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were carried out by the intraperitoneal route. At 6 h after the administration of APAP
or 50% PEG 400, the animals were sacrificed by decapitation and their blood col-
lected in heparinized tubes. Immediately thereafter the livers were removed using
the freeze-clamp technique of Wollenberger et al. ( 1960 ). From each blood sample,
a portion was set aside for the assay of reduced (GSH) and disulfide (GSSG) gluta-
thione, and the remaining portion was centrifuged at 3,000 rpm (500 ´ g ) and 4°C for
10 min to obtain the corresponding plasma fraction. For each liver, a 500 mg portion,
kept cold on an ice bath, was mixed in a 1:20 (w/v) ratio with Tris buffer pH 7.0
containing 1 mg of phenylmethylsulfonyl fluoride and homogenized using a handheld
electric blender. The resulting suspension was centrifuged at 12,000 rpm (8000 ´ g )
and 4°C for 30 min to isolate the supernatant, which was kept on ice until needed.
20.2.3
Assay of Liver Malondialdehyde
Malondialdehyde (MDA) was determined as thiobarbituric acid-reactive substances
(TBARS) by the endpoint assay method of Buege and Aust ( 1978 ) . The amount of
TBARS in the sample was derived from a calibration curve of MDA prepared from
serial dilutions of a stock solution of 1,1,3,3-tetraethoxypropane which had been
treated in the same manner as the sample preparation, and the results were reported
as nM of MDA/mg of tissue.
20.2.4
Assay of the Plasma and Hepatic
Levels of GSH and GSSG
The concentration of GSH in plasma and liver samples was measured by the method
of Hissin and Hilf ( 1976 ), after reaction with ortho -phthalaldehyde to form a highly
fluorescent product. The concentration of GSSG was measured in another aliquot of
the same sample, following removal of any preexisting GSH upon reaction with
N -ethylmaleimide. The concentrations of GSH and GSSG in the sample preparation
were determined by reference to standard curves of these compounds prepared on the
day of the assay and were reported either as nM/mL of plasma or as mM/g of tissue.
20.2.5
Assay of Plasma and Liver Antioxidant Enzymes
The catalase (CAT) activity was measured by the spectrophotometric method of
Aebi ( 1984 ), the glutathione peroxidase (GPX) activity was measured as described
by Flohé and Günzler ( 1984 ), and the activity of CuZn superoxide dismutase (SOD)
was measured using the spectrophotometric method of Misra ( 1985 ) . These activi-
ties were expressed as U/min/mg of protein.
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