Moderate hyperhomocysteinaemia and immune activation in patients with rheumatoid arthritis

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1 Clinica Chimica Acta 338 (2003) Moderate hyperhomocysteinaemia and immune activation in patients with rheumatoid arthritis Katharina Schroecksnadel a, Barbara Frick a, Sabine Kaser b, Barbara Wirleitner a, Maximilian Ledochowski b, Erich Mur b, Manfred Herold b, Dietmar Fuchs a,c, * a Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Fritz Pregl Strasse 3, A-6020 Innsbruck, Austria b Department of Internal Medicine, Leopold Franzens University of Innsbruck, Innsbruck, Austria c Ludwig-Boltzmann Institute of AIDS Research, Innsbruck, Austria Received in revised form 22 August 2003; accepted 5 September 2003 Abstract Background: Moderate hyperhomocysteinaemia related to folate deficiency has been described in patients with cardiovascular risk and also in patients with autoimmune diseases including rheumatoid arthritis (RA). Methods: In 33 patients with RA, serum concentrations of homocysteine and cysteine, of B-vitamins folate and vitamin B 12, and of immune activation markers neopterin and soluble 75-kDa TNF-receptor (stnf-r75) were measured. Results: A significant proportion of patients presented with elevated homocysteine and cysteine concentrations in comparison to reference ranges of healthy control persons. Moderate hyperhomocysteinaemia coincided with decreased serum folate and with higher concentrations of stnf-r75 and neopterin, but it was rather independent from methotrexate (MTX) therapy. Conclusions: The coincidence of higher homocysteine and lower folate concentrations with increased concentrations of immune activation markers in patients with RA suggests that immune activation could be involved in the development of hyperhomocysteinaemia. D 2003 Elsevier B.V. All rights reserved. Keywords: Rheumatoid arthritis; Homocysteine; Immune activation; Neopterin; Oxidative stress; Vitamin deficiency 1. Introduction * Corresponding author. Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Fritz Pregl Strasse 3, A Innsbruck, Austria. Tel.: ; fax: address: dietmar.fuchs@uibk.ac.at (D. Fuchs). Moderate hyperhomocysteinaemia has been established as an independent risk factor for atherosclerosis and other vascular diseases [1]. Moderate hyperhomocysteinaemia is also common in autoimmune diseases, e.g., rheumatoid arthritis (RA) [2,3], Behcet s disease [4], and Raynaud s phenomenon [5]. Hyperhomocysteinaemia in patients with RA is often associated with folate and vitamin B 12 deficiency [2]. Methotrexate (MTX), a cytostatic drug frequently used for the treatment of RA, could increase homocysteine by interfering with folate metabolism and thereby blocking methionine synthesis from homocysteine [6,7]. Folate supplementation in patients with RA being treated with MTX is able to decrease homocysteine concentrations [8,9] /$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi: /j.cccn

2 158 K. Schroecksnadel et al. / Clinica Chimica Acta 338 (2003) A relationship between increased homocysteine concentrations, antiphospholipid antibodies and thrombotic events was demonstrated in patients with RA [10]. Although the pathogenesis of RA is still unclear, the activation of T-lymphocytes and macrophages and the production of cytokines seem to play a crucial role in the initiation and perpetuation of the disease [11]. Recently, it has been demonstrated that serum soluble markers of immune activation like soluble cytokine receptors, e.g., stnf-r [12 14] and sil-2r [14 18], or neopterin [19,20], are raised in patients with RA, correlating well with disease activity. On the other hand, homocysteine was found to accumulate in supernatants of stimulated peripheral blood mononuclear cells [21] indicating that immune activation can be responsible for the development of hyperhomocysteinaemia. In this study, serum concentrations of homocysteine and cysteine were measured in patients with RA and were compared to B vitamin status and to concentrations of immune activation markers stnf-r75 and neopterin. 2. Materials and methods Thirty-one women and two men (mean age: 56.9 years F 9.2, range: years) with RA were recruited from the University Hospital of Innsbruck. Diagnosis was based on the Steinbrocker criteria. According to these criteria, 15 patients suffered from RA stage 2, the other 18 patients were classified as stage 3. All 33 patients with RA were under therapy (see details in Table 1). Of 21 patients treated with MTX, 10 were supplemented with 6 or 7.5 mg/week folate, all other patients did not receive vitamin supplementation within their treatment regimen. Patients were asked to keep a record of their diet (normal diet, no adaptation of their normal eating habits), and according to these records, vitamin intake was calculated. Patients gave informed consent to participate in this study, which was approved by the local ethics committee. Pregnant women and women in the puerperium, patients with malignant diseases or clinical relevant gastrointestinal, renal, hepatic, cardiorespiratoric, haematological, neurological, or psychiatric diseases as well as patients with metabolic disorders or chronic infections were excluded from the study. Blood samples from patients were obtained within the scope of routine blood examinations and centrifuged immediately, sera were stored at 20 jc for later analysis. Total homocysteine and cysteine were measured by reversed phase high-performance liquid chromatography (HPLC). Sixty microliters of specimens was used, and after reduction with tris-(2- carboxylethyl)-phosphine (TCEP) and derivatization with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4- sulfonate (SBD-F), separation was performed using a 55-mm cartridge, RP 18 LiChroCART 55-4 and RP 18 precolumns (Merck, Darmstadt, Germany). Homocysteine and cysteine concentrations were monitored by fluorescence detection at 385 nm excitation wavelength and 515 nm emission wavelength [22]. For the determination of concentrations of folate and vitamin B 12 double-labelled radioimmunoassay Table 1 Treatment of 33 patients with rheumatoid arthritis No therapy Therapy on demand NSAIDs (Diclofenac: mg/day; Meloxicam/Lomoxicam: 4 15 mg/day; Rheumotrop: mg/day; Ibuprofen: mg/day) Steroids (Methyl-prednisolone: 1 8 mg/day) Immunosuppressants (Methotrexate: mg/week; Cyclosporin A: mg/day; Leflunomide: 20 mg/day; Azathioprin: 100 mg; Sulfasalazine: mg/day) Opioids: (Tramadol mg/day) NSAIDs = non-steroidal anti-inflammatory drugs. Regular therapy

3 K. Schroecksnadel et al. / Clinica Chimica Acta 338 (2003) (Chiron Diagnostic, Walpole, MA, USA) was used. Concentrations of neopterin (BRAHMS Diagnostica, Berlin, Germany) and stnf-r75 (R&D Systems, Minneapolis, MN) were measured by ELISA. For statistical comparisons between subgroups of patients, non-parametric Mann Whitney U test was employed. Spearman rank correlation analysis was applied to assess correlations. p-values < 0.05 were considered to indicate statistical significance. 3. Results A significant percentage of patients with RA presented with elevated homocysteine and cysteine concentrations (Table 2) in comparison to reference ranges of healthy control persons [23,24]. In 14 of 33 patients, homocysteine concentrations above 15 AM (upper limit of normal) were observed. Concentrations of vitamin B 12 and folate were within the normal range in most of the patients (Table 2). Also, neopterin concentrations were elevated in a significant percentage of patients (Table 2). In the 14 patients with homocysteine concentrations above 15 AM, the concentrations of immune activation markers stnf-r75 and neopterin were higher than in the other 19 patients with normal homocysteine concentrations (Fig. 1). Cysteine concentrations were also higher, whereas folate concentrations were lower in the patients with hyperhomocysteinaemia. Vitamin B 12 concentrations showed only a tendency towards lower concentrations in patients with elevated homocysteine concentrations compared to the other 19 patients ( p < 0.06; Fig. 1). There existed correlations between concentrations of homocysteine and folate and between homocysteine and stnf-r75 (Fig. 2). Concentrations of vitamin B 12 and folate were associated as well (r s = 0.462; p < 0.01). Cysteine correlated with homocysteine (r s =0.713; p < 0.001) and stnf-r75 (r s = 0.487; p < 0.01); stnf-r75 concentrations also correlated with neopterin concentrations (r s = 0.607; p < 0.001). Patients treated with non-steroidal anti-inflammatory drugs (NSAIDs) had higher ( p < 0.01) and those treated with MTX had lower ( p < 0.05) vitamin B 12 concentrations. Furthermore stnf-r75 concentrations were lower in patients treated with MTX ( p < 0.05). There was no such difference of neopterin concentrations between groups with different treatment regimens. Dosage of MTX did not correlate with any of the parameters investigated. The median calculated dietary intake of vitamin B 12 was 5.0 mg/day (range: ) and 202 Ag/ day for folate, respectively (range: ), which was much lower than the recommended daily intake of 300 Ag/day. Only three patients had a folate intake higher than this recommendation. Patients with a higher folic acid intake also had a higher vitamin B 12 intake (r s =0.448, p < 0.05). Dietary intake of vitamins C and E was also below recommendations. No association was found between calculated dietary intake and blood concentrations of B vitamins. Ten of 21 MTX-treated patients were supplemented with folate, and two other patients had an calculated dietary intake of folate much higher than the recommended daily intake of 300 Ag. In patients treated with Table 2 Median and range of serum concentrations of homocysteine, cysteine, folate, vitamin B 12, stnf-r75, and neopterin in 33 patients with RA with reference ranges of these parameters and the percentage of patients with concentrations above or below the reference range Medians Range Reference limits (95th percentile) Outside reference limit Homocysteine (AM) [23] 42% increased* Cysteine (AM) [24] 85% increased** Folate (nm) [25] 12% decreased Vitamin B 12 (pm) [25] 21% decreased stnf-r75 (ng/ml) [26] 0% increased Neopterin (nm) [27] 36% increased* * p < **p <

4 160 K. Schroecksnadel et al. / Clinica Chimica Acta 338 (2003) Fig. 1. Boxplots of serum concentrations of folate and vitamin B 12 (upper) and of immune activation markers stnf-r75 and neopterin (lower) in 33 patients with RA (divided into two groups patients with homocysteine (HCy) concentrations V 15 AM and >15 AM). MTX and supplemented with folate, serum folate concentrations were higher ( p < 0.02) and homocysteine concentrations tended to be lower ( p < 0.06) in comparison with unsupplemented patients. In supplemented patients, serum folate concentrations weakly correlated with the amount of folate supplemented (r s = 0.371, p < 0.05). Inverse relationships between homocysteine and serum folate concentrations were of similar strength in MTX-treated patients with (r s = 0.612, p < 0.06) and without (r s = 0.700, p < 0.02) folate supplementation. Concentrations of stnf-r 75 and homocysteine correlated in MTXtreated patients without folate supplementation (r s = 0.718; p < 0.02) but not in supplemented patients (r s = 0.45; p>0.1). The correlation between neopterin and stnf-r75 was somewhat stronger in MTX-trea-

5 K. Schroecksnadel et al. / Clinica Chimica Acta 338 (2003) Fig. 2. Correlations of homocysteine with immune activation marker stnf-r75 (r s = 0.508; p < 0.01) and serum folate (r s = 0.519; p < 0.01) in 33 patients with RA. ted patients with folate supplementation (r s = 0.821, p < 0.01) compared to those without (r s = 0.685, p < 0.03). 4. Discussion This study confirms that patients with RA may present with increased serum homocysteine and cysteine concentrations in comparison with healthy control persons. Homocysteine concentrations in patients with RA were demonstrated to depend on vitamin availability [2,3]. In our patients, a significant inverse correlation existed between homocysteine and folate concentrations, although concentrations of B vitamins were well above the lower limit of normal in most patients. Twelve of 33 patients even presented with serum folate concentrations above 20 nm, the upper limit of normal in humans. In the 14 patients with hyperhomocysteinaemia, significantly lower concentrations of serum folate were found in comparison with the other 19 patients with low homocysteine concentrations. Also, vitamin B 12 tended to lower concentrations in patients with higher homocysteine. Interestingly, differences in marker concentrations were rather small between patients with and without MTX therapy and between MTX-treated patients with and without folate administration. In MTX-treated patients supplemented with folate, homocysteine concentrations were only slightly lower, folate concentrations slightly higher than in unsupplemented patients. These results, however must be regarded with care, because numbers in the subgroups of MTX-treated patients with/without folate were low. No significant relationship was found between calculated folic acid intake and concentrations detectable in the circulation. Interestingly, Houcher et al. [28] reported earlier that an association found between calculated folic acid intake and circulating concentrations in healthy controls could not be detected in patients with cardiovascular disease. Discrepancy between dietary intake of vitamins and circulating concentrations may relate to an increased consumption of vitamins in inflammatory conditions. Elevated concentrations of homocysteine and cysteine occurred concomitantly with increased concentrations of immune activation marker stnf-r75 and neopterin, indicating a link between immune activation and moderate hyperhomocysteinaemia. Concentrations of immune activation markers were higher in patients with lower vitamin concentrations. This association as well as the high percentage of hyperhomocysteinaemia (42%) in patients with RA per se suggests a relationship between the autoimmune

6 162 K. Schroecksnadel et al. / Clinica Chimica Acta 338 (2003) process and homocysteine metabolism. This link between higher homocysteine concentrations and immune activation was observed also in other autoimmune disorders [4,5]. Cysteine concentrations, which correlated well with homocysteine concentrations, also showed a strong association with concentrations of stnf-r75, but there was no evident association with vitamin availability. The positive correlation between homocysteine and cysteine may indicate that transsulfuration pathway of homocysteine is intact. The concentrations of homocysteine and immune activation markers did not differ between patients with RA stage 2 or stage 3. However, most patients were under treatment while blood specimens were taken, which will have influenced disease activity. In agreement, concentrations of immune activation markers usually much better correlate with the activity than with stage of RA [16 20]. Interestingly, we did not find a difference in homocysteine and folate concentrations in patients with or without MTX treatment, which contrasts earlier findings of others [6,8]. Concentrations of vitamin B 12 were lower in patients with MTX therapy and also concentrations of stnf-r75 were markedly lower in those patients indicating an influence of MTX on immune activation on the one hand, and possibly also on homocysteine metabolism on the other hand. Under regular treatment with NSAIDs, concentrations of vitamin B 12 were higher than in untreated patients ( p < 0.01), anti-inflammatory treatment therefore seems to influence vitamin B 12 availability in a positive manner. In parallel to the raised homocysteine concentrations, elevated blood concentrations of immune activation markers stnf-r75 and neopterin are found, and also the strong correlation between homocysteine and stnf-r75 indicates a link between homocysteine metabolism and immune system activation. A relationship between immune activation and hyperhomocysteinaemia was already demonstrated in patients with peripheral vascular disease [29], atherosclerosis [30,31], and also in patients with neurodegenerative disorders like dementia and Parkinson s disease [32,33]. Interestingly, oxidative stress appears to be deeply involved in the pathogenesis of all the diseases listed. Enhanced immune activation associated with production of reactive oxygen species (ROS) and developing oxidative stress may enhance consumption of antioxidant vitamins [34]. In this way, also blood concentrations of oxidation-sensitive tetrahydrofolate could be diminished by oxidative stress developing during immune activation, leading to raised homocysteine and cysteine concentrations [35,36]. Since B vitamin cofactors, necessary for the conversion of homocysteine, are oxidation sensitive, the observation accords well with the assumption that an enhanced consumption of these vitamins is likely during conditions going along with prolonged immune activation and oxidative stress. Similarly, in patients with Alzheimer s disease McCaddon et al. [37] recently suggested an enhanced consumption of vitamin B 12 as a consequence of oxidative stress impairing the metabolism of homocysteine. We conclude that immune activation, involved in the pathogenesis of RA, could be the reason for the increased requirement of B vitamins and the development of hyperhomocysteinaemia in patients. Further studies will be necessary to support the possible role of oxidative stress to increase the demand for B vitamins including folate in RA patients. Acknowledgements This work was supported by the Austrian Funds Zur Förderung der wissenschaftlichen Forschung, project 14942, and by the Austrian Federal Ministry of Social Affairs and Generations. References [1] Boushey CJ, Beresford SA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA 1995;274: [2] Pettersson T, Friman C, Abrahamsson L, Nilsson B, Norberg B. Serum homocysteine and methylmalonic acid in patients with rheumatoid arthritis and cobalaminopenia. J Rheumatol 1998;25: [3] Roubenoff R, Dellaripa P, Nadeau MR, et al. Abnormal homocysteine metabolism in rheumatoid arthritis. Arthritis Rheum 1997;40: [4] Aksu K, Turgan N, Oksel F, et al. Hyperhomocysteinaemia in Behcet s disease. Rheumatology 2001;40: [5] Marasini B, Casari S, Bestetti A, et al. Homocysteine concen-

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