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ANALYTICAL

BIOCHEMISTEY

27, 502-522 (1969)

Enzymic Method for Quantitative


Nanogram
Amounts of Total
Glutathione:
Applications

to Mammalian

FRANK

Determination
and Oxidized

Blood and Other

of

Tissues

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 involvement 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 nonprotein 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
&7.

reviews on methods
502

of glutathione

analysis see references

ENZYMIC

GSSG

,PNH

ASSAY

H+ &

OF

503

GIJJTATHIONE

ZGSH

+lPN+

coo-

(1)

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) (Ellman reagent) (15). Since the chromophoric product resulting from reaction 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 concentration 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


Calbiochem. The GSH was found to be contaminated

were obtained from


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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TIETZE

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 according to Ellmans 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 manner 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

505

GLUTATHIONE

ether. Residual traces of ether were removed by vigorous shaking under


a water pump.
The GSSG content of blood and tissue extracts was determined following 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 arrangement 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 components 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
GSH or GSSG
Glutathione reductase
TPNH

0.6 pmole
l-100 ng
10 Pg
0.2 rmole

Blank

DTNB
Glutathione
TPNH

ouvet

reductase

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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

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 concentrations
of the yeast enzyme when measured at 34-O mp in the absence

ENZYMIC

ASSAY

OF

507

GLUTATHIONE

TABLE 2
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.
Comparative

Expt.

1
2
3
4
5
6
7
8

Reaction

TPNH + DTNB
DPNH + DTNB
TPNH + DTNB
DPNH + DTNB
TPNH + DTNB
DPNH+DTNB+GR
TPNH + DTNB
DPNH + DTNB

GSSG concn.,
w/ml

mixture

+
+
+
+
+

GR
GR
GR
GR
GR

+
+
+
+

GSSG
GSSG
GSSG
GSSG

50
50
100
100
-

AOD2

mp/6 min

0.23
0.16
0.49
0.25
0.12
0.13
0
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 concentrations 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 inhibition 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 nonenzymic reaction of DTNB with thiol components other than GSH (e.g.,

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1
I

04

06

I
plo12hi
I
/Lmol~~20TNs

I
1.2

1.6

I
I8

2.4

FIR 2. Dependenceof the rate of reduction of DTNB by catalytic quantities of


GSSG on the concentration of componentsof the standard assay system. The
concentration of each component of the standard system was varied as shown
on the abscissa
in the presenceof the constantamountsof the other two components
aa given in Table 1. GSSG was presentthroughout 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 lO100 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 assumes high proportions at such low levels of GSSG concentration, requiring considerable care in the preparation of the respective reaction mixtures. It is nevertheless evident from the insert of Figure 3 that the rate
of color formation maintains approximately
the same rectilinear dependence 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

509

GLUTATHIONE

04

GSH

GSSG

03L
E
f
z
I
*
z

oz-

:
-2
4
olr

//

,
20

40
ng

y 2; :;
60
GSH

or

60
GSSG

FXJ. 3. Dependence of rate of reduction of DTNB in the standard assay system


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 spectraphotometer sensitivity with the use of a scale expansion accessory.

on

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
l:o
3:l
1:l
1:3

0:l

ratio

AODU

=f/tZ min
0.41
0.40
0.39
0.37
0.38

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procedure with respect to the presence of other thiols in the assay mixture 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 usefulTABLE 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

100 rig/ml

ooncentration

(3.3 X lo- pmolelml)

10 rig/ml (3.3 X lO+ pmole/ml)

Molar

ratio

CySH:

0
1:l
1O:l
25:l
5O:l
100: 1
0
5O:l
100: 1
500: 1
1000:

GSH

AOIW

=+/S min

0.37
0.39
0.41
0.35
0.33
0.33
0.033
0.025
0.040
0.034
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

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511

GLUTATHIONE

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 importance 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 findings, the GSSG levels of normal erythrocytes are exceedingly low, possibily less than 0.5% of the total glutathione content of the cells. Before
undertaking this study it was considered desirable to ascertain the reliability 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
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 erythrocyte volume using an assumed hematocrit of 0.47.
Literature

Tim3

Human blood

Rat blood

Tiwue

Bat kidney

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)
GSH, mg/gm tissue

0.92-1.34
0.67-1.08

.Rat liver
1.18-2.25
1.36-3.37
1.34-2.97
1.16-2.36
1.64~1.75

GSSG. #g/ml whole blood

Ref.

(<1.4)
(13; 16)
O-22 (7)

14
18
12
23
24
25
8
9
26
27
28
29
30
25

GSSG, mg/gm

(1.09)
t-01
(1.34)
(0.53)
(1.59; 2.13)
(1.89)
(1.12)
(1.76)
(2.0)
(2.18)
(1.77)

(4
(4

(1.74)

tissue

Ref.

29
25
8
31
26
27
28
29
30
25
8
10
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

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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 absenceof 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 immediately 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.

Mixture

2
3
4
II

2
3
4

GS:Hp~$hd
-

500
500
200
200
-

(1Corrected for initial contamination

GSS$adkd,

NEM
(0.02 M)

Glutat~;~a&.mnd.~

1.0
1.0
1.0
1.0

+
+

1.1
510.
0.9
0.9

0.2
0.2
0.2

+
+

230
0.24
0.80
0.45

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 homogenate (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 describedin Table 5. For determinationof GSSG content,,

bloodWLW
incubatedwith 0.02M NEM for 1 hr at,25 eitherasa 1:2 snspcnsio
[I of cells
in 0.9% NaCl or as a 1: 10 hemolyzatein 0.01M sodiumphosphate/O.005
M EDTA
buffer, pH 7.5, as indicated. Followingprecipitation of proteins with 5% TCA the
suspensions
were centrifugedand the supcrnatantsolutionsextracted 10 times with
ether. Glutatbionecontentof solutionswasdeterminedin the standardass&ysystemof
Table 1, with a control mixture containing50 ng GSSGservingasreferencestandard.
Conditions
of
NEM inoubation

Gluta;om,

% GS8G

322

a
b

NaCl suspension

1.7

0.52

Hemolyzate

1.7

0.52

aa
b
cb

+
+
+
+
++

439
0.60
0.57
361

0.14
0.13

NaCl suspension

NaCl suspension

:+

NaCl suspension
Hemolyzate
Hemolyzate

;
C

L
:
a
b
cd
d
ed

NaCl suspension

NaCl suspension
NaCl suspension

0.88

0.24
0.24

0.85
328

391
NaCl suspension
Hemolyzate

0.47
0.56

0.14
0.17

374
;;
:;

.
. I

A =
A =

2.8
3.2

0.21
0.29

0 Assayof TCA supernatant

of a 1: 2 suspension
of bloodin 0.9% NaCl.
b,Cellswashedtwice by centrifugationin 0.9% NaCl beforeincubationwith NEM.
c&ssayof TCA supemstantof a 1: 10hemolyzate.
d.2ag,GSSG/mlbloodaddedat beginningof 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

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515

GLUTATHIONE

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

Kidney

Expt.

homogenate

1
2

Liver

/&nl

4
5
1
2

50 (GSH,,
2.5 (GSSG)
-

60
115
4
8

A = 55
A=4

EEdpdgm t&e

pe%t%ssue

70 GSSG

896
883
764

38

4.7

915
881
2020
1930

35
39
61
78

3.6
4.2
2.9
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 experiments 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. Although 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 extracellular 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

516

FRANK

TIETZE

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

Plasma (rat)
Saliva (human)
Urine (human)

Sample

volume,

25
25
100
25
100
100
100
100

&l

0.15
0.15
0.33
0.17
0.45
0.025
0.33
0.22

1.5
1.5
0.7
0.5
1.0
0.06
0.72
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 particularly 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

517

GLUTATHIONE

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, however, both forms were found to disappear at approximately
equal rates
(Fig. 4).

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 spectrophotometry, 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 heretofore 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
GSH -+ GSSD G GSSG + DSH

(3)
(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+

DSSD + TPNH

+ Hf + 2 DSH + TFN+

(8)
(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 reactant. Also to be considered, although less likely, is the alternative

ENZYMIC

ASSAY

OF

519

GLUTATHIONE

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 Ellman-type aromatic disulfides such as DTNB or 2,2-dithiopyridine
themselves 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 backgroundreduction of DTNB wm caused by contamination 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
possiblepresenceand participation of a more strongly bound or covalently linked
speciesof glutsthione such as has been suggestedin previous studies on the
mechanismof 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 extracellular 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 mixture. In general, it appeared that the quantitaive results agreed satisfactorily 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 wellknown 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), possibly 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 possible 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 obtained by previous investigators.
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