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*
Division of Nutritional Sciences,
Department of Animal Sciences and
**
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: • vitamin A • all-trans ß-carotene • 9-cis ß-carotene • 13-cis ß-carotene • bioavailability • gerbils
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The actual contribution of ßC to the VA requirement may be influenced in part by the proportions of cis isomers of ßC in the diet. Although all-trans ßC (atßC) is accompanied by small amounts of 9-cis ßC (9cßC) and 13-cis ßC (13cßC) in raw fruits and vegetables (2
), the proportions of cis isomers in these foods may be greatly increased during processing (2
,3
).
The dietary intake of cis isomers of ßC may be substantial, but their metabolic fate is poorly understood. Cis isomers of ßC have been assigned a retinol equivalency that is 50% that of atßC (4
). Yet, the efficiencies of 9cßC and 13cßC as VA precursors in rats were >50% using a storage assay of liver VA (5
) and <50% using a growth assay (6
). Although we previously observed dramatic differences in the quantities of ßC in tissues of gerbils 6 h after a single dose of atßC, 9cßC and 13cßC, this length of time did not allow for estimation of the relative VA value (VAV) of each isomer (7
). Thus, the objective of this study was to determine the relative VAV of atßC, 9cßC and 13cßC. Total liver VA was quantified from groups of gerbils orally administered doses of oil containing increasing quantities of each ßC isomer for 7 d. VAV was then estimated by comparing slopes of dose-response lines using atßC as the reference.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Male Mongolian gerbils (Meriones unguiculatus, n = 57, age 28 d, 23 ± 2 g body weight, Charles River Laboratories, Raleigh, NC) were given free access to water and a modified AIN-93G diet formulated without VA or ßC (7
) for 56 d. All procedures were approved by the University of Illinois Laboratory Animal Care Advisory Committee. Ten gerbils were killed after depletion to establish baseline tissue VA. The remaining gerbils were assigned to one of 10 groups and repleted for 7 d by gastric intubation with a daily, oral dose of crystalline ßC solubilized in cottonseed oil or cottonseed oil alone. Nine groups (n = five) were divided equally among three ßC dosing treatments; atßC, 9cßC or 13cßC was provided at 141, 275 and 418 nmol/d. A control group (n = 3) received oil without ßC. To ensure solubility of the ßC crystals in the oil, 10 oils (3 oils/ßC isomer, plus a control oil) were freshly prepared every 2 d during the treatment period. Total ßC intake for each gerbil was the 7-d intake of ßC isomer from each oil. The gerbils were killed 24 h after the last dose, followed by collection of blood and organ tissues. Total VA and ßC were quantified by HPLC in serum, liver and lung tissue from each individual gerbil. Concentrations of VA and ßC were quantified in adrenal glands, spleen and kidneys from two pooled groups of tissue (2 gerbils/pooled group).
Data were analyzed using the Statistical Analysis System (version 6.12, SAS Institute, Cary, NC). Data for each ßC isomer oil were fitted to linear and quadratic models evaluating total liver retinol as a function of total ßC intake. If the quadratic model was significant, the data points for the highest dose were omitted, and the analysis was repeated. If the quadratic model was not significant, all data points were used to fit the data to a regression line. The ratio of slopes of the lines, using atßC as a reference, provided estimates of the relative VAV of ßC cis isomers expressed as a percentage. All values presented in the text are means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The baseline tissue VA levels were as follows: 1) liver, 36 ± 4 nmol (12 ± 2 nmol/g); 2) serum, 0.6 ± 0.1 µmol/L; 3) lung, 25 ± 3 nmol; 4) adrenal, 22 ± 2 nmol/g; 5) spleen, 18 ± 2 nmol/g; 6) kidney, 4 ± 1 nmol/g. Although the liver VA status suggests a high risk for VA deficiency, the gerbils did not exhibit ill health or physical signs of VA deficiency during the 56-d depletion period. Liver ßC content was 0.9 ± 0.7 nmol (0.3 ± 0.2 nmol/g). ßC was not detected in other tissues.
Total liver VA content increased linearly (P < 0.05) in gerbils administered atßC- (Fig. 1
A), 9cßC- (Fig. 1
B) and 13cßC- (Fig. 1
C) containing oils. The quadratic (P = 0.006) response to the data was significant for the atßC oil, but not for the cis ßC oils. Thus, the data points for the highest doses of atßC were omitted, thereby isolating the linear phase of the response. All data points were used to fit the linear regression line for the cis ßC oils. The equations for the lines were: atßC oil: y = 0.089x + 63.5 (r2 = 0.80); 9cßC oil: y = 0.034x + 44.8 (r2 = 0.65); and 13cßC oil: y = 0.055x + 48.0 (r2 = 0.81). A comparison of the slopes of these lines resulted in a relative VAV of 38% for 9cßC and 62% for 13ßC, compared with atßC.
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). The isomeric profiles of retinol in the livers of gerbils after the 7-d dosing period of atßC, 9cßC and 13cßC were similar (i.e., 88–93% at, 5–9% 13c, and 2–3% 9c).
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). Although liver VA was higher than liver ßC, we observed a similar linear increase (P < 0.05) in liver ßC in gerbils given ßC isomers. Relative to gerbils given atßC (i.e., 100%), total liver ßC was 67% in gerbils administered 9cßC and 42% in those given 13cßC. The major ßC isomer in tissues was atßC in gerbils administered atßC or 13cßC, but the principal isomer in gerbils gavaged with 9cßC was 9cßC.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Previous work investigating the VA biopotencies of cis isomers of ßC used either a growth or a liver VA storage assay (6
,8
,9
). Deuel and co-workers (6
) fed small graded doses of isomers of ßC for 28 d to rats using a growth assay. Guggenheim and Koch (8
) found that the liver storage assay was less time consuming and provided more precision than the growth assay, but required the administration of much larger doses of ßC. In our study, the use of gerbils provided the advantages of both assays in that we achieved increasing storage of liver VA using physiologic doses of ßC isomers according to Pollack and co-workers (10
) given over 7 d.
The depletion of tissue VA before carotene dosing has also been used in evaluating the VA biopotency of carotene isomers. Johnson and Baumann (9
) depleted rats (24–30 d) until they failed to gain weight over a 7-d period before repletion with carotenes. These rats exhibited the classic eye symptoms of VA deficiency. Because the liver may be substantially depleted of VA before any weight loss occurs, it has been suggested that carotene should be given before VA depletion results in a weight plateau (5
). During the depletion period in our study, gerbils were still gaining weight before ßC repletion.
Our VAV estimates of 38% for 9cßC and 62% for 13cßC suggest that 9cßC may have less and 13cßC more than the previously assumed retinol equivalency of 50% for cis isomers of ßC. Our data strongly suggest that both cis isomers have lower VA biopotencies than atßC, and also that 9cßC has a lower VAV than atßC, a lower VAV than 13cßC, and it has a VAV lower (relative to atßC) than 50%. The results are less persuasive that 13cßC has a VAV >50% of atßC. Indeed, calculation of slope values (
y/x) for responses between the control (no ßC dosing) and the first increment of ßC isomers yielded slopes (nmol total liver VA/nmol total ßC intake) of 0.872, 0.156 and 0.383 for atßC, 9cßC and 13cßC, respectively. Translating these slopes to VA biopotencies gives VAV (relative to atßC) of only 18% for 9cßC and 44% for 13cßC. Thus we conclude that the relative biopotency of 9cßC is both lower than that of 13cßC and <50% that of atßC, whereas the relative biopotency of 13cßC is 
50% that of atßC.
It is clear from rat studies and this gerbil study that the VA biopotencies are lower for cis isomers of ßC than atßC and that 9cßC is consistently ranked lower than 13cßC. However, the magnitude of difference from the assigned retinol equivalency of 50% that of atßC for these ßC cis isomers varies among studies. In rats, Deuel and co-workers (6
) and Johnson and Baumann (9
) reported a VAV notably <50% for 9cßC (i.e., 33 and 38%, respectively), and, in both studies, the VAV for 13cßC was essentially 
50% that of atßC (i.e., 48 and 53%, respectively). Estimates of the VAV of 9cßC reported by Weiser and co-workers (11
) were also consistently <50% that of atßC regardless of whether they used ovariectomized females in a growth assay (23%), a vaginal epithelial protection assay (26%) or VA-deficient males in a growth assay (23%).
In contrast to these studies, Sweeney and Marsh (5
) reported a VA biopotency much >50% of atßC for both 9cßC (61%) and 13cßC (74%) in rats (5
). They also observed a wide variation in liver VA in rats of similar weight fed the same ßC isomer at the same level. We observed similar variation in liver VA in gerbils, but lower relative VA biopotencies for 9cßC and 13cßC than did Sweeney and Marsh. Yet, our gerbil study is the only one reporting relative VA biopotencies of 9cßC and 13cßC, which appear to be notably lower and higher, respectively, than 50% that of atßC. In addition, the difference between the relative VA biopotencies of 9cßC and 13cßC is greater in gerbils than that reported in rats, which may be a result of differences in ßC metabolism between rats and gerbils. Differences in ßC metabolism are common among animals and between humans and animals. Thus, the applicability of our data in gerbils, as with other animal models, may be limited when comparing it with humans.
The lower VA biopotencies of 9cßC and 13cßC relative to atßC could be because of destruction in the digestive tract before absorption or slower absorption leading to greater loss in the feces (12
,13
). Yet, high recoveries of trans and cis ßC have been reported in feces of rats regardless of the isomer fed (5
,12
), suggesting little destruction but a high extent of isomerization of ßC in the digestive tract. Previously, we observed preferential absorption, transport and accumulation of atßC in tissues of gerbils 6 h after a dose of atßC vs. 9cßC and 13cßC (7
). Similar preferential accumulation of atßC has been reported in human serum (14
) and chylomicrons (15
). It is possible that the VA biopotency attributed to the cis isomers of ßC depends on the extent to which these isomers are converted to atßC before or after absorption, and for the most part, only that portion isomerized to atßC is cleaved to form VA. We have suggested that atßC appears to be the preferred substrate for ßC cleavage in gerbils (7
).
A few studies support in vivo isomerization of cis isomers of ßC. You and co-workers (13
) reported that 13C-labeled 9cßC yielded an equivalent amount of 13C-labeled all-trans retinol in human serum compared with the same oral dose of 13C-labeled atßC, suggesting that isomerization occurred during digestion, uptake and/or absorption. Kemmerer and Fraps (12
) reported progressive, nonspecific isomerization of 9cßC in the digestive tracts of rats after a dose of 9cßC. We also reported nonspecific isomerization of ßC isomers in the digestive tracts of gerbils (7
). A 50:50 cis:trans ratio of ßC was observed in the contents of the small intestine in gerbils administered 13cßC compared with a 70:30 cis:trans ratio of ßC in gerbils given 9cßC and a 20:80 cis:trans ratio in gerbils given atßC. Thus, the proportion of the all-trans isomer of ßC in the small intestinal contents of gerbils gavaged with 13cßC and 9cßC was 63 and 38% that contained in the small intestinal contents of gerbils administered atßC (7
). The presence of more atßC in the small intestine of gerbils given 13cßC compared with 9cßC (7
), combined with the results from the current study showing a higher relative VAV for 13cßC than 9cßC, suggests that isomerization before cleavage plays a major role in the VA biopotency of 13cßC and 9cßC.
The demonstration that the relative VAV of 9cßC was substantially less and that of 13cßC was somewhat more than the assigned value of 50% that of atßC may justify a revised retinol activity equivalent (RAE) for these isomers. On the basis of the current RAE (4
), 1 µg retinol = 12 µg of atßC from foods. Rather than the currently assigned RAE of 24 µg for provitamin carotenoids other than atßC, the RAE of 13cßC and 9cßC can be calculated to be 19 and 32 µg, respectively, on the basis of the relative VAV of these isomers (62 and 38%, respectively).
When vegetables are thermally processed, 30–50% of atßC may be converted to cis isomers (2
,3
). The proportions of atßC, 13cßC and 9cßC in canned carrots were reported to be 73, 19 and 8%, whereas those of canned spinach were 58, 15 and 25%, respectively (2
). On the basis of these proportions of isomers and the revised RAE values for 13cßC and 9cßC, the actual VA activity of canned carrots (with higher proportions of 13cßC than 9cßC) is higher and that of canned spinach (with higher proportions of 9cßC than 13cßC) is lower than expected using the current RAE. Although there are many factors that affect ßC bioavailability, the actual contribution of ßC to the VA requirement may be strongly influenced by the proportions of cis isomers of ßC consumed in the diet. Thus, isomers of ßC, especially from cooked foods, have to be considered in evaluating the VA biopotencies of ßC-rich vegetables consumed by marginally VA-deficient populations around the world.
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FOOTNOTES |
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3 Abbreviations used: atßC, all-trans ß-carotene; ßC, ß-carotene; 9cßC, 9-cis ß-carotene; 13cßC, 13-cis ß-Carotene; RAE, retinol activity equivalent; VA, vitamin A; VAV, vitamin A value.
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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1. de Pee, S., West, C. E., Permaesih, D., Martuti, S. & Muhilal & Hautvast, J. G. (1998) Orange fruit is more effective than are dark-green, leafy vegetables in increasing serum concentrations of retinol and ß-carotene in school children in Indonesia. Am. J. Clin. Nutr. 68:1058-1067.[Abstract]
2. Chandler, L.A. & Schwartz, S. J. (1987) HPLC separation of cis-trans carotene isomers in fresh and processed fruits and vegetables. J. Food Sci. 52:669-672.
3. Lessin, W. J., Catigani, G. L. & Schwartz, S. J. (1997) Quantification of cis-trans isomers of provitamin A carotenoids in fresh and processed fruits and vegetables. J. Agric. Food Chem. 45:3728-3732.
4. National Academy of Sciences, Institute of Medicine (2001) Dietary Reference Intakes: Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc 4:1-61 National Academy Press Washington, DC. .
5. Sweeney, J. P. & Marsh, A. C. (1973) Liver storage of vitamin A in rats fed carotene stereoisomers. J. Nutr. 103:20-25.
6. Deuel, H. J., Jr., Johnson, H. J., Meserve, E. R., Polgar, A. & Zechmeister, L. (1945) Stereochemical configuration and provitamin A activity IV. Neo-
-carotene B and neo-ß-carotene B. Arch. Biochem. 7:247-255.
7. Deming, D. M., Teixeira, S. R. & Erdman, J. W., Jr. (2002) All-trans ß-Carotene appears to be more bioavailable than 9-cis, or 13-cis ß-carotene in gerbils given single oral doses of each isomer. J. Nutr. 132:2634-2642.
8. Guggenheim, K. & Koch, W. (1944) Liver storage test for the assessment of vitamin A. Biochem. J. 38:256-262.[Medline]
9. Johnson, R. M. & Baumann, C. A. (1947) Storage and distribution of vitamin A in rats fed certain isomers of carotene. Arch. Biochem. 14:361-367.
10. Pollack, J., Campbell, J. M., Potter, S. M. & Erdman, J. W., Jr. (1994) Mongolian gerbils (Meriones unguiculatus) absorb ß-carotene intact from a test meal. J. Nutr. 124:869-873.
11. Weiser, H., Riss, G. & Biesalski, H. K. (1993) Uptake and metabolism of ß-carotene isomers in rats. Canfield, L. M. Krinsky, N. I. Olson, J. A. eds. Carotenoids in Human Health 1993:223-225 New York Academy of Sciences New York, NY. .
12. Kemmerer, A. R. & Fraps, G. S. (1945) The vitamin A activity of neo-ß-carotene and its steric rearrangement in the digestive tract of rats. J. Biol. Chem. 161:305-309.
13. You, C.-S., Parker, R. S., Goodman, K. J., Swanson, J. E. & Corso, T. N. (1996) Evidence of cis-trans isomerization of 9-cis-ß-carotene during absorption in humans. Am. J. Clin Nutr. 64:177-183.[Abstract]
14. Stahl, W., Schwarz, W. & Sies, H. (1993) Human serum concentrations of all-trans ß-carotene and -carotene but not 9-cis ß-carotene increase upon ingestion of a natural isomer mixture obtained from Dunaliella salina (Betatene). J Nutr. 123:847-851.
15. Stahl, W., Schwarz, W., van Laar, J. & Sies, H. (1995) All-trans ß-carotene preferentially accumulates in human chylomicrons and very low density lipoproteins compared with the 9-cis geometrical isomer. J. Nutr. 125:2128-2133.
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