The inhibitory effects are investigated of d,1-alpha-tocopherol and d,1-alpha-tocopheryl acetate on the free radical deterioration of cellular membranes. The level of toxicity of d,1-alpha-tocopherol and d,1-alpha-tocopheryl acetate in mice is determined

Richardson, D.

English

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FINAL TECHNICAL REPORT
Title of Grant: "Investigations of the Inhibitory Effects of Tocopheroi (Vitamin E)
on Free Radical Deterioratioa of Cellular Membranes"
"r=ust No. NSG 8017
June 1, 1974 - May 31, 1975
(NASA-CR- 146071) INVISTIGATI09S OF THE	 976-15770
INHIBITORY EFFECTS CF TOCOPHEROL (VITAMIN E)
ON FREE RADICAL DETERIORATION OF CELLULAR
MEMBRANES Final Technical Report, 1 Jun.	 Unclas
1974 - 31 bay 1975 (Oakwood Coll.) 11 p HC 63/51 08517
Dr. David Richardson
Principal Investigator
RECEWED
NASA Sn FMW
mm81m
OakNVood College
Huntsville, Alabama
35806
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Studies toward the inhibitory effects of d,l-alpha-tocopheryl acetate
on the Free Radical Deterioration of Cellular Membranes.
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ABSTRACT
Objectives:
The objectives of this research endeavor are: (1) to establish
I	 the inhibitory effects of d,l-alpha-tocopherol and d,l-alpha-
tocopheryl acetate on the free radical deterioration of cellular
membranes; and (2) the possible level of toxicity of d,l-alpha-
tocopherol and d, 1-alpha-tocopheryl acetate in mice.
« G -
Experimental:
Sixty-four mice were divided into five groups with approximately
sixteen mice in each group. Each group was again divided into sub-
groups; two cages in each group contained four three month old mice
and the third cage in each group contained approximately eight mice
which were five months old. All the mice lived in 11.5" X 5" X 7.25"
plastic cages which were cleaned daily.
The groups were again divided as follows:
Group A - received 2 grams of vitamin E (d.1-alpha-tocopheryl acetate)
per 1 gram of 170290 vitamin E deficient test diet powder.
Group B - received .8 grams of vitamin E (d,l-alpha-tocopheryl acetate)
per 1 gram of 170290 vitamin E dificient test diet powder.
Group C - received .8 grams of vitamin E (d,l-alpha-tocopherol) per
1 gram of 170290 vitamin E deficient test diet.
Group D - received 170290 vitamin E deficient test diet powder.
Control - received commercial Purina laboratory chow pellets.
Group
The mice ingested the vitamin E orally. The individual weights
of the mice were determined by averaging the weights of the entire
group and the required amount of vitamin E, agar and water were added
to powdered ARS/Sprague-Dowley 170290 vitamin E deficient test diet.
Noticeable differences in weights were compensated for by increasing
or decreasing the amount of vitamin E added to the deficient test diet.
The necessary precautions were taken to insure that the various
preparations remained pure.
The methodologies employed to measure physiological parameters
were:
J1. Heart rate and blood pressure recordings via a DMP-48
physiograph.
2. Blood clotting and hematocrits.
Discussion
Kutsky (3)
 reported that large quantities of vitamin E possibly
results in an increase in blood pressure; and work in our laboratory
supports Kutsky's statement. It was observed that mice in Groups A
and B showed systolic physiograph recordings that indicated high
blood pressure (Figure I). Figure I indicated blood pressure readings
for mice selected at random to be approximately 200 mm Hg. However,
the other groups including the control group have readings considerably
less than 200 mm Hg (Figure I). When blood pressure was compared to
blood clotting time (Figure II) it was noted that mice with high blood
pressure showed low clotting time; likewise mice with high clotting
time showed low blood pressure readings.
At present, there is not sufficient information to report on
hematocrits due to the unavailability of the necessary equipment for
this measurement.
Current literature indicates that mice on a vitamin E deficient
diet have difficulty reproducing. (4 )
	 Group D reproduced but the
offsprings were premature and never lived any longer than 24 hours.
The offsprings had a very peculiar color which was especially notice-
able in their hind legs (observations made in three different litters).
There has also been instances where mice in Group D appeared to be
pregnant (visual observations), but never completed their pregnancies.
It is believed that the fetuses were possibly re-absorbed into the
blood stream by some mechanism which was , not pursued.
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4 -
The offsprings of Group A were exceptionally large compared to
those of the other groups.
In Figure III, individual food consumption, it was noted that
mice in Group A consumed the least (excluding the control group).
Mice in Group A showed considerable weakness and developed
partial immobility.
The mice in Group C were small in size but active. Their food
consumption was lower than any other group except that of Group A.
During the blood clotting time test, blood is drawn from a vein
in the tail by a syringe. At the point where the needle punctures
the skin an infection occurs on the tail of the Group D mice.
(Illustrations not available.) If the mice in this group were injured,
the healing time is slow, even with medication. Apparently, vitamin
E deficiency has an effect on test animal's clotting mechanism and
there appears to be an inhibition to the healing process.
Studies with vitamin E at other laboratories indicated the lack
of vitamin E effects the pituitary glands, reproductive organs, kidneys,
muscles and other parts of the body. (2 & 3) Our research team has
shown that vitamin E deficiency effects the reproductive system and
the life expectancy of the test animals. Work in our laboratory appears
to indicate that large quantities of vitamin E results in high blood
pressure, muscular problems and eventually termination of life in mice.
However, at present, no conclusive evidence in experimental pro-
cedures have been employed to directly indicate that free radical
deteriorates fatty tissue. This was observed directly because of the
lack of continual support for the project and the unavailability of
of an electron spin resonance spectrometer (ESR). However, this is
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the subject of future experimentation if funds become available via
the present supporting institution in some alternate funding source.
The present research endeavor's are far from being conclusive, and
much more study in this virgin area of research must be done. From
consultation and re-examination of the scope of the research project
it was concluded that the work attempted through very meaningful was
much too broad.
Consequently, future endeavors will attempt to narrow the research
objectives to one specific problem, i.e., the mechanism of free radical
inhibition by vitamin E. This study will utilize ESR techniques for
detecting free radicals in fatty tissue.
Future Endeavors
Electron Spin Resonance (ESR). Objectives - Studies on the
inhibitory effects of d,1-alpha-tocopherol on the deterioration of
fatty tissues by ozone.
The inhibitory effects of tocopherol on free radical deterioration
of fatty tissue needs to be narrowed to some specific objective. This
narrowing would better contribute to the knowledge of free radical
destruction, in an area in which little is known concerning the mechan-
ism of radical scavenging via auto-oxidant.
Therefore, we purpose to study the electron spin resonance spectra
of fatty tissue which has been exposed to d,l-alpha-tocopherol and
ozone as compared to spectra of fatty tissue and ozone.
Samples of fatty tissues which have been exposed to ozone (in vivo)
and d,l-alpha-tocopherol extraced by butonal, will be prepared from
sacrificed mice and will be analyzed by ESR spectroscopy.
Ten groups will be studied. Fatty tissue will be extracted from
sacrificed mice in the following groups:
i
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Group A - 2 grams of vitamin E (d,l-alpha-tocopheryl acetate) per 1
gram of 170290 vitamin E deficient test diet powder.
Group B - .8 gram of vitamin E (d,l-alpha-tocopheryl acetate) per 1
gram of 170290 vitamin E deficient test diet powder.
Group C - .8 gram of vitamin E (d,l-alpha-tocopherol) per 1 gram of
170290 vitamin E deficient test diet.
Group D - 170290 vitamin E deficient test diet powder.
Cootrol - commercial Purina laboratory chow pellets.
Group
ESR Spectra of fatty tissue and the blood of these animals will
be investigated to determine any positive or negative occurances in
their spectra. From the ESR spectra it is anticipated that a mechanism
will be proposed which will possible help to delineate a mechanistic
pathway to explain the deterioration of fatty tissue by free radicals
and the inhibition of free radicals to the deterioration of fatty
tissue by tocopherol.
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Figure 1
BLOOD PRESSURE
A	 B	 C	 D	 Control
GROUPS	 I
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Figure II
BLOOD CLOTTING TIME
A	 B	 C	 0	 Control
GROUPS
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References:
1 1chiharo, Ichiro. Microscopic Studies of Anterior Pituitar Gla nds
of Vitamin E e centTe—Fiice.Nagor ya, Japan: 1969.
2Kutsky, Roman J. Handbook of Vitamins and Hormc,nes. New York;
Nastrand Rein'^o d^ompany, 1M.
3Pryor, William A.	 Chemical and Engineering News, 34, 1971.
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Investigations of the inhibitory effects of tocopherol (vitamin E) on free radical deterioration of cellular membranes

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