Vol.2, No.1, 45-50 (2010)
doi:10.4236/health.2010.21008
SciRes Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
Health
The effect of high fat food on erythrocyte phospholipids,
fatty acids composition and glutathione redox-system of
rats with alimentary dyslipidemia
Tatyana P. Novgorodtseva1, Yulia K. Karaman1, Natalia V. Zhukova2
1Institute of Medical Climatology and Rehabilitative Treatment SB RAMS, Vladivostok, Russia;
2Institute of marine biology of name A.V. Zhirmunskogo FEB RAS, Vladivostok, Russia
Received 6 November 2009; revised 7 December 2009; accepted 9 December 2009.
ABSTRACT
To evaluate the effects of high fat food consisted
of tallow (19% of total diets) and cholesterol (2%)
on modification of erythrocyte phospholipids,
fatty acids composition and glutathione redox-
system of male Wistar rats with alimentary dysli-
pidemia. The results demonstrated that after 30
and 180 days of high-fat feed erythrocyte phos-
phatidylinositol and phosphatidylcholine levels
were reduced, phosphatidylserine were in-
creased. Only on the 90 days of the experiment
phosphatidylinositol level increased. In all grow-
ups the erythrocyte 18:0 saturated fatty acids
and 20:4n6, 22:4n6 polyunsaturated fatty acids
(PUFA) were increased. Deficit of n3 PUFA-
20:5n3 and 22:6n3 after 90 and 180 days high fat
feed promoted compensatory synthesis from
18:1n9 on 20:3n9. Erythrocyte maleic dialde-
hyde increased, glutathione level decreased in
all groups of rats after fed with high-fat feed.
Glutathione reductase and glutathione peroxi-
dase activity decreased in erythrocytes after 30
and 180 days of high-fat feed. In conclusion:
high-fat diet during 30-90 days started adaptive
answer in lipids of membrane and glutathione
redox-system. Important mechanism of adapta-
tion of a cellular membrane to high-fat diet is
increase major, structuring a membrane phos-
phatidylethanolamine and minor, most meta-
bolic significant fractions phospholipids (phos-
phatidylinositol), keeps homeostasis of 18:2n6
and 22:6n3, 20:3n9 compensatory synthesis,
decrease in activity of processes lipid peroxi-
dation, activation of enzymes of redox-system
glutathione. But prolonging the high-fat feeding
(180 days and more) formed failure compensa-
tory processes (dysadaptation). It is a risk factor
of developmening atherosclerosis, diabetes,
steatogepatitis and other diseases.
Keywords: Fatty Acids; Phospholipids; Adaptation;
Dyslipidemia; GSH; Glutathione Reductase;
Glutathione Peroxidase
1. INTRODUCTION
Clinical, experimental and epidemiological researches
have proved that the high-fat feeding is a risk factor of
development of atherosclerosis, diabetes, steatogepatitis
and other diseases [1-4]. Insufficient or superfluous con-
sumption of separate components of food (deficiency of
fiber, surplus of fat, carbohydrates etc.) at the initial
stages forms the cascade of the stressful reactions di-
rected on activation of mechanisms of adaptation in an
organism.
In cellular processes of adaptation one of leading roles
is occupied with a plasma membrane and forming it
phospholipids (PL) and fatty acid composition [5,6]. The
fatty acid composition of phospholipids affects the phys-
icochemical properties of the membrane and thus influ-
ences conformation and function of membrane-bound
proteins, such as receptors, ion channels, and transporters
and also influences cell function by serving as precursors
for prostaglandins and other signaling molecules and
modulating gene expression through the activation of
transcription factors [7-9]. Thanks to ability PL transform
each other and also to be redistributed in a membrane
between an internal and external layer realize important
adaptable mechanism supporting the structural organiza-
tion and functional properties of a plasma membrane [10]
is carried out. The lipid peroxidation initiate modification
membrane PL and it fatty acid composition. The impor-
tant role in realization antioxidant protection from lipid
peroxidation is played by glutathione redox-system
which regulation of NF κβ (nuclear factor-κβ), ARE
(antioxidant response element) [11-14]. The urgent fast
adaptive answer of glutathione redox-system, consisting
of activation of enzymes glutathione reductase, glu-

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SciRes Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
46
Table 1. Daily allowance of rats (g/kg of animal weight).
Ingredients Experimental diet
General vivarium diet
Tallow 42.5
5
Cholesterol 4.3
-
Sunflower oil 5 5
Grain mixture 50 50
Bread 20 20
Grits 13 13
Beef 20 20
Skim cheese 8 8
Carrot 33 33
Greens 33 33
Table 2. Effect of high-fat diet on serum lipids.
Days of fed high-fat diet
Parameter Control group,
n=10
30 days
(group 1),
n=10
90 days
(group 2),
n=10
180 days
(group 3),
n=10
TC, mmol/l 1.57±0.04 3.34±0.04*** ***1.68±0.08 2.04±0.17*
TG, mmol/l 1.12±0.04 1.95±0.06*** ***0.51±0.05*** 1.17±0.08
HDL-C, mmol/l 0.67±0.04 0.26±0.02*** ***0.50±0.08 ***0.5±0.15
IA 1.43±0.15 11.87±1.55*** ***2.46±0.35* ***7.4±0.8***
in Table 2, 3, 4 (*) left – statistic significance of differences in to control group; right – group 1;
* р < 0.05; ** – р < 0.01; *** – р < 0.001.
tathione peroxidase and synthesis GSH prevents oxida-
tion phospholipids and membrane infringement. Data on
the specific features of membrane lipids behavior and
adaptive answer by high-fat diet are scarce. Identification
of adaptive response to the nutrition factor through the
investigation of membrane forming lipids and glutathione
redox-system will help to understand mechanism of cel-
lular adaptation and dysadaptation, how predictor of
pathogenesis many dietary-induced diseases.
The aim of the current study was to evaluate the effects
of high fat food on serum lipid, erythrocyte phospholipids,
fatty acids and oxidative stress in rats with experimental
dyslipidemia and establish the possible adaptation mech-
anism by high-fat diet.
2. METHODS
2.1. Animals and Diets
Subject to the research were 40 pubescent white male
Wistar rats with initial weight 173 ±5.6 g. Alimentary
dyslipidemia was induced in rats by high-caloric diet
(Table 1) [15].
The animals were divided into 4 groups, 10 rats in each:
normal, control group, fed with basic feed; and groups
comprising animals kept on experimental high-fat diet
(30 days – group 1; 90 days – group 2; and 180 days –
group 3). Experimental high-fat diet consisted on 19%
tallow and 2% cholesterol. Euthanasia was performed by
in conformity with the requirements of Directive 86/609
EEC on the protection of animals used in scientific re-
search [16].
2.2. Material
Venous blood samples of rats were drawn after decapita-
tion.
2.2.1. Blood Serum Lipid Investigation
Lipid spectrum of blood serum was investigated by Lab-
sistems biochemical analyzer FP-901 (Finland). Pa-
rameters to be determined included level of total choles-
terol (TC), triglyceride (TG), and high density lipopro-
teins (HDL). Index of atherogenesis (IA) was calculated
by formula: (TG - HDL)/HDL.
2.2.2. Erythrocyte Phospholipids and Fatty
Acid Investigation
The erythrocytes were washed three times with 0.9%
NaCl solution. Lipids were extracted from erythrocytes
by Bligh and Dyer method [17]. Bidirectional micro thin
layer chromatography on glass plates 6x6 with silica gel
and plaster suspension was used to separate polar lipids.
Quantitative analysis of certain phospholipids (PL)
classes after thin layer chromatography was made ac-
cording to V.E. Vaskovsky et al method [18,19]. Content
of each component was represented as percentage of total
PL. FA methyl ethers were received by Carreau and Du-
back method [20], analyzed on Shimadzu GC17A
gas-liquid chromatographer equipped with flame ioniza-

T. P. Novgorodtseva et al. / HEALTH 2 (2010) 45-50
SciRes Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
47
tion detector and capillary column (0.25 mm х 30 m)
with implanted phase Supelcowax 10. Temperature of
column and detector was 210С, temperature of evapo-
rator was 240С. Media gas was helium. Gas discharge
ratio in the evaporator was 1:30 at 1.8 atm. Z-Chrom
station was used for calculating area of chromatographic
peaks and processing the results. FA methyl ethers were
identified by retention time using standards and carbon
numbers [21]. Results were represented in relative per-
centage of total FA.
2.2.3. Determination of Srythrocyte Pro-Oxidant
and Anti-Oxidant
Erythrocyte maleic dialdehyde (MDA) was estimated
spectrophotometrically using thiobarbituric acid assay
[22]. GSH was measured according to the Ellman method
[23]. Glutathione reductase activity was measured ac-
cording to the method described [24], glutathione per-
oxidase activity – [25].
2.2.4. Statistic Methods
All data were analyzed by ANOVA using computer pro-
gram Statistika 6.1 (series 1203С for Windows). Data
present as means ± SEM. (М). Differences between
means were assessed by Student's significance test.
3. RESULTS AND DISCUSSION
Analysis result of serum lipids indexes of rats in different
stages are shown in Table 2. Serum TC, TG levels, index
of atherogenesis increased (р < 0.001) of rats in the group
1. Serum HDL-C levels reduced (р < 0.001). After 90
days of the experiment, serum TG, TC levels of rats in the
group 2 decreased compared with of rats in group1. At the
end experiment, no significant difference was present
between TG levels of group 3 and control group, neither
between HDL-C levels. Compared with the result of
group 2, the TG, TC levels were increased; the index of
atherogenesis of group 3 was lower than that of group 1
and group 2.
3.1. Erythrocyte Phospholipids
Separation of erythrocyte PL of rats identified six com-
ponents including phosphatidylcholine (PC), phosphati-
dylethanolamine (PE), phosphatidylserine (PS), sphin-
gomyelin (SM), phosphatidylinositol (PI) and phos-
phatidic acid. Phosphatidic acid was found as traces
(Table 3).
Erythrocyte phospholipids fraction of phosphatidy-
linositol and phosphatidylcholine were reduced in the
group 1 (р < 0.001). Levels of phosphatidylserine and
phosphatidylethanolamine were increased in erythrocyte
membrane. Onset of the deficit PI and PC which form
outer layer of membrane, indicate activation of specific
phospholipases and intensification of lipid peroxidation
processes contributing to plasma membrane destruction
[26,27]. A quick-response was increase in PE level ac-
cumulating in inner layer of membrane lipid fraction,
where most of the active centers of membrane bonded
enzymes are located. Research results prove that on the
30 day of high-fat diet membrane forming lipids promptly
responded to the stress to prevent deep structural and
functional damage to cell.
Changes in erythrocyte membrane at PS, PC and PE
level in rats of group 2 was of the same direction as in rats
of group 1. Decrease PC lever in outer layer of erythro-
cyte membranes was compensated by SM maintenance
within the range pertinent to control group animals. Such
condition can be described as a compensatory response of
cell to long-term exposure to stress alimentary factors.
Moreover, due to the high saturation of PE the cholesterol
is not hardly building into the inner monolayer. This helps
to preserve hydrophilic surrounding of cell membrane
integral proteins and, therefore, their function [28].
Depletion of erythrocyte PC which forms outer shell of
cell lipid matrix was evidenced at a longer exposure to
high-caloric diet (180 days) as well. PC reduction in
group 3 was accompanied by reliable increase in PS and
SM level as compared to control group, meaning that
erythrocyte cell was structurally and functionally inade-
quate. Due to high saturation with SM, the clusters
forming phospholipids in a membrane receive large
quantity of cholesterol, and this results in lower perme-
ability of cell membrane and interference in active
metabolic processes [28]. Thus, on the 180 day of the
experiment cell membrane became unable to resist the
continuous flow of alimentary stress factors, and the stage
of cell compensatory protection depletion occurred that
had been formed by the 90 day of high-caloric diet.
3.2. Erythrocytes Fatty Acids
Qualitative composition of erythrocytes FA in rats was
represented by components with carbon chain length
from С10 to С24, both even and odd, of normal and isomeric
structure, saturated, monounsaturated and polyunsatu-
rated (Table 3). Certain FA with content below 0.1% were
not included into Table 3. These mostly include saturated
FA with normal structure, (10:0, 19:0, 20:0, 22:0), some
monounsaturated (14:1, 18:1n5, 20:1, 22:1), 18:2n5/9 and
20:3n3 FA.
Analysis of the qualitative composition of FA of
erythrocyte lipids showed that rats with dyslipidemia
have considerable changes in FA composition as com-
pared to control group. Myristic (14:0), stearic (18:0) FA
share was observed to increase in group 1. Unsaturated
FA demonstrated minor increase of monoenoic oleic FA
(18:1n9, р<0.01) level, reliable accumulation of n6 FA –
20:4n6, 22:4n6, 22:5n6 and decrease in essentially li
group 1 is related to the specific features of experimental-
noleic FA (18:2n6). Share of n3 polyunsaturated fatty
acids (PUFA) slightly reduced due to the identified de

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SciRes Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
48
Table 3. Effect of high-fat diet on phospholipids and fatty acids percentage in the erythrocyte of rats.
Days of fed high-fat diet
Phospholipids and fatty acids
components
Control group,
n=10 30 days
(group 1),
n=10
90 days
(group 2),
n=10
180 days
(group 3),
n=10
Phospholipids, %
PS 6.800.85 13.000.67*** 11.10.5*** *13.011.04***
PI 3.900.00 1.140.17*** ***4.690.19* 1.660.29***
SM 14.420.97 15.772.09 14.50.39 ***17.80.52***
PC 55.881.14 42.380.96*** 47.70.66** 46.42.37**
PE 21.500.75 28.820.99*** *22.010.34 **20.41.05
Fatty acids, %
12:0 0.25±0.02 tr 0.100.01*** ***0.400.07***
14:0 0.52±0.04 0.83±0.08* **0.5±0.06
***1.010.12***
15:0 0.74±0.11 0.58±0.07 0.43±0.02** *0.560.04
15:1 0.24±0.08 0.18±0.02 tr **0.300.05
16:0 23.72±0.84 24.38±0.57 **28.6±0.56*** 27.61.4***
16:1n7 1.90±0.25 1.83±0.12 ***0.83±0.05***
2.040.21
17:0 0.94±0.11 0.65±0.10* 0.7±0.01**
0.580.05
17:1 0.96±0.16 0.32±0.02*** ***0.18±0.03* 0.260.04
18:0 10.1±0.5 14.68±0.48** 17.3±0.57***
13.92.77***
18:1n9 7.66±0.57 11.45±1.22** 10.5±0.31 11 . 2 1.38
18:1n7 3.90±0.25 2.95±0.38 3±0.08 2.590.13**
18:2n6 14.02±0.64 9.78±0.23*** *12.6±0.4 **11.30.97**
18:3n3 1.45±0.32 tr tr 0.30.08***
20:2n6 0.27±0.02 0.43±0.03 **0.78±0.06*** ***0.290.02**
20:3n9 0.35±0.12 tr 0.17±0.03 ***0.950.09***
20:3n6 0.6±0.13 0.88±0.09 *1.23±0.06** 0.90.08**
20:4n6 6.78±1.72 21.27±1.92*** *16.3±0.5*** **16.30.94***
20:4n3 0.17±0.03 0.38±0.02*** tr 0.320.08***
20:5n3 0.9±0.23 0.73±0.29 0.58±0.05* 0.680.05**
21:5n3 0.67±0.09 tr 0.3±0.01***
**0.780.18
22:4n6 0.58±0.07 1.58±0.09** ***0.85±0.05** ***0.660.08
22:5n6 0.23±0.15 0.55±0.05*
***0.17±0.03
***0.530.13***
22:5n3 1.32±0.16 1.62±0.27 1.68±0.09** 1.150.27
22:6n3 5.53±0.92 3.93±0.71 *2.22±0.08** *2.850.53***
Total n6 36.4±1.1 34.32±1.86 31.9±0.26** *27.11.93***
Total n3 8.77±0.22 6.47±1.19 4.55±0.1** *3.750.68***
UI 183.5±20.8 167.92±3.14* *140±1.35* ***11811.3***
U
I – unsaturation index (sum of products of double bond and FA relative percentage in each FA); tr – less 0.1 %.
crease in eicosapentaenoic (20:5) and docosahexaenoic
(22:6) FA. FA saturation index in rats of group 1 was low.
The reduction of general erythrocyte lipids unsaturation
revealed by the experiment is mostly conditioned by
decrease in relative level of essential linoleic FA and
redistribution between n6 and n3 family acids [29,30].
Metabolism of n6 FA is known to originate from linoleate
consumed with food. Low linoleate content in rats of diet,
poor in PUFA. Further metabolic transformations of es-
sential FA resulted in accumulation of arachidonic
(20:4n6) FA, which is a predecessor of synthesis of
anti-inflammatory leukotriens and thromboxanes with
their strong aggregation and vasoconstriction properties
[31, 32]. Growing share of 22:4n6 and 22:5n6 in eryth-
rocyte lipids can be deemed a compensatory response to
the deficiency of docosahexaenoic FA.

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Table 4. Effect of high-fat diet on parameter of erythrocytes pro-oxidant and anti-oxidant.
Days of fed high-fat diet
Parameter Control group,
n=10
30 days
(group 1),
n=10
90 days
(group 2),
n=10
30 days
(group 1),
n=10
MDA, mkmol/g Hb 4,6±0,3 5,3±0,3* ***8,1±0,2** 9,1±0,2***
GSH, nmol/g Hb 5,30,2 3,50,3*** **4,10,1* **2,00,2***
Glutatione reductase, mkmol
NАDPН/mgHb/min 75,11,5 68,01,5** *73,31,6 **50,12,4***
Glutathione peroxidase, mkmol
GSH /mgHb/min 44,5±0,8 32,51,3** **40,1±2,2 ***21,4±1,2***
In group 2 unsaturated fatty acids (12:0, р<0.001) in-
crease in comparison to the control group. On the contrary,
rats of group 3 demonstrated growth of 12:0 level
(р<0.001). Accumulation of saturated 14:0, 16:0 FA in
erythrocyte lipids group 3 was more evident than in rats
of group 2. Modification of n6 composition in PUFA
was distinguished by reducing 18:2n6 (р<0.01) share in
comparison to the control group, reliable figures being
obtained from rats of group 3; and growth of 20:2n6
(р<0.001), 20:3n6 (р<0.01) and 22:4n6 (р<0.01) content,
more considerable in rat of group 2. Increase in 22:5n6
(р<0.001) share was noticed for rats of group 3 only. Rats
subject to long-term high-caloric diet demonstrated defi-
cit of n3 PUFA – 20:5n3 (р<0.01) and 22:6n3 (р<0.001)
FA. 20:3n9 compensatory synthesis from 18:1n9 FA was
a natural consequence of n3 deficit in rats with prolonged
dyslipidemia. The change in FA composition profile, as
revealed by the experiment indicates higher risk of car-
diovascular pathology occurrence [30].
3.3. Erythrocytes Pro-Oxidant and
Anti-Oxidant
MDA erythrocytes content increased in all groups of rats
with dyslipidemia in comparison to the control group
(Table 4). GSH level decreased in all groups after fed
with high-fat feed in comparison to the control group (р <
0.001). GSH differences of rats in group 1 and group 2
had no statistical significance. Glutathione reductase
activity decreased in erythrocytes in group 1 (р < 0.01)
and group 3 (р < 0.001) in comparison to the control
group (р < 0.001). Glutathione reductase activity in group
2 was higher then in group 1 (р < 0.05) and no statistical
significance in comparison to the control group. Glu-
tathione peroxidase activity decreased in 1 group and 3
group of rats with dyslipidemia. Glutathione reductase
activity in group 2 had no statistical significance the
control group.
4. CONCLUSIONS
Thus, generalizing results of researches it is possible to
conclude that the important mechanism of adaptation of a
cellular membrane to high-fat diet loading is increase
major, structuring a membrane PL (PE) and minor, most
metabolic significant fractions PL (PI), keeps homeosta-
sis of 18:2n6 and 22:6n3 FA, 20:3n9 compensatory syn-
thesis, decrease in activity of processes lipid peroxidation
at the expense of activation of enzymes of glutathione
redox-system and glutathione synthesis. Active formation
and realization by a cage of the adaptable answer at
high-fat diet loading occurs during the period from 30 till
90 days. Apparently, at the expense of exhaustion com-
pensatory mechanisms in glutathione system and inten-
sifications of processes lipid peroxidation for 180 days of
fatty loading occur more essential deep reorganizations of
lipids at biological membrane. This is one of the impor-
tant mechanisms of pathogenesis heart diseases, meta-
bolic syndrome. The received results of research expand
knowledge of mechanisms of adaptation and dysadapta-
tion cages at stressful loadings and can be a basis for
diagnostics and treatment of a cellular pathology.
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