726
H. UTOꢀKONDO ET AL.
purchased from Japan SLC, Inc. (Shizuoka, Japan). All
rats were housed in plastic cages and fed a solid feed
Determinations of ethanol, acetaldehyde, and
acetate concentrations in plasma
(
CE-2) for 5–8 days prior to commencement of exper-
All blood samples were collected in heparinized cap-
illary tubes. Whole blood samples were immediately
mixed with 1 mol/L perchloric acid and kept on ice.
Af er 10 min, the tubes were centrifuged at 10,000 × g
for 10 min at 4 °C, and the pH of supernatant fractions
was adjusted to 7.5–8.0 by adding 0.7 mol/L K PO .
iments under controlled temperature (22 ± 1 °C) and
humidity (55 ± 15%) conditions, and 12-h light/dark
cycle (light period 7:00–19:00). All rats were given ad
libitum access to water.
3
4
Animal experimental designs
Af er incubation for 10 min on ice, the deproteinized
plasma was isolated by centrifugation at 2,300 × g for
First, to measure the concentrations of ethanol, acetal-
dehyde, and acetate in plasma, 36 male, 7-week-old SD
rats were randomly assigned to four groups (n = 9 in
each group). Af er fasting overnight, they were orally
administered 10 mL of 20% ethanol/kg body weight
1
0 min at 4 °C. e supernatant fractions were stored at
-
80 °C for analysis of acetate. Concentrations of ethanol
and acetaldehyde in plasma were determined immedi-
ately using F-kit Ethanol and F-kit Acetaldehyde (Roche
Diagnostics K.K, Tokyo, Japan), respectively. e quan-
titative analysis of acetate was conducted by the same
method as that of pyruvate.
(
Control group), garlic extract with 500 μmol ACSO
in 10 mL of 20% ethanol/kg body weight (Garlic-H
group), garlic extract of the same amount as Garlic-H
without ACSO in 10 mL of 20% ethanol/kg body weight
Activities of ADH and ALDH
(
Garlic-N group) or 500 μmol ACSO in 10 mL of 20%
ethanol/kg body weight (ACSO group). Before and
.5, 1, 2, 4, 6 h af er administration, 70 μL of blood
To determine the activities of ADH and ALDH in cyto-
solic/microsomal and mitochondrial fractions indi-
vidually, harvested livers were homogenized at 4 °C in
0
was collected by nicking the tail vein, and used to
determine plasma ethanol, acetaldehyde, and acetate
concentrations.
Second, to investigate the suppressive effect of
each sample on ethanol absorption from the gut, 18
male, 7-week-old SD rats were randomly assigned to
1
00 mmol/L K HPO -KH PO buffer (pH 7.4) contain-
2 4 2 4
ing 0.25 mol/L sucrose and 0.1 mmol/L EDTA, and the
homogenates were centrifuged at 600 × g for 10 min at
4
°C to remove nuclei and cell debris. e supernatant
fractions were then centrifuged at 10,000 × g for 15 min,
and the supernatant fractions were further centrifuged
at 105,000 × g for 60 min to obtain the cytosolic/micro-
somal fractions as supernatants that were used for the
measurement of ADH and ALDH activities. e pellets
were homogenized at 4 °C in 100 mmol/L K HPO -
3
groups: Control group, Garlic-H group, and ACSO
group (n = 6 in each group). Stomach and small intes-
tine ligation was performed as previously described
[
15]. Af er fasting overnight, the abdominal cavity was
opened, and the upper and lower regions of stomach or
jejunum were ligated twice with 5–0 surgical silk. en,
2
4
KH PO buffer (pH 7.4) containing 0.25 mol/L sucrose
2
4
1
0 mL of 20% ethanol/kg body weight (Control group),
and 0.1 mmol/L EDTA and centrifuged at 10,000 × g for
garlic extract with 500 μmol ACSO in 10 mL of 20%
ethanol/kg body weight (Garlic-H group), or 500 μmol
ACSO in 10 mL of 20% ethanol/kg body weight (ACSO
group) was injected into the ligated loop of the stomach
and small intestine. Before and 5, 10, 30, and 60 min
af er injection, 70 μL of blood was collected from the
portal vein, and used to determine plasma ethanol
concentration.
1
5 min at 4 °C. Next, the precipitates were sonically dis-
rupted in a sonicator at 4 °C for 2 min. e supernatant
fractions were then centrifuged at 105,000 × g for 60 min
to obtain the mitochondrial fractions as supernatants
that were used for the measurement of ALDH activity.
ADH activity in the cytosolic/microsomal fractions
and ALDH activity in the cytosolic/microsomal and
mitochondrial fractions were measured fluorometri-
cally according to the modified method of Eriksson et
al. [16]. For the measurement of ADH activity, the assay
ird, to investigate the activities of ADH and ALDH
in the liver, 30 male, 7-week-old SD rats were randomly
assigned to 3 groups: Control group, Garlic-H group,
and ACSO group (n = 10 in each group). Af er fast-
ing overnight, they were administered orally by gavage
+
mixture (1.2 mL) containing NAD (1.6 mg) and ALDH
(
(
0.32 U) in potassium pyrophosphate buffer (pH 9.0)
F-kit Ethanol, Roche Diagnostics K.K, Tokyo, Japan)
1
0 mL of 20% ethanol/kg body weight (Control group),
was added to a cuvette at 37 °C and incubated for 3 min.
e reaction was started by the addition of 100 μL of
garlic extract with 500 μmol ACSO in 10 mL of 20%
ethanol/kg body weight (Garlic-H group) or 500 μmol
ACSO in 10 mL of 20% ethanol/kg body weight (ACSO
group). Af er 60 min, rats were euthanized and their
livers were harvested. e collected livers were imme-
diately homogenized for the measurement of ADH and
ALDH activities.
ethanol as a substrate of ADH and the cytosolic/micro-
somal fractions. In this reaction, 2 mol of NADH were
generated from 1 mol of ethanol because acetaldehyde
produced from ethanol was further converted to acetate
by ALDH added in the reaction mixture. For the meas-
urement of ALDH activity, the assay mixture (1.2 mL)