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Department of Nutritional Sciences, University of Wisconsin, Madison, WI 53706
* To whom correspondence should be addressed. E-mail: jhowe{at}nutrisci.wisc.edu.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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3 µg ß-carotene to 1 µg retinol (1.5 mol ß-carotene to 1 mol retinol). The liver ß-carotene content was greater in the high-ß-carotene maize group (26.4 ± 6.0 nmol) than in the ß-carotene supplement group (14.1 ± 6.0 nmol; P < 0.05). In study 2, the gerbils' VA status improved with increasing dietary ß-carotene. Liver VA in gerbils fed orange maize was greater than in those fed yellow maize, regardless of maize percentage (P < 0.05). Biofortified maize adequately maintained VA status in Mongolian gerbils and was as efficacious as ß-carotene supplementation. In populations consuming maize as a staple food, using orange instead of white maize could dramatically affect VA status.
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Maize is a staple food for many VA deficient populations. In typical maize, provitamin A carotenoids include 
-carotene, ß-carotene, and ß-cryptoxanthin, but concentrations are low and range from 0 to 1.3, 0.13 to 2.7, and 0.13 to 1.9 nmol/g, respectively (4). Ongoing efforts to breed maize for increased provitamin A have resulted in varieties with 9–17 nmol/g total provitamin A carotenoids, primarily as ß-carotene (4). These concentrations are low compared with carrot (240 nmol ß-carotene/g fresh weight) (5). Low predicted bioconversion rates of ß-carotene, -carotene, and ß-cryptoxanthin to VA, i.e., 12, 24, and 24 µg to 1 µg all-trans retinol, respectively (6), and generally poor bioavailability of provitamin A carotenoids from food (7) question the bioefficacy of biofortified maize.
The bioavailability of provitamin A carotenoids from specific foods is not well understood. Before breeding efforts continue, it is essential to assess whether maize biofortification with provitamin A carotenoids can positively contribute to VA status. Many factors influence carotenoid absorption and bioconversion (8). Lack of methodology to directly measure bioavailability complicates the issue. Measuring the change in serum carotenoid concentrations following intervention has been used (7). Numerous factors affect results from this approach, including carotenoid and VA regulation in the blood and carotenoid bioconversion to VA preceding entry into the bloodstream (9). The conversion of ß-carotene to VA is tightly regulated and dependent on VA status and the amount administered in the dose or meal (10).
Other methods to evaluate bioavailability include postprandial chylomicron response (11), Caco-2 cells (12–15), stable isotope tracers (16,17), and animal models (18–21). Results from the postprandial chylomicron response model are highly variable among subjects, limiting their use (11). Caco-2 cells investigate bioaccessibility at the intestinal level, but do not reflect influences by the liver or other organs regulating enzyme activity and altering conversion factors. Isotope tracer studies in humans are the best method (17), but their expense is limiting and factors such as diet and VA status are difficult to control. Appropriate animal models provide a lower-cost alternative with greater experimental control. In addition, the use of animals permits direct measurement of liver VA, the best indicator of VA status (22). For carotenoids, rats and mice are not appropriate because, unlike humans, they absorb very little ß-carotene intact (18–20), but Mongolian gerbils absorb and metabolize ß-carotene similarly to humans (21,23–26).
The objective of this research was to investigate the bioefficacy of ß-carotene in biofortified maize in VA-depleted Mongolian gerbils. The first study compared the bioefficacy of ß-carotene from maize with VA and ß-carotene supplements. The second study investigated the effect of 2 types of maize at 2 dietary levels, yielding 4 different carotenoid concentrations, on VA status.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Four near-isogenic maize lines, generously provided by Dr. Torbert Rocheford, University of Illinois at Champaign-Urbana, were analyzed for carotenoid concentration (Table 1) and were used to prepare 6 pelleted Mongolian gerbil feeds with variable maize (Table 2) and carotenoid compositions (Table 3). The enhanced provitamin A content of maize was developed using traditional breeding approaches. For purposes of this study, maize lines are distinguished by color (i.e., white, yellow, orange, and dark-orange). Upon receipt, maize kernels were stored at –20°C (white and yellow maize) and –80°C (orange and dark-orange maize). Prior to feed preparation, maize kernels were ground to pass a 1-mm screen (particles <0.7 mm) using a C&N hammer mill 8 (Christy-Norris).
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Carotenoid composition of maize and feeds
Maize and diets were analyzed for carotenoid concentrations using a modified procedure (28). Maize flour or diet was ground with mortar and pestle and weighed (0.6 g) for analysis. Carotenoids were extracted according to the published procedure, except 500–750 µL of 80% (w:v) potassium hydroxide was added instead of 120 µL, due to the high oil content of the maize. Samples were reconstituted in dichloroethane:methanol (500 µL, 50:50, v:v), and injected (50–100 µL) into the HPLC system. ß-Apo-8-carotenal was used as an internal standard and was added post-saponification to account for mechanical losses. Preliminary experiments showed the greatest ß-carotene extraction at 85°C, but a 20–30% additional loss of internal standard occurred when added presaponification (data not shown).
Animals and procedures
Male 40-d–old Mongolian gerbils (n = 40/study) were obtained from Charles River Laboratories. Gerbils were individually housed in plastic shoebox cages and given free access to food and water. Gerbils were weighed daily and monitored for health until thriving, and thereafter weighed every 2 d. After the 4-wk depletion phase, gerbils were sorted into weight-matched treatment groups (n = 10/group) and placed on their respective diets. After 8 wk, gerbils were killed by exsanguination while under isoflurane anesthesia. Blood samples were centrifuged 2200 x g for 15 min in BD Vacutainer Gel and Clot Activator tubes (Becton Dickson) for serum isolation. Livers were excised and stored at –80°C until analysis. All animal handling procedures were approved by the University of Wisconsin-Madison's Research Animal Resource Center.
Experimental design
Study 1.
Dietary treatment groups included 60% dark-orange maize dosed with cottonseed oil, 60% white maize supplemented with ß-carotene in oil, 60% white maize supplemented with VA (as retinyl acetate) in oil, and 60% white maize dosed with oil as a negative control (Table 3). Each supplement was calculated from ß-carotene consumed from the dark-orange maize on the previous day, assuming 100% bioefficacy (i.e., 1 mol ß-carotene provides 2 mol VA). Equalization of the doses was based on ß-carotene because it is the primary provitamin A carotenoid in dark-orange maize contributing 80% of the theoretical VA in the maize. Dosing was performed twice daily, 6 h apart, to expand the absorption period for VA and ß-carotene.
Study 2. Treatment groups received 30 or 60% maize feed containing either orange or yellow maize (Table 3).
Preparation of ß-carotene and VA supplements for study 1
Oil doses of ß-carotene were prepared by dissolving ß-carotene supplements (GNC) into hexanes and then into cottonseed oil using sonication. Hexanes were removed by rotary evaporation and by flushing with argon; complete removal was verified by weight analysis. Retinyl acetate (Sigma) was dissolved directly into cottonseed oil using sonication. Final concentrations of ß-carotene and VA in oil were determined by dissolving an aliquot in hexanes and calculating the concentration using the 
[2592 for ß-carotene (29) and 1810 for VA (30)] at 450 and 325 nm, respectively; 40 µL oil delivered 26 nmol ß-carotene (14.2 µg) and 56 nmol VA (15.9 µg).
Serum and liver preparation for HPLC
All samples were analyzed under gold fluorescent lights to prevent photo-oxidation and isomerization. Retinyl butyrate (38 µmol/L methanol) was synthesized and used as an internal standard to determine extraction efficiency in serum (97 ± 6%) and liver (74 ± 10%). It was also used externally for quantification of retinol and retinyl esters. Modified published procedures were used for VA and ß-carotene analysis of serum (31–33) and liver (32). Serum (500 µL) was extracted 3 times with hexane (1 mL) and dried under argon. Liver (0.5–0.9 g) was ground with 3–5 g anhydrous sodium sulfate, extracted repeatedly with dichloromethane, and filtered into a 50-mL volumetric flask. An aliquot (5 mL) of the liver extract was dried under argon. Serum and liver samples were reconstituted in methanol:dichloroethane (100 µL, 50:50, v:v) and injected (50 µL) into the HPLC system described below using a Resolve C18 column (5 µm, 3.9 x 300 mm; Waters). Total liver VA reserves were calculated by summing retinol and all identifiable retinyl esters.
HPLC analysis
Analyses of carotenoids in maize and feeds were adapted from published procedures (34,35). A Waters HPLC system consisting of a guard column, C30 YMC carotenoid column (4.6 x 250 mm, 3 µm; Waters), 1525 binary HPLC pump, 717 autosampler, and either a 996 or 2996 photodiode array detector was used. Solvent A consisted of 92:8 methanol:water (v:v) with 10 mmol/L ammonium acetate. Solvent B was 100% methyl-tertiary-butyl ether. Gradient elution was performed at 2 mL/min with the following conditions: 29-min linear gradient from 83 to 59% A, 6-min linear gradient from 59 to 30% A, 1-min hold at 30% A, 4-min linear gradient from 30 to 83% A, and a 4-min hold at 83% A. Positive identification of lutein, zeaxanthin, ß-cryptoxanthin, and ß-carotene was determined using standards. Chromatograms were generated at 450 nm.
Statistical analysis
Values are means ± SD. Data were analyzed using Statistical Analysis System software (SAS, version 8.2). Outcomes of interest (i.e., gerbil weights, serum retinol concentration, and liver VA and ß-carotene concentrations) were evaluated using 1-way ANOVA. Differences between treatment groups were determined using least significant differences (LSD) at < 0.05.
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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No provitamin A carotenoids were detected in the 45% white maize feed used for the VA-depletion phases. Provitamin A carotenoid concentrations of the treatment maize feeds ranged from undetectable in the white maize diets to 8.85 ± 0.57, 0.99 ± 0.04, and 3.53 ± 0.02 nmol/g for ß-carotene, -carotene, and ß-cryptoxanthin, respectively, in the 60% dark-orange maize diet (Table 3). Food intake did not differ among groups in either study and ranged from 5.5 ± 1.5 g in the dark-orange maize group to 6.6 ± 1.0 g in the VA group in study 1 and from 6.1 ± 0.9 g in the 60% yellow maize group to 7.0 ± 0.7 g in the 30% yellow maize group in study 2.
Gerbil weights
Gerbil weight gain began to reach a plateau at 5 wk. For both studies, the final gerbil weights did not differ among groups and ranged from 72.9 ± 5.7 g in the negative control group to 76.3 ± 6.7 g in the VA group for study 1 and from 72.8 ± 4.8 g in the 30% yellow maize group to 77.8 ± 7.5 g in the 60% orange maize group for study 2.
Serum vitamin A and carotenoid concentrations
In both studies, serum retinol did not differ among treatment groups and carotenoids were not detected. The range of serum retinol concentrations in study 1 was 1.22 ± 0.16 µmol/L in the dark-orange maize group to 1.25 ± 0.22 µmol/L in the ß-carotene supplement group. In study 2, serum retinol concentrations ranged from 1.23 ± 0.16 µmol/L in the 30% yellow maize group to 1.32 ± 0.28 µmol/L in the 60% yellow maize group.
Liver VA and carotenoid concentrations
Study 1. As expected, liver VA concentration (Fig. 1A) and content (Fig. 1B) were greater in the VA supplement group than in other groups (P < 0.05). Hepatic VA in the negative control group was significantly lower than in the other groups due to continued VA depletion during the treatment phase. Hepatic VA in the ß-carotene supplement and dark-orange maize groups did not differ and was approximately half that of the VA group (P < 0.05). The only carotenoids present in livers in measurable quantities were cis- and trans-ß-carotene in the dark-orange maize and ß-carotene groups (Fig. 1C). The dark-orange maize group had a greater ß-carotene content than the ß-carotene supplement group (P < 0.05).
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Study 2. Liver VA reserves in the second study were positively correlated with increasing dietary provitamin A carotenoid concentration in the diet (r = 0.85, P < 0.001). The hepatic VA concentration was higher in both groups that consumed orange maize than those that consumed yellow maize (Fig. 2A, P < 0.05). On a total liver VA basis, the 60% orange maize group had more VA than either of the yellow maize groups, but the 30% orange maize group only had a greater concentration than the 30% yellow maize group (Fig. 2B, P < 0.05). Consumption of the maize feed did not differ among the groups (i.e., 6.5 ± 1.0 g feed/d). No carotenoids in measurable quantities were observed in the liver.
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130% higher from the 30% orange maize diet (Table 3). These results further support the roles of provitamin A carotenoids and ß-carotene in contributing to VA status.
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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-carotene comprise 20% of the total provitamin A in the diet. Depending on their bioavailability, these carotenoids may affect the conversion factor.
In a prior study, in which freeze-dried carrots were fed to Mongolian gerbils with an adequate VA status (33), conversion factors were calculated (36) using a daily VA utilization rate (i.e., 2.5 µg retinol/gerbil) from another study in gerbils (32). Conversion factors were estimated to be 9–11 µg ß-carotene to 1 µg retinol for typical orange carrots and 23 µg ß-carotene to 1 µg retinol for dark-orange carrots containing twice as much ß-carotene. Carrots, which are a rich source of ß-carotene, have very little oil and contain crystalline ß-carotene (37). Comparatively, the oil content in maize is much higher than in carrots. Because carotenoids are highly fat-soluble compounds, the higher oil content in maize could enhance carotenoid absorption. Effects of VA status (10), the food matrix (3,37), and the provitamin A concentration explain the differences between conversion factors.
The conversion factor for ß-carotene dissolved in oil, 2.9 µg ß-carotene to 1 µg retinol, is greater than that currently proposed by the Institute of Medicine (i.e., 2 µg ß-carotene to 1 µg retinol) (6) and assumes that all gerbils had similar utilization rates. The conversion factor for ß-carotene in oil in this study is also slightly greater than the values determined in a prior study of gerbils (i.e., 2.8 µg ß-carotene to 1 µg retinol) (32) and was obtained in Indonesian children using 13C-ß-carotene dissolved in oil (i.e., 2.6 µg ß-carotene to 1 µg retinol) (10). Conversion rates may be dependent on VA status (10) and the amount of carotenoid administered (8). Because both gerbil studies used a VA-depleted model and the VA status of Indonesian children is often depleted (38), VA status partially explains the similarities in these conversion factors. The gerbils received 3 times more supplemental ß-carotene than in the prior study (32), which is reflected in greater ß-carotene liver storage.
The percentages of maize selected in the second study encompass the contribution of staple cereal foods to typical diets of developing countries, as well as providing a range of provitamin A intakes for the gerbils. One difference between gerbil and human diets is the preparation of the maize. In a human diet, the maize is often soaked and cooked. Although the gerbil diets were not cooked, the pelleting process required the addition of water followed by drying at 60°C for 3 h, and then air drying until room temperature was reached (B. Mickelson, Harlan Teklad, personal communication). Although heat destroys carotenoids, especially in their pure form (39), preliminary studies found that heat (85°C) was required to release carotenoids from the maize and feed matrix. Additional studies are needed to determine the effect of various cooking methods on carotenoid retention and bioavailability.
In both studies, serum retinol concentrations did not differ among groups. In healthy individuals, serum retinol is homeostatically controlled (17,40). Only under severe VA deficiency would serum retinol values decrease. Liver VA is considered the best measure for VA status (22) because the liver is the primary storage site for VA. In study 1, the control group (0.1 µmol/g liver) was on the verge of VA deficiency, defined as 0.07 µmol/g liver. Thus, a difference in serum retinol concentration would not be expected. Carotenoids were not detected in the serum as observed (32) and discussed (33,41) in prior studies.
In the first study, the only carotenoid detected in the liver was ß-carotene, which was found in the cis- and trans- form in the dark-orange maize and ß-carotene supplement groups. The liver ß-carotene content of the maize group was 100% greater than that of the ß-carotene supplement group. Because the maize diets were consumed ad libitum, absorption of ß-carotene was likely incremental, allowing better storage than the larger, twice daily doses of ß-carotene. ß-Carotene supplements are quickly cleared in gerbils with 45 ± 19% reaching the cecum, and are thus no longer available for absorption by 3 h (41). In the second study, no carotenoids were identified in the livers of any treatment group, indicating that provitamin A carotenoids were preferentially converted to VA rather than stored. Storage of large amounts of ß-carotene in the liver might indicate adequate VA status. Although lutein and zeaxanthin are present in large quantities in maize, no lutein or zeaxanthin were detected in livers. This observation supports a prior study in gerbils showing that lutein is not efficiently absorbed and stored (42).
The theoretical percentage of VA stored in the liver that is attributable to the maize diet decreased as dietary carotenoids increased and as VA status improved (study 2). Only 16% of additional provitamin A carotenoid in the 60% orange maize group was converted to VA and stored in the liver, indicating that the VA requirements of the gerbils in this group were met. The 60% dark-orange maize group (study 1) and the 60% orange maize group (study 2) had similar liver VA, even though the dark-orange maize group received twice the provitamin A carotenoids (Table 3). The presence of ß-carotene in the liver of the ß-carotene supplement and dark-orange maize groups (study 1) further supports an adequate VA status and suggests that bioconversion might depend more on VA status rather than on the dietary amount. This partially explains the similar liver VA stores of the dark-orange maize and ß-carotene supplement groups in the first study.
In conclusion, these studies show that provitamin A carotenoids in maize are as bioavailable as ß-carotene supplements in a VA-depleted gerbil model. The positive effect of biofortified maize on VA status demonstrates the feasibility of this dietary approach. The bioconversion ratio, based on ß-carotene (2.8 µg:1 µg) for provitamin A carotenoids from maize to VA in gerbils, is much lower than that proposed by the Institute of Medicine (6) for dietary sources (12 µg:1 µg). Perhaps the ß-carotene in maize is located in or associated with oil droplets in the kernel and absorbed with the fat leading to better absorption rates than with carrots or other vegetables. VA status had a large effect on VA and ß-carotene storage, signifying that a single conversion factor may not accurately reflect bioconversion under all circumstances. This study demonstrates the potential for positively altering or maintaining VA status using maize biofortified with provitamin A carotenoids and indicates that evaluation in humans should be pursued.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 5 May 2006. Initial review completed 10 June 2006. Revision accepted 12 July 2006.
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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 J. P. Mills, G. A. Tumuhimbise, K. M. Jamil, S. K. Thakkar, M. L. Failla, and S. A. Tanumihardjo Sweet Potato {beta}-Carotene Bioefficacy Is Enhanced by Dietary Fat and Not Reduced by Soluble Fiber Intake in Mongolian Gerbils J. Nutr., January 1, 2009; 139(1): 44 - 50. [Abstract] [Full Text] [PDF]
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J. P. Mills, P. W. Simon, and S. A. Tanumihardjo Biofortified Carrot Intake Enhances Liver Antioxidant Capacity and Vitamin A Status in Mongolian Gerbils J. Nutr., September 1, 2008; 138(9): 1692 - 1698. [Abstract] [Full Text] [PDF]
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 C. E. Harjes, T. R. Rocheford, L. Bai, T. P. Brutnell, C. B. Kandianis, S. G. Sowinski, A. E. Stapleton, R. Vallabhaneni, M. Williams, E. T. Wurtzel, et al. Natural Genetic Variation in Lycopene Epsilon Cyclase Tapped for Maize Biofortification Science, January 18, 2008; 319(5861): 330 - 333. [Abstract] [Full Text] [PDF]
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 R. L Surles, J. P Mills, A. R Valentine, and S. A Tanumihardjo One-time graded doses of vitamin A to weanling piglets enhance hepatic retinol but do not always prevent vitamin A deficiency Am. J. Clinical Nutrition, October 1, 2007; 86(4): 1045 - 1053. [Abstract] [Full Text] [PDF]
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S. K. Thakkar, B. Maziya-Dixon, A. G. O. Dixon, and M. L. Failla {beta}-Carotene Micellarization during in Vitro Digestion and Uptake by Caco-2 Cells Is Directly Proportional to {beta}-Carotene Content in Different Genotypes of Cassava J. Nutr., October 1, 2007; 137(10): 2229 - 2233. [Abstract] [Full Text] [PDF]
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J. P. Mills, P. W. Simon, and S. A. Tanumihardjo {beta}-Carotene from Red Carrot Maintains Vitamin A Status, but Lycopene Bioavailability Is Lower Relative to Tomato Paste in Mongolian Gerbils J. Nutr., June 1, 2007; 137(6): 1395 - 1400. [Abstract] [Full Text] [PDF]
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