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Mineral Bioavailability Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University (R.J.W., J.C.F., K.C.), Boston, Massachusetts 02111; University of Virginia Medical School (M.E.B.), Charlottesville, Virginia 22908; and the University of Wisconsin (H.D.), Madison, Wisconsin 53706
Address all correspondence and requests for reprints to: Richard J. Wood, Ph.D., Mineral Bioavailability Laboratory, USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, Massachusetts 02111. E-mail: wood_mb{at}hnrc.tufts.edu
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Abstract |
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Introduction |
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Suboptimal vitamin D status is quite common among older people (12) and may be an important factor reducing plasma 1,25(OH)2D and intestinal calcium absorption efficiency. Exogenous administration of 1,25(OH)2D is known to increase calcium absorption (13). There is also evidence in both humans (14) and animals (15) that intestinal VDR is reduced, although this is controversial (16). Thus, the pathogenesis of calcium malabsorption in the aged could be due to a primary hormone deficiency because of lower circulating levels of 1,25(OH)2D, or to reduced target tissue responsiveness to 1,25(OH)2D (14), which could be mediated by a reduction in intestinal VDR (14, 15). Both situations, which could coexist, would lead to lower intestinal calcium absorption through a common mechanism, such as by reducing the expression of calbindin D (17), a mediator of intestinal calcium transport.
We have attempted to address the hypothesis that lowered intestinal responsiveness to 1,25(OH)2D is an important factor that reduces intestinal calcium absorption in aged rats. To test this hypothesis, we measured intestinal calcium absorption in young and old rats after achieving equivalent levels of circulating 1,25(OH)2D by continuous infusion of 1,25(OH)2D over several days.
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Materials and Methods |
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Study design
We report on the findings of four separate studies that were
conducted in young and old vitamin D-replete rats to investigate the
effects of 1,25(OH)2D status on calcium absorption in the
senescent animal. In two of the studies, 1,25(OH)2D was
administered as a continuous infusion from an implanted osmotic
minipump to measure the response to 1,25(OH)2D. In the
first study, unoccupied intestinal vitamin D receptor was measured in
36 male rats aged 1, 2, 3, 6, 12, and 24 mo (n = 6/group). In the
second study, we measured total duodenal vitamin D receptor protein and
calbindin D in 24 female rats aged 7 weeks, 4 months, and 17 months
(n = 8/group). In the third study, the response to
1,25(OH)2D infusion was determined in 22 young (3 months)
and 13 old (21 months) male rats following either days 0, 3, or 6 of
treatment (n = 4–8 rats/group). In the fourth study, a
dose-response of intestinal calcium absorption to
1,25(OH)2D treatment was determined in 25 young (3 months)
and 19 old (23 months) male rats by administering different doses of
1,25(OH)2D (0, 4, and 14 ng/100 g BW/day) over 9 days
(n = 3–5 rats/group). All research protocols were approved by the
Animal Care and Use Committee at Tufts University, and animals were
maintained in accordance with the NIH Guide for the Care and Use of
Laboratory Animals.
Surgical technique
Plasma 1,25(OH)2D levels were modified by continuous
infusion of 1,25(OH)2D. Each animal was anesthetized with
methoxyflurane inhalant and, under sterile conditions, surgically
implanted with a miniosmotic pump (Model 2002, Alzet Corp., Palo Alto,
CA). Pumps were implanted sc through a small intrascapular incision and
provided a constant infusion of either propylene glycol vehicle (Sigma
Chemical Co., St. Louis, MO) or 1,25(OH)2D (courtesy of Dr.
M. Uskokovic, Hoffman-LaRoche, Nutley, NJ) at either 4 or 14 ng/100 g
BW/day for up to 9 days. The lower dose of 1,25(OH)2D was
chosen because Lee et al. (18) had reported that 4 ng/100 g
BW/days for 9 days caused a significant increase in duodenal calcium
uptake in 4-month-old male rats. The higher 1,25(OH)2D dose
level was chosen because this dose increases the plasma
1,25(OH)2D concentration in treated animals to an average
value of 
400 pg/ml, which is markedly elevated but still in the
physiological range. Goff et al. (19) reported that this
level of plasma 1,25(OH)2D was found in 1-month-old rats
fed a very low (0.02%) calcium diet for 7 days.
1,25(OH)2D measurements
Blood was collected from the exposed heart and centrifuged at 4
C and 3000 rpm for 10 min in a Sorvall centrifuge (Model RT6000B).
Plasma or serum was harvested and stored at -20 C until measurement of
1,25(OH)2D, using a calf thymus receptor-binding assay
(20).
Duodenal calcium absorption
Intestinal calcium absorption rates were measured in the
duodenum by an in situ ligated loop technique. We used a
tied off duodenal intestinal loop because it had been previously shown
that calcium absorption in this intestinal segment is particularly
responsive to vitamin D status (21). After an overnight fast, animals
were anesthetized with sodium pentobarbital (24 µmol/100 g BW). The
abdominal cavity was opened by a midline incision, and the bile duct
was ligated. The intestine was flushed with warm saline. The proximal
10-cm of the duodenum was ligated, warmed in the body cavity for 1 min,
injected with 1 ml of a solution containing (in mM): 140
NaCl, 5.8 KCl, 0.34 Na2HPO4, 0.44
KH2PO4, 0.8 MgSO4, 2.5 glutamine, 2
CaCl2, 20 HEPES (pH 7.4), and 0.74 mBq 45Ca.
The duodenal loop was replaced in the abdominal cavity, and absorption
of 45Ca was allowed to proceed for 10 min. Preliminary
studies had shown that the amount of 45Ca left in the
tissue was negligible compared with total unidirectional
45Ca flux. Thus disappearance of 45Ca from the
loop was considered to represent unidirectional calcium absorption.
Animals were killed by exsanguination, and the intestinal segment was
cut out and flushed of residual 45Ca. The length of the
segment was measured after a 1 g weight was suspended from the
bottom of the segment. Aliquots of luminal fluid from the intestinal
loop were counted for 45Ca by a liquid scintillation
counter. The difference between total 45Ca injected in the
loop at time 0 and recovered after 10 min from the intestinal washing
was used as an indicator of unidirectional lumen-to-plasma calcium
absorption. The data was expressed as percent disappearance of total
45Ca dose administered per 10 cm loop in 10 min.
Net calcium absorption
Net dietary calcium absorption was measured as the difference
between dietary calcium intake and fecal calcium output in rats housed
in plastic metabolic cages. Details of the dietary calcium absorption
technique have been previously described (22).
Vitamin D receptor
Vitamin D receptor was measured in the duodenum of male rats
using a 3H-1,25(OH)2D binding assay, as
previously described (23), that estimates the unoccupied vitamin D
receptor. VDR in the experiment using female rats was assayed by an
immunoradiometric assay (IRMA) that estimates total vitamin D receptor
protein concentration (24).
Calbindin D protein
Calbindin D9k protein concentration in the proximal
duodenum was measured using an ELISA with bovine calbindin
D9k as the standard (17).
Statistical analysis
Treatment effects were compared by one-way ANOVA or linear
regression analysis using SYSTAT statistical software (SYSTAT 6.0,
Chicago, IL). Mean differences between groups with P <
0.05 were considered statistically significant. Comparisons of multiple
group means were done using the Tukey HSD multiple comparisons
procedure. Data are expressed in the text and tables as mean ±
SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the results of a cross-sectional
study of plasma 1,25(OH)2D and duodenal unoccupied VDR
levels, measured as 3H-1,25(OH)2D binding
activity, in male rats aged 1, 2, 3, 6, 12, and 24 months. As expected,
we found a marked drop in plasma 1,25(OH)2D with aging.
Surprisingly, however, we observed no significant age-associated
reduction in unoccupied VDR level in the duodenum.
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. As expected, we found that circulating
plasma 1,25(OH)2D, duodenal calbindin D protein, and net
dietary calcium absorption decreased with age. However, consistent with
the previous study, we found no age-associated change in the intestinal
VDR protein level.
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). At baseline, before the
administration of hormone, despite similar dietary calcium intakes,
plasma 1,25(OH)2D in the old animals was significantly
lower than in young animals. Likewise, duodenal calcium absorption
rates at baseline were also significantly reduced by 40% in the old
rats. Infusion of 1,25(OH)2D for 3 or 6 days elevated
plasma 1,25(OH)2D by up to 20-fold (21 vs. 445
pg/ml) in senescent rats and resulted in similar plasma
1,25(OH)2D in both young and old treated rats. We observed
that exogenous 1,25(OH)2D treatment increased intestinal
calcium absorption in both young and old rats. However, despite the
markedly elevated and equivalent plasma 1,25(OH)2D levels
on either day 3 or day 6 of 1,25(OH)2D treatment in the
young and old rats, duodenal calcium absorption in the older animals
was still significantly lower than in younger animals. This apparent
relative resistance in vitamin D-dependent Ca absorption response in
the duodenum of the senescent rat is most dramatically evident at the
earlier 3-day time point. Continuous exogenous infusion of
1,25(OH)2D resulted in an average increase in plasma
1,25(OH)2D by 333 pg/ml in young rats and by 375 pg/ml in
old rats; however, the vitamin D-induced increase in the rate of
duodenal Ca absorption was only about one-third as pronounced (7
vs. 19% Ca dose/10 cm · 10 min) in the old compared
with the young rat. A similar, but less marked, age-dependent
difference in intestinal absorption response to 1,25(OH)2D
was also observed following 6 days of exogenous 1,25(OH)2D
treatment.
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As shown in Fig. 1, the relationship
between plasma 1,25(OH)2D and duodenal calcium absorption
could be fitted to a nonlinear curve with an apparent plateau of the
effect of 1,25(OH)2D on calcium absorption occurring around
a plasma 1,25(OH)2D concentration of 400 pg/ml. However,
inspection of the individual data points representing young and old
animals clearly suggests that duodenal calcium absorption rate was
markedly lower on average in the old animals at all plasma
1,25(OH)2D concentrations. To further investigate this
association, we examined the relationship between duodenal calcium
absorption and plasma 1,25(OH)2D at concentrations below
400 pg/ml by linear regression analysis (Fig. 2).
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). The y-intercept of the
regression line represents the extrapolated rate of duodenal calcium
absorption when plasma 1,25(OH)2D is theoretically zero.
Presumably, this would represent duodenal calcium absorption that was
independent of vitamin D status. Although the intercept of the
regression line for the younger group was 68% higher than the old
group (14 vs. 8), statistical analysis of these regression
parameters did not indicate that they were significantly
(P < 0.05) different. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The relatively low circulating levels of 1,25(OH)2D consistently observed in older rats are probably related to reduced activity of the renal 1,25(OH)2vitamin D: 25(OH)vitamin D hydroxylase (29). Because 1,25(OH)2D is the primary hormonal regulator of intestinal calcium absorption (30), lower plasma 1,25(OH)2D or an intestinal resistance to the action of 1,25(OH)2D in the old animals would be significant factors contributing to the reduced calcium absorption. Our observation that duodenal calcium absorption is lower in old rats compared with young rats at all plasma 1,25(OH)2D within a wide physiological range strongly supports the notion of tissue-level resistance to the hormonal action of 1,25(OH)2D. This observation is in contrast, however, to the findings of Armbrecht et al. (31), who previously found no indication of intestinal resistance to 1,25(OH)2D in the old rat because intestinal calcium pump activity could be stimulated by 1,25(OH)2D. The reason for this apparent contradiction is unknown but could represent variable sensitivities of various vitamin D-dependent genes to 1,25(OH)2D stimulation.
Because old rats do respond to 1,25(OH)2D by increasing
calcium absorption, sufficient elevations of plasma
1,25(OH)2D, brought about by exogenous administration of
this steroid hormone, could overcome the age-related defect in
intestinal calcium absorption. However, based on the derived
relationship between plasma 1,25(OH)2D and calcium
absorption in the old rat as found in our studies (y = 8.38 +
0.062 x, where y is duodenal Ca absorption and x is plasma
1,25(OH)2D), it would be necessary to increase circulating
levels of 1,25(OH)2D in the old rat by about 13-fold (from
21 pg/ml to 284 pg/ml) to normalize duodenal calcium absorption in the
old rat to basal levels of absorption observed in the young rat (26%
dose/10 cm·10 min, see Table 3). An additional implication of our
derived relationship between plasma 1,25(OH)2D and calcium
absorption in the duodenum is that probably very little of the
difference in basal calcium absorption between young and old rats is
due to the usual difference in baseline levels of plasma
1,25(OH)2D concentration. For example, an increment of
plasma 1,25(OH)2D of 37 pg/ml (from 21 pg/ml to 58 pg/ml)
would be enough to overcome the basal difference in plasma
1,25(OH)2D between young and old rats (Table 3); however,
based on the regression equation (Fig. 2), it would be predicted (37
x 0.06) to increase calcium absorption in the old rat by only about
2%, i.e. about one-quarter of the observed differential (17
vs. 26%/10 cm·10 min) in calcium absorption in the
nontreated groups. Thus, based on these calculations, intestinal
resistance to 1,25(OH)2D action appears to be a major
determinant of the age-associated difference in vitamin D-dependent
duodenal calcium absorption and may have a strong influence on the
observed differences in net intestinal absorption of dietary calcium.
However, because calcium absorption efficiency in response to
1,25(OH)2D was investigated only in the duodenum and not
the whole intestine, these findings strictly pertain only to this
segment of the small intestine and should be interpreted cautiously
until additional information on whole gut net calcium absorption in
young and old rats in response to 1,25(OH)2D treatment is
available.
The most intriguing aspect of our findings was that intestinal resistance to 1,25(OH)2D effects on calcium absorption in our older animals was evident in the face of apparently equivalent intestinal vitamin D receptor levels in our young and old rats. The reduced sensitivity to 1,25(OH)2D action in the intestine of the old rat would be more consistent with the observations of others who reported lower vitamin D receptor levels in aged rats (15, 28). We do not have a ready explanation for the discrepancy in vitamin D receptor levels between our studies and those of others. Our measurement of total VDR protein and unoccupied VDR, which makes up the vast majority of the total vitamin D receptor pool, in two separate studies increases our confidence in this finding. However, the issue of whether aging is associated with a reduction in vitamin D receptor content warrants further study. Nevertheless, there are also other alternative explanations for the reduced responsiveness of the duodenum of the aged rat to 1,25(OH)2D that could be explored, such as a possible age-dependent difference in the ability of the vitamin D receptor to form heterodimers with RXR to activate gene transcription. Despite an earlier report to the contrary (14), a recent report by Kinyamu et al. (16) has suggested that aging in adult humans is not associated with a marked change in intestinal vitamin D receptor level (16). In that study there was also no effect of age on plasma 1,25(OH)2D, although intestinal calcium absorption was lower in older compared with younger women, suggesting that some other factors must be responsible for the age-associated difference in calcium absorption in humans. Our present findings of a marked difference in vitamin D-dependent calcium absorption in senescent animals, despite equivalent levels of plasma 1,25(OH)2D and intestinal vitamin D receptor content, would be consistent with these recent observations in humans.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Food, Nutrition and Food Services
Management, University of North Carolina at Greensboro, 310 Stone
Building, Greensboro, North Carolina 27403.
3 Visiting Scientist in the Mineral Bioavailability Laboratory. His
current address is Department of Nutrition, University College Cork,
Cork, Ireland.
Received December 23, 1997.
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