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*
Division of Nutritional Sciences,
Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801
2To whom correspondence should be addressed. E-mail: jwerdman{at}uiuc.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • all-trans ß-carotene • 9-cis ß-carotene • 13-cis ß-carotene • vitamin A • gerbils
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–4
). Human serum contains largely atßC with only small or negligible amounts of 13cßC and 9cßC (5
–10
) after the ingestion of ßC isomers. However, considerable amounts of cis isomers of ßC are present in various human (11
,12
) and animal tissues (13
–17
). These observations support the suggestion that ßC isomers may possess isomer-specific biological actions (6
). All-trans and 9-cis ßC, in particular, have received the most attention because they can be metabolized to their respective retinoic acid isomers, all-trans retinoic acid and 9-cis retinoic acid (18
,19
), both of which are active in gene regulation (20
,21
).
Consumption of cis isomers of ßC from foods may be substantial, but their bioavailability is poorly understood. Results from human studies consistently report elevated serum/plasma responses after ingestion of atßC, whereas the 9cßC response is negligible even after ingestion of large amounts of this isomer (5
–9
). A similar trend was reported in chylomicrons of humans (22
), suggesting a preferential enrichment of the all-trans isomer in the small intestinal mucosa. There is also some plausible evidence that ßC isomerizes at some point during the digestive, uptake or absorption processes (23
,24
), and that the rates of cleavage of ßC isomers to vitamin A (VA) and the composition of the respective isomer metabolites vary (18
,25
). Despite low serum and chylomicron concentrations, 9cßC is present in substantial quantities in liver tissue from humans (12
); it accumulates in liver tissue of chicks (14
), rats (15
) and ferrets (17
) after ingestion of mixtures of atßC and 9cßC. The 9-cis and 13-cis ßC isomers also appear to be less efficient precursors of liver retinol than the all-trans isomer (26
,27
). The contrasting serum responses and tissue accumulation of ßC isomers and their metabolites may be explained by differences in intestinal absorption, conversion to VA, rates of uptake of ßC from the circulation by tissues and/or isomerization in tissues.
The objective of this study was to investigate some of these factors by comparing the relative bioavailabilities of atßC, 9cßC and 13cßC in gerbils 6 h after an oral dose of each isomer in oil. Because gerbils absorb ßC intact at physiologic doses (28
), it is possible to determine tissue levels of ßC and VA in a relatively short time period after a single dose. We examined events in the early stages of digestion by comparing the ßC isomer composition of the purified dose to that in the residual contents of the stomach and the small intestine. We also estimated potential differences in the uptake of the ßC isomers by quantifying their levels in mucosal scrapings from the small intestine. Serum responses and tissue accumulation of ßC isomers were evaluated to explore whether differences in tissue uptake are determinants of serum responses. Finally, we quantified total VA in liver to evaluate the value of each isomer as a precursor of VA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Standards of crystalline ßC isomers (a generous gift from Dr. Regina Goralczyk, Hoffmann-La Roche, Basel, Switzerland) were evaluated photometrically after dissolution in hexane. Concentrations were calculated using E(1%, 1 cm) = 2590 at 450 nm for atßC, E(1%, 1 cm) = 2550 at 445 nm for 9cßC, and E(1%, 1 cm) = 2090 at 443 nm for 13cßC (29
). Echinenone (gift from Hoffmann La-Roche), was also dissolved in hexane and used as an internal standard for quantification of ßC isomers. Standards of all-trans retinol [E(1%, 1 cm) = 1850 in ethanol at 325 nm, Sigma Chemical, St. Louis, MO], 13-cis retinol (Sigma Chemical, St. Louis, MO) and 9-cis retinol (Hoffmann-La Roche) were dissolved in ethanol. The purity of all standards was verified using HPLC (atßC and 9cßC were 
99–100%; 13cßC was 95% with impurities of atßC; all-trans retinol was 99–100%).
Animals, diet and study design.
Male Mongolian gerbils (n = 26; Meriones unguiculatus), 28 d of age, with a body weight of 30 ± 2 g were obtained from Charles River Laboratories (Raleigh, NC). Upon arrival, gerbils were individually housed in plastic shoebox cages and given free access to water and a pelleted depletion diet, i.e., AIN93G diet (30
) formulated with vitamin-free casein, cottonseed oil and without any known source of VA or ßC for 49–52 d. The duration of the depletion period was based upon the amount of time required to reduce initial liver VA status, which was estimated to be 60 d according to Thatcher and co-workers (31
). The goal for this depletion period was to reduce liver VA stores in gerbils to a "marginally sufficient" status (
20 µg/g or 70 nmol/g liver). The advantages of a depletion period include decreasing experimental variance by equalizing liver VA levels before treatment and increasing the ability to detect treatment effects by reducing liver VA levels to a "marginally sufficient" status. The overall health of the gerbils was monitored daily and body weight was recorded once each week. Room temperature was constant and lighting was provided on a diurnal cycle of 12-h light:12-h dark for the duration of the study. The University of Illinois Laboratory Animal Care Advisory Committee approved all animal handling procedures.
After depletion, the gerbils were assigned to one of four treatment groups, and subsequently repleted with a single, oral dose of cottonseed oil with or without ßC. Three groups of gerbils (n = 7) received atßC, 9cßC or 13cßC in oil by gastric intubation. We calculated a ßC dose of 387 nmol on the basis of the daily requirement of VA from the full formula AIN93G diet, assuming an intake of 6–10 g food/(d · 100 g body) and between 6:1 and 13:1 molar conversion efficiency of ßC isomer to VA. The gerbils in the control group (n = 5) received a dose of oil without ßC. Each gerbil was anesthetized before gastric intubation.
Briefly, the gerbil was placed in an induction chamber connected to a precision vaporizer, and isoflurane (2.5–3%) was administered with oxygen (1.5%) by inhalation until loss of consciousness. The oil dose was administered using a 1-mL syringe inserted into a stainless steel gastric feeding needle (20-gauge x 3.8 cm) with a ball tip (1 mm). The gerbil was then returned to its cage and regained consciousness within 15–30 s. The syringe was weighed before and after gavage to determine the actual oil dose delivered to each gerbil. In a pilot study, we observed that gerbils practiced coprophagy to a greater extent when deprived of food overnight and after treatment (unpublished). Thus, in this study, gerbils were given free access to water and food before and after treatment, which substantially decreased the amount of feces and food observed in the upper gastrointestinal tract at the time of killing.
After dosing (6 h), the gerbils were anesthetized by CO2, and blood was collected immediately by cardiac puncture, followed by cervical dislocation. We chose the 6-h time point on the basis of the amount of time required to simultaneously optimize serum and liver responses of ßC after a single oral dose of atßC according to Pollack and co-workers (28
). Blood samples were centrifuged for 10 min at 2400 x g at 4°C and serum was collected. Samples of residual stomach contents and small intestinal contents were removed from each gerbil. The small intestine was removed and thoroughly rinsed with ice-cold, 9 g/L NaCl solution. A 15-cm section of small intestine was sliced longitudinally and mucosal cells were collected by scraping the tissue with a glass slide. Liver, lungs, adrenal glands, kidneys and spleen were removed from all gerbils, rinsed with saline, dried and weighed. All tissues were snap-frozen in liquid nitrogen, stored at -80°C and analyzed within 4 mo. A partial dose recovery from the upper gastrointestinal tract was performed in two gerbils from each treatment group on two separate days. Hemostats were used to clamp off the stomach and small intestine to prevent loss of contents before excision. Each of these tissues were then excised, rinsed twice with 20 mL ice-cold 9 g/L NaCl solution into preweighed plastic bags, stored in ice at 4–5°C and analyzed within 24–48 h. Experimental procedures were performed in 4 d. Each treatment was represented across the 4 d to avoid any bias.
Oil dose preparation and analyses.
Crystals of atßC, 9cßC and 13cßC were solubilized in equal amounts of cottonseed oil. Briefly, stock solutions of each ßC isomer were prepared by dissolution in hexane (100 mg ßC/L). Specific volumes of the stock solution were then pipetted into an amber-colored vial containing an appropriate volume of oil and placed under argon to evaporate the hexane. Oils were mixed by hand for 30 s between additions of stock solution. Complete evaporation of hexane was confirmed gravimetrically, and solubility of ßC in oil was confirmed by light microscopy using 100X magnification.
Preliminary storage studies of the oils were conducted at ambient temperature in our laboratory to monitor recrystallization of ßC in oil. Crystals were first visible only in the atßC oil on d 3 of storage, whereas no crystals were visible in the 9cßC or 13cßC oils at that time. Therefore, ßC isomer oils were prepared every 2 d during the 4-d dosing period. The purity of the ßC isomer oils and the molar concentration of the ßC isomer in the oils were evaluated by HPLC using the method of Yeum et al. (32
). Duplicate aliquots of each ßC isomer oil (225 mg) were extracted with hexane (1 mL) without saponification. A 20-µL portion of the hexane solution was collected and evaporated using an Automatic Speed Vacuum System, Model AES1010 (Savant Instruments, Farmington, NY). Extracts were flushed with argon and analyzed immediately.
Tissue extraction procedures.
The stomach contents, small intestinal contents, serum samples (300–800 µL) and mucosal scrapings of the small intestine were mixed with absolute ethanol containing 1 g/L BHT and extracted three times with excess hexane without saponification. Liver tissue was extracted in duplicate. Tissue samples (200–250 mg) were minced and mixed with 4 mL of BHT/ethanol to denature protein, and were saponified with 1 mL potassium hydroxide (500 g/L) in a water bath at 60°C for 25 min. Samples were immediately placed in ice and cooled to ambient temperature. Distilled water (3 mL) was added and this mixture was extracted three times with hexane (8 mL). Hexane layers were combined and evaporated as described above. Dried extracts were flushed with argon and stored at -20°C. A separate extraction of liver tissue was completed to evaluate liver retinol isomers. Extractions of the lungs, adrenal glands, kidneys and spleen were performed on groups of pooled tissues (n = tissues from 2 gerbils/group) from each treatment group. Each group consisted of tissue from the same two gerbils across tissue analysis. Pooled tissues were minced and extracted as described for liver tissue. Samples of the pelleted diet were also extracted in triplicate by methods described previously (33
). Analysis of retinol using HPLC was completed immediately after extraction, and that of ßC within 24 h of extraction.
The potential effects of extraction procedures on isomerization of ßC and retinol were considered and minimized by performing all procedures under yellow lighting, using BHT in solvents and securing test tubes in ice immediately after saponification and during hexane extraction. We did not observe extensive isomerization of ßC in tissues with the mild heating conditions of saponification (20–30 min, 60°C) used in our laboratory. In addition, carotenoid standards remained stable in our laboratory when dissolved in hexane and stored in amber glass vials flushed with argon for up to 2 wk at -20°C.
HPLC analyses.
ß-Carotene isomers in the oils and tissues were separated using a 250 mm x 10 mm YMC C30 column (YMC, Wilmington, NC) at 450 nm according to the gradient, reverse-phase HPLC method of Yeum and co-workers (32
). Individual isomers of ßC from tissues were identified using retention times of standards (13cßC at 13.7 min, atßC at 15.3 min, 9cßC at 15.9 min) and quantified relative to echinenone (internal standard) by determining peak areas calibrated against known amounts of the standards.
Retinol was separated isocratically using a Supelco LC-18 column (#58298 Supelco, Bellefonte, PA) and a mobile phase of methanol/acetonitrile/chloroform (47:47:6, v/v/v) at a flow rate of 1.5 mL/min with detection at 325 nm. Retinol was identified by retention time of the all-trans retinol standard (3.9 min) and quantified by determining peak areas calibrated against known amounts of this standard. Retinol isomers in liver tissue were evaluated according to the method of MacCrehan and Schonberger (34
). Briefly, retinol isomers were separated using a reverse-phase Vydac C18 column [Vydac 201 TP 54, 25 cm x 4.6 mm (i.d.); The Separations Group, Hesparia, CA] with an isocratic mobile phase of methanol/butanol/water (10 mmol/L ammonium acetate) (65:25:10, v/v/v) at 1 mL per min under detection at 325 nm. Retinol isomers in liver were identified using retention times of the standards (9c-retinol at 15.3 min, 13c-retinol at 16.0 min, at-retinol at 17.7 min).
The HPLC system for all analyses was comprised of a Dynamax Model SD200 Solvent Delivery System, a Dynamax Absorbance Detector UV D-I, and a Dynamax HPLC Methods Manager integrator (Rainin Instrument, Woburn, MA). The analytical column for each method was protected by a precolumn (Upchurch Scientific, Oak Harbor, WA) packed with ODS C-18 (Alltech Associates, Deerfield, IL). All samples were injected manually. Standards of ßC isomers and retinol isomers were injected periodically throughout the day of analysis to establish retention times of those compounds from tissues.
Statistical analyses.
Data analyses were completed using the Statistical Analysis System (SAS, 6.12, Institute, Cary, NC). The proportions of total ßC cis isomers in the oil doses, stomach contents, small intestinal contents, mucosal scrapings from the small intestine, serum and liver were analyzed using one-way ANOVA. If the F-statistic for the ANOVA was significant, means were further evaluated by least-square difference (LSD). The quantifiable levels of total ßC and retinol in the oil doses and tissues were analyzed using 1) one-way ANOVA and orthogonal contrast coding to test the effect of ßC isomer on levels of total ßC and total retinol in tissues, and 2) one-way ANOVA and LSD. Significance of differences was determined using a P-value < 0.05. All values are represented by means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The body weight of gerbils more than doubled from 30 ± 2 g to 67 ± 2 g during the 49- to 52-d depletion period. Liver VA of gerbils in the control group after VA depletion [10 ± 3 µg/g (34 ± 9 nmol/g liver)] was lower than we predicted [ 20 µg/g (70 nmol/g liver)]. HPLC analysis of the depletion diet revealed that it contained a small amount of vitamin A [0.5 ± 0.2 µg/g diet (1.7 ± 0.1 nmol/g diet)] and trace levels of ßC.
ßC isomer oils.
The dose of oil (185 ± 2 mg oil) and the total ßC in the oils did not differ. The purity of ßC isomers in the respective oils was relatively high compared with the crystalline isomer, indicating that isomerization was minimized during the oil preparation procedures. The atßC oil (97.8 ± 0.4% atßC, 1.2 ± 0.4% 9cßC, 1.0 ± 0.1% 13cßC) contained 2075 ± 7 nmol total ßC/g oil or 384 ± 3 nmol total ßC/dose of oil. The 9cßC oils (1.8 ± 0.6% atßC, 98.7 ± 0.6% 9cßC, 0% 13cßC) contained 2116 ± 5 nmol total ßC/g oil or 391 ± 2 nmol total ßC/dose of oil. The 13cßC oil (8.0 ± 0.9% atßC, 0% 9cßC, 92.0 ± 0.9% 13cßC) contained 2085 ± 6 nmol total ßC/g oil or 386 ± 2 nmol total ßC/dose of oil.
ß-Carotene isomer profiles in contents of the digestive tract.
The proportion of total ßC as cis isomers in the oil dose, the residual contents of the stomach and the small intestine, the mucosal scrapings from the small intestine, the serum and the liver collected from gerbils in each ßC isomer group is illustrated in Figure 1
.
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A). The residual contents of the stomach from this group contained a significantly higher proportion of cis ßC (21 ± 3%) with the same proportion in the residual contents of the small intestine (22 ± 3%). However, the small intestinal mucosal scrapings contained a significantly higher proportion of cis ßC (34 ± 1%) than these two fractions. In addition, the 13-cis isomer represented most of the total cis ßC contained in these fractions. Interestingly, the proportions of cis ßC in serum (13 ± 2%) and liver (14 ± 1%) were significantly lower than that in the other fractions and were closest in cis ßC content to the dose.
The dose given to the 9cßC group contained 98.7% cis ßC (Fig. 1
B). A progressive and significantly lower proportion of cis ßC was observed in the residual stomach contents (89 ± 3% cis ßC) and in the contents of the residual small intestine (69 ± 4% cis ßC). However, the scrapings from the small intestine contained a significantly higher proportion of cis ßC (80 ± 2% cis ßC) than that in the residual contents of the small intestine. The proportion of total cis ßC in serum (43 ± 2%) was significantly lower than that observed in any other fraction. In contrast, the proportion of cis ßC in liver (88 ± 3%) was high and not different from that in the residual stomach contents. Most of the total cis ßC in all fractions was the 9-cis isomer.
The extent of isomerization appeared to be greatest in the tissue fractions from the 13cßC group. The oil dose administered to this group contained 92.0% cis ßC (Fig. 1
C). The residual contents of the stomach from this group contained a significantly lower proportion of cis ßC (60 ± 3%). The residual contents of the small intestine (48 ± 5% cis ßC) and of the mucosal scrapings from the small intestine (39 ± 2%) were lower than in the stomach. Most of the total cis ßC contained in these fractions was the 13-cis isomer. As in serum from the 9cßC group, the proportion of cis ßC in serum from the 13cßC group (18 ± 7%) was significantly lower than that in any other fraction. In addition, the liver contained a significantly higher proportion of cis ßC (38 ± 2%) than in serum, but in this case, the proportion was not different from that observed in the mucosal scrapings.
The differences in extents of isomerization in the residual contents of the stomach and small intestine among the ßC isomer oils were investigated further by a quantitative recovery of ßC in the upper gastrointestinal tract of gerbils (n = 2) 6 h after the oil was gavaged (Table 1
). The total amount of ßC in the residual contents of the stomach and the small intestine represents a portion of the amount not taken up. The recovery of ßC from the dose after 6 h was low (atßC group, 4.1%; 9cßC group, 0.6%; 13cßC group, 1.8%), and most of the ßC was present in the contents of the stomach. We visually observed substantial quantities of oil in the large intestines from gerbils in all groups, suggesting that a large portion of the oil had passed through the small intestine within 6 h.
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Quantitative levels of total ßC in the mucosal scrapings from the small intestine in serum and in liver of all groups are presented in Figure 2
and Table 2
. All ßC isomer-treated groups had significantly more total ßC in these three tissues than the control group. Although atßC was the primary isomer in tissues of gerbils intubated with atßC (Fig. 2
A) and 9cßC was the primary isomer in tissues of gerbils given 9cßC (except serum) (Fig. 2
B), the same trend was not observed for gerbils administered 13cßC (Fig. 2C)
. Tissues from these gerbils contained > 50% atßC.
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. Low levels of ßC were detected in the mucosal scrapings from the control group. The atßC group had higher levels of total ßC than the 9cßC group and the 13cßC group (P = 0.03). Total ßC levels in the 9cßC group were also higher than those in the 13cßC group.
Table 2
also shows the ßC in serum from each group. ßC was not detected in serum from the control group. Serum ßC concentrations in the atßC group were greater than those in the 9cßC and the 13cßC groups (P = 0.04). Although the 9cßC group had significantly higher serum ßC than the 13cßC group, serum ßC levels from both cis-treated groups were lower than levels in the atßC group and in some cases almost undetectable. Notably, the primary ßC isomer in serum of both cis groups was the all-trans (Fig. 2
B, C).
Total ßC quantified in liver from each group is shown in Table 2
. Although endogenous levels of ßC were quantified in the livers of the control group, total ßC was 11-, 4- and 1-fold higher in livers from the atßC, 9cßC and 13ßC groups, respectively. The primary ßC isomers in livers from the atßC, 9cßC and 13cßC groups were the all-trans, the 9-cis and the all-trans, respectively.
VA levels in serum and liver from each oil group are shown in Table 3
. The liver VA stores in all three ßC isomer-treated groups tended to be higher than the control group (P = 0.063) and did not differ from one another. Additional HPLC analyses revealed the presence of all-trans, 9-cis and 13-cis retinol in livers from all groups, and interestingly, the composition of retinol isomers did not differ among the groups (Fig. 3
). The isomeric composition of liver retinol for the control group was 90 ± 2% at-retinol, 7 ± 1% 13c-retinol and 3 ± 2% 9c-retinol and for the atßC group, 92 ± 2% at-retinol, 6 ± 1% 13c-retinol and 2 ± 2% 9c-retinol. Proportions of liver retinol isomers for the 9cßC group were 87 ± 4% at-retinol, 8 ± 2% 13c-retinol and 5 ± 3% 9c-retinol, and for the 13cßC group, 91 ± 1% at-retinol, 7 ± 1% 13c-retinol and 2 ± 1% 9c-retinol.
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Concentrations of ßC in lung, adrenal, spleen, and kidney from each group are shown in Table 2
. ßC was not detected in these tissues from the control group. Levels of total ßC in lung, adrenal, spleen and kidney from the atßC group were dramatically and significantly higher than those from the 9cßC and 13cßC groups. ßC in lung from the atßC group was 64% all-trans and 36% 9-cis ßC (data not shown). Adrenal glands contained 79% atßC and 21% cis-ßC, of which 15% was 13cßC. Spleen tissue from the atßC group contained 94% atßC and 6% 9cßC. Levels of ßC in kidney from the atßC group were lower than those in other tissues from this group. The isomer profile in kidney was 50% atßC and 50% cis-ßC, of which 51% was the 13c isomer.
Levels of ßC in lung were higher in the 13cßC group than the 9cßC group (P = 0.04). ßC in lung from the 9cßC group was 54% all-trans and 46% 9-cis ßC, and that from the 13cßC group was 24% all-trans and 76% cis ßC, of which 67% was 13cßC. In contrast to lung, levels of ßC in spleen were higher in the 9cßC group than the 13cßC group (P = 0.001). Spleen from the 9cßC group contained 31% atßC and 69% cis-ßC, all of which was 9cßC. ßC in spleen from the 13cßC group was 100% all-trans ßC. Adrenal gland from the 9cßC group contained 14% atßC and 86% 9cßC, and that from the 13cßC group contained 100% atßC. The composition of ßC isomers in kidney from the 9cßC group was 41% atßC and 59% 9cßC. ßC was not detected in kidney from the 13cßC group.
VA concentrations in lung, adrenal, spleen and kidney from each group are shown in Table 3
. There were no differences in VA in any of these tissues except for kidney. However, although VA levels were quantifiable in this tissue, they were extremely low compared with other tissues.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–4
). However, little is known about the bioavailability of cis isomers of ßC. Elevated levels of all-trans (at) ßC and 13cßC in human serum have been reported in response to dietary and supplemental intake of atßC, but the normally low serum levels of 9cßC are not increased, even after high doses of the 9-cis isomer. Yet, relatively high levels of 9cßC have been reported in tissues of humans (11
,12
) and several species of animals (14
–17
). In rats, 9-cis and 13-cis ßC also appear to be less efficient precursors of liver VA than the all-trans isomer (26
,27
). It is not clear whether this anomaly surrounding 9cßC bioavailability is because of in vivo isomerization, less efficient uptake into the small intestine, a difference in conversion to VA, and/or more rapid tissue uptake after absorption.
We explored these factors by evaluating the relative bioavailability of atßC, 9cßC and 13cßc in gerbils 6 h after a single dose of each isomer solubilized in cottonseed oil. We chose gerbils to study ßC bioavailability because of their ability to absorb ßC intact at physiologic doses (28
) and to convert ßC to VA with an efficiency similar to that of humans (35
). Dietary ßC also reverses marginal VA status in gerbils (31
,35
). Therefore, we expected to observe serum and tissue responses to a dose of atßC and 9cßC similar to those in humans.
The growth rate of gerbils in our study was not different from those observed by House and co-workers (36
) who reported no difference in growth between gerbils fed the AIN93G diet and those fed a commercial pelleted rodent diet (Prolab RMH 1000 Rodent Diet, Agway, Syracuse, NY). Liver VA of gerbils in the control group after 49–52 d of VA depletion was lower than we predicted, i.e., 10 ± 3 µg/g (34 ± 9 nmol/g liver). An adult human with a liver vitamin A concentration of < 10 µg/g is considered to have a poor-to-marginal vitamin A status (37
). However, according to Olson (38
), individuals in the marginal state do not show clinical signs of deficiency but may have impaired physiologic responses. This may have been the case with the gerbils in our study because they did not exhibit ill health or any physical signs of compromised VA status.
Initially, we were interested in exploring whether the differences in bioavailability of atßC, 9cßC and 13cßC were due in part to steric rearrangement or isomerization of ßC isomers in the digestive tract. Kemmerer and Fraps (23
) observed a progressive increase in nonspecific isomerization of 9cßC in the digestive tract and feces of rats 4 and 6 h after feeding 9cßC solubilized in oil. Substantial nonspecific isomerization has also been reported in the gut washings and feces of chicks 4 h after a dose of different carotenoids dissolved in oil and incorporated into a diet (39
). It has been suggested that ßC isomerization in the digestive tract may be because of heat, gastric pH and/or intestinal microflora (40
).
Similar to the observations of Kemmerer and Fraps (23
), nonspecific isomerization of ßC occurred in the digestive tract of gerbils in each ßC isomer group 6 h after the oil dose. Although significant changes in the proportions of cis ßC were observed in the residual stomach contents relative to the dose in all three ßC groups, additional isomerization in the small intestine occurred only in gerbils administered the cis ßC oils. Moreover, the extent of isomerization appeared to be higher in those gerbils gavaged with the 13cßC oil than those administered the 9cßC oil. The ßC composition in the contents of the small intestine was approximately a 50:50 and a 70:30 ratio of cis:trans ßC for gerbils given the 13cßC and the 9cßC oils, respectively. These ratios are in striking contrast to the 20:80 ratio of cis:trans ßC in the contents of the small intestine of gerbils administered the atßC oil. In quantitative kinetic studies using 1H NMR, Doering and co-workers (41
) suggested that the rate of cis-to-trans isomerization decreases progressively as the location of the cis bond departs from the central position, which could explain the higher proportion of trans ßC in the small intestinal contents from gerbils gavaged with the 13cßC oil compared with those given the 9cßC oil. Although the reduction in the amount of cis ßC in the small intestine of gerbils administered the cis ßC oils may be due to isomerization of cis isomers of ßC to the all trans isomer, it is possible that the reduction is also due to a preferential uptake of cis isomers of ßC into the small intestine over the 6-h period after dosing.
Indeed, we observed a small, but significant, upward shift in the proportion of cis isomers between the small intestinal contents and the intestinal mucosal scrapings of gerbils given the atßC oil and the 9cßC oil. Boileau and co-workers (42
) reported a similar enhancement of cis lycopene isomers in mucosal tissue of ferrets, suggesting a selective incorporation of cis lycopene into bile acid micelles. Although a higher proportion of cis ßC in the mucosa could suggest selective incorporation of cis ßC into bile acid micelles before uptake, we did not observe this increase in cis ßC in the mucosa of gerbils administered the 13cßC oil. In fact, the levels of total ßC in the intestinal mucosal scrapings from gerbils given the atßC oil were higher than those of gerbils administered the cis ßC oils, suggesting a preferential uptake of the all-trans isomer.
This observation is consistent with a report by Stahl and co-workers (22
) in which a 10- to 50-fold higher accumulation of atßC in chylomicrons in humans was observed after ingestion of a 1:5 mixture of atßC and 9cßC. This group suggested that an isomer-selective mechanism may be operative in the intestinal mucosal cell, involving specific uptake or incorporation steps, which excludes the 9-cis isomer from accumulation into chylomicrons (22
). Of course, it is also possible that cis isomers are preferentially absorbed, perhaps 13-cis more than 9-cis, and then more rapidly taken up by tissues compared with the all-trans isomer. Johnson and co-workers (9
) reported that atßC reached a higher postprandial concentration in serum, but that 9cßC reached peak levels sooner, after a single dose of either atßC or a mixture of all-trans and 9-cis ßC in men. Their hypothesis was that these findings were due to either poor absorption or very rapid tissue uptake of 9cßC.
In addition to differences in intestinal uptake, there is evidence to support differential conversion of ßC isomers to their respective VA metabolites in the intestinal mucosa, resulting in different levels of ßC isomers and their metabolites at a given time point. Using in vitro enzyme preparations of rat intestine, Nagao and Olson (18
) reported that atßC was the preferred substrate for cleavage to retinal, the direct product of this reaction, compared with 9cßC and 13cßC. In addition, 9cßC was converted to a mixture of 9-cis, all-trans and 13-cis retinals, whereas atßC and 13cßC were converted to all-trans retinal. Moreover, the rates of cleavage of 9cßC and 13cßC relative to atßC, i.e., 6.8 and 1.8%, respectively, in rat small intestine reported by this group suggest that only small amounts of cis isomers of ßC are actually cleaved to retinal (18
) and thus may be poor precursors of VA compared with atßC.
In humans, elevated serum levels of atßC and 13cßC have been reported in response to intake of atßC (5
–9
). We found similar elevated levels of atßC and 13cßC in serum and liver from gerbils administered the atßC oil. The proportions of cis ßC in serum (13.4%) and liver (14.1%) were not different; although they are higher, they are most consistent with that in the atßC oil dose (2.2%) compared with other fractions. The predominant ßC isomer in livers from the atßC group was the all-trans, which was 86% of the total ßC. In rats, 9cßC contributed up to 10% of the total ßC in liver after a dose of pure atßC dissolved in soybean oil (22
). The accumulation of 9cßC in liver has also been reported in chicks and rats after feeding synthetic ßC (13
).
The observation of low serum levels and relatively high tissue levels of 9cßC in gerbils given the 9cßC oil in our study are also consistent with observations in tissues of humans and animals (14
,15
,17
). In fact, the total ßC in serum from gerbils given either of the cis ßC oils was significantly lower than that of those administered the atßC oil, and the predominant ßC isomer in serum from gerbils gavaged with the cis ßC oils was all-trans (57% in 13cßC oil, 83% in 9cßC oil). Total ßC in livers of gerbils given the 9cßC and 13cßC oils were significantly higher than that of gerbils given the control oil. 9-cis ßC was the predominant isomer in livers (79% of total liver ßC) from the 9cßC group. In contrast, the all-trans isomer was 63% of the total ßC in livers from 13cßC group, and the 9-cis isomer was 21%. This value is similar to that reported in human liver samples from autopsy patients, in which 9cßC was up to 25% of the total ßC (12
).
The use of a 6-h time point in our study resulted in measurable tissue accumulations of ßC isomers in all ßC groups. We estimated the bioavailability of the dose of ßC isomer absorbed and stored in the liver from each group on the basis of the gain in liver ßC and VA. The relative bioavailability was estimated to be 38% for the atßC oil group, 27% for the 9cßC oil group and 32% for the 13cßC oil group. Although these values assume that liver ßC is an available source of VA, the actual contribution of accumulated liver ßC to the VA needs of the body is unclear (31
).
We observed a trend for greater liver VA storage in all ßC isomer-treated groups compared with the control group 6 h after the dose was given (P = 0.63; Fig. 3
). It is particularly noteworthy that the isomeric composition of liver VA was consistent in gerbils given either of the ßC isomer oils, ranging from 87 to 90% all-trans, 6–7% 13-cis, and 2–5% 9-cis. Levin and Mokady (43
) observed a similar isomeric composition in livers from chicks fed diets containing a synthetic mixture of atßC and 9cßC for 1 wk. Retinol stereoisomers have also been reported in tissues of rats (44
) and fish (45
).
The predominance of the all-trans isomer of VA in the liver may be due to the fact that all-trans ßC is the preferred substrate for cleavage. Alternatively, isomerization may occur during cleavage. It is also possible that all-trans retinol is the preferred substrate for the lecithin retinyl acyl transferase enzyme, which esterifies retinol to fatty acids before storage. Straight-chain all-trans retinyl esters may provide greater storage efficiency than bent-shaped cis retinyl esters. Based on the relative VA biopotency of retinol isomers (100% for all-trans, 76% for 13-cis, 19% for 9-cis) estimated by Weiser and Somorjai (46
) using rat vaginal smear assays, the isomeric composition of liver VA should also be considered when evaluating the VA biopotency of ßC isomers.
Although we did not evaluate the isomeric composition of VA in serum, You and co-workers (24
) reported substantial levels of [13c]all-trans retinol in human serum after a dose of [13C]9cßC, suggesting that low serum levels of 9cßC may be because of 9cßC isomerization occurring in vivo at some undetermined point during the digestive, uptake or absorptive processes. Interestingly, this group further hypothesized that cis-to-trans isomerization of 9cßC before its secretion in the bloodstream may be a mechanism to limit the supply of a 9-cis retinoid precursor to tissues, while not totally sacrificing the VA value of this isomer.
In summary, because of its apparent preferential uptake, transport and tissue accumulation, atßC appears to be a more bioavailable ßC isomer than the 9cßC and 13cßC isomers 6 h after a dose in gerbils. Although the apparent bioavailability of ßC isomers was differentiated in tissues within 6 h, liver VA stores were not different among the groups. Yet, the mean total liver VA followed an increasing numerical trend for the groups: atßC > 13cßC > 9cßC. Although we can not assign a relative VA biopotency for atßC, 9cßC and 13cßC from these data, we evaluated their biopotencies in a subsequent study using a series of doses over a 7-d period (47
).
|
FOOTNOTES |
|---|
3 Abbreviations used: atßC, all-trans ß-carotene; ßC, ß-carotene; 9cßC, 9-cis ß-carotene; 13cßC, 13-cis ß-carotene; VA, vitamin A.
Manuscript received 17 December 2001. Initial review completed 26 February 2002. Revision accepted 15 May 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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