THE DISTRIRIJTION OF VITAMIN C IN PLANT AND ANIMAL TISSUES, AND ITS DETERMINATION*+ BY OTTO A. BESSEY AND C. G. KING (From the Department of Chemistry, University of Pittsburgh, Pittsburgh) (Received for publication, October 20, 1933) The study of vitamin C in relation to its functions in plant and animal tissues is greatly facilitated by its striking characteristic as a strong reducing agent. Isolation and identification of the vitamin as a single substance (l), which had been previously studied as a reducing substance in biological systems (2), has been verified in a number of laboratories (3) and has made possible a direct chemical approach to the study of vitamin C metabolism. The quantity of vitamin in most tissues is too small, and its isola- tion too intricate (4) for rapid progress based entirely upon its separation. Accordingly the development of a direct titration method for estimating the vitamin content of tissues is of great importance. Using the oxidation-reduction indicator, 2,6-dichlorophenol- indophenol, introduced by Mansfield Clark and associates (5), Tilhnans and associates (6) have introduced a method (supported by a considerable amount of animal assay work) which, with vari- ous minor modifications, has come into ext.ensive use (7, 8) for the direct titration of the vitamin. Studies in our laboratories during the past year have led to minor deviations from the method proposed by Tillmans, and have further demonstrated its value in physiological studies. * Contribution No. 274 from the Department of Chemistry, University of Pittsburgh. Much of the material upon which thii paper is based was presented before the meeting of the American Chemical Society at Chicago, August, 1933. t The authors are greatly indebted to Parke, Davis and Company, and the Abbott Laboratories, for a Research Fellowship Grant during the course of this investigation, and to the California Fruit Growers Ex- change for supplying the lemons used. 637 by guest on July 5, 2020 http://www.jbc.org/ Downloaded from
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THE DISTRIRIJTION OF VITAMIN C IN PLANT AND ANIMAL TISSUES, AND ITS DETERMINATION*+
BY OTTO A. BESSEY AND C. G. KING
(From the Department of Chemistry, University of Pittsburgh, Pittsburgh)
(Received for publication, October 20, 1933)
The study of vitamin C in relation to its functions in plant and animal tissues is greatly facilitated by its striking characteristic as a strong reducing agent. Isolation and identification of the vitamin as a single substance (l), which had been previously studied as a reducing substance in biological systems (2), has been verified in a number of laboratories (3) and has made possible a direct chemical approach to the study of vitamin C metabolism. The quantity of vitamin in most tissues is too small, and its isola- tion too intricate (4) for rapid progress based entirely upon its separation. Accordingly the development of a direct titration method for estimating the vitamin content of tissues is of great importance.
Using the oxidation-reduction indicator, 2,6-dichlorophenol- indophenol, introduced by Mansfield Clark and associates (5), Tilhnans and associates (6) have introduced a method (supported by a considerable amount of animal assay work) which, with vari- ous minor modifications, has come into ext.ensive use (7, 8) for the direct titration of the vitamin.
Studies in our laboratories during the past year have led to minor deviations from the method proposed by Tillmans, and have further demonstrated its value in physiological studies.
* Contribution No. 274 from the Department of Chemistry, University of Pittsburgh.
Much of the material upon which thii paper is based was presented before the meeting of the American Chemical Society at Chicago, August, 1933.
t The authors are greatly indebted to Parke, Davis and Company, and the Abbott Laboratories, for a Research Fellowship Grant during the course of this investigation, and to the California Fruit Growers Ex- change for supplying the lemons used.
Pwparation of Indicator--.The preparation of the dye indicator, 2 : 6dichlorophenolindopheno1, has been readily followed by sev- eral students in our laboratory, using essent.ially hhe met,hod given by Clark and associates (5). To 5 gm. of powdered 2,6dichloro- quinonechloroimide (Eastman Kodak Company) in a beaker cooled in an ice bath, add 2.25 gm. of phenol and stir thoroughly. Slowly, and with const,ant sbirring, add 18 cc. of 3 N sodium hy- droxide solution, keeping the temperature near 0”. After 30 minutes add 150 cc. of 15 per cent salt soWion, stir until precipi- tation is practically complete, and filter on a Ruchner funnel. The crude dye is then stirred with successive 300 cc. portions of warm distilled water until dissolved (2 to 3 liters), and again salted out by t,he slow addit,ion of salt with st,irring. The filtered and rinsed precipitate dries in 2 to 3 days in a vacuum desiccator, giv- ing a yield of 4 to 5 gm. An extraction of the dry powdered dye with ether is advantageous in that resalting out, alone may not remove a small amount of red-colored impurity which may mask the end-point, during titration.
Stmdardimtion of Indicator Solution-To prepare the indicator solution, dissolve 0.10 gm. of the dry dye with successive portions of warm water, cool, add wat,er to make the volume 206 cc., and filter. The keeping qua&y of the dye is improved by adding a small amount of phosphate buffer, pH 6.8, and storing in a dark bottle. Ahhough the dry dye is quite stable, aqueous solutions of the dye change slowly and should be restandardized daily. Solut,ions more than 5 days old should generally not be used as it is too difficult to obtain a satisfactory end-point, the color change being from red to brown instead of red to colorless.
Tilhnans’ method for the standardization of the indicator, by the use of a standard ferrous ammonium sulfate solution free from dissolved oxygen and carefully buffered wit,h sodium oxalate, was found to be inconvenient because of the extreme care necessary to exclude air and t,o have the solution properly buffered. For some time we used the pure vitamin for standardizing the reagent, but lat.er we found such concordant results with the titration of lemon juice that we now use this for routine standardization. This is due to the fact that the acid-iodine titration and the dye titration of good quality lemon juice give equivalent values for the vitamin content. Occasional check titrations against the
pure vitamin may be desirable, but in our experience the titra- t,ion of lemon juice witch the dye and t,hen wit.h standard iodine solution has provided a rapid and satisfactory procedure for stand- ardizat,ion.
A 5 cc. portion of freshly prepared, strained lemon juice is ti- trated with standard 0.01 N iodine solution comaining 15 gm. of KI per liter until a permanent bluing of t.he starch indicator results. Each cc. of 0.01 N iodine represents 0.88 mg. of vit,amin C (lac- tone form). A separate 5 cc. portion of the juice is then tit.rated with the dye solution to a permanent pink. The iodine titrat.ion permits calculating the vitamin C content of the lemon juice and from this the value of t,he dye solution can be calculated in t.erms of mg. of vitamin C. The dye and vitamin C react mole for mole, according to the equation:
C6HSOfi + O:C~H&ls:N~C,H,ON a + C6Hfi06 + HO.CaII2Cl*.NH.CBH1ONft Vitamin (Blue in alkali, (Colorless)
C red in acid)
Orange juice (and most ot,her plant juices) cannot be used for standardization because it contains appreciable amounts of sub- stances ot,her than vitamin C which titrate with iodine. This may be true with lemon juice to a very small degree, but the error so introduced is negligible. Using pure vitamin C as a standard (after establishing the purity by iodine titration) would be in- convenient for many laboratories at t,he present time. S band- ardizations made by the lemon juice method are in close agreement with ‘those made by the method of Tillmans.
Extraction and Titration of Plant and Animal TissuesEx- tract,s of plant and animal tissues for vitamin C titrations are best obtained by thoroughly rupturing the cellular structures in the presence of acid. In most cases the simple expression of juice is too incomplete to provide an accurate determination of the vita- min C cont,ent. The presence of acid is necessary to protect the vitamin from rapid oxidative reactions which occur upon the lib- eration of active enzymes from the macerat,ed cells. Trichloro- acet,ic acid solution is used for extracting animal Gssue (proteins may interfere with the titration). This reagent causes a slow fading of the indicator and allowance must be made for this effect on all titrations. Plant tissues may be satisfactorily extracted
with hot acetic acid, which does not react with 2,6-dichloro- phenolindophenol during the t,itration and permits a sharper end- point.
A representative portion of the tissue (5 to 10 gm.) is weighed and ground in a mort,ar with acid-washed white sand and 25 cc. of either 8 per cent trichloroacetic acid or 8 per cent hot acetic acid until a thin paste is formed. The solids are thrown down in a cent,rifuge tube and the clear extract decanted. Another portion (10 cc.) of the ext~racting liquid is used to wash the mort.ar and is then stirred into t.he solids which are again centrifuged. The washing and rinsing process is repeated a second time (5 cc.) and the decanted extracts, which contain practically all of the vitamin, are combined and made up to a volume (50 cc.) which should depend in part upon the vitamin potency of the tissue being tested. 10 cc. aliquots of the extract are diluted with 40 cc. of distilled water (from glass) or 8 per cent acid solution and titrated with standard 2,6dichlorophenolindophenol solution until a faint pink end-point is obtained. Vitamin C reacts very rapidly with the indicator, making only about a minute necessary for the titra- tion. The end-point with most plant materials is stable for some time, but with animal tissues and extracts containing substances such as glutathione and trichloroacetic acid a slow fading occurs which must not be confused with the titration of the vitamin. Such extracts should be titrated only to an end-point at which the rapid fading ceases. A correction for the amount of indicator required to titrate a solution of the same acid having the same strength and volume as used in the determination should be applied to all titrations.
Since the method is dependent upon the reduction of 2,6- dichlorophenolmdophenol by vitamin C, it is evident that any substance having a reduction potential lower than that of the dye is a possible source of interference. Many such substances (glu- tat&one, cysteine, phenolic compounds, etc.) occur in natural systems along with vitamin C. Although the reduction potential of vitamin C is not as low as that of many naturally occurring substances, the react,ion velocity is much greater than that dis- played by most of these compounds.
Several substances considered to be possible sources of inter- ference have been tested with 2,6-dichlorophenolindophenol.
Glutatcone, cysteine, tannic adid, resorcinol, tannin, pyrogallol, cakchol, glucose, glucose heabed a short time with alkali, heated acidified .glucose, heated acidified sucrose, heated alkaline sucrose, invert sugar, heated vit,amin-free orange juice, and glucic acid @Jottern, Nelson, and M7alker (7)) have been tested in aqueous acetic acid and trichloroacetic acid solution. Cysteine, pyro- gallol, heated alkaline sucrose and glucose, and glucic acid will fade the dye to an extent sufficient to cause serious interference in a vitamin C titration.
In the titration of vegetable tissues and extracts having a high vitamin C cont,ent, such as lemon juice, the error from interfering substances has been found to be very low, e.g. 2 to 3 per cent. The error tends t,o increase as the vitamin C content. of the material being examined decreases. In the titration of animal tissue ex- tracts the error reaches 6 to 8 per cent, owing to t.he use of tri- chloroacetic acid in the extra&ion and the presence of larger amounts of other interfering subst,ances. The 2,6dichlorophenol- indophenol titration is much more accurate than the biological assay method for investigating most plant and animal tissues in that it is sensit.ive to smaller differences. However, the dye titration method is valuable only when properly interpreted from a consideration of the chemical nature of the material being titrated and t,he possible sources of interference, It is evident from the examples cited above that a natural product or a product which has been subjected to laboratory or commercial processes may reduce 2,6dichlorophenolindophenol without being anti- scorbutic. It is also evident that the vitamin might be present in the reversibly oxidized form and not be indicated by the titra- t,ion, although it would respond when tested by animal assay (g), as cited in an earlier paper from our laboratory (1) in explanation of the finding by Zilva that there was not always a direct rela- tionship between the antiscorbutic activity of laboratory concen- trates and their reducing action.
Comparison of Dye Titrations with Animal AssaysIn Table I, it will be seen that t,he dye t.itrations correspond very closely with animal assays on a variety of foodstuffs as well as on the pure vitamin. Owing to the uncertain significance of relatively small variations in animal assays, however, it is evident that the dye titration method offers a t,echnique for studying changes in the
vitamin C content of foods, e.g. during processing, which could not readily be followed in animal assay studies. The marked in- stability of the vitamin in many pletnt and animal tissues, espe- cially when heated, exposed to air, or left in contact witch act,ive oxidative enzymes, makes the tit,ration method of study almost imperative for many types of physiological investigation and in food technological work. The data in Table I also provide an illustration of the advantage of using 0.5 mg. of vit,amin C as a protective feeding level for quantitative studies, in that it affords protection from de&rite scurvy symptoms, permits good survival during an assay period, and maintains a growth response which is
Milk, raw, 30 cc.. . . . . . . “ heated (30 min. at 1459, 30 cc...
Pea, cooked, 8 gm. . . . Tomato juice, 2.5 gm . .
* 29 day8 survival.
-
7
t
-
Uamin by
8itration
m7.
0.50 0.75 0.30 0.57 0.85 0.30 0.13 0.48 0.58
-
.-
-
Sm-JY More
O-2 0 9 O-2 0
10 15 o-2 0
Pm.
132 204
-87 160 212
-92 - 111*
160 210
-
.-
-
No. of animals
5 3 5 5 5 6 6
10 10
reasonably sensitive to fluctuations upward or downward with change in vitamin intake.
In most plant and animal tissues there is a significant amount of reducing material which reacts with iodine in acid solution but does not react rapidly with 2,f3dichlorophenolindophenol. A comparison of the two types of titration is given in Table II, sup- plemented by the most accurate data which we could find re- corded, from our own or other laboratories, indicative of the quantity of vitamin C present as shown by animal assay. It is evident that the dye titration tends to give a lower value than the iodine titration, and that the former corresponds with the animal
assay records within the limits of accuracy of the latter. Each tit.ration figure given is the average of several determinations.
In view of the apparent occurrence of vitamin C in relatively large quantities in all green leaves, and its association in many cases with the carotenoid pigments, it is almost certain that it plays a r61e in photosynthesis, acting as one of the many oxida- tion-reduction factors correlated with the function of chlorophyll.
TABLE II
Comparison of Animal Assays u&h Iodine and Dye Titrations
In a preliminary study there did not appear to be a marked change in the amount of reduced vitamin present in leaves at different times of the day, however. The average quantities found (by dye titration) in different leaves were as follows1 (mg. per gm.): maple 1.3, poplar 1.4, lilac 0.22, mullen 0.13, lawn grass 0.73.
r The authors are indebted to W. Bachrach and T. Gorsky for assistance with some of the titrations herein recorded.
A study of the distribution of vitamin C in the tissues of diifer- ent types of animals has provided striking evidence of its close relation to the normal respirat,ory processes. In addit.ion to (or what is perhaps a corollary) the relationship between respiratory activit,y and vitamin concentration, there is an evident close rela- tionship between the latter and the presence of complex lipids.2 The rapid depletion of vitamin content in the tissues of animals dependent upon a dietary supply is also well illustrated in Table III. The depletion is general for all tissues studied, but greatest in those having the highest normal concentration. Harris and
*Rolled oats and bran 59, milk powder 30, butter fat 10, salt 1 per cent, supplemented by cod liver oil.
associates and Svirbely and Szent-Gyorgyi have reported a similar drop in the vitamin content of the liver.
By comparing the data in Table IV with that in Tables III and V it will be noted that there is a fairly consistent relationship for the vitamin content in the corresponding tissues of different ani- mals and in human tissues. The adrenals are consistently high, with the brain, liver, ovaries, and testes in a second group with relatively high concentration. The heart and kidneys are in a third lower group, distinctly above the muscle t.issue and blood.
2 This relationship was first brought to our attention by Dr. M. L. Men- ten during histological studies carried out in collaboration with the above studies. A detailed account of this work will appear in another journal.
The quantity of vitamin in the latter is probably below the figures given, because the quantity present was very small for accurate titrations. According to Emmerie and associates (lo), a large part of the vitamin C in blood is in the reversibly oxidized form, a finding which is of part.icular interest in relation to the relatively high oxygen tension in blood compared to other tissues and also indicative of the general circulation of free vit,amin in all of the tissues in the body. It is of interest to note too that the vitamin content of the rat, rabbit, and chicken Gssues, where the vitamin is not a direct dietary factor but under physiological control, is
TABLE IV
Vitamin C in Tissues of Animals in Which It is under Physiological Control
higher than that found for guinea pigs receiving an apparently generous vitamin supply. In each of the types studied there was found a distinct tendency for the tissues of young, rapidly grow- ing animals to be higher in vitamin content than those of older animals on a similar diet, which is in harmony with the general relationship noted between the vitamin and tissue respiration.
The finding of as much vitamin C in the corpus luteum as in the adrenal cortex was of particular interest in corroborating the general physiological relationship of the vitamin to (a) a high respiratory rate, (b) complex lipids, and (c) rapidly growing tissue. It minimizes the probability of a direct specific relation to the
adrenal cortex, which has been considered frequently because of its high concentration there. Von Euler (11) has reported a simi- lar high concentration in the thymus gland. It appears probable that the vitamin function in the different tissues is similar, and fundament,ally correlated with respiration and metabolic rate rather than with specific functions in any part,icular tissue. Its general quantitative relation to metabolic rate in both plants (where it accumulates rapidly within a few hours when seeds start to germinate) and animals strongly indicates such a basic rela- tionship. The titration value for the vitamin content of the corpus luteum was verified by curat,ive test assay upon scorbutic guinea pigs, 1 gm. of pulped tissue per day serving to cure scurvy
and r&ore the growth rate to normal, after the animals had been on a vitamin C-free ration for 18 days (scurvy symptoms disap peared and weight increased from 227 gm. to 320 gm. (average) in 14 days).
Although the number of cases (Table V) for which we have been able to secure data is still very small, it is apparent that the vitamin C content of human tissues corresponds closely with that found for experimental animals. Presumably there would be a rapid depletion of the normal vitamin content of the tissues in the human body in contrast with the relatively slow onset of scurvy, analogous to the findings for guinea pig tissues. In the latter case the time required for the onset of scurvy is much greaterthan
that required for marked depletion of the normal vitamin reserve. The effect of maintaining animals or human beings in this sub- normal zone of vitamin supply has been very inadequately studied, but it would appear to be an important field of investigation, bot,h in relation to glandular function and in relation to general physiological welfare.
SUMMARY
The use of 2,6-dichlorophenolindophenol has been found rela- tively satisfactory for the direct titration of vitamin C in animal and plant tissues, and offers many advantages in relation to time and degree of accuracy. Acetic acid (8 per cent) has been found preferable for an extracting and tit,rating medium with most plant tissues, while trichloroacetic acid (8 per cent) is preferable with most animal tissues. Cysteine, glucic acid,a and heated sugar solutions may interfere seriously, however, if present. Gluta- thione and a number of other substances may interfere un- less special precautions are taken. Reservation on interpretation should be conditioned by consideration of possible animal assays and interfering substances.
The adrenals and corpus luteum contain nearly the same amount of vitamin C (approximately 1.4 to 2.3 mg. per gm.); the brain, liver, testes, ovaries, and other glandular tissues comprise a second group ranging considerably lower (approximately 0.1 to 0.4 mg. per gm.); the more active muscular tissue such as the heart comprises an intermediate group (e.g. 0.05 to 0.15 mg. per gm.), while lean muscle contains only about 0.04 mg. per gm.
There is a close relat,ionship to the complex lipid content of animal t,issues and also to the general rate of metabolism or res- piration in the individual tissues.
In younger animals the vitamin C content of the tissues tends to be higher than found for older animals.
In human tissues the distribution of vitamin C is similar to that in experimental animals. For those animals which require a direct dietary source of vitamin C, the time required for marked general tissue depletion is apparently much shorter than the time required for symptoms of scurvy to appear, indicating an impor- tant zone for investigation.
a The earlier papers on glucic acid by E. K, Nelson and E. M. Nelson have apparently been overlooked by von Euler and associates.
1. Waugh, W. A., and King, C. G., Science, 76, 357, 636 (1932); J. Biol. Chem., 97, 325 (1932).
2. Szent-Gyorgyi, A., Biochem. J., 22, 1387 (1928). 3. Svirbely, J. L., and Szent-Gyorgyi, A., Nature, 129,576 (1932); B&hem.
J., 26, 865 (1932). Cj. reviews by Sherman, H. C., American Public Health -4ssociation Year Book, 97 (1933). Harris, L. J., in Luck, J. M., Annual review of biochemistry, Stanford University, 2,253 (1933).
4. Waugh, W. A., Bessey, 0. A., and King, C. G., Proc. Sot. Exp. Biol. and Med., 30, 1281 (1933).
5. Gibbs, H. D., Cohen, B., and Cannan, R. K., Pub. Health Rep., U.S. P. H. S., 49, 649 (1925). Cohen, B., Gibbs, H. D., and Clark, W. M., Pub. Health Rep., U. 8. P. H. X., 39, 804 (1924).
6. Tillmans, J., Hirsch, P., and Hirsch, W., 2. Untersuch. Lebensmittel, 93, 1 (1932).
7. hfottern, H. H., Nelson, E. M., and Walker, R., J. Assn. 03. Agrie. Chem., 614 (1932).
8. Birch, T. W., and Dann, W. J., Nature, 131, 469 (1933). 9. Tillmans, J., Hirsch, P., and Jackish, J., 2. Untersuch. Lebensmittel,
