The Peter Attia Drive - #240 ‒ The confusion around HDL and its link to cardiovascular disease | Dan Rader, M.D.

Episode Date: January 30, 2023

View the Show Notes Page for This Episode Become a Member to Receive Exclusive Content Sign Up to Receive Peter’s Weekly Newsletter Dan Rader is a Professor at the Perelman School of Medicine at the University of Pennsylvania, where he conducts translational research on lipoprotein metabolism and atherosclerosis with a particular focus on the function of high-density lipoproteins (HDLs). In this episode, Dan goes in-depth on HDL biology, including the genesis of HDL, its metabolism, function, and how this relates to atherosclerotic cardiovascular disease (ASCVD). He explains why having high HDL-C levels does not directly translate to a low risk of cardiovascular disease and reveals research pointing to a better way to measure the functionality of HDL and predict disease risk. He also goes into detail on the role of HDL in reverse cholesterol transport and the benefits this has for reducing ASCVD. Additionally, Dan discusses the latest thinking around the association between HDL cholesterol and neurodegenerative diseases and ends the conversation with a discussion of how the latest research on HDL provides a promising outlook for ongoing trials and future therapeutic interventions. We discuss: The lipidology of apoB and apoA [4:00]; A primer on the high-density lipoprotein (HDL): genesis, structure, and more [9:30]; How the lipoprotein system differs in humans compared to other mammals [20:00]; Clarifying the terminology around HDL and apoA [25:30]; HDL metabolism [31:45]; CETP inhibitors for raising HDL-C: does it reduce CVD risk? [34:45]; Why it’s so important to have hard outcome trials in the field of cardiovascular medicine [42:30]; SR-B1: an HDL receptor important for cholesterol efflux [48:00]; The association between HDL levels and atherosclerosis: are they causally linked? [53:15]; How insulin resistance is impacting HDL, and how HDL-C provides insights into triglyceride metabolism [58:00]; Disappointing results from the studies of niacin—a drug that raises HDL-C and lowers apoB [1:08:15]; HDL lipidation, dilapidation, and reverse cholesterol transport [1:12:00]; Measuring the cholesterol efflux capacity of HDL: a better predictor of ASCVD risk than HDL-C? [1:22:00]; A promising new intervention that may promote cholesterol efflux and reverse cholesterol transport [1:32:45]; The association between HDL cholesterol and neurodegenerative diseases [1:34:00]; Challenges ahead, a promising outlook, and the next frontier in lipidology [1:44:45]; and More. Connect With Peter on Twitter, Instagram, Facebook and YouTube

Hey everyone, welcome to the Drive Podcast. I'm your host, Peter Atia. This podcast, my website, and my weekly newsletter, I'll focus on the goal of translating the science of longevity into something accessible for everyone. Our goal is to provide the best content in health and wellness, full stop, and we've assembled a great team of analysts to make this happen. If you enjoy this podcast, we've created a membership program that brings you far more in-depth content if you want to take your knowledge of this space to the next level. At the end of this episode, I'll explain

what those benefits are, or if you want to learn more now, head over to peteratia MD dot com forward slash subscribe. Now, without further delay, here's today's episode. I guess this week is Dan Raider. Dan is a professor of molecular medicine at the Perlman School of Medicine at the University of Pennsylvania where he conducts translational research on lipoprotein metabolism and atherosclerosis, with a particular focus on the metabolism and most importantly, function of high density lipoproteins, HDLs. Dan has received numerous awards and has been elected to the American Society of Clinical Investigation, the Association of American Physicians, and the National Academy of Medicine. He also currently serves on the Board of Directors

of the International Society of Atherosclerosis, the Board of External Experts at the National Heart, Lung, and Blood Institute, and the Advisory Board for the Clinical Research of the NIH. In this episode, we focus our entire conversation around high-density lipoprodenines or HDLs. Now, as any listener of this podcast will know, we have no shortage of content around lipids. And we focus a lot of that energy on the 8-O-B side of the family. That is, just to some extent, VLDLs and of course LP-Litelae, which is a subset of LDL. However, this is the first time we're doing a dedicated podcast on HDLs. Now, the reason for that, in other words, the reason for that

disparity is largely because HDL biology is so much more complex and we know so much less. I mean, at the highest level, I think people generally think of HDL as quote unquote the good cholesterol, but you've no doubt heard me rail on this stupidity of such a designation. But there is clearly something good about HDLs as in the LIPA proteins, not the cholesterol. And in this discussion, we really talk about everything from the biology of the HDL, its genesis, its origin, its metabolism, its life cycle, and of course, its function. And we also talk about why it has been so complicated to use pharmacologic interventions on the HDL side of the equation to impact atherosclerosis. Conversely, of course, it's been

the easiest thing, I would say, in medicine medicine and perhaps the greatest success of modern medicine, especially as it comes to cardiovascular disease has been our ability to manipulate the ApoB side of the equation as opposed to the ApoA side of the equation. That's one thing that's going to be important here and we get into the terminology when we talk about ApoA in the context of HDLs, we're talking about APO A as in a big A, which has no bearing whatsoever to APO A, the thing that of course defines an LP, little A. But I digress, that is simply one of the many details we get into. So as I said, we're going to talk here about the genesis of the HDL, the structure, its metabolism.

We talk about the differences between HDLs, LDLs, the difference between the HDL measurements, what exactly is HDL cholesterol versus APOA concentration versus HDL particle number. Talk about the idea that having a high HDL means you don't need to worry about cardiovascular disease and how that's obviously going to be bunk. I'm just going to let the cat out of the bag on that one. We speak about C-TEP inhibitors, which are a class of drug that have been repeatedly used to try to increase

HDL cholesterol with the hopes that that would reduce cardiovascular disease. And finally, we end the conversation around some of the new thinking around HDL and neurodegenerative disease, something again I learned a lot about. So without further delay, please enjoy my conversation with Dan Radar. Well, Dan, thank you so much for making time to join us on the podcast today. Tom Dayspring really recommended you highly and anytime Tom Dayspring says to me, you should have so and so on the podcast to talk about anything that has to do with lipids, I immediately pay attention.

Tom's an amazing guy. Now in particular, the subject matter I want to explore with you today is probably the area of lipidology that I personally am the least familiar with. And that has to do with kind of one half of the lipid family. You know, I explain this to patients as they're broadly speaking to families, the ApoB family and the ApoA family. We spend obviously so much more time talking about the ApoB family based on, I think, two families, the Apo B family and the Apo A family. We spend obviously so much more time talking about the Apo B family, based on I think two things. One is our clearer understanding of it and two, the direct and causal relationship to pathology.

But I often sort of waffle a bit when I'm talking about the Apo A side of the house. That's really why I'm excited to be sitting down with you today. But perhaps for the person listening to this who doesn't even understand what a lipoprotein is yet, never mind what the two families are that we're talking about, can you take it back to the beginning for where do these things fit into the broader architecture of our existence? Lipoproteins are these big complexes

that really are evolved to transport lipids within the blood. You know, lipids are like oil. They don't mix well with water. You know, oil droplets float to the top of a puddle. We wouldn't be able to transport lipids in the blood if we hadn't evolved a mechanism to do that. And lipoproteins are basically the mechanism to do that. As their names suggest, they're lipoproteins.

They have lipid in the core, and then they have proteins that dot the surface. That allows them to be transported in very complex and sophisticated ways within the bloodstream in terms of how they're metabolized, in terms of the receptors they bind to, and in terms of the specific proteins and lipids that they carry. You've talked a lot about apolipoproteins. I've listened to several of those podcasts. They're really fabulous. And of course, the apobelipoproteins really primarily transport or evolve to transport triglycerides as a source of energy, both from the gut to adipose and muscle and heart, as well as from the liver during times when we're fasting. You've talked a lot about that. And as you pointed out, they are very important in terms of causal relationship to

arthroscopic cardiovascular disease. HDL, which we're going to be talking about today, is also a lipoprotein. It's a very complex lipoprotein. It doesn't have this key protein apoby. That's what differentiates them. That's why we often refer to the apoby containing lipoproteins, and then HDL is the other half as you said. And HDL really is characterized by a different protein called A-Buy-1. HDL also transports lipids, especially cholesterol and some other complex lipids, and we'll be getting into that more. But essentially, lipo proteins are lipid transport vehicles within the blood and really evolved to do that function. A couple of things. I'll just throw in for folks. Again, maybe new to some of this, which is all of this stuff we're talking about is so important in terms of these light

proteins because unlike glucose, electrolytes, things that we kind of take for granted that are water soluble, we use our circulatory system as the freeway to move these things around. And unfortunately, as you said, because of the insolubility of both cholesterol, triglyceride lipids, et cetera, we have to come up with this more complicated system. I think the second thing I'll throw in just for folks because it could get confusing with the nomenclature,

when you talk about APOA, I wanna make sure people understand we're not talking about LP, Little A, which has another APOA, Little A. So maybe just clarify for folks the difference between those two, because we'll do our best to not talk about APO, Little A, today at all. Yeah, APO, Little A, or LP, Little A, is really important, but not the topic of today's discussion.

I think it's Peter already said, the main thing to remember is that that A is lower case, whereas the A we're talking about today is uppercase. I know it sounds crazy. It's just the way things evolve historically, but we're talking about a APOA uppercase A, and the most important one has a one after it, So, APO A1. So, you're going to hear us refer to APO A1 a lot totally different than the LP Little A, which is also an important topic, but not for today. Yeah, it's a shame that the lipid community back in the 60s, 70s and 80s didn't hire kind of a PR firm to sort of help with naming of this stuff because it is hands down some of the most complicated nomenclature. And if they realized back then, I'm half joking,

that it wouldn't be a bad idea for patients to actually understand this stuff. I think we could have done better. Okay, so you've already alluded to kind of different APOAs and APOA1. Again, that's APO capital A dash Roman numeral one. It's the most important, but let's go back even further than that. So again, not to draw the APO Bs into this again, but we kind of technically now have these two lineages of APO Bs, right? We have the APO B100, and virtually all of the time that we say APO B, we really mean APO B100. And that's to contrast it with APO B48, the one that you kind of alluded to very briefly as transporting

chile microns from the gut for the use of energy. Can you talk a little bit about the, or not a little bit, maybe a lot a bit, about the genesis of the high density like a protein? What is its parent, where is it formed, and how does it move through its evolution, such that, you know, when we're measuring HDL cholesterol something we'll talk about in a minute we have a sense of where it came from in the same way that we understand the LDL's ideals and LDL's. With all due respect to the APOB community and I'm part of that community too I would say the metabolism of HDL is perhaps an order of magnitude even more

complex and that's obviously what we're going to be working our way through today. So I'll start relatively simplistically, and then Peter, I know you'll lead me to increasingly more detail about the process. And by the way, I don't think anyone's going to disagree with that, Dan. Many, I don't think anybody who spent even a modicum of time looking at this literature, it is hands down the most confusing stuff in the lipids base. Again, I'll start with APO A1 as the key protein with HDL. The analogy is to APO B as Peter alluded to. Although one big difference is APO B, as your listeners know, APO B stays with that particle throughout its lifetime.

So it gets made by the intestine as B48 or the liver as B100. It stays with that particle. There's one molecule of this huge apobet protein and that it basically then ultimately gets taken up, mostly by the liver after it's time in the blood. Apob1 is also made by the intestine and the liver. There's some parallelism there. It also is a core protein, essentially, of HDL.

But unlike apob, there are several molecules of APOA1 and any given HDL particle. We'll probably talk more about that, but anywhere from one to four, maybe in some cases, a little bit more. And also, APOA1 doesn't stay with the particle. It can exchange onto other types of mostly HDL particles, but even sometimes onto APOB containing

like lipoproteins. What we've really learned in the last decade or so is that APOA1 is put into the blood by either the intestinal enterocyte or the hepatocyte in the liver as more or less of free protein. So it's secreted as a protein. One of the first steps that has to happen for HDL to be formed is that HDL, once it comes out of the cell, engages with a key transport protein called ABCA1. And Peter, stop me if I'm already getting too detailed.

ABCA1, we'll come back to that. Which its role, ABCA1's role, is to take lipid, cholesterol, and other types of lipids called phospholipids from the cell and export them to the newly secreted APOI1. So APOI1 has to acquire lipid, particularly phospholipid and cholesterol, soon after it's been secreted in order for the so-called nascent or early HDL to start forming. We'll come back to this when we talk about genetics, but we know this because humans that lack ABCA1

have virtually undetectable HDL. And that is because they cannot, they make plenty of A-Boy 1, but they cannot protect that A-Boy 1 with lipid once it's secreted and it goes out like a rocket from the blood. Basically, can't almost, can't measure it in the blood. So that's the first step in terms of HDL biogenesis, if you will. What other phenotype do those patients have? So in the absence of cholesterol accumulating in what would be an HDL, does it end up where more of it ends up in the LDL or back in the liver?

Where does it go? This is the value of studying these very rare human genetic disorders. It just tells us a lot about what specific proteins or genes are doing. This disorder, which we can talk about the history, it's a fasting history, it's called Tanger disease, it's named for a small flat bizarre island in the middle of the Chesapeake Bay, where the first patient was discovered. This disorder is primarily associated with accumulation of cholesterol in macrophages throughout the body. So tonsils spleen, places where there are a lot of macrophages,

the cholesterol really builds up. And they have huge sort of orange colored tonsils. They have big spleens that sometimes rupture. They have big livers because there are a lot of macravages and macravages like cells in the liver. And they also have some neurologic type issues, especially neuropathy, which maybe we'll leave to later when we start talking about HDL

and its relationship to the nervous system. They may or may not have some increased risk of atherosclerotic cardiovascular disease, but as we'll talk about that relationship between HDL and atherosclerosis is quite complex. This disorder has helped to inform that a little bit. Another sort of structural question, Dan. If you were to take a quasi-mature garden variety HDL particle, and again, it's more complicated there because of what we're talking about,

and put it next to a comparable LDL particle. What's the difference in size and can you explain a little bit why they come up with this different name of high versus low density? What specifically is that referring to? That's a great question. Again, this is a historical artifact of how light proteins were discovered and named. First of all, HDLs are much smaller. I would say you know, in the neighborhood of one fifth to one tenth the size of an LDL and of all, HDLs are much smaller, I would say, you know, in the neighborhood of one fifth to one tenth the size of an LDL, and of course much yet smaller than triglyceride rich lipoproteins. You know, lipoproteins have lipid, as I mentioned earlier, lipid floats, so lipids create buoyancy to particles. And the way lipoproteins were discovered is that when you subject plasma to spinning, the lipoprotein spin to the top. And they spin to the top at different rates under different forces.

The low density lipoproteins have more lipid or bigger, more lipid. They spin to the top more easily. The high density lipoproteins still have lipid. They do spin, but they don't spin quite as quickly or easily, so they're higher density than the low. And then in another artifact of sort of the history, Peter, after low density was discovered, something even lighter that spun up even more easily was discovered. And of course, if low was already taken, so that had to be called very low. And so there's the very low density lipoproteins that you've also talked about at length.

And then finally, the chala microns, the huge ones that come from the intestine, they're just so light and so buoyant that instead of calling them very, very, very low, they were just given the name chala microns. How long does an HDL particle last? And does it mean the same thing given that it's transferring its APOA in a way that makes it a little different from, as you said, the way the VLDL to IDL to LDL always stay with one lipoprotein APOB that allows us to kind of track its life? Before we get into kind of how HDL is removed from the blood, could I take a little bit more time to explain what happens after that? Absolutely. APOA1, quote unquote, nascent HDL particle gets formed because there's actually a bunch of stuff that has to happen first before we start talking about how the HDL then gets removed from the blood. So the APOA1 or then what we call the nascent HDL has, you know, APOA1 has phospholipids and it has what I'm going to call free cholesterol. And this is a concept that you may or may not have addressed before, I can't remember,

which is cholesterol itself, as a molecule, can have a fatty acid attached to it. When you attach a fatty acid to a hydroxyl moiety on the cholesterol molecule, you create what we call a cholesterol ester. And the cholesterol ester is even more hydrophobic or oily than the free cholesterol because it has this additional fatty acid sticking off it which is obviously oil. It's absolutely critical for the HDL maturation process to have this fatty acid attached to the cholesterol after the nascent HDL has been formed. And there's another key enzyme that is responsible for that so-called leceton cholesterol acyl transferase. It's the last time I'm going to say that. I'm just going

to call it LCAT. That's its nickname, LCAT, LCAT. So LCAT rides on the HDL particle, on that nascent particle. It takes a fatty acid from one of the phospholipids on the particle, and it transfers that fatty acid to the cholesterol and creates the cholesterol ester. Remember, that cholesterol ester is more oily. It actually then moves to sort of the center of the particle because it wants to be by itself and not interacting with the water. And it starts to form the core of the HDL particle. So the core of the mature, what I'll call the mature HDL particle, is basically cholesterol esters that are entirely dependent on LCAT for their formation. Another rare condition are people who lack LCAT genetically.

And as you might maybe expect from what I just said, people who lack LCAT, who can't do that process of esterifying the cholesterol, have extremely low levels of HDL, not quite as low as Tanger disease, but very low, and simply can't form the mature HDL particles that we see in most people. So again, we learn very important things about the key role of that enzyme. That ultimately leads to the mature particle that then is really what we're measuring as HDL cholesterol in people,

is this mature HDL particle where most of the cholesterol we're measuring is the cholesterol ester in the core. Just to make sure we're clear on semantics, when you say that those folks have very low HDL, you mean low HDL cholesterol or a low concentration of APOA1 or any of the APOAs? They have low HDL cholesterol.

So again, Tanger disease, HDL cholesterol is one. Wow. Virtually undetectable. L-cat deficiency, HDL cholesterol is 10. Also, as we'll talk about, quite low compared to normal. They also have low AP-way 1 levels. And part of that is because part of the metabolism of 8-way 1, which is where you were going, is that it's affected by the mature HDL and the cholesterol and the core. So when you don't make that cholesterol ester,

that 8-way 1 is much more rapidly removed from the blood. It's not the normal metabolism, and it basically the kidney sees that 8-Boy-1, which doesn't have much cholesterol on it and actually filters it and degrades it. Not surprisingly, the biggest issue with L-Cat deficient patients is that get renal disease, than a serious progressive chronic kidney disease in these patients that leads usually to kidney transplant related to something around the lipid metabolism and the kidney as a result of the lack of L-cat activity and a starification of cholesterol. Before we go deeper on this, I just want to ask if there are any insights we have

or how parallel is this system in other mammals, whether it be primates, you know, dogs, mice, etc. Because obviously a lot of these animals have a very different apob side of the equation. I'm curious as to how similar they are on this front. No, it's a great question. Apoy one is pretty highly conserved among, certainly among mammals. In many lower level mammals, the primary lipoprotein is HDL, that clearly includes apoy one. I'll just take mice. Obviously a major model system for human, for biomedical research. Mice, I'd say 90% of

cholesterol in the blood of mice is in HDL, even more than 90% in a normal mouse. That is the primary lipoprotein in mice. It's true for dogs, it's true for really all mammalian species. I mean, once you start getting below mammals, things change a little bit in terms of the lipoprotein metabolism. Probably we won't get into that today. But there's even HDL-type particles in much lower species leading to one of the kind of thoughts that HDL is kind of like the primordial lipoprotein, and that the APOB system kind of evolved subsequently, but that HDL probably very early evolved as a transport mechanism for lipids within circulatory

systems in lower level species. You know, this, to me, is one of the most interesting kind of teleologic slash evolutionary questions, which is, was God just pissed off at us? And is that why we as humans have ApoB? Because I can't, I've never really heard a convincing explanation for why humans have ApoB when it seems that every other species gets away without it, right? As you said, I mean, if a mouse or a primate or a dog who clearly have similar energetic requirements can use their APOAs, IEHTLs, for lipid transport and energy mobilization. Why did we need to evolve with this particle that in the short term really doesn't cause us any trouble, of course, but long enough,

it doesn't. And so, you know, one answer might simply be that evolution had no concern for the longevity of our species. That's for sure. That's already true. But there is some other short-term benefit from APOB that may be outweighs the long-term death that it brings our species via ASCVD. I do want to be clear. My stogs, pigs, rabbits, they all make APOB in both the gut and the liver.

So it's not that these species don't have APOB. The differences in terms of looking at the steady-state plasma levels is that their APOB system is just really markedly revved up. They take care of their business. They just must clear it up. And they make it. And it serves a similar role, you know, in terms of transporting triglycerides as a source of energy to tissues in the body

that need energy and to adipose tissue for storing it. It's just that the rate at which they do that is much more efficient, whereas in humans, for reasons we could talk about, but aren't totally clear, but certainly have to do partly with our lifestyle. Humans are not nearly as efficient at clearing the apobie containing lipoproteins, and they hang around, especially as LDL, as a byproduct of this metabolism in a way that then ultimately leads to, of course, arthroscopic vascular disease. We do need apobie, honestly. Mammals evolved apobie because one, we have to be able to efficiently capture fats in the diet for energy purposes.

Very efficiently, that's what the colonel microuns allows to do. And during fasting, which of course in evolution sometimes lasted a long time, we have to be very parsimonious about parsing out that available fat we have in our adipose stores, back out to heart and muscle to be able to have as a source of energy. That's what April B 100 and the liver does. We need these, especially in evolution, the latter. We need it very substantially. But now, unfortunately, in a modern times, the April B system is mostly a problem, not something we need. And I think the way you stated it earlier is obviously more accurate and perhaps I

want to just make sure I'm restating it without being quite as tongue in cheek. It's really not that we don't need April B. It's that we could be clearing it at a much greater and more efficient rate than we are, and we could more mimic primates and other mammals and walk around with an apoB concentration of, call it, 20 milligrams per desoliter instead of 100 milligrams per desoliter, a five full difference, which is about, that's the difference between getting atherosclerosis in our lifetime and never getting atherosclerosis in our lifetime. I agree with that.

And that's the interesting thing because even absent kind of the quote unquote lifestyle things that will raise APOB, it just occurs with aging. You know, Peter Libby has written about this very eloquently and children, Neonates, have APOB concentrations that are just like those of these other animals. And all things equal as we age, APOB just goes up. That concentration goes up as you say. We're less efficient in the way we clear APOB from circulation. Anyway, it's a fascinating topic and it's, I know not what we're going to talk about today

because it's one of speculation and we could speculate, but there are actually lots of things today that we don't have to speculate about as it comes to APOA and HGL. I want to go back to kind of the nomenclature a little bit because I know I'm still kind of confused on some of this. And I think it's going to be a little challenging because we're sometimes talking about Roman numerals and we're sometimes talking about numbers. But is there just an APOA1 and 2 in the Roman numeral, lipoprotein, APO lipoprotein cover? No. So it goes to 2,3.

Two other APO A's that at least we know about, or we talk about our APO A4 and APO A5. Don't ask me what happened to APO A3. That's a mystery I've never been able to solve. Okay. But anyway, it's another one of those. APOA4, we don't really know what it does. APOA5, I can't remember if you've talked about, but APOA5 has a very important role in stimulating lipoprotein lipase and in the metabolism of triglyceride-rich lipoproteins. It rides on HDL, but it also rides on triglyceride-rich lipoproteins.

And people who lack APOA5 have really high triglycerides and are also at major risk for atherosclerotic vasoport胎. So anyway, again, maybe not the topic for today, but it does illustrate one point, Peter, maybe that'll make, which is that HDL as a molecule, as a lipoprotein, I think of as a platform. It's a platform for all sorts of proteins and lipids that get transported by HDL in the blood, and then transfer off HDL to other things, to able-be container lipoprotein. So my example of April A5, it's named AAA because it was discovered on HDL, but its primary

role in metabolism is triglyceride-richlyperprotein metabolism. APO A5 is basically kept within the blood and stored, if you will, on HDL. But then when we eat a fatty meal, it transfers off to the triglyceride-richlyperproteins and it serves a role of promoting the hydrolysis of the triglyceride-rich lipoproteins, and it serves a role of promoting the hydrolysis of the triglycerides. So, I think that's a general concept for HDL that maybe we'll keep coming back to is this platform for all sorts of things that are doing different things that involve not only lipometabolism, but host defense, and other things that it evolved to do as a platform

for transporting things within the blood. Which I think explains at least in part the complexity of this system is it's much more dynamic than what we see on the apobeside. The apobeside as you said, it's a very monogamous relationship, right? I mean, you sort of get your apob in the liver and it's with you for life and you're not swapping, you're not trading, you're not increasing or decreasing, you're just marching through life. And the HDL is maybe the monogamy wasn't the best example because now I don't know what to come up with here because it's not just polygamy, it's even more complicated than that.

It's as you said, it's swapping, it's moving things around, it's carrying things that it doesn't really use, but loaning them out. It is indeed a complicated system. The next thing that seems to add a lot of confusion, at least to me, is some of the nomenclature around the numbering of the HDL particles. And I bring this up in part because various commercial labs, someone listening to this goes out and gets a really fancy, fancy blood test. We use Boston Heart Labs with our patients.

I know that this is, you know, something you see with a number of other labs. It might say things like HDL1, HDL2, HDL3. What's the relationship between that nomenclature and the APO-A1, APO-A2, APO-A4, etc? This is ridiculously complex. The short answer to your question is actually, believe it or not, there's no relationship. So the numbering of the apoliper proteins, or the apoliper proteins, one, two, four, five, is totally independent of the numbering

of the HDL particles, like especially HDL2 and HDL3, which are the classic two main, quote, subclasses of HDL. These are a different sizes and different densities of HDL that, again different sizes and different densities of HDL that again were isolated by centrifugation and by their flotation properties. HDL2 is bigger, HDL3 is a little smaller. HDL2 has mostly four molecules of APOA1.

HDL3 has mostly three molecules of APOA1, but those threes don't have anything to do with each other. And then to make things more complicated, there are other ways of sub-fractionating lipoproteins. I know you've discussed, like the NMR type of methodology that don't use HDL2 and HDL3, which frankly is a little outmoded now, but use things like very large HDL, large HDL, medium HDL, small HDL, very small HDL. HDL certainly comes in a whole series of sizes and densities that can be isolated by these different methodologies. There's been a cottage industry, as you know, Peter, of trying to relate these different fractions of HDL

to cardiovascular risk in a way that might give us some advantages in terms of trying to predict risk. We can come back to that, but I would say in general, I'm not very compelled that fractionating HDLs gives much clinically valuable information that allows us to predict risk. In contrast, I just want to say, in contrast to the APOB containing lipoproteins, where, you know, whether it's APOB or whether the, but I think the smaller denser LDL particles do have a relationship to increase risk, because you've discussed in the past. But I think the HDL fractionation is fascinating for understanding HDL biology and metabolism, but is relatively unimportant from a clinical relevance standpoint.

I'm actually glad to hear you say that because I was wondering if I need to be changing our clinical practice. In about 2016, we basically stopped paying attention to any of the HDL fractionation metrics. And really, it had to go out of our way to make sure that the lab was not running those because in some areas, we're running complicated labs. And we were sort of saying, look, we don't want any of this stuff. Hey, we don't want the additional cost of it. But more importantly, we don't think it's clinically actionable. We still get labs from other doctors who might have seen our patients in the past and they're chalk full of these things to which I don't

know what to do with them. But while we're on this topic of HDL's fractionation, unless you want to ask me another question, I wonder if I could use this as a springboard to continuing this complex saga of the metabolism of HDL's once they're formed and once they have cholesterol ester. And the reason we have this whole And the reason we have this whole sort of set of different size and density HDLs is because of the complex metabolism that occurs on the HDLs once they're in the blood and once they've kind of been made mature HDLs. I want to give you maybe a couple examples of that. One is that HDLs are acted upon by so-called lipases. And you've talked a lot about upon by so-called lipases.

And you've talked a lot about liproprotein lipase. It's critically important for triglycerid metabolism and energy metabolism. Liproprotein lipase has two other very close cousins that I don't think you've talked nearly as much about, appropriately, because they're more HDL-related. One is called, quote, hepatic lipase, and what is called, quote, endothelial lipase. We actually first reported a long time ago endothelial lipase as a member of this family. So both of these lipases, they're made in different places as their name suggests, but both of them fundamentally chew on HDL, specifically the phospholipids on HDL,

and result in modification of the phospholipid composition of the HCl particle in a way that does different things. It certainly changes the sizes of the HCls and they contribute to this distribution of different sizes. It changes the protein composition of the HCls in ways that we don't fully understand. And almost certainly has other important biological effects that we don't fully understand, but of course these evolve for some reason, one of which I'm going to get back to later when we talk about the central nervous system. So that's one. The lightpaces are really critical for HDL metabolism.

There's also this protein that I know you've had some discussion about cholesterol et cetera transfer protein. Happy to go into more detail. I'm sure we will because it's very relevant to HDL, but just for the metabolism part of it, this so-called CETP cholesterol transfer protein, essentially transfers cholesterol esters between able-be containing lipropotines and HDL.

And it's a major modifier of the HDL particle in terms of its size and composition. We know this because people who lacked CETP have hugely elevated HDL cholesterol levels, big time, like A over 100. That really told us that cholesterol ester transfer protein siphons cholesterol out of HDL. And when you don't have it,

you have a lot more cholesterol in HDL and just point back to our previous discussion of mice. Mice don't have CTP. So among the differences, one of the things is that mice have a lot more HDL cholesterol relative to APOB because they lack this CTP protein. I mean, I know we're going to come back to this, but I just, I worry that for the listener, this would be an opportunity missed if we don't dive into C-TEP a little bit because of the following. If we take a step back, a listener is going to be saying, Peter Dan, what the hell are you guys talking about?

Like I am so lost. The only thing I know is when I go to the doctor, the doctor says, my good cholesterol is high. I'm in good shape. So let's do this. Let's hit pause for one sec and acknowledge that a standard lipid panel spits out a bunch of numbers. Total cholesterol, LDL cholesterol, HDL cholesterol. And if you're lucky, VLDL cholesterol, non-HDL cholesterol and triglycerides, that's basically the standard metric.

If the lab is competent and they're using direct measurements, your HDL cholesterol, LDL cholesterol, and VLDL cholesterol should sum to your total cholesterol. And of course, your non-HDL cholesterol should be the same number as your total cholesterol, less your HDL cholesterol. I want to pause and also insert that with nomenclature, HDL is not a laboratory metric,

it is a lipoprotein, the laboratory metric is HDL, cholesterol HDL C, or if using NMR HDL P, HDL particle number, or APOA1, an analogy to measuring APOB. APOB, exactly. The units that you've been throwing around, of course, are the HL cholesterol. So a moment ago, you said, Hey, people with who are deficient in C-TEP could have HDLs over 100. Of course, that means HDL cholesterol over 100 milligrams per

desk liter. Now, there is an observation that goes back probably to the late 70s, right? I mean, it probably goes back to late 70ss early 80s, which is in some of the earliest framing camp cohorts, which observed the risk of ASEVD in five cities. We don't need to go into what framing him did in the first cohort. But what came out of that was higher HDL cholesterol was better than lower HDL cholesterol. In fact, that was four times greater if my memory serves correctly as a predictor of ASCVD than high LDL cholesterol was a negative predictor. Am I remembering that correctly? Yeah, that's about right. So by now we're talking about 82, 83, 84.

This has been a long time since I've looked at this stuff, but it emerges that hey, high HDL cholesterol seems to be positively associated with outcomes. And obviously, that's a big part of why HDL cholesterol became known as good cholesterol and LDL cholesterol became known as bad cholesterol. We'll obviously talk about why those terms are inaccurate. But it wasn't long until companies, drug companies were saying, hey, wait a minute, we know that if CTEP is inhibited, this enzyme is inhibited,

HCL cholesterol goes up, that must be a good thing, right? That led directly from this observation I mentioned that people who lack CTP genetically have these hugely elevated levels of HDL. One directly from the other, well, gosh, that must mean that if we could pharmacologically inhibit CTP, it would be a way to raise HDL. And of course, as you know, that absolutely turned out to be the case. But the story goes beyond that.

So the first company to do this was Pfizer, right? Wasn't the first C-TEP inhibitor. The first C-TEP inhibitor, Torsetropib, was from Pfizer, correct. And that was, you could take a skeptical view of the trial, which was, Lippitor was about to go off patent. And so they came up with a trial that was Lippitor by itself versus Lippitor combined with the CTAP inhibitor. And I remember actually, this was probably early 2000s, right, thinking, oh, this is super

M at this, this was, you know, back when I was in my surgical residency. So I was only tangentially interested in this, right? It wasn't my field. But interesting in that I knew my family history for ASCVD was high. So I was paying attention. And I remember thinking, well, this has got to be great, right? You got one drug that's going to lower LDLC, one that's going to raise HDLC. This is a can't mistrug. And then in September of 2006, low and behold, not only did it not get better,

it was slightly worse, right? Yeah, bombshell. So first, I want to emphasize, the genetics did predict what happened. That is, pharmacologic inhibition of CTP is extraordinarily effective at raising HDL a lot. In fact, putting people kind of over 100 HDL cholesterol like the folks who lack CTP. But you're absolutely right. There's a lot of excitement about that. A lot of excitement about HDL is the good cholesterol. And that trial, the first of several with different CTP inhibitors, not only didn't show benefit, but showed an

actual adverse effect. Now, it has to be said that in retrospect, we're pretty sure that adverse effect, more people, literally more people died. That adverse effect was due to off-target effects of the drug, not due to CTP inhibition itself. So that drug, of course, was discarded, but it didn't kill the field because the idea was, well, that was just a bad drug. Let's get a clean CTP inhibitor and see what that really does. I don't know if you want to ask me questions or I can continue the story. Yeah, let's go down that path because it's still a little, it got progressively less murky as time went on.

But let's go down the path of the evolution of C-TEP inhibitors, which let's be honest, it's for what we're almost fifth, but actually 16 years post the halting of that trial and the discarding of that drug, and we still don't have a C-TEP inhibitor on the market, right? We don't. There is still a C-TEP inhibitor that's in development, which I'll get to, but there is certainly not one on the market, right? We don't. There is still a CTP inhibitor that's in development, which I'll get to, but there is certainly not one on the market. As you know, Peter, and certainly probably many of our listeners do, three additional CTP

inhibitors were then taken into late-stage clinical development, including large cardiovascular outcome trials. To summarize the results of that, one of them was just flat, didn't do anything, didn't hurt people that didn't help. A second one was stopped early because it really didn't look like it was doing anything, helping. And the third one was followed through and did show about a disappointing 9% reduction in cardiovascular events. But I also want to point out that it lowered LDL and APOB pretty well also. 9% given that you also lowered LDL and APOB was not exactly exciting

and that drug was not taken further in terms of approval. I have to say one other thing that's just fascinating for sort of our field and maybe in general. One of these drugs, DelSetropib, that failed in its clinical trial. A detailed genetic study was done post-hoc. And there were individuals who were found to have a particular genetic variant that looked like on post-hoc analysis that group actually benefited from the drug. And that led to a subsequent attempt to do another trial focused specifically on people of that genotype with the CTP inhibitor, which was just reported out really within

the last year as a negative trial. That CTP inhibitor actually had two different trials, one in kind of a very focused precision medicine way, but it still didn't produce any benefit. This has been a long saga of using CTP inhibition to raise HDL as a way to reduce risk, and as we're going to be talking about more, it really is one of the key planks that has led to, I think, what is now rock solid, which is HDL cholesterol itself. The HDL cholesterol itself is not directly and causally protective against a throuschroidic cardiovascular disease. There's a lot of nuances there, which we'll come back to, but I feel pretty confident making that statement. And I just think this is a great opportunity to also talk about why it's so important to have hard outcome trials in the field of cardiovascular medicine.

I think it's true in all medicine. But let's go back and think about the first lipid lowering drug introduced in the United States in about 1950, right? Maybe 1959, something like that, right? So try Panerol, which I can never remember the name of the enzyme, but it's the enzyme that converts Desmosterol to cholesterol in the cholesterol synthetic pathway. Believe it or not, this is the name of the enzyme, but it's the enzyme that converts Desmosterol to cholesterol in the cholesterol synthetic pathway. Believe it or not, this is a sign of my aging, Dan.

I used to know the names of these enzymes. It was like, and it's not Delta 24 desaturates, but it's a cousin or derivative, isn't it? But anyway, so you had this drug, try Paranol that inhibited that enzyme, and low and behold, it lowered cholesterol. Now, this was for the listener. This is back in the day before we knew anything about the sub fractions of cholesterol.

So we just knew from some of the early work of Ansel Keys that if you looked at very, very high levels of total cholesterol and compared that to people who had very, very low levels of total cholesterol, there was a difference in outcome, cardiovascular outcomes. This was an observed finding. There was no intervention to test that. So this drug came along and it really lowered total cholesterol and it likely would have been lowering LDL cholesterol and APOB. It basically got approved on the basis of that without, of course, the hard outcome. So it gets approved. It goes into circulation and it's really only after it was approved, gosh, probably what eight

to 10 years later, that they had enough post surveillance data to say, you know what, this drug is lowering cholesterol, all right? But it's actually increasing mortality, cardiovascular mortality. Drug was pulled off the market. I don't know what is known about the Y, but I know Tom Despring and I have speculated that it was probably the Desmastral spike that was causing the problem. So you might have been trading one problem for a worse problem. Are you familiar with that drug or that story? Only peripherally, but of course, there's been a ton of research in Desmastral really

over the last several years. We now, based on that, the plausibility of the fact that the adverse effects were related to increasing this monster. I think is quite real. That very well, maybe the explanation. By the way, this is a totally tangent off topic point, but it's become quite invoked to use clomaphine or clomed for testosterone replacement in men. And the reason for it is, A, it's quite effective. So if you give clomaphyn, you are telling the pituitary to make a lot of LH and FSH, which of course is telling the testes

to make a lot of testosterone. This would certainly in the short term have the advantage of preserving testicular function unlike giving exogenous testosterone, which suppress preserving testicular function, unlike giving exogenous testosterone, which suppresses testicular function. And I think for short-term use, it probably isn't a bad thing. It's an off-label use, of course, for clomophine.

And we used to do it for this purpose, right? So if a guy was still considering reproduction or we were considering this a bridge treatment between testosterone, we would use it. But because we always measure desmosterol, the testosterone, the cholesterol, the cholesterol, these steriles, when we measure our patient's cholesterol levels, we noticed how high the desmosterol levels were getting in those patients. And we figured out it was pretty quickly, it was the clomad that was doing this.

And it would reverse, but it could take a year to return does Mosterol levels to normal after you stopped the clomad. So about four years ago, once we put two and two together, we stopped using clomad. But it's interesting to me that the use of that hormone has gone through the roof. There are now like clomad clinics opening up. And I don't understand why someone and we've done a lit search, I don't understand we just need to write this up. I think at some point, it's fascinating because I don't understand why someone has and put the two and two together, especially long term use. Again, I don't think using clomid for a couple of years is going to be problematic. And I certainly don't

think it's problematic for women using it for IVF when I think about a guy being on this for 10 years, and I'm talking to Desmastral levels going up by 20 fold. That would be concerning. We went back and tried to see if we could find out how high the Desmastral levels were in the not trials, but in the levels that if anybody had pulled serum or saved serum from those, because we couldn't find it. So we don't really know how high it was, but the assumption has to be that that drug and clomaphine both interfere with the same enzyme. Really fascinating. I would encourage you to write that up. So what's the point of that whole long-winded story? The point of that long-winded story is things can make a lot of sense until they don't, right?

That's for sure. Before we leave CTP inhibition, though, I do want to just remind your listeners that there is still one CTP inhibitor, Ovisetropib, that is still in clinical development. What differentiates it, it's much more effective at lowering LDL and APOB. We now no longer think that raising HDL cholesterol with CTP inhibition is going to help you. We don't think it hurts you, but we don't think it's going to help you. But we do think that there's still merit. Of course, we know there's merit to raising LDO cholesterol and APOB. You mean lowering.

I'm sorry, lowering. And perhaps the CTP inhibitor could be part of the armamentarium to do that. So we'll see. Stay tuned. Now Dan, maybe this was naive of me, but after the third failure of the C-TEP inhibitor, my very crude interpretation is not unique. I'm sure a lot of people have speculated this is that if at least part of the benefit of HDL involves stuff we're going to talk about, right, delipidation, reverse cholesterol,

transport, all of those things. And you slap a C-Tep inhibitor on one of those particles, thereby making it harder for the particle to E-flux its cholesterol, to get rid of its cholesterol, which is why you now measure much higher HDL cholesterol, which on the surface looks good. That could actually be problematic. In other words, if you see a lot of people in a room, it might be tempting to conclude that there's something awesome going on in that room. But what you don't know, if you can't measure all the ins and outs, is it

might be that those people are all stuck in the room because the door is locked. I know that's a bit of a crude analogy, but is there any merit to that sort of thinking around the different ways in which one might boost HDL cholesterol and how some of those could actually be deleterious if they prevent function? There actually is, but I think it's probably not relevant to CTP for reasons we can return to, but it is relevant to another protein that I think now is a good time to introduce, which is another key protein, a receptor that is basically the major HDL receptor.

The essentially the equivalent of the LDL receptor, but for HDL. And it's known as SRB1, another unfortunate nomenclature. But SRB1 is a receptor that basically is on a lot of cells, but the liver is the most important with regard to HDL. And it essentially binds the HDL particle, basically sucks the cholesterol ester out of the HDL particle, and then releases the cholesterol depleted HDL back into the circulation to go back around and do whatever it's doing. So the analogy I like to use, Peter,

is that of garbage trucks. So a very simplistic view of HDL, which we're going to return to because it's much more nuanced, is that HDL, to a certain extent, functions like garbage trucks that are picking up things, trash, in places where you don't want it, and returning it to the liver, dumping it off by S or B1, and then going back now empty to do more of its role. Again, it's more complicated than that, but that's an analogy. So this brings me back to SRB1. So there are humans who lack SRB1. What do you think the problem is? Very high HDLs. They have very high HDLs because they can't unload the dump trucks,

but they actually have increased risk of heart disease because they are not efficiently unloading that HDL. So very similar to your analogy of the locked room, you're not clearing the HDL and doing that normal process of recycling the trucks back to the periphery to do what they need to do. And so that's, I think, our best example of this concept of constipation of the system leading to high HDL but paradoxically to increase risk. And of course, the analogy would be that not the analogy, but the interpretation that

would be you would never want to develop an SRB1 inhibitor. Yes, it would raise HDL quite a lot, but it wouldn't protect against heart disease and probably would hurt people. Yeah, I remember the first time Tom shared a case study with me of a patient with presumably defective, not necessarily completely absent, but the SRB1 and this was one of those things where you could tell just reading the journal it must have been 40 years old,

but her LDL cholesterol was whatever it was, 100 milligrams per desk liter. Her HDL cholesterol was like 150 milligrams per desk leader. So she had quote unquote high cholesterol, but initially, you know, people assumed it was not of concern because the fraction that was HDL cholesterol was what was contributing to it. And of course, on further exam, I mean, she had very, very advanced atherosclerosis for woman her age.

Yep. Exactly. We don't see this often. I can tell you that I have not yet seen a case of that. Now, admittedly, my practice is not large, but we're certainly looking for it when we see people with high HDL cholesterol, typically, you know, north of 90. I guess we should talk about this as well, but we can do that in a moment after this question, which is why is there a sex difference between these two?

Because there is quite a significant sex difference between men and women in terms of HDL cholesterol. But at what point, Dan, as a clinician, do you start to worry about that? And if there are doctors or patients listening to this, when would you recommend that somebody go and get checked out for a genetic deficiency there? S&B1 deficiency is not common.

I completely agree with you. Huge project we've had for a long time now is essentially collecting people or consenting people with extreme high HDL with the goal of trying to find what the underlying genetic causes. I will tell you that that's how we found a few of these S-O-B-1 folks, but most of them don't have any identifiable gene that we can point to and say, here's the smoking gun, here's why your HGL is high. HGL is very heritable, meaning that there's a lot of genetic determinants of HGL,

but it's more about so-called polygenic inheritance, where multiple different genes, including SRB1, but not major genetic defects, just sort of more common variants, but many others, like some of the things we've been talking about, including a CTP and ABC1, all contribute to the heritability of the HDL cholesterol. But to get back to the clinical implications, my view at this point, we can talk more

about the data. It's pretty clear that high HDL is not uniformly associated with protection. And there are certain circumstances where that's true, especially if it's extremely high. Perhaps we'll get back to this, but there's a fair amount of evidence now, including a recent paper that individuals of African ancestry who tend to have Irish deals anyway, that the high-h deals are not as protective or maybe not even H-D HDL is not, you know, go get yourself sequenced to find out if you're deficient in S or B1. The odds of that are extremely low. But never use a high HDL as a reason for not using a statin or some other LDL lowering or preventive therapy.

That is, you should never be dissuaded from doing what you would have otherwise done in that patient just because their HDL is high. Does that make sense? Yeah, absolutely makes sense. And I have a very short paragraph in my book that'll be coming out in a while where I cite two Mendelian randomizations that, you know, really look at this in some detail. I think I've explained Mendelian randomization before in the podcast, but it's always worth, I think, a short explanation again in case you haven't heard it. This is basically a technique where you look for genes that impact traits that you are interested in. So in this case, because as you pointed out, HDL is very genetic.

That means that there are sets of genes that we would identify that predispose people to having very high versus very low HDL cholesterol. And because those genes are randomly occurring, you can look at that as though it's a natural experiment. And you can look at based on the natural occurring spreading or scattering of those genes, how our outcomes affected. And it turns out that low HDL cholesterol is not causally linked to atherosclerosis, and high HDL cholesterol, genetically high, is not causally linked to protection from ASCVD. So I think those are very important findings findings and I think it speaks to why, as you say, you can't use high HDL cholesterol as a reason to not treat in the presence of other

risk factors. I completely agree. It's interesting though that that persists, isn't it? I do find this to be probably top three most vexing discussions I have with other physicians, which is, I know as LDL cholesterol is 140 milligrams per desaliter, but God, I mean, as HDL is 80, I mean, you know, his ratio, I mean, that's when they say the ratio of total cholesterol to HDL or LDL to HDL, is such and such, and therefore, I don't need to treat. I mean, it makes me wish I had hair to pull out. You're absolutely right. And I'll just reiterate, high HDL is never a reason need to treat. I mean, it makes me wish I had hair to pull out. You're absolutely right. And I'll just reiterate, high HDL is never a reason not to treat someone who would have otherwise married a treatment.

I do want to make clear though, there are lots of people who are on the fence about whether to start a statin who have LDLs that are kind of borderline or even not that terribly high. And of course, we look at the whole patient, we look at the risk, we look at their calculated risk, other risk factors. But I'll just say that a low HDL in the setting of someone who you're genuinely on the fence about treating, perhaps could contribute to tilting toward, yes, I'm going to treat this person. In other words, a low HDL, at least in most populations, again, I think individuals of African ancestry

perhaps we have to be a little bit more careful about using HDL as a predictor. Maybe we'll come back to that. Otherwise, I think a low HDL can be used not absolutely but relatively to tilt toward being more aggressive, but only in the context of the overall risk profile. And obviously part of that Dan has to do with the relationship between low HDL cholesterol, if my memory serves me correctly, it's the HDL2 fraction and the association with that in insulin resistance. So there's no question that a phenotype, and as you point out, and we should come back to this, we don't even do this calculation for African-American patients because we've

long observed it's not helpful. But in non-African-American patients, the ratio of triglyceride to HDL cholesterol when both are in milligrams per desoleter is reasonably associated with insulin resistance. And the higher the ratio, the more insulin resistant they are. And obviously, that ratio is driven up by an increase in triglycerides in a reduction in HDL cholesterol. Why is it that, I mean, we've spent hours on this podcast talking about why insulin resistance would lead to or be associated

with high triglycerides. We haven't done the opposite or the reverse of that. What is it about IR that drives down H.D.L. too? I'll just say first though that one of the other analogies I like to make is that H.L. cholesterol, while not causally related to disease, is sort of like an HBA1C for cardiovascular risk factors. It's an integrator of information related to insulin resistance, related to triglycerides, related to inflammation that in one number, in most people, when it's low, it's telling you something about cardiovascular risk, even though itself, it isn't directly impacting on risk. So, in answer to your question, I think one of the big issues with H.D.L. cholesterol is that it's an inverse barometer of triglyceride efficiency of triglyceride

metabolism. And again, we talked about efficiency earlier. You can only learn so much from measuring a fasting triglyceride after a 12-hour overnight fast. It's useful. It's the way we do it in terms of lipid panels. But a lot happens after a fatty meal and a lot of that action is basically over in most people by 12 hours.

Some people are extraordinarily effective at clearing their dietary fat and even their liver drive fat, like mice, others are not so effective, but their fasting triglycerides may not necessarily even reflect that. The HDL cholesterol does reflect that. There's a complex, as we discussed earlier, complex frequent interaction and exchange

between triglyceride-rich lipoproteins and HDL with the net effect being the higher the triglycerides at any given time, the lower the HDL is as a result of the complex interactions. So if you can picture, we do a lot of these experiments where we bring in people and we give them a high fat milkshake. We draw blood multiple time points after that milkshake and we measure triglycerides and all sorts of other things. People differ a lot in their response to that high fat milkshake challenge.

The higher that triglyceride goes, the area under the curve, the lower the HDLs are. That relationship is extraordinarily strong. So just like HB1C, that HDL cholesterol is sort of reading out the 24-hour triglyceride metabolism much better than the overnight fasting triglyceride measurement is. A sort of YHB1C is better than fasting glucose. You know, that's the analogy. First of all, I've never heard that before and that might be if I

learn nothing else on this podcast and that is hands-down the most amazing thing I have learned not just today, but I learn nothing else on this podcast and that is hands down, the most amazing thing I have learned, not just today, but I'm gonna go out in the limon state this week, potentially this month. That is super fascinating. First of all, people on this podcast have probably heard me gripe over the lack of integral functions in biology.

You know, my background, of course, in math and engineering, we love integrals, right? As imperfect as the A1C is, as you said, it is an integrator, and it's so hard to find integrators. You know, Faratin sort of does it a little bit for iron, but not nearly as well. And, you know, the Holy Grail, of course, would be to find an integral of something like M-Tor activity or something like that. But I've never before been presented with or confronted with this. I want to make sure we all understand this a little bit better. So there's a couple of things

that you've said there. One is, if you stick an IV in a person's arm, let's just do that to go easy on them, right? So we're going to just be able to draw blood continuously or call it every five minutes without poking them. You bring them into a lab fasted. You measure the HDL cholesterol, you measure their trig. So let's just say they show up in the morning fasted, their trig are 100 milligrams per desk liter, HDL's 50 milligrams per desk liter. So most people would look at that and say, oh, that's great, person looks super healthy.

You give them a high fat shake and 30 minutes in, you just start measuring every couple of minutes, the concentration of those two things. I think most people somewhat familiar with metabolism would not be surprised to learn that the triglycerides will go through the roof. As an aside, anybody who has accidentally done a blood draw with their doctor after eating a meal when it was supposed to be fasting will be familiar with this, right?

We see this from time to time when patients make mistakes, you know, they have a big fat breakfast before the blood draw and you get their trigs back in their four or five hundred milligrams per desolate. But highly variable from person to person just to be clear. And highly meal dependent as well. Exactly. Very much depends on what the meal is. So in this experiment though, you're specifically giving them something to elicit the biggest

triglyceride response. In the example I gave Dan, let's say triglycerides go 125, 150, all the way up to 400 before they start to come down. That generates an area. You could integrate that on the curve. Can you give realistic ideas or values for what the HDL cholesterol would do during that period of time? The HDL cholesterol definitely dips during that time in a way that's proportional to roughly proportional to the triglyceride levels, but not anywhere near the same degree of magnitude. Right, simply because it's starting lower and you have a constraint bottom versus no constraint top. Starting lower, but also the dynamics of

its turnover are very different. I think the key point here is HCL is not just an integrator sort of acutely over that meal, but chronically. In your example, the HCL cholesterol might go from 40 to 38 or 37, which doesn't sound like a lot, after that one meal. But the repeated meals that that individual is eating that are high fat and that repeated triglyceride excursion has a more chronic effect, a little like glucose in HBA1C, that keeps taking the HDL down over time until you get to kind of a new steady state. So the acute effect on the HDL is real.

It's modest. The chronic effect on the HDL of that abnormal post-prandial triglyceride metabolism is quite substantial and that's why HDL is a good, and that's why H.D.L.S. is a good, as you say, integrator of this effect. And actually, what you just said, Dan, is kind of what I was hoping to go to next, which is, if everything I said were true, that might not be enough to explain the full integral function, because it probably captures some of what's happening outside of the meal. Just as hemoglobin A1C doesn't only reflect what would be captured in an oral glucose tolerance test,

it captures what's happening over 90 straight days when you're eating, when you're not eating. Exactly. And that's also why there's a very strong statistical relationship between fasting triglycerides and low NLHDL. So the fasting triglycerides themselves are still affecting HL metabolism. It's just that the post-prandial part is also a key component. So let's then again, just one more time sort of reiterate the lagging nature of HDL cholesterol through HDL biology to what's happening with the efficiency of maybe for lack of a better word, lipid partitioning. The lagging nature, meaning the integrating over time. Yeah, exactly. Let's use an example.

So you've got a person who is exercising. And so you have two people who are similar, except that one is very insulin resistant, and one is quite insulin sensitive. The insulin resistant person is going to have a higher level of insulin. All things equal, they're going to have a more difficult time oxidizing free fatty acid. So as energy demand goes up from the muscle, they're going to be more likely to utilize glycogen as opposed to utilizing triglyceride. So at any point in time, you might measure differences in glucose, differences in lactate. You might, well, you would see lactate as well, but differences in triglyceride level, you might also notice more fat in the liver, or some fat in the liver of the insulin,

resistant person, none in the insulin sensitive person. In that situation, even when they're not eating, they're quite different physiologically. What is the HDL discerning or doing in those two scenarios that's being reflected in the ongoing integral function of it? The simplest, most direct model is that the HDL is reflecting the 24-hour excursions and triglycerides, like we were just talking about. I think what you may be getting at is there are almost certainly other components of metabolism

that the HDL is integrating and reflecting, particularly in the complex insulin resistance world. We know insulin resistance has a lot of effects other than affecting triglycerides, and insulin resistance I'm sure has effects that are affecting and are reflected by lowering of HTL. What I can't tell you is exactly what those are. That's an area that is still a topic of investigation. I will give you one hypothesis though, and one that maybe I think has some merit. As you know, dipinectin is an adipocaine secreted by fat that has an inverse relationship to insulin resistance.

So, you know, basically people who are insulin resistant have lower levels and people who are you know, more insulin sensitive. And adipinectin itself appears to have some direct effect on HDL metabolism in the right direction. We could talk about it possibly by tweaking the liver in ways that then the liver affects HDL. So where I'm really going with this, although I have to say the data still are not completely solid, is that another way that insulin resistance is impacting on HDL is through a dip inectin secretion affecting HDL metabolism in a way that's completely different than the triglyceride hypothesis that I just put for it.

You follow me? Yeah, I mean, it's funny. We used to measure adipinectin and leptin levels. Again, one of those things I sort of stopped measuring. Do you think that that's a helpful biomarker independently and should that be something that's back on our plate? Absolutely. Fascinating to try to put together these metabolic pathways in terms of its utility as a

clinical marker. I guess I'd have to be a little bit skeptical. I'm not quite sure how I would use it in terms of guiding clinical care. I mean, I think that was sort of where we ended up, which was, look, we get more actionable insight out of other metrics. Okay, that was really interesting. And again, before we leave the pharmacoside of this, can we talk for a moment about Niasin? Oh, sure. It's been a while since Niasin came up on a podcast, but this is an interesting drug

because it clearly raises HDL cholesterol. And it lowers APOB, doesn't it? It does. It lowers triglycerides. It lowers APOB. It lowers LDL, all modestly, but really. Peter, in Niasin, I just chuckled because Niasin is one of these areas

where we as lipidologists and me personally have to really eat a bit of crow. There was a time when I prescribed Niasin to a lot of patients, with primarily the idea that it was the only thing we had to raise HDL. This was in an era where HDL is the good cholesterol and raising it must be good, right? I also told myself, well, nice and does lower triglycerides at lower's APOB at lower's LDL. It also, as I'm sure you know, modestly lowers LP, little A as well. About 15% lowering of it. Yeah, 15, 20%.

It basically was like, this is a nice broad spectrum lipid lowering drug that, you know, not in place of, but on top of a statin for certain people who had certain lipid profiles, mostly high triglycerides low HDL, has to be providing some benefit, even in the absence of, as you pointed out, the clinical trials that really are a cornerstone of cardiovascular medicine. Well, the clinical trials, you know, got done. The high trial, which was a very well done trial, which frankly just didn't show much benefit of niacin, uploaded us, but the field comes up with reasons why that trial may not have been right. Bottom line is, one more trial run by Merck with a drug that

also helped to potentially address some of the issues with niacin was done, a very large trial, very well powered trial, and that also had really minimal or very disappointing effects on reducing cardiovascular events. Essentially, those two trials killed Niesin. So over the next year as patients would come back, I would have the discussions that maybe you've had discussions with patients too like this.

You know that Niesin, I put you on, you know, eight years ago that you've been taking religiously despite the question that it causes when you take it. I really think I have to tell you that I'm not sure you need to take it anymore. And it was a humbling experience to basically have a drug that I had prescribed quite a lot, basically to tell patients that I don't think that in retrospect, this is helping you much. I do have a subset of patients who are so wedded to their niacin that despite that, they haven't wanted to stop.

But the vast majority of my patients have stopped their niacin. And what's the mechanism by which niacin raised HDL cholesterol? I think one is certainly triglyceride lowering, like we were talking about earlier. But probably not just that, because the increases in HDO were somewhat disproportionate to what you'd predict from the triglyceride lowering,

although, again, with the complexity, it's little hard to make that calculation. So there probably is some other mechanism, and to this day, I don't think we really understand it. We tried for a while to try to figure that out. Nyson is a very complex drug. Of course, we're talking about Nyson here in pharmacologic dosing, not in the kind of vitamin dosing.

We don't really know, I think it's fair to say, how nice and raises HDL beyond its triglyceride lowering effect. It's not a drug we used much, if at all, but the few times we did, I was actually really amazed at how much it raised HDL cholesterol. I mean, it wasn't uncommon to go from 50 milligrams per deciliter to 90 milligrams per deciliter on a strong dose. Let's shift gears for a second and talk about something that we briefly touched on, but I think we now want to go into a little bit more detail. If everything we've talked about so far is like somewhat complicated, I actually think for me at least this next part, especially the RCT stuff gets complicated.

So let's talk about what HDL lipidation, delipidation, and reverse cholesterol transport are because this is really where we start to get into some of the sophistication of the HDL and even the interaction with other cells like macrophages and things like that. I'll start and then you can kind of lead me along because this can get quite complicated. And maybe just to orient you Dan, why don't we start with, unless you have a better way to do it, I'm totally open, but if you're looking for a goal post to start in, you want to start with a foam cell stuck in an artery wall or is there a different place you'd want to start the discussion? That's a good idea. So many of your listeners know that a whore concept in arthroscratic vascular disease is the macrophage that's taken up lipids and is now a so-called foam cell, meaning it looks foamy under a microscope because it has all this lipid and when you stain tissues, the lipids become like bubbles within the cells and they become look foamy.

The foam cell, the lipid-loaded macrophage, is a core pathologic feature of arthro-scratic vascular disease. It's also the first thing you see. So careful studies that have really looked at vascular tissues in children and teenagers and young adults on the way up. You basically see lipid-loaded macavages accumulating in the sub-entimal space and the intimal space in the large vessels before you start seeing

some of the more complex features of infiltration of other leukocytes and extracellular matrix and all the stuff that ultimately comes the complex atherosclerotic plaque. There's been a strong belief for a long time that this is one of the core initiators of the process. and extracellular matrix and all the stuff that ultimately comes to complex atherosclerotic plaque. There's been a strong belief for a long time that this is one of the core initiators of the process. And I think that's probably true.

An analogy would be like without timers, the A beta being kind of the core initiator of the process leading to much more complex pathology. So macrophages have very well established mechanisms for ridding themselves of cholesterol. Keep in mind, no cell except the liver cell has the ability to metabolize cholesterol to other sterile species. Only the liver can do that. Cells can make plenty of cholesterol, but the only way cells can deal with their cholesterol is to, quote, efflux the cholesterol, to push the cholesterol out of the cell and get rid of it. So if you think at the whole body level, all of the body is making cholesterol, but that cholesterol ultimately has to come out of those cells into something.

This is where HDL comes in and ultimately get back to the liver where the liver then can metabolize it or directly excrete it into the bile, then it goes out in the intestine and the feces. So all cells have to be able to do this, and all cells in fact have the ability to push cholesterol out of their cells, but macrophages really have to do it. That is because macrophages are like kind of the dump truck of the body. They're picking up not only LDLs and lipoproteins with cholesterol. They're scavenging cells, dead scales, apoptotic

cells, and of course, all those cells have lots of cholesterol. So macrophages need very, very effective ways to rid themselves of cholesterol, and they do have effective ways of doing that. When those pathways get overcome or less efficient, the macrophage then builds up cholesterol and becomes the kind of foam cell that we're talking about. Macrophages have transporters. They have a very abundant amount of this ABCA1 that we talked about earlier. Earlier we talked about it in the gut and the liver.

Now I'm saying that macrophages have ABCA1. ABCA1 is one of the, not the only, way that macrophages rid themselves of cholesterol. If you recall, the main acceptor of cholesterol via ABC-A1 transporting out of a cell is AP-A1. This led now quite a long time ago to the general paradigm that macrophage foam cells are building up cholesterol because they're not getting rid of it effectively, and that one of the best ways to get rid of it would be to promote these efflux pathways by ABC1 and other transporters that are being driven by APY1, 2HDL, which then by my garbage truck analogy, the garbage

truck, IEDHDL, is picking it up in the periphery like the blood vessel and returning it to the liver, dumping it off in the liver, and then going back and doing its job again. This process of, quote, efflux, cholesterol efflux from macravages and frankly other cells, particularly in the blood vessel wall, has been for a long time now, thought of as a key process that would help to protect against the early initiation and progression of the atherosclerotic plaque. Peter, that was long, but that's a start. Well, and I think that's, look, let's just make sure everybody understands what we're talking about here, which is when we really talk about the positive valence of HDL as

a particle, it's because of this, right? In large part, this is a big piece of the positive association, presumably, or negative association, depending on the direction of HDL. See, we might see. As we've been talking about it, it really now speaks to function. Part of the problem it would appear is that very crude metrics like the amount of cholesterol in an HDL, or even the number of HDL particles or the size of an HDL particle, those are basically the only things we can measure clinically, at least. Those are so crude and so far removed

from providing any quantification of the process you just described that I suspect in part that's why we are stuck, in a way that we are not on the APOB side of the ledger, because so much of the damage caused by APOB is simply captured in the number of them, given the stochastic nature with which they enter artery walls and get retained. Would you agree with that assessment? I would absolutely agree with that. So this process of cholesterol leaflux as the sort of ideas evolved is if you will, the first step in this broader physiologic process

that Peter briefly referred to that we call reverse cholesterol transport or RCT, different than a randomized controlled trial. If forward cholesterol transport is basically the cholesterol coming out of the liver into VLDL and LDL and then depositing in tissues like the artery, the reverse transport is the picking up of the cholesterol, punitively by a 8-way 1-n-HTL, and returning it back to the liver.

This process of reverse cholesterol transport plausibly, and I think some data, at least in animal models, is related to protection against atherosclerosis. That is, the more effective you are at the integrated process of not only picking up the cholesterol by e-flux, but effectively returning the cholesterol to the liver for excretion, the more you would protect against atherosclerosis as the theory goes. And I'll just remind you about our example with SRB1. That's the terminal, sort of one of the terminal steps where the HDL is dumping off the cholesterol.

If you interrupt that process, HDL goes up, but the process of reverse cholesterol transport is being constipated, and therefore there's increased risk of atherosclerotic cardiovascular disease. One of the great goals of the field has been, can we promote that first step of the process? Can we figure out a way to promote the driving of the E-Flux process from the macavages and maybe other cells in the atherosclerotic plaque to acceptors like HDL

in order to protect or maybe even regress atherosclerotic plaque, shrink it as a way of trying to reduce risk. And I'll just say that as we move into this next phase of complexity, I think what's pretty clear is it's not the mature HDL particle. You know, when we measure HDL cholesterol, that's what we're measuring is the cholesterol in the mature particle. That's almost certainly not the particle that is driving this first step of the efflux

of the cholesterol from the foam cell and the macrophage. And that's maybe why simplistically, raising HDL cholesterol, like with C to be inhibition, doesn't actually reduce the athletic credit cardiovascular disease events, but are there other ways more creatively that we might be able to drive that process and also to Peter's early or functional point? Might it be that different people,

even with identical HDL cholesterol levels, have different function of their HDL? One person with HDL cholesterol of 50 might have something about their HDL. One person with HDL cholesterol of 50 might have something about their HDL pathway that is like super functional at driving E-flux. Whereas another person with the same HDL cholesterol doesn't do nearly as well. And if we could measure function efficiently,

might we have a better way of assessing risk and maybe even targeting interesting therapies more so than just measuring this fairly not so dynamic measure of HDL cholesterol itself. There's more to say there, Peter, but I'll turn it back over to you. Well, no, I mean, I think you illustrate the very important distinction between static biomarkers and dynamic biomarkers. And we have very few dynamic biomarkers, you know, an OGTT and an old glucose tolerance system system dynamic biomarker in a sense. But most things are very static and static things allow us to miss flux.

So that example you gave of two people whose HDL cholesterol is both 50 milligrams per desolate or we have no clue what the velocity through the HDL is. In one of those cases, it could be a bump on a log and the HDL is not really doing a heck of a lot. And in the other, that could be the busiest beaver on the face of the earth, just transporting lipid out of foam cells, right back to the liver,

back and forth, back and forth, back and forth, and that could be the most industrious little guy on the block. And you could be in a totally different risk situation as a result of that. So going back to the RCT, how often is it happening that an APOB by bearing lipoprotein? Let's just call it an LDL is floating around. So not yet in artery wall,

but on his way there, presumably. And an HDL come along and they collide in the artery itself and delipidation takes place. So the HDL says, Hey, I'm going to take some of your cholesterol away and take it back to the liver now, which by the way, LDLs do as well. I mean, LDLs are obviously carrying cholesterol back to the liver out of. But not as a reverse cholesterol transport process, but yeah. The scenario I described, does that occur often? No, I don't think so.

In fact, as we discussed earlier with CTP, the directional flux of cholesterol in the blood is more from HDL to able to be containing lipoproteins rather than the other way around. So I'm pretty comfortable in saying, I don't think the way you just outlined is a way that HDL doesn't directly dilipidate LDL. No, no, I think it's more about, if it's happening at all, I want to say, all this is couched in, this is the paradigm that we're dealing with, but we're there's still a lot of uncertainty about it. But if it's happening at all, it's really more that something about HDL or something, 8-1 is more interacting with cells to promote e-flux of

cholesterol rather than with other lipoproteins, like apobet container lipoproteins. What do you think the future could look like here in terms of commercial assays to measure HDL function? I know that we're a long way away from that, so maybe I should start with something even more basic, which is what would it take in the lab to measure HDL functionality if resources were unconstrained? No, that's where I wanted to go.

So first, I want to say that several years ago, with this concept of reverse cholesterol transport as a dynamic process, we developed an assay in mice that simply speaking, involved taking cholesterol-loaded macravages with labeled cholesterol, injecting them into the mice, and then following that label all the way through the HDL, to the liver, to the gut, to the feces, and we called that integrated reverse cholesterol transport. And we showed in a variety of different genetic and pharmacologic approaches that when you tweak that process, either up or down, it mirrored the effect of that process on atherosclerosis.

What I'm really trying to say is something that promoted that process and made it more efficient also reduced atherosclerosis, something that const that promoted that process and made it more efficient, also reduced atherosclerosis, something that constipated that process, increased atherosclerosis. It gave us a lot of confidence. Frankly, it gave the feel, a lot of confidence, that integrated measure of reverse cholesterol transport is actually relevant to atherosclerotic cardiovascular disease, at least in mice. So that brings me to humans. We can't do the type of experiment I just described in humans. It's not feasible as a research or as a clinical assay. So we thought long and hard about how can we start to think about doing this in people and develop what I'll call an

NXVVO cholesterol-leaflux assay where the concept is we took someone's blood or plasma, we bicelated the HDL specifically. We got rid of the it will be contained lipoproteins. And then put that on cells that were labeled with cholesterol and we measured the effectiveness by which that HDL removed cholesterol from cells. What kind of cells did? And what we found, as I think you know, is

we were really struck with how different individuals HDLs were at their ability to extract cholesterol from cells, even with the same HDL cholesterol level. And it's sort of affirmed this concept that HDL cholesterol is not really telling us or informing us on the efficiency of the function of HDL, at least in this case, the function defined as the ability to extract cholesterol from cells. And so we went on to use that in larger numbers of individuals and showed that actually,

that was much more predictive of risk of coronary heart disease than just measuring HDL cholesterol, even when you controlled for HDL cholesterol statistically. And many others have shown the same thing. So I think it's pretty well established at this point that this so-called cholesterol-eflux capacity measurement of HDL is a better marker or predictor of risk than just measuring HDL cholesterol, consistent with the idea that function is important and that perhaps if we could increase function, we could maybe

have a mechanism for reducing risk. Now Peter, you asked about the clinical applicability of that essay. I get asked all the time by patients and referring doctors, can you measure my patient's HDL function? There's a lot of effort in this regard. Full disclosure, I was part of starting a little company now several years ago called VASOR strategies.

And VASOR strategies does do this measurement in a very reproducible sort of way, mostly related to biomedical biofarmer research, but there is a lot of interest in trying to bring this clinically. And there are other assays that are being developed. When you're using radioactivity, it's a little, it's cumbersome for a chythroughput clinical assay. And others are developing assays that are cleaner and simpler and faster and cheaper. I think there's a chance we could have a more widely available clinical assay to us for this purpose within the next two to three years.

Right now, it's still under development. Two follow up questions there, Dan. One is just a technical question on the assay. What type of cell are you using to measure the E-flux capacity? Well, when we first sort of pioneered this work, we used a mouse macrophage cell line called the J774 cell. We have done it with human macrophages as well. And so have others.

We like to think that the macrophages the most relevant cell type for reasons we discussed. Yeah, that's the answer. And then I guess the second question is using the hypothetical, but probably somewhat real example of, we'll take a hundred people that have an HDL cholesterol that's about the same. We do this assay and we rank order them in effectiveness.

You're saying that that rank order of effectiveness correlates directly to risk. Are you saying directly related to risk by proxy, i.e. other measurements like insulin resistance, apob, or you saying actual outcomes? The first study we did in published was cross-sectional with clinical coroner disease. So basically it was calcium scores or or things like that. Association cross-sectionally with prevalent disease. Then we basically said, we need to know if this is predictive of incident disease, events that occur. So we went to colleagues who did the Eric Norfolk study,

which you're probably familiar with, a very large prospective study in the UK, in Europe. They provided samples, and we measured this in a very large number, many thousands of samples, in a UK, in Europe, they provided samples, and we measured this in a very large number, many thousands of samples in a baseline in people who had been followed for 10 plus years. And we showed even in that setting that the EFLUX capacity was predictive of incident cardiovascular events.

So yes, these are hard events, not just proxies like measurement of corner calcium, for example. Which is again, fascinating. How well does that prediction hold up if you corrected for other things that could be measured in the blood, such as insulin resistance, triglyceride, or APOB? We did do these statistical analyses correcting for clinical risk factors, including, of course, H.O. cholesterol itself. We corrected for things like BMI and presence of diabetes, dichotomL. cholesterol itself. We corrected for things like BMI and presence of diabetes, dichotomous presence of diabetes. If memory serves, we didn't attempt

to correct for like a sophisticated marker of insulin resistance like home or anything because I'm not sure we even had that data. So your point is well taken. At some point, is this causal or is it still associative but just even better associative because it correlates with things even better than H.J.O. cholesterol? And I think that's why we need, of course, interventional studies that actually test the hypothesis. Well, or even could we use it, there could be a hybrid there, right? Which is, it's absolutely causal, but because it's so difficult and complex to initiate at scale. What if we use AI to figure out that it's equivalent to a new metric that is a composite metric of things that we can measure as biomarkers, if that makes sense? I hear what you're saying.

That would be a great thing to think about. In our paper in Epic Norfolk, we did correlations of the EFLEX capacity with lots of different things. I frankly have to go back and look at that and remind myself if we had like fasting insulin or anything, because I think that starts to get more at the insulin resistance component. But you're absolutely right. We may be able to find markers that in composite would predict EFLEX capacity rather than having to measure the EFflux capacity itself.

I'm not convinced we'll be able to do that, but I think that it's a good thought. Because the analogy that comes to mind is certain labs use a series of NMR metrics to predict insulin resistance, which I've always found not that interesting, frankly, because we can measure insulin resistance so easily that using NMR to add another insulin resistance metric, it seems a bit backwards. But the idea was they were able to basically look at the NMR spectra of all of the lipoproteins and impute a composite metric that came back. So if you could do that in the other direction, which would actually be the valuable direction,

it could be amazing. In other words, if there was an HDL function score built out of X, Y, and Z, anything else before we leave the topic of measurement, we haven't really talked about the NMR side of the equation. That's probably a bias because we don't use NMR, but clearly there are labs out there that will count the number of HDL particles and spit out an HDL P. Anything you want to say about HDL P or anything like that? Yeah, the overall number of particles is obviously correlated with HDL cholesterol, but is a different measure because HDL cholesterol is just that amount of cholesterol carried in the HDL,

whereas the particles are more analogous to measuring AP APOB, the meaning total particle number. I think the data on balance suggests that HL particle number is a little bit better than HL cholesterol at predicting risk. We haven't formally compared HL particle to cholesterol-eflux capacity, the functional measurement. Fundamentally, it's another static measure, though. And so, while

it has maybe some limited predictive value, I don't think it takes us that much further in terms of trying to get at where we're really trying to go, is better predictions of risk, particularly in a setting where we might want to intervene to actually try to reduce risk. Let's talk about something you mentioned kind of in passing a couple of times. Peter, before we leave this topic, though, I do think it's important to remind your listeners that this concept of promoting the E-flux and reverse cholesterol transport pathway is being tested with another intervention called CSL 112. It's a form of 8-1 that's been complex with lipids in a so-called recombinant

H-L particle is being tested for impact on cardiovascular outcomes in the setting of acute coronary syndromes. So people have come in, been randomized to four weekly injections of this 8-O-A1 containing recombinant particle, which is very effective at promoting cholesterol leaflux based on all the work that's been done, with the idea being that this may directly impact the plaque and impact on cardiovascular events. So this is, I would say, the closest thing we have to a formal test of the cholesterol efflux hypothesis as an intervention in terms of whether it will reduce risk. Those of us in the field are on the edges of our seats, you know, it'll be a little while

to really see what this trial shows. I do think it's gonna be interesting either way. So earlier, Daniel mentioned kind of briefly the association of plasma APOA1 and HCL cholesterol with neurodegenerative diseases, such as dementia or Alzheimer's disease. I don't know if it extends to Parkinson's or Lewy Body. What do we know about that?

Well, this is a fascinating new area of HL biology that has a number of components and that's still being investigated. I want to maybe start by bringing in APOE. We haven't talked about APOE. APOE clearly has a role in ApoB containing lipoprotein metabolism and mediating the uptake of those remnant particles into the liver. Can you actually explain this a bit more because most people hearing this are going to think of ApoE

the gene. And of course, the gene for ApoE, people are going to be familiar with it exists in three isoforms, two, three, and four. But of course, these things combine. So you have six combinations of an APOE genotype, and of course, the gene codes for a protein. So I mean, I assume everybody has the same APOE protein. You're going to have different amounts of it and different potentially functionality, depending on the combination of which gene produced it? Exactly. ApoE is possibly one of the most fascinating genes and proteins in human biology in terms

of its roles and its and the isoform issue and its relationship to disease. So as you point out, there are three common isoforms of ApoE, ApoE3 being the so-called wild type or the most common. In lipidology, we've focused a lot on ApoE2, because if you inherited two copies of ApoE2, that form of ApoE is defective in binding to the LDL receptor and the other receptors that mediate uptake of remnant lipoproteins, both chylimachin remnants and BLDL remnants. And therefore, if you homozygous for ApoE2, you're at risk for so-called, again, the terminology is bad.

Type III hyperliplidemia, which is basically a remnant clearance disorder. These individuals can't clear their remnants appropriately. They get high triglycerides in cholesterol. They also are at increased risk of ethylscarotocardic cardiovascular disease. It's a classic lipidology thing. And these patients require phenophybrates to bring down the VLDL in triglyceride? They respond to a lot of the standard LDL lowering therapies,

like statin and acetymide. They even respond to PCISC and I and inhibition. But sometimes we have to add on fibrates as well to maximally control them, depends on how severe they are. And yet those people, and by the way, that's the only genotype I've never seen. It's much more rare than the 4-4 homozygots, but the 2-2 would come with about a 20% risk reduction, relative risk reduction in Alzheimer's disease, but paradoxically comes at this higher

risk of ASCVD. Exactly. So, comes at higher risk of lipid disorder and ASCVD, but that's exactly why I brought up APOE in response to your comment about neurodegenerative disease. So, as probably a lot of people know who are listening, the APOE4, as you just pointed out, the APOE4 form, which is present in about 25% of people.

So, it's quite common. And even in the homozygous form, certainly plenty of people out there who have both two copies is, in a dose-dependent way, the major genetic risk factor for Alzheimer's disease. No question about it. And as you said here,

April E2, which is bad for your lipids in the blood, is actually protective against Alzheimer's. It's absolutely fascinating biologically. There has been so much work to try to understand how APOE is interfacing with Alzheimer's disease. So APOE is made in the brain. It's made by cell types like microglia in the brain. And it's a lipid binding protein that transports lipids.

And now we know from genetics of Alzheimer's that there are lots of other lipid genes that are relayed Alzheimer's. So the story starts with the fact that apoe made in the brain is somehow impacting on Alzheimer's risk. And if you have this form of apoe for, and especially if you have two forms of apoe for, you are at very substantially increased risk of Alzheimer's. For reasons that we still don't fully understand, but as a lipidologist, I think it has something to do with lipid transport in the brain. So that brings me to HDL. The APA-E and the CSF and in the brain is mostly made there. The newer developments that are relevant to APA-1 and HDL are that

there is clearly APA-1 in the brain and in the CSF. APOA1 is not made in the brain. So APOA1 gets there somehow through the blood, which I'll come back to. But there's now quite a lot of observational data that strongly suggests that APOA1 is protective against neurodegenerative disease. Now, this is associative data. It doesn't prove causality. But the reason I think it's plausible is that two of the major genetic risk factors

for Alzheimer's that are expressed in the brain are ABCA1. Remember that lipid transporter, that red cells of cholesterol and that APOA1 interacts remember that lipid transporter, that red cells of cholesterol, and that APOA1 interacts with and promotes, and ABCA7, a very close relative of ABCA1 that structurally looks very similar, and that we don't know exactly what it does,

but I think it's a safe bet that it's transporting some sort of lipid to something in the extracellular space, whether that's 8-1 or 8-F levels of A-poi-1, and the relatively small number of studies that have measured both in the same people are not that well correlated. So it's not simply a matter of if you have a high level of A-poi-1 in the blood, that's going to generate a high level of A-poiI one in the CSF. So what that probably means is the processes that are happening that get the APOI one across the blood brain barrier are highly regulated processes that a little like the cluster leaflux story probably differ from

person to person and aren't directly being driven by the level of APOI one in the blood. Where I'm really going with this is this concept that if we can figure out how that happened, if we could somehow promote more Apoi 1 going into the brain into the CSF, the data suggests that that might be a very interesting opportunity to blunt or reduce risk of Alzheimer's.

I find it fascinating, as you can tell. Dan, do we know if this relationship is stronger or weaker as a function of ApoE genotype? Is the effect more pronounced? Do we have enough data to parse out that type of relationship? So I'll give you an example. Yeah, that's a really super question. And I don't think we have big enough data sets yet to be able to parse it out in terms of by apoe genotype to figure that out. But I have to go back and look for that, but I don't think we know that yet. Do you have any idea why labs have not developed what would be very easy to do,

which is just a commercial assay for apoe concentration? There's certainly some data to suggest that apoe concentration might be more relevant than apoe genotype, although of course it's highly influenced by apoe genotype, but you could almost think of it as a gene expression measurement, a crude measurement of gene expression for apoe. Is that something that you've seen or used, even in the lab?

You're talking about apoe in the blood, right? Not in the CSF, just to be clear. There are automated assays for apoi. We run one routinely in our lab. I think it's not a clinically used assay. No, I've never even seen a clea-based assay for it. Right.

I think mostly because it isn't that helpful in the limited studies that have been done, which admittedly maybe aren't as voluminous as they should be, it isn't that really helpful in terms of predicting cardiovascular risk. Total plasma apoe. Well, I was thinking more for neurodegenerative disease. Has that been looked at?

Well, I didn't say it, but the levels of apoe in the CSF and the levels of apoe in the blood for the few studies that have measured both in the same people also do not correlate much at all. You would need CSF apoe if you wanted to do anything with this. Exactly. That's what I'm getting at. You would need to be able to do it. I would not be surprised if someone develops an assay for APOE concentration in the CSF as a clinical tool because there I think that might be highly relevant. Interesting. So here we have basically HDL potentially being protective in the brain and

presumably doing so via APO A1, potentially offsetting some of the APOE problem, APOE being as I think one of our previous guests referred to is kind of the general contractor of cholesterol in the brain. Does HDL do anything with nitric oxide? I think I remember reading something about promoting sort of endothelial, NOS activity, nitric oxide synthase activity. Is that a major issue? There's been a variety of other, quote, functions that HDL has been reported to do, which I think are absolutely real. They're relevance to human disease and pathophysiology, I think, are less clear. But one example is what you just said. So a couple of investigators, most notably Phil Schall and Dallas, has shown very clearly

and beautifully in cells and mice that HDL can promote nitric oxide production in a way that would be expected to be beneficial, both in terms of blood pressure lowering and maybe protection against atherosclerosis, and that S-O-B-1, the receptor we've been talking about, is also present on antithylocels and mediates at least part of that effective HDL. It's a fascinating observation that is absolutely, I think, solid, translating that observation to relevance in humans I think is challenging, but I think plausible. You know, another, we've been talking about insulin resistance.

I'm sure you're aware of the data that suggests that HDL can interact directly with skeletal muscle in a way that promotes insulin sensitivity. No, I'm not. Fascinating work. Again, I put it in the same category of solid in terms of what it's been able to show mechanistically, a little bit unclear and relevance to human disease and physiology, I think, still a little bit unclear. These are just two of the other types of things that HDL has been shown to do that would

be putatively beneficial, but are of uncertain relevance to real human disease. It's even more sort of removed than that because even if we knew mechanistically that this were sound, we're still back in the same area we are with ASCVD. In ASCVD, there's really very little ambiguity about the utility of HDL, the beneficial utility of HDL. And yet we're standing here with our hands in front of us saying, well, what can we do about it clinically as a physician, as a patient? What do I get to do about this knowledge that HDLs

are helpful particles when I can't measure the manner in which they do their job? And the only things that I can measure are as useless to me as my eye color, basically. And then we're expanding into and look what they can do in muscles and look what they can do in insulin sensitivity and look what they can potentially do in the brain. It really comes back full circle to how we open the discussion, Dan, which is this is such a complicated area of biology that I would guess it has to be considered the next frontier of the lipid space. I mean, when we take a step back and put ASEVD in the context of cancer and

neurodegenerative diseases, right? So these are the big three killers in the modern world. We know so much more about ASEVD and we have so many more tools to effectively treat it. We know a bit about these other diseases, but in the case of Alzheimer's disease, we don't have a single tool to do anything about it. In the case of cancer, most of our tools do nothing. 90% of the tools do nothing, i.e. they barely extend median survival, but don't cure people. But in ASCBD, we can really move the needle. And yet, you could argue half of the field we still know nothing about.

And is there going to be a renaissance you think, or are we up against some technical limitation on this inability to measure function? And I say that in a practical way. I mean, yes, I think you're already measuring function in the lab. But is this a bridge too far for the clinician? Well, I'm going to separate that into using a measurement of function for risk prediction and then the implications for intervening therapeutically. Well, I'm going to separate that into using a measurement of function for risk prediction and then the implications for intervening therapeutically.

I think that HDL function, say, the cluster leaflux, has the ability to allow us to more specifically assign risk better than just measuring HDL cholesterol. I think realistically we need a good reproducible, easy-to-run, automatable assay that then is tested in large numbers of people and shown to predict risk better than H.J.L. cholesterol itself. I feel like knowing what I know is going on in terms of developing those assays, that there's a decent chance that we're going to get there. But it's not just the development of the assay, which actually there are several assays now that are, but it's the proving that the measurement actually enhances risk prediction

enough, not just incrementally, but enough to make it actually worth doing in clinical practice. I think there's a, I'm not going to say 100%, but I think there's a more than 50% chance that we're going to see at least some assay come out that will allow us to do that. And then it will be, frankly, physicians like yourself and like Tom Despring who pick up that assay and start using it in their patients and start trying to get a sense for whether it's useful in the context of a sophisticated preventive clinical practice. I think with regard to intervention, the study I talked about is huge in terms of this. Let's just assume that that study is positive.

For infusions, weekly infusions, significantly reduces cardiovascular events in patients with ACS after 90 days. If that study is positive, if you think about that, the implications for that product, but more importantly, for the field, that showing that actually triggering cholesterol efflux via infusion of that particle, that type of particle, that will rejuvenate the field of cholesterol efflux and reverse cholesterol transport as a therapeutic target for intervention.

I think if that trial is not positive, I think the concept of intervening around HDL and reverse cholesterol transport for purposes of AACVD is probably past recovery. That would be my view. And even the CTP inhibitor that I mentioned that's still in development, it's really more about reducing APOB than it is about raising HCl. When does that trial read out the function trial? I should have refreshed my memory on that. I just can't remember.

Where is it being done? How many centers? Roughly. It's a global study. There's centers in, you know, US and Australia and Europe. So a classic global large cardiovascular outcome trial. I think the leaders of the trial, I think, are in Boston. Remind me, does it have a fancy acronym yet? Yes.

And I'm embarrassed to say that I'm not quite coming up with that either. You will figure that out, and it'll be in the show notes. If I could just follow up on your next frontier comment, I don't think H.C. I'll per se is the next frontier, but I do think lipid metabolism in the brain is one of the next frontiers. I really do. With regard to neurodegenerative disease, but also other brain function, I say that as long-term lipidologist who has focused primarily on the blood.

So I admit my biases and looking for things that we can apply as lipidologists our expertise to. But I do think understanding lipid metabolism in the brain and its relationship to disease, you know, the brain is, of course, the most lipid-rich organ of any organ in the body has been an under-investigated area that we have a lot to do, but where I think we're going to be uncovering some very interesting things that with luck, have implications for therapeutic intervention to prevent neurodegenerative disease. Dan, it's hard to go anywhere from here.

That is, I mean, both exciting and important in ways that are rarely captured, even in this subject matter. So I want to thank you for taking a subject matter that is so complicated that I've largely shied away from really doing anything on it in the AMA series that we do and so eloquently describing it. And really using, if someone's listening to this and this is the first podcast that we've listened to of ours, they might think, oh my god, that podcast is too technical.

But I think for regular listeners, they will appreciate that you really did a great job simplifying a very complicated subject matter. And certainly did a better job than I would have ever been able to do, not simply because I don't know as much, but because the ease with which you were able to speak about this is impressive. So, thank you for taking the time. Thank you for doing that, and most importantly, of course, thank you for your work in this field.

We, I think, collectively look forward to seeing how things shake out over the next decade. Thanks very much, Peter. I really enjoyed it. Thank you for listening to this week's episode of The Drive. If you're interested in diving deeper into any topics we discuss, we've created a membership program that allows us to bring you more in-depth exclusive content without relying on paid ads. It's our goal to ensure members get back much more than the price of the subscription.

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