Vat. 86, No. 2241 DiSCUSSION CRYSTALLIZED VITAMIN C AND of oxidation with special regard to vitamin C and the HEXURONIC ACID fumarie acid catalyses" is erroneously interpreted in TFIRI well-merited award to Albert von Seent- many quarters solely as an award for the identification Gyiirgyi of the 1937 Nobel Prize in Ph~iolo~ and of vitamin C. Reports on crystal&e vitamin C are Medicine "for his discovery of the biological processes sufficiently clear that no suoh interpretation hoda DECXUBEB 10, 1937 BCIENCE 541 be made, but numerous careless or erroneous state- ments have appeared in scientific journals, reviews and books and in lay publications, which tend to obscure the priority of Dr. C. G. King and W. A. Waugh, of the University of Pittsburgh, in estab- lishing the identity of vitamin C with a pure crys- talline substance. In 1927 Szent-Gysrgyil made the first statement concerning a reducing substance which he had not yet isolated in crystalline form. He designated it &II, an apparently fortuitous we of the letter "C" and with no implication that it was related to vitamin C. Again in 1927 Szent-Gyiirgyi stated:* To exclude any anticipation of function and chenlical structure, the substance giving this reduction was named by its protocol1 number "&," being the twelfth sub- etance prepared and examined in my work on tissue oxida- tion and the function of the adrenal cortex. Having been enabled to resume this work at the Biochemical Labora- tory, Cambridge, the eubstance haa been further inveeti- gated and tially isolated in crystals, which on recrystal- lization showed a constant melting point. And Preliminary experiments tend to show that the sub- stance ia not devoid of biological activity. My earlier experiments; showing that Cxu ia not the hormone of the adrenal cortex, seem in the light of Banting's and Rogoff and Stewart's recent work to be inadequate. Analysie of the biological significance and chemical con&tution haa been started. Ziivaa on May 7,1932, wrote: Ever eince Prof. A. Szent-Gy6rgyi euggeated the poe- sible identity of hepuronic acid4 with the "reducing principle ' ' present in all active antiscorbutic eolutions . . . The paper* to which Zilva defers contains the follow- ing sole reference to vitamin C: The reducing properties of plant juice have repeatedly attracted attention, especially from atudenta of vitamin C. Bezseonoff has applied Folin's phosphomolybdic acid reagent. The reducing substances of lemon juice have been made the object of a thorough study by Zilva, who eetabliahed interesting relations between vitamin C and the reducing propertiee of the plant juice. The main reagent employed by Zilva was phenolindophenol. Indo- phenol blue ie readily reduced by the hexuronic acid, 80 that it is probable that it wa8 thie substance which has been etudied by Zilva. The title of Zilva's paper5 (received for publication on August, 25, 1932), "The Non-Specificity of the 1 Biochem. Zeits., 181: 433,1927. *Nature, 119 : 782, 1927. * Matwe, 129: 690, 1932. 4 Bioohem. Jour., 22: 1387, 1928. 6 Bioohem. Jour., 26: 1625, 1932. Phenolindophenol Reducing Capacity of Lemon Juice and Its Fractions az a Measure of Their Antiscorbutic Activity," succinctly reveals that Zilva did not believe that the reducing substance with which he had dealt in a series of investigations .was identical with vitamin C but a substance. associated with it,. Sinee Szent- Gyargyi, as quoted above, believed "that it is probable that it was this substance which was studied by Zilva," then hexuronic acid was identified by Szent-Gy6rgyi in 1928 with a substance associated with vitamin C but not vitamin C itself. Tillmans, Hirsch and Hirsch6 in January, 1932, stated (in a note added at proofreading) in regard to Szent-GyBrgyi's 1928 paper : Die Frage der etwalgen tl`bereinstimmung dieser Sub- etanz [hexuronia acid] mit dem vitamin C wird von Szent-Gyiirgyi nicht aufgeworfen. Er iet vielmehr der Aaeicht, dasz dieter Stoff in der Oxidationsmechania des Peroxydase-Systems eingeachaltet ist. This nob expresses an independent opinion that Szent-Gyiirgyi did not consider hexuronic acid to be vitamin C. M&in& and King' in 1930 established the acidia nature of vitamin C, and in discussing the acidity said : It ia also of interest in relation to the close aeeociation of vitamin C with the actively reducing uranic acid iso- lated by Szent-Gy3rgyi.4 That the idea was current among King's students that vitamin C was a uranic acid is further evidenced by the statement in 1931 by Smith and King.e In view of the fact that the active preparations were always characterized by a high content of strongly reduc- ing substances and acidic material analogous to the " hexuronic acid" ieolated by Scent-QyBrgyi, it was thought desirable to find out whether the we of a strongly reducing, weak acid, eueh aa formic, would prove advan- tageous in protecting the concentrates. In a review of the work on vitamin C during 1932, Harris9 contributes a parenthetical statement of sig- niflcance : Early in 1932 Szent-GySrgyi, in collaboration with Svirbely (who, it may be recalled, had lately been a8ao- ciated with King in work on vitamin C concentration at the University of Pittsburgh), announced . . . & April 1, 1932, as the culmination of a series of studies ooncerned directly with vitamin C, King and WaughlO announced the isolation of vitamin C in crystalline form. They said: 6 Zeits. Unterauoh. Lebensm., 63: 1, 1932. ' Jour. Biol. Chem., 87: 616, 1930. 8 Jour. Biol. Chem., 94: 491,1931. 0 Am. Rev. of Biochem., 2: 264, 1933. 10 SCIENCE, 75: 357, 1932. 542 BCIENCE VOL. 86, No. 2241 The recrystallized substance corresponds in chemical and physical properties to a hexuronie acid, and is appar- ently identical with the hexuronic acid described by Szent- Gyorgyi and reported as a reducing factor in adrenal cortex, cabbage and other sources. Feeding approxi- mately 0.5 mg. daily protects growing guinea pigs from scurvy and permits normal vitality in the animals when on a vitamin C free diet. A detailed account of the experimental work will be published in the near future, but this involves only a few steps beyond the work pre- viously published. On April 16, 1932, Svirbely and Szent-Gytirgyirl announced that they had, by means of 1 mg of hex- uranic acid daily, protected guinea pigs from scurvy in an experiment lasting 56 days, Because of the unavailability of milk powder for making the basal diet this test was unfortunately marred by loss of weight of all animals. A current experiment with a satisfactory diet was in progress, and three animals which had received 1 mg of hexuronic acid for 55 days were found to be free of scurvy. However, it was not until May 7, 1932, that Svirbely and Szent- GyGrgyP stated that hexuronio acid is vitamin C. They said: This allows us to conclude that vitamin C is a single substance and identical with hexuronic acid. Simultaneously with our previous note, C. G. King and W. A. Waughls reported that they have obtained, from lemon juice, crystals which showed antiscorbutic activity and were apparently similar in chemical and physical properties to hexuronic acid. The duration of the test period was not stated, and apparently no chemical analysis was made. Until this is done, the nature of their product remains in doubt. The results of Waugh and King13 and those of Svirbely and Szent-GyGrgyP were published in full in 1932. Subsequent studies by others have shown that vitamin C had indeed been identified. The following facts in chronological order are evi- dent from the above ana.lysis of the statements appear- ing in the literature : (1) Szent-Gyorgyi first isolated "hexuronic acid' ' but viewed it only as a reducing substance associated with vitamin C (1928). (2) McKinnis and King first published a positive sug- gestion that hexuronic acid is vitamin C (1930). (3) King and his students carried out a sustained study of vitamin C resulting in the isolation of the active sub- stance in the crystalline form and first stated that the crystalline substance was apparently identical with Szent- Gyorgyi `a hexuronic acid (April 1, 1932). 11 Nature, 129: 576, 1932. l*Nature, 129: 690, 1932. 1'3 Jour. Biol. Chem., 97: 325, 1932. ~4Biochem. Jaw., 26: 865, 1932. (4) Fifteen days after King and Waugh published, Svirbely and Szent-Gyorgyi gave their first evidence that hexuronic acid haa anti-scorbutic properties, but it was not until May 7, 1932, that they stated that "Vitamin C is a single substance and identical with hexuronic acid. ' `1s GERALD J. Cox NUTRITION FELLOWSHIP OF THE BUHL FOUNDATION, MELLON INSTITUTE