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Original Research

Calcium Absorption Varies within the Reference Range


for Serum 25-Hydroxyvitamin D
Robert P. Heaney, MD, FACN, M. Susan Dowell, PhD, Cecilia A. Hale, PhD, and Adrianne Bendich, PhD, FACN
Creighton University, Omaha, Nebraska (R.P.H., M.S.D.), GlaxoSmithKline, Parsipanny, New Jersey (C.A.H., A.B.)
Key words: vitamin D, calcium absorption, 25-hydroxyvitamin D, vitamin D status, parathyroid hormone, vitamin D
requirement
Background: Calcium absorption is generally considered to be impaired under conditions of vitamin D
deficiency, but the vitamin D status that fully normalizes absorption is not known for humans.
Objective: To quantify calcium absorption at two levels of vitamin D repletion, using pharmacokinetic
methods and commercially marketed calcium supplements.
Design: Two experiments performed in the spring of the year, one year apart. In the first, in which
participants were pretreated with 25-hydroxyvitamin D (25OHD), mean serum 25OHD concentration was 86.5
nmol/L; and in the other, with no pretreatment, mean serum concentration was 50.2 nmol/L. Participants
received 500 mg oral calcium loads as a part of a standard low calcium breakfast. A low calcium lunch was
provided at mid-day. Blood was obtained fasting and at frequent intervals for 10 to 12 hours thereafter.
Methods: Relative calcium absorption at the two 25OHD concentrations was estimated from the area under
the curve (AUC) for the load-induced increment in serum total calcium.
Results: AUC9 ( SEM), was 3.63 mg hr/dL 0.234 in participants pretreated with 25OHD and 2.20
0.240 in those not pretreated ( P 0.001). In brief, absorption was 65% higher at serum 25OHD levels averaging
86.5 nmol/L than at levels averaging 50 nmol/L (both values within the nominal reference range for this analyte).
Conclusions: Despite the fact that the mean serum 25OHD level in the experiment without supplementation
was within the current reference ranges, calcium absorptive performance at 50 nmol/L was significantly reduced
relative to that at a mean 25OHD level of 86 nmol/L. Thus, individuals with serum 25-hydroxyvitamin D levels
at the low end of the current reference ranges may not be getting the full benefit from their calcium intake. We
conclude that the lower end of the current reference range is set too low.

INTRODUCTION

as normal if their serum 25OHD concentration falls within


the reference range.
Studies that have evaluated serum parathyroid hormone
(PTH) concentration as a function of 25OHD level have generally found that PTH is higher at low 25OHD levels than at
higher 25OHD values. The curve flattens out above 25OHD
values that range, in various reports, from 75 to 110 nmol/L
[2 4]. The tendency for higher PTH levels below such inflection points is usually interpreted to indicate a physiological
adaptation to reduced calcium entry into the body, and there are
arguments on both sides of the question whether such physiological adaptation is conducive to or indicative of optimal
health [5 6]. What is lacking in the data available to date is
evidence of quantitative variation in function at 25OHD levels

In 1997 the Food and Nutrition Board of the Institute of


Medicine accepted serum 25-hydroxyvitamin D concentration
(25OHD) as the functional indicator of vitamin D status [1], but
data were insufficient at that time to characterize fully the
physiological normal range for this indicator. Most reference
laboratories cite lower limits varying from 37.5 to 50 nmol/L
(15 to 20 ng/mL), but this lower limit is empirically based on
measurements in ostensibly normal individuals. Since there
is a growing consensus that vitamin D insufficiency is more
common than previously thought, such empiric estimates may
well be circular; furthermore, individuals who would benefit
from higher vitamin D status may be inappropriately classified

Address reprint requests to: Robert P. Heaney, M.D., Creighton University, 601 N. 30th St., Suite 4841, Omaha, NE 68131. Email: rheaney@creighton.edu
Work supported in part by contracts with GlaxoSmithKline.

Journal of the American College of Nutrition, Vol. 22, No. 2, 142146 (2003)
Published by the American College of Nutrition
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Vitamin D Status and Calcium Absorption


in the disputed zone between the lower end of the nominal
reference range and the level where PTH becomes constant.
In an attempt to provide such data, we present in this paper
the results of paired studies of calcium absorption in healthy
postmenopausal women performed under different conditions
of vitamin D repletion.

METHODS
Protocol
Two studies were conducted in Omaha, Nebraska at 41.3
N. latitude, approximately one year apart, in the spring of the
year at the time of the seasonal nadir for serum 25OHD. Each
was a randomized, cross-over study, designed to test the relative absorbabilities of two calcium supplement sources, ingested at single, 500 mg loads taken as part of a standard low
calcium breakfast. Results from the first study, comparing the
two calcium sources, have been published previously [7]. The
protocol for the first study included pre-dosing with 25OHD
(Calderol, Organon, West Orange, NJ), at a dose of 20 g
given on alternate days for an average of three weeks prior to
the absorption measurements, a stratagem designed to bring all
participants rapidly into a state of vitamin D sufficiency. By
contrast, in the second study, there was no pretreatment with
vitamin D or 25OHD. The test substances in both studies were
commercially marketed preparations of Os-Cal and Citracal
in doses providing 500 mg of calcium (as calcium carbonate for
Os-Cal) and 515 mg of calcium (as calcium citrate for Citracal).
Both sources also provided 200 IU of vitamin D, which the
participants received one time only, on the day of the test.
Subjects were fed a low calcium lunch five hours after the test
breakfast.
In both studies the two marketed products tested were found
to be bioequivalent, with nearly identical (and nonsignificantly
different) indices of absorption. Hence, for purposes of this
analysis, within-subject averages of calcemia for the two calcium sources in each study were taken as the best estimates of
each participants absorptive performance under the then prevailing vitamin D status.

Participants
Participants were 34 postmenopausal women, 14 of whom
took part in both studies. Average age at time of study was
56 7 years in the first study and 64 9 years in the second.
Body mass index (kg/m2) was 29.2 5.2 for the study with
25OHD supplementation and 28.8 3.8 for the study without.
Twelve of the 24 women in the first study were receiving
estrogen replacement therapy, and 10 in the second study.
Individuals with digestive disorders, antibiotic use within five
days or unstable medical conditions of any sort were excluded.
The study was approved by the Creighton University Institutional Review Board, and each subject gave written consent.

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION

Absorption Measurement
Relative absorption was estimated from the area under the
curve over intervals ranging from 9 to 12 hours after dosing
(AUCt). Blood was drawn immediately prior to the test breakfast (time zero) and at frequent intervals thereafter out to 24
hours in the first study and to 12 hours in the second. AUC was
calculated for 9, 10 and 12 hours by the trapezoidal method
using the increment above each individuals baseline serum
calcium value, and the individual participant AUCt values were
aggregated across each study. Comparative absorption was
expressed as the ratio of the means of the two AUCt values.
Since the timing of the blood samples was not identical for the
two studies, AUCt was calculated for each using values confected at 9 or 10 hours (as the case may be) by linear interpolation between measurements on either side of the desired time
point. As the values were close to baseline by nine hours, this
assumption of linearity can have introduced at most only a
trivial error. Because AUC9 has been shown elsewhere [8] to be
better correlated with true absorption than AUC values at
earlier or later times, the primary comparisons we report will be
based on AUCt calculated over nine hours. Fractional absorption was calculated from the AUC9 values using the following
formula [8]:

AbsFx 51.1 AUC9)/Ingested load.

Analytical Methods
Serum calcium was measured by atomic absorption spectrophotometry (AAnalyst 100, Perkin-Elmer, Norwalk, CT).
Serum 25OHD was measured once at baseline on each subject
in each study (Nichols Institute Diagnostics, Catalog No. 402135, San Juan Capistrano, CA). Serum immunoreactive parathyroid hormone (iPTH) was measured as the intact molecule
by IRMA (Nichols, San Juan Capistrano, CA).

Statistical Analyses
Data were analyzed in two ways. Since approximately half
of the subjects in each group were not common to the two
studies, the two sets of values were analyzed as independent
samples, using ANOVA and testing for period and treatment
effects. For the 14 women common to each study, a repeated
measures ANOVA was performed, also testing for treatment
and period effects. In both instances we used SAS (SAS Institute, Cary, North Carolina), as well as the various descriptive
statistics provided by Excel (Microsoft, Redmond, WA).

RESULTS
Table 1 sets forth the numerical values for the principal
findings in this study. Serum 25OHD at baseline was 36
nmol/L higher in the study in which participants had been

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Vitamin D Status and Calcium Absorption


Table 1. Serum Ca AUC and Related Variables*

N
Serum 25(OH)D (nmol/L)
Baseline serum Ca (mg/dL)
Incremental AUC9 Ca (mg hr/dL)
Baseline serum PTH (pg/mL)
Incremental AUC10 PTH (pg hr/mL)

With 25OHD

Without 25OHD

24
86.5 24.0
9.64 0.33
3.63 1.15
36.9 13.7
116.0 79.5

24
50.2 15.7
9.31 0.34
2.20 1.18
43.0 13.3
103.1 79.9

0.001
0.002
0.001
NS
NS

* Error terms are 1 standard deviation.

pretreated with 25OHD. The fact of this difference is not


surprising, since that was the intent of the pretreatment. The
size of the difference is of greater interest. The mean withinindividual difference in the 14 participants common to both
studies was 34.3 nmol/L 3.7 (SEM), virtually the same as the
difference between the two group means. The primary outcome
was the incremental AUC9 for serum calcium, which was 65%
higher in the 25OHD treated study (D) than in the untreated
study (D). This difference was highly significant.
Fig. 1 shows the calcemia time course for each calcium
source in the two studies (with and without vitamin D repletion). The figure shows graphically not only that baseline
vitamin D status significantly influenced calcium absorption
but that the effect of D-status was equivalent for the two
calcium salts.
Fig. 2 presents the full time course of the mean serum
calcium increments for the two salts combined over the 10 to
12 hour periods following the test calcium loads. Not only was
the AUCt different, but at most of the time points from 3 to 10
hours, the serum calcium increment was significantly greater in
the D study than in the untreated. Additionally, as Table 1
shows, even the baseline serum calcium values, while all within

Fig. 1. Time course of the incremental calcemia following ingestion of


12.5 mmol Ca loads from two different sources and with and without
pretreatment with 25OHD. The top two lines are the calcemia curves
observed in participants pretreated with 25OHD, and the bottom two,
those measured without vitamin D. Error bars are 1 SEM. ( &
represent Citracal and { & E represent Oscal.) (Copyright Robert
P. Heaney, 2002. Used with permission.)

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Fig. 2. Time course of the mean increment in serum total calcium in


two studies, in one of which vitamin D status was elevated (D), and
in the other, it was not (D). Error bars are 1 SEM. (Copyright
Robert P. Heaney, 2002. Used with permission.)

the reference normal range (8.8 to 10.2 mg/dL), were significantly higher in the D participants (by 0.33 mg/dL; P
0.002). While basal serum PTH was higher in the D group,
the difference was not statistically significant, largely because,
unlike calcium, which is tightly regulated, PTH values exhibited broad dispersion with a coefficient of variation more than
10 greater. Similarly, the AUC for PTH showed less of a drop
in the study without supplemental vitamin D, but the difference
was not statistically significant. Finally, there was no difference
in AUC9 in either study between those receiving estrogen
replacement therapy and those not so treated (data not shown).
Even within each of the two treatment groups there was a
broad range of 25OHD values; in fact, 25OHD values were
continuously distributed across both groups. Hence we pooled
the two studies and regressed AUC9 on 25OHD concentration
(Fig. 3). As expected there was a significant positive association (P 0.05). The coefficient of correlation improved substantially if the regression were confined to 25OHD values
below 90 nmol/L (above which level the correlation was effectively zero).
Subjects in the second study, without supplemental 25OHD,
were significantly older than those in the first study. Because of
this age difference and the possibility that some or all of the
absorptive difference was due to age rather than to vitamin D
status, we separately evaluated the within-individual differences in AUC9 in the 14 participants common to both studies.

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Vitamin D Status and Calcium Absorption

Fig. 3. Correlation of AUC9 with serum 25OHD concentration in 48


measurements of calcium absorption in 34 postmenopausal women.
The lines represent the least squares regression line through the data
and its 95% confidence limits. (Copyright Robert P. Heaney, 2002.
Used with permission.)

In these women, the mean difference in AUC9 was 1.132 mg


hr/dL 0.339 (SEM; P 0.01), a value 45% greater than
without supplemental 25OHD and, in absolute magnitude,
nearly as large as the intergroup difference. Using the formula
for conversion of AUC9 to true fractional absorption values
(Eq. 1), mean calcium absorption under vitamin D repletion
was 35.3% of load ( 11.8) and, without supplemental vitamin
D, 22.5% of load ( 12.0).

DISCUSSION
Many nutrients exhibit what has been termed threshold
behavior, that is, the values for the physiological response
change directly with intake up to some threshold value, above
which the response does not change with further increases in
intake. Calcium, iron and ascorbic acid are well recognized
examples. Vitamin D is usually considered to exhibit similar
behavior, and the limited evidence available is consistent with
that interpretation. Threshold behavior in this instance would
mean that, in a state of vitamin D sufficiency, variations in
vitamin D intake within the physiological range would not alter
calcium absorption efficiency, while absorption would vary
with intake at subthreshold values for vitamin D status.
As noted earlier, the Food and Nutrition Board in its 1997
recommendations [1] was not able to specify the serum 25OHD
value at which vitamin D status reaches the response threshold.
Mortensen and Charles [9] had shown an improvement in
absorption in healthy Danish subjects given short-term pretreatment with vitamin D, but unfortunately their study provides no
data on serum 25OHD concentrations. Scotti et al. [11] more
recently have shown very similar results in Italian male subjects, in this case with accompanying serum 25OHD values.
Specifically, the index of calcium absorption used was higher at

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION

a serum 25OHD level of 67.5 nmol/L than at 55.5 nmol/L.


Unfortunately both studies used, as their measure of calcium
absorption, the induced rise in urine calcium excretion, which,
while convenient, is a very insensitive indicator.
In this study, using classical pharmacokinetic methods, we
found that healthy, postmenopausal women with serum 25OHD
levels averaging 86.5 nmol/L had calcium absorptive efficiencies from 45% to 65% greater than those with mean 25OHD
levels of 50.1 nmol/L. Aside from a general congruence with
the findings of Scotti et al. [11], this is the first such evidence
of which we are aware. Presumably it is precisely this reduced
calcium absorption at 25OHD levels below 80 nmol/L which
is the stimulus to the higher PTH secretion reported by others
[2 4] at 25OHD values below 80 nmol/L.
We have recently shown that a drop in serum 25OHD from
122 to 74 nmol/L did not produce a significant difference in
calcium absorption [10], in contrast to the findings in this study
where a drop from 86.5 to 50.1 nmol/L produced a large drop
in absorption. In the context of the threshold model, that means
that a serum 25OHD of 50 nmol/L is on the ascending limb of
the response curve, while 86 nmol/L is close to or above the
threshold. Consistent with placement of the threshold near
80 90 nmol/L is not only the PTH behavior alluded to above
[2 4], but our finding in this study that the correlation between
25OHD and AUC9 was tightest for 25OHD values below 90
nmol/L, and effectively non-existent above that level.
A limitation of this study is that the data were obtained
solely in postmenopausal women. Additional work needs to be
done in men and in women at other ages. A further limitation
is the pharmacokinetic method itself. While more sensitive and
specific than urine calcium-based methods, the pharmacokinetic approach presents formidable analytic challenges. The
degree of peak calcemia produced by a single 500 mg load,
especially in the face of lower vitamin D status, is, as Figs 1
and 2 show, on the order of only 5% above basal values. Of
necessity, some individuals will have below average peak elevations, and earlier and later values will be lower still. The
error in quantifying the calcemic rise in individual subjects is,
therefore, unavoidably large. While sample size can help overcome this limitation, it is of little help in evaluating associations, in which individual values must be employed. Thus,
while we found a significant positive correlation between
AUC9 and serum 25OHD concentration, the dispersion of the
data around the regression line was large, mainly, we believe,
because of the unavoidably poor precision of the individual
AUC estimates for the load-induced calcemia.
Since calcium absorption is critical to the ability to maintain
calcium balance, it follows that reduced absorptive performance at 25OHD levels between 50 and 80 nmol/L must be
considered suboptimal, and, accordingly, 25OHD values in that
range ought to be considered subnormal. While the precise
location of the threshold remains uncertain, the evidence presented here points to a value closer to 80 or 90 nmol/L,
consistent with the studies of PTH concentration [2 4]. In any

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Vitamin D Status and Calcium Absorption


event, it seems more certain now that the lower boundaries of
the reference ranges (i.e., 37.5 to 50 nmol/L) are incorrect, i.e.,
such levels of serum 25 hydroxyvitamin D are associated with
suboptimal calcium absorption, thereby exacerbating the negative effects of the low calcium intakes that are today found
across most population segments.

4.

5.
6.

Note Added to Proof


7.

A very recent publication corroborates our conclusion about


the inadequacy of 25OHD values in the lower half of the
reference range. Trivedi et al. [12], in a population-based,
randomized controlled trial of 2,686 individuals, aged 65 to 85,
showed that treatment with vitamin D3 in a dose sufficient to
raise serum 25OHD from 53 to 74 nmol/L, decreased fracture
risk at hip, forearm, or spine by 33% (P 0.02). This effect
occurred over almost exactly the 25OHD range for which
we here report a substantial difference in calcium absorption
efficiency.

8.
9.

10.

11.

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Received November 1, 2002; revision accepted January 27,


2003.

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