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*
Section for International Maternal and Child Health, Department of Women’s and Children’s Health, University Hospital, Uppsala, Sweden;
Ministry of Health and Child Welfare, Nutrition Unit, Bulawayo, Zimbabwe; and
**
University of Zimbabwe, Harare, Zimbabwe
2To whom correspondence should be addressed. E-mail: Mehari.Gebre-Medhin{at}ich.uu.se.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • vitamin A • ß-carotene • supplementation • lactating women • Zimbabwe
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The plan of
action of the International Conference of Nutrition in 1992 prioritizes
diet-based approaches for low income countries. However, recent
reports have raised doubts about the effectiveness of this approach,
particularly the increased consumption of green leafy vegetables
(2
3
4)
. Several reasons may have contributed to this lack
of improvement of the vitamin A status with plant food–based
interventions, i.e., the relatively satisfactory vitamin A status at
baseline, the dose of ß-carotene given and the bioavailability of the
plant food source.
It has also been suggested that vitamin A supplementation increases the
packed-cell volume and hemoglobin
(Hb)3
concentration in subjects with low vitamin A status and low Hb
(5)
. However, data regarding lactating women in this
respect are scarce. No such information is available in Zimbabwe.
The bioavailability of carotenoids is influenced by a number of factors
such as the carotenoid species ingested, the amount of carotenoid in
the diet, the matrix of the food source, the presence of absorption
enhancers or inhibitors, other host-related factors and
interactions among these factors (6)
. The uptake and
absorption of carotenoids may be inhibited by factors such as the
particle size and the location of the carotene in the plant
(7)
. Processing the food or reducing particle size makes
the carotenoids more available. The bioavailability of ß-carotene
from dark green leafy vegetables is lower than that from fruits
(8)
and both are lower than that from pure ß-carotene
(2
,8
,9)
. Furthermore, cooked or processed vegetables have
been shown to have more bioavailable ß-carotene than raw vegetables
(10)
.
Although most vitamin A research has focused on children because of
this vitamin’s demonstrated role in child health, growth and survival,
inadequate attention has been paid to the health and nutrition of women
in low income countries in which vitamin A deficiency may be prevalent.
As awareness of the importance of vitamin A deficiency in women
increases (11)
, it is important to focus on lactating
women whose vitamin A requirements exceed even those in pregnancy. If
the vitamin A intake is not sufficient to replace the amount
transferred to the infant through breast milk, the maternal vitamin
stores may become depleted (12)
. It is estimated that
lactating women in low income countries have an average daily intake of
vitamin A that is less than half that of lactating women in high income
countries (13)
. In a recent report on the vitamin A status
of lactating women in the arid area of Makhaza in Zimbabwe, we found
that 40% of the women were deficient in vitamin A from their serum
retinol levels and 76% were deficient based on relative dose response
(RDR) (14)
. These findings stress the urgency of the need
to redress the vitamin A situation in this area. The purpose of this
study was to examine the effect of supplementing lactating women with
grated carrots, puréed papaya and ß-carotene in oil capsules on
their vitamin A status and iron status.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study was a 60-d placebo-controlled supplementation trial undertaken to examine changes in the vitamin A and iron status of lactating women after they had been randomly assigned to one of the following treatment groups: group 1 received 6 mg ß-carotene in oil capsules; group 2 received 650 g puréed papaya; group 3 received 100 g grated carrots; and group 4 received a placebo, which was a teaspoonful of pink-colored water.
Supplements.
The daily papaya and carrot portions were measured to approximate 6 mg
of ß-carotene, using the conversion factor of 6 and the South African
Food Composition Tables (15)
. The carotinoid content of
carrots was 1100 µg; that of papaya was 175
µg/100 g edible portion. The ß-carotene in oil was
supplied in 6-mg brown capsules from Lancaster Industries (Cape Town,
South Africa); the placebo was pink-colored water, and
each woman assigned to the placebo group was given one teaspoonful
daily. Papayas were washed, cut in half and all seeds were removed; the
inner pulp was then scooped out into a bowl and puréed, and
650 g was weighed out for each woman in the papaya group. Carrots
were washed and grated and 100 g was weighed out for each woman in
the carrot group. The supplements were provided daily between 1000 and
1200 h from a central point within each village conveniently
selected by the women themselves. The women were trained in the daily
preparation and portioning of the supplements and were supervised by
nurses from the local health center and by the principal investigator
(T.N.). A meal containing at least 10 g of vegetable oil and no
vitamin A was supplied daily to all of the women in the study
throughout the 60-d supplementation period.
The puréed papaya and grated carrots supplements were both well accepted by the women. The large glass of ripe puréed papaya was a welcome refreshment on the hot September and October summer days during which the study was conducted. The supplements were given before the corn/bean meal was given when the women were still hungry and thirsty; no wastage was recorded. Toward the end of the study, the carrot group in one of the villages wanted to chew whole carrots, but when the purpose of grating and the need for uniformity was explained to them, they complied with no problem.
Subjects.
The study population consisted of lactating women with infants aged between 2 and 12 mo and living in the twelve villages comprising the Makhaza area of Tsholotsho district in Zimbabwe. After a house-to-house registration of such women, 211 were found eligible. Of these, two declined to participate and two others were excluded due to illness. One of the latter two required medical attention and was referred to the local clinic; the other had Bitot’s spots and was treated and excluded from the study.
The sample size required for each of the four treatment groups was
calculated to be 46 at 95% confidence interval, providing 90% power
to be able to detect a difference of 0.4 µmol/L with a
SD of 0.2 µmol/L in serum retinol between
the supplementation and placebo groups. This sample size was rounded up
to 50. After the above 4 exclusions, 207 eligible women gave written
consent and were enrolled. The women were then randomly assigned to the
four supplementation groups. The four groups were labeled A, B, C and D
before registration; as the women were registered, they were
systematically assigned to each group in the order in which they
arrived. After registration, when each woman had a group, the groups
were then assigned supplements. Group A was assigned ß-carotene,
referred to as medicine A; group B was assigned placebo (pink colored
water, designed to match the ß-carotene capsule, and referred to as
medicine B); group C was assigned papaya, referred to as food A; and
group D was assigned carrots, referred to as food B. Figure 1
shows how the sample of 207 women was selected and allocated to the
four treatment groups. The study was approved by the Ethical Committee
of the Medical Research Council of Zimbabwe and the Ministry of Health
and Child Welfare in Zimbabwe.
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The weight of the mothers was measured to the nearest 100 g, using SECA ALPHA 770 electronic adult scales. The scales were standardized and checked each day against a known weight. Height was measured to the nearest 0.5 cm, using a mounted tape measure. Body mass index was calculated as weight (kg)/height (m)2. Mid-upper arm circumference was measured to the nearest 0.1 cm with a tape measure, on the left arm, with the arm in an extended position, mid-way between the elbow and the acromion. Height was measured only at baseline, and the other anthropometric measurements were taken before and after supplementation.
Blood sample collection.
Blood samples were collected before and after supplementation. Venous
blood (
5 mL) was taken from the cubital vein in each mother. A
senior qualified medical laboratory technologist from Uppsala
University together with two nurses collected the blood under the
supervision of a physician. The blood samples were taken from
nonfasting subjects between 0800 and 1000 h, placed on ice and
protected from light by wrapping them in aluminum foil. All blood
samples were transported within 8 h to the laboratory and
centrifuged (300 x g, 5 min); the serum was
separated, frozen and kept at -20°C for the 7 d during which
fieldwork was done. The serum samples were subsequently stored at
-70°C at the University of Zimbabwe for 2 mo until transported on
dry ice to Sweden and stored at -70°C until analyzed 6–8 mo later.
A RDR test was conducted on every fifth woman selected systematically
from the 207 subjects. A subsample of 43 women was selected, and a dose
of 1.75 µmol of retinol palmitate in an oil suspension
was administered to them orally immediately after the baseline blood
sample had been taken. A follow-up blood sample was taken 5 h
later. While waiting for the follow-up blood sample, the women were
provided with a vitamin A–free snack of plain white bread and a cup of
black tea with sugar. The RDR was calculated as a percentage: RDR
= (A5 - A0)/A5 · 100
where A5 is the serum retinol concentration
5 h after the dose and A0 is the
concentration immediately before the dose was given (16)
.
Analytical procedures.
HPLC was used to determine the serum retinol levels. Serum proteins
were denatured by addition of acetonitrile, and 
-tocopherol acetate
was added as the internal standard for retinol. Retinol was
subsequently extracted into an organic matrix consisting of ethyl
acetate/butanol (1:1), and eluted by reversed-phase chromatography
from a C18 column with an isocratic mobile phase of methanol/water
(95:5). UV detection at 325 nm was used to identify analytes. The
method gave same-day and between-day CV of 3.5 and 4.8,
respectively.
Serum ferritin was assayed with a double monoclonal antibody technique
with a CV of 4.2% at 135 mg/L and 7.8% at 14.9 µg/L.
Serum ferritin assays were performed on a subsample of 163 women; the
other 39 samples were insufficient for analysis. C-reactive protein
(CRP) was assayed by a turbidometric method. The measuring
interval was 0–175 µg/L, with a reference interval of
<10 µg/L and a CV of 10% at 20
µg/L. Levels 
10 µg/L were
considered an indication of ongoing acute infection and were dealt with
separately in the data analysis.
Statistical analysis.
For analysis of data, the SPSS/PC statistical package for the social
sciences, version 10, was used. Means, medians and SD were
calculated for all normally distributed data; medians and the
25th–75th percentiles were calculated for data that were not normally
distributed. Independent sample t test and ANOVA were
used to test differences between groups. The paired
t test was used to test differences between
baseline and endpoint values, and Dunnett’s test was used to compare
supplementation groups with the control groups. Dunnett’s test is a
pairwise multiple comparison t test that compares a set
of treatments against a single control mean (17)
. The
differences between variables were considered significant at
P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. At endpoint, 5 women were away and not available for any tests.
Endpoint data were thus collected from 202 supplemented women 5 d
after the end of the supplementation period. Women were requested not
to change their diet during this period.
|
The effects of the different supplementations on serum retinol
concentrations are shown in Figure 2
and Table 2
. Serum retinol increased in the ß-carotene, papaya and carrot groups
(P < 0.001), but there was no increase in the placebo
group. A clear shift of the distribution to higher values as a result
of the intervention was evident. A subsample of 43 women was tested for
liver stores, using the RDR method. In the ß-carotene and papaya
groups, the mean RDR decreased significantly from baseline to endpoint
compared with the placebo group (P < 0.05). The carrot
and the placebo groups showed no improvement in vitamin A status. The
proportion of women with low liver stores (RDR >20%) decreased from
78% at baseline to 33% at endpoint in the ß-carotene group, and
from 58 to 33% in the papaya group. The mean differences in serum
retinol in the three supplemented groups compared with the placebo
group were all significant (P < 0.05). The mean
differences in RDR compared with the placebo group were significant for
the ß-carotene and papaya groups, but not for the carrot group
(P < 0.05).
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The effect of supplementation on the Hb levels is shown in Figure 3
and Tables 2 and
3. Hemoglobin levels increased in all groups including the placebo group,
but the mean increase was greater in the ß-carotene and
papaya groups than in the placebo group. Of 15 women with anemia at
baseline in the ß-carotene group, only 2 (13%) remained anemic after
supplementation; in the papaya group, 5 of 13 remained anemic, whereas
only 3 and 2 improved in the carrot and placebo groups, respectively.
The mean differences in the three supplemented groups compared with the
mean difference in the placebo group were not significant (P
> 0.05).
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and 3
. However, there was an increase in the median serum
ferritin concentration (25th–75th percentile) from baseline to
endpoint in the three supplemented groups but not in the placebo group.
The median (25th–75th percentile) serum ferritin level in all women
with normal CRP (n = 158) increased from 22 (16–29)
µg/L at baseline to 28 (20–38) µg/L at
endpoint (P < 0.001). The proportion of women with
iron deficiency (serum ferritin <12µg/L) decreased from
13% at baseline to 8% (n = 158) at endpoint. All
women in the three supplemented groups who had iron-deficiency
anemia (serum ferritin <12 µg/L and Hb < 120 g/L)
at baseline improved to a normal status after supplementation, but the
two women who had iron-deficiency anemia in the placebo group did
not improve and two more in that group became deficient.
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The effect of papaya appeared to be approximately equivalent to that of ß-carotene capsules. There were no significant differences in their effectiveness for any variable measured. Furthermore, there were no significant differences between the carrot and the papaya groups. However, the effect on serum retinol was significantly poorer in the carrot group than in the groups that received ß-carotene capsules.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The supplements effectively increased the mean serum retinol concentrations in all treatment groups, but not in the placebo group. The mean decrease in the RDR in the carrot group was not significant (P > 0.05), nor was it different from that in the placebo group. Not only were the mean values of the different indicators improved in the treatment groups, but the proportion of women below the WHO cut-off points for all indicators was also reduced.
Of great interest regarding the effect of diet-based approaches is
the type of carotene-containing food used, as well as the type of
meal eaten with it. In our study, we chose papaya and carrots because
both are high in carotene, they can be grown by and are readily
acceptable to the rural population of Zimbabwe, and they represent a
fruit and a root vegetable, respectively. Our study showed that grated
carrots and puréed papaya do improve the vitamin A and iron
status of lactating women. It is likely that the partial processing,
i.e., the grating of the carrots and the puréeing of the papaya,
enhanced the bioavailability of the carotenes in these two supplements.
Both supplements used in our study were processed by methods familiar
and acceptable to the population studied. Recent studies have shown
that processing of carrots might improve the bioavailability of their
ß-carotene. Brown et al. (8)
provided 29 mg of
ß-carotene in a single meal of cooked carrots and reported a
bioavailability of 21%; Huang et al. (18)
provided 12 mg
of ß-carotene in stir-fried shredded carrots and reported a
bioavailability of 33%; and Torronen et al. (19)
provided
12 mg of ß-carotene in carrot juice daily for 6 wk and reported a
bioavailability of 45%. In all of these studies, carrots in different
forms were used as supplements and their bioavailability seemed to
increase with processing, with carrot juice showing the highest
bioavailability.
All subjects in our supplementation trial, including those in the
placebo group, were given a daily meal consisting of corn, beans and
vegetable oil after taking the supplement. The meal was given to
minimize the effects of a low fat diet. Fat added to dark green leafy
vegetables has been shown to enhance serum retinol levels after
children were supplemented for 12 wk (20)
. Shiau et al.
(21)
reported that 35–71% labeled ß-carotene was
recovered as fecal losses when ß-carotene was taken with a meal,
compared with 83% without a meal. The dosage of ß-carotene (6 mg) in
our study approximated the recommended daily requirements for lactating
women (22)
and was given daily for 60 d. Repeated
doses of ß-carotene give less variable results than large single
pharmacologic doses. Constantino et al. (23)
and dePee et
al. (24)
found that the serum retinol response to
ß-carotene from fruits was four times that from green leafy
vegetables. The enhanced response could be due to the fact that
carotenoids in yellow-to-orange fruits are dissolved in oil droplets in
chromoplasts and can be readily extracted during digestion. In green
leafy vegetables, the ß-carotene molecules are in pigment-protein
complexes located in chloroplasts and are more difficult to extract.
Carrots may be intermediate between these extremes in their carotenid
bioavailability. The ß-carotene in carrots exists in crystalline
form, making it less bioavailable than that in orange and yellow fruits
(25)
.
The addition of fat to the diet greatly improves the absorption of
carotenoids (21
,26
,27)
. The meal that was given with the
supplements in our study provided 10 g of fat. The severely vitamin
A–deficient women in our study showed a significantly greater response
than women with a more normal vitamin A status. Similar observations
were made in two other studies (28
,29)
in which the
vitamin A status of malnourished children improved after consumption of
green leafy vegetables.
Our findings show a reduction in the proportion of women with anemia in the supplementation groups but not in the placebo group. Similarly, all of the subjects with iron deficiency anemia in the supplemented groups had normalized levels at the endpoint but this was not the case in the placebo group. The general improvement in serum ferritin could have been a result of the meal given to all groups and might also be a reflection of the hemo-concentration women experience after pregnancy and delivery.
In conclusion, our study showed that ß-carotene supplementation with yellow-to-orange fruits and vegetables improves the vitamin A and iron status of lactating women. In each of the supplemented groups, those who were severely malnourished showed significant improvements in all indicators, i.e., serum retinol, relative dose response, hemoglobin and serum ferritin.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CRP, C-reactive protein; Hb, hemoglobin; RDR, relative dose response.
Manuscript received July 18, 2000. Initial review completed August 23, 2000. Revision accepted January 8, 2001.
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) are baseline means and 95% confidence interval and
circles (
) are endpoint means and 95% confidence interval. Endpoint
values are after a 60-d supplementation trial.