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BIOCHEMISTEY
to Mammalian
FRANK
Determination
and Oxidized
of
Tissues
TIETZE
reviews on methods
502
of glutathione
ENZYMIC
GSSG
,PNH
ASSAY
H+ &
OF
503
GIJJTATHIONE
ZGSH
+lPN+
coo-
(1)
coo-
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.
504
FRANK
TIETZE
ENZYMIC
ASSAY
OF
505
GLUTATHIONE
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
506
FRANK
TIETZE
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
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.,
508
FRANK TIEiTZE
1
I
04
06
I
plo12hi
I
/Lmol~~20TNs
I
1.2
1.6
I
I8
2.4
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
on
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
510
FRANK
TIETZE
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
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
ENZYMIC
ASSAY
OF
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
512
FRANK
TIETZE
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
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
ENZYMIC
ASSAY
OF
513
GLUTATHIONE
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
-
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
514
FRANK
TIETZE
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
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.
enzymio degradation
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,
ENZYMIC
ASSAY
OF
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
ENZYMIC
ASSAY
OF
517
GLUTATHIONE
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
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)
(71
GSSD + TPNH
+ H+FGSH
+ DSH + TPN+
DSSD + TPNH
+ Hf + 2 DSH + TFN+
(8)
(9)
ENZYMIC
ASSAY
OF
519
GLUTATHIONE
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
ENZYMIC
ASSAY
OF
GLUTATHIONE
521
522
FUNK
TIETZE