570th MEETING, CARDIFF 1361

Barrow, A. & Griffiths, L. A. (1972) Xenobiotica 2,575-586

Colvin, L. B. (1969) J. Pharm. Sci. 58,1433-1443
Gafney, G . W.,Schreier, K., Diferrante, N. &Altman, K. I. (1954)J. Biol. Chem. 206,695-698
Juchau, M. R. & Horita, A. (1973) Drug Metab. Rev.1,71-100
Mukhina, N. U., Gilev, A. P. & Klimenko, V. G. (1965) Chem. Abstr. 63,1602
Overberger, C. G. & Di Giulio, A. V. (1958) J. Am. Chem. SOC.80,6562-6568
Satoh, Y . & Moroi, K. (1971) Biochem. Pharmacol. 20,504-507
Smith, G . E. & Griffiths, L. A. (1976) Xenobiotica 6,217-236
Youdim, M. B. H. (1975) Biochem. Ser. One 12,177-181

Production in vitro of Paracetamol from Phenacetin and [*H]Acetanilide:

A Study with Stable Isotopes
J. DAVID BATY* and PHILIP R. ROBINSON?
Department of Medicine, University of Liverpool, Liverpool L69 3SX,U.K.
We have studied the oxidation of phenacetin and [2,3,4,5,6-2H]acetanilide when both
substrates are incubated with rat liver supernatant solutions. Phenacetin is converted
into paracetamol by side-chain oxidation (Kiese & Renner, 1968) and acetanilide is con-
verted into paracetamol by hydroxylation in the p-position of the 2H-labelled benzene
ring (Daly, 1970). By using gas chromatography-mass spectrometry we measured the
amounts of the two forms of paracetamol present after incubation, and can thus compare
the activity of the two enzyme systems. We have studied induction of these enzyme sys-
tems with phenobarbitone, and the inhibition produced by metyrapone and cysteamine.
Adult male Wistar rats (200-3OOg) were maintained on a commercial diet with free
access to water. For the induction studies they were given a 0.2% solution of sodium
phenobarbitone as drinking water for 4 days and were killed on day 5 . The livers were
immediately excised and washed in an ice-cold solution of 1.15% (w/v) KCI in 0.01 M-
phosphate buffer, pH7.4. Liver weights were recorded and the livers were homogenized
in 3vol. of buffer. The homogenates were centrifuged at 9000g at 4°C for 15min and
the supernatant was used immediately. Samples (1 ml) of the supernatant were used in
each of the incubations, which were carried out in wide-necked glass scintillation vials
at 37°C in an atmosphere of 02. A buffer of 79.4pmol of KH2P04,78.5pmol of KOH,
11pmol of MgClz and 29.4pmol of nicotinamide in 1 ml of water was used. A typical
incubation mixture consisted of NADPH (1.2pmol), glucose 6-phosphate (1.9pmol)
and equimolar amounts of the two substrates (0.063pmol) in 0.5ml of buffer. Substrate
and cofactors were added to the flasks at O'C, and 1ml of the supernatant solution was
added to initiate the reaction. The flasks were incubated at 37°C for 20min. Cysteamine
(1.5pmol) and metyrapone were used in the inhibition studies. The reaction was termi-
nated by heating the flasks for 15min at 75°C. After cooling, 0 . 2 d of 0.05~-sodium
acetate, pH5.2, and 50p1 of 8-glucuronidase (Sigma typeII) were added, and the solution
incubated for 18h at 37°C to hydrolyse any conjugates of paracetamol formed in the
reaction.
To measure the two forms of paracetamol we used an assay based on gas chromato-
graphy-mass spectrometry (Baty & Robinson, 1976). After conversion into their tri-
methylsilyl ethers, the two compounds co-elute on the g.1.c. column, but can be dis-
tinguished due to their difference in mass. A mass value of m/e 280 (M+-15) is
characteristic of paracetamol produced from phenacetin and m / e 284 (M+-15)
represents the trirnethylsilyl ether of paracetamol with four 2H atoms in the aromatic
ring. The ratio m/e 280/284 is thus a measure of aliphatic oxidation compared to aromatic
oxidation.
* Present address: Department of Biochemical Medicine, Ninewells Hospital, Dundee
DD2 IUD, Scotland, U.K.
t Present address: Metabolic Studies Section, Sterling Winthrop Laboratories, Fawdon,
Newcastle upon Tyne, U.K.

Vol. 5
1362 BIOCHEMICAL SOCIETY TRANSACTIONS

Table 1. Induction and inhibition of paracetamol production in vitro from phenacetin and
[2,3,4,5,6-’H]acetanilide
Three incubations were made with the supernatant from each rat. Four rats were used
in each experiment except the phenobarbitone experiment where three rats were used.
The amounts of paracetamol were measured in six incubations from two rats. The values
shown are the mean Va1UeSfS.E.M. between each rat.
Paracetamol/ lo-’ x Paracet- lo-’ x [2,3,5,6-‘H]-
[2,3,5,6-’H]- amol production* Paracetamol pro-
Incubation conditions paracetamol ratio bmol) duction? (pmol)
Control 6.58 f0.12 1.23 f 0.07 0.18
Phenobarbitone induction 3.57 f 0.16 2.85k0.21 0.80
Control plus metyrapone 1.71 f 0.39 0.14 f 0.01 0.08
Control plus cysteamine 8.5 rt0.2 1.21 k0.02 0.14
* Measured value.
t VaIue calculated from paracetamol value and ratio measurement.

The results we have obtained in our initial experiments are shown in Table 1. The
mle 2801284 ratio is reproducible for multiple incubations of a particular supernatant
and there is very little variation in the data obtained from four control rats. The data
shows that oxidation of phenacetin proceeds more rapidly than the aromatic oxidation of
acetanilide. We have shown in further experiments with labelled and non-labelled acet-
anilide that a ‘H-isotope effect is not present in aromatic hydroxylation, but is present
in the oxidative de-alkylation of phenacetin.
The actual amounts of paracetamol measured and the ratios obtained from the experi-
ments in phenobarbitone-treated rats suggest that aromatic hydroxylation is induced
to a greater extent than aliphatic oxidation.
Both aromatic hydroxylation and oxidative de-ethylation were inhibited by metyra-
pone and cysteamine. Metyrapone produced a marked decrease in the mle 2801284 ratio,
whereas cysteamine provoked an increase. The actual amounts of paracetamol produced
indicated that paracetamol decreased to approx. 11 %of control values when metyrapone
was present in the incubation mixture. The amount of [2H]paracetamol produced by
aromatic hydroxylation was decreased to approx. 40 % of control values, indicating a
preferential inhibition of de-ethylation under these conditions. Cysteamine produced
a significant increase in the mle 280/284 ratio. Since the amount of paracetamol pro-
duced from phenacetin was not changed, the data indicate a moderate inhibition of
aromatic oxidation. This could be due to binding of cysteamine to one of the reactive
intermediates involved in the formation of paracetamol from acetanilide, e.g. an arene
oxide.
Baty, J. D. & Robinson, P. R. (1976) Biomed. Mass. Spectrom. 3,60-63
Daly, J. (1970) Biochem. Pharmacol. 23,2979-2993
Kiese, M. & Renner, G. (1968) Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol. 252,
480-500

1977