Chem.-Biol. Interactions, 80 (1991) 89-97
89
Elsevier Scientific Publishers Ireland Ltd.
ORAL GLUTATHIONE INCREASES TISSUE GLUTATHIONE IN VIVO
TAK YEE AWa, GRAZYNA WIERZBICKAb and DEAN P. JONES b
aDepartments of Physiology and Biophysics, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130 and bBiochemistry and Winship Cancer Center, E~dory University School of Medicine, Atlanta, GA 30322 (U.S.A.) (Received March 5th, 1991) (Revision received June llth, 1991) (Accepted June 19th, 1991)
SUMMARY
Mice were given an oral dose of glutathione (GSH) (100 mg/kg) and concentrations of GSH were measured at 30, 45 and 60 min in blood plasma and after 1 h in liver, kidney, heart, lung, brain, small intestine and skin. In control mice, GSH concentrations in plasma increased from. 30 #M to 75 t~M within 30 min of oral GSH administration, consistent with a rapid flux of GSH from the intestinal lumen to plasma. Under these GSH-sufficient conditions, no increases over control values were obtained in GSH concentrations in most tissues except lung over the same time course. Mice pretreated for 5 days with the GSH synthesis inhibitor, a-buthionine-S,R-sulfoximine (BSO, 80 #mol/day) had substantially decreased tissue concentrations of GSH. Oral administration of GSH to these GSH-deficient animals gave statistically significant increases in GSH concentrations in kidney, heart, lung, brain, small intestine and skin but not in the liver. Administration of the equivalent amount of the constituent amino acids, glutamate, cysteine, and glycine, resu!ted in little change in GSH concentrations in all tissues in GSH-deficient animals. Thus, the results show that oral GSH can increase GSH concentrations in several tissues following GSH depletion, such as can occur in toxicological and pathological conditions in which GSH homeostasis is compromised.
Key words: Glutathione -- Mice -- Oral GSH supplementation -- Tissue GSH
Correspondence to: Dr. Dean P. Jones, Department of Biochemistry, 0 Wayne Rollins Research Building, Emory University School of Medicine, Atlanta, GA 30322, U.S.A. 0009-2797/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
90 INTRODUCTION Studies in rats have shown that GSH can be absorbed intact in isolated vascularly perfused small intestine [1] and from the intestinal lumen in vivo [2]. Other studies have provided evidence that orally administered GSH increases blood plasma GSH concentration [3] and protects against toxic and pathological processes in vivo [4]. In a variety of epithelial cells, e.g. enterocytes [5], type II alveolar cells [6], renal proximal tubular cells [7], endothelial cells [8] and retinal pigmented epithelial cells [9], exogenously supplied GSH was shown to be taken up via Na÷-coupled transport systems. In all instances, the uptake of intact GSH afforded protection of cells against reactive oxidants. These studies therefore suggest that oral GSH can be directly used in tissues for GSHdependent detoxication reactions. However, the physiological significance of GSH supplementation and the extent to which luminal GSH can be utilized to enhance tissue GSH concentrations in vivo have not been addressed. The current study was designed to determine whether oral GSH increases tissue GSH concentrations. GSH was administered to mice at a dose and over a time course that provided increased plasma GSH concentrations. Five groups of animals were studied; two of these were not pretreated and the other three were pretreated with the GSH synthesis inhibitor, L-buthionine-S,R-sulfoximine (BSO). The untreated animals were given normal saline or GSH in normal saline by gavage. The BSO-treated animals were given BSO for 5 days prior to receiving normal saline, saline with 0.5% (w/v) GSH or saline with an equivalent amount of the constituent amino acids. The results show that oral GSH increases plasma GSH but not tissue GSH in control, untreated animals. However, significant increases in GSH occur in plasma as well as in several tissues in BSOtreated animals. Tissue GSH concentrations were not increased in animals given the constituent amino acids. Because amino acid administration did not give a corresponding increase in tissue GSH concentrations in the BSO-treated animals, the results are consistent with the suggestion that oral GSH can directly be used to enhance tissue content under GSH-deficient conditions. MATERIALS AND METHODS
Materials L-Buthionine-S,R-sulfoximine, glutathione, glutamate, cysteine, and glycine were obtained from Sigma Chemical Corp (St. Louis). Other reagents were at least of reagent grade and were purchased locally. Deionized water was used for all analytical assays.
Animal treatments, oral administration of GSH, and tissue sampling Mice (outbred albino ICR, 3 0 - 4 0 g) were purchased from Sasco (Omaha, Nebraska), and were maintained on mouse chow and water ad libitum for I week in the vivarium prior to BSO treatment. In the experimental group, mice received 20 mM BSO in their drinking water for 5 days before the start of the experiments. From the volume of water consumed, BSO consumption was about
91
80 #mol/day per mouse. During the 24 h prior to experimentation, food was removed, but water was continued in both control and BSO-treated animals. Mice were given 0.3 ml normal saline by gavage either without or with GSH or amino acids 1 h prior to sampling of blood and tissues. Animals were anesthetized lightly with ether and blood was obtained by heart puncture into heparinized syringes. A 10-ml cold saline solution (0.9% NaC1) was then injected through the heart to wash the organs. Animals were sacrificed by cutting through the diaphragm and the inferior vena cava, and the relevant tissues were rapidly excised, weighed, and placed in ice-cold 30% trichloroacetic acid. Tissues were immediately homogenized and the acid-soluble fractions were assayed for total acid-soluble thiols or derivatized for HPLC analyses as described below.
Glutathione assays GSH content, measured as acid-soluble thiols, was determined colorimetrically by the method of Saville [10]. Plasma and tissue samples of BSO-treated controls and those supplemented with GSH and amino acids were also derivatized with iodoacetic acid and Sanger's reagent and quantified by HPLC [11]. The results showed that the quantitative values of GSH determined by both methods correlate well (Table I) and suggest that the Saville assay measured principally reduced GSH under these conditions. For all conditions, measurement by HPLC verified that most of the thiol detected by the Saville assay is GSH. In some experiments, GSH was also determined enzymatically according to Tietze [12] to confirm that GSH measurements in the BSO-treated animals largely reflected reduced GSH concentrations. Statistical analyses Analyses were performed using the Student t-test.
TABLE I COMPARISON OF TISSUE GSH DETERMINATIONS BY COLORIMETRIC ASSAY AND HPLC Tissue GSH concentrations were determined as the total acid-soluble thiols by the colorimetric assay of Saville [10] or as the dinitrophenyl derivative by HPLC [11]. The results given are the mean of two animals which had been pretreated with BSO. The data are comparable to previously reported values for these tissues following BSO treatment [14]. Organ
Colorimetric method GSH, #mo]/g tissue
Liver Kidney Heart Lung Brain Intestine Skin
2.5 1.0 2.30 0.92 0.98 2.38 1.14
92 R E S U L T S A N D DISCUSSION
Oral administration of GSH to control mice significantly increased the plasma GSH concentrations. The maximal plasma GSH concentration occurred within 30 rain at 75 ~M, a value that is 2.5-fold above the baseline level (Fig. 1). This level declined to 50 ~M by 1 h (Fig. 1). These results are consistent with previous results obtained with rats [3] and humans [13] which show that oral administration of GSH increases plasma GSH. To determine whether GSH is accumulated in tissues over a similar time course, we quantified the tissue GSH concentrations in several tissues. These tissues were selected based on our previous studies in cells in which we have found GSH to be transported, i.e. kidney, small intestine and lung. In addition, several other major tissues were also sampled, including brain, liver, skin and heart. A typical result is shown in Fig. 2. Treatment with BSO for 5 days decreases tissue GSH substantially, yet GSH is readily identifiable in extracts (middle left). Oral supplementation with the constitutive amino acids (100 mg/kg) 1 h prior to analysis did not significantly increase kidney GSH in this BSO-inhibited state (top right). However, administration of GSH (100 mg/kg) resulted in a 2 - 4-fold increase in tissue GSH (top left) relative to the BSO-treated animals. Thus, oral supplementation with GSH increases kidney GSH in BSO-treated animals. GSH concentrations in tissues from BSO-treated animals without GSH administration ranged from 1.2 ~mol/g in skin to 7.0 ~mol/g in liver (Table II) and were comparable to previously reported values [14]. Administration of oral GSH to control animals resulted in little change in tissue levels for most organs except lung (Table II) despite a 2.5-fold increase in plasma GSH (Fig. 1), indicating that under GSH-replete conditions, cellular GSH homeostasis is tightly controlled. The wide range in GSH concentrations in different tissues is, however, somewhat surprising because the feed back regulation is thought to be the same
100
l
0 tD O
E
0 0
30
60
Time, mln
Fig. 1. Time course of appearance of GSH in plasma following oral administration of 100 mg/kg GSH. Blood was obtained by cardiac puncture and GSH concentrations were determined by HPLC following derivatization with iodoacetic acid and Sanger's reagent [11]. Results represent the mean ± S.E.M. of 4 mice for 0 and 30 min and 3 mice for 45 and 60 rain.
I .l_f ~
}i ~[-,
e--
Sh-
Fig. 2. HPLC analysis of GSH in kidney following BSO treatment. Left and right panels were run on different instruments. Bottom tracing in each case was 25 ~1 of 59/~M 08}/star~dard. Left panel: Standard GSH, 28.27 rain peak, 5.63 × 10 ~ integra2 units; middle tracing is from kidney extract of mouse treated for 5 days with BSO {GSH, 4.69 × 104 integral units); top tracing is from kidney extract of BSO-treated mouse that bad been given 100 mg GSH/kg body weight by garage 1 h prior to removal of kidney (GSH, 17.2 × i(}~ integral units). Right panel: Standard GSH, 25.93 rain peak, 5.73 × 106 integral units; top tracing is from k}dney extract of BSO-treated mouse that had been given an equivalent of the constituent amino acids of GSH (GSH, 3.06 × 10 ~ integral units}. Each tracing is representative of data from 4 to 5 animals. Experimental tracings are equal volumes and extracted from equiva|e~t amounts of tissue. GSH from analyses of control kidneys gave 56 × 106 to 78 × l0 s integral units under these conditions.
r---. r
Oca
94 TABLE II T I S S U E GSH CONCENTRATIONS IN CONTROL AND BSO-TREATED RATS WITHOUT OR W I T H ORAL S U P P L E M E N T A T I O N OF GSH OR AMINO ACIDS Tissue GSH concentrations were determined as the total acid-soluble thiols [10]. The results represent the mean ± S.E.M. for 4 animals. In all cases, results were independently confirmed by HPLC analysis. Control vs. GSH, not significant for all tissues except lung at P < 0 . 0 0 5 . Control vs. BSO, significant for all tissues at P < 0.005. BSO vs. BSO + GSH, significant at P < 0.005 for all tissues except liver. BSO vs. BSO + amino acids, not significant at P < 0.05 for all tissues. BSO + GSH vs. BSO + amino acids, significant at P < 0.005 for kidney, heart, lung, intestine and skin; at P < 0.05 for brain; not significant with respect to liver. Organ
GSH, ~mol/g tissue Control
Liver Kidney Heart Lung Brain Intestine Skin
7.02 4.10 1.52 2.63 2.45 2.35 1.21
± ± ± ± ± ± ±
GSH
0.51 0.42 0.10 0.38 0.29 0.07 0.09
7.42 3.88 1.93 3.37 2.18 2.01 1.50
BS0
± ± ± ± ± ± ±
0.48 0.64 0.12 0.33 0.11 0.15 0.19
2.91 0.28 0.20 0.38 1.30 1.90 0.09
BSO + GSH ± ± ± ± ± ± +
0.26 0.03 0.02 0.05 0.13 0.21 0.01
2.95 0.86 0.36 0.78 2.16 2.98 0.20
± ± ± ± ± ± ±
BSO + amino acids 0.31 0.14 0.01 0.03 0.18 0.17 0.01
2.30 0.34 0.17 0.30 1.46 1.97 0.11
± ± ± ± ± ± ±
0.10 0.03 0.02 0.05 1.46 0.12 0.02
in different tissues. The varied GSH concentrations in these tissues could be explained by variations in transport activities, but the current results show that increased plasma GSH does not affect tissue GSH concentrations under GSH-replete conditions. Furthermore, animals fasted for 24 h would be expected to have decreased GSH concentrations because GSH functions as a cysteine store [15]. The lack of a change in tissue GSH concentrations therefore indicates that a rapid adjustment of concentration does not occur following GSH administration. Administration of BSO, the GSH synthesis inhibitor, in drinking water for 5 days resulted in dramatic decreases in tissue GSH concentrations (Table II). Again, a curiosity of these data is that the residual GSH concentrations vary so widely and considerably as the fraction of GSH concentration that remained, i.e. the values ranged from 0.1 ~mol/g in skin to 2.9 ~mol/g in liver (Table II). Administration of GSH to these animals by gavage resulted in significant increases in GSH in all tissues except the liver (Table II). The results are consistent with the uptake of plasma GSH to supplement cellular GSH stores as was previously found for a variety of epithelial cells [5 - 9]. The lack of an increase in liver GSH is consistent with the inability of this tissue to take up exogenous GSH and with the fact that the liver is a supplier of GSH to the plasma via carrier-mediated efflux mechanisms [16,17]. Of all the tissues examined, the GSH levels in brain and small intestine returned to normal values compared to the other tissues (Table II). The reasons for these differences among the various tissues are not
96
tor. Thus, uptake could involve ~-giutamylcysteine and an acceptor species that is formed by GSH and function as a precursor in the cell [18]. Third, GSH can react spontaneously and reversibly with electrophilic compounds. For example, S-hydroxymethyl glutathione, formed from the reaction of GSH and formaldehyde, may be more permeable to the plasma membrane than is GSH [19]. Hence, uptake of GSH by tissues could involve reversible formation of an adduct that is permeable. Fourth, GSH can be transported as a mixed disulfide with cysteine or with a plasma protein such as albumin. The net effect of any of the above reactions is that GSH is taken up into cells, but the present results do not distinguish among these mechanisms for the increase in tissue GSH concentrations following oral supplementation. Regardless of mechanism(s) of uptake, the physiological effect of increasing tissue GSH by oral feeding has important nutritional and toxicological implications, particularly as pertains to pathological and toxicological conditions. Several examples of medical conditions in which increasing GSH supply in cells could enhance protection and minimize injury are: (a) oxidative injury to the lung which occurs from oxygen therapy, cigarette smoke and atmospheric pollutants [20]; (b) oxidative injury to the skin from UV radiation [21]; (c) injury to the heart and lung from antitumor therapy [22,23]; and (d) injury to the kidney [24] and small intestine [25] from reperfusion following ischemia. Several studies have provided evidence that oral supplementation can provide beneficial results, e.g. against paraquat poisoning [26], chemical carcinogenesis [27,28] and decline of immune function with aging [29]. Additional studies are needed to assess the efficacy of GSH supplementation in protection against electrophilic and oxidative damage in these various tissues in vivo. REFERENCES 1 T.M. Hagen and D.P. Jones, Transepithelial transport of glutathione in vascularly perfused small intestine of rat, Am. J. Physiol., 252 (1987) G607-G613. 2 T.M. Hagen, G.T. Wierzbicka, B.B. Bowman, T.Y. Aw and D.P. Jones, Fate of dietary glutathione; disposition in the gastrointestinal tract, Am. J. Physiol., 259 (1990) G530- G535. 3 T.M. Hagen, G.T. Wierzbicka, A.H. Sillau, B.B. Bowman and D.P. Jones, Bioavailability of dietary glutathione: effect on plasma concentration, Am. J. Physiol., 259 (1990) G524-G529. 4 M.S. Paller and B.E. Hedlund, Role of iron in postischemic renal injury in the rat, Kidney Int., 34 (1988) 474- 480. 5 L.H. Lash, T.M. Hagen and D.P. Jones, Exogenous glutathione protects intestinal epithelial cells from oxidative injury, Proc. Natl. Acad. Sci. USA, 83 {1986) 4641-4645. 6 T.M. Hagen, L.A. Brown and D.P. Jones, Protection against paraquat induced injury by exogenous GSH in pulmonary alveolar type II cells, Biochem. Pharmacol., 35 (1986} 4537- 4542. 7 T.M. Hagen, T.Y. Aw and D.P. Jones, Glutathione uptake and protection against oxidative injury in isolated kidney cells, Kidney Int., 34 (1988) 74-81. 8 S.P. Andreoli, C.P. Mallett and J.M. Bergstein, Role of glutathione in protecting endothelial cells against hydrogen peroxide oxidant injury, J. Lab. Clin. Med., 108 (1986) 190-198. 9 T.M. Hagen, J.N. Hartman, D.P. Jones and P. Sternberg, Exogenous glutathione protects cultured human retinal pigment epithelial cells from oxidative injury, Invest. Ophthalmol. Vis. Sci., 28 (1987) 255. 10 B. Saville, A scheme for the colorimetric determination of microgram amounts of thiols, Analyst, 83 (1958) 670-672.
97 11 D.J. Reed, J.R. Babson, P.W. Beatty, A.E. Brodie, W.W. Ellis and D.W. Potter, Highperformance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide and related thiols and disulfides, Anal. Biochem., 106 (1980) 55-62. 12 F. Tietze, Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues, Anal. Biochem., 27 (1969) 502- 522. 13 D.P. Jones, T.M. Hagen, R. Weber, G.T. Wierzbicka and H.L. Bonkovsky. Oral administration of glutathione (GSH) increases plasma GSH concentration in humans, FASEB J., 3 (1989) A1250. 14 A. Meister, Metabolism and transport of glutathione and other -~-glutamyl compounds, in: A. Larsson, S. Orrenius, A. Holgren and B. Mannervik (Eds.),, Functions of Glutathione: Biochemical, Physiological, Toxicological and Clinical Aspects, Raven Press, New York, 1983, pp. 1-30. 15 N. Tateishi and Y. Sakamoto, Nutritional significance of glutathione in rat liver, in: Y. Sakamoto, T. Higashi and N. Tateishi (Eds.), Glutathione: Storage, Transport and Turnover in Mammals, Japan Sci. Soc. Press, Tokyo, 1983, pp. 13-38. 16 M. Ookhtens, K. Hobdy, M.C. Corvasce, T.Y. Aw and N. Kaplowitz, Sinusoidal efflux of glutathione in the perfused liver: Evidence of a carrier-mediated process, J. Clin. Invest., 75 (1985) 258 - 265. 17 T.Y. Aw, M. Ookhtens, C. Ren and N. Kaplowitz, The kinetics of glutathione effiux from isolated rat hepatocytes, Am. J. Physiol., 250 (1986) G236-G243. 18 A. Meister, Glutathione and related ~-glutamyl compounds: biosynthesis and utilization, Annu. Rev. Biochem., 45 (1976)559-604. 19 L. Eklow-Lastbom, P. Moldeus and S. Orrenius, On the mechanisms of glutathione depletion in hepatocytes exposed to morphine and ethylmorphine, Toxicology, 42 (1986) 13-21. 20 J. Goerke, Lung surfactant, Biochim. Biophys. Acta, 344 (1974) 241-261. 21 C. yon Sontag, U. Hagen, A. Schon-Bopp and D. Schulte-Frohlinde, Radiation-induced strand breaks in DNA: Chemical and enzymatic analysis of end group and mechanistic aspects, Adv. Radiat. Biol., 9 (1981) 109-142. 22 A.C. Smith and M.R. Boyd, Preferential effects of 1,3-bis(2-chloroethyl)-l-nitrosourea(BCNU) on pulmonary glutathione reductase and glutathione/glutathione disulfide ratios: possible implications for lung toxicity, J. Pharmacol. Exp. Ther., 229 (1984) 658-663. 23 H.P. Witschi and R.C. Lindensahmidt, Pathogenesis of acute and chronic lung injury induced by foreign compounds, Clin. Physiol. Biochem., 3 (1985) 135-146. 24 M.S. Paller, Hyperthyroidism protects against free radical damage in ischemic acute renal failure, Kidney Int., 29 (1986) 1162-1166. 25 M.B. Grisham and D.N. Granger, Neutrophil-mediated mucosal injury. Role of reactive oxygen metabolites, Dig. Dis. Sci., 33 (1988) 6S-15S. 26 B. Matkovics, K. Basabas, L. Szabo and G. Berencsi. In vivo study of the mechanism of protective effects of ascorbic acid and reduced glutathione in paraquat poisoning, Gen. Pharmacol., 11 {1980) 455-461. 27 A.M. Novi, Regression of afiatoxin Bl-induced hepatoceltular carcinomas by reduced glutathione, Science 212 (1981) 541- 542. 28 A.M. Novi, R. F15rke and M. Stukenkemper. Glutathione and aflatoxin-Bl-induced liver tumors: requirement for an intact glutathione molecule for regression of malignancy in neoplastic tissue, Ann. NY Acad. Sci., 397 (1982) 62-71. 29 T. Furukawa, S.N. Meydani and J.B. Blumberg. Reversal of age-associated decline in immune responsiveness by dietary glutathione supplementation in mice, Mech. Ageing Dev., 38 (1987) 107-117.
89
Elsevier Scientific Publishers Ireland Ltd.
ORAL GLUTATHIONE INCREASES TISSUE GLUTATHIONE IN VIVO
TAK YEE AWa, GRAZYNA WIERZBICKAb and DEAN P. JONES b
aDepartments of Physiology and Biophysics, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130 and bBiochemistry and Winship Cancer Center, E~dory University School of Medicine, Atlanta, GA 30322 (U.S.A.) (Received March 5th, 1991) (Revision received June llth, 1991) (Accepted June 19th, 1991)
SUMMARY
Mice were given an oral dose of glutathione (GSH) (100 mg/kg) and concentrations of GSH were measured at 30, 45 and 60 min in blood plasma and after 1 h in liver, kidney, heart, lung, brain, small intestine and skin. In control mice, GSH concentrations in plasma increased from. 30 #M to 75 t~M within 30 min of oral GSH administration, consistent with a rapid flux of GSH from the intestinal lumen to plasma. Under these GSH-sufficient conditions, no increases over control values were obtained in GSH concentrations in most tissues except lung over the same time course. Mice pretreated for 5 days with the GSH synthesis inhibitor, a-buthionine-S,R-sulfoximine (BSO, 80 #mol/day) had substantially decreased tissue concentrations of GSH. Oral administration of GSH to these GSH-deficient animals gave statistically significant increases in GSH concentrations in kidney, heart, lung, brain, small intestine and skin but not in the liver. Administration of the equivalent amount of the constituent amino acids, glutamate, cysteine, and glycine, resu!ted in little change in GSH concentrations in all tissues in GSH-deficient animals. Thus, the results show that oral GSH can increase GSH concentrations in several tissues following GSH depletion, such as can occur in toxicological and pathological conditions in which GSH homeostasis is compromised.
Key words: Glutathione -- Mice -- Oral GSH supplementation -- Tissue GSH
Correspondence to: Dr. Dean P. Jones, Department of Biochemistry, 0 Wayne Rollins Research Building, Emory University School of Medicine, Atlanta, GA 30322, U.S.A. 0009-2797/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
90 INTRODUCTION Studies in rats have shown that GSH can be absorbed intact in isolated vascularly perfused small intestine [1] and from the intestinal lumen in vivo [2]. Other studies have provided evidence that orally administered GSH increases blood plasma GSH concentration [3] and protects against toxic and pathological processes in vivo [4]. In a variety of epithelial cells, e.g. enterocytes [5], type II alveolar cells [6], renal proximal tubular cells [7], endothelial cells [8] and retinal pigmented epithelial cells [9], exogenously supplied GSH was shown to be taken up via Na÷-coupled transport systems. In all instances, the uptake of intact GSH afforded protection of cells against reactive oxidants. These studies therefore suggest that oral GSH can be directly used in tissues for GSHdependent detoxication reactions. However, the physiological significance of GSH supplementation and the extent to which luminal GSH can be utilized to enhance tissue GSH concentrations in vivo have not been addressed. The current study was designed to determine whether oral GSH increases tissue GSH concentrations. GSH was administered to mice at a dose and over a time course that provided increased plasma GSH concentrations. Five groups of animals were studied; two of these were not pretreated and the other three were pretreated with the GSH synthesis inhibitor, L-buthionine-S,R-sulfoximine (BSO). The untreated animals were given normal saline or GSH in normal saline by gavage. The BSO-treated animals were given BSO for 5 days prior to receiving normal saline, saline with 0.5% (w/v) GSH or saline with an equivalent amount of the constituent amino acids. The results show that oral GSH increases plasma GSH but not tissue GSH in control, untreated animals. However, significant increases in GSH occur in plasma as well as in several tissues in BSOtreated animals. Tissue GSH concentrations were not increased in animals given the constituent amino acids. Because amino acid administration did not give a corresponding increase in tissue GSH concentrations in the BSO-treated animals, the results are consistent with the suggestion that oral GSH can directly be used to enhance tissue content under GSH-deficient conditions. MATERIALS AND METHODS
Materials L-Buthionine-S,R-sulfoximine, glutathione, glutamate, cysteine, and glycine were obtained from Sigma Chemical Corp (St. Louis). Other reagents were at least of reagent grade and were purchased locally. Deionized water was used for all analytical assays.
Animal treatments, oral administration of GSH, and tissue sampling Mice (outbred albino ICR, 3 0 - 4 0 g) were purchased from Sasco (Omaha, Nebraska), and were maintained on mouse chow and water ad libitum for I week in the vivarium prior to BSO treatment. In the experimental group, mice received 20 mM BSO in their drinking water for 5 days before the start of the experiments. From the volume of water consumed, BSO consumption was about
91
80 #mol/day per mouse. During the 24 h prior to experimentation, food was removed, but water was continued in both control and BSO-treated animals. Mice were given 0.3 ml normal saline by gavage either without or with GSH or amino acids 1 h prior to sampling of blood and tissues. Animals were anesthetized lightly with ether and blood was obtained by heart puncture into heparinized syringes. A 10-ml cold saline solution (0.9% NaC1) was then injected through the heart to wash the organs. Animals were sacrificed by cutting through the diaphragm and the inferior vena cava, and the relevant tissues were rapidly excised, weighed, and placed in ice-cold 30% trichloroacetic acid. Tissues were immediately homogenized and the acid-soluble fractions were assayed for total acid-soluble thiols or derivatized for HPLC analyses as described below.
Glutathione assays GSH content, measured as acid-soluble thiols, was determined colorimetrically by the method of Saville [10]. Plasma and tissue samples of BSO-treated controls and those supplemented with GSH and amino acids were also derivatized with iodoacetic acid and Sanger's reagent and quantified by HPLC [11]. The results showed that the quantitative values of GSH determined by both methods correlate well (Table I) and suggest that the Saville assay measured principally reduced GSH under these conditions. For all conditions, measurement by HPLC verified that most of the thiol detected by the Saville assay is GSH. In some experiments, GSH was also determined enzymatically according to Tietze [12] to confirm that GSH measurements in the BSO-treated animals largely reflected reduced GSH concentrations. Statistical analyses Analyses were performed using the Student t-test.
TABLE I COMPARISON OF TISSUE GSH DETERMINATIONS BY COLORIMETRIC ASSAY AND HPLC Tissue GSH concentrations were determined as the total acid-soluble thiols by the colorimetric assay of Saville [10] or as the dinitrophenyl derivative by HPLC [11]. The results given are the mean of two animals which had been pretreated with BSO. The data are comparable to previously reported values for these tissues following BSO treatment [14]. Organ
Colorimetric method GSH, #mo]/g tissue
Liver Kidney Heart Lung Brain Intestine Skin
2.5 1.0 2.30 0.92 0.98 2.38 1.14
92 R E S U L T S A N D DISCUSSION
Oral administration of GSH to control mice significantly increased the plasma GSH concentrations. The maximal plasma GSH concentration occurred within 30 rain at 75 ~M, a value that is 2.5-fold above the baseline level (Fig. 1). This level declined to 50 ~M by 1 h (Fig. 1). These results are consistent with previous results obtained with rats [3] and humans [13] which show that oral administration of GSH increases plasma GSH. To determine whether GSH is accumulated in tissues over a similar time course, we quantified the tissue GSH concentrations in several tissues. These tissues were selected based on our previous studies in cells in which we have found GSH to be transported, i.e. kidney, small intestine and lung. In addition, several other major tissues were also sampled, including brain, liver, skin and heart. A typical result is shown in Fig. 2. Treatment with BSO for 5 days decreases tissue GSH substantially, yet GSH is readily identifiable in extracts (middle left). Oral supplementation with the constitutive amino acids (100 mg/kg) 1 h prior to analysis did not significantly increase kidney GSH in this BSO-inhibited state (top right). However, administration of GSH (100 mg/kg) resulted in a 2 - 4-fold increase in tissue GSH (top left) relative to the BSO-treated animals. Thus, oral supplementation with GSH increases kidney GSH in BSO-treated animals. GSH concentrations in tissues from BSO-treated animals without GSH administration ranged from 1.2 ~mol/g in skin to 7.0 ~mol/g in liver (Table II) and were comparable to previously reported values [14]. Administration of oral GSH to control animals resulted in little change in tissue levels for most organs except lung (Table II) despite a 2.5-fold increase in plasma GSH (Fig. 1), indicating that under GSH-replete conditions, cellular GSH homeostasis is tightly controlled. The wide range in GSH concentrations in different tissues is, however, somewhat surprising because the feed back regulation is thought to be the same
100
l
0 tD O
E
0 0
30
60
Time, mln
Fig. 1. Time course of appearance of GSH in plasma following oral administration of 100 mg/kg GSH. Blood was obtained by cardiac puncture and GSH concentrations were determined by HPLC following derivatization with iodoacetic acid and Sanger's reagent [11]. Results represent the mean ± S.E.M. of 4 mice for 0 and 30 min and 3 mice for 45 and 60 rain.
I .l_f ~
}i ~[-,
e--
Sh-
Fig. 2. HPLC analysis of GSH in kidney following BSO treatment. Left and right panels were run on different instruments. Bottom tracing in each case was 25 ~1 of 59/~M 08}/star~dard. Left panel: Standard GSH, 28.27 rain peak, 5.63 × 10 ~ integra2 units; middle tracing is from kidney extract of mouse treated for 5 days with BSO {GSH, 4.69 × 104 integral units); top tracing is from kidney extract of BSO-treated mouse that bad been given 100 mg GSH/kg body weight by garage 1 h prior to removal of kidney (GSH, 17.2 × i(}~ integral units). Right panel: Standard GSH, 25.93 rain peak, 5.73 × 106 integral units; top tracing is from k}dney extract of BSO-treated mouse that had been given an equivalent of the constituent amino acids of GSH (GSH, 3.06 × 10 ~ integral units}. Each tracing is representative of data from 4 to 5 animals. Experimental tracings are equal volumes and extracted from equiva|e~t amounts of tissue. GSH from analyses of control kidneys gave 56 × 106 to 78 × l0 s integral units under these conditions.
r---. r
Oca
94 TABLE II T I S S U E GSH CONCENTRATIONS IN CONTROL AND BSO-TREATED RATS WITHOUT OR W I T H ORAL S U P P L E M E N T A T I O N OF GSH OR AMINO ACIDS Tissue GSH concentrations were determined as the total acid-soluble thiols [10]. The results represent the mean ± S.E.M. for 4 animals. In all cases, results were independently confirmed by HPLC analysis. Control vs. GSH, not significant for all tissues except lung at P < 0 . 0 0 5 . Control vs. BSO, significant for all tissues at P < 0.005. BSO vs. BSO + GSH, significant at P < 0.005 for all tissues except liver. BSO vs. BSO + amino acids, not significant at P < 0.05 for all tissues. BSO + GSH vs. BSO + amino acids, significant at P < 0.005 for kidney, heart, lung, intestine and skin; at P < 0.05 for brain; not significant with respect to liver. Organ
GSH, ~mol/g tissue Control
Liver Kidney Heart Lung Brain Intestine Skin
7.02 4.10 1.52 2.63 2.45 2.35 1.21
± ± ± ± ± ± ±
GSH
0.51 0.42 0.10 0.38 0.29 0.07 0.09
7.42 3.88 1.93 3.37 2.18 2.01 1.50
BS0
± ± ± ± ± ± ±
0.48 0.64 0.12 0.33 0.11 0.15 0.19
2.91 0.28 0.20 0.38 1.30 1.90 0.09
BSO + GSH ± ± ± ± ± ± +
0.26 0.03 0.02 0.05 0.13 0.21 0.01
2.95 0.86 0.36 0.78 2.16 2.98 0.20
± ± ± ± ± ± ±
BSO + amino acids 0.31 0.14 0.01 0.03 0.18 0.17 0.01
2.30 0.34 0.17 0.30 1.46 1.97 0.11
± ± ± ± ± ± ±
0.10 0.03 0.02 0.05 1.46 0.12 0.02
in different tissues. The varied GSH concentrations in these tissues could be explained by variations in transport activities, but the current results show that increased plasma GSH does not affect tissue GSH concentrations under GSH-replete conditions. Furthermore, animals fasted for 24 h would be expected to have decreased GSH concentrations because GSH functions as a cysteine store [15]. The lack of a change in tissue GSH concentrations therefore indicates that a rapid adjustment of concentration does not occur following GSH administration. Administration of BSO, the GSH synthesis inhibitor, in drinking water for 5 days resulted in dramatic decreases in tissue GSH concentrations (Table II). Again, a curiosity of these data is that the residual GSH concentrations vary so widely and considerably as the fraction of GSH concentration that remained, i.e. the values ranged from 0.1 ~mol/g in skin to 2.9 ~mol/g in liver (Table II). Administration of GSH to these animals by gavage resulted in significant increases in GSH in all tissues except the liver (Table II). The results are consistent with the uptake of plasma GSH to supplement cellular GSH stores as was previously found for a variety of epithelial cells [5 - 9]. The lack of an increase in liver GSH is consistent with the inability of this tissue to take up exogenous GSH and with the fact that the liver is a supplier of GSH to the plasma via carrier-mediated efflux mechanisms [16,17]. Of all the tissues examined, the GSH levels in brain and small intestine returned to normal values compared to the other tissues (Table II). The reasons for these differences among the various tissues are not
96
tor. Thus, uptake could involve ~-giutamylcysteine and an acceptor species that is formed by GSH and function as a precursor in the cell [18]. Third, GSH can react spontaneously and reversibly with electrophilic compounds. For example, S-hydroxymethyl glutathione, formed from the reaction of GSH and formaldehyde, may be more permeable to the plasma membrane than is GSH [19]. Hence, uptake of GSH by tissues could involve reversible formation of an adduct that is permeable. Fourth, GSH can be transported as a mixed disulfide with cysteine or with a plasma protein such as albumin. The net effect of any of the above reactions is that GSH is taken up into cells, but the present results do not distinguish among these mechanisms for the increase in tissue GSH concentrations following oral supplementation. Regardless of mechanism(s) of uptake, the physiological effect of increasing tissue GSH by oral feeding has important nutritional and toxicological implications, particularly as pertains to pathological and toxicological conditions. Several examples of medical conditions in which increasing GSH supply in cells could enhance protection and minimize injury are: (a) oxidative injury to the lung which occurs from oxygen therapy, cigarette smoke and atmospheric pollutants [20]; (b) oxidative injury to the skin from UV radiation [21]; (c) injury to the heart and lung from antitumor therapy [22,23]; and (d) injury to the kidney [24] and small intestine [25] from reperfusion following ischemia. Several studies have provided evidence that oral supplementation can provide beneficial results, e.g. against paraquat poisoning [26], chemical carcinogenesis [27,28] and decline of immune function with aging [29]. Additional studies are needed to assess the efficacy of GSH supplementation in protection against electrophilic and oxidative damage in these various tissues in vivo. REFERENCES 1 T.M. Hagen and D.P. Jones, Transepithelial transport of glutathione in vascularly perfused small intestine of rat, Am. J. Physiol., 252 (1987) G607-G613. 2 T.M. Hagen, G.T. Wierzbicka, B.B. Bowman, T.Y. Aw and D.P. Jones, Fate of dietary glutathione; disposition in the gastrointestinal tract, Am. J. Physiol., 259 (1990) G530- G535. 3 T.M. Hagen, G.T. Wierzbicka, A.H. Sillau, B.B. Bowman and D.P. Jones, Bioavailability of dietary glutathione: effect on plasma concentration, Am. J. Physiol., 259 (1990) G524-G529. 4 M.S. Paller and B.E. Hedlund, Role of iron in postischemic renal injury in the rat, Kidney Int., 34 (1988) 474- 480. 5 L.H. Lash, T.M. Hagen and D.P. Jones, Exogenous glutathione protects intestinal epithelial cells from oxidative injury, Proc. Natl. Acad. Sci. USA, 83 {1986) 4641-4645. 6 T.M. Hagen, L.A. Brown and D.P. Jones, Protection against paraquat induced injury by exogenous GSH in pulmonary alveolar type II cells, Biochem. Pharmacol., 35 (1986} 4537- 4542. 7 T.M. Hagen, T.Y. Aw and D.P. Jones, Glutathione uptake and protection against oxidative injury in isolated kidney cells, Kidney Int., 34 (1988) 74-81. 8 S.P. Andreoli, C.P. Mallett and J.M. Bergstein, Role of glutathione in protecting endothelial cells against hydrogen peroxide oxidant injury, J. Lab. Clin. Med., 108 (1986) 190-198. 9 T.M. Hagen, J.N. Hartman, D.P. Jones and P. Sternberg, Exogenous glutathione protects cultured human retinal pigment epithelial cells from oxidative injury, Invest. Ophthalmol. Vis. Sci., 28 (1987) 255. 10 B. Saville, A scheme for the colorimetric determination of microgram amounts of thiols, Analyst, 83 (1958) 670-672.
97 11 D.J. Reed, J.R. Babson, P.W. Beatty, A.E. Brodie, W.W. Ellis and D.W. Potter, Highperformance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide and related thiols and disulfides, Anal. Biochem., 106 (1980) 55-62. 12 F. Tietze, Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues, Anal. Biochem., 27 (1969) 502- 522. 13 D.P. Jones, T.M. Hagen, R. Weber, G.T. Wierzbicka and H.L. Bonkovsky. Oral administration of glutathione (GSH) increases plasma GSH concentration in humans, FASEB J., 3 (1989) A1250. 14 A. Meister, Metabolism and transport of glutathione and other -~-glutamyl compounds, in: A. Larsson, S. Orrenius, A. Holgren and B. Mannervik (Eds.),, Functions of Glutathione: Biochemical, Physiological, Toxicological and Clinical Aspects, Raven Press, New York, 1983, pp. 1-30. 15 N. Tateishi and Y. Sakamoto, Nutritional significance of glutathione in rat liver, in: Y. Sakamoto, T. Higashi and N. Tateishi (Eds.), Glutathione: Storage, Transport and Turnover in Mammals, Japan Sci. Soc. Press, Tokyo, 1983, pp. 13-38. 16 M. Ookhtens, K. Hobdy, M.C. Corvasce, T.Y. Aw and N. Kaplowitz, Sinusoidal efflux of glutathione in the perfused liver: Evidence of a carrier-mediated process, J. Clin. Invest., 75 (1985) 258 - 265. 17 T.Y. Aw, M. Ookhtens, C. Ren and N. Kaplowitz, The kinetics of glutathione effiux from isolated rat hepatocytes, Am. J. Physiol., 250 (1986) G236-G243. 18 A. Meister, Glutathione and related ~-glutamyl compounds: biosynthesis and utilization, Annu. Rev. Biochem., 45 (1976)559-604. 19 L. Eklow-Lastbom, P. Moldeus and S. Orrenius, On the mechanisms of glutathione depletion in hepatocytes exposed to morphine and ethylmorphine, Toxicology, 42 (1986) 13-21. 20 J. Goerke, Lung surfactant, Biochim. Biophys. Acta, 344 (1974) 241-261. 21 C. yon Sontag, U. Hagen, A. Schon-Bopp and D. Schulte-Frohlinde, Radiation-induced strand breaks in DNA: Chemical and enzymatic analysis of end group and mechanistic aspects, Adv. Radiat. Biol., 9 (1981) 109-142. 22 A.C. Smith and M.R. Boyd, Preferential effects of 1,3-bis(2-chloroethyl)-l-nitrosourea(BCNU) on pulmonary glutathione reductase and glutathione/glutathione disulfide ratios: possible implications for lung toxicity, J. Pharmacol. Exp. Ther., 229 (1984) 658-663. 23 H.P. Witschi and R.C. Lindensahmidt, Pathogenesis of acute and chronic lung injury induced by foreign compounds, Clin. Physiol. Biochem., 3 (1985) 135-146. 24 M.S. Paller, Hyperthyroidism protects against free radical damage in ischemic acute renal failure, Kidney Int., 29 (1986) 1162-1166. 25 M.B. Grisham and D.N. Granger, Neutrophil-mediated mucosal injury. Role of reactive oxygen metabolites, Dig. Dis. Sci., 33 (1988) 6S-15S. 26 B. Matkovics, K. Basabas, L. Szabo and G. Berencsi. In vivo study of the mechanism of protective effects of ascorbic acid and reduced glutathione in paraquat poisoning, Gen. Pharmacol., 11 {1980) 455-461. 27 A.M. Novi, Regression of afiatoxin Bl-induced hepatoceltular carcinomas by reduced glutathione, Science 212 (1981) 541- 542. 28 A.M. Novi, R. F15rke and M. Stukenkemper. Glutathione and aflatoxin-Bl-induced liver tumors: requirement for an intact glutathione molecule for regression of malignancy in neoplastic tissue, Ann. NY Acad. Sci., 397 (1982) 62-71. 29 T. Furukawa, S.N. Meydani and J.B. Blumberg. Reversal of age-associated decline in immune responsiveness by dietary glutathione supplementation in mice, Mech. Ageing Dev., 38 (1987) 107-117.
