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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 32, No 2, March/April 2021
AFRICA
89
flush out the drugs and refilled to 25 ml. After another 30 minutes
of stabilisation, cumulative concentrations of phenylephrine
(to maximal vasocontraction), followed by titration with
acetylcholine (to induce vasorelaxation) were added. Thereafter,
the organ bath, string and steel hooks were thoroughly rinsed
with boiled distilled water.
Western blot analysis was used to determine the expression
and activation of selected proteins involved in endothelial
function, such as AMPK, PKB and eNOS. Frozen sections of
the aortic tissue (40–50 mg,
n
= 5 per group) were pulverised
in a liquid nitrogen pre-cooled mortar and pestle and then
transferred into a microcentrifuge tube [Scientific Group
(Pty) Ltd, Milnerton, Western Cape, RSA] filled with 600 µl
of lysis buffer [composition in mM: 20 Tris-HCl (pH 7.5),
1 EGTA, 1 EDTA, 150 NaCl, 1
β
-glycerophosphate, 2.5 sodium
pyrophosphate, 1 Na
3
VO
4
, 50 nM NaF, 10 μg/μl leupeptin, 10
μg/ml aprotinin, 0.1% SDS, 1% Triton X-100 and 50 µg/ml
PMSF, which was added last]. Samples were homogenised in a
bullet blender
®
24 (Next Advance, Inc, New York) using 1.6-mm
stainless steel beads at speed 8 for one minute at 4°C, for a
total period of three minutes, with five-minute rests in-between
cycles. Samples were allowed to stand on ice for 15 minutes and
centrifuged at 15 000 rpm for 20 minutes at 4ºC.
Protein concentration was determined using the Bradford
method.
22
The samples were diluted in Laemmli sample buffer,
boiled for five minutes and stored at –80ºC. Equal amounts of
protein were separated using 26-well Bio-Rad TGX stain-free
TM
5–20% gradient precast gels and transferred to polyvinylidene
fluoride (PVDF) membranes using a Bio-Rad midi-transfer
system. Proteins that were successfully transferred onto the
membrane were visualised utilising the Bio-Rad ChemiDoc
TM
MP system and a record thereof was stored.
The non-specific sites on all the membranes were blocked
with 5% fat-free milk in TBS-Tween (Tris-buffered saline and
0.1% Tween
®
20). Membranes were incubated overnight at
4°C in a 7.5-µl polyclonal primary antibody solution, diluted
in 5 ml TBS-Tween or in primary SignalBoost
TM
. Thereafter,
the membranes were incubated for one hour in 2.5 µl horse
radish peroxidase-coupled secondary antibody (Amersham Life
Science, Buckinghamshire, UK) in 5 ml TBS-Tween or in
secondary SignalBoost
TM
immunoreaction enhancer (Sigma-
Aldrich, St Louis, MO, USA), diluted in 5 ml TBS-Tween. The
primary and secondary SignalBoost
TM
were specifically used for
the incubation of the eNOS antibody.
The following antibodies from Cell Signaling Technologies
®
were used: total [catalogue number (cat #): 2532] and phospho-
AMPK (Thr172; cat # 2531), total (cat # 9272) and phospho-
PKB/Akt (Ser473; cat #9271) and total (cat #9572) and phospho-
eNOS (Ser1177; cat # 9571). The proteins were visualised by
incubating the membrane with enhanced chemiluminescence
(ECL) (AEC Amersham, Buckinghamshire, UK) and exposed
in the Bio-Rad ChemiDoc
TM
MP system. The band pixels from
the exposed bands of the blot were normalised towards the total
protein per lane that was transferred to the PDVF membrane for
equal protein loading.
Antioxidant enzyme analysis
For the lysate preparation, the liver tissue samples were
homogenised in 0.05 mM sodium phosphate buffer (pH 7.5)
in a Bullet blender
®
24 (Next Advance, Inc, New York) using
1.6-mm stainless steel beads, in a coldroom, at speed 8 for three
minutes and speed 9 for four minutes, with one-minute rest
periods in-between cycles. Samples were then allowed to stand
on ice for 30 minutes and centrifuged at 15 000 rpm for 20
minutes at 4°C. Protein concentration was determined by means
of a Bicinchoninic acid (BCA) protein assay kit (BCA1, Sigma
Aldrich), using bovine serum albumin (BSA) (1 mg/ml) used as
a standard, as supplied in the kit. The lysates were then frozen at
–80°C and used for the downstream antioxidant enzyme assays
and for the determination of lipid peroxidation.
Catalase (CAT) [enzyme commission number (EC) 1.11.1.6]
catalyses the conversion of two H
2
O
2
molecules into oxygen
and two water molecules mostly in aerobic cells
.23
Liver tissue
homogenates were diluted to 0.1 µg/µl protein in assay buffer (50
mM potassium phosphate, 0.5% Triton X-100, pH 7.0). From
the diluted tissue lysate, 5 µl was assayed in triplicate in a 96-well
ultraviolet (UV) plate, followed by 170 µl of assay buffer. To
initiate the reaction, 50 µl of H
2
O
2
stock solution was added to all
the wells and the absorbance was measured over five minutes to
measure the linear decrease over time at 240 nm in a FLUOstar
®
Omega microplate reader. The molar extinction coefficient of
H
2
O
2
(43.6 M/cm), adjusted for the well path length, was used
to determine catalase activity (µmole H
2
O
2
consumed/min/µg
protein).
Superoxide dismutase (SOD) (EC 1.15.1.1) catalyses the
dismutation of the reactive superoxide radical (O
2
-
) into H
2
O
2
and oxygen. Activity was determined according to the method
modified from Ellerby and Bredesen.
23
Liver tissue homogenates
were diluted to 0.1 ug/µl of protein in SOD assay buffer (50 mM
sodium phosphate, pH 7.4). 6-hydroxydopamine (6-OHD)
1.6 mM was freshly prepared as follows: a volume of 50 µl
of concentrated (70%) perchloric acid (HClO
4
) was added to
10 ml deionised water (deiH
2
O) and purged with nitrogen for 15
minutes to displace the oxygen. Thereafter, 4 mg of 6-OHD was
added to this solution, wrapped in foil and kept on ice.
The samples (10 µl) and the blank (15 µl SOD assay
buffer) were assayed in triplicate and 170 µl of 0.1 mM
diethylenetriaminepentaacetic acid (DETAPAC), prepared in
SOD assay buffer (1 mg in 25 ml), was added to all the wells.
SOD assay buffer (5 µl) was added to all the sample wells,
excluding the blank well and the reaction was initiated by adding
15 µl of 6-OHD to each well of the 96-well plate. The kinetics of
the auto-oxidation of 6-OHD was monitored at 490 nm for five
minutes at 25°C, using the FLUOstar Omega
®
microplate reader,
and the results are expressed as units/mg protein.
Glutathione peroxidase (GPx) (EC 1.11.1.9) catalyses the
dismutation of lipids and hydroperoxides, including H
2
O
2
, by
reduced glutathione, and the activity was determined according
to Ellerby and Bredesen.
23
Liver tissue homogenates were diluted
using a 2.5 × dilution (40 µl sample: 60 µl assay buffer) so that the
protein concentration fell between the required range (5–10 mg/
ml). A cocktail solution consisting of 210 µl assay buffer [50 mM
potassium phosphate, 1 mM of ethylenediaminetetraacetic acid
(EDTA), pH 7.0], 2.5 µl glutathione (GSH) solution (30.7 mg/
ml, in deiH
2
O), 2.5 µl glutathione reductase (1.6 mg/ml, diluted
in assay buffer), 2.5 µl of 0.1 M sodium azide to inhibit catalase
and lastly, 2.5 µl of nicotinamide adenine dinucleotide phosphate
(NADPH) [5 mg dissolved in 0.1% of sodium bicarbonate
(NaHCO
3
)] was prepared.