Laboratory Methods for Lipid /Lipoprotein Analysis

Cholesterol Assays

Enzymatic method is assay of choice

Commonly used 3 step procedure

Enzyme reagent + serum is incubated under

Controlled conditions and color development is measured via spectrophotometry

Interference from other colored compounds

(1)bilirubin

(2) ascorbic aid

(3) hemoglobin

Triglycerides

Many different procedures used but ALL start the same

Small amounts of circulating free glycerol present in serum

Will overestimate levels if this endogenous glycerol isn’t corrected for

Triglyceride Reference Range

Desirable: Less than 150 mg/dL

Borderline high: 150 to 199 mg/dL

High: 200 to 499 mg/dL

Very high: Greater than 500 mg/dL

HDL-Cholesterol Assays

Early methods use pre-treatment centrifugation and precipitation methods to remove all non-HDL lipoproteins

After pre-treatment, supernatant is analyzed for HDL

non-HDL=

LDL, IDL, VLDL,
chylomicrons & remnants

Non-HDL Plasma value =

Total cholesterol value – HDL value

HDL-CHOLESTEROL *DIRECT METHOD

AKA HOMOGENOUS METHOD

No physical separation or pre-treatment of HDL from non-HDL

Mask all the other lipoprotein molecules (non-HDL)

Antibodies
Polymers

Enzymes

Use with caution when extremely high plasma triglyceride levels

LDL-Cholesterol

DL is the key metric/measure in NCEP guidelines for assessing heart disease ( increased values = high risk )

Until recently LDL-C was almost always measured indirectly by using the Friedwald Equation

LDL Reference method known

As β-Quantification

Requires ultracentrifugation overnight

Direct (homogenous) LDL cholesterol method

All detergent based to facilitate selective reactivity with reagent enzymes

Same idea as direct HDL just mask everything except LDL in this case

Risk Factor Assessment of CVD

1) Run a lipid panel: Total Cholesterol, HDL, LDL & Triglycerides

Measurement of Apolipoproteins

Immunoturbidometric/nephelometric assays used

ELISA if available

Genotyping for ApoE is often performed----associated with increased cholesterol and risk for Alzheimer’s disease

Measuring lipoprotein(a)

Carry a small fraction of total cholesterol but very pro-atherogenic

Measured in individuals with normal lipid panel but strong family history of CVD

Elevated levels confer increased risk for premature CHD, atherosclerosis and stroke

Immunoturbidometric/ nephelometric & ELISA assays used

Additional Lipid Metrics/Risk Factors

1. LDL particle size and LDL particle number
2. Homocysteine?
3.High-sensitivity C-Reactive Protein (hs-CRP)
4. Lp-PLA2: a marker of inflammation

High-sensitivity C-Reactive Protein (hs-CRP)

Increased serum CRP associated with risk of future CVD events

Early positive APP (very dramatic increase with inflammation)