BIO211 Cell Biology

Determination of serum lipids

-----------------------------------------------------------------------------------------------------------INTRODUCTION Plasma lipids are composed of various classes of lipids such as triglycerides, phospholipids, cholesterol and cholesterol esters, and small amounts of free fatty acids. Lipids are transported in an aqueous environment by associating the less soluble lipids with the more polar ones such as phospholipids and then combining them with cholesterol and proteins to form a hydrophilic lipoprotein complex. In this way, the triglycerides derived from the intestinal absorption of fat or from the liver are transported in the blood. Lipoproteins are often classified on the basis of their hydrated densities and electrophoretic mobilities into four major classes and these are important in clinical diagnosis. These are chylomicrons, very low-density lipoproteins (VLDL), lowdensity lipoproteins (LDL), and high-density lipoproteins (HDL). The HDL to LDL cholesterol ratio represents the artherogenic index and can be calculated.

1) Total cholesterol in serum

MATERIALS Serum Glacial acetic acid Propan-2-ol Cholesterol stock standard (200 mg in 100ml propan-2-ol) Chlesterol working standard (dilute the stock to give 20 mg/100 ml) Stock colour reagent; Ferric chloride solution (2.5g FeCl3.6H2O in orthophosphoric acid) Working colour reagent Dissolve 8 ml of stock in 100 ml of conc. Sulphuric acid with continuous stirring METHODS Mix 0.2 ml serum with 1.8 ml propan-2-ol in centrifuge tube. Shake well. Centrifuge at 2000 rpm for 5 minutes. Remove the supernatant solution. Arrange the tubes as follows: Solution Acetic acid Propan-2-ol Supernatant Colour reagent Blank (ml) 3.6 0.5 2.9 Test 1 (ml) 3.6 0.5 2.9 Test 2 (ml) 3.6 0.5 2.9

Mix the above solutions by swirling gently and leave to stand at room temperature for 20 minutes. Read absorbance at 580 nm. Using the working cholesterol solution, prepare a standard curve for cholesterol as follows. Tube 1 Solution Acetic acid Cholesterol standard Propan-2-ol Colour reagent (ml) 3.6 0.0 0.5 2.9 Tube 2 (ml) 3.6 0.1 0.4 2.9 Tube 3 (ml) 3.6 0.2 0.3 2.9 Tube 4 (ml) 3.6 0.3 0.2 2.9 Tube 5 (ml) 3.6 0.4 0.1 2.9 Tube 6 (ml) 3.6 0.5 0.0 2.9

2) Estimation of HDL-cholesterol in serum

VLDL and LDL fractions are removed by polyanionic precipitation with phosphotugstic acidMg2+ mixture and the supernatant solution that contains the HDL fraction is used for the estimation of HDL-cholesterol.

MATERIALS
Phosphotungstic acid (8.0 g dodeca-Tungstophosphoric acid in water, adjust to pH 7.6 with NaOH and make up to 100 ml with water). Refrigerate. Magnesium chloride solution (Dissolve 9.6 g MgCl2 in water and make up to 100 ml) Just prior to the experiment, mix equal volumes of solutions A and B and use the mixture as the precipitating reagent. PROCEDURE Mix 1.0 ml serum with 1.0 ml of the precipitating reagent. Shake well and leave to stand at room temperature for 15 minutes. Centrifuge at 3500 rpm for 30 minutes. Remove the supernatant and proceed as in determination of total cholesterol.

RESULTS
From the calibration curve, determine the concentration of total cholesterol and HDL-cholesterol in the serum (mg/1000 ml). How do your values compare to the literature values? What is the role of HDL-cholesterol?