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Department of Nutritional Sciences, The Pennsylvania State University, University Park, PA
4To whom correspondence should be addressed. Email: mhg{at}psu.edu.
ABSTRACT
To further investigate the effect of dietary vitamin A (VA) intake on milk VA concentrations and pup VA status, female rats were fed 2 concentrations of VA [0 (n = 9) or 50 µmol/kg diet (n = 10)] during pregnancy and lactation. Plasma retinol concentrations were significantly higher (30–40%) during lactation than before pregnancy or after weaning but were not influenced by dietary VA. In rats fed VA, VA concentrations during lactation were significantly higher in milk (1.5–3 times), mammary tissue (>100%), liver (4 times), pup plasma (20–40%), and pup liver (1.1–6.7 times). In Expt. 2, when VA intake was switched on d 7 of lactation from 0 to 50 µmol/kg, milk VA concentrations (2.24 ± 0.42 µmol/L; mean ± SD, n = 6) increased significantly (1.7 times) by d 9 to the same level as in rats administered 50 µmol/kg (6.04 ± 0.60 µmol/L; n = 6). When VA was removed from the diet on d 7, concentrations declined significantly (by 50%) and by d 11 were the same as those in rats given 0 µmol/kg. We conclude that the rapid effect of changes in dietary VA intake are attributable to changes in the delivery of chylomicron VA to mammary tissue and milk.
KEY WORDS: • lactation • vitamin A supplementation • vitamin A status • milk vitamin A
Vitamin A (VA)5 is essential for growth, but VA stores are low at birth in humans and rodents (1,2). Thus, an adequate concentration of VA in maternal milk is critical for improving the vitamin A status of newborns. Several studies in humans (3–6) and in animal models (7,8) showed that VA supplementation during lactation increases VA concentrations in milk. For example, Stoltzfus et al. (3) found an increase in milk VA after a single dose of the vitamin 1–3 weeks postpartum in Indonesian women. Similar observations were made by Roy et al. (4). Rice et al. (5) reported that giving a single dose of VA to Bangladeshi women at the time of parturition led to an acute increase in milk VA concentrations at 3 mo; however, the higher levels were not sustained at 6 and 9 mo. In chronic studies in rats, increased VA intakes during pregnancy and lactation are associated with increased milk VA concentrations (7,8).
On the basis of experiments in rats, it was suggested that chylomicrons play an important role in the delivery of VA to the lactating mammary gland and thus to milk. A recent report by Ross et al. (9) indicated that the recovery of [3H]vitamin A in mammary tissue after administration of [3H]VA-labeled chylomicrons peaked shortly after dosing, and that concentrations decreased over time. It was suggested that the uptake of VA by the tissue increases as chylomicron triglycerides are hydrolyzed by mammary tissue lipoprotein lipase. In a recent kinetic study, we found that the contribution of chylomicron VA vs. holo retinol-binding protein (holo RBP) to milk increased as a function of dietary VA intake during lactation (10).
In this study, we further investigated the effects of dietary VA on plasma and tissue VA during pregnancy and lactation in rat dams and their pups, and we examined the acute effects of changes in VA intake on milk and tissue VA concentrations. Among our findings, we determined that acute changes in dietary VA intake have rapid effects on milk VA concentrations in rats, presumably reflecting the sensitivity of the chylomicron delivery system to vitamin A intake.
MATERIALS AND METHODS
Animals and diets. Sexually mature virgin female and adult male Sprague-Dawley rats were purchased from Harlan Teklad. Rats were housed individually in a room with controlled temperature (22–23°C), humidity (60%), and light:dark cycle (light from 0600 to 1800), and they were allowed free access to food and water. Rats were fed a modification (8) of the AIN-93G purified diet (11) containing various amounts of vitamin A (see below) in the form of retinyl palmitate (Sigma Chemical). All animal procedures were approved by the Institutional Animal Care and Use Committee of The Pennsylvania State University.
Experimental designs.
For Expt. 1, female rats (56–63 d of age) were fed the purified diet containing 10 µmol VA/kg for 12 d after arrival as well as during mating (Fig. 1). For mating, 2 females were housed with 1 male rat for 5 d. After mating, females were randomly assigned to be fed either a vitamin A–free purified diet (–VA, n = 9) or the same diet supplemented with 50 µmol of retinyl palmitate/kg (+VA, n = 10). Rats consumed these diets during pregnancy and until d 21 of lactation, at which time they were fed a diet containing 4 µmol VA/kg for 7 d. The –VA and +VA diets were chosen to produce a negative and positive vitamin A balance, respectively. Because rats in the –VA group consumed a VA-adequate diet before breeding, their livers had accumulated sufficient VA to support pregnancy and lactation, and no signs of VA deficiency were observed in dams or pups. The +VA diet provided 
750 nmol vitamin A/d [2.125 µmol/(kg0.75·d)], assuming food intake of 15 g/d and a body weight of 250 g, which is 8.4 times (relative to metabolic body size) the recommended intake for lactating women in the United States. Although this amount constitutes a high VA intake, it would be readily obtainable through dietary supplements. In our studies, no signs of vitamin A excess were observed.
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For Expt. 2 (Fig. 1), female rats (57–63 d old, n = 30) were fed the –VA diet for 5 d and then during mating. To determine whether rats were pregnant before supplementation began, body weights were monitored for 7 d after mating while rats continued to consume the –VA diet. Nonpregnant rats (n = 6) were killed, and blood samples and livers were obtained for baseline values. Among the pregnant rats, half were fed the +VA diet (50 µmol vitamin A/kg, n = 12) whereas the others continued to consume the vitamin A–free diet (–VA, n = 12) for the rest of pregnancy and until d 7 of lactation. After parturition, litter size was adjusted as indicated in Expt. 1. On d 7 of lactation, blood and milk samples were collected from all dams. Then, half of the rats in each group were fed the other diet, which yielded 4 treatments: –VA/+VA, +VA/–VA, –VA/–VA, and +VA/+VA. Blood and milk samples were taken from dams on d 9, 11, 13, 16, 19, and 21 of lactation. Pups were killed at 21 d of age and dams were killed 7 d later; blood samples, livers, and mammary tissues (dams only) were collected.
Sample collection.
For dams, a tail vein was nicked with a #10 scalpel blade and blood was collected into microcentrifuge tubes containing Na2EDTA as an anticoagulant. Samples were immediately centrifuged at 12,535 x g for 4 min, and plasma aliquots (80–100 µL) were placed into test tubes. Samples were purged with nitrogen gas and stored at –20°C until analyzed.
For milk collection, pups were removed from the mother for 30 min in the early to midmorning. Oxytocin (20 IU) was administered i.m. to the dam, mammary glands were gently massaged, and milk (100–250 µL) was collected. Dams were then immediately returned to their pups. Aliquots of milk (100–200 µL) were stored as described for plasma.
At the time of killing, rats were asphyxiated with CO2. A blood sample was collected from pups using open chest heart puncture and then livers were excised. Because of their small size, pup livers from each dam were pooled for analysis. In the case of the dams, bodies were perfused by pumping 100 mL of 0.25 mol/L (86 g/L) cold sucrose solution through the vascular system from the left ventricle to the right auricle. Livers were excised and mammary tissues were dissected using a # 10 scalpel (Bard-Parker). Tissues were blotted, weighed, and stored under nitrogen at –20°C for later analysis. Livers were lyophilized before analysis.
Vitamin A analyses. To protect samples from photooxidation, all vitamin A analyses were conducted under fluorescent lights shaded with a UV-blocking film (CLCH, Sydlin). For quantification, TMMP-retinol [all-trans–9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraen-1-ol; donated by Hoffmann-La Roche], a nonsaponifiable internal standard, was added to samples before extraction.
Vitamin A was extracted from plasma into hexane containing BHT (5 mg/L) (12). Samples of milk, lyophilized liver, and mammary tissue were saponified in ethanol containing 0.1% pyrogallol and 60% potassium hydroxide (13,14). Samples were incubated for 45 min at 60°C and then extracted with hexane containing BHT. Lipid extracts of plasma, milk, liver, and mammary tissue were concentrated under a stream of nitrogen in a water bath at 37°C. Sample residues were dissolved in methanol and analyzed by reversed phase HPLC as described by Green et al. (8).
Statistical analyses. Descriptive data are presented as means ± SD. Statistical analyses were performed using Minitab (version 12) and SPSS (version 11.5). For Expt. 1, the effects of diet, time, and the interaction of diet x time on milk, mammary tissue, dam plasma and dam liver VA concentrations, as well as on pup plasma and liver VA were tested using a general linear model. Post hoc tests used for within- and between-group comparisons were Tukey's test and 2-sample t tests. For Expt. 2, effects of diet, time, and the interaction of diet x time on milk VA were tested using a general linear model; then the Mann-Whitney U test was used to analyze milk VA at each time. Plasma and mammary tissue VA of dams, and plasma and liver vitamin A of pups after lactation, were compared between groups using a 1-way ANOVA and Tukey's post hoc test. Differences were considered significant at P < 0.05.
RESULTS AND DISCUSSION
Experiment 1. Neither maternal nor pup body weights were affected by diet (data not shown). The pregnancy success rate was 87%; litter sizes ranged from 7 to 16 pups/dam in rats fed the +VA diet and from 12 to 17 in the –VA group.
As expected based on current understanding of the regulation of plasma retinol, diet had no effect on plasma retinol concentrations in this experiment (Fig. 2). However, in both dietary groups, plasma retinol concentrations were 30–40% higher during lactation than during pregnancy or after weaning (P < 0.001). Related to these observations, it was reported in older literature (15 and references therein) that plasma retinol concentrations decrease during pregnancy, perhaps due to the expanded plasma volume, or increase after parturition in humans. In rats, higher plasma retinol concentrations during lactation may be important in providing a basal level of VA to milk (see below).
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1.6 µmol/L) is presumably derived from holo RBP because chylomicrons would contain almost no vitamin A (10).
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Not surprisingly, maternal liver VA concentrations were also affected by diet in Expt. 1 (Table 1). Concentrations were 3.8 times higher on d 13 of lactation and 6.5 times higher on d 28 in the +VA group than in dams fed the –VA diet. However, within each group, liver vitamin A concentrations on d 13 and 28 did not differ.
For pup plasma retinol and liver vitamin A concentrations, diet, time, and the diet x time interaction were all significant (P < 0.001) (Table 2). Vitamin A concentrations were higher at all times in both plasma and liver of offspring from the +VA compared with the –VA group. Pup liver VA concentrations increased with time in the +VA group. Maternal diet may have contributed to this increase because we observed that pups were both nursing and consuming their mother's diet from d 13 of lactation. Our findings provide further evidence for the critical role of increased milk VA concentrations in improving VA status of offspring as well as the usefulness of milk VA concentration as an indicator of VA status in the nursling (16).
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50% lower than on d 7 (3.30 ± 0.82 vs. 6.70 ± 1.86 µmol/L). In that group, milk VA concentrations did not differ from those in the –VA/–VA group on d 11 of lactation or thereafter. In all groups, milk VA concentrations were lower toward the end of lactation (i.e., from d 19 to 21 of lactation) than earlier (i.e., d 7 to 13 of lactation) (P < 0.05).
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Results from this study extend the evidence that increased concentrations of vitamin A in milk are a direct effect of dietary vitamin A intake (i.e., chylomicrons). The increase in maternal VA intake resulted in a 4 µmol/L difference in milk VA, indicating that at least 60% of the vitamin A in milk is supplied by sources other than holo RBP (presumably chylomicron vitamin A). Based on the assumption that the volume of milk produced is 40 mL/d (17) in lactating rats and that VA intake of dams was 750 nmol/d, we estimate that a minimum of 28% of maternal VA intake [assuming a 75% absorption efficiency (18)] was directed toward milk production and therefore delivered to pups. The rapid change in milk VA when vitamin A was added to the diet indicates a high transfer rate of the vitamin to milk, a process that is dependent upon maternal vitamin A intake.
In an approach supported by the WHO (19), it is recommended that women at risk for low VA status be administered a single high-dose supplement of VA soon after parturition to improve concentrations of vitamin A in milk and thus the infant's VA status. However, the increase in milk VA concentration in response to such supplementation is not maintained throughout lactation (5). Although it would undoubtedly be much more expensive and difficult to provide on-going supplements of preformed vitamin A to lactating women at risk, our results show that, in rats, a higher intake of VA throughout lactation maintains increased concentrations of VA in milk and improves VA status of the young. By extension, it is likely that long-term VA supplementation of lactating women at risk of VA deficiency would provide an optimal strategy for improving VA status of the young.
FOOTNOTES
1 Presented in part at Experimental Biology 03, April 2003, San Diego, CA [Akohoue SA, Green JB, Green MH. Acute contributions of dietary vitamin A to milk vitamin A in the rat (abstract). 2003; FASEB J.17:A313], and as part of a doctoral thesis [Akohoue SA. Direct contributions of dietary vitamin A to mammary tissue and milk vitamin A in the rat (dissertation), University Park, PA: The Pennsylvania State University; 2003]. 
2 Supported by National Institutes of Health grant RO1HD32500 to M.H.G. and by funds from the College of Health and Human Development at The Pennsylvania State University.
3 Present address: Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232.
5 Abbreviations used: holo RBP, holo retinol-binding protein; TMMP-retinol, all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraen-1-ol; VA, vitamin A.
Manuscript received 13 July 2005. Initial review completed 15 August 2005. Revision accepted 28 September 2005.
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