540 SCIENCE Von. 86, No. 2241

DISCUSSION

CRYSTALLIZED VITAMIN C AND
HEXURONIC ACID
Tue well-merited award to Albert von Szent-
Gyorgyi of the 1937 Nobel Prize in Physiology and
Medicine “for his discovery of the biological processes

of oxidation with special regard to vitamin C and the
fumarie acid catalyses” is erroneously interpreted in
many quarters solely as an award for the identification
of vitamin C. Reports on crystalline vitamin C are
sufficiently clear that no such interpretation should
Decemserr 10, 1937

be made, but numerous careless or erroneous state-
ments have appeared in scientific journals, reviews
and books and in lay publications, which tend to
obseure 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-Gyérgyi! made the first statement
concerning a reducing substance which he had not yet
isolated in crystalline form. He designated it Cxn, an
apparently fortuitous uge of the letter “C” and with
no implication that it was related to vitamin C.

Again in 1927 Szent-Gyérgyi stated :?

To exclude any anticipation of function and chenlical
structure, the substance giving this reduction was named
by its protocoll number ‘‘Czn’’ being the twelfth sub-
stance 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 substance has been further investi-
gated and finally isolated in crystals, which on recrystal-
lization showed a constant melting point.

And

Preliminary experiments tend to show that the sub-
stance is not devoid of biological activity. My earlier
experiments, showing that Cxn is not the hormone of the
adrenal cortex, seem in the light of Banting’s and Rogoff
and Stewart’s recent work to be inadequate. Analysis
of the biological significance and chemical constitution
has been started.

Zilva® on May 7, 1932, wrote:

Byer since Prof. A. Szent-Gyérgyi suggested the pos-
sible identity of hexuronie acid¢ with the ‘‘reducing
principle’’ present in all active antiscorbutic solutions ...

The paper‘ to which Zilva refers contains the follow-
ing sole reference to vitamin C:

The reducing properties of plant juice have repeatedly
attracted attention, especially from students of vitamin
C. Bezssonoff 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
established interesting relations between vitamin C and
the reducing properties of the plant juice. The main
reagent employed by Zilva was phenolindophenol. Indo-
phenol blue is readily reduced by the hexuronic acid, so
that it is probable that it was this substance which has
been studied by Zilva.

The title of Zilva’s paper’ (received for publication
on August 25, 1932), “The Non-Speeificity of the

1 Biochem. Zeits., 181: 433, 1927.
2 Nature, 119: 782, 1927.
® Nature, 129: 690, 1932.
+ Biochem. Jour., 22: 1387, 1928.
5 Biochem. Jour., 26: 1625, 1932.

SCIENCE

’ © but a substance associated with it.

541

Phenolindophenol Reducing Capacity of Lemon Juice
and Its Fractions as a Measure of Their Antiscorbutic
Activity,” succinetly reveals that Zilva did not believe
that the reducing substance with which he had dealt
in a series of investigations was identical with vitamin
Since Szent-
Gyérgyi, 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-Gyérgyi
in 1928 with a substance associated with vitamin C but
not vitamin C itself. ;

Tillmans, Hirsch and Hirsch® in January, 1932,
stated (in a note added at proofreading) in regard to
Szent-Gyérgyi’s 1928 paper:

Die Frage der etwaigen tibereinstimmung dieser Sub-
stanz [hexuronic acid] mit dem vitamin C wird von
Szent-Gyérgyi nicht aufgeworfen. Er ist vielmehr der
Ansicht, dasz dieser Stoff in der Oxidationsmechanismus
des Peroxydase-Systems eingeschaltet ist.

This note expresses an independent opinion that
Szent-Gyérgyi did not consider hexuronic acid to be
vitamin C.

McKinnis and King’ in 1930 established the acidic
nature of vitamin C, and in discussing the acidity
said: ,

It is also of interest in relation to the close association
of vitamin C with the actively reducing uronic acid iso-
lated by Szent-Gyorgyi.4

That the idea was current among King’s students
that vitamin C was a uronic acid is further evidenced
by the statement in 1931 by Smith and King®

In view of the fact that the active preparations were
always characterized by a high content of strongly redue-
ing substances and acidic material analogous to the
‘‘hexuronie acid’’ isolated by Szent-Gydrgyi, it was
thought desirable to find out whether the use of a strongly
reducing, weak acid, such as formic, would prove advan-
tageous in protecting the concentrates.

In a review of the work on vitamin C during 1932,
Harris® contributes a parenthetical statement of sig-
nificance :

Early in 1932 Szent-Gydrgyi, in collaboration with
Svirbely (who, it may be recalled, had lately been asso-
ciated with King in work on vitamin © concentration at
the University of Pittsburgh), announced . . .

On April 1, 1932, as the culmination of a series of
studies concerned directly with vitamin C, King and
Waugh? announced the isolation of vitamin C in
crystalline form. They said:

8 Zeits. Untersuch. Lebensm., 63: 1, 1932.

? Jour. Biol. Chem., 87: 615, 1930. .

8 Jour. Biol. Chem., 94: 491, 1931.

9 Ann. Rev. of Biochem., 2: 264, 1933. -
10 ScrencE, 75: 357, 1932.
542

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-
Gyérgyi 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-Gyérgyi2!
announced that they had, by means of 1 mg of hex-
uronic acid daily, protected guinea pigs from seurvy
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 hexuronie acid for 55
days were found to be free of scurvy. However, it
was not until May 7, 1932, that Svirbely and Szent-
Gyoérgyi’? stated that hexuronic 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. Waugh! 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 King™ and those of
Svirbely and Szent-Gyérgyi'* 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 analysis 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 © (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
erystalline substance was apparently identical with Szent-
Gyérgyi’s hexuronic acid (April 1, 1932).

11 Nature, 129: 576, 1932.
12 Nature, 129: 690, 1932.

18 Jour. Biol, Chem., 97: 325, 1932.
14 Biochem. Jour,, 26: 865, 1932.

SCIENCE

Vou. 86, No. 2241

(4) Fifteen days after King and Waugh published,
Svirbely and Szent-Gyérgyi gave their first evidence that
hexuronie acid has anti-scorbutie properties, but it was
not until May 7, 1932, that they stated that ‘‘ Vitamin C
is a single substance and identical with hexuronic acid.’ ’12

GeraLp J. Cox
Nutrition FELLOWSHIP OF
THE Buu. Founparion,
MELLON INSTITUTE