Low urinary 6-sulphatoxymelatonin levels in patients with coronary artery disease

A decrease in nocturnal serum melatonin levels was reported in patients with clinically uncharacterized coronary artery disease. To assess whether there was a correlation between melatonin production and disease stage, we measured the nocturnal urinary excretion of 6-sulphatoxymelatonin (an index of blood melatonin concentration) in patients with chronic stable or unstable coronary disease and in a group of age-matched controls. Three groups of individuals were studied: a) 24 healthy subjects (mean age: 63 +/- 13 yr); b) 32 patients with chronic, stable, coronary disease (62 +/- 11 yr); and c) 27 patients with unstable angina (62 +/- 12 yr). For 6-sulphatoxymelatonin measurement, urine was collected from 18:00 to 06:00 hr, within 48 hr of hospitalization in the case of unstable angina. 6-Sulphatoxymelatonin was measured by a specific radioimmunoassay. Urinary 6-sulphatoxymelatonin excretion was significantly lower in unstable angina patients than in healthy subjects or in patients with stable angina. 6-Sulphatoxymelatonin correlated negatively with age in healthy subjects, but not in coronary patients. 6-Sulphatoxymelatonin excretion in patients treated with beta-adrenoceptor blockers did not differ significantly from coronary patients not receiving beta-blockers. The results indicate that patients with coronary disease have a low melatonin production rate, with greater decreases in those with higher risk of cardiac infarction and/or death.     

The role of granulocyte-macrophage-colony stimulating factor, cortisol, and melatonin in the regulation of the circadian rhythms of peripheral blood cells in healthy volunteers and patients with breast cancer

The circulating blood cells show highly reproducible circadian rhythms. However, the factors that regulate these rhythms are not well understood. In the current study, we examined the diurnal variations of peripheral blood cells (white blood cells, neutrophils, lymphocytes), granulocyte-macrophage-colony stimulating factor (GM-CSF), and melatonin levels, and considered the role of melatonin on these rhythms in healthy volunteers and in patients with early breast cancer. Fourteen premenopausal patients with early stage breast cancer (T2, N1 tumors) and 10 premenopausal healthy volunteers were included in the study. Blood samples were taken every 4 hr for a period of 24 hr. Peripheral blood cells were counted by automated analyser and also from peripheral blood films. GM-CSF levels were measured by ELISA and melatonin levels by radioimmunoassay (RIA). Serum melatonin, cortisol, and GM-CSF levels, and peripheral blood cell counts showed significant circadian rhythms in healthy volunteers. Except for GM-CSF, these circadian rhythms were found not to be suppressed in early breast cancer patients. While there were significant correlations of serum GM-CSF and cortisol levels with peripheral blood cell counts in healthy volunteers, only lymphocyte counts were found to be significantly correlated with serum GM-CSF and cortisol levels in patients with breast cancer. Serum melatonin levels were found to be significantly correlated with lymphocyte counts in both groups. Our results suggest that peripheral blood cells show significant circadian rhythms in both healthy volunteers and in patients with stage II (T2, N1) breast cancer, and GM-CSF, cortisol, and melatonin may have a role in the regulation of peripheral blood cell counts.     

N-acetyltransferase is not the rate-limiting enzyme of melatonin synthesis at night

Abstract:  Circadian melatonin production in the pineal gland and retina is under the control of serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase. Because NAT activity varies diurnally, it has been considered both the melatonin rhythm-generating enzyme and the rate-limiting enzyme of melatonin synthesis. In rats with dramatically reduced NAT activity due to a H28Y mutation in NAT, melatonin levels remained the same as in wildtype controls, suggesting that NAT does not determine the rate of melatonin production at night. Using a combination of molecular approaches with a sensitive in vivo measurement of pineal diurnal melatonin production, we demonstrate that (i) N-acetylserotonin (NAS), the enzymatic product of NAT, is present in vast excess in the night pineals compared with melatonin; (ii) the continuous increase in NAT protein levels at late night does not produce a proportional increase in melatonin; and (iii) an increase in NAS in the same animal over several circadian cycles do not result in corresponding increase in melatonin output. These results strongly suggest that NAT is not the rate-limiting enzyme of melatonin formation at night.     

Melatonin counteracts lipid peroxidation induced by carbon tetrachloride but does not restore glucose-6 phosphatase activity

Abstract: Carbon tetrachloride (CC1₄) exerts its toxic effects by the generation of free radicals. In this study we investigated whether melatonin, a potent free radical scavenger, could prevent the deleterious effects of CC1₄. Liver homogenates and liver microsomes were incubated with CCI4 in the presence of melatonin and lipid peroxidation and glucose-6 phosphatase (G6Pase) activity were determined. All doses of CC1₄ (1, 0.5, 0.1 raM) produced significantly high levels of lipid peroxidation, as reflected by increased levels of malonaldehyde and 4-hydroxyalkenals, in both liver homogenates and liver microsomes. These doses of CC1₄ concommitantly reduced the activity of microsomal G6Pase. Co-incubation with melatonin dose-dependently (2, 1, 0.5 raM) inhibited the production of lipid peroxidation, but it was unable to restore the activity of G6Pase. In in vivo studies, rats were also treated with melatonin (10 mg/kg, i.p.), given 30 min before and 60 min after the administration of CC1₄ (5 ml/kg, i.p.). Significantly elevated levels of lipid peroxidation were measured in the liver and kidney. Melatonin prevented the CCl₄-induced lipid peroxidation in the kidney, but not in the liver. These data suggest that melatonin may provide protection against some of the damaging effects of CCI4, possibly due to its ability to scavenge toxic free radicals.     

Effects of Melatonin on Ischemia and Reperfusion Injury of the Rat Heart

Effects of melatonin on various manifestations of ischemia/reperfusion injury of the isolated perfused rat heart were examined. Ischemia- and reperfusion-induced ventricular arrhythmias were studied under constant flow in hearts subjected to 10, 15 or 25 min of regional ischemia (induced by LAD coronary artery occlusion) and 10-min reperfusion. Melatonin was added to the perfusion medium 5 min before ischemia at concentrations of 10 mol/l or 10 nmol/l and was present throughout the experiment. Recovery of the contractile function was evaluated under constant perfusion pressure after 20-min global ischemia followed by 40-min reperfusion. Hearts were treated with melatonin at a high concentration (10 mol/l) either 5 min before ischemia only (M1) or 5 min before ischemia and during reperfusion (M2) or only during reperfusion (M3). At the high concentration, melatonin significantly reduced the incidence of reperfusion-induced ventricular fibrillation and decreased arrhythmia score (10% and 2.2 ± 0.3, respectively) as compared with the corresponding untreated group (62% and 4.1 ± 0.3, respectively); the low concentration had no effect. This substance did not affect the incidence and severity of ischemic arrhythmias. Melatonin (M2, M3) significantly improved the recovery of the contractile function as compared with the untreated group; this protection did not appear if melatonin was absent in the medium during reperfusion (M1). Our results show that melatonin, in accordance with its potent antioxidant properties, effectively protects the rat heart against injury associated with reperfusion. It appears unlikely that melatonin is cardioprotective at physiological concentrations.     

Melatonin, mitochondria, and cellular bioenergetics

Aerobic cells use oxygen for the production of 90-95% of the total amount of ATP that they use. This amounts to about 40 kg ATP/day in an adult human. The synthesis of ATP via the mitochondrial respiratory chain is the result of electron transport across the electron transport chain coupled to oxidative phosphorylation. Although ideally all the oxygen should be reduced to water by a four-electron reduction reaction driven by the cytochrome oxidase, under normal conditions a small percentage of oxygen may be reduced by one, two, or three electrons only, yielding superoxide anion, hydrogen peroxide, and the hydroxyl radical, respectively. The main radical produced by mitochondria is superoxide anion and the intramitochondrial antioxidant systems should scavenge this radical to avoid oxidative damage, which leads to impaired ATP production. During aging and some neurodegenerative diseases, oxidatively damaged mitochondria are unable to maintain the energy demands of the cell leading to an increased production of free radicals. Both processes, i.e., defective ATP production and increased oxygen radicals, may induce mitochondrial-dependent apoptotic cell death. Melatonin has been reported to exert neuroprotective effects in several experimental and clinical situations involving neurotoxicity and/or excitotoxicity. Additionally, in a series of pathologies in which high production of free radicals is the primary cause of the disease, melatonin is also protective. A common feature in these diseases is the existence of mitochondrial damage due to oxidative stress. The discoveries of new actions of melatonin in mitochondria support a novel mechanism, which explains some of the protective effects of the indoleamine on cell survival.     

Protective effects of chronic melatonin treatment against renal injury in streptozotocin-induced diabetic rats

The aim of this study was to investigate the effects of melatonin as an antioxidant, on prevention and treatment of streptozotocin (STZ)-induced diabetic renal injury in rats. Male Wistar rats were divided into four groups: (1) untreated, (2) melatonin-treated, (3) untreated diabetic (UD), (4) melatonin-treated diabetic (MD). Experimental diabetes was induced by single dose (60 mg/kg, i.p.) STZ injection. For 3 days prior to administration of STZ, melatonin was injected (200 microg/kg/day, i.p.); these injections were continued until the end of the study (4 weeks). Malondialdehyde (MDA) levels as a marker of lipid peroxidation were significantly increased in the renal homogenates of UD animals and decreased after melatonin administration. The activity of the antioxidative enzyme glutathione peroxidase (GSH-Px) was significantly reduced in UD rats. Melatonin treatment reversed STZ-induced reduction of GSH-Px activity without having an effect on blood glucose. Upon histopathological examination, it was observed that the melatonin treatment prevented the renal morphological damage caused by diabetes. Upon immunohistochemical investigation, glomerular anti-laminin beta1 staining decreased in MD rats. Additionally, no tubular anti-IGF-1 staining was observed in melatonin-treated rats. In conclusion, chronically administered melatonin reduced renal injury in STZ-induced diabetic rats and thus it may provide a useful therapeutic option in humans to reduce oxidative stress and the associated renal injury in patients with diabetes mellitus.     

Melatonin promotes survival of nonvascularized fat grafts and enhances the viability and migration of human adipose‐derived stem cells via down‐regulation of acute inflammatory cytokines

Nonvascularized fat grafting is a valuable technique for soft tissue reconstruction but poor survival of fat in the host environment remains a problem. A process known as cell‐assisted transfer is used to enhance fat graft retention by adding stromal vascular fraction, an adipose‐derived stem cell (ASC) rich content to lipoaspirate. We have recently shown that the use of melatonin, a reactive oxygen species scavenger, protects human ASCs from hydrogen peroxide‐induced oxidative stress and cell death in vitro but its role as a pharmacological adjunct in clinical fat grafting has not been studied. Herein, the effect of melatonin was examined on human ASCs in vitro using survival and functional assays including the MTT assay, CellTox Green assay, monolayer scratch assay as well as a human cytokine chemoluminescence, and tumour necrosis factor‐α assay. Further, the effect of melatonin‐treated fat grafts was tested in vivo with a murine model. Haematoxylin and eosin staining, perilipin and CD31 immunostaining were performed with morphometric analysis of adipose tissue. The results demonstrate that, in vitro, the addition of melatonin to ASCs significantly improved their cell‐viability, promoted cell migration and preserved membrane integrity as compared to controls. In addition, it induced a potent anti‐inflammatory response by downregulating acute inflammatory cytokines particularly tumour necrosis factor‐α. For the first time, it is demonstrated in vivo that melatonin enhances fat graft volume retention by reducing inflammation and increasing the percentage of adipose volume within fat grafts with comparable volumes to that of cell‐assisted lipotransfer. Based on these novel findings, melatonin may be a useful pharmacological adjunct in clinical fat grafting.