The Journal of Nutrition Vol. 128 No. 10 October 1998, pp. 1807-1810

A High Sucrose Diet Decreases the Mechanical Strength of Bones in Growing Rats^1,2

Leo Tjäderhane^{*, 3} and Markku Larmas^*,

^* Institute of Dentistry, University of Oulu, 90220 Oulu, Finland and  Oulu University Hospital, 90220 Oulu, Finland

	ABSTRACT

Abstract Introduction Methods Results Discussion References

High sucrose diets alter mineral metabolism in humans and animals. We examined the effect of a high sucrose diet on bone growth, composition and mechanical strength in growing rats. Weanling Wistar rats received a high sucrose (43 g/100 g) diet (9 males, 11 females). In the control diet (8 males, 8 females), sucrose was replaced with potato starch, providing an equal energy value. At the onset of the experiment, bones were marked by tetracycline. After 5 wk, the tibias and femurs were weighed, and maximum breaking strengths were determined. Tibias were cut at the tibia-fibular junction; the widths of the bone at the start of the experiment, the periosteal bone formation during the experiment, the widths of the medullary cavity and the final bone width were determined from tetracycline lines. Bone ash weight, Ca and P contents were determined. The breaking strengths of both bones were significantly lower in the sucrose-fed groups of both sexes. In females, the weight of both bones and the final width of the tibias were significantly lower in the sucrose-fed group. The Ca concentration in both bones and the P concentration in tibias were significantly lower in the sucrose-fed group. It was concluded that the metabolic interference induced by sucrose was the reason for the differences. The alterations were more pronounced in females, but independent of body weight.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

A high sucrose diet may induce alterations in calcium balance in humans (Ericsson et al. 1990, Lemann et al. 1970, Lindeman et al. 1967, Thom et al. 1978) and in bone composition in animals (Li et al. 1990, Saffar et al. 1981, Saffar and Makris 1982, Salem et al. 1992, de Tessiers and Saffar 1992). Our previous studies on the effect of a high sucrose diet demonstrated that the diet itself reduces dentin formation (Bäckman and Larmas 1997, Huumonen et al. 1997, Larmas and Tjäderhane 1992, Tjäderhane et al. 1994 and 1995a) and the degree of dentin mineralization (Tjäderhane 1996) during the primary dentinogenesis in rats. Because the dentin and bone formations have considerable similarities (Linde and Goldberg 1993), osteoblasts may also be affected by the high sucrose diet. Primary bone formation during adolescence is believed to determine its resistance against osteoporotic changes in the elderly (Dempster and Lindsay 1993, Heaney 1993, Lindsay 1993). Therefore, the effects of dietary alterations during juvenile osteogenesis merit detailed analysis.

In previous experiments, high dietary sucrose was often combined with high fat content (Li et al. 1990, Zernicke et al. 1995). Our findings of the dietary alterations in dentin metabolism were seen with a high sucrose/low fat diet. To specifically demonstrate the effect of high carbohydrate diets on metabolism and mechanical properties of the bones in growing rats, we conducted a study in which diets containing 43% of either a refined carbohydrate (sucrose) or isocaloric complex carbohydrate (starch) were fed. The differences between the groups in growth, calcium and phosphorus concentraion as well as mechanical strength of the long bones of the hind leg were determined. The working hypothesis was that the diet containing a high percentage of refined carbohydrate has a deleterious effect on bone growth during the active growing phase of young rats.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Wistar rats (n = 36), born and reared in the Experimental Animal Center (Oulu, Finland) were weaned at the age of 21 d, weighed, marked and randomly assigned to groups. The rats were housed and treated as described previously (Tjäderhane et al. 1993). Briefly, an intraperitoneal injection of oxytetracycline hydrochloride (30 mg/kg, Terramycin, Pfizer, Brussels, Belgium) was given to all rats on d 22 to mark the area of the bone formation during the experiment. The rats were divided into two groups. The high sucrose (refined carbohydrate) diet group (9 males, 11 females) received a modified Stephan-Harris high sucrose diet (Stephan 1966), containing 43 g/100 g sucrose, 22 g/100 g wheat flour, 32 g/100 g skim milk powder, 1 g/100 g vegetable oil and 2 g/100 g liver powder (diet described in Huumonen et al. 1997). The control (complex carbohydrate) group (8 males, 8 females), received the same diet except that sucrose was replaced with starch (potato flour) (detailed nutritional values of the diets are given in Table 1). The diets were mixed in our laboratory to ensure uniform constitution of the food. The rats had a free access to diet and tap water. The rats were weighed weekly during the experiment. After 5 wk of the experiment (on d 57), the animals were weighed, anesthetized and killed by decapitation. All experimental procedures were approved by the Experimental Animal Committee of the Medical Faculty, University of Oulu, Oulu, Finland.

The tibias and the femurs were defleshed and preserved in absolute ethanol. The left tibias and femurs of both sexes were used for the following analyses. The lengths of the bones were measured by a digital caliber (Mitutoyo CD-15s, Japan). In femurs, the femoral neck was used as a reference point to avoid the effect of the anatomy of the femoral head on the measurements. The weights of the whole tibias and femurs were determined (Mettler A30, Mettler Instrumente Ag, Zürich, Switzerland), followed by determination of the volume by pycnometric measurements. The weight and volume were used to calculate the bone densities. The breaking strength was determined by using a three-point bending test, in which the bones were set on a rack (13-mm wide) and increasing pressure was applied to the midsection of the bone in the standard fashion (Peng et al. 1994). The maximum pressure at the breaking point of the bone was recorded by a plotter (Perkin-Elmer 165, Hitachi, Tokyo, Japan). The maximal load in newtons was used for the comparison of bone strength.

The tibias were then cut transversally with a diamond saw, with the distal junction of the tibia and fibula as the reference point. The cross sections of the distal parts were examined under an Orthoplan Ploemopak (Leitz, Westlar, Germany) fluorescence microscope (detector wavelength 460 nm), which clearly revealed tetracycline lines marking the bone formed during the experiment. The measurements were done by using a hairpiece scale with ×40 magnification. The following were measurements: 1) the width of the periosteal bone formation at three points of the bone; 2) the width of the medullary area in the anteroposterior and mediolateral directions; 3) the distance between the tetracycline lines in the anteroposterior and mediolateral directions over the medullary cavity, revealing the width of the bone at the beginning of the experiment; and 4) the final width of the bone in the mediolateral direction. For the first three measurements, the average value was used to represent the variable of the bone.

The right tibias and femurs of the female rats were used for the determination of bone ash weight and calcium and phosphorus concentration. The bones of the female rats were chosen to be ashed for the chemical analyses because the tibial weights, final bone width, density, and breaking strengths of both tibias and femurs exhibited more pronounced differences. The whole bones were dried at 60°C for 24 h and pulverized with a micromill (Pulverisette 0, Fritsch, Oberstein, Germany). The bone ash weight was determined by ashing 25 mg of the pulverized bone at 900°C for 24 h. Bone calcium was determined by atomic absorption spectrophotometry (Perkin Elmer model 2380, Perkin Elmer, Norwalk, CT), using samples dissolved in a solution of HCl and HNO₃ (2:1). Phosphorus was measured by the colorimetric determination method (Fiske and Subbarow 1925).

Statistical analyses were performed with SPSS statistical software package (SPSS MS WINDOWS Release 6.1, SPSS, Chicago, IL). Means and SD were calculated. An independent-samples t test was used to determine whether differences existed between the diet groups within the sexes.

	RESULTS

Abstract Introduction Methods Results Discussion References

The mean initial body weights in the sucrose and control diet groups were 42.8 ± 9.4 and 51.6 ± 10 g for females and 44.6 ± 11.1 and 46.3 ± 13.0 g for males, respectively. The respective mean body weights at the termination of the experiment were 156.2 ± 18.6 and 165.0 ± 9.7 g for females, and 209 ± 29.4 and 216.7 ± 29.6 g for males. No significant differences in the body weights were found in either sex between the groups at the beginning or end of the experiment.

The weights of the tibias and the femurs in the female rats fed sucrose were significantly lower than those of the control group (P < 0.05). In males, no differences in bone weights were observed between the groups. There were no differences in bone lengths between the groups within either sex (Table 2).

There were no differences in the width of the tibias at the beginning of the experiment, as marked with the tetracycline label (Table 2). Periosteal bone formation during the experiment, the size of the medullary cavity and the width of the bone at the beginning of the experiment did not differ between groups (Table 2). The final width of the tibias in the female rats fed sucrose was significantly smaller than in the control group (P < 0.05), but no differences were observed in males (Table 2).

The densities of both the tibias and femurs of the female rats were significantly lower in the sucrose diet group (P < 0.001) (Table 2). A similar finding was observed also in males, with significantly different values for the tibia (P < 0.01) (Table 2). The breaking strengths of both the tibias and the femurs (Table 2) were dramatically lower in the high sucrose group in both sexes. The effect was proportionately greater in female rats , but also occurred in males.

There were no differences between the female groups in the bone ash weights (Table 2). The calcium and phosphorus concentrations were significantly lower in the tibias of the sucrose-fed group (P < 0.001); the concentration of calcium was also significantly lower in the femurs of the sucrose-fed group (P < 0.05).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Both diets met well the nutritional requirements of the NRC (1972). The amount of fat was below the recommendation, but equal in both diets. Thus the differences observed were not due to the low amount of dietary fat. Neither diet caused any evident nutritional deficiency. The absence of differences in the final body weights between the groups proves that the findings on bone variables in this study are independent of weight. Although both diets were slightly below the recommended energy level, no deprivation of energy resulted because rats increased food intake to meet energy demands (NRC 1972) and food was freely available.

The lower density and mechanical strength found in both the tibias and the femurs of the sucrose-fed groups demonstrates that the sucrose diet alters the mechanical properties of the bone. This finding is supported by previous studies (Li et al. 1990, Salem et al. 1992, Zernicke et al. 1995). The mechanism by which the sucrose diet affects bone is not clear. The effect may be mediated via the alterations in urinary calcium (Ericsson et al. 1990, Holl and Allen 1987, Lang et al. 1981, Lemann et al. 1970, Lennon et al. 1970, Thom et al. 1978). The loss of calcium in urine may be due to reduction in tubular reabsorption (Ericsson et al. 1990, Lemann et al. 1970). An increase in serum insulin (Holl and Allen 1987) and a direct effect of glucose (Lang et al. 1981) have been proposed as reasons for the reduced reabsorption. A substantial increase in urinary calcium caused by sucrose intake has been suggested to increase risk of developing bone loss associated with aging (Ericsson et al. 1990, Holl and Allen 1987, Lang et al. 1981). Recently, high glucose concentration has also been shown to have a direct inhibitory effect on osteoblast growth in vitro (Terada et al. 1998).

Both the short- and long-term experiments in young female rats fed a high fat/sucrose diet have results comparable to ours (Li et al. 1990, Zernicke et al. 1995). Our findings here in rats fed a low fat diet signify the importance of sucrose to the changes observed in bone. The adverse effect of sucrose on mineralizing tissue is also demonstrated as a reduction in dentin formation (Bäckman and Larmas 1997, Huumonen et al. 1997, Larmas and Tjäderhane 1992, Tjäderhane et al. 1994 and 1995a) and mineralization (Tjäderhane 1996) in rat molar teeth. Comparable effects of the calcium-deficient diet (Tjäderhane et al. 1995b) and calcium antagonist medication (Larmas and Tjäderhane 1992) on dentin formation suggest that the effect of a sucrose diet on calcium homeostasis may underlie the alterations in dentin formation.

Glucose and glucose polymers enhance the intestinal absorption of calcium in humans (Bei et al. 1986, Norman et al. 1980, Steinhardt and Adibi 1984). However, the urinary loss of calcium is not dependent on the amount of calcium ingested at the same time because sucrose alone increases urinary calcium (Ericsson et al. 1990, Holl and Allen 1987). Absorption of calcium from the lumen is not highly dependent on a specific fraction of the food or the form of the calcium, but is increased when administered with a meal (Heaney et al. 1989 and 1990, Pointillard and Guéguen 1993). Therefore, we suggest that in our experiment, the reduction in the amount of calcium absorbed was not the reason for the decreased bone strength or calcium concentration in the sucrose-fed groups. Starch, a complex carbohydrate, provides an energy value comparable to that of sucrose, but has been shown not to induce insulin resistance or glucose intolerance, as does sucrose (Grimditch et al. 1988).

Saffar et al. (1981) reported sucrose-induced changes characteristic of an osteoporotic state in the femurs of young golden hamsters. They further suggested that the diet-induced osteoporosis affects only endosteal structure, and an additional effect probably related to the bone remodeling process may be involved (Saffar and Makris 1982). Those results are supported in part by our findings because the width of the periosteal bone formation during the test period was not affected. However, we also did not observe any significant differences in the endosteal bone formation, when measured as the size of the medullary cavity in the tibias. This agrees with the suggestion by Heaney (1993) that in general, the effect of low calcium intake on the growing skeleton is mediated through modulation of the balance between bone formation and bone resorption and is confined to an effect on bone density. In our experiment, the bone calcium concentration was markedly lower in the sucrose diet groups, with only modest effects on bone size.

The reason for an outcome different from the results of Saffar et al. (1982) can be explained by the different ages of the rats. In our experiment, young growing animals with rapid rates of body weight gain and bone growth were used. Genetic regulation of bone size is responsible for the growth of the bones (Heaney 1993), but the availability of dietary calcium affects bone mass (Heaney 1993, Johnston et al. 1992). Saffar and co-workers (1981 and 1982) used young adult male hamsters with much slower growth rates.

The rats in this study were exposed to the diet at the age of 3 wk, right after weaning. Therefore, these results reveal the deteriorating effect of the refined carbohydrate diet on mineral composition and mechanical strength in rapidly growing bones. One of the most important preventive factors in osteoporosis is the peak bone mass achieved during the preteen years (Dempster and Lindsay 1993, Heaney 1993, Lindsay 1993), and the long-term experiment with a high fat/sucrose diet demonstrates that the adverse effects on bone are not recoverable (Zernicke et al. 1995), at least not without dietary change. These results further suggest that a difference exists in the response to the dietary alteration between sexes. In particular, young females consuming a high amount of refined sugars, a diet widely consumed by adolescents in Western countries (Bull 1992, Crawley 1993, Hackett et al. 1984, Lewis et al. 1992, Sivaneswaran and Barnard 1993, Summerbell et al. 1995) may be at future risk of developing osteoporosis.

	FOOTNOTES

Manuscript received 8 January 1998. Initial reviews completed 6 March 1998. Revision accepted 2 June 1998.

	ACKNOWLEDGMENTS

We thank Päivi Moilanen for her competent care of the animals and Eeva-Maija Kiljander for her skillful laboratory work.

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