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Evaluation of Hematological, Serum Biochemical and Tissue Antioxidant Changes in Experimentally Induced Hyperlipidemia in Wistar Albino Rats

Srinivasa Naik H. Srilatha Ch.1 Sujatha K. Sreedevi B. Prasad T. N. V.K. V.
Vol 8(2), 84-91
DOI- http://dx.doi.org/10.5455/ijlr.20170512080116

Hyperlipidemia is the disorder of lipid metabolism, characterized by elevated serum total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-C) and very low density lipoproteins cholesterol (VLDL-C) and decreased high density lipoprotein cholesterol (HDL-C). The present study was carried out by procuring 24 Wistar albino rats, divided into two groups consisting of 12 rats in each group. Hyperlipidemia was induced by hyperlipidemic diet consisting of 1% cholesterol and 15% saturated oil added to the 1000g of standard rat diet and given to group II rats. Group I kept as control and fed with standard rat diet. Sacrifice of the rats was done on 45 and 90 day of the experiment and six rats from each group were randomly selected for sacrifice. Group II rats clinically showed obesity with significant increase (p<0.05) in the body weight. Rats were sluggish with poor hair coat. Total leucocyte count (TLC), TC, TG, LDL-C, VLDL-C were significantly (p<0.05) higher, whereas HDL-C was significantly reduced in group II. Oxidative damage indicators like catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx), reduced glutathione (GR) and glutathione-S-transferase (GST) were significantly (p < 0.05) reduced, whereas lipid peroxidation products of thiobrarbituric acid reactive substance (TBARS) level was non-significantly (p < 0.05) increased in liver and heart tissues of hyperlipidemic diet fed group (Group II) compared to group I. Clinical pathological parameters like total erythrocyte count (TEC), total leukocyte count (TLC), packed cell volume (PCV) and hemoglobin estimation was carried out and results of TEC, PCV and Hb% were normal and non-significant (P < 0.05) throughout the experimental period in both the groups I and II, where as TLC in group II was non significantly higher when compared to group I.


Keywords : Biochemical Parameter Hyperlipidemia Hemotology Tissue Antioxidants Wistar Rats

Introduction

Hyperlipidemia is accompanied by elevated serum TC, TG, LDL-C and VLDL-C and decreased HDL-C levels (Rahaman et al., 2013). It is associated with cardiovascular diseases (CVD) including coronary heart disease and stroke and is the leading causes of mortality in both developed and developing countries, accounting 30% of all worldwide deaths per year (Eman et al., 2011). Current reports suggest that by the year 2020, India will have the largest cardio vascular disease (CVD) burden in the world (Munisankar et al., 2015). Hyperlipidemia is considered as the primary mediator of a cascade of atherosclerosis (Balakumar et al., 2007), pancreatitis, renal injury (Attia et al., 2002) and hereditary familial hypercholesterolemia (Frederick, 2009). Atherosclerosis (hardening of the arteries) is a cardiovascular and fibroproliferative inflammatory disease commonly associated with age and ‘dietary-related factors’ in humans. In animals, atherosclerosis is rarely noticed. Establishment of hyperlipidemia and understanding of various lipid, pathological and tissue antioxidants levels in the rat model helps in the formulation of various therapeutic agents against hyperlipidemia and its associated conditions by targeting these parameters. Hence the Present study has been carried out to evaluate the clinical, serum biochemical and tissue antioxidant levels in experimentally induced hyperlipidemia.

Materials and Methods

Procurement of Experimental Animals

Male Wistar albino rats weighing around 200g were obtained from Sri Venkateswara Agencies, Bangalore. Rats were acclimatized to the experimental conditions for one week, after acclimatization the animals were grouped and housed in standard poly propylene rat cages (three rats per cage) during the experiment. They were maintained at 25±10c and a 12:12 hour interval light / dark cycle throughout the experimental period for 90 days by taking necessary precautions and providing standard laboratory hygienic conditions and  laboratory animal feed and water adlibitum. The approval of the institutional animal ethical committee was obtained prior to commencement of the experiment.

Source of Cholesterol

Cholesterol extra pure, AR grade with product code No: 97900 was procured from the SRL fine chemicals, Indian Scientific, Tirupati, Andhra Pradesh.     

 

experimental Design

A total of 24 healthy Wistar albino male rats were distributed into 2 groups contains 12 rats in each group. Rats were maintained for 90days with adlibitum provision of feed and water. Hyperlipidemia and atherosclerosis were experimentally produced by 1% cholesterol and 15% hydrogenated oil in 1000 g  standard rat chow diet (high cholesterol diet).

Clinical Observations

Health condition, behavior, feed and water intake of all the rats was monitored every day, throughout the experimental period. The health conditions and clinical signs of the animals were recorded.

Body Weight

Body weights of all the rats were recorded on 45th and 90th day of the experiment.

Hematology

Blood samples were collected in 10% EDTA at each sacrifice from all the sacrificed rats and used for the estimation of TEC, TLC, PCV by micro hematocrit method (Jain,1986) and Hb by Sahli’s method (Coles, 1986).

Biochemical Parameters

At each sacrifice, blood samples from all the groups were collected in to the sterile test tubes. After the blood clots formation, clear serum samples were separated without RBC and stored at 4˚C. Estimation of TC, LDL-C, VLDL-C, HDL-C, and TG were carried out by using commercially available biochemical kits (Auto Span diagnostics, Bangalore).

Tissue Oxidative Stress

At each sacrifice, liver and heart tissue pieces were collected and stored at –200C in the deep freezer until use. Tissue pieces of liver and heart were minced separately and homogenized in 0.05 M ice cold phosphate buffer (pH 7.4) by using a virtis homogenizer to make 10% homogenate. For lipid peroxidation assay, 0.2 ml of the homogenate was used. The remaining part of homogenate was mixed with 10% trichloroacetic acid in the ratio of 1:1, centrifuged at 5000 g for 10 min at 4 0 C and supernatant was used for estimation of reduced glutathione (Moron et al., 1979). The remaining part of the homogenate was centrifuged at 15,000 g for 60 min at 4­­0 C and the supernatant obtained was used for SOD (Marklund 1974), catalase (Caliborne, 1985) and GPx (Rotruck et al., 1973) in liver and aorta of all rats in all groups.

Statistical Analysis

The results were analyzed statistically by performing one-way ANOVA (Snedecor and Cochron, 1967).

Table 1: Mean values of hematological and serum biochemical parameters of rats in different experimental groups at 45th and 90th day of experiment

Parameters Group I Group II
45th day of study 90th day of study 45th day of study 90th day of study
Body weight (grams) 240±11.7 309.16±10.2 262.83±13.34 393.33±21.6a
Total erythrocyte count (Million/mm3) 5.78±0.9 6.30±0.2 6.6±0.45 6.83±1.3
Total leukocyte count(xmm3µl) 8.81±1.12 10.22±0.58 12.4±0.76 16.3±0.73
Packed cell volume (%) 34.6±5.7 37.83±1.3 39.6±2.6 32.5±2.1
Haemoglobin (Hb) (g%) 11.55±1.92 12.61±0.4 13.2±0.8 10.8±0.7
Total cholesterol (mg/dl) 46.03±7.8 50.67±7.04 113.58±11.75a 155.3±10.58a
Triglycerides (mg/dl) 68.70±50.0 67.17±11.08 150.58±33.27a 178.2±39.84a
Low density lipoprotein cholelsterol (mg/dl) 17.44±4.7 20.13±2.78 64.88±5.86a 95.9±9.10a
Mean value of VLDL cholesterol (mg/dl) 10.45±1.8 13.43±2.22 21.12±6.65a 31.0±8.78a
High density lipoprotein cholesterol (mg/dl) 28.16±2.8 27.10±5.15 17.58±5.35a 18.4±3.96a
Atherogenic index (TC/HDL-C) 2.54±0.33 3.11±0.65 4.2±1.71 5.56±0.89a

Mean values with different superscripts differ significantly (P < 0.05)

Results and Discussion

Abnormal clinical symptoms were not observed in control (Group I) rats, whereas obesity, sluggishness with poor hair coat was observed in the hyperlipidemic diet fed group (Group II) rats and it is in concurrence with Prabha et al. (2013). Hyperlipidemic diet fed group showed a non-significant increase in the body weight in comparison with control rats by 45th day and significant increase by the end of experiment period. It is in accordance with the reports of Olubukola et al. (2012), Pande et al. (2012), and Faheemuddin et al. (2013). It might be due to high calorie fat (1% cholesterol and 15% saturated oils) present in the hyperlipidemic control compared to standard normal rat chew of control. Clinical pathological parameters like TEC, TLC, PCV and Hb estimation was carried out and results of TEC, PCV and Hb% of group I rats was normal and non-significant (P < 0.05) throughout the experimental period. Total leukocyte count in hyperlipidemic diet fed group were non-significantly higher when compared to control group (Group I). Similar reports were made by Mohamed Anwar et al. (2008) who observed increased WBC and lymphocytes levels in rabbits that were fed with high cholesterol diet. Increased leukocytes count in the present study might be due to increased level of LDL cholesterol which is responsible for increased viscosity of the blood and thereby resulted into highest TLC (Huang et al., 2001).

Table 2: Mean values of tissue antioxidants of rats in different experimental groups at 45th and 90th day of experiment

Parameters Group I Group II
45th day of study 90th day of study 45th day of study 90th day of study
TBARS (nmoL TBARS/ g tissue) in the liver 1.38±0.42 1.36±0.27 1.8±0.32 2.02±0.23
TBARS (nmoL TBARS/ g tissue) in the heart 1.98±0.26 1.84±0.32 3.4±0.23 4.1±0.17
Catalase activity (nM of H2O2 decomposed /min/mg of protein) in the liver 0.25±0.020 0.28±0.020 0.15±0.03 0.14±0.02
Catalase activity (nM of H2O2 decomposed /min/mg of protein) in the heart 0.3±0.032 0.35±0.024 0.19±0.02 0.14±0.012
SOD activity (U/min/mg of protein) in the liver 18±1.1 16±1.8 14±1.02 12±2.2
SOD activity (U/min/mg of protein) in the heart 15±1.2 14±1.7 10±1.52 9±1.8
GPx activity (U/min/mg of protein) in the liver 28±1.1 26±1.6 22±0.9 20±1.2
GPx activity (U/min/mg of protein) in the heart 24±1.3 23±0.8 19±1.4 15±1.4
Glutathione reductase  (nmol of GSSG  utilized/min/mg protein) in  the liver 7.54±0.23 6.9±0.32 4.5±0.34 3.5±0.19
Glutathione reductase  (nmol of GSSG  utilized/min/mg protein) in  the liver 9.77±0.34 8.8±0.24 5.5±0.28 4.8±0.21

Mean values with different superscripts differ significantly (P < 0.05)

Rats on hyperlipidemic diet showed a significant (P < 0.05) increase in serum TC, TG, LDL-C, VLDL-C and significant decrease in HDL-C compared with control rats that received standard basal diet throughout the experimental period. These observations are in consistent with the reports of Naiel Abbass et al. (2012) and Rahaman et al. (2013). Increased serum lipid parameters in the present study might be due to inclusion of 1% cholesterol and 15% saturated oils added to the 1000g of rat diet (Hyperlipidemic diet) compared to standard rat chew diet of control group and it indicated that the diet under trial has established a hyperlipidemia in this group of rats. Rise in serum cholesterol can be attributed to the reduced catabolic rate of serum TC or reduced activity of hepatic cholesterol -7-alpha-hydroxylase, the rate limiting enzyme in bile acid synthesis from cholesterol (Farjat and Hussan, 2002). Increased HMG-CoA reductase activity in the liver of animals fed on fat enriched diet and the reduced rate of the clearance of LDL from circulation due to defective LDL receptors is associated with increase of plasma TC concentrations (Zulet et al., 1999). Low density lipoprotein is a lipoprotein that transports lipids from the liver to the peripheral (extra heaptic) and is often called “bad” cholesterol and constitutes a half to two thirds of cholesterol (Murry et al., 1996) and high levels of LDLs are highly atherogenic lipoproteins. High density lipoprotein (HDL) cholesterol is often called “good” because it is a lipoprotein that transports lipids from the periphery to the liver. Because the molecules are relatively small compared to other lipoproteins, HDL can pass through the vascular endothelial cells and into the intima to bring back the accumulated cholesterol in macrophages (Sakia and Lama, 2011). HDL particles enhance the net removal of cholesterol from a variety of cells, such as smooth muscle cells, fibroblasts and cholesterol laden macrophages (Wolfgang and Antonio, 1995). HDLs also prevent the oxidation of LDL by virtue of its antioxidant and anti-inflammatory properties (Chiozie and Chidinma, 2009). The low levels of HDL in the blood will increase the risk of atherosclerosis and coronary heart disease (Moeliandari and Wijaya, 2002).

Hyperlipidemic diet significantly (p < 0.05) increased atherogenic index by the end of our experiment in hyperlipidemic diet fed group II rats compared to control rats fed on standard diet. It is in accordance with the earlier reports of Rahman et al. (2013) and Harini et al. (2016). It indicates that, HDL-C (Good cholesterol) is decreased in proportion to increased TC, LDL-C (Bad cholesterol) and it is significantly (p < 0.05) evidenced by all serum biochemical parameters. Increased the liver and heart (includes aorta) thiobabutric reacting substances (TBARS) and decreased all other antioxidant enzymes in hyperlipidemic diet fed group II rats compared to control group throughout the experimental period. Hyperlipidemic diet of present study established the hyperlipidemia evidenced by increased TC, TG, LDL-C, VLDL-C and low HDL-C and it has elevated the total leukocyte count and tissue TBARS levels along with decrease in other cellular anti-oxidant enzymes. Effect of hyperlipidemia on various tissues, especially in the liver, kidney, heart and aorta needs to be studied in order to know the atherosclerotic and fatty changes in these organs.

Conclusion

Hyperlipidemic diet of present study established the hyperlipidemia evidenced by increased TC, TG, LDL-C, VLDL-C and low HDL-C and it has elevated the total leukocyte count and tissue TBARS levels along with decrease in other cellular anti-oxidant enzymes. Effect of hyperlipidemia on various tissues, especially in the liver, kidney, heart and aorta needs to be studied in order to know the atherosclerotic and fatty changes in these organs.

Acknowledgement

Author is thankful to the authorities of College of Veterinary Science, Tirupati and Sri Venkateswara Veterinary University, Tirupati for extending all the support for carrying experiment.

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