

Our data demonstrate the value of measuring tissue vs. serum concentrations of BC. Compared with serum, buccal mucosa cells and breast milk concentrations of all-trans and 9-cis BC after the discontinuation of the BC supplement showed slower decreases to pre-study values. Buccal mucosa cells and breast milk are better indicators of long-term storage than serum. Also, we found that after 2 wk of low carotenoid diet consumption (pre-study phase), concentrations of both isomers decreased significantly in serum but not in breast milk (a pre-study measure in buccal mucosa cells was not made). This could mean that serum concentrations reflect short-term intakes of BC and breast milk concentrations reflect longer-term intakes. However, in the present study, we were able to measure changes in concentrations of BC in breast milk within 1 wk (d 1-8) because of the high supplemental doses that were used. However, given the small number of subjects and a tendency for a decline in all-trans BC during the pre-study period, this observation needs to be confirmed with a larger sample size.

ACKNOWLEDGMENTS

We express our gratitude to Ruth Lackie in her efforts in recruitment of volunteers for La Leche League, the women who participated in this study, and Barbara Golner for her help in blood drawing. We express our appreciation to Henkel Corporation for the samples of Betatene©, the carotenoid complex of Dunaliella bardawil, and to Hoffmann-La Roche for the sample of echinenone.

FOOTNOTES

1   Supported in part by federal funds from the U.S. Department of Agriculture, Agriculture Research Service under contract number 53-3K06-5-10 and a gift from Henkel Corporation, LaGrange, IL.
2   The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products or organizations imply endorsement by the U.S. government.
3   The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
4   To whom correspondence and reprint requests should be addressed.

Manuscript received 13 February 1997. Initial reviews completed 29 July 1997. Revision accepted 17 June 1997.

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0022-3166/97 $3.00 ©1997 American Society for Nutritional Sciences



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