Friday, March 20, 2009

Wherefore Art Thy Protection, O HDL?

by Chris Masterjohn

Hey everyone! It's great to be back. I got way behind with things after slipping and falling and dislocating my shoulder at the end of January, but I hope to be back to blogging regularly now.

Many of you may remember the drug torcetrapib, aimed at increasing HDL-cholesterol. It failed miserably, and killed a lot of people. Remarkably, there is a new drug, anacetrapib, aimed at doing the exact same thing, that is now going through the same trial process. Will it kill people?

An eight-week trial of under 600 people published in February found that anacetrapib had no more adverse effects than the placebo. This would be comforting, except that an eight-week trial of under 200 people published in 2006 found the exact same thing with torcetrapib, causing the lead author to publicly wonder, "Will torcetrapib be the next big thing in coronary heart disease risk reduction?" It was big, alright, but it didn't reduce any risks. A large-scale trial with over 15,000 people was stopped early at just over two years instead of more than 4.5 years because the drug was killing people. All-cause mortality was increased by 58 percent, representing an increase in cardiovascular deaths of 25 percent and a doubling of non-cardiovascular deaths.

These drugs are based on the reverse cholesterol transport theory, a theory that was tested for the first time in the torcetrapib trial and should have been at least tentatively rejected.

HDL binds to cells in the artery wall and takes up free cholesterol. It then esterifies the cholesterol, by which I mean it binds the cholesterol to a fatty acid, and moves it down into the core of the HDL particle. It can do two things with this cholesteryl ester. It can be taken up by the "LDL receptor" (the name is deceptive, I know) in the liver, thus delivering the cholesteryl ester to that organ, or it can deliver the cholesteryl ester directly to LDL. LDL, according to reverse cholesterol transport theorists, can deliver it to the liver too, but unfortunately can also deliver it right back into the artery wall.

What determines which action HDL takes? Cholesteryl ester transfer protein (CETP) facilitates an exchange between HDL and LDL (or especially VLDL, which is later transformed into LDL) wherein the HDL loses a cholesteryl ester and picks up a triglyceride.

Thus, let's block CETP! This is the reasoning of the drug manufactures. By blocking CETP, we lower LDL-cholesterol and boost HDL-cholesterol.

Epidemiological evidence suggests that the total-to-HDL cholesterol ratio, which is basically the same thing as the LDL-to-HDL cholesterol ratio, is the best blood lipid marker for heart disease. The word "marker" is key. Reverse cholesterol transport theorists assume this marker is causal. I believe it is a marker for the amount of time the LDL particle spends in the blood. If LDL receptor activity is low due to a) genetics, b) low thyroid status, or c) oxidative stress, LDL (or its VLDL precursor) will spend more time in the blood, interact more and more with CETP, and "steal" away cholesterol from HDL. LDL-cholesterol increases and HDL-cholesterol decreases. At the same time, it interacts with free radicals more and more, its limited supply of antioxidants runs out, the polyunsaturated fatty acids in its membrane oxidize, and it becomes atherogenic. This is causal.

How would reverse cholesterol transport theorists show that high HDL-cholesterol is causally protective? The perfect way to do this would be to use a drug that specifically boosts HDL-cholesterol and show that it reduces cardiovascular disease. The problem is that they just did this with torcetrapib and it increased cardiovascular disease. Reverse cholesterol transport theorists do not want to reject or modify their theory. They would prefer to believe there was something toxic about torcetrapib that had nothing to do with its CETP-blocking effects. There is one problem with this. In December, 2008, some scientists published a study finding no relationship between the degree to which torcetrapib boosted HDL-cholesterol and the change in the degree of atherosclerosis. They concluded the following:

The absence of an inverse relationship between high-density lipoprotein cholesterol [HDL-C] change and cIMT [carotid intima-media thickness, a measure of the degree of atheroslcerosis] progression suggests that torcetrapib-induced high-density lipoprotein cholesterol increase does not mediate atheroprotection [protection against atherosclerosis].

The medical field has created enormous confusion by conflating "HDL" with "HDL-cholesterol." When you go to the doctor and get bloodwork, they do not test your "LDL" and your "HDL." They test the amount of cholesterol contained in these lipoproteins. But they call your LDL-cholesterol your "LDL" and they call your "HDL-cholesterol" your "HDL."

When I say I do not believe that the epidemiological associations with HDL-cholesterol are causal, this does not mean that I do not believe that the HDL particle is protective. It is. But the evidence suggests that it is protective because of its highly specific role in delivering vitamin E to endothelial cells, not becuase of its role in reverse cholesterol transport. And guess what? CETP blockers directly undermine this process!

As Daniel Steinberg relates here and in his book, The Cholesterol Wars, researchers first discovered what came to be known as the oxidative modification of the LDL particle in 1979. When LDL was incubated for a long time with endothelial cells, the cells that line the inside of the blood vessel wall, something about it changed that made it toxic. Blood serum and HDL both prevented this change. In 1981, this "endothelial cell-modification" of LDL was shown to confer on the LDL particle the ability to be taken up by macrophages and transform those macrophages to the foam cells that populate atherosclerotic plaques. Further research showed that the polyunsaturated fatty acids in the membrane phospholipids of the LDL particle were oxidizing (going "rancid") during this modification, and that serum, HDL, or vitamin E could prevent the effect.

HDL is contained in serum and vitamin E is contained in HDL. This suggests that the protective effect of serum was largely due to its HDL content, and that the protective effect of HDL was largely due to its vitamin E content.

As it turns out, there is a very specific transport system designed to transfer vitamin E from the intestines and liver to endothelial cells, and it involves HDL. Vitamin E is originally released from the intestines into the lymph in chylomicrons, which then travel from the lymph into the blood. It is also recycled by the liver in the VLDL particle, which is the precursor to LDL. The liver secretes "nascent" HDL particles, meaning particles that have membrane proteins and phospholipids but not much of anything else. These HDL particles pick up vitamin E from the chylomicrons and the VLDL and LDL particles in the blood and then deliver it to endothelial cells.

HDL, as shown here, is three to five times more effective than LDL at delivering vitamin E to endothelial cells. LDL appears to deliver vitamin E to these cells simply by being taken up as a whole particle, whereas HDL interacts with what could be called the "HDL receptor" but is instead for the sake of confusing non-scientists called the scavenger receptor, class B, type I (SR-BI), and delivers vitamin E to the endothelial cell at between eight and twenty times the rate it is itself taken up by those same cells.

Once delivered to the endothelial cell, vitamin E not only prevents the oxidation of LDL particles as described above, but decreases the expression of "adhesion molecules" involved in the formation of atherosclerotic plaque, such as ICAM-1, VCAM-1, and E-selectin, and boosts the synthesis of nitric oxide, which protects against atherosclerosis at multiple levels. All of these actions have been attributed to the HDL particle, but since vitamin E accomplishes the exact same results, the actions of HDL appear to be attributable to its role in vitamin E transport.

Now how does HDL acquire the vitamin E from the other lipoproteins in the first place? One of the proteins involved appears to be... *drumroll*... CETP!

A test tube study published in May of last year found that a CETP-specific inhibitor decreased the transfer of vitamin E from other lipoproteins to HDL by 45 percent!

Perhaps torcetrapib killed people because of its "off-target toxicity." But why did its HDL-cholesterol-boosting effect fail to proportionally reduce atherosclerosis? Is it because the reverse cholesterol transport theory is wrong? If HDL's protective effect is due to its role in vitamin E transport, is it possible that CETP was killing people at least in part because it was undermining the transport of vitamin E from other lipoproteins to HDL, and thus undermining the delivery of vitamin E to the endothelial cells where it inhibits the oxidation of LDL, decreases adhesion molecule expression, and boosts nitric oxide synthesis, all the main components of the early atherosclerotic process?

If so, will large-scale long-term trials of the new CETP inhibitor anacetrapib show that this drug also kills people?

Read more about the author, Chris Masterjohn, PhD, here.


  1. Chris,

    What do you make of the mixed results of controlled trials on the effect of vitamin E supplementation on CHD risk?

    I read a study in the Lancet from 1996 (Stephens NG et al) where vitamin E supplements reduced non-fatal MI but not MI deaths. The effect on non-fatal MI was actually striking. Then there was the massive Physicians' Health trial that showed 400 IU plus vitamin C every other day had no effect. Epi studies seem to support a protective effect but I don't put much faith in those. Any thoughts on this?

  2. Stephan,

    I think the main thing is that alpha-tocopherol alone causes gamma-tocopherol deficiency by saturating alpha-tocopherol transport protein and thus preventing gamma-tocopherol's transport in the blood. Since a major portion of the oxidative stress vitamin E is needed to prevent is nitrative in nature and alpha-tocopherol is useless against nitrative stress, alpha-tocopherol can worsen oxidative stress.

    400 IU of vitamin E is a pharmacological, not nutritional, dose.

    Here, however, I am arguing that local vitamin E deficiency would worsen atherosclerosis, not that current vitamin E intakes need to be increased to prevent atherosclerosis. Perhaps the latter is true but it's a separate question.


  3. Chris:

    What do you mske of the Total:HDL ratio if a patient is on a statin and thus lowering the Total chol. artificially?

  4. Chris,

    I see. It would be interesting to see a trial using mixed tocopherols, don't you think?

    One of the things that's interesting is it seems like the gamma-tocopherol intake has been increasing in the US due to an increase in seed oil consumption. Apparently gamma is the main tocopherol in the US diet now. Maybe it's not enough to cover the increase in linoleic acid?

  5. Stephan,

    Statins mainly affect plasma cholesterol by increasing LDL receptor expression, due to a reduction in free cholesterol in the liver, which increases the clearance of LDL in plasma, thereby decreasing the level of LDL-cholesterol and increasing the level of HDL-cholesterol (because there is less apoB-containing lipoproteins that would take cholesterol from HDL), so the significance of the marker is the same for someone on statins.

    Trials should use a combo of mixed tocopherols and CoQ10, the main antioxidant of the LDL particle and possibly the limiter of the initiatory ROS in most cells. The antioxidants should be aimed at the LDL particle as well as at the endothelial cells.

    I definitely do not believe that increasing the intake of vitamin E is a substitute for decreasing PUFA intake.


  6. I mistakenly thought both of those comments were from Stephan. My apologies, Dr. Steven!



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