677
were taken by heart puncture from anesthetized rats; three or more
animals were killed and dissected immediately. Bile was collected
for 48 h from cannulated rats that had access to drinking water
containing 0.9% w/v sodium chloride in a Bollman cage. Bile
collected at each time period was weighed, and the radioactivity of
a portion of the sample was determined by LSC.
Materials and methods
Chemicals and instruments
[
14C-methyl]-p-cresol (Lot no. 1221-248, 185 MBq/mmol) was
In the blood concentration study, the rats were lightly anes-
thetized with ethyl ether, and 50-ll blood specimens were collected
from the tail vein into heparinized microtubes at designated times
after an oral administration of 14C-NPC. In a blood-level profile,
the rapid- and slow-phase slopes were calculated by the least-
squares method, and the half-lives (T1/2) were determined.
purchased from NEN (Boston, Mass., USA) and diluted with
187 mg of non-radioactive p-cresol. 14C-labeled-p-cresol was dis-
solved in 575 mg of benzene and nitrated with d-HNO3 (1:1)
according to the method reported by Schultz (1907).
Crude 14C-NPC (300 mg) was purified by column chromato-
graphy with a silica gel column (20·0.8 cm). The radiochemical
purity of each fraction eluted with dichloromethane was examined
by radiochromatography to obtain, in total, 208 mg of chemically
and radiochemically pure 14C-NPC. The Rf value of synthetic
14C-NPC was 0.64, identical with that of the authentic specimen,
with a developing solvent of benzene:dioxane:acetic acid (30:5:1) by
TLC. Chemical and radiochemical yields of 14C-NPC were 70.0%
and 75.0%, respectively.
Radioactivity measurement
Radioactivity was counted with a liquid scintillation counter
(Aloka LSC-651). Urine samples were diluted and an aliquot of the
diluted urine was counted in a dioxane scintillator. Dried feces were
powdered and portions (60–100 mg) combusted to carbon dioxide
with an auto-oxidizer (Aloka, ASC-113). The evolved radioactive
gas was trapped in a scintillator cocktail, which consisted of a
mixture of methanol, monoethanolamine and toluene (Tanaka et al.
1978).
Approximately 100 mg of organ or tissue sample were dissolved
in 1 ml of Soluene 350 (Packard) and counted in a toluene scint-
illator. Colored samples such as blood, liver, kidney and spleen
were decolored with some drops of 30% hydrogen peroxide solu-
tion before the addition of Soluene 350. Intestine samples were
totally digested in 0.5 M NaOH solution by being heated, and an
aliquot of the dissolved samples was counted in the dioxane
scintillator (Tanaka et al. 1978).
Mass spectra were measured on a JEOL OISG-2 high-resolu-
tion mass spectrometer with a direct inlet system. 13C-NMR mea-
surements were carried out at 50.1 MHz on a JEOL FX 200
Fourier transform NMR spectrometer. Chemical shifts are given as
d values (ppm) in a solution of CDCl3 or CD3OH with tetra-
methylsilane as an internal standard.
Assignments of 13C-NMR signals were based on the data from
1H-gated decoupled spectrum with nuclear Overhauser effect
(NOE) and H-selective decoupled spectrum.
1
Commercially available NPC (melting point: 36.5 ꢁC) was used
in all experiments. The 13C-NMR spectrum of NPC was as follows:
153.2, 138.7, 133.3, 130.1, 124.4, 119.7 and 20.2 ppm.
2-Acetylamino-p-cresol (AAPC) was obtained by acetylation of
2-amino-p-cresol with acetic anhydride. The 13C-NMR spectrum of
AAPC was as follows: 172.1, 147.3, 130.2, 127.3, 126.8, 124.3,
117.5, 23.4 and 20.7. Its mass weight showed: m/z 165.0754 (Cal-
culated m/z: 165.0790).
Identification of metabolites
We extracted a portion of urine with ether to isolate free metabo-
lites, which we confirmed by comparing them with authentic
specimens. Conjugates were separated by extraction of the urine
with butanol. An aliquot of urine extract was spotted on the thin-
layer plate (silica gel 254) containing a fluorescence indicator, and
the developing solvent systems used for TLC were as follows: A,
dichloromethane and B, chloroform:methanol (7:3). After being
developed, the TLC plates were scanned for radioactivity with a
radiochromatogram scanner (Aloka, TRM-1B). The exact portions
of the radioactive areas on the TLC plates were also located by
autoradiography with X-ray film (XTL-5, Kodak). For the iden-
tification of urinary conjugate metabolites, we treated the butanol
fractions of the labeled urine for 1 h with 800 units of b-glucu-
ronidase in 0.2 ml sodium acetate buffer (pH 6.0), or for 4 h with
13 units of sulfatase with 5 mM D-saccharic acid-1,4-lactone, to
inhibit b-glucuronidase activity in 0.2 ml sodium acetate buffer
(pH 6.0). After incubation, small portions (20 ll) of the hydroly-
sates were analyzed directly on TLC.
NPC sulfate was prepared by reaction of NPC with chloro-
sulfonic acid in the presence of pyridine and it showed an Rf value
of 0.71 on the thin-layer silica gel plate in a mixture of chloroform
and methanol (7:3) as a developing solvent. The 13C-NMR spec-
trum of the synthetic sulfate was as follows: 144.3, 143.7, 137.6,
135.7, 126.1, 125.4 and 20.8 ppm. Further, the existence of the
sulfate group was identified as the precipitate of barium sulfate by
addition of barium chloride.
b-Glucuronidase (type B-1), sulfatase (type V), and D-saccharic
acid-1,4-lactone were purchased from the Sigma Chemical Com-
pany (St. Louis, Mo., USA). Other chemicals and solvents used
were reagent grade.
Animal experiments
Female Sprague-Dawley rats (Japan SLC, Shizuoka, Japan)
weighing 168–215 g were used for the experiments after 1 week’s
acclimatization to the laboratory environment. They were starved
overnight before application of the labeled compound, and 14C-
NPC was given orally at a dose of 250 mg/kg as a 3.3% dimethyl
sulfoxide solution. They were housed individually in metabolic
cages. Water was freely given during experiments, but food was
given 6 h after they were dosed with 14C-NPC. Excretion studies
were carried out for 5 days on five rats per group after dosing.
In order to study metabolites in the expired gases, we placed
each rat in a glass metabolic chamber (KN-450, Natsume Com-
pany Tokyo) immediately after administration of the 14C-NPC. Air
was drawn through the chamber at approximately 300 to 400 ml/
min and then through a silica-gel trap connected in series with a
monoethanolamine trap to collect volatile compounds and respi-
ratory carbon dioxide, respectively. This expiration experiment was
carried out for 3 days on three rats.
Results and discussion
The time course of blood levels in female rats is shown in
Fig. 1. After an oral dose of 14C-NPC, blood levels of
14C-NPC reached the maximum concentration (39.4 lg-
equivalents/g) within 1 h, and decreased bi-exponen-
tially. The apparent half-lives of 14C-NPC were 3.8 h for
the rapid phase and 37 h for the slow phase. The data
show that 14C-NPC is absorbed very rapidly from the
alimentary canal in the rat after an oral dose of 250 mg/
kg given as 3.3% NPC solution in dimethyl sulfoxide.
Table 1 shows that approximately 90% of the
In the distribution study, radioactivities in organs and tissues
were determined at 1.5, 6, 24, 48, 72 and 120 h after oral admin-
istration of 14C-NPC. At 1.5, 6, 24, 72 and 168 h, blood samples radioactivity given was excreted into the urine and below