0021-972X/00/$03.00/0
The Journal of Clinical Endocrinology & Metabolism
Copyright © 2000 by The Endocrine Society
Vol. 85, No. 11
Printed in U.S.A.
Relationship of Intestinal Calcium Absorption to 1,25Dihydroxyvitamin D [1,25(OH)2D] Levels in Young Versus
Elderly Women: Evidence for Age-Related Intestinal
Resistance to 1,25(OH)2D Action*
S. PATTANAUNGKUL, B. L. RIGGS, A. L. YERGEY, N. E. VIEIRA,
W. M. O’FALLON, AND S. KHOSLA
Endocrine Research Unit (S.P., B.L.R., S.K.), Mayo Clinic and Mayo Foundation, Rochester,
Minnesota 55905; Section of Metabolic Analysis and Mass Spectrometry (A.L.Y., N.E.V.), National
Institute of Child Health and Human Development, Bethesda, Maryland 20892; and Section of
Biostatistics (W.M.F.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
ABSTRACT
Intestinal calcium absorption decreases with aging, but it is unclear whether this is attributable to an age-related intestinal resistance to 1,25-dihydroxyvitamin D [1,25(OH)2D] action. Thus, we assessed the in vivo dose response of active intestinal calcium
absorption to a broad range of circulating 1,25(OH)2D levels in elderly
[age (mean ⫾ SD), 72.5 ⫾ 3.0 yr] vs. young women (age, 28.7 ⫾ 5.3 yr;
n ⫽ 20 per group), who were stratified into 5 subgroups: group 1 was
given a high calcium intake of 75 mmol/day, suppressing 1,25(OH)2D
levels; group 2 was given a normal calcium diet of 15–30 mmol/day,
representing basal 1,25(OH)2D levels; group 3 was given a low-calcium diet of 5 mmol/day to stimulate endogenous 1,25(OH)2D production; group 4 was given the low-calcium diet plus 1 ␮g/day
1,25(OH)2D; and group 5 was given a low-calcium diet plus 2 ␮g/day
1,25(OH)2D. After 7 days of diet and/or 1,25(OH)2D treatment, fasting
fractional calcium absorption (FCA) was assessed by a double-tracer
S
ERUM PTH levels increase with age (1– 8), and this increase correlates with the age-related increase in bone
turnover (9 –12). Moreover, suppression of PTH secretion in
elderly vs. young women results in a disproportionately
greater decrease in bone resorption in the elderly women
than in premenopausal women (13). This indicates that bone
resorption is more dependent on circulating PTH levels in
elderly (compared with young) women and is consistent
with a causal role for PTH in mediating the age-related
increase in bone resorption (13). An important cause of the
secondary hyperparathyroidism in elderly individuals is an
age-related impairment in intestinal calcium absorption (14 –
16). However, the mechanism(s) for this decrease in calcium
absorption in aging women is not well understood.
Several indirect lines of evidence indicate that elderly
women may have intestinal resistance to 1,25-dihydroxyvitamin D [1,25(OH)2D] action. For example, we previously
found that, whereas serum 1,25(OH)2D levels increased in
Received December 8, 1999. Revision received May 12, 2000. Rerevision received July 13, 2000. Accepted July 26, 2000.
Address all correspondence and requests for reprints to: Sundeep
Khosla, M.D., Endocrine Research Unit, Mayo Clinic, 200 First Street SW,
5-194 Joseph, Rochester, Minnesota 55905. Email: khosla.sundeep@
mayo.edu.
* Supported by NIH Grants AG-04875 and RR-00585.
method using stable calcium isotopes. Serum 1,25(OH)2D and vitamin
D-binding protein levels were measured concurrently, and the free
1,25(OH)2D index [molar ratio of 1,25(OH)2D to DBP] was calculated.
FCA was significantly correlated with the free 1,25(OH)2D index
in the young (R ⫽ 0.63, P ⫽ 0.003) but not in the elderly women (R ⫽
0.27, P ⫽ 0.25). Moreover, the slope of the relationship between FCA
and free 1,25(OH)2D index (representing intestinal sensitivity to
1,25(OH)2D) was significantly greater in the young (compared with
the elderly) women [mean ⫾ SEM, 0.15 ⫾ 0.04 (young) vs. 0.03 ⫾ 0.02,
elderly, P ⫽ 0.03]. Thus, using an experimental design that allowed
us to assess FCA over a wide range of 1,25(OH)2D levels, we demonstrate that elderly women have a resistance to 1,25(OH)2D action
that may contribute to their negative calcium balance, secondary
hyperparathyroidism, and bone loss. (J Clin Endocrinol Metab 85:
4023– 4027, 2000)
women up to age 65 yr, intestinal calcium absorption remained unchanged (8), consistent with an impaired intestinal responsiveness to 1,25(OH)2D action. Moreover, studies
in humans (17) and in rats (18) have found an age-related
decrease in intestinal vitamin D receptor (VDR) concentrations, although this remains controversial (19, 20).
To date, human studies assessing intestinal calcium absorption with aging have been made primarily under basal
conditions, over a relatively narrow range of circulating
1,25(OH)2D levels (8, 17, 21–24), although Ireland et al. (25)
did find that the intestinal adaptation to a low-calcium diet
was impaired in elderly (compared with young) subjects.
However, serum 1,25(OH)2D levels were not measured in the
latter study, so it was unclear whether the impaired intestinal
adaptation to a calcium-deficient diet was attributable to
smaller increases in circulating 1,25(OH)2D levels or to reduced intestinal responsiveness to 1,25(OH)2D action in the
elderly (compared with the young) subjects. Thus, in the
present study, we used a study design that greatly expanded
circulating 1,25(OH)2D levels, thereby providing the necessary dynamic range in circulating 1,25(OH)2D levels to allow
us to test for a possible reduction in intestinal sensitivity to
1,25(OH)2D action in elderly (compared with young)
women.
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PATTANAUNGKUL ET AL.
Subjects and Methods
Experimental subjects
We studied 20 young premenopausal women (age range, 20 –35 yr;
mean ⫾ sd, 28.7 ⫾ 5.3 yr) and 20 elderly postmenopausal women (age
range, 65– 80 yr; mean ⫾ sd, 72.5 ⫾ 3.0 yr). None of the study subjects
had any diseases such as renal failure (creatinine ⬎ 133 ␮mol/L), malabsorption, or congestive heart failure, or was taking any drugs known
to affect calcium metabolism (calcium supplements ⬎ 12.5 mmol/day,
vitamin D supplements ⬎ 1,000 U/day, sodium fluoride, calcitonin,
bisphosphonates, glucocorticoids, diuretics, calcium channel blockers,
or anticonvulsants). All participants had a bone mineral density, of the
lumbar spine and hip, within the age-adjusted 95% confidence levels for
normal women. None of the elderly participants had vertebral fractures
on thoracic and lumbar spine radiographs. The studies were approved
by the Mayo Institutional Review Board. Signed informed consent forms
were obtained from all subjects. All studies were carried out in the Mayo
General Clinical Research Center (GCRC).
Study protocol
To expand the circulating range of 1,25(OH)2D levels, the young and
elderly subjects were divided into 5 groups of 4 subjects each: group 1
was given a high calcium intake of 75 mmol (3000 mg)/day [15 mmol
of calcium in the diet and 60 mmol of oral calcium supplement as calcium
citrate (Citracal) in 3 divided doses]; group 2 was given a normal calcium
intake [15–30 mmol (600 –1200 mg)/day]; group 3 was given a low
calcium intake [5 mmol (200 mg)/day]; group 4 was given a low calcium
intake (5 mmol/day) and received oral 1,25(OH)2D (Rocaltrol), 1 ␮g/
day; group 5 was given a low calcium intake (5 mmol/day) and received
1,25(OH)2D (Rocaltrol), 2 ␮g/day. Subjects were started on all dietary
and 1,25(OH)2D interventions 7 days before the study. All subjects were
ambulatory, and consumed diets were prepared by the dietitians in the
GCRC during the 8-day study period. Each subject was admitted into
the GCRC on the evening of day 7 of the study and stayed overnight for
2 nights. The intestinal calcium absorption study was performed in the
morning on day 8, after an overnight fast.
Assessment of fractional calcium absorption (FCA)
FCA was assessed as previously described (26), except that the oral
tracer dose was given only once (in the morning after an overnight fast)
with a fixed calcium carrier rather than being mixed with each of the
meals. Stable isotopes of calcium (42Ca for iv administration and 44Ca for
oral administration) were purchased from Oak Ridge National Laboratories (Oak Ridge, TN) and prepared as sterile solutions of calcium
chloride (26). At 0800 h on day 8, a blood sample was drawn through
an indwelling catheter for measurement of serum calcium, phosphate,
PTH, 25-hydroxyvitamin D [25(OH)D], 1,25(OH)2D, and vitamin D
binding protein (DBP). The serum was separated and stored at ⫺70 C
until analysis. Subjects in groups 4 and 5 took their assigned doses of
1,25(OH)2D, and then all subjects received an iv dose of 0.075 mmol (3
mg) 42Ca and an oral dose of 0.045 mmol (18 mg) 44Ca in a carrier of 2.05
mmol (82 mg) of calcium, as CaCl2 [total oral dose of calcium, 2.5 mmol
(100 mg)]. Previous studies (27) have shown that, at this oral calcium
load, calcium adsorption is mainly active [1, 25(OH)2D-mediated]. Each
subject remained fasting for 4 h after the oral dose of 44Ca, and then a
second blood sample was drawn for measurement of 1,25(OH)2D. The
mean of the two 1,25(OH)2D values in each subject was used to relate
to intestinal FCA. A 24-h urine was collected, beginning at 0730 h on day
8, for measurement of the 44Ca/42Ca ratio. Subjects were dismissed from
the GCRC on the following morning after completion of the 24-h urine
collection.
An aliquot of the 24-h urine specimen was prepared for mass spectrometric analysis by an oxalate precipitation procedure, as previously
described (28). Stable isotopic ratios were measured by thermal ionization mass spectrometry with a Finnigan MAT Thermoquad (THQ) Quadruple mass spectrometer (Bremen, Germany). All ratios were measured
relative to 48Ca. The relative precision of all determinants was 0.3– 0.5%.
Each sample was assayed in duplicate. FCA was calculated from the
urine ratio of 44Ca/42Ca, as previously described (26).
Biochemical methods
Serum calcium was measured by atomic spectroscopy, and serum
phosphate and creatinine were measured by kinetic centrifugal analyzer
methods. Serum PTH was determined by two-site immunochemiluminometric assay [Diagostics Systems Laboratories, Inc., Webster, TX; intraassay coefficient of variation (CV), 6.3%]. Serum 25(OH)D and
1,25(OH)2D were measured by competitive protein binding assays [Nichols Institute Diagnostics, San Juan Capistrano, CA; intraassay CVs,
9.2% for 25(OH)D and 7.3% for 1,25(OH)2D]. DBP was measured by
immunonephlometric assay (29) (intraassay CV, 2.9%).
Statistical methods
Statistical analyses were carried out by using the Statistical Analysis
System software program (30). Paired t tests were used to determine
significance, between the young and elderly subjects in each group, for
each of the measured variables. Results were considered significant for
P ⬍ 0.05. Correlation among variables was assessed through the calculation of Pearson’s product moment correlation coefficient. Linear
regression was used to assess the relationship between 1,25(OH)2D and
the free 1,25(OH)2D index [molar ratio of 1,25(OH)2D to DBP] and FCA
in the young and elderly women. Bivariate linear regression models
were used to simultaneously assess the impact of 1,25(OH)2D or the free
1,25(OH)2D index and 25(OH)D on FCA.
Results
Table 1 shows the baseline characteristics of the study
subjects. Body mass index was significantly higher, and creatinine clearance was significant lower, in the elderly (compared with the young) women.
Figure 1 shows serum 1,25(OH)2D levels and the molar
ratios of 1,25(OH)2D to DBP [free 1,25(OH)2D indices] in the
young and elderly women in the different groups. As evident, our study design did expand both values over a wide
range in the young and elderly women. Both 1,25(OH)2D and
the free 1,25(OH)2D indices tended to be higher in the elderly
women in groups 4 and 5, compared with the younger
women, although these differences did not reach statistical
significance.
Table 1. Baseline characteristics of the study subjects
Young women
(n ⫽ 20)
Age, years
Body mass index, kg/m2
Serum
Calcium, mmol/L
Phosphorus, mmol/L
Creatinine, ␮mol/L
25-Hydroxyvitamin D, nmol/L
Creatinine clearance, mL/min
NS, Not significant.
Elderly women
(n ⫽ 20)
P-value
29.2 ⫾ 1.2
24.2 ⫾ 0.9
72.6 ⫾ 0.7
26.9 ⫾ 0.8
⫺
0.04
2.20 ⫾ 0.01
1.19 ⫾ 0.03
79.6 ⫾ 1.8
79.9 ⫾ 8.5
98.7 ⫾ 5.3
2.23 ⫾ 0.02
1.19 ⫾ 0.03
79.6 ⫾ 3.5
67.4 ⫾ 4.7
78.0 ⫾ 3.2
NS
NS
NS
NS
0.002
AGING AND 1,25-DIHYDROXYVITAMIN D ACTION
4025
FIG. 1. Serum 1,25(OH)2D (A) and free 1,25(OH)2D index (⫻105) (B)
in the young (f) and elderly (䡺) women.
FIG. 3. Relationship between FCA and serum 1,25(OH)2D levels in
the young (A; r ⫽ 0.50, P ⫽ 0.02, slope ⫽ 0.002 ⫾ 0.001) and elderly
(B; r ⫽ 0.22, P ⫽ 0.35, slope ⫽ 0.001 ⫾ 0.001) women.
FIG. 2. Serum PTH levels in the young (f) and elderly (䡺) women.
*, P ⬍ 0.05.
Figure 2 shows the serum PTH levels in the different
groups of the young and elderly women. Serum PTH levels
were higher in the elderly women (compared with the young
women) in group 2, which represents basal conditions (although this did not achieve statistical significance because of
the small number of subjects per group). The major difference between the young and elderly women, however, was
in group 3, where the low-calcium diet induced significantly
higher PTH levels in the elderly.
To better assess the relationship between intestinal calcium absorption and 1,25(OH)2D levels, we took advantage
of the expanded range for serum total 1,25(OH)2D levels and
free 1,25(OH)2D indices achieved with our interventions, and
we plotted these against FCA (Figs. 3 and 4). As shown in Fig.
3, FCA was significantly correlated with 1,25(OH)2D levels
in the young (but not in the elderly) women. FCA was even
more strongly related to the free 1,25(OH)2D index than to
total 1,25(OH)2D levels in the young women but again was
not significant in the elderly women (Fig. 4). In addition, the
slope of the relationship between FCA and free 1,25(OH)2D
index was significantly greater in the young women [0.15 ⫾
0.04 (young) vs. 0.03 ⫾ 0.02 (elderly), P ⫽ 0.03]. To test for any
impact of circulating 25(OH)D levels on the relationship
between FCA and 1,25(OH)2D and/or the free 1,25(OH)2D
index, we also entered 25(OH)D levels as a covariate in the
analyses in Figs. 3 and 4, but it was not significant.
FIG. 4. Relationship between FCA and free 1,25(OH)2D index (⫻105)
in the young (A; r ⫽ 0.63, P ⫽ 0.003, slope ⫽ 0.15 ⫾ 0.04) and elderly
(B; r ⫽ 0.27, P ⫽ 0.25, slope ⫽ 0.03 ⫾ 0.02) women.
Discussion
Although calcium absorption is impaired in elderly
women (8, 17), it is unclear whether this is caused by an
intestinal resistance to 1,25(OH)2D action or to some other
mechanism. Using dietary and pharmacologic interventions
to expand the dynamic range of circulating 1,25(OH)2D levels, we demonstrate here that the relationship between FCA
and the free 1,25(OH)2D index is clearly different between
young vs. elderly women. Thus, whereas we found a strong
correlation between FCA and the free 1,25(OH)2D index in
the young subjects, this correlation was not significant in the
elderly subjects. Moreover, the slope of this relationship, a
measure of intestinal responsiveness to 1,25(OH)2D action,
was markedly reduced in the elderly women. Taken to-
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PATTANAUNGKUL ET AL.
gether, these data indicate a fundamental breakdown in the
relationship between FCA and free 1,25(OH)2D levels in
elderly women. This is further reflected by the greater dispersion of data points in the elderly subjects (Fig. 4B) vs. the
young subjects (Fig. 4A). Of note, several elderly subjects had
relatively high FCA values, even at low 1,25(OH)2D and free
1,25(OH)2D index values (Figs. 3 and 4). The reasons for this
are unclear, but this could be attributable simply to biologic
variability, because FCA tends to be fairly variable, even in
inbred strains of rat (19). Alternatively, because calcium absorption has both a passive (1, 25(OH)2D-independent) and
an active (1, 25(OH)2d-dependent) component (27), our findings in Fig. 4 may be attributable to a greater contribution of
passive vs. active calcium absorption in some of the elderly
subjects. Thus, while our 100-mg calcium load should have
led mainly to active [1, 25(OH)2d-mediated] calcium transport (27), this may not have been true in some of the elderly
subjects. A third possibility for these findings is that some
elderly subjects were unable to modulate (up or down) FCA
in response to changes in circulating 1,25(OH)2D levels. Indeed, these possibilities are not mutually exclusive, and all
could, in part, explain the data in Fig. 4. We should stress,
however, that when the entire data set is analyzed as a whole,
the fundamental observations are a breakdown of the relationship between FCA and 1,25(OH)2D levels in elderly subjects, with a reduced slope of this relationship.
In a recent study, Wood et al. (19) performed a similar
regression analysis between intestinal calcium absorption
and circulating 1,25(OH)2D levels in young vs. aged rats,
with results that were virtually identical to our findings in
humans. Although the reason(s) for the breakdown in the
relationship between FCA and free 1,25(OH)D2 levels in
elderly humans and animals is unclear, we have previously
reported that intestinal VDR levels were reduced in elderly
(compared with young) women (17). However, other studies
in humans (20) failed to demonstrate a reduction, and the
data in animals is also conflicting (18, 19). Thus, a postreceptor defect in 1,25(OH)2D action may also be present.
Because the elderly women were also estrogen deficient,
our study cannot dissociate the effects of aging from those of
estrogen deficiency. However, several lines of evidence suggest that estrogen may enhance intestinal calcium absorption. Thus, in perimenopausal women, before and 6 months
after oophorectomy, Gennari et al. (31) found that the increase in calcium absorption, in response to treatment with
1,25(OH)2D, was blunted in the presence of E deficiency,
suggesting a direct enhancing effect of estrogen on the intestinal response to 1,25(OH)2D. More recently, Liel et al. (32)
demonstrated that estrogen increased VDR levels and the
response of 1,25(OH)2D target genes to 1,25(OH)2D treatment in ovariectomized rats. In contrast, Bolscher et al. (33)
found that estrogen directly stimulated intestinal calcium
absorption in the rat, independent of 1,25(OH)2D action, and
the presence of estrogen receptors has been demonstrated in
rat intestine (34). Clearly, further studies, using the present
study design in estrogen-treated vs. untreated elderly
women, will be needed to resolve the issue of whether the
impaired intestinal sensitivity to 1,25(OH)2D that we observed in elderly women is, at least in part, caused by estrogen deficiency.
Though our data indicate that intestinal sensitivity to
1,25(OH)2D is reduced in elderly women, clearly there are
other factors that may contribute to the age-related decline
in calcium absorption. These include vitamin D deficiency
(35), although baseline 25(OH)D levels were similar in the
young and elderly women in our study, and 25(OH)D was
not a significant covariate in the analyses in Figs. 3 and 4. In
addition, age-related reductions in renal mass may result in
a defect in 1-␣-hydroxylation of 25(OH)D (36), resulting in
low 1,25(OH)2D levels in some elderly individuals. Our studies suggest, however, that (on average) there is intestinal
resistance to 1,25(OH)2D action in elderly women, and these
other abnormalities may be superimposed on this defect.
We also found, as expected, that baseline serum PTH levels
tended to be higher in the elderly (compared with the young)
women. With the induction of calcium deficiency in Group
3, however, serum PTH levels were markedly increased in
the elderly women, consistent with our previous observations that elderly women have a functional defect in parathyroid function consistent with parathyroid hyperplasia
(37).
In summary, we conclude that elderly women have an
impaired intestinal response to 1,25(OH)2D. This defect may
contribute to the negative calcium balance, secondary hyperparathyroidism, and bone loss in aging women. Further
studies are needed to define whether this abnormality is
caused by a primary impairment in calcium absorption associated with aging or is secondary to estrogen deficiency.
Acknowledgments
We thank Ms. Joan Muhs for help with recruitment of the study
subjects, Ms. Roberta Soderberg for handling of the samples, and Ms.
Kathleen Egan and Ms. Cindy Crowson for assistance with the statistical
analyses.
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