DOES GLUTATHIONE IS A SUFFICENT AGENT TO DEFENCE BRAIN AGAINST LIPID PEROXIDATION AFTER FEEDING RATS WITH DIFFERENT FATS

 

Stępień Tomasz, Dziedzic Barbara, Świątek Elżbieta, Walczewska Anna

 

Cell-to-Cell Communication Department, Medical University of Lodz, Mazowiecka 6/8,

92-215 Lodz, Poland

 

Abstract

The aim of our study was to investigate the effect of high-fat diet containing different fats on lipid peroxidation, and glutathione (GSH) and GSH disulfide (GSSG) in rat brains. Animals were fed the low fat and high fat diets prepared with the same fat. Lard, as a source of saturated and monounsaturated fatty acids, sunflower oil, as a source of linoleic acid (ω6 PUFA), and fish oil, as a source of long-chain ω3 PUFAs. We determined concentrations of GSH, GSSG by Tietze method and lipid peroxidation (LPO) products, malonodialdehyde plus 4-hydroxyalkenes (MDA + 4-HDA), in brain homogenates. Six-week feeding with high amount of lard and fish oil resulted in decrease in GSH and GSSG concentrations compare to the corresponding low fat diets. High sunflower oil fed rats had equal level of GSH compare to the low sunflower oil fed group. Oxidized-GSH had only tendency to decrease in high sunflower oil fed rats. Fish oil in the low and high amount in the diet was the most effective in lowering both GSH and GSSG concentrations. However, the GSH-to-GSSG ratio in rats fed three different high fat diets did not differe each other despite the differences in GSH and GSSG concentrations in these groups. The GSH-to-GSSG ratio was enhanced in rats fed high lard compare to the low lard diet.  The level of LPO products was a not compatible to the GSH-to-GSSG ratio in the brains. The both diets rich in PUFAs, equally in the low and high amount in the diet, increased brain LPO compare to the diet prepared with the same amount of lard. These results suggest that lowering glutathione antioxidant efficiency, in part, may be responsible for increased brain LPO in rats fed with fish oil but not in rats fed sunflower oil diets. The underactivity of some other antioxidant enzymes or to low concentration of other antioxidants may be a reason of a luck sufficient brain lipid defense against an attack of oxygenic agents in these rats.


Introduction

A common Western diet characterizes a high amount of dietary fats. An amount and mostly used a type of fat in a diet depends on the world part, eating habits, social and family customs.  A diet may be rich in saturated fatty acids (SFA), and monounsaturated fatty acids (MUFA) present in animal fats, like lard or beef tallow or in di- and tetra-unsaturated essential fatty acids, linoleic acid (LA; C18:2 ω6), and a-linolenic acid (ALA; C18:3 ω3) that cannot be formed de novo and should be ingested from a diet. These fatty acids are present manly in plant oils obtain e.g. from soybeans, olives or sunflowers. The essential fatty acids, and the complex lipids formed from them, are important constituents of biological membranes and contribute to maintain the structural and functional integrity of cells, and intracellular structures. Over the past decades grew up evidences of diverse beneficial effects of fish oil rich in omega-3 polyunsaturated fatty acids (ω3 PUFA) on heath (1). A dietary supplementation with eicosapentaenoic acid (EPA; C20:5 ω3) and docosahexaenoic acid (DHA; C22:6 ω3) is recommended for prevention of cardiovascular diseases (2) and cancer (3). However, unbalanced and high dietary fats may change metabolic pathways and affect many biological functions of tissues and organs. The type of dietary fat differentially affects the level of oxidative stress. High-saturated fat diet evokes hypercholestrolemy and enhances oxidative LDL modification (4). Omega-3 PUFAs in diet are incorporated in cell membranes, increasing the polyunsaturation of plasma membranes and their susceptibility for lipid peroxidation (5).

Reduced glutathione (GSH), a linear tripeptide of L-glutamine, L-cysteine, and glycine is a relatively small ubiquitous molecule in living cells (6). Its high electron-donating capacity (high negative redox potential) combined with high intracellular concentration (millimolar levels) generate great reducing power. (7) This characteristic underlies its potent antioxidant capacity and defense neurons against oxidative stressors include ultraviolet and other radiation (8), viral infections (9), environmental toxins, chemicals, and heavy metals (7), inflammation, burns and septic shock (10). Deficiencies in brain glutathione metabolism appear to be connected with several neurodegenerative diseases and brain aging (11). In Alzheimer's disease a decrease in gluthatione level has been reported and in Parkinson's disease the substantia nigra becomes greatly depleted of GSH (12).

In the present study, we investigated the concentration of malonodialdehyde plus            4-hydroxyalkenes (MDA + 4-HDA), as an index of lipid peroxidation and the GSH status in brain homogenates after six weeks rat feeding with three high-fat diets composed of the different fats. Lard, as a source of SFA and MUFA, sunflower oil, as a source of LA, (ω6 PUFA), and fish oil, as a source of long-chain ω3 PUFA.

 

 

 

Materials and Methods

Animals and diets

Male Wistar rats (150 – 160 g) were housed at plastic boxes in the animal facility on        a 12 h light-dark cycle (lights off 1800 h) and at 23 ± 1 °C. They were initially fed standard commercial rat chow (Motycz, PL) and water ad libitum until the beginning of the experiment. The protocol of the experiment was reviewed and approved of by the Ethical Committee for Animal Care of the Medical University in Lodz. After habituation, rats were randomized into six dietary groups (n = 12 per dietary group). Three control groups were fed low fat diet (10% energy from fat) prepared with the same fat as three high fat diet (40% energy from fat). The diets contained lard, sunflower oil or fish oil. The fish oil diet was supplemented with soybean oil to maintain adequate intake of essential ω6 PUFA. The composition of the experimental diets is given in table 1, and fatty acid composition of fats used to prepare the purify diets, in table 2. All diets were prepared weekly and kept in daily rations in sealed bags at –20 °C. The diets and tap water were provided ad libitum daily at 15.00 h. Food intake was recorded daily and corrected for spillage (± 0.1 g). Body weight was monitored every three days. After 6 weeks feeding purified diets and overnight fast, rats were anesthetized with ketamine and xylazine      (20 and 10 mg/kg, respectively), and then killed by decapitation.

 

Table 1.  Composition of the experimental diets

 

Low-fat diet

     High-fat diet

                         g/kg

 

Casein    

194.4

228.6

Corn Starch  1

Sucrose

364.4

291.5

252.6

201.1

Fat 2

  52.5

          203.4

Cellulose  3

  48.6

 57.1

AIN-93 Mineral Mix  4

  34.0

 40.0

AIN-93 Vitamin Mix 4

   9.7

 11.4

Choline bitartrate  5

   1.9

   2.3

L-Cystine  5

 

Energy density

    2.9

                       kcal/g

    3.92

   3.4

diet

    4.62

 

1 – Stobimyl XMH 042 (Stockmeier Food, Germany);  2 – Lard, (Pamso S.A., Poland), Menhaden Fish Oil (Omega Protein, Inc. Hammond, LA, USA), Sunflower Oil, (Fat Processing Com. Inc, Warsaw; Poland);  3 – Arbocel â (J. Rettenmaier & Söhne Gmbh + Co Faserstoff-Werke, Rosenberg, Germany); 4 –  Research Diets, Inc (NJ, USA); 5 – Sigma-Aldrich Sp z o.o

 

Table 2. Fatty acid composition of dietary fats

 

Total fatty acids

Lard

Sunflower oil

Fish oil

% by weight

Saturated

42.6

  9.2

29.0

Monounsaturated

50.3

30.5

26.0

n-6 PUFA   

6.3

59.5

  2.6

n-3 PUFA  

0.3

  0.6

30.7

 

Glutathione assay

The brains were immediately removed from the sculls, washed carefully in ice-cold PBS and homogenized in ice-cold 5% 5-sulphosalicilic acid (1 ml/100 mg of tissue). Than homogenates were centrifuged at 10000xg for 10 min at 4°C, supernatants were collected and aliquots were stored at -70°C. The Tietze`s GSH recycling method was used for GSH determination (13). This method is based on an enzymatic recycling procedure in which GSH is sequentially oxidized by 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) and reduced by glutathione reductase in the presence of  NADPH. The rate of formation of 2-nitro-5-thiobenzoic acid was spectrophotometrically measured at 412 nm and GSH was quantitated by reference to the standard curve. For total GSH, the samples were added to the buffered solution (115 mM phosphate buffer, pH 7.4) containing EDTA (5 mM), NADPH (0.2 mM) and DTNB (0.6 mM). The reaction was started by addition of glutathione reductase (830 U/L). Oxidized GSH (GSSG) was titrated by the same method after derivatization of reduced glutathione with 2-vinylpiridyne (14). 

Lipid peroxidation products measurement

The brains were homogenized in ice-cold 20 mM phosphate buffer, pH 7.4 containing     5 mM butylated hydroxytoluene. Homogenates was centrifuged at 3000xg for 10 min. at 4°C, supernatants were collected and aliquots for assays were stored at -70°C. The concentration of malonodialdehyde + 4-hydroxyalkenes (MDA + 4-HDA), as an index of lipid peroxidation, was determined using commercial CalBiochem kit (USA).

Chemicals and protein determination

All chemicals used in the assays were supply by Sigma-Aldrich Co. Protein content was measured using the Bradford method (15), with bovine serum albumin as the standard.

Statistical analysis

Data are shown as means ± SEM. ANOVA test followed by Turkey post-hock test was used for comparisons the effects of three different fats in low- and high fat diet and t-test to compare the effect of high fat diet with the corresponding low fat diet. 

 

 

Results

Lipid peroxidation

Rats fed both the low and high amount of lard revealed the lowest concentration of lipids peroxidation products (p<0.05; fig. 1). Any of the corresponding low fat and high fat fed groups did not differ significantly each other in brain lipid peroxidation. There were no significant differences in LPO level between rats fed with sunflower oil and fish oil in the low fat and high fat diet groups, as well.

Fig.1.  Effect of six weeks feeding low fat (LF) and high fat (HF) diets composed of lard, sunflower oil and fish oil on concentration of lipid peroxidation products in rat brains (n=12). Values are means ± SEM. a p<0.05 vs. LF sunflower and fish oil diets; b p<0.05 vs. HF sunflower and fish oil diets.

 

Glutathione status                                              

            The comparison of the low fat and high fat diets showed differences within lard and fish oil fed rats. High lard and fish oil fed animals had a significantly lower concentration of GSH and GSSG than the corresponding low fat fed rats (p<0.05; fig.2). In turn, the high fat diet composed of sunflower oil did not alter the concentration of GSH compare to the corresponding low fat diet. A tendency to decrease in GSSG level by high sunflower oil was only noted. The highest concentration of GSH among the high fat diets fed rats was find in animals fed with high sunflower oil (p<0.01). Evaluating glutathione status in the brains of rat fed three different fats in low and high fat diet, GSH/GSSG ratio was increased significantly only in rats fed high lard compare to the corresponding low fat diet (p<0.05). The lowest brain GSH/GSSG ratio reviled rats fed the low lard diet (p<0.01).

 

Fig. 2. Effect of six weeks feeding low fat (LF) and high fat (HF) diets composed of lard, sunflower oil and fish oil on glutathione (GSH) and oxidized glutathione (GSSG) concentrations in rat brain homogenates, and GSH/GSSG ratio (n=12). Values are means ± SEM.

a p<0.05 vs. the corresponding LF diet; b p<0.01 vs. the other LF or HF diets.

 

Discussion

            Fatty acids are essential structural components of the central nervous system and play a role in its function, as well (17). However, grater amount of dietary PUFA causes their increased incorporation into bilayer membranes and enhances susceptibility to oxidation (5). The neuronal components are protected against its damage by enzymatic and non-enzymatic antioxidants. The enzymes include CuZn-superoxie dismutase and Mn-superoxide dismutase (SODs), GSH peroxidase and catalase, as well as a small antioxidant molecules, like glutathione, ascorbic acid, vitamin E, and a number of dietary flavonoids (18).

Glutathione is an extremely important antioxidant cell protector. It directly quenches reactive hydroxyl free radicals, other oxygen-centered free radicals, and radical centers on DNA and other biomolecules (2). Deficiency in brain glutathione metabolism, which suggests an appearance of oxidative stress in neurons, is connected with several neurodegenerative diseases (11). Moreover, a shifting the GSH-to-GSSG ratio towards the oxidizing state activates several signaling pathways including protein kinase B, calcineurin, nuclear factor kappa-B, c-Jun and mitogen-activated protein kinase, may result in changes of brain function, reducing cell proliferation and increasing apoptosis (16). Gluthathione is also a cofactor of  (1) multiple peroxidase enzymes that detoxify peroxide products generated from oxygen radical attack on biological molecules; (2) transhydrogenases that reduce oxidized centers on DNA, proteins, and other biomolecules; and (3) glutathione S-transferases that conjugate GSH with endogenous substances and diverse xenobiotics.

Our study showed that both ω3 and ω6 PUFAs independent on amount in the diets increased the concentration of the lipid peroxidation products in brain compare to the lard diet but the GSH-to-GSSG ratio in brains of rats fed three high fat diets was similar. However, fish oil fed rats, especially in high amount in the diet, reviled significantly lower both GSG and GSSG concentrations although the GSH-to-GSSG ratio in these rats was comparable to sunflower oil fed rats. It means that a GSH-to-GSSG ratio may be a not specific index of intracellular antioxidant capacity. The present study clearly demonstrates that, at least in dietary ω6 PUFA, the lowering of glutathione level was not responsible for increased LPO in brain. It arise a question which other antioxidant system may defense brain lipids against their oxidation in case of increased fatty acid polyunsaturation of their cell structure membranes. Based on our study we cannot answer on this question. However we speculate two possibilities. First, any of normal working intraneuronal antioxidant systems is not able sufficiently defense plasma membranes against an attack of free radicals and oxidative agents without an increase in other antioxidant concentration e.g. vitamins E and C. Second, one or more other intraneuronal antioxidant systems were affected by enhanced level of dietary PUFA and it was less productive in cell detoxification from an oxidative attack on lipids. An underactivity of GSH peroxidase, catalase and/or SODs that could increase in brain oxidative agents concentration should be consider.

 

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