2.16: Cholesterol (Total and HDL)
- Page ID
- 120234
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:
- Determine serum cholesterol values with 90% accuracy.
- Separate HDL from other lipoprotein particles.
- Determine HDL cholesterol values with 90% accuracy.
- 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.
- $$\text{Cholesterol - esters} \xrightarrow{\text{Cholesterol esterase}} \text{Cholesterol} + \text{Fatty acids}$$
- $$\text{Cholesterol} + O_{2} \xrightarrow{\text{Cholesterol oxidase}} \text{Cholest -4 - en -3-one} + H_{2}O_{2}$$
- $$H_{2}O_{2} + 4 - \text{aminophenazone (or some other dye)} \xrightarrow{\text{Peroxidase}} \text{Oxidized dye}\; (A_{max}, 500nm) + H_{2}O$$
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.)
- Label 13 x 100 mm test tubes for a total cholesterol standard, Level 2 control, and total cholesterol for each serum sample.
- Pipet 2.5 mL of cholesterol reagent into each tube.
- Add 30 \(\mu\)L of sample to appropriate tubes for total cholesterol standard, Level 2 control, and each serum sample total cholesterol tube.
- Mix well and Incubate at 37°C for 15 minutes.
- Adjust a spectrophotometer to 500 nm and blank it with distilled water.
- Following incubation, read the absorbance of each standard, control, and serum sample using appropriate cuvettes. Record your results on the data sheet.
- 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
- Pipet 1 mL of each control and sample into labeled 10 x 75 mm test tubes.
- 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.
- Centrifuge at 2000 g for 30 minutes at 4°C.
- 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
- Follow steps 1 and 2 of total cholesterol procedure.
- 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.
- Follow steps 4 through 7 of total cholesterol procedure.
- 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
- What additional lipid calculations can be made that are clinically useful?
- If chylomicrons are present, what steps are necessary to determine HDL cholesterol?
- What substance(s) will interfere with cholesterol determinations using this method?
- If the triglyceride level of your sample was >4000 mg/L, could you calculate the LDL cholesterol?
- For each of the serum samples tested, briefly discuss the lipid picture illustrated by your results. See Chapter 33 in Kaplan and Pesce
- Why were two cholesterol standards employed in this exercise?