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LIBRARY

Michigan State
University

This is to certify that the

thesis entitled
THE RELATIONSHIP OF THE GLUTATHIONE PEROXIDASE
SYSTEM TO PHYSICAL STRESS

presented by
Paul Scott Brady

_ has been accepted towards fulfillment
of the requirements for

PhD Animal Husbandry

degree in

23mm &
Major professor

 

Date 20 Ju1y 1978

 

0—7639

 

THE RELATIONSHIP OF THE GLUTATHIONE PEROXIDASE SYSTEM TO

PHYSICAL STRESS

BY

Paul Scott Brady

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Animal Husbandry
and

Institute of Nutrition

1978

ABSTRACT

THE RELATIONSHIP OF THE GLUTATHIONE PEROXIDASE SYSTEM TO
PHYSICAL STRESS

BY

Paul Scott Brady

There are some data which suggest a relationship between
lesions of selenium (Se) and vitamin E (E) deficiency and
strenuous exercise. To investigate this phenomenon, a series
of four studies was conducted. The first two studies involved
exercising horses by running on a soft sand track, and monit—
oring blood parameters before and subsequent to exercise. In
the second of the equine studies, one—half of the horses were
provided with supplemental Se in the trace mineral salt. In
all of the equine studies, plasma enzymes and erythrocyte
malondialdehyde (MDA) rose immediately subsequent to exercise.
Both MDA and plasma enzymeshave been used as indicators of
tissue damage and peroxidation in Se/E deficiency. Parameters
of the glutathione peroxidase system were generally unrespon—
sive to exercise, with the exception of glutathione reductase
(GR). Total GR activity was consistently elevated subsequent
to exercise. The active GR activity also tended to be elevated;
however, the response was more variable. Se supplementation
had no effect on any of the parameters measured, including

gluathione peroxidase (GSH—Px) activity.

Paul Scott Brady

Thethird study was conducted with male weanling Holtz—
man rats fed torula yeast—based diets supplemented and un—
supplemented with Se and E. The rats were killed after
four weeks on the diet either prior to exercise, immed—
iately after exercise or 24 hr after exercise. Rats were
exercised by swimming to exhaustion. MDA was measured in
liver and muscle as an indicator of lipid peroxidation.
Enzymes of the glutathione peroxidase system were also
determined. Liver and muscle MDA rose subsequent to exercise
in liver and muscle among all dietary groups. Liver MDA
values returned to baseline within 24 hr subsequent to
exercise. Muscle values remained somewhat elevated. In
liver, MDA response was reduCed by dietary E, but not Se.
Muscle values were uneffected by diet. None of the glutath-
ione system enzymes responded to exercise, although Se
supplementation resulted in reduced hepatic GR and NADP-
linked dehydrogenase activities, and markedly increased GSH-
Px activity.

The forth study dealt with the response of white-
taileddeer to dietary Se and E. Adult female deer were fed
diets supplemented and unsupplemented with Se and E for two
years. Mortality of offspring was substantially increased
among deer fed diets not supplemented with E. This response
related well with in vivo and in vitro indicators of per-

oxidation among the adults. That is, while E supplementation

Paul Scott Brady
reduced mortality among the young, Se supplementation of
the adults' diet had no influence on mortality or peroxidation

at the levels used.

 

ACKNOWLEDGEMENTS

I would like to thank the members of my committee,

Drs. D.R. Romsos, W.W. Wells, G.D. Riegle and E.R. Miller,
for assistance, advice and patience. My special thanks

to DRR who put in more than his share of time listening to
unsubstantiated theories, preliminary data and hare—brained
ideas. To D.E. Ullrey, who had the hazardous duty of serving
as chairman for my program, my thanks for your understanding,
low boiling point, and friendship...

My deepest appreciation must go to my wife, Linda, who
now undoubtedly holds the record for most GSH—Px assays by
Hafeman's modification.

I wish to acknowledge the support of NIH. My tenure as
a predoctoral trainee has been both enjoyable and rewarding.

As always, with any piece of work, there are many people
who aid in the work in the course of their jobs, out of
friendship or just to experience different techniques.

I won't try to name these poor fellows...I'm almost certain
to forget someone. 80, let me take the safe route and offer
a blanket "THANK YOU" (So, who can accuse me of forgetting

anyone?).

ii

TABLE OF CONTENTS

Page
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . V
LISTOFFIGURES...................vii
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1
REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . 3
The Glutathione Peroxidase System . . . . . . . . . 3
Vitamin E . . . . . . . . . . . . . . . . . . . . . 7
Exercise and Peroxidation . . . . . . . . . . . . . 8
RAPID CHANGES IN EQUINE ERYTHROCYTE GLUTATHIONE
REDUCTASE WITH EXERCISE . . . . . . .lO
Introduction . . . . . . . . . . . . . . . . . . . .10
Materials and Methods . . . . . . . . . . . . . . .11
Results . . . . . . . . . . . . . . . . . . . . . .12
Discussion . . . . . . . . . . . . . . . . . . . . .12
LACK OF EFFECT OF SEIENIUM SUPPLEMENTATION ON THE
RESPONSE OF THE ERYTHROCYTE GLUTATHIONE SYSTEM AND
PLASMA ENZYMES TO EXERCISE . . . . . .16
Introduction . . . . . . . . . . . . . . . . . . . .16
Materials and Methods . . . . . . . . . . . . . . .16
Experiment 1 . . . . . . . . . . . . . . . . . . .16
Experiment 2 . . . . . . . . . . . . . . . . . . .17
Statistical analysis . . . . . . . . . . . . . . .18
SELENIUM, VITAMIN E AND THE RESPONSE TO SWIMMING
STRESS IN THE RAT . . . . . . . . . . .25
Introduction . . . . . . . . . . . . . . . . . . . .25
Materials and Methods . . . . . . . . . . . . . . .25

Experiment 1 . . . . . . . . . . . .
Experiment 2 . . . . . .
Blood . . . . . . . . . . . . .
Liver . . . . . .
Muscle . . . . . . . . .
Statistical Analysis . . . . . . . . . .
Results and Discussion .
THE EFFECT OF DIETARY SELENIUM AND VITAMIN E

BIOCHEMICAL PARAMETERS AND SURVIVAL OF YOUNG
WHITE—TAILED DEER (Odocoileus virginianus) .

Introduction . . . . . . . . . . . . . . .
Materials and Methods . . . . . . . . . .
Blood . . . . . . . . . . . . . .
Tissues . . . . . . . . . . . . .
In vitro hemolysis . . . . . . . . . . .
Statistical analysis . . . . . . . . . .
Results . . . . . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . .
CONCLUSIONS . . . . . . . . . . . . . . .
LITERATURE CITED . . . . . . . . . . . . . .
iv

ON
AMONG

Page
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.27
.28
.28

.28

-39
-39
39
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42
A2
A2
A3
52
50

53

 

LIST OF TABLES

Table Page
1 Blood variables in the exercised horse (n=6) . . . . l3

2 Whole blood, erythrocyte (RBC) and plasma variables
in exercised horses (Experiment 1) . . . . . . . . . l9

3 Whole blood, erythrocyte (RBC) and plasma variables
in exercised horses (Experiment 2) . . . . . . . . . 21

4 Composition of basal torula yeast—based diet . . . . 26

5 Significant linear correlations among rats fed diets
supplemented and unsupplemented with Se and E . . . 33

6 Composition of basal (low Se/E) diet for white—
tailed deer . . . . . . . . . . . . . . . . . . . . 40

7 Initial and 12 month body weight and blood variables
among adult female white-tailed deer fed diets
supplemented and unsupplemented with Se and E . . . 48

8 In vitro hemolysis among Se and E supplemented and
unsupplemented white-tailed deer after 12 months fed
the respective diets . . . . . . . . . . . . . . . . 49

9 Pooled (first and second year) mortality data for
offspring of Se and E supplemented and unsupplemented
white—tailed deer . . . . . . . . . . . . . . . . . 50

10 Body weight and blood variables among male offspring
of female white-tailed deer fed diets supplemented and
unsupplemented with Se and E, at weaning . . . . . . 51

ll Hepatic and muscle variables among adult female white—
tailed deer fed diets supplemented and unsupplemented
with Se and E . . . . . . . . . . . . . . . . . . . 53

12 Hepatic and muscle variables among male offspring of
of white-tailed deer fed diets supplemented and
unsupplemented with Se and E . . . . . . . . . . . . 54

Table Page

13 Linear relationships of Se concentration (ug/ml
plasma or ug/g tissue) and GSH-Px activity (U/mg
Hb or U/mg protein), where GSH—Px = bSe + a, among
white-tailed deer adults and offspring . . . . . . 55

 

vi

LIST OF FIGURES

Figure Page
1 The glutathione peroxidase system . . . . . . . . 4
2 Erythrocyte glutathione reductase (active) activity

as a function of time after exercise. Each point is

the mean of 8 observations: 0 = initial values; a =
after 2 weeks fed diets; A = after 4 weeks fed
diets . . . . . . . . . . . . . . . . . . . . . . 22

Erythrocyte enzymes as a function of time after
exercise. Number per mean is given in parentheses:
o = -Se-E; A = -Se+E; o = +Se—E; A = +Se+E . . . 30

Hepatic enzyme activities. Number per mean is given
in parentheses. Stippled bar represents —SeiE diets.
The error bars are SEM. NS = no significant effect

of dietary Se; * = P<0.05; ** = P<0.01 . . . . . 32

Hepatic fat soluble antioxidant concentration as a
function of time after exercise. Numbers per mean
are per Fig. 3. o = —Se-E; A = -Se+E; o = =Se—E;

A = +Se+E . . . . . . . . . . . . . . . . . . . 5

Hepatic TBARS (MDA) concentration as a function of
time after exercise. Each value is the mean of 4
observations. 0 = -Se-E; A = -Se+E; o = +Se-E;

A = +Se+E . . . . . . . . . . . . . . . . . . . 36

Muscle TBARS (MDA) concentration as a function of
time after exercise. Numbers per mean are per Fig.

3. o=-Se-E;A=-Se+E;o=+Se—E;A=+Se+E. . 7
Plasma selenium among white-tailed deer as a
function of months fed diets. o = —Se:E; A =
+SeiE......................45
Plasma a—tocopherol among white-tailed deer as

a function of months fed diets. o = iSe—E;

A = iSe+E . . . . . . . . . . . . . . . . . . . 47

 

INTRODUCTION

Lesions of selenium-vitamin E deficiency have been
reported in a number of species subsequent to physical
exertion (Young & Keeler, 1962; Young et al, 1975; Ullrey,
1973; Harthoorn & Young, 1974; Muth, 1963). Degeneration
of the muscle fibers, or "white muscle disease", is the
most common lesion, although liver and kidney necrosis may
occur (Harthoorn & Young, 1974). Muscle lesions are
generally symmetrical; however, Young and Keeler (l962)
produced asymmetrical lesions in lambs by restraining one
limb. Lesions in the restrained limb were absent or greatly
reduced. Thus, it appears that physical exertion may
contribute to the development of selenium-vitamin E def-
iciency lesions at least in muscle.

The metabolic roles of selenium (Se) and vitamin E
(E) are now reasonably well defined. This represents
recent work. Still, with the growing knowledge of the
function of these nutrients, the present series of studies
represents an attempt to relate the known metabOlic effects
of Se and E to the phenomenon of stress-induced deficiency
signs. The horse, rat and white-tailed deer were used in
these studies. Hartley and Grant (1961) and Hill (l963)
have suggested that various exercise myopathies of the
horse may be responsive to Se or E. No such suggestions
have been made for the rat. Yet, unlike the horse, this

species is inexpensive as a laboratory animal and conditions

are more easily controlled. The white-tailed deer like the
horse appears more sensitive to Se or E deficiency in the

face of physical stress.

 

REVIEW OF LITERATURE

Early studies demonstrated that dietary Se and B were
mutually sparing (Calvert et a1, 1962; Schwarz & Foltz,
1957). At least one definable function of E was as an
antioxidant (Green, 1972). Primarily on this basis, an
antioxidant function was also postulated for Se. It was
not until 1972 that Rotruck and coworkers (1972) demon-
strated the mode of Se's antioxidant effect. Their finding,
that Se is an essential component of glutathione peroxidase
(EC 1.11.1.9), served to confirm not only the antioxidant
role of Se, but also that the role of E was also as an
antioxidant.

The Glutathione Peroxidase System. Glutathione per—

 

oxidase and glutathione reductase (EC 1.6.4.2) serve as the
basis of an enzyme system for the reduction of various
peroxides. The peroxidase (GSH—PX) will reduce a wide

range of lipid peroxides (Christopherson, 1968 & 1969;
Flohe et a1, 1974), hydrogen perox1de (Cohen & Hochstein,
1963) and, perhaps, hwhoxyl free radical (McCay et al,
1976). The basic system is presented in figure 1. Electrons
are transferred via the GSH-Px from reduced glutathione
(GSH) to the peroxide. The enzyme is quite specific in its
requirement for GSH (Flohe et a1, 1974). Two moles of GSH
are conjugated to form one mole of oxidized glutathione

(GSSG). The glutathione reductase (GR) then catalyzes the

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Figure 1. The glutathione peroxidase system.

5

reduction of GSSG using NADPH for the reducing equivalents
(Beutler & Yeh, 1963; Rieber et al, 1968). However, GSH

may serve functions other than peroxide reduction. GSH
serves as a reservoir for cysteine in rat liver (Tateishi
et a1, 1977) and functions in amino acid transport (Meister,
1976). Thus, to consider GSH and GR solely committed to

the process of peroxide detoxication is an oversimplifica—
tion.

In the mature erythrocyte, NADPH for the GR is provided
solely via the hexose monophosphate shunt. NADPH competi-
tively inhibits glucose-6-phosphate dehydrogenase, and
provides a major control of glucose flux through this path-
way. However, Jacob and Jandl (1966) working with the ery-
throcyte model and Eggleston and Krebs (1974) working with
rat liver showed that GSSG decreased inhibition of glucose-
6-phosphate dehydrogenase (G6PD) by NADPH. Therefore, not
only does the ratio of NADPH/NADP modulate flux through
the hexose pathway, but also the ratio of GSSG/GSH.

Because the erythrocyte glutathione peroxidase system
has been extensively studied, a tendency has developed to
assume the hexose shunt to be the major source of reducing
equivalents for this system in all tissues (Chow & Tappel,
1974). Stark et a1 (1975) have suggested that under certain
dietary regimens malic enzyme may serve as the primary
source of reducing equivalents for GR.

In the erythrocyte, then, G6PD may be thought to be

linked directly to the glutathione pathway. A source of

6
glucose is essential for the erythrocyte to maintain NADPH
concentration. Rotruck et a1 (1971) found that dietary Se
protected RBC from in vitro hemolysis only when glucose was
included in the incubation medium. Conversely, G6PD defic—
iency, a genetic anomaly extensively studied in man, results
in hemolysis only upon oxidant stress (Beutler, 1972).

GR require an FAD (flavin adenine dinucleotide) co-
factor; indeed, GR activity has been used as an indicator
of riboflavin status (Glatzle et a1, 1970). Riboflavin
deficiency may lead to a moderate normocytic anemia in
swine (Wintrobeet a1, 1944; Brady et al, 1978). This
anemia is probably not associated with impairment of the
glutathione peroxidase system, however. Beutler (1974) has
reported that reduction of GR activity by 50% did not
influence GSH levels.In fact, GR activity may fall by
75% in the swine erythrocyte without decrease in the level
of GSH (Brady et a1, 1978). Both increased activity of GR
(Gaetani et a1, 1973) and enhanced binding of FAD (Flatz,
1970) has been reported among G6PD-deficient erythrocytes.
Erythrocyte GR and G6PD activities may also snow a close
relationship, even where overt deficiency of one enzyme
or the other is not involved (Brady et al, 1978b).

The GSH which GR spends its time trying to keep redu—
ced, is a simple tripeptide (y-glutamylcysteinylglycine).
Its distribution is ubiquitous (Beutler, 1974). GSH is

synthesized in two steps:

y-glutamylcysteine
synthetase

 

l) glutamate + cysteine y—glutamyl—

cysteine,

 

 

7
2) y-glutamylcysteine + glycine + ATP lutathione GSH.

synthetase
This process is caried out in all tissues including
erythrocytes (Beutler, 1975). Turnover of GSH is very
rapid; in the mature erythrocyte, half-life is about four
days (Dimant et a1, 1955). The erythrocyte concentration
of GSH is also quite high, about 2mM in most species
(Beutler et al, 1955). It has been calculated that the
erythrocyte could replace its store of GSH within minutes,
based on the Vmax for the synthesizing enzymes (Williams et
a1, 1975). Why then is GR needed? There are two probable
reasons. First, the process of continual synthesis of
GSH would prove costly in terms of substrate consumption.
Further, GSSG is quite toxic to the cell (Srivastava &
Beutler, 1975). It is important for the erythrocyte and
other cells to keep intracellular GSSG concentrations
low. In addition to reducing GSSG via GR, cells will
actively dump GSSG to the extracellular space (Srivastava
& Beutler, 1975; Chance et a1, 1977).

Vitamin E. Vitamin E, while not directly linked to
the glutathione peroxidase system, does serve a parallel
function as a free radical quenching agent (Tappel, 1970;
Urano et a1, 1977). It terminates the free radical perpet-
uation of peroxidation. However, E is lipid soluble, while
GSH-PX and associated enzymes and metabolites artawater soluble.
This has caused McCay et a1 (1976) to suggest that the
peroxide on which E and GSH—Px act is not the same. This

is still a question of active interest.

8

Exercise and Peroxidation. The evidence linking

 

exercise and the generation of peroxides is minimal. To
begin, lesions of Se/E deficiency have been reported subse-
quent to exercise in a broad range of species (Harthoorn
& Young, 1974; Young et al, 1975; Ullrey, 1973; Muth, 1963).
A number of these reports have suggested that dietary Se
and E were adequate, prior to the production of the stress-
induced lesions (Young et a1, 1975; Ullrey, 1973; Harthoorn
& Young, 1974). The problem with these reports is that the
lesions produced by Se/E deficiency are not pathognomonic.
The white muscle disease, hepatic necrosis, etc. are
suggestive of a deficiency of one or both of these nutrients
but do not provide conclusive proof of such a deficiency.
Elevation of various plasma enzymes has been shown
to occur with Se/E deficiency (Olson, 1974; VanVleet, 1975;
Whanger et a1, 1977). Such elevations are used as indicators
of preclinical deficiency, and are thought to reflect mem-
brane damage with subsequent leaking of the enzymes into the
plasma. Similarly, exercise may result in transient, but
very substantial, increases in plasma enzymes (Cardinet
et al, 1963; Milne et a1, 1976; Thompson, 1962; King et
al, 1976). Still, it is unclear whether the apparent
change in membrane permeability associated with muscular
exertion occurs via the same process as that found with Se/
E deficiency.
There are very limited data relating exercise to

lesions of nutritional muscular dystrophy or to oxidative

 

attack. Young and Keeler (1962) were able to produce
asymmetrical muscle lesions by restraining one limb of
lambs on dystrophogenic diets. These workers did not
determine Se or E content of their diets. Stokinger
(1963) produced ozone toxicity at reduced ozone levels
(subtoxic) in rats by exercising the animals intermit—
tently during exposure. "March hemoglobinuria", a hemo—
lytic disorder brought on by exercise (hence,."march"),
has been shown to be the result of a genetic deficiency

of erythrocyte GSH-PX (Bernard et al, 1975).

The sum of these findings suggest that Se/E deficiency

may be exacerbated by exercise. Two questions remain:

what is the effect of Se/E supplementation on the response

to exercise and 2) can strenuous exercise induce lesions

(biochemical or otherwise) of Se/E deficiency where these

nutrientswould otherwise be present in the diet in ade-

quate concentration to prevent deficiency?

RAPID CHANGES IN EQUINE ERYTHROCYTE GLUTATHIONE

REDUCTASE WITH EXERCISE

§NTRODUCTION. The erythrocyte glutathione per-

 

oxidase system has been presented (fig.l). Considerable
interest has centered on this pathway with the identif-
ication of Se as a component of GSH-PX (Rotruck et al,
1972). This system is of interest because lesions of Se/
ISdeficienC$.including muscle degeneration (white muscle
disease) and hepatic necrosis, have been reported in a
wide range of species subsequent to exercise, as discussed
previously. Because the only known role for Se is as a
component of GSH-PX, it seems likely that lesions in animals
subjected to exercise might be related to insufficient GSH
relative to the rate of peroxidation.

Work has been done with the production of muscle
lesions by running zebra, a relatively close relative
of the domestic horse, to exhaustion (Harthoorn & Young,
1974). Further, exercise-induced myopathies of the
domestic horse (azoturia, myositis, etc.) have been reported
(Siegmund, 1973). In fact, it has been suggested that the
equine myopathies respond favorably to Se supplementation
(Siegmund, 1973; Hartley & Grant, 1961).

The response of the erythrocyte system was determined
10

11

in the hOpe of defining a metabolic limiting factor to GSH
availability in this biochemically simple tissue; however,
in view of the ubiquitous distribution of this system, the
erythrocyte might in many respects serve as a reasonable

model for other tissues.

 

MATERIALS AND METHODS. Six mature horses (5 Quarter—
horses and 1 Arabian) were used. These animals had been
occasionally exercised, but had not been regularly trained.
Horses were run on a circular sand track until they
refused to maintain their pace. An average of 2 km in
7.4 min was run before this point. Heparinized jugular
blood samples were taken before (base line), immediately
after and 1 hr after exercise.

Blood GSH was determined by the method of Beutler
et a1 (1963), with the modification that blood was pre—
cipitated directly, without prior hemolysis, to reduce
oxidation. Blood lactate (Hohorst, 1963) and pyruvate
(Bucher et al, 1963) were determined enzymatically. The
hematocrit (packed cell volume) and hemoglobin (Crosby et
al, 1954) were also determined.

Erythrocytes were washed twice with cold isotonic
saline (0.9% NaCl) and lysed with cold distilled water.
Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.44)
(Langdon, 1966), GSH-PX (Hafeman et a1, 1974), and active
and total GR (Sauberlich et al, 1972) were determined at
37°.

Data were analyzed by analysis of variance using a

 

12

randomized complete block design. Where a significant
(P<0.05) exercise effect was found, data were further
analyzed by Tukey's w-procedure (Steel & Torrie, 1960).

RESULTS. The data are presented in table 1. Hemo-
globin (Hb) and hematocrit (PCV) were markedly increased
immediately after horses were exercised, but returned to
base line within one hr after exercise. The mean corpuscular
hemoglobin concentration (MCHC) remained unchanged.

Lactate and pyruvate also increased significantly
immediately after exercise with return to near base line
values after one hr. The ratio of 1actate:pyruvate was
increased with exercise. GSH/d1 blood was also increased
with exercise. However, GSH/d1 erythrocyte did not change
with exercise. GSH-Px and G6PD activities were not affected
by treatment.

GR did exhibit an exercise effect. Total GR increased

by over 30% immediately after exercise. Within one hr after
exercise total GR had not declined significantly. Active GR
also increased immediately after exercise and remained unch-
anged one hr after exercise. The percent of total GR that
was active actually was depressed immediately subsequent to
exercise, increasing somewhat by one hr after exercise.

DISCUSSION. Changes in Hb and PCV can be attributed

 

to splenic release of erythrocytes with exercise (Turner &
Hodgetts, 1959). The MCHC remained constant, as would be
expected were spleen release involved. Splenic storage of

erythrocytes is a dynamic process (Turner & Hodgetts, 1959).

13

Table 1. Blood variables in the exercised horse (n=6).

 

Base line (before Immediately after At 1 hour

 

 

Variable . . . SEM
exerClse) exerc1se after exerCise

Hemoglobin

(g/dl of blood) 11.7a 15.8b 11.2a 0.2
Hematocrit (a) 35.0a 47.1b 33.0a 0.7
Mean corpuscular

hemoglobin

concentration (%) 33.3 33.7 33.6 0.4
Reduced glutathione b

(mg/d1 of blood) 15.4a 23.6 17.3a 1.6
(mg/dl of RBC) 43.5 50.2 52.3 3.7
Lactate (mg/d1

of blood) 3.0a 26.0b 5.7a 3.6
Pyruvate (mg/d1

of blood) 0.8a 1.4b 0.8a 0.1
Lactate/pyruvate

(molar ratio) 3.6a 18.2b 6.7a 2.3
Glutathione peroxidase

(-AloglGSH '103-min'1-mg

hemoglobin'l) 19.8 17.8 16.8 1.4
Glucose—6-phosphate

dehydrogenase (IU/g

hemoglobin) 6.3 6.1 6.5 0.3
Glutathione reductase

Total (IU/g of ab

hemoglobin) 1.3a 1.8b 1.6 0.1
Active (IU/g of a
hemoglobin) 1.1 1.2b 1.2b 0.04
Percent active 81.0a 66.6b 72.6C 1.4

 

a-CMeans with different superscripts are significantly (P<0.05) different.

SEM = standard error of the mean; this value is generated via the analysis

of variance.

14

Because the stored cells are constantly being exchanged
with circulating cells, changes in GR activity should not
be due to release of younger cells. While G6PD activity has
been shown to decrease with increasing cell age, GR is not
influenced by the age of the erythrocyte (Oski, 1970). For
these reasons, it is likely that the increase in total GR
was due to activation of a pre-existing inactive form of
the enzyme.

Increased GR activity has been reported in the G6PD
deficient erythrocyte (Gaetani et al, 1973). It has been
suggested that synthesis of GR is increased in response to
inadequate NADPH. In the present study, the time was too
short to allow for protein synthesis, even if it were assum—
ed that a large pOpulation of reticulocytes were released
into the circulation (for which evidence is also lacking).
There are several problems with the assumption that NADPH
supplies had become limiting and that latent GR was act-
ivated in response. For one, erythrocyte G6PD is over 90%
inhibited by NADPH under most conditions (Holten et a1,
1976). If NADPH supplies declined, inhibition of G6PD would
be removed and the rate of NADP reduction increased. Further,
NADPH inhibition of G6PD is removed by GSSG (Jacob & Jandl,
1966).

Increased GR activity in G6PD deficiency has been
linked to concentrations of NADPH below the GR Km for NADPH
(Gaetani et al, 1973). Changes in the cell membrane assoc-

iated with early stages of peroxidation may modulate GR

15

activity. GR activity has been increased in vitro by
incubation of lysate with stroma at 50°. It is possible
that GR exists in an inactive, membrane-bound form and
in an active soluble form. Such a situation exists for
human erythrocyte glyceraldehyde-3-phosphate dehydro-
genase (McDaniel & Kirtley, 1974).

The majority of variables associated with the
GSH-Px system showed no response to exercise in the
erythrocyte. While blood GSH increased, GSH is known to
be restricted to the erythrocyte (Ray & Prescott, 1975).

GSH/d1 erythrocyte did not increase with exercise.

 

 

 

LACK OF EFFECT OF SELENIUM SUPPLEMENTATION ON THE RESPONSE
OF THE EQUINE ERYTHROCYTE GLUTATHIONE SYSTEM AND PLASMA

ENZYMES TO EXERCISE

INTRODUCTION. As mentioned in the previous study,

 

Hartley and Grant (1961) and Hill (1963) have suggested that
various exercise myopathies of the horse may be Se/E
responsive. The previous study demonstrated that GR increased
rapidly subsequent to exercise, while the other measured com-
ponents of the glutathione peroxidase system were not
altered. The present study was designed to determine 1)

if exercise, like Se/E deficiency, might result in increased
peroxidation and 2) if Se supplementation could alter this

response.

MATERIALS AND METHODS

 

Experiment 1. Six adult Quarterhorses (ave. wt. 510kg) were

 

run on a soft sand track an average of 2.2 km in 10 min.
Heparinized venous blood samples were taken before (base
line), immediately after, one hr after and 24 hrs after
exercise.

Aliquots were deproteinized immediately for GSH
determination (Beutler et a1, 1963, as modified previously),
lactate (Gutman & Wahlefeld, 1974) and pyruvate (Czok &

Lamprecht, 1974). Hb and PCV were determined as before.
16

 

17

Erythrocytes and plasma were separated, washed and lysed
as before. In addition an aliquot of erythrocytes was used
for determination of malondialdehyde (MDA), an indicator of
lipid peroxidation (Placer et a1, 1966), after Mengel et a1
(1967).
The lysed erythrocytes were used for determination
of G6PD (Kornberg & Horecker, 1955), 6-phosphogluconate
dehydrogenase (6PGD; EC 1.1.1.49)(Horecker & Smyrniotis,
1955), and total and active GR, as described before. All
activities were assayed at 370 and expressed per g Hb.
Plasma was assayed for glutamic oxalacetic trans-
aminase activity (GOT; EC 2.6.1.1)(Sigma Tech. Bull. No.
505, Sigma Chemical Co., St.Louis). Plasma Se was deter-
mined after Whetter and Ullrey (1978).

Experiment 2. Eight adult horses (4 Arabians and

 

4 Quarterhorses; ave. wt. 528 kg) were assigned to two
dietary treatments. Both groups were fed on pasture,
essentially ad libitum, plus 450 g/ day cornzoat concentrate
(1:1, by volume). The concentrate was supplemented with
30g trace mineral salt daily. This amount of concentrate
plus salt was readily consumed. The salt was either
supplemented or unsupplemented with 60ppm Se as sodium
selenite, calculated to provide 0.15ppm supplemental Se
daily on a whole diet basis. Blood samples were taken
before and after exercise, as in the previous experiment,
at 0, 2 and 4 weeks on the dietary regimens. PCV, Hb, GSH,

MDA and erythrocyte enzymes were determined as in Experiment

l8
1. In addition, erythrocyte GSH-PX was determined (Hafeman
et al, 1974).

Plasma GOT, creatine phosphokinase (CPK; EC 2.7.3.2)
(Sigma Tech. Bull. No. 520), alkaline phosphgtase (AP; EC
3.1.3.1 )(Sigma Tech. Bull. No. 104) and lactate dehydro—
genase (LDH; EC 1.1.1.28)(Sigma Tech. Bull. No. 500). Retic—
ulocyte counts were made at week 4 (Ham, 1956). Plasma,
forage, trace mineral salt, corn and cats were analyzed for
Se (Whetter & Ullrey, 1978).

Statistical analysis. Data were analyzed by analysis

 

of variance using a split plot design for Experiment 1 and
a split-split plot design for Experiment 2 (Steel & Torrie,
1960).

RESULTS AND DISCUSSION. Data from the first experiment

 

are presented in Table 2. Both Hb and PCV rose immediately
after exercise, but returned to base line within 1 hr. This
response was similar to the previous study and is consistent
with splenic release of erythrocytes (Persson & Bergsten,
1976). MDA and plasma GOT showed a similar response, rising
immediately after exercise and returning to base line within
1 hr.

Blood lactate and pyruvate were also highest im-
mediately after exercise, returning to base line within
24 hrs. Similar increases have been reported previously
(Milne et al, 1976). Erythrocyte G6PD and 6PGD activities
did not exhibit any immediate effect of exercise; however,
G6PD activity doubled between 1 and 24 hrs after exercise.

Total and active GR rose immediately after exercise and

19

Table 2. Whole blood, erythrocyte (RBC) and plasma variables in exercised
horses (Experiment 1).

 

Hours after exercise

 

 

 

variable Base line 0 l 24 SEM
Whole blood
Hemoglobin
(g/dl of blood) 13.4 17.3 13.1 14.0 0.5**
Hematocrit (%) 35.3 48.2 33.1 35.1 l.l**
Mean corpuscular hemoglobin
concentration (%) 38.0 35.9 39.6 39.8 1.6ns
Lactate (umol/ml) 0.8 3.4 1.6 0.8 0.3**
Pyruvate (umol/dl) 6.6 11.9 7.8 7.0 1.1**
Egg
MDA (10A/m1) 7.6 14.9 8.3 7.8 1.5*
GSH (umol/ml) 1.4 1.5 1.9 1.8 0.1*
G6PD (IU/g Hb) 0.9 0.9 0.9 2.1 O.2**
6PGD (IU/g Hb) 0.3 0.3 0.3 0.3 0.04ns
Total GR (IU/g Hb) 3.9 5.2 4.7 4.6 0.3*
Active GR (% of
total) 103 100 100 98 2.3nS
Plasma
GOT (S-F U/ml) 165 184 164 NA 2**

 

Each value is the mean of 6 animals.
SEM = standard error of the mean; ns
:_0.05; * = P<0.05; ** = P<0.01).

NA = not available.

= no significant exercise effect (P

 

20

fell toward base line within 24 hrs, basically the same
response as in the first equine study. However, in the pre-
sent study, the percent active GR remained constant at about
100%. Plasma Se was found to be 0.16 i 0.02 ug/ml. Stowe
(1967) has reported very similar values for Se-adequate
horsefiin Kentucky.

The data for the second experiment are presented in
Table 3. None of the measured parameters showed significant
effects of Se, weeks-fed-diet or Se x exercise interaction;
therefore, values were pooled to demonstrate the exercise
effects. Only GR showed a significant effect of weeks-fed-
diet x exercise interaction (fig. 2). While GR activity was
always elevated immediately after exercise, subsequent
response varied by week of sampling. Active GR was essential-
ly equal to total at all points during this experiment. The
reason for the 15 to 30% increase in erythrocyte GR act-
ivity immediately after exercise remains unclear. However,
when reticulocytes were determined at week 4, no increase
was seen with exercise. This would suggest that the erythro-
cyte age profile, which might alter enzyme activity, was
not altered with exercise.

No attempt was made to separate G6PD and 6PGD act-
ivities in this experiment. Pooled activity corresponded
well with G6PD + 6PGD activities from EXperiment 1; however,
the increase in G6PD at 24 hrs was not seen.

Notably, erythrocyte GSH-PX, which requires Se for

activity, showed no effect of supplemental Se, although a

21

Table 3. Whole blood, erythrocyte (RBC) and plasma values in exercised
horses (Experiment 2).

 

Hours after exercise

 

 

 

 

Variable Base line 0 1 24 SEM
Whole blood

Hematocrit (%) 36.5 46.8 35.3 36.1 1.0**
Reticulocytes/103 RBC 7.9 7.9 10.9 9.3 1.4ns
Egg

MDA (10A/m1) 7.2 8.4 7.8 7.6 0.2ns
GSH (umol/ml) 2.2 2.1 2.2 2.0 0.1ns
G6PD + 6PGD (IU/g Hb) 1.7 1.4 1.3 1.3 0.2nS
GSH—PX (U/mg Hb) 22.1 20.8 19.5 17.4 0.9**
Plasma

AP (Bessey-Lowry U/ml) 5.0 5.6 5.1 5.1 0.1**

CPK (Sigma U/ml) 11.2 13.3 11.6 11.8 0.4*

GOT (Sigma-Frankel U/ml) 168 189 173 170 4**

LDH (Berger-Broida U/ml) 725 800 733 717 27**

 

Each value is the mean of 24 observations with the exception of retic-
ulocytes which are means for 8 animals taken at week 4.
SEM = standard error of the mean; ns = not significant; * and ** indicate

significance at the 0.05 and 0.01 levels, respectively.

’

 

 

 

n
Iii
7i

 

 

 

3‘.— 1

J1

 

0.
o

ugqolboweq b/n1 ‘asolonpal euogqlolnlg

Figure 2.

Erythrocyte glutathione reductase (active)
Each

as a function of time after exercise.
point is the mean of 8 observations. 0
initial values;¢: = after 2 weeks fed diets;

after 4 weeks fed diets.

I,

 

Hours after exercise

 

Baseline

 

23

slight decline in activity after exercise was noted. Further,
plasma Se was not altered by Se supplementation of the diet.
Mean (1 SEM) plasma Se was 0.15 i 0.004 ug/ml. These data
again suggest that dietary Se was adequate prior to sup—
plementation (Stowe, 1967). Michigan feeds are reported
to be low in Se (Ku et al, 1972) and analysis of the feeds
available to these animals would support this (corn, oats
and forage contained 0.035, 0.025 and 0.030 ppm Se, resp-
ectively). Whether the horses were able to maintain ade-
quate Se stores because of a unique feature of their
physiology or because of an unidentified source of Se was
available to them is unclear. Horses had been maintained
in Michigan on essentially this diet for at least 12
months. While overt Se deficiency has not been unequivocally
demonstrated in the horse, Stowe (1967) reduced plasma Se
to 0.035 ppm on semi-purified diets on a torula yeast—
based diet.

As in the previous study, plasma enzyme activity (AP,
CPK, GOT and LDH) as well as erythrocyte MDA increased after
exercise. Stowe (1967) has associated increased plasma GOT
with subclinical Se deficiency in the horse. Further,
Cardinet et a1 (1963) have shown a relationship between
large increases in plasma CPK and the probability that a
horse will "tie—up", a syndrome characterized by stiffness,
difficulty of movement, swelling of legs and myoglobinuria.
Increased plasma LDH, GOT and CPK have been reported in

many species after exercise (Milne et al, 1976; Thompson,

 

 

24

1962; King et al, 1976), and with Se/E deficiency (Olson,
1974; VanVleet, 1975; Hayes et a1, 1969; Whanger et al,
1977; 1977b). Increased plasma enzymes are associated with
increased membrane permeability or destruction (Cardinet
et al, 1963; Thompson, 1962).

Increased MDA has been reported with Se deficiency
(Whanger et a1, 1976; Noguchi et a1, 1973; McCay et al,
1976) and is thought to reflect tissue peroxidation (Placer
et a1, 1966). If the basic process resulting in increased
plasma enzymes and erythrocyte MDA with exercise was re-
lated to peroxidation of muscle and other tissues, sup-
plementation of the diet with Se did not alter this response.
Since Se supplementation did not result in increased plasma
Se or erythrocyte GSH-PX, it appears that the horses in these
experiments were adequate in Se, even though identified
dietary sources were quite low in Se. The possibility remains
for a different response to Se and exercise in Se—deficient

animals.

SELENIUM, VITAMIN E AND THE RESPONSE TO SWIMMING STRESS

IN THE RAT

INTRODUCTION. To this point, data have been presented

 

which suggest that exercise does result in increased per-
oxidation, at least in the erythrocyte. Increased plasma
enzymes might indicate damage to various tissue membranes.
In the Se-adequate horse, Se supplementation does little to
alter the response to exercise. The present study with the
laboratory rat was undertaken to 1) determine if perox—
idation could be measured in liver and muscle subsequent to
exercise and 2) investigate this response in Se/E deficient-
as well as adequate-animals.

MATERIALS AND METHODS

 

Experiment 1. Eighty male weanling Holtzman rats

 

(The Holtzman Co., Madison, WI), ave. initial wt. 66 i lg
(Mean 1 SEM), were assigned to four torula yeast-based

diets (Table 4): 1) unsupplemented with Se or E (-Se-E);

2) supplemented with 0.5 ppm Se as sodium selenite (+Se-E); 3)
supplemented with 50 ppm E as Dgfd-tocopheryl acetate

(-Se+E); or 4) supplemented at the above levels with both

Se and E (+Se+E). Diets and water were provided to
individually housed rats for four weeks, a point just prior

to the time when mortality might be expected on this diet
25

26

Table 4. Composition of basal Torula yeast-based diet.

 

 

Ingredient Percent of diet
Torula yeast ’ 30.0
Sucrose 55.7
Lard (Vitamin E—free) 5.0
Cod liver oil 3.0
Salt mix1 5.0
Vitamin mix2 1.0
DL-methionine 0.3

 

100.0

 

lSalt mix (g/kg): CaC03, 543.0; MgC03, 25.0; MgSOt, 16.0;

NaCl, 69.0; KCl, 112.0; KHZPOt, 212.0; ferric ammonium
citrate, 20.5; KI, 0.08; MnSOi, 0.35; NaF, 1.0; A1K(so.)2o
12H20, 0.17; CuSOt, 0.9.
2Vitamin mix (per 1009): glucose monohydrate, 88.58 g;
thiamin-HCl, 40 mg; riboflavin, 25 mg; pyridoxineoHCl,
20 mg; calcium-ngpantothenate, 200 mg; choline chloride,
10 g; niacin, l g; menaquinone, 10 mg; folic acid, 20 mg;
biotin, 10 mg; vitamin B12 triturate (0.1% B12 in mannitol),
100 mg; retinyl acetate, to provide 2500 IU/lOOg diet;
ergocalciferol, to provide 200 IU/lOOg diet.

 

27
with this strain of rat (Hafeman et al, 1974). At the end

of four weeks, the rats were assigned to three exercise
groups: an unexercised control (base line), which was
decapitated at the same time as exercised rats; a second
group, exercised by swimming to exhaustion and killed im-
mediately by decapitation; and a third group, exercised as
before, but killed 24 hrs later. Rats were swum in groups of
five to six, with 2% of their body weight in lead weight

attached to their tails, until exhausted, a period of

 

10 to 15 min. All animals were killed within a three hr
span of time on three consecutive days. Blood, liver and
left hind limb were taken for analysis.

Experiment 2. Forty-eight male weanling Holtzman rats

 

were assigned to diet and exercise groups as in the previous
experiment. Only liver was taken in this study.

Blggd. Heparinized blood samples were collected on
decapitation (Experiment 1 only) and assayed for GSH.
PCV was used to correct this value to an erythrocyte basis.
Washed erythrocytes were prepared as in previous studies
and assayed at 370 for GSH-PX, G6PD, active and total GR
activities as before. All activities were expressed per
unit Hb.

£1325. Liver was removed, washed and homogenized in
0.15M KCl. Cytosol was prepared by centrifugation at
48,000 x g for 90 min. Hepatic GSH-Px, active GR (which is
equal to total in this tissue) and G6PD activities were
determined as above. In addition, "malic enzyme" (ME;

EC 1.1.1.40) and NADP-isocitrate dehydrogenase (ICDH;

 

28

EC 1.1.1.42) activities were assayed (Frenkel et al, 1972).
A11 hepatic enzyme activities were expressed per unit
protein (Lowry et a1, 1951).

Hepatic fat soluble antioxidant (FSAO) concentration
was determined using an d-tocopherol standard (Glavind,
1963). FSAO was expressed as ug d-tocopherol equivalents
per g liver, wet weight. Hepatic MDA was determined in
the second experiment and expressed as a relative index
of absorbance at 532nm per g liver, wet weight (Placer
et al, 1966).

Muscle. Left hind limb was assayed for MDA, GSH—PX
and active GR as above. As for hepatic GR, muscle GR showed
complete saturation with FAD.

Statistical analysis. Data were analysed as a 2 x 2 x

 

3 factorial design with unequal replications. Linear cor-
relations of the various enzymes were also run (Gill, 1978).
The level required for statistical significance was set as
P<0.05.

RESULTS AND DISCUSSION. Weight gain was not altered
by diet. Animals gained approximately 95 g or an average
of 3.8 g/day. While a longer period fed Se or E deficient
diets might be expected to depress gain, a lack of diet—
ary effect in a period as short as these experiments would
be anticipated (Hafeman et a1, 1974).

Erythocyte GSH concentration was not altered by diet.
However, exercise resulted in a slight but significant
decline, from 2.3 : 0.2mM at base line to 2.0 : 0.2mM

at 0 and 24 hrs after exercise. This observation is in

29

contrast to the horse, where GSH is unchanged or slightly
elevated subsequent to exercise. RBC enzymes are presented
(fig. 3). Active and total GR activities fell subsequent to
exercise. Since the percent active GR remained constant with
exercise, only active GR is presented. G6PD activity also
fell following exercise. GR and G6PD activities were directly
correlated (table 5). A probable reason for the concomittant
decline in activities of both enzymes could be change in the
erythrocyte age profile toward older cells (Oski, 1970).

G6PD activity is known to decline with increasing cell age.

 

Such an age dependency has not been reported for GR.
Neither of these enzymes showed a response to dietary Se
or E.

RBC active GR as a percent of total was 95% or greater
among —Se-E, +Se-E and -Se+E rats, but only 86% among rats
fed the +Se+E diet. Although percent active GR does respond
to G6PD activity (Flatz, 1970; Ajmar et a1, 1972; Brady et
al, 1978), the relationship of Se and E to FAD binding of
this enzyme has not been previously investigated. Indeed,
the mechanism for alteration in GR affinity for FAD is not
known.

Erythrocyte GSH-Px activity did, of course, respond to
dietary Se (but not to dietary E). Values were quite com-
parable to those reported for this strain of rat after four
weeks fed this diet (Hafeman et a1, 1974). Unlike erythro—
cyte GR and G6PD, erythrocyte GSH-Px activity was not af—

fected by exercise. Equine GSH—PX may show a slight decline

 

30

 

 

 

 

 

 

Glucose-G— Phosphate Dehydrogenose
,5 SEM=I.6
.0
2 20 (7)
c» .
O
E
a)
.c I 5
8’
2
Lo
j (8)
r Significant effects= Ex
Active Glutathione Reductase
4-0 - SEM = 0.3
.5
.0
E; 30 -
E
m
.c 20
Q .
2 \A
LOJ-
’ Significant effects= Ex

A

 

Glutathione Peroxidase

 

 

 

 

55 ~ SEM s 5.4 /
.E
n
.9
05
O 45 >-
E I: a:
a)
.c
O
35 -
E
.1:
c
D
25 ;
( Significant effects = Se
f. l l
Base line 0 24

 

Hours after exercise

Figure 3. Erythrocyte enzymes as a function of time after
exercise. Number per mean is given in parentheses:
e = -Se€E; A = -Se+E; o = +Se-E; )3: +Se+E.

 

nth

 

31

after exercise.

To facilitate discussion of hepatic enzyme response,
only data from the first experiment are presented (fig.4).
However, the responses in both experiments were quite com-
parable. Since none of the enzymes showed dietary E or exer—
cise effects, data were pooled to show the Se effects. Of
the hepatic enzymes measured only ICDH did not respond to
dietary Se. Although ICDH was slightly lower among Se-sup—
plemented rats in Experiment 1, this effect was reversed
in Experiment 2. In neither experiment was the difference
statistically significant.

Hepatic GSH-PX activity was markedly depressed when
Se was not added to the diet. At the same time, hepatic ME,
G6PD and GR activities were reduced by addition of Se to
the diet. In fact, GR exhibited a significant negative
correlation with GSH-Px in both experiments (table 5). Still,
a relatively large number of animals was necessary to demon—
strate this relationship. The relationship of the NADP—
1inked dehydrogenases to GSH-Px or GR was not as easily
defined. While G6PD correlated with GSH-Px in the first
experiment, this relationship did not appear in the second
(table 5). ME did not correlate with GSH-PX, but did show
a relationship to GR in the second study. Stark et a1
(1975) have reported a high correlation of hepatic ME and
GR activities among rats on certain dietary regimens.

This relationship was variable in the present circumstance.

Although not presented, G6PD, ME and ICDH activities were

 

 

32

IU/ unit protein

 

 

 

 

 

 

 

 

 

 

 

 

 

r' F“ (A
0 V0 9 o
g .:O:O:O:O:O:O:O:O:O.O.O.O.O.O.O.O.....O...O.O....A
m A
3 up
6?
‘i’
.0
o
5
o
:1:
O
o O O O 0.0
m N
“
o O
U) o .
t }-i"‘
as
x TA“ A ' l
O - N u ‘ st a)
0 o o o o

GSH -Px, Ell/mg protein

Figure 4. Hepatic enzyme activities. Number per mean is
given in parentheses. Stippled bars represent
—SeiE diets. The error bars are SEM. NS = no
significant effect of dietary Se; * = P<0.05;
** = P<0.01.

33

Table 5. Significant linear correlations among rats fed
diets supplemented and unsupplemented with Se
and E.

 

 

t-<
ll

Slope (X) + Intercept ; r

 

Erythrocyte (Experiment 1) df=682
G6PD = 0.79 GR + 0.65 ; 0.66**

Liver (Experiment 1) df=74

 

G6PD = -21.0 GSH-Px + 39.4 ;-0.30**
GR = — 4.7 GSH-Px + 77.0 ;-0.34**
GR = -27.2 ME + 314.5 ;-0.02

Liver (Experiment 2) df=44

 

G6PD = -1425 GSH-PX + 2934 ;-0.07
GR = -82.6 GSH-PX + 1094 ;-0.30*
GR = 0.28 ME - 0.42 ; O.43**

 

l I 0 I i I I I
r is the linear correlation coeff1c1ent; * and ** indicate

significance at 0.05 and 0.01 levels, respectively.

de indicates the degrees of freedom for the linear corre-

lation.

34

all interrelated. While the response of GR, ME and G6PD
to Se deficiency suggests an interrelation with GSH-PX
activity, a simple linear correlation does not clarify
this relationship. Hepatic FSAO concentration increased
with dietary E supplementation (fig.5), but did not
respond to dietary E or exercise. FSAO has been shown
previously to reflect dietary E intake (Trostler et a1,
1978).

Muscle GSH-PX activity was quite similar to the

 

hepatic enzyme in its response to dietary Se, and its lack
of response to dietary E and exercise. Activity was in-
creased roughly 25-fold in response to Se supplementation
(4.2 i 5.0 versus 91.2 i 5.0 U/lOOmg protein). GR in this
tissue did not respond to diet or exercise. Mean (i SEM)
activity was 1.7 i 0.3 IU/lOOmg protein.

The MDA concentration or index has been used as a
measure of lipid peroxidation (Placer et al, 1966). Hepatic
(fig.6) and muscle (fig.7) MDA increased immediately after
exercise. Hepatic values returned to base line within 24 hrs
while muscle values did not. Dietary E reduced the response
in liver but not in muscle. Dietary E also reduced the base
line level of peroxidation. Dietary E had no effect on the
base line level of MDA nor on the subsequent response to
exercise. While Se supplementation would be expected to
result in reduced levels of MDA (Noguchi et a1, 1973; McCay
et al, 1976; Whanger et al, 1976), the duration of the
present studies may have been too short for this response

to manifest itself. I had expected to induce mortality among

 

35

SEM = 25

6':
o

('5

 

(O
O

 

.4
O

8

 

0|

0
ii
4

 

Fat-Soluable antioxidant [lg/g wet weight
5

% Significant effects: E

l 1

Base line 0 24
Hours after exercise

 

Figure 5. Hepatic fat soluble antioxidant concentration
as a function of time after exercise. Numbers
per mean are per Fig.3. 0 = -Se-E; A = -Se+E;
o = +Se-E; A = +Se+E.

 

.0-
.I:
,9 5.0
Q)
3
“
(D
3 4.0
9
<
9- 3.0
m“
a:
33
2.0
.—

Figure 6.

36

SEM: 0.3

 

A/

_

 

)' Significant effects E, Ex
IL 1 1

l
O 24

Hours after exercise

Baseline

 

Hepatic TBARS (MDA) concentration as a function
of time after exercise. Each value is the mean
of 4 observations. 0 = -Se-E; A = -Se+E; 0 =
+Se-E; A = +Se+E.

 

 

 

TBARS, IOA/g wet weight

37

SEM=O.25
l.5 r

 

'0
1

 

 

 

L
0'5) Significant effects: Ex
J 1 4
Baseline O 24

 

Hours after exercise

Figure 7. Muscle TBARS (MDA) concentration as a function
time after exercise. Numbers per mean are per
Fig.3. 0 = -Se-E; A = -Se+E; o = +Se-E; A =
+Se+E.

38

Se/E deficient animals subsequent to exercise. However, no
deaths occurred before, during or within 24 hrs of exercise
among any of the dietary treatments in either experiment.

At the same time, marked reduction in muscle, liver and
erythrocyte GSH-PX activity on the Se deficient diets was
observed; and depressed FSAO concentration in liver of

E deficient animals was found. The animals of these exper-
iments showed responses to Se deficiency similar to reported

values for GSH-Px. The report of Hafeman et a1 (1974) also

 

indicates no mortality with four weeks on the Se deficient
diet. Clearly, there is more to the development of lesions
and death from Se/E deficiency than the reduction of GSH-PX
activity and E stores.

From the data collected in these experiments, it
is unlikely that even extreme physical exertion will induce
Se/E deficiency signs in a Se or E-supplemented animal.
Even with a combined deficiency of Se and E lesions of
deficiency were not produced with exercise. At the same
time, peroxidation was apparently increased as a result
of exercise. Thus, a relationship to Se and E nutriture
is suggested, but exercise may induce lesions only when
lesions are already an imminent concern. Then, the possibi-
lity exists for a different response with a more prolonged
deficiency. It may also be of interest to determine the

response to training of the exercise-induced peroxidation.

THE EFFECT OF DIETARY SELENIUM AND VITAMIN E ON BIOCHEMICAL
PARAMETERS AND SURVIVAL OF YOUNG AMONG WHITE-TAILED DEER

(Odocoileus virginianus)

 

 

INTRODUCTION. Up to this point the various studies

 

have considered the effect of exercise on the response of
rats and horse glutathione system parameters and peroxi-
dation. While this study does not deal directly with the
problem of exercise, the white-tailed deer is of some
interest because deer have been reported to respond to
physical stress with lesions of Se/E deficiency. This

is particularly true of the young. Stuht et a1 (1971)
have reported mortality among young fed a diet containing
0.2 ppm Se but not supplemented with E. The present

study addresses the question of Se and E effects on bio-
chemical parameters and reproduction in this species.

.MATERIALS AND METHODS. Thirty-two adult female white-

 

tailed deer were assigned to a complete pelleted diet'

(table 6), supplemented with 0 or 45 IU vitamin E (as EL:
d-tocopheryl acetate), and 0 or 0.15 mg Se (as sodium
selenite) per kg diet, for two years. The basal diet con-
tained 0.04 ppm Se and 5.5 ppm E. Animals had been maintained

39

 

 

 

40

Table 6. Composition of basal (low Se/E) diet for white-
tailed deer (Brady & Ullrey, 1975)l.

 

Ingredient Percent (%)

 

2

 

Corn cob product 35.0
Corn grain 18.7
Soybean meal (49% crude protein) 23.95
Alfalfa meal (17% crude protein) 5.0
Cane molasses 5.0
Wheat, soft winter 10.0
Soybean oil 1.0
Limestone (38% calcium) 0.4
Trace mineral salt3 0.5
Vitamin A & D premix4 0.25
Calcium propionate 0.2
100.0

 

lContained0.04 ppm Se and 5.5 ppm d-tocopherol (Horwitz,
1975) by analysis.

2Material remaining after hard cylinder of cob is removed.
Cell wall constituents comprised 81.2%; acid detergent
fiber, 37.5%; lignin, 6.5%, by weight. A product of The
Anderson's, Maumee, OH.

3A product of Diamond Crystal Salt Co., Akron, OH.

4Supplied 3300 IU vitamin A as retinyl acetate and 220 IU

ergocalciferol per kg of mixed diet.

41

 

in captivity throughout their lives. Prior to this study,
deer were fed a pelleted diet identical to the +Se+E diet.
Mean age of the female deer was 3.1 i 0.5 years (mean i
SEM) at the start of the study. Deer averaged 59.3 i 2.2
kg. The animals were individually housed in outdoor pens.
Pen floors were of packed earth and individual shelters
were provided. Diet and water were available ad libitum.
Animals were weighed, restrained using CI-744 (Parke-

Davis Co., Ann Arbor, MI) and blood samples taken bimonthly

 

for the first year and quarterly for the second. Deer were
mated in the fall of both years and survival of the offspring
followed to weaning at about 4 months of age. All deaths
were evaluated clinically and histologically for evidence
of white muscle disease (WMD), the primary lesion of Se/
E deficiency reported for this species (Stuht et a1, 1971).
At weaning in the second year, twelve male young (three per
treatment) and the surviving adult females were killed.
Blood, liver and muscle were taken for analysis.

Blpgd. Blood samples were taken by jugular venipuncture
using heparin as an anticoagulant. Hemoglobin (Crosby et
al, 1954) and hematocrit were determined. Aliquots of
whole blood were deproteinized immediately and transported
on ice to the laboratory (3 to 5 hrs) for lactate (first
year only)(Gutman & Wahlefeld, 1974), MDA (Hunter et al,
1963) and GSH (Beutler et a1, 1963) determination. GSH was
once again expressed per ml erythrocyte.

Erythrocytes were separated from plasma by

 

42

centrifugation. Washed erythrocytes were lysed and assayed
for G6PD, total and active GR and GSH-PX activities as
previously described. All activities were assayed at 370
and expressed per unit hemoglobin. Serum was frozen (-200)
and susequently analyzed for Se (Whetter & Ullrey, 1978)
and a-tocopherol (Desai, 1968).

Tissues. Muscle and liver samples were taken for
ME, ICDH, G6PD, GR and GSH-Px assay as described for the

rat. MDA and FSAO were also determined.

 

In vitro hemolysis. Erythrocytes from the 12 month

 

sample were used for time-dependent (Draper & Csallany,

1969) and hydrogen peroxide—dependent (Rotruck et al,

1971) hemolysis tests. Time-dependent hemolysis was carried
out for 24 hrs, using erythrocytes from 3 deer per diet at 37°.
Peroxide—dependent hemolysis was also performed at 370 using
erythrocytes from 5 deer per treatment. Both tests were run
with and without exogenous glucose (150 mg/dl, final con-
centration) to provide reducing equivalents needed for the
function of the glutathione peroxidase system (Rotruck et

a1, 1972; Rotruck et al, 1971).

Statistical analysis. Data were analyzed as a 2 x 2

 

factorial design with unequal replication (Gill, 1978).
Mortality data for the two years were pooled and analyzed
by chi-square analysis (Steel & Torrie, 1960). Simple
linear correlations for the relationship of Se and GSH-Px

were determined (Steel & Torrie, 1960).

43

RESULTS. Both plasma Se (fig.8) and E (fig.9) had
essentially stabilized within 10 to 12 months fed the res-
pective diets. To facilitate discussion, only 0 and 12
month values are presented (table 7). Plasma Se, erythrocyte
GSH-Px and blood lactate showed significant effects of diet—
ary Se, while plasma E was the only measured blood variable
to respond to dietary E. Neither GR nor G6PD in the ery-
throcyte showed a significant response to diet. Percent
active GR was also unresponsive to diet.

Although blood MDA did not respond to diet, in vitro
hemolysis was altered by dietary E (table 8). In the absence
of glucose, dietary E reduced hemolysis 30 to 90%. In the
presence of glucose, the E effect was lost for the time-
dependent hemolysis. Glucose addition did not significantly
reduce hemolysis. The hemolysis data are generally consis-
tent with mortality data for the young (table 9). Mortality,
and mortality with WMD lesions was reduced when the parental
diet contained E, but did not vary in response to dietary
Se. Even when adults were fed the +Se+E diet there was some
mortality with WMD.

Blood variables for the offspring are presented (table
lQ)and are quite comparable to the adult values, showing
significant effects of dietary Se on plasma Se and erythro-
cyte GSH-PX. Dietary Se did, however, result in reduced
erythrocyte MDA among the young, unlike the adults. MDA and

plasma E were responsive to dietary E in the young. Again,

 

44

Figure 8. Plasma selenium among white—tailed
deer as a function of months fed
diets. o = —SeiE; A = +SeiE.

45
Plasma selenium , pp m

E
l

'0
no
I

5
I

'o
00
l

 

_
m

 

_ _ _
_O _N 3

2.02.2.5 mmo 0_m._.

_
No

mm Nb

 

 

46

Figure 9. Plasma d-toc0pherol among white-tailed
deer as a function of months fed diets.
0 = :Se-E; A = iSe+E.

   

   

13K] 03:1 SHlNOW

8| 9| l7| 2| Ol

02

172 22

47
Plasma a -tocopher0|, pg/ ml

 

 

 

o S o
i ii).
. .
. .
. .
. .
.
. .
.
l)

 

I“
o
l

 

48

Table 7. Initial and 12 month body weight and blood variables among adult
female white-tailed deer fed diets supplemented and unsupplemented
with Se and E.

 

Months

 

 

 

 

, Diet 1 Significant
Variable féd -Se-E +Se-E —Se+E +Se+E SEM effects2
diet
Body weight, kg 0 57.33 60.4 59.0 60.0 2.2 ns
12 60.1 61.9 60.6 61.5 5.1 ns
Plasma
Selenium, ug/ml 0 0.11 0.12 0.12 0.12 0.005 ns
12 0.07 0.10 0.07 0.11 0.005 Se
Vitamin E, ug/ml
0 1.81 1.83 1.58 1.80 0.12 ns
12 1.03 0.90 2.10 2.20 0.13 E
Whole blood
Lactate, mM 0 7.2 8.4 7.3 6.9 1.8 ns
12 10.6 4.9 7.2 4.1 1.5 Se
MDA, lOA/ml 0 2.8 2.6 3.3 3.1 1.5 ns
12 4.2 4.0 3.3 2.9 2.0 ns
Erythrocyte
GSH, mM 0 0.24 0.21 0.22 0.25 0.08 ns
12 0.21 0.15 0.16 0.14 0.03 ns
G6PD, IU/g Hb 0 2.4 2.3 2.0 2.4 0.2 ns
12 2.2 2.2 2.1 2.4 0.2 ns
Active GR, IU/g Hb
0 2.1 1.8 2.0 2.4 0.3 ns
12 2.8 2.2 2.2 3.0 0.2 ns
Percent active GR, %
0 92 87 88 94 4 ns
12 84 86 84 83 3 ns
GSH-PX, U/mg Hb 0 42.0 38.4 36.5 35.6 3.1 ns
12 15.3 30.4 15.6 30.2 2.3 Se

 

1Standard error of the mean.

2ns = no significant effect of diet (P>0.05); Se = significant effect of
dietary Se; E = significant effect of dietary E.

3Each value is the mean of 8 deer at 0 months. At 12 months, -Se-E represents
8 deer; +Se-E, 7 deer; -Se+E, 7 deer; and +Se+E, 5 deer.

 

49

Table 8. In vitro hemolysis among Se and E supplemented and
unsupplemented white-tailed deer after 12 months fed
the respective diets.

 

 

 

 

Diet 1 Significant
Test -Se—E +Se-E -Se+E +Se+E SEM effects2
% hemolysis

Time-dependent (24 hr)3
-Glucose

28 32 4 10 7 E
+G1ucose”

20 23 6 4 8 ns
Hydrogen peroxide-dependent5
-Glucose

32 33 3 3 7 E
+G1ucose”

23 13 3 3 6 E

 

1Standard error of the mean.

2ns = no significant effect of diet (P>0.05); E = significant effect
of dietary B.

3Each value is the mean of three deer.
”Glucose added to provide final concentration of 150 mg/dl.

5Each value is the mean of five deer.

 

50

Table 9. Pooled (first and second year) mortality data for
offspring of Se and E supplemented and unsupple—

mented white-tailed deer.

 

 

 

Variable Diet Significant
-Se-E +Se-E -Se+E +Se+E effects1
Young born 27 30 19 20 ns
Young surviving to weaning
9 10 9 11 ns

Young dying prior to weaning
18 20 10 9 E

Young dying with WMD lesions
16 16 5 4 E

 

1ns = no significant dietary effects (P>0.05); E = significant

effect of dietary E by chi-square analysis.

 

51

Table 10. Body weight and blood variables among male offspring of
female white—tailed deer fed diets supplemented and un-
supplemented with Se and E, at weaning.

 

 

 

 

 

 

. Parent's diet 1 Significant
V
ariable -Se-E +Se—E -Se+E +Se+E SEM effects2
Body weight, kg
21.23 24.9 27.4 22.6 2.7 ns
Plasma
Se, ug/ml 0.04 0.08 0.05 0.07 0.006 Se
E, ug/ml 0.34 0.34 0.88 0.83 0.12 E
Whole blood
MDA, 10A/ml 3.1 1.2 0.9 1.3 0.3 Se, E,
Se x E
Erythrocyte
GSH, mM 0.56 0.70 0.62 0.45 0.07 ns
G6PD, IU/g Hb
2.3 2.3 2.2 2.5 0.5 ns
Active GR, IU/g Hb
2.1 2.4 2.2 2.3 0.4 ns
Percent active GR, %
90 89 86 94 4 ns
GSH-PX, U/mg Hb
17.8 35.7 27.7 38.3 3.5 Se

 

1Standard error of the mean.

2ns = no significant effect (P>0.05); E = significant effect of dietary

E; Se = significant effect of dietary Se; Se x E = significant inter-
action.

3Each value is the mean of three deer.

 

52

GR, percent active GR and G6PD activities were not altered
by diet.

Hepatic and muscle variables for adults (tableljj
and young (tableJJZ) are presented. The adults showed
elevated hepatic FSAO concentrations with dietary E sup-
plementation. This effect was lacking among the young. Diet-
ary Se significantly affected hepatic GSH-Px activity, and
hepatic and muscle Se concentration among adults and young.
Muscle GSH-Px activity was significantly increased among
the young of Se-supplemented adults. Se supplementation
also resulted in reduced hepatic MDA levels among the
young.

Hepatic and muscle G6PD, GR, ME and ICDH activities
were not affected by diet. Measurable ME activity was
found only in liver of young. ICDH activity was also too
low to measure in muscle of both young and adults.

The relationship of tissue Se concentration and GSH—PX
activity is presented for erythrocytes, liver and muscle
(table13 ). GSH-PX activity in all tissues showed a sign—
ificant linear relationship to tissue Se concentration.

DISCUSSION. Initially, an interesting question is

 

how wild species in general, and white-tailed deer, in
particular, survive in a Se-deficient environment such as
that of Michigan (Ku et al, 1972; Kubota et a1, 1967).
Clearly, on the basis of plasma, muscle and liver Se and

erythrocyte, muscle and liver GSH-PX activity, white-tailed

 

 

53

Table 11. Hepatic and muscle variables among adult female
white-tailed deer fed diets supplemented and
unsupplemented with Se and E.

 

 

 

 

 

 

 

 

 

 

. Parent's diet 1 Significant
Variable -Se-E +Se-E -Se+E +Se+E SEM effects2
Number of animals

8 7 7 5
Liver
Weight, kg 1.2 1.1 1.1 1.4 0.2 ns
MDA, U/g 4.0 3.6 3.5 3.4 0.3 ns
FSAO, ug/g 2.8 3.0 6.4 4.9 1.1 E
G6PD, IU/g protein
7.3 6.8 5.4 6.8 2.0 ns
ICDH, IU/g protein
111 115 130 131 2 ns
ME, IU/g protein
<0.5
GR, IU/g protein
46 44 46 45 3 ns
GSH-PX, U/mg protein
2.8 10.7 2.9 16.1 1.8 Se
Se, ug/g 0.24 0.36 0.24 0.34 0.02 Se
Muscle
G6PD, IU/g protein
1.8 1.8 1.6 1.3 0.5 ns
ICDH, IU/g protein
<0.5
ME, IU/g protein
<0.5
GR, IU/lOOmg protein
1.4 1.0 1.1 1.0 0.2 ns
GSH-PX, U/mg protein
6.1 6.4 4.4 5.9 0.8 ns
Se, ug/g 0.07 0.09 0.06 0.08 0.01 Se

 

1Standard error of the mean.
2ns = no significant effect of diet (P>0.05); Se and E =
significant dietary effects of Se and E, respectively.

54

Table 12. Hepatic and muscle variables among male offspring
of white-tailed deer fed diets supplemented and

unsupplemented with Se and E.

 

 

 

 

 

 

 

 

. Parent's diet 1 Significant
Variable -Se-E +Se-E -Se+E +Se+E SEM effects2
Liver
Weight, g 5393 536 505 529 51 ns
MDA, U/g 1.7 0.7 2.1 0.6 0.5 Se
FSAO, ug/g 1.8 2.9 2.0 1.6 0.8 ns
G6PD, IU/g protein
0.5 1.1 0.5 1.2 0.5 ns
ICDH, IU/g protein
65 95 75 75 21 ns
ME, IU/g protein
0.8 2.4 2.3 1.4 0.2 ns
GR, IU/g protein
51 43 97 70 29 ns
GSH-Px, U/mg protein
2.0 11.2 7.2 13.0 0.6 Se
Se, ug/g 0.21 0.31 0.19 0.29 0.02 Se
Muscle
G6PD, IU/g protein
2.5 1.8 2.0 2.0 1.3 ns
ICDH, IU/g protein <0.5
ME, IU/g protein <0.5
GR, IU/g protein
44 22 28 21 10 ns
GSH-PX, U/mg protein
1.4 6.4 2.0 6.1 1.7 Se
Se, ug/g 0.05 0.07 0.05 0.07 0.004 Se
1Standard error of the mean.
2ns = no significant diet effect (P>0.05); Se = significant

effect of dietary Se.
3Each value is the mean of three deer.

 

 

 

55

Table 13. Linear relationships of Se concentration (ug/ml
plasma or ug/g tissue) and GSH-Px activity (U/
mg Hb or U/mg protein), where GSH-PX = bSe + a,

among white-tailed deer adults and offspring.

 

 

a b r

Adult

Erythrocyte -l.8 287.2 0.71**
Liver -13.5 71.0 0.72**
Muscle 1.1 63.0 0.51**
Young

Erythrocyte 2.0 464.0 0.92**
Liver -8.2 66.4 0.82**
Muscle -9.7 227.5 0.95**

 

1r is the linear correlation; ** designates a highly

significant (P<0.01) linear correlation.

56

deer fed ona defined diet were susceptible to Se depletion.
Equally clearly, Se depletion, per se, did not result in any
untoward effects. There was no evidence of increased lipid
peroxidation, as reflected by MDA and in vitro hemolysis,
nor was there increased preweaning mortality of young.

At the same time, others have reported that GSH—Px act-
ivity does reflect Se status (Hafeman et al, 1974; Oh et a1,
1976; Reddy & Tappel, 1974; Chow & Tappel, 1974), that Se
deficiency will result in increased erythrocyte hemolysis
(Rotruck et al, 1972b), and that increased lipid perox-
idation, as reflected by increased MDA, is a sequel to
Se deficiency (Noguchi et el, 1973).

Although GSH-PX activity in liver, muscle and erythro-
cytes did parallel Se concentration, other gross and meta-
bolic responses were lacking among the adult deer fed the
deficient diets for 2 years. Although little is known of the
specific requirement of deer for Se, the requirement for
domestic ruminants is now thought to be in excess of 0.1
ppm (Oh et al, 1976; 1976b; Whanger et al, 1977; Ullrey et
al, 1977). The 0.04 ppm provided by the -Se diets is typical
of values for forage and grains from most areas of Michigan
(Oldfield et a1, 1971; Muth & Allaway, 1963; Ullrey, 1974).

Blood and liver MDA was also unresponsive to dietary
E. Again, others have shown this (Noguchi et al, 1973) and
other parameters of lipid peroxidation (Dillard et al, 1977)
to increase with E deficiency. The prolonged period (3 to

5 hrs) before analysis may have led to increased variability

57

of MDA values with subsequent loss of sensitivity for this
indicator of peroxidation. In vitro hemolysis, hepatic
FSAO and plasma E among adults, and plasma E and mortality
among the young were responsive to dietary E. Hemolysis
(Draper & Csallany, 1969), hepatic FSAO (Trostler et al,
1977) and plasma E (Hayes et al, 1969; Machlin et a1, 1977)
responses to dietary E have been reported previously for
other species.

Although evidence of dietary influence on tissue Se
and E was demonstrated among adults, the only mortality
resulted from trauma during and after handling. This was
not true of the young. Mortality was E-related among the,
young. However, even when Se and E were included in the
parents' diet, some mortality with lesions of WMD was
found. It is uncertain why this is so. Others have reported
mortality in zebra from acidosis with lesions identical to
WMD (Harthoorn & Young, 1974). The WMD lesions are, then,
not pathognomonic for Se/E deficiency but represent the
end product of several means of muscle destruction. But
blood lactate was high among the adults when compared to
other species (Beutler, 1975; Harthoorn & Young, 1974;
Brady et al, 1978; Harvey & Kaneko, 1975). Lactate was
further elevated among deer fed the low Se diets after 12
months. Thus, there apparently is some relationship between
acidosis and Se.

Mortality, even from +Se+E parents ran as high as 50%,

rising to 67% when E was not present in the diet. Other

. -1);

 

 

58
sources of mortality included parasitism, congenital defect,
nephritis, heat stress, and unknown causes. While mortality
was significantly lower when E was included in the parents'
diet, tissue variables among the young contribute little
toward an explanation of the observed effect. Plasma E and
blood MDA responded to the parents' diet, hepatic FSAO and
MDA did not. It is certainly possible that the surviving
fawns were those least susceptible to E (and Se) deficiency.
Biochemical parameters were not determined for young dying
prior to weaning.

From these data, it appears that dietary E is important
for survival of young, at least to weaning. E did not appear
important to the survival of adults. Dietary Se had no
measurable influence on survival of young or adults during
the course of this study. A "non-Se glutathione peroxi-
idase" has been described (Lawrence & Burk, 1976; Prohaska
& Ganther, 1976) and identified as glutathione-S-trans-
ferase (EC 2.5.1.18)(Prohaska & Ganther, 1977) in the rat.
Non—Se GSH-PX has also been described in other species
(Lawrence & Burk, 1977). While it is possible that Se-
dependent GSH-PX is not critical to survival of deer, it
remains uncertain if any GSH-Px activity is needed, since
non-Se dependent activity was not measured.

A number of other comments may be of comparative
interest. While G6PD and the hexose monophosphate shunt is
the only source of NADPH for GR in the erythrocyte

(Beutler, 1975), a number of NADP-dependent dehydrogenases

 

 

 

 

 

59

are available in liver and other tissues. These would
include ME and ICDH, as well as G6PD. ME activity was
essentially absent from muscle of adults and young, and
liver of adults. Hanson and Ballard (1968) have also
reported low hepatic ME activity in the adult bovine.
Hepatic ICDH is also quite high in the bovine, much as
observed for the deer. G6PD was the only measurable dehy-
drogenase in muscle of adults and young.

There were some differences between the young and
adults. Young tended to have lower hepatic G6PD and ICDH
activities than the adults, but measurable ME. Otherwise
hepatic, muscle and erythrocyte enzyme activities were
comparable.

Erythrocyte GSH was low among young and adult deer
when compared to the l to 2 mM reported for other species
(Beutler, 1975; Harvey & Kaneko, 1975; Reid et al, 1948;

Agar & Stephens, 1975). The young had higher GSH concentra-
tions than the adults. Also, erythrocyte percent active GR
was on the order of 85% among adults at 12 months. Less than
83% would be considered indicative of a riboflavin deficiency
in man and rat (Sauberlich et al, 1972; Glatzle et al, 1970).
We do not have percent active GR values for other ruminants.
Thus, these low values could be characteristic of ruminants
in general or a transient effect of handling stress in white-
tailed deer, much like the shift observed in the first

horse study.

CONCLUSIONS

In the initial studies with the horse, it was demon-
strated the erythrocyte MDA, an indicator of lipid perox-

idation, increases subsequent to exercise. This increase is

 

accompanied by elevations of a number of plasma enzymes.
These data suggest that exercise results in 1) an increased
level of in vivo lipid peroxidation and 2) an increase in
permeability of muscle membrane. Addition of Se to a diet
apparently adequate in Se did nothing to alter these
responses. The effects of dietary B were not investigated.

The subsequent work with rats indicated that exercise
also yields increased lipid peroxidation in liver and
muscle, a response which again was independent of dietary
Se (even where Se deficiency was pronounced) and only
slightly altered by dietary E.

On the basis of these studies, it seems likely that
exercise could exacerbate Se/E deficiency, if an animal
were on the borderline of deficiency to begin with. No
support is lent to the suggestion that Se- or E—adequate
animals could spontaneously develop lesions of deficiency
subsequent to strenuous exercise. The species employed may

not have provided adequate models for this problem, however.
60

61

Exercise-induced myopathies have not been reported in the
rat. Such myopathies have been reported for the horse, and,
indeed, linked to Se deficiency. However, controlled data
are lacking.

The final study with white-tailed deer, a "sensitive
species", was not in any sense an exercise study. It is a
difficult problem to obtain resting samples from deer. Still,
as a first step, a knowledge of Se/E contributions to the

survival of this species would be of interest.

 

Its GSH-PX activity (erythrocyte, liver or muscle;
young or adult) is quite low, even on Se—supplemented
diets. However, dietary Se had no effect on subsequent
mortality of the young. E, on the other hand, did alter
the mortality of the young. The one clearly documented
instance of stress-induced mortality in this species
followed accidental omission of E from the diet (Stuht et
al, 1971).

This suggests a possible relationship to stress-
induced lesions in swine, a Species not investigated in this
series of studies. E is a relatively expensive ingredient
and it is generally kept to a minimum in commercial diets.
Further, with the legalization of Se supplementation of
swine diets and the known sparing effect of Se on E, there
may be a tendency to reduce the dietary E concentration.
It is possible that the reports of stress-induced lesions
result when swine, particularly young swine, are fed diets

low in E and subjected to some physical stress, such as

62

comingling of litters.

Certainly, a great deal of work is yet to be done
to confirm this possibility. The data presented herein
merely strengthen the likelihood of a relationship of
exercise to increased lipid peroxidation, a property

shared with Se/E deficiency.

 

’-

 

 

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