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S¸.G. Ku¨c¸u¨kgu¨zel et al. / Il Farmaco 55 (2000) 624–630
development. A dark red color was obtained. The
HPLC chromatograph consisted of an isocratic system,
a Rheodyne syringe loading sample injector valve
(model 7725) fitted with a 20 ml sample loop, a model
481 Waters wavelength UV detector and a Unicam
4880 Chromatography Data Handling System integra-
tor. The HPLC column (Spherisorb, m-Bondapak C18
5
mm, 25 cm length×4.6 mm and Novapak C18 5 mm, 15
cm length×3.9 mm) was purchased from Phase Sepa-
rations Limited, UK. The guard column packing mate-
rial (Whatman Pellicular ODS) was purchased from
Whatman International Ltd., Maidstore, Kent, UK.
The composition of the mobile phases, flow rates and
the retention times of the CBBAH and its potential
metabolites were given in Table 1. All these compounds
were detected by their absorbance at 254 nm.
2.4.1. Biological studies
Glucose-6-phosphate dehydrogenase (G6PH) was
purchased from the Boehringer Mannheim Corporation
(London) Ltd. Nicotinamide adenine dinucleotide
phosphate mono sodium salt (NADP) and glucose-6-
phosphate (G6P) disodium salt were obtained from
Sigma Ltd. The animals used in this investigation were
male rats. Hepatic washed male rat microsomes were
prepared at 0°C using the calcium chloride precipita-
tion method [12]. Incubation was carried out in a
shaking water-bath at 37°C using a standard co-factor
solution consisting of NADP+ (disodium salt, 1 mmol),
G6P (disodium salt, 5 mmol), G6PDH (0.5 unit) and
MgCl2 (50% w/w, 10 mmol) in 1 ml of phosphate buffer
(0.2 M, pH 7.4) for each flask. Co-factor solutions were
pre-incubated for 5 min before the addition of substrate
(2 mmol) in methanol (50 ml) and microsomes equiva-
lent to 0.25 g original liver. Incubation was continued
for 30 min, terminated and the incubates were extracted
with dichloromethane (2×5 ml). Extracts were evapo-
rated to dryness under a stream of nitrogen at 20°C.
Dry organic residues were reconstituted in 200 ml of
methanol for HPLC and 50 ml of methanol for TLC
analysis.
Fig. 3. TLC chromatogram obtained following extraction from male
rat microsomal incubation mixture with CBBAH as substrate. 1,
BAH; 2, CBDBAH; 3, CBBAH; 4, CB; 5, test; 6, incubation of CB
and BAH in the presence of denatured microsomes; 7, control
(denatured microsomes); 8, control (omission of co-factors); 9, con-
trol (omission of the substrate).
accordance with the findings reported by Low et al.
[13]. Since the chemical oxidation of CBBAH using
m-CPBA yielded CBDBAH as the major product, it
could be proposed that this hydrazone would also arise
from a direct oxidation of CBBAH via dehydratation
of an unstable N-hydroxy intermediate (Fig. 4) [14]. CB
was further oxidized to 4-chlorobenzoic acid (CBA).
BA was found as a metabolite which may arise either
from the hydrolysis of CBBAH or alternatively from
the hydrolysis of BAH. All these metabolites were not
observed with control incubates with denatured micro-
somes or incubates lacking co-factors. This indicates
that these metabolites formed via enzymatic reactions.
Metabolic and chemical reactions possibly involved in
the biotransformation of CBBAH are shown in Fig. 4.
N-Dealkylated and hydrolytic metabolites were de-
tected together with a hydrazone function formed. Ben-
zoic acid hydrazide produced by the N-dealkylation
reaction is possible to form hydrazone with reactive
carbonyl groups, i.e. pyruvic acid and a-ketoglutaric
acid. If this occurs, all a-keto acid resources needed for
the pyruvate oxidation by tricarboxylic acid cycle
would be depleted. This may interrupt the carbohydrate
3. Results and discussion
Following the metabolic experiments using CBBAH
as a substrate, the N-dealkylation products, i.e. BAH
and CB were observed by RP-HPLC (Fig. 2) and TLC
(Fig. 3). The formation of the corresponding hydrazone
(CBDBAH) as a major in vitro metabolic product was
also demonstrated. The control experiments were per-
formed to investigate the mechanism of formation of
this metabolite by co-incubation of BAH with CB in
the presence of denatured microsomes. The result indi-
cated that the formation of CBDBAH was most proba-
bly via condensation of CB and BAH. This was in