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Division of Clinical Biochemistry and Hanson Institute, Institute of Medical and Veterinary Science, Adelaide, South Australia and School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia
1To whom correspondence should be addressed. E-mail: peter.oloughlin{at}imvs.sa.gov.au.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • oophorectomy • rat • calcium balance • calcium excretion.
The temporal relationship between estrogen deficiency–induced bone loss and the calcium fluxes involved in calcium balance is yet to be elucidated. The tight regulation of plasma calcium requires similar regulation of the major calcium fluxes, including intestinal absorption, intestinal secretion and renal tubular reabsorption (1). When bone mineral resorption is markedly stimulated, such as occurs with the rapid decrease in estrogen after oophorectomy, the flow of calcium into plasma is markedly increased without hypercalcemia (2,3). Estrogen deficiency in postmenopausal women is associated with decreases in both renal tubular reabsorption and intestinal absorption of calcium although their relationship with bone loss is controversial (2). Some have argued that these changes contribute to postmenopausal bone loss, whereas others have concluded that they are the result of postmenopausal bone loss (4,5).
In the young growing oophorectomized rat model of postmenopausal osteoporosis there is a transient rise in intestinal calcium secretion at 3 and 6 wk postoophorectomy, associated with impaired calcium balance, with no evidence of intestinal calcium malabsorption until 9 wk postoophorectomy (6). Reduced intestinal calcium absorption, as the major component of impaired calcium balance, is detectable in adult rats from 10 wk postoophorectomy (5).
A study of the temporal relationship between the changes in calcium fluxes and bone loss immediately after oophorectomy may provide information regarding the contribution of such changes to estrogen deficiency–induced bone loss. Thus we have assessed the effects of oophorectomy on calcium balance and its components including intestinal calcium absorption, intestinal calcium secretion and urine calcium excretion within the first 9 wk after oophorectomy in adult rats to identify the acute effects of estrogen deficiency on calcium fluxes.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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On arrival in the laboratory, 20 adult (28 wk old) female Sprague-Dawley rats (Gilles Plains Animal Research Centre, Gilles Plains, South Australia) were separated into individual cages and allowed unrestricted access to a modified AIN-76A-starch diet (5) (Table 1) and tap water. The diet contained 0.4 g Ca/100 g diet, 0.3 g P/100 g diet and 0.1 µg cholecalciferol/kg diet. At 30 wk of age calcium balance and its components, intestinal calcium absorption, intestinal calcium secretion and urine calcium excretion were determined. Immediately after the initial balance study, rats were randomly allocated to either sham or oophorectomy operations and calcium balance studies were repeated at 3, 6 and 9 wk postoperation (postop). The experimental protocol was approved by the institutional animal ethics committee.
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Calcium balance was measured by a previously published method (5). Rats were placed in individual metabolic cages (Techniplast, Buguggiate, Italy) for a 5-d acclimation period, during which food was freely available and consumption was monitored. On d 4, 2 MBq of 45Ca (CaCl2, 83.6GBq/L: Amersham, Little Chalfont, UK) was administered intramuscularly to monitor the secretion of endogenous calcium into the gut. From d 5 to the end of the balance study on d 11, rats were fed 90% of the mean food consumption of the ovary-intact rats during the first 4 d of the acclimation period. Calcium balance was determined over the 6-d period between d 6 and 11. Urine samples were collected into 10 mol/L HCl. Any food remaining at the end of each 24-h period was collected, weighed and the exact consumption recorded. On completion of the 6-d balance study, urine and feces samples were collected and weighed.
Triplicate 10-g samples of the diet and entire 6-d fecal samples were charred in porcelain crucibles in a muffle furnace (Tetlow, Melbourne, Vic, Australia) by raising the temperature gradually to 400°C over a period of 4 h after which they were ashed at 800°C for 18 h. The ash was dissolved in 5mL of 5 mol/L HCl, warmed to 
75°C for 5 min and made up to exactly 10 mL with 5 mol/L HCl. The pH of the urine samples was adjusted to <2 with HCl. Fecal 45Ca and urine 45Ca were determined by liquid scintillation counting (Packard Minaxi, Tri-carb 4000, Downers Grove, IL). Fecal 40Ca, urine 40Ca and diet 40Ca were determined by atomic absorption spectrometry (model 3030, Perkin-Elmer, Norwalk, CT). Calculation of the components of calcium balance were as previously described (6). The data for calcium balance and intestinal calcium absorption are presented as mean changes compared with the values obtained preoperatively at 30 wk of age.
Statistical analyses.
The data are expressed as the mean or mean change ± SEM. The effects of oophorectomy and time were analyzed by two-way ANOVA. Specific differences were determined by the Tukey’s post-hoc test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Calcium balance data indicated a net loss of whole-body calcium stores in oophx rats, with maximal loss occurring at 3 wk postop. This finding is consistent with histological studies that have demonstrated bone loss commencing within 10 d of oophx in rats (9,10) and is similar to our findings of calcium balance in young rats (6) and adult rats at >10 wk postoophorectomy (5). The findings of the present study indicate that oophx-induced bone loss in adult rats occurs throughout the skeleton within 3 wk of the operation.
The negative calcium balance between 3 and 9 wk postoophorectomy was due to a significant increase in calcium excretion. Urine calcium excretion was elevated postoophorectomy compared with the ovary-intact group. There is some controversy concerning the effect of oophx in rats on urine calcium excretion. We reported that there was no effect of oophx on 24-h urine Ca excretion within 9 wk of surgery (11), but by 18 wk, urine calcium excretion after overnight withdrawal of food was increased compared with ovary-intact controls (7). Similar to the findings of the present study, Draper et al. (12) reported transiently increased calcium excretion at 1 wk postop in oophx rats compared with ovary-intact controls.
Endogenous fecal calcium and intestinal calcium secretion data indicated that there was a greater excretion of calcium via the intestine at 3 wk postop in the oophx rats compared with ovary-intact rats. At later times, there was no difference between the groups. This finding is consistent with the transient increase in intestinal calcium secretion after oophx in young rats (6), although in young rats, this condition persisted until 6 wk after oophx. When dietary calcium is low, Draper et al. (12) also found greater excretion of calcium via the gastrointestinal tract compared with the kidney, but did not demonstrate its transient nature.
The ratio of intestinal calcium secretion to urine calcium excretion was 3:1 preoperatively (Table 2). Thus, the intestinal route of calcium excretion is of greater physiologic importance in rats than urine calcium excretion. Because the excretion of calcium by both the gastrointestinal tract and the kidney were affected in the same manner, it is likely that they arise from a common mechanism. This increased excretion of calcium after oophx occurs in concert with increased resorption of bone calcium flowing into the extracellular fluid (9,10). A small but significant decrease in serum ionized calcium is detectable 15 d after oophx in rats. This returns to preop values after estrogen supplementation (3). The fall in ionized calcium is not mediated by changes in serum 1,25 dihydroxyvitamin D or parathyroid hormone levels. Thus it appears that oophx has a rapid effect on at least three calcium fluxes, increased bone resorption and increased intestinal and renal excretions.
The present study did not demonstrate an effect of oophx on true calcium absorption in adult rats fed a normal calcium diet within the first 9 wk after oophx. The time of onset of intestinal malabsorption of calcium after oophx and the pathology of such malabsorption in rats are controversial. We previously demonstrated calcium malabsorption at >10 wk after oophx by calcium balance studies (5). Decreased levels of vitamin D receptor (VDR) (13,14) and VDR mRNA (14) in the duodenal enterocyte have been reported within 21 d postoophorectomy. Although an earlier report (15) demonstrated a slight decrease in so-called villous enterocyte VDR between 5 and 7 wk postoophorectomy, Colin and co-workers (16) more recently demonstrated by immunoassay that VDR levels are unaffected in duodenal enterocytes at 28 d postoophorectomy.
On the basis of previous findings and those from the present study, we conclude that the bone loss in adult rats within 14 d of oophx is associated with a rapid increase in bone resorption, (9,10), accompanied by a transient increase in calcium excretion via both kidney and gut. In the long term, impaired intestinal calcium absorption is a major determinant of the negative calcium balance and a significant contributor to continued bone loss (5).
Manuscript received 21 January 2003. Initial review completed 13 February 2003. Revision accepted 7 May 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. O’Loughlin, P. D. & Morris, H. A. (1998) Calcium homeostasis and osteoporosis. Clin. Biochem. Rev. 19:3-17.
2. Nordin, B.E.C. (1997) Calcium and osteoporosis. Nutrition 13:664-686.[Medline]
3. Schulz, S. R. & Morris, H. A. (1999) Ionized calcium and bone turnover in the estrogen-deficient rat. Calcif. Tissue Int. 65:78-82.[Medline]
4. Nordin, B.E.C., Need, A. G., Horwitz, M. & Morris, H. A. (1996) Perspectives on osteoporosis: understanding and managing osteoporosis in the elderly. Clin. Geriatr. 4:73-88.
5. O’Loughlin, P. D. & Morris, H. A. (1998) Oestrogen deficiency impairs intestinal calcium absorption in the rat. J. Physiol. 511:313-322.
6. O’Loughlin, P. D. & Morris, H. A. (1994) Oophorectomy in the young rat impairs calcium balance by increasing intestinal calcium secretion. J. Nutr. 124:726-731.
7. Baldock, P. A., Morris, H. A., Moore, R. J., Need, A. G. & Durbridge, T. C. (1998) Pre-pubertal oophorectomy limits accumulation of cancellous bone in the femur of growing rats with long term effects on metaphyseal architecture. Calcif. Tissue Int. 62:244-249.[Medline]
8. Kalu, D. N. (1984) Evaluation of the pathogenesis of skeletal changes in ovariectomized rats. Endocrinology 115:507-512.
9. Dempster, D. W., Birchman, R., Xu, R., Lindsay, R. & Shen, V. (1995) Temporal changes in cancellous bone structure of rats immediately after ovariectomy. Bone 16:157-161.[Medline]
10. Sims, N. A., Morris, H. A., Moore, R. J. & Durbridge, T. C. (1996) Increased bone resorption precedes increased bone formation in the ovariectomised rat. Calcif. Tissue Int. 59:121-127.[Medline]
11. Morris, H. A., Porter, S. J., Durbridge, T. C., Moore, R. J., Need, A. G. & Nordin, B. E. (1992) Effects of oophorectomy on the biochemical and bone variables in the rat. Bone Miner. 18:133-142.[Medline]
12. Draper, C. R., Dick, I. M. & Prince, R. L. (1999) The effect of estrogen deficiency on calcium balance in mature rats. Calcif. Tissue Int. 64:325-328.[Medline]
13. Chen, C., Noland, K. A. & Kalu, D. N. (1997) Modulation of intestinal vitamin D receptor by ovariectomy, estrogen and growth hormone. Mech. Ageing Dev. 99:109-122.[Medline]
14. Liel, Y., Shany, S., Smirnoff, P. & Schwartz, B. (1999) Estrogen increases 1, 25-dihydroxyvitamin D receptor expression and bioresponse in the rat duodenal mucosa. Endocrinology 140:280-285.
15. Chan, S.D.H., Chiu, D.K.H. & Atkins, D. (1984) Oophorectomy leads to a selective decrease in 1, 25-dihydroxycholecalciferol receptors in rat jejunal villous cells. Clin. Sci. (Lond.) 66:745-748.[Medline]
16. Colin, E. M., Van Den Bemd, G.J.C.M., Van Aken, M., Christakos, S., De Jonge, H. R., DeLuca, H. F., Prahl, J. M., Birkenhager, J. C., Buurman, C. J., Pols, H.A.P. & Van Leeuwen, J.P.T.M. (1999) Evidence for involvement of 17ß-oestradiol in intestinal calcium absorption independent of 1, 25-dihydroxyvitamin D3 level in the rat. J. Bone Miner. Res. 14:57-64.[Medline]
17. American Institute of Nutrition (1977) Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 107:1340-1348.
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Different from oophx at 6 wk postoperation, P < 0.05. 
Different from ovary-intact at 6 wk postoperation, P < 0.05.