ANALYTICAL BIOCHEMISTEY 27, 502-522 (1969)

Enzymic Method for Quantitative Determination of Nanogram Amounts of Total and Oxidized

Glutathione:

Applications to Mammalian Blood and Other Tissues

FRANK TIETZE

National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service, United States Department of Health, Education

and Welfare, Bethesda, Maryland M014

Received June 7, 1968

The widespread distribution of glutathione and its apparent involve- ment in a multitude of biological functions (l-4) have generated a continual interest in methods of analysis o,f this cellular component ever since its discovery and isolation 40 years ago.l Although the tripeptide can exist in both a reduced (sulfhydryl) and an oxidized (disulfide) form it is maintained in viva predominantly in the former state through the action of the equally ubiquitous enzyme glutathione reductase (1). Since the reduced form comprises in most instances the bulk of cellular non- protein sulfhydryl groups, measurement of acid-soluble thiol has been commonly employed for the estimation of GSH levels of tissue extracts in addition to a limited number of more or less specific enzymic (8) or chemical (6) procedures. In contrast, accurate measurement of tissue GSSG levels has proved more difficult, both because of the much lower amounts of this form normally present within cells and because of the absence of a convenient chemical feature such as that possessed by the reduced peptide. Procedures for the estimation of the oxidized form have, therefore, relied generally on its estimation as GSH following its chemical (9)) electrolytic (lo), or enzymic (11, 12) reduction or, preferably, on measurement of the change of absorbancy at 340 rnp following its enzymic reduction by DPNH or TPNH in the presence of glutathione reductase (reaction 1) (13, 14).

The method of glutathione assay described here initially grew out of the need for a procedure for GSSG more sensitive than those obtainable with previously published methods. In accordance with this requirement

l For comprehensive reviews on methods of glutathione analysis see references &7.

502

ENZYMIC ASSAY OF GIJJTATHIONE 503

GSSG + ,PNH + H+ & ZGSH +lPN+ (1)

coo- coo-

an attempt was made to increase the sensitivity of the spectrophotometric procedure illustrated by reaction 1 by incorporating into the reaction mixture the sulfhydryl reagent 5,5’-dithiobis- (2-nitrobenzoic acid) (Ell- man reagent) (15). Since the chromophoric product resulting from re- action of the reagent with GSH, viz., 2-nitro-5-thiobenzoic acid (reaction 2)) possesses a molar absorption at 412 rnp approximately twice that of TPNH at 340 rnp and since, in addition, two moles of GSH are formed per mole of reduced nucleotide utilized in GSSG reduction (reaction 1)) it was anticipated that the inclusion of Ellman reagent in the reaction mixture and subsequent measurement of absorbancy change at 412 rnp should result in a 4-fold increase in sensitivity relative to that obtainable by simple measurement of the disappearance of reduced nucleotide at 340 mp. Contrary to this expectation, the addition of a known amount of GSSG to a model reaction mixture containing DTNB, TPNH, and yeast glutathione reductase resulted in a color yield at 412 rnp greatly in excess of that calculated from the foregoing stoichiometry. Further experiments showed that the rate of excess color development, which also occurred following addition of GSH, depended on the concen- tration of glutathione in the reaction mixture and was still detectable at concentrations as low as 10 nanograms/ml. These observations have been exploited in the development of a highly sensitive and specific procedure for glutathione analysis which is similar in principle to that described recently by Grassetti and Murray (16)) whose published report appeared during the concluding phases of this study. The present paper describes in detail the composition and characteristic properties of this assay system together with some typical applications to the estimation of total and oxidized glutathione contents of blood and other tissues. A preliminary report has appeared (17).

EXPERIMENTAL

Reduced pyridine nucleotides, GSH, and NEM,2 were obtained from Calbiochem. The GSH was found to be contaminated with approximately

‘Abbreviations used: NEM, N-ethylmaleimide; DTNB (Ellman reagent), 5$- dithiobi&-nitrobenzoic acid) ; TCA, trichloroacetic acid; GSH and GSSG, reduced and oxidized glutathione; DPNH and TPNH, reduced di- and triphosphopyridine nucleotide.

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0.8% GSSG (by weight), as determined by titration with TPNH in the presence of glutathione reductase, as described below. Cysteine hydrol chloride and the disodium salt of oxidized glutathione were products of Nutritional Biochemicals Corp. and Sigma Chemical Co., respectively. DTNB2 was purchased from Aldrich Chemical Co. The majority of experiments employing yeast glutathione reductase was carried out with. the crystalline suspension (ca. 100 I.U./mg) distributed by Calbiochem; occasional use was made of preparations possessing similar properties obtained from Sigma and Boehringer. Unless otherwise specified, stock solutions of the various reagents used in the enzymic assays were made up in 0.1 M sodium phosphate/O.005 M EDTA buffer, pH 7.5, referred to. hereafter simply as phosphate-EDTA buffer. Solutions of GSH and cysteine were prepared immediately before use in cold 0.01 N HCl.

Where necessary, the concentrations of GSH and GSSG stock solutions. were established as follows: (1) GSH by titration with DTNB accord- ing to Ellman’s procedure (15) ; (6) GSSG by the change in absorbancy at 340 rnp following its addition to a mixture of excess TPNH and yeast glutathione reductase (13). Rat kidney and liver homogenates were prepared by grinding the blotted and weighed tissues at 0’ for l-2 min at 1009 rpm in a Potter-Elvehjem apparatus with Teflon pestle. Tissue or protein suspensions in trichloroacetic acid were routinely centrifuged at 17,009 g for 15 min at 2O. All spectrophotometric measurements were carried out at 25’ in a Cary model 14 recording spectrophotometer equipped with double-beam optics and with provision for scale expansion.

Rat blood and tissues were obtained from male animals (150-300 gm) of the Sprague-Dawley stain fed ad libitum. The blood was withdrawn by cardiac puncture and maintained in a heparinized condition at 0”. Human saliva was collected following stimulation of secretion by chewing with paraffin wax.

The procedure for determining the total glutathione content (GSH + GSSG) of whole blood was as follows: 10 pl of blood, obtained from the rat as described or from normal human subjects by finger puncture, was hemolyzed in 0.99 ml cold 0.01 M phosphate/O.005 M EDTA buffer, pH 7.5, For analysis, 25 ~1 of the resulting hemolyzate was added to the standard glutathione assay mixture described in the following section. Preliminary experiments showed that hemolyzates prepared in this man- ner maintained constant levels of glutathione for at least 4 hr when kept at 0”.

Determination of the total glutathione content of liver and kidney homogenates prepared in TCA was carried out following removal of the protein precipitant from the supernatant solutions by extraction with

ENZYMIC ASSAY OF GLUTATHIONE 505

ether. Residual traces of ether were removed by vigorous shaking under a water pump.

The GSSG content of blood and tissue extracts was determined follow- ing preliminary reaction of the GSH contained therein with excess NEM essentially according to the method of Giintherberg and Rost (18). The rapid and complete reaction of NEM with GSH (19) to form a stable complex prevents participation of t,he reduced form in the enzymic assay as well as its possible oxidation to GSSG. Following incubation with NEM (final concentration 0.02 M) for 40-60 min at 25’ the solution was extracted at least 10 times with ether to ensure complete removal of the unreacted sulfhydryl reagent, which is an inhibitor of yeast glutathione reductase (20). Further details are given in the legends of Tables 7-9.

RESULTS

Characteristics of the assay system. A preliminary investigation of the dependence of DTNB reduction on the components of the glutathione reductase system resulted in the adoption of the experimental arrange- ment shown in Table 1 for the routine assay of glutathione. This protocol, which will be referred to hereafter as the standard assay system, makes use of two reaction mixtures balanced with respect to all com- ponents except glutathione. The employment of such a balanced system was dictated by the necessity for correction of the background rate of reduction of DTNB by the TPNH-glutathione reductase pair alone, a type of reaction which has been noted elsewhere (16)) coupled with the availability of a spectrophotometer equipped with double beam optics in which this correction could be carried out automatically. Typical photometric tracings resulting from the reduction of DTNB in reaction mixtures containing 10, 50, and 100 ng GSSG are shown in Figure 1 (curves l-3). Initiation of the reaction by the addition of TPNH results

TABLE 1 Protocol for Standard Glutathione Assay System

Components were dissolved in phosphate-EDTA buffer, pH 7.5, and were added in the amounts and in the order indicated. Final volumes were 1.0 ml. The rate of reaction at 25’ was usually expressed as the change in absorbancy per 6 min at 412 mN.

Test cuvet Amount

DTNB 0.6 pmole GSH or GSSG l-100 ng Glutathione reductase 10 Pg TPNH 0.2 rmole

Blank ouvet

DTNB -

Glutathione reductase TPNH

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in a rate of color development at 412 rnp that is linear for well beyond 6 min, the period of time generally employed as the basis of DTNB reduction. Curve 4 of Figure 1 shows the rate of color development, relative to buffer alone, which occurs under these conditions in the blank cuvet, i.e., the rate of reduction of DTNB in the absence of GSSG.

GSSG DTNB

-GSSG -

0 MINUTES

Fro. 1. Typical spectrophotometric tracing obtained during reduction of DTNB by catalytic quantities of GSSG in the standard assay system containing TPNH and yeast glutathione reductaae (GR). Tracings l-3 were produced with the balanced system of Table 1 at the GSSG levels indicated at the left. Tracing 4 shows the course of reduction of DTNB in the blank cuvet, measured against buffer, during the same interval of time. In all cases the reactions were initiated by the addition of TPNH (second arrow).

Although yeast glutathione reductase is generally regarded as specific for TPNH, the replacement of this nucleotide in a standard system containing 50 and 100 ng GSSG by an equivalent amount of DPNH resulted in only a 50% decrease in the rate of DTNB reduction (Table 2, Expts. l-4). The corresponding basal rate was, however, unaffected (Expts. 5 and 6). As expected, there was no reduction of DTNB by either DPNH or TPNH alone in the absence of both glutathione and enzyme (Expts. 7 and 8). That the ability of DPNH to serve as hydrogen donor in Expts. 2, 4, and 6 is related to the very high levels of enzyme employed in these reaction mixtures (10 rg/ml) was shown by additional experiments, not recorded here, in which this nucleotide was found to participate in the enzymic reduction of GSSG by similar high concen- trations of the yeast enzyme when measured at 34-O mp in the absence

ENZYMIC ASSAY OF GLUTATHIONE 507

TABLE 2 Comparative Rates of Reduction of DTNB in Standard Glutathione Assay System

with DPNH or TPNH as Hydrogen Donor The balanced reaction mixtures of Expts. 14, containing either DPNH or TPNH

(0.2 rmole/ml) and the amounts of GSSG indicated, were made up according to the protocol of Table 1. The corresponding blank rates of reduction of DTNB by TPNH or DPNH in the presence and absence of yeast glutathione reductase (GR) measured against buffer are shown in Expts. 5-8.

Expt. GSSG concn.,

Reaction mixture w/ml AOD”2 mp/6 min

1 TPNH + DTNB + GR + GSSG 50 0.23 2 DPNH + DTNB + GR + GSSG 50 0.16 3 TPNH + DTNB + GR + GSSG 100 0.49 4 DPNH + DTNB + GR + GSSG 100 0.25 5 TPNH + DTNB + GR - 0.12 6 DPNH+DTNB+GR 0.13 7 TPNH + DTNB 0 8 DPNH + DTNB 0

of DTNB. In this case, however, the resultant specific activity was only about 1% of that observed with TPNH as hydrogen donor.

The ultimate choice of conditions listed in Table 1 was based upon a study of the dependence of the rate of DTNB reduction on the con- centrations of the individual components comprising the reaction mixture. The results of this study are summarized in Figure 2. The reaction rate does not appear to be strikingly dependent upon TPNH concentration up to about 1 pmole/ml, beyond which point a falling off in rate is indicated. Similar inhibition of rat liver glutathione reductase at high TPNH concentration has been observed previously (21). The observed nonproportionality between rate of reduction and concentration of glutathione reductase (GR) may well be a reflection of the extremely high enzyme:glutathione substrate molar ratios (ca. 1: 1) prevailing in these reaction mixtures. The inhibition of the rate of color development noted at concentrations of DTNB exceeding 0.546 pmole/ml may be due in part to the susceptibility of yeast glutathione reductase to in- hibition by sulfhydryl reagents in general (22) or to diminution in the amount of GSSG formed cyclically in the reaction mechanism to be considered later (see “Discussion”).

Systematic experiments showed that the rate of reduction of DTNB in the presence of catalytic quantities of GSH or GSSG was not influenced by the order of addition of the components listed in Table 1. However, it was considered desirable to follow the specific order indicated in the foregoing protocol so as to allow a sufficient period of time for the non- enzymic reaction of DTNB with thiol components other than GSH (e.g.,

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1 I I I I 04

plo12hi 1.2 1.6

I I I I I 06 /Lmol~‘~20TNs I8 2.4

FIR 2. Dependence of the rate of reduction of DTNB by catalytic quantities of GSSG on the concentration of components of the standard assay system. The concentration of each component of the standard system was varied as shown on the abscissa in the presence of the constant amounts of the other two components aa given in Table 1. GSSG was present throughout at a fixed level of 60 &ml.

cysteine, protein SH) in the mixture undergoing analysis, prior to initiation of the enzymic reaction.

The rate of reduction of DTNB in the standard assay system as a function of the concentration of GSH or GSSG is shown in Figure 3. The rates are rectilinear with respect to glutathione concentration and are identical for the two relevant forms within the concentration range lO- 100 rig/ml. With the use of scale expansion it is possible to extend the method down to the range l-10 rig/ml, as shown in the insert of Figure 3. Under these latter conditions the background reduction of DTNB as- sumes high proportions at such low levels of GSSG concentration, requir- ing considerable care in the preparation of the respective reaction mix- tures. It is nevertheless evident from the insert of Figure 3 that the rate of color formation maintains approximately the same rectilinear depend- ence on GSSG concentration as would be anticipated from the data obtained within the higher range of concentration. Although sensitivity of the kind illustrated in the insert of Figure 3 has not generally been required for routine assay of total glutathione of blood or tissue extracts, it has proved of value in certain circumstances, e.g., in the determination of the very low levels of GSSG present in mammalian erythrocytes (see below).

ENZYMIC ASSAY OF GLUTATHIONE 509

04

:

03L

E f z I *

z oz-

:

-2 4

olr

// , y 2; :; 20 40 60 60

ng GSH or GSSG

FXJ. 3. Dependence of rate of reduction of DTNB in the standard assay system on GSH and GSSG concentrations. Reaction mixtures were made up according to Table 1. Data shown in the insert were obtained by ten-fold increaee of spectra- photometer sensitivity with the use of a scale expansion accessory.

Table 3 shaws the results of experiments designed to assess the degree of dependence of DTNB reduction on various GSH:GSSG ratios at constant total glutathione concentration. It is clear from these results that the rate of DTNB reduction in the standard assay system is an additive function of the individual amounts of GSH and GSSG present in the reaction mixture.

In order to obtain some indication of the specificity of the enzymic

TABLE 3 Dependence of Rate of Reduction of DTNB in Standard Assay System

on GSH: GSSG Ratio Rates of DTNB reduction were measured in reaction mixtures made up as in Table 1

and containing a constant total concentration of glutathione (GSH + GSSG) of 100 m/ml.

GSH:GSSG weight ratio AODU =‘f’/tZ min

l:o 0.41

3:l 0.40

1:l 0.39

1:3 0.37

0:l 0.38

0 GSH

. GSSG

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procedure with respect to the presence of other thiols in the assay mix- ture the effect of varying quantities of cysteine on the rate of reduction of DTNB by catalytic quantities of GSH was determined. The results, shown in Table 4, indicated that apart from a slight inhibition (ca. 11%) produced at the highest concentrations of cysteine employed, there was no notable effect of excess amounts of this thiol on the rate of color development. These data, in conjunction with those shown in Figure 3 and Table 3, confirm the impression that the enzymic procedure described here constitutes a sensitive and specific method for the determination of the total glutathione (GSH + GSSG) content of unknown mixtures.

Application to blood and other tissues. In order to evaluate the useful-

TABLE 4 Effect of Cysteine on Rate of DTNB Reduction in Standard Glutathione Assay

System Containing Catalytic Quantities of GSH Cysteine (CySH) was added in the amounts shown to the test cuvet of the standard

assay system (Table 1) immediately before GSH.

GSH ooncentration Molar ratio CySH: GSH AOIW =+/S min

100 rig/ml (3.3 X lo-’ pmolelml) 0 0.37 1:l 0.39

1O:l 0.41 25:l 0.35 5O:l 0.33

100: 1 0.33 10 rig/ml (3.3 X lO+ pmole/ml) 0 0.033

5O:l 0.025 100: 1 0.040 500: 1 0.034

1000: 1 0.030

ness of the foregoing analytioal procedure the glutathione contents of a limited number of selected mammalian tissues were determined. No attempt was made in these studies to conduct a comprehensive or critical examination of the various experimental conditions requisite for reliable analytical results, but rather to gain a general impression of the range of applioability of the present method to different types of tissues and to compare the quantitative results with existing analytical data.

It was found in initial experiments that the characteristic sensitivity of the assay could be used to particular advantage in the determination of the total glutathione content of whole human blood. In the procedure employed for this purpose (see “Experimental” section) no preliminary treatment of the sample was necessary other than the preparation of a 1:lOO hemolysate from as little as 10 ~1 of blood obtained by finger puncture. The results of a series of determinations on 8 normal non-

ENZYMIC ASSAY OF GLUTATHIONE 511

fasting human adults, using the equivalent of 0.25 JLI of whole blood per assay, are shown in Table 5. The range of values shown in this Table overlap broadly those for human blood reported previously and which have been tabulated in abbreviated form in Table 6. A somewhat higher average glutathione content was obtained in this study (396 pg/ml) as compared with the average of the means (328 pg/ml) listed in Table 6. These differences may be related to the fact that the data obtained in this investigation are uncorrected for hematocrit variation while the values cited in Table 6 have been derived from data based, for the most part, on erythrocyte volume. Furthermore, the results reported here were derived from analyses of whole blood hemolyzates in contrast to the cited data, which were obtained primarily with protein-free filtrates.

TABLE 5 Total Glutathione Content of Adult Human Whole Blood

10 ~1 of blood was obtained from each of eight nonfasting human adults by finger puncture and hemolyxed in 0.99 ml of cold 0.01 M sodium phosphate/O.005 M EDTA, pH 7.5. For assay, 25 J of hemolyzate was added to the test cuvet of the standard assay mixture of Table 1 and the rate of reduction of DTNB followed for 6 min. Calculation of total glutathione content was made by reference to the rate of color formation at 412 w in a standard mixture containing 50 ng GSSG.

Subject Glutathione, ,~g,‘ml whole blood

288 388 380 652 297 498 350 304

Because of the interest currently focused on the role of GSH in the maintenance of intact erythrocyte structure and of the presumed im- portance of a high GSH:GSSG ratio in this function (32) it seemed appropriate to extend the preceding analyses of total glutathione to the individual forms of this substance present in whole blood. Srivastava and Beutler (14) recently showed that, contrary to most previous find- ings, the GSSG levels of normal erythrocytes are exceedingly low, pos- sibily less than 0.5% of the total glutathione content of the cells. Before undertaking this study it was considered desirable to ascertain the reli- ability of the method to be employed by conducting a number of model assays of GSSG under conditions approximating those expected to obtain during actual analysis of blood or other tissues, viz., very low levels of GSSG in the presence of a several hundred-fold excess of GSH and

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TABLE 6 Literature Values for Glutathione Contents of &man Blood and Rat Blood,

Kidney, and Liver Mean values are given in parentheses. Values for human and rat blood marked with

an asterisk (*) have been recalculated from data expressed originally in terms of eryth- rocyte volume using an assumed hematocrit of 0.47.

Tim3

Human blood

Rat blood

GSH. &ml whole blood

245-340 (303) * (324)*

230431 (316)* 195-517 (298)* 277-324 (303) * 310470

(357; 320) * 250410 (345)

(233; 255) * (277) * (340)

222-550 (370) (400; 410)

380480 (400)

GSSG. #g/ml whole blood

(<1.4) (13; 16)

O-22 (7)

Ref.

14 18 12 23 24 25

8 9

26 27 28 29 30 25

Tiwue GSH, mg/gm tissue GSSG, mg/gm tissue Ref.

Bat kidney

.Rat liver

0.92-1.34 (1.09) 29 0.67-1.08 t-01 25

(1.34) 8 (0.53) 31 (1.59; 2.13) 26 (1.89) 27 (1.12) 28

1.18-2.25 (1.76) 29 1.36-3.37 (2.0) 30 1.34-2.97 (2.18) (4 25 1.16-2.36 (1.77) 8 1.64~1.75 (4 10

(1.74) 31

following treatment of the mixture with excess NEM, precipitation with trichloroacetic acid, and extraction of excess reagents with ether. The results of two such model analyses are shown in Table 7. In Expt. I of this table the components of mixtures 1-4 were dissolved initially in phosphate-EDTA buffer in the presence and absence of NEM, while in Expt. II the initial solutions of GSH and GSSG were made up in 5% TCA/O.Ol N HCl in order to reproduce the conditions employed for similar analyses performed on rat kidney and liver (see below). In the latter experiment a preliminary extraction of TCA with ether at 0” was

-conducted prior to reaction with NEM. The recoveries recorded in the

ENZYMIC ASSAY OF GLUTATHIONE 513

TABLE 7 Recovery of Glutathione from GSH-GSSG Mixtures Treated with NBM

The reaction mixtures of Expt. I were made up in phosphate-EDTA buffer and incubated in the presence and absence of 0.02 M NEM for 1 hr at 25’ es indicated. All solutions were then treated with equal volumes of 10% TCA and extracted 10 times with ether, residual traces of which were removed by vigorous shaking under a water pump. The extracted solutions were assayed for glutathione according to the protocol of Table 1, with a control mixture containing 50 ng GSSG serving ss reference standard. In Expt. II the glutathione mixtures were dissolved initially in 5’% TCA/lO n~I4 HCl and im- mediately extracted 5 times at. 0” with equal volumes of ether. Extracted mixtures 3 and 4 were then treated with equal volumes of 0.04 M NEM in phosphate-EDTA buffer and incubated 1 hr at 25’. After 10 extractions with ether to remove unreacted NEM the solutions were assayed as above.

Expt.

I

II

Mixture

1 2 3 4 1 2 3 4

GSS$adkd, NEM GS:Hp~$hd (0.02 M)

Glutat~;~a&.mnd.~

- 1.0 - 1.1 500 1.0 - 510. - 1.0 + 0.9

500 1.0 + 0.9 200 - - 230 - 0.2 - 0.24

200 0.2 + 0.80 - 0.2 + 0.45

(1 Corrected for initial contamination of GSH with 0.8% GSSG.

last column of Table 7 indicate the general reliability of the procedures used.

The application of the foregoing procedures to the determination of total and oxidized glutathione of whole rat blood resulted in the data shown in Table 8. Although it again appears that the mean of the whole blood values obtained here (369 @g/ml) is slightly greater than that calculated from the literature data summarized in Table 6 (336 pg/ml), the results of the GSSG assays are in good accord with the findings of Srivastava and Beutler (14) in indicating, in all cases but one, that less than 0.5% of the total glutathione content of rat blood is normally in the oxidized form. Expt. 5 of Table 8 also includes the results of assays of control mixtures of NEM-treated blood to which had been added known amounts of GSSG (2 pg/ml). The recovery of somewhat greater than the added amounts of GSSG following precipitation with TCA and extraction with ether may be due in some measure, in this experiment as well as in a similar recovery experiment carried out with liver homoge- nate (see Table 9)) to neglect of the volume of precipitate resulting from the addition of TCA and to some enrichment of the aqueous fraction thereby.

Extension of the preceding analyses to kidney and liver required

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TABLE 8 GSH-GSSG Contents of Whole Heparinized F&t Blood

Except, where noted, total blood glutathione (-NEM) was determined by direct, assay of 1: 100 hemolyzates as described in Table 5. For determination of GSSG content,, blood WLW incubated with 0.02 M NEM for 1 hr at, 25“ either as a 1: 2 snspcnsio [I of cells in 0.9% NaCl or as a 1: 10 hemolyzate in 0.01 M sodium phosphate/O.005 M EDTA buffer, pH 7.5, as indicated. Following precipitation of proteins with 5% TCA the suspensions were centrifuged and the supcrnatant solutions extracted 10 times with ether. Glutatbione content of solutions was determined in the standard ass&y system of Table 1, with a control mixture containing 50 ng GSSG serving as reference standard.

Conditions of NEM inoubation

Gluta;om, % GS8G

a b C

aa b cb

; C

L :

a b cd d ed

- + + - + + - + + - - + +

- +

: +

NaCl suspension Hemolyzate

NaCl suspension NaCl suspension

NaCl suspension NaCl suspension

NaCl suspension Hemolyzate

NaCl suspension NaCl suspension Hemolyzate Hemolyzate

322 1.7 0.52 1.7 0.52

439 0.60 0.14 0.57 0.13

361 0.88 0.24 0.85 0.24

328 391

0.56 0.47 0.14 0.17

374 0.21

;‘; A = 2.8 .

0.29 :‘; . I A = 3.2

0 Assay of TCA supernatant of a 1: 2 suspension of blood in 0.9% NaCl. b,Cells washed twice by centrifugation in 0.9% NaCl before incubation with NEM. c&ssay of TCA supemstant of a 1: 10 hemolyzate. d.2 ag,GSSG/ml blood added at beginning of NEM incubation.

modification of the procedures employed in the preparation of tissue extracts and their reaction with NEM so as to preclude the extensive enzymio degradation of the peptide known to take place in these tissues at, neutral pH (33). Such degradation was found to occur in the rat kidney in the present investigation even in the presence of excess NEM, as evidenced by the failure to recover any added GSSG from neutral homogenates prepared in buffers containing the sulfhydryl reagent,. Initial extraction of total glutathione was therefore carried out under the stabilizing conditions employed by previous investigators (28, 31), viz., homogenization of the tissue in an acidic protein precipitant, followed by reaction of a portion of the aqueous solution with NEM at, neutral

ENZYMIC ASSAY OF GLUTATHIONE 515

TABLE 9 GSH-GSSG Contents of Rat Kidney and Liver

Weighed amounts of tissues were homogenized in 5% TCA/O.Ol N HCP for l-2 min at 0”. After centrifugation the supernatant solutions were extracted 5 times at 0” with equal volumes of ether and divided into two portions, one of which was used without further treatment for the determination of total glutathione. The second portion was incubated for 1 hr at 25” with an equal volume of 0.04 M NEM in phosphate-EDTA buffer. After removal of unreacted NEM by 10 extractions with ether the solution was assayed for GSSG in the usual manner. In kidney experiments 2 and 3 the amounts of GSH or GSSG shown were added to measured volumes of the tissue suspension in TCA immediately after homogenization and subjected to the treatments described above.

Tissue Expt.

GS=i%.z?SG Total GSH or GSSG recovered

EEd- /&nl homogenate pdgm t&e pe%t%ssue 70 GSSG

Kidney 1 896 2 - 60 883

50 (GSH,, 115 A = 55

3 - 4 A=4 764 38 4.7 2.5 (GSSG) 8

4 - 915 35 3.6 5 - 881 39 4.2

Liver 1 - 2020 61 2.9 2 - 1930 78 4.0

D HCl was included in the extraction medium in order to maintain acidity of the solution during subsequent extraction of TCA with ether and to minimize the oxidation of endogenous GSH.

pH. The resulting levels of total and oxidized glutathione found in rat kidney and liver in a limited number of assays are shown in Table 9. Total glutathione contents are well within the range of values obtained for these tissues by previous investigators (cf. Table 6). Results of assays carried out on NEM-treated extracts further demonstrated that in all cases over 95% of the total glutathione of these tissues was in the reduced state. Also included in Table 9 are the results of two experi- ments on the recovery of GSH and GSSG which had been added to aliquots of TCA homogenates of kidney in amounts approximately equal to those present endogenously, as determined in preceding assays. Al- though good recovery of added GSH was achieved the result with GSSG was somewhat greater than expected due, possibly, to some oxidation of endogenous GSH during the initial manipulations (see “Discussion”).

Although it is known that glutathione is virtually absent from extra- cellular tissue fluid (34) the sensitivity of the present method appeared to be such as to allow its possible quantitative estimation in rat plasma and in human saliva and urine essentially without further treatment of the sample. Data from a limited number of assays conducted on these

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fluids are recorded in Table 10 and serve to emphasize the trace nature of the peptide therein. It should be pointed out, in connection with these results, however, that no account was taken in these determinations of the possible extent of degradation or disappearance of glutathione from the samples during the necessary periods of storage at 0”. Furthermore, the interpretation of the results obtained with saliva was complicated by the as yet, unexplained observation that rates of reduction of DTNB in the standard assay system were not linear but tended to increase markedly during the course of assay. A further study of this phenomenon is in progress.

TABLE 10 Total Glutathione Content of Some Mammalian Tissue Fluids

Plasma was obtained by centrifugation of hepariniaed rat blood at 1500 rpm and 2’. Human urine and saliva from three normal adults were centrifuged for 15 min at 27,000 g (2”) to remove suspended matter. The clear fluids were added without further treatment directlv to the assay cuvet in the amounts shown.

Tiue fluid Sample volume, &l

Plasma (rat) 25 0.15 1.5 25 0.15 1.5

Saliva (human) 100 0.33 0.7 25 0.17 0.5

100 0.45 1.0 Urine (human) 100 0.025 0.06

100 0.33 0.72 100 0.22 0.47

That, reduced glutathione is not stable in some extracellular fluids has been indicated previously, most, notably in the recent study of Beutler et al. (24) in which its rapid disappearance from plasma was observed. Although the assay method employed by the authors measured only the reduced form of the peptide it was concluded that the observed loss was not, the result, of a simple metal-catalyzed oxidation to the disulfide form since the rate was not affected by the presence of EDTA. However, the precise route of elimination was not ascertained. Since comparable information on the disposition of oxidized glutathione in plasma appeared to be lacking it seemed worthwhile to apply the present analytical method to its behavior, as well as that of GSH, in this medium partic- ularly in view of the recent finding that GSSG is rapidly eliminated from normal, intact, erythrocytes (14, 35). Preliminary to this study the stability of both forms of glutathione in bovine albumin solution was ascertained to clarify the possible influence of thii plasma component on the levels of added peptide. In contrast to the essentially constant level maintained by GSSG throughout the period of incubation in

ENZYMIC ASSAY OF GLUTATHIONE 517

albumin solution, GSH was found to disappear rapidly from solution (Fig. 4), most probably as a result of mixed disulfide formation with the protein via a disulfide-sulfhydryl exchange reaction. In plasma, how- ever, both forms were found to disappear at approximately equal rates (Fig. 4).

2 3 4 5 2 3 4 5

HOURS

FIQ. 4. Disappearance of GSH and GSSG from bovine serum albumin (BSA) solution and from rat plasma. GSH or GSSG was incubated at concentrations of 10 ag/ml in either 4.5% BSA in phosphate-EDTA buffer (pH 7.5) at 25” or in rat plasma at 37”. Open symbols represent corresponding control incubations carried out at 37” in buffer alone. At the intervals shown, 10 pl samples of the incubation mixtures were added without further treatment to the test cuvet of the standard assay system.

DISCUSSION

Aside from the difference in the nature of the reagent disulfides employed, the procedure for glutathione assay published recently by Grassetti and Murray (16) and that developed independently in this laboratory (17) are similar. In addition, it has been shown by the former authors that the same assay system can also be utilised as a sensitive procedure for the determination of TPN by coupling with a suitable TPNH-generating system such as the glucose 6-phosphate: glucose g-phosphate dehydrogenase pair. However, the application of the enzymic method to the determination of the GSH-GSSG levels of tissues was not reported by these authors. One possible advantage of the method described here lies in the use of Ellman reagent (DTNB) as an indicator disulfide in place of 2,2’-dithiopyridine as employed in the procedure of Grassetti and Murray. Since reduction of the latter disulfide yields a product (2-thiopyridone) whose maximum absorbance at 343 rnp coincides with that of TPNH, some limitation is placed upon the

518 FRANK TIETZE

sensitivity of the method which is not encountered with the use of DTNB, whose reduced form absorbs maximally at a wavelength (412 rnp) considerably removed from that of the reduced nucleotide. For this reason, and in conjunction with the use of double-beam spectrophotom- etry, it has proved possible to extend the useful lower limits of the present assay to optical density changes corresponding to glutathione contents of l-10 rig/ml of assay mixture, a level of sensitivity which is at least an order of magnitude greater than that obtainable with the previous methods of analysis that have been commonly employed. More-- over, the present method of assay is unique among those described here- tofore in that it effectively measures the total content of oxidized and reduced forms of the peptide.

In considering the underlying mechanism of the assay itself it is probable that the catalytic action of glutathione resides in its continual regeneration following a series of reactions similar to those which have been proposed by Eldjarn and Pihl (36) and by Pihl et at. (37) to explain the possible role of the glutathione : glutathione reductase system in the cellular reduction of disulfide bonds. The possible mechanisms under consideration are shown in reactions 3-6 and 7-9, in which DSSD and DSH represent the oxidized and reduced forms, respectively, of Ellman reagent, GSSD the mixed disulfide with glutathione, and GR. refers to glutathtine reductase.

GSH + DSSD ti GSSD + DSH (3) GSH -+ GSSD G GSSG + DSH (4)

GSSG + TPNH + H+z2 GSH + TPN+ (5)

DSSD + TPNH + H+ + 2 DSH + TPN+ (6)

GSH -+ DSSD Q= GSSD + DSH (71

GSSD + TPNH + H+FGSH + DSH + TPN+ (8)

DSSD + TPNH + Hf + 2 DSH + TFN+ (9)

Taking into account the presumed intermediate formation of the mixed disulfide GSSD (reaction 3), the GSH-catalyzed reduction of DTNS may be represented, following Pihl et al. (37), as proceeding according to reactions 3-5, which together constitute a more elaborate statement of the mechanism pictured by Grassetti and Murray (16). The net reaction (reaction 6)) obtained by summation of reactions 35, takes the form of a simple over-all reduction of DSSD by TPNH in which, consonant with its catalytic action, glutathione does not appear as re- actant. Also to be considered, although less likely, is the alternative

ENZYMIC ASSAY OF GLUTATHIONE 519

mechanism pictured in reactions 7 and 8, which invokes the possibility of a direct, enzyme-catalyzed reduction of the intermediate mixed disulfide GSSD. Although the enzymic reduction of mixed disulfides containing glutathione was ruled out, in the studies of Pihl et al. (37) on kinetic grounds, a number of considerations call attention to this possibility in the present, instance. The first of these is the extremely high DSSD : GSH molar ratios (ca. lo*) which characterize these reaction mixtures and which would be expected to result in the formation of the mixed disulfide as the predominate form involving glutathione. Second is the unusually high concentration of glutathione reductase (10 pg/ml; ca. 1 I.U.) present in the reaction mixture. This amount, of enzyme is several hundred- to a thousand-fold greater than that, usually required in reaction mixtures prepared for the purpose of measuring the rate of GSSG reduction spectrophotometrically. Under these circumstances an “inert” substrate of the mixed disulfide type such as that under consideration might, well be reduced at, a rate commensurate with that observed in these studies. In this connection it is pertinent, to point out that three laboratories have reported on the ability of the TPNH-glutathione reductase system to catalyze at a low rate the reduction of at least one mixed disulfide involving glutathione, viz., that with coenzyme A (38-40). Moreover, there remains the observation, made here and elsewhere (16)) that Ell- man-type aromatic disulfides such as DTNB or 2,2’-dithiopyridine them- selves appear to function as substrates of glutathione reductase at these high enzyme Ievels .3 Arguing against direct enzymic reduction of the mixed disulfide, however, is the observation, noted in Figure 2, that the rate of reduction proceeds through a maximum with increasing DTNB concentration, a behavior reminiscent, of that. observed previously by Pihl et al. (37) in their studies on the enzymic reduction of disulfides by GSH in the presence of the TPNH-glutathione reductase system and ascribed by them to the continuous rise in the level of inactive mixed disulfide (see reaction 3) at the expense of GSSG formation (see reaction 4).

The utility of the analytical method employed here has been explored by conducting a number of glutathione assays of selected tissues. Its application to the determination of the total glutathione concentration of whole blood appears to be particularly convenient because of the

“The possibility that the background reduction of DTNB wm caused by con- tamination of glutatbione reductaze. with trace quantities of glutathione appeared unlikely since exhaustive dialyaiz of the enzyme preparation against buffer wm without effect on this activity. This treatment would not, however, eliminate the possible presence and participation of a more strongly bound or covalently linked species of glutsthione such as has been suggested in previous studies on the mechanism of action of this enzyme (41-43).

,529 FRANK TIETZE

small sample volume required (<lo ~1) and the avoidance of preliminary treatment other than the preparation of a 1: 190 hemo1yzat.e. Moreover, the sensitivity is such as to permit glutathione estimations in extra- cellular fluids, e.g., saliva, plasma, and urine, normally containing ca. 1 pg/ml of the peptide, by direct addition of the sample to the assay mix- ture. In general, it appeared that the quantitaive results agreed satis- factorily with those obtained in previous studies employing a variety of other analytical methods. Measurement of the glutathione content of’ blood and other tissue extracts pretreated with NEM confirmed the well- known fact that the overwhelming proportion of the peptide is present. in the reduced state. In erythrocytes in particular the level of oxidized: glutathione was well below 0.5% of the total and in this respect agreed~ well with a recent estimate (14) of this ratio. The higher proportions. (ca. 3-5s) observed in liver and kidney were greater than those that have been reported in some previous studies (10, 25) (cf. Table 6), pos- sibly as a result of some oxidation of GSH in the TCA extracts prior to reaction with NEM. Indeed, precautions against the use of TCA as. an extraction medium for this reason have been voiced (5, 9). Although the model recovery experiments recorded in Table 7 did not demonstrate any unusual tendency for GSH to undergo oxidation in TCA, it is pos- sible that other acid-soluble components of the tissue extracts, such as metal ions, could have catalyzed this reaction. The use of extractants other than TCA (5) may therefore be preferable for the determination. of the GSSG:GSH ratio in such tissues.

Insofar as the specificity of the enzymic procedure is concerned it seems reasonable to assume that it parallels that possessed by the pure yeast enzyme itself. Although a comprehensive study of this aspect was not carried out, the noninterference of cysteine at high relative ratios (Table 4) suggests a corresponding reliability in the presence of other nonglutathione thiol components. Conceivably, hydrolytic products or analogs of glutathione might interfere in the reaction, either as substrate or as inhibitor. It is of interest to note, however, that at least one such product, viz., bis+cystinylglycine, was found to be totally inert in the .enzymic reaction, even at concentrations as high as 1 ,umole/ml.

SUMMARY

A method for the analysis of nanogram quantities of glutathione has been developed which is based on the catalytic action of GSH or GSSG in the reduction of Ellman reagent (DTNB) by a mixture of TPNH and yeast glutathione reductase. Unlike previous methods of analysis the procedure described here effectively measures the total glutathione (GSH + GSSG) content of unknown mixtures and is not subject to appreciable

ENZYMIC ASSAY OF GLUTATHIONE 521

interference by the presence of other thiol components. It is suggested that the catalytic action of glutathione in this system resides in the continual enzymic regeneration of GSH, present initially or formed enzymically from GSSG, following its interaction with the sulfhydryl reagent.

The sensitivity of the method is such as to permit the determination of total glutathione in extracellular tissue fluids such as plasma, saliva, and urine normally containing very low levels of this material, essentially without pretreatment of the sample. The same is true for glutathione determinations of whole blood, in which the preliminary procedure is confined to the preparation of a 1: 100 hemolyzate from as little as 10 ~1 of sample.

Following published procedures, the pretreatment of tissue extracts with NEM to form an enzymically inactive complex with free GSH allowed the determination of the low levels of oxidized glutathione normally present therein. The use of the foregoing analytical method in the determination of total and oxidized glutathione contents of rat blood, kidney, and liver gave values in good agreement with those ob- tained by previous investigators.

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