# Comparative Tissue Responses of Marine Mollusks on Seasonal Changes in the Northern Adriatic Sea
## 2 Materials and Methods
### Analytical Methods  
Tissues for biochemical analyses and lipid peroxidation were homogenized on ice (Ultra-Turrax, IKA, Germany) in an isotonic solution of 0.9% NaCl with 0.1 mM protease inhibitor PMSF (1 g of tissue: 3 mL of isotonic solution). Obtained homogenates were subsequently centrifuged for 20 min at 4 \({}^{\circ}\)C (12,000 g) and supernatants were used for analyses of biochemical and antioxidative parameters. Concentrations of glucose (GLU), triglyceride (TRIG), cholesterol (CHOL), magnesium (Mg) and calcium (Ca) in supernatants were determined with Abbott commercial kits on Abbott Architect c4000 clinical chemistry analyzer (Abbott, Germany). The GLU concentration was determined in 2.0 \(\mu\)L of supernatant with the enzymatic method based on hexokinase/glucose-6-phosphate dehydrogenase catalytic reaction. The TRIG and CHOL concentrations were determined in 2.4 \(\mu\)L of supernatant using enzymatic methods with glycerol-phosphate oxidase and cholesterol esterase/cholesterol oxidase, respectively. The Mg concentration was measured in 3.2 \(\mu\)L of supernatant, with the isocitrate dehydrogenase enzymatic method, while the Ca concentration was measured in 2.6 \(\mu\)L of supernatant using the arsenazo-III method. All biochemical parameters were expressed as mg/g of tissue. Activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and total antioxidant status (TAS) were measured using Randox commercial kits (Dublin, Ireland) on Abbott Architect c4000 (Abbott, Germany) according to the manufacturers' instructions. The SOD activity was measured by \begin{table}
\begin{tabular}{l c c c c c c} \hline \hline  & \multicolumn{3}{c}{**Ormer Sampling (Measured at 7 m Depth)**} & \multicolumn{3}{c}{**Scallop Sampling (Measured at 15 m Depth)**} \\ \cline{2-7}  & **Fall** & **Winter** & **Spring/Summer Fall** & **Winter** & **Spring/Summer** \\ \hline Temperature (\({}^{\circ}\)C) & 19.30 \(\pm\) 3.21 & 11.33 \(\pm\) 2.11 & 21.20 \(\pm\) 1.41 & 20.90 \(\pm\) 1.98 & 12.55 \(\pm\) 4.88 & 16.87 \(\pm\) 3.05 \\ Salinity (psu) & 37.97 \(\pm\) 0.25 & 38.30 \(\pm\) 0.30 & 34.90 \(\pm\) 1.56 & 38.10 \(\pm\) 0.14 & 38.15 \(\pm\) 0.64 & 36.07 \(\pm\) 2.02 \\ Dissolved oxygen (mg/L) & 5.15 \(\pm\) 0.31 & 6.02 \(\pm\) 0.26 & 5.99 \(\pm\) 1.31 & 4.99 \(\pm\) 0.17 & 5.89 \(\pm\) 0.60 & 5.41 \(\pm\) 0.32 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Water temperature, salinity and dissolved oxygen concentrations at the sampling sites and respective depths for green ormer (_Haliotis tuberculata_ L.) and Mediterranean scallop (_Pecten jacobeus_ L.). the degree of inhibition of superoxide radical formation in 5.0 \(\mu\)L of supernatant. The GPx method is based on the oxidation of glutathione by cumene hydroperoxide and its activity was measured in 4.0 \(\mu\)L of supernatant. The TAS concentration in samples was measured in 4.0 \(\mu\)L of supernatant by suppression of the radical production, which was visualized with specific chromogen. All enzyme activities were expressed as U/g of protein, and TAS was expressed as mmol/g of protein. The reaction between malondialdehydedehyde (MDA) and thiobarbituric acid (TBA) under acidic conditions resulted in the formation of thiobarbituric acid reactive substances (TBARS), as indicators of lipid peroxidation. Lipid peroxidation was measured in the entire soft body of animals, in accordance with our previous study [20]. Tissues were weighed, homogenized and centrifuged, and the supernatant was added to the reagent mixture of 10% TCA and 0.01% BHT. Samples were vortexed for 15 sec, cooled for 15 min and centrifuged for 10 min at 12,000 g (all at 4 \({}^{\circ}\)C). The supernatant (750 \(\mu\)L) was mixed with 500 \(\mu\)L TBA and heated at 99 \({}^{\circ}\)C for 30 min. The reaction stopped by cooling at 4 \({}^{\circ}\)C, when the absorbency of supernatant was read at 535 nm using a FLUOstar OPTIMA plate reader (Infinite M200, Tecan, Austria).