Effect of antioxidant capacity on blood lipid metabolism and lipoprotein lipase activity of rats fed a high-fat diet

Nutrition 22 (2006) 1185–1191 www.elsevier.com/locate/nut

Basic nutritional investigation

Effect of antioxidant capacity on blood lipid metabolism and lipoprotein lipase activity of rats fed a high-fat diet RuiLi Yang, M.S., Guowei Le, Ph.D.*, Anlin Li, M.S., Jianliang Zheng, M.S., and Yonghui Shi, M.S. Key Laboratory of Food Science and Security, Ministry of Education, Southern Yangtze University, Wuxi, Jiangsu, China

Abstract

Objective: The present study explored the effect of antioxidant capacity on blood lipid metabolism and lipoprotein lipase (LPL) activity of rats fed with a high-fat diet (HFD). Furthermore, the relation of the atherosclerotic index (AI) and LPL activity to total antioxidant capacity (TAC) was studied. Methods: Thirty-two Sprague-Dawley rats were randomly assigned to one of four groups (n ⫽ 8). The control group consumed an ordinary diet (5.1% fat, w/w). The other three experimental groups were fed with an HFD (14.1% fat, w/w), an HFD plus 0.1% lipoic acid (LA), or an HFD plus 0.1% N-acetylcysteine (NAC). After 4 wk, serum levels of triacylglycerol, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol and LPL activity were examined. To evaluate rats’ antioxidant status, TAC and superoxide dismutase activities and malondialdehyde level were measured. Results: The HFD induced abnormal increases in lipid peroxidation, serum concentrations of total cholesterol, triacylglycerol, and low-density lipoprotein cholesterol, and a decrease in high-density lipoprotein cholesterol concentration. Decreased activity of LPL, accompanied by a depressed antioxidant defense system, was observed in HFD-fed rats. These changes were partially restored in the NAC- and LA-treated groups. There was a negative correlation between AI and TAC (r ⫽ ⫺0.969, P ⬍ 0.05). In addition, a significant positive correlation between LPL activity and TAC was found (r ⫽ 0.979, P ⬍ 0.05). Conclusion: Oxidative injury and lipid abnormalities were induced by an HFD. Administration of LA and NAC can improve the antioxidant capacity and activity of LPL and reduce blood lipid significantly. Antioxidant capacity is correlated with AI and LPL activity. © 2006 Elsevier Inc. All rights reserved.

Keywords:

Antioxidant capacity; Hyperlipidemia; Lipoprotein lipase; Atherosclerotic index; High-fat diet

Introduction Atherosclerosis, the underlying cause of heart attack, stroke, and peripheral vascular disease, is a main cause of morbidity and mortality worldwide. The disease can generally be viewed as a form of chronic inflammation that is induced and perturbed by lipid accumulation [1,2]. One of the initial events in the development of atherosclerosis is the accumulation of cells containing excess lipids within the arterial wall. Hyperlipidemia or high levels of serum triac-

This project is based on work supported by the National Natural Science Foundation of China (grant 30571347). * Corresponding author. Tel./fax: ⫹86-510-586-9236. E-mail address: [email protected] (G. Le). 0899-9007/06/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2006.08.018

ylglycerol (TG) and cholesterol is a risk factor for premature atherosclerosis [3,4]. During the past decade, a great deal of interest has focused on the effect of lipoprotein lipase (LPL) on lipid metabolism and atherogenesis. LPL is produced mainly by the adipose, heart, and muscle tissues and to some extent by macrophages. It plays an important role in lipid metabolism by hydrolyzing core TGs from circulating chylomicrons and very low-density lipoproteins (VLDLs) [5]. Decreased LPL activity leads to increased TG and decreased high-density lipoprotein cholesterol (HDL-C) levels, which are risk factors for the development of atherosclerosis [6,7]. In addition, it has been demonstrated that increased intracellular generation of reactive oxygen species (ROS) plays an important role in chronic inflammatory responses to atherosclerosis [8 –10].

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It is well known that ROS can bring about damage to proteins by site-specific damage at metal binding sites and disulfide bonds in addition to fragmentation, cross-linking, and aggregation, which may damage or change the functions of proteins. Moreover, it has been recognized that ROS are widely used as secondary messengers to influence gene expression [11,12]. Therefore, the antioxidant may affect the activity of LPL directly and/or indirectly by scavenging ROS, which in turn influences lipid metabolism. Different studies have shown that compounds that can scavenge free radicals can improve lipid metabolism [13–15]. However, little is known about whether there is a relation between LPL activity, the atherosclerotic index (AI), and antioxidant capacity. Lipoic acid (LA) is unique, among antioxidants because it retains powerful antioxidant properties in its reduced (dihydrolipoic acid) and oxidized (LA) forms [16]. It not only scavenges free radicals directly but also provides the reducing medium for the regeneration of the antioxidant from oxidized form. LA has been recognized as a universal antioxidant [17]. The antioxidant activity of N-acetylcysteine (NAC) has been well documented in vivo and in vitro. It is a precursor of glutathione and may decrease cell oxidative stress directly, as a source of sulfhydryl groups that neutralize ROS, or indirectly, by restoring the glutathione content [18,19]. Thus in the present study, hypothesizing that the antioxidant may influence the activity of LPL directly and/or indirectly by changing the cellular redox state, we investigated the effect of the two antioxidants on the blood lipid metabolism and the activity of LPL. In addition, the relation of the AI and LPL activities with total antioxidant capacity (TAC) was evaluated.

Materials and methods Animals The experiment was conducted with male Sprague-Dawley rats (3 wk old, 75 ⫾ 8 g). The animals were housed under conditions of controlled temperature (23 ⫾ 2°C) and humidity (60%) with natural light. The experimental protocol was developed according to the institution’s guideline for the care and use of laboratory animals. Experimental design Test animals were fed initially, before the study, standard diets for 1 wk for adaptation. Then they were assigned to one of four groups with eight rats in each group. Group I (control) received only a normal diet containing 5.1% fat (1.41% saturated fatty acid and 2.92% unsaturated fatty acid), 18.7% protein, and 4.8% fiber. Group II (high-fat diet [HFD]) received an HFD containing 14.1% fat (5.03% saturated fatty acid and 7.81% unsaturated fatty acid), 18.4% protein, and 4.0% fiber. Group III (LA) was fed with the HFD in conjunction with 0.1% LA. Group IV (NAC) was

Table 1 Composition of diet Ingredient

Cornmeal* Soybean meal† Lard‡ Fishmeal§ Corn bran Wheat flour Lysine

Content (%) Normal diet

High-fat diet

34.5 21.5 2.0 2.0 11.0 26.1 0.12

34.0 20.0 11.0 4.0 3.0 25.3 0.12

Ingredient

Methionine CaCO3 CaHPO4 NaCl Mineral mix Vitamin mix Choline

Content (%) Normal diet

High-fat diet

0.13 1.3 1.0 0.2 0.03 0.02 0.10

0.13 1.1 1.0 0.2 0.03 0.02 0.10

* Cornmeal contains 9.2% protein, 73.8% carbohydrate, and 3.5% fat. Soybean meal contains 41.5% protein, 35% carbohydrate, and 5% fat. ‡ Lard provides the following (g/100 g lard): 14:0, 2.0; 14:1, 0.3; 15:1, 0.1; 16:0, 26.5; 16:1, 3.7; 17:0, 0.5; 17:1, 0.4; 18:0, 12.1; 18:1, 42.5; 18:2(␻-6), 9.8; 18:3(␻-3), 0.7; 20:0, 0.2; 20:1, 0.6; 20:4(␻-6), 0.3. § Fishmeal contains 58% protein, 12% carbohydrate, and 10.5% fat. †

fed with the HFD in conjunction with 0.1% NAC. Compositions of the animal diets are listed in Table 1. All rats were allowed free access to the test diets and deionized water throughout the test period. At the end of the experimental periods, rats were deprived of food for 12 h and then they were slightly anesthetized with diethyl ether and injected with 150 IU of heparin per kilogram of body weight. Exactly 15 min after that, the rats were sacrificed by decapitation. Blood was collected from the neck into glass tubes. Serum was obtained from blood samples after centrifugation (1000g for 10 min at 4°C) then frozen and stored at ⫺80°C until analysis. Analytical procedures Estimation of serum lipids. For determination of serum total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), HDL-C, and TG concentrations, the corresponding diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, PR China) were used according to the instructions of the manufacturer. The lipoproteins LDL-C and HDL-C were fractionated by a dual precipitation technique [20]. After fractional precipitation, lipoprotein cholesterol was estimated. The AI was calculated as (TC ⫺ HDL-C)/HDL-C. TAC determination. The antioxidant defense system consists of enzymatic and non-enzymatic antioxidants, which are able to reduce Fe3⫹ to Fe2⫹. TAC was measured by the reaction of phenanthroline and Fe2⫹ using a spectrophotometer at 520 nm. At 37°C, a TAC unit is defined as the amount of antioxidants required to make absorbance increase 0.01 in 1 mL of serum [21]. A TAC detecting kit was obtained from Nanjing Jiancheng Bioengineering Institute.

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Table 2 Initial and final body weight, body weight gain, food intake, and food efficiency ratio* Parameter

Group I (control)

Group II (HFD)

Group III (LA)

Group IV (NAC)

Initial body weight (g) Final body weight (g) Body weight gain (g/d) Food intake (g/d) Food efficiency ratio†

115.37 ⫾ 5.13 336.05 ⫾ 9.49 7.88 ⫾ 4.29 24.15 ⫾ 1.52储 32.62 ⫾ 1.08储

115.32 ⫾ 3.66 348.14 ⫾ 8.86 8.32 ⫾ 3.45 20.52 ⫾ 1.02‡ 40.54 ⫾ 0.80‡

115.47 ⫾ 5.12 337.91 ⫾ 7.12 7.94 ⫾ 5.01 19.67 ⫾ 1.13‡ 40.36 ⫾ 1.02‡

115.37 ⫾ 4.94 340.60 ⫾ 10.65 8.04 ⫾ 5.05 20.03 ⫾ 1.32‡ 40.14 ⫾ 0.54‡

HFD, high-fat diet (14.1% fat, w/w); LA, high-fat diet plus 0.1% lipoic acid; NAC, high-fat diet plus 0.1% N-acetylcysteine * Values are expressed as mean ⫾ SD for eight animals. † Food efficiency ratio ⫽ (body weight gain [g]/food intake [g]) ⫻ 102. ‡ P ⬍ 0.05. 储 P ⬍ 0.05.

Superoxide dismutase assay. Total superoxide dismutase (SOD) activity was measured at 412 nm by testing the inhibition degree of nitrite formation [22]. The unit of enzyme activity is defined as the enzyme required to produce 50% inhibition of pyrogallol auto-oxidation.

Analysis was done with SPSS 11.5 (SPSS, Inc., Chicago, IL, USA).

Glutathione assay. Total reduced glutathione (GSH) was estimated by the procedure of Moron et al. [23]. In this procedure reduced GSH reacts with 5,5dithiobis-(2nitrobenzoic acid) to produce a compound that absorbs at 412 nm.

Food intake, weight gain, and food efficiency ratio

Lipid peroxidation determination. Free radical damage was determined by specifically measuring malondialdehyde (MDA). MDA formed as an end product of lipid peroxidation was treated with thiobarbituric acid to generate a colored product that was measured at 532 nm (MDA detecting kit from Nanjing Jiancheng Bioengineering Institute) [24]. LPL assay. Lipoprotein lipase catalytic activity was measured according to the method of Iverius and OstlundLindqvist [25], with modifications. The assay for LPL activity used a 3H-triolein– containing substrate emulsified with lecithin and normal human serum as a source of apoprotein CII. After incubating the samples with substrate for 45 min at 37°C, the reaction was stopped by the addition of a mixture of chloroform-methanol-heptane and the liberated 3H-free fatty acids were separated and quantified by liquid scintillation. The activity of LPL in post heparin serum was expressed as micromoles of free fatty acid released per hour per milliliter (serum). Statistical analysis Data were reported as mean ⫾ standard deviation for eight rats per group. Comparisons across groups were performed by one-way analysis of variance. Pearson’s correlation coefficient (r) was used to determine the relation of AI and LPL activity to TAC. A difference of P ⬍ 0.05 was considered statistically significant.

Results

The HFD and control diets met the dietary requirements of rats [26]. The HFD-fed groups exhibited significantly lower food intake compared with the control group (Table 2). When the HFD groups (HFD, LA, and NAC) were compared, food intake was not significantly different. There was no significant difference in weight gain across the four groups. The HFD caused at most a small increase in body weight gain compared with the control diet (5.5%, P ⫽ 0.10). The food efficiency ratio was significantly higher in the HFD, LA, and NAC groups than in the control group. The HFD, LA, and NAC groups exhibited a similar food efficiency ratio. Effect of LA and NAC on serum lipid concentrations of HFD-fed rats Feeding of the HFD for 4 wk resulted in the development of hyperlipidemia in experimental rats, as is evident from Table 3. There were significant (P ⬍ 0.01) increases in TC (137%), TG (136%), and LDL-C (193%), whereas lower HDL-C (70.3%) concentrations were observed in HFD-fed compared with control rats. All these abnormalities were considerably reduced on treatment with LA and NAC. Effect of LA and NAC on the AI of HFD-fed groups versus control group Table 4 presents the effect of LA and NAC on the AI of HFD-fed groups. There was a significant (P ⬍ 0.01) increase in the AI of HFD-fed compared with control rats. Treatment with LA or NAC brought about a significant decrease in the AI of HFD-fed rats.

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Table 3 Effect of LA and NAC on serum lipid status of HFD-fed groups versus control group* Groups Group Group Group Group

TG (mmol/L)

TC (mmol/L)

储

0.58 ⫾ 0.06 0.79 ⫾ 0.09‡ 0.63 ⫾ 0.08储 0.67 ⫾ 0.09§

I (control) II (HFD) III (LA) IV (NAC)

1.69 ⫾ 0.38储 2.31 ⫾ 0.11‡ 1.88 ⫾ 0.19储 2.02 ⫾ 0.18†§

LDL-C (mmol/L) 储

HDL-C (mmol/L) 1.08 ⫾ 0.07储 0.76 ⫾ 0.01‡ 0.98 ⫾ 0.05储 0.96 ⫾ 0.05†储

0.58 ⫾ 0.23 1.12 ⫾ 0.19‡ 0.83 ⫾ 0.22§ 0.88 ⫾ 0.12§

HDL-C, high-density lipoprotein cholesterol; HFD, high-fat diet (14.1% fat, w/w); LA, high-fat diet plus 0.1% lipoic acid; LDL-C, low-density lipoprotein cholesterol; NAC, high-fat diet plus 0.1% N-acetylcysteine; TC, total cholesterol; TG, triacylglycerol * Values are expressed as mean ⫾ SD for eight animals. † P ⬍ 0.05. ‡ P ⬍ 0.01 versus control. § P ⬍ 0.05. 储 P ⬍ 0.01 versus HFD.

Effect of LA and NAC on serum antioxidant status of HFD-fed groups versus control group

the AI was high in groups with a reduced antioxidant capacity.

The status of antioxidants is presented in Table 5. There was a decrease in the activities of TAC (P ⬍ 0.01) and SOD (P ⬍ 0.05) and a decrease in the concentration of the non-enzymatic antioxidant molecule GSH (P ⬍ 0.01) in HFD-fed rats. Treatment with LA or NAC brought about a significant improvement in antioxidant defenses of HFD-fed rats. There was an increased (P ⬍ 0.01) concentration of lipid peroxidation in HFD-fed rats. Treatment with LA or NAC partially restored the lipid peroxidation concentrations to that of control levels but remained significantly (P ⬍ 0.01) higher than in the controls.

Correlation of TAC and LPL activity The correlation between TAC and LPL activity is shown in Figure 3. A significant positive correlation was found between TAC and LPL activity (r ⫽ 0.979, P ⬍ 0.05). The correlation of TAC and LPL activity suggested that the change in redox state has a role in the change in LPL activity.

Discussion Effect of LA and NAC on LPL activity of HFD-fed groups versus control group

The present study explored the effect of antioxidant capacity on the metabolism of blood lipid of rats fed an HFD. We found that LPL activity was positively correlated with TAC, whereas the AI was negatively correlated with TAC. There was a close relation of TAC to LPL activity and the AI. The protective role played by the two antioxidants in a hyperlipidemic condition is highlighted. Results from the serum lipid status of HFD-fed rats for 4 wk showed increased concentrations of serum TC, TG, and LDL, whereas HDL was decreased. The elevations in serum total TG and TC levels observed in our study on HFD feeding is in agreement with those reported in several studies [27,28]. Treatment of HFD-fed rats with LA and NAC showed a considerable restoration of these parameters to that of control levels. The AI, defined as the ratio of TC-

Figure 1 shows the effect of LA and NAC on the LPL activity of HFD-fed groups. There was a significant decrease (P ⬍ 0.01) in the activity of LPL in the serum of HFD-fed rats. Treatment with LA or NAC ameliorated the change induced by HFD feeding. Correlation of TAC and AI These data suggested that the AI was directly related to oxidative stress. The relation between the AI and TAC was determined by correlation analysis. The analysis revealed a negative correlation (r ⫽ ⫺0.969, P ⬍ 0.05; Fig. 2). Thus,

Table 4 Effect of LA and NAC on the AI of HFD-fed groups versus control group* Groups

Group I (control )

Group II (HFD)

Group III (LA)

Group IV( NAC)

AI

0.70 ⫾ 0.27

2.02 ⫾ 0.14

0.93 ⫾ 0.20

1.17 ⫾ 0.24†‡

†

‡

†

AI, atherogenic index; HFD, high-fat diet (14.1% fat, w/w); LA, high-fat diet plus 0.1% lipoic acid; NAC, high-fat diet plus 0.1% N-acetylcysteine * Values are expressed as mean ⫾ SD for eight animals. † P ⬍ 0.01 versus HFD. ‡ P ⬍ 0.01 versus control.

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Table 5 Effect of LA and NAC on serum antioxidant status of HFD-fed groups versus control group* Groups Group Group Group Group

TAC (U/mL)

I (control) II (HFD) III (LA) IV (NAC)

储

3.26 ⫾ 0.34 2.23 ⫾ 0.40‡ 2.97 ⫾ 0.24储 2.66 ⫾ 0.42‡储

SOD (U/mL)

GSH (mg/L)

MDA (nmol/mL) 储

150.32 ⫾ 14.21 114.17 ⫾ 26.71† 144.30 ⫾ 27.03§ 137.96 ⫾ 10.05§

3.14 ⫾ 0.30储 4.79 ⫾ 0.47‡ 3.97 ⫾ 0.29‡储 4.33 ⫾ 0.33‡§

55.32 ⫾ 6.67 43.94 ⫾ 7.19‡ 56.63 ⫾ 7.50储 52.00 ⫾ 8.98储

§

GSH, reduced glutathione; HFD, high-fat diet (14.1% fat, w/w); LA, high-fat diet plus 0.1% lipoic acid; MDA, malondialdehyde; NAC, high-fat diet plus 0.1% N-acetylcysteine; SOD, superoxide dismutase; TAC, total antioxidant capacity * Values are expressed as mean ⫾ SD for eight animals. † P ⬍ 0.05. ‡ P ⬍ 0.01 versus control. § P ⬍ 0.05. 储 P ⬍ 0.01 versus HFD.

HDL-C and HDL-C, is believed to be an important risk factor of atherosclerosis. Our data clearly demonstrate that LA and NAC significantly decrease the ratio. It has shown that abnormally high serum levels of LDL-C and low serum levels of HDL-C are associated with an increased risk for atherosclerosis [29 –31]. Increasing the HDL-C concentrations and decreasing the LDL-C concentrations in HFD-fed rats indicates the antiatherogenic property of LA and NAC. Oxidative stress is one of the causative factors that link hyperlipidemia with the pathogenesis of atherosclerosis [32]. An imbalance between free radical production and antioxidant level leads to oxidative stress, which is obvious from the depressed antioxidant defense system in the HFD group of our study. Administration of LA and NAC to HFD-fed rats prevented the buildup of oxidative stress by restoring normal activities of the enzymatic antioxidant SOD and normal levels of the non-enzymatic antioxidant

2.5

LPL activity(U/ml)

2.0

1.5

GSH in the serum; the untreated HFD-fed rats showed diminished concentrations of these antioxidants. The diminished antioxidant defense system in HFD-fed rats leads to damage to so-called lipid peroxidation. We have observed increased concentration of thiobarbituric acidreactive substances, indices of lipid peroxidation, in the circulation of HFD-fed animals. Administration of LA and NAC caused a significant decrease in the peroxidation concentration. The LPL activity in the serum of rats fed an HFD in our study was found to be decreased. The administration of LA and NAC caused increased LPL activity, which may play a key role in the lowering of AI. Our study suggests that the decrease in serum lipid concentration might result, at least to some extent, from an increased LPL activity. LPL is a glycoprotein involved in the transformation of dietary lipids into sources of energy for peripheral tissues. It separates free fatty acids from TGs present in chylomicrons and very low density lipoproteins. The hydrolysis of TG-rich lipoproteins also releases apolipoproteins and phospholipids that are precursors of high density lipoproteins (HDLs). HDLs are key components of reverse cholesterol transport, bringing cholesterol from peripheral cells to the liver, where it is excreted in the bile. As a result, an efficient LPL is associated with lower TG and LDL levels but higher

1.0 2. 5 2

0.5 AI

1. 5 1

0.0

Group I (control) Group II (HFD)

Group III (LA) Group IV (NAC)

groups

r =- 0. 969 p<0. 05

0. 5 0 2

Fig. 1. Effect of LA and NAC on LPL activity of HFD-fed groups compared with control group. Values are expressed as mean ⫾ SD (n ⫽ 8/group), with SEMs indicated by vertical bars. ⫹P ⬍ 0.05, ⫹⫹P ⬍ 0.01 versus control, * P ⬍ 0.05, **P ⬍ 0.01 versus HFD. HFD, high-fat diet (14.1% fat, w/w); LA, high-fat diet plus 0.1% lipoic acid; LPL, lipoprotein lipase; NAC, high-fat diet plus 0.1% N-acetylcysteine.

2. 5

3

3. 5

TAC(U/ml)

Fig. 2. Scatterplots show correlation between TAC and the atherosclerotic index. Squares indicate the values of atherosclerotic index ⫾ SD, paired with TAC for each group. Sample sizes were eight for each data point. LPL, lipoprotein lipase; TAC, total antioxidant capacity.

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LPL activity(U/ml)

2. 5

Acknowledgement

2

The authors are grateful for support and contributions made by Prof. YongHui Shi and Prof. GuoWei Le.

1. 5 1

r =0. 979 p<0. 05

0. 5

References

0 2

2. 5

3

3. 5

TAC(U/ml)

Fig. 3. Scatterplots show correlation between TAC and lipoprotein lipase activity. Squares indicate the values of lipoprotein lipase activity ⫾ SD, paired with TAC for each group. Sample sizes were eight for each data point. AI, atherosclerotic index; TAC, total antioxidant capacity.

HDL levels and is therefore potentially atheroprotective [33,34]. Accumulating evidence has revealed that LPL is essentially an antiatherogenic enzyme [35–39]. Increased oxidative stress causing decreased LPL activity was implicated in the disturbance of lipid metabolism. Because ROS not only can damage or change the functions of proteins directly but also act as subcellular messengers to influence the gene expression, it is reasonable to assume that the change in redox state accounts for the change of LPL activity. The past decade has seen a rapid if not explosive growth in the number of genes shown to be influenced by redox changes and in those that exert downstream effects through increased ROS formation [11,12]. Thus, impaired LPL activity may be due to decreased expression of LPL and/or by a direct effect of ROS on LPL. This effect was opposed by the two antioxidants. However, specific studies must be performed to explain how NAC and LA might interfere with the expression or the activities of LPL, or both, in cellular lipid metabolism. The latter proposal is under current study in our laboratory. Our study demonstrated that LPL activity is decreased and AI is increased in HFD-fed compared with control rats. TAC is negatively correlated with AI and it is positively correlated with LPL activity. These findings suggest that the increase in AI may occur due to the increase in the imbalance between the production of ROS and the capacity of antioxidant defenses accompanied by disturbed LPL activity.

Conclusion The present results indicate that there is a close relation of TAC to LPL activity and AI. LA and NAC prevent the negative biochemical changes related to oxidative stress and exert a hypolipidemic effect in hyperlipidemia. It is possible to try concentrations other than 0.1% LA or NAC.

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