www.elsevier.com/locate/clinchim

Clinica Chimica Acta 338 (2003) 157–164

Moderate hyperhomocysteinaemia and immune activation in

patients with rheumatoid arthritis

Katharina Schroecksnadela, Barbara Fricka, Sabine Kaserb, Barbara Wirleitnera,Maximilian Ledochowskib, Erich Murb, Manfred Heroldb, Dietmar Fuchsa,c,*

a Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Fritz Pregl Strasse 3, A-6020 Innsbruck, AustriabDepartment of Internal Medicine, Leopold Franzens University of Innsbruck, Innsbruck, Austria

cLudwig-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 B12, 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

Moderate hyperhomocysteinaemia has been estab-

lished as an independent risk factor for atherosclerosis

and other vascular diseases [1]. Moderate hyperho-

mocysteinaemia is also common in autoimmune dis-

0009-8981/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.cccn.2003.09.003

* Corresponding author. Institute of Medical Chemistry and

Biochemistry, University of Innsbruck, Fritz Pregl Strasse 3, A-

6020 Innsbruck, Austria. Tel.: +43-512-507-3519; fax: +43-512-

507-2865.

E-mail address: dietmar.fuchs@uibk.ac.at (D. Fuchs).

eases, e.g., rheumatoid arthritis (RA) [2,3], Behcet’s

disease [4], and Raynaud’s phenomenon [5]. Hyper-

homocysteinaemia in patients with RA is often asso-

ciated with folate and vitamin B12 deficiency [2].

Methotrexate (MTX), a cytostatic drug frequently

used for the treatment of RA, could increase homo-

cysteine by interfering with folate metabolism and

thereby blocking methionine synthesis from homo-

cysteine [6,7]. Folate supplementation in patients with

RA being treated with MTX is able to decrease

homocysteine concentrations [8,9].

K. Schroecksnadel et al. / Clinica Chimica Acta 338 (2003) 157–164158

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 macro-

phages 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 homocys-

teine and cysteine were measured in patients with RA

and were compared to B vitamin status and to con-

centrations of immune activation markers sTNF-R75

and neopterin.

2. Materials and methods

Thirty-one women and two men (mean age: 56.9

yearsF 9.2, range: 37–77 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

Table 1

Treatment of 33 patients with rheumatoid arthritis

NSAIDs (Diclofenac: 25–100 mg/day;

Meloxicam/Lomoxicam: 4–15 mg/day;

Rheumotrop: 60–90 mg/day;

Ibuprofen: 300–600 mg/day)

Steroids (Methyl-prednisolone: 1–8 mg/day)

Immunosuppressants (Methotrexate: 2.5–15 mg/week;

Cyclosporin A: 50–100 mg/day; Leflunomide: 20 mg/day;

Azathioprin: 100 mg; Sulfasalazine: 1000–1500 mg/day)

Opioids: (Tramadol 50–200 mg/day)

NSAIDs = non-steroidal anti-inflammatory drugs.

(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, hae-

matological, 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 centri-

fuged 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 speci-

mens 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, RP18 LiChroCART 55-4 and RP18precolumns (Merck, Darmstadt, Germany). Homocys-

teine and cysteine concentrations were monitored by

fluorescence detection at 385 nm excitation wave-

length and 515 nm emission wavelength [22].

For the determination of concentrations of folate

and vitamin B12 double-labelled radioimmunoassay

No therapy Therapy on

demand

Regular

therapy

13 12 8

6 23 4

6 0 27

27 5 1

K. Schroecksnadel et al. / Clinica Chimica Acta 338 (2003) 157–164 159

(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 pre-

sented with elevated homocysteine and cysteine con-

centrations (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. Concen-

trations of vitamin B12 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 con-

centrations (Fig. 1). Cysteine concentrations were also

higher, whereas folate concentrations were lower in the

patients with hyperhomocysteinaemia. Vitamin B12

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).

Table 2

Median and range of serum concentrations of homocysteine, cysteine, folate

reference ranges of these parameters and the percentage of patients with c

Medians Range

Homocysteine (AM) 13.9 4.7–31.3

Cysteine (AM) 416 268–701

Folate (nM) 16.8 1.8–49.5

Vitamin B12 (pM) 244 129–979

sTNF-R75 (ng/ml) 2.73 1.6–4.7

Neopterin (nM) 8.08 3.5–27.7

*p< 0.01.

**p< 0.001.

There existed correlations between concentrations

of homocysteine and folate and between homocys-

teine and sTNF-R75 (Fig. 2). Concentrations of

vitamin B12 and folate were associated as well

(rs = 0.462; p < 0.01). Cysteine correlated with homo-

cysteine (rs = 0.713; p < 0.001) and sTNF-R75

(rs = 0.487; p < 0.01); sTNF-R75 concentrations also

correlated with neopterin concentrations (rs = 0.607;

p< 0.001).

Patients treated with non-steroidal anti-inflamma-

tory drugs (NSAIDs) had higher ( p < 0.01) and those

treated with MTX had lower ( p < 0.05) vitamin B12

concentrations. Furthermore sTNF-R75 concentra-

tions were lower in patients treated with MTX

( p < 0.05). There was no such difference of neopterin

concentrations between groups with different treat-

ment regimens. Dosage of MTX did not correlate with

any of the parameters investigated.

The median calculated dietary intake of vitamin

B12 was 5.0 mg/day (range: 1.77–25.5) and 202 Ag/day for folate, respectively (range: 69.2–933.0),

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 B12 intake (rs = 0.448, p < 0.05). Dietary

intake of vitamins C and E was also below recom-

mendations. No association was found between cal-

culated 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 recom-

mended daily intake of 300 Ag. In patients treated with

, vitamin B12, sTNF-R75, and neopterin in 33 patients with RAwith

oncentrations above or below the reference range

Reference limits

(95th percentile)

Outside reference limit

15.0 [23] 42% increased*

347 [24] 85% increased**

4.0 [25] 12% decreased

150 [25] 21% decreased

4.8 [26] 0% increased

8.7 [27] 36% increased*

Fig. 1. Boxplots of serum concentrations of folate and vitamin B12 (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).

K. Schroecksnadel et al. / Clinica Chimica Acta 338 (2003) 157–164160

MTX and supplemented with folate, serum folate

concentrations were higher ( p < 0.02) and homocys-

teine concentrations tended to be lower ( p < 0.06) in

comparison with unsupplemented patients. In supple-

mented patients, serum folate concentrations weakly

correlated with the amount of folate supplemented

(rs = 0.371, p < 0.05). Inverse relationships between

homocysteine and serum folate concentrations were

of similar strength in MTX-treated patients with

(rs =� 0.612, p < 0.06) and without (rs =� 0.700,

p < 0.02) folate supplementation. Concentrations of

sTNF-R 75 and homocysteine correlated in MTX-

treated patients without folate supplementation

(rs = 0.718; p < 0.02) but not in supplemented patients

(rs = 0.45; p>0.1). The correlation between neopterin

and sTNF-R75 was somewhat stronger in MTX-trea-

Fig. 2. Correlations of homocysteine with immune activation marker sTNF-R75 (rs = 0.508; p< 0.01) and serum folate (rs =� 0.519; p< 0.01) in

33 patients with RA.

K. Schroecksnadel et al. / Clinica Chimica Acta 338 (2003) 157–164 161

ted patients with folate supplementation (rs = 0.821,

p < 0.01) compared to those without (rs = 0.685,

p < 0.03).

4. Discussion

This study confirms that patients with RA may

present with increased serum homocysteine and cys-

teine concentrations in comparison with healthy con-

trol 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 concen-

trations of serum folate were found in comparison

with the other 19 patients with low homocysteine

concentrations. Also, vitamin B12 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 con-

centrations were only slightly lower, folate concen-

trations 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 detect-

able in the circulation. Interestingly, Houcher et al.

[28] reported earlier that an association found between

calculated folic acid intake and circulating concen-

trations in healthy controls could not be detected in

patients with cardiovascular disease. Discrepancy be-

tween dietary intake of vitamins and circulating con-

centrations may relate to an increased consumption of

vitamins in inflammatory conditions.

Elevated concentrations of homocysteine and cys-

teine occurred concomitantly with increased concen-

trations of immune activation marker sTNF-R75 and

neopterin, indicating a link between immune activa-

tion and moderate hyperhomocysteinaemia. Concen-

trations of immune activation markers were higher in

patients with lower vitamin concentrations. This

association as well as the high percentage of hyper-

homocysteinaemia (42%) in patients with RA per se

suggests a relationship between the autoimmune

K. Schroecksnadel et al. / Clinica Chimica Acta 338 (2003) 157–164162

process and homocysteine metabolism. This link

between higher homocysteine concentrations and

immune activation was observed also in other auto-

immune disorders [4,5]. Cysteine concentrations,

which correlated well with homocysteine concentra-

tions, also showed a strong association with concen-

trations 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 homocys-

teine 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 agree-

ment, 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 B12 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 B12 were higher than in

untreated patients ( p < 0.01), anti-inflammatory treat-

ment therefore seems to influence vitamin B12 avail-

ability in a positive manner.

In parallel to the raised homocysteine concentra-

tions, elevated blood concentrations of immune acti-

vation markers sTNF-R75 and neopterin are found,

and also the strong correlation between homocys-

teine and sTNF-R75 indicates a link between homo-

cysteine 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 Par-

kinson’s disease [32,33]. Interestingly, oxidative

stress appears to be deeply involved in the patho-

genesis 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 vita-

mins [34]. In this way, also blood concentrations of

oxidation-sensitive tetrahydrofolate could be dimin-

ished by oxidative stress developing during immune

activation, leading to raised homocysteine and cys-

teine concentrations [35,36]. Since B vitamin cofac-

tors, 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 oxida-

tive stress. Similarly, in patients with Alzheimer’s

disease McCaddon et al. [37] recently suggested an

enhanced consumption of vitamin B12 as a conse-

quence 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 develop-

ment 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 Forderung der wissenschaftlichen Forschung’’,

project 14942, and by the Austrian Federal Ministry

of Social Affairs and Generations.

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