Vegetal Reduction of Dehydroascorbic Acid E. F. KOHMAN’ AND N. H. SANBORN National Canners Association, Waehington, D. C.
M
The juice of legume seeds contains glutathione and additional substance or substances which reduce dehydroascorbic acid. These substances therefore act as a protection against the oxidation of ascorbic acid by oxygen of the air. The reduction of dehydroascorbic acid by these substances is catalytically accelerated by an enzyme. After the dehydroascorbic acid is reduced, an enzyme catalyzes the reduction of the oxidized glutathione and perhaps other substances oxidized by the dehydroascorbic acid. These substances seriously interfere with the estimation of ascorbic acid by titration with 2,6-dichlorophenolindophenol. Copper does not affect the reduction of dehydroascorbic acid by these reducing substances as it does the oxidation of ascorbic acid by oxygen. I t has not been determined whether iodide added with the dehydroascorbic acid is responsible for the lack of effect of copper on the reduction. I t is not known that the marked reducing value toward 2,6-dichlorophenolindophenol that develops in raw pea juice upon the addition of dehydroascorbic acid represents biologically active ascorbic acid (vitamin C) Until these above questions can be answered, definite limitations must be placed upon present chemical methods for evaluating the vitamin C content of certain plant materials.
UCH remains to be desired in the chemical determination of ascorbic acid. I n a recent issue of the Journal of Biological Chemistry alone, two
papers criticized the technic commonly employed. Mack and Tressler (16)state that it “may involve large constant errors.” Lyman, Schultze, and King (13) indicate that the determination might be significantly influenced even though the same pH is observed, depending upon whether metaphosphoric, orthophosphoric, sulfuric, hydrochloric, or hydrobromic acid is used in the extraction necessary for the chemical determination. It is rather generally accepted that the urine contains substances that interfere with the chemical methods for the determination of ascorbic acid. It would be surprising not to find a similar situation in at least some vegetables or fruits. Stress has been laid on the possibility that results are affected by oxidation during the extraction process as well as any subsequent necessary manipulation. Obviously there is need for more information with reference to factors that may influence ascorbic acid.
Destruction of Vitamin C by Hot Acid Extraction The original method of Bessey and King (3) was recently employed by Daniel, Kennedy, and Munsell (6) in a comparison of the vitamin C content of orange and tomato juices. This consists of extracting 10 cc. of the juice (containing the usual suspended pulpa) three successive times with 25, 10, and 10 cc. of hot 8 per cent acetic acid, centrifuging, respectively, 5, 3, and 3 minutes, and decanting after each centrifuging process. Heating after acidification is generally destructive of vitamin C. To determine its effect in this process, a number of determinations3 were made. The tomato juice obtained by passing tomatoes through a seprosive (a household size of one type of commercial tomato juice extractor) was diluted with an equal volume of solution consistPresent address, Campbell Soup Company, Camden, N. J. Tomato juice contains only about 0.6 per cent insoluble solids, and orange juice even less, and therefore a true extraction process is unnecessary. I t is necessary only t h a t the diluent come t o equilibrium with the juice occluded in the insoluble solids. This occurs a t least within a minute when the insoluble solids are as finely divided as they are in commercial tomato juice. If the tomatoes have been cooked or canned, no ordinary home screening process requires a materially longer time for equilibrium t o be established with the diluent. With raw tomatoes more care must be given to the degree of fineness with which the insoluble solids are divided. As has been suggested by others, 2 per cent metaphosphoric acid added to the acetic acid solution is also desirable. 8 Incidentally in all cases, except where otherwise specified in this paper, for ascorbic acid determinations the juice was acidified with an equal volume of a solution containing 6 per cent acetic and 2 per cent metaphosphoric acid. The acidified mixture was then centrifuged and the supernatant liquid pipetted o f f for titration with 2,6-dichlorophenolindophenolas promptly as possible. Filtration instead of centrifuging a t this point frequently requires considerable time and thus overexposes the juice t o oxygen of the air and entails loss in titration value. This determination constitutes ascorbic acid aa spoken of here.
.
1
1
ing of 6 per cent acetic and 2 per cent metaphosphoric acid. It was filtered almost immediately and then titrated, in contrast to the hot extraction process consisting of three centrifugings employed by Daniel, Kennedy, and Munsell, who used 8 per cent acetic acid in some cases and in others, 6 per cent acetic plus 2 per cent metaphosphoric acid solution. The cold dilution gave consistently higher results ranging from 3.2 to 7.9 per cent. The effect of heating after acidification is brought out in Table I. The tomatoes were passed through a seprosive, and the juice was heated (200 cc. in 300-cc. Erlenmeyer 1195
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INDUSTRIAL AND ENGINEERING CHEMISTRY
VOL. 29, NO. 10
Effect of pH on Ascorbic Acid Stability
flasks) after acidification with an equal volume of 6 per cent acetic plus 2 per cent metaphosphoric acid, in comparison t o heating a similar volume of tomato juice under similar conditions without acidification, and then cooling, acidifying, and filtering it for titration with 2,6-dichlorophenolindophenol. Any loss due to evaporation was corrected by replacement with water. In every instance the loss incurred by heating after acidification was greatest, Attention is called also to the much greater destruction resulting from actual boiling over a direct flame for 45 minutes for 30 minutes by placing the Erlenmeyer water bath.
There is an enormously wide variation in the relative stability of ascorbic acid in different vegetables and fruits. Although pH is a factor in this connection, its effect has been grossly exaggerated. Acidification to prevent loss of vitamin C in cooking has a t times been recommended, when really the effect is generally adverse rather than beneficial. Table I1 shows that the effect of pH is only moderate and in some cases negligible compared with other factors. The data were obtained as follows : Oranges, tomatoes, and grapefruit were juiced in the usual fashion. The other products were first ground and then pressed between canvas pads at a pressure of 15,000 pounds per square inch (1055hg. per sq. om.). Possibilities of copper contamination were avoided. To BEFORE AND AFTER ACIDIFICATION TABLEI. EFFECTOF HEATING 100-cc. samples of the raw fruit or vegetable juice, suffiAscorbic Acid cient 5 per cent hydrochloric acid or sodium hydroxide was Heated Heated before after added to bring the original pH near 3.7 and 6.5, respectively. Sample AcidiAcidiWater was added t o make the final volume the same in all No. fication fication Source of Juice Treatment Mg. per 100 CC. cases. The juices were all held a t room temperature in Boiled 45 min. over 1 11.1 8.6 Canned tomatoes 250-cc. beakers containing 25-cc. portions and making a direct flame depth of 0.68 em. The beakers were uncovered but were not 2 11.2 8.3 Boiled 45 min. over direct flame in a direct draft. There was only slight evaporation, which 3 21.7 19.9 Heated 30 min. in water bath was corrected for sampling in all cases by the addition of 4 24.0 22.4 Heated 30 min. in enough water to restore the initial weight. Adjusting the water bath 5 14.6 4.4 Raw ripe (picked green) Boiled 30 min. over pH to the desired point required a little time during which direct flame the ascorbic acid naturally present was lost to a large extent 6 11.3 7.0 Raw (vine ripened) Boiled 30 min. over direct flame in some products. Therefore ascorbic acid was added to some of the juices so that appreciable quantities would be present a t the beginning of the experiment; the pH was not Destruction of Ascorbic Acid by Oxidation adjusted in every instance because it was sufficiently close to that desired. Numerous publications have dealt with the loss of vitamin In some of the products the ascorbic acid is practically as C due to oxidation in vegetables and fruits under conditions stable a t the higher as a t the lower pH. To a varying degree of handling: ‘LPeasin the pod held at ordinary room temperatures lose their vitamin C content rather rapidly” (16). this is true of oranges, grapefruit, tomatoes, and peas in a descending order. In one group of products, such as apple, “Shipment or storage of peas in the pods for 1 or more days potato, and carrot, although the pH is not without effect, the has a marked destructive effect on their ascorbic acid conascorbic acid disappears a t a rapid rate a t both pH values. tent” (7). “Spinach held a t room temperature lost approxiIn another group of products, notably spinach, turnip, and mately one-half of its ascorbic acid in 3 days, and practically cabbage, lowering the pH has a more marked effect in preall of it in 7 days)’ (20). “Snap beans, peas, and spinach venting loss of ascorbic acid. rapidly lose ascorbic acid if held a t room temperature” (19). Bezssonoff (4), early in the history of vitamin C, found that Systems Which Reduce Dehydroascorbic Acid its content in pressed potato juice was so fugitive that it was All this leads to the question as to why growing vegetables lost within the time required to feed his animals. Generally and fruits constantly exposed t o the oxygen of the air, and the ascorbic acid in freshly pressed juices is far more unstable than in the unbruised plant cell, and enzyme activity seems to be involved in its destructioh. The authors have found that vitamin C loss is greatly accelerated by shredding carTABLE11. VITAMIN C STABILITY AB AFFECTED BY PH rots as for salad (11); on the other hand, the losses that occur Initial Ascorbic during storage of raw produce are arrested by the canning Acid --Per Cent Loss in:-Product pH Content 1 hr. 3 hr. 6 hr. process (IO). My./100 CC. Szent-Gjorgyi (18) demonstrated enzymic activity destrm46.1 0 0 6.2 Orange 6.42 tive to hexuronic (ascorbic) acid in minced cabbage leaves, 46.6 0 0 7.2 3.72” Qrapefruit 6.24 39.4 1.3 2.7 4.1 and Kertesa, Dearborn, and Mack (9) said that such activity 3.61 39.6 0 0 0 in peas is due to an “ascorbic acid oxidase.” The claim of 8.1 13.6 Tomato 6.00 64.0b 0 1.8 1.9 3.0 3.66 64.3, McHenry and Graham (14) that some vegetables show an Pea 6.48” 27.9 10.0 16.0 27.1 3.80 33.0 3.2 8.6 13.3 increased titration value for ascorbic acid after heating is now 6.21 39.8b 46.8 100 Apple . generally explained on the basis that the heating inactivates 3.66 40.4b 95.3 72.2 96:s 6.30“ 37.0b 85.0 100 Potato the enzymes that accelerate oxidation during the manipula3.76 36.8b 21.7 49.4 70:O tion necessary for the chemical determination, although 45.1 100 6.77 39.W Carrot 3.76 39.6) 7.5 23.6 35:3 McHenry and Graham explain this on the hypothesis that 6.36 42.4b 37.2 100 Spinach some of the ascorbic acid occurs in a combined form that is 2.8 6.0 9:3 3.80 42.0 Turnip 6.66 18.6 21.6 56.8 81.6 hydrolyzed during the heating process. McHenry and 0 2.2 4.4 3.83 18.4 Graham also make the statement that vegetable tissues do 6.10 63.Ob 6.8 15.5 27.1 3.70 63.0) 1.0 1.7 2.6 not appear to contain a mechanism to prevent the aerobic Cabbage 6.71“ 71.46 16.0 47.6 81.8 oxidation of ascorbic acid. Moreover Saent-Gjorgyi (18) 3.72 71.4) 4.6 7.8 8.7 No acid or alkali added to adjust pH. failed in an attempt to demonstrate the reduction of reversi“ 6 0 mg. ascorbic acid per 100 cc. added just before taking sample for bly oxidized hexuronic (ascorbic) acid in minced cabbage “initial ascorbic acid content” determination. leaves. 0
~
OCTOBER, 1937
INDUSTRIAL AND ENGINEERING CHEMISTRY
constantly using oxygen in the respiratory process, retain their vitamin C. The assumption seems warranted that every vegetable and fruit must contain some sort of reducing mechanism that serves as a protection against the oxidation of ascorbic acid. In a recent note (12)preliminary evidence to justify such an assumption was given. Pfankuch (17) made claims of an enzyme in potatoes which catalyzed the reduction of dehydroascorbic acid if cysteine is present. Hopkins (8) suggested in cauliflower and similarly related plants (Brassica) an enzyme which will reduce dehydroascorbic acid in the presence of glutathione. Hopkins is of the opinion, however, that Brassica does not contain glutathione. Convinced by all available data that the stability of ascorbic acid is by no means as simple a matter as oxidation, acidity, and pH, and that the very existence of ascorbic acid in the plant kingdom is dependent upon some reducing system, the writers have been watching for such a system. To their knowledge the reversible oxidation of ascorbic acid and reduction of dehydroascorbic acid are the only characteristics of this system so far demonstrated to be capable of biological functions. The data to be presented demonstrate a reducing system in peas, lima beans, and navy beans, indicating that it is present in all legumes although it does not function under similar conditions in other vegetables or fruits tested. When freshly pressed pea juice was exposed to air during the pressing operation it had a lower titration value if titrated immediately than if titrated a short time later. For example, juice titrating 44 mg. per 100 cc. when first pressed titrated 48.8 somewhat later. Pouring this juice back and forth several times after it was freshly pressed reduced the titration value to 36.0 mg. per 100 cc.; later it again titrated 48.3 mg. per 100 cc. A comparable rise in reducing value to 2,6-dichlorophenolindophenol was observed in pea juice after adding dehydroascorbic acid obtained by the action of 2,6-dichlorophenolindophenol on ascorbic acid dissolved in an acid mixture consisting of 6 per cent acetic and 2 per cent metaphosphoric acid. For this oxidation 25 mg. of ascorbic acid were added to the 10-cc. acid mixture, and to this slightly less dry 2,6dichlorophenolindophenol powder was added than was required to oxidize all the ascorbic acid. After filtering, this was neutraKzed to phenolphthalein with sodium hydroxide and added to the pea juice. In subsequent experiments it was found more convenient to oxidize ascorbic acid with iodine before adding it to the pea juice. It was convenient to work with 100-cc. portions of pea juice. Therefore an iodine solution was prepared, 5 cc. of which would exactly oxidize 100 mg. of ascorbic acid. The iodine solution was then dropped slowly a t first into the dry ascorbic acid. As soon as there was sufficient liquid to maintain an excess of ascorbic acid in solution, the iodine could be run in as rapidly as it was decolorized, and in this manner any possible effect of excess iodine on dehydroascorbic acid was avoided. The hydriodic acid thus generated was insufficient to effect appreciably the pH of the pea juice when 5 CC. of dehydroascorbic acid solution, equivalent to 100 mg. of ascorbic acid, were added to 95 cc. of pea juice. The juice pressed from soaked pea seeds has a reducing effect to dehydroascorbic acid, although merely by soaking overnight for 16 to 18 hours it is less marked than the juice from green peas. To obtain the soaked pea juice of Table 111,200 grams of Thomas Laxton pea seeds were spread in a shallow pan with 500 cc. of water so that they would still be only one layer deep after they had absorbed the water while soaking for 16 hours. The solids content of such peas is then approximately 25 per cent, which is in the neighborhood of that of the green peas of Table 111. The green peas were the California variety purchased in the Washington market.
1197
It is likely that greater activity would be manifested by freshly harvested peas. To study the effect of heat, the pea juice was heated in an Erlenmeyer flask set in a sugar solution bath whose boiling temperature was around 105' C. By agitating the pea juice somewhat, a temperature of 80" C. was reached in approximately 4 minutes, 98" C. in approximately 10 minutes, and 100" C. in approximately 14 minutes, when the pea juice was cooled and the original volume restored by adding water that evaporated during heating. Just prior to starting the experiment to determine the reducing effect on dehydroascorbic acid, ascorbic acid equivalent to 100 mg. per cent was oxidized with iodine and then added to the juice. Samples were immediately taken for both ascorbic acid and glutathione. In some instances this required 4 or 5 minutes before the sample for ascorbic acid was diluted with an equal volume of acid mixture containing 6 per cent acetic and 2 per cent metaphosphoric acid, and the sample for glutathione with three times its volume of 131/a per cent trichloroacetic acid, giving a final concentration of 10 per cent. Apparently this acidification completely inhibits the action in question. Glutathione was determined after acidification with trichloroacetic acid and filtration, by precipitation with cadmium lactate according to the method of Benet and Weller ($2') which applies only to reduced glutathione. The figures for glutathione are given with the realization that there is not sufficient information to appraise the accuracy of this method. Except in systems of known components, the difference between iodine titration and dye titration as a measure of glutathione is utterly useless because it is not specific. TABLE111. EFFECT OF HEATON SOARED AND (MARKET)PEAJUICD
-
-
ON
-Soaked Pea Juice--Green Pea -UnheatedHeated -UnheatedAs.a G1. As. G1. As. GI. cMilligrama per 100 cc. 3.5 79.2 2.0 66.4 23.0 61.3 0.6 13.8 6.0 49.9 3 29.5 33:6 12.1 56:6 99.7 62'0 4 As. = ascorbic acid: G1. glutathione.
Time Hr. 0
-
GREEN
-
-
J u i c w
Heated As. GI.
19.0
23.7 29.9
61.3 36:9
Since the equivalent of 100 mg. of ascorbic acid per 100 cc. was added to these pea juices as dehydroascorbic acid, there was apparently a recovery in 3 hours of 26.0 and 10.1 per cent, respectively, in the unheated and heated soaked pea juice and 76.3 and 10.9 per cent, respectively, in the unheated and heated green pea juice of Table 111. The glutathione figures do not lend themselves to logical deduction except that, if the method for glutathione determination is of any value whatsoever, it is apparent that the amount of glutathione present is inadequate to account for the amount of dehydroascorbic acid reduced. Incidentally, to convert the glutathione figuresto ascorbic acid equivalent, they must be divided by 3.49. It must be borne in mind that the glutathione might be acting as an intermediate in the reducing system, the glutathione in turn being reduced by some other substance in the pea juice as it is oxidized by the dehydroascorbic acid. Such a condition would serve to explain the lack of regularity in the glutathione values, since we would expect that the proportion of reduced and oxidized glutathione present a t any moment would be a matter of chance. In a comparable experiment with green lima bean juice, the ascorbic acid values changed from 7.1 mg. per 100 cc. a t zero time to 24.7 mg. after 3 hours, whereas the corresponding glutathione values were 6.6 and 7.9 mg. per 100 cc. These small glutathione values are further evidence that the reduction cannot be accounted for by glutathibne. The fact that heating the juice has such a marked effect indicates that the reduction of dehydroascorbic acid is catalyzed by an enzyme. In other
1198
INDUSTRIAL AND EN(SINEERING CHEMISTRY
experiments merely bringing the temperature to 98" C. in about 7 minutes was as effective as prolonging the heating period several fold, although in no case was the reducing effect completely destroyed by heating.
Effect of Copper The catalytic effect of copper on the oxidation of ascorbic acid by elementary oxygen is well known. Barron, Barron, and Klemperer (I) advanced the theory that biological fluids possess inhibitory mechanisms that protect the ascorbic acid from oxidation in varying degrees of efficiency, and that fluids of animal origin and some vegetable fluids containing TABLEIV. LACKOF INHIBITING EFFECT OF 12 P. P. M. OF COPPER ON DEHYDROASCORBIC ACIDREDUCTION AB. in Spaked -Pea JuiceWithout With Time Cu Cu Hr. , 0 6 7 5.7 1 29.9 27.6 8 3 8 . 9 37.2
.
Soaked Navy Bean Juice -HeatedWithout Cu With Cu Without Cu With Cu As. G1. As. G1. As. As. C1. Milligrams per 100 cc. 2.7 28.0 2.7 28.0 1 . 3 1 . 3 11.3 18.1 419 14:6 i:O g.*l 3'.'6 f.'s -Unheated-
..
appreciable quantities of ascorbic acid constitute a group with the most efficient inhibitory mechanism. Protective mechanism and ascorbic acid content cannot becorrelated however. The vitamin C potency-that is, ascorbic acid content -of spinach and potatoes is of the same magnitude as the vitamin C potency of citrous fruits. As is evident from the data in Table 11,there is a vast difference in the stability of the ascorbic acid in these vegetables and citrous fruits even though their pH is brought together. These investigators explain the protection from oxidation on the basis that such substances as glutathione, proteins, and amino acids inhibit copper catalysis. Their data seem to show that glutathione does function in this manner when copper is the catalytic agent. If glutathione is the active principle causing the reduction of dehydroascorbic acid in the juice of legume seeds, then copper in turn should inhibit the activity of glutathione. However, copper has very little effect in this respect, as indicated in Table IV. The changes in ascorbic acid content in the raw navy bean juice are roughly twice as great as the glutathione changes expressed in terms of ascorbic acid equivalent. Unfortunately no determination of the glutathione content of the heated juice was made before adding dehydroascorbic acid. Assuming that heating did not affect the glutathione content, the change in glutathione content was somewhat more than enough to account for the small change in ascorbic acid values.
VOL. 29, NO. 10
and supplied with similar fritted glass filters to make aeramost effective. The large flasks made provision for the profuse frothing that occurred. Aeration was very rapid so that the second flask in the series would not receive a lower concentration of oxygen due to its being depleted by the juice in the first flask. The copper was added to the second flask in the series so that, if any such effect did occur, the greater concentration of oxygen in the current of air would be in the first flask containing no added copper. Table V shows that,when 25 p.p.m. of copperwere added, the ascorbic acid content dropped from 76.2 to 18.7 mg. per 100 cc. in a total time of 20.5 minutes; 7.5 minutes were spent in active aeration, and the remainder of the time was required to take samples between aeration periods. During similar aeration, the ascorbic acid in the pea juicewithout added copper dropped only from 76.2 to 71.5 mg. of ascorbic acid per 100 cc. Table VI gives data on another sample of the same pea juice that was also divided into two portions, aftei. adding to it dehydroascorbic acid equivalent to 50 mg. of ascorbic acid per 100 cc. To one portion were then added 25 p. p. m. of copper, and the reduction of the dehydroascorbic acid was determined. The reducing effect was almost as marked in the presence of copper as in its absence. In this connection the idea must be considered that the copper may be inactivated by the hydriodic acid resulting from the oxidation of the ascorbic acid and added to the pea juice with the dehydroascorbic acid.
. tion
TABLE VI. LACKOF EFFECT OF 25 P. P. M. OF COPPERON REDUCTION OF DEHYDROASCORBIC ACID^ ADDEDTO GREENP B h JUICE -ME. As./100 Co.Without Cu With Cu 0 44.0 44.0 1 86.7 83.1 3 87.7 83.7 Equivalent to 50 mg. ascorbic acid per 100 O C . Time, Hr.
5
The data in Table VI1 indicate that glutathione reduces dehydroascorbic acid and that thereafter the oxidized glutathione is reduced. Green pea juice to which had been added dehydroascorbic acid equivalent to 100 mg. of ascorbic acid per 100 cc. was divided into three portions. Sixty and 120 mg., respectively, of glutathione per 100 cc. were added to two of the portions. As quickly as possible after the addition of dehydroascorbic acid and glutathione, samples were taken. For ascorbic acid determinations, these were diluted as quickly as possible with an equal volume of a mixture of acid containing 6 per cent acetic acid and 2 per cent metaphosphoric. For glutathione determination, samples were diluted per cent trichlorowith three times their volume of 13 acetic acid. The sample treated with the mixture of acetic and metaphosphoric acid was centrifuged and then titrated 2,6-dichlorophenolindophenoland also with iodine in the with EFFECT OF 25 P. P. M. OF COPPER ON OXIDATION TABLE V. form of a standard potassium iodate solution. The sample OF ASCORBIC ACIDDURING AERATION OF GREENPEAJUICE treated with trichloroacetic acid was filtered, and the filtrate Asoorbic Aoid Sample Sampling WithWith was used for cadmium precipitation of the glutathione. NO. Aeration Time Time out Cu Cu The precipitate was titrated with the same potassium iodate Min. Min. M g . per 100 CC. solution used for titrating the clear centrifuged liquor of the 1 Control immediately on adding Cu 7 6 . 2 76.2 2.6 5 73.3 53.2 2 other sample. The sampled juices were then held in small 2 3 72.1 34.1 3 bottles filled to the brim and capped with a cover glass. I t 3 5 71.5 18.7 4 should be borne in mind that a few minutes (5 to 7) were required to take samples and acidify them. If any rapid changes More speciftc data were desired on the relative effect of were occurring, this would be reflected in the results obtained copper in the oxidation of ascorbic acid during aeration as a t zero time. According to Hopkins (8) the enzymic reducagainst its effect upon the reduction of dehydroascorbic tion of dehydroascorbic acid by means of glutathione is a acid. After the addition of 50 mg. of ascorbic acid per 100 rather rapid reaction. This idea is confirmed in Table VII. cc., green pea juice was divided into two portions and aerated The ascorbic acid content as determined by the dye titrain series in the necks of two 6-liter flasks that were inverted tion is progressively higher at zero time in portions 2 and ~
OCTOBER, 1937 TABLE VII.
INDUSTRIAL A N D ENGINEERING CHEMISTRY
COMPARISON OF RESULTS OF GLUTATHIONE DETERBY CADMIUM PRECIPITATION METHOD WITH DIFFER-
MINATIONS ENCE BETWEEN IODINE AND ~,~-DICHLOROPHENOLINDOPHENOL
TITRATION
---Ascorbic
-GlutathioneKIOa minus dye, KIOs X 3.49 Cd. ppt. MilliQrams per 100 cc. Hr. 1. 100 M g . Dehydroascorbic Acid Added per 100 Cc. 0 34.4 59.0 85.9 17.7 101.4 48.9 9.9 1 87.4 3 114.3 132.7 64.3 36.1 2. 100 Mg. Dehydroascorbic Acid 60 Mg. Glutathione Added per 100 Cc. 0 44.7 69.4 86.2 43.1 1 101.3 111.0 33.8 10.3 98.4 84.5 3 117.5 145.7 100 M g . Dehydroascorbic Acid 120 M g . Glutathione Added per 100 Cc. 72.9 67.4 60.7 0 53.0 116.5 22.0 14.7 1 110.2 144.1 128.4 3 122.7 164.0 Time
Acid-
Dye
7
+
+
3 to which glutathione had been added. Moreover, the sample taken a t zero time for glutathione revealed a much lower glutathione content in portions 2 and 3 than had been added. After an hour it was apparent that there was a marked increase in ascorbic acid and further decrease in glutathione content. After 3 hours the proportionate increase of ascorbic acid was small, but a large increase in glutathione content was apparent. This indicates not only that the dehydroascorbic acid was reduced in the pea juice, the reduction being accompanied by an oxidation of glutathione, but that subsequently the oxidized glutathione was again reduced.
Discussion The peas from which the juice of Tables V and VI was obtained were the most succulent of all encountered in this work; the juice for these experiments was pressed out a t the same time and subsequently divided into two lots. Since 50 mg. of ascorbic acid per 100 cc. were added to the portion represented in Table V, the pea juice must have contained a t least the difference between 76.2 and 50, or 26.2 mg. per 100 cc. The experiment in Table V required about 30 minutes. Then dehydroascorbic acid, equivalent to 50 mg. of ascorbic acid per 100 cc., was added to a second portion of juice, after which titration indicated 44.0 mg. of ascorbic acid per 100 cc. The difference between 26.2 and 44.0 is due in large part to the fact that dehydroascorbic acid formed from exposure to air in the pressing operation was reduced during subsequent standing for 30 minutes when the experiment in Table VI was begun. In part it was due to the fact that a small amount of added dehydroascorbic acid was reduced before a sample could be taken for analysis. For these reasons a n accurate estimate of the yield of ascorbic acid obtained from the dehydroascorbic acid added is not possible. The difference between 87.7 and 44.0 mg. per 100 cc., or 87.4 per cent recovery, is probably conservative. It has been intimated that there are insufficient data to justify an appraisal of the cadmium lactate precipitation method for glutathione determination, although the method probably does give approximate results. It has also been intimated that the difference in titration with iodine minus
1199
titration with 2,6-dichlorophenolindophenolis of no value in evaluating glutathione content, although many have used this technic in various connections. All the data presented here indicate some glutathione in legume seeds, but the quantity present is far from adequate to account for the amount of dehydroascorbic acid that may be reduced. There appears to be another substance, or substances, however, that the dehydroascorbic acid oxidizes, as indicated by a comparison of the figures in the last two columns of Table VII, calculated as glutathione, respectively. from the difference between iodine (potassium iodate) and 2,6-dichlorophenolindophenoltitration and from iodine titration of the cadmium precipitate. That the dye is also reduced by this unknown factor more slowly than by ascorbic acid but so rapidly as to make it unsatisfactory for ascorbic acid determination is apparent if the juice from soaked peas to which no dehydroascorbic acid has been added is titrated with the dye. Such soaked pea juice slowly reduces 2,6-dichlorophenolindophenol, and as a result a sharp, satisfactory exid point is impossible. The same pea juice to which dehydroascorbic acid had been added sometime previously gives, a much sharper and more satisfactory end point. Finally, the reducing value to the dye that develops upon the addition of dehydroascorbic acid may not represent biologically active antiscorbutic value but instead a substance or substances of the nature referred to by Borsook and associates (6) as arising from the spontaneous decomposition of dehydroascorbic acid. The writers are not in a position to determine this point as was originally planned.
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