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Effect of vitamin C on paracetamol hepatotoxicity
175
Toxicology Letters, 2 (1978) 175-178 o Elsevier/North-Holland Biomedical Press
EFFECT
OF VITAMIN C ON PARACETAMOL
T.C. RAGHURAM,
D. KRISHNAMURTHI
HEPATOTOXICITY
and R. KALAMEGHAM
National Institute of Nutrition, Indian Council of Medical Research, Hyderabad - 500 007 (India) (Received February lst, 1978)
SUMMARY
Vitamin C and paracetamol are known to compete for the sulphate pool in the body. Hence the effect of vitamin C on paracetamol hepatotoxicity was studied. In therapeutic doses, simultaneous administration of Vitamin C and paracetamol did not result in liver dysfunction in undernourished subjects as judged by serum GOT and r-GT levels. On the other hand, even after toxic doses of paracetamol, Vitamin C may have a protective role in mice possibly through its antioxidant property.
INTRODUCTION
Paracetamol, also known as acetaminophen is commonly used either alone or in combination with other drugs as a mild analgesic an3 antipyretic since when given in therapeutic doses, it has fewer side effects as compared to salicylates. In large doses, however, the drug can lead to acute centrilobular liver necrosis. Paracetamol poisoning is now one of the commonest causes of hepatic failure in Britain [ 61. Hence its metabolism and mechanism of hepatotoxicity has, in recent years, been extensively studied in man and animals [ 1,7] . In therapeutic doses most of the paracetamol is conjugated with glucuronide or sulphate and excreted in the urine, although some is metabolised by cyt. P-450 dependent mixed-function oxidases to form its active arylating metabolite [ 131. It is only at high doses when the amounts of this active metabolite can overwhelm the GSH protective mechanism that hepatic necrosis occurs. If GSH is depleted to about 20% of normal in animals, then necrosis is believed to occur: sulphation and glucuronidation are limited and saturable [ 9, lo], and in overdosage hepatic glutathione is depleted. In the absence of hepatic glutathione, the arylating metabolite binds covalently with hepatocellular macromolecules and thereby causes hepatic necrosis [ 121. Vitamin C, which is also sulphated, has been shown to prolong the biological half-life of paracetamol in humans by competing for available sulphate in the body; concomitant administration of sodium sulphate has been shown to pre-
176
vent this effect [ 51. It is common practice to prescribe vitamin C in megadoses along with analgesics and antipyretics such as paracetamol during infections. Recent reports [2,7, 141 indicate that in some of the patients paracetamol may produce liver damage after chronic administration, even in therapeutic doses. In view of these findings, it was considered important to study the effect of vitamin C on paracetamol hepatotoxicity after high doses to animals and therapeutic doses to man. MATERIALS
AND METHODS
Animal study: 75 weanling male albino Swiss mice were divided into 3 groups and given the following treatment: Group I: Control - Normal saline intraperitoneally (i.p.) Group II: Paracetamol .- 200 mg/kg body weight in normal saline (i.p.) Group III: Paracetamol - 200 mg/kg body weight in normal saline (i.p.). In addition, vitamin C - 200 mg/kg body weight - was given i.p. in two divided doses 1 h before and 1 h after paracetamol injection. All animals were sacrificed at 24 h after treatment, the liver was removed and fixed in 10% buffered neutral formalin. Paraffin sections of the liver were stained with haematoxylin and eosin, and examined under a light microscope. Human study For the clinical study, 6 undernourished male volunteers aged between 25 and 45 years were given 500 mg of vitamin C three times a day for 5 days to decrease the sulphate pool in the body [ 51 and were then given paracetamol 500 mg three times a day in addition to vitamin C for a further period of 5 days. [Ascorbic acid at a level of 500 mg/tablet is incorporated in one of the paracetamol preparations marketed locally - Ed.] Blood samples were collected and SGOT [3] and serum r-GT [15] levels were determined as indicators of liver dysfunction before and after vitamin C and paracetamol treatment. RESULTS
AND
DISCUSSION
Of the 25 mice which received paracetamol alone, 8 died within a few hours following the injection, while only one of the 25, which received vitamin C with paracetamol died. For the purpose of comparison of hepatic damage between the two groups of mice, only those animals which survived for 24 h, were considered since some animals died very early, much before the morphological features of hepatic necrosis could become evident. Mice in both groups exhibited centrilobular congestion and variable degree of centrilobular liver cell necrosis often extending to the midzones. The incidence and extent of hepatic necrosis was not however appreciably different between the groups. These data suggest that vitamin C may in fact have a protective role against mortality arising from acute paracetamol toxicity.
177
TABLE
I
CLINICAL DATA AND SERUM GOT AND r-GT LEVELS IN 6 SUBJECTS (MEAN * SEM) -_ After After Vitamin C Enzymes Basal Weight (kg) Sei-Ulll + Paracetamol Vitamin C albumin (g %)
46.7 + 3.63
SGOT
U/L
13.02 + 2.22
17.62 f 3.71
15.84 f 2.35
r-GT
U/L
32.05 + 5.87
32.06 f 5.40
34.77 f 5.56
3.32 + 0.025
Statistical significance
between
different groups paired ‘t’-test - Not significant
The results of the clinical study are presented in Table I. Undernourished subjects were selected in this study since in them the hepatic glutathione levels are likely to be low and the risk of liver damage is therefore greater. It has been shown that a protein-deficient diet reduces hepatic glutathione levels [4] and greatly potentiates paracetamol hepatotoxicity [ 111. However, even in undernourished subjects, paracetamol in combination with vitamin C did not produce any liver damage in therapeutic doses, as judged by serum GOT and r-CT levels. It is known that vitamin C is involved in the oxidation and reduction of glutathione. Hence, vitamin C, even though it competes for sulphate with paracetamol, may have preserved the hepatic glutathione levels thereby’ helping in detoxication of the reactive metabolite of paracetamol. Alternatively,vitamin C per se might have acted as an antioxidant and influenced the attack of hepatic macromolecules by the active metabolite of paracetamol. Such a mechanism is supported by the finding that in the animal experiment vitamin C administration reduced the mortality due to paracetamol. ACKNOWLEDGEMENTS
We thank Dr. S.G. Srikantia, Director, his keen interest and helpful suggestions.
National
Institute
of Nutrition,
for
REFERENCES 1 B. Ameer and D.J. Greenblatt, Acetaminophen,
Ann. Int. Med., 87 (1977) 202-209. 2 J.D. Barker Jr., D.J. de Carle and S. Anuras, Chronic excessive acetaminophen use and liver damage, Ann. Int. Med., 87 (1977) 299-301. 3 KU. Bergmeyer and E. Bernt, Glutamate-oxaloacetate transaminase, UV-assay manual method, in H. U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Verlag Chemie, Weinheim, 1974, pp. 727-733. 4 S. Edwards and W.W. Westerfeld, Blood and liver glutathione during protein deprivation, Proc. Sot. Exp. Biol. Med., 79 (1952) 57-59. 5 J.B. Houston and G. Levy, Drug biotransformation interactions in man, IV. Acetaminophen and ascorbic acid, J. Pharm. Sci., 65 (1976) 12191221. 6 0. James, M. Lesna, S.H. Roberts, L. Pulman, A.P. Douglas, P.A. Smith and A.J. Watson, Liver damage after paracetamol overdose, Lancet, 2 (1975) 579-581.
178
7 G.K. Johnson and K.G. Tolman, Chronic liver disease and acetaminophen, Ann. Int. Med., 87 (1977) 302-304. 8 J. Koch-Weser, Drug therapy - Acetaminophen, N. Engl. J. Med., 295 (1976) 1297-1300. 9 G. Levy and T. Matsuzawa, Pharmocokinetics of salicylamide elimination in man, J. Pharmacol. Exp. Ther., 156 (1967) 285-293. 10 G. Levy and H. Yamada, Drug biotransformation interactions in man, III. Acetaminophen and salicylamide, J. Pharm. Sci., 60 (1971) 215-221. 11 A.E.M. McLean and P.A. Day, The effect of diet on the toxicity of paracetamol and the safety of paracetamol-methionine mixtures, Biochem. Pharmacol., 24 (1975) 37-42. 12 J.P. Mitchell, D.J. Jowlow, W.Z. Potter, J.R. Gillette and B.B. Brodie, Acetaminopheninduced hepatic necrosis, IV. Protective role of glutathione, J. Pharmacol. Exp. Ther., 187 (1973) 211-217. 13 J.E. Mrochek, S. Katz, W.H. Christie and S.R. Dinsmore, Acetaminophen metabolism in man, as determined by high-resolution liquid chromatography, Clin. Chem., 20 (1974) 1086-1096. 14 J.R. Mitchell, Host susceptibility and acetaminophen liver injury, Ann. Int. Med., 87 (1977) 377-378. 15 S.B. Rosalki and D. Tarlow, Optimized determination of y-glutamyl-transferase by reaction-rate analysis, Clin. Chem., 20 (1974) 1121-1124.
Toxicology Letters, 2 (1978) 175-178 o Elsevier/North-Holland Biomedical Press
EFFECT
OF VITAMIN C ON PARACETAMOL
T.C. RAGHURAM,
D. KRISHNAMURTHI
HEPATOTOXICITY
and R. KALAMEGHAM
National Institute of Nutrition, Indian Council of Medical Research, Hyderabad - 500 007 (India) (Received February lst, 1978)
SUMMARY
Vitamin C and paracetamol are known to compete for the sulphate pool in the body. Hence the effect of vitamin C on paracetamol hepatotoxicity was studied. In therapeutic doses, simultaneous administration of Vitamin C and paracetamol did not result in liver dysfunction in undernourished subjects as judged by serum GOT and r-GT levels. On the other hand, even after toxic doses of paracetamol, Vitamin C may have a protective role in mice possibly through its antioxidant property.
INTRODUCTION
Paracetamol, also known as acetaminophen is commonly used either alone or in combination with other drugs as a mild analgesic an3 antipyretic since when given in therapeutic doses, it has fewer side effects as compared to salicylates. In large doses, however, the drug can lead to acute centrilobular liver necrosis. Paracetamol poisoning is now one of the commonest causes of hepatic failure in Britain [ 61. Hence its metabolism and mechanism of hepatotoxicity has, in recent years, been extensively studied in man and animals [ 1,7] . In therapeutic doses most of the paracetamol is conjugated with glucuronide or sulphate and excreted in the urine, although some is metabolised by cyt. P-450 dependent mixed-function oxidases to form its active arylating metabolite [ 131. It is only at high doses when the amounts of this active metabolite can overwhelm the GSH protective mechanism that hepatic necrosis occurs. If GSH is depleted to about 20% of normal in animals, then necrosis is believed to occur: sulphation and glucuronidation are limited and saturable [ 9, lo], and in overdosage hepatic glutathione is depleted. In the absence of hepatic glutathione, the arylating metabolite binds covalently with hepatocellular macromolecules and thereby causes hepatic necrosis [ 121. Vitamin C, which is also sulphated, has been shown to prolong the biological half-life of paracetamol in humans by competing for available sulphate in the body; concomitant administration of sodium sulphate has been shown to pre-
176
vent this effect [ 51. It is common practice to prescribe vitamin C in megadoses along with analgesics and antipyretics such as paracetamol during infections. Recent reports [2,7, 141 indicate that in some of the patients paracetamol may produce liver damage after chronic administration, even in therapeutic doses. In view of these findings, it was considered important to study the effect of vitamin C on paracetamol hepatotoxicity after high doses to animals and therapeutic doses to man. MATERIALS
AND METHODS
Animal study: 75 weanling male albino Swiss mice were divided into 3 groups and given the following treatment: Group I: Control - Normal saline intraperitoneally (i.p.) Group II: Paracetamol .- 200 mg/kg body weight in normal saline (i.p.) Group III: Paracetamol - 200 mg/kg body weight in normal saline (i.p.). In addition, vitamin C - 200 mg/kg body weight - was given i.p. in two divided doses 1 h before and 1 h after paracetamol injection. All animals were sacrificed at 24 h after treatment, the liver was removed and fixed in 10% buffered neutral formalin. Paraffin sections of the liver were stained with haematoxylin and eosin, and examined under a light microscope. Human study For the clinical study, 6 undernourished male volunteers aged between 25 and 45 years were given 500 mg of vitamin C three times a day for 5 days to decrease the sulphate pool in the body [ 51 and were then given paracetamol 500 mg three times a day in addition to vitamin C for a further period of 5 days. [Ascorbic acid at a level of 500 mg/tablet is incorporated in one of the paracetamol preparations marketed locally - Ed.] Blood samples were collected and SGOT [3] and serum r-GT [15] levels were determined as indicators of liver dysfunction before and after vitamin C and paracetamol treatment. RESULTS
AND
DISCUSSION
Of the 25 mice which received paracetamol alone, 8 died within a few hours following the injection, while only one of the 25, which received vitamin C with paracetamol died. For the purpose of comparison of hepatic damage between the two groups of mice, only those animals which survived for 24 h, were considered since some animals died very early, much before the morphological features of hepatic necrosis could become evident. Mice in both groups exhibited centrilobular congestion and variable degree of centrilobular liver cell necrosis often extending to the midzones. The incidence and extent of hepatic necrosis was not however appreciably different between the groups. These data suggest that vitamin C may in fact have a protective role against mortality arising from acute paracetamol toxicity.
177
TABLE
I
CLINICAL DATA AND SERUM GOT AND r-GT LEVELS IN 6 SUBJECTS (MEAN * SEM) -_ After After Vitamin C Enzymes Basal Weight (kg) Sei-Ulll + Paracetamol Vitamin C albumin (g %)
46.7 + 3.63
SGOT
U/L
13.02 + 2.22
17.62 f 3.71
15.84 f 2.35
r-GT
U/L
32.05 + 5.87
32.06 f 5.40
34.77 f 5.56
3.32 + 0.025
Statistical significance
between
different groups paired ‘t’-test - Not significant
The results of the clinical study are presented in Table I. Undernourished subjects were selected in this study since in them the hepatic glutathione levels are likely to be low and the risk of liver damage is therefore greater. It has been shown that a protein-deficient diet reduces hepatic glutathione levels [4] and greatly potentiates paracetamol hepatotoxicity [ 111. However, even in undernourished subjects, paracetamol in combination with vitamin C did not produce any liver damage in therapeutic doses, as judged by serum GOT and r-CT levels. It is known that vitamin C is involved in the oxidation and reduction of glutathione. Hence, vitamin C, even though it competes for sulphate with paracetamol, may have preserved the hepatic glutathione levels thereby’ helping in detoxication of the reactive metabolite of paracetamol. Alternatively,vitamin C per se might have acted as an antioxidant and influenced the attack of hepatic macromolecules by the active metabolite of paracetamol. Such a mechanism is supported by the finding that in the animal experiment vitamin C administration reduced the mortality due to paracetamol. ACKNOWLEDGEMENTS
We thank Dr. S.G. Srikantia, Director, his keen interest and helpful suggestions.
National
Institute
of Nutrition,
for
REFERENCES 1 B. Ameer and D.J. Greenblatt, Acetaminophen,
Ann. Int. Med., 87 (1977) 202-209. 2 J.D. Barker Jr., D.J. de Carle and S. Anuras, Chronic excessive acetaminophen use and liver damage, Ann. Int. Med., 87 (1977) 299-301. 3 KU. Bergmeyer and E. Bernt, Glutamate-oxaloacetate transaminase, UV-assay manual method, in H. U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Verlag Chemie, Weinheim, 1974, pp. 727-733. 4 S. Edwards and W.W. Westerfeld, Blood and liver glutathione during protein deprivation, Proc. Sot. Exp. Biol. Med., 79 (1952) 57-59. 5 J.B. Houston and G. Levy, Drug biotransformation interactions in man, IV. Acetaminophen and ascorbic acid, J. Pharm. Sci., 65 (1976) 12191221. 6 0. James, M. Lesna, S.H. Roberts, L. Pulman, A.P. Douglas, P.A. Smith and A.J. Watson, Liver damage after paracetamol overdose, Lancet, 2 (1975) 579-581.
178
7 G.K. Johnson and K.G. Tolman, Chronic liver disease and acetaminophen, Ann. Int. Med., 87 (1977) 302-304. 8 J. Koch-Weser, Drug therapy - Acetaminophen, N. Engl. J. Med., 295 (1976) 1297-1300. 9 G. Levy and T. Matsuzawa, Pharmocokinetics of salicylamide elimination in man, J. Pharmacol. Exp. Ther., 156 (1967) 285-293. 10 G. Levy and H. Yamada, Drug biotransformation interactions in man, III. Acetaminophen and salicylamide, J. Pharm. Sci., 60 (1971) 215-221. 11 A.E.M. McLean and P.A. Day, The effect of diet on the toxicity of paracetamol and the safety of paracetamol-methionine mixtures, Biochem. Pharmacol., 24 (1975) 37-42. 12 J.P. Mitchell, D.J. Jowlow, W.Z. Potter, J.R. Gillette and B.B. Brodie, Acetaminopheninduced hepatic necrosis, IV. Protective role of glutathione, J. Pharmacol. Exp. Ther., 187 (1973) 211-217. 13 J.E. Mrochek, S. Katz, W.H. Christie and S.R. Dinsmore, Acetaminophen metabolism in man, as determined by high-resolution liquid chromatography, Clin. Chem., 20 (1974) 1086-1096. 14 J.R. Mitchell, Host susceptibility and acetaminophen liver injury, Ann. Int. Med., 87 (1977) 377-378. 15 S.B. Rosalki and D. Tarlow, Optimized determination of y-glutamyl-transferase by reaction-rate analysis, Clin. Chem., 20 (1974) 1121-1124.