J. Nutr. Sci. Vitaminol.,
EFFECTS
OF
VITAMIN
SELENIUM
23, 273-280,
DEFICIENCY
E METABOLISM
IN
1977
ON
RATS1
W. C. FISCHER2 and P. D. WHANGER Department of Agricultural Chemistry Oregon State University Corvallis, Oregon 97331 (Received February 21, 1977)
Summary The metabolism of radioactive vitamin E in selenium defi cient rats has been compared to the metabolism of this vitamin in rats supplemented with 0.1ppm selenium as sodium selenite in the diet. After dosing with tritiated a tocopherol, the plasma level of radioactivity remained elevated through 48 hours in deficient rats. In contrast, a decline to nearly background levels occurred within 12 hours in the sup plemented rats. The uptake and release of radioactivity in the erythro cytes and liver were found to be more rapid in deficient rats than in sup plemented ones. A 65% greater increase in excretion of radioactivity oc curred in the urine from deficient rats as compared to supplemented ones. These data indicate that vitamin E is metabolized more rapidly in selenium deficient rats than in supplemented ones. The addition of vitamin E or selenium to the diets of experimental animals has been shown to protect against a number of pathological conditions. These include liver necrosis and carbon tetrachloride poisoning in rats (1-6), muscular degenera tion and exudative diathesis in chicks (7-11) and carbon tetrachloride poisoning in sheep (12). This similarity of action has been attributed to vitamin E func tioning as a free radical scavenger in the lipid phase of the cell and selenium as a component of glutathione peroxidase in the aqueous phase (13). A metabolic interrelationship between selenium and vitamin E has been known for several years (14), and the metabolism of one of these is influenced by the other. The hair coat and growth rate in second generation selenium deficient rats have been shown to respond to selenium supplementation but not to vitamin E (15, 16). 1 Published
with the approval
of the Oregon
State Agricultural
Experiment
Station
as Techni
cal Paper No. 4477. This research was supported by Public Health Service Research Grant No. NS 07413 from the National Institute of Neurological Disease and Stroke. 2 This communication is part of a thesis submitted by the senior author to the Graduate Facul ty of Oregon Present
address:
State
University Ciba-Geigy
in partial Corporation,
fulfillment
of the requirements
P. O. Box 11422, Greensboro, 273
for the Ph. D. degree. North
Carolina
27409.
274
W. C. FISCHER and P. D. WHANGER
Poor absorption of dietary vitamin E has been found in selenium deficient chicks (17). In selenium deficient rats, an alteration of the distribution of vitamin E has been reported (18). The objective of the present study is to determine the effect of selenium deficiency on the metabolism of vitamin E in rats and further elucidate the complex relationship between these two nutrients. MATERIALS The
rats
Second
used
for
generation
deficient
diet
given
the
the
study
were
selenium
from
deficient
throughout
selenium
AND
the
deficient
our
closed
animals
course
ration
METHODS
of
colony
were
of O.
S. U.
maintained
the
experiment
supplemented
with
on
(16)
.
Browns
a
Control
0.1ppm
.
selenium rats
selenium
as
were sodium
selenite. Alpha
tocopherol,
tritiated
Amersham/Searle, obtained were
used
prior
to
and
by
heart
tative
24
48
hours
group,
England,
were by
obtained
the
grade
redistilled
passing
from
Omni-Flor
Reagent
solvents purified
was
and
California.
was
blood
ml
portions of
diluted with
the
of
digested
was
chemicals
in
it through
was
diluted were
a
glass
still
a column
of
of
sealed
in
a clinical
25ml the All
70%
for
to and
were screw shaken
centrifuge
volumetric last
flask
partitioning,
samples
hour
at
similarly
were
to
diluted cap for to
the
the
aqueous
diluted
the
buffy KCl.
vials.
to
The one
hour
the
cells
Liver
with
phase
volume
was
respective
taken
the
washed volumes
cells
were of
were
di
vials,
The
decolorized
with
100•}10mg
the
red
and
cells a
3 .0ml
was 10min
top
layer
four
more
2 ,500
removed
times
either
added, at
was
a 10ml with
were
blood
benzene for
.
The
portions
plasma
water
into
minutes
vials. The
of for
of
repeated
ten
in sealed
distilled
separation,
phase
was
ml
were
centrifugation
partitioning
body 4 , 12,
a represen
for
and
samples used
equal
After
their
cells
for
An
and
Half
and
50ml
blood
layer.
75•Ž
procedure
to
of at
scintillation
H2O2
75•Ž.
5min. insure
in
0.9%
the
the KCl
centrifuge
placed
of
tubes.
and
a clinical
with
at
250g
counting.
along
of 30%
%
was 3H-a
chloroform
After 0.9
volumes
acid
0.2ml
one
for
and
scintillation
perchloric with
samples
removed
three in
E.
rats 12.5 ƒÊCi
per
with
with
collected in
tube
deoxycholate
vitamin
removed
supplemented
stomach
killed
perfused
removed
with
placed
H2O2
tube
were
by
were
tritiated rapidly also
selenium
sodium
group
at 2,500rpm
plasma
decolorized
urine
portion
was
decolorized 30%
The
Urine
plasma
and
and
with were
16 given
0.5%
dietary
livers
and was
and
each
the
0.2ml
0.4ml
ethanol
centrifuged
of
rats
animal
dosing
the
cells
was
5%
after
was
twice
plasma
deficient
from
taken.
The
gested
in
puncture,
washed
lowing
Centre,
Organic
Each
rats
sample
a
5 methyl
Richmond,
which
selenium
acetate
remainder
flask.
toluene
Four
24,
to
investigation.
experiment.
tocopherol
rpm
Inc.,
this
of
this
weight.
the
number
Radiochemical
Rad
except
total
in
Half
the
silica.
A
the
Bio
in use
activated
used
The
from
in
.
Fol
volumetric benzene
.
SELENIUM
or
water.
and
One
15ml
were
of
decolorized
heated
to
for
cooling,
H2O2
the
scintillation
of
allowed
to
stand were
the
scintillation one
the
ml
of
AND
benzene
phases
added.
Half
cocktail3
by
Fifteen
samples
samples
in
75•Ž
After with
the
ml
scintillation
DEFICIENCY
vials
by
the
VITAMIN
were ml
placed
samples
addition
E
275
in of
of
scintillation
the
0.2ml
vials
aqueous
of
30%
phases H2O2
and
hour. chemiluminescence
addition
of
was
25ƒÊl
of
cocktail
overnight
was
at room
counted
in
4%
quenched
ascorbic
added
to
temperature
a Packard
Tri
each in
Carb
in
acid
the
and of
the
dark.
liquid
all
samples
7,500 vials
decolorized
units
of
which
After
catalase. were
cooling
scintillation
counter
then
to
5•Ž, .
RESULTS
Figure 1 shows that selenium supplemented ity from tocopherol into the plasma which
Fig,
1.
Uptake
of radioactivity
with tritiated tocopherol ed and 6 deficient rats.
After 8 changes
in plasma
acetate.
of selenium
rats rapidly subsequently
deficient
accumulate radioactiv disappears quickly .
and supplemented
Each time point represents
an average
hours, the level decreased until 24 hours were reached were not noted. In contrast, the level of radioactivity
selenium deficient rats did not reach decline over the next 36 hours.
a peak until
12 hours
rats dosed
of 4 supplement
in which further in the plasma of
afterwards,
with a slow
Figure 2 shows a different pattern of tocopherol uptake by erythrocytes than was seen for plasma. The selenium supplemented rats reach a peak at 12 hours, a time coincident with the selenium deficient animals. This is followed by a dec line to a steady
level by 24 hours.
In contrast,
the selenium
deficient
rats show a
3 Hundred ml a 10% solution of Omni-flor in toluene was diluted with 534ml of Triton X-100 and 1,360ml of toluene.
276
W. C. FISCHER and P. D. WHANGER
Fig. 2. Uptake of radioactivity in erythrocytes of selenium deficient and supplemented dosed with tritiated tocopherol acetate. Each time point represents an average supplemented
Fig.
and 6 deficient
rats of 4
rats.
3. Percentage of radioactivity in plasma of blood from selenium deficient and supple mented rats dosed with tritiated tocopherol acetate. Each time point represents an average
of 4 supplemented
and 6 deficient
rats.
SELENIUM
DEFICIENCY
AND
VITAMIN
E
277
more rapid uptake of the vitamin the first 12 hours followed by a rapid decline over the next 36 hours. A comparison of the percentage of radioactivity which is recovered in the plasma of selenium supplemented and deficient rats is shown in Fig. 3. This figure verifies the trends suggested by Figs. 1 and 2. Initially the supplemented rats incorporated and average of nearly 75% of the radioactivity into the plasma. At 12 hours after dosing, however, this decreased to about 15% and by 24 hours an equilibrium was reached at approximately 11%. The is in contrast to the selenium deficient rats in which only 24% of the radioactivity was in the plasma at 4 hours. This rapidly declined to 11% and no changes are apparent at the 12, 24, or 48 hour periods. Figure 4 shows the uptake of vitamin E by the liver. Although not to as great a degree, a trend similar to that seen in the erythrocytes is evident. Up to the 24 hour sample, the supplemented and deficient rats paralleled each other in the uptake of radioactivity, but the deficient rats took up a greater percentage (p
EFFECTS
OF
VITAMIN
SELENIUM
23, 273-280,
DEFICIENCY
E METABOLISM
IN
1977
ON
RATS1
W. C. FISCHER2 and P. D. WHANGER Department of Agricultural Chemistry Oregon State University Corvallis, Oregon 97331 (Received February 21, 1977)
Summary The metabolism of radioactive vitamin E in selenium defi cient rats has been compared to the metabolism of this vitamin in rats supplemented with 0.1ppm selenium as sodium selenite in the diet. After dosing with tritiated a tocopherol, the plasma level of radioactivity remained elevated through 48 hours in deficient rats. In contrast, a decline to nearly background levels occurred within 12 hours in the sup plemented rats. The uptake and release of radioactivity in the erythro cytes and liver were found to be more rapid in deficient rats than in sup plemented ones. A 65% greater increase in excretion of radioactivity oc curred in the urine from deficient rats as compared to supplemented ones. These data indicate that vitamin E is metabolized more rapidly in selenium deficient rats than in supplemented ones. The addition of vitamin E or selenium to the diets of experimental animals has been shown to protect against a number of pathological conditions. These include liver necrosis and carbon tetrachloride poisoning in rats (1-6), muscular degenera tion and exudative diathesis in chicks (7-11) and carbon tetrachloride poisoning in sheep (12). This similarity of action has been attributed to vitamin E func tioning as a free radical scavenger in the lipid phase of the cell and selenium as a component of glutathione peroxidase in the aqueous phase (13). A metabolic interrelationship between selenium and vitamin E has been known for several years (14), and the metabolism of one of these is influenced by the other. The hair coat and growth rate in second generation selenium deficient rats have been shown to respond to selenium supplementation but not to vitamin E (15, 16). 1 Published
with the approval
of the Oregon
State Agricultural
Experiment
Station
as Techni
cal Paper No. 4477. This research was supported by Public Health Service Research Grant No. NS 07413 from the National Institute of Neurological Disease and Stroke. 2 This communication is part of a thesis submitted by the senior author to the Graduate Facul ty of Oregon Present
address:
State
University Ciba-Geigy
in partial Corporation,
fulfillment
of the requirements
P. O. Box 11422, Greensboro, 273
for the Ph. D. degree. North
Carolina
27409.
274
W. C. FISCHER and P. D. WHANGER
Poor absorption of dietary vitamin E has been found in selenium deficient chicks (17). In selenium deficient rats, an alteration of the distribution of vitamin E has been reported (18). The objective of the present study is to determine the effect of selenium deficiency on the metabolism of vitamin E in rats and further elucidate the complex relationship between these two nutrients. MATERIALS The
rats
Second
used
for
generation
deficient
diet
given
the
the
study
were
selenium
from
deficient
throughout
selenium
AND
the
deficient
our
closed
animals
course
ration
METHODS
of
colony
were
of O.
S. U.
maintained
the
experiment
supplemented
with
on
(16)
.
Browns
a
Control
0.1ppm
.
selenium rats
selenium
as
were sodium
selenite. Alpha
tocopherol,
tritiated
Amersham/Searle, obtained were
used
prior
to
and
by
heart
tative
24
48
hours
group,
England,
were by
obtained
the
grade
redistilled
passing
from
Omni-Flor
Reagent
solvents purified
was
and
California.
was
blood
ml
portions of
diluted with
the
of
digested
was
chemicals
in
it through
was
diluted were
a
glass
still
a column
of
of
sealed
in
a clinical
25ml the All
70%
for
to and
were screw shaken
centrifuge
volumetric last
flask
partitioning,
samples
hour
at
similarly
were
to
diluted cap for to
the
the
aqueous
diluted
the
buffy KCl.
vials.
to
The one
hour
the
cells
Liver
with
phase
volume
was
respective
taken
the
washed volumes
cells
were of
were
di
vials,
The
decolorized
with
100•}10mg
the
red
and
cells a
3 .0ml
was 10min
top
layer
four
more
2 ,500
removed
times
either
added, at
was
a 10ml with
were
blood
benzene for
.
The
portions
plasma
water
into
minutes
vials. The
of for
of
repeated
ten
in sealed
distilled
separation,
phase
was
ml
were
centrifugation
partitioning
body 4 , 12,
a represen
for
and
samples used
equal
After
their
cells
for
An
and
Half
and
50ml
blood
layer.
75•Ž
procedure
to
of at
scintillation
H2O2
75•Ž.
5min. insure
in
0.9%
the
the KCl
centrifuge
placed
of
tubes.
and
a clinical
with
at
250g
counting.
along
of 30%
%
was 3H-a
chloroform
After 0.9
volumes
acid
0.2ml
one
for
and
scintillation
perchloric with
samples
removed
three in
E.
rats 12.5 ƒÊCi
per
with
with
collected in
tube
deoxycholate
vitamin
removed
supplemented
stomach
killed
perfused
removed
with
placed
H2O2
tube
were
by
were
tritiated rapidly also
selenium
sodium
group
at 2,500rpm
plasma
decolorized
urine
portion
was
decolorized 30%
The
Urine
plasma
and
and
with were
16 given
0.5%
dietary
livers
and was
and
each
the
0.2ml
0.4ml
ethanol
centrifuged
of
rats
animal
dosing
the
cells
was
5%
after
was
twice
plasma
deficient
from
taken.
The
gested
in
puncture,
washed
lowing
Centre,
Organic
Each
rats
sample
a
5 methyl
Richmond,
which
selenium
acetate
remainder
flask.
toluene
Four
24,
to
investigation.
experiment.
tocopherol
rpm
Inc.,
this
of
this
weight.
the
number
Radiochemical
Rad
except
total
in
Half
the
silica.
A
the
Bio
in use
activated
used
The
from
in
.
Fol
volumetric benzene
.
SELENIUM
or
water.
and
One
15ml
were
of
decolorized
heated
to
for
cooling,
H2O2
the
scintillation
of
allowed
to
stand were
the
scintillation one
the
ml
of
AND
benzene
phases
added.
Half
cocktail3
by
Fifteen
samples
samples
in
75•Ž
After with
the
ml
scintillation
DEFICIENCY
vials
by
the
VITAMIN
were ml
placed
samples
addition
E
275
in of
of
scintillation
the
0.2ml
vials
aqueous
of
30%
phases H2O2
and
hour. chemiluminescence
addition
of
was
25ƒÊl
of
cocktail
overnight
was
at room
counted
in
4%
quenched
ascorbic
added
to
temperature
a Packard
Tri
each in
Carb
in
acid
the
and of
the
dark.
liquid
all
samples
7,500 vials
decolorized
units
of
which
After
catalase. were
cooling
scintillation
counter
then
to
5•Ž, .
RESULTS
Figure 1 shows that selenium supplemented ity from tocopherol into the plasma which
Fig,
1.
Uptake
of radioactivity
with tritiated tocopherol ed and 6 deficient rats.
After 8 changes
in plasma
acetate.
of selenium
rats rapidly subsequently
deficient
accumulate radioactiv disappears quickly .
and supplemented
Each time point represents
an average
hours, the level decreased until 24 hours were reached were not noted. In contrast, the level of radioactivity
selenium deficient rats did not reach decline over the next 36 hours.
a peak until
12 hours
rats dosed
of 4 supplement
in which further in the plasma of
afterwards,
with a slow
Figure 2 shows a different pattern of tocopherol uptake by erythrocytes than was seen for plasma. The selenium supplemented rats reach a peak at 12 hours, a time coincident with the selenium deficient animals. This is followed by a dec line to a steady
level by 24 hours.
In contrast,
the selenium
deficient
rats show a
3 Hundred ml a 10% solution of Omni-flor in toluene was diluted with 534ml of Triton X-100 and 1,360ml of toluene.
276
W. C. FISCHER and P. D. WHANGER
Fig. 2. Uptake of radioactivity in erythrocytes of selenium deficient and supplemented dosed with tritiated tocopherol acetate. Each time point represents an average supplemented
Fig.
and 6 deficient
rats of 4
rats.
3. Percentage of radioactivity in plasma of blood from selenium deficient and supple mented rats dosed with tritiated tocopherol acetate. Each time point represents an average
of 4 supplemented
and 6 deficient
rats.
SELENIUM
DEFICIENCY
AND
VITAMIN
E
277
more rapid uptake of the vitamin the first 12 hours followed by a rapid decline over the next 36 hours. A comparison of the percentage of radioactivity which is recovered in the plasma of selenium supplemented and deficient rats is shown in Fig. 3. This figure verifies the trends suggested by Figs. 1 and 2. Initially the supplemented rats incorporated and average of nearly 75% of the radioactivity into the plasma. At 12 hours after dosing, however, this decreased to about 15% and by 24 hours an equilibrium was reached at approximately 11%. The is in contrast to the selenium deficient rats in which only 24% of the radioactivity was in the plasma at 4 hours. This rapidly declined to 11% and no changes are apparent at the 12, 24, or 48 hour periods. Figure 4 shows the uptake of vitamin E by the liver. Although not to as great a degree, a trend similar to that seen in the erythrocytes is evident. Up to the 24 hour sample, the supplemented and deficient rats paralleled each other in the uptake of radioactivity, but the deficient rats took up a greater percentage (p
