点击显示 收起
1 From the Program in International Nutrition, Department of Nutrition, University of California, Davis (MJH, JMP, and KHB), and the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B): Centre for Health and Population Research, Dhaka, Bangladesh (KMJ, FH, MIH, and GJF)
2 Supported by the US Department of Agriculture (grant no. 98-35200-6099) and the Micronutrient Initiative (grant no. 5600-0001-04-300. Retinyl palmitate and ß-carotene were donated by Roche Vitamins (Parsippany, NJ), and sweet potatoes were donated by the Allen Canning Company (Siloam Springs, AR). 3 Reprints not available. Address correspondence to MJ Haskell, University of California, Program in International Nutrition, 3217A Meyer Hall, One Shields Avenue, Davis, CA 95616. E-mail: mjhaskell{at}ucdavis.edu.
ABSTRACT
Background: Recent evidence suggests that the vitamin A equivalency of ß-carotene from plant sources is lower than previously estimated.
Objective: We assessed the effect of 60 d of daily supplementation with 750 µg retinol equivalents (RE) of either cooked, puréed sweet potatoes; cooked, puréed Indian spinach (Basella alba); or synthetic sources of vitamin A or ß-carotene on total-body vitamin A stores in Bangladeshi men.
Design: Total-body vitamin A stores in Bangladeshi men (n = 14/group) were estimated by using the deuterated-retinol-dilution technique before and after 60 d of supplementation with either 0 µg RE/d (white vegetables) or 750 µg RE/d as sweet potatoes, Indian spinach, retinyl palmitate, or ß-carotene (RE = 1 µg retinol or 6 µg ß-carotene) in addition to a low–vitamin A diet providing 200 µg RE/d. Mean changes in vitamin A stores in the vegetable and ß-carotene groups were compared with the mean change in the retinyl palmitate group to estimate the relative equivalency of these vitamin A sources.
Results: Overall geometric mean (±SD) initial vitamin A stores were 0.108 ± 0.067 mmol. Relative to the low–vitamin A control group, the estimated mean changes in vitamin A stores were 0.029 mmol for sweet potato (P = 0.21), 0.041 mmol for Indian spinach (P = 0.033), 0.065 mmol for retinyl palmitate (P < 0.001), and 0.062 mmol for ß-carotene (P < 0.002). Vitamin A equivalency factors (ß-carotene:retinol, wt:wt) were estimated as 13:1 for sweet potato, 10:1 for Indian spinach, and 6:1 for synthetic ß-carotene.
Conclusion: Daily consumption of cooked, puréed green leafy vegetables or sweet potatoes has a positive effect on vitamin A stores in populations at risk of vitamin A deficiency.
Key Words: Deuterated retinol dilution • stable isotope • bioavailability • vitamin A status • ß-carotene • vitamin A stores • green leafy vegetables • sweet potatoes • Bangladesh
INTRODUCTION
Vitamin A deficiency is a serious public health problem in low-income populations in less-industrialized populations (1). Young children and women of childbearing age are considered to be at greatest risk of deficiency. In these populations, supplementation with vitamin A has been shown to reduce childhood mortality by 23% (2) and to reduce maternal mortality by 44% (3). The World Health Organization recommends supplementing infants and children <5 y of age with high doses of vitamin A to improve their vitamin A status (4). However, because vitamin A is potentially teratogenic, high-dose vitamin A supplements can be given safely to women of childbearing age only within the first 6 wk postpartum, when the likelihood of becoming pregnant is very low.
The provision of small daily doses of vitamin A from food may be an alternative strategy for improving vitamin A status in populations at risk of deficiency. Appropriate foods provide safe amounts of vitamin A and can be given to all population groups at risk of deficiency, including women of childbearing age. Animal source foods, such as dairy foods, eggs, and liver, contain preformed retinol, which is readily absorbed in the human intestine; however, these foods are generally not affordable for populations at risk of deficiency (5). In less-industrialized countries, 65–85% of vitamin A in the diet is estimated to be supplied by provitamin A carotenoids in vegetables and fruit (5). However, recent evidence indicates that the bioavailability of provitamin A carotenoids from plant sources is lower than previously assumed and suggests that plant sources of vitamin A may not be efficacious for improving vitamin A status (6, 7). In the most recent edition of the Dietary Reference Intakes (8), the vitamin A equivalency factors for provitamin A carotenoids from foods were increased from 6:1 to 12:1 for ß-carotene [12 µg ß-carotene = 1 µg retinol = 1 retinol acitivity equivalent (RAE)] and from 12:1 to 24:1 for -carotene, -carotene, and ß-cryptoxanthin (24 µg other provitamin A carotenoids = 1 µg retinol = 1 RAE). The equivalency factors were increased because recent evidence indicates that the efficiency of absorption of ß-carotene from foods is lower than previously estimated (16% compared with 33%) (8–10).
The effect of supplementation with plant sources of vitamin A on vitamin A status in humans has been assessed in placebo-controlled trials by examining changes in plasma retinol concentration in response to supplementation with plant or synthetic sources of vitamin A (6, 7). However, because of homeostatic regulation, plasma retinol concentrations are not likely to change in response to supplementation, unless subjects are moderately or severely vitamin A deficient at the onset of the intervention. Even when plasma retinol concentrations respond to supplementation in depleted persons, the magnitude of increase may not be directly proportional to the vitamin A bioavailability from a particular food source. In contrast, the deuterated-retinol-dilution (DRD) technique is an indirect method for quantitatively estimating total body stores of vitamin A in humans (11), and the technique has been validated in 2 sets of surgical patients with adequate to large (11) or small to adequate hepatic vitamin A reserves (12). Moreover, the paired-DRD technique (estimation of vitamin A pool size before and after supplementation) provides expected quantitative estimates of change in total-body vitamin A stores in response to supplementation with different amounts of vitamin A (13). The purpose of the present study was to assess quantitative changes in total body stores of vitamin A by using the paired-DRD technique before and after 60 d of supplementation with an orange tuber (sweet potatoes), a green leafy vegetable [Indian spinach (Basella alba); local name: pui sak], or an equivalent amount of synthetic vitamin A, which was provided as either retinyl palmitate or ß-carotene in oil, to determine the relative efficacy of plant sources of vitamin A for improving vitamin A status.
SUBJECTS AND METHODS
Subjects
The study was conducted at the outpatient facility of the International Centre for Diarrhoeal Disease Research in Dhaka, Bangladesh. Subjects attended the facility daily from 0730 to 1930 and consumed all of their meals and snacks under supervision. The study was conducted during 3 separate cycles because of space limitations at the study facility. Before each cycle, 75 men (18–35 y of age) were screened for plasma retinol concentration. From this group, we selected those who had the lowest plasma retinol concentrations; no clinical evidence of vitamin A deficiency, intestinal malabsorption, or other conditions that might interfere with vitamin A absorption or metabolism; a serum albumin concentration > 35 g/L; and a serum C-reactive protein concentration < 10 mg/L. Written informed consent was obtained from each of the participants. The study protocol was approved by the Institutional Review Board of the Universtiy of California, Davis, and the Ethical Review Committee of the International Centre for Diarrhoeal Disease Research, Bangladesh.
We chose to study adult males in Bangladesh because we know from a previous study (13) that it was possible to identify persons in this population with low to adequate total body stores of vitamin A on the basis of their plasma retinol concentrations. Although women and preschool-aged children are likely to be at greater risk of vitamin A deficiency than are men, enrollment of women into the study was not possible because it is not culturally acceptable in Bangladesh for women to spend the time required to complete the study protocol (12 h/d for 113 d) at the study facility. Preschool-aged children were not enrolled because at the time the study was conducted the plasma kinetics of [2H4]retinol had not been described in young children, and it was not known whether the isotope dilution equation that is used for estimating total-body vitamin A stores in adults would be appropriate for use in that age group. Moreover, children in Bangladesh are scheduled to receive periodic large-dose supplementation as part of a national intervention program.
The subjects were ranked according to their initial plasma retinol concentration and randomly assigned in blocks of 5 to 1 of 5 treatment groups. The subjects in each treatment group received a low–vitamin A diet that was supplemented twice per day, at the noon and evening meals, with either 1) low–vitamin A vegetables (white potato, cauliflower) and a corn oil capsule [0 µg retinol equivalents (RE)/d], 2) sweet potato (80 g to provide 2.25 mg ß-carotene, or 375 µg RE/meal) and a corn oil capsule, 3) Indian spinach (75 g to provide 2.25 mg ß-carotene, or 375 µg RE/meal) and a corn oil capsule, 4) low–vitamin A vegetables and a vitamin A capsule (375 µg RE/meal as 685 µg retinyl palmitate in corn oil), or 5) low–vitamin A vegetables and a ß-carotene capsule (375 µg RE/meal as 2.25 mg ß-carotene in corn oil). Thus, a total of 750 µg RE/d was provided in the vitamin A–supplemented groups. The subjects who were assigned to the low–vitamin A control group, hereafter referred to as the control group, received a capsule containing 60 mg vitamin A on completion of the study protocol.
A vitamin A equivalency factor of 6:1 was used to calculate the portion sizes of the vegetables (6 µg ß-carotene = 1 µg retinol = 1 RE) because this was the recommended vitamin A equivalency factor (14) at the time the study was conducted. Using the new recommended vitamin A equivalency factor of 12:1 (12 µg ß-carotene = 1 µg retinol = 1 RAE) (8), the estimated vitamin A equivalency of the ß-carotene–containing food or capsule supplements was 188 µg RAE/meal, for a total of 375 µg RAE/d.
Study design: paired-DRD technique
One week before beginning the study procedures, the subjects received 800 mg albendezole (Smith Kline Beecham Pharmaceuticals, Philadephia) for treatment of intestinal helminths. Three days before beginning the study procedures, the subjects began receiving the basal low–vitamin A diet (described below) (Figure 1). On study day 1, the subjects received an oral dose of 10 mg [2H4]retinyl acetate, which was followed by a high-fat, low–vitamin A breakfast (fried curried potato pastries, tea). Twenty days later, a blood sample was drawn for measurement of plasma retinol and carotenoid concentrations (referred to as "initial" concentrations) and of the plasma isotopic ratio of [2H4]retinol to retinol for estimation of initial vitamin A pool size. For the next 60 d, the subjects received their assigned dietary treatment (described below). At the end of the 60-d supplementation period, a blood sample was drawn for measurement of postsupplementation plasma concentrations of retinol and carotenoids (referred to as "final" concentrations). Immediately thereafter, the subjects received the basal low–vitamin A diet for a period of 10 d to allow the vitamin A that was consumed during the 60-d supplementation period to equilibrate with endogenous vitamin A stores. After the 10-d stabilization period (study day 91), a blood sample was drawn for measurement of the plasma isotopic ratio of [2H4]retinol to retinol, and a second oral dose of 10 mg [2H4]retinyl acetate was administered to the subjects. Blood was drawn 20 d later (study day 113) for measurement of the plasma isotopic ratio of [2H4]retinol to retinol for estimation of the final vitamin A pool size.
FIGURE 1.. Study protocol.
Diets
The basal low–vitamin A diet consisted of rice, wheat flat bread, lentils, small amounts of curried chicken or mutton, and pale vegetables and fruit, such as cauliflower, cabbage, white potatoes, white squash, and banana, all of which are very low in vitamin A content. On the basis of food-composition tables (15), the basal diet provided 200 µg RE/d (100 RAE/d). The subjects were allowed to consume selected low–vitamin A and low-fat foods, such as wheat flat bread, lentils, and white fruit and vegetables ad libitum at the breakfast meal and at the midafternoon snack to allow for differences in individual caloric requirements. The meals were standardized to contain the same amount of fat (16 g/meal) and similar amounts of fiber (29 g/meal; references 15–17).
Canned sweet potatoes (from the same production lot) were donated by the Allen Canning Company (Siloam Springs, AR) and shipped to Bangladesh. Canned sweet potatoes were used because suitable local orange tubers and fruit were seasonal and were not available in local markets during the full study period. Indian spinach is a low-cost green leafy vegetable that is available year round in Bangladesh. The Indian spinach was obtained daily from the same supplier.
The sweet potatoes and Indian spinach were prepared to optimize the bioavailability of ß-carotene by following standardized recipes. The sweet potatoes were puréed by using an electric food processor and sautéed in corn oil for 5 min with onion, salt, cardamom, and ground chili seeds. The Indian spinach was steamed for 10 min, puréed, and sautéed in corn oil for 5 min with garlic, salt, and ground chili seeds. Each portion of vegetables contained 6.8 g corn oil. The carotenoid content of the cooked, puréed vegetables was measured by using HPLC (18) to determine the portion sizes required to supply 2.25 mg (375 µg RE) as all-trans-ß-carotene.
Portions of sweet potatoes or Indian spinach were weighed onto tared plates, along with the other foods, at the noon and evening meals. Subjects were supervised during mealtimes and were asked to consume all of the food provided. Groups receiving the vitamin A–containing foods received a placebo capsule (corn oil) with each meal. Subjects in the vitamin A and ß-carotene groups received capsules containing 375 µg RE as either retinyl palmitate or all-trans-ß-carotene (Roche Vitamins, Parsippany, NJ). It was not possible to mask the sweet potatoes and Indian spinach, but the placebo, vitamin A, and ß-carotene capsules were identical in appearance.
Estimation of vitamin A pool size
Total body stores of vitamin A were estimated before and after the 60-d supplementation period by using plasma isotopic ratios that were measured 20 d after oral administration of [2H4]retinyl acetate and the isotope dilution equation described by Furr et al (11). Because [2H4]retinyl acetate was used for the pool size estimates both before and after the period of dietary supplementation, the plasma isotopic ratio measured on study day 113 (to estimate final pool size) had to be adjusted for the contribution of isotope remaining from the first dose of [2H4]retinyl acetate that was administered for estimation of the initial pool size. This was accomplished by subtracting the plasma isotopic ratio of [2H4]retinol to retinol measured on day 92 from the isotopic ratio measured on day 113. The subjects were provided the basal low–vitamin A diet for 10 d before measurement of the plasma isotopic ratio on day 92 to allow the supplemental nonlabeled vitamin A that was provided during the 60-d supplementation period to equilibrate with vitamin A stores. Thus, it is very unlikely that the plasma ratio of [2H4]retinol to retinol on day 92 was affected by any residual nonlabeled vitamin A that was consumed during the supplementation period. The subjects were given the low–vitamin A diet throughout the rest of the study period to minimize the effect of nonlabeled dietary vitamin A on the plasma isotopic ratios that were measured on study day 113 to estimate the final vitamin A pool size.
Evaluation of abbreviated method for assessing bioavailability of ß-carotene
On the first day of the dietary supplementation period (study day 22), the subjects received an oral dose of [2H8]retinyl acetate (0, 5, or 10 mg) to determine whether the plasma isotopic ratios of [2H8]retinol to retinol on days 3, 5, or 7 during the supplementation phase differed between the dietary treatment groups and whether these plasma isotopic ratios could be used to predict changes in vitamin A pool size in response to supplementation. The results of this component of the study will be presented separately. Final vitamin A pool sizes were estimated by using the plasma isotopic ratio of [2H4]retinol to unlabeled retinol; thus, any [2H8]retinol that remained in the body was not included in the final estimate of total body stores of vitamin A. The final pool size estimates reflect only the amount of unlabeled vitamin A in the body.
Laboratory methods
Plasma concentrations of retinol, lutein, -carotene, ß-carotene, and -tocopherol were measured by using HPLC (18) on a Shimadzu Class VP (Shimadzu, Columbia, MD) equipped with a photo-diode array detector and autosampler. Pre- and postsupplementation plasma samples for each subject were analyzed together during the same set of HPLC analyses. For quality control, a plasma pool was prepared and calibrated by using control serum (fat-soluble vitamins) from the National Institute of Standards (Gaithersburg, MD). Three aliquots of the plasma pool were analyzed with each set of study samples. The within-day CV for the measurements of retinol, lutein, -carotene, ß-carotene, and -tocopherol concentrations in the plasma pool samples were 5%, 10%, 10%, 7%, and 6%, respectively. To assess accuracy, control plasma from the National Institute of Standards was analyzed. The measured concentrations of retinol, lutein, -carotene, ß-carotene, and -tocopherol were within 3.1%, 3.4%, 9.1%, 4.3%, and 2.6% of the certified values for the control plasma. The all-trans-ß-carotene content of the cooked, puréed foods (Indian spinach and sweet potato) was determined by using HPLC (Class VP; Shimadzu) (18). Three ß-carotene standards (Fluka Chemical Co, Buchs, Switzerland) were saponified, extracted, and analyzed with each set of food samples according to the same procedures (18). The interday CV for the ß-carotene concentration of the standards was <8%, and the interday CV for the ß-carotene concentration of the food samples was <12%. The plasma isotopic ratios of [2H4]retinol to retinol and [2H8]retinol to retinol were determined by using gas chromatography–mass spectrometry as previously described (19). Briefly, retinol was isolated from plasma by using HPLC, and the tert-butyldimethylsilyl derivative of retinol was formed. Isotopic ratios were measured by using gas chromatography–mass spectrometry on a Shimadzu QP 5000 quadrupole mass spectrometer with 1.12 x 10–17 (70 eV) electron ionization. Standards with known weight ratios of [2H4]retinol to retinol and [2H8]retinol to retinol were analyzed with each set of study samples. The interday CV for the [2H4]retinol:retinol and [2H8]retinol:retinol standards was <6%.
Statistical analysis
Descriptive statistics were calculated for each variable. Variables that were not normally distributed were transformed to natural logarithms for the statistical analyses. Analysis of covariance was used to compare mean changes in outcome variables (total-body vitamin A stores, plasma concentrations of retinol and carotenoids) between treatment groups, with treatment group as the main effect and the initial value of the outcome variable as a covariate. For the comparison of mean changes in plasma retinol concentration between treatment groups, initial values and the dose of [2H8]retinyl acetate administered to subjects were used as covariates. In addition, a main effect for study cycle and an interaction between treatment group and study cycle were included in each analysis to test whether pooling the study cycles was statistically justified. Linear regression was used to examine the relation between estimated change in vitamin A pool size and initial vitamin A pool size, the relation between estimated change in vitamin A pool size and estimated change in plasma ß-carotene concentration, and the relation between estimated change in plasma retinol concentration and initial plasma retinol concentration. Treatment group assignments were unmasked only after all of the statistical analyses were completed. All statistical analyses were performed by using SAS software (release 6; SAS Institute Inc, Cary, NC).
RESULTS
Subjects
A total of 70 subjects who had plasma retinol concentrations ranging from 0.52 to 1.25 µmol/L at the time of the screening procedures were enrolled in the study (14 subjects/group). The mean age, mean values for anthropometric characteristics, and mean plasma retinol concentrations of the study participants by treatment group at the time of screening are shown in Table 1. There were no significant differences in mean age, height, weight, body mass index, or plasma retinol concentration between the treatment groups.
View this table:
TABLE 1. Initial characteristics of study participants by treatment group1
ß-Carotene concentration in Indian spinach and sweet potatoes
On the basis of the HPLC analyses, the mean (±SD) all-trans-ß-carotene concentration in cooked Indian spinach was 30.5 ± 2.1 µg/g, and the mean concentration of cis isomers of ß-carotene was 8.1 ± 0.4 µg/g (21% of total ß-carotene). The mean lutein content of the Indian spinach was 70.7 ± 5.0 µg/g. The mean all-trans-ß-carotene concentration in cooked sweet potatoes was 28.3 ± 1.3 µg/g, and the mean concentration of cis isomers of ß-carotene was 6.0 ± 0.3 µg/g (17% of total ß-carotene); lutein was not detected in sweet potatoes. -Carotene and ß-cryptoxanthin were not detected in Indian spinach or sweet potatoes. Portion sizes of cooked Indian spinach (75 g/meal; 375 µg RE) and cooked sweet potatoes (80 g/meal; 375 µg RE) were determined by using the estimated all-trans-ß-carotene concentration in each vegetable. The vitamin A activity of cis isomers of ß-carotene was not included in the determination of portion sizes because of uncertainty about the bioavailability of cis-ß-carotene relative to all-trans-ß-carotene. If the vitamin A activity of cis isomers of ß-carotene is estimated to be one-half that of ß-carotene, the total vitamin A activity would be 426 µg RE (213 RAE) in a serving of Indian spinach and 415 µg RE (208 RAE) in a serving of sweet potatoes.
Plasma retinol concentrations
The overall initial mean (±SE) plasma retinol concentration was 1.27 ± 0.15 µmol/L. Initially, 13 (18.6%) of the subjects had plasma concentrations < 1.05 µmol/L. One subject (1.4%) had a plasma concentration < 0.70 µmol/L. After supplementation the mean plasma retinol concentration decreased significantly in the control group (P < 0.0001) and increased significantly within the synthetic ß-carotene group (P < 0.0001). There were no significant changes in mean plasma retinol concentration in the other treatment groups (P 0.12). The final mean plasma retinol concentrations in the sweet potato, Indian spinach, vitamin A, and ß-carotene groups were significantly higher than the final mean concentration in the control group (P < 0.004) (Table 2). The final mean plasma retinol concentration in the ß-carotene group was significantly higher than the final mean concentrations in the control, sweet potato, and vitamin A groups (P 0.03) but was not significantly different from the final mean concentration in the Indian spinach group (P = 0.17). (Note that the subject with an initial plasma retinol concentration < 0.70 µmol/L was in the vitamin A group. His plasma retinol concentration increased from 0.64 to 0.87 µmol/L, and his estimated vitamin A pool size increased from 0.028 to 0.070 mmol.) For the comparison of final mean plasma retinol concentrations between treatment groups, the initial plasma retinol concentrations and the dose of [2H8]retinyl acetate that was administered on day 1 of the supplementation period were used as covariates for adjustment of any potential effect of these variables. (Any uncertainty regarding the effect of [2H8]retinyl acetate on final mean plasma retinol concentrations is not critical because vitamin A equivalency factors were estimated on the basis of relative changes in vitamin A pool size, as described below. Plasma retinol concentrations were not used to estimate vitamin A pool size or vitamin A equivalency factors.)
View this table:
TABLE 2. Initial and final plasma concentrations of retinol, carotenoids, and -tocopherol by treatment group1
Plasma carotenoid and -tocopherol concentrations
The overall initial mean plasma ß-carotene concentration was 0.07 ± 0.01 µmol/L, and initial concentrations did not differ significantly between treatment groups. The final mean ß-carotene concentrations in the sweet potato, Indian spinach, and ß-carotene groups were significantly higher than those in the control and vitamin A groups (P < 0.002) (Table 2). The overall initial mean lutein concentration was 0.08 ± 0.02 µmol/L. The final mean lutein concentration in the Indian spinach group was significantly higher than the final mean concentrations in the other treatment groups (P < 0.0001). The overall initial mean -carotene concentration was 0.04 ± 0.01 µmol/L. The final mean -carotene concentration in the Indian spinach group was significantly higher than the final mean concentrations in the other treatment groups (P < 0.0001). The overall initial mean plasma -tocopherol concentration was 13.4 ± 1.5 µmol/L. The final mean -tocopherol concentration in the Indian spinach group was significantly higher than the final mean concentrations in the other treatment groups (P < 0.0001).
Mean change in estimated vitamin A pool size
The overall initial mean vitamin A pool size was 0.108 ± 0.067 mmol. The raw initial and final geometric mean vitamin A pool sizes and mean changes in pool size are shown by treatment group in Table 3. The adjusted mean change in vitamin A pool size was estimated for each of the treatment groups by using analysis of covariance with control for initial values as described previously. The adjusted mean changes in vitamin A pool size in the Indian spinach (0.022 mmol; P = 0.034), vitamin A (0.046 mmol; P < 0.001), and ß-carotene groups (0.043 mmol; P < 0.002) were significantly larger than the adjusted mean change in the control group (–0.019 mmol). The adjusted mean change in pool size in the sweet potato group (0.010 mmol) was larger than that in the control group, but the difference was not significant (P = 0.21) (Table 3).
View this table:
TABLE 3. Initial and final vitamin A pool sizes, changes in vitamin A pool size, and estimated vitamin A equivalency factors by treatment group1
Estimation of vitamin A equivalency factors
Vitamin A equivalency factors for ß-carotene from the vegetable or synthetic sources were estimated by comparing the mean change in vitamin A pool size in each of these groups with that in the group who received vitamin A as retinyl palmitate (Table 3). As shown in Table 3 and as described above, the mean vitamin A pool size in the control group decreased in response to consumption of the low–vitamin A diet during the supplementation phase of the study. Thus, to estimate the net mean change in pool size in the groups who received a source of supplemental vitamin A relative to the change in the control group, the adjusted mean change in pool size in the control group (–0.019 mmol) was subtracted from the adjusted mean change in pool size in each of the supplemented groups (Table 3). Relative to the control group, the adjusted mean changes in pool size were 0.029 mmol for the sweet potato group, 0.041 mmol for the Indian spinach group, 0.065 mmol for the retinyl palmitate group, and 0.062 mmol for the ß-carotene group. These estimates of the net mean change in pool size in the vegetable and ß-carotene groups were compared with the net mean change in pool size in the retinyl palmitate group to estimate the relative vitamin A equivalency of the vegetables and synthetic ß-carotene according to the following equation:
DISCUSSION
The paired-DRD technique was used to assess quantitative changes in vitamin A pool size in response to supplementation with sweet potato, Indian spinach, retinyl palmitate, or ß-carotene to determine the relative efficacy of plant sources of vitamin A for improving vitamin A status. The mean changes in vitamin A pool size in the Indian spinach, vitamin A, and ß-carotene groups were significantly larger than the mean change in the control group. The mean change in vitamin A pool size in the sweet potato group was larger than that in the control group, but the difference was not significant. For the retinyl palmitate group, the expected theoretical change in vitamin A pool size was identical to the observed change in vitamin A pool size, which indicates that the paired-DRD technique provides reasonably good estimates of the change in pool size in response to supplementation, as was seen in a previous study (13).
Final mean plasma retinol concentrations in the supplemented groups were also significantly higher than the final mean concentration in the control group. Although plasma retinol concentrations responded to supplementation, the magnitude of change in plasma retinol concentration was less than was observed for mean changes in vitamin A pool size (Figure 4). Thus, in this study population, change in vitamin A pool size was a more sensitive indicator for detecting a change in vitamin A status than was change in plasma retinol concentration.
FIGURE 4.. Mean (± SEM) percentage changes in plasma retinol concentration () and estimated vitamin A pool size () in response to supplementation by treatment group. n = 68. The percentage change in plasma retinol concentration and the percentage change in vitamin A pool size were significantly different, P = 0.0002 (nonparametric signed-rank test). The percentage changes in plasma retinol in the groups who received a vitamin A source were significantly different from the percentage change in the control group (P < 0.004), and the percentage change in plasma retinol in the ß-carotene group was significantly different from the percentage changes in the vitamin A, sweet potato, and control groups (P 0.03) but was not significantly different from the percentage change in the Indian spinach group (P = 0.17) (analysis of covariance with initial value and [2H8]retinyl acetate dose as covariates). The percentage changes in vitamin A pool size in the Indian spinach, vitamin A, and ß-carotene groups were significantly different from the percentage change in the control group, P < 0.04 (analysis of covariance with initial value as the covariate).
Vitamin A equivalency factors were estimated as 13.4:1 for sweet potato and 9.5:1 for Indian spinach. In Indonesian schoolchildren, a vitamin A equivalency factor of 12:1 was reported for orange and yellow fruit and vegetables, and a factor of 26:1 was reported for green leafy vegetables (7). The value of 13:1 for sweet potato in the present study is similar to the value of 12:1 for orange fruit and vegetables; however, the value of 10:1 for Indian spinach is much less than the factor of 26:1 for dark green leafy vegetables in the Indonesian study. There are several factors that may account for the different estimates of vitamin A equivalency factors for dark green leafy vegetables. In particular, the studies differed in 1) the methods that were used for assessing change in vitamin A status, 2) food preparation techniques, and 3) the treatment of intestinal helminths.
In the present study, vitamin A equivalency factors were estimated on the basis of relative quantitative changes in vitamin A pool size, whereas in the Indonesian study (7), these factors were estimated on the basis of relative changes in serum retinol concentration in response to supplementation with plant or synthetic sources of vitamin A. As mentioned earlier, serum retinol is not an optimal indicator for assessing change in vitamin A status in response to supplementation. This is illustrated in the present study by the greater magnitude of response to supplementation for vitamin A pool size than for plasma retinol concentration.
In the present study, Indian spinach was prepared to optimize the bioavailability of ß-carotene by steaming, puréeing, and then sautéeing the spinach in oil (6.8 g oil/serving). This food-preparation method probably enhanced the bioavailability of ß-carotene by lessening food matrix effects, which are known to reduce the bioavailability of carotenoids from green leafy vegetables (21, 22). In the study in Indonesian schoolchildren (7), the food-preparation techniques were not described, and the fat content of the ß-carotene–containing food supplements was not specified.
Intestinal helminths (Ascaris lumbricoides) can have an adverse effect on ß-carotene absorption when the intensity of infection is high [>3200 eggs/g (epg) feces] (23). In the present study, the subjects were treated with an antihelminthic drug before beginning the study procedures. In the Indonesian study, the incidence of ascaris infection was 60%, the median intensity of infection was 4720 epg (25th–75th percentiles: 610–16990 epg), and the children were not treated with an antihelminthic. There was a significant negative correlation between serum ß-carotene concentrations and intensity of ascaris infection in children who received ß-carotene from fruit but not in children who received ß-carotene from vegetables (7). Nevertheless, it is conceivable that the presence of roundworms reduced the overall absorption of ß-carotene from dark green leafy vegetables.
In a related study, lactating Indonesian women were supplemented for 12 wk with 3.5 mg ß-carotene/d (0.583 RE/d, 0.292 RAE/d) as a mixture of green leafy vegetables and carrots or as a ß-carotene–enriched wafer in a placebo-controlled trial. The vegetable supplement contained 7.8 g fat/serving (6); however, there was almost no change in the serum ß-carotene concentration (0.03 µmol/L; 15% increase above initial value) in the women who received the vegetables. By contrast, serum ß-carotene concentrations increased significantly (0.73 µmol/L; 384%) in women who received the ß-carotene–enriched wafer containing the same amount of ß-carotene and 4.4 g fat. This suggests that ß-carotene absorption was lower in the vegetable group because of a food matrix effect. However, the incidence of intestinal helminths in the Indonesian women was 80% [median intensity: 13020 epg (25th–75th percentiles: 1580–40540 epg)]. The adverse effect of intestinal helminths on ß-carotene absorption may be greater when ß-carotene is in a complex food matrix than when it is in a simpler matrix. In the present study, plasma ß-carotene concentrations increased in response to supplementation with sweet potato (0.21 µmol/L; 300%) and Indian spinach (0.27 µmol/L; 385%), which suggests that the food-processing techniques used or the treatment for intestinal helminths enhanced ß-carotene absorption in the study subjects.
In the present study, a vitamin A equivalency factor of 6.3:1 was estimated for synthetic ß-carotene in corn oil, which is higher than previous estimates of 2:1 to 4:1 (24–27). The earlier estimates (2:1 and 3.3:1) were derived from comparisons of the amount of ß-carotene or vitamin A that was required to reverse abnormal dark adaptation in a small number of vitamin A–depleted subjects (24, 25). More recent estimates (3.8:1 and 2.4:1) are based on stable-isotope methods for estimating absorption and bioconversion of ß-carotene to retinol (26, 27). One possible explanation for the higher estimate of 6:1 in the present study is that the daily doses of ß-carotene were given with a meal that contained 29 g dietary fiber, which may have reduced ß-carotene absorption.
Relation between percentage change in vitamin A pool size and initial vitamin A pool size
In the present study, the percentage change in vitamin A pool size was negatively related to initial vitamin A pool size in the groups who received synthetic vitamin A or ß-carotene, which suggests that subjects with low vitamin A status may be more responsive to treatment with vitamin A than are subjects with higher vitamin A status. It has been suggested previously that vitamin A status may affect the absorption and bioconversion of ß-carotene to vitamin A. A few animal studies have shown that bioconversion of ß-carotene to vitamin A decreases when vitamin A intake increases (28–30). In contrast, the activity of intestinal ß-carotene 15,15-oxygenase in rats is not affected by depletion or excess feeding of ß-carotene or retinol (31). Filipino children with the lowest vitamin A status before intervention showed the greatest improvement in vitamin A status in response to supplementation with provitamin A–rich fruit and vegetables. The inverse correlation was much stronger when vitamin A status was assessed by using serum ratios of [2H4]retinol to retinol 3 d after the isotope dose than when serum retinol concentrations were used (32). This suggests that absorption and bioconversion of ß-carotene to vitamin A are greater when the initial vitamin A status is low. However, the disposal rate of vitamin A is known to vary with vitamin A status in rats (33). The lower retinol disposal rate in rats with small vitamin A reserves presumably conserves the vitamin, whereas the higher disposal rate in rats with large reserves prevents too much vitamin A from accumulating in the body. Thus, in the present study, subjects with small initial vitamin A reserves who received synthetic ß-carotene may have absorbed and bioconverted more ß-carotene to vitamin A and disposed of vitamin A to a lesser extent than did subjects with large initial vitamin A reserves. In the vegetable groups, a relation between percentage change in vitamin A pool size and initial vitamin A pool size was not observed. One possible explanation for this is that less ß-carotene was absorbed from the vegetables than from the synthetic ß-carotene capsules because of food matrix effects. This seems likely because the plasma ß-carotene responses and mean changes in vitamin A pool size in the vegetable groups tended to be lower than those in the synthetic ß-carotene group. Because stores increased to a lesser extent in the vegetable groups, and the range of changes in pool size was smaller, it may not have been possible to detect a differential response in percentage change in vitamin A pool size by initial pool size in these groups.
Relation between estimated vitamin A pool size and plasma ß-carotene concentrations
Changes in plasma ß-carotene concentration were significantly related to changes in vitamin A pool size. Estimates of vitamin A equivalency factors based on relative changes in plasma ß-carotene concentration between the synthetic ß-carotene group and the vegetable groups were similar to those based on relative changes in vitamin A pool size between the retinyl palmitate group and the vegetable groups. This suggests that absorption and bioconversion of ß-carotene to retinol are directly related within the range of pool sizes observed in the present study. The relative mean changes in plasma ß-carotene concentration in response to consumption of equivalent amounts of synthetic ß-carotene or ß-carotene from food sources may be an alternative method for estimating vitamin A equivalency factors in populations with low to adequate initial vitamin A pool sizes; however, this requires further investigation.
In summary, the results of the present study indicate that daily consumption of green leafy vegetables, prepared as described, has a positive effect on vitamin A status in Bangladeshi men. Further research is needed to assess the effects of food-preparation techniques, intestinal parasites, and initial vitamin A status on the efficacy of plant sources of vitamin A for improving vitamin A status.
ACKNOWLEDGMENTS
We thank the study participants for their efforts in completing the study protocol.
MJH contributed to the study design, the training of field personnel, data collection and analysis, and the writing of the manuscript. KMJ managed the clinical phase of the study and contributed to the training of field personnel, data collection, and the writing of the manuscript. FH and MIH each contributed to data collection during the clinical phase of the study. JMP contributed to the study design, statistical analyses, and the writing of the manuscript. GJF contributed to the management of the clinical phase of the study. KHB contributed to the study design, data analysis, and the writing of the manuscript. None of the authors had any financial or personal interests in either of the 2 agencies that supported this study.
REFERENCES