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Original Studies |
Mineral Metabolism, Jerry L. Pettis Memorial Veterans Administration Medical Center (K.Å., K.-H.W.L., D.J.B.), Loma Linda, California 92357; and the Departments of Medicine (K.Å., K.-H.W.L., D.J.B.), Biochemistry (K.-H.W.L., D.J.B.), and Nutrition (P.J., E.I.), Loma Linda University, California 92350
Address all correspondence and requests for reprints to: K.-H. William Lau, Ph.D., Mineral Metabolism (151), Jerry L. Pettis Memorial Veterans Administration Medical Center, 11201 Benton Street, Loma Linda, California 92357. E-mail: laub{at}llvamc.va.gov
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The effects of dietary calcium restriction (i.e. calcium depletion) and the recovery process (i.e. calcium repletion) on bone metabolism have been extensively investigated in a weanling rat calcium depletion/repletion model (3, 4, 5, 6, 7, 8, 9). Accordingly, the restriction of dietary calcium intake in weanling rats led to the development of hypocalcemia (4, 5, 8), which then caused a marked increase in serum PTH (4). The elevated PTH level, in turn, increased the number and activity of osteoclasts (3, 9), which then resulted in a stimulation of bone resorption. Under normal conditions, an increase in bone resorption is coupled to a compensatory increase in bone formation in an equal magnitude to ensure that no net bone mass is lost. However, during depletion when there is a demand to mobilize calcium from bone to counteract the hypocalcemia, the normal bone coupling process becomes compromised. Despite a significant increase in bone resorption, bone formation not only did not increase, but was significantly inhibited in these weanling rats (4). Consequently, the combined actions of calcium depletion on bone resorption and formation led to a significant loss of bone mass (4). Conversely, when the dietary calcium was reinstituted in these weanling rats, the depletion-associated hypocalcemia and secondary hyperparathyroidism were rapidly reversed (3, 7). Thus, the bone resorption rate returned to normal, and the bone formation rate was acutely increased. This repletion response (i.e. increased bone formation during repletion) is responsible for rapid replacement of the bone mass that was lost during depletion (4, 7).
Although the effects of dietary calcium depletion and repletion on bone metabolism have been fairly well characterized in growing rodents, the skeletal responses to calcium depletion and repletion in humans have been poorly defined. Therefore, the present study sought to determine the effects of calcium depletion and repletion on bone turnover in young adult humans. Accordingly, we evaluated the acute effects of a short term (i.e. 22 days) dietary calcium depletion and repletion on bone turnover in 15 young Caucasian women. To assess the acute effects of calcium depletion and repletion on the bone turnover rate, changes in biochemical markers of bone turnover during the early phase (i.e. the first 2 days) of depletion and repletion were determined. Because different serum biochemical markers may reflect different aspects of the bone turnover process, we evaluated two bone formation markers to obtain additional information on the repletion response parameters that would allow us to assess whether there was an increase in the number of osteoblasts occurring during depletion and then an increase in their activity occurring during repletion, as observed in rodents (3, 4, 5, 6, 7). Accordingly, serum osteocalcin and type I C-terminal procollagen peptide (PICP) were measured as markers of bone formation, and urinary deoxypyridinoline was measured as an index of bone resorption. Serum levels of calcium, PTH, and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and urinary calcium level were also determined to monitor the low calcium stress. In this study, we found evidence that humans respond to calcium stress in a manner similar to that in rodents. Hence, information gathered from the rodent calcium depletion and repletion model could shed light on human pathophysiology.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Fifteen Caucasian college women, from 21–30 yr of age, were
recruited for this study. The body weight of these women was within
20% of their ideal body weight according to the 1959 Metropolitan Life
Insurance Height-Weight Table
(10). All subjects were in excellent
health and had been consuming at least 800 mg/day calcium (dietary or
supplemental) for at least 6 weeks before enrollment in the study. None
of the subjects was performing excessive exercise, was a smoker, or had
a history of drug and/or alcohol abuse. None of them was taking oral
contraceptives or any medications known to affect calcium and bone
metabolism. The study protocol and written consent were reviewed and
approved by the institutional review board of Loma Linda University.
Signed written informed consent was obtained from each study subject
before participation.
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Figure 1 illustrates the study
protocol schematically. The study was an out-patient protocol and
contained three phases: 1) the baseline phase (study days -4 to 0), 2)
the depletion phase (study days 1–22), and 3) the repletion phase
(study days 23–29).
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On the morning of day 1, the study subjects were shifted to a diet containing less than 300 mg/day calcium (the depletion phase). During the depletion phase, each subject was instructed to take 5 g of a calcium binder, Calcibind (cellulose sodium phosphate, a gift from Mission Pharmacal Co., San Antonio, TX) 30–60 min after each meal to further lower dietary calcium absorption. Because phosphorus deficiency, like calcium deficiency, affects bone turnover and bone mass (5, 12), each subject was asked to take daily phosphorus supplements (Neutra-phos-K, provided free of charge by Willen Drug Co., Baltimore, MD) throughout the depletion phase to ensure sufficient phosphorus intake during depletion. The remaining Neutral-phos-K and Calcibind at the conclusion of the depletion phase were counted to ascertain that the subjects had taken the supplements. The depletion phase lasted for 22 days. Fasting blood and 24-h urine samples were obtained from each study subject on days 2 and 3 of the low calcium diet and also on the last day (day 22) of the depletion phase.
The repletion phase was initiated on the morning of day 23. The subjects resumed a normal diet containing more than 800 mg/day calcium. Neutral-phos-K and Calcibind supplementations were discontinued, and the repletion phase lasted for 7 days. On days 25 and 29 (the third and last day of repletion), fasting blood and 24-h urine samples were obtained from each subject.
Detailed daily dietary intake records were kept by each subject throughout the entire study and were analyzed for daily intakes of calories, protein, sodium, calcium, and phosphorus, using the 1989 version of the Nutritionist III computer program (N-Squared Computer, Silverton, OR). Compliance was determined from diet records. The subjects were contacted regularly to encourage compliance with the study protocol. Each subject was weighed before (i.e. day 0) and after (i.e. day 22) the low calcium diet and also at the end of the study (i.e. day 29).
Serum and urine chemistries
All blood samples were drawn between 0700–0800 h. Serum samples were collected after a 90-min blood clotting at 4 C. All serum samples were stored in aliquots at -70 C until assay. Twenty-four-hour urine samples were collected in a 3-L 24-h urine specimen bottles (VWR Scientific, Los Angeles, CA) containing 1 g boric acid as the preservative. Aliquots of urine samples were stored at -20 C until analyses. Levels of calcium, phosphorus, 1,25-(OH)2D3, PTH, osteocalcin, and PICP were measured in each serum sample. Levels of calcium, deoxypyridinoline, and creatinine were measured in the 24-h urine samples.
Biochemical assays
Serum and urinary calcium levels were determined using the StanBio Total Calcium Procedure (Fisher Chemical Co., Los Angeles, CA). Serum phosphorus was assayed with a colorimetric assay (13). 1,25-(OH)2D3 was measured by a commercial RRA (Incstar Corp., Stillwater, MN). PTH was determined with an immunoradiometric assay kit for intact PTH from Nichols Institute (San Juan Capistrano, CA). Osteocalcin was measured by an in-house competitive RIA (14). Weighed regression of the standard curve was constructed according to the log-logit method of Rodbard (15), and the amount of osteocalcin in the unknown serum was determined from the standard curve. PICP was determined by a commercial RIA kit, as previously described by Melkko et al. (16). The urinary deoxypyridinoline level was assessed with a high performance liquid chromatography assay method (17) and was standardized against urinary creatinine level, which was assayed according to the method of Heinegard and Tiderstroem (18). Each reported assay passed quality control testing.
Statistical analyses
The results in this report are shown as the mean ± SEM (n = 15). Statistical significance of the data was evaluated with two-tailed Student’s t test and one-way ANOVA. Correlation between groups was analyzed by the Pearson’s correlation matrix method, and multiple linear regression analyses were performed with the Sigma Stat Statistical program (Jandel Scientific Software, San Rafael, CA). Differences are considered significant when P < 0.05.
Because there was no statistically significant difference between the two separate baseline samples (i.e. days -4 and -3) for each test parameter, the averages of these two sets of values were used as the baseline values for calculation of changes and for comparison.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Table 1
shows the characteristics
and dietary intakes of the study subjects before and after the calcium
depletion and repletion phases. The mean age and weight of the study
group were 24 yr and 58 kg, respectively. There was no significant gain
or loss in body weight during the entire study period. The mean daily
dietary calcium intake of this group of young women was approximately
1200 mg during both the baseline and repletion phases and approximately
250 mg during the depletion phase (i.e. an approximately
80% decrease from baseline values). During the depletion phase, these
women also received significantly fewer daily calories and had
significantly lower protein and sodium intakes.
Effects of calcium depletion and repletion on serum calcium, PTH, and 1,25-(OH)2D3 levels
Figure 2 shows the effects of a
short calcium depletion/repletion cycle on serum and urinary calcium
levels of these young women. Calcium depletion significantly reduced
the level of serum calcium (top panel) and 24-h urinary
calcium excretion (bottom panel) after 1 day. However, in
contrast to the urinary calcium level in which the reduction was
sustained throughout the depletion phase, the decrease in serum calcium
appeared to regress with time, as it was no longer significantly
different from the baseline value at the end of the depletion phase.
Upon resumption of a regular diet containing more than 800 mg/day
calcium (i.e. repletion), the serum calcium level (top
panel) and 24-h urinary calcium excretion (bottom
panel) each returned to their respective baseline value after 3
days. Due to the phosphorus supplementation, the serum phosphorus level
was increased during the depletion phase (data not shown). There was no
correlation between the serum calcium level and the 24-h urinary
calcium excretion in any phase of the study (data not shown).
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) and
1,25-(OH)2D3 (bottom panel of Fig. 3) in these young women were significantly elevated after 1 day of
calcium depletion. The increase in serum levels of these calcitropic
hormones, sim-iliar to that of serum calcium, was significantly
less at the end of the depletion phase than during the early depletion
phase. A week of repletion returned the serum levels of PTH and
1,25-(OH)2D3 to their respective baseline
levels.
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Effects of calcium depletion and repletion on biochemical markers of bone turnover
Effects of the short term calcium depletion and repletion on bone
turnover were evaluated by measuring the levels of a urinary bone
resorption marker (deoxypyridinoline) and two serum bone formation
markers (osteocalcin and PICP). Consistent with the premise that
depletion increased bone resorption, dietary calcium restriction
significantly (P < 0.01 compared to baseline, by
ANOVA) and rapidly (after 1 day of depletion) increased urinary
deoxypyridinoline excretion (top panel of Fig. 4). This bone resorption marker remained
elevated throughout the entire depletion phase and rapidly returned to
the baseline level upon dietary calcium repletion (P <
0.01 compared to the level at the end of depletion, by ANOVA).
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), it significantly
(P < 0.05 compared to baseline, by ANOVA) decreased
the serum PICP level (bottom panel of Fig. 4). The effects
of repletion on these bone formation markers were also complex.
Accordingly, the serum PICP level was significantly (P
< 0.02 compared to the level at the end of depletion, by ANOVA)
increased after 1 week of repletion. In contrast, the serum osteocalcin
level was elevated during depletion and remained elevated even after 1
week of repletion (not significant compared to the level at the end of
depletion).
To assess the relative effects of depletion and repletion on these bone
turnover markers, the results are also expressed as the percent change
from each respective baseline value (Fig. 5). During repletion, calcium depletion
appeared to cause a bigger increase in the bone resorption parameter
(i.e. urinary deoxypyridinoline; increased by 55%) than in
the bone formation parameter (i.e. serum PICP; by
<15%).
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), but not with the serum PICP level
(r = 0.200; P = NS). Additional correlation
analysis revealed that neither the level nor the relative change in the
level (i.e. percent change from basal) of urinary
deoxypyridinoline correlated with the level of or the relative change
in serum PTH or 1,25-(OH)2D3 during either
depletion or repletion phase (data not shown). Multiple linear
regression analysis also revealed no significant partial correlation
coefficient between urinary deoxypyridinoline and serum PTH,
1,25-(OH)2D3, or calcium (data not shown).
There was also no reliable association between the level of or change
in level of serum PTH or 1,25-(OH)2D3 and those
of the serum bone formation markers in depletion or repletion (data not
shown). Multiple linear regression analysis confirmed the lack of a
significant correlation between the serum level of PTH or
1,25-(OH)2D3 and that of the two serum bone
formation markers (data not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Previous studies in both premenopausal (21, 22) and postmenopausal (22, 23) women as well as in animal models (4, 5) have shown that calcium depletion would result in an increase in bone resorption. Consistent with the premise that calcium deprivation in young subjects could cause a rapid increase in bone resorption, the dietary calcium restriction in young women also significantly and rapidly (after only 1 day) increased urinary deoxypyridinoline excretion. Past studies have suggested that the depletion-associated increase in the bone resorption rate is related to the secondary hyperparathyroidism (4, 5, 21, 22, 23, 24). Accordingly, it is surprising that we did not observe a significant correlation between the level (or the change in level) of urinary deoxypyridinoline and the level (or the change in level) of serum PTH or serum calcium in this study. However, we should note that we had only three measurements of each parameter per patient during the depletion period. Because PTH is known to be secreted in a pulsatile manner, it is possible that the limited number of measurements per patient did not provide a sufficient statistical power to demonstrate a correlation. In addition, the variations in urinary deoxypyridinoline assay were relatively high compared to those in other biochemical marker assays. The lack of a significant correlation between serum PTH and the bone resorption marker provides an example of the importance of multiple sampling when determining the effects of dietary factors on serum parameters, a concept previously advanced by Smith and Nordin (25).
Two findings in this study are noteworthy and intriguing. First, calcium depletion appeared to have contrasting effects on the circulating levels of the two serum bone formation markers (osteocalcin and PICP). Accordingly, calcium depletion significantly increased the serum level of osteocalcin by as much as 10%, but it significantly decreased the serum PICP level by as much as 15%. Although there may be several possible explanations for the differential effects of depletion on these serum bone formation markers, we favor the possibilities that calcium depletion/repletion might have differential effects on different aspects of bone formation, and that these two serum markers may represent different aspects of the bone formation process. In this regard, because PICP is a product of bone collagen synthesis (26), it is presumed that the PICP concentration may reflect the total amount of bone matrix synthesized. On the other hand, it is not entirely clear what exactly the serum osteocalcin level will reveal. However, as osteocalcin is secreted by mature osteoblasts (27), it is possible that the serum level of osteocalcin may reflect the number and/or activity of mature osteoblasts.
Our tentative interpretation that serum osteocalcin and PICP may reflect different aspects of bone formation is, in a large part, based on our previous bone histomorphometric findings in a weanling rat calcium depletion/repletion model (3, 4, 5, 6, 7, 8, 9). In essence, we found evidence in the rodent model that during the depletion phase, there was a marked reduction in collagen synthesis, as indicated by a reduction in bone formation measured by tetracycline labeling using histomorphometric methods (4). Despite this decrease in bone formation, there was an increase in osteoblast number (5). This increase in osteoblast number was associated with an increase in serum osteocalcin (8). Thus, in this particular rodent model, there was a dissociation between collagen synthesis and osteoblast number as well as osteocalcin synthesis during depletion. During the repletion phase, osteocalcin remained elevated (8), and at that time, there was a large increment in bone formation (3, 7), indicating that there was an elevation of both collagen synthesis and serum osteocalcin. The increase in bone formation during the repletion period in rats was documented histomorphometrically (3, 7). Although we recognize that the serum level of biochemical markers represents changes in bone turnover of the entire skeleton, whereas bone histomorphometry may indicate changes at a local bone site, the biochemical marker findings in humans in this study along with the previous bone histomorphometric data in rodents led us tentatively to conclude that during depletion, serum osteocalcin reflected osteoblast cell number and not bone formation, whereas serum PICP represented collagen synthesis and bone formation. In contrast, during repletion, osteoblast number remained high, as again reflected by the serum osteocalcin level, and bone collagen synthesis was stimulated, as shown by the increase in serum PICP.
The rapid increase in serum PICP (and collagen synthesis) during repletion is consistent with the premise that bone formation is rapidly increased upon repletion. We should also note that the increase in bone formation in our past rodent studies occurred immediately upon the reinstitution of dietary calcium regardless of the length of the calcium deprivation period (3, 4, 5, 6, 7, 8, 9). Consequently, although we cannot rule out the possibility that the increase in bone formation observed during repletion is merely a consequence of the completion of a normal bone-remodeling cycle, we believe that the increase in bone formation during repletion is most likely the consequence of an activation of osteoblasts in response to increased availability of calcium.
The second noteworthy observation is that there was a strong positive correlation between serum osteocalcin and the bone resorption marker, urinary deoxypyridinoline, during depletion. In this regard, we would expect that the normal bone coupling process (i.e. an increase in bone resorption is followed by a compensatory increase in bone formation) would become temporarily impaired during depletion in order to mobilize calcium from bone to counteract hypocalcemia (3, 4, 5, 6, 7, 8, 9), leading to the dissociation between the bone resorption and formation processes. Accordingly, it is surprising to note a strong positive correlation between the formation marker, serum osteocalcin, and the bone resorption marker, urinary deoxypyridinoline, during calcium depletion. However, past bone histomorphometric studies in weanling rats have shown a strong positive correlation between the number of osteoblasts and the osteoclast nuclei during calcium depletion (5). Thus, if our hypothesis that the serum osteocalcin level reflects the number of mature but inactive osteoblasts rather than the de facto bone formation rate is indeed correct, the observed positive correlation between serum osteocalcin and urinary deoxypyridinoline may reflect the apparent association between the increase in osteoblast number and osteoclast number during depletion.
These observations led us to advance an interesting concept that in rats bone may have an inherent fail-safe mechanism to replenish bone that is lost as a result of using the bone mineral reservoir during periods of calcium deficiency. In this regard, we speculate that during bone loss in depletion, there is the manufacture of new osteoblast line cells that will eventually completely repair the bone volume deficit during a subsequent repletion period. Moreover, this same mechanism seemed to be operative in the young adult females. Accordingly, during the depletion phase, there was an increase in serum osteocalcin, and as in our rat model, there was a dissociation between bone collagen metabolism and serum osteocalcin, in that the PICP level decreased when osteocalcin was increased. We postulate that the increase in osteocalcin during the depletion phase reflected the increase in osteoblast number generated during this phase. This is consistent with our previous correlative findings between serum osteocalcin and histomorphometric results in our weanling rat model. During repletion in the young female adults, there was a significant increase in serum PICP along with a continued increase in serum osteocalcin. Again, these results in young adult females are similar to those previously found in our rat model and suggest that during the repletion phase, there is a higher number of osteoblasts than at the basal point, as indicated by osteocalcin, and an increase in bone formation, as indicated by the increase in serum PICP. Although much additional work will be required to confirm this concept, it nevertheless is attractive and warrants further attention.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Orthopedics, Malmo University
Hospital, Lund University, S-205 02 Malmo, Sweden.
Received November 13, 1997.
Revised February 2, 1998.
Revised March 4, 1998.
Accepted March 11, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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