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Service de Radioanalyse, Hôpital Neuro-Cardiologique and * Laboratoire de Physiologie de l’Environnement, Faculté de Médecine, Lyon, France
3To whom correspondence should be addressed. E-mail: bruno.claustrat{at}chu-lyon.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • melatonin • folate • deficiency • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). MLT synthesis in the pineal gland undergoes large daily changes, with a peak occurring at night. This day/night rhythm of MLT synthesis is generated at the serotonin acetylation step by a large nighttime increase in the activity of a specific aryl-alkylamine-N-acetyltransferase (NAT, EC 2.3.1.87). The activation of NAT depends on the activity of the sympathetic nerves originating from the superior cervical ganglia (2
). By contrast, hydroxy-indole-O-methyltransferase (HIOMT, EC 2.1.1.4), the enzyme that catalyzes the final step of MLT synthesis from N-acetyl-serotonin, with S-adenosylmethionine (SAM) as a methyl donor, displays little or no day/night change.
SAM is provided by a metabolic pathway involving sulfur-containing amino acids (homocysteine and methionine) (Fig. 1
). Homocysteine is at the intersection of two pathways, i.e., remethylation to methionine, which requires folate and vitamin B-12 (or choline via betaine in an alternative reaction), and transsulfuration to cystathionine, which requires pyridoxal 5'-phosphate as a cofactor (3
). These pathways are coordinated by SAM, which acts as an allosteric inhibitor of the methylenetetrahydrofolate reductase and as an activator of cystathionine ß-synthase.
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). Although sulfhydryl radicals are considered to be strong reducing groups, some authors have suggested that homocysteine is a prooxidant that produces H2O2 (3
).
MLT displays marked protective effects against oxidative stress, as a free radical scavenger. This action is aided by its ability to cross all biological membranes without a receptor (5
). Moreover, MLT increases gene expression for various antioxidant enzymes in rat brain (6
). The present study was undertaken to address the question whether folate deficiency could alter MLT secretion in rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The study was conducted in accordance with the guidelines approved for animal experimental procedures by the French Ethics Committee (decree 87–848). Male Sprague-Dawley rats (n = 24; 190 ± 10 g, 6 wk old; IFFA CREDO, L’Arbresle, France) were housed in a room with controlled temperature (21 ± 2°C) and a 12:12-h light:dark (lights on at 0700 h, 300 lx at the animal level). They had free access to food (nonpurified diet A04, U.A.R., Epinay sur Orge, France) and water. After a 2-wk adaptation period, they were randomly assigned to two groups (3 rats/cage). The control group (n = 12) received a synthetic diet (HICG) (7
) containing 20% casein, 8% gelatin, 8 mg folic acid + 10 g succinylsulfathiazole/kg diet for 4 wk, whereas the folate-deficient group (n = 12) was fed an identical diet with the folate omitted for the same time. Diets were supplied by INRA-UPAE (Jouy en Josas, France).
Sample collection.
The rats were kept individually in metabolic cages (U.A.R.) to collect urine for 1 d before diet administration and at the end of wk 2 and 4 of treatment. Urine samples were collected every 3 h over a 24-h period, using a fraction collector maintained at 7°C, then weighed after collection to determine their volume and stored at -20°C until assay. Blood (0.2 mL) was collected weekly by incision of the tail for hematological evaluation. At the end of the experiment, the rats were killed by decapitation under anesthesia with diethylether at a precise time, between 0900 and 1200 h. Trunk blood was collected into plastic tubes containing EDTA and centrifuged at 4°C (2000 x g for 10 min). Plasma was stored at -20°C until assay. Brains were removed immediately after killing and stored at -70°C for further studies. Pineal glands were dissected separately.
Analyses.
Body weight was measured twice weekly. Hematological variables including leukocyte and erythrocyte counts, blood hematocrit and hemoglobin concentration were determined using an autoanalyzer (Minos STX, ABX, Montpellier, France). Erythrocyte folate concentration was determined with an 125I folate kit (ICN Pharmaceuticals, Costa Mesa, CA). Plasma total homocysteine (tHcy) was measured by HPLC using the fluorometric method of Ubbink et al. (8
).
The levels of 6 sulfatoxymelatonin (aMT6S), the main hepatic MLT metabolite, and/or MLT were determined in urine and/or pineal samples, using RIA developed in our laboratory (9
,10
). All pineal glands and urine samples from each rat were assayed in the same series to eliminate the interassay variability. Plasma epinephrine (E) and norepinephrine (NE) were determined after alumina extraction using HPLC and electrochemical detection. Metanephrines (metepinephrine, ME and normetepinephrine, NME), which represent methylated catabolites of catecholamines, were determined in urine by HPLC with electrochemical detection after ion-exchange solid phase extraction (11
).
Statistical analysis.
All results are presented as means ± SEM. Student’s t test was used to detect significant group differences in data obtained at wk 4. Urine data were tested by multiple- or two-way ANOVA for repeated measures to evaluate the influence of diet and time on MLT, aMT6S and catechol compound levels. Post-hoc comparisons were performed with Bonferroni’s multiple range test to identify the significant differences. Differences were considered significant at P < 0.05 (two-tailed).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). At no time point did the mean weights differ between the two groups (data not shown). Erythrocyte folate concentrations were significantly lower and plasma tHcy concentrations were significantly higher in the folate-deficient rats than in the controls. Figure 2
displays the 24-h urine MLT (panel A) and aMT6S (panel B) excretions in control and folate-deficient rats. Multiple ANOVA showed daily MLT and aMT6S variations as well as effect of time (wk) and a time x treatment interaction. Multiple comparisons performed on 3-h blocks showed that the alterations in MLT and aMT6S excretions occurred in the evening and the night and were maximal at wk 2 in the folate-deficient group. MLT and aMT6S excretions peaked at the same time. In addition, a two-way ANOVA performed on day, night and 24 h MLT and aMT6S excretions showed an effect of time and a time x treatment interaction. Post-hoc comparisons confirmed a treatment effect that was more pronounced at wk 2 (Table 2
). The 24-h urine aMT6S excretion was lower in the folate-deficient rats at wk 2 and 4 than at wk 0 (more marked at wk 2), whereas night and 24-h urine MLT and night urine aMT6S excretions were lower only at wk 2 vs. wk 0. Night and 24-h urine MLT and aMT6S excretions differed between the two groups at wk 2 and 4. Pineal MLT concentration in the folate-deficient group was lower than in the control at wk 4 (Table 3
). Urinary methoxylated catechol (NME and ME) excretions were lower in the folate-deficient group at wk 2 and 4 than at wk 0. By contrast, plasma NE and E levels did not differ between groups when measured at wk 4 (Table 3)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1% of the total secretion, appears to be a sensitive marker for our purpose. Nocturnal MLT secretion is very sensitive to folate deficiency because reduced MLT excretion was observed from wk 2 of folate deficiency. The slight rise of urine MLT and aMT6S levels at wk 4 could be related to an alternative methylation of N-acetyl-serotonin with choline as a donor because folate and choline metabolism are interdependent. Severe folate deficiency causes secondary depletion of choline and phosphocholine in rat liver (12
). By contrast, in the control rats, which were 10 wk old at the end of the experiment, there was a slight (P = 0.07) increase in MLT excretion with time, which could be related to growth. Indeed, in young adult rats, MLT secretion increases with maturation (13
).
The decreases in MLT and aMT6S excretions in the folate-deficient rats, which were more marked during the first part of the night, were probably the consequence of impaired methylation of N-acetyl-serotonin, the last step of MLT synthesis, rather than an alteration of the release of NE from sympathetic nerve terminals in the pineal gland. In the absence of abnormal plasma E and NE concentrations, the decrease in urine methoxylated catechol excretions strengthens this hypothesis. Further, the inappropriate presence of disulfide-containing compounds (circulating tHcy is the sum of free, disulfide and protein-bound Hcy) could inactivate HIOMT, with the consequence of a reinforced alteration of MLT synthesis (14
).
Our present data could explain, at least in part, why MLT secretion declines progressively with age in mammals, especially in humans where folate deficiency can occur in the elderly (15
). Results of case studies and population-based studies have suggested that low blood folate concentrations are related to dementia and to poor cognitive function in older adults (16
). Recent data indicate that serum or cerebrospinal fluid folate levels have a strong negative association with the risk of Alzheimer’s disease (17
,18
). In addition, a decrease in the amplitude of the MLT rhythm has been described in senile dementia and in elderly people with insomnia (19
,20
). It would be of interest to evaluate folate status in these patients and to determine MLT levels after folate supplementation because a beneficial effect of folate treatment of insomnia has been suggested (21
).
In conclusion, our findings show a close interaction between folate and MLT metabolism. They suggest the possible benefits of testing the hypothesis that folate supplementation could be an indirect and safe way to increase MLT secretion in humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported in part by European grant BIOMED BMH4 CT97 2327.
4 Abbreviations used: aMT6S, 6 sulfatoxymelatonin; E, epinephrine; HIOMT, hydroxy-indole-O-methyltransferase; ME, metepinephrine; MLT, melatonin; NAT, N-acetyltransferase; NE, norepinephrine; NME, normetepinephrine; SAM, S-adenosylmethionine; tHcy, total homocysteine.
Manuscript received 15 January 2002. Initial review completed 13 February 2002. Revision accepted 30 May 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Arendt, J., Deacon, S., English, J., Hampton, S. & Morgan, L. (1995) Melatonin and adjustment to phase shift. J. Sleep Res. 4:74-79.[Medline]
2. Klein, D. C. (1993) The mammalian melatonin rhythm generating system. Wetterberg, L. eds. Light and Biological Rhythms in Man 1993:55-71 Pergamon Press Oxford, UK. .
3. Selhub, J. (1999) Homocysteine metabolism. Annu. Rev. Nutr. 19:217-246.[Medline]
4. Graham, I. M., Daly, L. E., Refsum, H. M., Robinson, K., Brattstrom, L. E., Ueland, P. M., Palma-Reis, R. J., Boers, G. H., Sheahan, R. G., Israelsson, B., Uiterwaal, C. S., Meleady, R., McMaster, D., Verhoef, P., Witteman, J., Rubba, P., Bellet, H., Wautrecht, J. C., De Valk, H. W., Sales Luis, A. C., Parrot-Rouland, F. M., Tan, K. S., Higgins, I., Garcon, D. & Andria, G. (1997) Plasma homocysteine as a risk factor for vascular disease. The European Concerted Action Project. J. Am. Med. Assoc. 277:1775-1781.[Abstract]
5. Reiter, R. J. (1988) Oxidative damage in the central nervous system: protection by melatonin. Prog. Neurobiol. 56:359-384.
6. Kostler, M., Rodriquez, C., Sainz, R.M., Antolin, I. & Menendez Pelaez, A. (1998) Melatonin increases gene expression for antioxidant enzymes in rat brain cortex. J. Pineal Res. 24:83-89.[Medline]
7. Walzem, R. L. & Clifford, A. J. (1988) Folate deficiency in rats fed diets containing free amino acids or intact proteins. J. Nutr. 118:1089-1096.
8. Ubbink, J. B., Vermaak, W.J.H. & Bissbort, S. (1991) Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. J. Chromatogr. 565:441-446.[Medline]
9. Brun, J., Claustrat, B. & David, M. (1987) Urinary melatonin, LH, oestradiol, progesterone excretion during the menstrual cycle or in women taking oral contraceptives. Acta Endocrinol 116:145-149.
10. Harthé, C., Claustrat, B., Brun, J. & Chazot, G. (1991) Direct 6-sulfatoxy-melatonin radioimmunoassay in plasma with use of an iodinated tracer. Clin. Chem. 37:536-539.
11. Revol, A., Comoy, E., Forzy, G., Garnier, J. P., Gerhard, M. F., Hirth, C., Jacob, N., Mathieu, P., Patricot, M. C., Peyrin, L., Piton, A. & Ruedas, E. (1994) Recommended methods for the measurement of catecholamines and their metabolites in urine. Ann. Biol. Clin. 52:625-637.
12. Kim, Y. I., Miller, J. W., Da Costa, K. A., Nadeau, M., Smith, D., Selhub, J., Zeisel, S. H. & Mason, J. B. (1994) Severe folate deficiency causes secondary depletion of choline and phosphocholine in rat liver. J. Nutr. 124:2197-2203.
13. Yie, S. M., Liu, G. Y., Johansson, E., Brown, C. & Brown, G. M. (1992) Age-associated changes and sex differences in urinary 6-sulphatoxymelatonin circadian rhythm in the rat. Life Sci. 50:1235-1242.[Medline]
14. Sugden, D. & Klein, D.C. (1987) Inactivation of rat pineal hydroxyindole-O-methyltransferase by disulfide-containing compounds. J. Biol. Chem. 262:6489-6493.
15. Nilsson, K., Gustafson, L., Fäldt, R., Andersson, A., Brattstrom, L., Lindgren, A., Israelsson, B. & Hultberg, B. (1996) Hyperhomocysteinaemia—a common finding in a psychogeriatric population. Eur. J. Clin. Invest. 26:853-859.[Medline]
16. Riggs, K. M., Spiro, A., III., Tucker, K. & Rush, D. (1996) Relations of vitamin B-12, vitamin B-6, folate, and homocysteine to cognitive performance in the Normative Aging Study. Am. J. Clin. Nutr. 63:306-314.
17. Snowdon, D. A., Tully, C. L., Smith, C. D., Perez Riley, K. & Markesbery, W.R. (2000) Serum folate and the severity of atrophy of the neocortex in Alzheimer disease: findings from the Nun Study. Am. J. Clin. Nutr. 71:993-998.
18. Serot, J. M., Christmann, D., Dubost, T., Béné, M.C. & Faure, G. C. (2001) CSF-folate levels are decreased in late-onset AD patients. J. Neural Transm. 108:93-99.
19. Touitou, Y., Fevre, M., Bogdan, A., Reinberg, A., De Prins, J., Beck, H. & Touitou, C. (1984) Patterns of plasma melatonin with ageing and mental condition: stability of nycthemeral rhythms and differences in seasonal variations. Acta Endocr. 106:145-151.
20. Haimov, I., Laudon, M., Zisapel, N., Souroujon, M., Nof, D., Shlitner, A., Herer, P., Tzischinsky, O. & Lavie, P. (1994) Sleep disorders and melatonin rhythms in elderly people. Br. Med. J. 309:167.
21. Kelly, G.S. (1998) Folates: supplemental forms and therapeutic applications. Altern. Med. Rev. 3:208-220.[Medline]
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