α1-Microglobulin Protects Against Bleeding-Induced Oxidative Damage in Knee Arthropathies

Abstract

Knee injury increases the risk of developing knee osteoarthritis (OA). Recent evidence suggests involvement of oxidative stress induced by inflammation and bleeding in the joint. This study investigates the role in this process of α₁-microglobulin (A1M), a plasma and tissue antioxidant protein with reducing function, and heme- and radical-binding properties. We studied matched knee synovial fluid (sf) and serum (s) samples from 122 subjects (mean age 40 years, 31% females): 10 were knee healthy references, 13 had acute inflammatory arthritis (AIA), 79 knee injury 0-10 years prior to sampling, and 20 knee OA. Using immunoassays, we measured sf-A1M and s-A1M, sf-hemoglobin (sf-Hb), sf-total free heme (sf-Heme), and sf-carbonyl groups (sf-Carbonyl). We explored associations by partial correlation, or linear regression models with adjustments for age, sex and diagnosis, and evaluated diagnostic capacity by area under the receiver operator characteristics curve (AUC). The AIA group had 1.2- to 1.7-fold higher sf-A1M and s-A1M concentrations compared to the other diagnostic groups; other biomarkers showed no between-group differences. sf-A1M and s-A1M were with AUC of 0.76 and 0.78, respectively, diagnostic for AIA. In the injury group, the amount of bleeding in the joint was inversely correlated to time after injury when measured as sf-Heme (r = -0.41, p < 0.001), but not when measured as sf-Hb (r = -0.19, p = 0.098). A similar inverse association with time after injury was noted for sf-A1M (r = -0.30, p = 0.007), but not for s-A1M and sf-Carbonyl. Linear regression models showed that sf-Heme was more strongly associated with sf-A1M and sf-Carbonyl than sf-Hb. Independent of diagnosis, sf-Heme explained 5.7% of the variability in sf-A1M and 3.0% in the variability in sf-Carbonyl, but appeared unrelated to s-A1M. High sf-A1M and low sf-Heme or sf-Hb were independently associated with low sf-Carbonyl. In conclusion, our results demonstrate that independent of disease, Hb and heme within a knee joint correlates with an increased sf-A1M concentration that appears to be protective of oxidative damage, i.e., a reduction in carbonyl groups. High concentrations of A1M in synovial fluid and serum was further diagnostic for AIA.

Keywords: heme; hemoglobin; hemolysis; knee injury; knee osteoarthritis; oxidative stress; synovial fluid; α1-microglobulin.

FIGURE 1

A1M by diagnostic groups. Concentrations of A1M in (A) synovial fluid and serum and (B) as a ratio in concentrations synovial fluid to serum. Group means and 95% confidence intervals are plotted as circles and triangles with error bars for the injury, acute inflammatory arthritis (AIA) and osteoarthritis (OA) groups, and as a dashed line and green area for the reference group. (Plotted data and statistics are reported in full in Supplementary Table S1).

FIGURE 2

Oxidative stress in synovial fluid by diagnostic groups. Log₁₀ transformed concentrations in synovial fluid (sf) of (A) hemoglobin (Hb), (B) total heme, and (C) carbonyl groups. Group means and 95% confidence intervals are plotted as circles with error bars for the injury, AIA and osteoarthritis (OA) groups, and as a dashed line and green area for the reference group. sf-Carbonyl values are given relative to total protein concentration. (Plotted data and statistics are reported in full in Supplementary Table S1).

FIGURE 3

Temporal variations in biomarkers of oxidative stress after knee injury. Bivariate scatterplots of biomarker concentrations against weeks after injury with regression lines (solid) and 95% confidence intervals of the regression lines (dashed). (A, red) Blood in the joint measured as hemoglobin (Hb) or total heme in synovial fluid (sf). (B, green) Concentrations of A1M in synovial fluid and serum, and the ratio of A1M in synovial fluid to serum. (C, yellow) Oxidative damage measured as protein carbonyl groups in synovial fluid. Partial correlation coefficients (r) indicate correlations adjusted for age and sex.

FIGURE 4

Major findings in summary. (1) Having AIA in a knee joint was associated with increased A1M concentrations locally in synovial fluid (sf) and systemically in serum (s). With area under the ROC curve of close to 0.8, A1M was a biomarker for AIA in both synovial fluid and serum. (2) Independent of disease, cell-free Hb and heme within a knee joint triggered an increased synovial concentration of A1M that appeared to be protective of oxidative damage measured as sf-Carbonyl. The net amount of local oxidative damage (sf-Carbonyl) was dependent not only on the absolute concentrations of oxidative Hb and heme, or antioxidative A1M, but the ratio between pro- and antioxidative molecules appeared as important. (3) In knee injured there was a similar temporal pattern in the synovial concentrations of both oxidants (Hb and heme) and antioxidants (A1M), which were highest early after injury, without a corresponding temporal change in oxidative damage (sf-Carbonyl).