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Br J Clin Pharmacol. Oct 2007; 64(4): 517–526.
Published online May 15, 2007. doi:  10.1111/j.1365-2125.2007.02911.x
PMCID: PMC2048559

Inflammatory status and kynurenine metabolism in rheumatoid arthritis treated with melatonin

Abstract

What is already known about this subject

  • There is good evidence that oxidative stress, associated with the generation of free radicals, is a major contributor to joint damage in rheumatoid arthritis.
  • It is also well established that melatonin is one of the most powerful, endogenous free radical scavengers, and it is very safe for human use.
  • We have therefore examined whether melatonin might be a useful adjunctive compound with which to treat arthritis.

What this study adds

  • Once-nightly administration of melatonin increases concentrations of some inflammatory markers, but patients experience no significant improvement in symptoms and no changes of proinflammatory cytokine concentrations.
  • Melatonin is an effective antioxidant, but because it is either not sufficiently effective, or it has some proinflammatory activity, it is not likely to prove beneficial in patients.

Aim

Since melatonin is antioxidant and has some anti-inflammatory actions, we have tested it as adjunctive treatment in patients with rheumatoid arthritis, to determine whether it can improve patients' symptoms.

Methods

A total of 75 patients were allocated randomly to receive melatonin 10 mg at night in addition to ongoing medication, or a placebo of identical appearance. Monthly blood samples were taken and disease severity assessed over 6 months, plasma being analysed for inflammatory indicators [C-reactive protein, erythrocyte sedimentation rate (ESR), neopterin], proinflammatory cytokines [interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α], lipid peroxidation products and the kynurenine pathway metabolites of tryptophan.

Results

An increase of ESR (two-way anova F(1,127) = 5.24, P = 0.024) and neopterin concentrations (F(1,136) = 4.64, P = 0.033) was observed in treated patients compared with controls, reflected also in a significant trend for both to decline in placebo-treated patients (P= 0.022), but not the melatonin-treated group. Peroxidation products showed a significant trend to decrease in placebo- but not melatonin-treated patients. These results suggest a proinflammatory action, but there were no significant effects of melatonin treatment on clinical assessments of patient symptoms or the concentrations of three proinflammatory cytokines, IL-1β, IL-6 and TNF-α. Melatonin significantly increased plasma kynurenine concentrations (F(1,124) = 4.24, P = 0.041), again suggesting proinflammatory activity.

Conclusion

A daily dose of 10 mg melatonin shows a slowly developing antioxidant profile in patients with arthritis and increases the concentrations of some inflammatory indicators, but these effects are not associated with any change of proinflammatory cytokine concentrations or clinical symptoms.

Keywords: arthritis, cytokines, kynurenine, melatonin, neopterin, tryptophan

Introduction

There is an increased concentration of oxidative stress, associated with greater lipid peroxidation, in patients with rheumatoid arthritis [15], with a positive correlation between markers of oxidant status and disease activity [6]. One possible approach to treating arthritis might be to use adjunctive therapy with an antioxidant, since antioxidant vitamins can reduce the indicators of oxidative stress to the extent of inducing a reduction of patients' symptoms [7].

Melatonin is the primary neurohormone of the pineal body, responsible for the regulation of circadian rhythms and changes of physiology with alterations in day length. It is also one of the most efficacious antioxidant compounds in mammals [8, 9], being at least as active as vitamin E in its ability to scavenge hydrogen peroxide [10] and the highly destructive hydroxyl and peroxynitrite anions [9, 1114]. As little as 1 nm melatonin is sufficient to inhibit significantly the activity of nitric oxide synthase, leading also to the reduced formation of nitrogen free radicals [1517].

Melatonin exhibits several additional anti-inflammatory actions, such as reduction of the translocation of nuclear factor-κB, and has been reported to inhibit the production of proinflammatory cytokines [9, 18, 19]. It may also possess the ability to modulate cell interactions by affecting factors such as adhesion molecules that could be relevant to the progression of diseases such as arthritis [14].

The administration of melatonin to humans has a long record of safety. It has been used as adjunctive therapy in the treatment of cancer [20], and its additional widespread use as a prophylactic against jet-lag supports the impressive level of acceptability of this compound [21]. In the present study we have sought to determine whether the administration of melatonin would be of value in the treatment of patients with rheumatoid arthritis, on the premise that several of these actions, most particularly the antioxidant activity, could ameliorate tissue damage and disease progression. In addition, since melatonin is a tryptophan metabolite and we have previously noted abnormal aspects of tryptophan metabolism in rheumatoid arthritis [22] and the fact that tryptophan can promote oxidative stress [23], we have also sought changes of tryptophan metabolism during the melatonin treatment.

Methods

Seventy-five patients were recruited from a busy rheumatology clinic in a large district hospital. Since all patients were experiencing active disease, it was not ethically possible to examine the effects of melatonin alone. The patients were therefore recruited as consecutive patients whose drug regime had been unchanged for at least 2 months before examination, and in whom no change of drug profile was anticipated over the 6 months of this study. The patients were then allocated randomly to receive either melatonin or placebo. Randomization was performed according to a table of random numbers produced by our statistical adviser. The entire study, including both clinical and laboratory phases, was performed double blind. Patients were allocated to receive melatonin 10 mg day−1, to be taken before retiring to bed each evening, or a placebo tablet which was specifically manufactured for this study and was identical in appearance to the active drug tablets, including the supplier's logo. The patient groups included 38 allocated to receive placebo (10 male, 28 female, mean age 60.0 ± 1.8 years) and 37 allocated to receive melatonin (12 male, 25 female, mean age 65.11 ± 2.1 years). There was no significant difference between the group mean ages (t-test, P = 0.063). All patients gave their informed consent to participate in this study, which was approved by the Ethical Committee of the Epsom and St Helier University Hospital NHS Trust.

As noted above, patients were included in this study only if they had no recent changes in drug use. The randomization process was successful in ensuring that similar proportions of patients in the melatonin and placebo groups were consuming different types of drug. Thus, when the treatment code was broken at the end of the study, it was noted that in patients taking melatonin there were 26 taking nonsteroidal anti-inflammatory drugs (NSAIDs), four taking disease-modifying antirheumatic drugs and 14 taking steroids. In the placebo group the comparable figures were similar: 16, 6 and 10, respectively. We are confident therefore that differences of drug usage between the two cohorts is not likely to have made a major contribution to the results obtained.

Patients attended the clinic at monthly intervals and on each occasion completed a modified Health Assessment Questionnaire (HAQ). In addition, at baseline and at the 3- and 6-month visits, patients were assessed clinically by the attending physician (L.G.D. or N.S.), who assessed disease severity on the basis of a physical examination, using the revised diagnostic criteria of the American Rheumatism Association [24].

Before the use of melatonin or placebo began, a 10-ml blood sample was obtained by venepuncture from each patient. This sample was taken by a professional phlebotomist into heparin/ethylenediamine tetraaceticacid (EDTA) tubes and was then centrifuged at 3000 g and stored at −80°C until biochemical analysis. Further blood samples were taken at monthly intervals, all beingtaken in the morning, approximately 12 h after consuming the test tablets.

The blood samples were analysed for a range of parameters generally recognized to reflect the severity of the arthritic disease and systemic inflammation. These included the acute response protein, C-reactive protein (CRP), which was measured using a Behring Turbitimer (Marburg, Germany), and the erythrocyte sedimentation rate (ESR) which was measured by a Starrsed Automated ESR machine (R&R Mechatronics An Zwaag, The Netherlands) using the method recommended by the International Council for Standardization in Haematology [25], based on the method of Westergren [26].

Melatonin assay

The concentration of melatonin in plasma collected in EDTA-containing tubes was measured commercially by Stockgrand Ltd (Guildford, UK) using a radioimmunoassay method [27].

Cytokine assays

Interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α were measured by commercial human ELISA kits obtained from Biosource (Nivelles, Belgium).

Neopterin

Neopterin concentrations were measured in 10-µl aliquots of heparinized plasma using an ELISA kit (Immunobiological Laboratories, Hamburg, Germany). All samples were analysed in duplicate.

Lipid peroxidation products

The lipid peroxidation products malondialdehyde and 4-hydroxynonenal were measured in 100-µl aliquots of EDTA-containing plasma using a commercial Bioxytech LPO-586 colorimetric assay kit (Bio-Stat, Stockport, UK). The reaction yields a stable chromophore with maximal absorbance at 586 nm. All samples were tested in duplicate.

Analysis of kynurenines by high-performance liquid chromatography

Samples of plasma were analysed by high-performance liquid chromatography (HPLC) to quantify the concentrations of tryptophan, kynurenine and kynurenic acid, as described previously in detail [28, 29]. The method was based on that presented by Hervé et al. [30]. Tryptophan and L-kynurenine were quantified by ultraviolet (UV) absorption and kynurenic acid by fluorescence detection.

During sample preparation, plasma samples were kept on ice. To 500 µl plasma, 100 µl of internal standard (240 µm 3-nitro-L-tyrosine) and 50 µl 2 mm ascorbic acid were added, followed by 50 µl 4 m perchloric acid. Samples were vortexed for 30 s immediately after acid addition, centrifuged at 5000 g for 10 min at 4°C, and the supernatant collected. The precipitated proteins were resuspended in 100 µl water and 50 µl 4 m perchloric acid, the mixture vortexed for 30 s and centrifuged at 5000 g for 10 min at 4°C. Again the supernatant was collected. This washing and centrifugation step was repeated and the supernatants combined. The combined supernatants were filtered in Whatman Vectaspin Micro Anopore tubes during a further centrifugation at 3500 g for 5 min at 4°C, prior to injection of 100 µl onto the HPLC column. Calibration curves were determined using various concentrations of standard compounds in solution.

Isocratic reversed-phase HPLC was performed at 37 °C, using a Waters HPLC system (Waters UK, Elstree, Hertfordshire, UK). Separation was achieved using a Kingsorb C18 column (250 × 4.6 mm i.d., particle size 5 µm; Phenomenex, Macclesfield, UK) and the detection system included both a Waters 2487 dual-wavelength UV detector (250 nm and 365 nm) and a Waters 474 fluorescence detector, connected in series. The mobile phase was 50 mm acetic acid, 100 mm zinc acetate containing 3% acetonitrile. Zinc acetate was included in the mobile phase as it significantly enhances the fluorescence of kynurenic acid [31]. Tryptophan was determined by UV detection at a wavelength of 250 nm and kynurenine was detected at 365 nm. Kynurenic acid was determined by fluorescence detection (excitation 344 nm, emission 390 nm). The internal standard, 3-nitrotyrosine, was quantified at 365 nm. The flow rate was 1 ml min−1.

The limits of detection, using an injection volume of 100 µl and a signal-to-noise threshold of 3, were tryptophan 20 pmol, L-kynurenine 5 pmol and kynurenic acid 0.2 pmol on the column. Recoveries from a test batch of nine samples were >90%.

Statistics

All data are expressed as mean ± SEM. The effects of treatment were assessed using two-way anova with repeated measures for drug treatment and time. Differences between the treated group and the placebo group at corresponding time points were assessed using an unpaired t-test or Mann–Whitney (nonparametric) test and taking P < 0.05 as the limit of significance.

Results

Melatonin concentrations

All patients showed resting blood melatonin concentrations around 14 pg ml−1. At baseline there was no difference between the two groups allocated to receive melatonin or placebo (Figure 1). After 6 months of treatment, the concentrations of melatonin in patients given placebo were not different from this basal concentration, whereas the plasma concentrations in patients receiving melatonin treatment were more than twofold greater than at baseline (P > 0.001; Figure 1).

Figure 1
Concentrations of melatonin in the plasma of patients with rheumatoid arthritis treated with placebo or melatonin (10 mg nocte). There was a highly significant between-group increase in concentrations produced by dosing. ***P < 0.001. Placebo ...

CRP

There was no significant difference between the concentrations of CRP in the treated and placebo groups (Figure 2a) and there was no evidence of a differential effect of melatonin treatment over the 6 months of the study.

Figure 2
Levels of (a) C-reactive protein, (b) erythrocyte sedimentation rate, (c) neopterin and (d) lipid peroxidation products in the plasma of patients with rheumatoid arthritis treated with placebo or melatonin (10 mg nocte). Between-group comparison: *P < ...

ESR

Two-way anova indicated an overall significant effect of melatonin treatment (F(1,127) = 5.24, P = 0.024), but no effect of time. There was no significant difference between the ESR values in the treated and placebo groups at the start of the study assessed by t-test, although there was a significantly greater ESR in the melatonin recipients at 6 months of treatment (Figure 2b). This difference was also reflected in the existence of a significant linear trend for a decline in the ESR of placebo-treated patients (P= 0.022), although this was not so for the melatonin-treated group.

Neopterin

The concentrations of neopterin at baseline were similar to those we have previously found in healthy control subjects [29]. There was a significant effect of melatonin treatment on neopterin concentrations (F(1,136) = 4.64, P = 0.033), but no variation with time. There was no difference between neopterin concentrations in the treated and placebo groups at the start of the study, although t-tests showed a significantly higher concentration in the melatonin recipients at 4, 5 and 6 months of treatment (Figure 2c). Linear trend analysis revealed a significant downward trend in the placebo group (P= 0.012), but not in the melatonin-treated group.

Lipid peroxidation products

There were no significant differences in the concentrations of lipid peroxidation products between the placebo and melatonin-treated groups (Figure 2d), although two-way anova revealed a significant effect of time (F(1,128) = 3.699, P = 0.011). A linear trend analysis indicated a significant trend towards lower concentrations in the placebo group (P= 0.035), but not in patients treated with melatonin.

Clinical parameters

Two-way anova indicated no effect of melatonin or time on the modified HAQ score (Figure 3a). The clinician's assessment of disease severity on physical examination also indicated no significant effect of melatonin treatment, although there was a highly significant effect of time (two-way anova, F(1,104) = 43.01, P < 0.001) (Figure 3b).

Figure 3
Levels of (a) mean Health Assessment Questionnaire score and (b) physician-assessed disease severity in patients with rheumatoid arthritis treated with placebo or melatonin (10 mg nocte). Placebo (An external file that holds a picture, illustration, etc.
Object name is bcp0064-0517-fu3.jpg); Melatonin (□)

Cytokines

None of the three cytokines measured (IL-1β, IL-6 and TNF-α) showed any significant effect of melatonin treatment or time (two-way anova) (Figure 4).

Figure 4
Concentrations of (a) interleukin (IL)-1β, (b) tumour necrosis factor (TNF)-α and (c) IL-6 in the plasma of patients with rheumatoid arthritis treated with placebo or melatonin (10 mg nocte). There were no significant differences within ...

Tryptophan and kynurenines

The concentrations of tryptophan in all the patients were low in comparison with those that we and others have observed previously in control, disease-free subjects. The mean levels in the present cohorts were around 40 µm, whereas healthy subjects generally have concentrations around 60 µm [28, 29, 32, 33]. Two-way anova indicated highly significant effects of melatonin (F(1,124) = 11.41, P < 0.001) and of time (F(1,124) = 4.12, P = 0.044), but no interaction between them (F(1,124) = 1.05, P = 0.31) (Figure 5a).

Figure 5
Concentrations of (a) tryptophan, (b) L-kynurenine, (c) the k : t ratio and (d) concentration of kynurenic acid in the plasma of patients with rheumatoid arthritis treated with placebo or melatonin (10 mg nocte). Between-group comparison: *P < ...

Kynurenine concentrations were increased by melatonin treatment (F(1,124) = 4.24, P = 0.041) (Figure 5b). When the ratios of the concentrations of tryptophan to those of kynurenine (the k : t ratio) were calculated, it was noted that, although patients were allocated randomly to the placebo or melatonin groups, those patients in the melatonin group exhibited a higher ratio compared with the placebo group (Figure 5c). However, this difference did not change over the course of the study. There was no overall effect of melatonin or time on the concentrations of kynurenic acid in this study (Figure 5d).

Discussion

The baseline concentrations of melatonin in these patients (approximately 14 pg ml−1) are similar to those we have reported previously in healthy control subjects (around 18 pg ml−1; [34]). An earlier study in rheumatoid arthritis patients suggested that blood melatonin concentrations were decreased [35], consistent with the possibility that the loss of its antioxidant activity could contribute to the disease. The concentrations quoted in that report were around 20 pg ml−1 in controls and 6 pg ml−1 in patients. The same study examined the effect of administering the cyclooxygenase inhibitor indomethacin to a group of healthy control subjects, with the result that the blood concentrations of melatonin were reduced to concentrations similar to those found in arthritic patients. It seems likely, therefore, that the lower values in patients may have been caused by the consumption of NSAIDs, which were, at the time, the mainstay of treatment for the condition.

It was extremely encouraging to find that a single daily dose of 10 mg of melatonin, taken before retiring, yielded substantially elevated concentrations in the blood that were maintained until at least the following morning, despite the fact that its half-life in blood is around 40 min [36]. We are confident, therefore, that we have produced a significant increase in the functional activity of this hormone over an extended period of time each day. It is not possible to say what the peak concentrations of melatonin would have been overnight, but those we have measured the morning after consuming the compound, around 280 pg ml−1 (Figure 1), are substantially greater than the peak nocturnal concentrations of around 50 pg ml−1 observed in normal subjects [37, 38], even though our blood samples were taken 10–12 h after the presumed nocturnal peak. The occurrence of these high concentrations testifies to the high rate of compliance of the melatonin-treated group.

For several of the parameters measured in this study it is clear that there is no effect of melatonin administration in the early period up to about 3 months. There are, however, several differences which develop between placebo and melatonin groups in the period between 3 and 6 months. Perhaps the most surprising of these is the finding that the chronic administration of melatonin, at least in the dose of 10 mg day−1 for several months, tends to increase, rather than decrease, the inflammatory indicators ESR and neopterin concentrations in the sense that melatonin prevents or reverses the significant downward trend which was seen in the placebo group. There is a general decrease in the concentration of peroxidation markers from their initial levels, at the beginning of this study, which were around fourfold greater in these patients than those we have seen in healthy volunteer subjects [28].

These results are counter to the view that the antioxidant activity of melatonin should help suppress the symptoms and progression of rheumatoid arthritis, although this view was based largely on the reportedly lower concentrations of melatonin in patients [35], a finding that we have been unable to reproduce, as discussed above. Indeed, on the contrary, the administration of melatonin has been claimed to exacerbate experimentally induced arthritis in mice [39], while pinealectomy improves the condition [40], results that would be consistent with the present data.

There is a further paradox in that, despite the decrease of tissue peroxidation and the rise in ESR and neopterin concentrations, there are no significant differences between placebo and melatonin groups either in the assessments of patients' symptoms, or in the concentrations of the proinflammatory cytokines IL-1β, IL-6 or TNF-α. There is a substantial volume of work favouring the view that melatonin can suppress the production of TNF-α [9, 18, 19, 4144]. However, many of these studies have use different doses or concentrations, different cell types in vitro including monocytes, Th-2 lymphocytes, macrophages or undefined cellular sources, or various in vivo systems, and different activating stimuli such as lipopolysaccharide or phytohaemagglutinin, which make it difficult to compare with the present study. Some of these same investigations have also noted increased production of IL-1β at doses or concentrations that suppressed TNF-α [19], while other studies have observed that melatonin has little or no effect on the release of either IL-1β or TNF-α, especially from macrophages [45, 46]. In contrast, there is good evidence that there are receptors for melatonin on synovial macrophages [47, 48] which promote the release of some Th-1-type proinflammatory cytokines such as IL-12 [49]. Indeed, it has been pointed out that the higher blood concentrations of melatonin in arthritic patients, especially in the early morning, may help to explain the morning stiffness and joint swelling experienced by patients [5052].

There are therefore several possible reasons for the complex effects of melatonin in arthritis, including the possibility that suppression of proinflammatory cytokines does not occur in human subjects with daily melatonin doses of 10 mg per subject, or that any effect of melatonin on cytokine release is sufficiently small to be lost in the group statistics, or that the net effects of melatonin result from changes in the balance of several cytokines, such as IL-1β, TNF-α and IL-12. It would be valuable to re-examine the effects of melatonin using multicytokine arrays to assess changes in the pattern of these and other inflammation modulating factors.

We have previously reported that tryptophan concentrations are decreased and kynurenine concentrations increased in patients with rheumatoid arthritis relative to healthy control subjects [22]. It was subsequently discovered that the administration of a tryptophan load to healthy subjects increased the production of lipid peroxidation products, indicating increased concentrations of oxidative stress [23]. Since melatonin is itself a tryptophan metabolite, the possibility was considered that indirectly induced changes of tryptophan metabolism could contribute to the effects of melatonin in vivo. However, the present results reveal no clear or consistent changes of tryptophan metabolism in these groups of patients, suggesting, first, that the dose of melatonin used here was not sufficient to modify tryptophan metabolism, and second, that the antioxidant, and possible proinflammatory effects, of melatonin seen here were not mediated by changes in the concentrations of the kynurenine pathway-oxidized tryptophan metabolites.

It seems likely, therefore, that there is a balance of activities mediated by melatonin, and the overall outcome of its administration on clinical and inflammatory parameters may depend on a wide range of factors. In particular, since the decrease in oxidation and increase of inflammatory markers develop over several months, the chronic administration of melatonin, as in the present work, may lead to the development of a biochemical profile that is not seen in acute studies.

The chronic administration of melatonin may, for example, allow an equilibrium to be reached between the concentrations and activities of the hormone itself and its metabolites, each acting differently on several leucocyte subtypes, and on pteridine and cytokine production. A major metabolite of melatonin, 6-hydroxymelatonin, can undergo a unique form of quinone reduction which leads to the oxidation of a variety of molecules [53]. One possibility is that with chronic administration, the accumulation or increased efficacy of 6-hydroxymelatonin compromises the antioxidant activity of melatonin itself. Another possible factor is that melatonin and several metabolites, including N-acetyl-5-methoxykynurenamine, are potent inhibitors of cyclooxygenase [5458] and it may be that chronic inhibition of this pro-oxidant enzyme contributes to the lowering of tissue peroxidation. At the same time, the complex and controversial effects of melatonin on cytokine production, noted above, appear not to be large or consistent enough to generate a clear overall anti-inflammatory action in rheumatoid arthritis.

Acknowledgments

We are grateful to the NHS R&D Levy, the Denbies Foundation and the Peacock Trust for financial support. We thank Ms Rosalind McMillan and Ms Gillian Harman for their most excellent technical assistance in quality and quantity.

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