Simultaneous Determination of Plasma Cortisol by High Pressure Liquid Chromatography and Radioimmunoassay Methods in Fish

Blahova J., R. Dob‰ikova, Z. Svobodova, P. Kalab: Simultaneous Determination of Plasma Cortisol by High Performance Liquid Chromatography and Radioimmunoassay Methods in Fish. Acta Vet Brno 76, 2007: 59-64. The aim of the study was to compare the sensitivity of High Performance Liquid Chromatography (HPLC) and Radioimmunoassay (RIA) analytical methods in the determination of blood plasma cortisol level of common carp (Cyprinus carpio L.). Cortisol, the most potent glucocorticoid, is widely used as an indicator of stress. The monitoring of plasma cortisol concentration was performed during the test focused on stress response caused by handling and long-distance transport of common carp. The samples were collected before, during and after transport. The correlation between blood cortisol level in fish measured by HPLC and RIA was determined using 66 samples. The correlation was HPLC = 0.9454 RIA + 0.40676 and correlation coefficient was 0.815. From the presented results it can be concluded that both methods (RIA and HPLC) can be used for the determination of plasma (serum) cortisol level. Transport, Cyprinus carpio L. , stress Cortisol is the most potent and abundant glucocorticoid secreted by the outer cortex of the adrenal gland. Its secretion is stimulated by the adrenocorticotrophic hormone (ACTH) produced in the pituitary in response to corticotropin-releasing hormone (CRH). In mammals, 90% of the secreted cortisol in circulation is bound to plasma proteins, mainly to cortisol-binding globulin (CGB). The rest of cortisol circulates in an unboud form, i.e. the physiological active form. In fish, cortisol is bound to plasma proteins in considerably lower amounts. Caldwell et al. (1991) cites that only 48.2%, 16% and 19% of cortisol is bound to plasma proteins in adult females, adult males and juvenile individuals of rainbow trout, respectively. Plasma cortisol levels are known to cycle diurnally and to change according to season. During the day the highest concentration is measured in the morning and then the level declines throughout the day (Kaneko 1997; McLeese et al. 1994; Zima et al. 2002). Plasma cortisol level is widely used as an indicator of stress (Mostl and Palme 2002). Stress is commonly defined as a state or condition in which the homeostasis of an individual is disturbed as a result of the actions of external or internal stimuli, termed stressors. The stressors elicit changes in the animal’s physiological state, which is interpreted as the stress response (Gregory and Wood 1999). In response to a stressful event, the hypothalamic portion of the brain stimulates the release of ACTH. ACTH circulates into the anterior kidney, where it stimulates the interrenal cells to produce cortisol and other corticosteroid hormones. Cortisol then plays an important role in mobilizing fuels such as glucose, lipids, and fatty acids for the maintenance of homeostasis and exerts direct and indirect effects on intermediary metabolism, particularly in response to stress. The fish are exposed to stressors in nature, as well as in artificial conditions such as in aquaculture, or in the laboratory. The increasing contamination of bodies of natural freshwater and marine ecosystem worldwide by anthropogenic substances is one