Clinical Laboratory Strategies: March 10, 2011

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Measuring Cholesterol Efflux Capacity
Method Opens Doors to Understanding HDL’s Protective Benefit
By Genna Rollins

Researchers have recognized for some time that high levels of high-density lipoprotein cholesterol (HDL-C) have a protective cardiovascular effect, but the exact mechanism of this benefit still is unclear. One promising theory involves HDL’s role in reverse cholesterol transport, a hypothesis explored in this issue of Strategies.

A body of evidence accumulated over many years indicates that high levels of high-density lipoprotein cholesterol (HDL-C) are associated with lower risk of cardiovascular disease. Robust research efforts have focused on understanding the mechanisms behind this protective benefit. The leading theory has been HDL’s role in maintaining cholesterol homeostasis, but until recently, researchers lacked a means of measuring this function, according to Alan Remaley, MD, PhD, section chief of the Lipoprotein Metabolism Section of the Pulmonary and Vascular Medicine Branch at the National Institutes of Health.

“The main reason HDL has been considered beneficial is that it mediates what’s called the reverse cholesterol transport pathway or cholesterol efflux—the removal of cholesterol from the peripheral tissue to the liver, where it’s excreted. This system is what maintains cholesterol homeostasis,” he explained. “The dilemma has been, while low-density lipoprotein cholesterol [LDL-C] is actually a good surrogate for measuring the proatherogenecity of LDL, the cholesterol in HDL is probably not a good measure of its ability to remove cholesterol.”

Now, however, University of Pennsylvania and Rutgers University researchers have reported not only measuring HDL’s ability to transport cholesterol, but also associating this cholesterol efflux capacity with both subclinical atherosclerosis and coronary artery disease, associations that persisted after adjustment for traditional cardiovascular risk factors (N Engl J Med 2011;364:127-35. Click here to link to the article).

“Measuring HDL similar to the way we measure LDL has been thought to be a good marker for what HDL does. In reality, though, HDL metabolism is so complex that just looking at the mass of what the cholesterol is that’s carried by the HDL is not enough to tell us about HDL’s function,” said co-author Marina Cuchel, MD, PhD, research assistant professor of medicine at the University of Pennsylvania in Philadelphia. “We decided if we wanted to have more insight into the protection of HDL—or not—we would need to set up a method that would measure the ability of HDL to efflux cholesterol from macrophages in the periphery. What we report is practically the first test of the reverse cholesterol transport mechanism.”

Cuchel and her colleagues developed an assay to measure cholesterol efflux capacity that involves radiolabelling and incubating individuals’ HDL in J774 cells derived from mice macrophage cell lines. This method quantifies the cholesterol efflux mediated by pathways known to be important in reverse cholesterol transport, including ATP-binding cassette transporter A1, ATP-binding cassette transporter G1, scavenger receptor B1, and aqueous diffusion. The researchers used the assay to test their hypothesis that cholesterol efflux capacity is a determinant of atherosclerotic burden and is independent of HDL-C level.

The study involved two independent cohorts, including 203 healthy subjects who had their carotid artery intima-media thickness assessed, and 793 case-control subjects who underwent cardiac catheterization. Among the healthy subjects, the researchers found no significant relationship between HDL-C level and carotid intima-media thickness in either unadjusted or adjusted models. However, they observed a significant inverse relationship between cholesterol efflux capacity and carotid intima-media thickness, a relationship that remained robust after adjustment for HDL-C and apolipoprotein A-I (Apo A-I) levels.

Similarly, in the case-control group, the investigators found that case patients with coronary artery disease had significantly lower levels of HDL-C, Apo A-I, and cholesterol efflux capacity. As cholesterol efflux capacity rose, the proportion of patients with coronary artery disease fell, a relationship that remained after adjustment for traditional cardiovascular risk factors, after addition of HDL-C level as a covariate, and after substitution of Apo A-I level for HDL-C level. The researchers also found that while HDL-C was the strongest predictor of efflux capacity, total phospholipids, Apo A-I, Apo A-II, and Apo E also were significantly associated with cholesterol efflux capacity.

“The results are rather striking,” said Remaley, who was not involved in the study. “The researchers showed that the biological measure of the ability of HDL to move cholesterol actually correlated better with the presence or absence of these than the HDL cholesterol assay, which is what everyone uses in clinical labs. This is very exciting, and it fits with our hypothesis for many years about how HDL really works.”

As important as the findings may be, both Remaley and Cuchel emphasized that they are only a starting point for better understanding HDL’s role. Most significantly, the researchers only assessed one aspect of cholesterol efflux, removal of cholesterol from lipid-laden cell macrophages. Their method does not evaluate the ability of serum to remove cholesterol. “This may not be the only story to tell about HDL,” said Remaley. “Recently it’s been shown to have many other effects, including being anti-inflammatory and beneficial to endothelial cells. Also, we may find, for example, that some individuals’ serum is good at removing cholesterol but their cells lack sufficient transporters to bring it to the cell surface.”

Both Remaley and Cuchel also noted that the assay the researchers used will not be seen in clinical labs anytime soon. “At least not at this stage of the development of the method is it something that clinical labs could perform so that a physician could use it to assess the risk of his patients,” said Cuchel. “It’s not difficult in the setting of a research lab that’s familiar with cell culture; it’s just very tedious and takes about 48 hours to get results back, so it’s not something that can be run in an autoanalyzer.” Remaley, who also uses the method in his lab, agreed. “It’s not that difficult per se to do for a research study, but it’s just not practical for routine clinical processing,” he said.

For now, both the assay and study findings may be most relevant for drug development. The pharmaceutical industry is quite interested in finding drugs that take advantage of HDL’s protective cardiovascular effect, but drug trials so far have not produced particularly promising results. Most notably, Pfizer stopped development of torcetrapib, a cholesterol ester transfer protein inhibitor that increased HDL levels, when phase III studies showed excess mortality in the treatment group. Those findings and others reflect HDL’s complexity, according to Remaley. “It’s a very complicated complex of both proteins and lipids. Recent studies have shown it has upwards of 40 different proteins and lots of different lipids, but right now, we don’t know what role they all have. Eventually, we might be able to have an assay that will be much better than just measuring HDL in predicting the absence or presence of disease, and that in turn will help drug companies target therapies to those different functions,” he said.

Even if a cholesterol efflux assay is not immediately in store for clinical practice, Cuchel and Remaley urged laboratorians to keep abreast of further developments in this area. Already, Cuchel and her colleagues have a series of further studies planned, including assessing the whole cholesterol efflux pathway in an in-vivo assay. Other researchers are certain to build on their findings, Remaley predicted. “This cleverly points out that what we’re doing now in terms of measuring the cholesterol content of HDL is an imperfect measure, and it will prompt people who develop biomarkers to look at other measures besides HDL-C that may correlate with cholesterol efflux or may even be superior.”

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