Identification of Vitamin C

Work now began in earnest to identify and isolate the antiscorbutic factor. The Medical Research Council’s 1932 publication Vitamins: A Survey of Present Knowledge may be referred to for a detailed account of the large number of papers published during the 1920s on what was by then known as “vitamin C.” Foremost in these early efforts was Zilva, working at the Lister Institute, London. The essential feature of Zilva’s procedure was the precipitation of the factor with basic lead acetate after removal of the bulk of the other organic acids with calcium carbonate. He applied this technique to a variety of sources, such as lemon juice and swede (rutabaga) tissues, and he succeeded in increasing the concentration of the antiscorbutic factor some 200 to 300 times (Hughes 1983).

Other workers were similarly occupied, notably N. Bezssonoff in France and C. G. King in the United States. King has stated that during this period “many investigators had abandoned or failed to publish their work for various reasons.” He referred, specifically, to Karl Link of Wisconsin, who had prepared several grams of crude calcium ascorbate during the 1920s but carried his work no further because of lack of financial support (King 1953).

The purest of these early “concentrates” still contained much impurity, and it was not until 1932 that W A. Waugh and King published their paper “The Isolation and Identification of Vitamin C,” which included a photograph of vitamin C crystals (Waugh and King 1932).

These attempts to isolate and characterize vitamin C were paralleled by two separate, but nevertheless highly relevant, developments in related areas. J. Tillmans and P Hirsch, German government chemists, extensively studied the capacity of lemon juice preparations to reduce the redox dye 2,6-dichlorophenolin-dophenol, and they claimed that the reducing power of their preparations was always in proportion to their antiscorbutic potency; the indophenol dye technique later became a standard method for the assay of vitamin C. Zilva, however, disagreed with the German findings and appears, at that point, to have been diverted from his main endeavor by an attempt to disprove them (Zilva 1932).

The other significant development at this time was Albert Szent-Gyorgyi’s isolation of hexuronic acid. Szent-Gyorgyi, a Hungarian biochemist working on plant respiration systems at Groningen in Holland, became interested in a reducing compound present in his preparations. Hopkins invited him to Cambridge to extend his studies, and in 1927, Szent-Gyor-gyi isolated his “Groningen reducing agent” in a crystalline, from oranges, lemons, cabbages, and adrenal glands (Szent-Gyorgyi 1928).

He proposed to name his crystalline sample “ignose” - thus indicating its apparent relationship to sugars while at the same time underlining his ignorance of its true nature. But Harden, the editor of the Biochemical Journal at the time, according to Szent-Gyorgyi, “did not like jokes and reprimanded me.” A second suggestion “godnose”was judged to be equally unacceptable. Szent-Gyorgyi finally agreed to accept Harden’s somewhat more prosaic suggestion “hex-uronic acid” - “since it had 6 Cs and was acidic” (Szent-Gyorgyi 1963:1-14).

Hexuronic acid was a strongly reducing compound. So, too, according to Tillmans and Hirsch, was the antiscorbutic substance (vitamin C). The suggestion that hexuronic acid and vitamin C were actually one and the same substance appeared in print in 1932 in papers by both J. L. Svirbely and Szent-Gyorgyi and by Waugh and King, but there can be little doubt that the idea had been mooted some years previously. Who first made the suggestion is, however, unclear, and even the main participants in the drama later appeared uncertain and confused. King (1953) claimed that it was E. C. Kendall in 1929, but according to Hopkins (reported by King) it was Harris in 1928 (King 1953) - and he had, in any case, already attributed the idea to Tillmans and Hirsch (Harris 1937: 95). But E. L. Hirst (a member of the team later involved in chemical studies on the structure of vitamin C) named Waugh and King (Hirst 1953:413).

Hopkins had already, in 1928, sent a sample of Szent-Gyorgyi’s hexuronic acid to Zilva for comments on its vitamin C potency. According to King, Hopkins was disturbed because Zilva (who, naturally perhaps, was reluctant to admit that his “antiscorbutic preparations” were in reality identical with hexuronic acid) had replied that the sample was not vitamin C, but did so without reporting the evidence of his tests (King 1953).

By 1932, however, evidence in favor of the identity of hexuronic acid as vitamin C was substantial. Waugh and King had shown that their “crystalline vitamin C” cured scurvy in guinea pigs (Waugh and King 1932), and earlier the same year, Svirbely and Szent-Gyorgyi (now working in his native Hungary) had described the antiscorbutic potency of a sample of “hexuronic acid” isolated from adrenal glands (Svirbely and Szent-Gyorgyi 1932).

In 1933, in a single-sentence letter in Nature, Szent-Gyorgyi and W N. Haworth drew attention to the chemical inaptness of the term “hexuronic acid” and suggested the term “ascorbic acid,” thus formally acknowledging the antiscorbutic nature of the compound. Harris and his colleagues at Cambridge demonstrated the positive correlation between the hexuronic acid content and the antiscorbutic potency in a wide range of foodstuffs and published a highly convincing “eight-point” proof of the identity of the two substances.

Their three most important points were as follows:

(1)  Hexuronic acid paralleled antiscorbutic potency;

(2)  destruction of hexuronic acid by heat or by aeration was accompanied by a corresponding fall in the antiscorbutic activity; and (3) hexuronic acid disappeared from the organs of scorbutic guinea pigs (Birch, Harris, and Ray 1933).There could now be little doubt that hexuronic acid (Tillmans and Hirsch’s reducing compound) and vitamin C were one and the same substance.

The situation was not without its human aspects, and even today the question of priority in the discovery of vitamin C still elicits discussion.“The identification of vitamin C is one of the strangest episodes in the history of vitamins,” wrote T. H. Jukes in commenting on the appearance in 1987 of a book by R. W. Moss that placed, in Jukes’s opinion, too great an emphasis on Szent-Gyorgyi’s contribution (Moss 1987; Jukes 1988: 1290). Moss had implied that King had rushed off his claim for the identity of vitamin C and hexuronic acid after it became clear to him that Szent-Gyorgyi intended making the same point in a note to Nature - a situation curiously reminiscent of the suggestion that Charles Darwin behaved similarly on learning in 1858 that Alfred Russel Wallace was about to publish his theory of evolution.

The emphasis now shifted to the elucidation of the structure of vitamin C. Haworth, a Birmingham (U. K.) chemist, had received from Szent-Gyorgyi a sample of his “hexuronic acid,” and in 1933, in a series of impressive papers, the Birmingham chemist, using both degradative and synthetic procedures, described the structure of the molecule (Hughes 1983). The molecule was synthesized simultaneously, but independently, by T. Reichstein in Switzerland and by Haworth and his colleagues in Birmingham, both groups using essentially the same method.

The synthesis - which, as it later emerged, was quite different from the biosynthetic pathway - was based on the production of xylosone from xylose and its conversion with cyanide to an imino intermediate that, on hydrolysis, gave ascorbic acid. The Swiss group published their results just ahead of the Birmingham workers (Ault et al. 1933, Reichstein, Gruss-ner, and Oppenheimer 1933). The picture was completed the following year when the Birmingham workers joined forces with Zilva to demonstrate that synthetic ascorbic acid produced at Birmingham had exactly the same antiscorbutic potency as a highly purified “natural” sample from the Lister Institute (Haworth, Hirst, and Zilva 1934).

The annual report of the Chemical Society for 1933, with perhaps unnecessary caution, stated that “although it seems extremely probable that ascorbic acid is vitamin C. . . it cannot be said that this is a certainty.” Other were less circumspect. A. L. Bacharach and E. L. Smith, addressing the Society of Public Analysts and Other Chemists in November 1933, said that “Vitamin C can now be identified with a sugar acid known as ascorbic acid. . . . Contrary to expectation, it is the first vitamin not merely to have assigned to it a definite molecular formula, but actually to be synthesised by purely chemical means” (Hughes 1983). Budd was correct in his 1840 prophecy that both physiologists and chemists could contribute to the identification of the “antiscorbutic factor.” But his “not too distant future” proved to be a period of 93 years!