[FBOS THE l3IOCHEMICAL JOURNAL, VOL. XXVI, No. 3, pp, 865-NO,19321
            [All Rip%2 reserved]

CV. THE CHEMICAL NATURE OF VITAIWN C.

BY JOSEPH LOUIS SVIRBELF AND ALBERT SZENT-GY~RGYI.

(Received Ape2 25th, 1932.)

HEXURONIC acid was discovered [1927] and isolated from plants and the
adrenal cortex [1928] by Szent-Gyiirgyi at the Biochemical Laboratory,
Cambridge, and was investigated in regard to its fun&ion and chemistry 11928,
1930, 19311. Its chemical config~ation has been studied by Haworth, Hirst
and Reynolds [1932]. The striking similarity of this substance with vitamin C
was evident from the beginning. As far 8s it is known, its distribution in plants
closely follows the distribution of the vitamin. The chemical properties of both
substances agree and both are biologically active in minute quantities.

As is known, hexuronic acid has strongly reducing properties and the
reducing power of many plant extracts is mainly due to this substance. The
close relation between vitamin content and reducing power has been empha-
sised by Tillmans et a?,. {1932], whose careful work strongly suggested that
the reducing power of plant extracts was in part due to the vitamin itself,
which was identical with hexuxonic acid,

Tillmans and his co-workers claim to have shown that the vitamin content
and the reducing capacity run on strictly parallel lines under many conditions.
Furthermore they have shown that the reducing factor can be oxidised in
two different ways, reversibly and irreversibly. They have succeeded in re-
versing the oxidation of the reversible type by the application of reducing
agents suoh as H&Z!. They observed that by the reversible oxidation the
reducing factor becomes very labile and prone to undergo irreversible changes.
The vitamin showed a closely analogo~ beha~o~. On treatment with oxidis-
ing agents, which produce a reversible oxidation of the reducing factor, the
vitamin did not lose its activity, but became very labile. Tillmans and his
collaborators were led to the conclusion that the reducing factor and the
vitamin were in fact identical.

As is known, hexuronic acid can be oxidised reversibly and irreversibly,
the reversible form being readily again reduced by HZS. The agents reported
by Tillmans to produce a reversible change in reducing factor also oxidise
hexuronic aoid reversibly. Agents, such as molecular oxygen, which produce
an ~reversible change in the reducing factor, also destroy hexuronio acid.
1 Holder of an American-Hungarian Exchange B'ellowBhip, 1931-32, from the Institute of
Xnternatiom$ $&c&ion, New York.


866     J. L. SVIRBELY AND A. SZENT-GYijRGYI

The strongly reducing properties of active fractions of lemon juice were
also emphasised by King and his co-workers [King and Waugh, 1932 ; Smith
and King, 1931; Svirbely and King, 19311.

In a series of his well-known publications also, Zilva [1927, 1928, 19301
has found many parallels in the distribution of vitamin and reducing factor.
Zilva, however, partly in collaboration with Connell [1924], was able to show
that reducing capacity and antiscorbutic activity can vary independently
during the course of fractionation. He succeeded in preparing fractions with
a high antiscorbutic activity and no reducing power as well as fractions with
a marked reducing capacity and no antiscorbutic activity. From this he justly
concluded that vitamin C and the reducing factor could not be identical.  Zilva
also observed that on oxidising the reducing factor the antiscorbutic activity
did not disappear, but the antiscorbutic principle became very labile, in which
observation he was corroborated later by Tillmans. Zilva explained this change
in stability by a protecting action of the reducing factor.
The reversible oxidation of hexuronic acid admits the possibility that in
Zilva's extracts, which were antiscorbutically active but showed no reducing
power, the hexuronic acid was present in a reversibly oxidised state. On the
other hand it is known that the reducing factor is of a complex nature, as
was also lately brought out by Guthrie and Wilcoxon [1932]. One of us
(A. Sz.) has spent much time in studying, a phenolic substance, found in
relatively great quantities in orange juice, which shows a marked reducing
power. It shows similarities to the flavonols but also gives reactions different
from this group of substances. This substance is also readily carried down by
lead acetate. Some years ago it was kindly tested by Zilva for antiscorbutic
activity with negative results. The presence of such a reducing substance,
not identical with hexuronic acid, might explain the reducing power of frac-
tions which show no antiscorbutic activity. Taking all this into account, the
authors think that Zilva's evidence against the identity of hexuronic acid and
vitamin C cannot be regarded aa conclusive.

Another objection raised recently by Zilva [1932] against the identity of
vitamin C and hexuronic acid is based on the magnitude of the protective
dose of hexuronic acid. The protective daily dose of lemon juice is 1.5 cc.,
which would keep growing guinea-pigs entirely free from scurvy. This quantity
of lemon juice contains approximately 0.5 mg. of hexuronic acid. The pro-
tective dose of this substance would be thus of this magnitude. Zilva points
out that with O-5 mg. of fractions of lemon juice, which were still grossly
contaminated, animals were protected against scurvy by himself and other
authors. Thus the vitamin must have a higher potency.

The authors are of opinion that this evidence is not conclusive. It is
difficult to judge to what extent these fractions were contaminated, and how
much hexuronic acid they contained. If, however, O-5 mg. of hexuronic acid
gives full protection against scurvy, it is natural that somewhat smaller doses
should have some activity, On the other hand 0.5 mg. of hexuronic acid for


CHEMICAL NATURE OF VITA&ITN C        867

1.5 cc. lemon juice gives only an approximate value, and it is possible that
the hexuronic acid content of lemon juice is somewhat lower. Owing to the
lack of a specific reaction it is impossible to state at present to what extent
the reducing activity of a plant extract is entirely due to hexuronic acid or
other substances and only rough estimates can be given.

We think that no circumstantial evidence can have much value either way
in a question which can be subject to direct experiment.  If it can be shown
that the hexuronic acid present in the lemon juice has the same activity as
the lemon juice itself, there can be little doubt about the identity of vitamin
and hexuronic acid.

The striking similarity of vitamin C and hexuronic acid has been evident
ever since the discovery of the acid. The possible identity of both substances
has been expressed repeatedly by one of us. The collaboration of the present
authors at the Institute of Medical Chemistry of the University of Szeged
offered the necessary facilities for the testing of the antiscorbutic activity
of hexuronic acid. The experiment was undertaken last autumn with a
positive result. The negative controls died with symptoms of severe scurvy,
while the animals receiving hexuronic acid were kept alive and free from
scurvy in a 56 days' test. Owing however to the small number of animals
used and certain deficiencies in the diet, which prevented normal growth,
the results of the test were not published. A short account of this test was
given in our first preliminary note [1932].

In the test to be reportedl, all shortcomings of the first experiment were
remedied. The authors think that the experiment shows clearly that the
hexuronic acid present in the lemon juice has the same activity as lemon juice
itself, and thus clearly brings out the identity of the two substances2.

EXPERIMENTAL.

The general procedure used in testing the antiscorbutic activity of hex-
uranic acid was that recommended by Sherman et al. [1922]. In addition to
the basal diet, O-5 cc. of cod-liver oil was given to each test animal every week.

The hexuronic acid used in the test was prepared in crystalline form from
ox suprarenal glands 2 years ago at the chemical department of the Mayo
Foundation at Rochester. We wish to emphasise the fact that our preparation
was derived from animal tissues. If our hexuronic acid had been prepared from
lemon juice or other plant sources the objection might arise that the activity
was not due to the acid itself but to a contamination by a more potent anti-
scorbutio substance.

1 A brief account of this experiment was given in our two preliminary notes in Nature.
* Simultaneously with our preliminary note King and Waugh [1932] reported that they had
obtained crystals from lemon juice, which showed antiscorbutic activity and were apparently
similar to a hexuronic acid. Since no chemical analysis w&s reported the chemical nature of their
product is left in doubt. Since no mention was made of weight curves, number of animals,
duration of the experiment and post mortem examinations, their claim of an antiscorbutic activity,
based on "vitality," cannot be judged.


J. L. SVIRBELY AND A. SZENT-GY6RGYI

A sufficient amount of the acid was weighed out every 8 days and dissolved
in water. Aliquot portions of the solution, adequate for daily feeding, were
put in small phials and kept at - 18" in an atmosphere of CO,. The solution
was administered to the animals by a pipette. The minimum protective dose
of lemon juice is generally regarded as 1.5 cc., and this quantity contains
approximately 0.5 mg. of hexuronic acid. In our test, however, 1 mg. of
hexuronic acid has been given daily to the animals since, owing to the age of
the preparation and the unavoidable exposure to air, some of the acid may
have become decomposed.

There was only one disturbing factor in our experiment, which however
did not essentially influence the result. Owing to the general economic con-
ditions we were unable to obtain milk powder until the 19th day of the experi-
ment, at which time it was introduced into the diet.  It was due to this fact
that until that day the animals did not show normal growth, which however
was promptly attained as soon as the milk powder was given. This growth was
not due to traces of vitamin present, since the skimmed milk powder was
heated in a tray in the drying-oven at 110" for 4 hours. It was specially tested
in two animals on the 43rd day of the main test (see Table I and Fig. 1).

Table I.

                  No. of
   Fed daily          animals
1 mg. of hexuronic acid        3
1 cc. of lemon juice
Basal diet only      ii
1 mg. of hexuronic acid        7
1 cc. of lemon juice
Basal diet only      x
* Highest possible score is 24.

Average
survival
(90 da&test)
  55t
2
~~
   30

      Average
Average    gain in
scurvy    weight
score*       6.
  0        41
  2
  15   -1:;
ii    281 147
  19      - 114
t Chloroformed.

I         I         I
30        60        90
Days
Fig. 1.


CHEMICAT, NATURE OF VITAMIN C         869

The following groups of test animals were used: (1) those receiving 1 mg.
of hexuronic acid daily; (2) the positive controls, which received 1 cc. of
lemon juice daily; (3) the negative controls, which received the basal diet
only.

The results of the test are given in Table I. In Fig. 1 the weight curves of
the animals receiving hexuronic a.cid are contrasted with those of the negative
controls; Pig. 2 gives the average weight curves of the three groups of animals
used in the test.

Average survival in days
   Fig. 2.

As seen from the Table and Pigs. all the negative controls died within the
period of 20-34 days with the usual loss in weight. At autopsy they all
showed symptoms of severe scurvy. The animals receiving hexuronic acid
showed normal growth. Three of these animals were killed at the end of
65 days and the rest at the end of 90 days' test period. At autopsy they were
found entirely free from symptoms of scurvy. The positive controls receiving
1 cc. lemon juice showed mild scnrvy at autopsy.

SUMMARY.

Details of an experiment are described in which hexuronic acid was tested
for antiscorbutic activity. The acid was administered in doses comparable
with the hexuronic acid content of the protective dose of lemon juice. Given in
this quantity (1 mg. daily), hexuronic acid affords complete protection against
scurvy in a 90 days' test. Sinoe it is generally assumed that animals kept free


870

J. I,. SVIRBELY AND A. SZENT-GYORGYI

from scurvy for 90 days would remain so indefini~ly under identical GOI~-
ditions, it is concluded that vitamin C is a single substance and identical with
hexuronic acid.

This research was sponsored by the Ella Sachs Plotz foundation.

REFERENCES.

Connell and Zilva (1924). &o&em. J. 18, 632.
Guthrie and Wilcoxon (1932). Contr. from Boyce Thompson Institute, 4, 99.
Haworth, Hirst and Reynolds (1932). Nature, 129, 576 (April 16).
King and Wangh (1932). Science, 75 (April 1). No. 1944.
Sherman, La, Mer and Campbell (1922). 6. Amer. Chem. SOG 44, 165.
Smith and King (1931). J. BaoZ. Chem. 94, 491.
Svirbely and King (1931). J. Biol. Chem. 94, 483.
- and Szent-GyBrgyi (1932). Nature, 129, 576;; 690 (April 16, May 7).
Szent-Gyijrgyi (1927). N&are, 129, 782 (Ma.y 28).
__ (1928). Biochem. J. 22, 1387.
- (1930). Science, 72, No. 1857.
- (1931). J. Biol. Chem. 90, 385.
Tillmans, Hirsch and Dick (1932). Z. Unters. Lebensmille.!, 63,267.
-- and Hirsch (1932). 2. Unters. Lebensmittel, 63, 1.
- - and Jakisch (193&l). 2. U&r& L~e~~~~e~, 63, 241.
__-- (1932, 2). Z. Uuters. LdensmitteZ, 63, 276.
__ - and Siebert (1932). 2. Unters. Lebensnzittd, 63, 21.
Zilva (1927). Biochem. J. 21,689.
__ (1928). Biochem. J. 22, 779.
- (1930). Beech. J. 24, 1687.
- (1932). Nature, 128, 690 (May 7).