A STUDY OF GLUTATHIONE.

II. THE DETERMINATION OF REDUCED GLUTATHIONE IN TISSUES.

BY HAROLD L. Me4SON.

(From the Division of Chemistry, The Mayo Foundation, Rochester, Minnesota.)

(Received for publication, February 10, 1930.)

The iodine titration proposed by Tunnicliffe (10) has been em- ployed by most workers for the estimation of reduced glutathione in tissues. On account of its importance this method will be dis- cussed in detail, but first it is desired to mention two methods which have appeared recently.

Flatow (3) has described a method in which an excess of ferri- cyanide is used to oxidize the glutathione in a strongly alkaline solution. The excess ferricyanide is titrated with indigo sulfonic acid. The method as described is applied to blood, in which case it is necessary to make corrections for uric acid and t’lioneine.

The other method, proposed by Gabbe (6), at The Thirteenth International Physiological Congress, but not yet published in full, is also based on the use of ferricyanide. The glutathione is treated with an excess of ferricyanide in hydrochloric acid solution. The excess ferricyanide is then allowed to react with potassium iodide and the free iodine is titrated with thiosulfate. It will be

shown that reduced glutathione is not oxidized by ferricyanide in a solution which is more than 0.01 N in hydrochloric acid (Table III). Consequently, oxidation of the glutathione does not occur until the iodine is liberated. Essentially, therefore, this method is an iodine titration in which iodine is added in excess and the excess is back-titrated with thiosulfate. The method appears to have no advantage over the usual titration with iodine.

Thompson and Voegtlin (9) adopted the iodine titration after some scrutiny. They preferred to continue the titration until the nitroprusside reaction disappeared rather than to use starch as an

623

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624 Reduced Glutathione

internal indicator. On the other hand, Blanchetikre and Melon (2) maintained that the nitroprusside reaction is not sufficiently sensitive to indicate the presence of a small quantity of glutathione. They used starch to show the end-point. Perlzweig and Delrue (8) recommended the use of starch provided there is sufficient iodide present to stabilize the starch-iodine color. Turner (11) reported a comparison of starch and nitroprusside as indicators. The use of nitroprusside led to figures which were 5 to 15 per cent

TABLE I.

Titration of Glutathione with Iodine.*

m7.

10.0 10.0 10.0 10.0 10.0 61.4 61.4 12.3 12.3 12.3 5.0 5.0

-

-

Initial VOlUIIX

cc.

50 50 50 50 50 50

200 50 50 50

100 100

-7- 7

KI added. GSH found with starch indicator.

gm. mg. per cent

0.00 10.8 108 0.01 10.5 105 0.05 10.4 104 0.10 9.9 99 0.50 9.8 98 0.50 58.7 96 2.0 60.2 98 0.5 12.1 99 0.5 12.3 100 0.5 12.3 100 1.0 5.2 104 1.0 5.2 104

-

GS%%und nitroprusside

indicator.

per cent

91 94 88 92

* A recrystallized sample of the tripeptide was used. By titration with potassium ferricyanide and by calorimetric comparison with cysteine it was found to contain 97 per cent of GSH.

The iodine used was approximately 0.01 N except for the sixth and seventh titrations when it was twice this strength.

lower than those obtained with starch. Potassium iodide was not added.

The question as to the validity of the results obtained with the iodine titration is of significance since this method has been widely used. Inasmuch as Okuda (7) found that the iodine uptake of cysteine varies with the concentration of acid and with the tem- perature, one might expect the same to be true of glutathione. An examination of the method as applied to crystalline glutathione has been made with due consideration of the various points men- tioned. Table I shows the results.

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H. L. Mason 625

Most striking is the effect of iodide on the consumption of iodine. This effect is to be attributed to the elimination of side reactions rather than to stabilization of the starch-iodine color. An experi- ment was designed to test this interpretation. An excess of iodine was allowed to act in the absence of iodide and then the excess was titrated in the presence of potassium iodide and starch. 30 per cent more iodine was taken up by the glutathione than when potassium iodide was added before the excess of iodine. Since in both instances there was sufficient iodide to stabilize the starch- iodine color, it is evident that the extra consumption of iodine is in no way concerned with this color. The effect of the iodide is on the course of the reaction between iodine and glutathione.

The contention of Blanchetiere and Melon and of Turner that the use of the nitroprusside reaction to indicate the end-point leads to low results, has been confirmed. The differences between the figures obtained by the two methods for the determination of this end-point agree well with Turner’s observations. However, the sensitivity of this reaction is so dependent on the procedure that it may possibly give better results in the hands of other workers. Starch is obviously the indicator of choice.

The effect of a decrease in the concentration of GSH is to in- crease the uptake of iodine although the effect is not marked. A dilution of 5 times brought about an increase of about 4 per cent; a dilution of 12 times, an increase of 7 per cent.

In contrast to cysteine, variations in the acidity from 0.05 to 3.0 cc. of 5 N hydrochloric acid in 50 cc. changed the titer little more than the experimental error. These figures are not included in Table I.

It is thus shown that in the absence of other tissue extractives reduced glutathione can be estimated to within a few per cent by titration with iodine in the presence of potassium iodide and with starch as an internal indicator. However, the presence of other tissue constituents may invalidate a method which is excellent for the pure substance. Although Tunnicliffe found that urea, uric acid, creatinine, glucose, and fructose are not attacked by iodine, there are present in some tissues substances other than glutathione which do react with iodine and therefore interfere with the method. The iodine titration of tissue extracts will be considered further in comparison with a new method for the determination of reduced glutathione.

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626 Reduced Glutathione

The new method is based on the oxidation of GSH within anar- rowrange of pH with potassium ferricyanide. Oxidation of sulf- hydryl to disulfide occurs with the formation of an equivalent amount of ferrocyanide. The ferrocyanide is estimated colori- metrically by conversion into Prussian blue. Certain details of the method were suggested by Folin’s ferricyanide method for blood sugar (4) and two of his reagents have been adopted.

As the acidity increases the oxidation potential of ferricyanide increases, but the reducing intensity of GSH decreases at such a rate that at pH values below 5.7 it is not completely oxidized by ferricyanide. Some tissue constituents act similarly but they cease to reduce ferricyanide at an acidity which is not sufficient to inter- fere with the complete oxidation of GSH. Accordingly, it is possible to select for the oxidation a pH at which glutathione is completely oxidized while other substances are not attacked or only partially so. A buffered solution of pH 5.9 was found to be satisfactory.

An important feature of the method is the blank in which form- aldehyde prevents the oxidation of glutathione without interfering with the oxidation of a number of other substances. Although this action of formaldehyde is not entirely specific for the sulfhydryl group, it affords a valuable correction for the reducing power of other substances which react with potassium ferricyanide.

Reagents.

Potassium Ferricyanide.-A 0.01 N solution of potassium ferri- cyanide, purified by the method of Folin (4), is used.

Bu$er.-This is Sorensen’s phosphate buffer. The stock solu- tions are ~/7.5 primary potassium phosphate and ~/7.5 secondary sodium phosphate. For use 1 volume of the secondary phos- phate is mixed with 9 volumes of the primary phosphate. The pH of the buffer is close to 5.9.

Stock Xolution of Cysteine.-Dissolve 157.5 mg. of cysteine hydrochloride in a little water containing 10 cc. of 5 N hydrochloric acid. Dilute to 100 cc. This solution is 0.01 N in cysteine. It will not lose an appreciable amount of reducing power in 6 to 8 hours if kept in the ice box, but it must be renewed or standardized daily. It should always be standardized even when freshly prepared.

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H. L. Mason 627

Standard Cysteine Solution.-Dilute 10 cc. of the stock solution to 50 cc. to make a solution 0.002 N in cysteine. This solution will keep for 3 or 4 hours at room temperature. 1 cc. is equivalent to 0.614 mg. of reduced glutathione.

Ferric Sulfate Reagent.-The reagent described by Folin is used with one change. The amount of gum arabic is doubled. No difficulty has been experienced in keeping the reagent 4 to 6 weeks.

Formaldehyde.-The u. s. P. 40 per cent solution is used.

Standardization of Cysteine.

All of the samples of cysteine hydrochloride which have been prepared have been found to be slightly impure. It is therefore necessary to determine the cysteine content of each sample. One method for this is the titration of a standard solution of iodine in alcohol with an alcohol solution of the cysteine hydrochloride.

A more satisfactory method is the titration with potassium ferri- cyanide at a pH of about 7.4. Measure 25 cc. of the stock solution of cysteine into an Erlenmeyer flask and add 10 cc. of phosphate buffer of pH 7.4 (a mixture of 200 cc. of the primary with 800 cc. of the secondary phosphate stock solutions). Now add sufficient sodium hydroxide to neutralize the hydrochloric acid present and titrate immediately with 0.01 N potassium ferricyanide until a permanent yellow remains. The end-point is determined by comparison with a blank which contains a volume of water equal to the volume of the titrated solution at the end of the titration and 0.2 cc. of the ferricyanide. This amount of ferricyanide is then subtracted from the titer. It is easily possible to duplicate titra- tions to within 0.1 cc.

Procedure.

To make clear certain steps in the procedure, two facts must be mentioned : (1) Cysteine and glutathione undergo autooxidation very slowly in acid solution but when the pH is brought to 5.9 appreciable loss of these substances may occur in a few minutes; and (2) it is important that the volumes of standard and unknown should be approximately equal at the time the Prussian blue is developed. When these facts are taken into account, the pro- cedure which follows has been found convenient in most cases.

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628 Reduced Glutathione

Measure into a 100 cc. flask a volume of tissue extract, prepared by the method of Folin and Wu (5), which contains between 1 and 2 mg. of glutathione. Titrate another portion with standard alkali. Congo red is used as the indicator. Dilute the sample to such a volume that when the alkali and buffer have been added the total volume will be 25 cc. Now add 5 cc. of buffer and, without delay, the requisite amount of alkali, and then the potassium ferricyanide which should be added a few drops at a time. The flask is shaken and not more than 3 or 4 drops of ferricyanide are added in excess

TABLE II.

Relation between the Amount of Glutatlaione Used and the Color Produced.*

Standard, co. 0.002 iv cyst&m

0.50 0.50 0.50 0.50

0.50 2.00 2.00

2.00 2.00 2.00

2.00 2.00 2.00

2.00

-

-

Glu~taon

m!J. mm. ml.

0.20 31.9 0.192

0.25 25.1 0.245

0.30 21.3 0.288 0.40 15.7 0.391

0.50 12.6 0.487

0.50 54.0 0.456 1.00 25.6 0.96

1.20 21.5 1.14

1.40 18.0 1.37 1.60 16.0 1.54

1.80 13.9 1.77

2.00 12.8 1.92 2.50 9.8 2.51

3.00 8.3 2.96

Reading. GSH found.

per cent

96

9s 96 97 97

91 96 95

98 96 98

96 100 99

* The standard was set at 20 mm.

so that the solution has a distinctly yellow color. Wait 15 min- utes. Because the reaction is slow an apparent excess of ferri- cyanide may disappear after a few minutes and necessitate the addi- tion of more. Now add 3 cc. of the ferric sulfate reagent and allow 10 minutes for the color to develop. Dilute to volume and compare in the calorimeter with the standard prepared from cysteine.

If there is a large difference in the intensity of the colors of standard and unknown, the comparison in the calorimeter is dilli- cult. The color of the excess ferricyanide appears more intense in

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H. L. Mason

the solution containing the less Prussian blue when the blue colors are matched. Folin has shown that this difficulty can be over- come by the addition of ferricyanide to the solution containing the more Prussian blue. For further details, reference should be made to Folin’s paper.

The standard is prepared as just described. The amount of cysteine is so chosen that the unknown reads between 15 and 30 with the standard set at 20 mm. The color of the excess ferri- cyanide is more likely to interfere if there is too great a difference between standard and unknown. Table II shows the limitations in this respect.

If a solution contains less than 1 mg. of glutathione in 15 cc., smaller flasks may be used. Less than 0.15 mg. of glutathione cannot be determined accurately for the color is too faint to be read in the calorimeter. With these small quantities the volume in which the color is developed should be kept as small as possible.

The volume of tissue extract for the blank may be doubled to advantage since the blanks are usually small. Place the sample in a 25 cc. flask, add 0.5 cc. of formaldehyde, and proceed as before. One precaution must be observed. After the addition of the ferric sulfate the blanks are immediately placed in the dark since the ferric ion sensitizes them to light. This does not cause difficulty in reading them in the calorimeter as the action is slow. An ex- posure of 15 minutes to the diffuse light of the laboratory may cause a definite color in what would otherwise be a colorless blank.

It is best to provide a series of standards which contain 0.1, 0.2, 0.3, and 0.5 cc. of the standard cysteine solution for comparison with the blanks. A blank which has less color than would match a 0.25 cc. standard cannot be read accurately. The blankis then so low that it is sufficient to compare it with the appropriate stand- ards with the unaided eye.

The procedure as outlined can be varied if the precautions noted are observed. A few words should be added concerning the vol- ume in which the Prussian blue is formed. A small difference between the volumes of standard and unknown is not of conse- quence but a larger difference (25 cc.) may cause considerable error. Development in a large volume with subsequent dilution yields less color in 100 cc. than development in a small volume with subsequent dilution. This is due to a latent period in the

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630 Reduced Glutathione

development of color in the larger volume since the colors will be the same after several hours. The color is inversely proportional to the volume when diluted after full development.

Large amounts of salts tend to retard the development of color. The quantity of salts encountered in tungstic acid filtrates is negligible.

If oxalates are present the solution becomes sensitive to light after the addition of the iron, but no difficulty is experienced when the solution is shielded from the light and read in the calorimeter without delay.

TABLE III.

Zn$uence of pH on Determination of Reduced Glutathione.*

PH

2.0 5.2

5.5

5.7 5.9 6.2

6.7 7.4

1

-

GSH in crystsl- line preparation,

mg. GSH found in 2.0 mg.

sample.

0.00 1.10

1.70 1.91

1.95

1.95 -

GSH in liver extract.

Total reducing power as GSH.

0.83 0.06

0.91 0.07 0.94 0.08 1.01 0.13

1.04 0.13 1.05 0.13

_- Blank as GSH.

-

--

-

GSH found.

ml.

0.77 0.84

0.86 0.88 0.91

0.94

* The extract was a tungstic acid filtrate of fresh rabbit liver. 5 cc. were used for each determination.

The straight line relation between the intensity of color and amount of glutathione is shown in Table II. The same relation holds for cysteine. Deviations from the straight line relation occur only when there is a wide divergence from the standard.

DISCUSSION.

Although the blank is relied on to eliminate the effect of some of the substances other than glutathione, the action of formalde- hyde is not specific. Thus, the oxidation of uric acid at pH 7.4 is partially inhibited by formaldehyde. Doubtless there are other substances which are affected similarly. In consideration of this point a number of compounds was examined as to their possible interference with the method.

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H. L. Mason 631

i. Cysteine.-This behaves in every way like glutathione. 2. Thioneine.-The oxidation of thioneine is completely in-

hibited by formaldehyde. However, at pH 5.9 it gives only one- tenth as much color as an equal weight of glutathione. About 8 mg. of thioneine is the average of the amounts found in human blood by Benedict and Newton (1). Such a quantity would in- crease the apparent glutathione values obtained by this method by 0.8 mg., a negligible amount. It is of course possible to deter- mine the amount of thioneine and apply the proper correction.

3. Uric Acid.-This is not oxidized at pH 5.9. 4. Phenols.-Quinol, pyrogallol, and epinephrine are readily

oxidized by potassium ferricyanide. Their oxidation is not affected by formaldehyde.

5. Hexuronic Acid of the Suprarenal Cortex.-This compound, obtained from Szent-GyGrgyi, is rapidly oxidized at pH 5.9 and its oxidation proceeds equally well in the presence of formaldehyde.

6. Glucose, Lactic Acid, and Urea.-These are not attacked. The values obtained by this method are maximal. All errors ex- cept those of manipulation are positive. If any other substances are oxidized, the apparent amount of glutathione will be increased unless this oxidation occurs to the same extent in the blank or unless some of the tissue extractives have an influence on the oxida- tion of the GSH. The oxidation of GSH is not affected by other tissue constituents since GSH added to tissue extracts can be quantitatively recovered. To a blood extract which contained 0.26 mg. of GSH were added 1.15 mg. of GSH. Found 1.37 mg. 0.77 mg. of GSH was added to a liver extract which contained 2.38 mg. Found 3.13 mg.

The influence of pH on the oxidation of GSH and on the values of the total reducing power and the blank of tissue extracts is well illustrated by Table III. It will be noted that a variation of 0.1 pH unit either side of 5.9 is of no consequence. The amount of buffer used can easily maintain the pH within this range when the solution has previously been made just alkaline to Congo red. The pH of 5.9 allows complete oxidation of glutat,hione with a minimum of oxidation of other substances.

Comparison of the amounts of glutathione found in yeast, liver, and kidney extracts by the iodine titration and by the new method (Table IV) reveals some striking differences. The iodine titrations

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632 Reduced Glutathione

indicate the presence of 20 to 100 per cent more glutathione than the new method. Although most of the titrations were performed before the influence of iodide was appreciated, consideration of the

TABLE IV.

Comparison of Iodz’ne Titrations with Calorimetric Estimations.*

I Tissue.

Yeast.

Blood (hog).

Blood (rabbit).

Liver (hog).

Liver (rabbit).

Kidney (rabbit).

Muscle (rabbit). 1.50 0.62 0.00 0.6:

Calorimetric estimation.

ai 2 ti 2 * .%j 75 4

;; -at

E P -- gm. w?. 1.33 1.62 1.60 3.2: 1.25 2.04

2.10 2.7s 1.20 2.1s

~~

1.50 0.34 1.50 0.31

~-

1.50 0.4:

--

1.00 2.1;

1.00 3.1: --

1.00 1.3: 1.00 3.OE

--

1.00 0.74

ti 8 2 % -8

2 3 w 19 F+ __- nag. mg. 0.06 1.6:

0.56 2.6 0.19 1.8! 0.08 2.7.

0.21 1.9 --

0.00 0.3‘ 0.00 0.3 __-

0.00 0.4:

0.66 1.41

0.65 2.4 --

0.49 0.8~ 0.63 2.4:

--

0.19 0.5,

-

3

7 5 1

7 . _

i 1

3

6

7 -_

4 3

5

-_

3

E‘ bo 8

.4 6 2

2.2 0

mg.

123

167 148

129 164

23 21

29

146

247

84

243

55

42

--

--

--

--

-_

Iodine titration.

Indicator.

w7. 227

216 174

156 204

Xitroprusside. “

I‘ “ “

42 Nitroprusside. <‘

26 33

.-

- -

_-

Nitroprusside. Starch.

327

410

Nitroprusside. “

154 319

368

Nitroprusside. “

Starch.

61 Nitroprusside. 88 Starch.

30 Nitroprusside.

46 Starch.

* Potassium iodide was added to those extracts which were titrated with iodine in the presence of starch. Potassium iodide was not added to the other extracts.

data of Table I shows that the omission of iodide cannot account for the large differences found? When applied to extracts of blood and muscle the results of the two methods are not far apart. The iodine titration, however, tends to give a high result when the

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H. L. Mason 633

indicator is starch, whereas the result is lower when the disappear- ance of the nitroprusside reaction is taken as the end-point. This is in full accord with the results of other workers. Further refer- ence to Table I shows that the lack of sensitivity of the nitroprus- side reaction is sufficient to account for the low values. It is apparent that the iodine titration cannot be used for the estima- tion of glutathione in extracts of yeast, liver, and kidneys. Its use with extracts of other tissues should be thoroughly investigated before reliance is placed on the results.

It is perhaps significant that when the iodometric and colori- metric values are close together the blank is very small or zero; when the two values are divergent, the blank is large. It appears that some reducing factors in the tissues which react with iodine are eliminated to a large extent in the ferricyanide oxidation by the use of the low pH and the blank. These reducing factors are particularly abundant in yeast, liver, and kidneys.

Thioneine as a constituent of blood is of particular interest since, like glutathione, it has reducing power associated with a sulfhydryl group. Accordingly the iodine titration of thioneine alone and in the presence of GSH was studied. It was found that a sample of thioneine obtained from T. B. Johnson used 0.9 as much iodine as an equal weight of glutathione. This consumption of iodine is 9 times the number of equivalents of ferricyanide reduced by thio- neine. It might very well account for the difference in the results obtained by the two methods with blood filtrates. It seems likely that the errors involved in the iodine titration of blood filtrates are those involved in the titration of GSH itself plus the error due to thioneine.

Aside from its greater specificity the new method has a further advantage over the iodine titration in that much smaller amounts of tissue are required. When small concentrations of glutathione are involved, it is more precise since glutathione uses more than an equivalent amount of iodine at low concentrations (Table I).

SUMMARY.

In the course of a study of the iodine titration of reduced glutathione it was found that iodide is necessary to make the reac- tion proceed in a straightforward manner. Starch is much to be preferred to nitroprusside as the indicator.

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Reduced Glutathione

A new method for the estimation of reduced glutathione in tissues is described.

The new method is compared with the iodine titration method. The values found for glutathione in extracts of yeast, liver, and kidneys by titration with iodine are much larger than those found by the new method. Since the latter gives maximal values, the iodine titration is considered as not trustworthy for these tissues. With blood and muscle there is fair agreement between the two methods. Any disagreement in the case of blood is probably due to thioneine.

BIBLIOGRAPHY.

1. Benedict, S. R., andNewton, E. B., J. Biol. Chem., 83,357 (1929). 2. Blanchetiere, A., and Melon, L., Compt. rend. Sot. biol., 97, 242 (1927).

3. Flatow, L., Biochem. Z., 194,132 (1928). 4. Folin, O., J. Riol. Chem., 77, 421 (1928). 5. Folin, O., and Wu, H., J. Biol. Chem., 38,81 (1919). 6. Gabbe, E., Am. J. Physiol., 90,354 (1929).

7. Okuda, Y., J. Biochem., Japan, 6,201 (1925). 8. Perlzweig, W. A., and Delrue, G., Biochem. J., 21, 1416 (1927).

9. Thompson, J. W., and Voegtlin, C., J. BioZ. Chem., 70,793 (1926). 10. Tunnicliffe, H. E., Biochem. J., 19, 194 (1925). 11. Turner, R. H., Proc. Sot. Exp. BioZ. and Med., 26,641 (1929).

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Harold L. MasonGLUTATHIONE IN TISSUES

DETERMINATION OF REDUCED A STUDY OF GLUTATHIONE: II. THE

1930, 86:623-634.J. Biol. Chem. 

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