Association of Apolipoprotein B and NMR Spectroscopy–Derived LDL Particle Number with Outcomes

Association of Apolipoprotein B and Nuclear Magnetic Resonance Spectroscopy–Derived LDL Particle Number with Outcomes in 25 Clinical Studies              

1. Thomas G. Cole¹,*,                      
2. John H. Contois²,
3. Gyorgy Csako³,
4. Joseph P. McConnell⁴,
5. Alan T. Remaley³,
6. Sridevi Devaraj⁵,
7. Daniel M. Hoefner⁴,
8. Tonya Mallory⁴,
9. Amar A. Sethi⁶ and
10. G. Russell Warnick⁴

                    

Abstract

 

BACKGROUND: The number of circulating LDL particles is a strong indicator of future cardiovascular disease (CVD) events, even superior to the concentration of LDL cholesterol. Atherogenic (primarily LDL) particle number is typically determined either directly by the serum concentration of apolipoprotein B (apo B) or indirectly by nuclear magnetic resonance (NMR) spectroscopy of serum to obtain NMR-derived LDL particle number (LDL-P).

                    

CONTENT: To assess the comparability of apo B and LDL-P, we reviewed 25 clinical studies containing 85 outcomes for which both biomarkers were determined. In 21 of 25 (84.0%) studies, both apo B and LDL-P were significant for at least 1 outcome. Neither was significant for any outcome in only 1 study (4.0%). In 50 of 85 comparisons (58.8%), both apo B and LDL-P had statistically significant associations with the clinical outcome, whereas in 17 comparisons (20.0%) neither was significantly associated with the outcome. In 18 comparisons (21.1%) there was discordance between apo B and LDL-P.

                    

CONCLUSIONS: In most studies, both apo B and LDL-P were comparable in association with clinical outcomes. The biomarkers were nearly equivalent in their ability to assess risk for CVD and both have consistently been shown to be stronger risk factors than LDL-C. We support the adoption of apo B and/or LDL-P as indicators of atherogenic particle numbers into CVD risk screening and treatment guidelines. Currently, in the opinion of this Working Group on Best Practices, apo B appears to be the preferable biomarker for guideline adoption because of its availability, scalability, standardization, and relatively low cost.

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Read the complete article here.

Shocking Cholesterol News - Suzy Cohen

Shocking Cholesterol News

Dear Pharmacist,

I saw Dr. Oz interview a doctor on television about cholesterol. The guest said your total cholesterol doesn’t matter and I read that in your book 6 years ago. Suzy, I take a statin, and do a “Lipid Profile” annually. Is this okay? –M.D., Austin, Texas

Answer: No, it’s not okay, and I’m about to shock everyone, unless you’ve read my books, then this will be review.

Recently I wrote a column about LDL and that we should not necessarily strive to lower it. We need to know the type and number of LDL particles. For example, Lipoprotein A  or “Lp(a)” and another called apolipoprotein B or “Apo B” are two subtypes of LDL particles. These particular scores directly affect your cardiovascular risk. Do you have those numbers on your lab test? I bet you don’t.

In my first book, The 24-Hour Pharmacist from 2007 and many syndicated columns I’ve explained that statins are not very effective in reducing LDL particle number or Apo B and usually do not increase the size of your LDL particles, that’s why I don’t encourage them.

It’s confusing for consumers (and physicians who unwittingly accept drug propaganda) because studies conclude statins reduce total LDL. And yes, they do reduce “total” LDL, they are also excellent anti-inflammatories so they are not completely without merit. But I’m bent on you reducing Lp(a) and Apo B, the dangerous subtypes of LDL known to raise risk for heart attack and stroke.  One day I’ll tell you which vitamin reduces those bad boys, since drugs can’t, but now, back to this testing dilemma.

I’ll never submit myself for a routine “Lipid Profile” because it would waste my money. Half the people who have heart attacks have normal total cholesterol. If your results shows a low LDL (considered the bad particle), then you may assume you’re okay but you see, a low total LDL score doesn’t say much. Your triglycerides might be through the roof! You may have a huge concentration of dangerous Lp(a) and Apo B, subtypes of LDL that are never measured in that basic lipid profile.

Likewise, you may be happy with your high HDL cholesterol score, (HDL is considered a good cholesterol), but what if you have the wrong kind of HDL particles? Yeah, some HDL is bad, you didn’t know that?!  You’re still at very high risk.  These basic “Lipid Profiles” don’t provide the crucial details. It’s like a car mechanic who you hire to fix your engine, but you only let him look at the hood of your car, he can’t open the hood to see inside!

The better tests, sometimes covered by insurance measure particle size, type and sometimes the actual number of LDL and HDL particles. I urge you to ask your physician to order tests from Berkeley HeartLab, a leader in this field. There’s also another one called the “VAP Test” by Atherotec Diagnostics and finally, the “NMR Lipoprofile” by LipoScience.

http://www.dearpharmacist.com/2013/01/16/shocking-cholesterol-news/

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The straight dope on cholesterol – Part IX - Attia

The straight dope on cholesterol – Part IX

 

Previously, across 8 parts of this series we’ve laid the groundwork to ask perhaps the most important question of all:

What should you eat to have the greatest chance of delaying the arrival of cardiovascular disease?

Before we get there, since this series has been longer and more detailed than any of us may have wanted, it is probably worth reviewing the summary points from the previous posts in this series (or you can just skip this and jump to the meat of this post).

What we’ve learned so far

1. Cholesterol is “just” another fancy organic molecule in our body but with an interesting distinction: we eat it, we make it, we store it, and we excrete it – all in different amounts.
2. The pool of cholesterol in our body is essential for life. No cholesterol = no life.
3. Cholesterol exists in 2 forms – unesterified or “free” (UC) and esterified (CE) – and the form determines if we can absorb it or not, or store it or not (among other things).
4. Much of the cholesterol we eat is in the form of CE. It is not absorbed and is excreted by our gut (i.e., leaves our body in stool). The reason this occurs is that CE not only has to be de-esterified, but it competes for absorption with the vastly larger amounts of UC supplied by the biliary route.
5. Re-absorption of the cholesterol we synthesize in our body (i.e., endogenous produced cholesterol) is the dominant source of the cholesterol in our body. That is, most of the cholesterol in our body was made by our body.
6. The process of regulating cholesterol is very complex and multifaceted with multiple layers of control. I’ve only touched on the absorption side, but the synthesis side is also complex and highly regulated. You will discover that synthesis and absorption are very interrelated.
7. Eating cholesterol has very little impact on the cholesterol levels in your body. This is a fact, not my opinion. Anyone who tells you different is, at best, ignorant of this topic. At worst, they are a deliberate charlatan. Years ago the Canadian Guidelines removed the limitation of dietary cholesterol. The rest of the world, especially the United States, needs to catch up. To see an important reference on this topic, please look here.
8. Cholesterol and triglycerides are not soluble in plasma (i.e., they can’t dissolve in water) and are therefore said to be hydrophobic.
9. To be carried anywhere in our body, say from your liver to your coronary artery, they need to be carried by a special protein-wrapped transport vessel called a lipoprotein.
10. As these “ships” called lipoproteins leave the liver they undergo a process of maturation where they shed much of their triglyceride “cargo” in the form of free fatty acid, and doing so makes them smaller and richer in cholesterol.
11. Special proteins, apoproteins, play an important role in moving lipoproteins around the body and facilitating their interactions with other cells. The most important of these are the apoB class, residing on VLDL, IDL, and LDL particles, and the apoA-I class, residing for the most part on the HDL particles.
12. Cholesterol transport in plasma occurs in both directions, from the liver and small intestine towards the periphery and back to the liver and small intestine (the “gut”).
13. The major function of the apoB-containing particles is to traffic energy (triglycerides) to muscles and phospholipids to all cells. Their cholesterol is trafficked back to the liver. The apoA-I containing particles traffic cholesterol to steroidogenic tissues, adipocytes (a storage organ for cholesterol ester) and ultimately back to the liver, gut, or steroidogenic tissue.
14. All lipoproteins are part of the human lipid transportation system and work harmoniously together to efficiently traffic lipids. As you are probably starting to appreciate, the trafficking pattern is highly complex and the lipoproteins constantly exchange their core and surface lipids.
15. The measurement of cholesterol has undergone a dramatic evolution over the past 70 years with technology at the heart of the advance.
16. Currently, most people in the United States (and the world for that matter) undergo a “standard” lipid panel, which only directly measures TC, TG, and HDL-C. LDL-C is measured or most often estimated.
17. More advanced cholesterol measuring tests do exist to directly measure LDL-C (though none are standardized), along with the cholesterol content of other lipoproteins (e.g., VLDL, IDL) or lipoprotein subparticles.
18. The most frequently used and guideline-recommended test that can count the number of LDL particles is either apolipoprotein B or LDL-P NMR, which is part of the NMR LipoProfile. NMR can also measure the size of LDL and other lipoprotein particles, which is valuable for predicting insulin resistance in drug naïve patients, before changes are noted in glucose or insulin levels.
19. The progression from a completely normal artery to a “clogged” or atherosclerotic one follows a very clear path: an apoB containing particle gets past the endothelial layer into the subendothelial space, the particle and its cholesterol content is retained, immune cells arrive, an inflammatory response ensues “fixing” the apoB containing particles in place AND making more space for more of them.
20. While inflammation plays a key role in this process, it’s the penetration of the endothelium and retention within the endothelium that drive the process.
21. The most common apoB containing lipoprotein in this process is certainly the LDL particle. However, Lp(a) and apoB containing lipoproteins play a role also, especially in the insulin resistant person.
22. If you want to stop atherosclerosis, you must lower the LDL particle number. Period.
23. At first glance it would seem that patients with smaller LDL particles are at greater risk for atherosclerosis than patients with large LDL particles, all things equal.
24. “A particle is a particle is a particle.” If you don’t know the number, you don’t know the risk.
25. With respect to laboratory medicine, two markers that have a high correlation with a given outcome are concordant – they equally predict the same outcome. However, when the two tests do not correlate with each other they are said to be discordant.
26. LDL-P (or apoB) is the best predictor of adverse cardiac events, which has been documented repeatedly in every major cardiovascular risk study.
27. LDL-C is only a good predictor of adverse cardiac events when it is concordant with LDL-P; otherwise it is a poor predictor of risk.
28. There is no way of determining which individual patient may have discordant LDL-C and LDL-P without measuring both markers.
29. Discordance between LDL-C and LDL-P is even greater in populations with metabolic syndrome, including patients with diabetes. Given the ubiquity of these conditions in the U.S. population, and the special risk such patients carry for cardiovascular disease, it is difficult to justify use of LDL-C, HDL-C, and TG alone for risk stratification in all but the most select patients.
30. To address this question, however, one must look at changes in cardiovascular events or direct markers of atherosclerosis (e.g., IMT) while holding LDL-P constant and then again holding LDL size constant. Only when you do this can you see that the relationship between size and event vanishes. The only thing that matters is the number of LDL particles – large, small, or mixed.
31. HDL-C and HDL-P are not measuring the same thing, just as LDL-C and LDL-P are not.
32. Secondary to the total HDL-P, all things equal it seems smaller HDL particles are more protective than large ones.
33. As HDL-C levels rise, most often it is driven by a disproportionate rise in HDL size, not HDL-P.
34. In the trials which were designed to prove that a drug that raised HDL-C would provide a reduction in cardiovascular events, no benefit occurred: estrogen studies (HERS, WHI), fibrate studies (FIELD, ACCORD), niacin studies, and CETP inhibition studies (dalcetrapib and torcetrapib). But, this says nothing of what happens when you raise HDL-P.
35. Don’t believe the hype: HDL is important, and more HDL particles are better than few. But, raising HDL-C with a drug isn’t going to fix the problem. Making this even more complex is that HDL functionality is likely as important, or even more important, than HDL-P, but no such tests exist to “measure” this.

Did you say “delay?”

That’s right. The question posed above did not ask how one could “prevent” or eliminate the risk cardiovascular disease, it asked how one could “delay” it. There is a difference. To appreciate this distinction, it’s worth reading this recent publication by Allan Sniderman and colleagues. Allan sent me a copy of this paper ahead of publication a few months ago in response to a question I had posed to him over lunch one day. I asked,

“Allan, who has a greater 5-year risk for cardiovascular disease, a 25 year-old with a LDL-P/apoB in the 99^th percentile or a 75-year-old with a LDL-P/apoB in the 5^th percentile?”

The paper Allan wrote is noteworthy for at least 2 reasons:

1. It’s an excellent reminder that age is a paramount risk factor for cardiovascular disease.
2. It provides a much better (causal) model for atherosclerosis than the typical age-driven models, and explains why age is an important risk factor.

What do I mean by this? Most risk calculators (e.g., Framingham) take their inputs (e.g., age, gender, LDL-C, HDL-C, smoking, diabetes, blood pressure) and calculate a 10-year risk score. If you’ve ever played with these models you’ll quickly see that age drives risk more than any other input. But why? Is there something inherently “risky” about being older?
Sniderman and many others would argue (and I agree) that the reason age is a strong predictor of risk has to do with exposure to apoB particles — LDL, Lp(a), and apoB-carrying remnants. Maybe it’s because I’m a math geek, but such models just seem intuitive to me because I think of most things in life in terms of calculus, especially integrals, the “area under a curve.”
[I once tried to explain to a girlfriend who thought I wasn’t spending enough time with her that my interest in her should be thought of in terms of the area under the curve, rather than any single point in time. That is, think in terms of the integral function, not the point-in-time function. Needless to say, she broke up with me on the spot (in the middle of a parking lot!), despite me drawing a very cool picture illustrating the difference, which I’ve re-created, below.]

The reason age is such a big driver of risk is that the longer your artery walls are exposed to the insult of apoB particles, the more likely they are to be damaged, for all the reasons we covered in Part IV of this series. [This paper also reviews the clinical situation of PCSK9 mutations which builds a very compelling case for the causal model of apoB particles in the development of atherosclerosis].

What does eating have to do with cardiovascular risk?

So now that everyone is on the edge of their seat in anticipation of this punch-line, let me provide two important caveats.

First, there are no long-term studies – either in primary or secondary prevention – examining the exact question we all want to know the answer to with respect to the role of dietary intervention on cardiovascular disease. There are short-term studies, some of which I will highlight, which look at proxies for cardiovascular disease, but all of the long-term studies (looking at secondary prevention), are either drug studies or multiple intervention studies (e.g., cholesterol-lowering drug(s) + blood pressure reducing drug(s) + dietary intervention + exercise + …).

In other words, the “dream” study has not been done and won’t be done for a long time. The “dream” study would follow 2 randomized groups for many years and only make one change between the groups. Group 1 would consume a standard American diet and group 2 would consume a very-low carbohydrate diet. Furthermore, compliance within each group would be excellent (many ways to ensure this, but none of them are inexpensive – part of why this has not been done) and the study would be powered to detect “hard outcomes” (e.g., death), instead of just “soft outcomes” (e.g., changes in apoB, LDL-C, LDL-P, TG).

Second, everything we have learned to date on the risk relationship between cardiovascular disease and risk markers is predicated on the assumption that a risk maker of level X in a person on diet A is the same as it would be for a person on diet B.

Since virtually all of the thousands of subjects who have made up the dozens of studies that form the basis for our understanding on this topic were consuming some variant of the “standard American diet” (i.e., high-carb), it is quite possible that what we know about risk stratification is that this population is not entirely fit for extrapolation to a population on a radically different diet (e.g., a very-low carbohydrate diet or a ketogenic diet). Many of you have asked about this, and my comments have always been the same. It is entirely plausible that an elevated level of LDL-P or apoB in someone consuming a high-carb diet portends a greater risk than someone on a ketogenic or low-carb diet. There are many reasons why this might be the case, and there are many folks who have made compelling arguments for this hypothesis.

But we can’t forget the words of Thomas Henry Huxley, who said, “The great tragedy of science is the slaying of a beautiful hypothesis by an ugly fact.” Science is full of beautiful hypothesis slayed by ugly facts. Only time will tell if this hypothesis ends up in that same graveyard, or changes the way we think about lipoproteins and atherosclerosis.

The role of sugar in cardiovascular disease

Let’s start with what we know, then fill in the connections, with the goal of creating an eating strategy for those most interested in delaying the onset of cardiovascular disease.

There are several short-term studies that have carefully examined the impact of sugar, specifically, on cardiovascular risk markers. Let’s examine one of them closely. In 2011 Peter Havel and colleagues published a study titled Consumption of fructose and HFCS increases postprandial triglycerides, LDL-C, and apoB in young men and women. If you don’t have access to this journal, you can read the study here in pre-publication form. This was a randomized trial with 3 parallel arms (no cross-over). The 3 groups consumed an isocaloric diet (to individual baseline characteristics) consisting of 55% carbohydrate, 15% protein, and 30% fat. The difference between the 3 groups was in the form of their carbohydrates.
Group 1: received 25% of their total energy in the form of glucose
Group 2: received 25% of their total energy in the form of fructose
Group 3: received 25% of their total energy in the form of high fructose corn syrup (55% fructose, 45% glucose)
The intervention was relatively short, consisting of both an inpatient and outpatient period, and is described in the methodology section.

Keep in mind, 25% of total energy in the form of sugar is not as extreme as you might think. For a person consuming 2,400 kcal/day this amounts to about 120 pounds/year of sugar, which is slightly below the average consumption of annual sugar in the United States. In that sense, the subjects in Group 3 can be viewed as the “control” for the U.S. population, and Group 1 can be viewed as an intervention group for what happens when you do nothing more in your diet than remove sugar, which was the first dietary intervention I made in 2009.

Despite the short duration of this study and the relatively small number of subjects (16 per group), the differences brought on by the interventions were significant. The figure below shows the changes in serum triglycerides via 3 different ways of measuring them. Figure A shows the difference in 24-hour total levels (i.e., the area under the curve for serial measurements – hey, there’s our integral function again!). Figure B shows late evening (post-prandial) differences. Figure C shows the overall change in fasting triglyceride level from baseline (where sugar intake was limited for 2 weeks and carbohydrate consumption consisted only of complex carbohydrates).

The differences were striking. The group that had all fructose and HFCS removed from their diet, despite still ingesting 55% of their total intake in the form of non-sugar carbohydrates, experienced a decline in total TG (Figure A, which represents the daily integral of plasma TG levels, or AUC). However, that same group experienced the greatest increase in fasting TG levels (Figure C). Post-prandial TG levels were elevated in all groups, but significantly higher in the fructose and HFCS groups (Figure B). The question this begs, of course, is which of these measurements is most predictive of risk?

Historically, fasting levels of TG are used as the basis of risk profiling (Figure C), and according to this metric glucose consumption appears even worse than fructose or HFCS. However, recent evidence suggests that post-prandial levels of TG (Figure B) are a more accurate way to assess atherosclerotic risk, as seen here, here, and here. One question I have is why did the AUC calculations in Figure A show a reduction in plasma TG level for the glucose group?
The figure below summarizes the differences in LDL-C, non-HDL-C, apoB, and apoB/apoA-I.

Again, the results were unmistakable with respect to the impact of fructose and HFCS on lipoproteins, and by extension, the relative lack of harm brought on by glucose in isolation. [Of course, removal of glucose and fructose/HFCS would have been a very interesting control group.]
One of the simultaneous strengths and weaknesses of this study was the heterogeneity of its subjects, who ranged in BMI from 18 to 35, in age from18 to 40, and in gender. While this provided at least one interesting example of age-related differences in carbohydrate metabolism (older subjects had a greater increase in triglycerides in response to glucose than younger subjects), it may have actually diluted the results. There were also significant differences between genders in the glucose group.
What was most interesting about this study was the clear difference between the 3 groups that was not solely a function of fructose load. In other words, the best outcome from a disease risk standpoint was in the glucose group, while the worst outcome was not in the all-fructose group, but in the 50/50 (technically 55/45) mixed group. This is a very powerful indication that while glucose and fructose alone can be deleterious in excess, their combination seems synergistically bad.

The role of saturated fat in cardiovascular disease

In the next week or two I’ll be posting an hour-long comprehensive lecture I gave at UCSD a few weeks ago on this exact topic. Rather than repeat any of it here, I’ll highlight one study that I did not include in that lecture. The study, Effect of a high saturated fat and no-starch diet on serum lipid subfractions in patients with documented atherosclerotic cardiovascular disease, published in 2003, treated 23 obese patients (average BMI 39) with known cardiovascular disease (status post coronary artery bypass surgery and/or stent placement) with a high-fat ketogenic diet. Because the study was free-living and relied on self-reporting, not all subjects had documented levels of elevated serum B-OHB. However, the subjects were instructed to avoid starch and consume 50% of their caloric intake via saturated fat, primarily in the form of red meat and cheese. There were no restrictions on fruits and vegetables, which may have accounted for the observation that not all subjects were ketotic during the 6-week intervention. In total, only 5 of the 23 patients achieved documented ketosis.
All of the subjects were on statins and entered the study at a goal LDL-C level target of 100 mg/dL, which may have been the only way the authors could get the IRB to approve such a study.
The table below shows the changes in lipoprotein fractions following the intervention (there was no control group):

This study was conducted during the height of the “outcry” over the Atkins diet. While most doctors reluctantly agreed that Dr. Atkins’ diet could reduce body fat, most believed it was still very dangerous. In the words of Dean Ornish, “Sure you can lose weight on a low-carb diet, but you can also lose weight on heroin and no one would recommend that!”

Fair point. In fact, the authors of this study acknowledged that they “strongly expected” this dietary intervention to increase risk for cardiovascular disease, which is why they only included subjects on statins with low LDL-C. However, as you can see from the table above, the authors were startled by the results. The subjects experienced a significant reduction in plasma triglycerides and VLDL triglycerides, without an increase in LDL-C or LDL-P. In fact, LDL size and HDL size increased and VLDL size decreased – all signs of improved insulin resistance. Furthermore, fasting glucose and insulin levels also decreased significantly. The mean HOMA-IR was reduced from 5.6 to 3.6 (normal is 1.0) and TG/HDL-C from 3.3 to 2.0 (normal is considered below 3, but “ideal” is probably below 1.0) in just 6 weeks. Taken together, these changes, combined with the dramatic change in VLDL size, suggest insulin resistance was dramatically improved while consuming a diet of 50% saturated fat!

As all of these patients were taking statins, we’re really robbed of seeing the impact of this diet on LDL-P, which did not change. Also, CRP levels rose (though not clinically or statistically significantly).

Putting it all together

It is very difficult to make the case that when carbohydrates in general, and sugars in particular, are removed or greatly reduced in the diet, insulin resistance is not improved, even in the presence of high amounts of saturated fats. When insulin resistance improves (i.e., as we become more insulin sensitive), we are less likely to have the signs and symptoms of metabolic syndrome. As we meet fewer criteria of metabolic syndrome, our risk of not only heart disease, but also stroke, cancer, diabetes, and Alzheimer’s disease goes down.

Furthermore, as this study on the Framingham cohort showed us, the more criteria you have along the spectrum of metabolic syndrome, the more difficult it becomes to predict your risk, due to a widening gap in discordant risk markers, as shown in this figure.

As I noted at the outset, the “dream” trial has not yet been done, though we (NuSI) plan to change that. Until then each of us has to make a decision several times every day about what we will and won’t put in our mouths. Much of this blog is dedicated to underscoring the impact of carbohydrate reduction on insulin resistance and metabolic syndrome.

The results of the trials to date, combined with a nuanced understanding of the lipoprotein physiology and their role on the atherosclerotic disease process, bring us to the following conclusions:

1. The consumption of sugar (sucrose, high fructose corn syrup) increases plasma levels of triglycerides, VLDL and apoB, and reduces plasma levels of HDL-C and apoA-I.
2. The removal of sugar reverses each of these.
3. The consumption of fructose alone, though likely in dose-dependent fashion, has a similar, though perhaps less harmful, impact as that of fructose and glucose combined (i.e., sugar).
4. The addition of fat, in the absence of sugar and starch, does not raise serum triglycerides or other biomarkers of cardiovascular disease.
5. The higher the level of serum triglycerides, the greater the likelihood of discordance between LDL-C and LDL-P (and apoB).
6. The greater the number (from 0 to 5) of inclusion criteria for metabolic syndrome, the greater the likelihood of discordance between LDL-C and LDL-P (and apoB).

I would like to address one additional topic in this series before wrapping it up – the role of pharmacologic intervention in the treatment and prevention of atherosclerotic disease, so please hold off on questions pertaining to this topic for now.

Related Posts

• The straight dope on cholesterol – Part VI
• The straight dope on cholesterol – Part V
• The straight dope on cholesterol – Part IV
• The straight dope on cholesterol – Part VIII
• The straight dope on cholesterol – Part III

Read the complete article here.

How do we measure cholesterol?

How do we measure cholesterol?

Posted by Peter Attia on May 10, 2012

Concept #5 – How do we measure cholesterol?

All this talk about cholesterol probably has some of you wondering how one actually measures the stuff. Much of the raw content I’m going to present here is actually material I’ve had to learn recently. One of the best resources I’ve found on this topic is the text book Contemporary Cardiology: Therapeutic Lipidology, in particular, chapter 14 by Tom Dayspring and chapter 15 by Bill Cromwell and Jim Otvos. Anyone aspiring to be a lipid savant like these three pioneers probably ought to get a copy. The other book that tells this story well is The Cholesterol Wars: The Skeptics versus the Preponderance of Evidence. For most folks, however, I’m hoping this series is sufficient and I’ll do my best to get the important points across.

As far back as the 1940’s scientists understood that cholesterol and lipids could not simply travel freely within the bloodstream without something to carry them and obscure their hydrophobicity, but it certainly wasn’t clear what these carriers looked like.

The initial breakthrough came during the Second World War when two researchers, E.J. Cohn and J.L. Oncley at Harvard developed a complex and elaborate technique to fractionate (i.e., separate) human serum (serum is blood, less the cells and clotting factors) into two “classes” of lipoproteins: those with alpha mobility and those with beta mobility. [“Alpha” versus “beta” mobility describes a pattern of movement seen by different particles, relative to fluid, under a uniform electric field, which is the essence of electrophoresis.]

You’ll recall that LDL particles are also called “beta” particles and HDL particles are also called “alpha” particles. Now you see why.

This work set the stage for subsequent work, by a physicist named John Gofman, using the techniques of preparative and analytic ultracentrifugation to fully classify the major classes of human lipoproteins. The table below summarizes what was gleaned by these experiments.


Cool, huh? Well, sort of. While this was an enormous breakthrough scientifically, it didn’t really have an inexpensive and quick test that could be used clinically the way, say, one could measure glucose levels or hemoglobin levels in patients routinely. What became crucial with Gofman’s discovery is that lipoproteins were now a recognized entity and they got their names according to their buoyancy: very low density, intermediate density, low density and high density.

There is more interesting history to this tale, but let’s fast-forward to where we are today. When you go to your doctor to have your cholesterol levels checked, what do they actually do?

Let’s start at the finish line. What do they report? The figure below is a representative result. It reports serum cholesterol (in total), serum triglycerides, HDL cholesterol (i.e., HDL-C), LDL cholesterol (i.e., LDL-C) and sometimes non-HDL-C (i.e., LDL-C + VLDL-C). But where do these numbers come from?

Blood is drawn into a tube called a serum separator tube (SST) and immediately spun in centrifuge to separate the blood from “whole blood” into serum (normally clear yellow, top) and blood cells (dark red, bottom). A gel film, from the SST, separates the serum and blood cells, as shown below. The tube is kept cool and sent from the phlebotomy lab to the processing lab.

As early as the 1950’s scientists figured out clever chemical tricks to directly measure the content of total cholesterol in the serum. The chemical details probably are not interesting to non-chemists, but I was able to find a great paper from 1961 that details the methodology. The point is this: initially it was only possible to measure the total content of cholesterol (TC), or concentration to be technically correct, in plasma. By that I mean it is the total mass (weight of all the cholesterol molecules) of cholesterol trafficked within all of the lipoprotein species that exist in a specified unit of volume: in the United States, we measure this in milligram of cholesterol per deciliter of plasma abbreviated as mg/dL, or in the rest of the world as mmol/Liter or mmol/L. Why? Think back to our analogy from last week:
Cholesterol is a passenger on a ship — the “ship,” of course, being a lipoprotein particle. The early methods of measuring cholesterol had to break apart the hull of the ship to quantify the cargo. The assays to do so, like the one described above, were pretty harsh. If you had a bunch of LDL ships, HDL ships, VLDL ships, and IDL ships, these assays ripped them all apart and told you the sum total of the cargo. Obviously this was a great breakthrough in the day, but it was limited. From this assay, one could conclude, for example, that a person had 200 mg/dL of cholesterol hiding out in all their lipoprotein particles.

Good to know, but what next? It turns out there were two other important factors that could be measured directly in blood: triglycerides and the cholesterol content within the HDL particle, HDL-C. Early on laboratories could easily separate apoA-I-containing particles (i.e., HDL) from the apoB-containing particles (i.e., VLDLs, IDLs and LDLs), but they could not easily and economically separate the various apoB-containing particles from one another. A full description of these methods is not necessary to appreciate this discussion, but for those interested, methodologies can be found here (TG) and here (HDL-C).

Important digression for context

What becomes critical to understand for our subsequent discussions is that the apoB particles have the potential to deliver cholesterol into an artery wall (the problem we’re trying to avoid), and 90-95% of the apoB particles are LDL particles. Hence, it is LDL particle number (LDL-P or apoB) that drives the particles into the artery wall. Thus, physicians need to be able to quantify the number of LDL particles present in a given individual. For decades there was no way of doing that. Then LDL-C (read on) became available and it served as a way (not entirely accurate, but nonetheless a way) of quantitating LDL particles.

Back to the story
How can one figure out the concentration of cholesterol in the LDL particle? As you may recall from last week, LDL is the “ship” that carries the most cholesterol cargo. More importantly, as I mentioned above, it is also the key ship that traffics cholesterol directly into the artery wall. Thus, there has always been an enormous interest in knowing how much cholesterol is trafficked within LDL particles.

For a long time it was not possible to directly measure LDL-C, the cholesterol content of an LDL particle. However, we did know the following had to be true:

TC = LDL-C + HDL-C + VLDL-C + IDL-C + chylomicron-C + remnant-C + Lp(a)-C
where X-C denotes the cholesterol content of a respective cholesterol-carrying particle. There are 2 particles in the equation above that I didn’t specifically mention last week, the remnant particle and the Lp(a) particle (pronounced “EL – pee – little – a,” which sounds less silly than, “Lip-a”). Lp(a) is an LDL-like particle but with a special apoprotein attached to it, aptly called apoprotein(a), which is actually “attached” to the apoB molecule of a standard LDL particle. Think of Lp(a) as a “special” kind of LDL particle. As we’ll learn later in this series, Lp(a) particles are bad dudes when it comes to atherosclerosis.

“Remnants” are nearly-empty-of-triglyceride particles of chylomicrons and VLDL. In essence they are larger TG-rich particles that have lost a lot of their TG core content as well as surface phospholipids and are thus smaller than, or remnants of, their “parent particles.” Hence,they are cholesterol-rich particles. Under fasting conditions, in a not-too-terribly-insulin-resistant person, IDL-C, chylomicron-C, and remnant-C are negligible. Furthermore, in most people Lp(a)-C does not exist or is not very high.

So we’re left with this simplification:
TC ~ LDL-C + HDL-C + VLDL-C
which is clearly an improvement in convenience over the first equation. But what to do about that pesky VLDL-C?

There are a number of variations, but essentially a breakthrough (mid 1970s) formula, called the Friedewald Formula, estimates VLDL-C as one-fifth the concentration of serum triglycerides (some variants use 0.16 instead of one-fifth, or 0.20). This assumes all TG are trafficked in one’s VLDL particles and that a normally composed VLDL contains five times more TG than cholesterol.
Rearranging the above simplified formula we have:
LDL-C ~ TC – HDL-C – TG/5
Let’s plug in the numbers from the above figure, as an example. TC = 234 mg/dL; HDL-C = 48 mg/dL, and TG = 117 mg/dL. Hence, LDL-C is approximately 234 – 48 – 117/5 = 163 mg/dL.
Kind of a long run for a short slide, huh? Perhaps, but it is important to understand that when you go to your doctor and get a “cholesterol test,” odds are this is exactly what you’re getting.
Therefore LDL-C can be estimated knowing just TC, HDL-C, and TG, assuming LDL-C matters (hint: it doesn’t matter much in many folks).

Furthermore, what if the LDL particle is cholesterol-depleted instead of its normal state of being cholesterol-enriched? Unfortunately, especially in an insulin resistant population (i.e., the United States), both TG content within lipoproteins and the exchange of TG for cholesterol esters between particles is very common, and using this formula can significantly underestimate LDL-C. Worse yet, LDL-C becomes less meaningful in predicting risk, as I will address next week.

What about direct measurement of LDL-C?

To chronicle the entire history of direct LDL-C measurement is beyond the scope of this post. Many companies have developed proprietary techniques to measure LDL-C directly, along with apoB, and ultimately LDL-P. I’ll address two “major players” here: Atherotech and LipoScience.

Atherotech developed an assay, called a VAP panel (VAP stands for Vertical Auto Profile) to do everything described above, but also to directly measure the amount of cholesterol contained within the LDL particle. Furthermore, they have developed assays to directly measure the cholesterol in IDL particles, VLDL particles, and even Lp(a) particles. Below is a snapshot of how VAP reporting looks.

A couple of things are worth mentioning:

1. Subparticle cholesterol content information is also generated, including 2 different classes of HDL particles (HDL-2, HDL-3) and 4 different classes of LDL particles (LDL-1, LDL-2, LDL-3, LDL-4).
2. LDL particles, based on the subparticle information, are classified as “pattern A,” “pattern B,” or “pattern A/B.” Pattern A implies more large, buoyant LDL particles, while pattern B implies more small, dense LDL particles.

Remember, though, while cholesterol mass concentration numbers may correlate with the number of particles, they often do not. They only convey the mass concentration of cholesterol molecules within all of the particle subtypes per unit of volume. VAP tests do not report the number of LDL or HDL particles, but they do attempt to estimate atherogenic particle number (apoB) using a proprietary formula based on subparticle cholesterol concentration and particle sizes. I should point out that the formula, to my knowledge, has not been validated in any study and not published in a peer reviewed journal.

A high estimate of apoB100 (i.e., what the VAP reports) is said to correlate with the actual measurement of apoB. Since apoB is found on each LDL particle, this serves as a proxy of LDL-P. The American Diabetic Associate and the American College of Cardiology Consensus Statement on Lipoproteins and the new National Lipid Association biomarker paper stipulates that apoB must be done using a protein immunoassay, not an estimate, such as that of VAP.

But how can one actually count the number of LDL particles and HDL particles?

There are several methods of doing this, but only one company, LipoScience, has the FDA approved technology to do so using nuclear magnetic resonance spectroscopy, or NMR for short. The other available methodologies are ion mobility transfer and ultracentrifugation (by Quest) and separation of LDL particles with particle staining (by Spectracell). Virtually all guidelines (e.g., ADA, ACC, AACC and NLA) only advise LDL-P via NMR at this time.

NMR, which is the basis for not only how to count lipoprotein particles, but also diagnostic tests such as MRI scans, is really one of my favorite technical topics. In residency I wrote a surgical handbook and on page145-146, if you’re interested, you can read a brief description of how MRI technology works, which will explain how NMR technology can actually count lipoprotein particles.

As an aside, and just to give you an idea of what a great sport my wife is, I wrote this surgical handbook over the course of a year while in residency. To do so, I had to read approximately 8,000 pages of surgical textbooks and try to distill them down to just this 160 page summary. Doing so required reading about 22 pages every day while working about 110 hours per week, typical of a surgical residency “back in the day.” Besides exercising, I spent every single moment of my “free” time reading for and writing this handbook. Finally, a few months into it, my wife asked, “Why the hell are you doing this? You never watch TV, you never go out, you never do anything else!” I responded that it was the best way I could learn this material, but also, that I wanted to have a legacy when I left residency. Half joking, I asked her, “What’s your legacy?” Blank stare. A few months later, for Valentine’s Day, she gave me this t-shirt. I think it’s safe to say not a single person has read this handbook. So much for my legacy…

A brief explanation of how NMR works to count (and measure) particles can also be found here.
Below is a snapshot of how NMR reporting looks. This particular report is from Health Diagnostics Laboratory (HDL), Inc. LipoScience performs the actual NMR test, but HDL, Inc. runs a number of complimentary biomarkers I will discuss in subsequent posts. I now use the HDL, Inc. test exclusively for reasons I will explain later.

In addition to counting the actual total number of LDL particles (LDL-P) and HDL particles (HDL-P) per liter, HDL, Inc. (not LipoScience) directly measures apoB and apoA-I. Furthermore, the size of each particle is measured using NMR in nanometers (to give you a sense of how small these things are, and why we need to use nanometers to measure them, about 1.3 million LDL particles stacked side-by-side would measure only one inch).

The final point I’ll make about the value of NMR reported subparticle sizes and diameters is particularly telling when it comes to insulin resistance. In the panel below, you can see that this person has small VLDL particles, small HDL particles, and LDL particles. Why is this interesting? The presence of increased large VLDL-P, large VLDL size, increased small LDL- P, small LDL size, reduced large HDL-P, small HDL size are early markers for insulin resistance, and such findings may actually precede more conventional signs of insulin resistance (insulin levels, glycemic abnormalities) by several years. In other words, the number and size of the lipoprotein particles is perhaps the earliest warning sign for insulin resistance.

In summary

1. The measurement of cholesterol has undergone a dramatic evolution over the past 70 years with technology at the heart of the advance.
2. Currently, most people in the United States (and the world for that matter) undergo a “standard” lipid panel which only directly measures TC, TG, and HDL-C. LDL-C can be measured directly, but is most often estimated.
3. More advanced cholesterol measuring tests do exist to directly measure LDL-C (though none are standardized), along with the cholesterol content of other lipoproteins (e.g., VLDL, IDL) or lipoprotein subparticles.
4. The most frequently used and guideline recommended test that can count the number of particles is the NMR LipoProfile. In addition to counting the number of particles – the most important predictor of risk – NMR can also measure the size of each lipoprotein particle, which is valuable for predicting insulin resistance in drug naïve patients, before changes are noted in glucose or insulin levels.
   

I know some of you are getting antsy. I thank you for your patience, and I hope you appreciate that it was a necessary step to get through this somewhat technical material and nomenclature. Next week we’ll get to the “fun” stuff – what does all of this cholesterol have to do with heart disease?

In addition, we’ll get further into the importance of using LDL-P as the best predictor of risk. If anyone wants to read up on another very important topic, especially for understanding why LDL-P is more important to know than LDL-C, get familiar with the concepts of discordant and concordant variables. You’ll be hearing a lot about these.
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Read the complete article here.

Be sure to read his complete series on cholesterol.

Previously, in Part I and Part II of this series, we addressed 4 concepts:
#1 — What is cholesterol?
#2 — What is the relationship between the cholesterol we eat and the cholesterol in our body?
#3 — Is cholesterol bad?
#4 – How does cholesterol move around our body?

Back to basics: Coronary calcium

Posted on January 30, 2012 by Dr. William Davis

After having my attentions pulled a thousand different directions these past 6 months, with the release of Wheat Belly and all the wonderful media attention it has attracted, I’ve decided to pick up here with a series of discussions about the fundamental issues important to the Track Your Plaque program and prevention and reversal of coronary atherosclerotic plaque.

I fear the discussions at times have drifted off into the exotic. This is great because this is how we learn new lessons, but we can never lose sight of the basics, else we risk losing control over this disease.

Imagine you’ve got a beautiful new car. You wax it, gap the spark plugs, rotate the tires, etc. and it looks brand-new, just like it came off the dealer’s lot. 50,000 miles pass, however, and you realize you’ve forgotten to change the oil. Ooops! In other words, no matter how meticulous the attention to transmission, tires, and paint job, neglect of the most basic responsibility can ruin the whole thing. We can’t let that happen with heart health.

If we propose to reverse coronary atherosclerotic plaque, we’ve got to have something to measure. First, it tells us whether we have atherosclerotic plaque in the first place, the stuff that accumulates and blocks flow and causes anginal chest pains, and ruptures like a little volcano and causes heart attacks. Second, it gives us something to track over the years to know whether plaque has grown, stopped growing, or been reduced. Without such a measure, you will be driving without a speedometer or odometer, just guessing whether or not you’ve gotten to your destination.

Of course, the conventional approach to heart disease and heart attack is not to track atherosclerotic plaque in your coronary arteries, but to track some distant “risk factor” for atherosclerotic plaque, especially LDL cholesterol. But LDL cholesterol is flawed at several levels. First, it is calculated, not measured. The nearly 50-year old Friedewald equation used to calculate LDL cholesterol is based on several flawed assumptions, yielding a value that can be 20, 30, or 50% inaccurate as a rule, only occasionally generating a value close to the real value. (No point in publicizing this problem, of course: Why compromise a $27 billion annual cash cow?) It also ignores the effect of diet. (No, cutting fat does not reduce LDL for real, only the calculated value. Cutting carbohydrates, especially wheat–”healthy whole grains”–slashes measured LDL values like NMR LDL particle number and apoprotein B.)

But all risk factors are, at best, snapshots of the situation at that moment in time. They change from day to day, week to week, month to month, year to year. If you do something dramatic in health, like lose 50 pounds, you can substantially change your risk factors values, like LDL cholesterol and HDL cholesterol. But you may not modify the amount of atherosclerotic plaque in your heart’s arteries.

Measuring the amount of atherosclerotic plaque in your heart’s arteries is, in effect, a cumulative expression of the effects of risk factors up until the moment of measurement.

There are several stumbling blocks, however, in the concept of measuring coronary atherosclerotic plaque. We cannot measure all the unique components of plaque, such as fibrous tissue like collagen, or degradative enzymes like collagenases, or inflammatory proteins like matrix metalloproteinase, or the debris of hemorrhage and inflammation. We struggle to contemporaneously mix in measures of bloodborne inflammation, coagulation and viscosity, and physiological phenomena of the artery itself, like endothelial dysfunction, medial (muscle) tone, and adventitial fat.

So we are left with semi-static measures of total coronary atherosclerotic plaque like coronary calcium, obtainable via CT heart scans as a calcium “score.” No, it is not perfect. It does not reflect that moment’s blood viscosity, it does not reflect the inflammatory status of the one nasty plaque in the mid-left anterior descending, nor does it reflect the irritating sheer effects of a blood pressure of 150/95.

But it’s the best we’ve got.

If anyone has something better, I invite you to speak up. Carotid ultrasound, c-reactive protein, ankle-brachial index, stress nuclear studies, myoglobin, skin cholesterol, KIF6 genotype . . . none of them approach the value, the insight, the trackability of actually measuring coronary atherosclerotic plaque. And the only method we’ve got to gauge coronary atherosclerotic plaque that is non-invasive and available in 2012? Yup, a good old CT heart scan calcium score.

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Why small LDL particles are the #1 cause of heart disease in the US

Posted on September 15, 2011 by Dr. William Davis

Ask your doctor: What is the #1 cause of heart disease in the US?

Let’s put aside smoking, since it is an eminently modifiable risk and none of those crazies read this blog anyway. What will your doctor say? Most like he or she will respond:

High cholesterol or high LDL cholesterol

Too much saturated fat

Obesity

Pfizer, Merck, AstraZeneca and their kind would be overjoyed to know that they can add your doctor to their eager following.

I’d tell you something different. I would tell you that small LDL particles are, by far and away, the #1 cause for heart disease. I base this claim on several observations:

–Having run over 10,000 lipoprotein panels (mostly NMR) over the past 15 years, it is a rare person who does not have a moderate, if not severe, excess of small LDL particles. 50%, 70%, even 90% or more small LDL particles are not rare. Over the course of a year, the only people who show no small LDL particles are slender, athletic, pre-menopausal females.

–In studies in which lipoproteins have been quantified in people with coronary disease, small LDL particles dominate, just as they do in my office. Here’s a 2006 review.

–Small LDL is largely the province of people who consume carbohydrates, such as the American population instructed to “cut fat and eat more healthy whole grains.” Conventional diet advice has therefore triggered an expllosion in small LDL particles.

–When fasting triglycerides exceed 60 mg/dl, small LDL particles increase as a proportion of total LDL particles. This includes the majority of the US population. (This ignores postprandial, or after-eating, triglycerides, which also contribute to small LDL formation.)

If you were to read the data, however, you might conclude that small LDL affects a minority of people. This is because in most studies small LDL categorize it as either “pattern B,” meaning exceeding some arbitrary threshold of percentage of small LDL particles, versus “pattern A,” meaning falling below that same arbitrary threshold.

Problem: There is no consensus on what percentage of small LDL particles should mark the cutoff between pattern A vs. pattern B. In many studies, for instance, people with 50% small LDL particles are called “pattern A.”

If, instead, we were to set the bar lower to identify this highly atherogenic (atherosclerotic plaque-causing) particle at, say, 20-30% of total, then the number or percentage of people with “pattern B” small LDL particles would go much higher.

I see this play out in my office and in the online program, Track Your Plaque, every day: At the start eating a low-fat, grain-filled diet with lots of visceral fat (“wheat belly”) to start, they add back fat and cut out all wheat and limit carbohydrates. Small LDL particles plummet

 

About Dr. William Davis

Dr. Davis is Medical Director of the Track Your Plaque program and advocate of early heart disease prevention and reversal. He practices preventive cardiology in Milwaukee, Wisconsin.

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