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National Institute of Nutrition, Indian Council of Medical Research, Jamai-Osmania Hyderabad - 500 007, India
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSExperimental protocolRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • vitamin A deficiency • protein deficiency • ß-carotene dioxygenase • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSExperimental protocolRESULTSDISCUSSIONREFERENCES |
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). Gronowska-Senger and Wolf (1970)
found reduced intestinal carotene cleavage enzyme activity in
protein-deficient rats. Vitamin A deficiency has been shown to
increase carotene cleavage activity (Villard and Bates 1986
). However, no data are available on in vivo conversion
either in protein- or vitamin A-deficient rats. Thus the importance
of the changes in ß-carotene dioxygenase activity in modifying the
availability and absorption of vitamin A has not been ascertained.
Unlike vitamin A, high doses of ß-carotene are not toxic. This is
evident in patients with erythropoietic protoporphyria where treatment
with large doses of ß-carotene for prolonged period did not result in
vitamin A toxicity (Mathews-Roth 1989
). This lack of
toxicity may have been due to the down-regulation of ß-carotene
dioxygenase activity. While considerable attention has been paid to the
qualitative and quantitative conversion of carotene to vitamin A and
the underlying mechanisms, there have been no attempts to correlate in
vitro enzyme activity with in vivo conversion of ß-carotene to
vitamin A. We have recently described a novel approach to obtain in
vivo conversion of ß-carotene to vitamin A in rats and children and
demonstrated that the efficacy is related to the vitamin A status in
rats (Parvin et al. 1999
). This is based on the ratio of
the area under the plasma vitamin A concentration vs. time curves
(AUC0–12h)3
for vitamin A obtained after oral doses of ß-carotene or vitamin A
and therefore referred to as relative conversion of carotene to vitamin
A. In an effort to validate the method and to obtain useful information
on carotene nutriture, differences in the in vivo conversion of
ß-carotene to vitamin A were compared with the in vitro ß-carotene
dioxygenase enzyme activity in vitamin A- and protein-deficient
rats, as well as in appropriate controls.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSExperimental protocolRESULTSDISCUSSIONREFERENCES |
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Male weanling WNIN rats (21-d-old) were supplied by the National Center for Laboratory Animal Sciences (NCLAS) at NIN. The rats were housed in individual cages in a well-ventilated room and fed their experimental diets. Free access to water was ensured. Individual body weights were recorded once each week, and food intake was monitored daily. Daily dark and light cycles were of 12-h duration. The experiments were approved by the Institutional Animal Ethical Experiments Committee.
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Experimental protocol |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSExperimental protocolRESULTSDISCUSSIONREFERENCES |
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Vitamin A deficiency.
Weanling rats were randomly assigned to three groups (n
= 30) and were fed a vitamin A-deficient basal diet
(John and Sivakumar 1989
). Vitamin A-deficient (AD)
and control (AA) rats had free access to the diet while the third group
(AR) received the diet restricted to the mean amount consumed by the AD
rats. AA and AR rats were fed twice weekly oral supplements of 40 µg
retinol/d as retinol palmitate, prepared in arachis oil. When the rats
attained the weight-plateau stage of deficiency at 8–10 wk, in
vivo and in vitro studies were conducted to assess the conversion of
ß-carotene to vitamin A.
In vivo studies.
Four different doses of either ß-carotene (50, 100, 200, 300 µg) or
retinol palmitate (equivalent to 25, 50, 100, 150 µg retinol) were
given orally to three rats in each group in 0.2 mL arachis oil. Blood
was drawn at 0, 4, 8 and 12 h. The doses used were fixed based on
the preliminary studies, wherein they resulted in significant increases
in plasma vitamin A from the basal value. The dose was administered
after depriving rats of food overnight. Rats were not fed until the
last blood sample was drawn. Plasma vitamin A was estimated by
spectrofluorimetry (Selvaraj and Susheela 1970
),
AUC0–12h were calculated using the trapezoidal rule
(Gibaldi and Perrier 1975
). The regression lines
for serum vitamin A AUC0–12h vs. ß-carotene doses or
vitamin A doses were computed and the slopes were calculated. The ratio
of slope of vitamin A from ß-carotene-treated rats to that of vitamin
A-treated rats provides an estimate of the effectiveness of
ß-carotene as a source of vitamin A.
In vitro studies.
Six rats from each group were killed by cervical dislocation after
withholding food overnight. The 100,000 x g
supernatant of intestinal duodenal mucosa was prepared for the assay of
ß-carotene dioxygenase (EC 1.13.11.21). The method was essentially as
described by Goodman and Olson (1969)
, and the products were assayed by
HPLC according to Wang et al. (1991)
.
Experiment 2.
Protein deficiency. Weanling rats (n = 42) were randomly assigned to three groups. One received a 5% protein diet (PD) and the other two control groups received a 20% protein diet. PD rats and one of the controls (PA) had free access to diet. The other control group (PR) was fed the 20% protein diet in an amount equal to the mean intake of PD rats.
The protein-deficient diet included (g/kg): wheat flour (55),
roasted chick pea (Cicer arietinum) powder (151), casein
(10), skimmed milk powder (10), arachis oil (60), vitamin mixture (10),
salt mixture (40), corn starch (600), cellulose (64). In the 20%
protein diet, the quantities of chickpea powder, casein and skimmed
milk were quadrupled at the expense of starch. The vitamin and salt
mixtures were described earlier (John and Sivakumar 1989
). After 6 wk, in vivo conversion of ß-carotene to
vitamin A using 100 µg oral ß-carotene or vitamin A and intestinal
ß-carotene dioxygenase activity was determined in these rats as
described for vitamin A-deficient rats. A single dose was employed
in this experiment because the linear dose response for
AUC0–12h with different doses of ß-carotene and vitamin
A doses was established in the vitamin A-deficiency experiment.
Plasma vitamin A was estimated by the fluorimetric method given earlier
and serum albumin was determined by a dye-binding method
(Gustafsson 1976
). Both methods have intraassay and
interassay variations < 6%.
Food restriction. Because we had two sets of data from food-unrestricted and food-restricted controls in the vitamin-A and protein-deficiency experiments, their in vitro and in vivo conversions of ß-carotene to vitamin A were compared also.
Statistical analyses. The differences in means for variables such as body weight, plasma vitamin A, AUC0–12h, serum albumin and enzyme activity were evaluated by ANOVA with least significant differences as the posthoc test. Changes in plasma vitamin A with time were evaluated by the repeated measures ANOVA.
For each rat, the AUC0–12h were calculated after correction for the initial plasma vitamin A concentration. All AUC0–12h in expt.1 were plotted against the doses administered and subjected to regression analysis for the three different groups. The statistical significance of correlations between the dose and AUC0–12h for ß-carotene or vitamin A were tested for their difference from 0. The correlation coefficients (r) were compared among the three groups and tested for homogeneity using ‘z ’ transformation. In the case of expt. 2, the ratio of vitamin A AUC0–12h for ß-carotene and vitamin A was derived from the mean values of AUC0–12h from four rats obtained with a single dose of ß-carotene or vitamin A.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSExperimental protocolRESULTSDISCUSSIONREFERENCES |
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Induction of deficiency.
Rats fed vitamin A-deficient diet (AD) became vitamin
A-depleted after 8 wk, and their body weight was less than in the
AR and AA rats (P < 0.01) (Table 1
). Their lower plasma vitamin A (P < 0.01) along with
no significant gain in body weight over the preceding week (data not
shown) indicated that the weight-plateau stage of vitamin A
deficiency had been attained. AR rats that received a restricted amount
of food had significantly lower body weight and plasma vitamin A than
AA rats (P < 0.01).
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). In general, AUC0–12h of plasma vitamin A in
rats given higher doses of both ß-carotene or vitamin A were greater
than in rats administered lower doses and showed no further change
after an optimum dose was reached (Table 3
). There was a significant positive correlation between the dose of
ß-carotene or vitamin A administered and the
AUC0–12h of plasma vitamin A in AD rats
(Fig. 1A
and B
). The results in AR and AA rats were similar to
those of AD rats when vitamin A dose was administered. However, in the
case of ß-carotene, the relationship was linear and significant only
up to the 200-µg dose (r = 0.714, P
< 0.05; n = 9) and not with the inclusion of the
300 µg dose (r = 0.405, P > 0.05;
n = 12).
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In AD group rats, the in vivo conversion of ß-carotene to
vitamin A derived from the slope ratio was higher than the values in AR
and AA rats. The ß-carotene dioxygenase activity in AD rats was
significantly greater than in AR and AA rats (Table 4
).
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. The
body weights of PR rats were significantly lower than those of PA rats.
The serum albumin concentrations did not differ among the three groups.
The AUC0–12h with vitamin A dose was
significantly higher in PD rats compared to other two groups
(Table 5
).
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.
The intestinal ß-carotene dioxygenase activity was also lower in PD
rats than in both the controls.
Food restriction.
The in vivo conversion of ß-carotene to vitamin A in AR and PR groups
was lower than in their corresponding food-unrestricted controls,
AA and PA rats (Table 4)
. The ß-carotene dioxygenase activity was
comparable in the two sets of control groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSExperimental protocolRESULTSDISCUSSIONREFERENCES |
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). In the present study we have compared the enzyme activity with in vivo conversion in vitamin A- and protein-deficient rats and in food-restricted rats.
Vitamin A deficiency.
The weight-plateau stage represents depletion of vitamin A stores
without any complications such as infection. The plateauing of weight
gains and a decline in the plasma level of vitamin A at 8 wk confirm
the attainment of the weight-plateau stage of deficiency in AD rats
(Table 1)
.
As expected, the plasma levels of vitamin A after ß-carotene or
vitamin A administration showed a peak after 4–8 h and subsequently
decreased by 12 h. However, in AD rats, the 12-h plasma vitamin A
concentrations were above the basal values. This is consistent with the
earlier findings of Loerch et al. (1979)
, who developed the relative
dose-response test. In vitamin-A deficiency, accumulation of
apo-RBP in liver was reported to occur, and a pulse of vitamin A
resulted in dramatic release of vitamin A into the circulation as the
holo-RBP complex. Thus, in vitamin A-depleted rats, the plasma
vitamin A levels at 5 h are expected to be higher than those in
the basal state, whereas in controls, there is no increase at 5 h.
The increase in efficiency of conversion of ß-carotene to vitamin A
in AD rats, as reflected by the AUC0–12h slope
ratio (Table 4)
may be an adaptive response that enhances the
availability of vitamin A to the system during adverse vitamin A
nutriture. Villard and Bates (1986)
made similar observations. There
have been few studies in which in vitro enzyme activity was compared
with in vivo conversion in the same set of rats.
Recently, an effort was made by van Vliet et al. (1996)
to correlate
the in vitro intestinal ß-carotene dioxygenase activity with the in
vivo ß-carotene conversion to vitamin A. In this study, a dose of
ß-carotene was administered to rats with three different levels of
vitamin A nutriture. The intestinal conversion was derived using the
molar ratio of retinyl esters and ß-carotene present in lymph. The
authors concluded that the activity of the intestinal enzyme is an
adequate indicator of in vivo cleavage activity. However, the validity
of the data of van Vliet et al. (1996)
is questionable because no
corrections were made for changes in vitamin A pool size in vitamin
A-deficient rats, and no quantitative data on conversion were
given. Also, the data pertain to only a small fraction of the total
vitamin A that was administered.
Protein deficiency.
In PD rats, there was a significantly lower body weight, though the
serum levels of albumin were not significantly affected. This may be
because 6 wk of feeding the 5% protein diet had not resulted in severe
enough protein deficiency to alter the serum albumin levels (Table 1)
.
One of the important observations is that both in vitro ß-carotene
dioxygenase enzyme activity as well as in vivo carotene conversion
reflected by AUC0–12h ratio were lower in PD
rats than in controls (Table 4)
. Earlier, Gronowska-Senger and Wolf (1970)
found lower intestinal ß-carotene dioxygenase activity in
protein-deficient rats. Our results confirm their finding. However,
there have been no reports in the literature on in vivo conversion of
ß-carotene to vitamin A in protein-deficient animals.
Food restriction.
Food-restricted rats had similar specific activity of ß-carotene
dioxygenase to controls in both experiments but relatively lower in
vivo conversion (about 25% in expt. 1, 50% in expt. 2) compared to
rats with free access to food (Table 4)
. The reasons for this
difference in the two variables are not understood. It is possible that
factors other than ß-carotene dioxygenase enzyme activity may be
involved in the conversion of ß-carotene to vitamin A.
As described by other investigators (Gronowska-Senger and Wolf 1970
, Villard and Bates 1986
), we found greater
ß-cartotene dioxygenase activity in vitamin A-deficient rats and
a lower activity of this enzyme in protein-deficient rats. The
variation in intestinal enzyme activity was thus found to broadly agree
with the in vivo conversion in each set of experiments, validating the
method for determining in vivo conversion values (Table 4)
. This is the
first time that differences in ß-carotene dioxygenase activity were
related to the physiologically important measure of in vivo conversion.
In contrast to what is generally accepted, that ß-carotene conversion
is a constant figure of 50% (WHO 1967
), the present
results indicate that it is flexible and varies with the nutritional
status.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AA, food-unrestricted control to AD group; AD, vitamin A- deficient group; AR, food-restricted control to AD group; AUC0–12h, area under plasma vitamin A time-curves during 0–12h; PA, food-unrestricted control to PD group; PD, protein-deficient group; PR, food-restricted control to PD group; RBP, retinol-binding protein.
Manuscript received May 10, 1999. Initial review completed May 28, 1999. Revision accepted November 5, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSExperimental protocolRESULTSDISCUSSIONREFERENCES |
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1. FAO/WHO (1967) Requirements of vitamin A, thiamine, riboflavine and niacin. FAO Ser. No. 8, WHO Tech. Rep. Ser. No. 362. FAO, Rome and WHO, Geneva.
2. Gibaldi M., Perrier D. Swarbrick J. eds. Drugs and Pharmaceutical Sciences 1975;Vol I:48 Pharmacokinetcs Marcel Dekker Inc., New York
3. Goodman D. S., Olson J. A. The conversion of all trans ß-carotene into retinal. Meth. Enzymol. 1969;15:462-475
4. Gronowska-Senger A., Wolf G. Effect of dietary protein on the enzyme from rat and human intestine which converts ß-carotene to retinal. J. Nutr. 1970;100:300-308
5. Gustafsson J. E. C. Improved specificity of serum albumin determination and estimation of acute phase reactants by use of the bromocresol green reaction. Clin. Chim. 1976;22:616-622
6. John A., Sivakumar B. Effect of vitamin A deficiency on nitrogen balance and hepatic urea cycle enzymes and intermediates in rats. J. Nutr. 1989;119:29-35
7. Loerch J. D., Underwood B. A., Lewis K. C. Response of plasma levels of vitamin A to a dose of vitamin A as an indicator of hepatic vitamin A reserves in rats. J. Nutr. 1979;109:778-786
8. Mathews-Roth M. M. ß-carotene: clinical aspects. New Protective Roles for Selected Nutrients Curr. Top. Nutr. Dis. 1989;22:17-38
9. Parvin, S. G., Bhaskaram, P. & Sivakumar, B. (1999) A novel pharmacokinetic approach to determine the relative efficiency of intestinal conversion of ß-carotene to vitamin A in rats and children. Nutr. Res. (in press).
10. Selvaraj R. J., Susheela T. P. Estimation of serum vitamin A by a micro fluorimetric procedure. Clin. Chim. Acta. 1970;27:165-170[Medline]
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Smith J. E., Muto Y., Milch P. O., Goodman D. S. The effects of chylomicron vitamin A on the metabolism of retinol-binding protein in the rat. J. Biol. Chem. 1973;248:1544-1549
12. van Vliet T., Vlissingen M. F., van Schaik F., van Den Berg H. ß-carotene absorption and cleavage in rat is affected by the vitamin A concentration of the diet. J. Nutr. 1996;126:499-508
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14. Wang X. D., Tang G. W., Fox J. G., Krinsky N. I., Russell R. M. Enzymatic conversion of ß-carotene into ß-apocarotenals and retinoids by human, monkey, ferret and rat tissues. Arch. Biochem. Biophys. 1991;285:8-16[Medline]
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