EFFECT OF COLD AND HEAT STRESS ON RAT ADRENAL , SERUM AND LIVER ASCORBIC ACID CONCENTRATION

Changes in ascorbic acid (AA) concentration were examined in the adrenals, serum and liver of Wistar rats exposed to cold (6 oC) and heat stress (38 oC) for 60 min. The exposure of animals to cold stress for 60 min did not change concentration of AA in the serum, adrenals and liver as compared to controls maintained at room temperature. After a 60 min heat exposure the concentration of AA in the adrenals decreased (***p<0.001), in the liver remained unchanged whereas it significantly increased in the serum (***p<0.001) in respect to control values. .


INTRODUCTION
Body tissues differ in the ascorbic acid (AA) concentrations.However, tissues of high metabolic need, such as the corpus luteum in the female, the testis in the male and the adrenal glands, generally have higher concentrations of this vitamin.Knockout genes preventing AA synthesis in pigs have demonstrated a poor skeletal and collagen formation and poor antioxidant protection (Mahan et al., 2004).Ascorbic acid is thought to have a role in the regulation of corticosteroid synthesis in the adrenal glands (Finnand Johns, 1980).The data obtained with bovine adrenocortical cells in vitro favor its role as an antioxidant that especially regulates aldosterone synthesis, possibly by protecting the cytochrome P450 11β from lipid peroxidation.Vitamin C may also act as a part of an auxiliary electron transport system for the last step of aldosterone synthesis (Redmann et al., 1995;Bahr et al., 1996).According to Bjorkhem et al. (1978) the adrenal ascorbate might be of some importance for the capacity to convert cholesterol into pregnenolone.Besides, stress and the associated rise in ACTH may reduce the level of AA in pigs adrenals, but the animals rapidly recovers to its resting state once the stressor agent is removed (Mahan et al., 2004).A rad et al. (1985) found a significant increase in AA level in plasma of rats and rabbits after hypoxia and the following asphyxia.The observed changes in plasma AA levels are obviously mediated through ACTH and the adrenal gland is the major source of ascorbate efflux into the circulation during oxygen deprivation.
Bearing in mind the facts mentioned above, we examined the ascorbic acid turnover e.g. the concentration of AA in the serum, adrenals and liver of rats exposed to physical -environmental stressors, cold and heat.

MATERIAL AND METHODS
The male rats of Wistar strain (Rattus norvegicus), 60-90 days old, weighing 180-220 g, were used for the experiments.The animals were acclimated to 22 ±1ºC, kept at a 12:12h light-dark cycle and given commercial rat food and tap water ad libitum.The rats were divided into three groups, each consisting of six animals.The first group represented intact controls.The rats from the second and third group were exposed to the extreme ambient temperatures 6 ºC for 60 min and 38 ºC for 60 min, respectively, in temperature controlled chambers.The animals were decapitated immediately after the stress exposure.Blood was collected from the trunk and serum frozen for ascorbate determination, while the adrenals and liver were quickly excised at +4 °C, weighed and stored at -20 °C prior to AA concentration measurement.The AA was determined by the method of Roe and Kuether(1943) based on the oxidation of ascorbic acid to dehydroascorbic acid (DHAA) which is irreversibly transformed to 2,3-diketogulonicacid (DKA).DKA is coupled with 2,4-dinitrophenylhydrazineand the resulting derivative is treated with H 2 SO 4 to produce color which is measured on LKB Micro Plate Reader, on 540 nm.
The values are expressed as μg/mg tissue (the adrenals or liver) or mg/l serum.Anova one way test was used to detect the differences among the groups.The values are presented as means ± S.E. of six animals and the level of significance was set at p<0.05.

RESULTS
The exposure of the animals to cold stress for 60 min did not change concentration of the ascorbic acid in the serum, adrenals and liver as compared to the controls maintained at room temperature.After a 60 min heat exposure the concentration of ascorbic acid in the adrenals decreased (***p<0.001), in the liver remained unchanged, whereas it significantly increased in the serum (***p<0.001), in respect to control values.

DISCUSSION
Stress is always accompanied with an increased ascorbate demand because of its increased consumption and/or decreased synthesis and uptake (Banhegyi et al., 1998).While species such as rodents produce ascorbic acid viahepatic synthesis (Grollman and Lehninger, 1957), primates, guinea pigs and fruit-eating bats must acquire this vitamin through dietary intake as theylack L-gulonolactone oxidase, the terminal enzyme in the L-ascorbicacid biosynthetic pathway.These animals survive because membrane transport processes allow dietary ascorbate to be absorbed as chime moves through the small intestine (Rose, 1991).Regardless of its acquisition route,theascorbic acid is accumulated in adrenocortical cells by membrane transportersagainst a large concentration gradient even in the animals in which ascorbate is not a dietary requirement as synthesis occurs onlyin the liver (Palludanand Wegger, 1984).
Our present results show that exposure of rats to extreme environmental temperature of 38 ºC for 60 min caused a significant decrease in the adrenal ascorbate concentration accompanied by its increase in circulation.Obviously, the source of the ascorbate efflux into the cir- culation are adrenals since there were no changes in AA content of the liver, which is the site of AA synthesis in rats.Besides, Arad et al. (1985) showed that adrenalectomy performed 24h before exposure of rats to stress abolished the rise of AA in blood.Our findings are in agreement with Sayersand Sayers(1972) who have shown the AA concentration decrement under stress when adrenal cortical hormone activity is high.The ascorbic acid is involved in the process of steroidogenesis since the adrenal cortex contains high levels of vitamin C which are depleted upon adrenocorticotropic hormone (ACTH) stimulation of the gland (Lahiriand Lloyd, 1962;Laney et al., 1990).However, some data suggesting that differing plasma concentrations of AA regulate in vivo steroidogenesis by altering the activity of the membrane-bound enzyme adenylate cyclase (Doulas et al., 1987).
In spite of the fact that heat stress affected changes of AA in serum and adrenals, the same duration of cold exposure did not alter the concentration of AA in the liver, serum and adrenals.It is not surprising since we have already shown that heat is the stronger stressor judging by the fact that it provokes a six fold greater plasma ACTH increment as compared to cold stress (Djordjević et al., 2003).
In conclusion, present results demonstrate that the exposure of rats to heat stress caused the ascorbic acid turnover e.g. the increase in the amount of ascorbate in blood due to the depletion of adrenals content, without de novo hepatic synthesis and support previous findings that heat is the stronger stressor in comparison to cold.

Fig 1 .
Fig 1.The effect of 60 min exposure of rats to cold (6 ºC) and heat (38 ºC) stress on ascorbate content of liver, serum and adrenal gland.The values represent the means ± SE of six animals and are expressed in μg/mg tissue (the adrenals or liver) or mg/l serum; ***p<0.001.