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International Journal of Marine Science 2014, Vol.4, No.44, 1-14
http://ijms.biopublisher.ca
4
sample; the final volume was 1.5 mL. The reactivity
was measured using OD at 560 nm.
Catalase (CAT) was measured using a previously
described method (Asatiani, 1969) that involves a
hydroperoxide reduction.
Peroxidase (PER) activity was detected by
spectrophotometry at 600 nm using benzidine reagent
(Litvin, 1981). Specifically, the reaction mixture
contained 1 mL acetate buffer pH 5,4, 0.4 mL 0.09%
benzidine, 0.2 mL 0.03% H
2
O
2
, and 0.2 mL sample.
Glutathione reductase (GR) activity was assayed
spectrophotometrically using a method modified after
Goldberg and Sparner (Goldberg and Sparner, 1987).
The reaction mixture contained 0.1 mL mM NADPH,
0.5 mL 7.5 mM oxidized glutathione, 0.2 mL mM
EDTA, and 2 mL 0.05 M phosphate buffer pH 8.0.
After incubation for 10 min (at what tempt), the OD of
the mixture was determined at 340 nm.
Aminotransferases activity: Aminotransferases (ALT
and
AST)
activity
was
also
determined
spectrophotometrically with 2,4-dinitrophenylhydrazine
using the standard kit (Filicit-Diagnosis, Ukraine).
Specifically, 0.2 mL substrate-buffer solution was
added to 0.04 mL of liver extract or serum and
incubated at room temperature for 1 h. The reaction
was stopped by 0.2 mL 1,4-dinitrophenylhydrazine
and the solution was incubated for 20 min. Then 2 mL
0.4 N NaOH was added to the mixture and the OD of
the sample was measured at 500~530 nm. The ratio
AST/ALT (de Rytis coefficient) was calculated also
(Catalogue, 2005).
Total soluble protein, albumin and hemoglobin
concentrations, Total soluble protein was quantified
spectrophotometrically
according
the
method
described by Lowry et. al. (1951). Albumin (Alb)
concentration was assayed spectrophotometrically on
the basis by Bromokresol green reaction, used the
standard commercial kit (Felicit-Diagnosis, Ukraine).
Hemoglobin (Hb) concentration was measured by
spectrophotometric method on the basis of cyanide
reaction (Young, 1997). The enzyme activities were
calculated per mg protein in liver extracts, RBC and serum.
2.4 Statistical analysis
Biochemical measurements were detected in duplicate
for each sample. The number of tested individuals
ranged from 5 to 12 animals, depending on species
(Table 1). Simple, descriptive statistics were
performed using an ANOVA to determine means (+/-
SE) (Halafyan, 2008). P value of 0.05 was used for
determination of statistical significance in all cases.
The graphs were made using the computer program
EXELL. Statistical correlations between studied
biochemical parameters in tested animals and tissues
were calculated by the least-squares method using the
computer program CURVEFIT (Version 2.10-L).
Table 1 Number of analyses of examined biochemical characteristics of elasmobranch species caught in Sevastopol coastal waters
(Black Sea, Ukraine)
Para-meters
Species
Dogfish
Buckler skate
Stringray
Liver
RBC
Serum
Liver
RBC
Serum
Liver
RBC
Serum
OP
7
7
7
5
5
5
7
7
7
SOD
12
10
-
6
5
-
6
10
-
CAT
12
10
-
9
5
-
9
10
-
PER
12
10
-
7
5
-
5
10
-
GR
12
10
-
6
5
-
6
10
-
ALT
7
-
7
8
-
8
7
-
7
AST
7
-
7
8
-
8
7
-
7
Alb
-
-
10
-
-
7
-
-
10
Hb
-
10
-
-
7
-
-
7
-
3 Results
3.1 Biometric characteristics
Biometric characteristics of examined individuals
are present in Table 2. They are varied in examined
animals depending on fish species (Black Sea,
Ukraine).
3.2 Hepatic biomarkers
Oligopeptide concentrations in the liver of tested
elasmobranch species were higher in buckler skate
than in dogfish (P<0.01) and stringray (P<0.05)
(Figure 2). No significant differences were shown in
the hepatic oligopeptides in shark and stringray.