We studied the ability of Kalsis, a food supplement that contains selenium, citric acid, and vitamin E, to prevent the effects of ovariectomy on bone loss. Six-month-old, Wistar female rats were studied. Groups (
Osteoporosis was defined as a skeletal disease characterized by low bone mineral density (BMD) and poor bone quality that reduces bone strength and increases the risk of fractures [
Recent animal studies have identified additional unexpected regulatory effects of estrogen centered at the level of adaptative immune response [
The antioxidants can be endogenous or obtained exogenously, for example, as a part of diet or as dietary supplements. The most efficient enzymatic antioxidants involve glutathione peroxidase and catalase. Nonenzymatic antioxidants include vitamins E and C (ascorbic acid), carotenoids, and other compounds [
Drugs used to prevent and treat postmenopausal osteoporosis have been designed to act directly on bone remodelling comprising their main intended effect to maintain or recover bone mass [
Kalsis (Catalysis Lab., Spain) is an antioxidant, a dietary supplement that contains, among others, vitamins C and E and an organic selenium compound. Previous studies in humans seem to demonstrate its beneficial effects on bone mass in osteoporotic patients (unpublished results). Due to the inherent difficulties associated with human investigation, the use of animal models is a helpful tool. The ovariectomized rat is a widely validated experimental model for studying postmenopausal osteoporosis and the effects produced by the different drugs used to prevent or treat the disease [
Thirty-six female Wistar rats from the stabulary of Instituto de Investigación Sanitaria Fundación Jiménez Díaz (Madrid, Spain) with 6 months of age and weighing
On the day following the last treatment, the experimental animals were weighed and killed by exsanguination under ether anaesthesia. Blood samples were obtained by cardiac puncture and serum samples were immediately frozen as aliquots at –80°C. The animals were frozen at –20°C and maintained thawed at 4°C for two days before the analysis.
All procedures were carried out in accordance with European Community Standards on the Care and Use of Laboratory Animals.
Kalsis was kindly supplied by Catalysis Laboratories, Spain. The exact composition of the supplement is (per 500 mg capsule): calcium gluconate (185 mg), magnesium gluconate (85 mg), citric fibre (70 mg),
Serum calcium levels were measured using an autoanalyzer method (modular Roche DDPP). The sensitivity of this method was 0.2 mg/dL. The intra- and interassay coefficients of variation were <0.9% and <1.5%, respectively.
Serum creatinine levels were measured using an autoanalyzer method (modular Roche DDPP). The sensitivity of this method was 0.1 mg/dL. Intra- and interassay coefficients of variation were <0.7% and <2.3%, respectively.
Serum bone Glaprotein (BGP) was measured by a specific ELISA for rat BGP (Rat-Mid Osteocalcin, IDS, UK). The sensitivity of this assay was 50.0 ng/mL. Intra- and interassay coefficients of variation were <5.0% and <5.5%, respectively.
Serum N-terminal propeptide of type I procollagen (PINP) was measured by a specific ELISA for both rat and mouse PINP (Rat/Mouse PINP EIA, IDS, UK). The sensitivity of this assay was 0.7 ng/mL. The intra- and interassay coefficients of variation were <7.4% and <8.0%, respectively.
Beta isomer of serum C-telopeptide of type I collagen (CTX) was measured by an ELISA specific for rat CTX (RatLaps ELISA, IDS, UK). The sensitivity of the assay was 2.0 ng/mL. Intra- and interassay variation coefficients were <5.6% and <10.5%, respectively.
Serum tartrate-resistant acid phosphatase form 5b (TRAP 5b) was measured by an ELISA specific for rat TRAP 5b (RatTRAP Assay, IDS, UK). The sensitivity of the assay was 0.1 U/L. The intra- and interassay variation coefficients were <4.1% and <3.0%, respectively.
Bone mineral density (BMD) was determined
The distal region of the right femur was thawed at room temperature and immersed in saline solution (0.9% NaCl) and analyzed, without further sample preparation, by micro-CT (Skyscan N.V., Aartselaar, Belgium), using an X-ray tube voltage of 100 kV and current of 100
Both trabecular and cortical bone regions were obtained by tracing regions of interest and then analyzed using the commercial software provided with the equipment (SkyScan CT-analyzer software, version 1.7.0). Morphometric indices of the trabecular bone region were determined from the microtomographic data sets (within a VOI) using direct 3D morphometry. Total volume of VOI (tissue volume TV, mm3) and trabecular bone volume (BV; mm3) were calculated based on the hexahedral marching cubes volume model of the VOI. Trabecular bone volume (BV/TV; %) was directly calculated. Trabecular thickness (Tb.Th; mm), trabecular separation (Tb.Sp; mm), and trabecular number (Tb.N; 1/mm) were measured directly with the use of 3D images using methods previously described [
The results of the experiments were expressed as the mean ± SD of the different parameters. A nonparametric method, the Mann-Whitney test (Medcalc Software Program, Belgium), was used to compare the different treatment groups. A
Three months after surgery, rats in the OVX group showed higher body weight when compared with SHAM group (
Table
Creatinine, calcium, CTX/TRAP index, BGP, and PINP serum levels in female Wistar rats at the end of the study. Six-month-old rats at the beginning of the study were ovariectomized (OVX) or sham-operated (SHAM) and randomized in the following groups: SHAM group (SHAM) treated with vehicle for 3 months; ovariectomized group (OVX) treated also with vehicle for 3 months; ovariectomized group treated with Kalsis at a dose of 25 mg/kg/day, for 3 months (OVX + K25). Statistical significance
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Creatinine (mg/dL) |
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Calcium (mg/dL) |
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CTX/TRAP index |
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BGP (ng/mL) |
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PINP (ng/mL) |
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As expected, 3 months after ovariectomy the OVX rats presented a significant decrease in femoral BMD and lumbar BMD. Daily treatment with Kalsis for 3 months avoided the loss in femoral and lumbar BMD due to ovariectomy, and the levels of BMD in the treated group were similar to those of the SHAM group. The decrease in femoral BMD in OVX group is due mainly to an increase in BA. Although there is also an increase in BA in the OVX + K25 group, the increase in BMC with respect to OVX group leads to a higher value of BMD than that of OVX group. In the case of lumbar BMD a similar feature is observed (Table
Bone Area (BA), bone mineral content (BMC), and bone mineral density (BMD) measured in femur and in lumbar vertebra in female Wistar rats at the end of the study. Six-month-old rats at the beginning of the study, ovariectomized (OVX) or sham-operated (SHAM) were randomized in the following groups: SHAM group (SHAM), treated with vehicle for 3 months; ovariectomized group (OVX) also treated with vehicle for 3 months; ovariectomized group treated with Kalsis at a dose of 25 mg/kg/day, for 3 months (OVX + K25). Statistical significance
BA (cm2) | BMC (g) | BMD (cm2/g) | |
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OVX |
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OVX + K25 |
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OVX + K25 |
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Figure
Representative images of bone trabecular 3D-microarchitecture in femur sections of all the study groups obtained by computed microtomography. The study groups of female Wistar rats, 6 months old at the beginning of the study, are ovariectomized (OVX) or sham-operated (SHAM) and randomized in the following groups: SHAM group (SHAM) treated with vehicle for 3 months; ovariectomized group (OVX) also treated with vehicle for 3 months; ovariectomized group treated with Kalsis at a dose of 25 mg/kg/day, for 3 months (OVX + K25).
As shown in Table
3D trabecular microarchitecture analysis of femur by microtomography in rats which were sham operated (SHAM), ovariectomized (OVX), or ovariectomized and treated with kalsis (OVX + K25). The parameters studied are bone volume/total volume (BV/TV; %), trabecular number (Tb.N; 1/mm), trabecular thickness (Tb.Th; mm), trabecular separation (Tb.Sp; mm), and trabecular pattern factor (Tb.Pf; 1/mm). Female Wistar rats, 6 months old at the beginning of the study, ovariectomized (OVX) or sham-operated (SHAM) and randomized in the following groups: SHAM group (SHAM) treated with vehicle for 3 months; ovariectomized group (OVX) also treated with vehicle for 3 months; ovariectomized group treated with Kalsis at a dose of 25 mg/kg/day, for 3 months (OVX + K25). Statistical significance
SHAM | OVX | OVX + K25 | |
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BV/TV (%) |
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Tb.N (1/mm) |
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Tb.Th (mm) |
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Tb.Sp (mm) |
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Tb.Pf (1/mm) |
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Kalsis treatment for 3 months (OVX + K25) partially avoided a decrease in BV/TV and in Tb.N, as evidenced by the ovariectomized rats (OVX). Also, the preventive Kalsis treatment administered immediately after ovariectomy (OVX + K25) avoided an increase in Tb.Sp, albeit not completely. Kalsis produced a significant increase in Tb.Th with respect to the SHAM and OVX groups.
The results of the trabecular pattern factor (Tb.Pf) show that ovariectomy produced a disconnection of the trabecular structure when compared with the SHAM group. Kalsis treatment attenuated this effect and partially maintained the trabecular connection.
In this study, we found that daily treatment of rats for 3 months using a dietary supplement (Kalsis) that contains vitamin C, vitamin E, and selenium prevented loss of FBMD and LBMD due to ovariectomy. Ovariectomized rats treated with Kalsis presented FBMD and LBMD levels similar to those of SHAM group. Untreated OVX rats showed a significant decrease both in LBMD and FBMD with respect to control animals. The loss of bone mass due to ovariectomy has been widely reported in previous works [
When studying variations in bone mass through trabecular microarchitecture analysis of the femur using microtomography, we did not find a complete preservation of microarchitecture with Kalsis treatment. The decrease in BV/TV and Tb.N and the increase in Tb.Sp with respect to the SHAM group were lower in the OVX group treated with Kalsis than in the untreated group. Moreover, treatment with Kalsis brought about a significant increase in Tb.Th with respect to the SHAM and OVX groups. Tb.Pf also was maintained in the OVX + K25 group values between the SHAM and untreated castrated rats. These results show that, used as a preventive treatment, Kalsis attenuates bone loss produced by ovariectomy to such an extent that differences in bone mass between OVX-treated and intact rats can only be detected by
When we analyzed the action of Kalsis on bone remodelling by examining biochemical markers of bone turnover, we observed the same observations as other authors had [
In this respect, there are recent works that show that association between selenium and vitamin E reduces prostate cancer incidence through changes in the oxidation-reduction balance of cells, decreasing expression of antiapoptotic and proinflammatory genes, that are associated with prostate cancer and enhancing DNA repair [
According to an internal publication edited by Catalysis laboratories [
Another interesting advantage of administering this compound may be weight gain. It is known that there is a net body gain weight after menopause, as can be observed in the group of OVX rats. Although the rats treated with Kalsis had a greater weight gain than the control group, this gain was significantly lower than that of the untreated OVX group.
In conclusion, administration of Kalsis, a food supplement containing ascorbic acid, vitamin E, and selenium which is submitted to a molecular activation process by the manufacturer, partially prevented bone loss in ovariectomized rats; bone loss was undetectable by classical densitometric methods like DEXA and could only be observed by using more sensitive methods like computed microtomography. This compound also partially prevented the weight gain produced by ovariectomy. The mechanism of action of this supplement is not the result of a decrease in the rate of bone remodelling, because levels of bone formation and resorption are similar to those of untreated animals. The antioxidant action of this compound as a cause of its beneficial effect is suggested. Further work will be necessary, measuring markers of oxidation stress in ovariectomized rats with and without Kalsis treatment.
Due to the fact that some risk factors for osteoporosis, such as smoking and diabetes mellitus, are associated with increased oxidative stress, human studies are needed to analyze the possible role played by Kalsis in preventing the development of osteoporosis in humans with multiple risk factors. It may be especially beneficial to implement this treatment in the periods in which drugs commonly used to treat osteoporosis need to be suspended due to the risk of negative effects due to their prolonged use.
This work was supported by a grant from Catalysis Laboratories (Spain).