Antioxidant Supplements Improve Profiles of Hepatic Oxysterols and ...

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Antioxidant Supplements Improve Profiles of Hepatic
Oxysterols and Plasma Lipids in Butter-fed Hamsters
Johanne Poirier1, Kevin A. cockell1,2,3, W.M. Nimal ratnayake2, Kylie A. scoggan2,3, Nick hidiroglou2,
claude gagnon2, hélène rocheleau2, heidi gruber2, Philip Griffin2, rené Madère2, Keith Trick2
and stan Kubow1
1school of Dietetics and human Nutrition, Macdonald campus of Mcgill University, ste-Anne-de-Bellevue, Quebec,
canada h9X 3V9. 2Nutrition research Division, Food Directorate, health Products and Food Branch, health canada,
Ottawa, Ontario, canada K1A 0K9. 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa,
Ottawa, Ontario, canada K1h 8M5. email: stan.kubow@mcgill.ca
Abstract: Hypercholesterolemic diets are associated with oxidative stress that may contribute to hypercholesterolemia by adversely
affecting enzymatically-generated oxysterols involved in cholesterol homeostasis. An experiment was conducted to examine whether
the cholesterol-lowering effects of the antioxidants selenium and α-tocopherol were related to hepatic oxysterol concentrations. Four
groups of male Syrian hamsters (n = 7–8) were fed high cholesterol and saturated fat (0.46% cholesterol, 14.3% fat) hypercholesterol-
emic semi-purified diets: 1) Control; 2) Control + α-tocopherol (67 IU all-racemic-α-tocopheryl-acetate/kg diet); 3) Control + selenium
(3.4 mg selenate/kg diet); and 4) Control + α-tocopherol + selenium. Antioxidant supplementation was associated with lowered plasma
cholesterol concentrations, decreased tissue lipid peroxidation and higher hepatic oxysterol concentrations. A second experiment exam-
ined the effect of graded selenium doses (0.15, 0.85, 1.7 and 3.4 mg selenate/kg diet) on mRNA expression of the oxysterol-generating
enzyme, hepatic 27-hydroxylase (CYP27A1, EC 1.14.13.15), in hamsters (n = 8–9) fed the hypercholesterolemic diets. Supplementation
of selenium at 3.4 mg selenate/kg diet was not associated with increased hepatic 27-hydroxylase mRNA. In conclusion, the cholesterol
lowering effects of selenium and α-tocopherol were associated with increased hepatic enzymatically generated oxysterol concentra-
tions, which appears to be mediated via improved antioxidant status rather than increased enzymatic production.
Keywords: selenium, lipid hydroperoxide, glutathione, sterol 27-hydroxylase mRNA, tocopherols, thiobarbituric acid-reading substances
Nutrition and Metabolic Insights 2010:3 1–14
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Nutrition and Metabolic Insights 2010:3


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Poirier et al
Introduction
Enzymatical y generated oxysterols are primarily
Numerous human and animal studies have demonstrated
synthesized via cytochromes P-450 enzymes39 acting on
that intake of high cholesterol and high saturated
cholesterol levels that are over and above cel require-
fat (HCHS) diets results in both increased oxidative
ments. An important cytochrome P-450 enzyme is
stress1–3 and elevated plasma concentrations of non-
27-hydroxylase that is present in both liver and extra-
high density lipoprotein cholesterol (non-HDL-C), a
hepatic tissues and is proposed to be involved in the
major risk factor for cardiovascular disease.4 The asso-
regulation of cholesterol metabolism.39 Se induces tissue
ciation noted between oxidative stress and plasma lip-
cytochromes P-450 content,11,40 which suggests that its
ids5 suggests that antioxidants such as selenium (Se)
supplementation may lead to both an upregulation of the
and α-tocopherol (α-Toc) might beneficially influence
enzyme 27-hydroxylase and its product 27-OHC which
plasma lipids.
would help to explain its hypocholesterolemic effects.
Dietary Se supplementation in various animal models
Similar to the enzymatically generated oxysterols
has been shown to be associated with decreased plasma
that are associated with diets high in cholesterol,41–43
levels of total cholesterol (TC), low density lipopro-
free radical generated oxysterols are produced via con-
tein cholesterol (LDL-C), low density lipoprotein
sumption of HCHS diets;44 however, they do not act as
cholesterol + very low density lipoprotein cholesterol
ligands for LXR. Moreover, recent evidence suggests
(LDL-C + VLDL-C) and triglycerides (TG).6–20 Sev-
that free radical generated oxysterols can impede the
eral studies also indicate that supplementation of Se
physiological actions of enzymatic oxysterols.45 The
can lower LDL-C and raise high density lipoprotein
oxysterol 7-ketocholesterol (7-keto) is one of the main
cholesterol (HDL-C) in human subjects.21–24 Human
free radical oxysterols produced during cholesterol
and animal studies have examined the effects of
oxidation46 and is used as a marker of oxidative stress.
α-Toc on plasma lipids with mixed results. In animal
There is little knowledge regarding the impact of
models, α-Toc supplementation is generally hypocho-
antioxidant supplements, provided either alone or in
lesterolemic25–30 whereas, in most human trials, α-Toc
combination, on either free radical or enzymatically
has typically shown no effect on plasma cholesterol
generated oxysterols. Antioxidants including Se have
concentrations except for certain circumstances such
been shown to inhibit the production of free radical
as in diabetes.31
produced oxysterols,47 which suggests that antioxi-
The cholesterol-lowering mechanisms of Se
dant supplements can lead to improved ratios of enzy-
and α-Toc are not well understood. Mechanisms
matic to free-radical generated oxysterols. Such ratio
underlying the cholesterol lowering effects of the
improvements might enhance the physiological actions
supplementation of both Se15–20 and α-Toc32,33 have
of enzymatic oxysterols including their cholesterol
been explored, however the antioxidants have not
modulating effects, which could lead to cholesterol-
been studied for their association with the liver X
lowering effects of Se and α-Toc supplementation.
receptor (LXR) pathway. Activation of the LXR
In the present work, two experiments were performed.
pathway has been shown to result in the lowering
In Experiment 1, the effects of non-toxic pharmacologi-
of whole body levels of cholesterol that, in turn,
cal supplementation of Se and α-Toc in concert with
can lead to blood cholesterol lowering action.34 The
HCHS feeding in adult male Golden Syrian hamsters
most important physiological activators for the LXR
were examined on: i) plasma lipid concentrations; i )
are the enzymatically produced oxysterols which
hepatic glutathione (GSH) content, hepatic Se-dependent
include the species 24(S)-hydroxycholesterol (24(S)-
GSH peroxidase (SeGSH-Px, EC 1.11.1.9) and non-
OHC), 25-hydroxycholesterol (25-OHC) and 27-
Se-dependent GSH peroxidase (non-SeGSH-Px, EC
hydroxycholesterol (27-OHC).35–37 The importance
2.5.1.18) activities; and i i) in vivo lipid peroxidation
of these three oxysterols as in vivo ligands for LXR
and enzymatical y and free radical produced hepatic
has been strongly implicated in a recent study, which
oxysterols. In Experiment 2, adult male Golden Syr-
showed that feeding a high cholesterol diet to triple-
ian hamsters were fed graded levels of Se for four
knockout mice that do not synthesize 24(S)-OHC,
weeks in order to examine the effect of Se on hepatic
25-OHC and 27-OHC failed to induce hepatic mRNA
cytochrome P-450 27-hydroxylase (Cyp27a1) mRNA
expression of LXR responsive genes.38
expression.

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Selenium improves hepatic oxysterol profiles
Materials and Methods
to Syrian hamsters for 25 wk.50 Subacute toxicity of
experiment 1—Animals, diets,
Se has been tested in previous work, which found
and sample preparation
levels of dietary Se up to 10 ppm to be non-toxic
The feeding and animal handling protocol was fol owed
to Syrian hamsters.51,52 The levels of α-Toc in the
as previously described.10 Adult hamsters were chosen
basal diet were chosen based on National Research
for the present study based on similarities with humans
Council (NRC) recommendations for the hamster
with regards to dietary cholesterol48 and dietary Se
of 27 IU all-racemic α-tocopheryl acetate/kg diet
metabolism.49 Following the acclimatization period,
(10 tocopherol equivalents = 10 mg all-racemic
hamsters were fed for three wk one of four diets
α-tocopherol = 15 IU).53 The α-Toc supplemented
(Table 1) which consisted of: i) Control (CT) con-
diet contained 67 IU all-racemic α-tocopheryl
taining formulated basal requirements of α-Toc and
acetate/kg diet which provided 2.5-fold higher levels
Se; ii) CT + α-toc (67 IU all-racemic α-tocopheryl
of α-tocopheryl acetate/kg diet NRC recommenda-
acetate/kg diet); iii) CT + Se (3.4 mg Se/kg diet);
tions for the hamster.53 Care and handling of the
iv) CT + α-Toc (67 IU all-racemic α-tocopheryl
Syrian hamsters conformed to the guidelines of the
acetate/kg diet) + Se (3.4 mg Se/kg diet). The phar-
Canadian Council of Animal Care54 and the protocols
macological supplemental level of Se (3.4 ppm/kg
for the animal experiments were approved by the
diet) was chosen to be equivalent to two-thirds of
McGill University Animal Care Committee. Samples
the non-toxic 5 ppm Se/kg diet dose fed previously
were prepared as previously described.10
Table . composition of experimental diets (g/kg)a
Ingredients
cTb
cTb + α-Toc
cTb + se
cTb + α-Toc + se
casein, vitamin-free
159.1
159.1
159.1
159.1
cornstarch
285.44
285.34
284.66
284.56
sucrose
175.3
175.3
175.3
175.3
Dextrose
99
99
99
99
cellulose
43
43
43
43
Butterc
157
157
157
157
Safflower oild
13.76
13.76
13.76
13.76
cholesterol, UsPe
4.5
4.5
4.5
4.5
Mineral mixf
43
43
43
43
Vitamin mixg
8.6
8.6
8.6
8.6
choline bitartrate
11.2
11.2
11.2
11.2
sodium selenate
0.046
0.046
0.8229
0.8229
Vitamin e acetate
0.052
0.134
0.052
0.134
Metabolizable energy, MJ/Kg
18.3
18.3
18.3
18.3
aAll diets were formulated at Mcgill University and prepared in pellet form by Dyets Inc. (Bethlehem, Penn).
bFatty acid composition of cT is as follows (% by weight) as provided by Dyets: c4:0, 3.4; c6:0, 2.0; c8:0, 1.2; c10:0, 2.7; c12:0, 3.0; c14:0, 10.7; c14:1,
1.6; c16:0, 28.0; c16:1, 2.5; c18:0, 13.0; c18:1, 26.8; c18:2, 2.5; c18:3, 1.5; c20:0, 1.1.
cButter contains 18% h O and therefore 157 g fat /kg diet provided 129 g fat/kg diet.
2
dSafflower oil was added to prevent essential fatty acid deficiency. α-Toc concentration of sAFF is 350 ppm of α-tocopherol, 180 ppm of other tocopherols.
Fatty acid profile of safflower oil included (% by weight): 14:0, trace; 16:0, 6.9; 16:1, trace; 18:0, 2.9; 18:1, 12.2; 18:2, 78.0; 18:3, trace.
echolesterol UsP was added to butterfat 4.5 g/kg.
fThe mineral mix was free of se and was composed of (g/kg): calcium carbonate 336.4; calcium phosphate, monobasic 285.0; magnesium oxide 2.985;
potassium iodate (10 mg KI/g) 0.76; potassium phosphate, dibasic 40.76; sodium chloride 11.45; cupric carbonate 0.084; cobalt chloride 0.133; sodium
fluoride 0.002; ferric citrate 25.45; manganous carbonate 0.229; ammonium paramolybdate 0.008; zinc carbonate 0.53; sucrose 296.209. Sodium selenate
(10 mg/g sodium selenate) was added separately to make the diets; for basal se diets 0.046; for high se diets 0.8229.
gThe vitamin mix was free of α-Toc and was composed of (g/kg): vitamin A palmitate (500,000 IU/g) 0.4263; vitamin D3 (400,000 IU/g) 0.9315; vitamin K1
premix (10 mg/g) 110.0; biotin 0.03; folic acid 0.3; niacin 13.5; pantothenate (Ca) 1.5; riboflavin 2.25; thiamin HCl 3.0; pyridoxine HCl 0.9; vitamin B12
(0.1%) 1.5; sucrose 865.6622. α-Toc acetate (500 IU/g) was added separately to make the diets; for basal α-Toc diets, 0.052; for high α-Toc, 0.129.
Abbreviations: cT, control; cT + α-Toc, control + α-tocopherol; cT + se, control + selenium; cT + α-Toc + se, control + α-tocopherol + selenium.
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Poirier et al
experiment 2—Animals, diets,
acid-reacting substances (TBARS) using a modified
and sample preparation
method of Asakawa and Matsushita55 and Wong et al;56
Forty adult Golden Syrian male hamsters,
c) tissue supernatants were assayed for total GSH-Px
aged 9–10 wks (approximate weight 110–120 g) were
and SeGSH-Px activity using an automated modifica-
purchased from Charles River. Hamsters were housed
tion57 of the coupled assay of Paglia and Valentine;58
in the Animal Resources Division, Food Directorate
d) the detection of GSH and GSSG was completed
of Health Canada (Ottawa, ON, Canada) in stainless
using a Cayman chemical kit (Ann Arbor, MI, USA);
steel wire-bottom cages and acclimatized to labora-
e) the concentrations of α-Toc in liver were analyzed
tory conditions for 10 days while being fed a standard
by HPLC; and f) protein concentrations of all superna-
commercial diet. At the end of the acclimatization
tants were determined using the Biuret assay (Abbott
period hamsters were weighed and randomized to
LN5A13-26) with BSA as a standard. For liver Se
four groups of ten animals each and fed their respec-
analysis, hepatic tissue was digested with nitric acid
tive diets for four wk. The dietary levels of Se were
and the Se content was measured using flame atomic
adjusted to four different levels which included: 1)
absorption spectrophotometry (Hitachi, Polarized
CT; 2) CT + 0.85 ppm Se; 3) CT + 1.7 ppm Se; and
Zeeman AAS, Z-8200, Mississauga, Canada).
4) CT + 3.4 ppm Se. As in Experiment 1, the mini-
mal level of Se of 0.15 ppm in the basal diet con-
hepatic oxysterol analysis
formed with NRC guidelines of 0.1 ppm53 (Table 1).
Oxysterol determination of hepatic tissues was per-
The semi-purified diet composition was similar to
formed by GC-MS as described previously.59 Briefly,
Table 1 except that cholesterol was lowered from
19-hydroxycholesterol was added to the samples as an
0.45% to 0.1% w/w and the basal level of α-Toc was
internal standard before lipid extraction. Artifactual
increased from 27 IU/kg diet to 45 IU/kg diet in CT
oxidation of cholesterol during sample processing was
diet. The feeding and animal handling protocol was
minimized by incorporation of L-ascorbic acid and
followed as per Experiment 1.10 At the end of the
sodium acetate to scavenge oxygen and acidic spe-
feeding period, hamsters were fasted overnight and
cies, respectively. The lipid extract was saponified and
sacrificed within 2 days in a treatment-blocked ran-
unsaponified lipids were extracted with diethyl ether
domized order. Immediately following removal, the
and free fatty acids were removed by KOH. Bulk cho-
liver pieces were weighed, frozen in liquid nitrogen
lesterol was removed by solid-phase extraction and
and stored at −80 °C until further use.
oxysterols were eluted with 2-propanol in hexane.
Samples were evaporated at room temperature under
Assays
nitrogen and converted to trimethylsilyl ethers for
Plasma cholesterol and triglyceride
GC-MS analysis (Agilent 6890 GC System with 5973
(Tg) analysis, measurement of tissue
Mass Selective Detector, Agilent Technologies, Wilm-
lipid peroxidation, analysis of hepatic
ington, DE, USA) using a J and W DB-1 capillary col-
total gsh-Px, segsh-Px and non-
umn with flow rate of helium carrier gas of 1.0 mL/min.
segsh-Px (gsh transferase) activities,
The injector was operated in splitless mode and with
an initial temperature of 290 °C. After injection, oven
hepatic gsh and oxidized gsh (gssg)
temperature began at 80 °C, and then programmed at
analysis, hepatic α-Toc and se analysis,
a rate of 30 °C/min to a final temperature 215 °C, held
and hepatic protein
for 2 min, followed by a rate of 2 °C/min to a final
The following assays were carried out as previ-
temperature of 280 °C held for 10 min. A volume of
ously described in greater detail:10 a) plasma lipids
1 µL per sample was injected. Oxysterol analysis was
were measured using automated enzymatic method-
carried out using selected ion monitoring. The mul-
ology using the Abbott VP Super System (Abbott,
tiple ion detector was focused on m/z 145, 353, and
Irving, TX, USA) with Abbott enzymatic reagent kits
366 for 19-OHC; m/z 367 and 472 for 7-keto; 145,
(Abbott, Irving, TX, USA; b) the extent of lipid per-
413, 456 for 24(S)-OHC; 131, 327, 456 for 25-OHC;
oxidation in plasma was determined as thiobarbituric
and m/z 129, 417, and 456 for 27-OHC.

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Selenium improves hepatic oxysterol profiles
hepatic rNA extraction and real-time
was omitted in the cDNA synthesis reaction. Standard
quantitative polymerase chain reaction
curves for Cyp27a1 and Gapdh were used to calculate
(rT-Pcr)
the relative levels of Cyp27a1 mRNA. The relative
Total RNA was extracted from frozen hamster liver
amounts of Cyp27a1 were normalized using Gapdh
samples using two passes of Trizol reagent (Invitro-
mRNA expression levels as an endogenous internal
gen Life Technologies, Burlington, ON, Canada) for
standard. Normalized values (Cyp27a1/Gapdh) were
each sample. The isolated RNA was then purified and
then calibrated to control group, namely, control rats
DNase I treated on RNeasy mini columns (Qiagen,
fed the control diet (set as 1.0).
Mississauga, ON, Canada) as per the manufacturer’s
RT-PCR was performed in a model Stratagene
recommended conditions. RNA integrity was assessed
Mx4000 detection system using Mx4000 software. The
spectrophotometrically by measuring the absorbance
thermal cycler parameters were as fol ows: 95 °C for
ratio of 260/280 nm and by a conventional agarose gel.
10 min 1 cycle to activate SureStart Taq, 40 cycles of
RNA was quantified using RiboGreen RNA Quantita-
denaturation (95 °C 30 sec), Annealing (60 °C 45 sec)
tion Reagent and Kit (Molecular Probes, Eugune, OR,
and Extension (72 °C 1 min), 1 cycle for denaturation
USA). 2 µg of RNA per sample was transcribed to
of amplicons (95 °C 1 min), 81 cycles for dissociation
complementary DNA with Retroscript Kit (Ambion)
curve (55 °C to 95 °C, 10 sec, 0.5 sec/cycle), 1 cycle for
using Oligo dT as per the manufacturer’s instructions.
end of assay (25 °C, hold).
Primers for Cyp27a1 were designed using Primer-
Quest software based on sequence alignment of regions of
statistical analysis
high homology between rat and human Cyp27a1 mRNA
Results are presented as means ± SEM. Data were
(Genbank NM_178847 and AY178622, respectively)
tested using the mixed model procedure using SAS
available through NCBI.60 The sequences were analyzed
software version 9.1.62 ANOVA was used to determine
using the Basic Local Alignment and Search Tool60 to
the effect of treatment and differences between treat-
verify that the primers were specific for Cyp27a1. For
ment means were identified by least square means.
glyceraldehyde-3-phosphate dehydrogenase (Gapdh),
Treatment effects and differences between treatments
appropriate Syrian hamster primers were identified
were considered significant when P  0.05. Data was
based on previously published rat primers.61 The for-
assessed for normality (univariate) and homogeneity of
ward primer exactly matched Syrian hamster Gapdh
variance using a mixed model procedure. Correlations
sequence (Genbank accession number U10983.1).
between biochemical measurements were examined
A single-base change was made to the reverse primer
using Spearman’s correlation coefficient by rank.
to make an exact match with the Syrian hamster. Primer
For Experiment 1, to determine whether the α-Toc
sequences were 5’-GGA TCC AAC ACC CAT TTG
and Se treatments had independent or interactive
GCT CTG-3’ and 5’-TGT ATC AGC CTT GAC AGC
effects on the dependent variables tested, a fixed
AGG AGT-3’ for Cyp27a1, and 5’-TCA AGA AGG
effect, 2 (Se levels) x 2 (α-Toc levels) analysis of
TGG TGA AGC AGG C-3’ and 5’-GCA TCA AAG
variance factorial design was used. P values are given
GTG GAA GAG TGG G-3’ for Gapdh.
as the least square means of the interaction effects
The quantification of Cyp27a1 was carried out using
(α-Toc x Se) and the main effects (α-Toc, Se) with
SYBR Green Core Reagent Kit (Sratagene, La Jolla,
pooled SEM. Main effects include the effect of basal
CA, USA). To a microtiter plate well, 5 µL volume of
α-Toc vs. supplemental α-Toc, and basal Se vs. sup-
cDNA was added per reaction up to 50 µL final volume
plemental Se. The four interaction effects included the
per well. Final primer concentration equaled 250 nM,
combinations of basal α-Toc x basal Se, basal α-Toc
magnesium chloride at 2.5 mM. Duplicate samples
x supplemental Se, supplemental α-Toc x basal Se,
were run and averaged. For Cyp27a1 and Gapdh primer
and supplemental α-Toc x supplemental Se. Blocking
sets, two types of negative controls were used: 1) No-
was included in the statistical analysis because it was
template control in which water was added instead
an integral part of the experimental design due to the
of cDNA template; and 2) No-Reverse Transcriptase
staggered feeding which occurred in distinct blocks
control, in which the Reverse Transcriptase enzyme
over time and included one hamster from each dietary
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Poirier et al
group to make a total of four hamsters per block. Final
experiment 1—Plasma cholesterol
weight was used in the model to analyze liver weight
and Tg concentrations
(P  0.05).
As shown in Table 2, a significant main effect of
For Experiment 2, a fixed effect of one factor with
Se treatment was observed with respect to plasma
4 levels factorial design was used. Blocking was con-
concentrations of TC (P  0.05), non-HDL-C
sidered to be four blocks of ten hamsters from each
(P  0.005) and the ratio of potentially atherogenic
of four dietary groups determined at time of death of
non-HDL-C/antiatherogenic HDL-C (P  0.005),
hamsters. The microtiter plate which was used for
which were 18%, 32% and 22% lower, respectively,
mRNA analysis was also considered to be a source
than the CT diet-fed hamsters. The hamster treatment
of variation. Neither blocking nor microtiter plate
group receiving α-Toc supplements also had signifi-
were used in analysis as they were found to be non-
cantly (P  0.05) lower plasma concentrations of
significant in the model. Final weight was included in
TC and non-HDL-C concentrations (Table 2). Sig-
the analysis of liver weight (P  0.005).
nificant interactive effects of α-Toc x Se on plasma
HDL-C concentrations were observed (P
Results
 0.05)
(Table 2). The hamsters fed the α-Toc and Se sup-
experiment 1—Final weight
plemented diet had significantly (P  0.05) higher
and liver weight
plasma HDL-C concentrations in comparison to the
Adult hamsters consuming CT, CT + α-Toc, CT + Se,
α-Toc-supplemented hamsters. In addition, hamsters
and CT + α-Toc + Se diets showed final weights (g) of
receiving the combined supplements of α-Toc and Se
121 ± 1.5, 122 ± 1.4, 121 ± 1.4, and 124 ± 1.4, respec-
showed a significantly lower ratio of non-HDL-C/
tively. Final liver weights (g) of adult hamsters consum-
HDL-C in comparison to all other treatment groups.
ing CT, CT + α-Toc, CT + Se, and CT + α-Toc + Se diets
Plasma TG concentrations did not differ among treat-
were 6.8 ± 0.1, 6.8 ± 0.1, 6.7 ± 0.1, and 7.1 ± 0.1, respec-
ment groups (Table 2).
tively. Neither α-Toc nor Se treatments were associated
with any significant effect on final body weight or liver
experiment 1—hepatic oxysterols, total
weight. The pharmacological dose of 3.4 ppm Se used
hepatic content of enzymatic oxysterols,
in the present study was wel tolerated by the hamsters
and hepatic oxysterol/7-keto ratios
as general health, survival, final body weight, and organ
A significant main treatment effect of Se supplementa-
weights of hamsters remained unaffected.
tion on hepatic concentrations of 25-OHC (P  0.05)
Table . ef ects of dietary α-Toc and se supplementation on plasma lipid concentrations (Tc, non-hDL-c, hDL-c, non-hDL-c/
hDL-c ratios and Tg) of adult male syrian hamsters fed hchs diets for 3 wk.1
Variable
Dietary treatment
Main and interaction effects
cT
cT + α-Toc
cT + se
cT + α-Toc + se
α-Toc
se
α-Toc x Se
Tc
5.8 ± 0.2a
4.9 ± 0.2b
4.8 ± 0.2b
4.9 ± 0.2b
P  0.05
P  0.05
Ns
(mmol/L)
Non-hDL-c
2.6 ± 0.2a
2.1 ± 0.2b
1.8 ± 0.2b
1.6 ± 0.2b
Ns
P  0.005
Ns
(mmol/L)
hDL-c
3.2 ± 0.2ab
2.8 ± 0.2a
3 ± 0.2ab
3.3 ± 0.2b
Ns
Ns
P  0.05
(mmol/L)
Non-hDL-c/ 0.83 ± 0.08a
0.84 ± 0.08a
0.65 ± 0.08b
0.50 ± 0.08b
Ns
P  0.005
Ns
hDL-c
Tg (mmol/L)
4.4 ± 0.4
4.1 ± 0.4
3.7 ± 0.4
4.1 ± 0.4
Ns
Ns
Ns
1Values are mean ± seM, n = 8. Means within rows without a common superscript letter differ, P  0.05. Diets and abbreviations are as indicated in
Table 1. 2Main effects include the effect of basal α-Toc vs. supplemental α-Toc and basal se vs. supplemental se. The four interaction effects included
the combinations of basal α-Toc x basal se, basal α-Toc x supplemental se, supplemental α-Toc x basal se, and supplemental α-Toc x supplemental se.
Blocking included in statistical model.
Abbreviations: hchs, high cholesterol and high saturated fat diet; hDL-c, high density lipoprotein cholesterol; Non-hDL-c, non-high density lipoprotein
cholesterol; Non-hDL-c/hDL-c, non-high density lipoprotein cholesterol/high density lipoprotein cholesterol ratio; Tc, total cholesterol; Tg, triglyceride.

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Selenium improves hepatic oxysterol profiles
Hepatic oxysterols
300
*
* *
*
250
*
200
150
100
(pmol/g wet weight)
50
* *
0
7-keto
24(S)-OHC
25-OHC
27-OHC
Total
Figure . effects of dietary α-Toc and se supplementation on liver 7-keto, 24(s)-Ohc, 25-Ohc, 27-Ohc and total oxysterols concentrations of adult male
Syrian hamsters fed high cholesterol and high saturated fat diets for 3 wk. (*) Significantly different from control (P  0.05). White bars, cT; Light grey bars,
cT + α-Toc; striped bars, cT + se; Dark grey bars, cT + α-Toc + se. Values are mean ± seM (n = 8). Diets and abbreviations are as indicated in Table 1.
Abbreviations: 24(s)-Ohc, 24(s)-hydroxycholesterol; 25-Ohc; 25-hydroxycholesterol; 27-Ohc, 27-hydroxycholesterol; 7-keto, 7-ketocholesterol; Total,
total oxysterols.
and 27-OHC (P  0.05) was observed (Fig. 1). In
observed. Hamsters consuming CT, CT + α-Toc, CT
terms of the liver content of 25-OHC, the CT + Se and
+ Se, and CT + α-Toc + Se diets showed ratios of
the CT + α-Toc + Se groups had higher (P  0.05)
6.63 ± 2.1, 4.95 ± 2.1, 9.30 ± 2.2, and 10.2 ± 2.1,
concentrations in comparison to the CT group. Both
respectively.
Se-supplemented diet groups had a significant (P  0.05)
increase of approximately 200% in the liver content of
experiment 1—Plasma TBArs
27-OHC as compared to livers of hamsters consuming
concentrations and hepatic LPO
the CT diet. Additional y supplementation of α-Toc
concentrations
alone showed a significant increase (P  0.05) in
A significant main effect of Se treatment (P  0.05) on
hepatic 27-OHC concentrations relative to the CT diet-
plasma TBARS concentrations was observed as ham-
fed hamsters. No significant effect of the dietary treat-
sters consuming Se supplemented diets showed approx-
ments on hepatic concentrations of 7-keto was observed
imately 19% lower plasma TBARS concentrations as
(Fig. 1). A main treatment effect of Se was noted on
compared to hamsters consuming CT diets (Table 3).
total enzymatic oxysterols (P  0.05) (Figure 2). Also,
In addition, α-Toc supplemented hamsters had signifi-
the combination of Se and α-Toc was shown to signifi-
cantly (P  0.05) lower plasma TBARS concentrations
cantly increase total enzymatic oxysterols as compared
as compared to the hamsters fed the CT diet (Table 3).
to controls (P  0.05) (Fig. 1).
A significant main effect of Se treatment on con-
A significant (P  0.05) main treatment effect of
centrations of LPO was observed in liver (P  0.05)
Se was noted on hepatic 24(S)-OHC/7-keto. Adult
(Table 3). Se supplementation resulted in a signifi-
hamsters consuming CT, CT + α-Toc, CT + Se, and
cant (P  0.05) lowering of LPO concentrations of
CT + α-Toc + Se diets showed 24(S)-OHC/7-keto
approximately 45% in liver tissue relative to ham-
oxysterol ratios of 1.76 ± 0.49, 1.34 ± 0.53, 2.96 ±
sters fed the CT diet. In addition, hamsters fed the
0.53, and 2.78 ± 0.57, respectively. A tendency (P =
CT + α-Toc or CT + α-Toc + Se diets had signifi-
0.07) for a significant main effect of Se treatment
cantly lower (P  0.05) liver concentrations of LPO
on the total enzymatic oxysterols/7-keto ratio was
relative to the CT-fed hamsters.
Nutrition and Metabolic Insights 2010:3


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Poirier et al
1.8
1
)
1.6
*
*
1.4
(
Cyp27a
1.2
1
0.8
0.6
mRNA Expression
0.4
Relative Hepatic
0.2
0
Figure . effects of dietary se supplementation on liver sterol 27-hydroxylase (cyp27a1) mrNA levels of adult male syrian hamsters fed high cholesterol
and high saturated fat diets for 4 wk. (*) Significantly different from control (P  0.05). White bar, cT (0.15 ppm); Light grey bar, cT + se (0.85 ppm); Dark
grey bar, cT + se (1.7 ppm); Black bar, cT + se (3.4 ppm). Values are seM (n = 8 or 9).
Abbreviations: cT, control; se, selenium; ppm, parts per million.
experiment 1—Tissue concentrations
hepatic total enzymatic oxysterols/7-keto ratios (r2 =
of gsh and gssg
0.45, P  0.05).
Significant main effects of Se supplementation were
seen with respect to hepatic concentrations of GSH
experiment 1—hepatic segsh-Px
(P  0.005) (Table 4). Hamsters receiving diets
and non-segsh-Px (gsh transferase)
containing Se supplements had significantly higher
activities
hepatic concentrations of GSH relative to non-
The hepatic SeGSH-Px activity was significantly
Se supplemented dietary treatments (Table 4). In
higher (P  0.05) in association with Se supplementa-
the liver, a main effect of Se supplementation was
tion as the CT + Se and the CT + α-Toc + Se groups
observed with respect to hepatic GSSG concentra-
had higher activity relative to the CT group (Table 4).
tions (P  0.05) (Table 3). Supplementation of α-
Hepatic SeGSH-Px activity was not affected by supple-
Toc did not affect hepatic concentrations of GSSG
mental α-Toc (Table 4). Non-SeGSH-Px activity was
(Table 3). Hepatic GSH/GSSG ratios were unaffected
unaffected by the dietary treatments in liver (Table 4).
by dietary treatments (Table 4). Hepatic content of
GSH was positively correlated with a) hepatic Se
experiment 1—hepatic content of α-Toc
content (r2 = 0.54; P  0.005); b) hepatic SeGSH-Px
and se
activity (r2 = 0.50; P  0.005); c) hepatic concentra-
As expected, significant (P  0.005) main effects
tions of 24(S)-OHC (r2 = 0.42; P  0.05); and d)
of α-Toc treatment on α-Toc concentrations were
Table . effects of dietary α-Toc and se supplementation on plasma TBArs and liver LPO of adult male syrian hamsters
fed hchs diets for 3 wk.1
Variable
Dietary treatment
Main and interaction effects
cT
cT + α-Toc
cT + se
cT + α-Toc + se
α-Toc
se
α-Toc x Se
TBArs
1.08 ± 0.06a
0.93 ± 0.06b
0.87 ± 0.06b
0.87 ± 0.06b
Ns
P  0.05
Ns
(µmol/L)
LPO
14.8 ± 1.5a
10.4 ± 1.5b
8.2 ± 0.5b
8.5 ± 1.5b
Ns
P  0.05
Ns
(µmol/g protein)
1Values are mean ± seM (n = 8). Means within rows with no common superscript roman letter differ significantly (P  0.05). Diets and abbreviations are
as indicated in Table 1. 2Main effects include the effect of basal α-Toc vs. supplemental α-Toc and basal se vs. supplemental se. The four interaction
effects included the combinations of basal α-Toc x basal se, basal α-Toc x supplemental se, supplemental α-Toc x basal se, and supplemental α-Toc x
supplemental se.
Abbreviations: hchs, high cholesterol and high saturated fat; LPO, lipid hydroperoxide; TBArs, thiobarbituric acid-reacting substances.

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Selenium improves hepatic oxysterol profiles
Table . effects of dietary α-Toc and se supplementation on liver gsh, gssg, gsh/gssg ratios, segsh-Px activity,
Non-segsh-Px activity of adult male syrian hamsters fed hchs diets for 3 wk.1
Variable
Dietary treatment
Main and interaction effects
cT
cT + α-Toc
cT + se
cT + α-Toc + se
α-Toc
se
α-Toc x Se
gsh
318 ± 2.4a
16 ± 2.0a
27 ± 2.0b
30 ± 2.0b
Ns
P  0.005
Ns
(µmol/g protein)
gssg
38 ± 1.0a
9 ± 0.9a
11 ± 0.9b
11 ± 0.9b
Ns
P  0.05
Ns
(µmol/g protein)
gsh/gssg
33 ± 0.55
2 ± 0.55
3 ± 0.45
3 ± 0.45
Ns
Ns
Ns
segsh-Px
208 ± 9a
198 ± 9a
238 ± 8b
232 ± 9b
Ns
P  0.05
Ns
(units/mg protein)
Non-segsh-Px
54 ± 5
41 ± 5
50 ± 5
49 ± 5
Ns
Ns
Ns
(units/mg protein)
1Values are mean ± seM (n = 8) except where noted, 3n = 7. Means within rows with no common superscript roman letter dif er significantly (P  0.05).
Diets and abbreviations are as indicated in Table 1.
2Main effects include the effect of basal α-Toc vs. supplemental α-Toc and basal se vs. supplemental se. The four interaction effects included the
combinations of basal α-Toc x basal se, basal α-Toc x supplemental se, supplemental α-Toc x basal se, and supplemental α-Toc x supplemental se.
Blocking included in statistical model except for gsh/gssg ratios.
Abbreviations: gsh, glutathione; gssg, oxidized glutathione; gsh/gssg, glutathione/oxidized glutathione ratio; hchs, high cholesterol and high
saturated fat diet; segsh-Px, se dependent glutathione peroxidase; Non-segsh-Px, non-se dependent glutathione peroxidase.
observed in liver with α-Toc-supplemented hamsters
experiment 2—hepatic cyp27a1 mrNA
showing 1.7-fold increases in α-Toc content in liver
expression
tissues, respectively, relative to the CT-fed hamsters
Hamsters consuming the HCHS + 0.85 ppm and
(Table 5). In liver, the CT + α-Toc and the CT +
HCHS + 1.7 ppm Se diets showed significant
α-Toc + Se treatment groups had significantly higher
(P  0.05) increases in hepatic mRNA expression of
(P  0.05) α-Toc concentrations relative to the other
Cyp27a1 as compared to livers of hamsters fed the
diet groups. Liver α-Toc was positively correlated
HCHS + 0.15 ppm Se diet (Fig. 2). No significant
with a) 25-OHC (r2 = 0.31; P  0.05); b) 27-OHC
effect on hepatic Cyp27a1 mRNA expression was
(r2 = 0.33; P  0.05); and c) total enzymatic oxyster-
observed in hamsters fed the HCHS + Se (3.4 ppm)
ols (r2 = 0.44, P  0.05).
diet associated with plasma cholesterol-lowering
A significant main effect of Se treatment (P  0.005)
effects.
on liver Se concentrations was observed as consump-
tion of Se was associated with a significant 209%
increase (P  0.005) in liver Se content in hamsters
experiment 2—Final weight, liver weight,
fed the CT + Se diet in comparison to the liver Se
liver weight/final weight ratio
content of hamsters fed the CT diet alone (Table 5). A
Adult hamsters consuming CT (0.15 ppm), CT
significant main effect of α-Toc (P  0.005) and a sig-
+ Se (0.85 ppm), CT + Se (1.7 ppm), and CT +
nificant interactive effect of α-Toc × Se (P  0.05) on
Se (3.4 ppm) diets showed final weights in grams
liver Se content were observed. Hamsters fed the CT +
of 116 ± 3, 109 ± 3, 111 ± 3, and 109 ± 3, respec-
α-Toc + Se diet showed significantly higher liver con-
tively. Final liver weights in grams of adult hamsters
centrations of Se as compared to hamsters consuming
consuming CT (0.15 ppm), CT + Se (0.85 ppm),
the CT (P  0.005), CT + α-Toc (P  0.005) and CT
CT + Se (1.7 ppm), and CT + Se (3.4 ppm) diets
+ Se (P  0.005) diets. Hepatic Se content was posi-
were 4.9 ± 0.12, 5.1 ± 0.12, 5.0 ± 0.12, and 5.1 ±
tively correlated with hepatic content of: a) 24(S)-OHC
0.12, respectively. Liver weight/final weight ratios
(r2 = 0.33; P  0.05); b) 25-OHC (r2 = 0.37; P  0.05);
of adult hamsters consuming CT (0.15 ppm), CT +
c) 24(S)-OHC/7-keto (r2 = 0.481, P  0.05); and
Se (0.85 ppm), CT + Se (1.7 ppm), and CT + Se
d) 25-OHC/7-keto (r2 = 0.482, P  0.05).
(3.4 ppm) diets were 0.044 ± 0.001, 0.045 ± 0.001,
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Poirier et al
Table . effects of dietary α-Toc and se supplementation on liver α-Toc and liver se of adult male syrian hamsters fed
hchs diets for 3 wk.1
Variable
Dietary treatment
Main and interaction effects
cT
cT + α-Toc
cT + se
cT + α-Toc + se α-Toc
se
α-Toc x Se
α-Toc (nmol/g protein)
321 ± 2a
334 ± 2b
324 ± 2a
36 ± 2b
P  0.005
Ns
Ns
se (nmol/g wet wt)
12 ± 2a
13 ± 2a
24 ± 2b
34 ± 2c
P  0.005
P  0.005
P  0.05
1Values are mean ± seM (n = 8) except where noted, 3n = 7. Means within rows with no common superscript roman letter differ significantly (P  0.05).
Diets and abbreviations are as indicated in Table 1.
2Main effects include the effect of basal α-Toc vs. supplemental α-Toc and basal se vs. supplemental se. The four interaction effects included the
combinations of basal α-Toc x basal se, basal α-Toc x supplemental se, supplemental α-Toc x basal se, and supplemental α-Toc x supplemental se.
Abbreviation: hchs, high cholesterol and high saturated fat.
0.045.0 ± 0.001, and 0.046 ± 0.001, respectively. Se
without an increase in expression levels of CYP27A1
treatment was not associated with any significant
mRNA.63
effect on final weight, liver weight or liver weight/
The improved hepatic ratios of enzymatic to free
final weight ratio.
radical generated oxysterols resulting from Se sup-
plementation could be the result of diminished break-
Discussion
down of the enzymatically-generated oxysterols by
The results from this study provide the first evidence
free radical-generated oxysterols. Previous work has
that antioxidants can enhance tissue concentrations of
shown that treatment of cultured human umbilical vein
important enzymatically-generated oxysterols as sup-
endothelial cells with oxidized LDL-C that contains
plementation of Se or combined Se and α-Toc sup-
the free radical-generated oxysterol, 7β-hydroxycho-
plementation increased hepatic 25-OHC and 27-OHC
lesterol, diminished cellular 27-OHC concentrations
content and α-Toc supplementation increased hepatic
by 78% within 24 h.45 Tissue content of the important
27-OHC content. The oxysterols 25-OHC and 27-OHC
cellular antioxidant GSH was inversely related to free
are considered to be the most important physiological
radical produced oxysterols as incubation of murine
activators for LXR receptors.35–38 Activation of
peritoneal macrophages with 7β-hydroxycholesterol
the LXR pathway results in hypocholesterolemic
for 24 h led to dose-dependent reduction in cellular
effects.34 Thus, the increase in hepatic oxysterol con-
GSH.64 Treatment with Se elevates cellular GSH lev-
centrations observed in the present study in concert
els,64 which was clearly observed in the present work
with Se and α-Toc supplementation could play a role
(Table 3). Hepatic GSH content correlated positively
in their hypocholesterolemic action.
with the total enzymatic oxysterol/7-keto ratios, which
As tissue oxysterol concentrations can be augmented
indicates that the tissue levels of enzymatically gener-
via increased synthesis, the effect of Se on hepatic
ated oxysterols were positively related to antioxidant
Cyp27a1 mRNA expression was examined. Although
activity. The positive correlations between hepatic Se
Se supplementation at lower levels (0.85 and 1.7 ppm)
and α-Toc levels with hepatic enzymatic oxysterol
was associated with significant increases in hepatic
content also support the notion that enhanced antioxi-
mRNA expression, the effective cholesterol-lowering
dant protection inhibited breakdown of hepatic enzy-
Se supplemental dose of 3.4 ppm was not associated
matically-generated oxysterols.
with increased expression of Cyp27a1 mRNA. Thus,
HCHS diets are associated with increased oxi-
it appears that this supplemental level of Se led to
dative stress and impaired antioxidant capacity,
enhanced tissue content of enzymatically-generated
particularly in terms of impaired status of tissue
oxysterols via decreased oxysterol catabolism, as
GSH and GSH-dependent antioxidant enzymes.1,65
opposed to increased synthesis. Analogous findings
The present study shows that pharmacological Se
have been observed from the cholesterol loading
supplementation can at least partially overcome the
of human macrophages, which showed cholesterol
suppression of both hepatic GSH-Px activity and
induced a dose-dependent increase in 27-OHC content
GSH content consistently noted with HCHS diets.1,65
0
Nutrition and Metabolic Insights 2010:3

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