Homocysteine and folate status in methotrexate-treated patients with rheumatoid arthritis

A. E. van Ede, R. F. J. M. Laan, H. J. Blom1, G. H. J. Boers2, C. J. Haagsma3, C. M. G. Thomas4, T. M. de Boo5 and L. B. A. van de Putte

Department of Rheumatology,
1 Laboratory of Pediatrics and Neurology,
2 Department of General Internal Medicine,
4 Department of Obstetrics and Gynaecology and Chemical Endocrinology and
5 Department of Medical Statistics, University Medical Center St Radboud, Nijmegen and
3 Department of Rheumatology, Medisch Spectrum Twente, Enschede, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To study (i) the influence of methotrexate (MTX) therapy on homocysteine and folate metabolism in patients with rheumatoid arthritis (RA), (ii) the influence of the C677T mutation in the methylenetetrahydrofolate reductase gene (MTHFR) on the change in plasma homocysteine levels during MTX treatment, and (iii) the interference of folate and homocysteine metabolism with the efficacy and toxicity of treatment with MTX.

Methods. The 113 patients enrolled in this study were participating in a 48-week, multicentre, double-blind, placebo-controlled study comparing the efficacy and toxicity of MTX treatment with and without folic or folinic acid supplementation. The MTX dose was 7.5 mg/week initially and increased to a maximum of 25 mg/week if necessary. Concentrations of total folate, 5-methyl tetrahydrofolate (in serum and in erythrocytes) and of homocysteine, cysteine and cysteine-glycine and the MTHFR genotype were determined before the start of the study, after 6 weeks, and after 48 weeks or on withdrawal from the study. Blood was drawn from fasting patients at a standardized time in the morning, 16 h after intake of MTX. The laboratory results were related to parameters of efficacy and toxicity of MTX treatment.

Results. Baseline values were distributed equally in the three treatment groups. The mean plasma homocysteine level (normal range 6–15 µmol/l) before the start of MTX was relatively high in all groups: 15.4 µmol/l [95% confidence interval (CI) 13.5 to 17.2] in the MTX plus placebo group (n=39), 14.3 µmol/l (95% CI 12.2 to 16.4) in the MTX plus folic acid group (n=35) and 15.9 µmol/l (95% CI 13.7 to 18.1) in the MTX plus folinic acid group (n=39). After 48 weeks of MTX therapy, the mean homocysteine level showed an increase in the placebo group (+3.6 µmol/l, 95% CI 1.7 to 5.6). In contrast, a decrease was observed in the groups supplemented with folic or folinic acid (folic acid, –2.7 µmol/l, 95% CI -1.4 to -4.0; folinic acid, -1.6 µmol/l, 95% CI -0.1 to -3.0). The differences in the change in plasma homocysteine level between the placebo group and each of the two folate-supplemented groups were statistically significant (P<0.0001), contrary to the difference between the folic and folinic acid groups (P=0.26). Linear regression analysis showed that the change in plasma homocysteine level was statistically significantly associated with folic or folinic acid supplementation (P=0.0001) but not with the presence or absence of the C677T mutation in the MTHFR gene. Homozygous mutants had a higher plasma homocysteine concentration at baseline. No relationship was found between the change in disease activity and the change in homocysteine concentration or the mean homocysteine concentration after 48 weeks of MTX therapy. Toxicity-related discontinuation of MTX treatment was not associated with the change in homocysteine concentration.

Conclusions. Low-dose MTX treatment in RA patients leads to an increased plasma homocysteine level. Concomitant folate supplementation with either folic or folinic acid decreases the plasma homocysteine level and consequently protects against potential cardiovascular risks. No relationship was found between the change in homocysteine concentration and the presence or absence of the C677T mutation in the MTHFR gene. Homocysteine metabolism was not associated with efficacy or toxicity of MTX treatment.

KEY WORDS: Homocysteine, Folic acid, Folinic acid, MTHFR C677T mutation, Methotrexate, Rheumatoid arthritis.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Methotrexate (MTX) has become one of the most widely used disease-modifying anti-rheumatic drugs (DMARDs) in the treatment of rheumatoid arthritis (RA). MTX acts relatively fast, has one of the best efficacy:toxicity ratios and is also cheap [12]. Toxicity remains an important reason for the discontinuation of MTX [3].

Despite extensive research, the precise mechanism of action of MTX is still unknown and we cannot predict which patients will benefit from MTX treatment and who will suffer adverse events [45]. MTX, a folic acid antagonist, influences several metabolic pathways, including the homocysteine–methionine pathway (Fig. 1Go). In addition to the inhibition of dihydrofolate reductase, resulting in reduced availability of reduced folates, MTX may also inhibit the conversion of 5,10-methylene tetrahydrofolate (5,10-CH2-THF) to 5-methyl tetrahydrofolate (5mTHF) [6,7]. Homozygosity of the C677T mutation of the methylene tetrahydrofolate reductase (MTHFR) gene also results in reduced activity of the MTHFR enzyme. The resulting decrease in the level of a methyl group donor causes impeded remethylation of homocysteine to methionine and, consequently, hyperhomocysteinaemia [8, 9]. Hyperhomocysteinaemia may be related to MTX toxicity in the short or long term. Several papers have drawn attention to the adverse effects of hyperhomocysteinaemia, especially increased cardiovascular risks [1013].



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FIG. 1.  Simplified metabolic scheme illustrating folate metabolism and its relationship to homocysteine–methionine metabolism. Known inhibition of enzymes by methotrexate is indicated by double bars through arrows. DHF, dihydrofolate; DHFR, dihydrofolate reductase; THF, tetrahydrofolate; 5,10-CH-THF, 5,10-methenyl tetrahydrofolate; 5,10-CH2-THF, 5,10-methylene tetrahydrofolate; 5-CH3-THF, 5-methyl tetrahydrofolate; 5-CHO-THF, 5-formyl tetrahydrofolate (folinic acid); 10-CHO-THF, 10-formyl tetrahydrofolate; Hcy, homocysteine; Met, methionine; ms, methionine synthetase; mthfr, methylene tetrahydrofolate reductase; R, methyl acceptor; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-homocysteine.

 
We investigated the effects of MTX treatment in RA patients on folate and homocysteine metabolism, the role of folic acid and folinic acid supplementation and the influence of the C677T mutation in the MTHFR gene. We also studied the relationship of homocysteine metabolism with the efficacy and toxicity of MTX treatment.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
One hundred and thirteen patients with RA, according to the American College of Rheumatology criteria, were included in the present study [14]. They comprised a sample from 411 participants in a 48-week, multicentre, double-blind, placebo-controlled, randomized clinical trial examining the effect of folic acid and folinic acid supplementation on the efficacy and toxicity of MTX treatment [15]. As the measurement of parameters of folate and homocysteine metabolism requires special conditions for drawing blood and special laboratory conditions, only patients from nine of 22 participating centres could be enrolled in this study. The assignment of patients to treatments was stratified for each centre. Patients were randomized between three treatment modalities: MTX plus placebo, MTX plus folic acid (1.0 mg/day) and MTX plus folinic acid (2.5 mg/week). The initial MTX dose was 7.5 mg/week and was increased to a maximum of 25 mg/week if necessary. The doses of folic and folinic acid were doubled when the MTX dose reached 15 mg/week or more [22].

Inclusion criteria
Entry criteria were as follows: active arthritis, defined as a Disease Activity Score (DAS) >=3.0 (see below); a wash-out period for other DMARDs of 2 weeks; corticosteroids and NSAIDs permitted in stable doses from at least 1 month before enrolment until the end of the study.

Exclusion criteria
Exclusion criteria were as follows: previous treatment with MTX; concomitant treatment with folic or folinic acid; pregnancy, breast-feeding; >=20 consumptions of alcohol per week; elevated liver transaminases (above the upper limit of normal); impaired kidney function [estimated creatinine clearance (according to Cockcroft) of less than <50 ml/min]; leucopenia [white blood cell (WBC) count <3.5x109/l] and thrombocytopenia (platelet count <120x109/l).

Efficacy
The efficacy of MTX treatment was measured as the DAS every 6 weeks [16]. The DAS is a composite outcome measure based on the Ritchie Articular Index, the number of swollen joints, the erythrocyte sedimentation rate (ESR) and a visual analogue scale for general health [17]. Response categories (good, moderate and none) were defined according to the European League Against Rheumatism (EULAR) response criteria [18].

Toxicity
Toxicity was assessed at intervals of 3 weeks using a standard toxicity form including 36 items designed by Fries et al. [19] and special forms for other symptoms and complaints. Routine laboratory measurements (made every 3 weeks) included ESR (mm 1st h), haemoglobin content (mmol/l), WBC count (x109/l), platelet count (x109/l), serum creatinine level (µmol/l) and alanine aminotransferase (ALT, IU/ml).

Gastrointestinal toxicity included all symptoms and complaints of the mouth and the upper or lower abdominal tract, except laboratory abnormalities.

Elevation of liver enzymes was defined as elevated ALT values less than three times the upper limit of normal levels occurring at least in two out of four consecutive (3-weekly) evaluations (mild) or as elevated ALT values at least three times the upper limit of the normal range (moderate). In case of adverse events, the protocol included the possibility of dose reductions. Mild elevation of ALT values was handled by a reduction in MTX dose by 2.5 mg/week. After resolution, subsequent increases in MTX dose were allowed if the response was insufficient. If mild elevation of ALT values persisted (tests not normalizing despite decreased MTX dose) or recurred (recurrence of mild elevation of ALT values after resolution of the first elevation), this was considered to be a moderate elevation of ALT values. Moderate adverse events were handled by temporarily withholding MTX. When the laboratory results returned to normal, rechallenge with MTX was allowed at the last tolerated dose. Again, if the response was insufficient, further increases in MTX dose were allowed. Recurrent or persisting moderate elevation of ALT values was considered severe. Severe adverse events were handled by definite discontinuation of MTX and study drugs.

Folate and homocysteine metabolism
For the present study, blood samples were taken at study entry (before the first MTX dose), after 6 weeks, and after 48 weeks (end of study) or on withdrawal of the patient from the study. Blood was drawn from fasting patients in the morning, 16 h after intake of MTX, for the following measurements. Vitamin B6 (reference values 35–107 nmol/l), measured only at baseline, was determined in plasma by high-performance liquid chromatography (HPLC) as described previously [20]. Vitamin B12 in serum (reference values 160–750 pmol/l), measured only at baseline, and folic acid in serum (reference values 7–39 nmol/l) and erythrocytes (reference values 150–250 nmol/l) were determined with the Dualcount SPB (solid phase boil) radioassay (Diagnostic Products Corporation, Los Angeles, CA, USA), as described previously [21]. The concentrations of 5mTHF were determined in serum (reference values 3–17 nmol/l) and erythrocytes (reference values 83–720 nmol/l) with a modified procedure based on reverse-phase ion-pair HPLC and a diode array detector described previously by Selhub [22].

Homocysteine (reference values 6–15 µmol/l), cysteine (reference values 150–250 µmol/l) and cysteine–glycine (reference values 40–60 µmol/l) were measured in plasma by the HPLC technique with fluorimetric detection [23, 24].

MTHFR gene mutation
Genotypic analysis of the MTHFR gene was done using DNA from WBC by the polymerase chain reaction and restriction enzyme analysis [9].

Statistical analysis
Baseline variables and changes in laboratory measurements were compared using Student's t-test. The independent influences of folic or folinic acid supplementation and of the C677T mutation in the MTHFR gene on homocysteine metabolism, corrected for MTX dose and duration of treatment, were studied by multiple linear regression analysis. The change in plasma homocysteine level was considered as the dependent variable; the independent variables were folic or folinic acid supplementation, the C677T mutation in the MTHFR gene (homozygous or heterozygous), MTX dose and duration of MTX therapy.

In order to study the hypothesis that the MTX-induced improvement in disease activity is mediated by changes in homocysteine metabolism, we performed linear regression analyses. First, univariate analysis was done with the change in DAS as the dependent variable and the change in plasma homocysteine as the independent variable. Subsequently, we performed multiple linear regression analysis. Again, the change in DAS was the dependent variable. The independent variables included the use of folate supplementation, MTX dose and duration of MTX treatment, and the change in plasma homocysteine. Finally, we substituted the change in plasma homocysteine for the plasma homocysteine level at the end of the study in these analyses.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
A total of 113 patients were included in the study: 39 in the placebo group, 35 in the folic acid and 39 in the folinic acid group. Patient characteristics and baseline variables were similar in all groups (Table 1Go). No patients suffered from deficiency of folic acid, vitamin B6 or B12 before MTX was prescribed.


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TABLE 1.  Demographics and disease characteristics of the patients studied (n=113) at baseline [mean (S.D.) unless indicated otherwise]

 
Homocysteine and cysteine
At the start of the study, the mean plasma homocysteine level was relatively high in all groups [placebo group, 15.4 µmol/l, 95% confidence interval (CI) 13.5 to 17.2; folic acid group, 14.3 µmol/l (95% CI 12.2 to 16.4); folinic acid group, 15.9 µmol/l (95% CI 13.7 to 18.1)]. After 6 weeks the mean plasma homocysteine level showed an increase in the placebo group (3.0 µmol/l, 95% CI 1.9 to 4.1), was unchanged in the folinic acid group (0.1 µmol/l, 95% CI –1.0 to 1.3) and decreased (–2.8 µmol/l, 95% CI –1.3 to –4.3) in the folic acid group. After 48 weeks of MTX therapy, the mean plasma homocysteine had increased by 3.6 µmol/l (95% CI 1.7 to 5.6) in the placebo group. In contrast, the group supplemented with folic acid and the group supplemented with folinic acid both showed a decrease, which was most pronounced in the folic acid group (folic acid, –2.7 µmol/l, 95% CI –1.4 to –4.0; folinic acid, –1.6 µmol/l, 95% CI –0.1 to –3.0). The difference in changes in plasma homocysteine level between the placebo group and both folate supplemented groups was statistically significant (P<0.0001), unlike the difference between the folic and the folinic acid group (P=0.26) (Fig. 2Go). Cysteine and cysteine–glycine levels in plasma showed an increase in the folic acid- and folinic acid-supplemented groups (most pronounced in the folinic acid group) and a decrease in the placebo group (Table 2Go).



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FIG. 2.  Homocysteine levels (with standard errors) during the 48 weeks of the study.

 

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TABLE 2.  Laboratory variables: mean (95% CI) values at baseline and changes from baseline at the end of the study

 

Folates and the MTHFR gene mutation
At the start of the study, the mean serum folate levels were in the normal range (placebo group, 13.8 nmol/l, 95% CI 11.7 to 16.0; folic acid group, 12.7 nmol/l, 95% CI 10.8 to 14.5; folinic acid group, 12.7 nmol/l, 95% CI 10.9 to 14.5). In the placebo group, serum folate decreased by 4.1 nmol/l (95% CI -7.6 to -0.6). In contrast, both folic and folinic acid supplemented groups showed an increase, most pronounced in the folic acid group (+47.0 nmol/l, 95% CI 30.8 to 63.2 vs +13.6 nmol/l, 95% CI 9.1 to 18.0, in the folinic acid group. The difference in folate increment between the folic acid- and folinic acid-supplemented groups was 33.4 µmol/l (95% CI 16.1 to 50.7).

The changes in mean levels of folate in erythrocytes and of 5mTHF in serum and erythrocytes during MTX treatment showed similar patterns: there was an increase in the folic acid- and folinic acid-supplemented groups, which was most pronounced in the folic acid group, and a decrease in the placebo group (Table 2Go).

Fifty patients (45%) showed the C677T mutation in heterozygous form (CT), 12 patients (10%) were homozygous for the mutation (TT) and in 51 patients (45%) the mutation was not present (CC). These figures were similar in the three treatment groups. Baseline plasma homocysteine level was higher in the homozygous (17.1 µmol/l, 95% CI 11.1 to 23.1) and heterozygous (15.2 µmol/l, 95% CI 13.5 to 16.9) patients than in the non-mutated group (14.9 µmol/l, 95% CI 13.2 to 16.6). The differences in baseline plasma homocysteine level between the homozygous group on the one hand and the heterozygous and non-mutated groups on the other were statistically significant (P=0.017 and P=0.014 respectively).

Linear regression analysis was used to study the independent effects of folic acid and folinic acid supplementation and the C677T mutation in the MTHFR gene on the change in plasma homocysteine level during MTX treatment, after correction for MTX dose and the duration of MTX therapy. Again, the use of folates was significantly associated with a decrease in homocysteine levels (ß estimate –5.7, 95% CI –3.7 to –7.6 µmol/l). The effect of folate supplementation on the plasma homocysteine level was similar in patients without the C677T mutation (677CC genotype) and in patients who were either homozygous (677TT) or heterozygous (677CT) for the mutation (data not shown). The C677T mutation itself did not influence the change in plasma homocysteine level during MTX treatment (ß estimate -1.0, 95% CI -2.8 to 0.9 µmol/l). MTX dose was not associated with changes in plasma homocysteine level (ß estimate -0.07, 95% CI -0.26 to 0.11 µmol/l), contrary to the duration of MTX therapy (ß estimate 0.13, 95% CI 0.03 to 0.22 µmol/l).

Efficacy
Univariate linear regression analysis showed no statistically significant relationship between the change in DAS and the change in plasma homocysteine level (P=0.30). In subsequent multivariate linear regression analysis, the change in DAS was statistically significantly associated with folate supplementation (P=0.04) and the duration of MTX therapy (P=0.01), but not with the changes in homocysteine concentration (P=0.69) and MTX dose (P=0.51). No association was found between the change in DAS and the mean homocysteine level at the end of the study.

According to the EULAR response criteria, 47 patients (42%) showed a good response, 60 patients (53%) a moderate response and six patients (5%) no response [25]. These results were similar in the groups of patients with and without folate supplementation.

Toxicity
In the present study, discontinuation of MTX due to toxicity of the drug occurred in 38 of 113 patients (34%). In 15 of these (39%), withdrawal was due to elevated ALT, in four (11%) it was due to gastrointestinal toxicity and in 19 (50%) to a variety of reasons. No statistically significant association was found between changes in plasma homocysteine level and toxicity-related discontinuation of MTX therapy. Also, no relationship was found between the baseline homocysteine level and the occurrence of adverse events.

The same analysis was performed in the subgroups of patients treated with MTX alone and MTX plus folic acid or folinic acid. In both subgroups, the change in plasma homocysteine concentration and the concentration itself did not differ between patients with and without toxicity.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The main result of the present study is the increase in plasma homocysteine concentration during MTX treatment and the prevention of this increase by folic or folinic acid supplementation. The decrease in homocysteine was more pronounced and more rapid in the folic acid group than in the folinic acid group, but the difference between these two treatment groups was not statistically significant. As in previous studies in RA patients, we found a relatively high mean baseline plasma homocysteine level (15.2 µmol/l, 95% CI 14.0 to 16.5; reference value 6–15 µmol/l) [13, 23, 2527]. Baseline values of folic acid in serum and erythrocytes and vitamin B6 and B12 were within the normal range and cannot explain these relatively high plasma homocysteine levels.

The results of our study confirm the findings of former studies, which report a trend towards elevation or a significant increase in plasma homocysteine concentration between the pretreatment level and the level after 6–12 months of MTX treatment [23, 28, 29]. One should consider that plasma homocysteine levels are dependent on the timing of blood sampling after MTX administration [30]. In the first 48–72 h there is an increase, and this is followed by a gradual decrease. In the above-mentioned studies, the time of blood sampling after MTX administration was different—it was after 5–7 days in the studies of Morgan et al. [28, 29] compared with 24 h in the study by Haagsma et al. [23]; in the present study it was after 16 h. Nevertheless, all studies showed an increase in plasma homocysteine level. The homocysteine increment was most pronounced in the present study: 3.6 µmol/l vs 2 µmol/l in the study of Morgan et al. [28] and 3.1 µmol/l in the study of Haagsma et al. [23].

The effect of supplementation with folic acid on plasma homocysteine concentration has been studied before in RA patients treated with MTX. Morgan et al. [28] measured plasma homocysteine levels 5–7 days after MTX administration and showed that the concentration decreased in patients supplemented with 5.0 or 27.5 mg folic acid per week. Our results indicate that supplementation with either folic acid (1–2 mg/day) or folinic acid (2.5–5.0 mg/week) also inhibits the early rise in plasma homocysteine, 16 h after MTX administration. This effect of folate supplementation occurred despite the mean dose of MTX at the end of the study being higher and more consistent with current practice then in the study by Morgan et al. (15.3 mg/week in the present study vs 9.5 mg/week in the study of Morgan et al. [28]).

In the long term, hyperhomocysteinaemia may be associated with considerable toxicity. Mild hyperhomocysteinaemia (above 12–15 µmol/l) is considered to be an independent risk factor for vascular disease [12]. The relationship between plasma homocysteine and the risk of cardiovascular disease seems to be graded and linear [31]. In a meta-analysis, Boushey et al. showed that an increment in homocysteine of 5 µmol/l is related to a 1.7-fold higher risk of coronary artery disease, a 1.5-fold higher risk of cerebrovascular disease and a 6.8-fold higher risk of peripheral arterial disease [32]. So even the modest increment of 3.6 µmol/l in our study may have an adverse effect on cardiovascular risks. Moreover, after 48 weeks of MTX therapy, the difference between the folic acid- and folinic acid-supplemented groups on the one hand and the MTX plus placebo group on the other was 6.3 µmol/l. In a retrospective study, Landewé et al. [33] found increased mortality in patients with cardiovascular disease who had started treatment with MTX without folic or folinic acid supplementation, in contrast to similar patients who had started another DMARD. They hypothesize that this increased mortality may be attributed to an MTX-induced increase in homocysteine concentration.

Theoretically, the presence of the C677T mutation in the MTHFR gene, which inhibits the reduction of 5,10-CH2-THF to 5mTHF, would lead to additional or synergistic alterations in folate and homocysteine metabolism when MTX is given. In contrast to previous studies, we showed a statistically significant higher mean baseline homocysteine level in patients homozygous for the mutation compared with heterozygous patients and patients without the mutation [23, 34]. However, in the present study the change in plasma homocysteine concentration during MTX treatment was not influenced by the presence or absence of the C677T mutation in the MTHFR gene. The number of patients with the C677TT genotype may have been too small for such an interaction to be observed.

The mechanism of action of the anti-inflammatory effects of MTX is not completely understood. MTX is a folate antagonist. The inhibition of the enzyme MTHFR is responsible for the increase in homocysteine level. Hyperhomocysteinaemia may therefore be considered as an antifolate effect. We found no association between the increase in homocysteine concentration and the improvement in DAS. In the multiple linear regression analyses, folate supplementation was associated with a small but statistically significant decrease in the efficacy of MTX. On the other hand, folate supplementation also prevented the increase in homocysteine concentration. Considered together, these results suggest that the antifolate activity of MTX is not the most important pathway contributing to its efficacy. Our data are consistent with the hypothesis, formulated by Cronstein [35], that the anti-inflammatory effects of MTX are mediated by adenosine release.

In conclusion, MTX treatment leads to hyperhomocysteinaemia, which can be prevented by supplementation with folic acid or folinic acid. The presence or absence of the C677T mutation in the MTHFR gene did not modify this effect. No relationship was found between homocysteine metabolism and the efficacy or toxicity of MTX treatment. As hyperhomocysteinaemia is related to increased cardio- and cerebrovascular disease, this forms another argument for folate supplementation during MTX treatment.


    Acknowledgments
 
This study was supported by a grant from the Dutch National Fund against Rheumatism. HJB was supported by a grant from the Netherlands Heart Foundation (D97-021).


    Notes
 
Correspondence to: R. F. J. M. Laan, Department of Rheumatology, University Medical Center St Radboud, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 6 April 2001; Accepted 28 December 2001