Pharrnaco/ogica/ Research Communications/Vo/. 9, No. 8, 1977
701
PORTAL VENOUS INFUSION OF PARACETAMOL AND ANTIPYP.INE IN THE P~T J. Kelleher +, M.S.F. McLachlan*, B.E. Walker +, M.F. Dixon ~ M. S. Losowsky +. +
University Dept. of Medicine, St. James's Hospital, Leeds, England Dept, of Pathology, University of Leeds.
~,.
Dept. of Radiology, McMaster University, Hamilton, Ontario, Canada.
Received 16 March 1977
SUMMARY I.
Antipyrine either alone or simultaneously with hepatotoxic doses of paracetamol was infused intrasplenically in rats, some of which had been treated with phenobarhitone to induce liver enzymes.
.
The extent of liver damage, assessed histologically and by the plasma level of aspartate aminotransferase at 24h after paracetamol, varied greatly in all groups, indicating that variation in response to large dosss of paracetamol is not due to differences in absorption.
.
In both induced and non-induced rats the blood levels of paracetamol at 2 and 4 hours were similar whether or not liver disease was present.
.
Paracetamol had a rapid and profound effect in prolonging antipyrine half-life.
A significant correlation existed
between antipyrine half-llfe and liver damage.
Though this
may suggest that liver damage caused by paracetamol is a factor on prolonging antipyrine half-life, this prolongation could also be explained by competition for metabolic sites.
Pharmacological Research Communications, VoL 9, No. 8, 1977
702
INTRODUCTION Paracetamol(p-hydroxy-
acetaminophen) metabolism in the live~
has been e~t~nsively investigateO (Mitchell, Jollo~, Potter, Davis, Gillette & Brodie, 1973a).
l~e most important metabolic pathway
is by conjugation with glucuronide and sulphate. A small proportion (~.%) is oxidised by liver microsomal enzs~nas to a potentially toxic intermediate alkylating metabolite whi,:;h is conjugated with glutathione
(Mitche!1, ~ - Tho roeirsson, Potter, Jo!lo~ & Kaiser 197L~)
~.~.~.n paracetamol is administered in toxic dosage, the supply of ~lutathione is exhausted, the metabo!ite binds to cell proteins and
O
liver necrosis results (Mitchell, Jollow, Potter, Gillette & Brodie, 1973b). Susceptibility to the hepatotoxic effects of paracetamo! varies greatly in man (Prescott, Wright, P.oscoe & Brown, 1971) and in animals )Walker, Kelleher, Dixon & Losowsky, 1974).
The reasons
for this variation are not known, but a possible explanation is that the rate or extent of gastrointestinal absor[)tion and hence of delive%~ of the drug to the liver may differ from one subject to another.
We have examined this by assessing the extent of liver
damage after administration of paracetamol directly into the portal venous system by intrasplenic infusion in rats, some of which had receive4 phenobarbitone to induce liver enz~nes.
A second more
likely reason for varying susceptibility is that the efficiency of liver microsomal drug matabolising enzymes may differ from sub~ect to subject.
The ~]rug antipyrine may bo~ used either for the study
of microsomal drug metabolism (Bakke, Bending, :~rbakke & Davies, 1974), or as an indizator of liver necrosis (Branch, Herbert & Read, 1973; Forrest, Roscoe, Prescott & Stevenson, 1974).
~e •
~;
have examined the early effects of paracetamol on antipyrine metabolls~ in induced and non-inducer] rats. MATERIALS AND ~ T H O D S
Fifty three male Wistar rats weighing 200 - 250g with free access to tap water and Oxoid Diet 41B were studied (Table i).
Pharmacological Research Communications, Vol. 9, No. 8, 1977
C
703
TABLE i I
_ _ [ I ~ '
No. OF ANIMALS I~ EACH EZPERIME~[TAL GROUP Intrasplenic Infusion of Parac.etamol and Ant ipyr.~~..,." Phenobarbitone induction
intrasplenic Infusion of Antipyrine alone
Totals
17
6
23
No Phenobarbitone induction 24
6
30
12
53
Totals
41
To attempt to standardise the amount of induction of microsomal liver enzymes phenobarnitone
(4.3umol/ml) ~as added to the drinking
water of 23 animals for seven days before studies were performed. Two solutions for intrasplenic infusion were prepared.
Both
contained 1.6umol antipyrine with 0.5uCi (N-methyl-14C) antipyrine (58mCi/mmol)
(Radiochemi~al Centre, Amersham, Bucks.) per ml. of
0.9% sodium chloride; one also contained 132umol paracetamol (Sterling Winthrop Ltd) per ml., added immediately before administration.
Intrasplenic infusion ~as performed in all 53 animals of
which 23 had received phenobarbitone, after the spleen had been transposed to a subcutaneous position (Blumgart, Leach, ~Lachlan, Seager & Ryan, 1971).
The spleen ~as exposed by re-opening the
cutaneous incision 48h after this initial operation. cavity was not re-entered.
The abdominal
Under direct vision 10ml of one or other
solution was injected by hand into the splenic pulp over 3 minutes through a size 23G needle (Portex scalp vain set).
The dose of
paracetamol was therefore approximately 6.6mmol(Ig)/kg body weight and of antipyrine 80umol/kg body ~eight.
For each procedure animals
were anaesthetised by intraperitoneal injection of veterinary
pentobarbitone (160umol/kg body weight). In all animals, heparinlsed blood samples were obtained from a tail vein at 45 rain. intervals for up to 5 hours, commencing
Pharmacological Research.Communications, Vol. 9, No. 8, 1977
704
30-45 mins. after the end of injection.
Paracetamol was measured
in o. lml blood (Prescott, 1971) and antipyrine in 0.2ml blood (Bakke et al. 1974).
Concentrations were plotted on semi-logarithmic
time-concentration graphs and half-lives calculated for the 5 hour period by the method of least squares.
Aspartate amino-transferase
(oE.C. 2.6 i.i; ASAT) values were determined by the method of Henry, Chiamori, Golub & B e r k m a n
( 1 9 6 0 ) i n s e p a r a t e d plasma in several of
these samples and also at 24 hours after injection when animals were killed by cardiac puncture under light ether anaesthesia.
Enzyme
assays were carried out at 37 ° using the following volumes and concentrations of reagents, 3ml phosphate buffer, pH7.4, containing 30umol 2-oxoglutarate, 140umol L-aspartic acid, 0.5umol NADH, 0.51 malic dehydrogenase and 30ul of serum.
Results are expressed
as international units (i.u)/l (i.u. a r e d e f i n e d as umol of substrate
converted/min.).
Livers were removed and the extent of necrosis
assessed "blind" by the arbitrary grading of multiple samples (Walker et al. 1974). RESULTS i
After simultaneous intrasplenic infusion of paracetamol and antipyrine, 32 of the 41 animals survived for 24 hours.
The nine
which died were from the non, induced group, 3 died within 3 hours and 6 within 5 hours of the infusion.
Though the mean plasma ASAT
values at 4 hours ~in survivors (173 SD 68 i.u./l) was significantly less (p
701
PORTAL VENOUS INFUSION OF PARACETAMOL AND ANTIPYP.INE IN THE P~T J. Kelleher +, M.S.F. McLachlan*, B.E. Walker +, M.F. Dixon ~ M. S. Losowsky +. +
University Dept. of Medicine, St. James's Hospital, Leeds, England Dept, of Pathology, University of Leeds.
~,.
Dept. of Radiology, McMaster University, Hamilton, Ontario, Canada.
Received 16 March 1977
SUMMARY I.
Antipyrine either alone or simultaneously with hepatotoxic doses of paracetamol was infused intrasplenically in rats, some of which had been treated with phenobarhitone to induce liver enzymes.
.
The extent of liver damage, assessed histologically and by the plasma level of aspartate aminotransferase at 24h after paracetamol, varied greatly in all groups, indicating that variation in response to large dosss of paracetamol is not due to differences in absorption.
.
In both induced and non-induced rats the blood levels of paracetamol at 2 and 4 hours were similar whether or not liver disease was present.
.
Paracetamol had a rapid and profound effect in prolonging antipyrine half-life.
A significant correlation existed
between antipyrine half-llfe and liver damage.
Though this
may suggest that liver damage caused by paracetamol is a factor on prolonging antipyrine half-life, this prolongation could also be explained by competition for metabolic sites.
Pharmacological Research Communications, VoL 9, No. 8, 1977
702
INTRODUCTION Paracetamol(p-hydroxy-
acetaminophen) metabolism in the live~
has been e~t~nsively investigateO (Mitchell, Jollo~, Potter, Davis, Gillette & Brodie, 1973a).
l~e most important metabolic pathway
is by conjugation with glucuronide and sulphate. A small proportion (~.%) is oxidised by liver microsomal enzs~nas to a potentially toxic intermediate alkylating metabolite whi,:;h is conjugated with glutathione
(Mitche!1, ~ - Tho roeirsson, Potter, Jo!lo~ & Kaiser 197L~)
~.~.~.n paracetamol is administered in toxic dosage, the supply of ~lutathione is exhausted, the metabo!ite binds to cell proteins and
O
liver necrosis results (Mitchell, Jollow, Potter, Gillette & Brodie, 1973b). Susceptibility to the hepatotoxic effects of paracetamo! varies greatly in man (Prescott, Wright, P.oscoe & Brown, 1971) and in animals )Walker, Kelleher, Dixon & Losowsky, 1974).
The reasons
for this variation are not known, but a possible explanation is that the rate or extent of gastrointestinal absor[)tion and hence of delive%~ of the drug to the liver may differ from one subject to another.
We have examined this by assessing the extent of liver
damage after administration of paracetamol directly into the portal venous system by intrasplenic infusion in rats, some of which had receive4 phenobarbitone to induce liver enz~nes.
A second more
likely reason for varying susceptibility is that the efficiency of liver microsomal drug matabolising enzymes may differ from sub~ect to subject.
The ~]rug antipyrine may bo~ used either for the study
of microsomal drug metabolism (Bakke, Bending, :~rbakke & Davies, 1974), or as an indizator of liver necrosis (Branch, Herbert & Read, 1973; Forrest, Roscoe, Prescott & Stevenson, 1974).
~e •
~;
have examined the early effects of paracetamol on antipyrine metabolls~ in induced and non-inducer] rats. MATERIALS AND ~ T H O D S
Fifty three male Wistar rats weighing 200 - 250g with free access to tap water and Oxoid Diet 41B were studied (Table i).
Pharmacological Research Communications, Vol. 9, No. 8, 1977
C
703
TABLE i I
_ _ [ I ~ '
No. OF ANIMALS I~ EACH EZPERIME~[TAL GROUP Intrasplenic Infusion of Parac.etamol and Ant ipyr.~~..,." Phenobarbitone induction
intrasplenic Infusion of Antipyrine alone
Totals
17
6
23
No Phenobarbitone induction 24
6
30
12
53
Totals
41
To attempt to standardise the amount of induction of microsomal liver enzymes phenobarnitone
(4.3umol/ml) ~as added to the drinking
water of 23 animals for seven days before studies were performed. Two solutions for intrasplenic infusion were prepared.
Both
contained 1.6umol antipyrine with 0.5uCi (N-methyl-14C) antipyrine (58mCi/mmol)
(Radiochemi~al Centre, Amersham, Bucks.) per ml. of
0.9% sodium chloride; one also contained 132umol paracetamol (Sterling Winthrop Ltd) per ml., added immediately before administration.
Intrasplenic infusion ~as performed in all 53 animals of
which 23 had received phenobarbitone, after the spleen had been transposed to a subcutaneous position (Blumgart, Leach, ~Lachlan, Seager & Ryan, 1971).
The spleen ~as exposed by re-opening the
cutaneous incision 48h after this initial operation. cavity was not re-entered.
The abdominal
Under direct vision 10ml of one or other
solution was injected by hand into the splenic pulp over 3 minutes through a size 23G needle (Portex scalp vain set).
The dose of
paracetamol was therefore approximately 6.6mmol(Ig)/kg body weight and of antipyrine 80umol/kg body ~eight.
For each procedure animals
were anaesthetised by intraperitoneal injection of veterinary
pentobarbitone (160umol/kg body weight). In all animals, heparinlsed blood samples were obtained from a tail vein at 45 rain. intervals for up to 5 hours, commencing
Pharmacological Research.Communications, Vol. 9, No. 8, 1977
704
30-45 mins. after the end of injection.
Paracetamol was measured
in o. lml blood (Prescott, 1971) and antipyrine in 0.2ml blood (Bakke et al. 1974).
Concentrations were plotted on semi-logarithmic
time-concentration graphs and half-lives calculated for the 5 hour period by the method of least squares.
Aspartate amino-transferase
(oE.C. 2.6 i.i; ASAT) values were determined by the method of Henry, Chiamori, Golub & B e r k m a n
( 1 9 6 0 ) i n s e p a r a t e d plasma in several of
these samples and also at 24 hours after injection when animals were killed by cardiac puncture under light ether anaesthesia.
Enzyme
assays were carried out at 37 ° using the following volumes and concentrations of reagents, 3ml phosphate buffer, pH7.4, containing 30umol 2-oxoglutarate, 140umol L-aspartic acid, 0.5umol NADH, 0.51 malic dehydrogenase and 30ul of serum.
Results are expressed
as international units (i.u)/l (i.u. a r e d e f i n e d as umol of substrate
converted/min.).
Livers were removed and the extent of necrosis
assessed "blind" by the arbitrary grading of multiple samples (Walker et al. 1974). RESULTS i
After simultaneous intrasplenic infusion of paracetamol and antipyrine, 32 of the 41 animals survived for 24 hours.
The nine
which died were from the non, induced group, 3 died within 3 hours and 6 within 5 hours of the infusion.
Though the mean plasma ASAT
values at 4 hours ~in survivors (173 SD 68 i.u./l) was significantly less (p
