HDL Cholesterol

Introduction

1. HDL cholesterol represents a fraction of total serum cholesterol carried by HDL particles.
2. HDL particles are small, dense, and protein-rich lipoproteins that facilitate cholesterol efflux from cells.
3. They are anti-atherogenic due to their role in cholesterol removal and their anti-inflammatory and antioxidant properties.
4. Determining HDL cholesterol is a standard part of lipid panel testing, including total cholesterol, LDL cholesterol, triglycerides, and HDL cholesterol.
5. Measuring HDL cholesterol is crucial for assessing cardiovascular risk, as reduced HDL levels are an independent risk factor for atherosclerosis and coronary artery disease.
6. This document provides a comprehensive overview of the determination of HDL cholesterol, including methods, principles, sample requirements, reagents, procedures, calculations, normal ranges, and clinical significance.

 

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Methods

(PTA, Mg++/CHOD-PAP)

Principles

1. Precipitation Methods:
   • Non-HDL lipoproteins (LDL, VLDL, chylomicrons) are precipitated from serum using reagents such as heparin-manganese chloride, phosphotungstate-magnesium, or dextran sulfate.
   • The supernatant, containing HDL cholesterol, is analyzed enzymatically using the cholesterol oxidase-peroxidase method.

Cholesterol Esters + Cholesterol Esterase → Free Cholesterol + Fatty Acids

Free Cholesterol + O2 →        Cholesterol Oxidase         Cholest-4-en-3-one+H₂O₂

H₂O₂ + Chromogen →                       Peroxidase                  Colored Compound

1. Direct HDL-C Assays:
   • Utilize specific enzymes or detergents to selectively isolate HDL cholesterol without precipitating other lipoproteins.

 

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Sample Collection and Handling

1. Sample Type:
   • Serum or plasma (heparin or EDTA-treated) can be used.
2. Patient Preparation:
   • A 12-hour fasting sample is preferred to minimize interference from triglycerides.
3. Storage:
   • Samples can be stored at 2–8°C for up to 7 days or frozen at -20°C for long-term storage.

 

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Reagents

1. HDL-Cholesterol (Enzymes-Chromogen Buffer)   
2. HDL-Cholesterol (Diluent) 
3. HDL – Cholesterol (Precipitating Reagent) Ready for use 
4. HDL – Cholesterol Standard. (50 mg/dl)      

 

Procedure

	Test	Standard	Blank
Reagents, ml	1 ml	1 ml	1 ml
Standard, ml	–	0.05 ml	–
Sample, ml	0.05 ml	–	–
Distilled water, ml	–	–	0.05 ml

 

Mix well, incubate for ten minutes at 37oC, and read the absorbance of test and standard against reagent blank at 505nm (500-540 Green filter).

Calculation

HDL cholesterol concentration is calculated using the following formula:

HDL C (mg/dL) = Absorbance of Sample − Absorbance of Blank/Absorbance of Standard − Absorbance of Blank × Concentration of Standard (mg/dl)

 

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Normal Range

The reference range for HDL cholesterol is:

• Men: 40–60 mg/dL (≥40 mg/dL is considered protective)
• Women: 50–60 mg/dL (≥50 mg/dL is considered protective)
• Optimal HDL-C: >60 mg/dL (associated with reduced cardiovascular risk)
• Low HDL-C: <40 mg/dL (associated with increased cardiovascular risk)

 

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Clinical Significance

1. Low HDL Cholesterol:
   • Associated with increased risk of atherosclerosis, coronary artery disease, and stroke.
   • Commonly observed in obesity, sedentary lifestyle, diabetes mellitus, metabolic syndrome, and smoking.
2. High HDL Cholesterol:
   • Generally protective against cardiovascular disease.
   • Exceptionally high levels (>100 mg/dL) may indicate rare conditions such as familial hyperalphalipoproteinemia or other metabolic disorders.
3. Risk Assessment:
   • HDL cholesterol is an integral component of the lipid panel used for calculating the atherogenic index and cardiovascular risk.
   • Low HDL-C levels are included as a criterion for diagnosing metabolic syndrome.