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The relation of plasma homocysteine to radiographic knee osteoarthritis
Osteoarthritis and Cartilage (2009) 17, 766e771 ª 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.joca.2008.11.015
International Cartilage Repair Society
The relation of plasma homocysteine to radiographic knee osteoarthritis1 M. Fayfmany, J. Niuy, Y. Q. Zhangy, D. T. Felsony, B. Sacky, P. Aliabadiz, J. Selhubx and D. J. Hunteryk* y Boston University School of Medicine, Boston, USA z Department of Radiology, Brigham and Women’s Hospital, Boston, USA x Jean Mayer USDA HNRCA at Tufts University, Boston, USA k New England Baptist Hospital, Boston, USA Summary Objective: Homocysteine has been implicated in multiple diseases that involve changes in structural tissue. In vitro studies have found that it alters the structure of collagen cross-linking thus affecting stability and mineralization such as that occurring in bone tissue. In the present study we considered the possible relationship between plasma homocysteine levels and the development and progression of knee osteoarthritis (OA). Methods: The study question was posed in 691 men and 966 women from the original and offspring cohorts of the Framingham Osteoarthritis Study. We divided individuals into three groups according to plasma homocysteine levels and compared their risk for the development of new and progression of existing OA. We adjusted for potential confounders including age, body mass index, weight change, and physical activity. Results: In the crude analysis, men in the middle homocysteine tertile were found to be at a greater risk than men in the lowest tertile for incident OA [odds ratios of 1.9 (1.1e3.5)]. This result persisted after adjusting for covariates [odds: 2.0, (1.1e3.8)]. No significant correlation was seen in women for the development of OA. In the evaluation of progression no significant trends were seen for both men and women. Conclusions: Although cellular and molecular studies of homocysteine-related pathophysiology suggest a possible correlation between plasma homocysteine levels and OA, the present clinical study did not conclusively demonstrate such an association. However, further research is needed to explore the role of homocysteine in specific aspects of OA etiopathogenesis. ª 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Homocysteine, Knee OA, Incidence, Progression.
High plasma homocysteine is a risk factor for a number of chronic illnesses including cardiovascular disease, osteoporosis, and impaired cognitive function as well as for pregnancy complications5,7e9. The role of homocysteine in disease can be at least partially explained by its cellular effects on various types of connective tissue10e12. High plasma homocysteine is seen in cardiovascular disease where it leads to increased production of collagen by the smooth muscle cells10. Homocysteine is also involved in pregnancy complications and adverse outcomes including placental abruption, low birth weight, neural tube defects, and spontaneous abortion9,13,14. Homocysteine has recently been seen to play a role in osteoporosis related bone damage. An animal model study found that rats with elevated serum homocysteine have reduced bone quality15. Population studies in Framingham found homocysteine to be a predictor of hip fracture in older individuals5. The effects of homocysteine may be linked to its involvement in collagen formation. Homocysteine inhibits the synthesis of insoluble collagen fibrils in vitro by interfering with normal cross-linking16. From the perspective of cartilage homeostasis, these changes in matrix organization interfere with chondrocyte-mediated mineralization potentially altering the function and properties of calcified cartilage17. This may be due to homocysteine mediated inhibition of lysil oxidase, which catalyzes the cross-linking of collagen molecules, a function necessary for its mineralization in bone tissue17,18. Since osteophytes, which are marginal
Introduction Homocysteine is a sulfur-containing amino acid involved primarily in the metabolic pathway of methionine as well as that of cysteine and adenosine1. Its levels in the blood are controlled by multiple enzymes, whose activity varies due to genetic or environmental factors. Its role in multiple pathologic processes is evident from studies of individuals with homocysteinurea who have extremely elevated levels of plasma homocysteine due either to genetic mutation in key regulatory enzymes or underlying conditions such as kidney disease2. The normal range of plasma homocysteine levels for healthy adults is 5e15 mmol/liter3. However, even moderately increased levels within the normal range have been found to be involved in multiple disease states4e6. 1
Supported by NIH AR43873 and AR47785 and NIH AG18393 from the Framingham Heart Study of the National Heart, Lung, and Blood Institute of the National Institutes of Health and Boston University School of Medicine. This work was supported by the National Heart, Lung, and Blood Institute’s Framingham Heart Study (Contract No. N01-HC-25195). *Address correspondence and reprint requests to: D. J. Hunter, New England Baptist Hospital, Division of Research, 125 Parker Hill Av., Boston, MA 02120, USA. Tel: 1-617-754-6655; Fax: 1-617754-6888; E-mail: [email protected] Received 11 January 2008; revision accepted 24 November 2008.
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Osteoarthritis and Cartilage Vol. 17, No. 6 osteocartilaginous outgrowths, emanate from osteoarthritic joint tissue, homocysteine mediated inhibition of mineralization may actually have a protective effect. Homocysteine may also exert effects on bone by means other than collagen reorganization. The presence of high homocysteine leads to decreased secretion of osteocalcin and increased osteopontin by osteoblasts19. It also stimulates osteoclast differentiation enhancing bone resorption15,20. Given the importance of homocysteine in bone and collagen metabolism, and chondrocyte function we proposed to explore the relation of homocysteine to osteoarthritis (OA). OA is a disorder of multiple joint tissues characterized by different stages of progression. Collagen is a large component of articular cartilage, which forms the lining of the joint, and its destruction contributes to OA. Cross-linking bands of collagen molecules are necessary to form the stable fibrils that compose the cartilage found on articular joint surfaces and homocysteine has been seen to disrupt the intermolecular bonds of these fibrils11. Changes in the collagen networks of joint fibers observed early in the development of OA are a potential mechanism by which homocysteine is involved in this disease. Homocysteine, its metabolic precursors and products have been shown to alter various components of connective tissue, however, no studies have directly looked at the relation between homocysteine levels and knee OA in humans. In the present study, we used population-based data from the Framingham OA Study to look at the predictive value of homocysteine on both the development and progression of knee OA and it association with prevalent knee OA.
Methods STUDY POPULATION (FRAMINGHAM OA STUDY) The Framingham Heart Study is a longitudinal population-based cohort study established in 1948 in Framingham, Massachusetts to examine risk factors for heart disease21. A study of the offspring of the original cohort and their spouses was initiated in 1971. The details of this cohort have been previously described22. The Framingham OA study, which includes participants of the original and offspring cohorts, was developed to study the inheritance of OA23. The present study included 691 men and 966 women from the two cohorts. For the original cohort, baseline and follow-up visits and radiographs were obtained at examinations 18 (1983e1985) and 22 (1992e1993), respectively3. Subjects from the offspring cohort were examined between 1992e1994 and 2002e200524. Eligibility criteria for both cohorts in the present analysis included: men and women aged 40e85 years; ambulatory (use of assistive devices such as canes and walkers was allowed). Exclusion criteria were: the presence of bilateral total knee replacements, and the presence of rheumatoid arthritis. The Framingham OA Study protocol involved multiple components, one of which was a radiographic exam including posteroanterior (PA) fixed flexion25 and lateral radiographs26 of both knees.
PLASMA HOMOCYSTEINE Nonfasting blood samples for the original cohort were collected at the 16th biennial examination between 1979 and 1982 and stored at or below 20 C. These samples were thawed in 1997 and plasma homocysteine was measured using high-performance liquid chromatography5. For the offspring cohort, fasting (>10 h) blood samples were collected on the fifth examination, from 1993e1994 and were frozen immediately and stored at or below 70 C27. In 1995, the samples were thawed and the total plasma homocysteine concentrations were measured by high-performance liquid chromatography with fluorometric detection28. The coefficient of variation for this assay was nine percent29.
KNEE RADIOGRAPHY During the baseline visits, a weight-bearing fully extended radiograph of both knees, using a standardized protocol that included outlines of the feet
to keep constant the rotation of the knee at follow-up was obtained. Films were obtained at 0 and at 6 caudad, and the best view (based on the optimal superimposition of the anterior and posterior margins of the medial tibial plateau) of these two was selected for comparison at the follow-up examination. After the subjects completed their follow-up examination, both baseline and follow-up radiographs were read independently by two study readers, one a bone and joint radiologist (PA), and the other a rheumatologist (BS). X-rays were read in a paired fashion, unblinded to sequence. Readers evaluated the Kellgren and Lawrence (K&L) grade30 of each knee at both time points and evaluated individual features and whether these had changed over the follow-up period. Each knee was evaluated for the presence of osteophytes and joint space narrowing (JSN) on a 0e3 scale using the OARSI atlas31. If there was a difference in K&L grade that led to a difference in the assignation of either prevalent or incident OA status the films were adjudicated by a panel of three readers including the first two and a third reader (DTF). All adjudicated readings were arrived at by consensus of three readers using methods previously described32. For the bone and joint radiologist, the intra-reader kappa for K&L grade was 0.82. Inter-reader kappa was 0.74 (both P < 0.001). Knee OA was defined using the K&L grade where a score of two or higher indicated the presence of OA. Two outcomes were considered for the present study. Incident knee OA was defined when a knee had a grade of less than two during the baseline visit and had a K&L grade 2 at the follow-up visit. Progression of knee OA was defined when a given knee had a K&L grade of 2 or above at baseline and at least one grade increase at the follow-up examination32. Because they were not eligible for progression, knees with K&L grade 4 at baseline were excluded from the study.
CONFOUNDERS Information about potential confounding variables was available from the Framingham OA Study database. Weight was measured in kilograms without shoes using a standard balance beam scale. Weight change was defined as the difference in weight between baseline and follow-up examination. Height was measured in centimeters, without shoes, to the nearest quarter inch. Levels of habitual physical activity were estimated based on the number of hours spent in a typical day at different levels of activity [Physical Activity Scale for the Elderly (PASE)]33. Information about knee injury was derived from the response to a standard question posed at baseline, ‘‘Have you ever had a fracture or injury to a knee requiring the use of crutches or a cane?’’ Information was obtained at the baseline for all confounders except ‘‘weight change’’, which involved measures at baseline and follow-up visits.
STATISTICAL ANALYSIS We combined data from Framingham original cohort and Framingham offspring cohort and created sex-specific tertiles of homocysteine. A total of 479 of the subjects available were removed from the final analysis. 10 subjects were excluded due to bilateral knee replacement, 10 because they had rheumatoid arthritis, 398 did not have homocysteine levels measured, and 61 were under 40 years old. The final study group included 638 individuals from the original and 1019 from the offspring group. Subjects were categorized by tertiles of homocysteine levels to account for some of the individual variability. We examined the relation of tertile groups of homocysteine (using the lower tertile as the referent category) to the risk of incident radiographic knee OA using the logistic regression model. In the multivariable regression model, we adjusted for continuous age, dichotomous gender and knee injury history, body mass index, weight change, and physical activity index. These potential confounding variables were matched between the three tertiles in an all variables in a concomitant fashion. We used generalized estimating equations to account for correlation between two knees. Results are presented as odds ratios (OR) with 95% confidence intervals of upper and lower intervals of statistical significance. We adopted the same approach to assess the relation of levels of homocysteine to the risk of progressive radiographic knee OA progression.
Results The baseline characteristics of the study participants are shown in Table I. The mean age among men and women was 60.1 and 61.2 years, respectively. Among the men, the mean homocysteine levels were 12 3.9 for the original cohort and 10.6 for the offspring cohort. Mean homocysteine levels for the women of the original and offspring cohorts were 10.9 3.4 and 9.0 3.1, respectively. The
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M. Fayfman et al.: Homocysteine and knee OA Table I Characteristics of study sample (n ¼ 1546 persons) Men (n ¼ 691)
Women (n ¼ 966)
Age in years Mean (SD) {Range}
60.1 (10.5) {38}
61.2 (10.7) {38}
BMI kg/m2 Mean (SD) {Range) Prevalence of Knee OA (%)*
27.3 (4.1) {18.0, 43.3} 15.4
25.9 (5.1) {14.3, 53.6} 17.3
Homocysteine mmol/liter Mean (SD) {Range}
11.1 (3.4) {4.6, 30.8}
9.8 (3.3) {3.5, 29.0}
*Prevalence of OA measured in knees with OA over total number of knees.
combined mean homocysteine levels among the men were 11.1 mmol/liter and among the women were 9.8 mmol/liter (Table I). Distribution of the exposure variable (homocysteine) was different between men and women so the analyses were performed in a gender specific manner. The incidence of knee OA in men for three homocysteine tertiles is shown in Table II. Before adjusting for covariates (age, sex, body mass index, weight change, physical activity index and knee injury), we found that men in the middle homocysteine tertile had an increased risk of developing knee OA over the low homocysteine tertile [OR for middle tertile, 1.9 (1.1, 3.5)]. After adjusting for the above listed covariates, men in the middle tertile, as in the crude analysis, showed an increased risk of knee OA [OR for middle tertile, 2.0 (1.1, 3.8)]. The risk for incident knee OA in women of three homocysteine levels is shown in Table III. In the crude analysis, the group in the middle and high homocysteine tertile had no increased risk of knee OA over the low tertile group. No significant associations were seen in the adjusted analysis as well. We analyzed the risk of progression of knee OA in the study subjects. Table IV shows the risk of knee OA progression in men. The OR for men in the middle and high homocysteine tertiles in the crude analysis were 0.6 (0.2,1.4) and 0.7 (0.3,1.6), respectively. The results were similar for the adjusted analysis. Table V shows the progression of OA data for women. In the crude analysis, the OR for the middle and high tertiles was 1.5 (0.7, 3.4) and 1.7 (0.8, 3.8), respectively. These findings were similar for the adjusted analysis. In order to assess the effect of combining the original and offspring cohorts, we did a stratified analysis of the two groups and found that the results differed significantly from the combined study for the incidence of OA among men. The offspring cohort shows a significantly increased
risk of OA for the high tertile [Crude OR: 2.6 (1.2, 5.8); Adjusted OR: 2.6(1.0, 6.4)] while both the original and combined analysis of this group show no significant association. The other groups were consistent between the stratified and combined analyses. A t test for mean homocysteine levels showed a statistically significant difference between the two cohorts, with slightly increased homocysteine in the original cohort 11.3 3.6 mmol/liter compared with the offspring 9.7 3.2 mmol/liter (P < 0.001). To further evaluate the possible threshold effect of homocysteine on OA, we did a separated analysis comparing the low tertile to the combined middle and high tertiles and found that the results are consistent with those presented in the paper. We also looked at prevalent OA and homocysteine levels finding no significant correlations.
Discussion In the present study, we found an association between moderately elevated homocysteine levels and increased risk of developing OA in men. Men whose plasma homocysteine levels ranged between 9 mmol/liter and 11.3 mmol/liter had a significantly increased risk for incident OA compared to those with homocysteine levels between 4.60 and 8.97 mmol/liter. The men in the highest tertile also appeared to have an increased risk but these findings were not statistically significant within 95% confidence interval. For the offspring cohort only, there did appear to be a statistically increased incidence of OA in both the middle and high homocysteine tertiles. Given the sample size we have and risk of disease in the low homocysteine group we observed, we have 80% power to detect odds ratio as small as 1.6e1.9 for incident knee OA. In the analysis for the women, we did not see a significant association between levels of homocysteine and incidence
Table II Risk for incident knee OA according to homocysteine tertile in men (n ¼ 1166 knees) Homocysteine (range mmol/liter) Total n Incident OA Crude OR (95% CI) Adjusted OR (95% CI)*
Low tertile
Middle tertile
High tertile
P value for trend
4.60e8.97 398 25 1.0 (1.0) 1.0 (1.0)
9.00e11.30 394 41 1.9 (1.1e3.5) 2.0 (1.1e3.8)
11.32e30.79 374 37 1.7 (0.9e3.3) 1.7 (0.8e3.3)
0.168
*In the Adjusted OR, potential confounding variable are matched between the three tertiles and calculated in a multivariable regression model. Potential confounders include age measured as a continuous variable, body mass index, weight change between baseline and follow-up visits, physical activity index (PASE)32, and history of knee injury based on response to the question posed at baseline, ‘‘Have you ever had a fracture or injury to a knee requiring the use of crutches or a cane?’’.
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Osteoarthritis and Cartilage Vol. 17, No. 6 Table III Risk for Incident Knee OA according to homocysteine tertile in women (n ¼ 1587 knees) Homocysteine (range mmol/liter) Total n Incident OA Crude OR (95% CI) Adjusted OR (95% CI)*
Low tertile
Middle tertile
High tertile
P value for trend
4.60e8.97 562 53 1.0 (1.0) 1.0 (1.0)
9.00e11.30 531 55 1.0 (0.7e1.7) 0.8 (0.5e1.4)
11.32e30.79 494 49 0.9 (0.6e1.5) 0.7 (0.4e1.2)
0.274
*For the Adjusted OR, confounding variables were controlled in manner identical to that described in Table II.
of OA. It is important to note that, due to the age range of the female subjects, the study group likely included both pre and postmenopausal women. Sex hormones have been seen to affect plasma homocysteine levels; estrogen and progesterone as well as hormone replacement therapy tend to lower levels of homocysteine34. Postmenopausal women not taking hormone replacements have significantly higher plasma homocysteine levels35. Other studies looking at the effect of homocysteine on pathology in women have used a predominantly older population and this may be why significant associations were found in those cases4,5. One limitation of the present study is that it used a single homocysteine measure as a representation of the homocysteine status of the subjects. This assumption was likely inaccurate for certain subjects, such as women who had gone through menopause shortly before or during the course of the study. The limitation of a single homocysteine measure extends to men who may have also had significant unaccounted for fluctuations during the course of the study. In our analysis of progression of knee OA, we found no conclusive results regarding the risk for both men and women. However, our results show an increased OR for progression of knee OA for women with elevated homocysteine levels in the middle and high tertiles (Table V). In contrast, for men we noted a decreased OR for both of these groups (Table IV). Given that these results were not statistically significant, we cannot include or exclude that they may be due to chance. With the sample size of the study population and risk of disease in the low homocysteine group, we have 80% power to detect OR of 2.4e2.5 for knee OA progression; therefore, our study may not have sufficient power to detect a relationship if the true OR is less than 2.4. Using the approach employed in this study, a larger sample size would be required to obtain sufficient power for which the relationship between homocysteine and knee OA could reach statistical significance. Diet is large component of the normal variability in homocysteine levels among individuals and certain substances that affect these levels are also important in pathology. Folate is one such dietary component that is closely involved in the metabolism of homocysteine. Folate deficiencies reduce the activity of betaine homocysteine methyltransferase, the enzyme that catalyzes the synthesis of methionine from homocysteine36. Dietary supplements
including folate and cobalamin have also been demonstrated to improve function in people with hand OA37. Future research should consider the effect of homocysteine on specific aspects of OA. Homocysteine has been found to change the collagen matrix of bone and increase the incidence of fracture, demonstrating an effect of weakening bone integrity. As osteophytes are a pathological growth of bone that develop late in the progression of OA, elevated plasma homocysteine may interfere with their development. The absence of statistically significant findings for OA progression may be explained by a mixed effect of homocysteine in later stages of the disease. However, a larger study population with improved power is warranted in order to make any strong conclusions about the effect of homocysteine on OA progression. In the present study, correlating plasma homocysteine levels to both incidence and progression of OA was further limited by the single plasma homocysteine measurement available. Furthermore, all the covariates except weight change are also taken only at baseline, and do not reflect actual real time exposure representing a possible source of error in the present study. There are many factors that influence homocysteine status and, while we accounted for some of these in the present study, the findings may be improved upon by incorporation of nutritional factors such as vitamin supplementation. The present study combined data from the original and offspring cohorts, which employed different protocols of obtaining and of storing blood samples that were used for analysis. The two cohorts differed in methodology with regards to fasting state of the subjects as well as the storage temperature and duration of the homocysteine levels, potentially introducing a significant source of error into the study. The finding of increased risk of knee OA in the high tertiles in the offspring cohort, which required a 10 h fast prior to blood draws, not seen either the original or combined groups may indicate a better control for the effect of diet on homocysteine levels. That significant findings were not seen across both cohorts may also suggest that they are due simply to chance. In sum the preliminary evidence does not suggest a strong relationship between homocysteine and OA. However, further research is needed to explore the relation of homocysteine to other aspects of OA etiopathogenesis.
Table IV Risk for knee OA progression according to homocysteine tertile in men (n ¼ 187 knees) Total n Progression OA Crude OR (95% CI) Adjusted OR (95% CI)*
Low tertile
Middle tertile
High tertile
P value for trend
57 23 1.0 (1.0) 1.0 (1.0)
57 16 0.6 (0.2e1.4) 0.4 (0.1e2.1)
73 22 0.7 (0.3e1.6) 0.6 (0.1e1.1)
0.397
*For the Adjusted OR, confounding variables were controlled in manner identical to that described in Table II.
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M. Fayfman et al.: Homocysteine and knee OA Table V Risk for knee OA progression according to homocysteine tertile in women (n ¼ 303 knees)
Total n Progression OA Crude OR (95% CI) Adjusted OR (95% CI)*
Low tertile
Middle tertile
High tertile
P value for trend
69 16 1.0 (1.0) 1.0 (1.0)
104 27 1.5 (0.7e3.4) 1.5 (0.7e3.5)
130 36 1.7 (0.8e3.8) 1.7 (0.8e3.8)
0.226
*For the Adjusted OR, confounding variables were controlled in manner identical to that described in Table II.
Conflict of interest None to declare. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.
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15.
Acknowledgments We would like to thank the participants and staff of the Framingham Osteoarthritis Study. Role of funding source: The study sponsor was not involved in study design; in the collection, analysis, and interpretation of data; in the writing of the report; or the decision to submit the paper for publication.
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Osteoarthritis and Cartilage Vol. 17, No. 6 osteoarthritis in the elderly. The Framingham Osteoarthritis Study. Arthritis & Rheumatism 1995;38(10):1500e5. 33. Washburn RA, Smith KW, Jette AM, Janney CA. The Physical Activity Scale for the Elderly (PASE): development and evaluation. Journal of Clinical Epidemiology 1993;46(2):153e62. 34. van Baal WM, Smolders RG, van der Mooren MJ, Teerlink T, Kenemans P. Hormone replacement therapy and plasma homocysteine levels. Obstetrics & Gynecology 1999;94(4):485e91.
771 35. Wouters MG, Moorrees MT, van der Mooren MJ, Blom HJ, Boers GH, Schellekens LA, et al. Plasma homocysteine and menopausal status. European Journal of Clinical Investigation 1995;25(11):801e5. 36. Pajares MA, Perez-Sala D. Betaine homocysteine S-methyltransferase: just a regulator of homocysteine metabolism? [Review] [112 refs]. Cellular & Molecular Life Sciences 2006;63(23):2792e803. 37. Flynn MA, Irvin W, Krause G. The effect of folate and cobalamin on osteoarthritic hands. Journal of the American College of Nutrition 1994;13(4):351e6.
International Cartilage Repair Society
The relation of plasma homocysteine to radiographic knee osteoarthritis1 M. Fayfmany, J. Niuy, Y. Q. Zhangy, D. T. Felsony, B. Sacky, P. Aliabadiz, J. Selhubx and D. J. Hunteryk* y Boston University School of Medicine, Boston, USA z Department of Radiology, Brigham and Women’s Hospital, Boston, USA x Jean Mayer USDA HNRCA at Tufts University, Boston, USA k New England Baptist Hospital, Boston, USA Summary Objective: Homocysteine has been implicated in multiple diseases that involve changes in structural tissue. In vitro studies have found that it alters the structure of collagen cross-linking thus affecting stability and mineralization such as that occurring in bone tissue. In the present study we considered the possible relationship between plasma homocysteine levels and the development and progression of knee osteoarthritis (OA). Methods: The study question was posed in 691 men and 966 women from the original and offspring cohorts of the Framingham Osteoarthritis Study. We divided individuals into three groups according to plasma homocysteine levels and compared their risk for the development of new and progression of existing OA. We adjusted for potential confounders including age, body mass index, weight change, and physical activity. Results: In the crude analysis, men in the middle homocysteine tertile were found to be at a greater risk than men in the lowest tertile for incident OA [odds ratios of 1.9 (1.1e3.5)]. This result persisted after adjusting for covariates [odds: 2.0, (1.1e3.8)]. No significant correlation was seen in women for the development of OA. In the evaluation of progression no significant trends were seen for both men and women. Conclusions: Although cellular and molecular studies of homocysteine-related pathophysiology suggest a possible correlation between plasma homocysteine levels and OA, the present clinical study did not conclusively demonstrate such an association. However, further research is needed to explore the role of homocysteine in specific aspects of OA etiopathogenesis. ª 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Homocysteine, Knee OA, Incidence, Progression.
High plasma homocysteine is a risk factor for a number of chronic illnesses including cardiovascular disease, osteoporosis, and impaired cognitive function as well as for pregnancy complications5,7e9. The role of homocysteine in disease can be at least partially explained by its cellular effects on various types of connective tissue10e12. High plasma homocysteine is seen in cardiovascular disease where it leads to increased production of collagen by the smooth muscle cells10. Homocysteine is also involved in pregnancy complications and adverse outcomes including placental abruption, low birth weight, neural tube defects, and spontaneous abortion9,13,14. Homocysteine has recently been seen to play a role in osteoporosis related bone damage. An animal model study found that rats with elevated serum homocysteine have reduced bone quality15. Population studies in Framingham found homocysteine to be a predictor of hip fracture in older individuals5. The effects of homocysteine may be linked to its involvement in collagen formation. Homocysteine inhibits the synthesis of insoluble collagen fibrils in vitro by interfering with normal cross-linking16. From the perspective of cartilage homeostasis, these changes in matrix organization interfere with chondrocyte-mediated mineralization potentially altering the function and properties of calcified cartilage17. This may be due to homocysteine mediated inhibition of lysil oxidase, which catalyzes the cross-linking of collagen molecules, a function necessary for its mineralization in bone tissue17,18. Since osteophytes, which are marginal
Introduction Homocysteine is a sulfur-containing amino acid involved primarily in the metabolic pathway of methionine as well as that of cysteine and adenosine1. Its levels in the blood are controlled by multiple enzymes, whose activity varies due to genetic or environmental factors. Its role in multiple pathologic processes is evident from studies of individuals with homocysteinurea who have extremely elevated levels of plasma homocysteine due either to genetic mutation in key regulatory enzymes or underlying conditions such as kidney disease2. The normal range of plasma homocysteine levels for healthy adults is 5e15 mmol/liter3. However, even moderately increased levels within the normal range have been found to be involved in multiple disease states4e6. 1
Supported by NIH AR43873 and AR47785 and NIH AG18393 from the Framingham Heart Study of the National Heart, Lung, and Blood Institute of the National Institutes of Health and Boston University School of Medicine. This work was supported by the National Heart, Lung, and Blood Institute’s Framingham Heart Study (Contract No. N01-HC-25195). *Address correspondence and reprint requests to: D. J. Hunter, New England Baptist Hospital, Division of Research, 125 Parker Hill Av., Boston, MA 02120, USA. Tel: 1-617-754-6655; Fax: 1-617754-6888; E-mail: [email protected] Received 11 January 2008; revision accepted 24 November 2008.
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Osteoarthritis and Cartilage Vol. 17, No. 6 osteocartilaginous outgrowths, emanate from osteoarthritic joint tissue, homocysteine mediated inhibition of mineralization may actually have a protective effect. Homocysteine may also exert effects on bone by means other than collagen reorganization. The presence of high homocysteine leads to decreased secretion of osteocalcin and increased osteopontin by osteoblasts19. It also stimulates osteoclast differentiation enhancing bone resorption15,20. Given the importance of homocysteine in bone and collagen metabolism, and chondrocyte function we proposed to explore the relation of homocysteine to osteoarthritis (OA). OA is a disorder of multiple joint tissues characterized by different stages of progression. Collagen is a large component of articular cartilage, which forms the lining of the joint, and its destruction contributes to OA. Cross-linking bands of collagen molecules are necessary to form the stable fibrils that compose the cartilage found on articular joint surfaces and homocysteine has been seen to disrupt the intermolecular bonds of these fibrils11. Changes in the collagen networks of joint fibers observed early in the development of OA are a potential mechanism by which homocysteine is involved in this disease. Homocysteine, its metabolic precursors and products have been shown to alter various components of connective tissue, however, no studies have directly looked at the relation between homocysteine levels and knee OA in humans. In the present study, we used population-based data from the Framingham OA Study to look at the predictive value of homocysteine on both the development and progression of knee OA and it association with prevalent knee OA.
Methods STUDY POPULATION (FRAMINGHAM OA STUDY) The Framingham Heart Study is a longitudinal population-based cohort study established in 1948 in Framingham, Massachusetts to examine risk factors for heart disease21. A study of the offspring of the original cohort and their spouses was initiated in 1971. The details of this cohort have been previously described22. The Framingham OA study, which includes participants of the original and offspring cohorts, was developed to study the inheritance of OA23. The present study included 691 men and 966 women from the two cohorts. For the original cohort, baseline and follow-up visits and radiographs were obtained at examinations 18 (1983e1985) and 22 (1992e1993), respectively3. Subjects from the offspring cohort were examined between 1992e1994 and 2002e200524. Eligibility criteria for both cohorts in the present analysis included: men and women aged 40e85 years; ambulatory (use of assistive devices such as canes and walkers was allowed). Exclusion criteria were: the presence of bilateral total knee replacements, and the presence of rheumatoid arthritis. The Framingham OA Study protocol involved multiple components, one of which was a radiographic exam including posteroanterior (PA) fixed flexion25 and lateral radiographs26 of both knees.
PLASMA HOMOCYSTEINE Nonfasting blood samples for the original cohort were collected at the 16th biennial examination between 1979 and 1982 and stored at or below 20 C. These samples were thawed in 1997 and plasma homocysteine was measured using high-performance liquid chromatography5. For the offspring cohort, fasting (>10 h) blood samples were collected on the fifth examination, from 1993e1994 and were frozen immediately and stored at or below 70 C27. In 1995, the samples were thawed and the total plasma homocysteine concentrations were measured by high-performance liquid chromatography with fluorometric detection28. The coefficient of variation for this assay was nine percent29.
KNEE RADIOGRAPHY During the baseline visits, a weight-bearing fully extended radiograph of both knees, using a standardized protocol that included outlines of the feet
to keep constant the rotation of the knee at follow-up was obtained. Films were obtained at 0 and at 6 caudad, and the best view (based on the optimal superimposition of the anterior and posterior margins of the medial tibial plateau) of these two was selected for comparison at the follow-up examination. After the subjects completed their follow-up examination, both baseline and follow-up radiographs were read independently by two study readers, one a bone and joint radiologist (PA), and the other a rheumatologist (BS). X-rays were read in a paired fashion, unblinded to sequence. Readers evaluated the Kellgren and Lawrence (K&L) grade30 of each knee at both time points and evaluated individual features and whether these had changed over the follow-up period. Each knee was evaluated for the presence of osteophytes and joint space narrowing (JSN) on a 0e3 scale using the OARSI atlas31. If there was a difference in K&L grade that led to a difference in the assignation of either prevalent or incident OA status the films were adjudicated by a panel of three readers including the first two and a third reader (DTF). All adjudicated readings were arrived at by consensus of three readers using methods previously described32. For the bone and joint radiologist, the intra-reader kappa for K&L grade was 0.82. Inter-reader kappa was 0.74 (both P < 0.001). Knee OA was defined using the K&L grade where a score of two or higher indicated the presence of OA. Two outcomes were considered for the present study. Incident knee OA was defined when a knee had a grade of less than two during the baseline visit and had a K&L grade 2 at the follow-up visit. Progression of knee OA was defined when a given knee had a K&L grade of 2 or above at baseline and at least one grade increase at the follow-up examination32. Because they were not eligible for progression, knees with K&L grade 4 at baseline were excluded from the study.
CONFOUNDERS Information about potential confounding variables was available from the Framingham OA Study database. Weight was measured in kilograms without shoes using a standard balance beam scale. Weight change was defined as the difference in weight between baseline and follow-up examination. Height was measured in centimeters, without shoes, to the nearest quarter inch. Levels of habitual physical activity were estimated based on the number of hours spent in a typical day at different levels of activity [Physical Activity Scale for the Elderly (PASE)]33. Information about knee injury was derived from the response to a standard question posed at baseline, ‘‘Have you ever had a fracture or injury to a knee requiring the use of crutches or a cane?’’ Information was obtained at the baseline for all confounders except ‘‘weight change’’, which involved measures at baseline and follow-up visits.
STATISTICAL ANALYSIS We combined data from Framingham original cohort and Framingham offspring cohort and created sex-specific tertiles of homocysteine. A total of 479 of the subjects available were removed from the final analysis. 10 subjects were excluded due to bilateral knee replacement, 10 because they had rheumatoid arthritis, 398 did not have homocysteine levels measured, and 61 were under 40 years old. The final study group included 638 individuals from the original and 1019 from the offspring group. Subjects were categorized by tertiles of homocysteine levels to account for some of the individual variability. We examined the relation of tertile groups of homocysteine (using the lower tertile as the referent category) to the risk of incident radiographic knee OA using the logistic regression model. In the multivariable regression model, we adjusted for continuous age, dichotomous gender and knee injury history, body mass index, weight change, and physical activity index. These potential confounding variables were matched between the three tertiles in an all variables in a concomitant fashion. We used generalized estimating equations to account for correlation between two knees. Results are presented as odds ratios (OR) with 95% confidence intervals of upper and lower intervals of statistical significance. We adopted the same approach to assess the relation of levels of homocysteine to the risk of progressive radiographic knee OA progression.
Results The baseline characteristics of the study participants are shown in Table I. The mean age among men and women was 60.1 and 61.2 years, respectively. Among the men, the mean homocysteine levels were 12 3.9 for the original cohort and 10.6 for the offspring cohort. Mean homocysteine levels for the women of the original and offspring cohorts were 10.9 3.4 and 9.0 3.1, respectively. The
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M. Fayfman et al.: Homocysteine and knee OA Table I Characteristics of study sample (n ¼ 1546 persons) Men (n ¼ 691)
Women (n ¼ 966)
Age in years Mean (SD) {Range}
60.1 (10.5) {38}
61.2 (10.7) {38}
BMI kg/m2 Mean (SD) {Range) Prevalence of Knee OA (%)*
27.3 (4.1) {18.0, 43.3} 15.4
25.9 (5.1) {14.3, 53.6} 17.3
Homocysteine mmol/liter Mean (SD) {Range}
11.1 (3.4) {4.6, 30.8}
9.8 (3.3) {3.5, 29.0}
*Prevalence of OA measured in knees with OA over total number of knees.
combined mean homocysteine levels among the men were 11.1 mmol/liter and among the women were 9.8 mmol/liter (Table I). Distribution of the exposure variable (homocysteine) was different between men and women so the analyses were performed in a gender specific manner. The incidence of knee OA in men for three homocysteine tertiles is shown in Table II. Before adjusting for covariates (age, sex, body mass index, weight change, physical activity index and knee injury), we found that men in the middle homocysteine tertile had an increased risk of developing knee OA over the low homocysteine tertile [OR for middle tertile, 1.9 (1.1, 3.5)]. After adjusting for the above listed covariates, men in the middle tertile, as in the crude analysis, showed an increased risk of knee OA [OR for middle tertile, 2.0 (1.1, 3.8)]. The risk for incident knee OA in women of three homocysteine levels is shown in Table III. In the crude analysis, the group in the middle and high homocysteine tertile had no increased risk of knee OA over the low tertile group. No significant associations were seen in the adjusted analysis as well. We analyzed the risk of progression of knee OA in the study subjects. Table IV shows the risk of knee OA progression in men. The OR for men in the middle and high homocysteine tertiles in the crude analysis were 0.6 (0.2,1.4) and 0.7 (0.3,1.6), respectively. The results were similar for the adjusted analysis. Table V shows the progression of OA data for women. In the crude analysis, the OR for the middle and high tertiles was 1.5 (0.7, 3.4) and 1.7 (0.8, 3.8), respectively. These findings were similar for the adjusted analysis. In order to assess the effect of combining the original and offspring cohorts, we did a stratified analysis of the two groups and found that the results differed significantly from the combined study for the incidence of OA among men. The offspring cohort shows a significantly increased
risk of OA for the high tertile [Crude OR: 2.6 (1.2, 5.8); Adjusted OR: 2.6(1.0, 6.4)] while both the original and combined analysis of this group show no significant association. The other groups were consistent between the stratified and combined analyses. A t test for mean homocysteine levels showed a statistically significant difference between the two cohorts, with slightly increased homocysteine in the original cohort 11.3 3.6 mmol/liter compared with the offspring 9.7 3.2 mmol/liter (P < 0.001). To further evaluate the possible threshold effect of homocysteine on OA, we did a separated analysis comparing the low tertile to the combined middle and high tertiles and found that the results are consistent with those presented in the paper. We also looked at prevalent OA and homocysteine levels finding no significant correlations.
Discussion In the present study, we found an association between moderately elevated homocysteine levels and increased risk of developing OA in men. Men whose plasma homocysteine levels ranged between 9 mmol/liter and 11.3 mmol/liter had a significantly increased risk for incident OA compared to those with homocysteine levels between 4.60 and 8.97 mmol/liter. The men in the highest tertile also appeared to have an increased risk but these findings were not statistically significant within 95% confidence interval. For the offspring cohort only, there did appear to be a statistically increased incidence of OA in both the middle and high homocysteine tertiles. Given the sample size we have and risk of disease in the low homocysteine group we observed, we have 80% power to detect odds ratio as small as 1.6e1.9 for incident knee OA. In the analysis for the women, we did not see a significant association between levels of homocysteine and incidence
Table II Risk for incident knee OA according to homocysteine tertile in men (n ¼ 1166 knees) Homocysteine (range mmol/liter) Total n Incident OA Crude OR (95% CI) Adjusted OR (95% CI)*
Low tertile
Middle tertile
High tertile
P value for trend
4.60e8.97 398 25 1.0 (1.0) 1.0 (1.0)
9.00e11.30 394 41 1.9 (1.1e3.5) 2.0 (1.1e3.8)
11.32e30.79 374 37 1.7 (0.9e3.3) 1.7 (0.8e3.3)
0.168
*In the Adjusted OR, potential confounding variable are matched between the three tertiles and calculated in a multivariable regression model. Potential confounders include age measured as a continuous variable, body mass index, weight change between baseline and follow-up visits, physical activity index (PASE)32, and history of knee injury based on response to the question posed at baseline, ‘‘Have you ever had a fracture or injury to a knee requiring the use of crutches or a cane?’’.
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Osteoarthritis and Cartilage Vol. 17, No. 6 Table III Risk for Incident Knee OA according to homocysteine tertile in women (n ¼ 1587 knees) Homocysteine (range mmol/liter) Total n Incident OA Crude OR (95% CI) Adjusted OR (95% CI)*
Low tertile
Middle tertile
High tertile
P value for trend
4.60e8.97 562 53 1.0 (1.0) 1.0 (1.0)
9.00e11.30 531 55 1.0 (0.7e1.7) 0.8 (0.5e1.4)
11.32e30.79 494 49 0.9 (0.6e1.5) 0.7 (0.4e1.2)
0.274
*For the Adjusted OR, confounding variables were controlled in manner identical to that described in Table II.
of OA. It is important to note that, due to the age range of the female subjects, the study group likely included both pre and postmenopausal women. Sex hormones have been seen to affect plasma homocysteine levels; estrogen and progesterone as well as hormone replacement therapy tend to lower levels of homocysteine34. Postmenopausal women not taking hormone replacements have significantly higher plasma homocysteine levels35. Other studies looking at the effect of homocysteine on pathology in women have used a predominantly older population and this may be why significant associations were found in those cases4,5. One limitation of the present study is that it used a single homocysteine measure as a representation of the homocysteine status of the subjects. This assumption was likely inaccurate for certain subjects, such as women who had gone through menopause shortly before or during the course of the study. The limitation of a single homocysteine measure extends to men who may have also had significant unaccounted for fluctuations during the course of the study. In our analysis of progression of knee OA, we found no conclusive results regarding the risk for both men and women. However, our results show an increased OR for progression of knee OA for women with elevated homocysteine levels in the middle and high tertiles (Table V). In contrast, for men we noted a decreased OR for both of these groups (Table IV). Given that these results were not statistically significant, we cannot include or exclude that they may be due to chance. With the sample size of the study population and risk of disease in the low homocysteine group, we have 80% power to detect OR of 2.4e2.5 for knee OA progression; therefore, our study may not have sufficient power to detect a relationship if the true OR is less than 2.4. Using the approach employed in this study, a larger sample size would be required to obtain sufficient power for which the relationship between homocysteine and knee OA could reach statistical significance. Diet is large component of the normal variability in homocysteine levels among individuals and certain substances that affect these levels are also important in pathology. Folate is one such dietary component that is closely involved in the metabolism of homocysteine. Folate deficiencies reduce the activity of betaine homocysteine methyltransferase, the enzyme that catalyzes the synthesis of methionine from homocysteine36. Dietary supplements
including folate and cobalamin have also been demonstrated to improve function in people with hand OA37. Future research should consider the effect of homocysteine on specific aspects of OA. Homocysteine has been found to change the collagen matrix of bone and increase the incidence of fracture, demonstrating an effect of weakening bone integrity. As osteophytes are a pathological growth of bone that develop late in the progression of OA, elevated plasma homocysteine may interfere with their development. The absence of statistically significant findings for OA progression may be explained by a mixed effect of homocysteine in later stages of the disease. However, a larger study population with improved power is warranted in order to make any strong conclusions about the effect of homocysteine on OA progression. In the present study, correlating plasma homocysteine levels to both incidence and progression of OA was further limited by the single plasma homocysteine measurement available. Furthermore, all the covariates except weight change are also taken only at baseline, and do not reflect actual real time exposure representing a possible source of error in the present study. There are many factors that influence homocysteine status and, while we accounted for some of these in the present study, the findings may be improved upon by incorporation of nutritional factors such as vitamin supplementation. The present study combined data from the original and offspring cohorts, which employed different protocols of obtaining and of storing blood samples that were used for analysis. The two cohorts differed in methodology with regards to fasting state of the subjects as well as the storage temperature and duration of the homocysteine levels, potentially introducing a significant source of error into the study. The finding of increased risk of knee OA in the high tertiles in the offspring cohort, which required a 10 h fast prior to blood draws, not seen either the original or combined groups may indicate a better control for the effect of diet on homocysteine levels. That significant findings were not seen across both cohorts may also suggest that they are due simply to chance. In sum the preliminary evidence does not suggest a strong relationship between homocysteine and OA. However, further research is needed to explore the relation of homocysteine to other aspects of OA etiopathogenesis.
Table IV Risk for knee OA progression according to homocysteine tertile in men (n ¼ 187 knees) Total n Progression OA Crude OR (95% CI) Adjusted OR (95% CI)*
Low tertile
Middle tertile
High tertile
P value for trend
57 23 1.0 (1.0) 1.0 (1.0)
57 16 0.6 (0.2e1.4) 0.4 (0.1e2.1)
73 22 0.7 (0.3e1.6) 0.6 (0.1e1.1)
0.397
*For the Adjusted OR, confounding variables were controlled in manner identical to that described in Table II.
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M. Fayfman et al.: Homocysteine and knee OA Table V Risk for knee OA progression according to homocysteine tertile in women (n ¼ 303 knees)
Total n Progression OA Crude OR (95% CI) Adjusted OR (95% CI)*
Low tertile
Middle tertile
High tertile
P value for trend
69 16 1.0 (1.0) 1.0 (1.0)
104 27 1.5 (0.7e3.4) 1.5 (0.7e3.5)
130 36 1.7 (0.8e3.8) 1.7 (0.8e3.8)
0.226
*For the Adjusted OR, confounding variables were controlled in manner identical to that described in Table II.
Conflict of interest None to declare. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.
14.
15.
Acknowledgments We would like to thank the participants and staff of the Framingham Osteoarthritis Study. Role of funding source: The study sponsor was not involved in study design; in the collection, analysis, and interpretation of data; in the writing of the report; or the decision to submit the paper for publication.
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19.
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