Mariana Gavazza

Melatonin preserves arachidonic and docosapentaenoic acids during ascorbate-Fe2+ peroxidation of rat testis microsomes and mitochondria

The pineal hormone melatonin (N-acetyl, 5-methoxytryptamine) was recently accepted to act as an antioxidant under both in vivo and in vitro conditions. In this study, we examined the possible preventive effect of melatonin on ascorbate-Fe(2+) lipid peroxidation of rat testis microsomes and mitochondria. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:5 n6. The lipid peroxidation of testis microsomes or mitochondria produced a significant decrease of C20:4 n6 and C22:5 n6. The light emission (chemiluminescence) used as a marker of lipid peroxidation was similar in both kinds of organelles when the control and peroxidized groups were compared. Both long chain polyunsaturated fatty acids were protected when melatonin was incorporated either in microsomes or mitochondria. The melatonin concentration required to inhibit by 100% the lipid peroxidation process was 5.0 and 1.0mM in rat testis microsomes and mitochondria, respectively. IC 50 values calculated from the inhibition curve of melatonin on the chemiluminescence rates were higher in microsomes (4.98 mM) than in mitochondria (0.67 mM). The protective effect observed by melatonin in rat testis mitochondria was higher than that observed in microsomes which could be explained if we consider that the sum of C20:4 n6+C22:5 n6 in testis microsomes is two-fold greater than present in mitochondria.

The effect of alpha-tocopherol on the lipid peroxidation of mitochondria and microsomes obtained from rat liver and testis.

The effect of intraperitoneal administration of α‐tocopherol (100 mg/kg wt/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of mitochondria and microsomes isolated from rat liver and testis was studied. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:6 n3 in liver and C20:4 n6 and C22:5 n6 in testis. The lipid peroxidation of liver mitochondria or microsomes produced a significant decrease of C20:4 n6 and C22:6 n3 in the control group, whereas changes in the fatty acid composition of the α‐tocopherol treated group were not observed. The light emission was significantly higher in the control than in the α‐tocopherol treated group. The lipid peroxidation of testis microsomes isolated from the α‐tocopherol group produced a significant decrease of C20:4 n6 , C22:5 n6 and C22:6 n3, these changes were not observed in testis mitochondria. The light emission of both groups was similar. The treatment with α‐tocopherol at the dose and times indicated showed a protector effect on the polyunsaturated fatty acids of liver mitochondria, microsomes and testis mitochondria, whereas those fatty acids situated in testis microsomes were not protected during non enzymatic ascorbate‐Fe2+ lipid peroxidation. The protector effect observed by α‐tocopherol treatment in the fatty acid composition of rat testis mitochondria but not in microsomes could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is 2-fold than that present in mitochondria.

Protective effect of N-acetyl-serotonin on the nonenzymatic lipid peroxidation in rat testicular microsomes and mitochondria

Abstract:  N‐acetyl‐serotonin, the immediate precursor of melatonin in the tryptophan metabolic pathway in the pineal gland, has been reported to be an antioxidant. The aim of this study was to test the in vitro protective effect of N‐acetyl‐serotonin on the ascorbate‐Fe++ induced lipid peroxidation of polyunsaturated fatty acids (PUFAs) located in testis microsomes and mitochondria. We assayed increasing concentrations (0–10 mm) of N‐acetyl‐serotonin in testis microsomes and (0–1 mm) of N‐acetyl‐serotonin in testis mitochondria. Control experiments were performed by incubating microsomal and mitochondrial membranes with N‐acetyl‐serotonin in the absence of lipid peroxidation‐inducing drugs. Special attention was paid to the changes produced on the highly PUFAs C20:4 n6 and C22:5 n6. The light emission (chemiluminescence) used as a marker of lipid peroxidation was similar in both organelles when the control and peroxidized groups were compared. N‐acetyl‐serotonin reduced lipid peroxidation in testicular microsomes or mitochondria for both C20:4 n6 and C22:5 n6. Both long chain PUFAs were protected when N‐acetyl‐serotonin was incorporated either into microsomes or mitochondria. The N‐acetyl‐serotonin concentration required to inhibit by approximately 70% lipid peroxidation process was 10 mm in microsomes and between 0.50 and 1 mm in mitochondria. IC 50 values calculated from the inhibition curve of N‐acetyl‐serotonin on the chemiluminescence rates were higher in microsomes (4.50 mm) than in mitochondria (0.25 mm). In these experimental conditions, N‐acetyl‐serotonin was about 18 times more potent in testicular mitochondria in inhibiting the oxidative processes than it was in testicular microsomes. These results suggest that the protective role of N‐acetyl‐serotonin in preserving the long PUFAs may be related to its ability to reduce lipid peroxidation.

Sensitivity of mitochondria isolated from liver and kidney of rat and bovine to lipid peroxidation: a comparative study of light emission and fatty acid profiles.

Much work has been carried out on non-enzymatic–induced lipid peroxidation of mitochondria obtained from different tissues of monogastric animals, but little information is available about this process in poligastric animals. Studies were carried out to determine the sensitivity of mitochondria isolated from liver and kidney of rat and bovine to lipid peroxidation (ascorbate-Fe2+ dependent) by comparison of light emission and fatty acid profiles. Mitochondria from both species were susceptible to lipid peroxidation. Measurements of chemiluminescence indicate that the lipid peroxidation process was more effective in mitochondria from rat liver than in the organelle obtained from bovine, whereas changes were not observed in mitochondria from rat and bovine kidney. The fatty acid composition of total lipids isolated from liver and kidney mitochondria of both species was substantially modified when subjected to non-enzymatic lipid peroxidation with a decrease of arachidonic and docosahexaenoic acids. The polyunsaturated fatty acid (PUFA) composition was higher in mitochondria obtained from rat liver (43.11± 4.16) than in bovine (15.78 ± 0.76). As a consequence, the unsaturation index (UI), was higher in mitochondria of rat liver than in bovine. Nevertheless, the PUFA composition of kidney mitochondria from both species was similar; therefore, statistically significant differences in the UI were not observed. The results suggest that mainly the PUFAs present in hepatic and kidney mitochondria were sensitive to oxidative damage. The lipid peroxidation process was more effective in rat liver mitochondria than in bovine. (Mol Cell Biochem xxx: 77–82, 2005)

Non-enzymatic and enzymatic lipid peroxidation of microsomes and nuclei obtained from rat liver

The present study investigates in a experimental system in vitrothe relationship between the non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation in rat liver microsomes and nuclei. Chemiluminescence was measured as cpm/mg protein during 180 min at 37 °C. Approximately 50–55% of the fatty acids located in rat liver microsomes and nuclei are polyunsaturated with a prevalence of C18:2 n6 and C20:4 n6. The values of total light emission during the non-enzymatic and enzymatic lipid peroxidation were highest in microsomes than in nuclei. A significant decrease of C20:4 n6 and C22:6 n3 in rat liver microsomes and nuclei was observed during the lipid ascorbate-Fe2+-dependent peroxidation, whereas a significant decrease of C20:4 n6 in rat liver microsomes was observed during enzymatic lipid peroxidation. Over the time course studies, analysis of chemiluminescence in microsomes and nuclei demonstrated that the lipid peroxidation in the presence of ascorbate-Fe2+ reach a maximum at 50 and 30 min, respectively, whereas in the presence of NADPH it reachs a maximum at 20 min in both organelles. In liver microsomes and nuclei the peroxidizability index (pi) which indicates the degree of vulnerability to degradation of a selected membrane showed statistically significant differences between control versus ascorbate-Fe2+ when microsomes or nuclei were compared. Our results indicate that non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation are operative in rat liver microsomes and nuclei but the sensitivities of both organelles to lipid peroxidation evidenced by chemiluminescence was greater in the presence of ascorbate-Fe2+ when compared with NADPH. (Mol Cell Biochem 265: 1–9, 2004)

Liver and heart mitochondria obtained from Adelie penguin ( Pygoscelis adeliae ) offers high resistance to lipid peroxidation

Lipid peroxidation is generally thought to be a major mechanism of cell injury in aerobic organisms subjected to oxidative stress. All cellular membranes are especially vulnerable to oxidation due to their high concentration of polyunsaturated fatty acids. However, birds have special adaptations for preventing membrane damage caused by reactive oxygen species. This study examines fatty acid profiles and susceptibility to lipid peroxidation in liver and heart mitochondria obtained from Adelie penguin (Pygoscelis adeliae). The saturated fatty acids in these organelles represent approximately 40-50% of total fatty acids whereas the polyunsaturated fatty acid composition was highly distinctive, characterized by almost equal amounts of 18:2 n-6; 20:4 n-6 and 22:6 n-3 in liver mitochondria, and a higher proportion of 18:2 n-6 compared to 20:4 n-6 and 22:6 n-3 in heart mitochondria. The concentration of total unsaturated fatty acids of liver and heart mitochondria was approximately 50% and 60%, respectively, with a prevalence of oleic acid C18:1 n9. The rate C20:4 n6/C18:2 n6 and the unsaturation index was similar in liver and heart mitochondria; 104.33 +/- 6.73 and 100.09 +/- 3.07, respectively. Light emission originating from these organelles showed no statistically significant differences and the polyunsaturated fatty acid profiles did not change during the lipid peroxidation process.

Effect of fish oil and vitamin E on sperm lipid peroxidation in dogs

The objective was to evaluate the effects of dietary fish oil (FO) and vitamin E (VE) supplementation on sperm sensitivity to lipid peroxidation (LP) in dogs. Using an incomplete replicate 3 × 3 Latin square design, five dogs were allocated into three groups. One of the squares was incomplete and had two dogs that were used with three treatments. The dogs were assigned to three different treatments, fed a control diet of balanced commercial food (control group; CG), control diet supplemented with 54 mg FO/kg body weight0·75 per d (FO group; FG) and FO plus 400 mg VE per d (FO and VE group; FEG) for 60 d. Semen samples were collected on days 0 and 60 and divided into two halves, peroxidised and control, with or without ascorbate–Fe2+, respectively. LP was measured in both halves by chemiluminescence as counts per min/mg protein. Fatty acid profile was determined by GC. Data were analysed using the mixed procedure (SAS). On day 0, LP increased in all groups in the peroxidised samples (P < 0·05). However, on day 60 LP decreased in peroxidised samples of both the FG and FEG (P < 0·05), but there were no differences between the FG and FEG (P > 0·1). Additionally, on day 60 total n-3 was higher in the FG and FEG compared with the CG (P < 0·05). Supplementation with FO alone or together with VE decreased LP in peroxidised samples. These results could indicate a protective effect of n-3 on sperm. More studies are needed to understand the mechanism whereby FO and/or FO plus VE decrease LP in dogs’ sperm.