Quantification of chondroitin sulfates and hyaluronan in synovial fluid using high performance liquid chromatography

• Purpose: Extracellular proteins such as aggrecan may be primed with specific glycan-patterns which result in their degradation, and hence may play a role in the pathogenesis of osteoarthritis (OA). To be able to use glycans as molecular biomarkers, the method of analysis of these molecules needs to be validated. The primary aim of this study was to validate quantitative high performance liquid chromatography (HPLC) of chondroitin sulfate (CS) and hyaluronan (HA) in synovial fluid samples. The secondary aim was to examine the glycan-pattern in different subject groups, and the correlation between age and the concentration of specific glycans.Methods: OA (n=25, age=36-86 years, 40% women) and recent knee injury patients (0-5 days from injury; n=13, age=36-64 years, 46% women) were selected from a cross-sectional convenience cohort. Individual synovial fluid samples, a synovial fluid pool (SF-control; n=7) and a CS quality control sample (CS-QC; Sigma #C2905) were digested with chondroitinase ABC overnight. Samples and glycan standards (CS [n=8] and HA [n=1] standards from Iduron) were labelled with 2-aminoacridone (AMAC) and analyzed using a quantitative HPLC assay. Aggrecan from synovial fluid samples (n=6) was purified using density centrifugation (D1 mini-prep). Sulfated glycosaminoglycans (sGAG) were quantified using Alcian blue precipitation. Since the CS, HA and sGAG data were not normally distributed, non-parametric analyses for group comparisons were done. P-values less than 0.05 were considered statistically significant.Results: Synovial fluid samples were digested with varying concentrations of chondroitinase ABC and analyzed with the HPLC assay; 5 mU chondroitinase ABC per μg sGAG gave the highest CS- and HA-signals and were chosen for the rest of the study (data not shown).The CS profiles in synovial fluid and on aggrecan purified from corresponding synovial fluids were assessed from six knee injury patients. Of the six CS-markers that were detected, uronic acid (UA)-N-acetylgalactosamine (GalNAc) was only present in aggrecan samples, while UA2S-GalNAc and UA2S-GalNAc6S were found only in the synovial fluids. Similar proportions of UA-GalNAc4S and UA-GalNAc6S were found in synovial fluids and aggrecan samples (Figure 1), and these CS-glycans accounted for 95% of all glycans in the SF-control sample (Table 1).The technical performance of CS- and HA-markers using HPLC-assay were evaluated (Table 1). Of the nine markers, five were present in the majority of the synovial fluid samples (N=20-38) and included in the investigation of the technical performance. The mean intra coefficient of variation (CV) for the synovial fluid samples was between 1.2 and 12.9%. For the SF-control sample, the mean intra CV was 3.3-12.1% and the inter CV was 11.0-18.5%. For the CS-QC sample, the mean intra CV was 2.5-9.5% and the mean inter CV was 3.3-31.7%. For the glycan standards, the mean intra CV was 0.2-7.0%. With dilution of the SF-control sample up to 1:10, the dilution recovery rate for the five CS- and HA-markers was mainly between 75 and 125%.The synovial fluid concentration of biomarkers UA-GalNAc6S and UA2s-GalNAc6s and sGAG were approximately 2 to 3 times higher for the recent injury group compared to the age-matched OA group, while the HA levels were 3.7 times lower for the recent injury group (data not shown). No difference in biomarker concentrations were found between the sexes in any of the patient groups (data not shown). For correlation assessments, the two patient groups were merged (total N=38, assessment N = between 20 and 38). Synovial fluid concentrations of HA and UA-GalNAc4S,6S correlated positively with age (rS=0.420 and 0.532, respectively) while UA-GalNAc6S and sGAG correlated negatively with age (rS=-0.333 and -0.528, respectively). HA correlated negatively with UA-GalNAc6S (rS=-0.462) and sGAG (rS=-0.472) and positively with UA-GalNAc4S,6S (rS=0.868).Conclusions: The technical performance of the HPLC-assay indicates that the method is suitable for analyzing CS and HA markers in synovial fluid samples. Our results suggest that: the vast majority of CS in synovial fluid derives from aggrecan, the glycan pattern differs between OA and knee injured subjects and that the concentrations of some of the CS-markers seem to be associated with HA and age.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Klinisk medicin -- Klinisk laboratoriemedicin (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Clinical Medicine -- Clinical Laboratory Medicine (hsv//eng)

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