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

Blahová J . , R. Dob‰íková, Z. Svobodová, P. Kaláb: 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 (Möstl 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 ACTA VET. BRNO 2007, 76: 59–64; doi:10.2754/avb200776010059 Address for correspondence: Ing. Jana Blahová University of Veterinary and Pharmaceutical Sciences Palackého 1-3 612 42 Brno, Czech Republic Phone: + 420 5 4156 2785 Fax: + 420 5 4156 2790 E-mail: blahovaj@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm source of environmental stressor. Various stressors, such as grading, handling, transportation and vaccination, are necessary components of modern intensive fish culture (Svoboda 2001; Kubilay and Ulokoy 2002; Jelínek and Koudela 2003). Plasma cortisol level can be measured by commercial radioimmunoassay (RIA), enzymelinked immunosorbent assay (ELISA) or chromatographic methods (HPLC or GC). All three methods of analysis have their advantages and disadvantages. The aim of the study was to determine the most appropriate method. The commercial availability of sensitive and specific antisera for cortisol has made RIA the method of choice in most laboratories (Ruder et al. 1972; Drast ichová et al. 2004). However, the advantage of using ELISA is that it does not require the use of any radioactive reagent, and therefore it is safer and more economical than standard RIA. Chromatographic methods might be more specific and accurate analytical methods, but they are also considerably more time consuming, and require much more complicated instrumentation (Nozaki 2001; Bartels et al. 2003; Dob‰íková et al. 2006). HPLC with UV, fluorescence or mass spectrometry detection is the most often used for cortisol determination. The simplest and most often used detection method is UV detection (wavelength cca 250 nm). The HPLC/UV disadvantage is its lower sensitivity in comparison to other detection methods. Mass spectrometry is more sensitive but too expensive and not available in all laboratories. The use of fluorescence detection is subjected to conversion of cortisol to fluorescent substance. The conversion is enabled by using various derivatisation reagents (9-anthroylnitrile, dansylhydrazine, sulphuric acid with ethanol, etc.). Fluorescence detection methods are limited by precisely controlled reaction conditions and the instability of the fluorescent product (Goto et al. 1983; Volin 1995; Shibata et al. 1998; Nozaki 2001). Mass spectrometry detection is the most used GC method for the plasma cortisol determination (Heckmann et al. 2005). The aim of this study was to compare the sensitivity of HPLC and RIA analytical methods in the evaluation of blood plasma cortisol level of common carp (Cyprinus carpio L.). For HPLC determination of plasma cortisol a chromatographic system Alliance 2695 was used with Photodiode Array Detector 2996 (Waters, USA). For RIA determination of plasma cortisol a Cortisol RIA kit was used (Immunotech Prague; A Beckman Coulter Company). Materials and Methods Sample handl ing and preparat ion of plasma The monitoring of blood plasma cortisol was made during the test focused on the stress load by handling and transport of common carp. The samples were collected before, during and after regular transport (April 29, 2005) of fish from storage ponds at a commercial fish farm in Hluboká nad Vltavou via Brno (Czech Republic) to a fishpond in Boheoov (Slovakia), 12 hours in total. Carp were held in a freshwater flow-through storage pond for three days prior to transportation, and then transferred to 2.2 m3 transport tanks (loading density of 364 kg body weight·m-3). In total, 66 individuals of common carp were tested. The blood samples were withdrawn by cardiocenthesis and collected in glass tubes containing heparin and centrifuged at 3,000 rpm for 10 min, then plasma was separated (Svobodová et al. 1991). The plasma samples were kept frozen at 80 °C in Eppendorf test-tubes until analysis. Chromatographic condi t ions and sample pret reatment For plasma sample preparation, the SPE (solid phase extraction) was used before HPLC analysis. Cortisol was extracted using SPEC C18AR columns (3 ml, 30 mg, Varian Inc.), which had been activated with 1 ml of methanol (Merck) followed by 1 ml of deionized water. After application of samples (1 ml), the cartridges were washed with 1 ml deionized water followed by 1 ml of 20% methanol in deionized water. Then the cartridges were air-dried under reduced pressure for 5 min. The analyte was eluated with 1 ml of acetonitrile (Merck). 20 μl of the eluted sample was then injected into the HPLC system. The SPE was used for standard preparation procedure, too. Stock calibration solution of cortisol (Sigma-Aldrich) was prepared by dissolving 4 mg pure substance in 200 ml of 60% acetonitrile in deionized water. The solution was stable for at least two months when stored at 4 °C. Standard solutions were obtained by diluting stock solution with deionized water before use. The HPLC system was equipped with a Waters Model 2695 Alliance Separation Module and Waters 2996 Photodiode Array Detector. Chromatographic separation was achieved on a Polaris C18-A column (3μ, 150 × 4.6 mm, Varian Inc.). The guard column was a MetaGuard Polaris C18-A (5 μ, 10 × 4.6 mm, Varian Inc.) The mobile phase was pumped at a flow rate of 1 ml/min and consisted of acetonitrile (60%) and water (40%) at a temperature 60