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Nutrition Department, The Pennsylvania State University, University Park, PA 16802
2To whom correspondence should be addressed. E-mail: mhg{at}psu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • chylomicrons • vitamin A • milk • mammary tissue • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
and experimental
animals (4
,5)
. However, the mechanistic relationships
between dietary vitamin A and milk vitamin A have not been studied
extensively. Thus, the quantitative contributions of the two
physiologic carriers of vitamin A, chylomicrons and retinol-binding
protein
(RBP),4
to milk vitamin A are not yet known.
Scow and colleagues (6
,7)
hypothesized that during
hydrolysis of chylomicron triglycerides by lipoprotein lipase (LPL), a
membrane continuum develops among chylomicrons, the endothelial cells
to which LPL is anchored and the underlying tissue parenchymal cells.
This continuum may facilitate transfer of fatty acids, partial
glycerides, and some unesterified cholesterol and fat soluble vitamins
to the parenchymal cells. Further, Blaner et al. (8)
showed that LPL can hydrolyze [3H]retinyl
esters in lipid emulsions and that the enzyme increases
[3H] uptake into cultured adipocytes. Because
LPL activity decreases in white adipose tissue and increases in mammary
tissue during lactation (9)
, dietary fatty acids and
presumably other lipid-soluble nutrients are directed into milk
fat. It is thus reasonable to hypothesize that higher vitamin A intakes
are associated with an increased contribution of dietary (chylomicron)
vitamin A to milk.
Here we used the kinetic technique of "continuous infusion"
(10)
to examine the contribution of newly absorbed dietary
vitamin A to milk vitamin A during lactation in rats. By preloading the
liver with unlabeled vitamin A and then labeling the ingested vitamin A
in rats fed two levels of vitamin A, we were able to estimate the
quantitative importance of chylomicrons vs. holoRBP to milk as a
function of dietary vitamin A intake.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female (60-d-old) and adult male Sprague-Dawley rats were purchased from Harlan Teklad, Madison, WI. Rats were housed individually in shoe-box cages in a room controlled for temperature (22–24°C), humidity (50%) and light (0700–1900 h). Animals had free access to food (see below) and water throughout the studies; females were weighed twice weekly. Animal procedures were approved by The Pennsylvania State University’s Animal Care and Use Committee.
Female rats were fed a modification of the AIN-93G diet
(11)
containing the following (g/kg): vitamin-free
casein, 200; cornstarch, 397; maltodextrin, 132; sucrose, 100;
cellulose, 50; mineral mix, 35 (AIN93GMX, Teklad); vitamin A–free
vitamin mix, 10 (TD94161, Teklad); L-cystine, 3; choline
bitartrate, 2.4; t-butylhydroquinone, 0.014; and soybean
oil, 70 to which had been added 10 µmol of retinyl
palmitate (Sigma Chemical, St. Louis, MO) per kg. Male rats were fed
the same diet when they were being used for breeding and a commercial
cereal-based diet (Laboratory Rodent Diet 5001; PMI Nutrition
International, St. Louis, MO) at other times.
Specific batches of purified diet were labeled with [3H]vitamin A. [10,11-3H]Retinyl acetate (sp. act. 3,168 TBq/mmol; generously donated by Hoffman-La Roche, Nutley, NJ) was dissolved in a small amount of soybean oil; this oil was premixed with the total amount of oil required to formulate the diet. Vitamin A–labeled diets were fed during lactation as indicated below.
Experimental design.
Female rats were mated beginning at 80 d of age by housing two
females with one male for 5 d. After mating, females either
continued to consume the diet providing 10 µmol
vitamin A/kg (LO) or were fed the same diet with a higher amount of
vitamin A (50 µmol/kg; HI). These diets provided
vitamin A intakes of 
150 and 750 nmol/d, respectively, when food
intake was 15 g/d at the beginning of pregnancy.
Three days after parturition, litter sizes were adjusted to 7 pups/dam.
At 0000 h on d 6 of lactation, dams were offered feed that
provided the same vitamin A concentration as in their previous diet
plus trace amounts of radioactive vitamin A. Samples of maternal blood
and milk were obtained at 8 h, 2.3 and 4.3 d after the start
of feeding the radioactive diet (d 6, 8 and 10 of lactation). At each
time, dams were separated from pups for 30 min. A blood sample
(200 µL) was collected from a tail vein of each dam
into microcentrifuge tubes containing Na2EDTA. Then
oxytocin (2 U; Sigma) was administered intramuscularly.
Milk (300 µL) was collected by gentle manual
massage of the mammary glands, and the dam was returned to her litter.
Aliquots of plasma and milk were frozen under nitrogen for subsequent
analyses of vitamin A and tritium (see below).
On d 13 of lactation (7.3 d after the start of feeding the radioactive diet), dams (n = 6 in the LO group and n = 4 in HI) were killed after collection of samples of blood and milk as described above. In the LO group, 4 dams/time were also killed 8 h and 3.3 d after the start of feeding the radioactive diet. We had planned to include the same groups in the HI study, but resources were limited. At the time of killing, dams were anesthetized with ketamine HCl/xylazine [100 mg ketamine/kg body (Aveco, Fort Dodge, IA) and 10 mg xylazine/kg body weight (Mobay, Shawnee, KS)]. Mammary tissue was dissected using a #10 scalpel and livers were removed. Tissues were blotted, weighed and stored under nitrogen at -16°C for later analyses of vitamin A and radioactivity (see below). Pups were individually weighed and asphyxiated with CO2; livers were excised, blotted, weighed and frozen until analyzed for vitamin A and radioactivity. Dam and pup livers were lypophilized before analysis.
Vitamin A analyses.
All procedures involving vitamin A were conducted under fluorescent
light shaded by a UV-blocking film (CLCH; Sydlin, Lancaster, PA) to
prevent the photooxidation of vitamin A. Aliquots of plasma were
extracted (12)
, and aliquots of diet, milk, mammary
tissue and liver were saponified (13)
after addition of
the nonsaponifiable retinoid,
all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraen-1-ol
(TMMP-retinol; donated by Hoffmann-La Roche, Basel, Switzerland) as
an internal standard. Aliquots of these extracts were analyzed for
retinol and TMMP-retinol by reverse-phase HPLC (series 1050,
Hewlett Packard, Wilmington, DE) using a Supelcosil
3-µm LC-18 column and guard column (Supelco,
Bellefonte, PA) with UV detection at 325 nm (HP 1050 diode array
detector) and a mobile phase of methanol/water (91:9, v/v) at a flow
rate of 1.5 mL/min (HP 1050 quaternary pump). Peak areas were
calculated using a Hewlett-Packard 1050 Chemstation, and retinol
mass was determined using an internal standard method. For
determination of radioactivity, retinol peaks were collected (Foxy Jr.,
ISCO, Lincoln, NE) and solvent was evaporated. After addition of
scintillation solution (Ready Organic, Beckman Instruments, Fullerton,
CA), samples were counted twice (model LS 3801, Beckman Instruments and
model 460C, Packard Instruments, Downers Grove, IL) to a final 2-
error of 1.0%. Sample net counts/min (cpm) were converted to
disintegrations/min (dpm) by an external standard method. Specific
activities were calculated as dpm/L (or g)/nmol retinol/L (or g).
Statistical analyses.
Descriptive data are presented as means ± the population estimate
of the SD. Statistical analyses were done using two-way
(diet and time) ANOVA and independent t tests in Minitab
(14)
. Relative specific activity and percentage
contribution data were statistically compared between dietary groups
using the nonparametric Kruskal-Wallis test in Minitab. An
-level of 0.05 was used as the significance limit for statistical
tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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65%; litter sizes ranged from 9
to 16 pups at parturition. Body weights at the time of mating averaged
255 ± 14 g in the LO group (n = 6) and 235
± 14 g in the HI (n = 4); rats in the latter
group were 12 d younger. On d 13 of lactation (111–132 d of age),
body weights were 348 ± 16 g in the LO group and 303 ± 21 g in the HI group (n = 3); liver weight as a
percentage of body weight averaged 5.3 ± 0.16 and 4.2 ± 0.42, respectively.
Plasma and milk vitamin A concentrations were not significantly
affected by time between d 6 and 13 of lactation in either dietary
group (Table 1
). Although plasma vitamin A concentrations were not significantly
influenced by diet, milk retinol levels were 1.5 times higher in
dams with the higher vitamin A intake.
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). Liver vitamin A concentrations in lactating dams in the HI group were
4 times those in the LO group; values were 3.6 times in pup livers.
Vitamin A concentrations in mammary tissue of dams in the HI group were
2.3 times those of the LO group dams. In the LO group on d 6 and 9,
liver and mammary tissue vitamin A concentrations were similar to the
values shown in Table 2
for d 13 as follows: for liver, concentrations
were 546 ± 47 (n = 4) on d 6 and 522 ± 74
nmol/g (n = 4) on d 9; for mammary tissue,
corresponding means were 3.89 ± 0.69 and 3.00 ± 0.34
nmol/g, respectively.
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, vitamin A specific activities in plasma and milk as a percentage of
diet specific activity ("relative specific activity") reached
apparent plateau values by 2.33 d after incorporation of
[3H]vitamin A into the diet on d 6 of
lactation (i.e., there was no significant effect of time from 2.33 to
7.33 d). At all times and in both groups, relative vitamin A
specific activities were higher in milk than in plasma. In fact, in the
HI group, relative milk specific activities were nearly double those in
plasma. Relative vitamin A specific activities in both milk and plasma
were significantly affected by diet, i.e., values for plasma were
65–73% higher in dams fed the HI compared with the LO diet, and those
in milk were 83–115% higher.
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50% to milk vitamin A (52
± 9.7 and 48 ± 9.7%, respectively). Statistical analysis
indicated that the contribution of chylomicron vitamin A to milk was
significantly higher in rats in the HI vs. LO group (P
= 0.014). In the LO group, relative vitamin A specific activity in liver increased slowly with time (P < 0.001) from 0.030 ± 0.008 on d 6 to 0.38 ± 0.067 on d 9 to 1.24 ± 0.15 on d 13 of lactation; in the HI group, liver relative specific activity on d 13 (3.64 ± 0.19) was significantly higher (P < 0.001) than that in the LO group on d 13. Relative specific activities in liver are low because incoming labeled vitamin A is diluted into a large, slowly turning-over vitamin A pool. Relative vitamin A specific activities in mammary tissue were also much lower than in plasma and milk, indicating that the relatively small pool of mammary tissue vitamin A turns over slowly and may not be contributing much to milk vitamin A. Mammary tissue relative specific activities were significantly higher in the HI vs. LO group on d 13 of lactation (4.81 ± 2.15 vs. 1.80 ± 0.50; P < 0.01). In the LO group, relative specific activities in mammary tissue were significantly higher (P < 0.001) on d 9 (4.74 ± 0.78) than on d 6 and 13 (1.07 ± 0.45 and 1.80 ± 0.50, respectively). Because of the slow turnover of mammary tissue vitamin A, we could not apply the logic used above to estimate the relative vitamin A contributions from chylomicrons vs. RBP.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
and
rats (4
5)
, our results confirm the observations that
increases in dietary vitamin A intake are associated with increases in
milk vitamin A. Although these data suggest that dietary vitamin A may
be directed to milk, they provide no information on the quantitative
contributions of chylomicrons vs. holoRBP to milk vitamin A. Presumably
holoRBP is able to deliver vitamin A to lactating mammary tissue
because vitamin A is present in milk even if rats are fed a vitamin
A–free diet (5)
. In such animals, all of the milk vitamin
A must be derived from holoRBP because chylomicrons would contain
almost no vitamin A.
Investigating the source of milk vitamin A in monkeys, Vahlquist and
Nilsson (15)
concluded that RBP is the primary vehicle for
delivery of vitamin A to milk. However, these investigators compared
isolated RBP and a plasma lipoprotein preparation that presumably did
not contain significant amounts of chylomicrons. They speculated that
if plasma retinyl ester levels were increased (as would occur
transiently during chylomicron metabolism), the contribution from
lipoproteins would increase. In a later study, Davila et al.
(4)
found that when rats were fed 105 µmol vitamin A/kg
diet, milk vitamin A on d 14 of lactation was 7 times that of rats fed
2 µmol/kg diet. These authors speculated that a higher
vitamin A intake resulted in an enrichment of retinyl esters in
chylomicrons and that these could be delivered to milk during lipolysis
of chylomicron triglycerides by lactating mammary tissue. In our
experiment, when intake was increased 4 times (from 10 to 50
µmol/kg), milk vitamin A on d 13 of lactation increased
1.5 times. Because plasma retinol concentrations did not differ in the
LO vs. HI groups, we conclude that in the HI group, all of the increase
in milk vitamin A was due to chylomicrons. In addition, our
calculations indicated that chylomicrons contributed at least 32% of
the milk vitamin A in rats fed 10 µmol/kg diet and 74%
in the HI group. That is, as dietary vitamin A was increased,
chylomicrons became an increasingly important source of milk vitamin A.
We hypothesize that the extra dietary vitamin A is delivered as
chylomicron vitamin A to mammary tissue alveolar cells, the site of
milk synthesis and secretion. Because relative vitamin A specific
activity in milk was substantially higher than that in plasma in both
dietary groups, we speculate that a large portion of incoming vitamin A
is directed to the mammary gland for secretion into milk rather than to
liver as occurs in the nonlactating state. In view of the differing
specific activity responses that we observed in milk vs. mammary
tissue, we postulate that the vitamin A secreted into milk comes from a
pool that is kinetically distinct from that which we measured in
mammary tissue.
Until now, little was known about the effects of vitamin A intake on
mammary tissue vitamin A levels. In this study and in related work
(5)
, we have shown that dietary vitamin A level positively
affects mammary tissue vitamin A concentrations in
pregnant/lactating/postlactating rats, even though plasma retinol
levels were not changed. The data indicate that the increase comes
either from the higher vitamin A level in chylomicrons or from an
increased rate of uptake of holoRBP. In a related study
(5)
, we also reported that in age-matched rats that
did not conceive, mammary tissue vitamin A levels were not affected by
diet, implying that reproductive state may influence vitamin A uptake
by mammary tissue.
Ross et al. (16)
evaluated the ability of lactating
mammary gland to take up chylomicron vitamin A by measuring recovery of
[3H]vitamin A in lactating rat mammary tissue
after injection of [3H]vitamin A–labeled
chylomicrons. Of the dose given, 15–30% was recovered in mammary
tissue 2–3 min after chylomicron injection (A.C. Ross, Penn State
University; personal communication). Further, tritium uptake increased
directly as chylomicron [3H]vitamin A was
increased over a 25-fold range. Because there was little uptake of
label in postlactating rats, the authors speculated that chylomicron
vitamin A uptake by lactating mammary tissue depends on local binding
of chylomicrons and lipolysis of chylomicron triglycerides. This effect
is likely related to the increase in LPL activity in mammary tissue
that occurs at parturition and during lactation (9
,17)
.
Analogous to the situation in cultured adipocytes studied by Blaner et
al. (8)
, in which cellular uptake of
[3H]retinoids was facilitated by LPL, it may be
that LPL in lactating mammary tissue is responsible for hydrolysis of
chylomicron-derived retinyl esters, thus allowing retinol uptake by
alveolar cells. Because lactating mammary tissue also contains acyl
CoA:retinol acyltransferase (18)
, an enzyme which
esterifies retinol, vitamin A delivered during hydrolysis of
chylomicron lipids could be reesterified for secretion into milk or
storage in the epithelial cells. The observation that increased vitamin
A intakes facilitate vitamin A secretion into milk has important
implications for improving the vitamin A status of neonates.
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FOOTNOTES |
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3 Current address: Genentech, Incorporated, 1 DNA Way, South San Francisco, CA 94080.
4 Abbreviations used: cpm, counts/min; dpm, disintegrations/min; HI, diet that provided 50 µmol vitamin A/kg; LO, diet that provided 10 µmol vitamin A/kg; LPL, lipoprotein lipase; RBP, retinol-binding protein; TMMP-retinol, all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraen-1-ol.
Manuscript received October 13, 2000. Revision accepted January 17, 2001.
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REFERENCES |
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1. Bulux J., de Groot S.D.W., Kramer L., Green M. H., Solomons N. W., West C. E. Pattern of early breastmilk response to vitamin A supplementation in Guatemalan mothers. FASEB J 1998;12:A201(abs.)
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4. Davila M. E., Norris L., Cleary M., Ross A. C. Vitamin A during lactation: relationship of maternal diet to milk vitamin A content and to the vitamin A status of lactating rats and their pups. J. Nutr. 1985;115:1033-1041
5. Green, M. H., Snyder, R. W., Akohoue, S. A. & Green, J. B. Increased mammary tissue vitamin A levels associated with increased vitamin A intake during lactation are maintained after lactation in female rats. J. Nutr. 2001 (in press).
6. Blanchette-Mackie E. J., Scow R. O. Movement of lipolytic products to mitochondria in brown adipose tissue of young rats: an electron microscope study. J. Lipid Res. 1983;24:229-244[Abstract]
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Blaner W. S., Obunike J. C., Kurlandsky S. B., Al-Haideri M., Pian-tedosi R., Deckelbaum R. J., Goldberg I. J. Lipoprotein lipase hydrolysis of retinyl ester. Possible implications for retinoid uptake by cells. J. Biol. Chem. 1994;269:16559-16565
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