Alterations of plasma glycosaminoglycan profile in patients with rheumatoid arthritis in relation to disease activity

Clinica Chimica Acta 433 (2014) 20–27

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Alterations of plasma glycosaminoglycan profile in patients with rheumatoid arthritis in relation to disease activity Agnieszka Jura-Półtorak a,⁎, Katarzyna Komosinska-Vassev a, Anna Kotulska b, Eugeniusz J. Kucharz b, Katarzyna Klimek c, Magdalena Kopec-Medrek b, Krystyna Olczyk a a b c

Department of Clinical Chemistry and Laboratory Diagnostics, Medical University of Silesia, Katowice, Poland Department of Internal Medicine and Rheumatology, Medical University of Silesia, Katowice, Poland Department of Statistics, Medical University of Silesia, Katowice, Poland

a r t i c l e

i n f o

Article history: Received 12 September 2013 Received in revised form 24 February 2014 Accepted 25 February 2014 Available online 7 March 2014 Keywords: Plasma glycosaminoglycans Extracellular matrix remodeling Rheumatoid arthritis Disease activity

a b s t r a c t Background: Qualitative and quantitative evaluation of plasma glycosaminoglycans (GAGs) of rheumatoid arthritis (RA) patients in relation to disease activity estimated by DAS28 score was evaluated. Methods: GAGs were quantified by hexuronic acid assay and electrophoretic fractionation. Keratan sulfate (KS) and hyaluronic acid (HA) were measured by immunoassay. Results: Chondroitin/dermatan sulfate (CS/DS) and heparan sulfate/heparin (HS/H) in plasma of healthy subjects and RA patients were stated. Total GAGs, CS, HS/H and HA levels were higher in patients with high and moderate disease activity than in controls. Total GAGs and CS levels in patients with high disease activity were elevated in comparison to patients with low disease activity. HS/H levels in patients with high and moderate activity were elevated in comparison to those with low disease activity. KS levels were increased in all patient groups in comparison to controls. Total GAGs, CS, HS/H and HA levels were positively correlated with DAS28 and CRP. Conclusions: Structural tissue damage/remodeling of the extracellular matrix occurs in RA, which is reflected in the qualitative and quantitative changes of plasma GAGs. The above changes depend on DAS28 and may contribute to systemic changes in the properties of the extracellular matrix. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Rheumatoid arthritis (RA) is the most common systemic autoimmune connective tissue disease, characterized by symmetric inflammation of joints, progressive degradation of articular cartilage and periarticular tissues [1–3]. The pathogenesis of RA is not fully understood. It is assumed that a significant role in the initiation of the pathological process is played by genetic predispositions, environmental factors, bacterial or viral infections, immunological disorders or reactive oxygen species [4–8]. Changes in the extracellular matrix (ECM) components, including glycosaminoglycans (GAGs) could also play a significant role in the RA pathomechanism. GAGs are linear polysaccharide chains that consist of repeated disaccharide units composed of an amino sugar and uronic acid or galactose residues. Depending on the sugar composition of the repeat, they are classified as chondroitin/dermatan sulfate (CS/DS), heparan sulfate/heparin (HS/H), keratan sulfate (KS) and hyaluronic acid (HA). These macromolecules, being constituents of proteoglycans ⁎ Corresponding author at: Department of Clinical Chemistry and Laboratory Diagnostics, Medical University of Silesia Jedności 8, 41-200 Sosnowiec, Poland. Tel.: +48 32 364 11 50; fax: +48 32 364 11 57. E-mail address: [email protected] (A. Jura-Półtorak).

http://dx.doi.org/10.1016/j.cca.2014.02.027 0009-8981/© 2014 Elsevier B.V. All rights reserved.

(PGs), can be found in the ECM, on the surface of cells and inside of them [9]. These glycans react with numerous cytokines, growth factors and their receptors, components of extracellular matrix, enzymes and their inhibitors, thus participating – as regulatory factors – in the processes of cell migration, adhesion, proliferation and differentiation as well as in creation of the ECM structures [9–15]. GAGs appear in the circulation as the main products of catabolic process taking place in the extracellular matrix, or they are partially produced by blood cells, such as lymphocytes. Disturbed balance between biosynthesis and catabolism of PGs/GAGs as a consequence of the inflammatory process accompanying the RA should be reflected by GAG blood concentrations. During the development of RA, the increase of proinflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-17 or IL18, may decrease the biosynthesis of GAGs, while the increased activation of proteolytic enzymes, including matrix metalloproteinases, may intensify the catabolism of tissue PGs [2,3,16–18]. On the other hand, the inflammatory side effects, including excessive reactive oxygen species (ROS) formation, may contribute to the PGs' core protein oxidation as well as to the partial depolymerization of GAG chains into fragments that appear in the circulation. Moreover, ROS affect PGs' core protein indirectly by activation of latent forms of matrix metalloproteinases. However, changes of the profile of GAGs in plasma and their association with disease activity of RA patients have not been fully elucidated.

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Thus, the aim of this work was the qualitative and quantitative evaluation of plasma profile GAGs in RA patients in relation to the disease activity.

2. Materials and methods 2.1. Study population Sixty-nine patients with RA (56 women and 13 men; 53.32 ± 13.21 years old) treated in the Department of Internal Medicine and Rheumatology of Medical University of Silesia in Katowice, Poland, were studied. All patients fulfilled the diagnostic criteria for RA according to the American College of Rheumatology (ACR) [19]. Patients were assessed according to a standard protocol, which included a complete history and physical examination, including recording of swollen joint counts and tender joint counts, Health Assessment Questionnaire (HAQ) and Visual Analogue Scale (VAS) for patient assessment of disease activity and pain. Additionally, patients were submitted to following laboratory tests, such as: complete blood count, erythrocyte sedimentation rate (ESR), electrolytes, fasting glucose, fasting lipid profile, creatinine, bilirubin, liver enzymes, protein electrophoretic profile, C-reactive protein (CRP), rheumatoid factor (RF), anti-cyclic citrullinated peptide antibodies (anti-CCP) and antinuclear antibodies (ANA). The rheumatoid factor was positive in 56/69 (81.16%) of the patients, whereas anti-CCP was positive in 50/69 (72.46%) of the patients. The disease activity in RA was estimated by the Disease Activity Score in 28 joints (DAS28). Patients were divided into three groups based on their disease activity score: those with DAS28 of 5.1 or more were considered to have high disease activity (DAS I); patients with DAS28 of 3.2 to 5.1 were considered to have medium disease activity (DAS II) and patients with DAS28 of 3.2 or less were considered to have low disease activity (DAS III) [20]. RA patients were treated with disease modifying anti-rheumatic drugs, i.e. methotrexate (61 patients) or sulfasalazine (8 patients). They were not given intraarticular injection of steroids, chondroitin polysulfate or hyaluronic acid for at least 3 months prior to this study. The demographic, clinical and laboratory data of the patients are shown in Table 1. Twenty-two age-matched healthy volunteers (18 women and 4 men) of Medical University of Silesia, Katowice, Poland were investigated as controls. Subjects were screened by means of medical history, physical examination and laboratory analyses. Exclusion criteria were the presence of medical or surgical illness (clinical evidence of arthritis or any other inflammatory disease, diabetes mellitus, cancer or kidney

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and liver diseases), hospital admissions during the previous 3 years, current smoking, alcohol abuse, treatment for hyperlipidemia, antihypertensive or vasoactive medication and non-steroidal anti-inflammatory drugs. Healthy subjects enrolled into this study had results of morphological and biochemical analyses (complete blood count, ESR, electrolytes, fasting glucose, fasting lipid profile, creatinine, bilirubin, liver enzymes, protein electrophoretic profile, CRP and RF) within the reference range. The venous blood samples were obtained after an overnight fasting and were collected into citrate-treated (extraction and determination of plasma GAGs) and heparin-treated (measurement of plasma KS and HA levels) tubes. Plasma samples were divided into portions and stored at −70 °C until being used for measurement of GAGs. The Pathozyme ElisaSure Kit (Ref. OD707, Omega Diagnostics Ltd, Scotland, UK) was used to control linearity and precision of a microplate reader (Infinite M200, Tecan), as well as efficiency and reproducibility of the HydroxFlex microplate washer (HydroFlex, Tecan), and the precision of automatic pipettes. During the entire investigation period we followed the guidelines and regulations of the Helsinki Declaration in 1975, as revised in 1983 and the experiments were approved by the Ethical Committee of the Medical University of Silesia in Katowice, Poland; all healthy volunteers and RA patients signed an informed consent form.

2.2. Extraction and determination of plasma glycosaminoglycans The determinations of total glycosaminoglycans and particular types of GAGs were performed on the isolated plasma samples. These glycans were isolated using the method of Volpi et al. [21] and Olczyk et al. [22]. GAGs were released from plasma PG core proteins by alkaline treatment after extensive papain digestion. The total of 25 mg of papain was added to 1 ml of plasma and samples were incubated for 24 h at 65 °C with stirring. After boiling for 10 min at 100 °C, the mixture was brought to pH 9.0 by adding 0.1 N NaOH. After 24 h at 40 °C, the mixture was neutralized with 100% (w/v) trichloroacetic acid (TCA) to a final concentration of 7%. The samples were incubated for 24 h at 4 °C. Then the mixture was centrifuged at 18,000 ×g, 20 min, 4 °C, and the pellet was washed twice with 7% TCA. The precipitate was discarded. To the combined supernatants, 3 volumes of 96% ethanol were added and GAGs were allowed to precipitate for 24 h at 4 °C. After centrifugation (18,000 ×g, 20 min, 4 °C), 1 ml of 0.5 M aqueous CH3COOK was added to the precipitate. The GAG solution obtained was treated with 3 volumes of 96% ethanol. The GAGs were allowed to precipitate for 24 h at 4 °C. After centrifugation (18,000 ×g, 20 min, 4 °C), the precipitate

Table 1 Clinical and laboratory characteristics of the patients with rheumatoid arthritis. Groups of patients with rheumatoid arthritis

Gender female/male (n) Age (yr)a Disease duration (yr)b Morning stiffness (h)b DAS28a ESR (mm/h)b CRP (mg/l)b RF (IU/ml)b % RF positive (n) Anti-CCP (IU/ml)b % Anti-CCP positive (n) ANA (IU/ml)b

Patients with high disease activity (DAS 28 N 5.1)

Patients with medium disease activity (5.1 N DAS 28 N 3.2)

Patients with low disease activity (DAS 28 b 3.2)

22 F/7 M 55.69 ± 11.67 8 (6–13.5) 2 (0.5–3.75) 6.17 ± 0.94 36 (23–65) 24.4 (6.8–59.7) 92 (36–575) 86.21 (25/29) 361.5 (105–1065.25) 79.31% (23/29) 10.6 (6.1–12.4)

21 F/4 M 53.44 ± 14.24 6.5 (3.25–16.75) 1 (0.5–4) 4.33 ± 0.43 20 (16–30) 11.9 (4–21.7) 30 (29–160.5) 80 (20/25) 73 (20–182) 68% (17/25) 17.9 (8.3–28.03)

13 F/2 M 52.26 ± 11.89 5.5 (4–10.25) b0.2 2.96 ± 0.17 16 (14–17.75) b5 29 (19–30) 73.33 (11/15) 82 (40–100) 66.66% (7/15) 17.45 (9.6–29.73)

F, female; M, male; DAS28, disease activity score using 28 joints; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; RF, rheumatoid factor; anti-CCP, anti-cyclic citrullinated peptide antibodies; ANA, antinuclear antibodies. a Values given as mean ± SD. b Values given as median and interquartile (25th–75th percentile) range.

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was dissolved in 1 ml of H2O and GAGs were isolated by precipitation after addition of 0.02 ml of 5% (w/v) cetylpyridinium chloride (CPC) according to Scotta [23]. After incubation (24 h at 24 °C) and centrifugation (15,000 ×g, 30 min, 24 °C), GAGs precipitated by CPC were finally washed with 2 ml of 96% ethanol containing NaCl. After incubation (2 h at 4 °C), the solution of GAGs was centrifuged (15,000 ×g, 30 min, 24 °C) and the GAG precipitate was washed with 2 ml of 99.8% ethanol. The mixture was incubated for 24 h at 4 °C and once again centrifuged (15,000 ×g, 30 min, 4 °C). The supernatant was discarded, the final GAG precipitate (isolated and cleared plasma GAGs) was dried in 21 °C and samples were stored at − 20 °C until being used for biochemical analysis. The total amount of GAGs was quantified by a hexuronic acid assay according to Filisetti-Cozzi and Carpita [24]. Testing of all samples was completed in 1 day, so inter-assay variation was insignificant. The intra-assay variability of total GAGs was less than 7%. 2.3. Assay of plasma glycosaminoglycans Samples of isolated plasma GAGs (6 μg of hexuronic acid) were submitted to electrophoresis on cellulose acetate, before and after the use of agents eliminating specifically particular GAG types. The following GAG digestion factors were used: chondroitinase AC, chondroitinase ABC— separately and in combination with heparinase I and heparinase III. Chondroitinase AC was obtained from Seikagaku Corporation (Japan). Chondroitinase ABC, heparinase I and heparinase III were obtained from Sigma Aldrich (USA). 2.3.1. Digestion with chondroitinase AC Cleavage with chondroitinase AC, which specifically leads to degradation of C-4/6-S, retained DS and HS/H. An aqueous solution of GAGs (20 μl) was mixed 20 μl of Tris–HCl buffer (0.4 M, pH 7.3, containing 0.4 M sodium acetate and 0.1% (w/v) bovine serum albumin (BSA)) and incubated for 2 h at 37 °C with 20 μl chondroitinase AC solution (0.015 U). Boiling for 3 min at 100 °C stopped the reaction and the mixture was centrifuged 15,000 ×g for 30 min at 4 °C. The supernatant was supplemented with 3 volumes of 99.8% ethanol. GAGs were allowed to precipitate for 24 h at 4 °C. After centrifugation (15,000 ×g, 30 min, 4 °C), the precipitate containing non-digested GAGs was dissolved in double-distilled water and subjected to electrophoresis. 2.3.2. Digestion with chondroitinase ABC Chondroitinase ABC was used to remove C-4/6-S, DS and HA and retain HS/H. An aqueous solution of GAGs (20 μl) was mixed with 20 μl of Tris–HCl buffer (0.05 M, pH 8.0, containing 0.06 M sodium acetate and 0.02% (w/v) BSA) and incubated for 24 h at 37 °C with 20 μl chondroitinase ABC solution (0.01 U). Boiling for 3 min at 100 °C stopped the reaction and the mixture was centrifuged 15,000 ×g for 30 min at 4 °C. The supernatant was supplemented with 3 volumes of 99.8% ethanol. GAGs were allowed to precipitate for 24 h at 4 °C. After centrifugation (15,000 ×g, 30 min, 4 °C), the precipitate containing non-digested GAGs was dissolved in double-distilled water and subjected to electrophoresis. 2.3.3. Digestion with chondroitinase ABC, heparinase I and heparinase III Heparinase I and heparinase III were used to remove HS/H. After the enzymatic depolymerization of GAGs with chondroitinase ABC, 20 μl aliquot of sample solution was carried out in 0.04 M Tris–HCl buffer, pH 7.5, containing 0.1 M sodium chloride, 0.008 M calcium chloride and 0.01% (w/v) BSA and incubated together with heparinase I solution (0.5 U) and heparinase III solution (0.06 U) for 24 h at 25 °C. Boiling for 3 min at 100 °C stopped the reaction and the mixture was centrifuged 15,000 ×g for 30 min at 4 °C. The supernatant was supplemented with 3 volumes of 99.8% ethanol. GAGs were allowed to precipitate for 24 h at 4 °C. After centrifugation (15,000 ×g, 30 min, 4 °C), the precipitate

containing non-digested GAGs was dissolved in double-distilled water and subjected to electrophoresis. 2.3.4. Electrophoresis of glycosaminoglycans Electrophoretic fractioning of GAGs was performed in 0.034 M aluminum sulfate buffer (pH 2.6), at 150 V and 6 mA per acetate plate for 2 h at 21 °C. The cellulose acetate strips were stained with 0.15% (w/v) Alcian blue in solution containing 96% ethanol, re-distilled water and glacial acetic acid (10:14:1, v/v/v), according to Hronowski and Anastassiadesa [25]. After 24 h, the strip was rinsed off with solution containing 96% ethanol, re-distilled water and glacial acetic acid (10:14:1, v/v/v), without Alcian blue. The strips were first exposed to 98% methanol for 5 min and then to the mixture consisting of 84 ml of 98% methanol and 16 ml of 99% acetic acid. The identity of electrophoretic bands was confirmed by comparison of the electrophoretic patterns of plasma GAG samples submitted to electrophoresis without any previous treatment (Fig. 1, lane 1) and depolymerized with agents specifically eliminating particular GAG types (Fig. 1, lanes 2–4). Treatment of isolated plasma GAGs with chondroitinase AC made it possible to destroy CS and HA and retain DS and HS/H. Comparison of electrophoretic patterns of intact material containing CS, DS and HS/H (line 1) with those obtained after chondroitinase AC digestion (line 2) allowed localizing CS in electrophoregrams (line 1–line 2 = CS). Digestion of isolated GAG samples with chondroitinase ABC was performed in order to remove CS and DS. Material resistant to chondroitinase AC but susceptible to chondroitinase ABC was identified as dermatan sulfates (DS) (line 2– line 3 = DS). Heparan sulfates and heparin (HS/H) were identified as material resistant to chondroitinase ABC but susceptible to heparinase I and heparinase III digestion (line 3–line 4 = HS/H). The electrophoregrams obtained were quantitatively analyzed by system for documentation and analysis of gels—G:BOX, Syngene Company (USA). 2.4. Measurement of plasma keratan sulfate level KS levels in plasma were measured in duplicate using an immunoassay kit (Wuhan EIAab Science Co., Ltd. China) according to the manufacturer's instructions. The minimal detectable KS levels were less than 0.078 ng/ml. Detection range was 0.312–20 ng/ml. All samples were tested in 1 day; thus, inter-assay was insignificant. The intra-assay coefficient of variation was less than 7%. 2.5. Measurement of plasma hyaluronic acid level HA levels were measured in duplicate using an enzyme-linked immunosorbent assay kit (Chugai, Reads Medical Products, Westminster CO) according to the manufacturer's instructions. The HA test kit uses a naturally occurring hyaluronic acid binding protein (HABP) from bovine cartilage to specifically capture HA and an enzyme-conjugated version of the HABP to detect and measure the HA captured from the human serum or plasma. The minimum detectable HA level was 10 ng/ml. Testing of all samples was completed in 1 day, so interassay variation was insignificant. The intra-assay variation of the HA levels was less than 6%. 2.6. Statistical analysis Data analyses were performed using the StatSoft, Inc. (2011), STATISTICA (data analysis software system), version 10. www.statsoft. com. The normality of the distribution was verified using the Shapiro– Wilk test. Variables are summarized as mean ± SD (for normal distribution) or median and interquartile ranges (IQR) (for abnormal distribution). Statistical differences between groups were determined by non-parametric Kruskal–Wallis ANOVA, followed by multiple post-

A. Jura-Półtorak et al. / Clinica Chimica Acta 433 (2014) 20–27 Control

DAS I

DS

CS, DS

CS, DS, HS/H

CS, DS, HS/H

CS, DS, HS/H

HS/H HS/H

Start

Start

1

2

3

1

4

2

3

4

DAS III

DAS II

CS, DS

CS, DS CS, DS, HS/H CS, DS, HS/H

CS, DS, HS/H HS/H HS/H

HS/H Start

Start

1

2

3

4

1

2

3

4

Fig. 1. Electrophoretic resolution of glycosaminoglycans isolated from plasma of healthy subjects and patients with rheumatoid arthritis. Control—healthy subjects, DAS I—patients with high disease activity, DAS II—patients with moderate disease activity, DAS III—patients with low disease activity. Lane 1, sample containing plasma GAGs submitted to electrophoresis without any previous treatment (CS, chondroitin sulfate; DS, dermatan sulfate; HS/H, heparan sulfate/heparin); lane 2, sample resistant to the action of chondroitinase AC; lane 3, samples resistant to the action of chondroitinase ABC; lane 4, resistant to the action of chondroitinase ABC and heparinase I and heparinase III. Comparison of electrophoretic patterns of intact material containing CS, DS and HS/H (line 1) with those obtained after chondroitinase AC digestion (line 2) allowed to localize CS in electrophoregrams (line 1– line 2 = CS). Material resistant to chondroitinase AC but susceptible to chondroitinase ABC was identified as DS (line 2–line 3 = DS). HS/H were identified as material resistant to chondroitinase ABC but susceptible to heparinase I and heparinase III digestion (line 3– line 4 = HS/H).

hoc comparisons for all pairs. P values of less than 0.05 were considered to indicate significant differences in the above analysis. Spearman rank correlation coefficient was used to evaluate correlation between plasma GAGs and disease activity indicators, inflammatory and autoimmune indices as well as duration of disease in RA patients. The significance in case of multiple comparisons was assessed against a reference p value obtained after applying Bonferroni correction (p b 0.05/7 possible comparisons).

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with low disease activity (Fig. 2A). Furthermore, for the patients with RA, there were significant positive correlations between plasma of total GAG levels with DAS28 (r = 0.48; p b 0.0001), ESR (r = 0.46; p b 0.0002) and CRP (r = 0.47; p b 0.0002), respectively (Table 2). The electrophoretic analysis of plasma GAGs allowed to identify: CS, DS and HS/H in plasma of healthy subjects and RA patients (Fig. 1). The majority of GAGs were susceptible to digestion by chondroitinase AC and chondroitinase ABC, respectively, and were identified as CS. These glycans were the predominant type of plasma GAGs in all the investigated subject groups (70.31%–82.53% of total GAGs); (Fig. 3). CS isolated from plasma of patients with RA were characterized by the increased structural heterogeneity and different mobility of these compounds as compared to healthy subjects. A lower amount of GAGs was resistant to chondroitinase AC but sensitive to chondroitinase ABC, and these were identified as DS. Two or three DS fractions of different mobility were found in plasma of the patients with RA in contrast to three migration fractions in healthy subjects. The relative contribution of DS to the total plasma GAGs pool was found to be from 6.10% to 7.76% in all the investigated subject groups (Fig. 3). GAGs resistant to chondroitinase ABC depolymerization and susceptible to heparinase I and heparinase III action were identified as HS/H. Plasma HS/H was expressed at approximately 7.76%–18.53% of total GAGs, representing the second quantitative fraction of GAGs in all the investigated subject groups (Fig. 3). Moreover, enhanced levels of HS/H in all RA patient groups were characterized by significant structural heterogeneity of these compounds. Three HS/H fractions of different mobility were found in plasma of the all RA patient groups in contrast to two HS/H fractions in plasma of healthy subjects. Moreover, there was a significant difference in plasma CS levels between RA patients with high and moderate disease activity in comparison to healthy subjects (Fig. 2B). In addition, significant differences in plasma CS levels existed between patients with high disease activity and patients with low disease activity (Fig. 2B). Significant positive correlations existed between plasma of CS levels and DAS28 (r = 0.31; p b 0.0017), and CRP (r = 0.40; p b 0.0018), respectively (Table 2). Plasma DS levels in all groups of RA patients were not significantly different from those in healthy subjects (Fig. 2C). There were no correlations between plasma of DS levels and any parameters evaluated (Table 2). Levels of plasma HS/H were significantly higher in patients with high and moderate disease activity in comparison to healthy subjects. In addition, significant differences in plasma HS/H level existed between patients with high disease activity and patients with moderate disease activity, on one side, and patients with low disease activity, on the other one; (Fig. 2D). HS/H plasma levels in RA patients were positively correlated with DAS28 (r = 0.65; p b 0.0000), ESR (r = 0.52; p b 0.0000) and CRP (r = 0.45; p b 0.0003), respectively (Table 2). Plasma KS was expressed at approximately 0.2% of total GAGs on healthy subjects to 0.64%–0.8% of total GAGs of patients with RA, representing the lowest quantitative fraction of GAGs in all groups (Fig. 3). Levels of plasma KS were significantly increased in all the disease activity groups of RA patients in comparison to healthy subjects (Fig. 2E). Plasma HA levels of patients high and moderate disease activity were significantly higher than those of healthy subjects (Fig. 2F). There were no significant differences between plasma HA levels of patients with low disease activity and healthy subjects (Fig. 2F). For the patients with RA, there was a significant positive correlation between plasma HA concentrations and DAS28 (r = 0.36; p b 0.0044), ESR (r = 0.23; p b 0.0067) and CRP (r = 0.33; p b 0.0061), respectively (Table 2). 4. Discussion

3. Results Total plasma GAG levels were significantly higher in patients with high and moderate disease activity in comparison to healthy subjects (Fig. 2A–F). In addition, significant differences in total plasma GAG levels existed between patients with high disease activity and patients

We have shown quantitative and qualitative changes in plasma GAGs isolated from RA patients. The rise in plasma total GAGs level resulted mainly from increased CS and HS/H levels and may be explained mainly as a consequence or a cause of tissue ECM remodeling; however, active processes related with structural tissue damage/remodeling

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tGAGs [g/ml]

ab

B

A a

CS [g/ml]

ab

Control

DAS I

DAS II

DAS III

a

Control

DAS I

DAS II

DAS III

D

C DS [g/ml]

HS/ [g/ml]

ab

ab

Control

DAS I

DAS II

DAS III

Control

DAS I

E

DAS II

a

DAS III

F

a a

Control

DAS I

DAS II

DAS III

HA [ng/ml]

KS [ng/ml]

a a

Control

DAS I

DAS II

DAS III

Fig. 2. A–F. Plasma glycosaminoglycans (GAGs) level and distribution pattern of particular types of GAGs in healthy subjects and patients with rheumatoid arthritis. Control—healthy subjects, DAS I—patients with high disease activity, DAS II—patients with moderate disease activity, DAS III—patients with low disease activity. CS, chondroitin sulfate; DS, dermatan sulfate; HS/H, heparan sulfate/heparin, KS, keratan sulfate; HA, hyaluronic acid. Results are expressed as median, interquartile (25th–75th percentile) range and minimum, maximum of all data in the groups. Data were analyzed using a non-parametric Kruskal–Wallis ANOVA, followed by multiple post-hoc comparisons for all pairs. ap b 0.05, compared to healthy subjects; bp b 0.05, compared to patients with low disease activity.

during inflammatory condition and disease activity are closely interrelated. Until now, only the one study has described changes of plasma GAGs in RA patients [26]. Friman et al. [26] have stated that in patients suffering from active, erosive form of RA, total plasma GAG content was not different as compared to controls. The above mentioned authors did not evaluate, however, the particular types of plasma GAGs in RA patients [26]. Our study showed the presence of CS, DS and HS/H in all RA patients. Additionally, structural diversity of sulfated types of GAGs was expressed by different electrophoretic mobility and dye affinity of analyzed molecules. The above changes are probably caused by different lengths of polysaccharide chains of these glycans as well as by their different content of sulfated, glucuronic and iduronic groups. Changes in sulfation of GAGs may result in disruptions of interaction of these glycans with many types of molecules, including enzymes and their inhibitors, growth factors and their receptors, transcription factors or ECM proteins leading to systemic changes of the matrix remodeling in the course of RA [10,11,27].

A major quantitative fraction of GAGs found in our study was CS, which additionally increased with disease activity. Murata et al. [28] stated that serum CS is elevated in inflammatory diseases and in the early stage of wound healing. Little information is available on plasma CS levels in joint diseases. In this study, CS plasma levels in patients with high and moderate diseases activity were significantly higher than in healthy subjects. Generally, increased amounts of metabolite indicate increased tissues turnover. Poole et al. [29] analyzing epitope CS-846 as a marker of CS synthesis reported that CS turnover in serum increase above 50% in RA patients. Thus elevated plasma CS levels in RA patients may indicate both an increased turnover in joint tissues and disease activity. KS was the subsequent analyzed plasma GAG type in our study and was found to be elevated in all RA patients. The above GAG fraction is present mainly in the aggrecan, a large PG of the hyaline, fibrous and elastic extracellular matrix of the cartilage. Only small amounts of KS antigens are present in the blood of healthy subjects [30,31]. It may indicate that most of KS found in plasma should come from the

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Table 2 Correlation between plasma glycosaminoglycans (GAGs) and disease activity indicators, inflammatory and autoimmune indices as well as duration of disease in rheumatoid arthritis patients.

Disease duration DAS28 ESR CRP RF Anti-CCP ANA

tGAGs

CS

DS

HS/H

KS

HA

0.05 NS 0.48 (0.0001) 0.46 (0.0002) 0.47 (0.0002) 0.19 NS −0.29 NS −0.01 NS

−0.03 NS 0.31 (0.0017) 0.32 NS 0.40 (0.0018) 0.11 NS 0.18 NS −0.14 NS

0.25 NS 0.15 NS 0.05 NS 0.12 NS −0.44 NS 0.12 NS 0.16 NS

0.09 NS 0.65 (0.0000) 0.52 (0.0000) 0.45 (0.0003) 0.22 NS 0.13 NS 0.06 NS

−0.14 NS −0.08 NS −0.02 NS −0.15 NS −0.02 NS −0.45 NS −0.20 NS

0.22 NS 0.36 (0.0044) 0.23 (0.0067) 0.33 (0.0061) 0.12 NS 0.21 NS 0.13 NS

Note: Spearman rank correlation coefficients. Correlations were considered significant at: p b 0.0071 by applying a Bonferroni correction; NS—not significant. tGAGs, total amounts of glycosaminoglycans; CS, chondroitin sulfate; DS, dermatan sulfate; HS/H, heparan sulfate/heparin; KS, keratan sulfate; HA, hyaluronic acid; DAS28, disease activity score using 28 joints; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; RF, rheumatoid factor; anti-CCP, anti-cyclic citrullinated peptide antibodies; ANA, antinuclear antibodies.

degradation of cartilage aggrecan [32]. Moreover, the lack of differences in KS plasma levels in patients with particular disease activities suggests that circulating KS is not useful in the monitoring of disease activity of joint inflammation and should be rather seen as a potential marker of structural ECM tissue remodeling. In contrast to KS, HS/H could be helpful in assessing RA activity. The increased levels of plasma HS/H in RA patients correlated with both DAS28 and inflammatory markers in our study. However, these relations have been evaluated yet in clinical studies. On the other hand, the evaluated urinary excretion of HS/H was analyzed in RA patients founding it as an early marker of renal involvement in the course of RA [33]. Increased plasma levels of HS/H in RA patients found in our study may presumably be related with increased catabolism of these glycans caused by greater activity of enzymes, i.e. heparanase, degrading the

9,71% HS/H

0,2% KS 0,27% HA

7,76% DS

polysaccharide chains of these glycans [27,34]. This suggestion is based on the results of examinations evaluating the effect of heparanase on the metabolism of PGs/GAGs in the course of inflammatory diseases [27]. Due to the universal presence and various roles played by HS/H in ECM, their increased degradation in the course of inflammatory processes caused by heparanase influences directly all the functions of these glycans [15,27,35,36]. It was demonstrated that the intensified stimulation of heparanase is an intermediary in degradation of syndecan 1 and HS/H chains, which constitutes an important mechanism of chronic inflammation [37]. Increased concentration of heparanase was also found in synovial fluid of RA patients, indicating the key role of this enzyme in the degradation of articular tissues and progress of RA [27,38]. A close correlation found in our study between increased plasma levels of HS/H and disease activity probably indicates important relations

0.64% KS

3,58% HA

18,67% HS/H

6,69% DS 82,06% CS

70,42% CS

DAS I

Control

0,8% KS

0,72% KS

3,42% HA

2,52% HA

8,13% HS/H 6,1% DS

18,53% HS/H

6,94% DS 70,31% CS

DAS II

82,53% CS

DAS III

Fig. 3. Percentage profile of glycosaminoglycans types in plasma of healthy subjects and patients with rheumatoid arthritis. Control—healthy subjects, DAS I—patients with high disease activity, DAS II—patients with moderate disease activity, DAS III—patients with low disease activity. CS, chondroitin sulfate; DS, dermatan sulfate; HS/H, heparan sulfate/heparin, KS, keratan sulfate; HA, hyaluronic acid.

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between transformations of those glycans with the ongoing inflammatory condition and progressive degradation of articular tissues and systemic complications in the course of RA. HA was another type of GAGs in our study closely related to both DAS28 and inflammatory markers, such as ESR and CRP. The outcomes of this study correspond to a significant extent with the results of Majeed et al. [39] and Emlen et al. [40]. They demonstrated an increase in HA concentration in the serum of RA patients [39,40]. Additionally, Emlen et al. [40] found a positive correlation between blood HA level in RA patients and the disease activity score, evaluated on the basis of the number of swollen joints, and observed that a high concentration of this glycan in blood was related with exacerbation of sickness and progression of pathological changes in joints. The observed changes in HA plasma levels may constitute an expression of increased enzymatic and non-enzymatic degradation of this glycan, accompanying the RA. The products of incomplete tissual depolymerization of HA, i. e. micromolecular fragments of this compound and revealing various molecular weight, reach the circulatory system through lymph vessels and create plasma HA pool [14,41]. This is probably the main cause of the increased level of HA in circulation observed in our study, especially since other studies have shown that among all the GAG types it is HA that undergoes to the largest extent the free radical degradation in the tissues [41–43]. Moreover, total GAGs as well as all types of these macromolecules not correlated with disease duration although tissue damage in RA generally progress over time. The possible explanation could be related with the fact, that RA patients were diagnosed in different disease activity stage. Various age at diagnosis, age at symptom development or symptom duration RA is probably related with different tissue PGs/GAGs turnover [44]. Older age at diagnosis of the disease is usually associated with greater disease activity and characterized by changes in systemic and extra-articular complications. Furthermore, RA is a chronic disease, and disease activity is a fluctuating process, with great variation even during 1 day and during longer time periods [45,46]. Thus, the relationship between tissue PGs/GAGs damage progression is highly individualized. Plasma GAGs levels seem to be more determined by disease activity expressed by means of the DAS28 than disease duration in RA patients. On the other hand, significant positive correlation between RA disease activity and plasma levels of total GAGs, CS, HS/H and HA found in our study could indicate that plasma GAGs could be used as biomarkers for disease activity. However, DS and KS, i.e. GAG fractions which come almost exclusively from ECM localized tissue PGs, did not correlate with RA activity. Moreover, differences between high and low disease activity were found only for total GAG, CS and HS/H levels. Summing up, plasma GAGs seem to reflect active processes related with structural tissue damage/remodeling during inflammatory condition; however, diseases activity and ECM remodeling are closely interrelated processes. Acknowledgments This study was supported by the grant number KNW-2-098/08 from the Medical University of Silesia in Katowice, Poland. The authors thank Jadwiga Jura for technical support in extraction and determination of plasma glycosaminoglycans. The authors have declared no conflicts of interest. References [1] Lee DM, Weinblatt ME. Rheumatoid arthritis. Lancet 2001;358:903–11. [2] Goldring SR. Pathogenesis of bone and cartilage destruction in rheumatoid arthritis. Rheumatology (Oxford) 2003;42:11–6. [3] Kopec-Medrek M, Kotulska A, Widuchowska M, Adamczak M, Więcek A, Kucharz EJ. Plasma leptin and neuropepitide Y concentrations in patients with rheumatoid arthritis treated with infliximab, a TNF-α antagonist. Rheumatol Int 2012;32:3383–9. [4] Onozaki K. Etiological and biological aspects of cigarette smoking in rheumatoid arthritis. Inflamm Allergy Drug Targets 2009;8:364–8.

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