Advanced Lipoprotein Testing: Strengths and Limitations


Standard lipid testing, represented by total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides, is a well-established platform for cardiovascular disease (CVD) risk prediction and management. Despite the proven clinical utility of standard lipid testing for risk management, a significant burden of CVD events remains unaddressed in primary and secondary prevention populations. Standard lipids are carried within lipoprotein particles that are heterogeneous in size, density, charge, core lipid composition, specific apolipoproteins, and function.1 A variety of lipoprotein assays have been developed that subfractionate lipoprotein particles according to properties such as size, density, or charge. These advanced lipoprotein testing (ALT) assays have been proposed for improving assessment of CVD risk and guiding lipid modifying therapies.

Despite the large amount of information derived from ALT assays, it is unclear how this should be contextualized in relation to standard lipids (e.g. complementing standard lipids or substituting for them). New biomarkers have to meet certain requirements prior to clinical usage, including standardization and comparability, accessibility, conveying new information without data overload, having clear indications, improving clinical management and patient outcomes, and lastly demonstrating cost-effectiveness. As lipoproteins metabolic pathways are highly interconnected, one of the main challenges of ALT is to find effective and applicable situations for ALT testing at a reasonable cost. The clinical consequences of a new test as compared with an older standard are best understood through situations where there is disagreement (discordance) between the two tests.2, 3 As this is a brief review, we will focus here on the most promising ALT tests for potential clinical application.

Alternative LDL Measures

Apolipoprotein B (ApoB) and LDL Particle Number (LDL-P)

Very-low-density lipoprotein, intermediate-density lipoprotein, and LDL particles (VLDL-P, IDL-P, and LDL-P, respectively) are atherogenic lipoproteins in an interrelated metabolic chain. Each of these particles carries one apolipoprotein-B100 (apoB) molecule, which makes them apoB-containing lipoproteins. VLDL-P secretion by the liver is the starting point for the production of the other particles downstream. Under sequential action of lipases and transferases, VLDL-P diminishes its triglyceride content, reduces its size, and increases its density, resulting in smaller VLDL-P, IDL-P, and LDL-P particles.

Among these particles, LDL-P has more robust and independent association with atherosclerosis development and CVD events. Furthermore, smaller LDL particle size appears to be positively associated with CVD, not necessarily because small LDL are inherently more atherogenic than large ones, but because individuals with predominantly small LDL size also have more LDL-P.4 As the amount of cholesterol carried by each LDL-P can vary more than two-fold between individuals,5, 6 estimating LDL-P concentration by LDL-C may be misleading in some circumstances. This is particularly evident in patients with diabetes, metabolic syndrome, hypertriglyceridemia and low HDL-C,7-10 as their LDL-P carry less cholesterol and more triglycerides, and hence LDL-C may underestimate LDL-P.

Currently, LDL-P can be quantified directly by nuclear magnetic resonance spectroscopy (NMR) or by differential ion mobility analysis.11 ApoB is mostly (~90%) carried within LDL-P in the fasting state;12 hence, apoB concentration is also a good estimator of LDL-P or total atherogenic particle concentration. On the other hand, non-HDL-C (calculated as total cholesterol minus HDL-C), which reflects the cholesterol in all the apoB-containing particles, is readily accessible from standard lipid testing, and is generally a better risk predictor than LDL-C.13, 14 Some studies have found better risk assessment for apoB versus LDL-C or non-HDL-C,15 but other studies found similar risk prediction for apoB and non-HDL-c.16 Similarly, LDL-P may provide better risk prediction than LDL-C and non-HDL-C,17 but this type of analysis does not address the overlapping information of the tests, which is better assessed by the net reclassification index or statistical tests of discrimination.

In general populations, the Emerging Risk Factors Collaboration found only slight (<1%) reclassification improvements with the addition of apoB, apoA-I, or lipoprotein(a) to risk scores containing conventional risk factors,18 similar to prior results with apoB or LDL-P in a population of healthy women.19 However, among the subgroup of individuals in whom the tests are discordant with LDL-C, CVD risk discrimination may be better with non-HDL-C, apoB, or LDL-P20, 21 (Figure1).


Lipoprotein(a) is a modified form of LDL whose apoB molecule forms a disulfide bridge with a large glycoprotein called apo(a). Lipoprotein(a) is a well-established and probably causal risk factor for CVD, independent of LDL-C or other risk factors, although it carries a modest incremental magnitude of risk.22 Some have suggested more aggressive LDL-c lowering when lipoprotein(a) is high, and statins are efficacious in reducing overall CVD risk when lipoprotein(a) is high.23

Are ALT Measures Useful for Detecting or Managing "Residual Risk"?

Figure 1: Alternative LDL Measures
Figure: Alternative LDL Measures
The risk among statin-treated individuals remains high and has been termed "residual risk" (Figure 1). In terms of relative risk, approximately 70-80% of the statin-treated patients who otherwise would have had CVD events still experience CVD events. In terms of absolute risk, results from a meta-analysis of statin trials calculated that the rate of a major CVD event occurring during five years of follow-up among statin-treated patients in randomized clinical trials was 21.7% (one in five) for individuals with prior CVD and 9.5% (one in 10) for individuals with no prior CVD.24 Residual risk is associated with on-treatment levels of LDL-C, non-HDL-C, apoB, lipoprotein(a), and the inflammatory biomarker high sensitivity C-reactive protein (hsCRP).23, 25, 26 In recent meta-analysis, the strength of association with CVD events was slightly greater for on-treatment non-HDL-C as compared with on-treatment apoB or LDL-C.27

Guideline Recommendations

The Canadian Cardiovascular Society and American Association of Clinical Endocrinologists recommend measuring apoB as an alternative to LDL-c for initiating statin therapy or as a goal of therapy in patients with higher risk.28, 29 The National Lipid Association suggests apoB, LDL-P, or lipoprotein(a) could be useful for clinical risk assessment or for statin treatment decisions in patients with intermediate risk for CVD, or who have coronary heart disease (CHD) or its equivalent, family history of early CHD, or recurrent events.30 On the contrary, the 2013 American College of Cardiology/American Heart Association (ACC/AHA) guidelines do not issue any recommendation for apoB, LDL-P, or lipoprotein(a) and cite the lack of randomized trial evidence for these measures as well as non-HDL-C as goals of therapy.31 The European Atherosclerosis Society recommended that lipoprotein(a) measurement should be considered in individuals with intermediate or high CVD risk.32 Finally, the Centers for Disease Control and Prevention include standard lipids, apoB, and apoA-I, but do not include lipoprotein(a) in its Lipid Standardization Program.33

Despite recommendations from some expert groups, the downstream consequences of a "real world" risk assessment and management based on apoB and LDL-P beyond LDL-c are uncertain. Therefore, apoB, LDL-P, and lipoprotein(a) are promising ALT biomarkers, but they are not currently recommended as routine tools for CVD risk assessment and management until specific indications, reference goals, and related cost- effectiveness issues have been addressed.

Alternative HDL Measures

While HDL-c is a strong inverse indicator of CVD risk, this has not yet translated into clinical benefit. Given the extreme heterogeneity of HDL structure and function, measuring only the cholesterol content of HDL (HDL-C) will at best only partially reflect the potential role of HDL in CVD risk assessment and therapeutic drug development. This has led to interest in developing HDL metrics that may better indicate the atheroprotective functions of HDL. Proposed measurements include apoA-I, HDL particle number (HDL-P), average size, subclasses, and functional assays.34

Apolipoprotein A-I (ApoA-I)

ApoA-I constitutes roughly 65% of the HDL protein mass and is measured by immunonephelometry or immunoturbidimetry assays. With the exception of a few studies such as the case-control AMORIS study,35 most studies have generally found apoA-I not to be associated with CVD risk after accounting for HDL-C.36-39 When used in conjunction with apoB, several studies support the apoB/apoA-I ratio to be a better predictor of CVD risk when compared to individual parameters of standard lipid tests.40 Nonetheless, the value of apo-B/apoA-I ratio in predicting CVD risk is comparable to that of total cholesterol/HDL-C ratio,19, 41 and did not substantially increase risk classification or discrimination beyond total cholesterol and HDL-C.18, 41

HDL Particle Number (HDL-P)

Of the alternate HDL metrics, the number of HDL particles (HDL-P) has potential utility in CVD prevention. At present it can be measured by only nuclear magnetic resonance (NMR) spectroscopy and ion mobility analysis. Most studies, except the Malmo Cancer and Diet Study,42 have been based on the NMR method. Of the nine studies to-date comparing HDL-P with HDL-C, all 42-49 but two19, 48 found HDL-P to be comparable to or better than HDL-C in predicting CVD. Data from the Multi-Ethnic Study of Atherosclerosis (MESA) and the placebo arm of the Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) show that HDL-C is no longer predictive of CVD after adjusting for HDL-P, while HDL-P remained inversely associated with CVD after adjusting for HDL-C.44, 46

Similar findings were also reported by investigators from the European Prospective Investigation into Cancer and Nutrition-Norfolk study.45 HDL-P may also be of potential value in targeting residual CVD particularly for primary prevention as indicated by data from the rosuvastatin arm of JUPITER, where HDL-P, but not HDL-C, was inversely predictive of CVD.44 However, in the secondary prevention Heart Protection Study, both HDL-P and HDL-C had similar magnitude of association with CHD in the simvastatin and placebo arms.47

HDL Size and Subclasses

HDL subclasses have also been proposed for risk stratification on the premise that the different subclasses are functionally heterogeneous, and a host of ALT subfractionation methods quantify HDL size and/or size subclasses based on distinct physical or chemical HDL subclass properties.34 Recently, the HDL3-C subfraction assessed by the vertical auto profile (VAP) method shows promise for improved risk stratification.50

A recent consensus statement proposed a new unifying nomenclature for HDL size subclasses that harmonizes HDL size data obtained from the various fractionating methods, thus enhancing the comparability of future studies. This nomenclature groups HDL particles based on their size and density into five distinct subclasses (very large, large, medium, small, and very small).34 These five subclasses appear to be differentially associated with CHD risk.51 Finally, the potential clinical relevance of HDL size to cardiovascular risk may depend on the mechanism by which HDL size is modified.


Current limitations of ALT include the lack of: 1) standardization and comparability of information provided by the various tests; 2) accessibility with particular reference to cost and availability of expertise; and 3) evidence and consensus regarding the metrics of ALT that offer proven clinical benefits; this latter point inevitably precludes demonstration of cost effectiveness over standard lipid tests.11


ALT assays have the potential to expand the utility of lipoprotein-targeted initiatives for CVD prevention, particularly within the context of discordance with LDL-C and residual risk after statin therapy (Figure 1). However, current prevention guidelines remain benchmarked against standard lipid tests owing to lack of Grade A level of evidence for ALT. Future studies should assess the range of potential for ALT in CVD prevention and management in addition to addressing the current limitations of ALT for clinical practice.


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Keywords: Apolipoproteins, Cardiovascular Diseases, Cholesterol, Cholesterol, HDL, Cholesterol, LDL, Lipids, Lipoproteins, Lipoproteins, LDL, Lipoproteins, HDL, Secondary Prevention

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