1 Thurston Arthritis Research Center, Schools of Medicine and Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC.
2 Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA.
3 School of Medicine, East Carolina University, Greenville, NC.
4 Craft Technologies, Inc., Wilson, NC.
5 Centers for Disease Control and Prevention, Atlanta, GA.
6 School of Medicine, University of Maryland, Baltimore, MD.
Received for publication March 17, 2003; accepted for publication December 4, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
alpha-tocopherol; antioxidants; gamma-tocopherol; osteoarthritis, knee; tocopherols; vitamin E
Abbreviations: Abbreviation: HPLC, high performance liquid chromatography.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dietary factors are receiving renewed attention in this regard in humans and in animal models of osteoarthritis (917). More than two decades ago, Schwartz and others (1821) observed that high doses of vitamin C could limit the progression of surgically induced osteoarthritis in guinea pigs, and Black et al. (22) demonstrated that incubation of rabbit articular cartilage with -tocopherol preserved cartilage load-carrying capacity and viability. More recently, Kurz et al. (17) reported that a diet containing vitamins E, C, A, B6, and B2 and selenium increased the expression of antioxidative enzymes and decreased the incidence of osteoarthritis in STR/1N mice.
Micronutrients might mediate the osteoarthritis process by blocking oxidative damage. Nitric oxide and reactive oxygen species inhibit collagen and proteoglycan synthesis, activate matrix metalloproteinases, increase the susceptibility of cartilage to injury by other oxidants, and induce apoptosis (2326). Tocopherols are the most potent lipid-soluble antioxidants in blood, breaking free-radical chain reactions of lipid peroxidation (27). The antioxidative roles and serum levels of the different tocopherol isoforms are highly interdependent and may be complementary in function (28, 29). There is some evidence suggesting that isoform concentrations relative to each other may be important in preventing specific types of oxidative damage (3033), with -tocopherol possibly being more important than
-tocopherol in removing nitrogen oxides and other electrophilic mutagens (2830).
Tocopherols may also mediate the osteoarthritis process by interfering with inflammation, which is increasingly recognized as important in osteoarthritis (23, 24). Here, too, recent evidence has suggested that -tocopherol and its metabolite may be more potent than
-tocopherol in inhibiting cyclooxygenase 2 in lipopolysaccharide-stimulated macrophages and in interleukin-1ß-treated human epithelial cells (30). In this study,
- and
-tocopherols demonstrated similar inhibition of inducible nitric oxide synthase and total reactive oxygen intermediates, and importantly, the antiinflammatory effects were independent of the antioxidant effects (30).
Epidemiologic studies examining the role of antioxidants, specifically tocopherols, in human osteoarthritis are few (10, 14). In the Framingham Osteoarthritis Study, McAlindon et al. (10) reported that intakes of vitamin C, ß-carotene, and possibly vitamin E were associated with a reduced risk of progression of radiographic knee osteoarthritis but were not protective against incident disease. Several methodologically limited clinical trials have suggested that vitamin E supplementation might be superior to placebo and equal in effectiveness to antiinflammatory medication in relieving osteoarthritis symptoms, but other studies have failed to show an effect (13, 15, 16, 34, 35).
To our knowledge, no studies have examined biomarkers of individual tocopherols in relation to radiographic knee osteoarthritis. Measurement of tocopherols in plasma or serum can provide estimates of dietary tocopherol intake, but a plasma or serum micronutrient measure may more accurately reflect a biologically active internal dose, avoiding the difficulties inherent in the measurement of dietary intake and differences in absorption and metabolism (36, 37). The ratio of serum -tocopherol to
-tocopherol, which represents the amount of circulating
-tocopherol and the degree to which its binding protein is saturated (31), has been proposed as a better reflector of vitamin E intake and tocopherol status in the body, partly because the ratio can be elevated by modest levels of supplementation that do not raise plasma
-tocopherol levels (38, 39) and because it obviates the need for lipid correction (31, 38).
No study of vitamin E and osteoarthritis has included African Americans, despite reports that African Americans have lower dietary intakes and/or biologic levels of to-copherols than do Whites (31, 4044). The Framingham Studys observation concerning vitamin E and progression of radiographic knee osteoarthritis was restricted to men; no further studies of possible sex differences in the effects of tocopherols on osteoarthritis have been conducted. We examined cross-sectional associations between radiographic knee osteoarthritis and serum biomarkers of -,
-, and
-tocopherol and the
:
-tocopherol ratio in the Johnston County Osteoarthritis Project, a community-based, biethnic study of osteoarthritis carried out in North Carolina. We hypothesized that these tocopherols would be associated with lower odds of radiographic knee osteoarthritis. Our second goal was to explore potential ethnic and sex differences in the relations between radiographic knee osteoarthritis and these tocopherol measures.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sample selection
Radiographic knee osteoarthritis was defined from weight-bearing bilateral anteroposterior radiographs of the knee, according to the Kellgren-Lawrence grading scheme: 0 = no osteoarthritis; 1 = questionable osteoarthritis; 2 = mild osteoarthritis; 3 = moderate osteoarthritis; and 4 = severe osteoarthritis (46). One hundred cases from each ethnic group were randomly selected from those participants having Kellgren-Lawrence grade 2 or higher osteoarthritis in either knee. Two hundred controls were randomly selected from those participants with Kellgren-Lawrence grade 0 in both knees, matched to cases by age (±1 year), sex, and ethnicity.
Measures
Questionnaires
Each participant completed two interviewer-administered home questionnaires. Relevant variables for this study included demographic factors, educational attainment, and current smoking and alcohol consumption status.
Radiographs
Knee radiographs were read for Kellgren-Lawrence grade by a single bone and joint radiologist (J. B. R.) without knowledge of participant clinical status, using the standard atlas (46). Interrater reliability (assessed by another trained radiologist) and intrarater reliability were high (interrater reliability: weighted = 0.859; intrarater reliability: weighted
= 0.886), as described previously (45).
Clinical procedures
Height was measured in centimeters and weight in kilograms. Five milliliters of nonfasting blood was collected by venipuncture. The samples were immediately spun, and 1-ml aliquots of serum were withdrawn into opaque cryotubes without preservatives for serum total cholesterol determination by modified Trindar reaction (47) and frozen storage at 86°C.
Nutrient analyses
Assessment of serum concentrations of -,
-, and
- tocopherol levels by high performance liquid chromatography (HPLC) was performed at Craft Technologies, Inc. in Wilson, North Carolina. For serum extraction and HPLC methods, a modification of the procedures described by Nomura et al. (48) was used. Briefly, after thawing, 150-µl aliquots of serum were diluted with 150 µl of water and deproteinated by vortexing with 300 ml of ethanol containing tocol as an internal standard and butylated hydroxytoluene as an antioxidant. The samples were extracted twice with 1 ml of hexane; the combined supernatant was evaporated under nitrogen. The residue was dissolved with vortexing in 35 µl of ethyl acetate, diluted with 100 µl of mobile phase, and ultrasonically agitated for 15 seconds prior to placement in the autosampler. A 15-µl volume was injected.
The HPLC system consisted of a computer data system, a solvent degasser, a Spherisorb ODS2 column (Keystone Scientific, Bellefonte, Pennsylvania) (3 µm, 4.6 x 150 mm with titanium frits), a guard column containing the same stationary phase, an autosampler maintaining samples at 20°C and the HPLC column at 29°C, and a programmable fluorescence detector for measurement of tocol and tocopherols at 296 nm excitation/340 nm emission. The separation was performed isocratically using a mobile phase of 83 percent acetonitrile:13 percent dioxane:4 percent methanol containing 150 mM ammonium acetate:0.1 percent triethylamine at a flow rate of 1.5 ml/minute.
Quality control
In-house quality control samples were analyzed at the beginning, at the end, and at 24-sample intervals. Linear calibration curves consisted of multiple concentrations of analytes spanning the physiologic levels of serum tocopherols. Serum quantitation was performed by internal standard calibration using peak area ratios. The relative standard deviation of analytes in quality control samples ranged from 3 percent to 10 percent.
The repeatability of the laboratory protocol for each to-copherol was evaluated by examining scatterplots of assay results for 40 "phantom" samples plotted against their 40 duplicates and calculating Pearson correlation coefficients. These coefficients ranged from 0.91 to 0.95.
Statistical analyses
Samples with nondetectable levels of any tocopherol were assigned a value of one half of the detection limit for that tocopherol. Geometric mean values for serum levels of -,
-, and
-tocopherol and the
:
-tocopherol ratio were compared across ethnic and sex subgroups with a t statistic. P values of 0.05 or lower were considered indicative of significant differences between subgroups. Geometric means were then similarly compared across case and control groups, for the overall sample and for ethnic and sex subgroups.
Odds ratios and 95 percent confidence intervals for each osteoarthritis-tocopherol association were estimated by multivariable conditional logistic regression using tertiles of tocopherol values, with the lowest tertile being used as the referent. We tested for trend by including one term for the tocopherol in which the median value of controls in each tertile was assigned to participants in that tertile; p values of 0.10 or lower indicated a trend.
Potential confounders of a priori interest included education (high school graduation or not), current smoking (yes or no), current alcohol consumption (yes or no), body mass index (weight (kg)/height (m)2, coded as a continuous variable), and serum total cholesterol level (coded as a quadratic spline term (49)). Confounding of each osteoarthritis-tocopherol relation by each covariate was defined by a 5 percent or greater change in odds ratios for the tocopherol effect when that covariate was removed from the model.
We previously observed associations between serum carotenoid levels and radiographic knee osteoarthritis (14). Accordingly, we checked each osteoarthritis-tocopherol association for potential confounding by individual tertiles of lutein, zeaxanthin, - and ß-cryptoxanthin, trans- and cis-lycopene,
-carotene, trans- and cis-ß-carotene, and the other tocopherols. Of these, the tocopherols,
-carotene, ß-carotene, cis-ß-carotene, and lutein changed at least one tocopherol estimate by more than 5 percent and defined our "set" of potential nutrient confounders.
Effect modification by ethnicity and sex was assessed in stratified analyses and in overall models that included interaction terms, with p values of 0.10 or lower indicating significant group-specific differences in osteoarthritis-tocopherol effects.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The major sources of tocopherols in food are fats and oils (33, 38, 54, 55). Average servings of most foods contain 1 percent or less of the Recommended Dietary Allowance of 15 mg/day of -tocopherol, which suggests that many people may have a diet that is suboptimal in levels of this nutrient (56, 57). Higher intake can be achieved through the use of vitamin E supplements, which contain mainly natural and synthetic forms of
-tocopherol (31, 38, 54, 55, 58). Although
-tocopherol is the principal tocopherol in the typical US diet, the preferential reincorporation of
-tocopherol into low density lipoproteins results in higher concentrations of
-tocopherol in plasma and most human tissues (27, 33, 58). Supplementation with
-tocopherol leads to saturated plasma levels of
-tocopherol and depression of plasma levels of
-tocopherol (31, 33, 38, 54, 55, 58), suggesting that intestinal uptake and plasma transport make more efficient use of
-tocopherol than the other isoforms. In addition, the tocopherol transfer protein prefers
-tocopherol to the other vitamin E forms and uses all
-tocopherol for antioxidative activity before using the other isoforms (31, 57). Even though in vitro studies have shown that
-tocopherol may be a more potent antioxidant and antiinflammatory agent than
-tocopherol in some situations (2830, 59), it may not be the more important isoform in vivo as long as adequate
-tocopherol is available (50). Since we observed a direct rather than an inverse relation between
- and
-tocopherol levels and radiographic knee osteoarthritis, one might conjecture that persons with high
-tocopherol levels (and perhaps high
-tocopherol levels as well) and a high level of oxidative stress in our study may have exhausted their
-tocopherol in an unsuccessful attempt to curtail the osteoarthritis process.
Observational studies have suggested that vitamin E supplementation may be protective against several chronic diseases whose etiologies involve oxidative stress, such as cancer, dementia, and heart disease (33, 6062), but randomized clinical trials have often failed to replicate these results (63, 64). Recent clinical and in-vitro studies have suggested that -tocopherol may be more important than
-tocopherol in protection from cardiovascular disease and other diseases (32, 53, 59), and this may be the reason why studies of
-tocopherol supplementation (as opposed to dietary food sources of vitamin E richer in
-tocopherol) may have had disparate results (6569). Our results might appear to be contrary to the findings of these studies. However, not all clinical studies of
- and
-tocopherols and their ratio have found high
-tocopherol levels to be beneficial (31, 33, 52, 70, 71). Ingles et al. (31) found that a high
:
-tocopherol ratio was associated with lower odds of large colonic adenomas, and other investigators have reported either no association or a direct association between
-tocopherol and colon cancer (33), invasive cervical cancer (71), lung cancer (70), and coronary heart disease death or nonfatal myocardial infarction (52).
Do the few studies of vitamin E and osteoarthritis in the literature help us to explain our findings? In the Framingham Osteoarthritis Study, dietary vitamin E intake was associated with lower odds of radiographic knee osteoarthritis progression in men only (10). Some of our observations were also most notable in, or restricted to, men, including an inverse association between radiographic knee osteoarthritis and serum -tocopherol and a positive association between
-tocopherol and this outcome. (The latter result should be interpreted cautiously because of the extremely wide confidence interval.) Although our study cannot be compared directly with the Framingham Study because of differences in design, study population, assessment of tocopherol status, and analysis, both studies suggest possible sex differences in the relations between vitamin E and radiographic knee osteoarthritis. Whether these sex differences are the result of hormonal differences associated with osteoarthritis metabolic processes or sex differences in tocopherol metabolism or exposure to other potentially important factors, such as use of lipid-modifying medications, deserves further study.
All clinical trials of vitamin E supplementation in osteoarthritis have utilized -tocopherol, and results have been mixed at best (13, 15, 16, 34, 35). Those trials showing a salutary effect of vitamin E on osteoarthritis symptoms were typically small and/or short-term and had various methodological problems (13, 34). In the first placebo-controlled structure modification trial of vitamin E in knee osteoarthritis, Wluka et al. (15) and Brand et al. (16) reported no effect of vitamin E supplementation on symptoms at 6 months or 2 years or on cartilage volume, measured by magnetic resonance imaging, at 2 years. Interestingly, women were slightly more likely to be randomized to vitamin E therapy in this trial (15). Whether men might be more likely to respond to vitamin E therapy in osteoarthritis is not known. If such a sex difference in the effectiveness of vitamin E in knee osteoarthritis exists, this might have contributed to the null result.
Furthermore, all trials of vitamin E in osteoarthritis have been done in Caucasian study populations. In our study, we observed ethnic differences in the associations between radiographic knee osteoarthritis and -tocopherol and the
:
-tocopherol ratio, with strong inverse associations being noted in African Americans. As was previously reported by others (31, 4044), African Americans in our study had lower geometric mean levels of
-tocopherol and
-tocopherol and
:
-tocopherol ratio. In addition, African Americans may take supplements less frequently than Whites (31, 41, 72). Whether African Americans with osteoarthritis may respond more favorably than Whites to supplements or other interventions aimed at improving tocopherol status has not been examined.
There are several other possible explanations as to why our hypothesized inverse associations between serum tocopherols and radiographic knee osteoarthritis were not observed (14). First, measurement of tocopherol status was done contemporaneously with the assessment of osteoarthritis status rather than before osteoarthritis onset, making it impossible to determine temporality. Dietary intake may change as a result of pain and osteoarthritis-associated disability. For example, persons taking medications for osteoarthritis may be more likely to take vitamin supplements than those who do not take such medications (72). However, it is unlikely that those with increased levels of -tocopherol and osteoarthritis in our study were taking vitamin E supplements, because supplementation increases serum
-tocopherol level and decreases serum
-tocopherol level (31, 38, 54, 55, 58).
Second, we used a single serum measure of tocopherol status. However, diurnal and day-to-day variations in -tocopherol levels are generally low (73). Although data are somewhat conflicting (74), several studies have shown no significant seasonal variation in
-tocopherol levels (7577). One study showed small differences in serum
- and
-tocopherol levels measured 15 years apart, and rank-order correlations were consistent and high (75). For these reasons, we do not think that our choice of tocopherol assessment had a serious adverse impact on our ability to observe associations between tocopherols and radiographic knee osteoarthritis. Indeed, most other studies of serum biomarkers of tocopherol status have used single measures (31, 33, 52, 70, 71).
However, we do not know how well serum tocopherol measurements reflect tocopherol levels in the joint. In a study of inflammatory joint disease, -tocopherol levels in knee synovial fluid were significantly lower than in the same patients serum, although mean serum
-tocopherol levels in patients did not differ significantly from those in controls (78). This suggests that
-tocopherol may be locally consumed at the site of oxidative and/or inflammatory activity. We did not have access to synovial fluid in our study, so we cannot determine whether and in what direction the tocopherol levels in the joint may have varied from those in the sera. It is intriguing to speculate that larger differences in synovial fluid tocopherol levels might have existed between cases and controls than were observed in serum. In future studies, investigators should also consider the most representative measure of antioxidant burden and total antioxidant capacity specific to the site of action.
It is not known whether tocopherol effects vary by radiographic severity. The fact that vitamin E intake protected against knee osteoarthritis progression but not incidence in the Framingham Study (10) suggests that this might be so. Our study had 85 percent power to detect an odds ratio of 0.4 using tertiles and 200 pairs. Consequently, we were not able to examine differences between grade 2 or mild radiographic knee osteoarthritis (137 cases) and grades 3 and 4 or moderate/severe radiographic knee osteoarthritis (63 cases). In addition, persons with grade 1 or "questionable" osteoarthritis (26 percent of participants in the Johnston County Osteoarthritis Project, from which this sample was selected) were excluded from the study. This precludes our evaluation of the possible transition to definite radiographic knee osteoarthritis. In future longitudinal studies, investigators should evaluate whether tocopherols act differently at different stages of the osteoarthritis process.
Finally, we also cannot exclude possible confounding by vitamin C, which was protective against progression of knee osteoarthritis in the Framingham cohort (10), or by selenium. Vitamin C, a potent antioxidant in vivo, may additionally protect against osteoarthritis by stimulating increased collagen and proteoglycan synthesis (19, 79, 80). Although food sources of vitamin C are quite different from those of tocopherols, intake of vitamin C has been positively associated with -tocopherol levels in blood and inversely associated with
-tocopherol levels (81, 82). Furthermore, synergism between antioxidant systems operating in the lipid and aqueous phase of the cell may be important, because after
-tocopherol acts to break the lipid-peroxidation free-radical chain reaction, the newly formed
-tocopheroxyl can react with vitamin C to regenerate
-tocopherol (27).
The results of this study are important. As the number of people affected by osteoarthritis increases and the impact of osteoarthritis-associated disability grows (3, 4, 6), identification of potentially modifiable risk factors and preventive factors will become increasingly relevant. Preventive measures might be aimed at persons at high risk of osteoarthritis incidence and progression, such as the obese and persons with prior joint injury or a positive family history (14). Dietary intervention may be particularly relevant among those with inadequate nutrition, such as older persons and African Americans (31, 4044, 83, 84). The results of this study, data from animal models, and preliminary human epidemiologic studies (10, 14, 1719, 21, 80) suggest that further study of the effects of tocopherols on the incidence and progression of osteoarthritis, particularly in ethnic and sex subgroups, is warranted.
![]() |
ACKNOWLEDGMENTS |
---|
The authors acknowledge Janice Woodard, Wilma Howard, Kyle McDermott, and Drs. Edwin Hartman and Kim Shaftner for their devotion to implementing the protocol of the Johnston County Osteoarthritis Project in the field.
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|