Encephalomalacia

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ACTA GHEMICA SCANDINAVICA 18 (1964) 2173-2178

Studies on the Effects of the Nutrition on Antioxidant
Levels of the Body
I. Tissue Antioxidants in Chicks on an Encephalomalacia-
producing Diet
J. GLAVIND and E. SQNDERGAARD
Department of Biochemistry and Nutrition, Polytechnic Institute, Copenhagen, Denmark
The content of water-soluble antioxidants in the brain is decreased
in e n c e p h a l o m a l a c i a. The antioxidant content of blood plasma of
chicks is larger than that of mammals. Uric acid occurs in higher
concentrations in chicken plasma and has been found to possess
free-radical scavenging properties.
The function of vitamin E as an in viva antioxidant was early postulated
and was established beyond doubt by the demonstration of lipoperoxides
in the adipose tissues of chicks and rats fed vitamin E-de?cient diets contain-
ing highly unsaturated fatty acids}
_ The study of the mode of action of vitamin E became complicated by the
discovery that several of the de?ciency symptoms could also be prevented
by factor 3 and selenium. One of the diseases not in?uenced by factor 3/sele-
nium is encephalomalacia in chicks. It is prevented by vitamin E and by
certain redox compounds and antioxidants, e.g., methylene blue, but so far
all attempts to demonstrate the presence of lipoperoxides in the encephaloma-
lacic brain have been without result.
A study on the antioxidant level in vitamin E-de?cient chicks was carried
out by Budowski and Mokadi.2 The method used was an adaption of the novel
principle for determination of antioxidants by means of a stable free radical
indicated by Blois.3 Antioxidants were determined in trichloroacetic acid
?ltrates of livers and brains of chicks reared on an encephalomalacia-pro-
ducing diet. N 0 difference was observed in the livers, but inextracts of the
cerebella the amount of antioxidants was decreased in comparison with
control animals receiving tocopherol. Soluble thiols as Well as non-SH antioxi-
dants Were decreased suggesting that the decrease was due to the oxidizing
action of free radicals.
Methods for determination of antioxidants, based on Blois' principle,
were also Worked out in this laboratory and used in a study on the occurrence
Acta Chem. Scand. 18 (1964) No. 9 11

2174 GLAVIND AND SQNDERGAARD . ,
of antioxidants in mammalian blood and livers. Three groups of substances
reacting with the stable free radical a,a-diphenyl-)9-picrylhydrazyl are present:
Fat-soluble antioxidants, water-soluble antioxidants, and proteins containing,
e.g., thiol-groups.
The methods have been used for the determination of the antioxidants
in blood plasma, liver and brain of 0 chicks given an e'ncephalomalacia-pro-
ducing diet and of a control group supplemented with vitamin E. Three sets
of determinations were carried out, 'viz., (1) directly on plasma and aqueous
organ extracts (total amount of antioxidants), (2) on . protein-free ?ltrates
(non-protein antioxidants), and (3) ?nally, fat-soluble antioxidants were
determined in lipid extracts of brains. 
EXPERIMENTAL
Animals. Day-old chicks were reared on a vitamin E-low starter ration for one week
and then allocated to two groups. One of the groups was given the encephalomalacia-
producing diet while the other received the same diet supplemented with D,L-m-tO00pherOl
acetate, 0.1 mg/g diet.
The eneephalomalacia-producing diet had the percentage composition: casein ("Vita-
min-test", Genatosan Ltd., Loughborough, England), 30; gelatine, 3; salt mixture,
5.17; vitamin mixture, 0.1; choline chloride, 0.2; corn starch, 31.53; and lard 30, and
was supplemented with 2-methyl-1,4-naphthohydroquinone diphosphoric ester dicalcium
salt, 10 pg/g, and selenium dioxide, 1.4 pglg. Vitamins A and D were given orally in
aqueous solution, 0. 1 ml twice a week, corresponding to vitamin A, 250 i.u. ,- and vitamin
D8, 20 i.u. per chick daily. ~
The chicks were inspected daily. As soon as a chick showed signs of e n c e p h a l o m a l a c i a,
a sample of blood was taken from the jugular vein into heparin. The chick was killed by
decapitation, and cerebrum and a portion of the liver removed for analysis. Control
samples from a vitamin E-supplemented chick were taken simultaneously. Encephalo-
malacia was diagnosed by the occurrence of the -gross symptoms of ataxia and by macro-
scopic examination of the brain. i
Preparation of blood plasma and organ extracts. The heparinized blood sample was
centrifuged and the plasma diluted with 4 parts of distilled water. A part of the solution
(0.8 ml) was used for the determination of total antioxidants while the rest was depre-
teinized with tungstic acid. After adjusting pH to 5.6 with saturated aqueous sodium
acetate, 1 ml was used for determination of non-protein antioxidants. . - . _ _
The cerebra were weighed, homogenized with 30 parts of water in a tissue homogenizer
and gently centrifuged. Two ml of the supernatant was taken for determination" of dry-
matter, 0.5 ml for determination of total antioxidants, and the " remainder deproteinized
in the same manner - as plasma. One ml of the protein-free ?ltrate -was used for the
determination of non-protein antioxidants. - . .
Weighed portions of liver were treated in exactly the same manner as the brains.
Antioxidant determinations were carried out on 0.3 ml of the homogenates and on 0.6
ml of the protein-free ?ltrates.   -
Fat-soluble antioxidants .were determined in the brains of chicks raised on the same
diets, but from a second experiment. The following procedure was used for the extraction:
One cerebrum or 2-3 cerebella were weighed and homogenized with 3 ml benzene; 5 ml
acetone was added, the mixture ground again, centrifuged, and 3 ml of the supernatant
used for the determination. '
Determination of antioxidants. The methods for determination of water-soluble anti-
oxidants were as described in the previous paper .4 with the only modi?cation that amore
concentrated solution of the reagent was used (optical density of the blanks about 1.0)
This was done in" order to cover a broader ?eld of antioxidant levels. The results were
calculated as pequiv. per ml blood plasma and per g dry matter of liver and brain.
Fat-soluble antioxidants were determined by the addition of 1 ml reagent of a
suitable strength to 3 ml of the extract mentioned above. The optical densities were
Acta Chem. Scand. 18 (1964) No. 9_


ANTIOXIDANT LEVELS I 2177
no difference is observed. No signi?cant difference is seen between the livers
or the blood plasmas, neither with respect to the antioxidant activity of the
total nor the non-protein fraction.
N o difference was found between the contents of fat-soluble antioxidants
of the brains of vitamin E-de?cient chicks and those supplemented with toco-
herol.
P Studies on the occurrence in brains of the most prominent fat-soluble
free radical scavengers, the tocopherols and the ubichromenols, are scarce.
Edwin et al.12 reported that the content of a-tocopherol and ubichromenol
in rat brains varied with diet and sex; a total content of 0.02-0.1 pequiv./g
brain can be calculated from their ?gures. We ?nd a higher level which is
the same even though tocopherol is added to the diet. Since the use of our
method for brain has not been controlled with other methods, the results
may not be entirely reliable.
The antioxidant activity of the total water-soluble and the non-protein
fractions of the livers was of the same order of magnitude (total activity about
25 pequiv./g fresh tissue) as that reported earlier for mammalian livers.
On the other hand, the activity of the plasma was much greater than that
of mammalian plasma (Table 3). The reason for the higher activity of chicken
plasma was found to be the much higher concentration of ascorbic acid and,
especially, uric acid.
Uric acid reacts quantitatively with diphenyl-picryl-hydrazyl. It seems
that the function of uric acid as a free-radical scavenger has not earlier been
reported in the literature. The substance has been used as a stabilizer for hydro-
gen peroxide, and its ability to react with oxidizing substances to form a great
variety of oxidation products is well-known. From a quantum-mechanical
approach, based on its very low energy coef?cient of the highest occupied
orbital, Pullman and Pullman 13 have calculated that uric acid should be a
very good electron donor.
It was suggested in our previous paper 4 that ascorbic acid is the most
abundant antioxidant in plasma. An inspection of Table 3 shows, however,
that uric acid accounts for a greater share of the free-radical scavenging
potency of plasma, of chicks, rats and calves, than does ascorbic acid.
The biological signi?cance of the free-radical combining power of uric
acid is dif?cult to evaluate. Wyngaarden and Stetten 14 established that
although the larger part of uric acid administered to man is excreted un-
changed in the urine, about 20 % of labelled uric acid is broken down. Peroxi-
dase, catalase, and methemoglobin have been shown to be capable of catalyz-
ing the oxidation of uric acid in the presence of hydrogen peroxide. The
special reactivity of uric acid observed in our studies opens for still more
speculation about possible ways for uricolysis.
It remains also to be studied whether uric acid has a function as a biologi-
cal antioxidant. The addition of 2.5 % uric acid to the diet to one group
of chicks in?uenced very little the onset of signs of encephalomalacia. Evi-
dently uric acid cannot be included in the group of substances which exhibit
vitamin E-mimetic activity in feeding experiments. On the other
hand, the dietary amount of uric acid should be compared with the amount
synthesized in the animal. Creek and Vaisaitis 15 found that approximately
Acta Chem. Scand. 18 (1964) No. 9