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

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).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.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.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

Concentrations of ceramide species in human knee synovial fluid.

<p>Synovial fluid was obtained from donors serving as controls and from patients with early osteoarthritis (eOA), late OA (lOA), and rheumatoid arthritis (RA). Cer species were determined by electrospray ionization tandem mass spectrometry as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091769#s2" target="_blank"><i>Material and Methods</i></a>. Values are presented as median and interquartile range. Significance was considered in the following way: a: p≤0.05: control vs eOA; b: p≤0.05: control vs lOA; c: p≤0.05: control vs RA; d: p≤0.05: eOA vs lOA; and e: p≤0.05: eOA vs RA. (<b>A</b>): Cer species, (<b>B</b>): HexCer and Hex₂Cer species. Cer- ceramide, HexCer- hexosylceramide (most likely glucosylceramide), Hex₂Cer- dihexosylceramide (most likely lactosylceramide).</p

Concentrations of sphingomyelin species in human knee synovial fluid.

<p>Synovial fluid was obtained from donors serving as controls and from patients with early osteoarthritis (eOA), late OA (lOA), and rheumatoid arthritis (RA). SM species were determined by electrospray ionization tandem mass spectrometry as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091769#s2" target="_blank"><i>Material and Methods</i></a>. Species annotation is based on the notion that 2 hydroxyl groups are linked to a sphingoid base. Values are presented as median and interquartile range. Significance was considered in the following way: a: p≤0.05: control vs eOA; b: p≤0.05: control vs lOA; c: p≤0.05: control vs RA; d: p≤0.05: eOA vs lOA; and e: p≤0.05: eOA vs RA. SM- sphingomyelin.</p

Concentrations of phosphatidic acid (PA) and lysophosphatidic acid (LPA) species in human knee synovial fluid.

<p>Synovial fluid was obtained from donors serving as controls and from patients with early osteoarthritis (eOA), late OA (lOA), and rheumatoid arthritis (RA). PA and LPA species were determined liquid chromatography coupled with tandem mass spectrometry as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091769#s2" target="_blank"><i>Material and Methods</i></a>. Species annotation is based on the notion that only ester bonds are present. Values are presented as median and interquartile range. Significance was considered in the following way: a: p≤0.05: control vs eOA; b: p≤0.05: control vs lOA; and c: p≤0.05: control vs RA. (<b>A</b>): PA species, (<b>B</b>): LPA species. PA- phosphatidic acid, LPA- lysophosphatidic acid.</p

Concentrations of bis(monoacylglycero)phosphate (BMP) species.

<p>Concentrations of bis(monoacylglycero)phosphate (BMP) species in knee synovial fluid obtained from donors serving as controls and from patients with early osteoarthritis (eOA), late OA (lOA), and rheumatoid arthritis (RA). BMP species were determined by electrospray ionization tandem mass spectrometry as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091769#s2" target="_blank"><i>Material and Methods</i></a>. Values are presented as median and interquartile range in the brackets. BMP-Bis(monoacylglycero)phosphate, eOA-early osteoarthritis, lOA-late osteoarthritis, RA-rheumatoid arthritis.</p

Characteristics of synovial fluid donors.

<p>Inclusion criteria: both genders, age 18–85 years inclusive, BMI<40, CRP≤3 mg/L, and all CRP levels for RA. Exclusion criteria: joint infection; severe liver or kidney disease; any surgery within the last 3 months; knee joint surgery within the last 6 months; diabetes mellitus (OA); drug abuse; intraarticular treatment with hyaluronate; or corticosteroid treatment within the last 3 months; HIV infection; tumor/cancer.</p

Postmortem stability of lipids extracted from human synovial fluid of healthy knee joints used as controls.

<p>Lipids were determined by electrospray ionization tandem mass spectrometry (ESI-MS/MS) or liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091769#s2" target="_blank"><i>Material and Methods</i></a>. Values are displayed as a scatterplot of the concentration of each lipid class and species by postmortem time. (<b>A</b>): Lipid classes, (<b>B</b>): Lipid species. SM-sphingomyelin, Cer-ceramide, HexCer-hexosylceramide (most likely glucosylceramide), Hex₂Cer-dihexosylceramide (most likely lactosylceramide), PA-phospatidic acid, LPA-lysophosphatidic acid, PG-phosphatidylglycerol, LPG-lysophosphatidylglycerol.</p

Concentrations of lipids in human synovial fluid as a function of the age of patients with late stage osteoarthritis.

<p>Lipids were determined by electrospray ionization tandem mass spectrometry (ESI-MS/MS) or liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091769#s2" target="_blank"><i>Material and Methods</i></a>. Values are displayed as a scatterplot of the concentration of each lipid class and species by age of donors. (<b>A</b>): Lipid classes, (<b>B</b>): Lipid species. SM-sphingomyelin, Cer-ceramide, HexCer-hexosylceramide (most likely glucosylceramide), Hex₂Cer-dihexosylceramide (most likely lactosylceramide), PA-phospatidic acid, LPA-lysophosphatidic acid, PG-phosphatidylglycerol, LPG-lysophosphatidylglycerol.</p

Relative distribution of HexCer and Hex₂Cer, PA, LPA, PG and LPG species in in human knee synovial fluid.

<p>Synovial fluid was obtained from donors serving as controls and from patients with early osteoarthritis (eOA), late OA (lOA), and rheumatoid arthritis (RA). The data show the percentage of the lipid species from the total corresponding lipid class ( = 100%). Values are presented as median and interquartile range. (<b>A</b>): HexCer and Hex₂Cer species, (<b>B</b>): PA and LPA species, (<b>C</b>): PG and LPG species. HexCer- hexosylceramide (most likely glucosylceramide), Hex₂Cer- dihexosylceramide (most likely lactosylceramide), PA- phosphatidic acid, LPA- lysophosphatidic acid, PG- phosphatidylglycerol, LPG- lysophosphatidylglycerol.</p

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).

<p>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.</p