Practical 5: Serum Triglyceride and Cholesterol: Effects of Different Dietary Components

Name: Hung Wing Tung (2009053464)Group No.: 3Date: 14/4/2011

BIOL2537 Laboratory in Nutritional Science

1

AbstractThe serum triglyceride and cholesterol levels of four 8 weeks old male rats were determined after each having a 3-week dietary consumption of 20% butter fat/ 0.5% cholesterol with either (a) cholestyramine, (b) 5% fiber, (c) 10% fiber or (d) 20% fiber.15 L of serum from each rat was mixed with 1500 L of Stanbio Triglyceride Liquicolor kit, #2200 430 for the calculation of serum triglyceride level after reading the absorbance at 500 nm. Same volume of rat serum was mixed with 1500 L of Stanbio Cholesterol assay kit, #1010 225 for the calculation of serum total cholesterol. The serum of the rat fed with 20% fiber was found to contain the least amount of triglyceride (72.92 mg/dL) and cholesterol (46.37 mg/dL), while that of the rat fed with 5% fiber was found to contain the highest amount of triglyceride (173.45 mg/dL) and cholesterol (103.01 md/dL). It is concluded that increasing dietary fiber consumption could lower serum triglyceride and cholesterol levels.

IntroductionDietary lipids, once enter human body, are subjected to digestion and absorption. There are two main categories of blood lipid: triglycerides and cholesterols. Due to their hydrophobic nature, they are carried by lipoproteins in blood1.

Although lipids are essential building blocks of cells and organelles1, a high level of blood lipid is the major risk factor for coronary heart diseases2.High blood lipid can be caused by genetic factors, or problematic diet habits, such as alcoholism and high intake of saturated fat3. While excessive saturated fat intake can raise blood lipid level, high intake of dietary fiber is known to reduce it4. Thus, increasing the intake of dietary fiber serves to be the first-line approach for blood lipid reduction5. Besides dietary intervention, pharmacological treatment can also lower the blood lipid profile6. For example, cholestyramine, a bile acid-binding resin, is often used to treat people with hypercholesterolemia7.

The specific aim of this study is to examine and compare the effect of cholestyramine and dietary fiber on the serum lipid profile after consumption of a high fat diet. On the course of experimental treatment, both triglyceride and cholesterol assays will be used to determine the amount of triglyceride and total cholesterol in the rat serum respectively.

The principle of the triglyceride assay is as follows8:LipaseTriglyceride + H2O Glycerol

Glycerol kinaseGlycerol + ATP G-3-P + ADP

Glycerylphosphate oxidaseG-3-P + O2 DAP + H2O2

H2O2 + 4-Aminoantypyrine + 4-ChlorophenolPeroxidase Quinoneimine + HCl + 4 H2O

And the following is the principle of cholesterol assay9:

Cholesterol esteraseCholesterol esters Cholesterol + Fatty acids

Cholesterol oxidaseCholesterol + O2 Cholest-4-en-3-en-one + H2O2

H2O2 + 4-Aminophenazone + PhenolPeroxidase H2O + Q-Quinoeimine dye

Materials and Methods

Collection of MaterialsA. ReagentsStanbio Triglyceride Liquicolor kit, #2200 430 and Stanbio Cholesterol assay kit, #1010 225 were purchased by the School of Biological Sciences, University of Hong Kong. The former contains 2.0 mM ATP, 15.0 mM Magnesium Salt, 0.5 mM 4-Aminoantipyrine, 4 mM 4-Chlorophenol, 1500 U/L Glycerylphosphate oxidase, 0.01 % Sodium Azide, 4000 U/L Lipase, 400 U/L Glycerol kinase, 2000 U/L Peroxidase, 50 mM Goods Buffer (pH 6.7 0.1). The latter contains 0.25 mmol/L 4-Aminophenazone, 25.0 mmol/L Phenol, > 5.0 U/mL Peroxidase, > 0.15 U/mL Cholesterol esterase, > 0.1 U/mL Cholesterol oxidase, buffers and stabilizers.

Triglyceride and cholesterol standards were also prepared by the School. Triglyceride standard contains glycerol with surfactant to yield 200 mg/dL triglycerides as triolein, with 0.01% cSodium Azide being added as preservative. Cholesterol standard contains buffered aqueous solution of cholesterol with stabilizers, surfactants and preservative.

B. Animals and diets4 male (8 weeks old) Sprague Dawley rats were obtained from the Laboratory Animal Unit, University of Hong Kong. Rats were housed individually and kept in a controlled environment at 22C under a 12-h-light/12-h-dark cycle with light on from 7 am to 7 pm. They consumed ad libitum water and laboratory food (Lab Diet, The Richmond Standard, PMI Nutritional International) for 1 week before receiving the experimental diets (refer to Annex 1 for the composition of the diets). All prepared diets were stored at 20 C.

Rats were randomly assigned to one of the four 20% butter fat/ 0.5% cholesterol diets: (i) 8% w/w cholestyramine, (ii) 5% fiber, (iii) 10% fiber, (iv) 20% fiber and allow free access for 3 weeks. On day 21, they were anaesthetized with pentobarbital (Abbott Laboratory) intraperitoneal (50 mg/kg) after a 12-hour food deprivation overnight. Blood was drawn from abdominal vena cava of the rats to get the serum.

Serum Samples PreparationAbdominal cavity of the rats was exposed after midline incision. Blood was drawn from abdominal vena cava into a syringe, and then transferred into glass centrifuge tube. Blood was allowed to stand at room temp for 30 minutes for blood clot formation. After that blood was centrifuged at 2000 rpm at 4C for 10 minutes. The supernatant (serum) was transferred to clean tubes and stored at 2 8 C for preparation of assays.

Assays PreparationA. Serum Triglyceride AssayBlank solution containing distilled water (B), triglyceride standard (S) and 4 serum samples (U) were pipetted into cuvettes with the following volumes (L) (shown in Table a.) and mixed well with the Triglyceride LiquiColor Reagent by pipetting up and down.

Table a. The required volumes for different assays for testing serum triglyceride levelBSU

Cholestyramine5 % fiber 10 % fiber 15% fiber

Reagent150015001500150015001500

Standard-15----

Serum--15151515

Water15-----

Duplicate of this assays set was done at the same time. The cuvettes were incubated at room temperature for 10 minutes. Absorbance was read at 500 nm. Blank solution was used to set the spectrophotometer to zero absorbance.

B. Serum Cholesterol AssayBlank solution containing distilled water (B), cholesterol standard (S) and 4 serum samples (U) were pipetted into cuvettes with the following volumes (L) (shown in Table b.) and mixed well with the Enzymatic Cholesterol Reagent by pipetting up and down.Table b. The required volumes for different assays after testing serum triglyceride levelBSU

Cholestyramine5 % fiber 10 % fiber 15% fiber

Reagent150015001500150015001500

Standard-15----

Serum--15151515

Water15-----

Duplicate of this assays set was done at the same time. The cuvettes were incubated at room temperature for 10 minutes. Absorbance was read at 500 nm. Blank solution was used to set the spectrophotometer to zero absorbance.

Results

The absorbance at 500 nm of both triglyceride and cholesterol assays was read and summarized in Table c. and Table d. respectively.

Table c. Absorbance of different triglyceride assaysAbsorbanceAverage

Standard0.2800.2850.283

SerumsampleCholestyramine0.1270.1590.143

5% fiber0.2100.2800.245

10% fiber0.1750.1960.186

20% fiber0.0650.1410.103

Table d. Absorbance of different cholesterol assaysAbsorbanceAverage

Standard0.3120.2530.283

SerumSampleCholestyramine0.1300.1020.116

5% fiber0.1480.1430.146

10% fiber0.1290.0950.112

20% fiber0.0680.0630.066

After obtaining the absorbance readings, serum triglyceride and serum cholesterol levels were calculated using the following equation:

Serum Triglyceride or Serum Total Cholesterol (mg/dL) = [ (AU AB)/ (AS AB) ] x 200 Where AU, AS and AB were the absorbance values of the serum sample with unknown concentration of triglyceride or cholesterol, standard and blank solution respectively; 200 was the concentration of triglyceride or cholesterol standard (mg/dL). The calculated serum triglyceride and cholesterol levels of the rats fed with different diets were illustrated in the following tables.

Table e. Calculated serum triglyceride levels in different rat serum samplesRat Serum SampleSerum Triglyceride Level (mg/dL)

Cholestyramine101.2389

5% fiber173.4513

10% fiber131.3274

20% fiber72.92035

Table e. Calculated serum cholesterol levels in different rat serum samplesRat Serum SampleSerum Cholesterol Level (mg/dL)

Cholestyramine82.12389

5% fiber103.0088

10% fiber79.29204

20% fiber46.37168

Bar charts below showed the comparison of serum triglyceride and cholesterol levels among the rats fed with different diets.

Discussion

Effect of Different Concentrations of Fiber on Serum Triglyceride Levels Compared to ChloestyramineReferring to the bar chart illustrated in Fig a, the serum triglyceride levels were lower in rats fed with increasing percentages of fiber. Rat which consumed 20 % fiber had the lowest serum triglyceride level (72.92 mg/dL), followed by that fed with 10 % fiber (131.32 mg/dL) and lastly 5 % fiber (173.45 mg/dL). A 20 % fiber diet also showed a greater reduction on serum triglyceride level compared to a diet with cholestyramine (101.24 mg/dL), while the effects of 10 % and 5 % fiber diets on serum triglyceride reduction were less potent than that of the cholestyramine diet.

Effect of Different Concentrations of Fiber on Serum Total Cholesterol Levels Compared to ChloestyramineReferring to the bar chart illustrated in Fig b, the serum total cholesterol levels were lower in rats fed with increasing percentages of fiber. Rat which consumed 20 % fiber had the lowest serum total cholesterol level (46.37 mg/dL), followed by that fed with 10 % fiber (79.29 mg/dL) and lastly 5 % fiber (103.01 mg/dL). Both 20 % and 10 % fiber diets showed a greater reduction on serum total cholesterol levels compared to a diet with cholestyramine (82.12 mg/dL), while the effect of a 5 % fiber diet on serum total cholesterol reduction was less potent than that of cholestyramine diet.

Serum Lipids Lowering Effect of Fiber and CholestryamineDietary fiber includes for a wide range of plant substances resistant to digestive enzymes in human gastrointestinal tract10. It can be classified into 2 major groups depending on the solubility in water. Structural fibers like lignin and cellulose are insoluble fibers, while the gel-forming fibers, such as oats, psyllium, pectin and guar gum are soluble fibers4. Several reviews have suggested that it is the soluble fibers carrying the serum lipid lowering effect11,12. Insoluble fibers, unless they displace the food supplying saturated fat and cholesterol, had no effect on serum lipid profile13.

There are many suggested mechanisms by which fiber lowers blood lipid level. First, evidence has shown that some soluble fibers can bind with bile acids or cholesterol during the formation of micelles14. This lowers the cholesterol content in liver cells and up-regulates the LDL (low density lipoprotein) receptors, clearing more LDL cholesterol for bile acid synthesis. Another suggested mechanism involves an inhibition of hepatic fatty acid synthesis by the production of short-chain fatty acids (e.g. acetate and propionate) from the fermentation of soluble fibers in the gastrointestinal tract15. Also, fibers may increase intestinal motility16, hinder absorption of macronutrients (which increases insulin sensitivity and promotes breakdown of fats)17. Intake of fibers also increases satiety and reduces the total energy intake18.

Cholestyramine, on the other hand, can also increase fecal excretion of bile acids. This up-regulates the hepatic LDL receptors and lowers the plasma LDL cholesterol19. The reduction in LDL is coupled with an increase in triglyceride in human and most animals blood, causing a mild hypertriglyceridemia19. It is because in these animals, enterohepatic circulation of bile acids down-regulates the hepatic triglyceride synthesis, while interference in this circulation by cholestyramine inhibits this down-regulation19. However, from the results, rat fed with cholestyramine showed a drop in serum triglyceride level, which was inconsistent with the theory. This may be due to the absence of such down-regulation of hepatic triglyceride synthesis by bile acids in this specific species of rat19.

Causes and Drawbacks of HyperlipidemiaHyperlipidemia is referred to high blood lipid level3. There are 3 major types of hyperlipidemia: (a) Hypertriglyceridemia, (b) Hypercholesterolemia and (c) Mixed hyperlipidemia20.

Causes of hyperlipidemia are multi-fold, which involves personal and family history of cardiovascular diseases (CVD), relevant medical history (e.g. hypertension, diabetes and HIV infection), lifestyle (exercise and diet), smoking and alcoholism20. Genetic factors of hyperlipidemia include a single dominant gene defect, deficiency in lipoprotein lipase, or other abnormalities in lipid metabolism3. Diet is another key factor for hyperlipidemia. Excess saturated fat intake can raise the blood cholesterol level, while a high carbohydrate diet can cause elevated triglyceride in blood3. Besides, alcohol can also contribute to hyperlipidemia owing to its energy-rich nature and the increase in availability of substrates for triglyceride synthesis in liver3.

Hypertriglyceridemia can cause pancreatitis (inflammation of the pancreas), but the major consequence of hyperlipidemia is linked to an increased risk of atherosclerosis (hardening of arteries), which may result in coronary heart diseases, stroke and peripheral vascular diseases. The pathology of atherosclerosis is characterized by atherogenesis3. When the endothelial lining of arteries are damaged from high blood pressure, local injury, or inadequate oxygen supply, the permeability into subendothelial space will increase, which enables lipoproteins to pass more easily. Since LDL and triglyceride-rich lipoprotein remnants are atherogenic, when they enter the subendothelial space, they undergo oxidation. Oxidized LDL will be scavenged by marcophages by expressing the acetylated LDL receptors. LDL will then become lipid-rich foam cells, which undergo apoptosis and produce a lipid-rich extracellular medium. In response to this process, smooth muscle cells migrate from the tunica media of the arteries and proliferate. This produces a connective tissue matrix containing collagen, elastin and proteoglycans, which is known as plaque. Presence of plaque in blood vessels can lead to significant reduction in blood flow, producing symptoms such as angina or claudication. The thin cap and a large lipid core of plaque are also unstable and susceptible to rupture. This causes an acute occlusion of the arteries. Owing to the blockage in arteries, nutrients and oxygen delivery to organs such as heart and brain are hindered, causing syndromes such as myocardial infarction and stroke3. Other Drug Treatments for HyperlipidemiaMany pharmacological agents are available for treatment of hyperlipidemia. For example, in a bid to treat hypercholesterolemia, apart from cholestyramine, colestipol also serves as a bile acid sequestrant which lowers blood cholesterol by binding with bile acids irreversibly and up-regulating LDL receptors21. Other than bile acid sequestrants, fibrates and HMG-CoA reductase inhibitors can also lower blood cholesterol by the formation of derivative clofibrate22 that decreases cholesterol absorption and inhibition of cholesterol synthesis that reduces the cholesterol content of enterocytes and ACAT (cholesterol acyltransferase) activity23. Some emerging drugs like synthetic saponins and 2-azetidinones have been shown to impair cholesterol absorption by their potential interaction with cholesterol transporter molecules24,25.

Tetrahydrolipstatin (generic name: orlistat) is a drug applied to treat hypertriglyceridemia. It works by inhibiting the activities of gastric and pancreatic lipases, which prevents dietary triglyceride hydrolysis in the intestinal lumen 26. Doseresponse studies in humans have shown that maximal inhibition of fat absorption of orlistat can be up to 30% of dietary fat with regular meals everyday27.

Possible Experimental Errors and ImprovementSeveral experimental errors might occur and contributed to inaccurate results. First, the rats used might show different sensitivity to the experimental diets, such as different rates of digestion, absorption and metabolism of nutrients or drug. As only one rat was used for each experimental diet, any of the above biological variations would greatly affect the accuracy of results. Thus, more rats should be used for each test diet to minimize the effect of biological variations.Not only did the amount of rats matter, the number of trials also played an important role in results accuracy. Reliability would be lower if the number of trials was too few. In this study, only two trials were performed in measuring the absorbance values of sample assays, which was insufficient to give reliable results. As such, more trials should be done by preparing more serum sample sets.

Technical errors might occur during the sample preparing process too. For example, the volumes of solution might not be exactly the same as the guideline given, since air bubbles were produced during the pipetting up and down process, leading to a leakage of solution from the mouth of cuvettes. Also, the reagent and samples might not be well mixed before the measurement of absorbance. Thus, improvement in experimenters skills is necessary.

ConclusionThe effects of fiber and cholestyramine on serum triglyceride and total cholesterol levels in rats were determined. It is concluded that increasing fiber content in diet could lower both serum triglyceride and total cholesterol levels. Oral administration of cholestyramine also showed a reduction in serum triglyceride and total cholesterol.

Compared to cholestyramine, a 20 % fiber diet could decrease the serum triglyceride level to a greater extent; 20 % and 10 % fiber diets also exerted a greater reduction in serum total cholesterol.

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