Lipid Profile Test

Components of a Standard Lipid Profile

LDL-C = Total Cholesterol - HDL-C - (Triglycerides ÷ 5)

• Textbook of Family Medicine 9e, p. 234

Reference Ranges (mg/dL)

• Biochemistry, 8th ed - Lippincott Illustrated Reviews, p. 1419
• UW Lab Medicine lipid panel interpretation (updated March 2026)

Key Facts About Each Component

LDL-C

• Carries ~60-70% of plasma cholesterol
• Direct association with coronary heart disease (CHD) risk
• Primary therapeutic target in dyslipidemia management
• For high-risk patients (prior ASCVD), target is typically < 70 mg/dL; statin therapy is recommended when LDL-C ≥ 190 mg/dL

HDL-C

• Carries ~20-30% of total cholesterol
• Functions in reverse cholesterol transport (removes cholesterol from tissues back to the liver)
• Every 1 mg/dL decrease in HDL raises CAD risk by 2-3%
• Low HDL can be caused by: elevated triglycerides, liver disease, amyloidosis, inborn errors of metabolism

Triglycerides

• Transported primarily in VLDL and chylomicrons
• Marker of TG-rich lipoproteins; causally related to ASCVD
• TG > 500 mg/dL carries very high risk of acute pancreatitis
• Measured in fasting state (postprandial rise is normal and expected)

When and How to Test

• Fasting: Standard test requires a 9-12 hour fast. Failure to fast elevates triglycerides and underestimates LDL-C.
• Non-fasting: Total cholesterol and HDL-C are reliable without fasting. If non-fasting non-HDL-C ≥ 220 mg/dL or TG ≥ 500 mg/dL, a fasting panel is required.
• Timing: Morning specimens are preferred since triglycerides have diurnal variation (lowest in the morning).
• Stable diet required: Dietary changes appear in lipid values within 1-2 weeks, so patients should maintain a stable diet for 3 weeks before testing.
• Avoid testing after: Recent illness, surgery, MI, or stroke - cholesterol drops within 24 hours of MI and stays depressed up to 12 weeks. Wait 2-3 months after major illness.
• Screening interval: Every 4-6 years for adults aged 40-75 without indications for therapy. Hospitalized patients with ACS should have a lipid panel within 24 hours.
• Washington Manual of Medical Therapeutics, p. 97

Limitations of the Friedewald Formula

1. Chylomicrons present (non-fasting sample)
2. Triglycerides > 400 mg/dL
3. Dysbetalipoproteinemia (Type III hyperlipidemia)

Common Causes of Secondary Dyslipidemia

• Hypothyroidism
• Diabetes mellitus
• Nephrotic syndrome
• Obstructive liver disease
• Medications: estrogens, progestins, anabolic steroids, corticosteroids, cyclosporine, retinoids, atypical antipsychotics, antiretrovirals (protease inhibitors)

Clinical Significance

• Risk-based LDL-C targets under the 2018 ACC/AHA guidelines are set per ASCVD risk category, not single cutoffs
• The greater the LDL-C reduction, the greater the cardiovascular risk reduction
• Non-HDL-C is a useful secondary target since it includes VLDL, IDL, and Lp(a) - all atherogenic particles
• Lipoprotein subfractions and particle size testing is not recommended for routine cardiovascular risk assessment in asymptomatic adults (AHA 2010)

Experiment No. 20 - Lipid Profile Tests

AIM

• Total Cholesterol (TC)
• Triglycerides (TG)
• High-Density Lipoprotein Cholesterol (HDL-C)
• Low-Density Lipoprotein Cholesterol (LDL-C) - calculated
• Very Low-Density Lipoprotein Cholesterol (VLDL-C) - calculated

Chemicals and Reagents

• Standard Total Cholesterol calibrator
• Standard Triglyceride calibrator
• Standard HDL calibrator
• Enzymatic reagent kits:
  • CHOD-PAP reagent (Cholesterol Oxidase - Peroxidase) for Total Cholesterol
  • GPO-PAP reagent (Glycerol-3-Phosphate Oxidase - Peroxidase) for Triglycerides
  • HDL reagent (precipitating agent for manual method, or direct HDL reagent for automated assays)
  • Lipase (for some TG methods)
• Distilled water
• Serum sample (fasted sample, preferably 8-11 hours fasting)
• Standards, controls, and blanks as provided by kit manufacturer

Principle

1. Total Cholesterol (TC) - CHOD-PAP Method

1. Cholesterol esters are hydrolyzed by cholesterol esterase to free cholesterol and fatty acids.
2. Cholesterol oxidase then oxidizes cholesterol to cholest-4-en-3-one with production of hydrogen peroxide.
3. In the presence of peroxidase, H₂O₂ reacts with a chromogen to form a colored quinoneimine dye.
4. Absorbance (measured at ~500 nm) is proportional to cholesterol concentration.

2. Triglycerides (TG) - Enzymatic GPO-PAP Method

1. Triglycerides are hydrolyzed by lipase to glycerol and free fatty acids.
2. Glycerol is phosphorylated and subsequently oxidized, producing hydrogen peroxide.
3. H₂O₂ reacts with a chromogen (peroxidase-coupled) to give a colored product.
4. Measured spectrophotometrically; intensity is proportional to TG concentration.

3. HDL-C

• Can be estimated by precipitation method: polyanions + divalent cations precipitate LDL, VLDL, and chylomicrons; cholesterol is measured in the supernatant = HDL-C.
• OR by direct homogeneous assays that selectively block non-HDL particles.

4. LDL-C & VLDL-C - Calculated (Friedewald Formula)

Valid only when TG < 400 mg/dL and patient is fasting.

Procedure (General Laboratory Steps)

1. Prepare reagents, standards, and controls as per manufacturer instructions. Bring to room temperature if required.
2. Calibrate spectrophotometer/autoanalyzer with blank and calibrator(s) at the appropriate wavelength (~500 nm) following CHOD-PAP and GPO-PAP kit instructions.
3. Total Cholesterol:
   • a. Pipette appropriate volume of reagent and sample/standard into cuvette or well.
   • b. Incubate at specified temperature (often 37°C or room temperature) for the stated time.
   • c. Read absorbance against blank and calculate concentration using standard or calibration curve.
4. Triglycerides:
   • a. Mix sample/standards with GPO-PAP reagent as per kit.
   • b. Incubate and read absorbance at specified temperature (often 37°C or room temperature) for stated time.
   • c. Read absorbance against blank and calculate concentration.
5. HDL-C (Precipitation Method):
   • a. To 0.5 mL serum add precipitating reagent (e.g., phosphotungstic acid + magnesium chloride) as per kit.
   • b. Vortex and centrifuge at 3000 rpm for 10 min.
   • c. Carefully collect supernatant (contains HDL) and perform cholesterol assay on supernatant using CHOD-PAP reagent.
   • d. For direct HDL assay, follow the kit protocol for reagent/sample volumes and incubation.
   • e. Run quality control samples with each batch and record results. Repeat any out-of-range control or sample after troubleshooting.
6. Calculate LDL-C using Friedewald formula (when TG < 400 mg/dL).
7. Record results in report format and interpret according to reference ranges.

Calculations (Example)

Note: Friedewald is valid only when TG < 400 mg/dL and patient is fasting. For TG ≥ 400 mg/dL, use direct LDL measurement.

Reference Ranges and Interpretation

Report Format (Sample)

Precautions

• Use non-hemolyzed serum - haemolysis can interfere with some assays.
• Ensure fasting status for triglyceride accuracy (8-11 hrs).
• Follow kit instructions for reagent preparation, incubation times, and wavelength.
• Use proper calibration and run controls with each batch.
• Avoid lipemic samples for some direct assays, or note limitations.
• Always correlate laboratory results with clinical findings and patient history.