Male Wistar rats aged 4 or 22 months, were used in the experiments. Animals were maintained under a 12:!2 LD photoperiod, at 22°C environmental temperature, with water and food provided ad libitum. Blood samples were obtained by venous puncture under light ether anesthesia. Diurnal sampling were made at the middle of the period of light. Nocturnal samples were obtained 4h after the onset of the darkness period, under red light of low intensity. A group of 4-month-old animals were pinealectomized and diurnal and nocturnal blood samples were obtained before and 10 days after surgery. In all cases blood samples were stored at 5-8°C until clothing and serum were aliquoted and stored at -75°C until processed (no more than 3 weeks). The STAA was assayed in an in vitro model of brain phospholipid and glycolipids liposome peroxidation induced by Fe⁺⁺/Fe⁺⁺⁺ ions. Liposomes were prepared by suspending bovine brain extracts (Type VII, Sigma) in nitrogen-purged NaCl 0.9% solution (5mg/ml) and subjecting the suspension to vigorous agitation with vortex and sonication. The suspension was stored for 1h at 6°C to allow the liposomes to swell. The assay were carried out by exposing 1 ml of liposome suspension (concentration 500 µg/ml) to peroxidation induced by a combination of Fe³⁺ (150 µM final concentration) and Fe²⁺ (50 µM) during 1h at 37°C in presence or not of a sample of the serum studied (20µl). Reactions were stopped by inversion of the tubes in ice and addition of 100 µl of butylated hydroytoluene 0.5% (Sigma). The extend of the lipid peroxidation was measured by the formation of thiobarbituric acid-reactive products (Buege and Aust, 1978) and quantified against a standard curve derived from a freshly prepared solution of malonaldehyde (Sigma) by reading absorbance at 532 nm. Differences between diurnal and nocturnal STAA in young and aged rats were analyzed by paired Student t test. Differences in STAA between young and aged rats either on diurnal or nocturnal blood samples were processed by one-way ANOVA followed by Student-Newman-Keuls multiple comparisons test.

The STAA in 4-month-old animals was significantly lower (8.5%, p < 0.001) in nocturnal than in diurnal blood samples. This diurnal rhythm in STAA disappeared in the aged rats. The younger animals (4-month-old) showed diurnal STAA higher than aged rats (12%, p < 0.05) whereas nocturnal STAA was similar in young and old animals. Pinealectomy abolishes the STAA daily rhythm in young rats.

Serum is endowed with an array of antioxidant defense mechanisms which included ascorbate, urate, alpha-tocopherol, albumin, melatonin, etc. Our results shows a diurnal rhythm in STAA with the lower antioxidant capacity being during night. Although differences between diurnal and nocturnal STAA are small (8.5%) they are very consistent since in all animals (n=12) the diurnal values were higher than the nocturnal ones, this explaining the high rate of statistical significance achieved. Recently, diurnal variations in the STAA have been described in rats (Benot et al., 1998) although with the highest values during night, coinciding with the nocturnal melatonin peak. However, other authors, found that the lipoperoxidative activity in rats is highest during night, concurrently with a decrease in glutathione peroxidase activity and an increase in superoxide dismutase activity (Diaz-Muņoz et al., 1985). A higher oxidative stress in the rat heart during the dark phase has been also observed (Lapenna et al., 1992). These discrepancies could be explained by the relation between the time of the blood sampling and the fasting and feeding periods, or because the existence of circaannual rhythms in STAA. Circadian as well as circaannual oscillations of tissue lipoperoxides in rats have been described (Solar et al., 1995) . Circadian oscillations were two-peaked, with zeniths at various times of the light/dark period. In summer and autumn, the maximal concentration of liver lipoperoxides were found at 2000h and 0500h, whereas in spring the peak of lipoperoxides were found at 1100 and 1700 and in winter at 0800 and 1700.

Pinealectomy abolishes the diurnal rhythm in STAA in young animals, thus indicating a clear pineal influence in the daily changes in the antioxidant defense mechanisms. Pinealectomized rats has been described exhibit a greater oxidative damage than intact animals (Manev et al., 1996).

The circadian production of melatonin is greatly attenuated with aging (Reiter, 1992). We found that the diurnal rhythm in STAA disappeared in the old rats. Interestingly, the diurnal and nocturnal values of STAA in aged rats were similar to the nocturnal values of the young animals.

Taken together, our results support a physiological role of the pineal gland in the control of the diurnal variations of the serum antioxidative status. Melatonin could be the mediator of these effects. However, melatonin actions should be considered related more with the modulation of the diurnal rhythms of the antioxidant defense mechanisms than with direct antioxidant properties.

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