Mechanisms of aluminium neurotoxicity in oxidative stress-induced ...

CHAPTER 3
used as chemical dosimeters for PCC and PTC determinations, respectively. For in vitro
experiments, AlCl3 was preincubated with brain mitochondria at a final Al 3+
concentration of 5 �M.
Measurement of SOD activity
SOD activity was spectrophotometrically determined by the method reported by
McCord and Fridovich (1969). The assay was performed at 25°C in 2.8 ml of 50 mM
potassium phosphate buffer (pH 7.8) containing 0.1 mM EDTA, 0.05 mM xanthine and
0.01 mM cytochrome c. Then, 100 �l xanthine oxidase (0.02 U) were added in order to
produce a rate of reduction of cytochrome c of 0.025 absorbance unit per minute,
followed by 100 �l of brain sample. Increase in absorption was monitored at 550 nm for
5 minutes. SOD activity was expressed in U/mg protein. To evaluate the potential in
vitro effect of aluminium on SOD, different concentrations of Al 3+ (10, 50, 100 �M)
were added, followed by 100 �l of SOD (1 U) instead of brain sample.
Measurement of GPx activity
GPx activity was spectrophotometrically measured by a modified version of the
method reported by Flohe and Gunzler (1984). Briefly, the reaction mixture consisted of
500 �l phosphate buffer (60 mM, 0.6 mM EDTA, 1 mM NaN3, pH 7.0), 100 �l GR (0.5
U), and 100 �l GSH (1 mM). Brain sample (100 �l) was added to the reaction mixture
and preincubated at 37°C for 10 min. Then, 100 �l NADPH (150 μM in 0.1% NaHCO3)
were added to reaction mixture and the hydroperoxide-independent consumption of
NADPH monitored for 3 min. The reaction was started by adding 100 �l H2O2 (150
μM) and the decrease in absorption at 340 nm monitored for 5 min. Molar extinction
coefficient of 6.22·10 3 M -1 ·cm -1 of NADPH at 340 nm was used to calculate GPx
activity which was expressed in mU/mg protein. When assessing the in vitro effect of
aluminium on GPx activity, different concentrations of Al 3+ (10, 50, 100 �M) were
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