Articular Joint Lubricants during Osteoarthritis and Rheumatoid Arthritis Display Altered Levels and Molecular Species

Abstract

Background: Hyaluronic acid (HA), lubricin, and phospholipid species (PLs) contribute independently or together to the boundary lubrication of articular joints that is provided by synovial fluid (SF). Our study is the first reporting quantitative data about the molecular weight (MW) forms of HA, lubricin, and PLs in SF from cohorts of healthy donors, patients with early (eOA)- or late (lOA)-stage osteoarthritis (OA), and patients with active rheumatoid arthritis (RA).

Methods: We used human SF from unaffected controls, eOA, lOA, and RA. HA and lubricin levels were measured by enzyme-linked immunosorbent assay. PLs was quantified by electrospray ionization tandem mass spectrometry. Fatty acids (FAs) were analyzed by gas chromatography, coupled with mass spectrometry. The MW distribution of HA was determined by agarose gel electrophoresis.

Results: Compared with control SF, the concentrations of HA and lubricin were lower in OA and RA SF, whereas those of PLs were higher in OA and RA SF. Moreover, the MW distribution of HA shifted toward the lower ranges in OA and RA SF. We noted distinct alterations between cohorts in the relative distribution of PLs and the degree of FA saturation and chain lengths of FAs.

Conclusions: The levels, composition, and MW distribution of all currently known lubricants in SF--HA, lubricin, PLs--vary with joint disease and stage of OA. Our study is the first delivering a comprehensive view about all joint lubricants during health and widespread joint diseases. Thus, we provide the framework to develop new optimal compounded lubricants to reduce joint destruction.

Fig 1. The concentrations of boundary lubricants in human SF.

The content of HA (A) and lubricin (B) in SF were determined by ELISA, and ESI-MS/MS was used to quantify phospholipids (C) in 16 control SF (shaded bars), 27 eOA SF (hatched bars), 22 lOA SF (open bars), and 20 RA SF (grey bars) as described in Material and Methods. Data are presented as median with interquartile ranges. P-values less than 0.05 were considered statistically significant: *0.01< p≤0.05, **0.001<p≤0.01, ***p≤0.001.

Fig 2. Characterization of hyaluronic acid (HA) and lipids in human SF.

The molecular weight distribution of HA (A) was determined by horizontal agarose gel electrophoresis, and ESI-MS/MS was used to quantify phospholipid classes (B) in 16 control SF (shaded bars), 26 eOA SF (hatched bars), 22 lOA SF (open bars), and 20 RA SF (grey bars) as described in Methods. The molecular weight distribution of HA was calculated as the percentage of total HA (= 100%), whereas the relative distribution of phospholipid classes is shown as the percentage of total lipid content (= 100%). Data are presented as the median and interquartile ranges. Significance was defined as follows: a: p≤0.05: control vs. RA; b: p≤0.05: control vs. eOA; c: p≤0.05: eOA vs. late OA; and d: p≤0.05: eOA vs. RA, e: p≤0.05: eOA vs. RA; and f: p≤0.05: lOA vs. RA. The lipids that we measured were phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), PE-based plasmalogens (PE P), phosphatidylserine (PS), phosphatidylglycerol (PG), and sphingomyelin (SM).

Fig 3. Scatterplot of concentrations of hyaluronic acid (HA) by levels of lubricin (A) and phospholipids (PL) (B) and concentration of lubricin by MW distribution of HA (C-F).

HA and lubricin content in SF was determined by ELISA, and ESI-MS/MS was used to quantify phospholipids in 16 control SF, 27 eOA SF, and 22 lOA SF samples as described in Methods. Molecular weight distribution of HA was calculated as the percentage of total HA (= 100%). Linear regression was performed, and Pearson correlation coefficients were calculated.

Fig 4. Percentage composition of phosphatidycholine (PC) species in human SF.

PC species were quantified by ESI-MS/MS in 16 control SF (shaded bars), 27 eOA SF (hatched bars), 22 lOA SF (open bars), and 20 RA SF (grey bars) samples as described in Methods. Species were assigned based on the assumption that only FAs with an even number of carbon atoms are present. Subsequently, the percentage was calculated, defining total PC as 100%. Data are presented as the median and interquartile ranges. Significance was defined as follows: a: p≤0.05: control vs. RA; b: p≤0.05: control vs. eOA; c: p≤0.05: eOA vs. late OA; d: p≤0.05: eOA vs. RA, e: p≤0.05: eOA vs. RA; and f: p≤0.05: lOA vs RA.

Fig 5. Percentage composition of lysophosphatidylcholine (LPC) species in human SF.

PC species were quantified by ESI-MS/MS in 16 control SF (shaded bars), 27 eOA SF (hatched bars), 22 lOA SF (open bars), and 20 RA SF (grey bars) samples as described in Methods. Species were assigned based on the assumption that only FAs with an even number of carbon atoms are present. Subsequently, the percentage was calculated, defining total LPC as 100%. Data are presented as the median and interquartile ranges. Significance was defined as follows: a: p≤0.05: control vs. RA; b: p≤0.05: control vs. eOA; c: p≤0.05: eOA vs. late OA; d: p≤0.05: eOA vs. RA e: p≤0.05: eOA vs. RA; and f: p≤0.05: lOA vs RA.

Fig 6. The degree of saturation and length of fatty acids from phosphatidylcholine (PC) and lysophosphatidylcholine (LPC).

Phospholipid species were quantified by ESI-MS/MS in 16 control SF (shaded bars), 27 eOA SF (hatched bars), 22 lOA SF (open bars), and 20 RA SF (grey bars) samples as described in Methods. Subsequently, PC and LPC species were grouped by degree of saturation and number of carbon atoms in the FA chains, respectively, and calculated as a percentage of total PC and LPC, defined 100%. Composition of total FA was determined by gas chromatography for 6 lOA SF samples as described in Methods. Data are presented as median and interquartile ranges (A-D) or mean ± SD (E). P-values less than 0.05 were considered statistically significant: *0.01< p≤0.05, **0.001<p≤0.01, ***p≤0.001. A, Relative distribution of PC species according to degree of FA saturation. B, Relative distribution of LPC species by degree of FA saturation. C, Relative distribution of PC species according to chain length of FAs. D, Relative distribution of LPC species by chain length of FAs. E, Degree of saturation and length of fatty acid of total FAs representing 100%.