Regulation of Hepatic Glutathione Turnover in Rats In

Vivo and Evidence for KineticHomogeneity of the Hepatic Glutathione Pool

BERNHARDH. LAUTERBURGand JERRY R. MITCHELL, Department of Medicine andInstitute for Lipid Research, Baylor College of Medicine, Houston, Texas 77030

A B S T RA C T The intracellular distribution of gluta-thione into kinetically distinct pools and the deter-minants of glutathione turnover were examined in vivo.Glutathione turnover was measured in individual, re-strained rats with a biliary fistula by administration ofacetaminophen to trap the previously labeled hepaticglutathione as an excretable acetaminophen adduct.Fasting for 48 h resulted in a decrease of hepatic gluta-thione from 4.7+0.9 to 3.6±0.8 ,umol/g liver and amarked increase in the fractional rate of glutathioneturnover from 0.19±0.04 to 0.43±0.07/h. Within 6 h fol-lowing refeeding, the rate of glutathione turnover andthe hepatic glutathione concentration returned to nor-mal. The simultaneously determined specific ac-tivities of free intrahepatic glutathione and theacetaminophen-glutathione adduct in bile were identi-cal, indicating that the hepatic glutathione pool iskinetically homogeneous. The synthesis of glutathionecould, therefore, be estimated from the rate constantand the intrahepatic glutathione concentration. Dur-ing fasting hepatic synthesis of glutathione increasedfrom 0.86±0.17 to 1.50±0.23 ,umol/g per h. In fed ani-mals the administration of dibutyryl cyclic adenosinemonophosphate and theophylline stimulated the rate ofhepatic glutathione turnover similar to fasting. In con-trast, glucose given intraduodenally to fasted animalsdecreased the rate of glutathione turnover. These dataare consistent with the view that the increased gluta-thione turnover that occurs during fasting results fromtwo mechanisms. Because of a decrease in the intra-hepatic free glutathione/mixed disulfide ratio, which isapparently mediated by cyclic adenosine monophos-phate, the free glutathione pool contracts and turns over

Dr. Lauterburg is a recipient of the Pharmaceutical Manu-facturers Association Foundation Faculty DevelopmentAward in Clinical Pharmacology. Dr. Mitchell is a BurroughsWellcome Scholar in Clinical Pharmacology.

Receivedfor publication 21 April 1980 and in revisedform29 December 1980.

more rapidly in order to maintain glutathione synthe-sis. In addition, glutathione consumption via thegamma-glutamyl cycle apparently is increased, whichmay be related to the increased uptake of amino acidsfor gluconeogenesis during fasting.

INTRODUCTION

More and more evidence accumulates proving thatglutathione plays a fundamental role in cellularmetabolism. As the most abundant intracellular sulf-hydryl, glutathione is a key factor in the detoxificationof electrophilic metabolites of xenobiotics (1, 2). As thecosubstrate for glutathione peroxidase, glutathione pre-vents peroxidation of membrane lipids and, togetherwith other antioxidants, plays an important role inmaintaining cellular integrity (3). As the determinantof the sulfhydryl/disulfide ratio (4), glutathione modu-lates the activity of a number of enzymes (5), and re-cently it has been proposed that glutathione may be in-volved in the transport of amino acids across cell mem-branes (6). Although this hypothesis is mainly based onin vitro experiments, a role for glutathione in aminoacid metabolism in vivo has been supported by recentfindings in Meister's laboratory (7) and in our own (8).

Despite the fundamental role of glutathione in suchcritically important cellular functions, however, rela-tively little is known about the control mechanismsmaintaining glutathione homeostasis in vivo. Moststudies have been limited to the measurement of gluta-thione concentrations under given experimental condi-tions and reveal little about the kinetic aspects of gluta-thione metabolism. For example, it has long beenknown that stress and fasting are associated with de-creased hepatic glutathione concentrations (9, 10). Thedepletion of glutathione during fasting is of interestbecause fasting is associated with an increase in thehepatic necrosis caused by some electrophilic drugmetabolites capable of alkylating nucleophilic sites onvital hepatic macromolecules (11).

J. Clin. Invest. c The American Society for Clinical Investigation, Inc. * 0021-9738/81/05/1415/10 $1.00 1415Volume 67 May 1981 1415-1424

The decreased glutathione concentrations duringfasting have generally been considered the result of de-creased glutathione synthesis due to a lack of precursoramino acids (12). Indeed, the hepatic concentration ofcysteine, which is normally (13) in the range of theMichaelis constant (Ki) of the first and rate-limitingenzyme in glutathione synthesis, gamma-glutamyl-cysteine synthetase, drops further during starvation(12). However, the decrease in free glutathione duringfasting has been shown to be associated with an in-crease in glutathione-protein mixed disulfides (14). Asimilar shift in the free glutathione to glutathione/mixed disulfide ratio has been proposed to be re-sponsible for the diurnal variation of free hepatic gluta-thione, and both the diurnal and the fasting-inducedchanges correlate with alterations in hepatic cyclicadenosine monophosphate (cAMP) concentrations (14,15). These observations suggest that a decreased gluta-thione synthesis may not be the main mechanism re-sponsible for the decreased free glutathione duringfasting. In fact, recent data on the possible role ofglutathione in the transport of amino acids would leadone to speculate that the synthesis of glutathione mightbe increased, rather than decreased, during fasting.Short-term starvation is associated with an increase ingamma-glutamyl transpeptidase activity, the major en-zyme involved in the catabolism of glutathione (12).Moreover, fasting results in the mobilization of aminoacids for gluconeogenesis with an increased uptake ofamino acids into the liver, and we have shown recentlythat administration of amino acids increases hepaticglutathione turnover and consumption (8). During fast-ing, therefore, the increased load of amino acids and theincreased gamma-glutamyl transpeptidase activityshould result in an increased glutathione consumption.Consequently, the increased consumption should leadto an increased synthesis of glutathione because, atleast in vitro, glutathione synthesis appears to be regu-lated by feedback inhibition (13).

Analysis of the control mechanisms maintainingglutathione homeostasis in vivo is further complicatedby the observation that glutathione concentrations donot drop further as fasting continues but rather reach anadir of -70% of control values within 24 h, which ismaintained (16, 17). This observation has been construedas evidence for the presence of two glutathione pools,one pool with a fast turnover that is virtually abolishedby fasting and a pool with a slow turnover that is not af-fected by starvation. This interpretation appeared to besupported by the finding of a biexponential declineof the specific activity of glutathione as measured inliver homogenates after administration of radiolabeledamino acid precursors of glutathione (18). However,these investigators failed to account for the delayedcontribution to glutathione synthesis madeby the recir-culation of tracer derived from protein breakdown. A

fraction of the radioactive amino acid used to label theglutathione pool will be incorporated into proteinswhich, as they are metabolized, will provide a radio-labeled amino acid pool for glutathione synthesis. Be-cause overall protein turnover is much slower thanglutathione turnover, the time-course of the specific ac-tivity of the protein will determine the decline of thespecific activity of glutathione during later timeperiods. Indeed, the specific activity of glutathionemore or less paralleled the specific activity of the liverproteins in the studies of Higashi et al. (18). Accord-ingly, the observed slowly exchangeable pool of gluta-thione in these studies was most likely an artifact due torecirculation of the radiolabeled amino acid duringprotein turnover.

Thus, current views on the regulation of glutathionesynthesis and the distribution of intracellular gluta-thione into different pools are difficult to reconcile withsome of the more recent experimental data that in-directly reflect aspects of glutathione homeostasis. Ac-cordingly, we have measured the synthesis of gluta-thione directly during fasting and have attempted tocharacterize determinants of glutathione turnover invivo. In addition, we have experimentally tested thehypothesis that several intracellular pools of gluta-thione exist. For these purposes we have developedan approach that is well suited for the study of acutechanges in glutathione kinetics and allows an assess-ment of the rate of hepatic glutathione turnover in indi-vidual animals (19). A small dose of acetaminophen isadministered as a pharmacologic probe and the specificactivity of previously radiolabeled hepatic glutathioneis determined by measurement of the specific activityof the glutathione-acetaminophen adduct in bile or theacetaminophen mercapturic acid in urine.

METHODS

Male Sprague-Dawley rats, weighing 180-230 g (TimcoBreeding Laboratories, Houston, Tex.) had free access to foodand water and were kept in an air-conditioned room with acontrolled 12-h dark-light cycle. For the determination ofglutathione turnover the common bile duct and a femoralvein were cannulated under light ether anesthesia and the ani-mals were then restrained. Each turnover study was started be-tween 10:00 and 12:00 a.m. at least 1 h after the animals hadawakened from anesthesia. Approximately 15 ,Ci 35S-L-cysteine (121 mCi/mmol, Amersham Corp., Arlington Heights,Ill.) were administered intravenously followed 10 min later by50 mgof acetaminophen per kilogram. Other animals receivedL-[G-3H]glutamic acid (27 Ci/mmol, Amersham Corp.) insteadof 35S-cysteine. Because of the short half-life of acetaminophenin rats, repeat doses of acetaminophen (20 mg/kg) were in-jected 2, 3, and 4 h after the radioactive cysteine. Bile wascollected in 20-30-min periods starting 60 min after the in-jection of the radioactive precursor. The specific activity ofthe glutathione-acetaminophen adduct appearing in bilewas determined by high-pressure liquid chromatography andscintillation spectrometry as described previously (19). Thefractional rate of glutathione turnover was calculated

1416 B. H. Lauterburg and J. R. Mitchell

from the monoexponential decline of the specific activity ofthe glutathione-acetaminophen adduct by least squareregression analysis. Extensive studies with different radioac-tively labeled precursor amino acids of glutathione for meas-urements of glutathione turnover have shown that this methodprovides a valid assessment of glutathione turnover in vivo andthat the small doses of acetaminophen used as a pharmacologicprobe do not stimulate glutathione turnover (19).

To study the influence of fasting on glutathione turnover,rats having free access to water were starved for 48 h prior tothe kinetic study. Additional fasted animals were refed for 4 hand glutathione turnover was determined 2 h later. The refedanimals ate an average of 6.9 g of Rat Chow prior to the turn-over study. At the end of the experiments the liver was re-moved and hepatic glutathione concentration was deter-mined. To ensure that the hepatic concentration of glutathioneremained stable during the determination of turnover, hepaticglutathione was measured in additional rats at the start of theexperiment and again 4 h later.

Because diurnal and fasting-induced changes in hepaticglutathione content have been attributed to alterations incAMP concentration, the effect of cAMP on glutathioneturnover was studied in another group of rats. Dibutyryladenosine 3',5'-cyclic monophosphoric acid, 50 mg/kg (SigmaChemical Co., St. Louis, Mo.), and theophylline, 20 mg/kg(Sigma Chemical Co.), were administered subcutaneously tofed rats 3 h after labeling the glutathione pool with 35S5cysteine. The specific activity of the glutathione-acetamino-phen adduct in bile was determined as in the previous ex-periments before and after the injection of cAMP.

In order to assess the physiologic role of cAMP in regulat-ing the turnover of glutathione during fasting, 1.0 g/kg of a-D-glucose dissolved in 2 ml of water was instilled through aduodenal catheter 2.5 h after labeling the glutathione pool ina group of fasted rats. The decreased glucagon/insulin ratio re-sulting from the administration of glucose decreases the he-patic cAMP concentration (20-22). The turnover of gluta-thione was measured in the same animal before and after theadministration of glucose.

In order to compare the fractional rate of hepatic glutathioneturnover of fasted animals with the rate constants of fed ratswith comparable hepatic glutathione concentrations agroup of fed rats received 1.0 g/kg of diethyl maleate subcu-taneously 1 h after labeling the glutathione pool with radio-active cysteine. Hepatic glutathione concentrations reach anadir 1 h after the administration of diethyl maleate, and thelow concentrations of glutathione are maintained for about2.5 h (23). The rate of hepatic glutathione turnover was, there-fore, determined between one and 2.5 h after the injection ofdiethyl maleate when steady-state conditions may be ex-pected. Indeed, the analysis of the time-course of the specificactivities of the glutathione-acetaminophen adduct in bileshowed a monoexponential decline of the specific activitiesduring this interval (19).

To determine the size of the glutathione pool probed by thedescribed approach, the specific activity of the glutathione-acetaminophen adduct in bile and the specific activity of thefree intracellular glutathione were determined simul-taneously in a group of rats. Identical specific activities wouldindicate that the acetaminophen probe analysis measures theturnover of the total free glutathione pool as measured bychemical analysis, whereas lower specific activities of thehepatic glutathione would indicate the presence of an addi-tional pool of free glutathione that turns over more slowly.The specific activity of the glutathione-acetaminophen adductin bile samples obtained immediately prior to death wastherefore compared with the specific activity of the gluta-thione in liver homogenates of the same animal.

Analytical methods. To determine the specific activity ofthe acetaminophen-glutathione adduct in bile, the biliarymetabolites of acetaminophen were separated on a C,8,uBondapak column (Waters Associates Inc., Milford, Mass.)with water/methanol/acetic acid (86.5:12.5:1), 1 ml/min, as themobile phase (24). The mass of acetaminophen-glutathionewas calculated from a standard curve obtained from [3H]-acetaminophen-glutathione collected in the bile of a rat thathad received [3H]acetaminophen of known specific activity.The radioactivity in the collected acetaminophen-glutathionepeak was determined by liquid scintillation spectrometry withquench correction by the channels ratio technique.

Because this system did not fully resolve the acetamino-phen-glutathione adduct from radiolabeled diethyl maleatemetabolites, the bile samples of diethyl maleate treatedrats were chromatographed with water/methanol/aceticacid (91:8:1) containing 0.005 M heptanesulfonic acid, 1.7ml/min, as the mobile phase. A comparison of bile from ratsthat had received either diethyl maleate or acetaminophenin addition to radiolabeled cysteine verified that the chro-matographic system resolved the glutathione or cysteine-containing diethyl maleate metabolites from the acetamino-phen-glutathione adduct (19).

To determine the specific activity of intrahepatic gluta-thione, a portion of the liver was homogenized in 5% per-chloric acid and the supernate was treated with iodoaceticacid to form S-carboxymethyl glutathione. This adduct wasconjugated with 1-fluoro-2,4-dinitrobenzene and isolated byhigh pressure liquid chromatography on a C,8g Bondapakcolumn using methanol/water/acetic acid (17.5:81.5:1) at aflow rate of 1 ml/min. This system separates the correspondingderivatives of cysteine, cystine, glutathione disulfide, and thecysteine and glutathione adduct of acetaminophen fromglutathione as determined by standard compounds deriv-atized with the identical procedure. Mass was calculated bycomparison of the glutathione derivative peak with a standardcurve obtained with the synthetic glutathione derivative. Theradioactivity of the glutathione derivative was measured byliquid scintillation spectrometry with quench correction bythe channels ratio technique.

The concentration of acid soluble sulfhydryls in liverhomogenates [20% in 0.1 Mphosphate buffer pH 7.4, and 2mMEDTA] was determined according to Ellman (25) follow-ing precipitation of proteins with an equal volume of 4%sulfo-salicylic acid. Glutathione disulfide was measured by themethod of Tietze following derivatization of glutathione with2-vinylpyridine (26, 27). The concentration of mixed disul-fides was calculated from the difference in acid soluble sulf-hydryls before and after incubation of liver homogenates withan equal volume of 2% sodium borohydride in water at 400for 30 min (28). Foaming was prevented by adding 50 IlIn-octanol.

Gamma-glutamyl transpeptidase activity was assessed withgamma-glutamyl-nitroanilide as the substrate. The incubationmixture [1 ml] contained 0.05 M Tris-HCl buffer [pH 8],75 mMNaCl, 2 mMgamma-glutamyl-p-nitroanilide, 20 mMglycyl-glycine [pH 8] and 100 gl liver homogenate (29).L-Serine [5 mM] and borate [5 mM] was added to somesamples to inhibit the activity of the enzyme. After 1 h at roomtemperature 1 ml of 1.8 N acetic acid was added to stop thereaction and the absorbance was determined at 405 nm. Todemonstrate that increased gamma-glutamyl transpeptidaseactivity is associated with increased degradation of gluta-thione the hydrolysis of glutathione catalyzed by liverhomogenates was measured in vitro. The assay mixture (2ml) contained 0.1 Mphosphate buffer, pH 7.4, 2 mMEDTA,3 mMglutathione, and 1 ml of liver homogenate. The gluta-thione concentration was measured before and after incuba-

Regulation of Hepatic Glutathione Turnover in Rats In Vivo 1417

tion at 370C for 3 h by the enzymatic method of Tietze (26).The proteins in liver homogenates were determined by themethod of Lowry (30).

Statistical analysis and calculations. All values areexpressed as mean±+SD. The statistical significance of dif-ferences between the experimental groups and between re-gression coefficients was calculated by Student's t test (31).A two-compartment model was used to simulate the time-course of the concentration of tracer in the hepatic glutathionepool following introduction of the tracer into the hepatic poolof one of the precursor amino acids of glutathione (Fig. 1).The tracer was introduced into the precursor compartment(cysteine) at time zero and numerical solution of the system ofdifferential equations were obtained by the Runge-Kuttamethod (32). Under steady-state conditions the amount ofcysteine entering the glutathione pool must equal theamount of cysteine leaving the glutathione pool. Thus, k2, (I)= (k32 + k12) (II); because the size of the glutathione pool is'-24 times larger than the size of the cysteine pool (12) k2l = 24(k32 + k12). The experimentally determined rate constants forthe turnover of glutathione in the fed and fasted state werechosen for (k32 + k12). In the recirculation model it wasassumed that 50% of the glutathione turnover is accountedfor by efflux of glutathione from the liver. The arbitrary choiceof k4l is based on the assumption that only a fraction of theavailable cysteine will be used for glutathione synthesis.

RESULTS

As shown for a group of refed and starved rats in Fig. 2,the specific activity of the glutathione-acetaminophenadduct in bile declined monoexponentially from 1 honward after the injection of radiolabeled cysteine orglutamic acid. The fractional rate of glutathione turn-over was calculated by linear regression analysis afterlogarithmic transformation of the data, as previouslyreported (19). During fasting the rate constant for he-patic glutathione turnover was much higher than in re-fed rats of the same age or in animals that had free accessto food prior to the experiment (Table I). Quantitatively

I (Precursor) k2l 11 (Glutathione)

k4l k32

FIcuRE 1 Compartment model used to simulate the time-course of the concentration of tracer in the hepatic glutathionepool (II) following introduction of the tracer into the hepaticpool of one of the precursor amino acids of glutathione (I).k2l is the fraction of the precursor pool entering the gluta-thione pool per unit time; k4l, the fraction entering other meta-bolic pathways of the precursor amino acid such as proteinsynthesis. k12 plus k32 equals the fractional rate of turnover ofthe glutathione pool. k12 is the fraction of the labeled aminoacid in the glutathione pool reentering the precursor pool fol-lowing the catabolism of glutathione; k32, the fraction of thelabeled amino acid in the glutathione pool lost to the precur-sor pool due to efflux of intact glutathione.

lb

s.2cx m

C

-0 =

E X0

Ii, <a

E

Refed

\

I

Fasting

0.5[-

\

2 \ \

2 4 h,

2 4 h

FIGURE 2 Time-course of the specific activity of the gluta-thione-acetaminophen adduct in bile of rats fasted for 48 h orrefed for 4 h after a 48-h fast. Each curve represents data ob-tained from an individual animal. The radioactively labeledglutathione precursor 35S-L-cysteine was administered at timezero and small doses of acetaminophen were injected intra-venously at intervals to trap the glutathione as an excretableadduct. The fractional rate of glutathione turnover was cal-culated by linear regression analysis after logarithmic transfor-mation of the specific activities.

similar increases in the rate constant were observed fol-lowing the administration of either labeled cysteine orglutamic acid, which indicates that the measured rateconstant indeed reflects the fractional turnover of gluta-thione itself and not the turnover of the administeredprecursor amino acid. Due to the increased rate of turn-over and the decreased size of the hepatic glutathionepool, fasting animals incorporated a higher fraction ofthe administered precursor amino acid per micromoleglutathione than refed animals, as shown by the higherspecific activities of the adduct. The incorporation ofthe precursor amino acid and the rate constant were notsignificantly different in the refed animals and in non-fasted animals receiving a regular diet (Table I).

A comparison of the simultaneously determined spe-cific activities of free hepatic glutathione and of theglutathione-acetaminophen adduct in bile is shown inFig. 3. If two kinetically distinct intrahepatic pools ofglutathione were present, the specific activity of theglutathione in each pool would be different except forone point in time when the two specific activity timecurves intersect. To ensure that the specific activityof the free glutathione and the glutathione-acetamino-phen adduct were not accidentally measured at thatparticular point in time the animals were killed at dif-ferent times after the administration of the labeled pre-cursor amino acid. This experimental design results inthe wide range of specific activities shown in Fig. 3.The specific activity of the free hepatic glutathioneaveraged 98+11% (n = 10) of the specific activity of the

1418 B. H. Lauterburg andJ. R. Mitchell

1

TABLE IEffects of Fasting and Refeeding on the Fractional Rate of Hepatic Glutathione Turnover and Glutathione Synthesis

Regular diet Refed for 4 h following 48-h fast Fasting for 48 h

GSH GSH GSHK GSH* Synthesis K GSH* Synthesis K GSH* Synthesis

h-' ,Amol/g umolIg per h h-' tLmolIg LmolIg per h h-I pmollg pmollg per h

4.8 1.243.5 0.844.8 1.103.6 0.713.9 0.733.9 0.724.9 0.885.3 0.925.8 0.876.1 0.775.6 0.69

0.3190.3170.2890.2040.1740.174

4.0 1.284.0 1.272.4 0.696.2 1.266.8 1.185.4 0.94

0.589t0.492t0.4740.4680.4440.444t0.4150.406t0.3570.3520.310

2.0 1.182.5 1.232.9 1.374.3 2.013.8 1.693.2 1.424.1 1.703.6 1.464.0 1.434.7 1.654.3 1.33

0.186±0.042 4.7±0.9 0.86±0.17 0.246±0.064 4.8±1.5 1.10±0.22 0.432±0.074§ 3.6±0.811 1.50±0.23¶

* GSH, hepatic glutathione concentration determined by the Ellman method prior to reduction of disulfides with borohydride.These animals received [3H]glutamic acid instead of cysteine to label the glutathione pool.

§ Significantly different (P < 0.001) from regular diet and refed groups.1" Significantly different (P < 0.01) from regular diet groups.

Statistically different from regular diet (P < 0.001) and refed (P < 0.01) groups.

glutathione-acetaminophen adduct in bile. This ob-servation provides strong evidence that our approachprobes the same glutathione pool as the chemical analy-sis of the free intrahepatic glutathione. During the 4-hinterval required to measure the rate constant of gluta-thione turnover, the glutathione concentration re-

mained virtually constant, decreasing by an average ofonly 0.38 and 0.28 ,umol/g in three pairs of fed andfasted animals, respectively. Therefore, the synthe-sis of glutathione can be estimated by multiplying thechemically determined free glutathione concentrationat the end of the experiment with the measured frac-tional rate of turnover. Based on these calculations(Table I) the estimated synthesis of glutathione was sig-nificantly higher during fasting than in control and re-

fed animals.The decreased concentration of glutathione during

fasting that occurs together with an increase in gluta-thione synthesis suggests an increased consumption ofglutathione in the fasting state. Because gamma-

glutamyl transpeptidase is the main enzyme involvedin the degradation of glutathione its activity was

measured in liver homogenates of fed and fasted ani-mals. Using gamma-glutamyl-nitroanilide as the sub-strate and glycyl-glycine as the acceptor for the glu-tamyl moiety, the gamma-glutamyl transpeptidase ac-

tivity increased from 3.47+0.80 [n = 6] umol nitro-anilide released/h per g liver in fed rats to 5.67±1.72,umol/h per g [P < 0.051 after a fast of 48-h dura-tion. Addition of serine-borate to the incubation

mixture abolished the release of nitroanilide. Whenliver homogenates were incubated with glutathionealone the homogenates of fed rats hydrolyzed 0.28+0.21 ,umol glutathione/h per g liver comparedto 2.29±1.06 ,umol/h per g [P < 0.051 in fasted animals.The relative difference in these activities between fedand fasted rats does not change if expressed per milli-

Cpm/'Pmnmol

.2

~ ~ ~

X 25

.ffi CpXlI/I25 50

Specific Activityof Intrahepatic Glutathione

FIGURE 3 Comparison of the simultaneously determinedspecific activities of free hepatic glutathione and the gluta-thione-acetaminophen adduct in bile. Samples were obtainedat different time intervals following the administration of thelabeled precursor amino acid. The virtual identity of the spe-cific activities of the adduct and the free glutathione over a

wide range of specific activities indicates that no sizeable ad-ditional pool of glutathione with a slow rate of turnover ispresent within the liver.

Regulation of Hepatic Glutathione Turnover in Rats In Vivo

0.2580.2400.229t0.198t0.1860.1850.1790.174t0.150t0.1260.124

1419

gram protein instead of per gram liver, because theprotein content per gram liver was identical in fed andfasted animals.

During fasting the percentage of the total gluta-thione (measured following incubation with boro-hydride) present as mixed disulfides increased from31.9+5.0% to 53.9±2.9% [P < 0.01]. The concen-tration of glutathione disulfide (0.072±0.015 mM),however, did not change. A similar shift in the free sulf-hydryl to mixed disulfide ratio during fasting has beenshown to correlate with increases in the intracellularconcentration of cAMPand has been reproduced by theadministration of exogenous dibutyryl cAMP (14).Moreover cAMP has been shown to increase the ac-tivity of the glutathione catabolic enzyme gamma-glutamyl transpeptidase (33). From these observations,cAMPwould therefore be expected to increase the rateof turnover of glutathione. To test this hypothesis,dibutyryl cAMPand theophylline were administered tofed animals and hepatic glutathione turnover was deter-mined (Fig. 4). This treatment promptly increased theslope of the specific activity time curve, indicating anincreased influx of unlabeled cysteine into the gluta-thione pool. The similar effect of exogenous cAMPandfasting on the rate at which the specific activity of gluta-thione declines, suggests that increased concentrationsof cAMP during fasting may be responsible for thealtered glutathione kinetics.

To test whether this effect of exogenous cAMPreflects a possible physiologic response to the in-creased concentrations of cAMP stimulated by gluca-gon release during fasting, an attempt was made to re-

I _

4.-

A :%

2 u

acpo at

.tc

.- oj 0

verse the fasting-induced increase in the measured rateconstant of glutathione turnover by decreasing he-patic cAMPconcentrations. To decrease the glucagon:insulin ratio and thus the hepatic concentration ofcAMP (Fig. 5), glucose was administered intraduo-denally 2.5 h after labeling the glutathione pool infasted rats. The glucose load promptly reduced theslope of the specific activity time curve of the gluta-thione-acetaminophen adduct.

The association of an increased fractional rate ofglutathione turnover with decreased levels of freeglutathione observed during fasting might be the resultof feedback regulation of glutathione synthesis. Fig. 6demonstrates that as the hepatic glutathione concentra-tion was reduced by the administration of diethylmaleate to fed animals, the fractional rate of hepaticglutathione turnover indeed increased. However, thefractional rate of turnover measured in fasted animalswas clearly higher than in fed, diethyl maleate-treatedanimals with comparable glutathione levels and thusresulted in a greater amount of glutathione synthesizedduring fasting.

The simulated data (Fig. 7) demonstrate that inagreement with the experimental data the peak specific

ICa'-C

o _

c

.2 I'S o

k-;A

i.'2cAMP

10OF Glucoei

50F

20

n =3

10

50s

n=3

?<2 4 h

FIGURE 4 Effect of dibutyryl cAMPand theophylline on thetime-course of the specific activity of the glutathione-acetaminophen adduct in bile. Each point represents mean-+SD of three studies in three individual animals. Because ofvariable doses of radioactivity the initial specific activitieswere not identical in all studies. The specific activities are,therefore, expressed as a percentage of the specific activity at1 h. Subcutaneous administration of cAMPand theophyllineresults in an increased slope of the specific activity time curve.The slopes before and after cAMPare significantly different(P < 0.05).

2I 42 4

\;

6 h

FIGuRE 5 Effect of intraduodenally administered glucose onthe time-course of the specific activity of the glutathione-acetaminophen adduct in bile of rats that had been fasted for48 h. Each point represents mean+SDof three studies in threeindividual animals. Because of variable doses of radioactivitythe initial specific activities were not identical in all studies.The specific activities are, therefore, expressed as a per-centage of the specific activity at 1 h. Administration ofglucose reduces the slope of the specific activity time curve.The slopes before and after glucose are significantly different(P < 0.05). The specific activity time curve obtained in a fastedrat with a similar initial rate of turnover is shown for com-parison (*).

1420 B. H. Lauterburg and J. R. Mitchell

DISCUSSION1.QQ

-I0

0Q3- .0

2 4 6pmol Glutathione/g Liv

FIGURE 6 Rate of hepatic glutathione turnover as a functionof the intrahepatic glutathione concentration. The fractionalrate of glutathione turnover increases with decreasing concen-trations of glutathione in fed rats (0) and fed rats pretreatedwith diethyl maleate (A). Fasting animals (0) have a higherfractional rate of glutathione turnover than animals with com-parable intrahepatic glutathione concentrations.

activity of the glutathione pool will be higher in thefasted than in the fed state and that the specific activityof cysteine will decrease at an only slightly higher ratethan the specific activity of glutathione after 1 h if a frac-tion of the tracer recirculates. The simulated data alsoshow that the experimentally determined rate constantof the turnover of glutathione will underestimate the ac-tual rate constant if a fraction of the tracer recirculates,such as might occur from recirculation of tracer originat-ing from catabolism of protein or glutathione.

1

'-aEa

C.0O- E

_ 08A UO X1

.2_ O

O-

c >

(C.

0.5 F

0.

FED

1

0> GSH witho.s\'\ recirculation

GSH withoutrecirculation

0.5

With recirculation k21 = 5.5k12 = 0.115k32 = 0.115k4l = 18

Without recirculation k21 = 5_\ ~~~~k12 = °,1 \ k32 = °-

\ ~~~k4l = 11

Cysteine withp I recirculation

1 3 h

;.5

).2318

0.1

The presented data demonstrate that hepatic gluta-thione synthesis during fasting is increased and not de-creased as generally assumed. For the determination ofthe rate of glutathione synthesis by the current ap-proach, several criteria must be fulfilled. First, the cal-culation of glutathione synthesis from the intrahepaticconcentration and from the fractional rate of turnoveris obviously valid only if the pharmacologic probeanalyzes the total glutathione pool measured by thechemical analysis of glutathione by the Ellman method;it would not be accurate if two hepatic pools with dif-ferent rates of turnover were present. Our experiments,however, failed to demonstrate the presence of twoglutathione pools. Because our acetaminophen probeanalysis yields glutathione half-lives corresponding tothe proposed glutathione pool with rapid turnover (18),the specific activity of the intrahepatic glutathioneshould be lower than the specific activity of the gluta-thione-acetaminophen adduct if a sizeable pool withslow turnover were present. However, the specific ac-tivities of the free glutathione in liver homogenatesand the glutathione-acetaminophen adduct in bile weresimilar, indicating that the total free glutathione pool iskinetically homogeneous and therefore the calcula-tion of hepatic glutathione synthesis from the turnover

FASTING

0

' \ \ GSH with\ o°S \ recirculation

With recirculation k21 = 10.5GSH without k 2 = 0.215recirculation

k32 = 0.215

k4l= 18

Without recirculation k21 = 10.5

k12= 0

k3= 0.43

k41\ ~~~~~~k4l= 18

Cysteine withrecirculation

1 3 hTIME

FIGuRE 7 Time-course of the relative concentrations of tracer in the cysteine and glutathionepool using the model shown in Fig. 1 and the rate constants indicated in the figure. The simu-lation, in agreement with the experimental data, demonstrates that the peak specific activity ofthe glutathione pool will be higher in the fasted than in the fed state and that the specific activityof cysteine will decrease at an only slightly higher rate than the specific activity of glutathioneafter I h if a fraction of the tracer recirculates. The simulated data also show that the experi-mentally determined rate constant of the turnover of glutathione will underestimate the actualrate constant if a fraction of the tracer recirculates, such as might occur from recirculation oftracer originating from catabolism of protein or glutathione. GSH, hepatic glutathione con-centration.

Regulation of Hepatic Glutathione Turnover in Rats In Vivo

h-1 I

Q6e

1421

rate constant and pool size is appropriate. In support ofthis, Griffith and Meister have recently come to similarconclusions (34) by a different approach. In theirstudies, inhibition of glutathione synthesis in vivo re-sulted in a monophasic decline of intrahepatic gluta-thione to 20% of control values, a finding that speaksagainst the presence of separate pools of glutathionewith different turnover rates.

The other major consideration for the determinationof glutathione synthesis is the precursor-product rela-tionship that exists between an amino acid precursorand glutathione, such that changes in the kinetics of theprecursor pool caused by fasting could artifactually af-fect the experimentally determined rate constant forturnover of the glutathione pool. Because fasting leadsto a decrease in the intrahepatic concentration of cys-teine (12), fasting may be particularly prone to alter thekinetics of the cysteine pool. To understand better theinfluence of changes in the precursor kinetics on the ex-perimental data obtained by acetaminophen probeanalysis, a model was designed for analysis of the time-course of the tracer through the precursor and gluta-thione compartments under various conditions (Figs. 1and 7). The simulated data show that the initial alphaphase of the biexponential decline of the specificactivity of cysteine can not affect the measured rate con-stant for glutathione turnover because it declines at amuch greater rate than the fractional rate of glutathioneturnover. On the other hand, the terminal beta phase,which is mainly determined by recirculation of tracer,can markedly affect the measured rate constant forglutathione turnover. Because the cysteine pool is de-creased during fasting, it is conceivable that increasedrecirculation of tracer resulting from increased break-down of glutathione by gamma-glutamyl transpepti-dase could occur during fasting and lead to an under-estimation of the actual fractional turnover of gluta-thione. Increased recirculation of cysteine resultingfrom increased protein turnover would have a similareffect. In contrast to cysteine, however, the size of theglutamic acid pool is much larger and does not decreaseduring fasting (12) and thus should be minimallyaffected by food deprivation. Accordingly, the quanti-tatively similar increase in the rate constant for gluta-thione turnover produced by fasting when labeledglutamic acid is used as the glutathione precursor indi-cates that changes in the kinetics of the precursor poolduring fasting do not significantly influence the frac-tional rate of glutathione turnover and therefore ourestimates of glutathione synthesis.

Studies in vitro have documented that glutathioneregulates its own synthesis by feedback inhibition ofthe rate limiting step, the formation of gamma-glu-tamyl-cysteine by gamma-glutamyl-cysteine synthe-tase (13). Our observation that a decrease in glutathioneconcentration is accompanied by an increase in the rateof turnover of glutathione demonstrates that a similar

feedback regulation of glutatione synthesis is operativein vivo (Fig. 6). It is, therefore, not surprising that thedecrease in free hepatic glutathione during fasting isassociated with an increased fractional rate of gluta-thione turnover.

Our data, however, also indicate that the measuredincrease in the fractional rate of glutathione turnoverand hence the synthesis of glutathione is greater infasted animals even than in fed, diethyl maleate-treatedrats with comparable glutathione concentrations (Fig.6). Under the steady-state conditions that may be ex-pected once the new plateau of glutathione is reachedduring fasting, this increased synthesis of glutathionemust reflect an increased consumption of glutathione.The major route of glutathione catabolism is throughthe gamma-glutamyl cycle, which may be involved inthe transport of amino acids across cell membranes(6). As shown above, the consumption of gluta-thione and the activity of the enzyme gamma-glutamyltranspeptidase, which is responsible for the degrada-tion of glutathione via the gamma-glutamyl cycle, aremarkedly increased in homogenates from fasted ani-mals, confirming the previous report by Tateishi et al.(12). These data obtained in vitro thus are consistentwith the interpretation from our in vivo kinetic data thatthe consumption of glutathione increases duringfasting.

The quantitative contribution of this catabolic path-way to the turnover of hepatic glutathione is not clear.However, we have recently shown that the intravenousadministration of methionine and several other aminoacids increase the rate of turnover and decrease the con-centration of hepatic glutathione, and this stimulationwas abolished by the specific gamma-glutamyl trans-peptidase inhibitor, gamma-glutamyl carboxy phenyl-hydrazine (8). Since fasting results in a mobilization ofamino acids for hepatic gluconeogenesis and stimulateshepatic amino acid uptake (35), an increased transpepti-dation of the amino acids via the gamma-glutamyl cyclemight well explain the increased consumption of gluta-thione observed in fasted animals.

The present data also provide strong support for thehypothesis (14, 33) that the fasting-induced decrease inthe hepatic free glutathione/mixed disulfide ratio andthe increase in glutathione consumption results from anincrease in cAMP content. The administration of di-butyryl cAMP and theophylline promptly stimulatesglutathione turnover (Fig. 4). A similar effect of cAMPcan be inferred from the data of Higashi et al. (33) whofound a lower specific activity of hepatic gluta-thione in liver homogenates after administration ofdibutyryl cAMP, a result that would be expected ifcAMPincreased glutathione turnover. That the effect ofcAMP is not purely pharmacologic but rather physio-logic is suggested by our experiments where glucosewas administered intraduodenally and promptlyslowed the rate of glutathione turnover. The glucose

1422 B. H. Lauterburg and J. R. Mitchell

load may be expected to decrease the glucagon/insulinratio, thereby decreasing the hepatic concentration ofcAMP(21, 22). Consequently, the fasting-induced de-crease in the glutathione/mixed disulfide ratio is re-versed, and the concentration of free glutathione in-creases. In order to maintain glutathione synthesis, theexpanded pool thus will have to be renewed lessrapidly. However, other mechanisms might possiblycontribute to the decrease in the slope of the specificactivity time curve, such as the release of glutathioneof a higher specific activity from mixed disulfides. Thiswould have the same effect on the measured fractionalrate of turnover as an increased recirculation of tracer(Fig. 7) and result in an underestimation of the actualrate constant.

These studies do not address the mechanisms bywhich cAMP decreases the concentration of freeglutathione and increases the activity of gamma-gluta-myl transpeptidase. Recent data indicate that cAMPinhibits catalase and, thus, could lead to an increasedoxidation of glutathione to glutathione disulfide byendogenous peroxides via glutathione peroxidase (14).As a consequence of its metabolic effects cAMPmayalso decrease the availability of reducing equivalentsrequired to reduce glutathione disulfide. Both mecha-nisms result in an increase in glutathione disulfide and,as a consequence of disulfide exchange, in a decreasedfree glutathione/protein-mixed disulfide ratio.

One other point emerges from the current experi-ments and merits discussion. The administration oflarge doses of drugs such as acetaminophen results inthe excretion of large quantities of the hepatotoxicmetabolite of the drug as a glutathione adduct, whichappears in the urine as a mercapturic acid congener.In fact, the quantity excreted can exceed by several-fold the amount of glutathione initially present in theliver (36, 37). Since necrosis occurs only after the avail-ability of glutathione is exhausted (38) the liver must beable to synthesize rapidly great quantities of gluta-thione in order to prevent cell damage. Therefore, fast-ing may be a double hazard for liver injury by suchcompounds. First, less free glutathione is present in theliver for detoxification of the reactive drug inter-mediates. Second, glutathione synthesis is alreadymarkedly stimulated during fasting. Thus, the abilityof the liver to synthesize the additional amounts ofglutathione required for detoxification of electrophilicmetabolites is compromised.

ACKNOWLEDGMENTSThis work was supported by grants GM-26611 and GM-13901of the National Institute for General Medical Sciences.

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1424 B. H. Lauterburg and J. R. Mitchell