Inflammation associated anemia and ferritin as disease markers in SLE

Abstract

Introduction: In a recent screening to detect biomarkers in systemic lupus erythematosus (SLE), expression of the iron storage protein, ferritin, was increased. Given that proteins that regulate the storage, transfer and release of iron play an important role in inflammation, this study aims to determine the serum and urine levels of ferritin and of the iron transfer protein, transferrin, in lupus patients and to correlate these levels with disease activity, inflammatory cytokine levels and markers of anemia.

Methods: A protein array was utilized to measure ferritin expression in the urine and serum of SLE patients and healthy controls. To confirm these results as well as the role of the iron transfer pathway in SLE, ELISAs were performed to measure ferritin and transferrin levels in inactive or active SLE patients and healthy controls. The relationship between ferritin/transferrin levels and inflammatory markers and anemia was next analyzed.

Results: Protein array results showed elevated ferritin levels in the serum and urine of lupus patients as compared to controls, which were further validated by ELISA. Increased ferritin levels correlated with measures of disease activity and anemia as well as inflammatory cytokine titers. Though active SLE patients had elevated urine transferrin, serum transferrin was reduced.

Conclusion: Urine ferritin and transferrin levels are elevated significantly in SLE patients and correlate with disease activity, bolstering previous reports. Most importantly, these changes correlated with the inflammatory state of the patients and anemia of chronic disease. Taken together, altered iron handling, inflammation and anemia of chronic disease constitute an ominous triad in SLE.

Figure 1

Serum and urine ferritin levels are increased in patients with lupus. Ferritin levels in serum and urine of healthy (N = 3), SLE (N = 5) and LN (N = 5) patients were determined using a protein array (Raybiotech, Norcross, GA, USA). Serum ferritin levels were increased by 2.5-fold in SLE patients as compared to healthy controls, and the expression was 18.7-fold higher in the urine of LN patients compared to healthy controls. An unpaired Student's t-test with Welch's correction was applied to compare the means between groups. Data represent the mean ± standard error of mean (SEM). * represents P = 0.02.

Figure 2

ELISA analysis of serum ferritin and its correlation with disease activity and serum creatinine levels. Ferritin levels in serum of healthy (N = 9), inactive SLE (N = 10), and active SLE (N = 18) patients were determined by ELISA (Raybiotech, Norcross, GA, USA). Serum ferritin levels were increased 6.3-fold in active SLE patients compared to healthy controls and 9.8-fold higher in active SLE patients (P = 0.014) when compared with inactive SLE patients. Serum ferritin levels correlated significantly with SLEDAI scores (R = 0.56, P = 0.001) and serum creatinine levels (R = 0.54, P = 0.001).

Figure 3

ELISA analysis of urine ferritin and its correlation with disease activity, urine protein/Cr levels, and complement levels. Ferritin levels in urine of healthy (N = 9), inactive SLE (N = 10), and active SLE (N = 17) patients were determined by ELISA (Raybiotech, Norcross, GA, USA). Expression of ferritin in the urine of active SLE patients were increased 12-fold as compared to healthy controls (P < 0.001), and by 3-fold as compared to inactive SLE patients (P = 0.02). Ferritin expression was 4-fold higher in inactive SLE patients as compared to healthy controls (P = 0.03). Data represent the mean ± SEM. Urine ferritin/Cr levels correlated significantly with urine protein/Cr levels (R = 0.43, P = 0.013), with SLEDAI (R = 0.25, P = 0.096), and with serum C3/C4 levels (R = 0.301, P = 0.067).

Figure 4

ELISA analysis of urine and serum transferrin levels. Transferrin levels in urine of healthy controls (N = 10), inactive SLE (N = 10) and active SLE (N = 10) patients were measured by ELISA (Genway Biotech, San Diego, CA, USA). Transferrin expression was increased by 87-fold (P = 0.002) in active SLE patients compared with healthy controls, and by 14-fold (P = 0.003) when compared with inactive SLE patients. Urine transferrin/UCr levels correlated significantly with urine ferritin/Cr levels (R = 0.45, P = 0.005), disease activity, (R = 0.41, P = 0.033), and SLEDAI (R = 0.418, P = 0.033). Data represent mean ± SEM.

Figure 5

Correlation of serum and urine ferritin levels with inflammatory cytokine levels. Serum and urine ferritin levels determined using ELISA were correlated with inflammatory cytokine levels. Urine ferritin levels correlated with urine IL-6 (r = 0.68, P < 0.001) and serum IL-6 (r = 0.56, P < 0.0001).

Figure 6

Correlation of serum and urine ferritin with hematocrit (Hct) and hemoglobin (Hgb). Serum and urine ferritin levels measured by ELISA were correlated with Hct and Hgb. Serum ferritin correlated negatively with Hct (R = -0.42, P = 0.012) and Hgb (R = -0.47, P = 0.006). Urine ferritin levels also correlated negatively with Hct (R = -0.37, P = 0.028) and Hgb (R = -0.25, P = 0.110), although the correlation with Hgb was comparatively weaker.