NMR spectroscopic studies on the metabolism and futile deacetylation of phenacetin in the rat

Article abstract of DOI:10.1080/004982597239930

1. ¹H-NMR spectroscopy of urine was used to determine the % deacetylation and re-acetylation of ²H-labelled (in the acetyl) phenacetin metabolites in the rat. 2. Male Sprague-Dawley rats were each dosed with either phenacetin or phenacetin-C²H₃, at 50 mg kg^-1. The total urinary recoveries for phenacetin and phenacetin-C²H₃, were 47·6±16·7 and 50·1±16·2% respectively (not significantly different, p > 0·05). Paracetamol sulphate and glucuronide are the major urinary metabolites of both protio and deuteriophenacetin. 2. The futile deacetylation given by the urinary recovery of protio-acetyl metabolites of phenacetin-C²H₃, was 29·6±0·9% for paracetamol sulphate and 36·6±3·1% for paracetamol glucuronide. These observations demonstrate a high level of futile deacetylation in the paracetamol conjugates formed by metabolism of phenacetin-C²H₃, and this may indicate a high metabolic flux through the nephrotoxic intermediate 4-aminophenol. 4. The level of futile deacetylation for phenacetin was significantly higher than that found previously in studies of labelled paracetamol in rat or man, and may be important in understanding the higher nephrotoxicity of phenacetin as compared with paracetamol.

Full text of DOI:10.1080/004982597239930

xenobiotica , 1997, vol. 27, no. 11, 1175±1186
NMR spectroscopic studies on the metabolism and
futile deacetylation of phenacetin in the rat
A. W . NICHOLLS‹, J. C. LINDON‹, S. CADDICKŒ,
R. D. FARRANT, I. D. W ILSONŽ and J. K. NICHOLSON‹*
‹
Department of Chemistry, Birkbeck College, 29 Gordon Square, London
WC1H 0PP, UK
ΠSchool of Molecular and Pharm aceutical Science, University of Sussex, Falmer
BN1, UK

Physical Sciences Research Unit, GlaxoWellcomeMedicines Research Centre,
Stevenage SG1 2NY, UK
Department of Safety of Medicines, Zeneca Pharmaceutical s, Alderley Park,
Ž
Maccles®eld SK10 4TG, UK
Received 5 May 1997
1
. "H-NMR spectroscopy of urine was used to determine the % deacetylation and
re-acetylation of #H-labelled (in the acetyl) phenacetin metabolites in the rat.
2
. Male Sprague-Dawley rats were each dosed with either phenacetin or phenacetin-
C#H at 50 mg kgÕ ". The total urinary recoveriesfor phenacetinand phenacetin-C#H were
$
$
7±6³16±7 and 50±1³16±2 % respectively (not signi®cantly diåerent, p 0±05). Parace-
"
tamol sulphate and glucuronide are the major urinary metabolites of both protio and
4
deuteriophenacetin.
3
. The futile deacetylationgiven by the urinary recovery of protio-acetyl metabolitesof
phenacetin-C#H was 29±6³0±9 % for paracetamol sulphate and 36±6³3±1% for parace-
tamol glucuronide.These observations demonstrate a high level of futile deacetylation in
$
the paracetamol conjugates formed by metabolism of phenacetin-C#H and this may
indicate a high metabolic ¯ux through the nephrotoxic intermediate 4-aminophenol.
$
4
. The level of futile deacetylation for phenacetin was signi®cantly higher than that
found previously in studies of labelled paracetamolin rat or man, and may be important in
understanding the higher nephrotoxicity of phenacetin as compared with paracetamol.
Introduction
The metabolism and toxicity of phenacetin (N-(4-ethoxyphenyl)acetamide)
have been the subject of much research and controversy for the last 40 years.
Although in widespread use as an analgesic from the end of the nineteenth century,
it was not until the 1950s that phenacetin consumption (as a component of certain
proprietary combination analgesics) was ®rst implicated as a cause of chronic
interstitial nephritis (Spu
classi®ed phenacetin in combination analgesics as a cause of analgesic nephropathy
Sandler et al. 1989, Buckalew Jr 1996), a condition involving renal papillary
$
hler and Zollinger1953, Gloor 1993). Subsequentstudies
(
necrosis with interstitial nephritis and a reduction in renal size (Duggin 1980).
Although phenacetin has not been proven to cause nephropathy in man, the
clinicallyperceived association with toxicity led to its eventual withdrawalfrom the
market (Buckalew Jr 1996). Other toxicologically important phenomena related to
phenacetin ingestion are methaemoglobinaemia (Smith 1958) and an increased risk
of bladder and}or colon cancer (Hinson 1983).
*
Author for correspondence.
049±8254}97 $12±00 ’ 1997 Taylor & Francis Ltd
0

1
176
A. W. Nicholls et al.
Phenacetin is extensively metabolised in the liver by ®rst-pass metabolism such
that small quantities appear in the circulation only following very large doses
Duggin 1980, 1993). The metabolites of phenacetin are as shown in ®gure 1. The
(
major metaboliteof the drug has been identi®ed as paracetamol (acetaminophen,4-
acetamidopheno l) formed from cytochrome P450 1A2-catalysed de-ethylation
(
Guengerich et al. 1993). The paracetamol so formed from phenacetin is further
metabolisedvia phase II conjugatingenzymesto the O-sulphate,the O-glucuronide
and to N-acetyl-l-cysteinyl metabolites derived from glutathione conjugations
(
Thomas 1993). Since ®rst-pass metabolism is so extensive it has been suggested
that phenacetin is actually a pro-drug with the true pharmacologicaly active agent
being paracetamol (Brodie and Axelrod 1949). A second metabolite of phenacetin
has been shown to be the deacetylation product 4-ethoxyaniline ( p-phenetidine;
®
gure 1) which can also undergo further metabolism (Brodie and Axelrod 1949,
Duggin 1993). Other minor metabolites that have been observed are 2-hydroxy-
phenacetin (Hinson 1983) and N-hydroxyphenace ti n (Nelson 1982), but parace-
tamol and its conjugates constitute the major proportion of phenacetin metabolites.
We have recently investigated the futile deacetylation of paracetamol in which
the acetyl moiety of the drug is lost and then subsequently replaced by an acetyl
group from endogenous sources (Nicholls et al. 1995). To investigate this `silent’
metabolic pathway isotopically labelled paracetamol was administered to rats and a
human volunteer (Nicholls et al. 1995, 1997). The labelling used consisted of either
full deuteriation of the acetyl group or replacementof the methyl "# C with "$ C. "H-
NMR spectroscopy was then used to analyse the urine to assess the level of protio-
acetyl content within the molecule. This work showed an extensive degree of futile
deacetylation whereby the isotopically labelled ("$ C and #H) acetyl moiety of the
drug was removed and subsequently replaced by a protio group from endogenous
sources. Quantitative NMR measurementsindicated a level of approximately10%
futile deacetylation for the sulphate and glucuronide conjugates in the rat and
approximately1±2% futile deacetylation in a single subject study in man (Nicholls
et al. 1997). These results suggested that there was a signi®cant transient ¯ux
through the metabolic intermediate, and potent nephrotoxin, 4-aminophenol.The
nephrotoxicity of 4-aminophenol has long been established, with the compound
causing severe and acute necrosis of the kidney proximaltubules(Green et al. 1969).
However, the exact mechanism of 4-aminophenol nephrotoxicity still remains
unclear (Goldstein and Schnellmann 1996).
The apparently high level of futile deacetylation in paracetamol,prompted us to
investigatethe level of futile deacetylationin the closely-related and more potentially
nephrotoxicdrug, phenacetin.If the nephrotoxicityof this drug were related to the
production of 4-aminophenol, then the level of futile deacetylation in phenacetin
might be expected to be higher than for paracetamol.Any large diåerencesin the %
futile deacetylation between phenacetin and paracetamol would suggest that
metabolitesother than those of paracetamolwere also contributingto the formation
of 4-aminophenol and hence phenacetin toxicity. Hence in the present studies
phenacetin-C#H$ (fully deuteriated in the acetyl moiety) was administered to rat
(
i.p.) and the metabolism of the drug followed by "H-NM R spectroscopy of urine
to quantify the level of futile deacetylation.

In vivo futile deacetylation of phenacetin in the rat
1177
Figure 1. Structures of phenacetinand its principalmetabolites.Bi-directionalarrows denote the futile
deacetylation reactions.
M aterials and m ethods
All compounds were obtained from Aldrich (Poole, UK) except for paracetamol-C#H (99­ atom
$
D, purity 99%), which was synthesizedas previously described(Nichollset al. 1995). The synthesis
"
%
of phenacetin was achieved using the literature method (Vogel 1989).

1
178
A. W. Nicholls et al.
Synthesis of N-(4-ethoxyphenyl )acetamide (phenacetin )
-Acetamidophenol (paracetamol) (1±071 g, 6±95 mol) was dissolved in 10 ml ethanol. To this was
4
added 21% w}v sodium ethoxide in ethanol (2±19 ml, 0±460 g, 6±74 mmol).A re¯ux condenserwas added
and iodoethane (0±77 ml, 1±5 g, 9±6 mmol) slowly added dropwise down the condenser to the stirring
mixture. The mixture was re¯uxed for 60 min. Water (20 ml) was added and the mixture cooledin an ice-
bath. The solid was ®ltered oå to give N-(4-ethoxyphenyl)acetamide (phenacetin) (m.p.¯ 131±2 °C,
"
99 %), 0 233 g, 1 25 mmol.
± ±
purity
d"H (DMSO-d , JEOL GSX 270 spectrometer operating at 270±1 MHz "H-NMR frequency); 9±76,
’
s, NH ; 7±45, m, 2H (H , H ); 6±83, m, 2H (H , H ); 3±95, q, CH ; 1±99,s, COCH ; 1±29, t, CH .
#
’
$
&
#
$
$
d"$ C (DMSO-d , JEOL GSX 270 spectrometer operating at 67±9 MHz "$ C frequency); 167±3 (CO),
’
1
54±2 (C4), 132±4 (C1), 120±5 (C2,C6), 114±3 (C3,C5), 63±0 (CH ), 23±7 (COCH ), 14±6 (CH CH ).
#
$
#
$
m}z 179 (37 %), 137 (35 %), 108 (100%), 91 (2%), 80 (14 %), 65 (4%), 53 (5 %).
Synthesis of N-(4-ethoxyphenyl )acetamide -C#H (phenacetin C#H )
4
$
$
-Acetamidopheno l- C#H (paracetamol-C#H ) (1±074 g, 6±98 mol)was dissolved in 10 ml ethanol.To
$
$
this was added 21 % w}v sodium ethoxide in ethanol(2±2 ml, 0±462 g, 6±79 mmol).A re¯ux condenserwas
added and iodoethane (0±80 ml, 1±56 g, 10±0 mmol) slowly added drop-wise down the condenser to the
stirring mixture. The mixture was re¯uxed for 60 min. Water (20 ml) was added and the mixture cooled
in an ice-bath. The solid was ®ltered oå to give N-(4-ethoxyphenyl)acetamide-C#H (phenacetin-C#H ),
$
$
9­atom %D (m.p.¯ 130±2 °C, purity
d"H (DMSO-d , JEOL GSX 270 spectrometer operating at 270±1 MHz "H-NMR frequency); 9±76,
"
99 %), 0 519 g, 2 85 mmol.
± ±
9
’
s, NH ; 7±45, m, 2H (H , H ); 6±84, m, 2H (H , H ); 3±94, q, CH ; 1±29, t, CH .
#
’
$
&
#
$
d"$ C (DMSO-d , JEOL GSX 270 spectrometer operating at 67±9 MHz "$ C frequency); 167±7 (CO),
’
1
54±3 (C4), 132±4 (C1), 120±5 (C2, C6), 114±5 (C3, C5), 63±0 (CH ), 14±7 (CH ).
#
$
m}z 182 (60 %), 138 (45 %), 110 (100%), 91 (2%), 81 (21 %), 63 (3%), 53 (5 %), 46 (17%).
Administration of phenacetin and phenacetin- C#H$ to rat
Eight male Sprague-Dawley (SD) rats (weight approximately 200 g) were housed individually in
metabolism cages, allowed free access to food (rat maintenancediet TRM9607, Harlan Teklab, Bicester,
UK) and tap water and were subjected to regular light cycles. Three of the animals received a single
intraperitoneal (i.p.) dose of phenacetin at 50 mg kgÕ ", whilst three others received an i.p. dose of
phenacetin-C#H at 50 mg kgÕ ". A DMSO-saline (75 :25 v}v) dosing medium was used for both
$
compounds.Two of the animalsacted as controls each receiving a single i.p. dose of the dosing medium.
Urine was collected over ice during the following periods: ®24±0, 0±8, 8±24, 24±48 h. After collection,
urinary volumeswere recorded and the urine was centrifuged at 1500 g for 20 min at 4 °C to remove solid
debris. All samples were stored at ®20 °C until required for NMR analysis.
NMR- monitored solid phase extraction (SPE ) of metabolites from urine samples
The urine containing the phenacetin metabolites (0±8 h post-dose, 50 mg kgÕ " i.p.) (1 ml) was
acidi®ed to pH 2 with 1 m HCl and extracted on to a C18 bonded cartridge (Bond-Elut, 100 mg, supplied
by Jones Chromatography, Hengoed, UK). The cartridges had previously been activated by washing
with methanol(2 ml) and acidi®edwater (1 ml, pH 2). The fractions were eluted with acidi®edwater and
methanol. All samples were reduced to dryness and reconstituted in #H O prior to NMR analysis.
#
"
H-NMR spectroscopic analysis of urine and SPE fractions
An aliquot (0±5 ml) of each of the untreated urine samples was placed in a 5-mm NMR tube, 0±05 ml
1
mm sodium 3-trimethylsilyl-[2,2,3,3-#H ]-l-propionate (TSP) in #H O was added to each to act as a
chemicalshift and quanti®cationreference (d 0±0), and "H-NMR spectra were measured at 600±14 MHz
on a BrukerAMX-600 spectrometerusingthe ®rst incrementof a NOESY2D experiment,NOESYPR1D
%
#
(
Bruker GmbH,Rheinstetten,Germany),pulse sequence.Sixty-four free induction decays (FIDs) were
collected into 131,072 data points using a spectral width of 18,518±5 Hz. The FIDs were multiplied by
an exponential weighting function corresponding to a line broadening of 0±3 Hz prior to Fourier
transform ation. Double-quantum ®ltered correlated spectroscopy (DQF-COSY) experiments were
acquired on a Varian VXR600spectrom eter operating at 600 MHz "H resonance frequency.The spectra
were acquired using a spectral width of 6000 Hz in both the F2 and F1 dimensions.Sixty-four scans were
collectedper incrementfor 512 incrementsinto 2K data points. The FIDswere multipliedby a sine-bell-
squared function prior to Fourier transform ation. The regions of the 1D 600 MHz "H-NMR spectrum
corresponding to the aromatic and acetyl signals of the metaboliteswere integrated following automatic
polynomial baseline correction. The signal intensities of the aromatic ring proton doublets for
paracetamol sulphate, glucuronide and parent were integrated relative to TSP and the urinary
concentrationsof each calculated.The side chainN-acetyl singletof the N-acetyl-l-cysteinylparacetamol

In vivo futile deacetylation of phenacetin in the rat
1179
metaboliteof phenacetinwas integrated relative to TSP to determinethe concentrationof this metabolite.
The % total recovery of the metabolites was calculated for the 0±8-h period taking into account the
urinary volumes collected over this period. Integration of the CH acetyl signal relative to the aromatic
$
H$ and H& protons of the metabolite concerned allowed calculation of the extent of futile deacetylation.
Results
"
H-NMR spectroscopy of urine from rats dosed with phenacetin
The 600 MHz "H-NMR spectrum of whole rat urine 0±8 h after dosing with
phenacetin at 50 mg kgÕ " is shown in ®gure 2. A number of the resonances from the
endogenous components in urine have been previously assigned and are labelled in
the Figure (Nicholls et al. 1995, Nicholson and Wilson 1987). Paracetamol
resonances were also assigned from previous studies (Bales et al. 1984). The singlet
resonance observed at d3±15 was assigned to dimethyl sulphone by addition of
authentic material.Figure 3 shows expansionsfrom the 600 M Hz "H-NM R spectra
shown in ®gure 2. As expected the predominantphenacetinmetaboliteswere related
to paracetamol, with aromatic resonances (AA« XX« spin system) for paracetamol
sulphate at d7±46 and 7±32, and the corresponding protio-acetyl singlet at d2±18
(
Bales et al. 1984, Nichollset al. 1995, 1997). Integration of the aromatic resonances
relative to an internalstandard (TSP) gave a mean % recovery of 37±7³8±6 % for the
paracetamol sulphate as a % of the administered dose.
Paracetamolglucuronidewas also present with the aromaticresonances(AA«XX«
spin system) partially obscured at d7±36, whilst visible at d7±15 (Bales et al. 1984,
Nicholls et al. 1995, 1997). The former signal was overlapped by a resonance from
an endogenous substance so that only the latter was used for quanti®cation. The
glucuronideacetyl (protio) signal was clearly visible as a singlet at d2±16. The mean
%
recovery of paracetamol glucuronide was 8±5³3±3 % as a % of dose.
One of the aromatic doublets for the N-acetyl-l-cysteinyl metabolite of
paracetamolwas initiallyassigned to the doubletat d6±95 and the acetyl singlet of the
N-acetyl-l-cysteinyl residue to the resonance d1±86 (Bales et al. 1984, Nichollset al.
1
995, 1997). However, the relative heights and integrals of these resonances
indicated that the signal at d6±95, which was also phenacetin-related, was not solely
due to the paracetamol N-acetyl-l-cysteinyl-metabolite. Quanti®cation of the N-
acetyl-l-cysteinyl paracetamol gave a % mean recovery of 1±7³0±6 % of the dose.
The mean % total recovery for the three paracetamol conjugate metabolites of
phenacetin was 50±7³12±4 % of the received dose.
To provide further data to enable assignment of the unidenti®ed metabolite at
d6±95 a 600 MHz "H-"H DQF COSY experiment was obtained of the 0±8-h whole
urine (®gure 4). The drug metaboliteand endogenousmetaboliteNM R signals were
assigned from previous studies (Bales et al. 1985). Assignment of the N-acetyl-l-
cysteinyl conjugate of paracetamol was made to the resonances at d6±93 and 7±23.
The resonances at d6±99 and 7±16 and those at d6±89 and 7±25 were assigned to
4
-aminophenol sulphate (Gartland 1988) and paracetamol (Bales et al. 1985)
respectively after comparison with existing data. Due to extensive overlap with
other metabolites in the 1-D NMR spectrum, these two metabolites could not be
quanti®ed directly. The resonance overlapping with the N-acetyl-cysteinyl parace-
tamol was coupled to another at d7±31 indicating a para-substituted aromatic
compound, but complete identi®cation was not possible from these data alone.
To verify whether any further metabolites could be identi®ed solid phase
Y
extraction chromatography (SPEC) on a C18 Bond-Elut column using acidi®ed

1
180
A. W. Nicholls et al.
Figure 2. 600 MHz "H-NMR spectrum of whole rat urine followingdosing of phenacetin(50 mg kgÕ ").
The expansion of the aliphatic region shows the major endogenouscomponents.The endogenous
and phenacetin metabolites are as labelled. TSP, sodium 3-trimethylsilyl-[2,2,3,3-#H ]-1-
%
propionate; DMSO, dimethyl sulphoxide; TMAO, trimethylamine -N -oxide.
Figure 3. Expansions from the 600 MHz "H-NMR spectra of whole rat urine pre- (A) and post-dose
B) with phenacetin (50 mg kgÕ "). The endogenous and phenacetin metabolites are as labelled.
(

In vivo futile deacetylation of phenacetin in the rat
1181
Figure 4. Partial (d6±0±8±0) 600 MHz "H-"H DQF-COSY NMR spectrum of whole rat urine following
dosing of phenacetin (50 mg kgÕ "). The endogenous and phenacetin metabolites are as labelled.
water and methanol was used to purify the whole urine. Shown in ®gure 5 is a
stackplot of expansions from the 600 MHz "H-NMR spectra of the column eluent,
acidi®ed water (pH 2) and methanol fractions of the 0±8-h post-dose whole urine.
The phenacetin metabolites and endogenous resonances are as labelled. The

1
182
A. W. Nicholls et al.
Figure5. 600 MHz "H-NMR spectra of SPE fractions of wholerat urine followingdosing of phenacetin
50 mg kgÕ "). (A) Column eluent, (B) acidi®ed water (pH 2), and (C) 100% methanol fraction.
The endogenous and phenacetin metabolites are as labelled.
(
resonancesat d6±95 and 7±25 were assigned to unconjugatedparacetamol(Bales et al.
1
984, Nicholls et al. 1995, 1997). The resonance at d6±99 was attributed to the
unidenti®ed phenacetin metabolite with the chemical shift variation caused by the
pH of the solution. An acetyl resonance at d2±13 was found to integrate with a 3 :2
ratio to that of the unidenti®ed at d6±99 indicating that the molecule was probably
an N-acetylated para-substituted aniline. Since resonances of an ethyl group were
absent, the unidenti®ed molecule could not be phenacetin itself and based on
the chemical shifts the unidenti®ed molecule was tentatively assigned as N-
hydroxyparacetamol or a conjugate of this.
The "H-NMR resonancesat d8±10 and d6±23 were assigned to the nucleotidebase
uracil and the resonancesat d8±12 and d6±24 to uridine (Lynch et al. 1994). Increases
in the intensity of signals from both substances were observed following dosing of
phenacetin.
"
H-NMR spectroscopy of urine from rats dosed with phenacetin- C#H$
Expansionsof the 600 MHz "H-NMR spectra for pre-dose and 0±8 h post-dose
whole urine following dosing of phenacetin-C#H$ at 50 mg kgÕ " are shown in
gure 6. The endogenous and phenacetin metabolite "H-NMR resonances were
®
assigned from previous studies (Bales et al. 1984, Nicholls et al. 1995, 1997). The
paracetamol sulphate aromatic resonances (AA« XX« spin system) were observed at
d7±46 and 7±32 with the corresponding acetyl singlet at d2±18 (Nicholls et al. 1995,
1
997). The acetyl singlet was integrated relative to the aromatic signals to give a

In vivo futile deacetylation of phenacetin in the rat
1183
Figure 6. Expansions of the 600 MHz "H-NMR spectra of whole rat urine, pre- (A) and post-dosing
(
B) with phenacetin-C#H$ (50 mg kgÕ "). Both the endogenous and phenacetin metabolites are
as labelled.
mean % futile deacetylationof 29±6³0±9 % (12±3³0±1 % of dose, 23±3³0±9 % of the
recovery) for the paracetamol sulphate metabolite. The mean % recovery for the
paracetamol sulphate was calculated to be 41±6³9±4 % of the received dose.
Paracetamol glucuronide was also detected with aromatic resonances (AA«XX«
spin system) at d7±36 and 7±14 and an acetyl singlet at d2±16. Integration of the acetyl
singlet was used to give a mean % futile deacetylation of 36±6³3±1 % (2±5³0±1 % of
dose, 4±7³0±4 % of the recovery). The mean % recovery for this metabolite was
6
±8³1±8 % of dose.
As with phenacetin, the "H resonance at d1±86 was assigned to the N-acetyl-l-
cysteinyl metabolite of paracetamol and the signal at d6±95 was attributed to N-
acetyl-l-cysteinyl paracetamol overlapped with a signal from the unassigned
phenacetin metabolite. The low concentration of this metabolite, which accounted
for only 1±9³0±3 % of dose, meant that quanti®cation of the level of futile
deacetylation was not possible in the present studies. No other metabolites were
identi®ed from the 0±8-h urine. Thus the mean % total recovery of the metabolites
of phenacetin-C#H was 52±8³11±5 % of dose. There was no signi®cant diåerence
$
"
(
Student’s t-test, p 0±05) between the total % urinary recoveries for the non-
labelled and labelled phenacetin.
Discussion
Futile deacetylation is potentially a very common in vivo metabolic trans-
formation for many N-acetylated arylamines,the extent of the process being depen-
dent on ring electronic and side-chain steric interactions with deacetylases and
N-acetyltransferases. However, relatively few studies have dealt speci®cally with

1
184
A. W. Nicholls et al.
Table 1. Percentage urinary recoveries of phenacetin, phenacetin-C#H , paracetamol-C#H and
$
$
paracetamol-"$ CH dosed to rat along with the % futile deacetylationfor the isotopicallylabelled
drugs. Data derived from * Nichollset al. (1995) and ** Nichollset al. (1997). Ð, Not observed.
$
Mean % futile Mean % futile
deacetylation of deacetylation of Mean % urinary
Dose
level
animals (mg kgÕ ")
No. of
paracetamol
sulphate
paracetamol
glucuronide
recovery of total
metabolites
Compound dosed
Phenacetin
Phenacetin-C#H
Paracetamol-C#H *
Paracetamol-"$ CH
3
3
3
3
50
50
40
40
Ð
Ð
47±6³16±7
50±1³16±2
61±4³8±8
29±6³0±9
10±0³1±2
8±9³0±7
36±6³3±1
Ð
Ð
$
$
102±7³3±7
$
Paracetamol-
"$ CO"$ CH **
3
100
9±2
9±1
47±4³11±3
$
these biochemically `silent’ processes. The importance of a futile deacetylation
reaction with regard to toxicity of arylamineshas been demonstrated previously for
paracetamol (Nicholls et al. 1995, 1997). The results of the present and previous
studies on futile deacetylation in paracetamol are compared in table 1. The earlier
studies (Nicholls et al. 1995) indicated that a proportion of the paracetamol,
approximately 6 % of the dose, underwent futile deacetylation probably via
4
-aminophenol. This extent of futile deacetylation was considered to be a
possible contributing factor to the nephrotoxicity of paracetamol, underlying the
importanceof metabolicreactionsthat are ` silent’ in non-labelledmaterial(Nicholls
et al. 1995).
Phenacetin, which is shown herein to undergo extensive futile deacetylation
(
15% of dose, i.e. twice that observed for paracetamol),has been shown to lead to an
increase in the predisposition to kidney damage when taken for long periods, and
nephrotoxicityis similarlyseen in patients who have experienced overdose (Sandler
et al. 1989). The extent of futile deacetylation for phenacetin supports previous
evidence from "% C-labellingstudies, which also showed a total deacetylationof 15%
for phenacetin (Smith and Griæths 1976). Since the level of % futile deacetylation
was signi®cantlyhigher than that found for paracetamol,this indicatesthat products
formed from the deacetylation of phenacetin itself need to be considered as major
metabolites of the drug. Thus 4-ethoxyaniline, which has previously only been
considered as a minor metabolic product of phenacetin (Brodie and Axelrod 1949,
Bernhammer and Krisch 1965, Smith and Griæths 1976), might be an important
metabolite formed by futile deacetylation.
The diåerence between the % futile deacetylation for paracetamol and
phenacetinsuggests two possibleroutes for paracetamolformation from phenacetin.
The ®rst route involves the deacetylation of phenacetin to 4-ethoxyanilinefollowed
by re-acetylation back to phenacetin with subsequent de-ethylation to paracetamol
(
®gure 1). Another route involvesthe deacetylationof phenacetinto 4-ethoxyaniline
that in turn is de-ethylated to form 4-aminophenol. This can then be either
acetylated to paracetamol followed by conjugation or conjugated with subsequent
acetylation (®gure 1). The latter of these two routes would allow for a potentially
greater quantity of 4-aminophenolto be formed, which might explain the increased
nephrotoxicity of phenacetin over paracetamol. Circumstantial evidence for the
formation of these compounds in addition to the nephrotoxicity of phenacetin has
been the occurrenceof methaemoglobinaemiafollowingmetaboliteingestion(Smith
1
958, Gloor 1993). This toxicological condition has also been shown to occur

In vivo futile deacetylation of phenacetin in the rat
1185
following ingestion of 4-ethoxyaniline (Timbrell 1992). If indeed 4-aminophenol
and 4-ethyoxyaniline are the main toxic species then this would indicate that such
`
silent’ metabolic routes represent an, as yet, little appreciated major route of
metabolism with regard to drug toxicity for compounds of this class.
In conclusion, this study has emphasised the potential importance of the futile
deacetylationreaction pathwayin drug metabolism.In addition,the use of "H-NMR
spectroscopy and isotopic labelling has provided a simple, readily interpretable
analytical method for the measurement of such `silent’ metabolic reactions.
Acknowledgem ent s
We thank the Engineering and Physical Sciences Research Council (EPSRC)
and GlaxoWellcomefor their funding(A.W .N.) and Dr J. Parkinson of the EPSRC
NMR service at Edinburgh for the acquisition of the 2D NM R spectra.
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