The Journal of Nutrition Vol. 128 No. 11 November 1998, pp. 2009-2013

All-trans -Carotene Is Absorbed Preferentially to 9-cis -carotene, but the Latter Accumulates in the Tissues of Domestic Ferrets (Mustela putorius puro)^1,2,3

John W. Erdman Jr.⁴, Angela J. Thatcher, Nicolle E. Hofmann, Janine D. Lederman, Stephanie S. Block, Christine M. Lee, and Shoshana Mokady^*

Department of Food Science and Human Nutrition and Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801 and ^* Department of Food Engineering and Biotechnology, Technion, Haifa, Israel

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The algae Dunaliella bardawil and Dunaliella salina naturally contain large concentrations of all-trans and 9-cis -carotene (C). The purpose of this study was to compare the relative serum and tissue accumulation of all-trans and 9-cis C in ferrets fed different ratios of all-trans/9-cis C derived from two commercial sources, D. bardawil or D. salina (Betatene). Male ferrets (7 wk old) were fed carotene-free, pelleted diets for 27 d. Beginning on d 18, groups of ferrets (n = 6 or 7) received daily, one of six oral supplements varying in ratios of 9-cis and all-trans C mixed with ~1.0mL of Ensure. Four supplements containing 5.2-8.3 µmol total C were prepared from a 20% Betatene preparation, D. bardawil, a high-cis Betatene preparation, and Betatene further enriched in 9-cis C with all-trans C/9-cis C ratios of 2.2, 1.5, 0.6 and 0.4, respectively. Two control supplements, high and low C, were prepared from commercial C beadlets. The high control supplement had an all-trans/9-cis ratio of 19.0, whereas 9-cis C was not detected in the low supplement. On d 27, serum and tissues were obtained for HPLC analysis of C and its isomers. Analysis of livers showed that all-trans C was the primary isomer present, but 9-cis and other isomers were also detected in all groups. The hepatic all-trans/9-cis ratios were 5.9, 4.9, 2.5, 1.4, 52.2 and 47.5, respectively, for the groups listed above. Lower amounts of all-trans and 9-cis C were found in kidneys compared with the liver, but ratios of all-trans/9-cis were not different among groups. Only trace amounts of 9-cis C were found in serum. These results demonstrate that the algae D. bardawil and D. salina provide a bioavailable source of C isomers, but, as in humans, absorption of 9-cis C is poor and any 9-cis C absorbed is apparently cleared by the liver.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

There are ~600 naturally occurring carotenoids, although it is not clear how many have biological value in humans. -Carotene (C),⁵ the carotenoid that gives carrots their orange color, is the most commonly studied carotenoid due to its abundance in foods, its antioxidant properties and its nutritional value as a precursor of vitamin A (VA). C exists primarily with all of its double bonds in the trans configuration. However, light and heat can cause isomerization of one or more double bonds to cis configurations. There are numerous possible isomers of each carotenoid; however, the number of stable geometric isomer forms is limited. The primary isomers of C found in foods are all-trans, 9-cis and 13-cis (Chandler and Schwartz 1987).

Retinoid binding to nuclear receptors is important for growth, reproduction and maintenance of the skin and mucous membranes. In in vitro studies, 9-cis retinoic acid was determined to be a ligand for the human nuclear retinoic acid receptor, RXR- (Heyman et al. 1992, Levin et al. 1992). 9-cis Retinoic acid can be formed by the oxidation and isomerization of dietary retinol. In addition, 9-cis C can also be a source of 9-cis retinoic acid (Nagao and Olson 1994, Wang et al. 1994).

The potential conversion of 9-cis C to the ligand 9-cis retinoic acid has led to increased interest in the absorption and metabolism of 9-cis C. Stahl and co-workers (1993) found that when subjects ingested an isomer mixture of carotenoids, serum levels of -carotene, all-trans C, and other isomers of C such as 13- and 15-cis increased, but 9-cis C did not. Other researchers have also noted discrimination in absorption and transport of 9-cis vs. all-trans C (Gaziano et al. 1995, Stahl and Sies 1994). Even though humans have very low serum 9-cis C concentrations when fed a dose of 9-cis C, relatively high levels are found in the tissues (Stahl et al. 1992 and 1993). It is unclear whether this is due to rapid clearing of absorbed 9-cis C into tissues, tissue isomerization of all-trans C, or some other unknown mechanism. The study of 9-cis C metabolism in humans is difficult, although a human study with ¹³C-9 cis C suggested substantial isomerization of 9-cis C to all-trans C in the gastrointestinal tract before absorption (You et al. 1996). Animal models that absorb C similarly to humans should provide tissue data not achievable in human studies.

Many species, such as rats, do not accumulate C in tissues when C is fed at physiologic levels (Erdman et al. 1993). Like humans, domestic ferrets (Mustela purorius furo) have been shown to absorb substantial portions of ingested C intact and to accumulate C in tissues (Gugger et al. 1992, Lederman et al. 1998, Ribaya-Mercado et al. 1989). Previous studies in this laboratory have found that the ferret is an appropriate animal model to study the absorption and tissue uptake of C and its isomers (Lederman et al. 1998, White et al. 1993, Zhou et al. 1996). The purpose of this study was to compare the relative serum and tissue accumulation of all-trans and 9-cis C in ferrets fed different ratios of all-trans/9-cis C derived from two commercial sources, Dunaliella bardawil or Dunaliella salina (Betatene).

	METHODS AND MATERIALS

Abstract Introduction Methods Results Discussion References

Animals and diets.  Male, descented 8-wk-old ferrets (n = 40) were purchased from Marshall Farms (North Rose, NY). Upon arrival, all ferrets were individually housed in stainless steel rabbit cages. Ferrets had free access to food and water. Food intake, body weight and health were monitored.

Ferrets were fed a semipurified, pelleted diet (Research Diets, New Brunswick, NJ), which has been used successfully in previous ferret studies (Lederman et al. 1998, White et al. 1993, Zhou et al. 1996). The diet contained 5.4 mg/kg diet vitamin A (as retinyl palmitate) and undetectable levels of C or other carotenoids (+VA/-C diet). Oral doses of various all-trans and 9-cis C supplements were prepared from two different sources, D. bardawil and D. salina (Betatene). All diets were stored at 4°C until use.

Experimental design.  Upon arrival, ferrets (n = 40) were fed the basal (+VA/-C) diet for 17 d to reduce body stores of C (White et al. 1993) and to allow acclimation to the new environment. Vitamin A was included in the diet to maintain adequate VA status. After acclimation, ferrets were separated into six experimental groups (n = 6 or 7) and continued to receive the basal diet. Beginning on d 18, each group received a daily oral C supplement containing C mixed with lecithin in ~1.0 mL Ensure (Ross Laboratories, Columbus, OH). The doses, containing from 5.2 to 8.3 µmol total C, were prepared from the following: Betatene (Betatene A), high-cis Betatene (Betatene B) (both gifts from Henkel, LaGrange, IL), high-cis Betatene further enriched in 9-cis C (Betatene C), D. bardawil, or C beadlets (a gift from Hoffmann LaRoche, Nutley, NJ) (high C control) with ratios of all-trans C/9 to cis C of 2.2, 0.6, 0.4, 1.5 and 19.0, respectively. The final group received a low C control dose, prepared from C beadlets, which contained 0.8 µmol all-trans C and no detectable 9-cis C (Table 1).

View this table: [in this window] [in a new window]   Table 1. Sources and -carotene (C) composition of daily oral supplements fed to ferrets for 9 d1

After 9 d of dosing, the ferrets were anesthetized with a mixture of ketamine hydrochloride (Vetalar, Parke-Davis, Morris Plains, NJ) 11-15 mg/kg body weight and xylazine (Rompun, Miles Laboratories, Shawnee, KS) 0.3-1.0 mg/kg body weight administered by intramuscular injection. Blood samples were collected by cardiac puncture, and the ferrets were killed by severing the brachial vessels between the pectoralis major and the latissimus dorsi. The liver, kidneys and adrenal glands were then dissected and weighed. Tissue and serum samples were stored at 20°C until analysis, which was completed within 6 mo. All procedures were approved by the University of Illinois Laboratory Animal Care Advisory Committee.

All procedures were carried out under yellow lighting.

Dose preparation and administration.  The C doses were prepared daily and isomeric content verified by HPLC. Mean values of this analysis are presented in Table 1. The samples were mixed with Ensure because previous studies in this laboratory have shown that ferrets will readily drink this mixture (White et al. 1993). Lecithin (~1.0 mL) was added to 12 mL of Ensure before addition of the various preparations to allow emulsification of the C in the Ensure mixture. Water bottles were removed each morning and returned immediately after the dose was administered (early afternoon). The 1.0-mL dose was administered in a disposable pipet placed directly into the mouth. Doses were consumed quickly, without difficulty.

Betatene A, Betatene B (both gifts from Henkel), and D. bardawill (spray dried algae powder, a gift from NBT Eliat, Israel) were used as obtained from the supplier. To prepare the Betatene C dose, ~0.3g of Betatene B was vortexed with 10 mL ethanol (with 1.0 g/L BHT) for 1min. The mixture was centrifuged in a benchtop centrifuge for 5 min to pellet the unsolubilized portion and the ethanol was removed and placed into a glass tube. One milliliter of KOH (600 g/L water) was added to the ethanol and the mixture was saponified for 20 min at 70°C. Deionized water (1 mL) and 200 µL of 1mol/L KCl were added and the mixture was placed on ice to cool. Once cooled, 15.0 mL of hexane (with 1.0 g/L BHT) was added, the mixture was vortexed for 1 min and the hexane layer was removed. The hexane extraction was repeated three times. The hexane extracts were combined and evaporated under vacuum.

Serum and tissue preparation.  Tissues were saponified and C was extracted from tissues and serum as previously described by our laboratory (Lederman et al. 1998). Saponification does not alter the all-trans/9-cis C ratio in tissues (data not shown).

Preparation of intestinal mucosal samples.  The small intestine of one ferret supplemented with Betatene C was removed and thoroughly rinsed with ice-cold saline (9.0 g/L NaCl) before being sliced longitudinally. The mucosal layer was then scraped with a glass slide in 20-cm increments from duodenum to ileum. The weight of each increment was recorded. Mucosal scrapings were extracted and analyzed by HPLC for C as described for tissue samples.

HPLC analysis.  Quantification of C by HPLC was performed as described by Lederman et al.(1998).

Statistical analysis.  All analyses were compared between groups using one-way ANOVA and Fishers Protected Least Significant Difference analysis (StatView 512⁺, Brain Power, Calabasas, CA). Differences were considered significant at P < 0.05. Data shown represent group means ± SD. Linear regression was performed using CA-Cricket Graph III version 1.0 (Computer Associates International, Islandia, NY).

	RESULTS

Abstract Introduction Methods Results Discussion References

There was no effect of carotenoid dosing on food intake, body weight or apparent health of the ferrets (data not shown). Because the liver is the major site of accumulation for dietary C and vitamin A in ferrets (Ribaya-Mercado et al. 1989, White et al. 1993), liver stores of all-trans and 9-cis C were used as the primary indicator of C absorption and storage. Under similar experimental conditions, we found low conversion efficiency of C to VA (< 15:1) (Lederman et al. 1998). Thus, accumulated hepatic C should account for most of the C absorbed from diet.

The predominant C isomer in the livers was all-trans for all groups (Table 2). Liver concentrations of all-trans C were significantly higher in the Betatene A group than in all other groups except the C beadlet high control group. The concentration of 9-cis C in the liver was significantly higher in the Betatene C group compared with any other group, whereas both the C beadlet control groups had significantly lower hepatic 9-cis C than all other groups. For all groups except the low C control, the liver had a higher all-trans/9-cis C ratio than that of the supplement provided (Tables 1 and 2). Liver stores of all-trans and 9-cis C were both positively correlated with the amount in the daily supplemental dose; however, the liver stores of all-trans C increased at a substantially higher rate than 9-cis as the amount in the oral dose increased (Fig. 1). The percentage of C provided in the 9 d of supplemental doses recovered in the liver was between 2.6 and 8.2% for all-trans C, but only 0.7-1.2% for 9-cis C (Table 2).

View this table: [in this window] [in a new window]   Table 2. Hepatic concentrations of all trans (a-t) and 9- cis (9-c) -carotene (C), and percentage recovery of all-trans and 9-cis C from liver after 9 D of oral supplementation of ferrets with algal extracts of all-trans and 9-cis -carotene1,2

View larger version (15K): [in this window] [in a new window]   Fig 1. The amount of all-trans and 9-cis -carotene (C) in the liver of ferrets is positively correlated with the amount consumed in a daily supplement.

Serum from ferrets fed Betatene A, D. bardawil or high C control had higher concentrations of C than the other groups (Table 3). As with the liver, there was a linear relationship between the amount of all-trans C in the diet and the amount in the serum (r² = 0.77) (data not shown). Serum levels of 9-cis were below detection levels or very low. Only four ferrets from the Betatene C and two ferrets from the D. bardawil groups had detectable levels of 9-cis C (data not shown). The lower limit of detection of 9-cis C was 0.004 nmol/L serum.

View this table: [in this window] [in a new window]   Table 3. Concentration of all-trans (a-t) -carotene (C) and 9-cis C in the serum and tissues of ferrets after 9 d of oral supplementation with algal extracts of all-trans and 9-cis -carotene1

The ferret kidneys and adrenals had a pattern of all-trans and 9-cis C concentrations similar to that seen in the livers (Table 3). However, in most groups, the ratio of all-trans/9-cis C in these tissues was substantially higher than the ratios in the diet or in the liver.

The small intestine of a single ferret, dosed with Betatene C with a ratio of all-trans to 9-cis C of 0.4, was removed at the termination of the study. Nine sequential segments (section one beginning at the duodenum and section nine ending at the ileum) were analyzed for the concentration of all-trans and 9-cis, C (Fig. 2). There was substantially more all-trans than 9-cis C in the mucosal cells obtained from every segment of the intestine. Mucosal cells from the first four segments contained higher amounts of C than the latter five. The 9-cis C isomer was not detectable in the last two segments of the intestine.

View larger version (24K): [in this window] [in a new window]   Fig 2. The concentration of all-trans and 9-cis -carotene (C) in the mucosal cells from intestinal segments of a ferret supplemented for 9 d with a C dose consisting of 1.9 µmol all-trans and 5.4 µmol 9-cis C.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Several studies have analyzed human serum for isomers of -carotene. Stahl and co-workers (1993) reported that the predominant C isomer in human serum is all-trans and that only negligible amounts of 9-cis C are found, suggesting that there may be preferential uptake and absorption of all-trans C compared with the 9-cis C isomer. In contrast, this same group analyzed the concentrations of C and lycopene isomers in selected human tissues and found considerable levels of 9-cis C along with the other C isomers (Stahl et al. 1992 and 1993). The cis isomers of C are reported to comprise 15-30% of the C in tissues, but constituted only ~5% of the C in serum (Stahl and Sies 1994).

Gaziano et al. (1995) found that humans supplemented with a 50/50 mixture of 9-cis and all-trans C had serum increases in both isomers; however, the change in serum 9-cis C accounted for only a small portion of the total C change. They observed the same changes in subjects supplemented with only all-trans C, suggesting an absorption discrimination between C isomers. The presence of 9-cis C in the tissues of animals fed a diet containing little 9-cis C may also be due to isomerization of the all-trans isomer to 9-cis before or during absorption, or within tissues.

Very low levels of 9-cis C are seen in the serum of individuals fed pure 9-cis C, possibly due to isomerization of 9-cis C into all-trans C. This isomerization may occur in the gastrointestinal tract, in the mucosal cells or after absorption. Tamai and co-workers (1995) fed either a mixture of 9-cis C or pure all-trans C to subjects for 44 wk and found that serum levels of 9-cis C were highest in the group fed only the all-trans C. Stahl et al. (1993) suggested that a tissue isomerase that converts 9-cis C into the all-trans form may be present. The most supportive evidence of isomerization of 9-cis C to all-trans C is provided by You and co-workers (1996). In that study, humans were fed a dose of highly purified (99.9%) ¹³C-labeled 9-cis C. Analysis of the ¹³C-C in the blood revealed substantial quantities of ¹³C all-trans C but only negligible amounts of ¹³C 9-cis C. It is possible that some 9-cis C is converted to all-trans C in the gastrointestinal tract or, more likely, within mucosal cells. In a study by Kemmerer and Fraps (1945), rats were fed 9-cis C; at 4 and 6 h after feeding, increased levels of all-trans C and 13-cis C were recovered in the feces, providing evidence of C isomerization. Tang and Serfaty (1995) evaluated the effects of gastric pH in humans on the isomerization of C. They provided evidence that the high acidity in the stomach can result in the isomerization of 9-cis C into all-trans C, although the extent of this isomerization in the stomach is unknown.

Because many of the previous studies have used only one source of 9-cis C (Betatene) and only one ratio of all-trans/9-cis C, it was the objective of this study to determine if uptake and tissue accumulation of 9-cis C, derived from two algal sources, Dunaliella baradwil and Dunaliella salina, differed in domestic ferrets.

The data indicate that there is a preferential uptake of all-trans over 9-cis C in domestic ferrets. Mucosal cell concentration of C isomers was evaluated in only one ferret, but segmental analysis (Fig. 2) supports the preferential uptake of all-trans over 9-cis C by the intestinal mucosal cells. The ferret used for this study had been supplemented with the Betatene C, which had over twice as much 9-cis as all-trans C, but the concentration of all-trans C in the mucosa was ~10-fold higher than the concentration of 9-cis C. The existence of substantial isomerization of 9-cis C to all-trans C before, during and/or after uptake into the mucosal cells cannot be ruled out.

Serum of ferrets fed 9-cis C was essentially free of 9-cis C, which suggests poor absorption of this isomer. However, all of the solid tissues examined in this study contained detectable levels of 9-cis C. The liver had the largest concentration of both all-trans and 9-cis C. In the liver, 2.6-8.2% of the administered dose of all-trans C was recovered compared with ~1% of the administered 9-cis C. Thus, the 9-cis C that was absorbed must have been quickly cleared by the liver and other tissues. The adrenal glands had high concentrations of all-trans C, but very little 9-cis C. The kidneys had low amounts of both isomers. All three tissues appeared to have concentrations of all-trans C that increased as the dose increased. Only the liver had a high enough concentration of 9-cis C to determine a dose dependency. 9-cis C was detected in tissues of ferrets fed the low all-trans C supplement containing undetectable levels of 9-cis C, suggesting that 9-cis C may be a normal metabolite of all-trans C in tissues.

This laboratory has also evaluated the absorption of 9-cis C in preruminant calves (unpublished data). That species also demonstrated substantial tissue levels, of 9-cis C, particularly in the liver, but an absence of serum 9-cis C after a dose with pure 9-cis C or mixtures of 9-cis and all-trans C.

The presence of 9-cis C in the tissues implies that some dietary 9-cis C is absorbed and that the isomer travels through the blood. However, detectable levels of 9-cis C in serum were found only in four of seven ferrets from the Betatene C and two of six ferrets from the D. bardawil group. One explanation may be that the liver and other tissues clear the 9-cis more quickly than the all-trans from the blood. Another explanation may be that the 9-cis C is isomerized to all-trans C before being absorbed, and isomerized back to 9-cis C once in the tissues.

Groups of ferrets were fed doses of 9-cis C from two commercial sources, Betatene and D. bardawil, to determine whether the source of 9-cis C affected utilization. There were no differences in tissue concentrations of 9-cis C between the two sources or in the percentage of the dose found in the liver.

In summary, the results of this study suggest the following: 1) all-trans C is preferentially absorbed compared with 9-cis C; 2) 9-cis C is not retained in the serum; 3) there is either preferential clearance of 9-cis C from the blood by liver and other tissues compared with all-trans C and/or isomerization of all-trans C to 9-cis C in the tissues; and 4) the specific Dunaliella source of 9-cis C does not substantially affect its uptake and tissue distribution in domestic ferrets. The results of this study with domestic ferrets support and expand the findings in humans (Jensen et al. 1987, Stahl et al. 1993, Tamai et al. 1995, You et al. 1996) and preruminant calves (unpublished data).

The differential absorption and tissue deposition of 9-cis C compared with all-trans C may not be important metabolically. However, because 9-cis C may be converted to 9-cis retinoic acid, limitation of 9-cis C absorption and rapid clearance of 9-cis C from serum may be a regulatory mechanism to reduce the unwanted production of the RXR ligand, 9-cis retinoic acid.

	FOOTNOTES

Manuscript received 1 May 1998. Initial reviews completed 1 June 1998. Revision accepted 29 June 1998.

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