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1
University of Southern California and the Orthopaedic Hospital, Los Angeles, CA 90089-9317 and * Department of Orthopaedic Surgery and Department of Biostatistics, Carolinas Medical Center, Charlotte, NC 28232-2861
2 To whom correspondence should be addressed. E-mail: rrude60075{at}aol.com.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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because immunocytochemical localization of TNF- in osteoclasts was 199% greater than in controls at 2 mo, 75% at 4 mo and 194% at 6 mo. The difference in TNF- may be due to substance P, which was 250% greater than in controls in mononuclear cells at 2 mo and 266% at 4 mo. These data demonstrated that a Mg intake of 10% of NR in rats causes bone loss that may be secondary to the increased release of substance P and TNF-.
KEY WORDS: • magnesium • osteoporosis • substance P • tumor necrosis factor- • rats
Epidemiologic studies have demonstrated a positive correlation between dietary Mg intake and bone density and/or an increased rate of bone loss with low dietary Mg intake (1–6). The recommended daily allowance (RDA)2 for Mg for adult men and women is 420 mg/d and 320 mg/d, respectively (7). The usual dietary Mg intake, however, falls below this recommendation in a large proportion of the population (8). Mg exists in macronutrient quantities in bone (0.5–1% bone ash) (9), and dietary Mg deficiency has been implicated as a risk factor for osteoporosis (1–6).
Previous studies demonstrated that severe Mg deficiency [0.04% of nutrient requirement (NR)] results in osteoporosis in rodent models. Impaired bone growth, decreased bone formation, increased bone resorption, osteoporosis and increased skeletal fragility were observed (10–16). The cause of this effect of Mg depletion on bone is unclear, although increased amounts of inflammatory cytokines were found in the bone of Mg-deficient mice and elevated serum concentrations of inflammatory cytokines were observed in Mg-deficient rats (14,15,17). This degree of Mg depletion, however, likely is rare in humans. We assessed the effects of a more moderate dietary Mg restriction, 10% of NR, on bone and mineral metabolism in rats and utilized this animal model to determine whether this degree of Mg depletion alters the presence of inflammatory cytokines in bone.
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MATERIALS AND METHODS |
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Biochemical determinations. Blood samples were allowed to clot for 30 min and then centrifuged at 6000 x g in a Labnet Hermle Centrifuge, Model MR-2 for 10 min at room temperature. The serum was separated from the clot and frozen at -70°C. Serum Ca and Mg concentrations were determined by atomic absorption spectrophotometry (Perkin Elmer 560, Norwalk, CT).
Serum parathyroid hormone (PTH). PTH was determined with a two-site ELISA kit (Rat Intact PTH ELISA Kit by Immutopics, San Clemente, CA). This assay has a sensitivity of 0.17 pmol/L, an intra-assay precision of 2.1–2.4% and an interassay precision of 5.1-6.0% at low to high serum PTH concentration.
Serum 1,25-dihydroxy-vitamin D [1,25(OH)2-vitamin D]. A competitive equilibrium RIA was used for quantitative determination of 1,25(OH)2-vitamin D (DiaSorin, Stillwater, MN). This assay consists of a two-step procedure involving a preliminary extraction and subsequent purification of 1,25(OH)2-vitamin D from serum using C18OH "Extra Clean" cartridges. After extraction, the treated sample is assayed using a competitive RIA method based on a polyclonal antibody that is 100% specific for both 1,25(OH)2-ergocalciferol and 1,25(OH)2-cholecalciferol. This assay measures 1,25(OH)2-vitamin D in the range of 12–480 pmol/L and has a sensitivity of <4.8 pmol/L and a CV of 13.7–15.3%.
Serum Osteocalcin. Rat serum osteocalcin was determined utilizing a sandwiched ELISA Kit (Biomedical Technologies, Stoughton, MA). This assay measures osteocalcin in the range of 0.06–3.49 pmol/L and has a sensitivity of 0.09 pmol/L, an intra-assay CV of 4% (95% limits) and an interassay CV of 7% (95% limits).
Serum alkaline phosphatase. Alkaline phosphatase activity in rat serum was measured using an end-point colorimetric spectroscopy (Sigma-Aldrich, St. Louis, MO). The procedure was modified to accommodate the small volume of samples available. Enzymatic activity was determined by measuring the release of p-nitrophenol from substrate p-nitrophenylphosphate. The spectrophotometric reading was made at 405 nm (Spectra MAX 250, Molecular Devices, Sunnyvale, CA), and the unknown was compared with p-nitrophenol standards. Enzymatic activity is expressed in U/L.
Serum pyridinoline (Pyd) assay. Pyd crosslinks in rat serum were determined utilizing the Metra Serum PYD EIA kit (Quidel, Mountain View, CA). This competitive enzyme immunoassay utilizes a rabbit polyclonal anti-Pyd antibody to measure Pyd in serum in a microassay stripwell format. Sample filtering is required before assaying. This assay has a detection limit of 0.4 nmol/L, a 0–12 nmol/L range of detection, an intra-assay CV of 6.3–14.8% and an interassay CV of 8.7–11.6%.
Skeletal studies
Mineral content of bone. To assess the effect of Mg deficiency on bone mineral content, the right femur, stripped of soft tissue, was frozen in liquid nitrogen and stored at -70°C before bone ashing (14). Mg, Ca, and phosphorus contents were determined as described previously (15).
Microcomputed tomography. Analyses were performed using the SkyScan 1074 X-ray Microtomograph (Skyscan, Aartselaar, Belgium; Micro Photonics, Allentown, PA) and associated 3D-Calc, cone reconstruction and ANT model-building software. Left tibias were fixed in formalin for 24 h, stored in 70% ethanol, cleaned of soft tissue, dissected so that the proximal tibia section was 1.2 cm in length and dried overnight. Specimens were placed in the chamber, oriented with the proximal end up such that two thirds of the specimen’s upper segment fell within the X-ray field. An exposure time of 1440 ms was used with a step value of 0.9. A reconstruction of the bitmap data set (consisting of 400–500 sections) was obtained and used to build the 3-D model. The model was bisected with a plane down the anterior/posterior midline and a separate data set was obtained with 15 cross sections on either side of the plane. Each final data set was based on a standardized analysis of a tissue volume that averaged 3.255 mm3. Care was taken so that the region of interest contained only trabecular bone.
Quantitative bone histomorphometry. At the 2, 4 and 6 mo time points, femurs were harvested and prepared and histomorphometry performed as previously described (15,18,19). Quantitative bone histomorphometry utilized the OsteoMeasure software of OsteoMetrics (Atlanta, GA) and standard nomenclature (20). The following histomorphometric variables were examined: the percentage of trabecular bone volume (BV/TV), the percentage of bone volume occupied by osteoid (OV/BV), number of osteoblasts per mm bone surface (NOb/BPm), total proportion of bone surface involved in resorption (ES/BS), the percentage of bone surface covered by osteoclasts (OcS/BS), number of TRAP-positive osteoclasts per mm bone surface (NOc/BPm) and mean trabecular width in µm (TTh).
Immunohistochemical localizations.
The tibia was isolated and prepared for immunohistochemical staining as previously described (15). Indirect immunohistochemistry was used to localize substance P, TNF- and IL-1ß. The antibody sources were rabbit (substance P) and goat (TNF-, IL-1ß) in origin and obtained from R&D Systems, Minneapolis, MN (TNF- and IL-1ß) and Chemicon, Temecula, CA (substance P) (21). Biotin-labeled antibodies to the species in which the antibody was made was the second antibody.
Localization was visualized in the light microscope using the substrate containing red dye Nova Red (Vector Labs, Burlingame, CA) and counterstained with hematoxylin (blue) (Zymed Laboratories, South San Francisco, CA). The results were photographed in a Zeiss photo microscope (Carl Zeiss, Thornwood, NY) using a X40 objective.
Evaluation of cytokine localizations. Background localization was minimal compared with positive and negative controls. No difference in background localization was observed between Mg-deficient and control rats. Cells and tissues stained specifically as described for the antigen in the literature (21,22). Intensity was graded as 0 = no localization, 1 = weak localization, 2 = moderate and 3 = strong localization. The quantitative estimate of numbers of cells staining was a = <20%, b = 20–60%, and c = >60%. The mean relative positivity was <1b = 0; 2a and 2b = 1; and 3b and 3c = 2 (20,21).
Statistical analysis. Data were analyzed using SAS version 8.2 (SAS Institute, Cary, NC). A P-value of < 0.05 was considered significant. Standard statistical methods were used. Descriptive statistics, including means and standard deviations were calculated. When data were not normally distributed, the Wilcoxon rank sum test or Kruskal-Wallis test was employed. Spearman’s correlation coefficients were calculated to determine relationships among the outcome variables. Two-way ANOVA was used to assess differences between low Mg–treated rats and control rats at 2, 4 and 6 mo. In two-way ANOVA, an interaction between the two factors signifies that the effect of one factor is dependent on a particular level of the other factor. In this study, a significant interaction means that the difference between the low magnesium diet and the control diet was not uniform across the time points. For instance, the difference between the two dietary groups may increase over time.
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RESULTS |
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Serum Mg, Ca, PTH and 1,25(OH)2-vitamin D. In rats fed the low Mg diet, marked hypomagnesemia had developed at mo 2, which persisted throughout the experimental period (Table 3). Serum Ca was slightly, but significantly higher in Mg-deficient rats at all time points. At 2 mo, serum PTH was higher in Mg-depleted rats despite the slightly higher serum Ca levels. Thereafter, there was a progressive fall in serum PTH in Mg-depleted rats, whereas the concentration increased in control rats. Although serum 1,25(OH)2-vitamin D declined significantly in both groups, Mg depletion resulted in a greater reduction in serum 1,25(OH)2-vitamin D concentrations.
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Skeletal assessment
Bone mineral content. The percentage of ash Mg was significantly lower by up to 51% in Mg-deficient rats (Table 4). There was a slightly greater Ca bone content, which developed over time, perhaps reflecting the larger more perfect crystal formation previously described in Mg deficiency (23). Ash phosphorus did not differ between the diet groups.
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immunocytochemical localization in osteoclasts was 199% greater than controls at 2 mo, 75% at 4 mo and 194% at 6 mo. TNF- was also greater in megakaryocytes at 2 mo (146%), 4 mo (145%) and 6 mo (122%) and in macrophages at 2 mo (147%) (Fig. 3). No differences were observed for IL-1ß localization.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Although this level of Mg intake is still quite low, there may be some relevance regarding human osteoporosis. According to the U.S. Department of Agriculture, males and females over age 9 y, at the 5th percentile of intake, have a Mg intake that is 42% of the RDA; at the 25th percentile of Mg intake, the mean intake for males is 59% of RDA and for females 55% of RDA (7). In addition, many individuals have coexisting disorders that impair intestinal Mg absorption and/or are associated with renal Mg loss, such as malabsorption syndromes, alcoholism and diabetes mellitus [for review see (24)]. Individuals with such disorders have a higher than expected incidence of osteoporosis. It is thereby conceivable that dietary Mg insufficiency may play a role in the development of osteoporosis.
Bone loss is due to an imbalance of bone formation and bone resorption. In earlier studies of severely Mg-depleted animals, a reduction in osteoblast number was observed in Mg-deficient rats and mice as shown by histomorphometry (14,15). In other studies, serum and bone alkaline phosphatase, osteocalcin and bone osteocalcin mRNA were reduced, suggesting a decrease in osteoblastic function (12,13,25,26). Decreased collagen formation, sulfation of glycosaminoglycans (27) and decreased tetracycline labeling (13) were also observed. These data suggest that impaired osteoblastic bone formation is an important contributing factor in Mg deficiency–associated osteoporosis. In the current study of less severely depleted rats, osteoblast number did not differ from the control group. However markers of osteoblastic bone formation, serum alkaline phosphatase and osteocalcin, were reduced in the Mg-depleted rats; these findings suggest the presence of impaired osteoblast function.
A number of mechanisms may contribute to a decrease in bone formation in Mg deficiency. Mg is mitogenic for osteoblasts (28), and Mg depletion causes growth inhibition in cells in vitro (29); therefore, Mg deficiency may directly result in a decrease in osteoblastic bone formation. In most species, including humans, Mg deficiency causes impaired PTH secretion and/or PTH end organ resistance (30). In this study, serum PTH fell progressively over the 6-mo experimental period in Mg-deficient rats compared with the control rats. It is unclear why this opposite effect occurred; however, in the control rats, aging may have resulted in higher serum PTH values. The decline in Mg-deficient rats may represent the effect of Mg deficiency on PTH secretion. Serum 1,25(OH)2-vitamin D levels are also low in Mg-deficient humans and rats (14,31). In the present study, we again observed a marked effect of Mg depletion because serum 1,25(OH)2-vitamin D was profoundly low at 4 and 6 mo. Because both hormones influence osteoblast activity, these changes in PTH and 1,25(OH)2-vitamin D levels may contribute to impaired bone formation.
Our earlier studies of severely Mg-deficient rats and mice demonstrated an increase in osteoclast number (14,15), which was also observed in this study. Osteoclast number, the surface area covered by osteoclasts and eroded surface were significantly higher in Mg-deficient rats; this finding suggests that increased bone resorption was a major factor causing the decrease in bone mass. It is of interest, however, that we observed no increase in serum Pyd. In earlier studies, Mg-deficient young (26) and mature (32) rats had a decrease in urinary hydroxyproline and/or deoxypyridinoline. A study in humans, however, found that dietary Mg intake was the strongest predictor of urinary deoxypyridinoline excretion; that is, a low Mg intake was associated with elevated urinary deoxypyridinoline (3).
The potential mechanism(s) of the increase in osteoclastic bone resorption is unclear at present. Mg has been shown to inhibit the N-methyl-D-aspartate (NMDA) receptor (33); reduction of extracellular Mg lowers the threshold level of excitatory amino acids necessary to activate this receptor. Activation of the NMDA receptor induces the release of neurotransmitters such as substance P (17). Dietary Mg deficiency produces elevated serum levels of neuropeptides such as substance P within 1–3 d in rodents (16,17) and is followed by release of proinflammatory cytokines (IL-1ß and TNF-) within wk 1 of dietary Mg depletion (17). Substance P is released at nerve endings in bone and enhances osteoclastic bone resorption (34,35). We previously observed localized increased levels of substance P, TNF- and IL-1ß in cellular elements of the bone marrow microenvironment of Mg-deficient mice using immunohistochemical localizations (15). In this study, we again found increased substance P and TNF- in rats fed a diet with 10% NR. The significant increase of substance P at 2 and 4 mo but not at 6 mo, may reflect an earlier effect of the Mg-deficient state. This increase in substance P and TNF-, however, may contribute to increased osteoclast numbers and bone resorption, thus explaining the uncoupling of bone formation and bone resorption observed in Mg-deficient rats (14) and mice (15). These cytokines have also been proposed to contribute to an increase in osteoclastic bone resorption in postmenopausal women (36).
The results of this study have demonstrated a profound effect of Mg depletion on bone utilizing a diet that was 10% NR. The changes were characterized by evidence of decreased osteoblast function as well as increased osteoclastic activity. Increased substance P and TNF- in bone from Mg-deficient rats provides a possible explanation for the increased osteoclastic bone resorption accompanying Mg depletion. Similar mechanisms may explain lower bone mass in humans who have chronic dietary Mg deficits. The results support the hypothesis that dietary Mg deprivation is a risk factor for osteoporosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: 1,25(OH)2-vitamin D, 1, 25-dihydroxy vitamin D; BV/TV, % trabecular bone volume; ES/BS, total proportion of bone surface involved in resorption; NMDA, N-methyl-D-aspartate; Nob/BPm, number of osteoblasts per mm bone surface; NOc/BPm, number of TRAP-positive osteoclasts per mm bone surface; NR, nutrient requirement; OcS/BS, % of bone surface covered by osteoclasts; OV/BV, % of bone volume occupied by osteoid; PTH, parathyroid hormone; Pyd, pyridinoline; RDA, recommended daily allowance; TTh, mean trabecular width in µm.
Manuscript received 7 August 2003. Initial review completed 11 September 2003. Revision accepted 15 October 2003.
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