The Journal of Nutrition Vol. 128 No. 3 March 1998, pp. 633-639

Calcium Metabolism and Bone Calcium Content in Normal and Oophorectomized Rats Consuming Various Levels of Saline for 12 Months^1,2

Ellen Lai-Ping Chan and R. Swaminathan^*

Department of Chemical Pathology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong and ^* United Medical & Dental Schools, Guy's Hospital, London, SE1 9RT, United Kingdom

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The effect of different intakes of salt for 12 mo on bone calcium content and urinary excretion of calcium and hydroxyproline were examined in sham operated and oophorectomized (OX) rats to determine the long term effects of high sodium intake and its interaction with estrogen deficiency. Sham operated (n = 24) and OX (n = 24) rats were divided into groups of six rats in a 2 × 4 design. One group of sham and one of OX rats were given 0, 2, 6 or 18 g/L sodium chloride to drink. Urine samples were collected at 0, 2, 4, 6, 10 and 12 mo for the measurement of sodium, calcium, creatinine and hydroxyproline. At the end of 12 mo, blood was taken for measurement of calcium, albumin, alkaline phosphatase and creatinine and the left femur was removed and analyzed for calcium and phosphate. Body weights of the OX rats were higher than the sham operated controls. At the start of the experiment (10 d after OX) urinary excretions of calcium and hydroxyproline were significantly higher in OX rats. However, after 4-6 mo, they were significantly lower in OX rats. Calcium excretion and hydroxyproline excretion were increased by high salt intake, and there was a significant correlation between sodium and calcium excretion (r = 0.962). Bone calcium content of OX rats was lower than their corresponding sham-operated controls. Sodium intake also had a significant effect on bone calcium content. Multiple regression analysis showed that OX and sodium intake explained 7.6% and 1.5% of the variation in bone calcium content. We conclude that high sodium intake causes increased loss of calcium and reduces bone calcium content in sham-operated as well as OX rats.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Osteoporosis has become a major health problem, and the incidence of osteoporosis-related fractures is increasing in many countries (Lau et al. 1990). Peak bone mass and rate of bone loss are key determinants for the development of osteoporosis and osteoporosis-related fractures. One of the important factors determining the rate of bone loss in women is estrogen deficiency. Estrogen deficiency has been shown to cause rapid loss of bone in women as well as in experimental animals (Kalu 1984, Kalu et al. 1989, Riggs and Melton 1986, Riis et al. 1986, Sherman et al. 1989, Wronksi et a. 1985). In addition to estrogen, other factors including diet are important in determining the rate of bone loss (Riggs and Melton 1986). Of the dietary factors, sodium intake is important (Massey and Whiting 1996, Swaminathan 1991). In healthy subjects there is a strong positive correlation between urinary sodium excretion and calcium excretion (Chan et al. 1992, Law et al. 1988, Shortt and Flynn 1990). Increased sodium intake causes increased loss of calcium via the urine in humans (Chan et al. 1992, Nordin et al. 1993, Shortt and Flynn 1990, Shortt et al. 1988, Zarkades et al. 1989) and in experimental animals (Chan and Swaminathan 1993 and 1994, Chan et al. 1993, Goulding and Campbell 1983, Shortt et al. 1987, Shortt and Flynn 1990). Loss of calcium in urine may lead to substantial loss of bone if high salt intake is continued for a prolonged period of time. There have not been any long term studies on the effect of sodium intake on bone mineral density in human subjects. Short term studies in humans showed that change in sodium intake is associated with change in calcium excretion and markers of bone metabolism (Chan et al. 1992, Massey and Whiting 1996, McParland et al. 1989). In a cross-sectional study of postmenopausal women a correlation between sodium excretion and decrease in bone density was observed (Devine et al. 1995).

In experimental animals, high salt intake has been shown to cause an increase in calcium excretion and a decrease in bone mineral content (Chan et al. 1993, Goulding 1980, Goulding and Campbell 1983 and 1984, Goulding and Gold 1986, Gold and Goulding 1995). However, in most of these studies the dietary intake of salt was very high (80 g/kg diet) and the duration of study relatively short (10-84 d). In one study the effect of moderate salt intake (21 g of sodium chloride/per kg diet) and calcium metabolism were studied (Greger et al. 1987). In another study, which lasted 8 wk, the magnitude of increment in urinary calcium excretion induced by high sodium intake decreased with time (Goulding and Campbell 1983). This suggests that if animals are fed a high salt diet over a prolonged period of time, there may be some renal adaptation to reduce the excretion of calcium. However, there have not been many studies looking at long term effects of moderate to high sodium intake. Furthermore, the effect of estrogen withdrawal on the calciuric effect of high sodium intake has not been documented.

We have therefore investigated the effect of different amounts of salt intake for 12 mo on calcium metabolism and calcium content of bone. A preliminary report of this study on the effect of high salt intake on the excretion of calcium and hydroxyproline at 4 mo has already been published (Chan and Swaminathan 1993).

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

The experimental protocol has been described in detail previously (Chan and Swaminathan 1993). Briefly, 2-3-mo-old female Sprague Dawley rats were either ovariectomized (OX)³ or sham-operated. Ten days after the surgery, OX and sham-operated rats were divided into eight groups each consisting of six rats in a 2 × 4 design. One group each from the OX and sham-operated rats were given either tap water, 2, 6 or 18 g NaCl/L of drinking water (34, 102 and 307 mmol/L sodium, respectively). Rats were given free access to the appropriate saline solution and nonpurified diet (protein 20%, carbohydrate 54%) which had a calcium concentration of 0.80% (Purina Rat Chow 5012). Protocol for the study was approved by the local research committee.

The 24-h urine samples were collected at 0 (10 d after surgery and before starting the experimental drinks), 2, 4, 6, 10 and 12 mo. One aliquot was stored at 20°C for hydroxyproline assay, and another aliquot was acidified with concentrated HCl (Ng et al. 1984) and used for the measurements of calcium, sodium, creatinine and phosphorus.

Sodium, calcium, phosphorous and creatinine concentrations were determined by flame photometry, the cresolphthalein complexone method, the phosphomolybdate method and the Jaffe reaction, respectively. Alkaline phosphatase (EC 3.1.3.1) activity was determined using p-nitrophenol phosphate as substrate. An automated analyzer (Parallel, American Monitor Corp., Indianapolis, IN) was used for all these assays. Urine hydroxyproline was assayed by the method of Ho and Pang (1989).

At the end of 12 mo, rats were bled by cardiac puncture and killed by cervical dislocation. The left femur was removed, cleaned of muscle and connective tissue and dried at 100°C in an oven for 2 d. The dry weight was recorded, and the length was measured on the axis between the greater trochanter and the top of the external condyle. The bone was digested overnight in 5 mL of 10.5 mol/L nitric acid (Sherman et al. 1989). The mixture was then heated to 80°C in a water bath and 0.3 mL of 19.7 mol/L hydrogen peroxide was added. The mixture was adjusted to 10 mL and filtered through Whatman number one filter paper before measurement of the concentration of calcium and phosphorous after appropriate dilution.

Urinary excretion was expressed in relation to creatinine excretion to correct for any errors in urine collection.

Statistical analysis.  Results were expressed as mean and SEM. Urinary values were log transformed before analysis. At each time two way analysis of variance for repeated measures was used to examine the effect of oophorectomy and intake of salt. The effect of sodium and oophorectomy on bone mineral content was also analysed by two way ANOVA. Stepwise multiple regression analysis was done to examine the contribution of salt intake and OX on bone calcium content. All statistical analyses were done using a commercial software package, Abstat^TM (Winstar, Anderson Bell Corporation, Arvada, CO).

	RESULTS

Abstract Introduction Methods Results Discussion References

As previously noted (Chan and Swaminathan 1993), at the start of the experiment the body weights of OX groups tended to be slightly lower (P = 0.08) than that of the sham-operated groups (Table 1). At 2 mo, body weights were not available. At 4 and 6 mo, OX rats were heavier than their corresponding control groups and sodium intake had no effect. At 10 and 12 mo, OX had no significant effect on body weight, but high salt intake resulted in significantly lower body weights (P < 0.001, Table 1).

Urine sodium excretion in rats that drank saline was high in both OX and sham-operated groups (Table 2). There was no significant effect of oophorectomy on sodium excretion at any time after time 0. 

At the beginning of the experiment, calcium excretion in the OX rats (510 ± 46 µmol/mmol creatinine, n = 24) was higher than that in the control rats (371 ± 43 µmol/mmol creatinine, P < 0.05, n = 24). To determine whether the effect of OX on calcium excretion changed with time, calcium/creatinine ratios in groups given tap water or saline, (2 g/L) were examined. As shown in Figure 1a, the calcium/creatinine ratio in the OX rats was higher at the start (mo 0) and at 2 mo. From mo 4 onwards it was lower than in their sham operated controls. When all groups were considered, the effect of OX was significant at 6 and 12 mo (Table 3). Sodium intake had a significant effect on calcium excretion throughout the study with Ca excretion generally increasing with increased sodium consumption.

View larger version (42K): [in this window] [in a new window]   View larger version (31K): [in this window] [in a new window]   Fig 1. The effect of oophrectomy (OX) on a) urinary calcium (Ca)/creatinine (CR) ratio (µmol/mmol) and b) hydroxyproline (OHP)/creatinine (CR) ratio (µmol/mmol) at 0, 2, 4, 6, 10 and 12 mo in rats drinking saline (2 g/L). Data are means ± SEM, n = 6.

The hydroxyproline/creatinine ratio in OX rats (59.1 ± 11.7 µmol/mmol, n = 24) at the beginning of the experiment was higher than in the sham operated rats (40.2 ± 6.9 µmol/mmol, n = 24, P < 0.01). When the effect of OX was examined in groups given tap water or 2 g/L saline, the hydroxyproline/creatinine ratio was significantly lower in OX groups than in the sham controls from mo 4 onwards (Fig 1b). At higher intakes of sodium, hydroxyproline excretion was strongly influenced by the sodium intake with excretion being greater in rats with higher sodium consumption. (Table 4).

After 12 mo, plasma total calcium concentration was lower in the OX groups than in sham operated groups (Table 5) while salt intake had no effect. This effect of OX on plasma calcium concentration was likely due to differences in albumin concentration which was also lower in OX rats (Table 5). Plasma alkaline phosphatase activity was higher in OX rats irrespective of the intake of sodium. Plasma creatinine concentration was not affected by OX or by sodium intake.

Weight of the femur was significantly lower in OX rats, and increased salt intake had a significant lowering effect as well (Table 6). The calcium content of femur in OX rats were lower than their corresponding sham-operated controls and oophorectomy had a significant effect on total calcium (Figure 2) and phosphate. There was a significant effect of salt intake on bone calcium content, and there was a significant interaction between OX and salt intake (Table 6). The effects of body weight, femur weight, OX and salt intake were examined by stepwise multiple regression analysis, and the results showed significant contributions of femur weight (79.4%), OX (7.6%) and salt (1.5%) to the total variation in bone calcium content. Body weight did not contribute significantly.

View larger version (38K): [in this window] [in a new window]   Fig 2. The effect of feeding 0, 2, 6 or 18 g/L of saline for 12 mo on bone calcium content in sham-operated and oophorectomized (OX) rats. Data are means ± SEM, n = 6.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Previous studies on the effect of sodium intake on calcium metabolism and bone mineral content have been of relatively short duration (<84 d), and the sodium intake has always been very high. Typically, the sodium excretions in the high salt groups were 35- to 50-fold higher than the control groups (Gold and Campbell 1986, Goulding and Campbell 1983, 1984). In this study we have attempted to address two issues: the long term effects of different salt intakes and the interaction of high salt intake and oophorectomy on calcium metabolism and bone calcium content. In a preliminary report we showed that even moderately high intake of salt causes a persistent elevation in calcium excretion in normal and OX rats (Chan and Swaminathan 1993); we now show that the effect lasts for at least 12 mo.

Immediately after oophorectomy (i.e. 10 d after the operation), excretion of calcium and hydroxyproline in OX rats were significantly higher than the sham operated controls. However from mo 4 onwards excretion of hydroxyproline and calcium were lower in OX rats given no salt or 2 g/L saline (Table 4, Figure 1). These results suggest that the rapid bone loss seen immediately after OX does not persist.

Body weight of OX rats was significantly higher at 4 and 6 mo. However this effect was no longer seen at 12 mo. The increase in body weight caused by oophorectomy has been reported previously (Aitken et al. 1972, Wronski et al. 1986, Yamazaki and Yamaguchi 1989). Animals given very high salt intake had significantly lower body weight at 12 mo. The food intake (measured at 12 mo) was not different between the high salt groups and low salt groups (e.g. 45.9 ± 2.0 g/d vs 43.9 ± 3.2 g/d). However, fluid intake in saline groups was significantly higher.

OX rats had approximately 20% less bone calcium content than their corresponding controls and high sodium diet decreased this further (Figure 2); there was a negative linear relationship between sodium chloride in drinking water and total bone calcium content. Multiple regression analysis showed a small but significant effect of sodium intake (1.5%) on bone calcium content. OX had a much greater effect (7.6%). These results show the overriding effect of oophorectomy on bone calcium content and that high salt intake has a small but significant effect in OX rats, confirming a recent report (Gold and Goulding 1995).

There was a significant correlation between calcium excretion and sodium excretion (r = 0.962) and the correlations between these two variables in sham and OX rats were similar (r = 0.938 and 0.950 respectively). There was also a significant correlation between sodium/creatinine ratio and hydroxyproline/creatinine ratio (r = 0.578, P < 0.001).

The hypercalciuric effect of high sodium intake is primarily due to the effect on the renal reabsorption of calcium since the reabsorption of calcium and sodium are linked (Shortt and Flynn 1990). The associated increase in hydroxyproline excretion (Table 4) suggests that at lease some of the calcium is coming from bone. Measurement of a more specific marker of bone resorption such as deoxypyridinoline (Swaminathan 1997) would have been preferable. However, at the time of this study the method was not available to us. Previous studies using radioisotope labelling has shown increased bone resorption rather than a decrease in bone formation in animals given sodium chloride (Goulding and Campbell, 1983).

The mechanism of increased bone resorption induced by high salt intake is not clear. In parathyroidectomised animals the effect of high sodium on hydroxyproline is abolished (Goulding 1980) suggesting that increased bone resorption is mediated by parathyroid hormone (PTH). However, there have been few studies in which PTH was measured directly. In human studies, the effect of high sodium diet on serum PTH concentration has produced conflicting results (Breslau et al. 1982, Coe et al. 1975, McCaron et al. 1981, Zemal et al. 1986). These conflicting results could be due to poor assay for PTH. Using a sensitive and specific chemiluminescent assay for PTH, we could not demonstrate a change in serum PTH in response to an increase in 200 mmol of sodium per day in healthy volunteers (Chan et al. 1992) and a recent study confirms this (Lietz et al. 1997). Thus the role of PTH in the sodium induced bone resorption needs further studies.

As shown previously, the calcium excretion in rats consuming a high level of salt was very high (Chan et al. 1993). For example in the sham operated group given 18 g/L sodium chloride, the calcium excretion at the end of the study was at least 10 times that in the corresponding control rats. The decrease in bone calcium content was moderate in comparison. Thus, it is likely that intestinal absorption of calcium is increased in high salt-fed groups. Increased calcium absorption was seen in some studies (Goulding and Gold 1986) but not in others (Goulding and Campbell 1983, Goulding and Campbell 1984). These discrepant results could be due to the duration of high salt feeding (Chan et al. 1993). Young subjects given a high salt diet were found to have increased intestinal absorption of calcium (Breslau et al. 1982) while in elderly subjects this adaptation is not well marked (Breslau et al. 1985). Furthermore, results in this study and others suggest that in spite of possible increases in intestinal absorption, there is negative calcium balance leading to loss of bone calcium (Table 6, Goulding and Gold 1986).

The findings in this study have implications to human osteoporosis. This study shows that the loss of bone calcium is proportional to dietary intake of sodium and that even at moderate intakes of sodium there was significant loss of bone. Previously we have shown that high salt diet caused significant loss of bone calcium after 8 weeks in rats given low calcium diets (Chan et al. 1993). Short term human studies and cross sectional studies in humans have shown that high salt intake is associated with increased calcium loss (Chan et al. 1992, Lietz et al. 1997, Massey et al. 1996, Shortt and Flynn 1990). A significant negative correlation between sodium intake and bone mineral density has been reported (Nordin and Polley 1987). Recent studies also show a correlation between sodium excretion and rate of bone loss in post menopausal women (Devine et al. 1995). In prepubertal women a very close relationship between urinary calcium and sodium has also be reported (Matkovic et al. 1995). Estimates of calcium loss in response to sodium intake vary from 0.95-1.5 mmol/d for every 100 mmol of sodium (Massey and Whiting 1996). This degree of calcium loss can lead to substantial bone loss, especially if the calcium intake is low as in the elderly in whom the absorption mechanism may not be adequate.

We conclude that high sodium diet increases urinary calcium excretion and causes loss of bone calcium and this effect is seen in addition to effects of oophorectomy.

	ACKNOWLEDGMENTS

We would like to thank C. S. Ho for help with the assays and C. Morgan for preparing the manuscript.

	FOOTNOTES

Manuscript received 5 May 1997. Initial reviews completed 16 June 1997. Revision accepted 30 September 1997.

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