Comparison of healthy and normative aging reveals a metabolic component in hand oa and oa biochemical marker profiles

s / Osteoarthritis and Cartilage 20 (2012) S54–S296 S88 summed KL 2 The Netherlands Genomics Initiative-sponsored Netherlands Consortium for Hlth.y Aging, Leiden, NETHERLANDS Purpose: Epidemiological studies have shown associations between osteoarthritis (OA) and age, as well as between OA and metabolism, which are not completely understood. It can be questioned whether intrinsic metabolic programming affects OA risk and circulating levels of biochemical markers applied in OA research such as COMP and CTX2. These biomarkers measured in serum and urine reflect cartilage turnover and degradation, respectively. Insight in interactions between age, metabolism and OA will allow for better clinical classification of patients and for a more reliable assessment of disease activity or progression using biochemical markers. Such insights may be obtained from studies focused on aging and metabolism, independent of disease. To such end, we have analyzed levels of serum COMP and urinary CTX2 and hand radiographic OA (ROA) scores in healthy aging subjects from long-lived families, their partners as population controls and OA patients. We analyzed age related aspects of the biochemical markers and hand ROA scores and compared the marker and ROA score profiles in healthy and normative aging and in disease. Next we tested whether glucose levels influenced the levels of biochemical markers and hand ROA scores in the healthy and normative aging subjects. Methods: The Leiden Longevity Study (LLS) consists of long-lived siblings, their offspring and partners of the offspring as controls. In middle age the familial longevity trait is reflected by a 30% increased survival, low prevalence of metabolic disease and healthy metabolic profile (such as lower glucose levels). We measured uCTX2 and sCOMP levels in a subset of the females of the LLS (N1⁄4151 healthy agers and N1⁄4181 controls) and scored hand radiographs (20 joint sites) according to Kellgren and Lawrence (N1⁄4107 healthy agers and N1⁄4128 controls). The same scores were available for the OA affected females of the GARP study (N1⁄4315). Furthermore, these were previously typed for the biochemical marker levels sCOMP and uCTX2. Linear mixed models were applied testing for relationships between age and hand ROA data or marker levels, whilst correcting for BMI, familial dependencies and potential batch effects between the GARP and LLS study. Results: sCOMP shows a comparable age related increase in healthy and normative agers as well as in OA patients. Healthy agers, however, had a significantly higher mean sCOMP level as compared to the controls. Remarkably, uCTX2 levels showed no increase with age in healthy agers whereas a significant and equal age related increase was observed amongst the controls and OA patients. Healthy ageing women have the lowest age related increase of hand ROA score, whereas this has a significant linear increase via controls to the OA patients (study groups coded as 0, 1 and 2 for healthy agers, controls and GARP patients respectively, BMI and age adjusted). When we additionally selected the healthy agers with glucose levels below the median within their group, the interaction of study group with hand ROA scores became more pronounced between healthy agers and controls. Conclusions: When interpreting OA biomarkers, age is a major factor to consider. Furthermore, a healthy aging metabolic profile as reported for the Leiden longevity family members associated to a lower age-related increase in hand ROA scores and uCTX2 levels as compared to controls. This may indicate that metabolic health should also be considered in the assessment of the risk for hand ROA onset and monitoring of progression on the basis of these biomarkers. Comp as a new potential biomarker for osteoarthritis. 163 20KDa FRAGMENT OF OSTEOGLYCIN: AN INNOVATIVE TOOL FOR OA PATIENTS CHARACTERIZATION A. Farran, L. Tio, P. Benito, J. Monfort. IMIM-Hosp. del Mar, Barcelona, Spain Purpose: Osteoarthritis is the most common rheumatologic disease and is characterized by increased protease activity resulting in cartilage degradation. Nowadays, radiographic measures are the traditional study for diagnosis, but the abnormal metabolic process begins in the articular cartilage several years before destruction of the tissue can be detected radiologically. Our aim in this study is the search for new osteoarthritic molecular biomarkers, by the screening of different ECM proteins and their fragments in osteoarthritic (OA) and normal cartilage (CT). Methods: Normal samples were obtained from femoral cartilage of patients who underwent hip replacement due to osteoporotic fracture. OA cartilage samples came from knee and hip arthrosic patients undergoing arthroplasty. ECM proteins from these samples were extracted using Guanidinium chloride (4 mol/l) method. After the extraction, proteins and glycosaminoglycans (GAG's) were quantified by Bradford and dimethylmethylene-blue (DMB) assay respectively. Proteoglycans studied from the ECM protein extracts of knee and hip cartilage were normalized by Bradford concentration and analyzed by Western blotting. The bands intensity was quantified by Quantity One software. In order to study the activity of the protease in front of each proteoglycan studied and to find out specific fragments from OA patients, the extracts were digested with recombinant matrix metalloproteinase 13 (MMP-13) at 2, 4, 6, 8, 16 and 24h and analyzed with western blot. Results: Osteoglycin (OGN), Matrilin3 (MATN3), and Cartilage oligomeric matrix protein (COMP) were identified by Western Blotting. Our results showed that MATN3 expression did not present any difference between CT, OA hip and OA knee cartilage. We could observe digestion of MATN3 by MMP-13 after 6 hours of incubation and it was nearly completely digested at 24h. In the case of COMP expression, the analysis showed a different band pattern in OA knee samples compared with CT and OA femoral samples. Interestingly, a COMP fragment of approximately 60 KDa was observed at 16h and 24h of incubation in all the samples. Regarding the OGN results, we surprisingly found that it was more abundant in OA knee samples than in CT ones, contrary to other previously described proteoglycans observations. Concerning to OGN digestion, we could also see differences between samples; while OGN from CT samples was completely digested at 16h, OGN from OA femoral samples showed still a weak band at 16h of digestion that was completely digested at 24h. Finally, in OA knee samples, at 24 hours the digestions was not still