MELATONIN LEVELS ARE DECREASED IN RHEUMATOID ARTHRITIS Stanley K. West 1 and Jan M.C. Oosthuizen 2
department of Medical Physiology and Biochemistry, Faculty of Medicine, University of Stellenbosch, and 2Department of Physiology, Faculty of Medicine, University of the Orange Free State, Republic of South Africa
ABSTRACT
The hormone, melatonin, is a product of the pineal gland. This methoxy-indole, also known as N-acetyl-5-methoxy-tryptamine, is structurally related to indomethacin, a derivative of methylated indole. The fact that indomethacin has been used successfully in patients suffering from certain chronic inflammatory conditions (such as rheumatoid- and osteoarthritis) gives rise to the question as to whether melatonin also possesses anti-inflammatory virtues. This hypothesis has been tested by determining melatonin concentration levels by means of a radioimmunoassay in patients with rheumatoid arthritis. The daytime melatonin levels of untreated patients were significantly lower (mean concentration = 5.76 pg/ml) than the normal value (mean concentration = 15-33 pg/ml). The second part of this study showed that the administration of indomethacin (100 mg/day) to normal healthy subjects led to a 14.7 pg/ml decrease in plasma melatonin levels. This may indicate that melatonin and indomethacin act synergistically.
KEYWORDS
methoxy-indole, N-acetyl-5-methoxy-tryptamine, anti-inflammatory, cyclo-oxygenase inhibitor
indomethacin,
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33
Vol. 3, No. 1, 1992
Melatonin in Rheumatoid Arthritis
INTRODUCTION
Inflammation is coupled to the production of various mediators at the site of inflammation. These mediators include bradykinin, histamine, acetylcholine as well as prostaglandins. The immune response involves a complex series of cellular events each of which is initiated by antigen-specific and -nonspecific stimuli /I/. One of the antigen-nonspecific responses results directly from the activity of metabolites of arachidonic acid. PGE 2 inhibits antigen and mitogen stimulated T-cell proliferation /2/, as well as the production of lymphokines, interferon, and leukocyte migration inhibiting factor /3/. Indomethacin - a derivative of methylated indole - inhibits cyclo-oxygenase and therefore the production of prostaglandins. The modus operandi of this process is particularly complex and involves a chemical site on the enzyme other than the one that is acetylated during inhibition by aspirin. The methoxy-indole, melatonin (N-acetyl-5-methoxy-tryptamine), is structurally related to indomethacin (Fig. 1). Considering their molecular configurations, indomethacin and melatonin might be expected to have overlapping activities. It has been found, for instance, that melatonin inhibits the synthesis of thromboxane /4/, confirming the involvement of melatonin in inhibiting the synthesis of prostaglandins. It was therefore hypothesized that low plasma melatonin levels might be expected in patients suffering from inflammatory diseases, particularly if that process can readily be suppressed by indomethacin.
MATERIALS AND METHODS
Plasma melatonin concentrations were measured in 34 patients suffering from rheumatoid arthritis. None of these patients had been on any treatment for 3 weeks. In order to be included in this study, a patient had to fulfil the following criteria: 1. 2. 3. 4. 5.
34
Be a postmenopausal woman; Have an elevated erythrocyte sedimentation rate; Be rheumatic factor positive; Be antinucleus-antibody positive; Have rheumatoid arthritis as an absolute indication for indomethacin therapy.
Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
S.K. West and J.M.C. Oosthuizen
Journal of Basic & Clinical Physiology & Pharmacology
Melatonin
Indomethacln
Fig. 1:
Biochemical structures of melatonin and indomethacin.
All these patients were seen at the Arthritis Clinic, National Hospital, Bloemfontein. The data collected from these patients were compared with those of a control group of 100 women (Fig. 2) in the same age category, in whom criteria 2-5 were negative. Melatonin secretion is extremely dependent on light exposure. The aim was to get the subjects of the different groups to submit to the same standards of light exposure. One way of overcoming this problem was to ensure that sampling of the blood specimens of each group took place at 0800 h each time. This is when, under physiological conditions, daytime low melatonin levels should not yet have been reached. Whereas light in the common intensity range of artificial illumination (0-1500 lux) does not show a significant effect on free-running circadian rhythms of man, bright light with intensities above 3000 lux has been considered to show such an effect. Our patients, however, had not been subjected to such high intensities at that time of day. Heparinized venous blood was collected from an arm vein at 0800 h in both patients and controls. This was immediately transferred to polypropylene test tubes, which were then centrifuged at 1000 r.p.m. at 4°C for 10 minutes. The clear Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
35
Vol. 3, No. 1, 1992
30
Melatonin in Rheumatoid
/I
0
Patients if = 5.76 p g / m l SD = 1.68
0
5
10
IS
20
Arthritis
Control χ = 24.35 p g / m l SD = 9.28
25
30
35
40
45
Ψ 50
[MELATONIN ] ( p g / m l ) Fig. 2:
The
distributions
of
plasma
melatonin
concentrations
in
rheumatoid arthritic patients (shaded bars) and a control group (open bars).
supernatant plasma was removed and stored at -20 °C in total darkness. In a second study we investigated whether the plasma melatonin levels of normal postmenopausal women could be manipulated by the administration of indomethacin. Ten subjects were given 100 mg oral indomethacin per day for seven days. Their 0800 h plasma melatonin levels were measured before and after treatment (Fig. 3). The ages and weights of all three groups are shown in Table 1. Informed consent was obtained from each subject, and the protocol was approved by the Ethics Committee of the institution. Extraction procedure The heparinized plasma (1.0 ml) was pipetted into glass test tubes; it was then mixed with 5.0 ml dichloromethane and vortexed for 30 seconds in order to extract the lipid soluble fraction. The tubes containing the plasma-dichloromethane mixture were then 36
Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
Journal of Basic & Clinical Physiology & Pharmacology
S.K West and J.M.C. Oosthuizen
TABLE 1 Mean ages and weights of untreated rheumatoid arthritic patients (A), the control group (B) and healthy subjects who received indomethacin therapy for one week (C)
A
Β
χ AGE (years)
62.32 + SD
χ Weight (kg)
67.94 + SD 13.95
pre
8.90
C
9.39
67.70 + SD
8.62
66.02 + SD 11.39
-61.80 + SD
6.58
56.30 + SD
pre:
32.60 p g / m l SD = 12.69
post:
17.90 p g / m l SD = 5.57
post
INDOMETHACIN ADMINISTRATION FOR ONE WEEK
Fig. 3:
Plasma melatonin concentrations of normal subjects before and after administration of indomethacin for one week.
Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
37
Vol. 3, No. 1, 1992
Melatonin in Rheumatoid Arthritis
centrifuged for 20 minutes at 4000 r.p.m. and 4°C. The aqueous phase as well as the lipid interphase were aspirated by vacuum suction in a fume cupboard. Care was taken not to disturb the dichloromethane meniscus. The dichloromethane mixture (containing the lipid soluble fraction) was then transferred to clean glass tubes. The dichloromethane was subsequently evaporated by means of a nitrogen stream in a water-bath at 37 °C. The residue was resuspended in 500 μΐ 0.1% gelatine phosphate buffer and analysed for melatonin content, using ultraspecific rabbit anti-melatonin antibody as suggested in the radioimmunoassay described by Brown /5/.
RESULTS
The mean plasma melatonin concentration of untreated, rheumatic, postmenopausal patients was 5.76 ± 1.86 (SD) pg/ml. This is statistically significantly lower than the mean concentration of normal subjects: 24.4 ± 9.28 (SD) pg/ml (2-tailed Student's ttest; ρ < 0.001) (Fig. 2). The mean plasma melatonin concentration of the healthy subjects showed a significant decrease after one week of indomethacin therapy: 17.9 ± 7.57 (SD) pg/ml, compared to the initial value of 32.6 ± 12.69 (SD) pg/ml prior to administration of indomethacin (paired Student's t-test; ρ < 0.019) (Fig. 3). To eliminate the possibility that the decreased melatonin concentrations were merely a result of ageing, the plasma concentrations of the hormone in the rheumatoid arthritic group were age-matched to those in the control group. Statistical analysis indicates that no significant difference exists between the melatonin concentrations of the five intervals within each group (paired Student's t-test; ρ > 0.05), while the difference still remains obvious between the experimental and control groups (Fig. 4). This study shows that the low melatonin levels in rheumatic disease do not result from normal reduced secretion, sometimes associated with ageing.
DISCUSSION
We have shown that the plasma melatonin concentrations of untreated rheumatic patients are considerably lower than those of age-matched controls. The reason is uncertain. The lowered plasma melatonin levels could conceivably be a primary, 38
Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
S.K. West and J.M.C. Oosthuizen
Journal of Basic & Clinical Physiology & Pharmacology
40 35 30 __ 25
c c ο 20 φ 2
15 10 5
0
Fig. 4:
40-49
50-59 60-69 70-79 Age intervals
80-89
Plasma melatonin concentrations at different age intervals in the rheumatoid arthritic group (shaded bars) and the control group (open bars).
contributory factor in the pathogenesis of rheumatoid arthritis, which could lead to inappropriate accumulation of prostaglandins in arthritic tissue. Assuming, however, that indomethacin has at least the same, if not more pronounced, antiprostaglandin activities as does melatonin, then it is unlikely that a reduction in plasma melatonin concentrations is the direct cause of the inflammation in rheumatoid arthritis: indomethacin only partially relieves the symptoms of this disease. A second possibility is that the reduced plasma melatonin levels and the arthritic process may be caused by the same underlying defect. The reduced melatonin concentrations then merely aggravate the symptoms. The final possibility is that the melatonin concentrations in these patients had been suppressed by medication. We have shown that indomethacin therapy can dramatically lower the concentrations of melatonin in the plasma of normal subjects. Our patients had, to the best of our knowledge, not had any therapy during the 3 weeks prior to being tested, but we cannot exclude the possibility that some of them may nevertheless have taken analgesics which also have a melatonin lowering effect. The salicylates are structurally related to indomethacin and also have an antiprostaglandin action. 39 Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
Vol. 3, No. 1, 1992
Melatonin in Rheumatoid Arthritis
This could have been taken in any of many forms, some of which the patients may have regarded as mere "tonics". However, we did question the patients very closely about any medication they might have taken during the 3 weeks before the test. We therefore consider it very unlikely that recent medication could have influenced the results. Normal people also ingest analgesics for many reasons, which would then also have a melatonin suppressing effect, which we did not find. O u r conclusion is therefore that rheumatoid arthritis itself may probably be associated with reduced melatonin levels. However, more detailed studies concerning the diurnal secretion pattern of melatonin in rheumatic arthritis are essential in order to obtain a better understanding of the role this hormone plays in the symptomatology of the disease.
REFERENCES 1. Stenson WF, Parker CW. Prostaglandins and the immune response. In: Lee JB, ed, Prostaglandins. New York: Elsevier, 1982; p. 30. 2. Goodwin JS, Bankhurst AD, Messner ΑΡ. Suppression of human T-cell mitogenesis by prostaglandin. J Exp Med 1977; 146:1719-1734. 3. Rapaport RS, Dodge GR. Prostaglandin^ inhibits the production of human interleukin-2. J Exp Med 1982; 155:943-948. 4. Leach CM, Thorburn GD. A comparison of the inhibitory effects of melatonin and indomethacin on platelet aggregation and thromboxane release. Prostaglandins 1980; 20: 51-56. 5. Brown GM. Document: Chemistry Immunology Diagnostics. CIDtech Research Inc., Hamilton, Ontario, Canada, October 1985.
40 Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
department of Medical Physiology and Biochemistry, Faculty of Medicine, University of Stellenbosch, and 2Department of Physiology, Faculty of Medicine, University of the Orange Free State, Republic of South Africa
ABSTRACT
The hormone, melatonin, is a product of the pineal gland. This methoxy-indole, also known as N-acetyl-5-methoxy-tryptamine, is structurally related to indomethacin, a derivative of methylated indole. The fact that indomethacin has been used successfully in patients suffering from certain chronic inflammatory conditions (such as rheumatoid- and osteoarthritis) gives rise to the question as to whether melatonin also possesses anti-inflammatory virtues. This hypothesis has been tested by determining melatonin concentration levels by means of a radioimmunoassay in patients with rheumatoid arthritis. The daytime melatonin levels of untreated patients were significantly lower (mean concentration = 5.76 pg/ml) than the normal value (mean concentration = 15-33 pg/ml). The second part of this study showed that the administration of indomethacin (100 mg/day) to normal healthy subjects led to a 14.7 pg/ml decrease in plasma melatonin levels. This may indicate that melatonin and indomethacin act synergistically.
KEYWORDS
methoxy-indole, N-acetyl-5-methoxy-tryptamine, anti-inflammatory, cyclo-oxygenase inhibitor
indomethacin,
Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
33
Vol. 3, No. 1, 1992
Melatonin in Rheumatoid Arthritis
INTRODUCTION
Inflammation is coupled to the production of various mediators at the site of inflammation. These mediators include bradykinin, histamine, acetylcholine as well as prostaglandins. The immune response involves a complex series of cellular events each of which is initiated by antigen-specific and -nonspecific stimuli /I/. One of the antigen-nonspecific responses results directly from the activity of metabolites of arachidonic acid. PGE 2 inhibits antigen and mitogen stimulated T-cell proliferation /2/, as well as the production of lymphokines, interferon, and leukocyte migration inhibiting factor /3/. Indomethacin - a derivative of methylated indole - inhibits cyclo-oxygenase and therefore the production of prostaglandins. The modus operandi of this process is particularly complex and involves a chemical site on the enzyme other than the one that is acetylated during inhibition by aspirin. The methoxy-indole, melatonin (N-acetyl-5-methoxy-tryptamine), is structurally related to indomethacin (Fig. 1). Considering their molecular configurations, indomethacin and melatonin might be expected to have overlapping activities. It has been found, for instance, that melatonin inhibits the synthesis of thromboxane /4/, confirming the involvement of melatonin in inhibiting the synthesis of prostaglandins. It was therefore hypothesized that low plasma melatonin levels might be expected in patients suffering from inflammatory diseases, particularly if that process can readily be suppressed by indomethacin.
MATERIALS AND METHODS
Plasma melatonin concentrations were measured in 34 patients suffering from rheumatoid arthritis. None of these patients had been on any treatment for 3 weeks. In order to be included in this study, a patient had to fulfil the following criteria: 1. 2. 3. 4. 5.
34
Be a postmenopausal woman; Have an elevated erythrocyte sedimentation rate; Be rheumatic factor positive; Be antinucleus-antibody positive; Have rheumatoid arthritis as an absolute indication for indomethacin therapy.
Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
S.K. West and J.M.C. Oosthuizen
Journal of Basic & Clinical Physiology & Pharmacology
Melatonin
Indomethacln
Fig. 1:
Biochemical structures of melatonin and indomethacin.
All these patients were seen at the Arthritis Clinic, National Hospital, Bloemfontein. The data collected from these patients were compared with those of a control group of 100 women (Fig. 2) in the same age category, in whom criteria 2-5 were negative. Melatonin secretion is extremely dependent on light exposure. The aim was to get the subjects of the different groups to submit to the same standards of light exposure. One way of overcoming this problem was to ensure that sampling of the blood specimens of each group took place at 0800 h each time. This is when, under physiological conditions, daytime low melatonin levels should not yet have been reached. Whereas light in the common intensity range of artificial illumination (0-1500 lux) does not show a significant effect on free-running circadian rhythms of man, bright light with intensities above 3000 lux has been considered to show such an effect. Our patients, however, had not been subjected to such high intensities at that time of day. Heparinized venous blood was collected from an arm vein at 0800 h in both patients and controls. This was immediately transferred to polypropylene test tubes, which were then centrifuged at 1000 r.p.m. at 4°C for 10 minutes. The clear Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
35
Vol. 3, No. 1, 1992
30
Melatonin in Rheumatoid
/I
0
Patients if = 5.76 p g / m l SD = 1.68
0
5
10
IS
20
Arthritis
Control χ = 24.35 p g / m l SD = 9.28
25
30
35
40
45
Ψ 50
[MELATONIN ] ( p g / m l ) Fig. 2:
The
distributions
of
plasma
melatonin
concentrations
in
rheumatoid arthritic patients (shaded bars) and a control group (open bars).
supernatant plasma was removed and stored at -20 °C in total darkness. In a second study we investigated whether the plasma melatonin levels of normal postmenopausal women could be manipulated by the administration of indomethacin. Ten subjects were given 100 mg oral indomethacin per day for seven days. Their 0800 h plasma melatonin levels were measured before and after treatment (Fig. 3). The ages and weights of all three groups are shown in Table 1. Informed consent was obtained from each subject, and the protocol was approved by the Ethics Committee of the institution. Extraction procedure The heparinized plasma (1.0 ml) was pipetted into glass test tubes; it was then mixed with 5.0 ml dichloromethane and vortexed for 30 seconds in order to extract the lipid soluble fraction. The tubes containing the plasma-dichloromethane mixture were then 36
Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
Journal of Basic & Clinical Physiology & Pharmacology
S.K West and J.M.C. Oosthuizen
TABLE 1 Mean ages and weights of untreated rheumatoid arthritic patients (A), the control group (B) and healthy subjects who received indomethacin therapy for one week (C)
A
Β
χ AGE (years)
62.32 + SD
χ Weight (kg)
67.94 + SD 13.95
pre
8.90
C
9.39
67.70 + SD
8.62
66.02 + SD 11.39
-61.80 + SD
6.58
56.30 + SD
pre:
32.60 p g / m l SD = 12.69
post:
17.90 p g / m l SD = 5.57
post
INDOMETHACIN ADMINISTRATION FOR ONE WEEK
Fig. 3:
Plasma melatonin concentrations of normal subjects before and after administration of indomethacin for one week.
Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
37
Vol. 3, No. 1, 1992
Melatonin in Rheumatoid Arthritis
centrifuged for 20 minutes at 4000 r.p.m. and 4°C. The aqueous phase as well as the lipid interphase were aspirated by vacuum suction in a fume cupboard. Care was taken not to disturb the dichloromethane meniscus. The dichloromethane mixture (containing the lipid soluble fraction) was then transferred to clean glass tubes. The dichloromethane was subsequently evaporated by means of a nitrogen stream in a water-bath at 37 °C. The residue was resuspended in 500 μΐ 0.1% gelatine phosphate buffer and analysed for melatonin content, using ultraspecific rabbit anti-melatonin antibody as suggested in the radioimmunoassay described by Brown /5/.
RESULTS
The mean plasma melatonin concentration of untreated, rheumatic, postmenopausal patients was 5.76 ± 1.86 (SD) pg/ml. This is statistically significantly lower than the mean concentration of normal subjects: 24.4 ± 9.28 (SD) pg/ml (2-tailed Student's ttest; ρ < 0.001) (Fig. 2). The mean plasma melatonin concentration of the healthy subjects showed a significant decrease after one week of indomethacin therapy: 17.9 ± 7.57 (SD) pg/ml, compared to the initial value of 32.6 ± 12.69 (SD) pg/ml prior to administration of indomethacin (paired Student's t-test; ρ < 0.019) (Fig. 3). To eliminate the possibility that the decreased melatonin concentrations were merely a result of ageing, the plasma concentrations of the hormone in the rheumatoid arthritic group were age-matched to those in the control group. Statistical analysis indicates that no significant difference exists between the melatonin concentrations of the five intervals within each group (paired Student's t-test; ρ > 0.05), while the difference still remains obvious between the experimental and control groups (Fig. 4). This study shows that the low melatonin levels in rheumatic disease do not result from normal reduced secretion, sometimes associated with ageing.
DISCUSSION
We have shown that the plasma melatonin concentrations of untreated rheumatic patients are considerably lower than those of age-matched controls. The reason is uncertain. The lowered plasma melatonin levels could conceivably be a primary, 38
Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
S.K. West and J.M.C. Oosthuizen
Journal of Basic & Clinical Physiology & Pharmacology
40 35 30 __ 25
c c ο 20 φ 2
15 10 5
0
Fig. 4:
40-49
50-59 60-69 70-79 Age intervals
80-89
Plasma melatonin concentrations at different age intervals in the rheumatoid arthritic group (shaded bars) and the control group (open bars).
contributory factor in the pathogenesis of rheumatoid arthritis, which could lead to inappropriate accumulation of prostaglandins in arthritic tissue. Assuming, however, that indomethacin has at least the same, if not more pronounced, antiprostaglandin activities as does melatonin, then it is unlikely that a reduction in plasma melatonin concentrations is the direct cause of the inflammation in rheumatoid arthritis: indomethacin only partially relieves the symptoms of this disease. A second possibility is that the reduced plasma melatonin levels and the arthritic process may be caused by the same underlying defect. The reduced melatonin concentrations then merely aggravate the symptoms. The final possibility is that the melatonin concentrations in these patients had been suppressed by medication. We have shown that indomethacin therapy can dramatically lower the concentrations of melatonin in the plasma of normal subjects. Our patients had, to the best of our knowledge, not had any therapy during the 3 weeks prior to being tested, but we cannot exclude the possibility that some of them may nevertheless have taken analgesics which also have a melatonin lowering effect. The salicylates are structurally related to indomethacin and also have an antiprostaglandin action. 39 Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
Vol. 3, No. 1, 1992
Melatonin in Rheumatoid Arthritis
This could have been taken in any of many forms, some of which the patients may have regarded as mere "tonics". However, we did question the patients very closely about any medication they might have taken during the 3 weeks before the test. We therefore consider it very unlikely that recent medication could have influenced the results. Normal people also ingest analgesics for many reasons, which would then also have a melatonin suppressing effect, which we did not find. O u r conclusion is therefore that rheumatoid arthritis itself may probably be associated with reduced melatonin levels. However, more detailed studies concerning the diurnal secretion pattern of melatonin in rheumatic arthritis are essential in order to obtain a better understanding of the role this hormone plays in the symptomatology of the disease.
REFERENCES 1. Stenson WF, Parker CW. Prostaglandins and the immune response. In: Lee JB, ed, Prostaglandins. New York: Elsevier, 1982; p. 30. 2. Goodwin JS, Bankhurst AD, Messner ΑΡ. Suppression of human T-cell mitogenesis by prostaglandin. J Exp Med 1977; 146:1719-1734. 3. Rapaport RS, Dodge GR. Prostaglandin^ inhibits the production of human interleukin-2. J Exp Med 1982; 155:943-948. 4. Leach CM, Thorburn GD. A comparison of the inhibitory effects of melatonin and indomethacin on platelet aggregation and thromboxane release. Prostaglandins 1980; 20: 51-56. 5. Brown GM. Document: Chemistry Immunology Diagnostics. CIDtech Research Inc., Hamilton, Ontario, Canada, October 1985.
40 Brought to you by | University of Arizona Authenticated Download Date | 7/7/15 6:33 AM
