FEEDING EXPERIME1ITS WITH A DIET LOW IR TYROSINE

Thesis

Submitted to the Faculty of
Michigan State College in partial
fulfillment of the requirements for the
degree of Master of Science

by

Marjorie Beatrice Kenyon
June 1, 1928

ProQuest Number: 10008729

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whose assistance and helpful criticisms made the
completion of this, work possible.

FEEDING EXPERIMENTS WITH A DIET LOW IN TYROSINE

Introduction
Abderhalden

(Z. Physiol. Chem.

83,444,1913) fed

an adult dog a diet in which the nitrogen was supplied
hy fully hydrolyzed casein which had been freed from
tyrosine,

as completely as possible, by crystallization.

He found that the dog rapidly lost weight.
when tyrosine was restored

to the diet,

However,

the dog gained

in weight and recovered in health.
Abderhalden

(Z. Physiol. Chen.

96,1,1925) found

that when an amino-acid mixture free from 1-tyrosine
was fed to rats the nitrogen balance immediately
became negative.

Upon the addition of tyrosine or

phenylalanine to the diet the nitrogen balance rose,
although equilibrium was not reached.

Apparently

either tyrosine or phenylalanine are capable of sup­
plying some hemocylic compound which is required by
the animal.

Unsuccessful attempts were made to replace

tyrosine and phenylalanine 'with their oxi Qctiuion

o*•

ducts, p-hydroxyphenylpyruvic and phenylpyruvic acids,
indicating that the animal cannot synthesize the homocylic amino-acids.

- 2 Totani

(Biochem.

J. 10,382,1916) fed rats

several diets consisting of a basal mixture of
acid.-hydrolyzed gelatin,

fat,

the form of asheu dog biscuit,
which

sugar, salts in
and vitamins

were supplied by small portions of dried

alcoholic extract of milk,
ferent amino-acids.

to which was added dif­

He found that all of the rats

fed on this diet plus tryptophane,

could maintain

themselves for the experimental period of thirtyone days.

Two rats even gained in weight.

The

addition of tyrosine to the basal mixture plus
tryptophane had scarcely any effect.

Totani states

that this must be explained in one of two ways,
either phenylalanine is capable of replacing tyrosine
or the animal has the power to synthesize the benzene
ring.

Becua.se of the small amount of phenylalanine

in gelatine,

Totani thought the latter explanation

the more probable.
Shambaugh and Lewis

(J. Biol. Chem. 67,XXX,1926)

administered phenylalanine to rabbits subcutaneously
in doses of one gram per kilo and studied the changes
which took place in the blood and urine.

After the

administration of phenylalanine the phenols of the

- 3 "blood doubled and those of the urine increased so
that ten percent of the aromatic nucleus of the
phenylalanine could be accounted for as eliminated
in the urine as phenols.

There was no increase in

the urinary amino-nitrogen.

The increase in phenols

was partly due to the presence of an o-diphenol de­
rivative which gave the typical o-diphenol reaction
with ferric chloride.

The administration of tyrosine

increased the blood and urine phenols, but gave no
traces of o-diphenol.

This, then,

is evid.ence that

phenylalanine and tyrosine do not follow the sane
path of intermediary metabolism in the rabbit.
The indefiniteness of proof and the lack of
agreement made it desirable to attempt to obtain
further information regarding the effects of s. diet
low in tyrosine.
determination

With an improved method of tyrosine

(Folin and Giocalteu,

J. Biol. Chem.

73,627,1927) which permits a more accurate knowledge
of tyrosine in food components, we
protein mixture

hoped to obtain a

for our diet which vrould be even lower

in tyrosine

and phenylalanine than is hydrolyzed casein

or gelatin,

and to supply adequate salts

from undoubted sources.

and vitamins

This was accomplished by using

a mixture of hair hydrolysate,

digested gelatin,

trypto-

-

phane, histidine,

4 -

and cystine.

By feeding such a

diet to a larger number of rats, for a longer
period of time, we hoped to obtain more definite
information regarding the well-being of a rat on
a diet extremely low in tyrosine.

Experimental

Two diets Ila and lib having the following
composition were prepared:

Ila
Hydrolyzed gelatin
Hair hydrolysate
Tryptophane
Histidine
Cystine
Tyrosine
Yeast
Salt mixture (Osborn & Mendel)
Butter
Dextrine

8.8Q%
4.50
.20
.20
.10
.20
3.00
4.00
15.00
64.00

lib
9.063?
4.50
.20
.20
.10
.00
3.00
4.00
15.00
64.00

Preparation of the Protein Constituents of the Pood
One kilo of gelatin was dissolved in five liters
of solution buffered at Ph 7 by the addition of di ­
potassium phosphate and potassium dihydrogen phosphate.
Ten grains of pancreatin

(Digestive Ferments) were added

and the mixture digested for four days at 3 8 3 C .

The

fourth day, more pancreatin was added and the digestion

_ 5 allowed to proceed for four more days.

It was

then removed to the refrigerator for two days, at
the end of which time,

it was concentrated in vacuum

to a thick paste and then dried in a vacuum oven at
60° C until brittle.
Hair was used as a source of nitrogen because,
while it is low in tyrosine,

it does contain some

amino-acids that would appear to complement gels tin
as arginine,
content,

and to be very low in phenylalanine

(Kossel, Z. Physiol. Chen. 44,347,1905).

the preparation of the hydrolysate,

In

one kilo of human

hair was washed in gasoline, dried, washed in warm
water and again dried.

It was then hydrolyzed four

hours under a reflux condenser, with two liters of
concentrated hydrochloric acid,

filtered, and evaporated

to a thick rubberlike mass in vacco at 50° C,
moving most of the acid.

thus re­

The residue was then dissolved

in 500 cc of water by warming in a water bath at a
temperature not to exceed 60° C.

The solution was

cooled in a freezing mixture and adjusted to a Ph of
5.4 with 50% sodium hydroxide.

This point was deter­

mined when

five drops of

the solution in o cc of

water plus

five drops of methyl red gave the same

color as 5

cc of the Ph 5.4 buffer and five drops of

methyl red.

when compares,

in a. Coologe co operator.

The

- 6 -

solution was allowed to stand in the refrigerator
forty-eight hours and then filtered on a Buchner
to remove the tyrosine and a. large amount of sodium
chloride which precipitated.

The precipitate was

washed with small quantities of ice water.

All

washings that did not give a positive test with
Millon's reagent were added to the original filtrate.
This solution was concentrated in vacco nearly to
dryness and finally dried to brittleness in a vacuum
oven at 60° G.
The tryptophane was prepared from casein as
described by Cole,

(Practical Physiological Chemistry,

135) and extracted with butyl alcohol using Ons low’s
method,

(Biochem.

<T. 15,397,1921).

A much better

yield was obtained when fresh gland was used to digest
the casein.

Histidine dichloride was obtained from

fresh corpuscle paste by a method outlined by Morrow,
(Biochem. Lab. Methods,
Lab. Methods,
from raw silk.

142).

Morrow's method

(Biochem.

140) was used in preparing 1-tyrosine

-

7 -

Preparation of the Salt Mixture

Because of the high sodium and chlorine content
of the hydrolysate,

Osborn and M e n d e l ’s salt mixture

(j. Biol. Chem. 37,572,1919) was modified,

in that

the sodium carbonate and hydrochloric acid was omitted.
This made the sodium chloride content of the food
practically that fed by Sherman
1925)

(J. Biol. Chem. 62,331,

8

Tables of Analysis

Gelatin

Method of Analysis

mil g/g

Tyro sine

Folin & Ciocalteu
J. Biol. Chem.
73,627,1927

.48

Total nitrogen

Seales & Harrison
J. Ind. & T5ng. Chem.
12,350,1920

Amino nitrogen

Northrop, J. Biol.
Chem. 9,767,1926

162.4

43.3

Hair Hydrolysate
Tyro sine

Folin & Ciocalteu
J. Biol. Chem.
73,627,1927

.22

Cystine

Folin & Loony
J. Biol. Chem.
51,427,1922

44.5

Sulphur

Benedict
«T. Biol. Chem.
6,363,1909

13.5

Total nitrogen

Kjeldahl, Seales & Harrison
J. Ind. & Bug. Chem.
95.6
12,350,1920

Amino nitrogen

Northrop
J. Biol. Chem.
9,767,1926

Sodium

Hav/k
356.0
Practical Physiol. Chem.
786

Chlorine

Official & Tentative
Methods of Analysis
of A. D. A. C.

88.9

418.5

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9 -

Feeding

Seventeen rats at twenty-eight days of age
were divided into two groups in such a manner that
litter-mates were equally divided between the two
groups.

These groups were fed diets Ila and lib

for a period of eight v/eeks.
The yeast as a source of vitamin B appeared
to be insufficient, as indicated by the food intake
the first three weeks,

so one milligram of Vegex was

fed daily beginning the fourth week.
The growth of the rats is shown in tables I
and II and by the average growth curve I„

Discussion of Results

From these experiments it is evident that rats
can survive and grow slowly for a period of eight
weeks on a diet containing less than .04% tyrosine.
This is in agreement with the results obtained by
Totani.

We believe the conditions under which our

experiment was conducted afford better control of
the following experimental factors:

(l) less

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M IC H IG A N

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DEPARTMENT

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M A T H E M A T lO

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phenylalanine, (2) known amount of tyrosine,
(3) salt mixture known to be adequate,
vitamins from trustworthy sources,
number of animals,

(4 )

(5) greater

and (6) a longer feeding period.

Conclusion

For a period of eight weeks,

the growth

of rats receiving diets as described above is
independent of tyrosine content of the food.

Bibliography
Abderhalden, Z. Physiol. Chem. 83,444,1913
Abderhalden, Z. Physiol. Chem. 96,1,1925
Benedict,

J. Biol. Chem. 6,363, 1909

Cole, Practical Physiol. Chem. 135
Polin and Ciocalteu,
Polin and Loony,

J. Biol. Chem. 73,627,1927

J. Biol. Chem. 51,427,1922

Kossel, Z. Physiol. Chem, 44,347,1905
Morrow, Biochem. Lab. Methods 140
Morrow, Biochem. Lab. Methods 142
Northrop,

J. Biol. Chem. 9,767,1926

Onflow, Biochem.

J. 15,397,1921

Osborn and Mendel, J. Biol. Chem. 37,572,1919
Seals and Harrison,
Shambaugh and Lewis,
Sherman,

J. Ind. and Eng. Chem. 12,350,1920
J. Biol. Chem. 67,XXX,1926

J. Biol. Chem. 62,331,1925

Totani, Biochem.

J. 10,382,1916