●   size and number of lipoprotein particles in the various subclasses

●   other lipoprotein species such as Lp(a)

Both LDL and HDL are heterogeneous lipoprotein classes consisting of lipoprotein particles varying in size, composition, and atherogenicity.

Small and dense LDL (sdLDL) particles, an LDL subclass, are more atherogenic than the larger, more buoyant LDL particles because they are smaller (penetrate easier into the arterial wall), more prone to modification by oxidation, and therefore more rapidly taken up by the scavenger receptors of the macrophages, which then turn into foam cells, a characteristic feature of atherosclerotic lesions. sdLDL is now recognized as a strong risk factor for CHD both in Western and Japanese populations. This observation is becoming more and more evident that it is important to pay attention not only to LDL-C, but also to sdLDL and its significance in the evaluation of a patient's risk for CHD and in the prevention of CHD.

The protective HDL is also a heterogeneous lipoprotein class, the subclasses of which have been ascribed different atheroprotective potentials. Attention is directed toward the larger HDL, particularly the large HDL2 subclass, which appears to be the most protective HDL subclass. HDL2 is actively engaged in reverse cholesterol transport, the process by which cholesterol can be transported away from atherosclerotic lesions to the liver for excretion from the body via the bile. Lp(a) is a distinct genetic form of LDL that contains apo(a) in addition to apo B. Apo(a) has a structural similarity to plasminogen . Lp(a) is a strong risk factor for clinical atherosclerosis. Unlike LDL, Lp(a) does not respond to treatment with diet or statins. However, its concentration is markedly reduced by nicotinic acid.

Several methods are available for the determination of lipoprotein subclasses, and these methods are still mostly used in research or clinical trials. However, with the increasing awareness of the importance of various subclasses in the reverse cholesterol transport of atherosclerosis and as risk factors for CVD, these methods will undoubtedly be used more frequently in clinical practice. This development will be in response to clinicians' demands to obtain more advanced lipid profiles in which lipoprotein subclasses are also determined. Since the groundbreaking work of John Gofman with analytical ultracentrifugation in the 1990s, human plasma lipoproteins have been well established as a very heterogeneous group of proteins or particles, greatly varying in size, density, and composition. The different characteristics of the subclasses have made it possible to separate and quantify these proteins or particles by physicochemical methods, such as electrophoresis and ultracentrifugation.

Polyacrylamide gradient gel electrophoresis has been used for 25 years as a practical method for the separation and analysis of lipoproteins of different sizes, such as HDL and LDL particles that vary in diameter (7'12 nm and 21'28 nm, respectively). Polyacrylamide gels have pores of decreasing size in the direction of the electrophoretic mobility, preventing lipoproteins of a certain size from moving further on in the gel in the electric field.

To analyze HDL subfractions, a gradient in the range of 4%'30% polyacrylamide concentration is used. With this technique, HDL is separated into five distinct subclasses.

To determine LDL subclasses, 2%'16% polyacrylamide gel is generally applied. Gradient gel electrophoresis of LDL has identified seven subclasses. There is, however, no standard nomenclature for these subclasses. At present, the most useful terms for LDL subclasses are the two phenotypes of LDL described by Austin. Phenotype A contains large LDL, while phenotype B consists of sdLDL. The determination of sdLDL is the most significant measurement of LDL subclasses for the evaluation of a person's risk for CHD.

In ultracentrifugation, the density of lipoproteins is due to their lipid content; that is, the lipid/protein ratio determines the separation and identification of the various subclasses. Analytical ultracentrifugation is the standard for a quantification of lipoprotein classes, but it is too laborious and expensive for routine use. The recently developed SVAP method is a single vertical spin density gradient ultracentrifugation combined with cholesterol analysis of the separated lipoprotein classes. The advantages of SVAP are the use of small plasma samples, short centrifugation time, and determination of an advanced quantitative lipoprotein subclass spectrum.

The determination of plasma lipoprotein subclasses by nuclear magnetic resonance (NMR) spectroscopy started in the 1990s. A plasma sample is placed in the NMR analyzer, and a spectrum is recorded within a minute. By computer analysis of the spectrum, the amounts of large, medium, and small particle subclasses of VLDL, LDL, and HDL are estimated.

Studies of the subclasses of both LDL and HDL have revealed important clinical and metabolic differences between the subclasses. Several prospective epidemiological studies have shown that sdLDL is a very important risk factor for the development of atherosclerotic CVD. According to some studies, sdLDL is an even better indicator of CHD risk than LDL-C. For example, in a prospective Quebec study comprising more than 2000 men free of CHD, the subjects with high levels of sdLDL but normal concentrations of LDL-C had a fourfold increased risk for new events of CVD. High levels of sdLDL are common in hypertriglyceridemia, which is often present in type 2 diabetes and metabolic syndrome. The concentration of sdLDL can be reduced by lifestyle improvements and by pharmacological treatment with statins, nicotinic acid, and fibrates.

Both progression and severity of coronary atherosclerosis are inversely proportional to the large HDL2b subclass, which appears to have a more protective effect against atherosclerosis than other HDL subclasses. There is a pronounced difference in the distribution of HDL subclasses between men and women. Particularly noteworthy is that men have lower levels of protective HDL2, which partly explains the higher incidence of CHD in males than in females at a younger age. Fibrates and statins increase HDL-C by about 5%'20%, and nicotinic acid increases HDL-C by 20%'40%. The pharmacological treatment with statins, nicotinic acid, and fibrates also decreases the HDL2 subclass by about 100% and the HDL3 subclass by 20%, which further illustrates the importance of evaluating the subclasses of the major lipoprotein classes by advanced lipid testing.

Given the prevalence of CHD and CVD, tests to obtain detailed profiles of lipid subclasses are a necessity. Advanced lipid testing, though not recommended for routine screening, is important in identifying individuals at high risk of CHD or CVD, especially those with a family history of premature CHD, individuals with an intermediate risk for CHD, and postmenopausal women. These tests will facilitate early identification, more effective treatments, and an overall reduction in the occurrence of atherosclerotic CVD.