The importance of circadan rhythm alterations in erythrocyte deformability.

The hormone melatonin, secreted from the pineal gland at night and suppressed during the day, provides a circadian and seasonal signal to the organism. The impact of pharmacological doses of melatonin on erythrocyte deformability was investigated by our group in several studies in both in vitro and in vivo conditions. The aim of this study was to investigate the effects of alterations in the physiological melatonin levels via the circadian rhythm on erythrocyte deformability. 50 male rats weighing 250-300 g were divided in 5 groups. The rats were subjected to 12/12, 24/0, 0/24, 16/8 and 8/16 h of Light/Dark (L/D) cycle, respectively. The elongation indexes (EI) of the erythrocytes were measured by a laser diffractometer (Myrenne Rheodyne SSD) by using 30 microl of whole blood suspended in 2 ml of Dextran 60. There was no significant difference in the EI of the 24/0 h L/D group compared to the control (12/12), whereas the decrease of EI was statistically important in the 0/24 h L/D group (p = 0.009). This decrease in EI was also significant when this group was compared to the 24/0 h L/D group (p = 0.05). Furthermore, the EI was affected significantly by alterations in the circadian rhythm, compared to control (16/8, 8/16 h L/D; p = 0.05 and p = 0.007, respectively). As a result, the alterations in physiological melatonin levels via different circadian rhythms have significant impacts on the deformability of erythrocytes, which therefore may cause important cardiovascular implications in the people who are exposed to different light dark cycles. Furthermore, these data represents a new and a quite crucial open-field to be investigated and taken into account in in vivo hemorheological studies.

Melatonin and nitric oxide.

Melatonin is a product of the amino acid tryptophan in the pineal gland. Once synthesized, the specific mechanisms governing the release of melatonin from the pineal gland and its functions are largely unknown. Besides its regulatory role in circadian rhythms in mammals, because of its widespread subcellular distribution, melatonin contributes to the reduction of oxidative damage in both the lipid and the aqueous environments of the cell. This postulate is widely supported by the experimental observations showing that melatonin protects lipids in membranes, proteins in the cytosol, and DNA in the nucleus and mitochondria from free radical damage. Melatonin thus reduces the severity of disease conditions where free radicals are implicated. The direct free radical scavenging effects of melatonin are receptor independent. It has recently been shown that it has an ability to scavenge free radicals, including hydroxyl radicals, hydrogen peroxide, peroxyl radicals, singlet oxygen and nitric oxide (NO) and peroxynitrite anion. An excessive amount of NO, a free radical which is generated by the inducible form of NO synthase, is known to cause cytotoxic changes in cells. Hence, NO synthase is considered a pro-oxidative enzyme, and any factor that reduces its activity would be considered an antioxidant. Recent studies have shown that melatonin inhibits the activity of NO synthase, beside its NO and peroxynitrite scavenging activity. Thus, inhibition of NO production may be another means whereby melatonin reduces oxidative damage under conditions, such as ischemia-reperfusion, sepsis, etc, where NO seems to be important in terms of the resulting damage.

The in vivo antioxidant effectiveness of alpha-tocopherol in oxidative stress induced by sodium nitroprusside in rat red blood cells.

Reactive oxygen species avidly reacts with nitric oxide (NO) producing cytotoxic reactive nitrogen species capable of nitrating proteins and damaging other molecules which leads to the reduction of erythrocyte deformability. The aim of this investigation was to assess the importance of alpha-tocopherol (Vit-E) in the total antioxidant status of the erythrocytes in sodium nitroprusside (SNP), a nitric oxide donor, induced oxidative stress and its relation to erythrocyte deformability. Male Swiss Albino rats were used in 4 groups, comprising of 10 animals in each group. The first group was the control, and the other groups were administered SNP (10 mg/kg, i.p.), Vit-E (10 mg/kg, i.p.) + SNP, and SNP + L-NAME (10 mg/kg, i.p.), respectively. Relative filtration rate (RFR), relative filtration time (RFT) and relative resistance (Rrel) were determined as the indexes of erythrocyte deformability. In addition, malondialdehyde (MDA, as an index of lipid peroxidation) and nitric oxide levels and the antioxidant activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) were also determined in the red blood cells of all groups revealing the oxidant-antioxidant activity. RFT and the Rrel of the erythrocytes of the SNP-treated rats increased significantly (p<0.05) whereas the RFR of the erythrocytes decreased (p<0.05) in comparison to all groups reflecting the impaired deformability. This reduction in RFR was prevented with both L-NAME or Vit-E incubation. Vit-E has also reduced the Rrel of the erythrocyte which reveals that it has improved the erythrocyte deformability. Lipid peroxidation was suppressed by Vit-E and L-NAME significantly, where the red blood cell deformability was improved. Furthermore, SOD and CAT activities were significantly stimulated with SNP treatment (p<0.05), where as GSH-Px remained unchanged. In the contrary, GSH-Px activity was triggered significantly by Vit-E administration, whereas the SOD and CAT activities were reduced (p<0.05). As a result, these data reveal that Vit-E improves the erythrocyte deformability in SNP-induced oxidative stress by its antioxidant effects on the lipid peroxidation and antioxidant enzyme activities.

In vitro effects of melatonin on the filtrability of erythrocytes in SNP-induced oxidative stress.

Erythrocyte deformability is one of the most important charactheristics of erythrocytes for an effective microcirculatory function and is affected from a number of factors, including the oxidative-damage-induced by nitric oxide (NO). This study was performed to investigate the effects of in vitro melatonin incubation on the antioxidant status and deformability of erythrocytes in sodium nitroprusside (SNP), a nitric oxide donor, induced oxidative stress. 40 blood samples taken from the adult healthy people were divided into 4 groups randomly and incubated with saline, SNP (1 mM), melatonin (MEL, 1 mM), MEL + SNP and SNP + L-NAME (5 mM) respectively. Relative filtration rate (RFR), relative filtration time (RFT) and relative resistance (Rrel) were determined as the indexes of erythrocyte filterability. In addition, malondialdehyde (MDA, as an index of lipid peroxidation) and the antioxidant activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) were also determined in the red blood cells of all groups revealing the oxidant-antioxidant activity. RFT and the Rrel of the erythrocytes incubated with SNP increased significantly (p<0.05) whereas the RFR of the erythrocytes decreased (p<0.05) in comparison to all groups. This reduction in RFR was prevented with both L-NAME or MEL incubation. Furthermore, MEL was found to be significantly efficient in preventing the erythrocytes from lipid peroxidation in these groups. In addition, GSH-Px and SOD activities were elevated with SNP incubation reflecting the oxidative stress in erythrocytes, whereas the CAT activity remained unchanged. Melatonin has no significant effect on the GSH-Px and CAT activity but, it caused a significant decrease in SOD activity (p<0.05). These results reveal that, melatonin can protect the erythrocytes from impaired deformability in SNP-induced oxidative stress due to antioxidant effects as revealed by lipid peroxidation and antioxidant enzyme activities.

Lipid peroxidation and deformability of red blood cells in experimental sepsis in rats: The protective effects of melatonin.

Sepsis has been associated with a lipopolysaccharide (LPS) induced bacterial infection and causes biochemical, hemodynamic and physiological alterations in a system. Erythrocyte deformability is very critical for a microcirculatory system to function effectively. Hence, we were interested in examining the effects of a potent antioxidant, melatonin (Mel), on lipid peroxidation and deformability of eythrocytes in LPS-induced experimental sepsis. Male Swiss Albino rats were used in 6 groups, each group comprising of 10 animals. The first group was the control, and the other groups were administered LPS (10 mg/kg, i.p.), Mel (10 mg/kg, i.p.), LPS + L-NAME (5 mM, i.p.), Mel + LPS and Mel + LPS + L-NAME, respectively. Deformability of the RBCs decreased significantly (p < 0.05) in the LPS group in comparison to all other groups. This reduction was prevented with both L-NAME and Mel, but was not as significant as when administering L-NAME or Mel alone. This result was adversely seen in nitric oxide levels, i.e. RBCD was reduced when the NO levels were higher. Therefore in the Mel group the NO levels were reduced while the RBCD enhanced. In addition to these, as an index of lipid peroxidation, the Malondialdehyde levels were elevated in LPS groups whereas the deformability was reduced. This lipid peroxidation was suppressed by Mel and/or L-NAME significantly, where the RBCD was enhanced. These results show that, Melatonin can elevate the RBCD in experimental sepsis due to its nitric oxide scavenging activity and antioxidant effect as revealed by lipid peroxidation.