Nutttt

43

LAB 3: LIPID METABOLISM

PURPOSE: To employ proper laboratory technique (e.g., pipetting, spectrophotometry) to examine blood lipid levels using a laboratory triglyceride and cholesterol assay. BACKGROUND:

Lipids are commonly referred to as "fats" in our bodies. The major types of lipids present in the body are: fatty acids, glycerides, phospholipids, and sterols. Lipids function to add flavor to food and to provide a concentrated source of energy. Triglycerides are composed of 3 fatty acid chains attached to a glycerol backbone and are the major source of "dietary fat". Phospholipids are composed of 2 fatty acid chains attached to a glycerol backbone containing a phosphate group. They function primarily in cell membranes. Cholesterol is a sterol both consumed from the diet and naturally produced by the body. Cholesterol is mostly found in cell membranes (90%) and in myelin sheaths on nerve cells and is also used to synthesize vitamin D.

Lipids do not dissolve in an aqueous medium (i.e., body fluids) therefore they require a special method of transportation to reach their destinations throughout the body. They accomplish this by using lipoproteins. Lipoproteins are the transport vehicles for lipids in the circulation. They are distinguished by way of buoyant density so that they may be put into different classes. The major classes of lipoproteins are: chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). The chylomicrons transport dietary lipids from the intestine to the liver. The pathway of transporting dietary lipids is known as the exogenous pathway. VLDLs, IDLs, and LDLs are mainly involved in transport of lipids synthesized in the liver. This pathway is known as the endogenous pathway.

Very low density lipoproteins (VLDL) are responsible for the transport of triglycerides from the liver to the adipose tissue. The triglycerides are released from VLDL after being broken down by an enzyme appropriately named lipoprotein lipase (as you learned from class, a lipase hydrolyzes ester bonds between the glycerol backbone and fatty acids). The fatty acids are subsequently released and stored in adipose tissue, while the glycerol backbone is release into the bloodstream. The loss of triglycerides from VLDL reduces the percentage of triglyceride and would increase the percentage of cholesterol. This new particle is now higher in density and therefore, is no longer very low in density but is now just low density lipoproteins (LDL). LDL cholesterol has been coined the name "bad cholesterol" by researchers because it has the ability to deposit cholesterol into arteries that leads to the development of atherogenic lesions that may block arteries and would lead to a heart attack. A fasting LDL cholesterol reading below 130 mg/dl is considered normal, but if between 130 - 160 mg/dl, the risk for premature heart disease increases.

Unlike both chylomicrons and VLDLs, HDLs are small, dense particles synthesized in both the liver and the intestine. Basically, the HDL particle is known to pick up cholesterol derived from blood vessel walls and then transports this cholesterol to the liver. Since cholesterol is essentially cleared from the blood by this process, researchers consider HDL

44

cholesterol as “good cholesterol”. Therefore, high levels of HDL cholesterol are desired to decrease one’s risk for heart disease. Levels of HDL cholesterol less than 35 mg/dl for men and less than 45 for women increases the risk of premature heart disease.

Nutrition plays a major role in lipid metabolism and it is very well known that dietary fatty acids affect blood lipid levels. One factor that has been shown to have an impact on lipid metabolism is dietary fat. Dietary fat is mainly in the form of triglycerides (~98%). There are many different types of fatty acids and all foods contain different proportions. An increase in dietary saturated fatty acids consumption has been shown to elevate total cholesterol levels. Therefore it is important to monitor blood triglyceride and cholesterol levels in order to monitor cardiovascular health. Total cholesterol levels below 200 mg/dl and triglyceride levels below 150 mg/dl are desired to reduce risk for future heart disease. In this laboratory experiment, we will examine the methods used by clinical laboratories to determine the level of triglyceride and cholesterol from unknown samples.

PART I. Triglycerides determination Methods for triglycerides determination generally involve enzymatic or alkaline

hydrolysis of triglycerides to glycerol and free fatty acids followed by either chemical or enzymatic measurement of the glycerol released. The procedure described herein involves the enzymatic hydrolysis of triglycerides by lipase to glycerol and free fatty acids, as provided in a kit from Sigma Diagnostics�. The glycerol produced is then measured by coupled enzyme reactions catalyzed by glycerol kinase, glycerol-1-phosphate dehydrogenase and diaphorase as follows:

a) Triglycerides + lipoprotein lipase Æ glycerol + free fatty acids b) Glycerol + ATP + glycerol kinase Æ G-1-P + ADP c) G-1-P + NAD + G-1-PDH Æ DAP + NADH d) NADH + INT + Diaphorase Æ Formazan + NAD Triglycerides are first hydrolyzed by lipoprotein lipase to glycerol and free fatty acids.

Glycerol is then phosphorylated by adenosine-5-triphosphate (ATP) to form glycerol-1- phosphate (G-1-P) and adenosine-5-diphosphate (ADP) in the reaction catalyzed by glycerol kinase. The G-1-P is oxidized to dihydroxyacetone phosphate (DAP) with the concominant reduction of nicotinamide adenine dinucleotide (NAD) to NADH in the reaction catalyzed by glycerol-1-phosphate dehydrogenase (G-1-PDH). The NADH is oxidized with the simultaneous reduction of 2-(p-iodophenyl)-3-p-nitrophenyl-5- phenyltetrazolium chloride (INT) to formazan in the presence of diaphorase. The resulting formazan is a highly colored substance and has an absorbance maximum at 500nm. The intensity of the color produced is directly proportional to the triglycerides concentration of the sample. Procedures: 1. Turn on spectrophotometer and set the wavelength to 500 nm. 2. Set up 5 microtubes and label them as follows:

B (blank) TG1 (triglyceride 1) TG2 (triglyceride 2) S1 (standard 1) S2 (standard 2)

Note: You have 2 triglyceride samples and 2 standard samples because you will be performing the experiment in duplicate in order to control for human error.

3. Pipet 1.0 ml of Triglyceride reagent into each microtube and pre-warm at 37°C for at

least five minutes. 4. Add 10 Pl (0.01 ml) of saline into blank microtube (B) and mix.

46

5. Add 10 Pl (0.01 ml) of triglyceride standard (200 mg/dl) to the standard microtubes

(S1, S2) and mix. 6. Add 10 Pl (0.01 ml) of unknown sample to the triglycerides microtubes (TG1, TG2)

and mix. 7. Incubate all tubes for 5 minutes at 37°C. 8. Following incubation, add 2.0 ml of saline to 5 test tubes [properly labeled B, TG1,

TG2, S1, S2] and vortex.

Note: This step is necessary to raise the volume of the tubes so the spectrophotometer is able to read the samples.

9. Quickly transfer the solution from the microtubes to test cuvettes.

10. Adjust the spectrophotometer to 0.000 abs using the blank.

Note: When reading absorbance of each sample, make sure to use a cuvette designed for spectrophotometric use.

11. Read the absorbance of each sample making sure to blank the spectrophotometer between samples.

Note: Absorbance readings should be completed within 10 minutes of incubation time.

12. Record the absorbance readings in the table below.

47

Results: 1. Absorbance Reading

2. Determining unknown concentration of triglycerides samples (TG1, TG2)

Use the following formula to determine TG concentration: TG concentration (mg/dl) = Average TG absorbance−Blank absorbance

Average S absorbance x S (mg/dl)

Note: " S mg/dl" is the known concentration of the standard (S) as provided by the manufacturer.

Tube absorbance reading at 500 nm

average absorbance reading

B 0.00 TG1 (a) TG1 (b) TG2 (a) TG2 (b) S1 (a) S1 (b) S2 (a) S2 (b)

PART II. Total cholesterol determination

Similar to the triglycerides assay, methods for total cholesterol determination generally involve enzymatic hydrolysis of a substrate to form a product. The use enzymes to assay cholesterol have been studied by many investigators and the determination of total cholesterol will be performed using a kit provided by Wako Chemicals USA, Inc. The series reactions involved in the assay system is as follows:

a) Cholesterol esters + Cholesterol esterase Æ Cholesterol + free fatty acids

b) Cholesterol + O2 + Cholesterol oxidase Æ Cholestenone + H2O2

c) H2O2 + Chlorophenol + 4-AAP + peroxidase Æ Red quinone pigment + 2 H2O + HCl

Cholesterol esters are enzymatically hydrolyzed by cholesterol esterase to cholesterol and free fatty acids. Cholesterol, including that originally present, is then oxidized by cholesterol oxidase to cholestenone and hydrogen peroxide (H2O2). The hydrogen peroxide produced combines with chlorophenol and 4-aminoantipyrine (4-AAP) in the presence of peroxidase (POD) to form a chromophore (red quinone pigment) which may be quantitated at 500 nm.

Procedures:

1. Turn on spectrophotometer and set the wavelength to 520 nm. 2. Set up 5 microtubes and label them as follows: B (blank) C1 (Cholesterol 1) C2 (Cholesterol 2) S1 (standard 1) S2 (standard 2) Note: You have 2 Cholesterol samples and 2 standard samples because you will be performing the experiment in duplicate in order to control for human error. 3. Pipet 1.0 ml of Cholesterol reagent into each microtube and pre-warm at 37°C for at least five minutes. 4. Add 10 µl (0.01 ml) of saline into blank microtube (B) and mix. 5. Add 10 µl (0.01 ml) of Cholesterol standard (200 mg/dl) to the standard microtubes (S1, S2) and mix. 6. Add 10 µl (0.01 ml) of unknown sample to the Cholesterol microtubes (C1, C2) and mix.

49

Incubate all tubes for 5 minutes at 37°C. 8. Following incubation, add 2.0 ml of saline to 5 test tubes and vortex. Note: This step is necessary to raise the volume of the tubes so the spectrophotometer is able to read the samples. 9. Quickly transfer the solution from the microtubes to test cuvettes. 10. Adjust the spectrophotometer to 0.000 abs using the blank. Note: When reading absorbance of each sample, make sure to use a cuvette designed for spectrophotometric use. 11. Read the absorbance of each sample making sure to blank the spectrophotometer between samples. Note: Absorbance readings should be completed within 10 minutes of incubation time. 12. Record the absorbance readings in the table below.

50

Results: 1. Absorbance Reading

2. Determining unknown concentration of triglycerides samples (C1, C2)

Use the following formula to determine Cholesterol concentration: Cholesterol concentration (mg/dl) = Average Cholesterol absorbance−Blank absorbance

Average S absorbance x S

(mg/dl) Note: " S mg/dl" is the known concentration of the standard (S) as provided by the manufacturer.

3. Lab report 1.) Only 1 report for Lab 3. 2.) Need to follow the report format that described in assignment 3. 3.) Results should include part I and Part II’s absorbance reading and unknown

sample determination (using formula for Part I and Part II). 4.) Report the unknown sample’s value. 5.) Concepts to consider for discussion:

a. Discuss problems that were associated with the lab (if any). b.Interpret the lipid levels and any variations obtained. c. Explain how lifestyle and genetics plays a role in lipid levels. d.Discuss the importance of proper laboratory technique in ensuring accuracy

Tube absorbance reading at 520 nm average absorbance reading

B 0.00

C1 (a) C1 (b) C2 (a) C2 (b) S1 (a) S1 (b) S2 (a) S2 (b)