Liliana E. Albornoz

Melatonin increases oestradiol-induced bone formation in ovariectomized rats

Abstract: To assess the effect of melatonin on bone metabolism in ovariectomized rats, receiving oestradiol therapy or not, melatonin was administered in the drinking water (25 μg/mL water) and oestradiol (10 μg/kg body weight) or vehicle was given subcutaneously 5 days/week for up to 60 days after surgery. Urinary deoxypyridinoline (a marker of bone resorption) and circulating levels of bone alkaline phosphatase activity (a marker of bone formation), as well as serum calcium and phosphorus levels, were measured every 15 days. Bone area (BA), bone mineral content (BMC), bone mineral density (BMD) and total body fat (expressed as 100 g body weight) were measured by dual‐energy X‐ray absorptiometry at the end of the experiment. Body weight and total body fat were augmented after ovariectomy, and decreased after melatonin or oestradiol treatment. The effect of melatonin on body weight was seen in sham‐operated rats only. Ovariectomy augmented, and melatonin or oestradiol lowered, urinary deoxypyridinoline excretion. This effect of melatonin and oestradiol was seen mainly in ovariectomized rats. The efficacy of oestradiol to counteract ovariectomy‐induced bone resorption was increased by melatonin. Melatonin or oestradiol lowered serum bone alkaline phosphatase activity. Melatonin inhibition was seen mainly on the increase of bone alkaline phosphatase activity that followed ovariectomy. Serum phosphorus levels decreased after melatonin administration and were augmented after oestradiol injection; overall, melatonin impaired the increase of serum phosphorus caused by oestradiol. Ovariectomy decreased, and oestradiol increased, serum calcium levels while melatonin augmented serum calcium in sham‐operated rats only. On day 60 after surgery, BMD and content decreased after ovariectomy and were increased after oestradiol injection. Melatonin augmented BA of spine and BMC of whole of the skeleton and tibia. The highest values observed were those of rats treated concurrently with oestradiol and melatonin. The present results indicate that: (i) melatonin treatment restrained bone remodelling after ovariectomy; (ii) the effect of melatonin required adequate concentrations of oestradiol; (iii) melatonin augmented oestradiol effects on bone in ovariectomized rats; (iv) a counter‐regulation by melatonin of the increase in body fat caused by ovariectomy was uncovered. The melatonin doses employed were pharmacological in terms of circulating melatonin levels but not necessarily for some other fluids or tissues.

A multifactorial approach employing melatonin to accelerate resynchronization of sleep–wake cycle after a 12 time‐zone westerly transmeridian flight in elite soccer athletes

Rapid transmeridian translocation through multiple time zones has a negative impact on athletic performance. The aim of the present study was to test the timely use of three factors (melatonin treatment, exposure to light, physical exercise) to hasten the resynchronization of a group of elite sports competitors and their coaches to a westerly transmeridian flight comprising of 12 time‐zones. Twenty‐two male subjects were included in the study. They were professional soccer players and their coaches who travelled to Tokyo to play the final game of the Intercontinental Coup. The day prior to departure, urine was collected from each subject from 18:00 to 06:00 hrs to measure the melatonin metabolite 6‐sulphatoxymelatonin. Participants were asked to complete sleep log diaries from day 0 (preflight) to the day before returning to Buenos Aires (day 8). All subjects received 3 mg of melatonin p.o. daily at expected bedtime at Tokyo immediately after leaving Buenos Aires. Upon arrival at Tokyo the subjects performed a daily physical exercise routine outdoors at two restricted times of the day (from 08:00 to 11:00 hrs in the morning and from 13:00 to 16:00 hrs in the afternoon). Exposure to sunlight or physical exercise at other times of the day was avoided. Except for the number of awakenings (which increased on days 1 and 3) and sleep latency (which decreased on days 2, 6 and 8), there was an absence of significant changes in subjective sleep parameters as compared with preflight assessment. Sleep quality and morning alertness at Tokyo correlated significantly with preflight 6‐sulphatoxymelatonin excretion. Mean resynchronization rate of sleep–wake cycle to the 12 hr‐time shift was 2.13 ± 0.88 days, significantly different from the minimal resynchronization rate of 6 days expected after a 12‐time‐zones flight. The results indicate that the combination of melatonin treatment, an appropriate environmental light schedule and timely applied physical exercise can be useful to help elite athletes to overcome the consequences of jet lag.

Low urinary 6-sulfatoxymelatonin levels in patients with severe congestive heart failure

Objective: To assess urinary 6-sulfatoxymelatonin excretion in patients admitted to the hospital because of congestive heart failure (CHF). Methods: Urinary 6-sulfatoxymelatonin was measured by a specific radioimmunoassay in 33 hospitalized patients with CHF and in 146 healthy ambulatory volunteers. Individuals with hepatic or renal failure were excluded from the sample. Data were analyzed by the Mann-Whitney test and regression analysis. Results: 6-Sulfatoxymelatonin levels were significantly lower in CHF patients than controls (median 2.6 vs 6.02 µg, p<0.0001). This decrease was observed regardless of β-adrenergic blocker or benzodiazepine medication. A significant decrease in 6-sulfatoxymelatonin excretion occurred with age. There were no significant differences in urinary 6-sulfatoxymelatonin levels between chronic and acute CHF patients. Conclusions: The results suggest that circulating melatonin levels are low in patients with CHF. Such a decrease may precede aggravation of heart failure.

Gender-related differences in urinary 6-sulfatoxymelatonin levels in obese pubertal individuals

Abstract:  The objective of this study was to measure the urinary excretion of the main melatonin metabolite 6‐sulfatoxymelatonin in obese and normal weight (wt) boys and girls. The study included 94 subjects, aged 4–15.7 yr (50 obese and 44 normal wt; 48 boys) classified as: mid‐childhood (4–7.99 yr), late‐childhood (8–12 yr) and pubertal (10.1–15.7 yr, Tanner II–IV). Normal wt subjects were children with a body mass index (BMI) between the 25th and 75th percentiles, and the group of obese subjects included children whose BMI was above the 97th percentile. A 24‐hr urine sample was collected during two intervals: (i) 18:00–08:00 hr, and (ii) 08:00–18:00 hr. Analysis of urinary 6‐sulfatoxymelatonin levels was performed by radioimmunoassay. Excretion of 6‐sulfatoxymelatonin was expressed as: (i) total amount excreted (μg); (ii) μg excreted per time interval, nocturnal or diurnal; and (iii) the difference between nocturnal and diurnal samples (μg, estimated amplitude). A factorial analysis of variance indicated that nocturnal 6‐sulfatoxymelatonin excretion and amplitude were significantly higher in the obese individuals. A significant interaction ‘BMI × age’ was detected, i.e. the effect of BMI was significant in the pubertal group only. Total, nocturnal and diurnal 6‐sulfatoxymelatonin excretion was significantly higher in girls. The increase in 6‐sulfatoxymelatonin excretion found in obesity occurred only in boys and at the pubertal age. To what extent this increase in melatonin production contributes to a delayed puberty in some pubertal obese males remains to be established.

Vascular reactivity in diabetic rats: effect of melatonin.

The aim of the present study was to evaluate the in vitro contractile response of rat aorta in mild and severe type I diabetes and the effect of melatonin on it. Aortic rings were obtained from male Wistar rats injected with streptozotocin 8–12 wks earlier. Rats were divided into three groups: non‐diabetic rats (NDR), mildly diabetic rats (MDR) and severely diabetic rats (SDR). Dose–response curves for acetylcholine‐induced, endothelium‐related relaxation of aortic rings (after previous exposure to phenylephrine) and for serotonin‐induced vasoconstriction were conducted in the presence or absence of 10–5 mol/L melatonin. This protocol was repeated with rings preincubated in a high glucose solution (44 mmol/L). The contractile response to phenylephrine decreased in SDR, an effect counteracted by preincubation with high glucose. Melatonin decreased phenylephrine‐induced vasoconstriction in MDR and counteracted the effect of high glucose in SDR. Acetylcholine‐evoked relaxation decreased significantly after exposure to a high glucose in SDR, this effect being counteracted by melatonin. Serotonin‐induced vasoconstriction decreased in SDR and augmented in MDR, but only after exposure to high glucose. Melatonin reduced the maximal tension of aortic contraction after serotonin in MDR, both under basal conditions and after preincubation in a high glucose solution. The results support the existence of differences in vasomotor responses as a function of the diabetes state and of an improvement of contractile performance in diabetic rats after exposure to melatonin at a pharmacological concentration (in terms of circulating melatonin levels but not necessarily for some other fluids or tissues).

Effect of melatonin treatment on oxygen consumption by rat liver mitochondria

Summary.The objective of this study was to examine the in vivo effect of melatonin on rat mitochondrial liver respiration. Two experiments were performed: For experiment 1, adult male rats received melatonin in the drinking water (16 or 50 µg/ml) or vehicle during 45 days. For experiment 2, rats received melatonin in the drinking water (50 µg/ml) for 45 days, or the same amount for 30 days followed by a 15 day-withdrawal period. At sacrifice, a liver mitochondrial fraction was prepared and oxygen consumption was measured polarographically in the presence of excess concentration of DL-3-β-hydroxybutyrate or L-succinate. Melatonin treatment decreased Krebs’ cycle substrate-induced respiration significantly at both examined doses. The stimulation of mitochondrial respiration caused by excess concentration of substrate recovered after melatonin withdrawal. Basal state 4 respiration was not modified by melatonin. Melatonin, by curtailing overstimulation of cellular respiration caused by excess Krebs’ cycle substrates, can protect the mitochondria from oxidative damage.

Effect of Oral L-Arginine Administration for Three Weeks in Two Kidney-two Clip Hypertensive Rats

Nitric oxide (NO) has been identified as an effective vascular relaxant. This study analyses the contribution of the precursor L-arginine (L-arg) by oral administration in two kidney-two clip hypertension in the rat (2K-2C). Two groups were studied: sham (SH, n = 21) and hypertensive (HT, n = 15). After 4 weeks of surgery, a group of rats remained as controls (SHc and HTc, respectively), while others were supplemented with L-arg (1.25 g/L) in drinking water (SHa and HTa) for 3 weeks. Blood pressure was significantly increased in 2K-2C rats but remained unchanged after L-arg treatment. Plasma nitrite/nitrate concentrations were not different among groups. The contractile response of aorta to KCl, serotonin and the protein kinase C (PKC) stimulant, phorbol 12,13-dibutyrate (PDBu) was also evaluated. Higher contractile responses to PDBu (p < 0.001) and lower relaxation to acetylcholine (Ach 10−6M, p < 0.05 and 10−5M, p < 0.02) were observed in aortic rings of HTc vs SHc; L-arg supplementation significantly diminished tension development to all agonists (p < 0.05) but failed to modify the lower relaxation to Ach in HTa. Thromboxane (TxA2) – synthesis in rings of HTc was higher than in SHc under basal conditions (p < 0.05). In the groups with supplement of L-arg, PDBu significantly stimulated prostacyclin (PGI2) synthesis more in HTa rats than in SHa ones (p < 0.05). To conclude: 1) L-arg fails to modify hypertension development in 2K-2C rats; and 2) L-arg exerts a beneficial effect on the vascular wall, by reducing contractility in rings from HTa rats; it also improved PGI2 synthesis under PDBu stimulation. 3) greater PKC activation and TxA2 production rather than lower NO availability might result in systemic hypertension in 2K-2C rats.

Potential role of glycerol leading to rat fructose hypertension

A fructose-enriched diet promotes hypertension in rats. We thought that an enhancement of the glycolytic and/or lipid disorder (s) that raise blood pressure could be the cause. Therefore, we studied 4 groups of Sprague-Dawley rats (+/-200 g): (1) control rats received a standard diet and tap water; (2) the glycerol group of rats received a standard diet and 0.54 mol/L glycerol in tap water; (3) the fructose group was given a fructose-enhanced diet (chow had 55% fructose instead of dextrin) and tap water; and (4) the fructose-glycerol group was given the fructose-enhanced diet and 0. 54 mol/L glycerol in drinking water. At the end of the second week, the findings were as follows. Blood pressure was 149+/-2 mm Hg in the fructose-glycerol group versus 129+/-2 (P<0.001), 131+/-2 (P<0. 001), and 140+/-3 (P<0.005) mm Hg in the control, glycerol, and fructose groups, respectively. Insulinemia was higher in the fructose-glycerol group than the control (P<0.001), glycerol (P<0. 001), and fructose groups (P<0.001); triglyceridemia was higher in the fructose-glycerol (P<0.02), fructose (P<0.05), and glycerol groups (P<0.02) than the control group. Thoracic aorta rings showed a lower ED(50) to 12,13-phorbol dibutyrate in the fructose-glycerol group than in the control (P<0.001), glycerol (P<0.002), and fructose groups (P<0.001). In conclusion, glycerol-fructose administration resulted in hypertriglyceridemia, hyperinsulinemia, and increased vascular sensitivity to 12,13-phorbol dibutyrate (with respect to the control group), and significantly greater expression of protein kinase C alpha and betaII (with respect to the glycerol group).

Comparative effects of melatonin and vitamin E in restoring aortic relaxation in pancreatectomized rats

In a previous study we reported the efficacy of melatonin to restore the decreased relaxation response to acetylcholine (ACh) or to sodium nitroprusside (SNP) in aortic rings of rats turned hyperglycemic by subtotal pancreatectomy. The effect was amplified by pre-incubation in a high (44 mmol/l) glucose solution, a situation that resulted in oxidative stress. We hereby compare the effect of another antioxidant, vitamin E, with that of melatonin on ACh response in intact aortic rings or on SNP response in endothelium-denuded aortic rings obtained from pancreatectomized or sham-operated rats. Dose-response curves to ACh or SNP were performed in the presence or absence of melatonin or vitamin E (10-5 mol/l) in 10 or 44 mmol/l glucose medium. Melatonin was more effective than vitamin E in restoring Ach- or SNP-induced relaxation of aortic rings in a high glucose medium. The differences between the two antioxidants may rely on the ability of melatonin to diffuse readily into intracellular compartments.ResumenEn un estudio previo se describe la eficacia de la melatonina para restablecer la respuesta disminuida a la acetilcolina (ACh) o al nitroprusiato de sodio (SNP) de anillos aórticos de rata con pancreatectomía subtotal. Este efecto fue mayor en el grupo de anillos preincubados en solución de Krebs con elevada concentración de glucosa (44 mmol/l), lo que favorece la producción de estrés oxidativo. En el presente trabajo se comparan los efectos de la vitamina E y la melatonina sobre la respuesta a la ACh y al SNP de anillos aórticos con endotelio intacto o denudado, obtenidos a partir de ratas con pancreatectomía subtotal o con operación simulada (controles). Se realizaron curvas dosis-respuesta a la ACh o al SNP en medios de incubación con glucosa normal o alta con melatonina o vitamina E (10−5 mol/l). La melatonina fue más efectiva que la vitamina E para restablecer la relajación provocada por ACh o SNP en anillos aórticos expuestos a un medio con elevada concentración de glucosa. La diferencia entre el efecto de ambas sustancias antioxidantes podría deberse a la capacidad de la melatonina para difundir hacia el compartimiento intracelular.

Chronic administration of propylthiouracil ameliorates hyperdynamic circulation in portal hypertensive rats.

OBJECTIVE To evaluate the effect of a hypothyroid state, induced by chronic propylthiouracil administration, on splanchnic and systemic hemodynamic parameters in rats with portal hypertension due to portal vein ligation. METHODS Portal hypertension was induced by surgical stenosis of the portal vein. Cardiac index and portal blood flow were measured using radioactive microspheres. Measurements were performed after treatment with propylthiouracil (1 mg/ml in drinking water) for 5 days. RESULTS Propylthiouracil-treated portal hypertensive rats had a lower portal pressure (12.4 +/- 1.9 versus 16.3 +/- 0.7 mmHg; p < 0.05) and portal blood flow (11.6 +/- 0.7 versus 13.2 +/- 1.3 ml/min/100 g; p < 0.05) than non-treated animals. Splanchnic vasoconstriction in treated animals was associated with a higher peripheral vascular resistance (2.3 +/- 0.4 versus 1.8 +/- 0.3 mmHg/ml/min/100 g; p < 0.05) than controls. CONCLUSION These results suggest that portal pressure can be lowered by inducing a hypothyroid state by chronic administration of propylthiouracil.