November 2007: Volume 33, Number 11
The Changing Testing Paradigm
By Alan T. Remaley, MD, PhD, and G. Russell Warnick, MS, MBA
For more than 30 years, clinical laboratories have routinely measured high-density lipoproteins (HDLs), one of the major classes of lipoprotein particles, in order to assess patients’ risk of coronary heart disease (CHD) (1). The concentration of cholesterol carried on HDL particles (HDL-C) has been the main metric for this assessment, originating from early studies that first showed the link between cholesterol and atherosclerosis. Today, it is commonly accepted that low-density lipoproteins (LDLs) are “bad” in terms of promoting CHD and that some forms are protective or “good”, such as HDL (2). Although it took several decades for this approach to become accepted in routine clinical laboratory practice, measuring the cholesterol content of these major classes of lipoproteins has significantly improved CHD risk prediction.
But the significance of cholesterol in the development of CHD is now known to be even more complex and intriguing. The major lipoprotein classes are not homogeneous entities, but rather heterogeneous and polydisperse populations of particles with varying composition, physical characteristics, and pathophysiologic significance for the development of atherosclerosis. Researchers have found that some LDL subfractions, such as small dense LDLs or oxidized LDLs, are especially pro-atherogenic, suggesting that measurement of the underlying subfractions of the major classes of lipoproteins might improve prediction of CHD. More recently, it has been discovered that not all HDL particles are equivalent in terms of their anti-atherogenic properties and that sometimes even the “good” cholesterol can turn bad.
In this two-part review, we discuss the latest HDL research findings and their implications for the development of new diagnostic tests. In this first part, we describe the atherogenic properties of HDL, the current understanding of the composition of HDL particles, and the status of lab measurements of HDL. Part two of this review, to be published next month, will examine the various lab methods for measuring the subclasses of HDL and the current controversy over the use of these measurements to predict CHD.
The Push for Better Drug Therapies
The quest for new drugs to increase patients’ HDL levels has been a major driver of HDL research. Presently, statins, which largely work by lowering LDL-C, are the main therapeutic approach for preventing CHD. There is growing awareness, however, that lowering LDL merely slows the progression of atherosclerosis and only decreases CHD events by approximately a third. Emerging evidence suggests that combination therapies, such as adding niacin with a statin to simultaneously lower LDL and raise HDL, may be much more effective in achieving plaque regression and substantially decreasing event rates (3). Because the current formulations of niacin can cause unpleasant side effects, pharmaceutical manufacturers have a major interest in developing more effective and better tolerated HDL-raising drugs.
Due to these drug development efforts, as well as new research findings, researchers now question whether the traditional measurement of HDL in terms of cholesterol content is ideal or even sufficient for predicting CHD risk. For example, data from a recent phase III clinical trial showed that the cholesteryl-ester transfer protein (CETP) inhibitor Torcetrapib (Pfizer, New York, N.Y.) raised HDL-C approximately 50% by increasing the amount of large-sized HDL; however, a significant increase in cardiovascular deaths among participants caused the manufacturer to stop the trial. The reason for this unexpected finding is still not known and may be idiosyncratic to this particular drug or may represent an off-target effect. Nevertheless, the results highlight the fact that HDL represents a diverse population of discreet particle types that are not all equally atheroprotective. In fact, other recent studies have revealed that some forms of HDL may in fact be pro-atherogenic.
A Look at HDL Structure and Metabolism
As its name implies, HDL is composed of both proteins and lipids. Compared to other lipoproteins, HDL contains more protein and is denser. By weight, it is approximately 50% lipid and 50% protein, with a density ranging from 1.21 g/mL –1.063 g/mL. Not surprisingly, measures of both lipid and protein have been used for assessing patients’ HDL levels.
The main lipids on HDL are cholesterol, cholesteryl esters, phospholipids and triglycerides. The more amphipathic lipids with polar groups, such as unesterified cholesterol and phospholipids, form the outer lipid monolayer on HDL; whereas the more hydrophobic neutral lipids, cholesteryl esters and triglycerides, are in the hydrophobic core of the spherical forms of HDL. It is important to note there are other types of lipids on HDL that are not chemically defined entities; instead, they represent classes of lipids that exist in a wide variety of molecular forms. The potential diagnostic value of measuring these other specific lipids has not been thoroughly investigated. Recently developed mass spectrometry methods for performing global assessments of lipids in HDL particles, the so-called “HDL lipidome,” should help better define its structure in the near future.
In addition to the major lipids, HDL and all the other lipoproteins also contain a wide variety of low abundance lipids, which despite their low concentration may have a disproportionate effect on the function of HDL. For example, HDL is the main carrier of sphingosine-1-phosphate in plasma, a very potent bioactive lipid that may mediate many of the salutary effects of HDL on cell function (5). In fact, treating mice with a stable spingosine-1-phosphate analogue has been shown to reduce atherosclerosis, but whether measuring the sphingosine-1-phosphate content of HDL is useful for predicting its anti-atherogenic capacity has not been carefully investigated.
Figure 1 (below) depicts the various known mechanisms whereby HDL protects against the development of atherosclerosis. Researchers believe that the reverse cholesterol transport pathway is the main atheroprotective mechanism of HDL (1). Other mechanisms have been proposed, and this continues to be an active area of investigation.
The Reverse Cholesterol Transport Pathway and Other Sites of HDL Action on the Pathogenesis of Atherosclerosis
This diagram depicts the various known mechanisms whereby HDL protects against the development of atherosclerosis. The so-called “reverse cholesterol transport pathway” is believed to be the main protective effect of HDL (1). This pathway results in the transfer of excess cholesterol from peripheral cells—such as macrophages in atherosclerotic plaques, —to HDL, which eventually transfers its cholesterol to the liver for excretion. HDL is made in the liver and intestine where apoA-I, the main protein of HDL, is produced. Nascent HDL is formed when lipid-poor apoA-I, such as the small discoidal-shaped particle with pre-beta mobility on agarose gels, is secreted and interacts with the ABCA1 transporter on the surface of hepatocytes and enterocytes (4). This interaction results in the transfer of phospholipids and a limited amount of cholesterol to apoA-I to form a more lipid-rich, intermediate size form of HDL. Such HDL particles can then remove additional cholesterol from peripheral cells by ABCA1 and other transporters, such as the ABCG1 transporter, which primarily transfers cholesterol to only the larger, lipid-rich forms of HDL. Unesterified cholesterol on the particle surface is then moved to the hydrophobic core of HDL after its conversion to cholesteryl esters by lecithin:cholesterol acyltransferase (LCAT), creating a spherical-shaped HDL particle. HDL then returns cholesteryl esters to the liver via the SR-BI receptor, where apoA-I is recycled and returned to the circulation. Cholesterol is off-loaded and either converted into a bile salt by hepatocytes or is directly excreted into the bile. A significant fraction of cholesterol on HDL is also delivered to the liver by way of the LDL-receptor, after first being transferred from HDL to LDL by CETP.
Lab Measurements of Cholesterol
Cholesterol is the only lipid on HDL that has routinely been used as a measure of HDL concentration. Older precipitation-based assays for HDL-C have largely been replaced by fully automated direct or homogenous HDL-C tests. Several commercially available direct HDL tests work by either shielding cholesterol on the non-HDL fractions from reacting with the cholesteryl esterase and cholesterol oxidase enzymes used for measuring cholesterol, or by enzymatically consuming non-HDL cholesterol before the final cholesterol signal is generated from HDL-C. These tests work relatively well and show good correlation on normal samples with reference methods. But as discussed below, the complexity of the different forms of HDL raises the possibility that all these methods may not equally measure cholesterol in the minor subfractions of HDL that may be particularly pro- or anti-atherogenic, particularly in dyslipidemic samples. Interestingly, most existing epidemiologic data showing an inverse association between HDL-C and CHD is based on the older precipitation-based methods; the direct assays have not been thoroughly tested in large studies using clinical endpoints.
Another fundamental concern about all HDL-C assays that may limit their CHD- predictive ability relates to the HDL’s main anti-atherogenic mechanism—the reverse cholesterol transport pathway. If removing excess cholesterol from peripheral cells and delivering it to the liver for excretion is central to preventing the development of CHD, then it is the overall flux of cholesterol by this pathway that is relevant and not necessarily the pool size of cholesterol on HDL. Pool size is determined by both the input and egress of cholesterol from HDL, and under certain pathophysiologic circumstances, there may be a dissociation between HDL-C levels and the overall net flux of cholesterol by the reverse cholesterol transport pathway.
Therefore, one possible explanation for the results of the CETP inhibitor trial is that despite the observed increase in HDL-C achieved by preventing the transfer of cholesteryl esters from HDL, atherosclerosis may not decrease because the second pathway for returning cholesterol to the liver by the LDL-receptor is blocked by the drug. Another possible explanation is that the resulting larger apoE-assoicated HDL particles can be removed by other non-specific receptors that do not recycle the apoA-I (Figure 1, above).
The HDL Proteome: Protein Composition of HDL
Like the HDL lipidome, work on fully characterizing proteins of the HDL proteome has only just begun. ApoA-I, the main protein on HDL, comprises approximately 75% of HDL’s protein content, making it a natural choice as a measure of HDL. Besides apoA-I, however, HDL contains many other apolipoproteins, such as apoA-II, apoA-IV, apoA-V, apoE, apoCs, apoJ, and apoM , as well as new apolipoproteins that are still being discovered. The apolipoproteins on HDL all share a common structural motif, namely an amphipathic helix that enables them to bind to the lipids on HDL. The main role of these apolipoproteins is to stabilize the structure of HDL, but they also can modulate the activity of the various lipid-modifying enzymes that act on HDL and/or serve as ligands for cellular receptors. In contrast to apoA-I, some of these other apolipoproteins, such as apoA-II and apoE, may actually inhibit the anti-atherogenic function of HDL. In addition, there are a large number of proteins that show weak or only transient interaction with HDL. In one proteomic study of HDL by mass spectroscopy, 48 different proteins were found to be associated with HDL, and approximately half of these proteins were found to be more enriched in HDL relative to apoA-I in subjects with CHD versus controls (6). Researchers have also found proteins related to inflammation, acute phase response, anti-oxidation, proteolysis, and complement activation on HDL, which could potentially alter the ability of HDL to act as an anti-atherogenic agent.
Lab Measurement of HDL Proteins
Currently, the only protein constituent that labs routinely use to measure HDL is apoA-I. In fact, this test is the only widely available alternative to the HDL-C test. Similar to the situation with apoB versus LDL-C, there is growing evidence that apoA-I may be superior to HDL-C in predicting CHD risk. Several large epidemiologic trials have shown that apoA-I, and especially the apoA-I/apoB ratio, are superior to HDL-C and LDL-C in predicting CHD risk (7). Early assays for apolipoproteins were not as robust or as well standardized as the lipoprotein cholesterol assays, but these problems have largely been resolved in the newer generations tests. In fact, apoA-I and apoB have an inherent advantage in that they are chemically well-defined molecular entities, whereas HDL and LDL represent a diverse collection of particles, as discussed below. Simply measuring cholesterol in these major classes of lipoproteins may miss underlying biologic complexity and complicate method standardization. Unlike LDL and the other apoB-containing lipoproteins, which always have only one apoB molecule per lipoprotein particle, HDL contains a variable number of apoA-I molecules, which may limit its CHD-predictive ability.
Another important development in apoA-I assays has evolved from recognition of the importance of HDL as a potent anti-oxidant molecule. When apoA-I on HDL is oxidatively damaged, it loses its ability to promote cholesterol efflux and can be transformed into a pro-inflammatory molecule (8, 9). In fact, researches have identified the specific residues on apoA-I that are oxidized and are developing assays to detect these modified forms. Preliminary data suggests the oxidized forms may be useful as predictors of CHD (8, 9).
HDL-C Measurements: An Evolving Story
Accumulating data suggests that the historical focus on HDL-C measurement for the prediction of CHD may need refinement. Consequently, laboratorians will need to carefully evaluate emerging methods for measurement of other components of HDL particles. In part two of this article, we will describe the current knowledge of such assays and how they work.
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Alan T. Remaley, MD, PhD, is a member of the senior staff of the Department of Laboratory Medicine at the National Institutes of Health (Bethesda, Md.) and is the Section Chief of the Lipoprotein Metabolism Laboratory at the National, Heart, Lung and Blood Institute. He has served on the FDA’s Chemistry and Clinical Toxicology Panel since 2002.
G. Russell Warnick, MS, MBA, is Chief Scientific Officer and Vice President for Laboratory Operations at Berkeley Heartlab, Inc. (Burlingame, Calif.). Previously he directed the Core Lipoprotein Laboratory of the Northwest Lipid Research Center, associated with the University of Washington (Seattle) and founded Pacific BioMetrics, Inc. (Seattle, Wash.), a specialty service laboratory supporting pharmaceutical and diagnostic development. He serves as editor of The Fats of Life, a newsletter published by the Lipoproteins and Vascular Disease Division of AACC.