Risk of cardiovascular disease is assessed, in part, by laboratory measurements of the lipoproteins, VLDL, LDL and HDL. ß quantification uses ultracentrifugation to partially separate lipoprotein classes and is the basis for the reference methods for LDL- and HDL- cholesterol as practised by the CDC. This ‘gold standard’ method has been used to establish the concentrations of the major lipoprotein classes in most of the epidaemiological and clinical trials that have been used as guidelines for risk assessment.
The most useful aspect of ß quantification is the efficient removal of triglyceride-rich VLDL which often interferes with the measurement of LDL- and HDL- cholesterol by other methods. Estimation of LDL-cholesterol by the Friedwald equation is invalid for non-fasting samples containing chylomicrons and when triglyceride concentrations exceed 4.5 mmol/L. It is used in the diagnosis of type III hyperlipidaemia when the ratio of VLDL-C to total triglyceride exceeds 0.65.
It is important to note that the LDL fraction will also contain IDL and Lp(a).
Chylomicrons are removed by preliminary low speed centrifugation. Plasma or serum is centrifuged overnight at d=1.006g/ml to spin up VLDL. The apolipoprotein B containing lipoproteins in the bottom fraction (mainly LDL) are precipitated using heparin and manganese chloride, leaving HDL in solution.
LDL cholesterol is calculated by subtracting VLDL-C and HDL-C from total cholesterol. Alternatively, multiple fractions can be isolated by gradient ultracentrifugation.
Triglyceride <2.3 mmol/L
Cholesterol <5.0 mmol/L
LDL-C <3.0 mmo/L
HDL-C males >1.0 mmol/l
HDL-C females >1.2 mmol/l
Fasting (12-14h) EDTA plasma or serum, minimum volume 2ml.
Stable for one week at 4°C and one year at -70°C.
Transport – first class post.
Age, sex, NHS/Hospital No.
Lipid Research Clinics Manual of Laboratory Operations – DHEW Publication no. (NIH) 75 -628, 1974.