Illil'ln, . I'll! I, lit, ,1

 

THESIS

. _, J W-I-r-‘V vggsby if
:1

f
i' 7 (‘1‘?7’33; .'.'! Effi?‘ .n 1

  

This is to certify that the

thesis entitled

Some Aspects of Selenium
Metabolism in the Young Pig

presented by

Matthew J. Parsons

has been accepted towards fulfillment
of the requirements for

Ph.D. Anima] Science

degree in

f?%

 

 

 

Major professor

Date g//g/8/

0-7 639

 

MSU

LIBRARIES

 

 

 

RETURNING MATERIALS:
Place in book drop to
remove this checkout from
your record. FINES will
be charged if book is
returned after the date
stamped below.

 

 

 

 

 

SOME ASPECTS OF SELENIUM
METABOLISM IN THE
YOUNG PIG

By

Matthew James Parsons

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of ,

DOCTOR OF PHILOSOPHY

Department of Animal Science

1981

 

 

 

ABSTRACT
SOME ASPECTS OF SELENIUM METABOLISM
IN THE YOUNG PIG

By

Matthew James Parsons

The effects of dietary riboflavin supplementation, selenium source
and level of supplementation on the performance and selenium metabolism
of weanling pigs were studied. Pigs fed rfiboflavin-supplemented (10
mg/kg) casein based diets for l8 days gained faster than pigs fed the
riboflavin-unsupplemented diets. Percent active erythrocyte gluta-
thione reductase declined rapidly when pigs were placed on riboflavin-
unsupplemented diets and was obviously lower after l2 days of consuming
the riboflavin-unsupplemented diets than the erythrocyte percent active
glutathione reductase of pigs fed the riboflavin-supplemented diets.
Percent active erythrocyte glutathione reductase values of as low as
50% were needed before other riboflavin deficiency signs became evi-
dent.

Supplementation of diets with riboflavin (10 mg/kg) resulted in
increased liver and erythrocyte glutathione peroxidase activity and
decreased kidney and muscle glutathione peroxidase activity in trials
one and two. The selenium concentration of liver and heart were in-
creased, while plasma selenium levels were decreased by riboflavin-
supplementation.

Riboflavin-supplementation and selenium source did not affect

apparent selenium absorption. However, riboflavin supplementation did

Matthew James Parsons
decrease urinary selenium and concomitantly increased the apparent
retention of selenium. Urinary selenium excretion was decreased when
selenomethionine was used as the selenium source rather than sodium
selenite. As a result more selenium was apparently retained when
selenomethionine was fed.

In trial four riboflavin supplementation increased kidney, muscle,
heart and brain glutathione peroxidase activity when sodium selenite
was fed as the selenium source, but not when selenomethionine was fed
as the selenium source. Tissue selenium concentrations were not af-
fected by riboflavin supplementation of diets. Feeding selenomethi-
onine as the supplemental selenium source at 67% of the level of se-
lenium from sodium selenite resulted in higher muscle, heart and brain
selenium concentrations than feeding the higher level of selenium as
sodium selenite.

Serum glutathione peroxidase activity was more responsive to di-
etary selenium intake than erythrocyte glutathione peroxidase activity.
When pigs were reared under practical conditions, serum glutathione
peroxidase activity plateaued at 0.35 ppm selenium in a corn-soybean
meal diet at two weeks postweaning. Gains, feed intake and feed effi-

ciency were not affected by dietary selenium level.

ACKNOWLEDGEMENTS

I would like to thank Dr. E.R. Miller for providing the leadership
for my training as the committee chairman of my graduate committee and
for being so patient with me during this training. Drs. D.E. Ullrey
and H.D. Stowe have contributed greatly to my training by providing the
essential laboratory facilities needed to complete these studies and as
advisors to me for this graduate program. Dr. M.G. Hogberg deserves a
great deal of thanks as a source of advice and for making some opportu-
nities available to me which have helped me to develop as a scientist.
Dr. R.H. Dukelow has contributed some unique philosophy to this gradu-
ate comittee, which has helped to shed a different light on this
training program. I would like to thank Dr. R.H. Nelson as Chairman of
the Department of Animal Science for creating an atmosphere to study in
that is equalled by none. This manuscript would not have been possible
without the patience and devoted effort of S. Mileski as the typist.
My fellow graduate students have provided fellowship, thoughtful sug-
gestions and physical help throughout this program. Finally, I would
like to thank my wife for encouraging me to continue in this en-

deavour.

ii

TABLE OF CONTENTS
Page
LIST OF TABLES O O O O O O O O O O O O O O O O O O O O O O O O O 0 v

SECTION I. THE EFFECT OF DIETARY RIBOFLAVIN SUPPLEMENTATION
AND SELENIUM SOURCE ON SELENIUM RETENTION, TISSUE
SELENIUM LEVELS, AND TISSUE GLUTATHIONE REDUCTASE
AND GLUTATHIONE PEROXIDASE ACTIVITY. . . . . . . . . . l

IntrOdUCtI on. O O O O 0 0 0 O O O O O O O O O 0 0 O O O O O O 1
Materials and Methods . . . I . . . . . . . . . . . . . . . . 3
Tr1als I and II. 0 O O O O O O O O O 0 O O O O O O O 0 O 3
Tr1a] III. 0 O O O O O 0 O O O O O O 0 0 0 O O O O O O O 5
Tr1a1 IV C O O O O O O O O O O O O O O O O O O O O 0 0 O 6
Results and Discussion. . . . . . . . . . . . . . . . . . . . 7

Effect of riboflavin supplementation on tissue .
paramters O O O O O 0 O O O O O I O 0 O O O O O O 0 O O 7

Effects of riboflavin supplementation and selenium
source on selenium balance . . . . . . . . . . . . . . .l7

Effects of riboflavin supplementation and selenium
source on tissue parameters. . . . . . . . . . . . . . .21

sumry C O O O O O O O O I O O O O O O O O O O O O O O O O .29

SECTION II. THE RESPONSIVENESS OF SERUM GLUTATHIONE PEROXIDASE
ACTIVITY TO DIETARY SELENIUM LEVEL AND THE EFFECT
OF SUPPLEMENTAL DIETARY SELENIUM AND ENVIRONMENTAL
TEMPERATURE ON SERUM GLUTATHIONE PEROXIDASE
ACTIVITY OF PIGS REARED UNDER PRACTICAL CONDITIONS. .30

Introduction. . . . . . . . . . . . . . . . . . . . . . . . .30
Materials and Methods . . . . . . . . . . . . . . . . . . . .3l
Tria] v. 0 O O O O O O O O O O O O O O O O O O O O O O .3]
Trial VI 0 O O O O 0 O O O 0 O O O O 0 O 0 O O O O O O 033
Trial VII. 0 O O O O O O O O O O O O O O O O O O O O O .35
Results and Discussion. . . . . . . . . . . . . . . . . . . .38

The responsiveness of serum glutathione peroxidase
to dietary selenium level. . . . . . . . . . . . . . . .38

The effect of environmental temperature on performance .53

The effect of supplemental selenium on serum glutathione
peroxidase two weeks postweaning . . . . . . . . . . . .55

iii

Page

The effects of supplemental selenium during the first
week postweaning . . . . . . . . . . . . . . . . . . . .58

. .60
.61

sumaw O O O O O O O O O O O O O O O O O O O O O O O O O
conc1u510ns O O O O O O O O O O O O O O O O O O O O O O O 0

LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . .62

iv

LIST OF TABLES
Table Page
l Composition of the riboflavin-deficient diet . . . . . . . . 4

2 The effect of supplemental riboflavin on body weight
and gai "5 Of pi 950 O O O O O O O O O O O O O O O O O O O O O 8

3 The effects of supplemental riboflavin and days on trial
on active erythrocyte glutathione reductase activity
Of pigs. 0 O O O O O O O O O O O O O O O O O O I O O O 0 O .10

4 The effects of supplemental riboflavin and days on trial
on total erythrocyte glutathione reductase activity of

p195 O O O O O O O O O O O O O O O O O O O O O O O O O O O 0]]

5 The effect of supplemental riboflavin and days on trial
on erythrocyte percent active glutathione reductase of

pigs 0 O O O O O O O O O O O O 0 O O O O O O O O O O C O O .12

6 The effects of supplemental riboflavin on the actual weight
and percent of body weight of selected organs. . . . . . . .14

7 The effect of supplemental riboflavin on liver, kidney,
muscle, heart, brain and erythrocyte glutathione
peroxidase activity. . . . . . . . . . . . . . . . . . . . .15

8 The effect of supplemental riboflavin on liver, muscle,
heart, brain and plasma selenium concentration of pigs . . .16

9 The effect of supplemental dietary riboflavin for eighteen
days on liver, kidney, muscle, heart and brain glutathione
reductase activity and percent active erythrocyte
glutathione reductase. . . . . . . . . . . . . . . . . . . .18

10 The effect of dietary riboflavin supplementation and
selenium source on selenium balance. . . . . . . . . . . . .19

11 The effect of supplementing with equal available selenium
intakes from sodium selenite or selenomethionine and
riboflavin supplementation on body weight, gains and
selected organ weights of pigs . . . . . . . . . . . . . . .22

12 The effects of supplementing with equal available selenium
intake from sodium selenite or selenomethionine and
riboflavin supplementation of liver, kidney, muscle, heart
and brain glutathione reductase activity, and erythrocyte
percent active glutathione reductase of pigs . . . . . . . .24

13 The effects of supplementing with equal available selenium
intake from sodium selenite or selenomethionine and
riboflavin supplementation on liver, kidney, muscle, heart,
brain and serum glutathione peroxidase . . . . . . . . . . .27

V

Table Page

14 The effects of supplementing with equal available selenium
intake from sodium selenite or selenomethione and
riboflavin supplementation on liver, kidney, muscle, heart,
brain and plasma selenium concentrations of pigs . . . . . .28

15 Basal diet for trials V and VI . . . . . . . . . . . . . . .32
16 Selenium levels of diets fed in trials V and VI. . . . . . .34
17 Basal diet for trial VII . . . . . . . . . . . . . . . . . .36
18 Selenium levels of diets in trial VII. ... . . . . . . . . .37

19 The effect of selenium supplementation on average daily
gain in trials v and VI. 0 O O O O O O 0 O O O O O O O O O 039

20 The effect of selenium supplementation and days consuming
diets on serum glutathione proxidase activity in trial V . .40

21 The effect of selenium supplementation and days consuming
diets on serum glutathione peroxidase activity in trial VI .42

22 The effect of selenium supplementation for four weeks on
serum and erythrocyte pool of glutathione peroxidase
aCt‘I Vity 1n tria] v0 0 O O O O O O O O O O O O O O O O 0 O 043

23 The effect of selenium supplementation and days consuming
diets on erythrocyte glutathione proxidase activity in
tr1a] v. 0 O 0 O O O O O O O O O O O O O O 0 O O O O O O 0 045

24 The effect of selenium supplementation and days consuming
diets on erythrocyte glutathione peroxidase activity in
tr1a] VI 0 O O O O O O O O 0 0 I O O O O O 0 O O O O 0 O O .46

25 The effect of selenium supplementation and days consuming
diets on plasma selenium concentrations in trial V . . . . .48

26 The effect of selenium supplementation and days consuming
diets on plasma selenium concentrations in trial VI. . . . .49

27 The effect of selenium supplementation and days consuming
diets on serum tocopherol levels in trial V. . . . . . . . .5l -

28 The effect of selenium supplementation and days consuming
diets on serum tocopherol levels in trial VI . . . . . . . .52

29 The effect of selenium supplmentation and environmental
temperature on feed intake, gains and feed efficiency in
tria] VII. 0 O O O O O O O O O O O O O O I O O O O O O O O .54

vi

Table
30

Page

The effect of selenium supplementation for fourteen days
and environmental temperature on blood parameters. . . . . .56

31 The effect of level of selenium supplementation during

the first week postweaning on serum glutathione peroxidase
activity and blood reduced gluthathione levels . . . . . . .59

vii

seem;
Introduction

The glutathione peroxidase system requires the mineral selenium,
the sulfur amino acid cysteine, and the two water soluble vitamins
riboflavin and niacin. Glutathione peroxidase (EC 1.11.1.9) is a sele-
nium containing enzyme that has been isolated from erythrocytes
(Rotruck _e_t_ 31., 1972a and 1973), plasma (Burk and Gregory, 1980),
liver (Sunde _e_t__a_l_., 1978 and Burk and Gregory, 1980) and muscle (Sunde
g _a_l_., 1978). Glutathione peroxidase normally exists as a tetramer
(Awasthi e_t__aj_., 1975; Sunde _e_t_a_l_., 1978; and Ladenstein gal” 1979)
which contains four atoms of selenium per molecule of enzyme. The
active site of the enzyme appears to involve the selenocysteine moities
present in the molecule (Tappel gt a_l_.,' 1978a, and Ladenstein 93; 11.,
1979).

Glutathione is a cysteine containing tripeptide (Y -glutamy1cystei-
nylglycine) that provides reducing equivalents for the GSH-Px cata-
lyzed reduction of lipid peroxides or hydrogen peroxide to hydroxy
acids or water to protect tissues from oxidative damage (Mills, 1957;
and Mills and Randall, 1958). The sulfhydryl group of glutathione is
the active site (Styer, 1975). Niacin as a component of nicotinamide
adenine dinucleotide phosphate (NADPH) is essential to provide reducing
equivalents to reduce oxidized glutathione (Jacob and Jundl', 1966;
Eggleston and Krebs, 1974).

Glutathione reductase (EC 1.6.4.2) is a flavoprotein (Buzard and
Kopko, 1963) that contains two flavin adenine dinucleotide (FAD) mole-
cules per molecule of enzyme (Staal _e_t. _a__1_., 1969). It catalyzes the

reduction of oxidized glutathione using reduced nicotinamide adenine

2
dinucleotide phosphate as a source of reducing equivalents. The
erythrocyte glutathione reductase activity coefficient has been used as
an index of riboflavin status in rats (Glatzle 33 31,, 1970 and Til-
lotsen §t_al,, 1972), man (Glatzle, 1968 and Sauberlich g§_gfl,, 1972)
and pigs (Brady g§_al,, 1979).

Since riboflavin is a relatively unstable vitamin, and since un-
supplemented practical swine diets are generally deficient in ribofla-
vin (Pond and Maner, 1974), it is possible that a riboflavin deficiency
may exist. A riboflavin deficiency could cause an apparent selenium
deficiency by decreasing the glutathione reductase activity and conse-
quently lowering the supply of reduced glutathione available to the
glutathione peroxidase enzyme. In an attempt to test this hypothesis,
Brady 3!; a_1_. (1979) reported decreased in £13319. hepatic glutathione
peroxidase activity and a decreased hepatic selenium content in young
pigs on riboflavin-deficient diets. They did find the expected eryth-
rocyte glutathione reductase response to dietary riboflavin, but hepat-
ic and muscle lg_yltrg glutathione reductase activity was not affected
by riboflavin supplementation.

Studies by Groce gt al. (1973a) have shown that a lower proportion
of absorbed selenium from seleniferous corn was excreted in urine than
from sodium selenite. Increased tissue selenium levels have been re-
ported by Mahan and Moxon (1978b) and Mandisodza 11; _al. (1979) when
organic selenium sources were fed rather than sodium selenite.

Four trials were conducted to fUrther study the effects of ribo-
flavin supplementation and selenium source on the absorption and metab-

olism of selenium. Also the effects of riboflavin supplementation and

3
selenium source on the glutathione peroxidase system were examined.
Materials and Methods

Eight one-week old pigs weighing 2.84 :t .19 kg (mean 1 standard
error) were randomly allotted to either the riboflavin-deficient diet
shown in table 1 or a riboflavin-supplemented diet. The riboflavin-
supplemented diet (10 mg/kg of diet) was mixed by adding riboflavin to
the selenium chromium premix. In trial one sodium selenite was used as
the selenium source. The riboflavin-unsupplemented diet contained .17
ppm selenium, and the riboflavin-supplemented diet contained .20 ppm
selenium. Pigs fed the riboflavin-supplemented diet were pair fed to
pigs fed the riboflavin-deficient diet. Feeding was done twice daily
and pigs were trained to rapidly consume a mixture Of one part diet and
one part water.

Pigs were weighed and blood samples taken every three days for
twelve days. Erythrocytes were obtained by centrifugation and washed
three times in ice cold saline (.9% NaCl). Erythrocytes were used for
determination of percent active glutathione reductase. After eighteen
days on the diets all pigs were killed. Liver, kidney, heart, muscle,
brain and erythrocyte samples were frozen for glutathione peroxidase
and glutathione reductase activity assays. Liver, muscle, heart, brain
and plasma samples were taken for selenium analysis.

Liver, kidney, heart, muscle and brain samples were homogenized in
isotonic saline and centrifuged to obtain the cytosolic fraction for
enzyme activity determinations. Erythrocytes were hemolyzed in double
strength Drabkins Solution for enzyme assays.

Glutathione peroxidase activity was determined by the method of

Paglia and Valentine (1967) using hydrogen peroxide as the substrate.

TABLE I. COMPOSITION OF THE RIBOFLAVIN-DEFICIENT DIET

Internat'l.

 

Ingredient ref. no. g/kg diet
Casein, vitamin free1 5 01 162 300
Glucosez 4 02 125 471
a-ce11u1ose3 so
Mineral premix4 60
Lard 4 O4 790 so
Vitamin premix5 _3O
Corn oil 4 07 882 10
Selenium/chromium premix6 ._j§1
1000

 

1"Vitafree", United States Biochemical Corporation,
Cleveland, OH.

2"Dextrose 2001", CPC International, Englewood Cliffs,
NJ.

3“Solka-Floc", Brown Co., Berlin, NH.

4Provided per kg diet: 6.0 9 KCl, 1.2 mg KI, 630 mg
Fe504.7H20, 30 mg Cu504, 60 mg COC03, 60 mg

MnSO4.HzO, 240 mg ZnSO4.H20, I.2 g MgCOg,

15.0 g NaHC03, 21.6 g CaHP04.2H20, 7.5 g CaC03,

7.7 g cerelose.

5Provided per kg diet: 3 mg thiamin mononitrate, 40 mg
nicotinamide, 30 mg chalcium pantothenate, 2 mg
pyridoxine hydrochloride, 13 mg p-aminobenzoate, 80 mg
ascorbic acid, 130 mg mygyinositol, 1.3 g choline
chloride, 260 ug folate, 50 ug biotin, 100 ug
cyanocobalamin (0.1% triturate in mannitol), 20 mg D,
L-a-tocopheryl acetate, 40 ug menadione, 1.5 mg retinyl

palmitate, 12.5 mg ergocalciferol.

6Provided per kg diet: 2 mg Cr.

5
Glutathione reductase activity was determined by the method described
by Glatzle e; 31. (1970). Total glutathione reductase activity was
determined by saturating the reaction mixture with FAD and incubating
at 37°C for seven minutes, prior to the start of the reaction. Seleni-
um concentrations were determined by a modification of the Olsen method
(Whetter and Ullrey, 1978).

In trial two, eight one-week old pigs weighing 2.75 2 .15 kg (mean
t standard error) were randomly allotted to a riboflavin-deficient or a
riboflavin-supplemented diet containing 10 mg of supplemental ribofla-
vin per kg of diet. The diets used in trial two were the same as the
diets used in trial one, except the selenium source was DL-selenomethi-
onine (Calbiochem-Behring Corp., LaJolla, CA). The riboflavin-defi-
cient diet contained .50 ppm selenium and the riboflavin-supplemented
diet contained .54 ppm selenium. All other procedures in trial two
were the same as in trial one.

Statistical analysis of data from trials one and two was done as a
two by two factorial design. The main effects were trial and ribofla-
vin supplementation. Selenium source and trial were confounded. Sig-
nificance was determined using the F-test (Gill, 1978a). Erythrocyte
total, active and percent active glutathione reductase activities were
analyzed as a split-plot design (Gill, 1978b).

In a third trial twelve three-week old pigs weighing 5.6 t .13 kg
(mean 4.- standard error) were randomly allotted to a selenium balance
study. The main effects of the study were riboflavin supplementation
and selenium source. The pigs were fed three times per day in individ-
ual feeding cages and were returned to individual collection cages as

soon as they completed the meal. Seventy-five grams of a basal diet,

6
which was similar to the diet in table 1, but contained no supplemental
selenium or riboflavin, were fed each morning. A 25 g meal containing
0.11 mg of selenium as sodium selenite or selenomethione and 0 or 2 mg
of supplemental riboflavin was fed each day at noon. Treatment diets
were mixed by adding riboflavin and selenium to the chromium premix. A
third meal of 50 g of the basal diet was fed in the evening. This
approach was taken to minimize the problems associated with the de-
creased feed intake of pigs on the riboflavin-deficient diets that was
experienced in trials one and two. Each meal was mixed with an equal
part of water and pigs were trained to rapidly consume the slurry.
After a nine day adaptation period a three day total urine and fecal
collection was made on each pig. Feces were dried in a drying oven,
weighed and ground for selenium analysis. Urine was weighed and an
aliquot was taken for selenium analysis. All urine, feces and feed
selenium concentrations were determined by a modification of the Olson
method (Whetter and Ullrey, 1978).

In trial four, sixteen pigs weighing 4.74 t .50 kg (mean i stan-
dard error of treatment means) that had been weaned and started on a
corn-soybean meal diet for one week, were randomly allotted to a 2 x 2
factorial design. The main effects were riboflavin supplementation (0
or 2 mg/day) and supplemental selenium source. Since selenium reten-
tion results of pigs fed riboflavin supplemented diets in trial three
indicated the selenium from the sodium selenite supplemented diet was
only 67% as available as selenium from the selenomethionine supplement-
ed diet, the supplemental selenium intake as selenomethionine was 67%
of the supplemental selenium intake from sodium selenite in trial four

(.12 mg/day as selenomethionine or .18 mg/day as sodium selenite).

7

Total daily selenium intakes were .13 mg and .19 mg respectively for
pigs fed the selenomethione supplemented diet and the sodium selenite
supplemented diet, respectively. Pigs were fed for fourteen days by
the system described in trial three. 0n the fifteenth day all pigs
were bled, and serum samples were obtained for selenium analysis and
erythrocytes were obtained to determine the percent active glutathione
reductase. Pigs were then killed and liver, muscle, kidney, heart and
brain samples were frozen for selenium, glutathione peroxidase activity
and glutathione reductase activity determinations. The methods of
determination were the same as those described in trial one.

Data from trials three and four were analyzed as 2 x 2 factorial
designs (Gill, 1978a). The main effects were selenium source and ribo-
flavin supplementation.

Results and Discussion

Trials one and two. Pigs. fed diets supplemented with 10 mg of
riboflavin per kg of diet gained significantly faster (P<.01) and were
heavier than pigs fed the riboflavin-unsupplemented diets after eigh-

teen days on the trial (P<.08). Pigs fed the riboflavin-unsupplemented
idiets gained 74.3 g/day, while pigs fed the riboflavin-supplemented
diets gained 115.5 g/day (see table 2). Krider e_t_ 21. (1949) and
Forbes and Haines (1952) reported an increase in gains of starter pigs
with riboflavin supplementation of diets. Miller et_gl, (1954) report-
ed an increase in gain with increasing riboflavin supplementation up to
3 mg/kg. Feed efficiency was improved by riboflavin supplementation in
studies of Krider 2331. (1949) and Miller £21: (1954). Terrill e3;

a1. (1953), however, found a more variable response in feed efficien-

cy.

.—c.v¢ .uumuyo :.>e_mon_e poucmsmpaazmo
.mc.va .uommmo :_>epuonpg poucmEm—aasmn

.mcems acmEHemeu mo gouge ugeuceume

 

 

 

 

 

 

~.~ m.m__ m.¢~ o.-_ _.m~ o.mo_ e.c~ sae\a .o=_aa »_.ae aaaaa><

an. mm.a ~_.e mo.m No.4 no.4 m~.a as .auem_as Pae.a

AN. -.~ mm.~ aw.~ Ne.~ c~.~ ma.~ as .ugm.az .a.»_=_

a.z.m.m c_ o o. o c. Ammumsvc sag.
Haunt; ego—“woe... 556:9“: :26 :2. T.

.eucmEmpqqsm cum: paaemEo—aqam paucoeo—qaam

- pn—LH _ _u.sh

 

mama no mz~<w oz<
hzoumz >aom zc z—><4uom—a 4<hzuzm4¢m=m mo hummum uzh .N u4m<h

9

Erythrocyte active glutathione reductase values (table 3) and per-
cent active glutathione reductase values (table 5) were lower (P<.01)
for pigs fed the riboflavin-unsupplemented diets. This depression in
activity has been described in rats (Glatzle e3; a_1_., 1968), humans
(Glatzel et_gfl,, 1970) and pigs (Brady et_afl,, 1979). However, Brady
e1; 21. (1979) reported that erythrocyte percent active glutathione
reductase plateaued at 75% when pigs were fed 4 mg of riboflavin per
kilogram of diet. The values reported here for pigs fed the supple-
mented diet appear to be lower than that value and are considerably
lower than the 80% value suggested by Glatzle et_gfl, (1970) as a mini-
mum percent active erythrocyte glutathione reductase for human subjects
on riboflavin-sufficient diets.

There was a decline in total (P<.01) (table 4), active (P<.01)
(table 3) and percent active (P<.02) (table 5) erythrocyte glutathione
reductase activity, however, more importantly there was an interaction
of time and riboflavin supplementation for active (P<.01) and percent
active (P<.09) erythrocyte glutathione reductase activity. During the
course of the study active erythrocyte glutathione reductase activity
was maintained close to the initial activity for pigs fed riboflavin-
supplemented diets, but active erythrocyte glutathione reductase activ-
ity was lowered by feeding unsupplemented diets. The percent active
erythrocyte glutathione reductase generally declined throughout the
study for pigs fed the riboflavin-unsupplemented diets, while values
for pigs fed riboflavin-supplemented diets did not change or increased
slightly. These results were expected since erythrocyte active gluta-

thione reductase activity in the pig has been shown to be responsive to

10

TABLE 3. THE EFFECTS OF SUPPLEMENTAL RIBOFLAVIN AND DAYS ON TRIAL
ON ACTIVE ERYTHROCYTE GLUTATHIONE REDUCTASE ACTIVITYa OF PIGS

 

 

Trial I Trial II
Days Supplemental Supplemental Mean supplemental
on riboflavin riboflavin riboflavin effectb,e
triaibce O(mg/kg1 10 O 10 0 10 Mean
0 16.4 16.6 13.7 12.4 15.1 14.5 14.8
3 7.3 11.8 7.8 12.7 7.6 12.3 9.9
6 7.7 13.1 7.4 10.6 7.6 11.9 9.7
9 6.9 16.2 6.9 14.3 6.9 15.3 11.1
12 3.7 12.4 4.7 10.7 4.2 11.6 7.9
Mean 8.4 14.0 8.1 12.1 8.3 13.1

 

aExpressed as micromoles of glutathione reduced/minute/mg of
hemoglobin.

bStandard error of treatment means = .67; standard error of period
means a .66.

cTime on trial effect, P<.01.

dSupplemental riboflavin effect, P<.01.

eSupplemental riboflavin x time interaction, P<.01.

ll

TABLE 4. THE EFFECTS OF SUPPLEMENTAL RIBOFLAVIN AND DAYS ON TRIAL ON
TOTAL ERYTHROCYTE GLUTATHIONE REDUCTASE ACTIVITYa OF PIGS

 

 

 

Trial I Trial II
Days Supplemental Supplemental Mean supplemental
on riboflavin riboflavin riboflavin effectb
trialbic 0(mg/kg) 10 0 10 O 10 Mean
O . 26.2 26.3 25.3 21.6 25.8 24.0 24.9
3 14.8 19.5 17.3 17.5 16.1 18.5 17.3
6 . 19.6 18.9 17.6 14.0 18.6 16.5 17.5
9 17.6 28.6 16.8 21.4 17.2 25.0 21.1
12 20.0 21.9 25.1 19.5 22.6 20.7 21.6
Mean 19.6 23.0 20.4 18.8 20.1 20.9

 

aExpressed as micromoles of glutathione reduced/minute/mg of hemoglobin.
bStandard error of treatment means a 1.4; standard error of period
means a 1.3.

cTime on trial effect, P<.01.

12

TABLE 5. THE EFFECTS OF SUPPLEMENTAL RIBOFLAVIN AND DAYS ON TRIAL ON
ERYTHROCYTE PERCENT ACTIVE GLUTATHIONE REDUCTASEa OF PIGS

 

 

 

 

Trial I Trial II
Days Supplemental Supplemental Mean supplemental
on riboflavin riboflavin riboflavin effectbad’e
trialbicie 0(mglkg) 1O 0 10 0 10 Mean
0 62.9 65.0 54.9 57.7 58.9 61.4 60.2
3 50.9 62.3 46.7 72.9 48.8 67.6 58.2
6 41.9 69.5 42.2 75.3 42.1 72.4 52.3
9 39.9 68.2 45.3 67.7 42.6 68.0 55.3
12 19.2 57.2 18.0 57.0 18.6 57.1 37.9
Mean 43.0 64.4 41.4 66.1 42.2 65.3

 

aA11 values expressed as percent.

bStandard error of treatment means = 2.4; standard error of period
means a 4.2.

cTime on trial effect, P<.02.

dSupplemental riboflavin effect, P<.01.

eTime on trial x supplemental riboflavin interaction, P<.09.

13

dietary riboflavin (Brady et_gl., 1979). Furthermore, it is interest-
ing to note the speed with which the erythrocyte active glutathione
reductase activity responds to the lack of dietary riboflavin. Baker
3; al. (1966) reported that the clearance of circulating riboflavin
was very rapid. In subjects judged to have adequate circulating ribo-
flavin, the half-time for clearance of a 5 mg dose of riboflavin was
fourteen minutes, but subjects with low circulating riboflavin cleared
half of the dose in six unnutes. They indicated that the increased
clearance in deficient patients was due to greater avidity of tissues
for administered riboflavin.

Riboflavin supplementation significantly decreased actual liver
weight (P<.07) and liver weight as a percent of body weight (P<.01)
(table 6). Also, pituitary weight was decreased (P<.08) by riboflavin
supplementation. Heart (P<.03) and brain (P<.02) weights were increas-
ed by supplemental dietary riboflavin, but when expressed as a percent
of body weight there was no consistent trend.

Supplementation of diets with riboflavin resulted in a 22% in-
crease (P<.12) in liver glutathione peroxidase activity (table 7) and
an increase in erythrocyte glutathione peroxidase activity (P<.02).
However, riboflavin supplementation lowered kidney (P<.04) and nuscle
(P<.08) glutathione peroxidase activity. Glutathione peroxidase activ-
ity of heart and brain were not affected by level of riboflavin in the
diet. Plasma selenium concentration (table 8) was decreased (P<.05) by
riboflavin supplementation, while liver (P<.11) and heart (P<.01)
selenium concentrations were increased. The selenium concentration of
muscle and brain were not affected by riboflavin supplementation. It
is interesting to note the interaction (P<.08) of trials and dietary

riboflavin on heart selenium concentration. Pigs in trial two which

14

TABLE 6. THE EFFECTS OF SUPPLEMENTAL RIBOFLAVIN ON THE ACTUAL HEIGHT

ADD PERCENT OF BWY HEIGHT OF SELECTED GIGANS

 

 

 

 

Trial 1 Trial 11
Supplemental Supplemental Mean supplemental Slgnlflcance
riboflavin riboflavin riboflavin effect of rlboflavln
13:; MM 10 0 10 0 IO effect S.E.M.°i
leer
weight,g 112.0 88.2 117.0 101.0 114.5 94.6 .07 10.1
1 of body weight 2.72 1.90 2.92 2.03 2.82 1.97 .01 .32
Kidney
welght,g 31.3 30.6 28.5 35.2 29.9 32.9 . 18 2.2
1 of body weight .75 .66 .70 .71 .75 .69 -- .05
Heart '
weight,g 20.7 22.5 19.6 26.4 20.2 24.5 .03 1.8
1 of body "1911+ .49 .48 .48 .55 .49 .51 .11 .01
Brain
weight,g 43.6 46. 1 42.2 47.2 42.9 46.7 .02 1.5
1 of body «1911-1 1.05 1.00 1.05 .95 1.05 .98 .28 .06
emu-ow
welght,mg 55.8 38.8 51.3 50.5 ' 53.6 44.7 .08 4.8

 

8Standard error of treatment means.

15

TABLE 7. THE EFFECT OF SUPPLEMENTAL RIBOFLAVIN ON LIVER, KIDNEY, MUSCLE, HEART,

BRAIN AND ERYTHROCYTE GLUTATHIONE PEROXIDASE ACTIVITY

 

 

 

Trial 1 Trial 11

Supplemental Supplemental Mean supplemental Significance

riboflavin riboflavin riboflavin effect of riboflavin
L117. 0%) 10 0 10 0 10 effect S.E.M.'
Limb 1.68 2.06 1.71 2.07 1.70 2.07 .12 .25
Kidney” 1.55 1.26 2.59 1.19 1.96 1.25 .04 .54
Musclec 179.0 76.2 161.0 . 74.3 170.0 75.3 .08 16.4
Heartc 257.0 264.0 453.0 373.0 355.0 318.5 - 84.3
Brainc 497.0 285.0 567.0 611.0 532.0 448.0 - 62.8
Eryi-nroeyi-od 11.4 16.8 15.5 27.4 12.4 22.1 .02 5.7

 

5Standard error of treatment means.

bValues expressed as milllmoles of glutathione oxidized/minute/mg of protein.

cValues expressed as micromoles of glutathione oxidized/minute/mg of protein.

dValues expressed as micromoles of glutathione oxidized/minute/g of hemoglobin.

16

TABLE 8. THE EFFECT OF SUPPLEMENTAL RIBOFLAVIN ON LIVER, MUSCLE, HEART, BRAIN

AND PLASMA SELENIUM CONCENTRATION OF PIGS

 

 

Trial 1 Trial 11

Supplemental Supplemental _ Mean supplemental Significance

riboflavin riboflavin riboflavin effect of riboflavin
142 01mg) 10 O 10 o 10 effect 5.6.14.a
Liver, ug/gb 1.05 1.75 .77 1.18 .91 1.47 .11 .46
M08610, ug/gb .06 .06 .08 .08 .07 .07 - .07
Heart. 149/9" .25 .27 .25 .56 .24 .52 .O1 .02
Brain, pg/gb .21 .22 .24 .24 .25 .25 -- .09
Plasma ml .18 .15 .17 .15 .18 .15 .05 .01

 

aStandard error of treatment means.

bNet tissue basis.

17
were fed selenomethionine as a supplemental selenium source tended to
accumulate selenium in heart tissue when the diet was supplemented with
riboflavin.

Erythrocyte percent active glutathione reductase was increased
(P<.01) by feeding the riboflavin-supplemented diets for eighteen days.
Values at the end of the trial for supplemented pigs are similar to
values reported by Brady gt 21. (1979) for pigs on an adequately sup-
plemented diet (table 9). Also, brain glutathione reductase activity
was increased (P<.04) by dietary supplementation with riboflavin. In
trial one muscle glutathione reductase was not affected by riboflavin,
but in trial two when selenomethionine was fed, muscle glutathione
reductase activity was decreased (P<.05) by riboflavin supplementation.
Heart glutathione reductase activity was elevated when diets were sup-
plemented with riboflavin in trial one, but activity was depressed by
supplementation in trial two (P<.01).

The effects of riboflavin supplementation on liver selenium con-
tent, glutathione peroxidase activity and glutathione reductase activi-
ty are generally in good agreement with the trends reported by Brady et_
.11. (1979). They reported more variable results with these parameters
in muscle but these current data appear to give trends similar to those
which they reported.

Trial three. During the three day collection period selenium in-
takes ranged from 110 ug/day to 116 ug/day (table 10). There was no
effect of either dietary treatment (supplemental riboflavin or selenium
source) on fecal selenium concentration and fecal excretion, and con-
sequently apparent selenium absorption was not affected. Urinary

selenium concentration (P<.03) and urinary excretion (P<.01) were

18

TABLE 9. THE EFFECT OF SUPPLEMENTAL DIETARY RIBOFLAVIN FOR EIGHTEEN DAYS 0N LIVER,
KIDNEY, MUSCLE, HEART AND BRAIN GLUTATHIONE REDUCTASE ACTIVITY AND PERCENT

ACTIVE ERYTHROCYTE GLUTATHIONE REDUCTASE

 

 

 

Trial 1 Trial 11
Supplemental Supplemental Mean supplemental Significance
riboflavin riboflavin riboflavin effect of riboflavln

3;; 01m) 10 O 10 O 10 effect 5.13.11.a
Glutathione reductase activity, micromoles of glutathione reduced/mg protein/minute
Liver 307 323 265 305 286 314 .32 27
Kidney 300 342 316 275 308 309 - 28
Muscle 6.10 6.12 7.42 5.31 6.76 5.72 .06 .51
Heart 1 98 I34 131 101 115 118 - 11
Brain 42.1 50.2 38.8 48.4 40.5 49.3 .04 4
Percent active glutathione reducatase, S
Erythrocyte 41.6 70.4 23.7 71.8 32.7 71.1 .01 6.9

 

8Standard error of treatment means.

19

.233! ace-ice: .o .358 953.32.?

 

Q0530; a 28.35.?

essence a 23.39:.

a: 33.3.... tact:

a .055:

a: .8332: .63..

a .0085

 

 
 
 

 

5:88...

6.. .o.o ..S 6.6. 8.6 6.8 a... o... .8. o... 6.8 62...... 5...... 68.6... .6 n
~ .. .o.o . .6... .3: .o.o flan o... a... fie... . .3 6.2
..~ 3 .o 6.3 .4... 6. .o a .8 e... o... n... a... ~.8
3.: .o.o n. .8. 2.2. 8.6 2.... 8.6.... 8.63 8.2. 3.3. 8.2 6.. .635... 3...... .53....
3... a .o.o 6.... 3.2. .o.o 8.... a. .2. 8.... 2.8. 3.... o. .8...
6.6. 8.6 .8. 2.. 1 .8. ~66. .8. .8. 3.. 8.. 3.... 823.5668 3.6.... 2......
n .8. l e .: : N .68. c .o 6.8: 6.38 18.. 6.86. s ..-. p .ER
2.. 1 .o..o~ 2.2.... 8.6 via 2.68 8.8» 3.8... 8.2m :68 6.. .635... 63.6.... .33....
8.. 3.6 6. ... 2 .8 o. .o 8.3 8... .e... a. .9 8.8 o. .8
3. a... $.~ 66.. 8.6 8.. 8.~ a... 8.. 8.. 88 ox... .8.._......o..8 5...... .66..
6.. 1 6.3 «.2 I oi a...“ a..." ~..~ fig 13
2;: 8....» 3.3. 8.8. 63.3.2... 5...... .26.
a. n. a. 2 a .6368. .o.o 22......
a: n: a: a: o .3828 .o.o .3...
.33..
.88.:5: 8.8...6: ~ 6 n o a o 2...}... 52...... .3553...
.6 .2... £4.44.“ 1.1.4.3”. .6 .3... 6.... 5...: . 1431 .366. 3.5.2..
16...... 5.66... 3.8.3.. I... 6...... 3.66...

woe. .e e033. 3....

 

 

20
generally decreased when selenomethionine was used as the selenium
source, instead of sodium selenite. As a result more selenium was re-
tained (P<.Ol) when selenomethionine was used. It appears from these
data that more of the absorbed selenium from selenomethionine is in-
corporated into tissues than from sodium selenite.

Groce £5 £1. (l973a) reported that a smaller proportion of ingest-
ed selenium was excreted in urine when seleniferous corn was fed as a
selenium source rather than sodium selenite. Th1 both studies higher
fecal selenium losses were reported when seleniferous corn was fed
rather than sodium selenite. However, Groce gt al, (1973a) reported a
decrease in fecal selenium loss when the seleniferous corn diet was
supplemented with vitamin E.

The selenium content of muscular tissues was higher in pigs fed
diets in which the selenium sources were distillers grains or fish meal
than in pigs fed corn-soybean meal diets containing sodium selenite
(Mahan and Moxon, l978b). Mandisodza get 31. (1979) reported higher
kidney, liver and skeletal muscle selenium concentrations when seleni-
ferous white clover was fed rather than sodium selenite. The metabolic
significance of the higher retention of selenium from organic sources
is not clear, since Wang and Spallholz (l980) reported that selenometh-
ionine restored rat liver glutathione peroxidase activity much more
slowly than selenite or high selenium yeast in selenium depleted rats.
Yet, Schwartz and Foltz (1958) reported that sodium selenite and D-L
selenomethione had similar factor 3 activity in rats. Recently, Sunde
and Hoekstra (l980a) have shown with perfused rat liver that selenite
and selenide are metabolically closer than selenOCysteine to the

imnediate selenium precursor used for glutathione peroxidase synthesis.

2l
In another study Sunde _e_t_ g_l_. (l981) reported that low dietary methi-
onine levels decreased the biopotency of selenomethionine in rapidly
growing rats.

Riboflavin supplementation did not significantly affect the ap-
parent absorption of selenium. However, it did decrease the urinary
selenium loss (P<.09) and concomitantly increased the apparent reten-
tion of selenium (P<.06) (table l0). Riboflavin supplementation ap-
peared to improve the efficiency of selenium incorporation into body
tissues. Tappel (l978b) has proposed that glutathione reductase re- d-
uces selenite to hydrogen selenide, which is then incorporated into
protein to form glutathione peroxidase. Since FAD is a cofactor for
glutathione reductase, this mechanism may be responsible for the in-
crease in selenium retention when diets were supplemented with ribofla-
vin. However, since Tappel (1978b) has suggested that selenomethionine
is converted to selenocysteine, before being incorporated into glutath-
ione peroxidase and since there was no significant interaction of ribo-
flavin supplementation and selenium source on selenium retention, it s-
eems unlikely the increased selenium retention is due strictly to ribo-
flavin stimulated glutathione peroxidase synthesis.

Trial four. In trial four the pigs fed the riboflavin-unsupple-
mented diets did not show the expected growth depression (table ll) as
was reported by Krider _e_t_ ll, (l949),8Forb.es and Haines (l952) and
Miller g_t__a_l_. (1954). However, Terrill gta_l_. (1953) did report an
inconsistant growth response to riboflavin supplementation- Although
the erythrocyte percent active glutathione reductase of pigs fed the
riboflavin-unsupplemented diets was lower (P<.03) than that of pigs fed
the riboflavin-supplemented diets (table 12), the values for the

22

omCDOE #cflflfiflOLh *0 LOBBO fibflfl:fl#mfl

 

 

.o. .N. ... .o. .c. .o. .=._.. .eoa .o u

.N._ ._. N.. .... a... a... a... . ..z..o.
.. _ 95

No. e.. N.. co. 0.. .e. 0.. _e. .3.... x... .o u

..N .N. .N. ..NN ..NN ...N ..NN . .«g....
t3:

.o. .o. .n. N.. N.. .e. ... .z...x .eo. .o u

..N .o. .._. a... _..N N.NN . ..g...z
>ocv.x

«o... .c.N oo.N ..._ .o.N +5.... .eon .o u

0.. - .N. oN. .N. N_. e_. . ..g....
.a...
a... .N. N.N. h... o... N... . .c... >...e o....><
N.. .o.o o... n... o... .. .+..... .o.. .o...
o.. .N.. .s.. N... n... .x ..g.... .o.o .o...=.
..z.... =o_.o. .o.... +0.... N o N .ummxmr. o s...

1cohc_

cacao. =_>o..oa_¢

 

 

cocoa.._cu.m yo .o>o.

5.533.... 2.28.033

.>..\.. N... .=_=o.=..eo=o...

 

=—>fl_bOO—L .0fiCOIO_mm3m

.>.u\.a a... .+.=..o. 23....

 

 

oxohc. newlw0530mimm.co_om

 

 

.o.. .o .p:a.uz z<a¢c ou»0.... az< .z.<. .pzoqw: >oo. 2o zo.»<hzuxm..¢:. z.><..oo.¢ mz<

.z.zo.=..zczm... mo m..:.... 23.... sag. ...<»z. 25.2.... m..<._<>< .(20. :p.: .z.pzwxm...=. .o how... .1. ... m..<»

23
unsupplemented pigs were higher in this trial than in trials one and
two, thus, indicating that the pigs on the unsupplemented diet in this
study may not have been as depleted of riboflavin at the end of the
trial as pigs in trials one and two.

Liver and brain actual weight and as a percent of body weight were
not significantly affected by either dietary treatment '(table ll).
Kidney weight (P<.08) and as percent of body weight (P<.06) was in-
creased when selenomethionine was fed rather than sodium selenite.
Heart weight was not affected by treatments, but as a percent of body
weight heart was heavier for pigs fed the riboflavin-supplemented diets
(P<.06). These tissue weight results show a less severe effect of
feeding the riboflavin-unsupplemented diet than was reported in trials
one and two.

Erythrocyte percent active glutathione reductase was increased by
riboflavin supplementation (P<.03). Heart glutathione reductase activ-
ity tended to be increased by riboflavin supplementation (P<.l4), but
liver, kidney, muscle and brain activities were not affected (table
l2). This is similar to the results of Brady §t_gl, (1979) for eryth-
rocytes, liver and muscle. The selenium source fed did not affect
glutathione reductase activities in any of the tissues that were
assayed.

There was no significant effect of either dietary treatment on
tissue glutathione peroxidase activity. However, there were interac-
tions of selenium source and riboflavin supplementation for kidney
(P<.03), muscle (P<.03), heart (P<.08) and brain (P<.Ol) indicating

that riboflavin supplementation increases incorporation of selenium

24

.o~:c.l\vo~.v.xo .59.: *0 0.3.9.0.! . n a...

6:3... 20.5.3.3 .0 note ugoucmemo

 

 

o.n no. o.n~ ..~o n... _.oo u .o>_.oo u
3.60.213
~.. on. o.n. o... c.n_ n.e. ac.o.oca axau .=.ncm
w «n. «a. e.. n.an n.en o.vn o.nn a=_o.oca m\=u ..cooz
on. an.o n... no.0 an.o ac_o.oca mxzu .o.omaz
a. ”pm own man .mc ae.o.oca axam ..o=e_x
nu o~. om. ~o¢ man .c. ~.¢ ne.o.0ca mxzu .co>..
.136 .628 +8.... «8.... N o a 2.3de a 3:

aco.=. oocaom =.>a..oa_¢

 

 

00:3. . .cflw .o .o>o..

53:3... 350-833

.>oe\aa a... o=_=o_g.oeo=o.om

 

5.523... 320.33%

.xoe\ma o... o+_=o.om u:_eom

 

awry... 9.... 8.53 1:13:23

 

 

«0.... “.0 wm<h§u¢ 30.32.53 m>.ho< hzmomwn. 9:85....»5 g

.r...>....o< wgbgaz 9.0.1.2236 22$ 2 .529. £483. .>mzo.v. 33>... 98 8..p<...z!m..n.n5m z.><.....om.¢ g

3.5.ngdm m5 w.—.zw..wm 13.8w g... 9.3.2. Izzudm w§.<>< ..<...Om 1...... 9:23.; “.0 whommmm 9.... .N. w..m<.p

25
from sodium selenite into glutathione peroxidase (table l3). As dis-
cussed earlier, Tappel (l978b) has suggested that the flavoprotein
glutathione reductase is involved in glutathione peroxidase synthesis
from sodium selenite. The interaction observed here of selenium source
and riboflavin supplementation for kidney, muscle, heart and brain
tends to support the hypothesis that glutathione reductase is involved
in the incorporation of selenium from sodium selenite into glutathione

peroxidase, since riboflavin supplementation increased tissue 1 vitro

 

glutathione peroxidase activity when sodium selenite was used as the
supplemental selenium source, but did not increase _i_r_i_ vitro glutathione
peroxidase activity when selenomethionine was fed as the supplemental

selenium source. I vitro glutathione reductase activity of these

 

tissues was not greatly affected by riboflavin supplementation, how-
ever, Baker _e_t_ a_l.. (l966) reported that tissues avidly take up circu-
lating riboflavin when tissue levels are low and that tissues are very
slowly depleted of riboflavin when a deficient diet is fed. Conse-
quently, tissue riboflavin levels would be expected to decline more
slowly than circulating‘riboflavin levels when a riboflavin-deficient
diet is fed. This was observed in this study as the percent active
glutathione reductase was depressed (P<.03) when the riboflavin-unsup-
plemented diet was fed to the pigs, however, other tissue (liver, kid-
ney, muscle, heart and brain) glutathione reductase activities were not
affected by riboflavin supplementation. From these results it appears
that glutathione reductase may be involved in the incorporation of
selenium from sodium selenite into glutathione peroxidase.

Tissue selenium concentrations were not affected by riboflavin

supplementation (table l4). Muscle (P<.Ol), heart (P<.05) and brain

26

(P<.Ol) selenium levels were higher for pigs fed diets with selenometh-
ionine rather than sodium selenite as the selenium source. In trial
three the selenomethionine-supplemented diet with supplemental ribo-
flavin was 1.5 times more effective as a selenium source, based on
selenium retention, than the sodium selenite supplemented diet with
supplemental riboflavin. In this trial the selenium levels of the
sodium selenite-supplemented diets were l.5 times the selenium levels
of the selenomethionine supplemented diets. The selenium levels in
muscle (P<.05), heart (P<.Ol) and brain (P<.Ol) for pigs fed seleno-
methionine supplemented diets are higher than those fed sodium selenite
supplemented diets. Mahan and Moxon (l978b) and Mandisodza M.
'(l980) have also reported higher tissue selenium concentrations when
pigs were fed organic selenium sources rather than sodium selenite.

Since glutathione peroxidase activities for muscle, heart and
brain were not increased when selenomethionine was fed as the supple-
mental selenium source rather than sodium selenite, it appears that
selenomethionine must be incorporated into some other protein in these
tissues. Black _e_t_ _a_l_. (l978) have identified two selenium containing
proteins in ovine heart, muscle, kidney and liver, but have reported
that glutathione peroxidase activity was only associated with one of
these peaks. Beilstein gt g_l_. (l980) have separated three selenium
containing proteins from lamb heart and muscle. Based on chromato-
graphic results the selenium in these proteins appears to be seleno-
cysteine. Excess selenomethionine may be converted to selenocysteine
and incorporated into proteins other than glutathione peroxidase in

some tissues. This could account for the increased tissue selenium

27

.o...:.....\vo~.e.xo InE<z «0 0.3.9.0.! . n awn

amp—08. 53.08....ng *0 LOLLO 9L0930#w0

 

n.~ Nn. ..o~ ~.a~ n.e~ ~.a~ no=.o.0ca mxzm .sacom

 

 

 

n.c~ _o. n..~ o.no. n.ao o..n n=_o.oca mxnm .:_ocm

o.vn cc. n.oo o.~n. o.co. n.mn a=.o.oca mxaw ..coo:

_. me. .~. “.on o.°a n.c~ n..n ac_o.ota mxam .o.omaz

on. ma. ow. can ace. n~. nae ac_o.ota a\=u .>o=e_z

no. ope. sea a... cam nc_o.oca m\=m .co>_.

4...”..5 cozoo +0030 +0030 N c N 333 a so:
-to.=. outaom =.>o..oa.¢ c.>o..oa_c _mweoeaqmmmm. =_>a..oa_c .o.=oeo.mmmm.

 

ou:oo...mm.w .o .oso. .xooxas N... oc_=o.g«oeo=o.om .xoe\as a... o._=o.om ¢=.eom

 

3.3.... can 3.50» “3.3.3

 

no: “.0 >....>....Q<
mm<e§omwm 2.55.3.5 gwm 92 225 J»?! @409! £363. .¢m>... 3 3.h<hz!m..gm z.><.....om.¢ 92

w:.~5.:...§m..wm 8 m...zm...wm 3.8m 20¢". avg—.2. taxmamm w§.<>< 23m 3....) 9.32%.; no whommum. w...... .n. ugh

2&3

..a\a: c. mco..o.+:oo:ooo

.oamm.e eon .0 m\m: c. mco.+o.+:oo=oo:

.mcoae +c02+00e+ .o Lento acmvco+mo

 

 

.o. 2. t. 2. 2. enema...
.o. .o. .o. .N. mu. 2. t. .58..
No. 3. ow. on. E. ea. ate...
3. .c. 2.. c. . n. ._ N. . o. . .23....
8. 5.. z... 8.~ no.~ .1263.
8. No. no. 8. 2. A1.2,...
o.:.m.m 8:8 «8.... 8...... N o a 28%... c 8:

leoec.

8.8.... 53:8...

 

 

 

cocoo.*.:m.m .0 .o>o.

55.8.. 3.53%....

2.33... a. .. 2.8.5822...

 

c.>o.«on.. .oecoeo.mmmm

162...: a... 3.5.8 .533

 

030+:. ago oommwa 23.:o.om

 

mo.m mo

mzozéng 1:23.... .35.. 92 225 .23: .38.... .55... .52.. .8 3.25%....3 2:5..8... a...

mzo.:hmzozm.wm mo mh.zm.mm 2:.ocm 26mm mx<Hz. 1:.zwdwm wam<..<>< .(30m 1h.) mz.hzwxm.m¢:m mo mbOummm wzb .v— w4m<h

29
concentration without increased glutathionine peroxidase activity ob-
served in heart, muscle and brain of pigs receiving selenomethionine in
this study.

The lack of clear cut riboflavin deficiency signs in trial four
may be due in part to the fact that these pigs were fed a starter diet
containing 10% dried whey product for one week after weaning. The
calculated riboflavin content of this diet is 7.5 mg/kg, which is con-
siderably higher than the 3 mg/kg recommendation of NRC (l979). Fur-
thermore, the loss of riboflavin from the body once the animal is
placed on a riboflavin-deficient diet appears to be quite slow (Baker
‘gt.al:, l966).

In conclusion, erythrocyte percent active glutathione reductase
appears to be a sensitive indicator of riboflavin status of the pig.
Values below 50% are needed before other riboflavin deficiency signs
become evident. Riboflavin supplementation of diets can increase the
retention of absorbed selenium and it nay aid in the incorporation of
selenium from sodium selenite into glutathione peroxidase.

A higher percent of the selenium consumed as selenomethione was
retained and a larger proportion of the absorbed selenium was incorpo-
rated into tissues than selenium from sodium selenite. Feeding seleno-
methionine as the supplemental selenium source at 67% of the level of
selenium from sodium selenite resulted in higher muscle. heart and'
brain selenium concentrations than feeding the higher level of selenium

as sodium selenite.

SECTION II
Introduction

The metabolic role of selenium as a component of glutathione per-
oxidase was elucidated in 1972 by Rotruck gt_gl. Glutathione peroxi-
dase catalyzes the reduction of hydrogen peroxide and lipid peroxides
to water (Mills, 1959) and hydroxy acids (O‘Brien and Little, 1967),
respectively. The active center of the molecule contains four seleno-
cysteine moieties (Ludenstein _e_t_ 31., 1979). The major'portion of
erythrocyte selenium has been reported to be associated with the gluta-
thione peroxidase molecule (Rotruck g§_gl,, 1973; Oh g§_al,, 1974; and
Sunde §§.gl:, 1978). Sunde g§_3l, (1978) have shown that only 10% of
ovine liver selenium was associated with glutathione peroxidase.
Wegger‘gt.al, (1980) noted that there was a highly significant positive
correlation between selenium concentration and glutathione peroxidase
activity in pig liver, but not in pig kidney. Pig liver (Young gt 21,,
l977b; McDowel gt 21,, 1977; Mahan gt.gfl,, 1977; and Mahan and Moxon,
l978a), muscle (Groce e_t_ _a_l_., 1971; Ku _e_i; _a_l_., 1972; Mahan gt 31.,
1977; Young gt_gl,, l977a; and Mahan and Moxon, 1978a), kidney (Mahan
gt ad,, 1977; McDowell gt gfl,, 1977; and Mahan and Moxon, 1978a) and
blood (Mahan g 31., 1977; McDowell g _a_l_., 1977; and Meyer _e_t_ _al.,
1981) selenium concententrations have been shown to respond to dietary
selenium intake. Using rat repletion assays Hoekstra gt _a_l_. (1973)
reported that liver glutathione peroxidase activity responds to supple-
mental dietary selenium, but that enzyme activity plateaued at 0.1 ug/g
of selenium of diet. Also, they found that erythrocyte glutathione
peroxidase activity was restored at a rate consistent with the life

span of the erythrocyte. Using similar techniques Wang and Spullholz

3O

31
(1980) reported that rat liver glutathione peroxidase was responsive to
dietary selenium intake. Scott and Naguchi (1973) and Bunk gt a],
(1980) found that plasma glutathione peroxidase activity of chickens
responded to dietary selenium concentration. Chow and Tappel (1974)
and Smith gt 31, (1974) have shown plasma glutathione peroxidase to be
the most sensitive measure of selenium intake in rats. Gabrielsen and
Opstvedt (1980) have developed a selenium bioavailability assay in
chicks using plasma glutathione peroxidase as a measure of selenium
availability. This assay requires only nine days to complete because
of sensitivity of plasma glutathione peroxidase activity to dietary
selenium intake. However, little work has been done to study the re-
sponsiveness of pig serum and erythrocyte glutathione peroxidase to
supplemental selenium intake. The trials reported here were designed
to study the responsiveness of serum glutathione peroxidase to dietary
selenium levels. In addition, the effects of environmental temperature
and weaning stress on performance and serum glutathione peroxidase
activity were evaluated.
Materials and Methods

Trial V. Fifteen four-week old pigs which weighed 7.20 1' .46
kg (mean * standard error) were randomly allotted to three dietary
treatments. The pigs used in this study were farrowed and raised by
gilts which had received no supplemental selenium or vitamin E since
the gilts were weaned. The basal (B) selenium-unsupplemented diet is
shown in table 15. Additions of 0.1 (B+.1) or 0.2 (B+.2) ug of seleni-
um as sodium selenite per g of diet were made in place of cerelose.
The selenium levels of the diets are shown in table 16. The pigs were

housed in raised stainless steel decks in groups of five pigs per pen.

32

TABLE 15. BASAL DIET FOR TRIALS V AND VI

 

 

ingredient Amount
Ground shelled corn (IFN 4-02-931) 736.0
Soybean meal, dehulled (IFN 5-04-612) 200.0
Synthetic lysine (78.4% L-lysine) 2.0
Mono-dicalcium phosphate (18.5% Ca, 21% P) 14.0
Calcium carbonate (38% Ca) (IFN 6-01-069) 12.5
Vitamin trace-mineral premixa 5.0
Salt 3.0
Antibotic premixb 2.5
Vitamin E premixc 5.0
Glucosed (IFN 4-02-125) __2_o_,_q
1000.0
Calculated analysis _jL_
Lysine .96
Methionine-cystine .58
Tryptophan .17
Calcium .81
Phosphorus .60

 

aSupplies 3,300 IU vitamin A; 660 IU vitamin D; 3.3 mg
riboflavin; 13.2 mg d-pantothenic acid; 17.6 mg niacin; 19.8

ug vitamin 812; 126.7 mg choline chloride; 2.2 mg

menadione sodium bisulfate; 74.8 mg zinc; 59.4 mg iron; 37.4

mg manganese; 9.9 mg copper; and 2.75 mg iodine per kg of

diet.

bProvides 110 mg chlortetracycline, 110 mg sulfamethazine and

55 mg penicillin per kg of diet.
cSupplies 11 IU vitamin E per kg of diet.

d"Dextrose 2001," cpc International, Englewood Cliffs, NJ.

33
Feed was available free-choice from a self-feeder and water was
available at all times.

The pigs were weighed and blood samples taken at 0, 14 and 28 days
of the study. Erythrocytes were washed three times in ice cold saline
(.91 NaCl). Erythrocyte and serum glutathione peroxidase activity was
determined by the method of Paglia and Valentine (1967). Plasma selen-
ium levels were assayed by a modification of the Olson method (Nhetter
and Ullrey, 1978). Serum tocopherol and tocopherol acetate were
determined by the high pressure liquid chromatographic method of
Hidicus and Kirk (1979). On the twenty-eighth day of the trial hema-
tocrit and hemoglobin values were also determined. Using these values,
the body weight, enzyme activity values and blood volume values from
Kornegay g§_gl, (1964) serum pool and erythrocyte pool glutathione per-
oxidae values were calculated.

The statistical analyses of these data were done as a split-plot
design when time effects were considered. All other analyses were done
as a completely randomized design (Gill, 1978a and 1978b).

Trial VI. Nine pigs weighing 7.62 2 .36 kg (mean 1 standard er-
ror) were randomly allotted to the three dietary treatments described
in trial V. The selenium levels of the feeds are shown in table 16.
The pigs used in this study were farrowed and raised by sows that had
been fed corn-soybean meal diets that were supplemented with vitamin E
(11 IU/kg) and selenium (0.1 ppm). Blood samples were taken at 0, 14
and 28 days of the study for serum and erythrocyte glutathione peroxi-
dase activity and plasma selenium level determinations. The methods

used for laboratory and statistical analysis were described in trial V.

34

TABLE 16. SELENIUM LEVELS OF DIETS FED IN TRIALS V AND VI

 

Diet Trial V Trial VI
Basal (B) .066 L .06
B+0.l ppm selenium .16 .19
B+0.Z ppm selenium .29 .25

 

aing/9.

35

Trial VII. One hundred and two five-week old pigs were allotted
to two replicates of a 2 x 3 factorial design. The main effects were
environmental temperature and level of selenium supplementation. The
basal corn-soybean meal diet (table 17) contained 0.1 ppm of supple-
mental selenium. The basal diet (B) was supplemented with an addition-
al 0.2 ug/g and 0.4 ug/g of selenium as sodium selenite to fOrmulate
the B+.2 and B+.4 diets, respectively. The selenium levels of the
diets are shown in table 18. The daily high and low temperatures for
each environmental treatment were recorded. The average .daily high
temperature was 22.1 t 0.57°C (mean 2 standard error) and the average
daily low temperature was 20.5 : 1.01°C (mean 1 standard error) in the
cool room. The average daily high and low temperatures in the warm
room were 28.6 1 .69 (mean 1 standard error) and 25.3 i 1.64 (mean t
standard error), respectively. In the cool room there were nine pigs
per pen housed in 2.0 x 2.4 m pens on a totally slatted floor of alumi-
num slats. For the first two-weeks a 1.2 x 1.2 m solid plywood panel
was provided for the pigs to lay on. In the warm room eight pigs were
allotted per pen. The pens were 1.2 x 2.4 m with a 1.2 x 0.9 m solid
cement hovered area and the remainder of the pen being aluminum slats.
Feed and water were available ad libitum.

All pigs were weighed initially and at 14 and 28 days after the
start of the trial. Feed intakes were determined at 14 and 28 days.
Blood samples were taken from the pigs in one replicate initially and
at 14 days. Serum glutathione peroxidase activity was determined by
the method of Paglia and Valentine (1967). A modification of the Olson

method (Whetter and Ullrey, 1978) was used for analysis of serum for

36

TABLE 17. BASAL DIET FDR TRIAL VII

 

Ingredient Amount
Ground shelled corn (IFN 4-02-931) 756.0
Soybean meal, dehulled (IFN 5-04-612) 200.0
Mono-dicalcium phosphate (18.5% Ca, 21% P) 14.0
Calcium carbonate (38% Ca) (IFN 6-01-069) 12.5
Vitamin trace-mineral premixa 5.0
Selenium vitamin E premixb 5.0
Salt 3.0
Antibiotic premixc 2.5
Synthetic lysine (78% L-lysine) ___2_.£
1000.0

 

aSupplies 3,300 IU vitamin A; 660 IU vitamin D; 3.3 mg

riboflavin; 13.2 mg d-pantothenic acid; 17.6 mg niacin;

19.8 ug vitamin B12; 126.7 mg choline chloride; 2.2 mg

menadione sodium bisulfite; 74.8 mg zinc; 59.4 mg iron;

37.4 mg manganese; 9.9 mg copper, and 2.75 mg iodine per kg

of diet.

bSupplies 16.5 IU of vitamin E and 0.1 mg of selenium per

kg of diet.

CProvides 110 mg chlortetracycline, 110 mg sulfamethazine

and 55 mg penicillin per kg of diet.

37

TABLE 18. SELENIUM LEVELS OF DIETS IN TRIAL VII

 

Diet Selenium,#ug[g
Basal .17
B+.2 ppm selenium .35

B+.4 ppm selenium .64

 

38
seleniwm. Blood reduced glutathione levels were determined using the
method described by Beutler gt 31, (1963).

Blood samples were also taken on days 3 and 7 of the study from
eight pigs in the warm room allotted to the basal diet and eight pigs
in the same room allotted to the B+.2 diet. Serum glutathione peroxi-
dase activity and blood reduced glutathione levels were determined by
methods already described.

Statistical analysis of the main trial was done as a 2 x 3 factor-
ial design (Gill, l978a). The initial, 3 and 7 day serum glutathione
peroxidase activity and blood reduced glutathione levels, were analyzed
as a split-plot design (Gill, l978b).

Results and Discussion

The average daily gains of the pigs in trials five and six were
not affected by the dietary treatments (table 19). Wastell e_t_ 11.
(1972) and Glienke and Ewan (1977) reported a growth response to sup-
plementary selenium when added to vitamin E supplemented torula yeast
diets. However, Groce 33.3Q, (1971), Ku gt_al, (1973), Mahan et 31,
(l978a), Mahan et a1. (l978b) and Mandisodza et a1. (1979) have observ-
ed no growth response to selenium supplementation of corn-soybean meal
diets for pigs.

There was an interaction (P<.10) of time and supplementary selen-
ium level on serum glutathione peroxidase activity in trial five. Pigs
fed the unsupplemented diet exhibited a decline in serum glutathione
peroxidase activity during the trial. The decline was rapid and
reached a low point by 14 days of the trial. Pigs fed the B+.2 diet
-exhibited an increase in serum glutathione peroxidase activity, while

there was la much larger increase in serum glutathione peroxidase

39

TABLE 19. THE EFFECT OF SELENIUM SUPPLEMENTATION DN
AVERAGE DAILY GAIN IN TRIALS V AND VI

Supplemental selenium

 

(us/9)
Item 0 0.1 0.2 S.E.M.a
Trial V, average 428 412 450 43
daily gain, 9
Trial VI, average 352 367 517 77

daily gain, g

 

aStandard error of treatment means.

40

TABLE 20. THE EFFECT OF SELENIUM SUPPLEMENTATION AND DAYS CONSUMING DIETS
0N SERUM GLUTATHIONE PEROXIDASE ACTIVITYa IN TRIAL V

 

 

Day Supplemental dietary seleniumdae

of (us/9)

trialbecie o 0.1 0.2 Mean
0 19.8 13.5 5.3 13.2
14 3.4 11.5 10.5 8.5
28 3.5 70.1 23.5 32.4
Mean 8.9 31.8 13.5

 

aExpressed as umoles of glutathione oxidized/minute/g of protein.
bStandard error of treatment means a 6.4; standard error of period
means 8 6.6.

cTime effect (P<.05).

dDiet effect (P<.10).

eInteraction of time and diet (P<.10).

41

activity of pigs fed the B+.l diet during the study (table 20). Scott
and Naguchi (1973) reported plasma glutathione peroxidase activity of
chickens fed selenium deficient diets fell to less than 10% of the
normal activity within 5 days after being placed on the deficient diet.
Gabrielsen and Opstvedt (1980) observed a response in plasma glutathi-
one peroxidase of chicks within seven days after being placed on a re-
pletion or a depletion diet.

In trial six an interaction (P<.10) of time and supplementary se-
lenium level on serwm glutathione peroxidase was also observed (table
21). The trends in this trial were similar to those in trial five.
There was a significant diet effect in trial six with pigs fed supple-
mented diets having higher serum glutathione peroxidase activity values
than those fed the unsupplemented diet. Chavez (1979) has reported
higher plasma glutathione peroxidase activity in weaning pigs, fed
selenium-supplemented (0.1 ppm selenium as sodium selenite) torula
yeast diets than in pigs fed the unsupplemented diet. Meyer e_t_ 31.
(1980) observed greater plasma glutathione peroxidase activity at two
weeks post-weaning when corn-soybean meal diets were supplemented with
0.1 ppm of selenium as sodium selenite.

In trial five total serum pool glutathione peroxidase activity was
calculated using blood volume estimates from Kornegay et a1. (1964).
Pigs .fed the basal diet had lower serum pool glutathione peroxidase
activity (P<.Ol) than pigs fed the supplemented diets (table 22).

Total serum pool glutathione proxidase acitivity in trial five and
serum glutathione peroxidase activity in trial six appeared to plateau
at 0.1 ppm supplemental selenium. In both trials pigs fed 0.1 ppm

supplemental selenium were able to maintain serum glutathione

42

TABLE 21. THE EFFECT OF SELENIUM SUPPLEMENTATIDN AND DAYS CONSUMING DIETS
0N SERUM GLUTATHIONE PEROXIDASE ACTIVITYa IN TRIAL VI

 

 

Day Supplemental di etary sel eni umbsc ,d

of (09/9)

trialbac o 0.1 0.2 Mean
0 12.10 11.60 9.97 11.2
14 7.35 17.00 10.80 11.7
2a 5.35 13.60 15.00 11.5
Mean 8.59 14.07 11.92

 

aExpressed as umoles of glutathione oxidized/minute/g of protein.
bStandard error of treatment means a .94; standard error of period
means - 1.07.

cSignificant dietary effect (P<.05).

dSignificant interaction of time and diet (P<.10).

43

TABLE 22. THE EFFECT OF SELENIUM SUPPLEMENTATION FOR FOUR WEEKS ON SERUM
AND ERYTHROCYTE POOL OF GLUTATHIONE PEROXIDASE ACTIVITY IN TRIAL V
Supplemental dietary selenium
(Hg/9)

Item 0 0.1 0.2 S.E.M.a
Erythrocyte pool

glutathione peroxidase

activity, umoles of

glutathione oxidized/minute/pig 854.2 978.9 737.0 161.0
Serum pool

glutathione peroxidase

activity, umoles of

glutathione oxidized/minute/pig 66.3b 291.70 346.8c 50.5

 

aStandard error of treatment means.
b:CMeans with different superscripts are significantly different
(P<.01).

44

peroxidase activity throughout the study. Meyer _1_:_ __1_. (1981) reported
that plasma glutathione peroxidase plateaued at 0.35 ppm of supple-
mental selenium at 5 weeks postweaning. However, Chavez (1979) has
shown more variable results. In a trial with two week old pigs the
plasma glutathione peroxidase activity was maintained by supplementing
a torula yeast diet with 0.1 ppm selenium as sodium selenite, but in
another trial 0.1 ppm supplemental selenium did not maintain plasma
glutathione peroxidase activity of two week old pigs.

Erythrocyte glutathione peroxidase activity was not affected by
dietary selenium supplementation in trials five (table 23) and six
(table 24), however there was a decline in activity with increasing
time in trial five (P<.01). The erythrocyte pool glutathione peroxi-
dase activity at 28 days was not affected by dietary selenium in trial
five (table 22). Hoekstra g; _a_]_. (1973) noted that rat erythrocyte
glutathione peroxidase activity was restored (after depletion) by diet-
ary selenium at a rate consistent with the life span of the erythro-
cyte. The life span of the swine erythrocyte has been estimated to be
from 72 days (Hithrow and Bell, 1969) to 86 days (Bush _e_t_ _a_l_., 1955)
and Talbor and Swensen (1963) have suggested the swine erythrocyte has
a half-life of 28 days. Using these values approximately 30% to 50% of
the erythrocyte pool should have been turned over during a 28 day
trial. However, erythrocyte destruction in swine is due to b0th an age
dependent process and a random process. Bush _e_t_ fl. (1955) and Hithrow
and Bell (1969) concluded that random destruction accounts for the
larger portion of the cells which are destroyed. Tao (1973) observed
some random destruction of erythrocytes, but suggested that age-depen-

dent destruction was the primary destructive mechanism in pigs. It

45

TABLE 23. THE EFFECT OF SELENIUM SUPPLEMENTATIDN AND DAYS CONSUMING DIETS
0N ERYTHROCYTE GLUTATHIONE PEROXIDASE ACTIVITYa IN TRIAL V

 

 

Day Supplemental dietary seleniumb

of (HQ/9)

trialbec 0 0.1 0.2 Mean
0 8.33 ' 8.71 13.90 10.31
14 7.25 8.99 7.70 7.98
28 5.11 . 5.23 4.08 5.14
Mean 6.90 7.98 8.56

 

aExpressed as umoles of glutathione oxidized/minute/g of hemoglobin.
bStandard error of treatment means a .49; standard error of period
means a .88.

cSignificant time effect (P<.01).

46

TABLE 24. THE EFFECT OF SELENIUM SUPPLEMENTATIDN AND DAYS CONSUMING DIETS
0N ERYTHROCYTE GLUTATHIONE PEROXIDASE ACTIVITYa IN TRIAL VI

 

 

Day Supplemental dietaryseleniumb

of (no/g)

trial 0 0.1 0.2 Mean
0 9.74 7.85 7.72 8.44
14 2.92 3.15 1.54 2.54
28 4.70 4.28 5.37 4.78
Mean 5.79 5.10 4.88

 

aExpressed as umoles of glutathione oxidized/minute/g of hemoglobin.
bStandard error of treatment means a 2.61; standard error of period

emans a 2.38.

47
appears that newly formed erythrocytes that would reflect recent di-
etary selenium intakes may be destroyed by the random destruction pro-
cess. Therefore, a longer period of time may be needed in swine before
dietary induced changes in erythrocyte glutathione peroxidase activity
could be observed than in species such as sheep which exhibit primary
age dependent erythrocyte destruction (Hithrow and Bell, 1969).

Serum glutathione peroxidase activity appears to be a more sensi-
tive indicator of selenium intake than erythrocyte glutathione peroxi-
dase activity in pigs. Smith £11.91: (1974) reported that plasma gluta-
thione peroxidase activity was the most responsive to dietary selenium
followed in order by heart, kidney and erythrocyte glutathione peroxi-
dase activity in rats. Chow and Tappel (1974) have also shown similar
results with both depletion and repletion studies.

In trial five there was a significant (P<.Ol) effect of supple-
mental selenium on plasma selenium level. More importantly, there was
an interaction (P<.Ol) of supplemental dietary selenium and days on
treatment for plasma selenium concentrations in both trials five (table
25) and six (table 26). In both trials pigs fed the basal diet showed
a rapid decline in plasma selenium levels during the first fourteen
days of the study. In trial five, the plasma selenium level of pigs
fed the B+.1 diet increased slightly throughout the study, while plasma
selenium values increased greatly when pigs were fed the B+.2 diet.
Pigs fed the unsupplemented diets in trial six exhibited only a slight
rise in plasma selenium levels during the study.

Groce e_t _a_l_. (1973b) reported that serum selenium levels were
increased by selenium supplementation up to 0.1 ug/g. Young _e_t_ a_l_.

(1977b) observed that serum selenium concentration of six-week old

48

TABLE 25. THE EFFECT OF SELENIUM SUPPLEMENTATIDN AND DAYS CONSUMING DIETS
ON PLASMA SELENIUM CONCENTRATIONSa IN TRIAL V

 

 

Day Supplemental dietary seleniumbacad

0f (Mg/9)

trialbad 0 0.1 0.2 Mean
0 .05 .05 .05 .05
14 .02 .07 .10 .05
28 . .02 .07 .12 .07
Mean .03 .06 .09

 

aExpressed as ug/ml.

bStandard error of treatment means = .006; standard error of period
means a .021.

cSignificant diet effect (P<.01).

dSignificant interaction of diet and time (P<.01).

49

TABLE 26. THE EFFECT OF SELENIUM SUPPLEMENTATIDN AND DAYS CONSUMING DIETS
ON PLASMA SELENIUM CONCENTRATIONSa IN TRIAL VI

 

 

Day Supplemental dietary seleniumb:c

9f (09/9)

trialb 0 0.1 0.2 Mean
0 .11 .12 .10 .11
14 .05 .14 .14 .11
28 . .05 .15 .13 .11

Mean .07 .14 .12

 

9Expressed as ug/ml.
bStandard error of treatment means = .008; standard error of period
means - .004.

cSignificant interaction of time and diet (P<.01).

50

boars plateaued at 0.56 ppm dietary selenium, when a high moisture
corn-soybean meal diet was fed. The serum selenium values reported by
Young 33 gfl, (1977b) tended to be higher (.14 to .18 ug/ml) than the
plasma selenium concentrations in trials five and six. Mahan and Moxon
(1978a) observed that serum selenium concentrations declined rapidly in
the first seven days postweaning when pigs were fed unsupplemented
corn-soybean meal diets. Furthermore, they reported that pigs raised
by sows fed selenium-supplemented diets (0.1 ppm supplemental selenium)
had higher serum selenium levels at weaning than pigs raised by sows
fed unsupplemented corn-soybean meal diets. This trend is evident in
trials five and six. Pigs used in trial five were raised by gilts fed
selenium-unsupplemented diets, while pigs in trial six were raised by
sows fed selenium-supplemented diets. The initial serum selenium
values were .05 and .11 ug/ml, respectively, for trials five and six
(tables 25 and 26). Mahan and Moxon (l978a) reported that supplement-
ing starter diets with 0.1 ppm of selenium as sodium selenite resulted
in an increase in serum selenium levels of pigs raised by sows fed se-
lenium-unsupplemented diets, but did not affect serum selenium levels
of pigs raised by sows fed selenium-supplemented diets. Similar trends
were noted in trials five and six.

Initial serum tocopherol values were lower in trial five (table
27) than in trial six (table 28) (.32 and .50 ug/ml, respectively).
Pigs used in trial five were raised by gilts fed corn-soybean meal
diets that were not supplemented with vitamin E or selenium, while pigs
used in trial six were raised by sows fed vitamin E (11 IU/kg) and
selenium (0.1 ppm) supplemented corn-soybean meal diets. In trial five

there was an interaction (P<.lO) of supplemental selenium and days on

51

TABLE 27. THE EFFECT OF SELENIUM SUPPLEMENTATION AND DAYS CONSUMING DIETS
0N SERUM TOCDPHEROL LEVELSa IN TRIAL V

 

 

Day Supplemental dietary seleniumb»c

of . (u9/g) ’

trialbac 0 0.1 0.2 Mean
0 .48 .17 .31 .32
14 .19 .35 .30 .28
28 ..22 .19 .38 .26
Mean .30 .24 .33

 

9Expressed as 09 of tocopherol and tocopheryl acetate/m1.
bStandard error of treatment means = .05; standard error of period
means a .05.

cSignificant interaction of time and diet (P<.10).

52

TABLE 28. THE EFFECT OF SELENIUM SUPPLEMENTATION AND DAYS CONSUMING DIETS
ON SERUM TDCDPHEROL LEVELSa IN TRIAL VI

 

 

Day Supplemental dietary seleniumb

of (ug/g)

trial ‘ O 0.1 0.2 MeanC
D .53 .42 .54 .50

14 .48 .56 .46 .50

28 .81 .67 _ .59 .69

Mean .61 .55 .53

 

aExpressed as 09 of tocopherol and tocopheryl acetate/m1.
bStandard error of treatment means = .09; standard error of period
means a .04.

cTime effect (P<.01).

53

treatment on serum tocopherol level. Serum tocopherol levels of pigs
fed the basal diet declined during the first two weeks of the study and
remained low throughout the study. Pigs fed the B+.2 diet exhibited a
slight rise in serum tocopherol levels during the study. During the
first 14 days the serum tocopherol concentration of pigs fed the B+.1
diet rose, but then declined to a level similar to the pig fed the un-
supplemented diet. In trial six there was no significant effect of
selenium supplementation on serum tocopherol concentration. Both Meyer
3391. (1981) and McDowell e_t__a_1_. (1977) have reported no effect of
supplemental dietary selenium on circulating tocopherol levels.

In trials five and six, serum glutathione peroxidase activity was
more responsive postweaning than erythrocyte glutathione peroxidase
activity to dietary selenium levels during the four weeks. At least
0.1 ppm of supplemental selenium was needed to maintain serum glutath-
ione peroxidase activity and plasma selenium levels throughout the
four-week post weaning period. It appears that the intake of selenium
and vitamin E by the dam influences the weaning plasma selenium, and
serum tocopherol levels, as well as the response of pigs to supple-
mental selenium.

Supplemental dietary selenium did not affect rate of gain, average
daily feed intake or feed efficiency in trial seven (table 29). Simi-
lar results have been reported by Groce gtal. (1971), Ku 31331. (1973)
l. (1978b), Mandisodza _e_t_ a_l_. (1979)

Mahan £12].- (1978a), Mahan gt
and Meyer gt_a_1. (1981). Average daily feed intake was not signifi-
cantly affected by the environmental temperature. Pigs in the cool
room gained faster (P<.O4) than pigs in the warm room (.52 vs .46

kg/day) during the second two-week period of the trial. During the

54

 

 

 

 

 

.88. 88. 88.. 88.8 88.. 88.. 88.8 8..8 ...8 .8.8 8888 88-8
888. 88. 88.. 88.. .8.. 88.. .8.. 88.. 88.. 88.. 888888-..
88.. 88.8 88.8 .8.8 88.8 .8.8 88.8 88.8 88.8 8888 ...8
6.88. 8.8888888
888. 88. 88. 88. 88. 88. .8. 88. .8. 8888 88.8
.8.. .8. .8. 88. .8. 88. 88. .8.. 88. 8888 88-..
888. 88. 88. 88. 88. 88. 88. 88. .8. 8888 ...8
8. .88888. 888. 8..88 888.8><
8.8. 88. 88. 88. 88. .8. 88. 88. .8. 8888 88.8
888. .8. 8.. 8.. 8.. 8.. .8. 88. .8. .8. 8888 88-..
8.8. 8.. ... 8.. ... ... 8.. 8.. ... 8888 ...8
m... ...—cm >23 88.33.
88.8 8..8 88.8 88.8 8..8 ...8 ...8 88.8 8. ...8... .8.....
888.8. .68... 68:8 ea... ...8 8.8 8 888.. ...8 8.8 8 8:
:88.- 888. . >5 +0 _ o t. _ a 80 . a
..o....m 00:33:81.8 filed. €1.33 80.. .000
«o .26..

 

..>18c.88

z. Kim-0.83m awn. a! maze .9th— amm 3 35.26%... 1.55.36 2 ~6;<hz!w._m83m 3.2w4wm 85 .8me m2... .ON w4m<h

55

first two-week period, however, the gains were not affected by the
environmental treatment. Also, overall gains for the trial were not
affected by the environmental treatment. Feed required per unit of
gain was lower for pigs in the warm room for the second two-week period
(1.83 vs 1.91, P<.05) resulting in an improved overall feed efficiency
(1.98 vs 2.08, P<.02). Improved caloric efficiency with increasing
temperature has been reported (Nichols £3 31,, 1980) in growing pigs in
the same range of temperatures used in this study. Similar trends have
been observed in heavier pigs, also (Jensen §t_al,, 1969 and Sugahara
‘gthgl,, 1970).

The serum glutathione peroxidase activity (table 30) was higher
for pigs fed ‘the B+.2 and B+.4 ‘than for pigs fed the basal diet
(P<.05). The serum glutathione peroxidase activity appeared to be
maximum at 0.35 ppm selenium (B+.2 diet). Meyer _e_i; _a_l_. (1981) has
suggested that the point at which plasma glutathione peroxidase activif
ty peaks declines from one to five weeks post-weaning. At 14 days
postweaning, Meyer _e_t_ _a_l_. (1981) reported that plasma glutathione per-
oxidase activity plateaued at 0.4 ppm dietary selenium.

Plasma selenium level increased (P<.Ol) with each increment of
supplemental dietary selenium at two weeks postweaning (table 30). The
plasma selenium concentration did not plateau over a range of dietary
seleniwn used in this study (0.17 to 0.64 ppm). Meyer gt gfl, (1981)
observed that plasma selenium levels plateaued at 0.45 ppm dietary
selenium at two weeks post weaning. However, Young g _a_l. (1977b)
reported that serum selenium values plateaued at 0.56 ppm dietary

selenium in six-week old boars fed high moisture corn diets. Mahan and

56

...o.vm. 33.80.08 _o+:olo_aaam 80 88838.0:- gooo .0 800880 8=oo_8.8a_mo

..mo.vm. 80.8 .0888 .0 cool cog. co+ooem 080 880.0 808coao.88:m 388 v. van N. 80. 880018

.8.:e.e\888.8.xo :88... .8 8.2:. . u a. .8

 

 

 

 

.88. .8. 8. . 8. . 88. 8. . 8. . 88. 8.888..
.53—80.08 8388.8
o.on .88 «so 880 8.0 .no use .s\80.oe:
.o:o_sbn8:.m
800380; me_88.:uc_o
8.8. .8. 8.88 8.8 8.88 8.88 8.88 8.88 8.88.8.6... 8.5.8
. .>8_>_+oo omov.xocoa
oco_g8o«:_m [atom
0.8388 800.80 8008.0 v.+m ~.+m m ¢.+m ~.+m 0 I08.
:88: 8colco._>8w .o.o #o_o .o.o
80.8w cocoo_8_mm_a

.o .o>o.

3008 I883 :60. .ooo

 

 

88885.. 88.8

:8 188885.889. 2823.828 92 8.28 2888588 88.. 3.8.55.5... 28.28.88 “.8 8888.... g .88 39.8

57

Moxon (1978) observed that serum selenium values did not plateau in
weanling pigs over a range of 0 to 0.3 ppm supplemental selenium.

Circulating reduced glutathione levels of pigs were not affected
by their dietary or environmental treatment (table 30). Since the re-
duction of lipid peroxides by glutathione peroxidase is a first order
reaction with respect to reduced glutathione (O'Brien and Little,
1967), one could conclude that the reduced glutathione supply could

limit i vivo glutathione peroxidase activity, since there are dif-

 

ferences in ig_yltrg glutathione peroxidase activity, or one must con-
clude that reduced glutathione is available in excess of the needs for
.1_‘11!g_activity. Since glutathione is rapidly synthesized (Anderson
and Mosher, 1951) and since oxidized glutathione is a positive effector
of glucose-6-phosphate dehydrogenase, (Jacob and Jandl, 1966) it seems
likely that after fourteen days of the study ample reduced glutathione
would be available to support reduction of lipid peroxides.

During the second two-week period, one pig in the cool room being
fed the B+.4 diet died. Liver and muscle samples contained 2.24 and
0.51 09 of selenium/g of dry tissue, respectively. The liver selenium
concentration is clearly above the level of 0.52 ug/g reported by Young
35,3g, (l977b) for pigs that died of vitamin E-selenium deficiency, and
is similar to the value of 2.05 ug/g reported by K0 3; fl. (1973) for
pigs fed selenium supplemented diets (0.4 ppm supplemental selenium).
Survey work done by Young £E.£fl: (l977a) would indicate that the liver
selenium concentration is consistant with adequate supplemental dietary
selenium. The muscle selenium level of this pig was greater than that

of pigs fed deficient diets (0.24 ug/g) reported by Young 31; fl.

58
(l977b) and also higher than the level for pigs fed selenium supple-
mented diets (0.4 ppm supplemental selenium) of 0.46 ug/g reported by
K0 _e_t__a__1_. (1973). It appears that the cause of death was not related
to the dietary treatment.

During the first week of the trial (first week postweaning), pigs
fed the B+.2 diet had higher (P<.Ol) serum glutathione peroxidase act-
ivity than pigs fed the basal diet (table 31). Glutathione peroxidase
activity was also affected by time on the diets (P<.Ol), as the activi-
ty rapidly increased during the first three days after weaning. More
importantly, there was a significant interaction of days on treatment
and supplemental selenium (P<.05) for serum glutathione perixodase
activity. Pigs fed the B+.2 diet exhibited a greater rise in serum
glutathione peroxidase activity by three days and maintained serum
glutathione peroxidase activity higher throughout the remainder of the
week, than pigs fed the basal diet. These results are consistent with
the results reported by Meyer _e_t_al. (1981). Mahan e_t_ a_l_. (1977) has
suggested that there is a very short carryover of selenium from the dam
to the progeny.

Circulating reduced glutathione levels (table 31) were higher in
pigs fed the B+.2 diet than the B diet (P<.06). Reduced glutathione
concentrations declined throughout the study in pigs fed the basal diet
and increased during the study in pigs fed the B+.2 diet (P<.04).
Since reduced glutathione is the rate limiting substrate for glutathi-
one peroxidase (O'Brien and Little, 1967) and since glutathione has
been shown to be synthesized more rapidly in rat liver than most pro-
teins (Anderson and Mosher, 1951) the decline in reduced glutathione in
pigs fed the basal diet may reflect a lower _i_g_ y_i_v_9_ glutathione

peroxidase activity than for pigs fed the B+.2 diet. It is unlikely

S9

.00 a 0:005 0o.c08 .0 00.00 8800:088 888 a 8:008 #:0880088 .0 008.0 0000:0880
.0._ u 8:083 0o_.08 0o 00:00 8800:088 80.. n 8:085 #:0080088 .0 .0000 0008:0880

.0+==_E\v0n.3xo Ina—<2 ..o 0.8.: _ u :u .0

 

 

 

 

 

00. 00. 080 080 0.0 000 008 000 888 n.0 o.e\80_oez
.0:o_:808:.m
8003000 m:_+0.:ue_o
ma. .0. .o. 8.08 ..nn ..0n 0.0. 0.08 0.0. 0.08 8.0. 0:.08008 m\:m
.>+.>_8oo 0800_xo.08
0:o_:+0+:.m £0800
:o_+00 800880 800.80 :00: s n o :00: s n o 00+.
1008:. .0..8 «0.0 lam—:003+mom «>00 1w0.:003+mom «>00
.6 888 .888: 88.. .8..8 .888: 8. .. 8

 

 

000mm.8.mm.u .o _0>04

:o_80:8:0o:oo mm.:0_08 0:0 80.9

 

me>u4 mzo.zh<h:40 Quezon: ocean oz< >h_>.hu<

wm<a.xocwm mzo_xh<h:4@ 131mm 20 mz.z<m3hw0l xumx hm¢_m 01h az_m=o zo.h<hzmxm4¢m:m 25.20400 00 Jw>w4 no bowmmu m!» ..n wam<h

60

that changes in the reduced glutathione supply are due to slow regen-
eration of reduced glutathione from oxidized glutathione if all other
essential dietary nutrient requirements are met, since oxidized gluta-
thione releases the inhibition of glucose-6-phosphate dehydrogenase by
reduced nicotine adenine dinucleotide phosphate (Jacob and Jandl, l966
and Eggleston and Krebs, l974). This release would allow the hexose
monophosphate shunt to produce more reducing equivalents which could be
used to reduce glutathione.

The data from this study indicate that weanling pigs require at
least 0.35 ppm selenium to maximize serum glutathione peroxidase acti-
vity at two weeks postweaning. Serum glutathione peroxidase activity
responds to dietary selenium levels up to at least 0.35 ppm during the

first week post weaning.

6l

CONCLUSIONS
l. Erythrocyte percent active glutathione reductase is a sensitive
indicator of the riboflavin status of pigs. Values below 50% are
needed before other riboflavin deficiency signs become evident.
2. Riboflavin supplementation of swine diets increased selenium re-
tention and appeared to increase the incorporation of selenium from
sodium selenite into glutathione peroxidase.
3. A higher percent of the selenium from selenomethionine was re-
. tained and incorporated into body tissues than selenium from sodium
selenite. Muscle, heart and brain selenium concentrations indicated
that the retention of selenium from selenomethionine was l.5 times
greater than that from sodium selenite.
4. Serum glutathione peroxidase activity was more responsive to di-
etary selenium level than was erythrocyte glutathione peroxidase acti-
vity over a four week period. Serum glutathione peroxidase activity
appeared to peak at lower dietary selenium concentrations than did
plasma selenium levels.
5. Under routine rearing conditions, feeding a diet containing 0.35
ppm selenium resulted in a higher serum glutathione peroxidase activity
by three days postweaning than feeding a diet containing 0.17 ppm
selenium. This advantage was maintained throughout the remainder of
the first week after weaning.
6. At least 0.35 ppm dietary selenium was required for serum gluta-
thione peroxidase activity to become maximum at two weeks postweaning

when pigs were reared under routine management conditions.

LITERATURE CITED

Literature Cited

Anderson, 5.1. and w.A. Mosher. 1951. Incorporation of s35 from
DL-cystine into glutathione and protein in the rat. J. Biol. Chem.
188:51.

Awasthi, Y.C., E. Beutler and S.K. Srivastava. 1975. Purification and
properties of human erythrocyte glutathione peroxidase. J. Biol.
Chem. 250:5144.

Baker, H., 0. Frank, S. Feingold, R.A. Gellene, C.M. Leevy and S.H.
Hunter. 1966. A riboflavin assay suitable for clinical use and
nutritional surveys. Am. J. Clin. Nutr. 19:17.

Beilstein, M.A., M.J. Tripp and P.D. whanger. 1980. Evidence of
selenocysteine in low molecular weight selenium containing
proteins in ovine muscle. Fed. Proc. 39:338. (Abstr.).

Beutler, E., O. Duron and B.M. Kelly. 1963. Improved method for the
determination of blood glutathione. J. Lab. Clin. Med. 61:882.

Black, R.S., M.J. Tripp, P.D. Nhanger and P.H. Neswig. 1978. Selenium
proteins in ovine tissues: III Distribution of selenium and
glutathione peroxidase in tissue cytosol. Bioinorganic Chem.
8:161.

Brady, P.S., L.J. Brady, M.J. Parsons, D.E. Ullrey and E.R. Miller.
1979. Effects of riboflavin deficiency on growth and glutathione ,
peroxidase system enzymes in the baby pig. J. Nutr. 109:1615.

Bunk, M.J., M.S. Cupp and G.F. Combs, Jr. 1980. Relationship of
selenium dependent glutathione peroxidase activity to nutritional
pancreatic atrophy in selenium deficient chicks. Fed. Proc.

39:556.

62

63

Burk, R.F. and P.E. Gregory. 1980. 75$e incorporation into liver
and plasma fractions of Se-deficient (0 Se) and control (c). Fed.
Proc. 39:1678 (Abstr.).

Bush, J.A., N.I. Berlin, N.N. Jensen, A.B. Brill, G.E. Cartwright and
M.M. Nintrobe. 1955. Erythrocyte life span in growing swine as
determined by glycine-2-C14. J. Exp. Med. 101:451.

Buzard, J.A. and F. Kopko. 1963. The flavin requirement and some
inhibition characteristics of rat tissue glutathione reductase. J.
Biol. Chem. 238:464.

Chavez, E.R. 1979. Effect of dietary selenium on glutathione peroxidase
activity in piglets. Can. J. Anim. Sci. 59:67.

Chow, C.K. and A.L. Tappel. 1974. Response of glutathione peroxidase to
dietary selenium in rats. J. Nutr. 104:444.

, Eggleston, L.V. and H.A. Krebs. 1974. Regulation of the pentose
phosphate cycle. Biochem. J. 138:425.

Ewan, R.C., M.E. Nastell, E.J. Bicknell and V.C. Speer. 1969.
Performance and deficiency symptoms of young pig fed diets low in
vitamin E and selenium. J. Anim. Sci. 29:912.

Forbes, R.M. and w.T. Haines. 1952. The riboflavin requirement of the
baby pig 1. At an environmental temperature of 85°F and 70%
relative humidity. J. Nutr. 47:411.

Gabrielsen, Bjdrn 0. and J. Opstvedt. 1980. A biological assay fbr
determination of availability of selenium for restoring blood
plasma glutathione peroxidase activity in selenium-depleted
chicks. J. Nutr. 110:1089.

Gill, J.L. 1978a. Design and Analysis of Experiments in the Animal and
Medical Sciences, Volume I (lst Ed.). Iowa State University Press,

Ames.

64

Gill, J.L. 1978b. Design and Analysis of Experiments in the Animal and
Medical Sciences, Volume 2 (lst Ed.). Iowa State University Press,
Ames.

Glatzle, 0., F. Weber and 0. Miss. 1968. Enzymatic test for the
detection of a riboflavin deficiency. NADPH-dependant glutathione
reductase of red blood cells and its activation by FAD in vitro.
Experientia (Basel) 24:1122.

Glatzle, 0., R.F. Korner, S. Christeller and 0. Miss. 1970. Method
for the detection of a biochemical riboflavin deficiency
stimulation of NADPHz-depentent glutathione reductase from human
erythrocytes by FAD 13_yj§52, Investigations on the vitamin
82 status in healthy people and geriatric patients. Internat.

J. Vit. Res. 40:166.

Glienke, L.R. and R.C. Ewan. 1977. Selenium deficiency in the young
pig. J. Anim. Sci. 45:1334.

Groce, A.w., E.R. Miller, K.K. Keahey; D.E. Ullrey and D.J. Ellis.
1971. Selenium supplementation of practical diets for
growing-finishing swine. J. Anim. Sci. 32:905.

Groce, A.w., E.R. Miller, J.P. Hitchcock, D.E. Ullrey and w.T. Magee.
1973a. Selenium balance in the pig as affected by selenium source
and vitamin E. J. Anim. Sci. 37:942.

Groce, A.w., E.R. Miller, D.E. Ullrey, P.K. Ku, K.K. Keahey and D.J.
Ellis. 1973b. Selenium requirements in corn-soy diets for

growing-finishing swine. J. Anim. Sci. 37:948.

65

Hitchcock, J.P., E.R. Miller, K.K. Keahey and D.E. Ullrey. 1978.
Effects of arsanilic acid and vitamin E upon utilization of
natural or supplemental selenium by swine. J. Anim. Sci. 46:425.

Hoekstra, 0.6., 0. Hafeman, S.H. 0h, R.A. Sunde and H.E. Ganther. 1973.
Effect of dietary selenium on liver and erythrocyte glutathione
peroxidase in the rat. Fed. Proc. 32:885 (Abstr.).

Jacobs, H.S. and J.H. Jandl. 1966. Effects of sulfhydryl inhibition on
red blood cells. III Glutathione in the regulation of the hexose
monophosphate shunt. J. Biol. Chem. 24:4243.

Jensen, A.H., D.E. Kuhlman, D.E. Becker and 8.6. Harmon. 1969. Response
of growing-finishing swine to different housing environments
during winter seasons. J. Anim. Sci. 29:451.

Kornegay, E.T., E.R. Miller, B.E. Brent, C.H. Long, D.E. Ullrey and
J.A. Hoeffer. 1964. Effect of fasting and refeeding on body
weight, rectal temperature, blood volume and various blood
constituents in growing swine. J. Nutr. 84:295.

Krider, J.L., s.w. Terrill and R.F. VanPoucke. 1949. Response of
weanling pigs to various levels of riboflavin. J. Anim. Sci.
8:121.

Ku, P.K., w.T. Ely, A.H. Groce and D.E. Ullrey. 1972. Natural dietary
selenium, o-tocopherol and effect on tissue selenium. J. Anim.
Sci. 34:208.

Ku, P.K., E.R. Miller, R.C. Nahlstrom, A.w. Groce, J.P. Hitchcock and
D.E. Ullrey. 1973. Selenium supplementation of naturally high

selenium diets for swine. J. Anim. Sci. 37:501.

66

Ladenstein, R., O. Epp, K. Bartels, A. Jones and R. Huber. 1979.
Structure analysis and molecular model of the selenoenzyme
glutathione peroxidase at 2.8 A resolution. J. Mol. Biol.
134:199.

Mahan, 0.C., A.L. Moxon and Mariana Hubbard. 1977. Efficacy of
inorganic selenium supplementation to sow diets on resulting
carry-over to their progeny. J. Anim. Sci. 46:738.

Mahan, 0.C. and A.L. Moxon. 1978a. Effect of increasing the level of
inorganic selenium supplementation in the post-weaning diets of
swine. J. Anim. Sci. 46:384.

Mahan, 0.C. and A.L. Moxon. 1978b. Effects of adding inorganic or
organic selenium sources to the diets of young swine. J. Anim.
Sci. 47:456.

Mandisodza, K.T., H.E. Pond, D.J. Lisk, D.E. Hogue, L. Krook, E.E. Cary
and R.H. Gutenmann. 1979. Tissue retention of Se in growing pigs
fed fly ash or white sweet clover grown on fly ash. J. Anim. Sci.
49:535.

McDowell, L.R., J.A. Froseth, R.C. Piper, I.A. Dyer and G.H. Kroening.
1977. Tissue selenium and serum tocopherol concentrations in
selenium-vitamin E deficient pigs fed peas (Piggy sgglyum). J.
Anim. Sci. 45:1326.

Meyer, H.R., 0.C. Mahan and A.L. Moxon. 1981. Value of dietary selenium
and vitamin E for weanling swine as measured by performance and
tissue selenium and glutathione peroxidase activities. J. Anim.
Sci. 52:302.

Miller, E.R., R.L. Johnston, J.A. Hoefer and R.H. Luecke. 1954. The

riboflavin requirement of the baby pig. J. Nutr. 52:405.

67

Mills, G.C. 1957. Hemoglobin catabolism I. Glutathione peroxidase, an
erythrocyte enzyme which protects hemoglobin from oxidative
breakdown. J. Biol. Chem. 229:189.

Mills, G.C. and H.P. Randall. 1958. Hemoglobin catabolism II. The
protection of hemoglobin from oxidative breakdown in the intact
erythrocyte. J. Biol. Chem. 232:589.

Mills, G.C. 1959. The purification and properties of glutathione
peroxidase of erythrocytes. J. Biol. Chem. 234:502.

Morris, V.C. and 0.A. Levander. 1980. Metabolism of selenium (Se) fed
to rats as selenite or high selenium yeast. Fed. Proc. 39:338.
(Abstr.).

Nichols, 0.A., 0.R. Ames and R.H. Hines. 1980. Effect of temperature on
performance and efficiency of finishing swine. J. Anim» Sci.
51:(Supp1. 1) 63 (Abstr.).

NRC. 1979. Nutrient Requirements of Domestic Animals, No. 2. Nutrient
Requirements of Swine. Eighth Revised Ed. National Academy of
Science-National Research Council. Washington, DC.

O'Brien, P.J. and C. Little. 1967. An intracellular glutathione
peroxidase with a lipid peroxide as substrate. Biochem. J.
103:31P.

0h, S.H., H.E. Ganther and H.G. Hoekstra. 1974. Selenium as a component
of glutathione peroxidase isolated from ovine erythrocytes.
Biochemistry 13:1825.

Paglia, D.E. and H.N. Valentine. 1967. Studies on the quantitative and
qualitative characterization of erythrocyte glutathione
peroxidase. J. Lab. Clin. Med. 70:158.

68

Pond, w.G. and J.H. Maner. 1974. Swine Production in Temperate and
Tropical Environments. H.H. Freeman and Company, San Francisco.

Rotruck, J.T., H.G. Hoekstra, A.L. Pope, H. Ganther, A. Swanson and D.
Hafeman. 1972. Relationship of selenium to glutathione peroxidase.
Fed. Proc. 31:691 (Abstr.).

Rotruck, J.T., A.L. Pope, H.E. Ganther, A.B. Swanson, 0.G. Hafeman and
H.G. Hoekstra. 1973. Selenium: Biochemical role as a component of
glutathione peroxidase. Science 179:588.

Sauberlick, H.E., J.H. Judd, Jr., G.E. Nichoalds, H.P. Broquist and
H.J. Darby. 1972. Application of the erythrocyte glutathione
reductase assay in evaluating riboflavin nutritional status in a
high school student population. Amer. J. Clin. Nutr. 25:756.

Schwarz, K. and C.M. Foltz. 1958. Factor 3 activity of selenium
compounds. J. Nutr. 233:245.

Scott, M.L. and T. Naguchi. 1973. Metabolic function of selenium in
prevention of exudative diathesis in chicks. Fed. Proc. 32:885.
(Abstr.).

Smith, P.J., A.L. Tappel and C.K. Chow. 1974. Glutathione peroxidase
activity as a function of dietary selenomethionine. Nature
(London). 247:392.

Staal, G.E.J., J. Visser and C. Veeger. 1969. Purification and
properties of glutathione reductase of human,erythrocytes.
Biochem. Biophys. Acta, 185:39.

Stryer, L. 1975. Biochemistry. H.H. Freeman and Company, San

Francisco.

69

Sugahara, M., D.H. Baker, B.G. Harmon and A.H. Jensen. 1970. Effect of
ambient temperature on performance and carcass development in
young swine. J. Anim. Sci. 31:59.

Sunde, R.A., H.E. Ganther, and H.G. Hoekstra. 1978. A comparison of
ovine liver and erythrocyte glutathione peroxidase. Fed. Proc.
37:757. (Abstr.).

Sunde, R.A. and H.S. Hoekstra. 1980a. Selenium incorporation into
glutathione peroxidase in the isolated perfused rat liver. Fed.
Proc. 39:555 (Abstr.).

Sunde, R.A. and H.E. Hoekstra. 1980b. Incorporation of selenium into
liver glutathione peroxidase in the Se-adequate and Se-deficient
rat. Proc. Soc. Exp. Biol. Med. 165:291.

Sunde, R.A., G.E. Gutzke and H.G. Hoekstra. 1981. Effect of dietary
methionine on the biopotency of selenite and selenomethionine in
the rat. J. Nutr. 111:76.

Talbot, R.B. and M.J. Swenson. 1963. Survival of CR51 labeled
erythrocytes in swine. Proc. Soc. Exp. Biol. Med. 112:573.

Tao, R.V.P. 1973. Biochemistry and metabolism of mamnalian blood
glycosphingolipids. Ph.D. Thesis. Michigan State University, East
Lansing, MI.

Tappel, A.L., J.H. Forstrom, J.J. Zabowski, D.E. Lyons and V.C. Hawkes
l978a. The catalytic site of rat liver glutathione peroxidase as
selenocysteine and selenocysteine in rat liver. Fed. Proc. 37:706

(Abstr.).

7O

Tappel, A.L. l978b. Measurement of and protection from ig_yiyg_1ipid
peroxidation. Proc. of "Conference on Biochemical and Clinical
Aspects of Oxygen".

Terrill, S.H., C.B. Ammerman, D.E. Walker, R.M. Edwards, H.w. Norton
and D.E. Becker. 1955. Riboflavin studies with pigs. J. Anim. Sci.
14:593.

Tillotson, J.A., and E.M. Baker. 1972. An enzymatic measurement of the
riboflavin status in man. Am. J. Clin. Nutr. 25:425.

Hang, C.S. and J. Spallholz. 1980. Biological availability of selenium
from sodium selenite, selenium yeast, monoselenodiactic acid and
selenomethionine for glutathione peroxidase. Fed. Proc. 39:555
(Abstr.).

Hastell, M.E., R.C. Ewan, M.H. Vorhies and V.C. Speer. 1972. Vitamin E
and selenium for growing and finishing pigs. J. Anim. Sci.
34:969.

Hegger, 1., K. Rasmussen and P. F. Jdrgensen. 1980. Glutathione
peroxidase activity in liver and kidney as indicator of selenium
status in swine. Livestock Prod. Sci. 7:175.

Whetter, P.A. and D.E. Ullrey. 1978. Improved fluormetric method for
determnning selenium. J. Assoc. Off. Anal. Chem. 61:927.

Ridicus, H.A. and J.R. Kirk. 1979. High pressure liquid chromatographic
determination of vitamins A and E in cereal products. J. Assoc.
Off. Anal. Chem. 62:637.

Withrow, G. and M.C. Bell. 1969. Erythrocytic life span estimations in
growing sheep and swine using 75$e. J. Anim. Sci. 28:240.

Young, L.G., A.G. Castell and D.E. Edmeades. l977a. Influence of
dietary levels of selenium on tissue selenium of growing pigs in

Canada. J. Anim. Sci. 44:590.

71
Young, L.G., R.B. Miller, D.E. Edmeades, A. Lun, G.C. Smith and G.J.
King. 1977b. Selenium and vitamin E supplementation of high
moisture corn diets for swine reproduction. J. Anim. Sci.

45:1051.

   

iiiii1111111131111111:11:11i