Initial intraorgan formation of mercapturic acid

Article abstract of DOI:10.1248/bpb.24.1324

The disposition of S-benzyl-glutathione (BSG) in male Wistar rats was evaluated by the HPLC method to examine whether the kidney and liver contributed independently to the biosynthesis of S-benzyl-N-acetylcysteine (BNAc), a mercapturic acid (Chart 1). After intravenous injection, BSG was rapidly transported in both the kidney and the liver at a ratio of about 7:3. Simultaneously, a large amount of BNAc was found in both the kidney and the liver. In the kidney, S-benzyl-cysteine (BCys) reached a maximum concentration (C_max) at 2 min after BSG injection, whereas BNAc reached C_max within 3 to 5 min. The generation of BNAc was also observed in the liver. While renal BNAc reached C_max within 3 to 5 min, hepatic BNAc reached C_max around 5 min after BSG injection. Moreover, the elimination half-life of the BNAc after intravenous injection of the BSG was equivalent to that observed after intravenous injection of the BNAc itself. These results demonstrate that the kidney contributes to the initial intraorgan generation of BNAc and that this mercapturic acid is also synthesized in the liver and preferentially excreted into urine.

Full text of DOI:10.1248/bpb.24.1324

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324
Notes
Biol. Pharm. Bull. 24(11) 1324—1328 (2001)
Vol. 24, No. 11
Initial Intraorgan Formation of Mercapturic Acid
Kazumi SANO,* Yoji IKEGAMI, and Takashi UESUGI
Department of Drug Metabolism and Disposition, Meiji Pharmaceutical University, Kiyose, Tokyo 204–8588, Japan.
Received May 22, 2001; accepted August 25, 2001
The disposition of S-benzyl-glutathione (BSG) in male Wistar rats was evaluated by the HPLC method to
examine whether the kidney and liver contributed independently to the biosynthesis of S-benzyl-N-acetylcysteine
(
BNAc), a mercapturic acid (Chart 1). After intravenous injection, BSG was rapidly transported in both the kid-
ney and the liver at a ratio of about 7 : 3. Simultaneously, a large amount of BNAc was found in both the kidney
and the liver. In the kidney, S-benzyl-cysteine (BCys) reached a maximum concentration (Cmax) at 2 min after
BSG injection, whereas BNAc reached Cmax within 3 to 5 min. The generation of BNAc was also observed in the
liver. While renal BNAc reached Cmax within 3 to 5 min, hepatic BNAc reached Cmax around 5 min after BSG in-
jection. Moreover, the elimination half-life of the BNAc after intravenous injection of the BSG was equivalent to
that observed after intravenous injection of the BNAc itself. These results demonstrate that the kidney con-
tributes to the initial intraorgan generation of BNAc and that this mercapturic acid is also synthesized in the
liver and preferentially excreted into urine.
Key words S-benzyl glutathione; intraorgan; mercapturic acid; retrograde; renal excretion; hepatobiliary
Mercapturic acid biosynthesis is one of the important rivatizing reagent by the following modification of the
4
)
detoxification mechanisms for many xenobiotics. The mer- method of Sokolovsky et al. BSG, S-benzyl cysteine
capturic acid pathway is thought to involve interorgan coop- (BCys), N-acetyl-S-benzyl cysteine (BNAc), S-benzyl cys-
1
)
eration of the liver and kidney. Inoue et al. reported that in- teinylglycine (BCysGly) and S-benzyl glutamylcysteine
1
4
travenous injection of S-carbamido ( C) methyl glutathione (BGluCys) were synthesized as follows: Benzyl chloride
in mice resulted in the rapid accumulation of radioactivity in (1 mmol in 10 ml of methanol) was added slowly with stir-
the kidney. The subsequent decrease in renal radioactivity ring to a solution of 1 mmol of each mercaptide dissolved in
corresponded to an increase in hepatic radioactivity without 5 ml of 2.5% NH OH. The reaction was allowed to proceed
4
excretion of radioactivity into urine. Based on these results, for 15—20 min. The reaction mixture was concentrated in
they proposed that mercapturic acid is formed by a process vacuo and acidified with dil. HCl. A fine white precipitate
of interorgan cooperation involving these two organs. Lend- was collected by vacuum filtration and washed with ice cold
ing support to such a notion of interorgan cooperation in the water and methanol, then recrystallized from aqueous
formation of mercapturic acid is the established fact that glu- methanol. N-acetyl-S-benzyl glutathione (NAcBSG) was
tathione (GSH) conjugation occurs in the liver and GSH hy-
drolysis to cysteine takes place in the kidney before N-acety-
lation of cysteine occurs after transport back to the liver.
2
)
Hinchman et al., however, published findings that refuted
such a theory of mercapturic acid formation. They demon-
strated intrahepatic biosynthesis of mercapturic acids in iso-
lated perfused livers from rats and guinea pigs using the
model compound 1-chloro-2,4-dinitrobenzene (CDNB), which
has a high molecular weight and lipophilicity. We have also
confirmed the intra-hepatobiliary formation of mercapturic
3
)
acid using sulphobromophthalein (BSP).
In the present study, S-benzyl-glutathione (BSG) was used
as the model compound and its metabolites were determined
by an ion-paired HPLC method. Using whole bodies of rats
in vivo, we demonstrated that the liver and kidney have inde-
pendent intraorganic pathways of mercapturic acid synthesis.
MATERIALS AND METHODS
Chemicals Cysteinylglycine and glutamylcysteine were
purchased from Bachem Bioscience Inc. (Bubendorf,
Switzerland). Acetyl-CoA and acivicin (AT-125) were ob-
tained from Sigma Chemical Co. (St. Louis, MO, U.S.A.).
All other reagents obtained were of the highest quality avail-
able.
Chemical Synthesis of S-Benzyl Mercaptides S-benzyl
mercaptides were prepared from benzyl chloride as the de-
Chart 1
∗
To whom correspondence should be addressed. e-mail: ksano@my-pharm.ac.jp
© 2001 Pharmaceutical Society of Japan

November 2001
1325
5
)
synthesized according to the procedure of Zbarsky et al.
(t ) was obtained by dividing natural logarithm of 2 by K ,
1
/2
el
Each S-benzyl mercaptide was checked for purity by HPLC the apparent elimination rate constant, as obtained from the
and confirmed to be more than 99% pure.
amount remaining to be excreted in urine.
Animal Experiments Male Wistar rats (230—250 g,
Preparation of Microsomal Fractions After overnight
Tokyo Laboratory Animals Co., Ltd., Tokyo, Japan) were starvation, the animals were anesthetized with ether and then
anesthetized with sodium pentobarbital (50 mg/kg, i.p.). killed by exsanguination. Tissues were homogenized in 4 vol.
Through an abdominal incision, the bile duct and ureters of ice-cold 1.15% KCl in Tris–HCl buffer (pH 7.4). The ho-
were cannulated with polyethylene tubing (PE-10). Body mogenate was centrifuged at 20000ϫg for 20 min and then
temperature was maintained at 38Ϯ0.5 °C using a heating the supernatant was recentrifuged at 105000ϫg for 60 min.
lamp. For the continuous collection of the urine, mannitol The microsomal pellet resuspended in the homogenizing
(
20 mg/ml in saline) was infused into the femoral vein at a medium containing 20 (v/v) % glycerol was used as an en-
rate of 2.34 ml/h. BSG and BNAc were dissolved in saline zyme source of N-acetyltransferase. Protein concentrations
8
)
and each solution containing 10 mmol of drug (2 ml/kg) was were determined according to the method of Lowry et al.
injected into the opposite femoral vein over a 15-s period.
Animals were pretreated by retrograde intrabiliary infu-
using bovine serum albumin as a standard protein.
N-Acetyltransferase Activity Microsomal N-acetyl-
sion of acivicin as follows: Acivicin (20 mmol/kg) dissolved transferase activity towards BCys was assayed by the modi-
9
)
in saline (50 ml) was introduced into the bile duct cannula fied procedure of Green et al. in which cold acetyl-CoA
1
4
over a 30-s period and followed by a 50 ml wash of normal was used instead of [ C] acetyl-CoA. The addition of the
saline. After 2 min, bile was allowed to flow freely. Ten min- same volume of dimethyl sulfoxide (DMSO) as the incuba-
utes after the acivicin infusion, BSG solution was injected tion mixture terminated the enzyme reaction, and the mixture
into rats.
was centrifuged at 15000ϫg for 5 min. The BNAc formed
Preparations of Liver, Kidney and Plasma Samples was directly determined by HPLC analysis of the super-
At fixed time periods (1, 2, 3, 5, 10, 15 min) after administra- natant. BNAc could be quantitatively recovered from the
tion of BSG, the liver and kidneys were immediately excised assay mixture at concentrations ranging from 20—2000
and homogenized with 4 vol. of 0.1 M NaOH. An equal vol- nmol/ml by DMSO extraction.
ume of 4.5% ZnSO was added to the homogenates followed
4
by centrifugation at 15000ϫg for 10 min. S-Benzyl mercap- RESULTS AND DISCUSSION
tides in the supernatants were determined by HPLC. Blood
samples (0.1 ml) were collected from the femoral artery and
centrifuged to obtain the plasma fractions.
Figure 1 shows the elution pattern of authentic compounds
of BSG metabolites (A), chromatograms obtained from drug-
HPLC Analysis S-benzyl mercaptides were detected by free kidney homogenate (B) and a kidney homogenate sam-
6
)
the modified HPLC method of Uesugi et al. The HPLC sys- ple at 3 min after BSG injection (C). The metabolites of BSG
tem consisted of a Jasco model 2000 liquid chromatograph were separated and quantitated well, without disturbance to
system utilizing a Jasco RP spectrometric detector (Nippon the physiological components.
Bunko Inc., Tokyo, Japan) and a Shimadzu WX integrator
Shimadzu Bunko, Kyoto, Japan). Isocratic elutions were the dose over a 90-min period was excreted into urine,
performed using a Capcell Pak C18 SG column (5 mm, whereas only 6% was excreted in bile (Fig. 2). BNAc was the
ϫ150 mm; Shiseido Co., Ltd., Tokyo, Japan) with a mobile
phase of 0.1 M (pH 7.0) triethylamine–H PO buffer contain-
When BSG (10 mmol) was injected intravenously, 76% of
(
6
3
4
ing 1 mM tetra-n-butylammonium bromide : methanol : ace-
tonitrile (75 : 25 : 8, v/v/v) at a flow rate of 1.0 ml/min. Com-
pounds were detected at 240 nm and quantitated by the exter-
nal standard method using the area under the peaks. The bile
and urine samples were diluted with the mobile phase and in-
jected into the column directly. The limit of detection was
defined as the amounts of the compounds resulting in a
signal-to-noise ratio of 5. The detection limit of BSG was
ca. 20 pmol. The calibration lines for BSG resulting from
a least-squares analysis for best fit was yϭ53.45xϪ750
2
(
r ϭ0.998). Five aliquots of each sample was analyzed on
the same day, and the resulting coefficient variation (C.V.) in-
dicated within-day reproducibility. Aliquots of the same sam-
ples were tested once a day for 5 d, and the resulting C.V. in-
dicated between-day reproducibility. The C.V. of peak areas
for each compound was less than 2%.
Pharmacokinetic Analysis The plasma, hepatic and
Fig. 1. Chromatograms of HPLC Pattern of A; Standard Mixture, B;
renal concentration–time data (0—15 min) from each subject Drug-Free Kidney Homogenate, and C; Kidney Homogenate at 3 min after
was analyzed by a model-independent method using the Intravenous Injection of BSG (10 mmol)
7
)
HPLC conditions are given in Materials and Methods. Peaks: 1ϭS-benzyl cysteine
BCys); 2ϭS-benzyl glutamylcysteine (BGluCys); 3ϭS-benzyl-GSH (BSG); 4ϭS-ben-
zyl-(N-acetyl)-glutathione (NAcBSG); 5ϭS-benzyl cysteinylglycine (BCysGly); 6ϭS-
MULTI computer program. The area under the concentra-
(
tion–time curve (AUC) was calculated from the values ob-
tained (0—15 min) using the trapezoidal rule. The half-life benzyl-(N-acetyl)-cysteine (BNAc).

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Vol. 24, No. 11
Fig. 2. Time Course of S-Benzyl-mercaptide Excretion after Intravenous Injection of BSG (10 mmol) in Male Rats
Data are expressed as the meanϮS.E. of 4 animals.
Fig. 3. Time Course of S-Benzyl-mercaptide Concentrations in Plasma, Kidney and Liver after i.v. Intravenous Injection of BSG (10 mmol)
Data are expressed as the meanϮS.E. of 4 animals.
sole metabolite in urinary excretion. In biliary excretion,
The elimination rate of mercapturic acid was estimated
however, small amounts of unchanged BSG, BCys and from urinary excretion after the BSG injection. The t1/2 was
BNAc were detected simultaneously. Subsequent measure- 14.7 min, which was essentially equivalent to that observ-
ment of the concentration transitions of plasma, and the kid- ed after intravenous injection of mercapturic acid itself
ney and liver are shown in Fig. 3. Following the rapid disap- (14.4 min) (Fig. 4). This result strongly suggests that BSG
pearance of BSG from the blood, an efflux of BNAc from the was very rapidly converted to BNAc. Moreover, it suggests
liver into blood was observed. The elimination half-life (t ) that the BNAc generation rate in the liver and the efflux rate
1
/2
of BNAc was 38 min. The BSG was rapidly distributed into from the liver into plasma occurs more rapidly than the uri-
both the kidney and the liver at a ratio of about 7 : 3. This nary excretion rate. Given these results, we could not deny
distribution ratio was estimated from the amount of total that BCys was indeed shuttled from the kidney to the liver,
metabolites in the kidney and liver at 1 min postinjection.
but it was also clear that BNAc was generated in each organ
Although a small amount of BSG was detected in the kid- independently in the early term (1—5 min). However, we ob-
ney in the initial stage, it subsequently disappeared, and served an efflux of BNAc, and not BCys, into blood follow-
NAcBSG was also detected. BCys reached a maximum con- ing the rapid disappearance of BSG. The elimination half-life
centration (Cmax) at 2 min after BSG injection, whereas of BNAc was 38 min. The most efflux of BNAc to plasma
BNAc reached Cmax within 3 to 5 min. The peak value in the appears to be derived from the liver, because the elimination
kidney was about 1.7 times of that detected in the liver. In rate of BNAc estimated from urinary excretion was rapid
addition, a high concentration of BNAc was observed in the (t ,15 min) and BNAc was not excreted at all into bile.
1
/2
liver. The AUC (0—15 min) of BNAc in the liver
BSG transported in the liver may be rapidly secreted into
(
(
4.73 mmol/tissue/min) was two-thirds of that in the kidney the bile canaliculi and then broken down to a cysteine conju-
7.23 mmol/tissue/min). While renal BNAc reached C gate by biliary g-glutamyltranspeptidase (g-GT) and dipepti-
max
1
0,11)
within 3 to 5 min, hepatic BNAc reached Cmax around 5 min dases.
after BSG injection.
Rats pretreated with acivicin (an inhibitor of g-
1
2)
GT) demonstrated increased biliary excretion of BSG and

November 2001
1327
1
4
a concomitant decrease in the biliary excretion of mercap- bolic fate of S-carbamido( C)methyl glutathione (MW: 364)
1
)
turate in relation to that of the control animals (Fig. 5). This in mice. The authors found a rapid accumulation (within 1
is clear evidence that BSG distributed in liver is degraded to to 2 min) of radioactivity, accounted for by the presence of
BNAc by biliary g-GT and dipeptidases.
cysteine and cysteinylglycine conjugates, predominantly in
Next, we compared the enzymatic activity related to mer- the kidney, that subsequently decreased gradually with a con-
capturic acid generation in the liver and kidney to confirm comitant increase in hepatic radioactivity. After intravenous
1
4
that this generation occurs in these two organs independently. administration of S-carbamido( C)methyl cysteine (MW:
The kidney is known to have high g-GT activity
1
3,14)
and the 178), a rapid accumulation of radioactivity in the liver was
liver to have high N-acetyltransferase activity. Table 1 shows also observed. It is important to note that the shuttling of the
the specific activities of microsomal N-acetyltransferase of cysteine conjugates back to the liver is analogous to that of
the liver and kidney toward BCys. The renal activity of N- amino acids and dipeptides released after the renal degrada-
1
)
acetyltransferase was sufficient to synthesize BNAc in the tion of GSH, where the amino acids and dipeptides, such as
kidney.
glutamic acid and cysteinylglycine formed from GSH by
Mercapturic acid biosynthesis is believed to occur through renal g-GT and dipeptidases, can be rapidly transported back
1
5,16)
an interorgan process, with the liver as the major site for to the liver for synthesis of new GSH.
Since the kidney
GSH conjugation and the kidney as the primary site for con- has enough N-acetyltransferase activity (243 nmol/min/kid-
version of GSH conjugates into cysteine conjugates. The cys- ney towards BCys) to N-acetylation of cysteine conjugates
teine conjugates formed in the kidney appear to be trans- (BCys AUC after BSG administration; 160 nmol/min/kidney)
ported back to the liver for N-acetylation, and mercapturic and a specific activity nearly the same as (Table 1) or twice
1
)
9,17)
acids are ultimately excreted mainly in urine. This interor- that of the liver,
the shuttling of S-carbamidomethyl cys-
gan cooperation model originated from a study on the meta- teine back to the liver for acetylation is puzzling. Given these
findings, such a small cysteine conjugate as S-carbamido-
methyl cysteine would probably be incorporated into the in-
terorgan metabolism of GSH.
Other research groups have also suggested the intraorganic
1
8)
generation of mercapturic acid. Moldèus et al. used aceta-
minophen as a substrate to demonstrate that conversion of
the GSH conjugate to mercapturic acid was primarily cat-
alyzed by isolated rat kidney cells, whereas isolated liver
cells were quite efficient at catalyzing the formation of the
GSH conjugate. Moreover, intrahepatic conversion of dini-
Table 1. N-Acetyltransferase towards S-Benzyl Cysteine (BCys) in the
Microsomes of Male Wistar Rats
N-Acetyltransferase activity (nmol/min/mg protein)
a)
Liver
Kidney
16.5Ϯ2.0 (14.5Ϯ0.9 )
a)
16.2Ϯ0.6 (33.6Ϯ1.3 )
N-Acetyl-S-benzyl cysteine (BNAc) formed were determined by the HPLC method
as described in the text. Data are expressed as the meanϮS.E. of 4 experiments. a) Val-
ues determined by the radio-assay method are cited from the data of Green et al.
Fig. 4. Urinary Excretion of S-Benzyl Mercapturic Acid after i.v. Intra-
venous Injection of Drug (BSG: ᭺, BNAc: ᭹) (10 mmol, respectively)
9
)
Fig. 5. Time Course of Biliary Metabolites after i.v. Intravenous Injection of BSG (10 mmol)
(
A) Control animals, (B) animals treated by retrograde infusion of acivicin (20 mmol/kg) into bile duct. Data are expressed as the meanϮS.E. of 4 animals.

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Vol. 24, No. 11
trophenyl GSH (DNPSG) to mercapturic acid has been con- mentary pathways; urinary excretion is greater for conjugates
2
)
firmed in isolated perfused rat and guinea pig livers. Hinch- with lower molecular weights, and tends to decrease as mole-
man et al. indicated that cysteinylglycine, cysteine and N- cular weight increases. This finding suggests that a balance
acetylcysteine (NAC) conjugates were detected in bile and a of the orientation of distribution and excretion of each glu-
noticeably larger amount of mercapturic acids were found in tathione conjugate would decide the proportion of intraorgan
guinea pigs, which have higher g-GT activities than rats. and interorgan mercapturic acid synthesis. How to determine
Recently they reported that the liver plays a significant role the orientation of distribution of GSH conjugates is under
in the clearance of a representative mercapturic acid, DNP- current investigation.
1
9)
NAC, from the bloodstream. The study indicated that
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1
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