Thursday, September 3, 2009

Animal Models of Atherosclerosis: LDL

Researchers have developed a number of animal models of atherosclerosis (fatty/fibrous lesions in the arteries that influence heart attack risk) to study the factors that affect its development. In the next two posts, I will argue that these models rely on a massive increase in LDL, up to 10-fold, due to overloading the cholesterol metabolism of herbivorous species with excessive dietary cholesterol. This also greatly increases oxidized LDL, leading to atherosclerosis. I will discuss the role of saturated fat, which often receives the blame, in this process.

A reader recently sent me a reference to an interesting paper titled "Dietary Fat Saturation Effects on Low-density-lipoprotein Concentrations and Metabolism in Various Animal Models". It's a review of animal studies that have looked at the effect of different fats on LDL concentration as of 1997. 

When an investigator wants to study diet-induced atherosclerosis, first he selects a species that's susceptible to it. These are generally herbivorous or nearly herbivorous species such as rabbits, guinea pigs, hamsters, and several species of monkey. Then, he feeds it an "atherogenic diet". This is typically a combination of 0.1 to 1% cholesterol by weight, plus 20-40% of calories as fat. The fat can come from a variety of sources, but animal fats or saturated vegetable fats are typical. The remainder of the diet is processed grains, vitamin and mineral supplements, and often casein for protein.

Let's put that amount of cholesterol into human context. Assuming the average person eats about 2 pounds dry weight of food per day, 0.5% cholesterol would be 4.5 grams. That's the equivalent of:
  • 17.5 pounds of beef steak, or
  • 3.8 pounds of beef liver, or
  • 22.5 eggs
Per day. Now feed that to an herbivore that's not adapted to clearing cholesterol. You can imagine it doesn't do their blood lipids any favors. For example, in one study, compared to a low-fat, low-cholesterol "control diet", a diet of 20% hydrogenated coconut oil plus 0.12% cholesterol caused hamsters' LDL to increase by more than 7-fold. A polyunsaturated fat (PUFA) rich diet caused LDL to increase less. This study is typical, and the interpretation is typical as well: SFA raises LDL. But there's another possible explanation: in the absence of unnatural amounts of dietary cholesterol, PUFA reduces LDL in some species, and SFA has very little effect on it in most.

It's important to remember that the relevance of this hamster experiment to humans is unclear. No one is claiming that reducing saturated fat and cholesterol will reduce a human's LDL by 7-fold.  

But let's get back to the animal models. The hypothesis the paper addresses is that saturated fat raises LDL in animal models. If that is true, it should be able to raise LDL even in the absence of added cholesterol. So let's consider only the studies that didn't add extra cholesterol to the diets. And if saturated fat raises LDL, it should also do it relative to monounsaturated fat (MUFA- like olive oil), rather than only in comparison to PUFA, which has a known cholesterol-lowering effect. So let's narrow the studies further to those that compared SFA-rich fats, MUFA-rich fats and PUFA-rich fats. In Fernandez et al. (1989), investigators fed guinea pigs 35% of calories from corn oil (PUFA), olive oil (MUFA) or lard (MUFA-SFA). Here's what their LDL looked like:
The same investigators published two more studies showing similar results over the next five years. The next study was published by Khosla et al. in 1992. They fed cebus and rhesus monkeys cholesterol-free diets containing 40% of calories from safflower oil (PUFA), high-oleic safflower oil (MUFA) or palm oil (SFA-MUFA). How was their LDL?
None of the differences were statistically significant. Khosla and colleagues published another study with the same result in 1993. This is hardly supportive of the idea that saturated fat raises LDL in animal models. The most you can say is that PUFA lowers LDL in some, but not all, species. There is no indication from these studies that SFA raises LDL in the absence of excessive dietary cholesterol. I didn't cherry pick studies here; this is every study in the review paper that met my two criteria of no added cholesterol and a MUFA comparison group.

The bottom line is that experimental models of atherosclerosis appear to rely on overloading herbivorous species with dietary cholesterol that they are not equipped to clear. SFA does exacerbate the increase in LDL caused by cholesterol overload. But in the absence of excess cholesterol, it does not necessarily raise LDL even in species ill-equipped to digest these types of fats. Dietary cholesterol has a modest effect on LDL cholesterol in humans, and it has even less effect on LDL particle number, a more important measure. So there may not be a cholesterol overload for saturated fat to exacerbate in humans. 

PUFA vegetable oils do lower LDL in humans, and the effect appears to persist for at least a few years (probably indefinitely). But the evidence is not conclusive that lowering cholesterol in this way actually prevents heart attacks.

39 comments:

Erik said...

It'd be interesting if they'd do similar studies (and actually publish the findings!) on omnivorous/carnivorous species - dogs, cats, bears, etc. Alas, it'll probably never happen.

arnoud said...

This analysis puts the SFA - LDL relationship in a most transparent 'picture.' Thank you for clarifying the typically muddy waters around this topic.

One question: based on established research, is it reasonable to state that the reason that SFA typically raises serum LDL measurements in humans is due to SFA causing the lipoproteins to grow in size and weight? In the short term, increased SFA intake is not changing LDL particle count, but only increasing LDL weight. The longer term effect may be that the diet change reduces LDL lipoprotein particle count, explaining the longer term effect of insignificant change in LDL by weight. An important point would be the changing make-up of LDL (increased size, reduced number) which reduces the risk of oxLDL and thereby artherosclerosis).

Manne said...
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Manne said...

Excellent and very readable post on a complex matter, thank you!

I have been reading a lot about these things lately (just went on to eat LCHF), and one question keeps popping up in my mind:

If it is so simple to point out serious flaws in these types of studies, why do we keep seeing them being made, and their results taken seriously?

Jenny said...

We keep seeing these studies because a lot of established elder scientists have built their careers, and those of their students around these rodent studies.

And because their "findings" confirm entrenched beliefs which the health charity financial powerhouses, the American Heart Association and the American Diabetes Association, have been promoting for decades and continue to promote.

These groups cannot admit to the public that the dietary advice they are so closely identified with harm people. So they cling to these rodent studies that appear to back their theories.

The rodent models of type 2 diabetes are equally flawed. They use pure bred strains of mice whose genetic flaws do cause diabetes in the mouse, but are NOT the genes linked to human forms of diabetes.

The diabetic mice see their diabetes worsen when they eat fat for reasons similar to those explained in this post--and because of the nature of their broken gene. High fat/low carb diets greatly improve the long term health of humans with type 2 diabetes. Sadly, the ADA continues to fight against people with diabetes learning this.

Manne said...

Jenny,

you could even (quite easily) argue that high fat/low carb (LCHF) are good for anyone, diabetes or not.

As for the rest of your comment, yeah, that would be one obvious reason. I have seen that explanation before, and sort of believe myself that it rings true. We are all only human, after all.

It does however imply a bit of "conspiracy" and knowingly hiding the true facts. Which to me is very worrying.

If this is the state in the world of dietary science, surely that also goes on in other domains which in a way means that the whole peer review system and ways studies get funded are flawed.

And maybe this IS the case and everyone already know about it but to me it is an eye opener and not a little bit frightening.

Dr. B G said...

Great post and clarifications Stephan! I've wondered the same but I don't understand the GI system well in animals herbivores v. omni's. (Did you mean guinea pig in the first graph, not hamster?)

Some researchers like Volek believe guinea pigs are the best models. Do you come to similiar conclusions? I think rabbits and certain chimps are the WORSE.

GK said...

Date - TC - HDL - LDL
2005-06-15: 5.09 1.8 2.6
2007-10-15: 7.1 1.9 4.64
2008-11-11: 8.33 2.22 5.41

I have read that dietary SFA will raise both HDL and LDL cholesterol, but now you say otherwise. I personally found it raises TC a lot. The above table shows what happened to me after going paleo (à la Cordain) in 2006, followed by adding a lot more animal fats (post Taubes's GCBC 2007), I estimate my intake is now 60% fat by calories. So now I have "high cholesterol". The question is now, "So what?"

John Adam said...

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Phil said...

Manne,

Yeah, I think you've uncovered an unpleasant truth. what's happening here is hardly unique to dietary science. (Although, unlike this one, most bad ideas don't take 50 years to kill off!)

Personally, I think that if a cabal was at the heart of the trouble, it would have been exposed long ago. Confirmation bias and habitual set are worse enemies of science than any conspiracy, in part because the peer review process is blind to them. But what's the alternative?

Actually, the fact that so many papers are open to reinterpretation gives me hope. Writing in a way that admits no other interpretation but your own is not a difficult art form to master. Instead, at least some authors are toeing the line in their abstracts, but letting the data speak for itself.

Stephan Guyenet said...

Arnoud,

In short-term trials showing increased LDL-C upon saturated fat feeding, it appears to be an increase in LDL size, not number.

Jenny and Manne,

I think there's a bit of suspension of disbelief by certain researchers, but not a conspiracy. I think most of them just don't realize or don't care that their model system isn't directly applicable to humans.

G,

Yes, I did mean guinea pig, oops. And it looks like my link isn't working either. I'll fix that.

Guinea pigs are supposed to have similar lipoprotein metabolism to humans. But they're still herbivores. I agree that monkeys are not the best.

Hi GK,

What makes you think it's the saturated fat that raised your cholesterol?

GK said...

Hi Stephan,

You asked me why I think satfat raised my cholesterol. It could be from cutting carbs and sugars, or it could be from eating more fat. I assumed the latter because the literature says this usually happens.

Also, it says that cutting carbs usually decreases triglycerides. I found that this happened as well.

GK

Helen said...

Good to see you back!

Dave Moss said...

Ah the old 'stuffing unsuitable animals with huge amounts of foreign substances trick'!
Very glad to read this post, put me in mind of this article that I've been having to respond to lots of people about recently: http://www.pnas.org/content/early/2009/08/21/0907995106.abstract?sid=d9291635-8525-45ef-b007-53285e5247cf after it was very badly reported on the BBC. It might well have already been mentioned on here: 'Vascular effects of a low-carbohydrate high-protein diet'.

Basically (if I've read it right) they fed mice the equivalent of about 6 large steaks per day and were surprised when they got atherosclerosis (but didn't seem to get any worse markers for inflammation etc) and so posited that it was these controversial sounding endothelial progenitor cells that were behind it.

Primitivo said...

Dear all, if saturated fats are not a real problem, and I beleive they aren't, why do some epidemiological studies, like this one, find very unfavourable cardiovascular risks for consumption of these fats? Are there any major confounding factors here distorting reality? Stephan, need your help on this one! -> "Individual saturated fatty acids and nonfatal acute myocardial infarction in Costa Rica" - http://www.nature.com/ejcn/journal/v57/n11/pdf/1601709a.pdf

Robert Andrew Brown said...
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Robert Andrew Brown said...
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Robert Andrew Brown said...
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Robert Andrew Brown said...

My apologies for the deletions the system froze.

Primitivo

The study you cites states most foods were fried.

Does this include the meat and cheese.

Cholesterol intake rose with risk (Table 2)

Cholesterol (mg/1000 kcal) 91 112 122 141 175

Both oxidised oils and oxidised cholesterol in foods have both been associated with increased levels of oxidised cholesterol in the blood stream.

Interestingly "Intake of ice cream, sour cream and butter was also associated with increased risk of MI, although the association
was not statistically significant."

There is insufficient data on the Omega 3:6 intake. Soy contains some Omega 3, and soy intake falls with rising risk - but to what extent had the Omega 3 been removed by hydrogenation.

Increased oxidised cholesterol and much lower Omega 3 intakes are factors that could account for at least some of the differential in risk profiles.

Dave Moss said...

Ah the old 'stuffing unsuitable animals with huge amounts of foreign substances trick'!
Very glad to read this post, put me in mind of this article that I've been having to respond to lots of people about recently: http://www.pnas.org/content/early/2009/08/21/0907995106.abstract?sid=d9291635-8525-45ef-b007-53285e5247cf after it was very badly reported on the BBC. It might well have already been mentioned on here: 'Vascular effects of a low-carbohydrate high-protein diet'.*

Basically (if I've read it right) they fed mice the equivalent of about 6 large steaks per day and were surprised when they got atherosclerosis (but didn't seem to get any worse markers for inflammation etc) and so posited that it was these controversial sounding endothelial progenitor cells that were behind it.

**Edit**
Just noted that it's covered pretty extensively over at hyperlipid.

Primitivo said...

Dear Robert, thanks for those hints. Here is a nice article for everyone: "The questionable role of saturated and polyunsaturated fatty acids in cardiovascular disease." - http://www.nmsociety.org/App_Themes/Images/AboutFat/The%20Questionable%20Role%20of%20Saturated.pdf Regards, Ricardo.

Robert Andrew Brown said...

Primitivo

Thanks for the Ravnskov paper.

It is fascinating.

I googled him.

http://en.wikipedia.org/wiki/Uffe_Ravnskov

Courageous determined perceptive and where does he find the time.

Robert Andrew Brown said...
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Dr. T (Nephrologist) said...

Bravo, Stephan

Stephan Guyenet said...

GK,

My interpretation of the literature is that in the long term (one year or more), saturated fat has little or no influence on LDL in humans. The only studies that found that long-term saturated fat feeding leads to higher LDL were comparing it to PUFA oils. Of course, that's on average, so it's always possible that some individuals respond differently.

Primitivo,

I wouldn't worry about that. If you look at the observational studies as a whole, the overwhelming majority have found no connection between saturated fat consumption and the risk of heart attacks. Only a small fraction have found any connection at all, and some of those are questionable if you examine them in detail. That's the dirty little secret of the diet-heart hypothesis. I may post on it eventually; I've been lazy because the post will involve digging up 10+ studies to support my point. Anthony Colpo has a great table of the prospective trials in "The Great Cholesterol Con".

Oh, I just noticed you posted the Ravnskov article, that's a great one. He goes over a lot of the observational studies in that paper.

Primitivo said...

Thanks Stephan for your clarification. Here is another recent paper from the Paleodiet group I would like to share: "Dietary fat quality and coronary heart disease prevention: a unified theory based on evolutionary, historical, global, and modern perspectives." -
http://www.thepaleodiet.com/articles/Dietary%20Fat%20Quality%20%20CHD%20August%202009.pdf

Carl M. said...

If not monkeys, then what? Don't some monkeys eat insects to do with their fruit?

Or, how about a baboons?

I would agree that leaf-eating monkeys would be a bad model.

Of course, the beauty of rats is: they are cheap.

Senta said...

Welcome back, Stephan! Fantastic post, as usual.

Carl M, not only are rats (and mice) cheap but so far, they don't have a lot of fans to protect them. The closer animals get to being "human" the more people object to them being used in experimentation.

I think they should use humans. Properly informed, consenting humans.

Adolfo David said...
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Adolfo David said...

Everyday I am more convinced by high fat theory of Weston Price Foundation although I continue to think that Omega 3 and olive oil should be my main fats.

I already took Omega3-enriched eggs almost everyday, probably I will include organic semi-skimed milk instead of skimed. I am thinking still about organic butter.. At the end they are all calories, and no one can take calories with no limit.

Also I take everyday a lipid-soluble multivitamin and high dose vitamin D3 along with my fish oil. Supplements are not like drugs as I think you believe Stephan. Greetings!

Anna said...

Adolfo David,

I eat a fairly high fat diet (though I don't calculate calories or fat %). It's easy to overeat fat and excess calories if also consuming carb-rich foods (milk is a good example), but if carbs are restricted, it's hard to over-consume fat (like butter) for very long. Without the carbs and subsequent insulin rush, fat just satisfies faster, blunting and delaying hunger pangs and food cravings.

Adolfo David said...

Hi Anna, I go on years ago Dr Barry Sears Zone Diet, no glycemic food in my diet so.

Adolfo David said...

Milk has almost so much protein as carb. Maybe do you consider milk a glycemic food as some studies point? I take only a glass a day of skimed organic cow milk or organic soy milk.

Anna said...

Adolfo David,

I suppose it depends on how intact your glucose control system is. Mine isn't normal anymore and probably hasn't been for a long time (impaired first phase insulin response; sometimes delayed, then over-robust second phase insulin response).

So for me I find a Zone-type diet is often still too high in carbs and too low in fat to obtain the kind of BG control I aim for (with low fat high carb foods my BG goes too high quickly, and then either stays there or a long time or rapidly goes too low). A diet higher in fat and low in sugars and starches seems to work best for me to keep my BG, weight, mood, energy level, etc. in a steady, even range. I don't take diabetes meds, just carb control and lots of natural fat.

I don't drink as much whole milk as my son does (we use raw milk), but I do use a lot of cream. I've always disliked the taste and watery consistency of reduced fat milk, plus the sugars are fairly high for me to consume except rarely or in very limited quantities. And personally, I feel that industrially-produced reduced fat foods are too artificial and processed, including conventionally produced dairy. I'm trying to consume foods closer to their traditional forms. But YMMV. If it works for you, great.

From the Zone Diet website:
The Zone Diet can best be described as a moderate-carbohydrate, moderate-protein, moderate fat diet that has approximately one gram of fat for every two grams of protein and three grams of carbohydrates (the Zone 1-2-3 Method™). These ratios represent the newest dietary recommendations from the Joslin Diabetes Research Center at Harvard Medical School for the treatment of obesity and type 2 diabetes.

Robert McLeod said...

Carl M. wrote:If not monkeys, then what? Don't some monkeys eat insects to do with their fruit?

Dogs and pigs are better choices. Pigs eat a lot of food compared to rats, however, so they are very expensive.

Adolfo David said...
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Adolfo David said...

Zone is a paleodiet based on achieve your personal optimal insuline zone, not too hight but not too low. From its ratio 1-2-3 grams fats-protein-carbs, which is 40 30 30, you learn to increase a little and reduce more carbs to adjust your personal ideal ratio carbs/protein. Simply hormonal common sense.

Stephen Guy-Clarke said...

A great insight is that animals do not get atherosclerosis – a fact well known to vets for the past 50 years. Animals produce an enzyme that converts glucose into vitamin C in the liver. Could it be that we humans are suffering from a form of scurvy caused by insufficient amounts of vitamin C which in turn forces the body to use lipoproteins in cholesterol to repair damaged arteries? Thus, atherosclerosis in humans and guinea pigs (guinea pigs and humans need vitamin C in their diet whereas all other mammalian species make their own) is due to vitamin C deficiency. The body reservoir of vitamin C in people is on average 10 to 100 times lower than the vitamin C levels in animals.
www.beatingcholesterol.com

Aerialrose said...

What no one here is mentioning: herbivores are the only animals capable of developing atherosclerosis.

You can feed 200 X the amount of dietary cholesterol and animal saturated fat that a human normally eats to cats or dogs and they will never develop atherosclerosis.

What does this say about humans? The conclusion a 4 year old can figure out: humans are herbivores. And this why rabbits were used (and other herbivorous animals) in the studies, and why, though I don't support any animal testing, if I were to use an animal, it would be one that matches the human on all major accounts, like this comparative anatomy chart shows:

http://www.vegsource.com/news/2009/11/the-comparative-anatomy-of-eating.html

For those promoting the low carb, high fat diet from Weston A. Price's website:


Guess what? Weston A. Price died from a heart attack at age 68. He's not exactly someone I would take health longevity advice from.