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Therapeutic doses of acetaminophen stimulate the turnover of cysteine and glutathione in man
Journal of Hepatology, 1987; 4:206-211 Elsevier
206
HEP 00277
Therapeutic doses of acetaminophen stimulate the turnover of cysteine and glutathione in man
Bernhard H. Lauterburg 1 and Jerry R. Mitchell 2 1Department of Clinical Pharmacology, Universityof Bern (Switzerland) and 2Centerfor Experimental Therapeutics, Baylor College of Medicine, Houston, TX (U.S.A.) (Received 22 September, 1986) (Accepted 9 December, 1986)
Summary In spite of the importance of glutathione (GSH) in the detoxification of toxic metabolites of drugs, virtually nothing is known about the regulation of hepatic GSH homeostasis in man. In order to estimate the turnover of hepatic GSH and to assess the effect of different doses of acetaminophen (paracetamol) on the synthesis of GSH in man, [3H]cystine and varying doses of acetaminophen were administered to healthy volunteers, and the time course of the specific activity of the cysteine moiety of N-acetylcysteinyl-acetaminophen excreted in urine was followed. The fractional rate of turnover of the tracer in N=acetylcysteinyl-acetaminophen increased significantly from 0.031 + 0.007 h -t after doses of acetaminophen ranging from 50 to 300 mg to 0.045 + 0.011 and 0.121 + 0.027 h -I following 600 and 1200 mg of acetaminophen, respectively. The data indicate that therapeutic doses of acetaminophen markedly stimulate the rate of turnover of the pool of cysteine available for the synthesis of GSH, most likely due to an increased rate of synthesis of GSH which is required to detoxify the toxic metabolite of acetaminophen. Patients who are not able to respond to a similar demand on their stores of GSH by increasing the synthesis of GSH may be at higher risk of developing hepatic injury from drugs that require GSH for their detoxification.
Glutathione (GSH) plays an important role in the detoxification of certain toxic metabolites of drugs and of potentially toxic oxygen intermediates [1,2]. Although the metabolism of GSH and the regulation of GSH homeostasis have been extensively studied in
experimental animals, virtually nothing is known about the turnover of GSH in man. In view of the toxicologic significance of GSH it would be clinically relevant to know more about the kinetics of GSH in health and disease and about the response to a stress
Correspondence: Bernhard H. Lauterburg, Department of Clinical Pharmacology, University of Berne, Murtenstrasse 35, CH-3010 Bern, Switzerland.
0168-8278/87/$03.50© 1987 Elsevier Science Publishers B.V. (Biomedical Division)
GSH TURNOVER IN MAN on the GSH pool as it might be produced by a drug consuming GSH. With this goal in mind we have developed and validated an approach to estimate hepatic GSH turnover in intact rats [3,4]. Because GSH is not taken up to any significant extent by the liver, the GSH pool is labelled by administration of labelled cysteine, a precursor amino acid for GSH synthesis. Only minuscule quantities of GSH and no breakdown product that can be readily attributed to GSH are excreted in urine. Thus, in order to determine the specific activity of GSH, small quantities of acetaminophen are administered and the specific activity of N-acetylcysteine-acetaminophen (NACacetaminophen, acetaminophen mercapturic acid) excreted in urine is measured. Since NAC-acetaminophen is one of the metabolic endproducts of glutathionyl-acetaminophen (GSH-acetaminophen) [5], the specific activity of NAC-acetaminophen reflects the specific activity of intrahepatic GSH, and the analysis of the time course of the specific activity of NAC-acetaminophen provides an estimate of the turnover of hepatic GSH [4]. We have now applied this acetaminophen probe analysis of GSH turnover to normal volunteers and have studied the effect of varying doses of acetaminophen on the specific activity time course of NACacetaminophen excreted in urine following administration of labelled cystine.
Subjects and Methods
Human studies. Nineteen healthy volunteers gave informed consent to participate in the study that had been approved by the Institutional Review Board. The participants all had normal hepatic and renal function as judged from a normal serum creatinine and normal serum transaminases and alkaline phosphatase. None of the subjects had taken any medication during the week preceding the study, and none consumed ethanol in excess of 10 g/day. Following an overnight fast the subjects received 50 ~Ci of L-[3,3'3H]cystine (0.5 Ci/mmol, Amersham Corporation) p.o. followed 1 h later by an initial dose of acetaminophen ranging from 50 to 1200 mg. With the 1200 mg
207 dose sufficient quantities of NAC-acetaminophen were excreted for 8 h to determine accurately its specific activity. With the lower doses, where the specific activity time course was followed for 28 h due to the longer apparent half-life of the label in NACacetaminophen, repeat doses of 50 mg of acetaminophen were administered 6, 12, and 24 h after ingestion of the radioactively labelled precursor amino acid. Acetaminophen and the amino acid were both dissolved in sugar-free lemonade. During the study the subjects followed their regular daily activities and their regular diet. Urine samples were collected at intervals for 1 h. Prior to each collection period the subjects were asked to empty their bladder. Three female patients with a T-tube in place were studied 7 days after cholecystectomy for cholelithiasis and bile duct exploration. The same protocol as described above was followed, and timed samples of urine and bile were collected for 6 h. Animal studies. The acetaminophen probe analysis has been validated using the biliary excretion of the GSH adduct of acetaminophen [3,4]. Since we intended to use the urinary excretion of NAC-acetaminophen in man, the feasibility of that approach had to be demonstrated. For that purpose male Sprague-Dawley rats (Harlan Industries, Houston, TX) were studied. The animals had free access to food and water and were acclimatized in an air-conditioned room with a 12 h dark-light cycle. Under ether anesthesia the common bile duct was cannulated with PE10 polyethylene tubing and a PE50 polyethylene catheter was placed in the bladder and a femoral vein. The animals were then placed in a restraining cage and 1 h after awakening from the anesthesia 30 ~Ci of L-[35S]cysteine (121 mCi/mmol, Amersham Corporation) were injected intravenously. Thirty minutes later 50 mg/kg of acetaminophen in 0.9% saline were administered intravenously. Repeat doses of acetaminophen were injected every 2 h because of the short half-life of the compound in rats. Bile and urine were collected for periods of 30 min over 6 h.
Analytical methods The specific activity of NAC-acetaminophen in the urine of the volunteers was determined by high per-
208 formance liquid chromatography and liquid scintillation spectroscopy. Three ml of urine were passed over a washed ClS Sep-Pak column (Waters Associates) which was then rinsed with 3 ml of water to remove more polar components of urine. The NACacetaminophen was eluted with 1 ml of 50% methanol in water. Depending on the concentration of NAC-acetaminophen in the sample, the eluate was concentrated 2-5-fold under a stream of nitrogen at room temperature. NAC-acetaminophen was isolated using a C]8 Semipreparative column (Waters Associates) and water/methanol/acetic acid (86.5:12.5:1) at a flow rate of 3.5 ml/min as solvent [6]. The column effluent was monitored at 254 nm. The peak corresponding to synthetic NAC-acetaminophen was collected into a scintillation vial. At later time points, when the specific activity tended to be low, the peaks collected from 2 - 3 injections were pooled. The collected column effluent was lyophilized, redissolved in 0.5 ml of water, and the radioactivity was measured following addition of 10 ml of scintillation fluid (Aquasol, New England Nuclear) using the channels ratio technique for quench correction. The mass of the collected peak(s) was calculated by comparing the peak area with a standard curve obtained with urine of a rat that had received acetaminophen of known specific activity. Analysis of bile samples. Bile was acidified with an equal volume of 4% sulfosalicylic acid to precipitate proteins. Following centrifugation the supernatant was extracted with 4 volumes of chloroform, and the extracted aqueous phase was then analyzed by HPLC as described above. Rat bile was analyzed as described previously [3]. Rat urine was assayed without prior extraction using the same chromatographic system described above. Calculations. The fractional rate of turnover of the tracer in NAC-acetaminophen was calculated from the monoexponential decline of the specific activity by least-square regression analysis. Statistical differences between groups were assessed by Student's ttest.
B.H. LAUTERBURG and J.R. MITCHELL Results
In order to validate our approach we first had to confirm that the fractional rates of turnover of GSH calculated from the specific activity time course of GSH-acetaminophen in bile and NAC-acetaminophen in urine were identical. In the rat this is indeed the case, as shown in Fig. 1. The fractional rates of turnover calculated from biliary and urinary data are virtually identical, indicating that the urinary excretion of NAC-acetaminophen can be utilized for the assessment of hepatic GSH turnover in the rat. To confirm this correlation in man we studied three patients with indwelling T-tube. Interestingly, no GSH-acetaminophen could be identified in the bile samples of the three patients following administration of 1200 mg of acetaminophen. However, large peaks corresponding to the cysteine adduct of acetaminophen and smaller peaks corresponding to the cysteinylglycine adduct of acetaminophen were readily identified. Most likely, the higher hepatobil-
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Fig. l. Fractional rates of turnover of hepatic glutathione calculated from the specific activity-time curves of glutathionylacetaminophen excreted in bile and N-acetylcysteine-acetaminophen excreted in urine following the administration of [35S]cysteine and acetaminophen to rats. The rates calculated from biliary and urinary data are virtually identical, indicating that the turnover of hepatic glutathione may be estimated from urinary data.
GSH TURNOVER IN MAN Subject# 1
209
Subject# 2
Subject # 3
100. 80. °/o
60-
40.
o Bile • Urine
~o
Hours
Fig. 2. Specific activities (expressed as percent of peak specific activity) of cysteine-acetaminophen excreted in bile and of N-acetylcysteine-acetaminophenexcreted in urine in three patients with an indwelling T-tube. Labelled cystine was administered at time zero followed 1 h later by 1200 mg of acetaminophen. The slopes of the specific activity-time curves from bile and urine are similar, indicating that the same information is obtained from urinary and biliary data also in man.
iary activity of gamma-glutamyltransferase in m a n resulted in the metabolism of G S H - a c e t a m i n o p h e n to cysteinylglycine- and cysteinyl-acetaminophen. T h e radioactivity in the cysteinylglycine adduct was insufficient for an accurate d e t e r m i n a t i o n of the specific activity. Consequently, the specific activity of the cysteine adduct in bile was c o m p a r e d with the specific activity of N A C - a c e t a m i n o p h e n in urine. A s shown in Fig. 2, the time course of the two specific activity-time curves was similar in all three patients, suggesting that also in m a n the same information is obtained from bile and urine. W e subsequently investigated the effect of varying doses of a c e t a m i n o p h e n on the time course of the specific activity of N A C - a c e t a m i n o p h e n . A s shown in Fig. 3, the decline in specific activity with time was much faster in subjects receiving 1200 mg than in sub-
0.15-
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0 i
i
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i
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i
Fig. 3. Specific activities (expressed as percent of peak specific activity) of N-acetylcysteine-acetaminophen excreted in urine in four subjects receiving either 600 or 1200 mg of acetaminophen 1 h after ingestion of labelled cystine. The slope of the curves, reflecting the fractional rate of turnover of tracer in hepatic GSH, is steeper following the larger dose, this being consistent with a stimulation of glutathione turnover by high doses of acetaminophen.
|
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.6
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Fig. 4. Fractional rate of turnover of cysteine in glutathione in' 19 healthy volunteers following the administration of 50-1200 mg of acetaminophen. After ingestion of 600 or 1200 mg the fractional rates were significantly higher than after the lower doses, this being consistent with an increased turnover of hepatic glutathione resulting from the increased consumption of glutathione by higher doses of acetaminophen.
210 jects receiving 600 mg of acetaminophen. This dose dependency was confirmed in additional subjects (Fig. 4). The fractional rate of turnover of the administered tracer in NAC-acetaminophen was identical in subjects receiving 50, 100, and 300 mg of acetaminophen initially (0.0305 + 0.0065 h-l; mean + SD). After administration of 600 mg the fractional rate of turnover averaged 0.0445 + 0.0109 h -l and was significantly higher (P = 0.011) than with the lower doses. A dose of 1200 mg of acetaminophen increased the fractional rate of turnover to 0.1205 + 0.0273 h -I, which was significantly higher than in the groups receiving 300 mg or less (P < 0.001) and 600 mg of acetaminophen (P = 0.001).
Discussion Our data clearly demonstrate a dose-dependent decrease in the apparent half-life of tracer molecules in NAC-acetaminophen excreted in urine. Since NAC-acetaminophen is thought to be a metabolic endproduct of GSH-acetaminophen, the time course of the specific activity of the mercapturic acid should reflect the time course of the specific activity of the tracer in hepatic GSH. Indeed, in experimental animals the fractional rates of turnover calculated from the specific activities of GSH-acetaminophen excreted in bile and of NAC-acetaminophen excreted in urine are virtually identical (Fig. 1). This relationship could not be documented in man because no GSH-acetaminophen was detected in bile. Nevertheless, the identical time course of the specific activity of cysteinyl-acetaminophen in bile and of NAC-acetaminophen in urine (Fig. 2) suggests that also in man the time course of tracer in hepatic GSH can be followed by determination of one of its metabolites in urine. However, the apparent fractional rate of turnover of labelled cysteine in GSH does not necessarily refleet the turnover of GSH [4]. A complex precursorproduct relationship exists between cysteine and GSH. Not only does cysteine originating from the catabolism of GSH reenter the precursor pool, but labelled cysteine may also be incorporated into other
B.H. LAUTERBURG and J.R. MITCHELL compounds and reenter the precursor pool upon their catabolism. This recirculation may markedly influence the time course of the specific activity of hepatic GSH. As we [4] and others [7] have demonstrated, recircu[ation of tracer will result in a prolonged apparent half-life of hepatic GSH. In the rat the underestimation of the actual rate of turnover resulting from recirculation of tracer is not quantitatively important. Three different approaches to estimate hepatic turnover of GSH, acetaminophen probe analysis [4], determination of efflux of GSH from the liver [8], and estimation of hepatic production of GSH from the plasma clearance of GSH and its steady state plasma concentration [8], yield similar results in rats. In man, however, the underestimation of the rate of GSH turnover by acetaminophen probe analysis appears to be quantitatively more important. Based on the plasma clearance of GSH and the steady state concentration of GSH in plasma in healthy volunteers, we have recently estimated the input of GSH into the circulation to be approximately 28/~mol/min [9]. Assuming that most of this GSH originates in the liver as it does in rats [8], and assuming a liver weight of 1500 g, the hepatic production of GSH in man amounts to approximately 18 nmol/g liver.min. Maintaining this effiux and an intracellular concentration of approximately 4 j~mol/g [10,11] requires a fractional rate of turnover of 0.27 h -I corresponding to a half-life of 2.6 h. Evidently, the apparent halflife of hepatic GSH estimated by acetaminophen probe analysis in man is an order of magnitude longer. In view of the similar intrahepatic and plasma concentrations in man and rat, and lacking good reasons to doubt the validity of the clearance approach, the half-life of hepatic GSH estimated by acetaminophen probe analysis in man is probably too long. Most likely, recirculation of tracer accounts for the long apparent half-life of hepatic GSH determined by this method, and the actual half-life is much shorter, probably in the range found in experimental animals. In man, therefore, the acetaminophen probe analysis most likely reflects the turnover of the cysteine pool available for GSH synthesis rather than the turnover of hepatic GSH. The effects of increasing doses of
GSH TURNOVER IN MAN
211
a c e t a m i n o p h e n are consistent with this interpretation. With increasing doses of a c e t a m i n o p h e n the apparent half-life of the tracer in the G S H pool became progressively shorter. This may be explained either by a higher fractional rate of turnover of the G S H pool or an increased rate of turnover of the pool of cysteine serving as precursor for G S H synthesis. Since a p p r o x i m a t e l y 8% of a dose of a c e t a m i n o p h e n is metabolized to the cysteine- and N-acetylcysteineadducts [12], 1200 mg of a c e t a m i n o p h e n will consume over 6 0 0 / a m o l of G S H . This increased consumption of G S H will not only decrease the recirculation through the p r e c u r s o r pool of cysteine originating from the catabolism of G S H but also shunt more cysteine into G S H synthesis, because the decrease in hepatic G S H resulting from conjugation with the toxic metabolite of a c e t a m i n o p h e n will lead to a compensatory increase in G S H synthesis as long as sufficient quantities of cysteine are available [3,13,14]. In addition, a p p r o x i m a t e l y 30% of a therapeutic dose of a c e t a m i n o p h e n is sulfated [12]. A n increased rate of formation of sulfate from cysteine might be another
mechanism by which the turnover of cysteine is stimulated. We conclude that a c e t a m i n o p h e n administered in therapeutic doses to normal healthy volunteers m a r k e d l y stimulates the rate of turnover of the pool of cysteine available for the synthesis of GSH. The increase in turnover is at least in part due to an increased d e m a n d for G S H to detoxify the toxic metabolite of a c e t a m i n o p h e n and consequently an increased rate of synthesis of G S H . Patients who are not able to respond to a similar stress on their G S H stores with an increase in the synthesis of G S H might be at higher risk of developing hepatic injury from drugs such as a c e t a m i n o p h e n .
References
257: 3747-3753. 8 Lauterburg BH, Adams JD, Mitchell JR. Hepatic glutathione homeostasis in the rat: Efflux accounts for glutathione turnover. Hepatology 1984; 4: 586-590. 9 Burgunder JM, Lauterburg BH. Plasma kinetics of glutathione (GSH) in man: A non-invasive probe to assess hepatic GSH production. Gastroenterology 1986; 90 (part 2): 1717 (Abstract). 10 Jewell SA, Di Monte D, Gentile A, et al. Decreased hepatic glutathione in chronic alcoholic patients. J Hepatol 1986; 3: 1-6. 11 Lauterburg BH, Velez ME, Mitchell JR. Plasma glutathione as an index of intrahepatic GSH in man: Response to acetaminophen and chronic ethanol abuse. Hepatology 1984; 4:1051 (Abstract). 12 Forrest JAH, Clements JA, Prescott LF. Clinical pharmacokinetics of paracetamol. Clin Pharmacokin 1982; 7: 93-107. 13 Richman PG, Meister A. Regulation of gamma-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem 1975; 250: 1422-1426. 14 Lauterburg BH, Mitchell JR. Toxic doses of acetaminophen suppress hepatic glutathione synthesis in rats. Hepatology 1982; 2: 8-12.
1 Ketterer B, Coles B, Meyer DJ. The role of glutathione in detoxication. Environ Health Perspect 1983; 49: 59-69. 2 Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979; 59: 527-605. 3 Lauterburg BH, Vaishnav Y, Stillwell WG, Mitchell JR. The effects of age and glutathione depletion on hepatic glutathione turnover in vivo determined by acetaminophen probe analysis. J Pharmacol Exp Ther 1980; 213: 54-58. 4 Lauterburg BH, Mitchell JR. Regulation of hepatic glutathione turnover in rats in vivo and evidence for kinetic homogeneity of the hepatic glutathione pool. J Clin Invest 1981; 67: 1415-1424. 5 Boyland E, Chasseaud LF. The role of glutathione and glutathione S-transferases in mercapturic acid biosynthesis. Adv Enzymol 1969; 32: 173-219. 6 Buckpitt AR, Rollins DE, Nelson SD, Franklin RB, Mitchell JR. Quantitative determination of the glutathione, cysteine and N-acetyl cysteine conjugates of acetaminophen by high pressure liquid chromatography. Anal Biochem 1977; 83: 168-177. 7 Meredith MJ, Reed DJ. Status of the mitochondrial pool of glutathione in the isolated hepatocyte. J Biol Chem 1982;
Acknowledgements Supported by grant number 3.824.0.84 from Schweizerischer Nationalfonds zur Unterstiitzung der Wissenschaftlichen Forschung and grant n u m b e r G M 34120 from the National Institute of G e n e r a l Medical Sciences.
206
HEP 00277
Therapeutic doses of acetaminophen stimulate the turnover of cysteine and glutathione in man
Bernhard H. Lauterburg 1 and Jerry R. Mitchell 2 1Department of Clinical Pharmacology, Universityof Bern (Switzerland) and 2Centerfor Experimental Therapeutics, Baylor College of Medicine, Houston, TX (U.S.A.) (Received 22 September, 1986) (Accepted 9 December, 1986)
Summary In spite of the importance of glutathione (GSH) in the detoxification of toxic metabolites of drugs, virtually nothing is known about the regulation of hepatic GSH homeostasis in man. In order to estimate the turnover of hepatic GSH and to assess the effect of different doses of acetaminophen (paracetamol) on the synthesis of GSH in man, [3H]cystine and varying doses of acetaminophen were administered to healthy volunteers, and the time course of the specific activity of the cysteine moiety of N-acetylcysteinyl-acetaminophen excreted in urine was followed. The fractional rate of turnover of the tracer in N=acetylcysteinyl-acetaminophen increased significantly from 0.031 + 0.007 h -t after doses of acetaminophen ranging from 50 to 300 mg to 0.045 + 0.011 and 0.121 + 0.027 h -I following 600 and 1200 mg of acetaminophen, respectively. The data indicate that therapeutic doses of acetaminophen markedly stimulate the rate of turnover of the pool of cysteine available for the synthesis of GSH, most likely due to an increased rate of synthesis of GSH which is required to detoxify the toxic metabolite of acetaminophen. Patients who are not able to respond to a similar demand on their stores of GSH by increasing the synthesis of GSH may be at higher risk of developing hepatic injury from drugs that require GSH for their detoxification.
Glutathione (GSH) plays an important role in the detoxification of certain toxic metabolites of drugs and of potentially toxic oxygen intermediates [1,2]. Although the metabolism of GSH and the regulation of GSH homeostasis have been extensively studied in
experimental animals, virtually nothing is known about the turnover of GSH in man. In view of the toxicologic significance of GSH it would be clinically relevant to know more about the kinetics of GSH in health and disease and about the response to a stress
Correspondence: Bernhard H. Lauterburg, Department of Clinical Pharmacology, University of Berne, Murtenstrasse 35, CH-3010 Bern, Switzerland.
0168-8278/87/$03.50© 1987 Elsevier Science Publishers B.V. (Biomedical Division)
GSH TURNOVER IN MAN on the GSH pool as it might be produced by a drug consuming GSH. With this goal in mind we have developed and validated an approach to estimate hepatic GSH turnover in intact rats [3,4]. Because GSH is not taken up to any significant extent by the liver, the GSH pool is labelled by administration of labelled cysteine, a precursor amino acid for GSH synthesis. Only minuscule quantities of GSH and no breakdown product that can be readily attributed to GSH are excreted in urine. Thus, in order to determine the specific activity of GSH, small quantities of acetaminophen are administered and the specific activity of N-acetylcysteine-acetaminophen (NACacetaminophen, acetaminophen mercapturic acid) excreted in urine is measured. Since NAC-acetaminophen is one of the metabolic endproducts of glutathionyl-acetaminophen (GSH-acetaminophen) [5], the specific activity of NAC-acetaminophen reflects the specific activity of intrahepatic GSH, and the analysis of the time course of the specific activity of NAC-acetaminophen provides an estimate of the turnover of hepatic GSH [4]. We have now applied this acetaminophen probe analysis of GSH turnover to normal volunteers and have studied the effect of varying doses of acetaminophen on the specific activity time course of NACacetaminophen excreted in urine following administration of labelled cystine.
Subjects and Methods
Human studies. Nineteen healthy volunteers gave informed consent to participate in the study that had been approved by the Institutional Review Board. The participants all had normal hepatic and renal function as judged from a normal serum creatinine and normal serum transaminases and alkaline phosphatase. None of the subjects had taken any medication during the week preceding the study, and none consumed ethanol in excess of 10 g/day. Following an overnight fast the subjects received 50 ~Ci of L-[3,3'3H]cystine (0.5 Ci/mmol, Amersham Corporation) p.o. followed 1 h later by an initial dose of acetaminophen ranging from 50 to 1200 mg. With the 1200 mg
207 dose sufficient quantities of NAC-acetaminophen were excreted for 8 h to determine accurately its specific activity. With the lower doses, where the specific activity time course was followed for 28 h due to the longer apparent half-life of the label in NACacetaminophen, repeat doses of 50 mg of acetaminophen were administered 6, 12, and 24 h after ingestion of the radioactively labelled precursor amino acid. Acetaminophen and the amino acid were both dissolved in sugar-free lemonade. During the study the subjects followed their regular daily activities and their regular diet. Urine samples were collected at intervals for 1 h. Prior to each collection period the subjects were asked to empty their bladder. Three female patients with a T-tube in place were studied 7 days after cholecystectomy for cholelithiasis and bile duct exploration. The same protocol as described above was followed, and timed samples of urine and bile were collected for 6 h. Animal studies. The acetaminophen probe analysis has been validated using the biliary excretion of the GSH adduct of acetaminophen [3,4]. Since we intended to use the urinary excretion of NAC-acetaminophen in man, the feasibility of that approach had to be demonstrated. For that purpose male Sprague-Dawley rats (Harlan Industries, Houston, TX) were studied. The animals had free access to food and water and were acclimatized in an air-conditioned room with a 12 h dark-light cycle. Under ether anesthesia the common bile duct was cannulated with PE10 polyethylene tubing and a PE50 polyethylene catheter was placed in the bladder and a femoral vein. The animals were then placed in a restraining cage and 1 h after awakening from the anesthesia 30 ~Ci of L-[35S]cysteine (121 mCi/mmol, Amersham Corporation) were injected intravenously. Thirty minutes later 50 mg/kg of acetaminophen in 0.9% saline were administered intravenously. Repeat doses of acetaminophen were injected every 2 h because of the short half-life of the compound in rats. Bile and urine were collected for periods of 30 min over 6 h.
Analytical methods The specific activity of NAC-acetaminophen in the urine of the volunteers was determined by high per-
208 formance liquid chromatography and liquid scintillation spectroscopy. Three ml of urine were passed over a washed ClS Sep-Pak column (Waters Associates) which was then rinsed with 3 ml of water to remove more polar components of urine. The NACacetaminophen was eluted with 1 ml of 50% methanol in water. Depending on the concentration of NAC-acetaminophen in the sample, the eluate was concentrated 2-5-fold under a stream of nitrogen at room temperature. NAC-acetaminophen was isolated using a C]8 Semipreparative column (Waters Associates) and water/methanol/acetic acid (86.5:12.5:1) at a flow rate of 3.5 ml/min as solvent [6]. The column effluent was monitored at 254 nm. The peak corresponding to synthetic NAC-acetaminophen was collected into a scintillation vial. At later time points, when the specific activity tended to be low, the peaks collected from 2 - 3 injections were pooled. The collected column effluent was lyophilized, redissolved in 0.5 ml of water, and the radioactivity was measured following addition of 10 ml of scintillation fluid (Aquasol, New England Nuclear) using the channels ratio technique for quench correction. The mass of the collected peak(s) was calculated by comparing the peak area with a standard curve obtained with urine of a rat that had received acetaminophen of known specific activity. Analysis of bile samples. Bile was acidified with an equal volume of 4% sulfosalicylic acid to precipitate proteins. Following centrifugation the supernatant was extracted with 4 volumes of chloroform, and the extracted aqueous phase was then analyzed by HPLC as described above. Rat bile was analyzed as described previously [3]. Rat urine was assayed without prior extraction using the same chromatographic system described above. Calculations. The fractional rate of turnover of the tracer in NAC-acetaminophen was calculated from the monoexponential decline of the specific activity by least-square regression analysis. Statistical differences between groups were assessed by Student's ttest.
B.H. LAUTERBURG and J.R. MITCHELL Results
In order to validate our approach we first had to confirm that the fractional rates of turnover of GSH calculated from the specific activity time course of GSH-acetaminophen in bile and NAC-acetaminophen in urine were identical. In the rat this is indeed the case, as shown in Fig. 1. The fractional rates of turnover calculated from biliary and urinary data are virtually identical, indicating that the urinary excretion of NAC-acetaminophen can be utilized for the assessment of hepatic GSH turnover in the rat. To confirm this correlation in man we studied three patients with indwelling T-tube. Interestingly, no GSH-acetaminophen could be identified in the bile samples of the three patients following administration of 1200 mg of acetaminophen. However, large peaks corresponding to the cysteine adduct of acetaminophen and smaller peaks corresponding to the cysteinylglycine adduct of acetaminophen were readily identified. Most likely, the higher hepatobil-
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Fig. l. Fractional rates of turnover of hepatic glutathione calculated from the specific activity-time curves of glutathionylacetaminophen excreted in bile and N-acetylcysteine-acetaminophen excreted in urine following the administration of [35S]cysteine and acetaminophen to rats. The rates calculated from biliary and urinary data are virtually identical, indicating that the turnover of hepatic glutathione may be estimated from urinary data.
GSH TURNOVER IN MAN Subject# 1
209
Subject# 2
Subject # 3
100. 80. °/o
60-
40.
o Bile • Urine
~o
Hours
Fig. 2. Specific activities (expressed as percent of peak specific activity) of cysteine-acetaminophen excreted in bile and of N-acetylcysteine-acetaminophenexcreted in urine in three patients with an indwelling T-tube. Labelled cystine was administered at time zero followed 1 h later by 1200 mg of acetaminophen. The slopes of the specific activity-time curves from bile and urine are similar, indicating that the same information is obtained from urinary and biliary data also in man.
iary activity of gamma-glutamyltransferase in m a n resulted in the metabolism of G S H - a c e t a m i n o p h e n to cysteinylglycine- and cysteinyl-acetaminophen. T h e radioactivity in the cysteinylglycine adduct was insufficient for an accurate d e t e r m i n a t i o n of the specific activity. Consequently, the specific activity of the cysteine adduct in bile was c o m p a r e d with the specific activity of N A C - a c e t a m i n o p h e n in urine. A s shown in Fig. 2, the time course of the two specific activity-time curves was similar in all three patients, suggesting that also in m a n the same information is obtained from bile and urine. W e subsequently investigated the effect of varying doses of a c e t a m i n o p h e n on the time course of the specific activity of N A C - a c e t a m i n o p h e n . A s shown in Fig. 3, the decline in specific activity with time was much faster in subjects receiving 1200 mg than in sub-
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Fig. 3. Specific activities (expressed as percent of peak specific activity) of N-acetylcysteine-acetaminophen excreted in urine in four subjects receiving either 600 or 1200 mg of acetaminophen 1 h after ingestion of labelled cystine. The slope of the curves, reflecting the fractional rate of turnover of tracer in hepatic GSH, is steeper following the larger dose, this being consistent with a stimulation of glutathione turnover by high doses of acetaminophen.
|
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1.2 g
acetarninophen
Fig. 4. Fractional rate of turnover of cysteine in glutathione in' 19 healthy volunteers following the administration of 50-1200 mg of acetaminophen. After ingestion of 600 or 1200 mg the fractional rates were significantly higher than after the lower doses, this being consistent with an increased turnover of hepatic glutathione resulting from the increased consumption of glutathione by higher doses of acetaminophen.
210 jects receiving 600 mg of acetaminophen. This dose dependency was confirmed in additional subjects (Fig. 4). The fractional rate of turnover of the administered tracer in NAC-acetaminophen was identical in subjects receiving 50, 100, and 300 mg of acetaminophen initially (0.0305 + 0.0065 h-l; mean + SD). After administration of 600 mg the fractional rate of turnover averaged 0.0445 + 0.0109 h -l and was significantly higher (P = 0.011) than with the lower doses. A dose of 1200 mg of acetaminophen increased the fractional rate of turnover to 0.1205 + 0.0273 h -I, which was significantly higher than in the groups receiving 300 mg or less (P < 0.001) and 600 mg of acetaminophen (P = 0.001).
Discussion Our data clearly demonstrate a dose-dependent decrease in the apparent half-life of tracer molecules in NAC-acetaminophen excreted in urine. Since NAC-acetaminophen is thought to be a metabolic endproduct of GSH-acetaminophen, the time course of the specific activity of the mercapturic acid should reflect the time course of the specific activity of the tracer in hepatic GSH. Indeed, in experimental animals the fractional rates of turnover calculated from the specific activities of GSH-acetaminophen excreted in bile and of NAC-acetaminophen excreted in urine are virtually identical (Fig. 1). This relationship could not be documented in man because no GSH-acetaminophen was detected in bile. Nevertheless, the identical time course of the specific activity of cysteinyl-acetaminophen in bile and of NAC-acetaminophen in urine (Fig. 2) suggests that also in man the time course of tracer in hepatic GSH can be followed by determination of one of its metabolites in urine. However, the apparent fractional rate of turnover of labelled cysteine in GSH does not necessarily refleet the turnover of GSH [4]. A complex precursorproduct relationship exists between cysteine and GSH. Not only does cysteine originating from the catabolism of GSH reenter the precursor pool, but labelled cysteine may also be incorporated into other
B.H. LAUTERBURG and J.R. MITCHELL compounds and reenter the precursor pool upon their catabolism. This recirculation may markedly influence the time course of the specific activity of hepatic GSH. As we [4] and others [7] have demonstrated, recircu[ation of tracer will result in a prolonged apparent half-life of hepatic GSH. In the rat the underestimation of the actual rate of turnover resulting from recirculation of tracer is not quantitatively important. Three different approaches to estimate hepatic turnover of GSH, acetaminophen probe analysis [4], determination of efflux of GSH from the liver [8], and estimation of hepatic production of GSH from the plasma clearance of GSH and its steady state plasma concentration [8], yield similar results in rats. In man, however, the underestimation of the rate of GSH turnover by acetaminophen probe analysis appears to be quantitatively more important. Based on the plasma clearance of GSH and the steady state concentration of GSH in plasma in healthy volunteers, we have recently estimated the input of GSH into the circulation to be approximately 28/~mol/min [9]. Assuming that most of this GSH originates in the liver as it does in rats [8], and assuming a liver weight of 1500 g, the hepatic production of GSH in man amounts to approximately 18 nmol/g liver.min. Maintaining this effiux and an intracellular concentration of approximately 4 j~mol/g [10,11] requires a fractional rate of turnover of 0.27 h -I corresponding to a half-life of 2.6 h. Evidently, the apparent halflife of hepatic GSH estimated by acetaminophen probe analysis in man is an order of magnitude longer. In view of the similar intrahepatic and plasma concentrations in man and rat, and lacking good reasons to doubt the validity of the clearance approach, the half-life of hepatic GSH estimated by acetaminophen probe analysis in man is probably too long. Most likely, recirculation of tracer accounts for the long apparent half-life of hepatic GSH determined by this method, and the actual half-life is much shorter, probably in the range found in experimental animals. In man, therefore, the acetaminophen probe analysis most likely reflects the turnover of the cysteine pool available for GSH synthesis rather than the turnover of hepatic GSH. The effects of increasing doses of
GSH TURNOVER IN MAN
211
a c e t a m i n o p h e n are consistent with this interpretation. With increasing doses of a c e t a m i n o p h e n the apparent half-life of the tracer in the G S H pool became progressively shorter. This may be explained either by a higher fractional rate of turnover of the G S H pool or an increased rate of turnover of the pool of cysteine serving as precursor for G S H synthesis. Since a p p r o x i m a t e l y 8% of a dose of a c e t a m i n o p h e n is metabolized to the cysteine- and N-acetylcysteineadducts [12], 1200 mg of a c e t a m i n o p h e n will consume over 6 0 0 / a m o l of G S H . This increased consumption of G S H will not only decrease the recirculation through the p r e c u r s o r pool of cysteine originating from the catabolism of G S H but also shunt more cysteine into G S H synthesis, because the decrease in hepatic G S H resulting from conjugation with the toxic metabolite of a c e t a m i n o p h e n will lead to a compensatory increase in G S H synthesis as long as sufficient quantities of cysteine are available [3,13,14]. In addition, a p p r o x i m a t e l y 30% of a therapeutic dose of a c e t a m i n o p h e n is sulfated [12]. A n increased rate of formation of sulfate from cysteine might be another
mechanism by which the turnover of cysteine is stimulated. We conclude that a c e t a m i n o p h e n administered in therapeutic doses to normal healthy volunteers m a r k e d l y stimulates the rate of turnover of the pool of cysteine available for the synthesis of GSH. The increase in turnover is at least in part due to an increased d e m a n d for G S H to detoxify the toxic metabolite of a c e t a m i n o p h e n and consequently an increased rate of synthesis of G S H . Patients who are not able to respond to a similar stress on their G S H stores with an increase in the synthesis of G S H might be at higher risk of developing hepatic injury from drugs such as a c e t a m i n o p h e n .
References
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Acknowledgements Supported by grant number 3.824.0.84 from Schweizerischer Nationalfonds zur Unterstiitzung der Wissenschaftlichen Forschung and grant n u m b e r G M 34120 from the National Institute of G e n e r a l Medical Sciences.