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Gerontology and Geriatrics, University of Perugia, 06122 Perugia, Italy; Institute of Physiological Chemistry I, Heinrich Heine University of Dusseldorf (M.C.P.), 40225 Dusseldorf, Germany; and Division of Bone and Mineral Diseases, Washington University (R.P.), St. Louis, Missouri 63110
Address all correspondence and requests for reprints to: Dario Maggio, M.D., Department of Gerontology and Geriatrics, University of Perugia, Ospedale Regionale Monteluce, Via Brunamonti, 06122 Perugia, Italy. E-mail: dario_maggio{at}msn.com.
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Abstract |
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Our results reveal that antioxidant defenses are markedly decreased in osteoporotic women. The mechanisms underlying antioxidant depletion and its relevance to the pathogenesis of osteoporosis deserve further investigation.
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Introduction |
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Several epidemiological studies have found an association between dietary vitamin C intake and bone mineral density (BMD) in postmenopausal women (10, 11, 12). This evidence is sounder for early postmenopausal subjects who have never used estrogen and who have a daily intake of at least 500 mg calcium (10, 11). More importantly, it has been recently shown that lower dietary intake of vitamins C and E (another antioxidant vitamin) may substantially increase the risk of hip fracture in smokers (13).
Possible indications of a link between antioxidants and bone health derive from studies of isoflavones, weak bone-sparing agents that have been shown to possess significant antioxidant properties in studies conducted in vitro on sperm and lymphocytes and in vivo on low density lipoprotein oxidation in humans (14, 15, 16, 17).
Despite the experimental and epidemiological evidence relating antioxidant vitamins to osteoporosis and fracture risk, there are no data on vitamin C or other antioxidant plasma levels in osteoporotic subjects. The purpose of our study was to assess whether plasma antioxidant defenses are decreased in elderly osteoporotic women compared with controls. Both nonenzymatic (plasma vitamin C, vitamin A, vitamin E, and uric acid), and enzymatic [superoxide dismutase (SOD) in plasma and erythrocytes and plasma glutathione peroxidase (GPx)] antioxidants were measured in an adequate sample of postmenopausal women. In a parallel way, to detect any sign of increased oxidative stress in osteoporosis, we measured plasma levels of a byproduct of lipid peroxidation, i.e. malondialdehyde (MDA), in both groups.
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Materials and Methods |
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Inclusion criteria for the osteoporotic group were age of 60 yr or more, female gender, postmenopausal state, independent mobility, and a femoral neck T score of -3.5 or less. We chose this cut-off to select subjects with overt osteoporosis. The control group consisted of elderly women with identical inclusion criteria, but with a femoral neck T score -1 or more. All subjects were on a free diet.
Exclusion criteria for both groups were secondary osteoporosis; diseases known to be associated with increased oxidative stress (dementia, cardio- and cerebrovascular disease, diabetes, renal or hepatic insufficiency, and inflammatory diseases); malnutrition; previous or current treatment with hormone replacement therapy, bisphosphonates, or other antiresorptive drugs; and use of antioxidant vitamins in the 6 months before the enrollment. Based on these criteria, 197 (100 osteoporotic and 97 control subjects) were selected and asked to participate in the study. One hundred fifty women (75 osteoporotic and 75 control subjects, i.e. 76% of those selected) gave their informed consent and were finally enrolled. The project was approved by our institution ethics committee.
Study variables collected in all of these subjects included age, years since menopause, body mass index (BMI), self-reported fractures, smoking habit, nutritional status (evaluated by means of the Mini Nutritional Assessment questionnaire) (18), functional status (assessed through the Katz activities of daily living), number of drugs, and number of comorbidities (expressed as the total number of diseases in each subject). All pertinent information was collected through a questionnaire that was administered by a trained interviewer.
The BMD of the proximal femur and its subregions (total, femoral neck, intertrochanteric, greater trochanter, and Ward’s triangle) was measured in all subjects using dual energy x-ray absorptiometry (QDR 2000 densitometer, Hologic, Inc., Waltham, MA). A single, well trained operator performed all bone density measurements according to the manufacturer’s guidelines, using the array mode. The precision errors for the total femur and femoral neck in our laboratory are 1.1% and 1.3%, respectively.
Study subjects underwent a fasting blood withdrawal in 20-ml heparinized tubes on the day of the bone scan. Blood was kept on ice and centrifuged within 30 min. Plasma aliquots (500 µl) were frozen at -80 C until analysis. To preserve vitamin C, an aliquot of plasma was deproteinized with 10% metaphosphoric acid, and the supernatant was kept at -80 C. Vitamin C and uric acid were detected by HPLC with electrochemical detection according to the method of Kutnink et al. (19). Vitamin A and vitamin E were measured, after extraction with ethanol and hexane, by HPLC with UV detection at 280 nm (20). Levels of vitamins and uric acid are expressed as micromoles per liter. As the ratio between plasma vitamin E or vitamin A and total cholesterol did not alter results, data are expressed as plasma levels of these compounds.
Plasma SOD (units per milliliter) and GPx (micromolar concentrations of NADPH per minute per milliliter) activities were measured according to the methods of L’Abbé and Fisher (21) and Flohé and Gunzler (22), respectively. To measure SOD activity in erythrocytes, red blood cells were hemolyzed with cold distilled water, and extraction was performed with an ethanol/chloroform mixture (1:1). SOD activity (units per gram of hemoglobin) was measured in the supernatant according to the method reported by Winterbourn and colleagues (23). Plasma MDA levels were measured by HPLC with fluorescence detection (24).
Statistical analysis
Statistical analysis was carried out using STATISTICA version 5.1 (Stat-soft, Inc., Tulsa, OK). All data are reported as the mean ± SD or as a percentage. Demographic and clinical variables were compared by unpaired t test. Prevalences were compared by the 
2 test. Correlation analysis was performed by means of the Spearman test. Analysis of covariance was performed to compare femoral BMD as well as antioxidant and MDA levels between groups, with age and BMI as covariates. Statistical significance was defined as P < 0.05.
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Results |
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The only notable exceptions, besides BMD, which was discriminating for the attribution of the study subjects to either of the two groups, were the number of previous fractures and the BMI.
We counted 11 fractures in the osteoporotic group (4 wrist, 4 vertebral, 1 hip, 1 humerus, and 1 ankle fractures) and 5 in the control group (1 elbow, 2 wrist, and 2 ankle fractures). Confirmation of all fractures was obtained from revision of x-rays. All fractures except 1 (1 wrist fracture) in the control group were the result of significant trauma (2 motor vehicle accidents, 1 skiing trauma, and 1 fall from higher than standing height).
As far as the BMI is concerned, even if no specific dietary assessment aimed at determining single antioxidant intake was made, all subjects were on a free diet and obtained a score 23.5 or more, which is the threshold that indicates a risk for malnutrition in the elderly, according to the Mini Nutritional Assessment.
Mean plasma levels of vitamin C, vitamin E, vitamin A, and uric acid were consistently lower in osteoporotics than in controls (Table 2). Despite this, none of the subjects belonging to the two groups had levels below the normal vitamin C and vitamin E ranges. This reinforces the assumption of adequate nutritional status in both groups, at least in terms of dietary antioxidants. Also, the activities of antioxidant enzymes in plasma (SOD and GPx) and erythrocytes (only SOD) were significantly lower in osteoporotics than in controls (Table 2). Adjustment for BMI and age did not alter differences between the two groups.
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Femoral neck BMD showed a negative correlation with age in subjects with osteoporosis (r = -0.4; P < 0.01). Although all antioxidants were positively correlated with BMD, the association was stronger and statistically significant only for vitamin A, vitamin C, and plasma GPx (vitamin A: r = 0.36; P < 0.01; vitamin C and plasma GPx: r = 0.26, P < 0.05; Table 3).
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Discussion |
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Taken in aggregate, these data seem to point to a negative role for antioxidant deficit in age-related bone loss. This is in keeping with prior evidence deriving both from some epidemiological studies that suggested that higher dietary antioxidant intake has a protective role on bone health (10, 11, 12, 13) and from a single cohort study that found decreased activity of superoxide dismutase and glutathione peroxidase in postmenopausal osteoporotic women (25)
Vitamin C, a crucial cofactor in the maturation of collagen, whose triple helix stabilization depends on the hydroxylation of proline, a metabolic step requiring vitamin C (26), is the antioxidant with the most significant evidence for a possible influence on bone formation/bone loss. Data from various observational studies, albeit not consistently, seem to carve out a positive role for vitamin C in contrasting age-related bone loss of women in their early and midpostmenopausal years, especially if they were calcium repleted, but not estrogen repleted (10, 11). Furthermore, dietary intake of vitamin C and vitamin E has proven protective against hip fracture in a particular subset of female smokers selected from a large population of women followed prospectively for up to 5 yr (13).
Apart from data on vitamin C and vitamin E, evidence of other antioxidants’ protection against bone loss is scanty. In contrast, excess dietary retinol (vitamin A) intake has been recently recognized as a risk factor for hip fracture and has been associated with accelerated bone loss (27, 28, 29, 30, 31, 32) This nutrient, according to some researchers, could provide a reasonable explanation for the higher incidence of osteoporotic fractures in Sweden and Norway, where the diet is particularly rich not only in calcium and vitamin D compared with the rest of Europe, but also in vitamin A, contained in cod liver oil, dairy products, and milk, usually fortified with vitamins A and D (27, 28). Our data seem to contrast with this view, in that we found lower levels of vitamin A in osteoporotics. Moreover, vitamin A was the dietary antioxidant most strongly and positively correlated with bone mass in the osteoporotic population. Notwithstanding our results, it is tenable that very high levels of vitamin A exert a negative effect on bone, especially in light of the results of the Rancho Bernardo study (31). This prospective investigation, indeed, not only suggests that supplemental retinol users are at increased risk of femoral neck bone loss, but also attributes a potential advantage in terms of bone mass retention to subjects with high vitamin A intake not deriving from supplements. The researchers conclude by hinting at the existence of a delicate balance between ensuring adequate vitamin A intake and amplifying age-related bone loss due to excessive retinol supplementation. Any apparent discrepancy with inferences from the present investigation can be interpreted in light of the fact that subjects taking supplements were excluded from our study.
For any condition or disease in which a decrease in antioxidant defenses is demonstrated, the question has to be answered whether this is mainly due to an increased production of free radicals or to a poor dietary antioxidant intake. When free radicals are produced in excess to the capacity of the body to neutralize them, a condition of oxidative stress takes place. Oxidative stress, defined as an imbalance between antioxidants and prooxidants in favor of the former, potentially leading to damage, generally implies that antioxidants are low and markers of oxidative damage are increased (33, 34). In this study we measured plasma MDA as a marker of free radical-mediated lipid peroxidation and found no difference between groups. This finding contrasts with the results of a recent study, reporting increased levels of MDA in a limited sample of postmenopausal osteoporotic women (25). However, in the latter study the osteoporotic subjects were compared with a smaller sample of healthy younger controls, therefore precluding the possibility of discriminating the effects of aging from those of osteoporosis.
Possible alternative explanations for the absence of an MDA increase in our osteoporotic subjects compared with controls are 1) the decrease in antioxidants in osteoporotic women reflects an increased production of reactive oxygen intermediates (ROI), i.e. free radicals, to such an extent that they are unable to generate high levels of MDA; 2) the decrease in antioxidants in osteoporotic women reflects an increased production of ROI, leading to the formation of oxidized biological molecules different from MDA (e.g. F2 isoprostanes, another marker of lipid oxidation; or 8-hydroxydeoxyguanosine, a byproduct of oxidation of DNA; or protein carbonyls, byproducts of oxidation of proteins); and 3) poor antioxidant status is due to some nutritional deficit and not to an increased oxidative stress: inadequate antioxidant intake in some way amplifies age-related bone loss and/or is a signal for some other dietary deficiency.
Although the first mechanism is purely speculative and is not substantiated by any evidence, the second one is at least in part supported by the results of a recent study that found a negative relationship between urinary levels of 8 iso-prostaglandin 2
and BMD (35). MDA measurement, although performed by means of a reliable HPLC method, suffers from a number of limitations that might prevent its use as a marker of oxidative damage in osteoporotic subjects (36). The third mechanism, i.e. low dietary intake of antioxidants and metals, such as selenium, zinc, and copper, necessary for the activity of antioxidant enzymes, might also be supported by our data, because controls had higher BMI than osteoporotic subjects. Although all subjects were receiving a free diet, it is possible that the diet of osteoporotic women included a lower number of fruit and vegetable servings than that of controls and hence did not provide an adequate amount of antioxidants.
Whatever the cause of the low antioxidant levels, the results of this and previous studies suggest that antioxidant deficiency has a negative impact on bone mass. Several potential mechanisms might underlie this relationship. For instance, nuclear factor-
B, which is known to mediate some of the important actions of TNF, a cytokine synthesized in the bone microenvironment, on osteoclastogenesis, is activated in osteoblast-like cells by mitogens and cytokines through the generation of ROI (37, 38, 39, 40). In other words, intracellular free radical production might represent the final common mechanism of nuclear factor-B activation by a variety of factors (41). If this mechanism of action of osteoclastogenic cytokines satisfactorily fits most models of bone loss, it is conceivable that low intracellular and probably interstitial levels of antioxidants are a signal, i.e. a consequence, of increased osteoclastogenic activity and bone turnover. Alternatively, it is also tenable that low levels of intracellular antioxidants may amplify osteoclastogenesis through uncontrolled availability of excess ROI.
Not only might osteoclastic differentiation of bone precursors be modulated by ROI, but osteoblastic differentiation as well. Mody et al. (42) have shown that oxidative stress is able to inhibit bone cell differentiation of a preosteoblastic cell line (MC3T3-E1) and of a marrow stromal cell line that undergoes osteoblastic differentiation.
Quite recently, other evidence linking osteoporosis to increased oxidative stress has been produced, particularly for a severe osteoporotic syndrome in relatively young males (34). A small 3.7-kb deletion in mitochondrial DNA has been associated, through the consequent inefficiency in oxidative phosphorylation, defective electron transport chain, and increased oxygen free radical production, to increased oxidative stress (43). The presence of the latter, clearly signaled by hyperlactemia and elevated lactate/pyruvate ratio, could have brought about, with the mediation of chronic mild to moderate acidosis, a detrimental effect on bone metabolism.
Some important limitations of our study should be acknowledged. First, we did not measure dietary calcium. Different intakes in the two groups could have at least in part influenced BMD results, even because some of the positive effects of vitamin C on bone might not be realized in the absence of adequate calcium intake. Secondly, we did not directly measure dietary whole caloric or single antioxidant intake. Although this potential bias was mitigated by the fact that all subjects were well nourished, there is a possibility that minor or selective nutritional deficits might be more represented in the osteoporotic group. Thirdly, the results of our study may not apply to the general elderly population, because the frailest and oldest elders were not included. Finally, our study is limited by the fact that we did not verify any of the relationships between low levels of antioxidants and reduced BMD.
We conclude by suggesting that elderly osteoporotic women have lower antioxidant defenses compared with a normal age-matched reference population. The mechanisms underlying antioxidant depletion and its relevance to the pathogenesis of osteoporosis deserve further investigation.
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Footnotes |
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Abbreviations: BMD, Bone mineral density; BMI, body mass index; GPx, glutathione peroxidase; MDA, malondialdehyde; ROI, reactive oxygen intermediate; SOD, superoxide dismutase.
Received September 24, 2002.
Accepted January 7, 2003.
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B1 and NF-B2. Nat Med 3:1285–1289[CrossRef][Medline]
B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review). Free Radical Res Commun 17:221–237[Medline]
increases expression of IL-6 and ICAM-1 genes through activation of NF-B in osteoblast-like ROS17/2.8 cells. J Bone Miner Res 13:1290–1299[CrossRef][Medline]
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