2.16: Cholesterol (Total and HDL)

RELATED READING: Chapter 33. See Methods on CD-ROM for Cholesterol.

OBJECTIVES

Upon completion of this exercise, discussion, and appropriate readings,
the student will be able to:

1. Determine serum cholesterol values with 90% accuracy.
2. Separate HDL from other lipoprotein particles.
3. Determine HDL cholesterol values with 90% accuracy.
4. Using triglyceride, HDL—cholesterol, and total cholesterol values, calculate LDL-cholesterol concentrations.

PRINCIPLE

Cholesterol can be measured by a variety of methods. The most common of those currently in use utilizes a series of enzymatic pathways to produce a measurable product. The following series of reactions are widely used for point of care, automated and manual methods.

1. $$\text{Cholesterol - esters} \xrightarrow{\text{Cholesterol esterase}} \text{Cholesterol} + \text{Fatty acids}$$
2. $$\text{Cholesterol} + O_{2} \xrightarrow{\text{Cholesterol oxidase}} \text{Cholest -4 - en -3-one} + H_{2}O_{2}$$
3. $$H_{2}O_{2} + 4 - \text{aminophenazone (or some other dye)} \xrightarrow{\text{Peroxidase}} \text{Oxidized dye}\; (A_{max}, 500nm) + H_{2}O$$

This method can be used to determine both total cholesterol and HDL cholesterol values.

MATERIALS

• 10 x 75 mm test tubes
• Serum Samples
• Cholesterol reagent
• Pipets
• Spectrophotometer
• Controls
• Refrigerated Centrifuge
• Precipitating Reagent

If available a point of care analyzer may replace the cholesterol reagents, and the
spectrophotometer.

CHOLESTEROL ANALYSES PROCEDURE

(This may be replaced with a point of care instrument.)

1. Label 13 x 100 mm test tubes for a total cholesterol standard, Level 2 control, and total cholesterol for each serum sample.
2. Pipet 2.5 mL of cholesterol reagent into each tube.
3. Add 30 \(\mu\)L of sample to appropriate tubes for total cholesterol standard, Level 2 control, and each serum sample total cholesterol tube.
4. Mix well and Incubate at 37°C for 15 minutes.
5. Adjust a spectrophotometer to 500 nm and blank it with distilled water.
6. Following incubation, read the absorbance of each standard, control, and serum sample using appropriate cuvettes. Record your results on the data sheet.
7. Calculate the total cholesterol values for the control and each serum sample using the absorbance proportion method.

HDL CHOLESTEROL SAMPLE PREPARATION

PRINCIPLE

In the presence of Mn²⁺ and heparin, chylomicrons, VLDL, and LDL are precipitated, leaving only HDL in solution. The precipitated materials are sedimented by centrifugation, and the HDLcontaining supernatant can be removed.

PROCEDURE

1. Pipet 1 mL of each control and sample into labeled 10 x 75 mm test tubes.
2. Add 0.1 mL of precipitating reagent (1.06 M MnCl₂ with sodium heparin added) to each tube and mix well using a vortex type mixer.
3. Centrifuge at 2000 g for 30 minutes at 4°C.
4. Carefully remove the clear supernate into labeled 10 x 75 mm test tubes with Pasteur pipets and save it for subsequent analysis.

HDL CHOLESTEROL PROCEDURE

1. Follow steps 1 and 2 of total cholesterol procedure.
2. Add 50 \(\mu\)L of supernate (from precipitation procedure) to HDL control and HDL serum sample tubes, add 50 \(\mu\)L of HDL standard to appropriate tube.
3. Follow steps 4 through 7 of total cholesterol procedure.
4. Multiply the final value (from Step 7) by 1.1 to correct for the dilution made in the precipitation step.

DATA SHEET, EXERCISE #16

NAME: ___________ DATE: ___________

RESULTS

	ABSORBANCE (500 nm)	CHOLESTEROL LEVEL mg/L	CORRECTED HDL-C mg/L
Cholesterol Std
HDL Cholesterol Std			(target value)
Level II Control
HDL Control			(target value)
Sample # (Cholesterol)
Sample # (HDL-C Cholesterol)
Sample # (HDL-C)

CALCULATIONS

Calculate LDL and VLDL values for each serum sample and the controls using the following equations.

\[\text{VLDL cholesterol, mg/L} = \frac{\text{Triglyceride level (mg/L)}}{5}\]

\[\text{LDL cholesterol} = \text{total cholesterol} - \text{(VLDL cholesterol + HDL-C)}\]

Your instructor will provide you with the measured triglyceride level for your sample.

DISCUSSION QUESTIONS

1. What additional lipid calculations can be made that are clinically useful?
2. If chylomicrons are present, what steps are necessary to determine HDL cholesterol?
3. What substance(s) will interfere with cholesterol determinations using this method?
4. If the triglyceride level of your sample was >4000 mg/L, could you calculate the LDL cholesterol?
5. For each of the serum samples tested, briefly discuss the lipid picture illustrated by your results. See Chapter 33 in Kaplan and Pesce
6. Why were two cholesterol standards employed in this exercise?