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Effects of Estrogen and Folic Acid on High Fat Induced Hypercholesterolemic Mice

S. Akter
Department of Physiology, Bangladesh Agricultural University, Mymensingh-2202,Bangladesh.

M. K. Islam
Department of Physiology, Bangladesh Agricultural University, Mymensingh-2202,Bangladesh.

M. A. H. N. A. Khan
Department of Pathology, Bangladesh Agricultural University, Mymensingh-2202,Bangladesh.

M. A. Miah
Department of Physiology, Bangladesh Agricultural University, Mymensingh-2202,Bangladesh.

Abstract/ Executive Summary:

Aims: To elucidate the effect of estrogen and folic acid on high fat (butter) induced lipid profile (total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), and triglyceride (TG)) and on tissue texture changes in mice.

Designs: Randomized block design.

Place and Duration of Study: Department of Physiology and Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh between January 2012 and April 2012.

Methodology: 2 months old 50 male Swiss Albino mice (Mus musculus) were used for this study and divided into equal groups. Group A (control) was fed with normal rat pellet. Mice in the group B was fed with butter; group C was fed with butter and estrogen; group D was fed with butter and folic acid and group E was fed with butter, estrogen and folic acid. The atheroprotective effect of estrogen and folic acid was evaluated based on weight gain, biochemical parameters and histopathology.

Results: The highest body weight gain was detected in group B (P<0.001). In biochemical study, group B showed the increase in total plasma cholesterol, especially low density lipoprotein (LDL), and triglyceride (TG) (P<0.001) compared to other treated groups. Group C, D and E showed a lower level of total plasma cholesterol (P<0.001) compared to group B. Among them group E showed the lowest total plasma cholesterol level (P<0.001). In histopathological study, the aorta of butter treated group showed sloughing of lining endothelium, increased aortic wall thickness and loss of integrity of tunica intima. It also revealed fatty changes in liver in animal models fed with butter, compared with those on a normal diet.

Conclusions: 20% butter supplementation would be able to cause a rise in lipid profile and produce degenerative changes in aorta and liver and addition of estrogen or folic acid in butter supplemented diet counteracts the adverse effect.

Keywords: Mice; Butter; Estrogen; Folic acid; Hypercholesterolemia.

Location: Department of Physiology and Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202,

Start Date: 00-01-2012

End Date: 00-04-2012

Program Area: Animal Health and Management

Commodity: Rats

Objectives:

To investigate the application of high fat with or without estrogen and folic acid on biochemical profile of blood and on histo-texture of aorta and liver in mice.

Methodology:

Experimental Animals

Two months old 50 male Swiss Albino mice (Mus musculus) with mean initial body weight of 20±g were obtained from ICDDR, B, Dhaka, Bangladesh. All mice were maintained in the animal care facilities according to university animal care and use guidelines.

Experimental Protocol and Diets

After arrival, animals were allowed free access to a basal diet of commercial rodent ration and water for 3 days to allow adaptation to the environment. Baseline values of serum total cholesterol were measured at the end of two weeks in mice that were deprived of food overnight. The baseline serum total cholesterol level was almost similar in all mice and then they were randomly divided into five groups. The control group (A) continued to be fed the normal rodent diet. Group B were fed with 20% butter which added in the normal rodent diet. In addition to 20% butter in diet, mice of group C was fed with estrogen (Estracon® – 0.625 mg/tablet, Renata Limited) at a dose rate of 10 μg/kg/day with water; group D was fed with folic acid (Tablet Folison®-5 mg/tablet, Jayson laboratories) at a dose rate of 10 μg/kg/day with water and mice in group E was fed with estrogen at a dose rate of 10 μg/kg/day plus folic acid at a dose rate of 10 μg/kg/day with free access to water for 90 days. Diet consumption and body weight were measured on day 0 and day 90. Blood samples were collected on day 0 and day 90 from food-deprived mice (14 hours) anesthesized using 3 μL of 5 mg/mL acepromazine. Blood was collected from the lateral saphenous vein with a sterile 23 gauge needle, into Microtainer® serum separator tubes. At the end of the experimental period (90 days), the mice were euthanized by carbon dioxide asphyxiation and blood was withdrawn by cardiac puncture using a 22 gauge needle and a 5 mL syringe. Cardiac blood was transferred into serum separator tubes and the blood was allowed to clot at 23ºC for 30 minutes and subsequently placed at 4ºC until centrifugation. Serum was separated by low-speed centrifugation at 2000 g for 20minutes at 4ºC temperature. Serum was frozen at −85ºC until further analysis. Serum total lipids and glucose were assayed by conventional enzymatic methods on a Hitachi 911 automated analyzer from Roche Diagnostics (Laval, QC, Canada). The precision performance of these assays was within the manufacturer’s specifications. LDL cholesterol was calculated by the Friedewald equation. Blood glucose concentrations were measured using commercial kits (Labtest).

Histology

The aorta and whole liver were excised from each animal, immersed in chilled phosphatebuffered saline and blotted dry. A 4 mm section of the aorta and liver was placed into a histological cassette. The cassette containing the aorta and liver section of each animal were individually immersed in a 10% (v/v) buffered formalin phosphate solution for fixing and subsequent staining. The formalin-fixed, paraffin-embedded tissue samples were ultrasectioned (4–5 μm thickness), stained with hematoxylin and eosin and examined under a light microscope (LABOMED).

Statistical Analysis

All data were expressed as mean ± SD, and differences among the groups of animals were compared using one-way ANOVA with post-hoc Duncans test. Paired t-tests were used to compare pre-treatment and post-treatment value of different groups. Statistical significance was set at P< 0.05. Statistical analysis was performed using SPSS software version 17 (SPSS Inc., Chicago, IL, USA).

Source of Information: British Biotechnology Journal, 3(1): 39-53, 2013, SCIENCEDOMAIN international

Article Link (Online Journal): www.sciencedomain.org

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

We found the mice of butter treated group exhibited significant increase in body weight, change in lipid profile and increase blood glucose level. Histo-pathological analysis confirms the development of fatty liver and the changes in aorta. Addition of both folic acid and estrogen significantly reduce the lipid profiles, glucose level and prevent the butter induced fatty liver changes. Therefore, dietary intake of folic acid or estrogen along with butter rich diet reduces the risk of atherosclerosis, fatty liver disease and coronary heart disease. Although more confined studies are necessary, the inclusion of folic acid or estrogen in fat rich food might providean important means of reducing atherosclerotic burden. Our findings will provide the tools for further study and strength the previous findings as well.

Knowledge Management: Journal