THE EFFEU OF BETA AND GAMMA
IOMZNG BADMTEOR 0N. THi WHETNE
VALUE OF WHEAT PROTEIN

Thesis for flu chmo of M. 5.
MICHIGAN STATE UNEVERSITY

Joan Elaine Canaan
1959

THE".

  
     

  

LIBRARY '
Michigan State '

Univcmity

THE EFFECT or BETA AND GAMMA IONIZING RADIATION ON
THE NUTRITIVE VALUE OF WHEAT PROTEIN ' '

By

Joan Ela ine Cannon

A THESIS
submitted to the College of Home Economics
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

MASTER OF SCIENCE

Department of Foods and Nutrition

1959

Approved by W

!

 

ABSTRACT

This study describes the effects of beta
and gamma radiation at both low and high dosage
levels, on the nutritive value of wheat as fed to
rats for a ten week period. The dosage levels of
radiation ranfied from a low of 0.28 xloé rad
to a high of 9.3 X lo0 rad. Gross measurements of
growth, nitro:en balance, biological value, protein
efficiency ratio and apparent digestibility were
used in conjunction with more sensitive methods
at the cellular level, namely, an assay of liver
xanthine oxidase activity and the determination of
liver fat, to evaluate the nutritive value of the
irradiated wheat protein.

The highest dosa e level of 9.3 X 106 rad of
gamma radiation decreased significantly the nitrogen
balance and biological value of the wheat protein.
All other radiation dosage levels were not
significant with respect to these measurements.
ior did the remaining gross measurements: indicate
any alteration in the nutritive value of irradiated
wheat protein.

At the cellular level, however, the nutritive

value of the wheat appeared to be affected by each

dosa7e level of radiation. Liver fat was significantly
increased and liver Xanthine oxidase tended to

decrease with increasing dosages of radiation.

Wheat irradiated at all levels of gamma adiation
produCed the same dezree of change. With beta
radiation, the alteration, of the cellular

components increased with increasing dosages.

THE EFFECT OF BETA AND GAMMA IONIZING RADIATION ON
THE NUTRITIVE VALUE OF WHEAT PROTEIN

BY

Joan Elaine Cannon

A THESIS

Submitted to the College of Home Economics
Michigan State University of Agriculture and
Applied Science in partial fulfillment of

the requirements for the degree of

MASTER OF SCIENCE

Department of Foods and Nutrition

1959

AC:-€ZT01~ILEDGI Tn T S

The author gratefully acknowledges
the help and co-Operation given her
throughout this project by Dr. B.E. Rutherford.
Thanks are also extended to the rest of the
staff in the Department of Foods and
Nutrition for their interest and encouragement.
Too, the co-operation of the Department of
Agricultural Engineering, Michigan State
University, East Lansing and the Phoenix
Laboratory, University of Kichifan, Ann Arbor,

in irradiating the wheat, was greatly appreciated.

TABLE OF CONTENTS

INmODUCTIOquOOOO0.0.000000000000000000000l

REVIEW OF LITERATURE
Ionizing Radiation....................7

Irradiation effects on
amino aCidSOOOOOO0.0.0.00000000000000013

Irradiation effects on
proteins...OOOOOOOIOOOOOOOOOO0.00.00.018

The nutritive value of
prOteinSoooooo000.00.000.00...-000000.22

EXPERIMENTAL PROCEDURE....................3l
RESULTS AND DISCUSSION....................34
SUMMARY AND CONCLUSIONS...................50
LITERATURE CITED..........................52

APPEIqDI-XOOOOOOOOOO...OOOOOOOOOOOOOOOOOOOOOi

Table I
Table II

Table III

List of Tahlgg

Composition of Diet..........32

Average weight Gain in
Grams for Ten week Period....36

Average values of Protein
Efficiency Ratio, Nitrogen
Balance, Biological Value and
Apparent Digestibility.......39

Fig 1(a), (b)

List of Figures

Growth rate of rats fed
beta and gamma irradiated wheat....35

Xanthine oxidase activity and
percent fat in livers of rats fed
beta and gamma irradiat ed wheat . . . .h6

Table l

1!

fl

‘0 03 rd 0‘ U1 $' \9

ll
12

13

1h

15

16

17

List of Ta 8 - A en 1

'Wei ht gain in grams for the
per od of ten weeks........................iii

Feed intake in grams for the
period of ten weeks........................iv

Nitrogen intake in grams for period I......v
Nitrogen intake in grams for period II.....vi
Urine nitrogen in grams for period I.......vii
Urine nitrogen in grams for period II......viii
Feces nitrogen in grams for period I.......ix
Feces nitrogen in grams for period II......x

Protein.efficiency ratios, expressed
as weight gain in grams per gram of
nitrogen consumed, for period I............xi

Protein efficiency ratio, expressed
as weight gain in grams per gram of
nitrogen consumed, for period II...........xii

Nitrogen balance in grams for period 1.....xiii
Nitrogen balance in grams for period II....xiv

Biological value eXpressed.as the

percentage of nitrogen retained for

periOd IOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOiV
Biological value expressed as the

percentage of nitrogen retained for

perIOd I 00.0000....00...OOOOOOOOOOCOOOOOOOXV1
Apparent digestibility expressed as

percentage of dietary nitrogen apparently
absorbed for periOd O...OOOOOOOOOOOOOOOOOOXVii

Apparent digestibility expressed as

percentage of dietary nitrogen apparently
absorbed for period II.....................xviii

Liver xanthine oxidase activity expressed
as micromoles of xanthine per gram of
liver (wet weight) per 30 minutes..........xix

Table 18 - Percentage of fat in liver, based
on dry weight of liver...................xx

" l9 - Percentage of nitrogen in liver..........xxi

h 20 - AnalYSiS Of VariancezF values. 0 e e o o o o O 0 0 Oxxii

INTRODUCTION

Radiation has been proposed in recent years as a
means of destroying grain-infesting insects and molds.
Because of the availability of large quantities of
radioactive waste products, radiation would seem a
logical solution to the simplification of certain
food storage and shipping problems. The estimated
yearly damage to stored food products in the United
States alone is over one billion dollars (Hassett &
Jenkins, '52). It was also reported that the United
States government had an investment of $4.7 billion
dollars in surplus grain. Therefore any feasible method
which avoids spoilage of grain by insect infestation
merits some consideration. However if radiation
preservation is adopted, the irradiated product must be
acceptable for human consumption. It would appear
that acceptability of irradiated foods from the stand-
point of palatibility is a more serious problem than
is the alteration in nutritive value of the macro-
nutrients. I

The treatment of grain with ionizing radiations is
not only a means of controlling insect infestation but
it could also be used for preserving grain from fungal
growth which is the major source of respiration as well
as the cause of heating and chemical deterioration of
grain in storage. Yen, Milner and Ward ('56) reported

that the respiratory pattern, reflecting growth of

-2...

grain-spoilage fungi on freshly dampened grain at
ordinary temperatures, was eliminated in hard winter
wheat, with a 20% moisture content, by dosages slightly
beyond 125,000 rep* but that treatments greater than
625,000 rep were required to destroy viability in grain
containing 12% moisture. Radiation damage is more severe
in the presence of water due to the indirect action of
irradiation byproducts of water on the solute present.
In the absence of water or with a decreased amount of
water, the level of irradiation used has to be increased
to produce an alteration in solute and hence on the
organism.

Hassett and Jenkins ('52) indicated that the amount
of gamma radiation necessary for killing insects was much
less than that needed for bacterial sterilization. Doses
of 65,000 rep which were quickly and completely lethal
stopped the damage of heavy infestations. Light in-
festations however, could be controlled by doses of
lesser intensity which stopped reproduction. (Baker
et al., '53)

The following dosage levels for the radiation of
food products have been suggested: (Siu, '57; Baker
et al., '53; Yen et al., '56; Hassett & Jenkins, '52):

12,000 rep inhibits potatoes and onions
from Sprouting;

 

* rep = roentgen equivalent physical and is equivalent
to the absorption of 93 ergs per unit gram of density

-3-

10-20,000 rep sterilizes adult insects;
25-30,000 rep kills insects;
200,000 rep kills most micro-organisms;

250-625,000 rep preserves grain from
fungal growth;

2,000,000 rep kills the most resistant
of bacteria.

Radiant energy at high dosages will not only destroy
all micro-organisms by inducing changes in their chemical
structure, but it will also bring about similar chemical
changes in the food. Therefore, before this new food
processing method can be accepted, evidence is necessary
to demonstrate that these chemical changes result in
little or no deleterious effects on the nutritive value
of the food. The following is the recommendation from
the U.S. Food and Drug Administration (Vorhes & Lehman,
'56):

"The observed organoleptic and nutrient changes in
products subjected to cold sterilization confirm that
expected chemical reactions do take place. They signify
the possibility of a wide variety of reactions and hence,
a wide variety of end products. For all practical pur-
poses, these are food additives. Good practice in
toxicological investigation involves feeding of exper-
imental animals at a high level of a proposed food
additive, with the objective of discovering the nature
of definable injury, a middle level which may or may
not give evidence of injury and a lower level which

does not affect the animal. The data so obtained

permit an estimate of the margin of safety of the
additives in use."

There is evidence suggesting that radiation does
cause degradation of the protein molecule. Lloyd,
Milner and Finney ('57) found that the viscosity of
gluten sols decreased with increasing doses of gamma
radiation. Barron and Finkelstein ('52) showed that
X-irradiation of aqueous solutions of serum albumin,
serum globulin and egg albumin caused an increase in the
absorption of ultra-violet light and a slight increase
in the viscosity of the proteins. Alsup ('59) has also
shown by electrOphoretic studies that there was an
increased alteration of gluten with increasing levels
of radiation from 3 x 105 rep to 10 x 106. How-
ever, the yield of gluten appeared to increase up to
l x 106 rep and then decreased with 3 x 106 rep and at
10 x 106 rep no gluten was obtained. Viscosity studies
with irradiated flour followed the same pattern; namely,
an increase in viscosity with increasing dosages of
radiation to the level of l x 106 rep, followed by a
decrease in viscosity.

Metta and Johnson ('56) reported that the biological
value of milk and pea protein irradiated at a level of
3 x 106 rep was reduced by 8%. Teply and Kline C56)
found that there was a slight decrease in the growth
rate of animals fed chicken and gelatin dessert powder

6
irradiated at levels of 3 x 106 rep and 6x10 rep.

-5-

Melehy ('58) fed irradiated wheat to rats and found that
the biological value of the wheat did not alter with
radiation dosages up to l x 106 rep.

Further study of the effects of radiation on the
biological value of wheat protein is warranted,
eSpecially at higher levels to establish the level at
which the biological value of the protein could be
altered, since it is known that radiation can cause
degradative changes in the protein molecule. A study
of levels beyond the normal dosage anticipated for
commercial processing or sterilization should be includ-
ed to see if the higher dosage will augment the product-
ion of any harmful changes. Too, it has not been
definitely established that the nutritive value of wheat
protein at lower levels of radiation remains unchanged.
Thus if there were no deleterious change at either low
or high levels, then it could be assumed that the
processing method is harmless.

The study reported herein compares the effects of
two types of radiation, beta and gamma,at dosages

6 to 9.3 x 106 rad*, on the

ranging from 0.28 x 10
biological value of wheat protein for rats. An attempt
has been made to include more sensitive measurements of
the nutritive value of proteins than have hitherto been

reported, so that slight differences in nutritive value,

 

as:
rad is equivalent to the absorption of 100 ergs per
unit gram of density. 0ne rad unit = 93/100 rep.

if present, will be revealed.

REVIEW OF THE LITEB\TILE

Two ty pes of ray 5, beta and gamma, ave been
considered for the commercial application of irradiation
to increase the storage life of food products.

Cathode rays or high volta:e electrons are
artificially accelerated electrons or beta particles. The
terms electron beam, cathode ray and beta particle are
used interchangeably to designate the flow of electrons.
A beta particle is an electron emitted from an excited
nucleus. The term cathode ray is used to designate the
flow of electrons from some mechanical equipment.
Electrons accelerated by potentials of several million
volts are capable of penetrating a number of substances.
Penetrat ng electrons ionize living tissue so that the
chemical composition of the tissue is changed.

Gamma rays are electromagnetic waves which penetrate
relatively deeply into matter ant have essentially the
same effect on living tissue as accelerated electrons.
They are produced by the disintegration or ato:ic nuclei.
Cobalt60 , obtained by bombarding ordinary cobalt in a
nuclear-reacting pile and radioactive waste products are
sources of gamma rays suitable for irradiating food.

The penetrating and ionizing powers of gamma and
beta rays differ. tramia rays are able to pass through
as much as 20 centimeters of lead, whereas beta rays can

be stOpped by a few millimeterso aluminuz. (Friedlarder

& Kennedy, '56; Young, '57). Their relative
penetrating powers are approx mately one hundred to one,
whereas their relative ionizing p0wers are one to one
hundred.

In radiation chemistry there are two paths of
activation. Some of the molecules are electronically
excited whereas others are ionized. An atom consist
of Hi ively charged nucleus and a surrounding
constell fit on of negative electrons; the whole being
electrically neutra l The princ1_al means of ener gy
dissipation by an ionizing radiation in its passage
through matter is the ejection of electrons from atoms
throuéh whichit passes. An atom so ionized is left
positively charged and is referred to as an ion. This
action is known as ionization. The ne _ative electron

LL
attaches itself to a molecule, so that within lO'l'

seconds a positive ative ion pair is formed. The
ions of the ion pair in liquid media often dissociate
or interact with neutral molecules and form radicals

1

or more stable Chemical compounds. Ytowin: that the
chemical bonds which hold a molecule together are
constituted by electrons shared between the two atoms
joined by the bondg it is eXpected that the removal of
such a bonding electron from a molecule will lead to its
dissociation or oth er cl emical change. The re oval of

electrons other than boncing electrons may also be

eXpected to result in chemical change.

-9-

Excitation results in the raising of an electron in
an atom or molecule to a state of higher energy and is
a less drastic process than the complete ejection of an
electron. The molecule in which the excited atom is
located then, might dissociate, emit a light quantum,
interact with another molecule or degrade the excitation
energy by collisions with other molecules into vibrat-
ional and rotational energy.

Two theories have arisen to account for the effects
of radiation: one being the target theory or direct
action; the other being the indirect action or activated
water theory.

Direct action can be defined as the chemical change
a molecule will undergo when it is acted upon by radiat-
ion directly to cause ionization of that molecule.

This theory can be applied to the following classes of

action (Lea, '55):

l. The effect of radiation is due to a single
ionizing particle, that is, it is due to the change
produced in a single molecule by the ionization of that
molecule. For example, it has been applied to explain
the inactivation of viruses and production of gene
mutations.

2. The effect due to a single ionizing particle
which produces a sufficient number of ionizations as it
passes through the molecule, e.g. the production of

certain chromosome aberrations.

—-LV-

3. The effect due to a number of ionizing particles
which produces a large number of ionizations within the
molecule.

The result of direct action of ions on the molecule is
thought to cause Splitting or fragmentation of large
biological molecules (Tobias, '51).

Indirect action can be defined as the chemical
change a molecule will undergo due to the production
of radicals from the radiation of water, which then in
turn act upon the molecule. The primary ionization or
excitation occurs at some distance from the molecules
affected. It occurs when the short-lived ions disappear
and give rise to radicals and intermediate chemical
compounds in the liquid medium. In very dilute aqueous
suSpensions, the primary radiation effect is on water
and most of the effect is due to water decomposition
products. (Dahaet al., '43; Weiss, 'hh, 'té; Tobias,
'51; Proctor et al., '52). i

In the absence of oxygen, some hydrogen gas and
hydrogen peroxide is formed. When oxygen is also present,
dissolved in water, the radical H02 forms, which in
turn enhances H202 formation. Oxygen dissolved in the
cell medium is known to increase radiation sensitivity.
In the absorption of radiation by the hydroxyl radical,
it loses its electron which will be transferred to a
neighboring hydrogen ion represented as follows: (Weiss,
'44, v46).

(HO’) H+ + radiation -—.. HO + H (1)
or

-11..

(HO') H20 + radiation —-+ H0 + H + 0H“ (la)
Recombination of the decomposition products to restore
the initial state can still occur although this rarely
happens:

H + OH ---—-> H20 (2)

The actual decomposition of pure water can only occur
in so far as the subsequent reactions of hydrogen
formation

2H -——+ H2 (3)
and oxygen formation

20H -————+ H20 + o (A)

20 __..____) 02 (5)
can compete with the reverse reaction (2). Both
hydrogen atoms and hydroxyl radicals are extremely
reactive; hydrogen atoms are powerful reducing agents
and hydroxyl radicals are strong oxidizing agents.
Therefore substances dissolved in water will be
attacked by these powerful reagents and will thus act
as acceptors towards one or the other of the radicals
formed by the radiation.

The action of ionizing radiation on water can be
represented by the following equations: (Proctor
et al., '52)

H20 W H20 + e'

e' 4- H20 -—> H20“

e' + H+ -——-b H

1-120+ ____.. H” + OH

-12..

H20” -—e0H" + H
OH' ———’OH + e'

H + OH-—>H20 (Rarely happens)

The indirect action theory is more widely accepted
than the target theory. If the former accounts for
most of the radiation changes, then it might be expected
that wheat is not as sensitive to chemical alteration
by radiation as other food products with higher
moisture contents. However the flavor and odor changes
produced upon irradiation of wheat can perhaps best
be explained by the target theory since the moisture
content of wheat is relatively low. The result of
direct action would cause splitting or polymerization
or a combination of both, in the protein moiety of
the wheat. Any water that is present would further
accentuate any changes occurring due to the effect of

radiation by indirect action.

Irradiation Effects qggAmino Acids

Much work has been reported on the effects of
radiation on amino acids and it is known that most of
the amino acids do decompose on irradiation, some
perhaps, more readily than others. Stenstrom and
Lohmann ('28) exposed an aqueous solution of tyrosine
to radiation and found the phenol group was the one
most affected and that the amount of tyrosine that was
changed was proportional to the dose of radiation
absorbed. Allen et al. ('37) submitted solutions of
several amino acids and a large number of derivatives
of amino acids and dipeptides to the action of cathode
rays and ultra-violet light. By spectroscopic measure-
ments, it was found that in all cases, the compounds
underwent a change and that ammonia was liberated. This
same result was also substantiated by Stein and Weiss
('48) and further that the irradiation produced
aldehydes similar to those obtained from the same sub-
stances ia vitro by the action of hydroxyl radicals
produced chemically. Dale et al. ('49) found deaminat-
ion to occur when aqueous solutions of amino acids were
irradiated by X-rays and also that the deamination of
glycine by X-rays was a non-polymerizing reaction. How-
ever, Dale and Davies ('50) found that the deamination
of glycine and other amino acids by X-radiation was
dependent upon the concentration of the glycine solution.

The yield of ammonia from these amino acids showed a

-14-

continuous rise up to nearly saturated solutions and
then levelled off. This would be in agreement with
the fact that dilute solutions are more easily attacked.
Further work has indicated that ammonia is not the
only compound liberated. Investigations of the effects
of X-rays on aqueous solutions of alpha amino acids
have shown that, in vacuo, besides deamination,
decarboxylation occurred, with the subsequent formation
of the corresponding aldehyde (Johnson et al.,'51).
Some hydroxylamine was also produced under certain
conditions. Radiation in the presence of oxygen
resulted in the formation of both alpha keto acid and
the aldehyde e.g. alanine to pyruvic acid. Alanine
irradiated in vacuo yielded predominantly acetaldehyde.
Dale and Davies ('51) found that with cysteine, H28
production took place in preference to deamination.
Histidine monohydrochloride irradiated at levels up
to l x 106 rep produced a yellow pigmentation, the
intensity of color increasing with increased dosages.
(Bhatia & Proctor, '51). The decomposition of histidine
was accomplished through deamination of the alpha
amino group and fission of the imidazole ring. Again
Proctor and Bhatia ('52) showed that aqueous solutions
of D,L-tryptophan, L-tyrosine, D,L-phenylalanine and
L-cystine irradiated up to levels of 1 x 106 rep, were
decomposed, the amount being proportional to the dose.
They eXplained that the evolution of H23 from the cystine

solution indicated that cystine was probably decomposed

-15-

at the disulphide linkage. Further work by Proctor

and Bhatia ('53) indicated the order of deamination

to be histidine)cystine>phenylaline>tyrosine)tryptophan.
Also the cathode radiation of three aromatic amino
acids, tryptophan, phenylalanine and tryosine caused

a Split of the benzene ring.

Jayson, Scholes and Weiss, ('5h) have shown that
the presence of oxygen during radiation could have
a twofold effect, the formation of hydroperoxy
radicals H + 02-9pH02 and the reaction of oxygen
with an organic radical R to produce an organic peroxy
radical. 4Moreover these workers concluded that it
would not be unlikely that an organic hydroperoxide
ROZH would eventually be formed. Proof of this result-
ed when, in the case of tryptophan, the hydroperoxide
resulting from an initial attack by hydroxyl radicals
at the 2-3 bond of the indole ring could decompose to
give formylkynurenine.

The effect of X-rays on aqueous, oxygen-free
solutions of glycine produced nine identifiable compounds;
ammonia, methylamine, glyoxalic acid, formaldehyde,
acetic acid, formic acid, carbon dioxide, hydrogen and
hydrogen peroxide. (Maxwell, Peterson and Sharpless,
'5#). However, although Garrison and Weeks ('56) con-
firmed the fact that ammonia, carbon dioxide, hydrogen,
formaldehyde, glyoxalic acid and acetic acid were the

principal products of the indirect action of radiation

-10-

on glycine in oxygen-free aqueous solutions, these
workers also found in addition that succinic, amino
succinic and diamino succinic acids were formed. The
indirect action of radiation on aqueous glycine in-
dicated the intermediate formation of imino-acetic

acid (Jayko & Garrison, '56). This formation of
ammonia and aldehyde through the oxidation of primary
amine to imine by indirect action in oxygen - saturated
solutions is represented by the following series of
reactions:

11%.an + OH—-+ RCHZNH or (RCHNHg)

RCHZNH + 02 —_——) RCH=NH +H02

RCH=NH+H20 ————-y RCHO + NH3
Results of the experiment suggest the following
reactions may be involved in the radiation - induced
cleavage of the peptide chain.

RCO-NH-CHZ-R + OH -—§ R-CO-NH-CHvR

R-CO-NH-CH-R + 02 -——9 R-C-N=CH-R + H02

R-CO-N=CH-R + H20 -——+.RCONH2 + RCHO
The hydrolysis reaction provides a specific mechanism
for post irradiation effects observed in certain
protein systems.

The action of ionizing radiation on aqueous
solutions of alanine yielded different products in
the presence or absence 0f oxygen. If no oxygen
was present, ammonia, pyruvic acid, acetaldehyde,
propionic acid, carbon dioxide and ethylamine were

formed (Sharpless, Blair &.Maxwell, '55). In the

presence of oxygen and also with large doses of
irradiation, both acetic and formic acids were
formed. With both glycine and alanine, oxygen
suppressed the reductive deamination of the amino
acid to the parent fatty acid, while it enhanced.the
oxidative deamination to the fatty acid containing
one less carbon atom.

The effect of increasing levels of radiation
on amino acids then, is deamination, decarboxylation
with the subsequent formation of the corresponding
aldehyde, hydrogen sulfide production and the ruptun
of the aromatic rings. In the presence of oxygen,
irradiation causes the formation of the correspond-
ing keto acid in addition to the aldhyde. Also in
dilute aqueous solutions of amino acids the above
effects are greater. In a dry state, there is less

alteration of the amino acids.

-18-

Irradiatipn Effects on Protein

Arnow ('35) reported that radiation of egg albumin
with alpha particles resulted in the formation of a
visible coagulum if the initial pH was equal to the iso-
electric pH. The temperature of coagulation was lower-
ed by eXposure to alpha particles when the pH was equal
to or greater than the isoelectric point. At lower
values of pH the coagulation temperature was increased.
It was also shown that the coagulation of isoelectric
egg albumin solutions on exposure to ultra-violet
radiation involved three distinct processes (Clark, '35):

l) the light denaturation of the albumin molecule;

2) a reaction between the light denatured molecule
and water which may be similar to heat denaturation but
occurs at a lower temperature;

3) the flocculation of the denatured molecule to
form a coagulum.

Hemoglobin and serum.albumin exposed to ultra-
violet light and alpha radiation showed that radiation
caused the formation of low molecular weight substances
and also a general inhomogenization of the protein
(Svedberg & Brohult, '39). The ultracentrifugal test
revealed a very pronounced inhomogenization of the pro-
tein; a continuous series of molecules of both lower
and higher weight than the normal being formed. Radiat-
ion of 0.07% serum albumin solutions with 75,000
roentgens at 25° 0 caused precipitation (Barron &

Finkelstein, '52). No precipitation however, occurred

-19-

at 4° c. With 10 x :Lo‘+ and 20 x 104 rep a dimer

was formed. Radiation of fibrinogen solutions led to
fragmentation as well as polymerization of the omiginal
fibrinogen molecule (Scherage & Nims, '52). Barron ('55)
reported that increased sensitivity to heat and.loss ‘
in solubility of proteins occurred on relatively mild
radiation. Upon radiation of raw evaporated milk at

3 x 106 rep, coagulation.and precipitation of the milk
protein occurred, whereas only a darkening of the milk
occurred with heat sterilization (Johnson, '57). Nicholas
et al. (' 58) showed that loaf volumes were not'signif-
icantly different among treatments up to 50 x 10‘F rep
in.the case of bread made from irradiated flour. How-
ever, Milner and Ian ('56) reported a decrease in crumb
and loaf volume with flour milled from irradiated wheat
beyond 25 x 10“ rep. Losses in wheat protein solubility
were stimulated markedly by radiation treatment at the
high level, of l x 106 rep. (Yen et al., '56). Alsup
('59) has also shown by electrophoretic studies of gluten
sols that there was an alteration of this protein, with
the levels of radiation used, 3 x 105, l x 106, 3 x 106
and 10 x 106 rep and this alteration progressed

with each dosage.

Degradative changes of the protein molecule can be de-
tected by changes in.the absorption spectra. the ultra-
violet absorption spectrum of irradiated egg albumin
was changed quantitatively by dose (Arnow, '35). Barron
and Finkelstein ('52) showed that X-radiation of aqueous

-20..

solutions of serum albumin, serum globulin and egg
albumin also caused an increase in the absorption
spectrum of ultra-violet light. The absorption Spect-
rum changes seemed to be due to the oxidation of tyrosine
residues and of other oxidizable groups. Barron et al.
('55) observed that upon radiation of proteins with a
tyrosine to tryptOphan ratio greater than one, there was
an increase in light absorption, whereas those with a
ratio less than one showed a decrease. 0n X-radiation

of serum albumin with X-ray doses not high enough to
produce denaturation, there was a decrease in the combin-
ing power with anionic dyes and an increase in the
combining power with cuprous ions. These effects were
explained as the consequence of the loss of amino groups
in the protein.

Changes have also been observed in viscosity. The
viscosities of egg albumin solutions at or below the
isoelectric point were raised by radiation (Arnow, '35).
If the pH was greater than that at the isoelectric point,
viscosity was lowered. X-radiation reduced the viscosity
of nucleic acid and nucleoproteins in vitro (Feinstein,
'51). Barron and Finkelstein ('52) showed that the
viscosities of aqueous solutions of serum albumin, serum
globulin and egg albumin increased slightly on radiation
with 50 x 104 rep. X-radiation increased the viscosity
of fibrinogen solutions and there was an increased
viscosity with increased dosage (Scherage & Nims, '52). At
dosages greater than three hundred kilo-roentgens, the

solution gelled Spontaneously. Lloyd et al. ('57) found

 

-21-

that the viscosity of gluten sols decreased with increas-
ing doses of gamma radiation. These differences in the
effect on viscosities could be explained on the basis

of splitting of the protein molecule, thereby producing
a lower viscosity, or of polymerization of protein
molecules, producing a higher viscosity. Both reactions
are important nutritionally,as bonds in the protein
molecule might be produced which could not be hydrolyzed
during digestion and metabolism,or irregular fragmentat-
ion could render the products unahailable for tissue
synthesis.

When high-energy radiations strike the food particle,
off-odors and flavors are produced as a result of the
effect of ionizing radiations on the carbohydrate, fat, and
protein components of the food particle. In some foods
there may exist some protective compounds to prevent the
alteration of the protein or amino acids which might be
responsible for off-odors and flavors. The elimination
of off-flavors has been successfully accomplished in
several foods by using ascorbic acid, d-isoascorbic acid
and their salts (Proctor & Goldblith, '52). The mech-
anism of protection is not simply an oxidation-reduct-
ion effect but probably a competitive reaction as added
niacin protected food from flavor changes (Proctor et
al., '52) and niacin is resistant to oxidation.

However, most of the off-odors in irradiated food
appear to be due to the formation of hydrogen sulfide and
mercaptans (Littman, Carr & Clauss, '57). Feinstein 051)

and Barron and Finkelstein ('52) suggested masking the

-22-

sulfhydryl groups by Specific chemical reactions, rather
than protecting them by competing reactions. Cysteine
was best protected by glyoxal, formaldehyde, pyruvic
acid, pyruvic aldehyde diacetyl and glyceraldehyde
which reduced the amount of hydrogen sulfide formed to
10-15% of that formed in unprotected solutions. Glut-
athione was best protected'byglyoxal, pyruvic acid and
formaldehyde (Littman, Carr & Clause, '57). However
work by Proctor & Bhatia ('50) showed that when fish
fillets were radiated by cathode rays at three levels
up to 5.7 x 106 rep, there was no significant destruct-
ion of any one of ten amino acids.

Because of the complex nature of most foods, the
alteration of the protein or amino acids which might be
responsible for off-odors and flavors, could be lessened
due to the existence of protective compounds. Thus the
chemical changes produced by radiation of pure amino
acids and proteins might not be as drastic when the amino
acids are incorporated in the protein which in turn is
incorporated in the complex food.

Nutritive Value of Proteins

Since foods vary as to protein, amino acids and moist-
ure content, as well as other factors present, the
nutritive value of some foods could be altered by radiat-
ion to a greater extent than others.

Poling and coworkers ('55) reported that when raw
ground beef, radiated with 2 x 106 rep cathode rays, was fed
to rats there were no major differences between the control

and eXperimental groups over a life Span of two years.

-23-

The small and occasionally statistically significant
decreases in growth, food efficiency, reproduction, adult
body size and survival were considered to be due to
slightly decreased nutritimal quality similar to that
which occurs during heat sterilization. The results of
sterilizing a semi-synthetic diet, by steam or cathode
rays, showed that sterilization did not produce any
consistently harmful effects when fed to mice over three
generations (Lucky et al., '55). Vitamin losses were
mere severe in the steam sterilized diet than in the
cathode ray sterilized diet. The effect of feeding dogs
the flesh of lethally irradiated cows and sheep indicated
no differences in weight gain, hematological or patho-
logical changes, attributable to the ingestion of ground
meat from either source. (Wassernan 8c Trum, '55). Metta
and Johnson ('56a,b) reported that radiation sterilizat-
ion did not affect the apparent or trte digettibility of
beef, milk, pea or lima bean protein radiated at 3

x 106 rep and fed to rats. However, the biological value
of both the irradiated milk and pea protein was lowered
by 8% as compared to heat processing which lowered the
biological value by 6%. These authors stated that as high
levels of radiation could cause further polymerization in
the case of protein, it would seem there must be some
radiation level at which the energy value of a food
nutrient could be depressed, and therefore affect the
nutritive value of that food.

When a 3 x 106 rep gamma irradiated diet was fed
to chickens there was a slightly reduced rate of growth

-24-

and a slightly delayed maximization of hatchability of
eggs from the pullets fed the irradiated diet (Burns,
Brownell & Eckstein, '56). However, these workers
concluded that there was no evidence indicating the
presence of chronic or subacute toxicity or gross
nutritional losses other than vitamin losses. In a
study of thirteen foods Teply and Kline ('56) reported
there was no significant difference in growth of rats
fed the control or irradiated foods. In fact the
radiation of cauliflower, celery and white potatoes
produced a moderate increase in the growth rate.
However, there was a slight decrease in growth rate
produced by feeding irradiated apricots and chicken.
Rats fed organ meats radiated at 3 x 106 rep grew as
well as the control animals. (Bubl & Butts, '56).
Irradiated ground beef, fresh ham, sliced bacon,
haddock fillets, green beans, whole kernel corn, sliced
beets, frozen strawberries, sliced peaches, bread,
cereal and powdered whole milk fed to rats for eight
weeks, showed excellent growth and food consumption

at the levels of radiation used, 3 x 106 and 6 x 106
rep (Kraybill et al., '56:.Read et al., '58a). However,
these authors point out the need for more sensitive
criteria to establish the safety of using radiation to
sterilize food. Read et al. ('58b) fed the above -
mentioned foods to four succesSive generations of male

and female albino rats. No adverse effects of radiation

-25-

sterilization of the food were observed, in so far as
growth, reproduction, lactation, energy metabolism, or
average life Span of the animals were concerned.
Richardson and Brock ('58) in studying the nutritional

6 rad

value of a synthetic diet sterilized by 2.79 x 10
of gamma rays, as measured by reproduction and life
span of rats, noted that the average weight of young
from mothers receiving the control diet was approximately
three grams more than that of those from mothers
receiving the irradiated diets. The life Span in both
groups was essentially the same. In general, the data
showed that the nutritive value of the irradiated diet
was slightly less than that of the non-irradiated.
However this could posSibly be attributed to vitamin
losses in the irradiated diet.

Metabolic balance studies on humans were carried
out by three feeding experiments of nine subjects each,_
with 35%, 65%, and 80% of the caloric content made up
of irradiated food. White potatoes were radiated at
2 x 104 rep and all other food making up the diet was
radiated at 3 x 106 rep. No deleterious effect on the
subjedts was reported when the irradiated food was fed
for a duration of 15 days. (McGary & Shipman, '56)

Three levels of radiation, 1 x 104, l x 105 and l
x 106 rep were used to radiate a wheat diet fed to
three grain insects. (Hodges, '57). Evidence was present

throughout the experiments that there were differences

-26-

associated with the level of radiation; however these
differences were not consistent over all the experiments.
Furthermore, the effect appeared to be that of increasing
the rate of reproduction rather than decreasing it as
might be expected if radiation of the diet had a del-
eterious effect upon the insects. Melehy ('58) in-
vestigated the effect of high voltage cathode ray
ionizing radiation on the biological value of wheat
protein fed to rats at the radiation levels of 5 x log,
10 x 104, 50 x 10h, and l x 106 rep. No significant
differences were found among gross measurements of
nutritive value such as nitrogen balance, biological
value, protein efficiency ratios, apparent digest-
ibility and urinary omatinine nitrogen percentage.
Metta and Johnson ('59) fed 2.8 x 106 and 9.3 x 106
rad, gamma-irradiated corn and wheat gluten to rats.
Radiation did not affect the digestibility of the
wheat gluten but the radiation of corn at 9.3 x 106
rad lowered its digestibility by 5%. However the
biological values of corn and wheat gluten were not
affected. Microbiological assays of the limiting
amino acids, lysine in wheat gluten and lysine and
tryptophan in corn, showed there was no destruction
of these amino acids by radiation. Therefore due to
the small magnitude of the chemical changes accom-

panying radiation sterilization, the animal-feeding

experiments ordinarily employed should be expanded

-27-

to include more sensitive methods of measuring the
effects of radiation on foods.

The degree of activity of certain enzyme systems
appears to be directly related to the nutritive
value of foods. An assay of xanthine oxidase from
intestinal tissue of rats fed a diet containing
irradiated soybean oil, indicated a reduction in enzyme
activity. (Andrews, Mead, & Griffith, '56). However,
these workers also reported a corresponding decrease in
growth. Alterations in metabolism, too small to be
noticed by the usual techniques, were detected when
several enzymes were used.as indices of metabolism at in
cellular level (Kraybill, et al., '56). Assays of
liver xanthine oxidase, cytochrome oxidase, succinic
dehydrogenase and serum alkaline phosphatase were
carried out. An increase in the activity of liver
cytochrome oxidase was reported (Kraybill et al., '56;
Read et al., '58b). 5 ,

The possibility of using activities of enzyme
systems to determine the nutritive value of proteins
was stimulated by the work of Miller ('hB) who found
that fasting caused.a loss of the‘activities of cat-
alase, alkaline phosphatase, xanthine dehydrogenase,
and cathepsin.in the livers of rats. ‘Williams and
Elvehjem ('49) reported that liver xanthine oxidase
could be used as a senstive index of amino acid
availability in dietary proteins under conditions

in.which gross body changes were not in general

—~v—

sensitive enough to reflect small protein variations
in the animal body. Also there is ample evidence cor-
relating the loss of activity of xanthine oxidase with
loss in protein, with certain essential amino acid
deficiencies and with the nutritive value of dietary
proteins (Allison, '55; Younathan, '56; Litwack, '52;
'53a & 'SA; Williams, 'h9, '50; Chance, '52; wainio,
'53). ‘

Usually associated with the loss in cyt0plasmic
proteins is a reduction in water and an increase in the
fat content of the liver. (Maynard & Loosli; '56).
However the amount of lipid in the liver is also a
function of the amount of dietary choline, methionine,
cystine and possibly other amino acids. DeSphande,
Harper and.Elvehjem ('58) created amino acid imbalances
by supplementing a low protein diet containing 6%
‘fibrin. with various combinations of amino acids.
Supplementation of the 6 % fibrin diet with six amino
acids, calculated to be most limiting for growth,
stimulated growth yet caused fat to accumulate in the
liver. These fatty livers reSponded to lysine and to
threonine. A combination of the two was more effective
than either of the individual amino acids. Vennart,
Perna and Stewart ('58) found that when rats were fed
a corn diet a fatty liver was produced which was
completely reversed by the addition of lysine and tryp-

tophan.

A fatty liver does not occur in all cases of
amino acid imbalance; however if radiation causes an
alteration of the protein in wheat affecting its amino
acid distribution, an increase in the deposition of fat
in the liver could occur. The percentage of fat in the
liver, then could be used to corroborate other data
in the experiment or perhaps indicate alterations in
the nutritive value of the protein which are not seen
by the usual technigues employed.

The above evidence indicates that liver fat and
xanthine oxidase might be good measurements of subtle
differences in the nutritive value of proteins, produced
by radiation. Therefore in the present study the
determinations of liver xanthine oxidase activity and
fat were used as more sensitive indices of measuring
the nutritive value of irradiated wheat protein.

Two types of radiation, beta and gamma, were chosen.
to see if the resultant effects on the biological value
of wheat protein would be the same. The radiation levels
were chosen to include both low and extremely high levels
to see if there was a point at which the animals did
reSpond to the known changes that take place in
irradiated wheat. If a radiation level is reached at
which point, the wheat protein is rendered unavailable
to the animal, then a margin of safety could definitely
be established for this processing method in accordance with

the recommendations of the U.S. Food and Drug Administration

-30-

(Vorhes & Lehman, '56).

Therefore this study compared the effects of two
different types of radiation, each ranging in dose
from 0.28 x 106 to 9.3 x 106 rad, on the nutritive
value of the protein of irradiated wheat. Growth,
protein efficiency ratio, nitrogen balance, apparent
digestibility and biological value are defined as
gross techniques; the measurements of liver xanthine

oxidase activity and fat, as fine techniques.

-31-

EXPERIMENTAL PROCEDURE

Four samples of wheat were exposed to gamma
radiation produced from cobalt-601 and four samples
were radiated by cathode rays in a high energy electron
beam generatorz. A ninth sample was not radiated and
was used as a control. The do sage levels of both
types of radiation were the following:

0.28 x lo6

0.93 x 106 rad
6

6

rad

2.8 x 10 rad

9.3 x 10 rad.

Each sample representing one type of radiation at each
of the above dosage leVels was incorporated into an
experimental diet according to Table 1.

Each of the 9 diets was fed to a group of eight
weanling male albino rat 3, weighing approximately fifty
grams at the begim ing of the experiment. The animals
were allotted at random to individual wire-bottom
cages and given food and water ad libitum for a ten
week period. The weight of food eaten and the weight
of the animals were determined each week.

After a seven day ration-acclimatization period,

the rats were placed in metabolism cages for a further

seven days for nitrogen balance determinations. The

fl

1. Phoenix Laboratory, University of Michigan, Ann Arbor.

 

2. Dept. of Agricultural Engineering, Michigan State

University , East
Lansing .

3329

TABLE 1
COMPOSITION OF DIET

WHEAT 90.1.73
CORN OIL 5 .o%
MINERAL. MIXI" h .076?
VITAMIN 14wa o .676

 

* wesson's Salts - Nutritional Biochemicals Corporation
Cleveland, Ohio.

**Vitamin mix provides the following vitamins per
kilOgram of diet: 1 mgm thiamin hydrochloride, 2.8 mgm
riboflavin, l mgm pyridoxine hydrochloride, 20 mgm
nicotinic acid, 10 mgm calcium pantothenate, 0.08 mgm

folic acid, 210 mgm choline, 1930 I.U. vitamin A and
630 I.U. vitamin D.

-33..

metabolism period was repeated during the seventh week.
These two determinations are referred to as period I
and period II reSpectively. The metabolism cages were
sprayed with a hot 2% boric acid solution prior to the
experiment. 0n the termination of each seven day
experiment, the cages were rinsed with hot water, the
washings being added to the urine which was collected
in 0.1 1'1 HZSOA. Feces were collected daily, dried in a:
air oven at approximately 100°C and then ground to a
fine powder. Any food which was spilled, was washed with
hot water to renove any traces of urine, the washings
added to the urine and the amount of spilled food
subtracted from the total intake of food for that
period. Nitrogen determinations of the food samples,
urine and feces were made by the boric acid modificat-
ion of_the Kjeldahl-Gunning method. (A.0.A.C., '57)

At the termination of the experiment the animals
were killed by decapitation, their livers removed and
xanthine oxidase activity determined by the color-
imetric procedure of Litwack et al. ('53b). A
slight modification of the procedure was made. (See
Appendix) The liver was also analysed for total
nitrogen, by the boric -acid modification of the Kjeldahl-
Gunning nethod, (A.O.A.C., '57), fat, by ether .‘x-
tracticn in the Goldfisch apparatus, and moisture, by

difference after ov en-dryilg.

-34-

RESULTS AND DISCUSSION

Growth of an animal has been correlated with an
increase in body protein and so with this in mind, the
rates of growth of the experimental animals were
compared. However, it is understood that growth
curves can only serve as an index of the nutritive
quality of the diet as the protein content of the
gains in weight are not necessarily constant (Barnes
& Bosshardt, '59). Therefore data presented by growth
curves must be substantiated by other measurements.

The growth curves in Figures 1(a) and 1(b),
obtained by plotting the average gain in weight as the
ordinate and time in weeks as the abscissa, indicated
that the animals fed the irradiated diets up to the
level of 2.8 x 106 rad, grew at about the same rate.

It is apparent from these growth curves that the highest
level of both betaamd gamma irradiation, namely 9.3

x 106 rad, produced the slowest rate of growth.

However, whether the rats on these diets would have
reached the same level of growth as those on the re-
maining diets is unknown due to the fact that the
experiment was terminated at ten weeks before the
animals had reached their peak of growth. In the case
of 0.28 x 106 rad of gamma radiation, it appeared that
these animals grew at about the same rate as those on
the diet of highest radiation. However this could be

due to the fact that some of the animals in this group

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-35-

contracted an eye infection during the experiment

and as a result their growth rate was somewhat

slower. .Moreover the growth curves of the non-
infected animals fed the 0.28 x 106 rad gamma irradiat-
ed diet appeared almost equal to that of the control.
Therefore it is concluded that levels of irradiation

up to 2.8 x 106 rad did not significantly affect the
weight gain of the animals during the ten week study.
In all cases the smallest gains in weight, 112.0 g

and 11h.o g, (See Table II), although not significantly
different from the control, were found in the animals

fed the diets irradiated at 9.3 x 106 rad.
TABLE II

AVERAGE WEIGHT GAIN IN GRAws FOR TEN NEEK PERIOD

 

TYPE of RADIATION
BETA. "',"G'Amm"""" '

RADIATION DOSAGE (RAD)

 

 

G. G.
CONTROL 129 129
0.28 x lO6 132 11A
0.93 x lO6 123 119
2.8 x 106 125 128

9.3 x lo6 112 115

 

 

 

-37-

No significant difference in weight gains was
apparent between the two types of radiation used.
Whether the slight decrease in weight gain, occurring
as the result of ingesting the diet irradiated at the
highest level of radiation, is an indication of a
change in the biological value of wheat protein, remains
to be seen. It would appear that this was the case,
even though no Significance was found among the
weight gains, feed intake, nor weight gains corrected
for feed intake. (See Appendix, Table 20)

These data were similar to the findings reported by
Poling et al. ('55), Nasserman and Trum ('55), Bubl
and Butts ('56), Burns et a1. ('56), Richardson and
Brook ('58) and Read et al. ('58a,b), although none of
these workers used a level of radiation as high as
9.3 x 106rad. Burns et al. ('56) did observe that
slight differences in body weight, although not signif-
icant, persisted in chickens fed a diet which had
received three million roentgens 0f1§mma irradiation.
Teply and Kline ('56) fed rats several foods that had
been irradiated at levels of 3 x 106 rep and 6 x 106
rep. Again no significant differences in growth of the
animals fed the control or the irradiated foods was
found. But the radiation of cauliflower, celery and
white potatoes suggested a moderate increase in growth
rate. An apparent decrease in growth rate was produced

by the radiation of apricots and chicken. The above

-38-

results show very clearly that the effects of radiat-
ion on all foods are not necessarily the same and
emphasizes also the fact that growth alone cannot be
used as a criterion to measure the nutritive value of
a food, eSpecially when differences in growth are small.
A refinement of the Simple growth method is the
concept of protein efficiency ratio, ‘which in this
experiment is defined as the gain in weight per gram of
nitrogen eaten. No significant differences for these
ratios were found among the radiation levels used nor
between the types of radiation (See Table III). This
was probably due to the fact that there was quite a
variation among the ratios within any one irradiated
diet. (See Appendix, Tables 9 and 10). For example,
the protein efficiency Tratio‘. calculated for the 9.3 x
106 rad beta irradiated wheat was 1.303.56.
The method of measuring protein quality by an
efficiency ratio of growth to protein intake implies
that the protein content of the gains in body weight of
growing animals is constant regardless of the age, size
of the animal, the quality of the protein, or the rate
of growth (Mitchell, 'Ah). But there sometimes is a
difference in the composition of body weight gains.
Allison and wannemacher ('58) reported that protein
efficiency ratios may be changed by merely increasing
the intake of food. Therefore this method of assaying
food protein biologically may be expected to exaggerate

quality differences among proteins when these differences

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-40-

are considerable and to obscure them when they are
only minor because of the large experimental error
to which the method is subject as it is ordinarily
employed (Mitchell, 'AA).

Thus, on the basis of growth and protein efficiency
ratios, neither the level of radiation nor the type
of radiation used appeared to cause any alteration in
the quality of the dietary protein.

A determination of the nitrogen in the food and
excreta under controlled conditions provides a quan-
titative measure of the protein metabolism and
Specifically shows whether the body is gaining or
losing protein (Maynard & Loosli, '56). A positive
balance shows an increase in actual protein tissue
thus representing a more exact measure of growth
than increase in weight or protein efficiency ratio.
Nitrogen balance in this experiment was defined as the
difference between the dietary nitrogen intake and
total nitrogen excreted.

The data in Table III Show that the experimental
animals were able to retain approximately the same
amount of nitrogen, at all the beta radiation levels
of the ingested diet, namely 0.71-0.84 grams in the
first balance period and 0.54-0.65 grams in the second.
An analysis of variance failed to reveal any significant
differences (See Appendix, Table 20). However for

gamma radiation, the nitrogen balance determined at the

level of 9.3 x 106

rad was found to be significantly
different at the 5% level of Significance in the second
balance period, that is, 0.42 grams which was 28.8% low-
er than the control. In the first period, the nitro-
gen balances for the gamma irradiated diets were not
found to be significantly different although it was
apparent that the nitrogen balance obtained at 9.3
x 106 rad, 0165 grams, tended to be less than the
values obtained at other levels of gamma radiation.
However, again there were large individual variations
among the animals on the same diet. For example,
the standard error of the mean was 0.65 1 0.32 at
the highest level of gamma radiation.
The second period Of the nitrogen balance studies
in both the beta and gamma groups showed a lowered
nitrogen balance as compared to the first period. .Melehy
('58) also observed this and offered the explanation
that in the first period maximum use was made of the
protein ingested to meet the demands of growth and in
the second period when the animals were ingesting more
food, the demands for growth were met more readily and
so any excess nitrogen would then be excreted. This
would cause an increase in the amount of nitrogen
excreted thus resulting in the lowered nitrogen balance.
The highest level of gamma radiation, namely 9.3

6

x 10 rad, appeared to affect the composition of the

wheat protein more than that of the beta irradiated

-42-

wheat. The nitrogen balance for the 9.3 x 106 rad gamma
irradiated wheat was 0.42 grams as compared to 0.54 grams
for the 9.3 x 106 rad beta irradiated wheat. Perhaps
then, the penetrating power of the gamma ray at this
level has a greater effect on the wheat protein than
the ionizing power of the beta which is greater than

that of the gamma ray. Eisdlander & Kennedy '56)

The ability of a given source of protein to supply

amino acids in the relative amounts needed to form
the nitrogenous tissues and compounds required for
body functions is referred to as its biological value
(Maynard & Loosli, '56). This value expressed as a
percentage, usually takes into account the amount of
nitrogen excreted which results from the minimum
essential catabolism incident to the maintenance of
the vital processes of the body. This has been
measured by feeding a nitrogen-free energy-adequate diet
or by incorporating small amounts of egg protein into
the nitrogen-free diet. In the first instance there
is some question as to whether nitrogen excretion, during
a protein-free period measures the so-called
endogenous nitrogen under conditions of protein feeding.
Allison ('56) assumed that in the usual measurement of
the biological value, the animal is not subjected to
conditions that would cause the develOpment of protein
depletion and so that throughout the course of the

determination, the endogenous nitrogen would remain

constant. Therefore for comparing the apparent
biological values of irradiated wheat protein.a
formula, not involving the measurements of endogenous
and metabolic nitrogen was used. The biological value
was expressed as:

= Nitrogen Intake - Total Nitrogen Excreted

 

x 100
Nitrogen Intake - Total Fecal Nitrogen

The data for biological value, shown in Table III
followed the pattern Of nitrogen balance. There were
no significant differences found among the beta
irradiated diets in either balance period, although the
second period showed a lowered biological value than
the first. Again the highest level of gamma radiation,
9.3 x l06 rad, produced a significant decrease of 17.1%
of the control in the biological value in the second
period. This decrease in biological value was also
apparent in the first period, although it was not
found to be significant.

Therefore, based on the results of nitrogen balance
and biological value, it would appear that the gamma

6

radiation level of 9.3 x 10 rad caused a decrease in
nitrogen balance and biological value. There were no
significant differences among the remaining levels of
radiation nor between the types of radiation used.

Although Metta & Johnson ('59) found a decrease in

the digestibility of corn protein irradiated at 9.3 x

-44-

106 rad, the biological values of the irradiated corn
and the wheat gluten were not reduced. In the above
study the wheat gluten was irradiated at A0 C. and in
an atmOSphere of nitrogen, which could account for the
differences obtained between this report and the present
study.

From the data obtained on nitrogen balance, the
apparent digestibility of the wheat protein can also
be determined. It is defined as the percentage of
dietary nitrogen apparently absorbed from the
digestive tract. The data presented in Table III
Show that there were no significant differences in the
digestibility of the wheat protein among the radiation
levels used nor the types of radiation. These results
are in accord witthetta and Johnson ('59) who
reported that gamma radiation of corn protein and
wheat gluten at the level of 2.8 x 106 rad did not
affect the digestibility of corn protein or wheat gluten.
However the radiation of corn at 9.3 x 106 rad caused
a 5% decrease in its digestibility. The wheat gluten
was not affected at this level.

The gross measurements of growth rate, protein
efficiency ratio, nitrogen balance, biological value
and apparent digestibility showed that betaand gamma
radiation up to the level of 2.8 x 106 rad did not
alter the nutritive value of wheat protein significantly.

However gamma radiation at the level of 9.3 x 106 rad

caused, in the second balance period, a 28.8% decrease
in the nitrogen balance and a 17.1% decrease in the
biological value. Both values were found to be
Significant at the 5% level of Significance.

The more sensitive measurement of nutritive value
of proteins by xanthine oxidase activity (micromoles
of xanthine disappearing per gram of liver per 30
minutes) showed no significance among radiation levels
or types of radiation (See Table IV). The percentage
of liver fat, based on the dry weight of the liver, of
animals fed the irradiated diet was significantly
different from the amount of fat in the livers of
animals fed the non-irradiated diet (See Appendix,

Table 20). However even though no significance was

TABLE IV
AVERAGE LIVER XANTHINE OXIDASE ACTIVITY IN MICROMOLES
0F XANTHINE PER GRAM 0F LIVER (WET WEIGHT) PER

 

 

 

30 MINUTES
‘TYEE‘OE‘RIDIATION“
RADIATION DOSAGE (RAD)

BETA GAMMA
CONTROL 6 12.2 12.2
0.28 x 10 11.9 11.1
0.93 x 10% 10.6 9.1
2.8 1x 106 10.1 10.A
9.3 A 10 9.2 10.2

 

 

 

obtained for the xanthine oxidase activity, the data

illustrated in Figures 2a and 2b suggest that with

Micromoles xanthine / gram of liver / 50 minutes.

-46-

 

 

 

 

 

 

 

 

 

12. 0*;
P L.
10- I 8
//
8""‘ Xanthine oxidase activity /
/
/
eq-—-‘ '0 félt I, I- 6
n‘.“
’I‘
.-q ,9”
4 ,a
[I
I
2- /’ 2(a) P4
’/
"
T I .3 W 6 I 6 ' ‘6
Control 0.28x10 . 0.9tx10 2.8x10 9.3x10
1 ' is f b t radiati n (ran)
12-
1;]: . I 8
Xanthine oxidase activity
{3-4
. fi‘\
------5 f't / ‘\ __,.....-.._
d. / d- , ‘V’- ‘fi~~‘ . 6
/
/
/
4‘ /
I
/
d l c"
2 , 6(0) .4
d

 

 

I I -- U ,. I I

o | 6 u
,-. - . r . —\ b (s . w
Control p.25xlo C.uox10 c.8x10 9.5x10

levels of gamma radiation (rad)

fl

Fig. 2(a),(b)- Xanthine oxidase activity and percent fat
in livers of rats fed beta anc gamma irradiated wheat.

fat (dry weight)

:0
'./

/O

-47-

increasing levels of beta radiation, there is a slight
decrease from 12.2 to 9.2 micromoles of xanthine per
gram of liver per 30 minutes in the activity of

xanthine oxidase in the liver. Also accompanying this
decrease in xanthine oxidase activity was an increase in
fat deposition from 3.5 to 8.2% in the liver. (See

Table V & Figures 2a & 2b). This correlative trend was
not quite as apparent with the animals fed the gamma
irradiated diets. But both the xanthine oxidase activity
and fat deposition were different from the non-irradiated
diet. Gamma irradiation appeared to cause an initial
change with the first level of irradiation used, 0.28 x
106 rad, after which no further change occurred even

at the highest level of radiation. This trend toward
decreased activity of xanthine oxidase with increasing
levels of radiation indicates a possible alteration of
the wheat protein even at the lowest level of radiation
used, which was not apparent in the gross measurements
employed.

Carroll and Arata ('59) showed that a threonine
deficient diet caused a decrease in rat liver xanthine
oxidase activity accompanied by an increase in fat. This
significant trend disappeared the longer the animals were
on the diet due to the "adaptation" of the animals to the
diet. However it was noted that it took a longer time
for the mobilization of fat to a normal level than for
the return of xanthine oxidase activity to normal.

This adaptation theory could possibly explain the

-48-

difference between the significant fat deposition and
non-Significant xanthine oxidase activity obtained in
the present study. These animals had been on the
experimental diet for seventy days before killing and
it is quite possible that they had started to adapt to

whatever slight changes might have occurred in the

TABLE V
AVERAGE LIVER FAT AS PERCENTAGE OF DRY WEIGHT OF LIVER

 

 

TYPE OE RADIATION"
RADIATION DOSAGE (RAD) BETA G9MMA
CONTROL 6 5.5 3.5
0.28 x 106 A.8 0.8
0.93 x 106 . 5.A 6.1
2.8 x 106 5.7 6.3
9.3 x 10 8.2 6.0

 

 

 

NECESSARY DIFFERENCE FOR SIGNIFICANCE (P.05) = 1.6

 

_.__.—-

MEAN s CARE FOR ERROR
\/ N X ,/2 X t
NUMBER IN GROUP

 

wheat upon irradiation. The fact that the data showed
a slight decrease in enzyme activity and a significant
increase in fat deposition in the liver at the end of
seventy days strongly suggests that irradiation of
wheat does cause an alteration in its nutritive value
that can be detected by the animal body, although this
was not apparent when gross measurement techniques were
used.

It was found, however, that there was a lack of

-49-
correlation between the decreased enzyme activity and
increased fat deposition. This was probably due to
the large standard deviation among scores obtained for
the enzyme activities. For example the xanthine
oxidase activity at the beta radiation level of 0.28 x
106 rad was 11.9 i 5.0. If the theory of adaptation
does not apply in this instance, the increased fat
content of the liver could be explained on the basis
that this statistically significant increase in fat
was due to other than dietary protein. As.a result of
using these sensitive measurements, it was apparent
that radiation did cause a slight alteration of the
nutritive value of wheat even at the low levels of
radiation employed.

A fatty liver did oCcur from a lysine deficiency
when an 8% wheat protein diet was fed to rats (Harris
& Burress, '59). It was noted that fatty livers were
not observed in rats fed a 15% wheat protein diet.
The protein content of the experimental diet in the
present study was 14.25% (N x 5.7). The non-irradiated
diet produced a value of 3.5% liver fat whereas the diet
radiated at the highest level of beta radiation pro-
duced a value of 8.2% liver fat. The data presented by
Harris and Burress ('59) offers support to a theory
that the increased deposition of fat in the liver from
the irradiated wheat could be the result of an amino acid
imbalance; that radiation might cause an alteration in
one or more of the amino acids, thus decreasing slightly

their biological availability.

SUMMARY AND CONCLUSIONS

Sixty-four male albino rats were fed, for a
period of ten weeks, diets containing wheat that had
been irradiated with sufficiently high radiation dosages
using beta and gamma radiation, to see if the
nutritive value of the wheat protein would be changed
thereby affecting the tissues of the animals. The
radiation levels employed were 0.28 x 106 rad, 0.93 x
106 rad, 2.8 x 106 rad and 9.3 x 106 rad. The nutritive
value of the irradiated wheat was then ascertained by
employing both gross and sensitive methods of
measurement. The standard methods of measuring the
nutritive value of a protein, namely growth rate,
protein efficiency ratio, nitrogen balance, biological
value, and apparent digestibility were defined as
gross measurements. Liver xanthine oxidase activity
and percentage of fat were defined as sensitive
measurements.

The growth curves indicated that the animals fed

the 9.3 x 106

rad irradiated diet had a slower rate of
growth than the remaining animals. Only gamma radiation
at a level of 9.3 x 106 rad appeared to cause a
significant decrease in the nitrogen balance and
biological value. The types of radiation or levels of

radiation did not affect either the protein efficiency

ratio or the apparent digestibility of the diets.

-51-

There was a trend indicating that with increasing
levels of radiation there was a decrease in liver
xanthine oxidase activity. The percentage of fat
deposited in the liver was directly proportional to
the levels of beta radiation. With gamma radiation
the lowest dosage level produced approximately the
same amount of fat in the liver as that of the
highest dosage level.

Based on the gross techniques, it appears that
neither the type of radiation nor the level of
radiation affects the biological value of the wheat
protein with the exception of 9.3 x 106 rad of
gamma radiation. With the more sensitive techniques,
it would appear that the biological value of the
wheat protein is affected by each level and type of
radiation to some degree and that the effects of beta
and gamma radiation are slightly different.

A recommendation for future work then, would be
the determination of the xanthine oxidase activity and
fat deposition in the liver at specified time intervals
up to seventy days to see if the trend observed in
this experiment is more apparent in earlier stages of
the experiment and further the determination of the

amino acid content of the irradiated wheat.

-52-

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a

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-55-

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~

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APE‘EI\IDIX '

METHOD OF DETERMINING XANTHINE OXIDASE

The animals were stunned by a blow on the head,
decapitated and their livers removed. The livers

were placed immediately in cracked ice, chilled,
blotted and weighed, and then homogenized in 10 volume
of sodium potassium phosphate buffer (pH 7.4) and
strained through gauze. Five ml. of homogenate were
pipetted into two 50 m1. erlenmeyer flasks and incubahd
for forty minutes in a water bath at 37°C. At this
point 0.3ml.of buffer and 0.6m1 of water were added

to flask one and 0.3m1 of buffer and 0.6m1 of 0.038 M
xanthine to flask two. One ml. aliquots were taken

at zero time and then every 30 minutes for a total of
two hours. These samples were pipetted directly into
10 m1. volumetric flasks containing 1 m1. of 40%
sodium tungstate, 5 ml. of water, 1 m1. of 2 N H2301,

and then made to volume. The flasks were emptied

into centrifuge tubes and washed with 2 ml. of water.
The contents were centrifuged for a total of 25 minutes.
(In some cases the solution on centrifuging was
celloidal in appearance. This was overcone by filtering
the solution after carrying out the colorimetric pro-
cedure.) A 0.5 ml. aliquot was pipetted into test

tubes and 2.5 m1. of water and 1 ml. of diluted 1:1
Folin reagent added. The color was develOped by adding

5 ml. of saturated sodium carbonate solution, then

ii

filtered and read in the Bausch and Lamb Spectronic
20 calorimeter with a 660 mu filter.

3 standard curve, ranging in concentration from
14.45 micrograms to 93.94 micrograms of xanthine was
plotted. Values obtained for the control (flask I)

were subtracted from those obtained from the test

flask (flask II) to which xanthine had been added.
A plot was nade of the resulting data in which the
ordinate was the xanthine remainirg in the system.

(multiplied by a dilution factor of 120__-__ 0.455 which
was then equivalent to the micrograms of'xanthine per

gram of liver) and the abscissa, the time of incubation
in minutes. A _

The enzyme activity was calculated from the average
change in xanthine concentration over 30 minutes on
the curve. This value represented the micrograms of

xanthine disappearing per 30 minutes per gram of liver.

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.TABLE 20 - ANALYSIS OF VARIANCE : F VALUES

10:11

E VALUE
WW
‘WEIGHT GAIN .903 1.560
FEED INTAKE .903 3.690
PROTEIN EFFICIENCI RATIO -I .129 8.516 *.**
K x .. -II 1.719 0.853 *.**
NITROGEN BALANCE -I .939 A.57A *.**
, A -II 3.083 *,*# --
BIOLOGICAL VALUE -I .959 --
. ~ . - -11 3.330 *,** --
APPARENT DIGESTIEILITI -1 1.989 13.321 *.**
. . .+. -II .723 .hl?
IANTHINE OXIDASE AOTIVITI 1.2h3 .507
EAT CONTENT OF LIVER ’ 5.154 *,** .093

* Indicates statistical significance at 5% level
** Indicates statistical significance at 1% level

SOURCE a? VARIANCE
TOTAL
AMONG DIETS

AMONG GROUPS
REMA INDE R

DF.

70

f

7
1
62

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