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(Journal of Nutrition. 2001;131:2295-2299.)
© 2001 The American Society for Nutritional Sciences


Articles

Calcium Fractional Absorption and Metabolism Assessed Using Stable Isotopes Differ between Postpartum and Never Pregnant Women1

P. B. Moser-Veillon2, A. R. Mangels, N. E. Vieira*, A. L. Yergey*, K. Y. Patterson{dagger}, A. D. Hill{dagger} and C. Veillon{dagger}

Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742; * Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and {dagger} U.S. Department of Agriculture, Beltsville Human Nutrition Research Center, Beltsville, MD 20705

2To whom correspondence and reprint requests should be addressed. E-mail: pv6{at}umail.umd.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Determining the fractional absorption (FA) of calcium using the incorporation into urine of stable isotopes given intravenously (IV) and orally has become a routine procedure. We investigated the FA of calcium in two groups of (2–3 mo) postpartum women lactating (LACT) (n = 6) and nonlactating (PPNL) (n = 6), and in never pregnant (NP) women (n = 7). The women consumed a controlled diet containing 30–33 mmol/d calcium (Ca) for 21 d. On d 7 of the controlled diet, the women received 0.05 mmol of 42Ca IV and 0.25 mmol 44Ca orally in milk. Urine samples (24-h) were collected for the next 14 d and morning blood samples were collected from fasting subjects before dosing and at 24 and 48 h after receiving the isotopes. Milk samples from the LACT women were collected from each feeding beginning 24 h before to 72 h after dosing. There were no significant differences in the FA of calcium as measured by stable isotope incorporation into urine (23.8 ± 2.9%), serum (24.0 ± 3.4%) or milk (23.6 ± 3.6%) of LACT women. The fractional calcium absorption measured in urine of the postpartum women (LACT and PPNL, 23.8 ± 2.9% and 25.0 ± 3.3%, respectively) did not differ but was greater (P < 0.028) than that of the NP women (17.3 ± 1.3%). The postpartum LACT and PPNL women had a reduced urinary excretion of calcium (P < 0.01) compared with the NP women. There was a significantly greater incorporation (P < 0.001) by LACT women of the oral isotope dose into milk than into urine. Calcium FA can be determined from incorporation of stable isotopes into breast milk and serum as well as urine.


KEY WORDS: • calcium • postpartum • lactation • fractional absorption • stable isotopes • humans


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The absorption of calcium has been determined by the use of intravenous (IV)3 and oral doses of calcium stable isotope incorporation into urine. Compared with the balance method, this method was shown by Abrams et al. (1)Citation not to be significantly different at absorptions <25%. Smith et al. (2)Citation gave small amounts of calcium tracers and found that isotope incorporation into blood, urine and saliva yielded similar results for calcium absorption. Theoretically, the incorporation of Ca stable isotopes into breast milk can also be used to determine the absorption of Ca.

Calcium absorption using the incorporation of isotopes into urine has been used for studies of girls (3Citation 4Citation 5)Citation , pregnant women (6Citation 7Citation 8)Citation and lactating women (6Citation 7Citation 8Citation 9Citation 10Citation 11)Citation . One group that has not received attention is postpartum nonlactating women (PPNL).

This study was undertaken to compare the fractional absorption of Ca as measured by stable isotope incorporation into urine, serum and milk of lactating women. In addition, the incorporation of Ca from oral and IV stable isotopes into serum and urine was compared among postpartum lactating (LACT), PPNL and never pregnant (NP) women. This study is unique because of the controlled calcium intake of the subjects.


    SUBJECTS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Subjects.

This study was approved by the Institutional Review Boards (IRB) for human studies at the University of Maryland and the USDA. Written informed consent was obtained from each subject before beginning the study.

At 2–3 mo postpartum, LACT (n = 6) and PPNL (n = 6) as well as NP women (n = 7) were recruited from the greater Baltimore-Washington, DC area. None of the women used supplements containing minerals for >=2 mo before the study or oral contraceptives for >=6 mo before the study. All subjects and their infants were in good health. All infants exhibited normal growth with appropriate weights for age, lengths for age and weights for length. A description of the subjects was published previously (12)Citation .

Before beginning the study each woman kept a 7-d food and drink intake record. These records were analyzed using the University of Maryland database and Food 8 program (13)Citation . Figure 1Citation summarizes the study design.



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Figure 1. Study design.

 
Diet and stable-isotope doses.

Subjects remained free living but were fed a controlled diet for a 21-d study period. This diet was similar in nutrient composition to diets of healthy lactating women during previous studies (13Citation 14Citation 15)Citation . Energy intake was adjusted to maintain subject weight by supplementing the diet with mineral-free beverages. One of the lactating subjects also increased her intake by an additional glass of skim milk. All food was purchased in lots, prepared and packaged at the USDA, Beltsville, MD. Food was frozen until use and was delivered daily to subjects. A 3-d rotating menu was used. A composite for each of the 3-d menus, including all food and beverages, was weighed, digested and analyzed by atomic absorption spectrometry to determine calcium content (16)Citation . Mean daily calcium content for the LACT subjects averaged 32.8 mmol/d (1316 mg/d) with the inclusion of the one subject that consumed the extra milk and 30.1 mmol/d (1206 mg/d) when she was excluded, and 30.1 mmol/d (1206 mg/d) for the PPNL and NP women.

After consuming the diet for a 6-d equilibration period and after an overnight fast, subjects were brought to the University of Maryland Hospital, a blood sample was collected and they then received an injection of 0.05 mmol (2 mg) 42Ca as calcium carbonate. Immediately after the injection, the subjects consumed a standardized breakfast that contained 377 mg of Ca, and 90 mL of milk to which 0.25 mmol (10 mg) 44Ca as calcium carbonate had been added the previous day and allowed to equilibrate with the calcium in milk overnight at 4°C. The stable isotopes were obtained from Oak Ridge National Laboratory (Oak Ridge, TN). Sterile solutions of 42Ca and 44Ca were prepared by the NIH pharmacy and tested for sterility and pyrogenicity.

Sample collection.

Venous blood samples were collected from fasting subjects immediately before and 24 and 48 h after receiving the oral and IV calcium doses. Trace element–free syringes were used. Samples of serum were frozen at -80°C for later analysis.

Subjects collected all urine from 1 d before and for 2 wk after receiving the isotope doses. Urine collections (24-h) were made daily into opaque polyethylene containers (Fisher Scientific, Springfield, NJ), previously shown to be free from trace element contamination. Samples from each 24-h urine collection were frozen until analysis.

Lactating women hand-expressed milk samples into acid-washed plastic vials at the beginning of each feeding beginning 24 h before receiving the stable isotopes and for 48 h immediately after stable isotope dosing. Infants were fed on demand throughout the study. Milk samples were frozen at -80°C until analysis. Infants were weighed by their mothers with a portable infant balance (Toledo Scale, Columbus, OH) before and after each feeding on those days on which milk samples were collected. Time of feeding and weights were recorded and the difference between final and initial infant weight was used to estimate milk intake. The daily weight of milk consumed was averaged and adjusted for insensible weight loss (17)Citation .

Sample analysis.

Serum and milk samples and aliquots of the 24-h urine samples were collected from 1 d before receiving the isotopes and for 48 h after dosing and were analyzed for 44Ca and 42Ca enrichment.

Urine samples were prepared using a method similar to that described by Turnlund et al. (18)Citation and described in more detail elsewhere (19)Citation . Briefly, small urine samples were centrifuged (6,500 x g for 5 min) to remove precipitates and the calcium separated from the supernatant using a saturated solution of ammonium oxalate (pH 8). Serum and milk samples (0.2 and 0.05 mL, respectively) were dry-ashed at 480°C and the calcium separated using ammonium oxalate. The calcium oxalate precipitate was dissolved in 0.1 mol/L nitric acid and diluted to the desired concentration. The isotope ratios were determined using a PlasmaQuad II+ (VG Elemental) modified to operate in the cool plasma mode (19)Citation . Total calcium was determined by atomic absorption spectrometry (Model 5000, Perkin-Elmer, Norwalk, CT).

Calculations.

The amount of the tracer was determined using the following equation, based on the equations of Turnlund et al. (20)Citation

where T is the total amount of calcium in the sample; mt is the amount of enriched calcium tracer; Wn is the atomic weight of natural or unenriched calcium; Wt is the atomic weight of the enriched calcium tracer; A is used to designate atomic abundance with the subscripts indicating the isotope and the source of the isotope; i is the tracer (42Ca or 44Ca) isotope; x is the reference (43Ca) isotope; n is the natural element; t is the enriched stable isotope tracer; and Ri/x is the ratio of reference to tracer isotope.

Calculation of fractional absorption.

The fractional absorption (%FA) of the oral calcium dose was determined by the relative amounts of the two tracers in the IV and oral doses, and in the samples using the following equation:

where 42Caiv is the IV dose, 44Caoral is the oral dose, and 42Ca and 44Ca are the amounts of the two tracers in the biological sample, all expressed in molar or mass terms.

Statistical analysis.

ANOVA was used to compare fractional absorption determined from urine, serum and milk as well as to compare calcium fractional absorption and calcium losses among the three groups of women. Unpaired t test was used to compare the postpartum women (lactating and nonlactating) and NP women. The Least Significant Difference procedure was performed to determine differences among group means. Data are reported as means ± SEM. Differences were considered significant when P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The subject profile of the three groups of women is presented in Table 1Citation . The three groups of women did not differ in height, weight or body mass index. The two groups of postpartum women did not differ in the length of the postpartum period. Before the controlled feeding study, the LACT women consumed significantly more (P < 0.05) energy and calcium than the PPNL and NP women.


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Table 1. Subject profile1

 
There were no differences in Ca fractional absorption as determined by stable isotope incorporation into urine, serum or milk of the LACT women. Ca fractional absorption of PPNL and NP women did not differ when determined by stable isotope incorporation into urine or serum. (Table 2Citation ).


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Table 2. Fractional absorption of calcium as measured by oral (44Ca) and IV (42Ca) incorporation into urine, serum, and milk of lactating and nonlactating postpartum and never pregnant women1

 
The fractional calcium absorptions of the postpartum LACT and PPNL women measured in urine did not differ and therefore were combined. The mean fractional absorption of the postpartum women was significantly greater (P < 0.028) than that of the NP women (Table 2)Citation . The postpartum women who had resumed menses (n = 7) tended to have a greater (P < 0.07) fractional calcium absorption, 27.5 ± 2.8%, than the postpartum women who were amenorrheic (n = 5), 21.0 ± 1.2%. When the two postpartum groups were separated and compared with the NP group, there was a significant difference (P < 0.046) in the NP women compared with the PPNL, but not with the LACT women (P < 0.085) (Table 2)Citation .

There was no difference in the cumulative incorporation of the IV and oral stable isotope doses into either milk or urine when the oral dose was corrected for absorption (data not shown). There was significantly more (P < 0.001) of the 44Ca oral dose incorporated into the milk of LACT women compared with urine (Table 3Citation ). The two groups of postpartum women, LACT and PPNL, had significantly less (P < 0.01) of the oral dose excreted into urine than the NP women.


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Table 3. Cumulative incorporation of the oral (44Ca) isotope dose into urine and milk 48 h after dosing in lactating and nonlactating postpartum and never pregnant women1

 
Figure 2Citation shows the pattern of IV and oral isotope incorporation into milk 48 h after isotope dosing for a typical subject. The IV dose was detected in the first feeding after the dosing and the oral in the second feeding after dosing. Both the oral and IV doses appeared to peak ~10 h after dosing, with the IV dose showing a more rapid decline than the oral dose over the next 30 h.



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Figure 2. Pattern of incorporation of 42Ca (IV) and 44Ca (Oral) doses into milk of subject 101, 48 h after dosing.

 
Urinary calcium excretion over 24 h was significantly lower (P < 0.002) for both LACT and PPNL women (1.1 ± 0.26 mmol/d; 43 ± 10.3 mg/d) and (2.3 ± 0.52 mmol/d; 90 ± 20.8 mg/d, respectively), compared with the NP women (4.1 ± 0.63 mmol/d; 164.3 ± 25.3 mg/d) (Fig. 3Citation ). The total calcium loss from urine and milk for the lactating women was 6.7 mmol/d (268 mg/d). Using the analyzed Ca intake for the controlled feeding and the percentage of Ca fractional absorption, the lactating women were absorbing 7.8 ± 0.94 mmol/d (313 ± 37.8 mg/d) of Ca. This Ca intake would more than cover the total urinary and milk losses of these women. An estimated mean calcium balance, which did not include endogenous Ca losses, was calculated for the three groups of women (Table 4Citation ). The PPNL women had a significantly higher (P < 0.002) positive estimated Ca balance (5.2 ± 1.21 mmol/d; 212 ± 48.6 mg/d) than the LACT women (1.1 + 1.50 mmol/d; 45 ± 60.1mg/d) or the NP women (1.1 + 0.51; 45 ± 20.4 mg/d).



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Figure 3. Urinary and milk Ca losses of lactating (LACT), postpartum nonlactating (PPNL) and never pregnant, nonlactating women (NP) women. Values are means ± SEM; those with different letters differ, P < 0.002.

 

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Table 4. Absorbed calcium intake and losses in lactating and nonlactating postpartum and never pregnant women1

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Smith et al. (2)Citation showed that fractional calcium absorptions determined from isotopic ratios in blood, urine and saliva were similar. They concluded that saliva samples could be used to determine calcium kinetic parameters for compartmental modeling and fractional absorption, thereby eliminating the need to collect blood and urine samples. We found that milk samples also gave values similar to those of blood and urine for isotopic ratios of Ca enrichment. Thus, during lactation, milk samples can be used to determine fractional calcium absorption and eliminate the need for the collection of urine or blood.

The fractional calcium absorptions for the lactating and nonlactating women in our study were in the same range as those reported when the oral isotope was given in milk (9)Citation and slightly lower than what has been reported when the isotope is given in a water solution (6Citation ,8)Citation . Heaney et al. (21)Citation reported that the oral calcium load influences fractional calcium absorption measurements. Thus, if the oral dose of stable isotope is given with milk, which has a high calcium content, it would be expected that the fractional calcium absorption measurement would be lower than when the oral isotope is given in a water solution.

Our observation of no significant difference in the FA of calcium between LACT and PPNL is consistent with the results of longitudinal studies of calcium absorption during reproduction that have not shown an increase in calcium absorption during lactation (6Citation ,7Citation ,8)Citation . Our observation is also consistent with studies that compared lactating women with nonlactating postpartum controls (11)Citation and found no difference in the FA of calcium. However, increases have been observed postlactation after weaning and the resumption of menses (8Citation ,11)Citation . We also observed a nonsignificant increase in calcium FA with the resumption of menses. Our result that postpartum women have a higher calcium FA than the NP women is surprising given the results of the longitudinal and controlled studies of FA during lactation (6Citation 7Citation 8Citation 9Citation 10Citation 11)Citation . The main difference in our study compared with these other investigations of calcium absorption during lactation is that ours was a controlled feeding study in which all subjects received 30–33 mmol/d Ca from food sources for 6 d before the measure of absorption. The differences in FA detected in our study could be due to the fact that the women had been adapted to a standard calcium diet; this allowed us to measure a difference that was not seen in other studies because the women in previous studies did not consume a controlled diet. The NP women absorbed less calcium, indicating that they did not need as much calcium, whereas the postpartum women did not show this adaptation, indicating they had a greater need for calcium.

The controlled calcium intake during the study was similar to what the lactating group had been consuming before the study, 30 mmol/d (1209 mg/d), but was significantly greater than the 19.9 mmol/d (798 mg/d) consumed by the NP women and 15.9 mmol/d (636 mg/d) by the PPNL women. Another possible explanation for the lower FA of the NP group is that it reflects an increased calcium intake compared with their usual intake. Although the PPNL women also had a lower Ca intake before the study period, their FA was not reduced compared with the LACT group. Possibly they had only recently reduced their intake of energy and calcium compared with what it had been during pregnancy and had not adjusted to a lower intake before beginning the greater intake of the study period.

The significantly greater (P < 0.046) FA of the PPNL women compared with the NP group could reflect a greater calcium need by the PPNL group to restore bone calcium. The lack of a significant difference (P < 0.085) between the LACT and the NP groups could be related to the sample size.

The LACT women incorporated ~3 times more of the oral 44Ca dose into milk than was excreted in urine. Also, the orally ingested 44Ca was detected in breast milk <2 h after dosing, suggesting that there is a relatively rapid incorporation of calcium into milk. The amount of calcium incorporated into milk was equivalent to the urinary calcium excretion of NP women consuming the same amount of calcium. The observation of reduced urinary calcium excretion by LACT and PPNL women is consistent with the findings of many other investigators (7Citation 8Citation 9Citation ,15Citation ,22)Citation and has been suggested as a means for providing more calcium for incorporation into breast milk for the lactating women. Klein et al. (15)Citation estimated that this reduced urinary excretion of calcium could provide 30–40% of calcium incorporated into breast milk. On the basis of the difference found between urinary calcium excretion in NP and LACT women, an additional 3.0 mmol/d Ca was conserved. This amount represents ~50% of the milk calcium for these women. The calcium conserved by a reduced urinary excretion of the PPNL women was reflected in the higher positive calcium balance compared with the LACT or NP group. On the basis of the difference observed between the urinary excretion in NP and PPNL women, an additional 1.8 mmol/d of Ca would be conserved.

Our estimated balances of the three groups did not include endogenous fecal or integumental losses. Data for these losses in postpartum women are limited. There is one earlier study by Heaney and Skillman (23)Citation that estimated the fecal endogenous loss for four postpartum women, two of whom were lactating, using injections of 48Ca and its appearance in feces. The mean estimated fecal endogenous loss of these women was 3.42 mmol/d (137 mg/d). Using this estimate of fecal endogenous loss for our two postpartum groups, the PPNL women would still be in a positive calcium balance, whereas the LACT women would be in a negative balance. This increased calcium retention by the PPNL group may be used to increase bone calcium stores. Hopkins et al. (24)Citation recently reported that in PPNL women, bone mineral content increased within the first 3 mo postpartum. This would be expected to be the result of increased calcium retention in these postpartum women. Numerous studies (8Citation ,24Citation 25Citation 26Citation 27Citation 28Citation 29Citation 30)Citation have shown that bone mineral density decreases in the first 6 mo of lactation with recovery within 6 mo of weaning (8Citation ,25Citation 26Citation 27Citation 28Citation 29Citation 30)Citation . Thus, lactation delays the bone mineral recovery that occurs earlier in PPNL women. The more positive calcium balance in the PPNL group compared with the LACT women reflects this earlier bone mineral recovery.

Fractional calcium absorption during lactation can be determined from the incorporation of stable isotopes into milk samples, eliminating the need for the collection of urine or blood samples. During lactation, orally ingested calcium is preferentially incorporated into breast milk rather than being excreted in urine, providing a mechanism for calcium redistribution to meet maternal needs. PPNL women metabolize calcium in the same way as LACT women with similar absorption and urinary excretion but with no calcium loss through milk, resulting in a greater calcium retention than that of LACT women.


    FOOTNOTES
 
1 Supported in part by the Maryland Agriculture Experiment Station (project number MD-Y-23) and the U.S. Department of Agriculture (specific cooperative agreement 58–32U4–6-167). Back

3 Abbreviations used: FA, fractional absorption; IV, intravenous; LACT, lactating; NP, never pregnant, nonlactating women; PPNL, postpartum nonlactating women. Back

Manuscript received February 12, 2001. Initial review completed April 24, 2001. Revision accepted June 12, 2001.


    LITERATURE CITED
 TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Abrams S. A., Yergey A. L. & Heany R. P. (1994) Relationship between balance and dual tracer isotopic measurements of calcium absorption and excretion. J. Clin. Endocrinol. Metab. 79:965-969.[Abstract]

2. Smith S. M., Wastney M. E, Nyquist L. E., Chi-Yu S., Wiesmann H., Nillen J. L. & Lane H. W. (1996) Calcium kinetics with microgram stable isotope doses and saliva sampling. J. Mass. Spectrom. 31:1265-1270.[Medline]

3. O’Brien K. O., Abrams S. A., Liang L. K., Ellis K. J. & Gagel R. F. (1996) Increased efficiency of calcium absorption during short periods of inadequate calcium intake in girls. Am. J. Clin. Nutr. 63:579-583.[Abstract/Free Full Text]

4. Abrams S. A., Copeland K. C., Gunn S. K., Stuff J. E., Clarke L. L. & Ellis K. J. (1999) Calcium absorption and kinetics are similar in 7- and 8-year old Mexican-American and Caucasian girls despite hormonal differences. J. Nutr. 129:666-671.[Abstract/Free Full Text]

5. O’Brien K. O., Abrams S. A., Liang L. K., Ellis K. J. & Gagel R. F. (1998) Bone turnover response to changes in calcium intake is altered in girls and adult women in families with histories of osteoporosis. J. Bone Miner. Res. 13:491-499.[Medline]

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20. Turnlund J. R., Michel M. C., Keyes W. R., King J. C. & Margen S. (1982) Use of enriched stable isotopes to determine zinc and iron absorption in elderly men. Am. J. Clin. Nutr. 35:1033-1040.[Abstract/Free Full Text]

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A. B Beck, S. Bugel, S. Sturup, M. Jensen, C. Molgaard, M. Hansen, O. W. Krogsgaard, and B. Sandstrom
A novel dual radio- and stable-isotope method for measuring calcium absorption in humans: comparison with the whole-body radioisotope retention method
Am. J. Clinical Nutrition, February 1, 2003; 77(2): 399 - 405.
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