Designed By Nature

When looking at the risk calculators for cardiovascular disease, your risk of coronary atherosclerosis is calculated based on several factors. Today we already see that LDL alone doesn’t create much of a risk when using the industry top ranked tools. It looks like science has moved on while medical care and general public is still focused heavily on reducing LDL, the ‘bad’ cholesterol.

Personally I’m not worried about it either but there is one thing I’m left with that I’m just genuinly curious about and that is oxidized LDL (oxLDL), oxidized and glycated.

Why oxidized and glycated? Because it seems to be a pretty good proxy for your risk. Research also shows us that glycated LDL is easier oxidized. Using glucose 6-phosphate they show a 73.77% higher oxidation. It makes me think, is oxidized LDL in general bad or is it more the glycated oxidized LDL? After all, according to Joseph Kraft: “Those with cardiovascular disease not identified with diabetes are simply undiagnosed“. That would give us a hint on the importance of glycation and oxidation versus oxidation alone.

“Study on the levels of glycosylated lipoprotein in patients with coronary artery atherosclerosis” https://onlinelibrary.wiley.com/doi/full/10.1002/jcla.22650

“Why is glycated LDL more sensitive to oxidation than native LDL? A comparative study.” https://www.ncbi.nlm.nih.gov/pubmed/11049692

There are people who go on a low carb diet and tend to develop high LDL cholesterol (LDLc) with levels of > 180 reaching >400 or even >500. That is huge! I personally have >200. My research to understand the high levels has led me to conclude that this happens due to a reduced production combined with a lowered clearance. You can trace back my work starting here.

So naturally the question comes up, are such people at higher risk? You could argue that the longer residency of LDLc in the pool gives it more chance of getting glycated and oxidized. If there is any causality to oxLDL then for sure having such high LDLc numbers must mean higher chance of CAD right?

Let’s see where the research leads us to.

First to further understand the importance of glycation, the following paper summarizes what the effects are:

Metabolic abnormalities associated with glycation of LDL include diminished recognition of LDL by the classic LDL receptor; increased covalent binding of LDL in vessel walls; enhanced uptake of LDL by macrophages, thus stimulating foam cell formation; increased platelet aggregation; formation of LDL-immune complexes; and generation of oxygen free radicals, resulting in oxidative damage to both the lipid and protein components of LDL and to any nearby macromolecules. Oxidized lipoproteins are characterized by cytotoxicity, potent stimulation of foam cell formation by macrophages, and procoagulant effects. Combined glycation and oxidation, “glycoxidation,” occurs when oxidative reactions affect the initial products of glycation, and results in irreversible structural alterations of proteins. Glycoxidation is of greatest significance in long-lived proteins such as collagen. In these proteins, glycoxidation products, believed to be atherogenic, accumulate with advancing age: in diabetes, their rate of accumulation is accelerated.

“Glycation and oxidation: a role in the pathogenesis of atherosclerosis.” https://www.ncbi.nlm.nih.gov/pubmed/8434558

The main question now that stands out is the following.

Do people on a low carb diet, with very high elevated LDLc, have higher glycated LDL equaling the levels of diabetics and thus potentially equaling the CAD risk?

To answer this I would like to understand what causes glycation of LDL in the first place. Secondly I would also like to understand how clearance of glycated LDL works under non-diabetic conditions.

sdLDL

A first paper shows us that the density of LDL makes a difference in its susceptibility to glycation. Small dense LDL (sdLDL) is much more affected by glycation (The percentage of apo B present in LDL1 and 2 which was glycated was 1.8+/-1.8% whereas in LDL3 it was 17.4+/-18.5% (P<0.001)). As you go on a low carb, your LDL profile shift to the large buoyant size, away from the sdLDL. If you double the amount of LDL1 and LDL2 sized particles, you just reach the same volume of sdLDL but we also know that such a shift goes in hand with a reduction of sdLDL so overall your glycated particles are less in total (Of the glycated apo B in LDL 67.8+/-21.9% was in small dense LDL (LDL3; D1.044-1.063g/ml) whereas only 32.2+/-21.9% was in more buoyant LDL subfractions (LDL1 and 2; D1.019-1.044g/ml)).

“Glycation of LDL in non-diabetic people: Small dense LDL is preferentially glycated both in vivo and in vitro.” https://www.ncbi.nlm.nih.gov/pubmed/18511055

AGE’s

What actually causes the glycation? One such thing appears to be Advanced Glycation End products (AGE)-peptides which are found at increased levels in diabetic patients.

“Modification of low density lipoprotein by advanced glycation end products contributes to the dyslipidemia of diabetes and renal insufficiency.” https://www.ncbi.nlm.nih.gov/pubmed/7937786

These AGE-peptides are formed when breaking down AGE’s and once those AGE-peptides are formed they are normally excreted through urine. For this reason we have a higher independent risk of CAD for patients with end-stage renal disease. This confirms glycation is an important factor.

https://en.wikipedia.org/wiki/Advanced_glycation_end-product#Clearance

“Cardiac risk assessment for end-stage renal disease patients on the renal transplant waiting list” https://academic.oup.com/ckj/article/12/4/576/5479991

AGE’s are created through the binding of glucose with proteins or lipids. There is no way to avoid this, it is part of our system so what is the normal way of clearing glycated LDL (gLDL)?

Lipoprotein lipase (LPL)

The gLDL are increasingly taken up, mediated by LPL, according to the level of glycation. This is independent of LDL receptors of which we know the numbers go down on low carb. Apart from the number of receptors, also the affinity for the receptors is lowered. It looks like the greater the LPL, the greater the affinity for gLDL enhancing the binding, uptake and degradation.

“Lipoprotein Lipase Mediates the Uptake of Glycated LDL in Fibroblasts, Endothelial Cells, and Macrophages” https://diabetes.diabetesjournals.org/content/50/7/1643

So how is LPL influenced on a low carb diet? Jeff Volek speculates a reduction of lipase in adipocytes, hepatic lipase but an increase in muscular lipase so that energy is diverted to where it is needed. This is not just simple guess work. We see that just shifting from a 43% to a 54% dietary intake of fat for 4 weeks increases the muscle lipoprotein lipase with 80% (During the high-fat diet period, the muscle lipoprotein lipase activity (LPLA) increased from 59 +/- 8 to 106 +/- 12 mU/g (mean +/- SE) (P less than 0.05)).

“Modification of Lipoproteins by Very Low-Carbohydrate Diets” https://academic.oup.com/jn/article/135/6/1339/4663837

“Lipoprotein lipase activity and intramuscular triglyceride stores after long-term high-fat and high-carbohydrate diets in physically trained men.” https://www.ncbi.nlm.nih.gov/pubmed/3545651

Exercise

There are many benefits to exercise but exercise may raise glucose levels during exercise. Does that cause any issues like increased AGE’s?

A study on the effects of exercise in a HIV study group, who are more susceptible to AGE’s due to increased insulin resistance (a CAD risk factor) shows that exercise reduces AGE’s to the same level as a healthy control group. The active HIV group even had lower AGE levels than the healthy control group.

“Influence of Physical Exercise on Advanced Glycation End Products Levels in Patients Living With the Human Immunodeficiency Virus” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6291474/

We also see this in rats, children and athletes and a correlation with increased skin AGE depending on western diet (FFQ data) in youth.

“Regular moderate exercise reduces advanced glycation and ameliorates early diabetic nephropathy in obese Zucker rats” https://www.ncbi.nlm.nih.gov/pubmed/19608208

“Influence of physical fitness and activity on advanced glycation end-products accumulation in children” https://esc365.escardio.org/Congress/EuroPrevent-2018/Young-investigator-award-session-IV-Prevention-Epidemiology-Population-Scie/169407-influence-of-physical-fitness-and-activity-on-advanced-glycation-end-products-accumulation-in-children

“Effects of Long-Term Physical Activity and Diet on Skin Glycation and Achilles Tendon Structure” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627972/

Conclusions

As a first dive into this world it is enough for now. What do we have so far?

Glycation seems to be the major risk factor. Considering diabetes and end-stage renal disease it looks like our system, when overwhelmed with AGE-peptides gets into trouble.

What does this mean for people on a low carb diet with elevated LDLc?

1. They have stable glucose levels due to the low carb diet so there are rarely elevated glucose levels (hyperglycemia) which would intensify the glycation.
2. They have increased circulation time of LDLc raising accumulation of glycation
3. However, skeletal muscle lipoprotein lipase is highly increased creating a beneficial sink of gLDL avoiding large uptake by macrophages.
4. Being active further reduces the AGE’s leading to lower glycation of LDL and likely a diversion of uptake to muscle away from fibroblasts (as the achilles tendon shows) and macrophages.

A few words on oxidized LDL in general though because we have been putting our attention on glycation so far.

Oxidized LDL

Let’s first get a bit of an idea what oxLDL is. There are different levels of oxidation, affecting the properties and behavior of the LDL particle. The lipids can be oxidized, the cholesterol content can be oxidized and also the lipoprotein itself can be oxidized(1). These all change the properties every time and they all reflect together the degree of oxidation that was undergone. This makes research on it very difficult and to make it even more difficult, there are different ways to oxidize the LDL particle (UV radiation, iron ions, copper ions etc), creating inconsistent lab results as the method also influences the properties.

Below is a picture of the different forms of oxLDL.

There is also a difference in when this oxidation happens. Due to processing, fatty acids and cholesterol can become oxidized by the time they end up on our plate so exogenous oxidation. That sets it apart from endogenous oxidation which is the oxidation that happens in our body. The distinction here is made because when we eat oxidized fatty acids or cholesterol, they end up in our body already oxidized and therefor directly increase the level of oxidized LDL. For endogenous oxidation, it will depend on the antioxidant activity on one side and the oxidation activity on the other side.

Resources

(1) “In vitro oxidative footprinting provides insight into apolipoprotein B-100 structure in low density lipoprotein” – Sourav Chakraborty, Yang Cai, and Matthew A. Tarr – 2014 – https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320993/

(2) “Oxidized Low-Density Lipoprotein” – Sampath Parthasarathy, Achuthan Raghavamenon, Mahdi Omar Garelnabi, and Nalini Santanam – 2010 – https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3315351/

Degree of oxidation

To immediatly make things complex, there is evidence suggesting a completely different role for oxLDL depending on its level of oxidation. ‘mild’ oxidation appears to prevent macrophage uptake and foam formation (1). That is interesting, now we have to be cautious about the level of oxidation per particle when looking into research and see if they control for that in experiments.

What exactly the right level is is hard to find but we can see from observation that people who are diagnosed with coronary artery disease (CAD) have an increased chance of cardiac events(2). Non-events had a mean of 17.6 U/ml while CE had 20.3 U/ml of oxLDL. The HR of the highest quartile was 3.15 versus the lowest quartile.

However, when looking at the totality of evidence you’ll find different results ranging from no association to very high association(3). I believe this is due to either measurement method and/or what is detected as oxidized. Are they only looking at the oxidized cholesterol or oxidized ApoB? Are they looking at a certain degree, like a threshold level? As you could see there are varying degrees of oxidation and even apparent protective effect when there are mild forms of oxidation(1).

Resources

(1) “Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation” – Jason M. Meyer, Ailing Ji, Lei Cai, and Deneys R. van der Westhuyzen – 2014 – http://www.jlr.org/content/55/8/1648

(2) “Circulating oxidized low-density lipoprotein is an independent predictor for cardiac event in patients with coronary artery disease” – Kazunori Shimada, Hiroshi Mokuno, Eriko Matsunaga, Tetsuro Miyazaki, Katsuhiko Sumiyoshi, Katsumi Miyauchi, Hiroyuki Daida – 2004 – https://www.sciencedirect.com/science/article/abs/pii/S0021915004000905

(3) “Association between circulating oxidized low-density lipoprotein and atherosclerotic cardiovascular disease” – Shen Gao, Jing Liu – 2017 – https://www.sciencedirect.com/science/article/pii/S2095882X16301244

Protective factors

Vitamin E

It seems that the fat-soluable vitamin E has the ability to suppress (2) and delay (9) oxidation and prevent macrophages from taking them up. When looking at vitamin E we want to make sure we look at its most bioavailable form which is α-tocopherol (aT) .

aT is preferentially bound to α-Tocopherol Transfer Protein (aTTP) in the liver through which it is transferred onto ApoB particles (3).

Hypoxia seems to induce an increase in aTTP (4). Could this potentially lead to deficiency of vitamin E over time if this hypoxia is chronic? It would take us too far to investigate but COPD patients can suggest us there is a link (5).

An experiment in rats also gives us an indication that insufficient protein in the diet may lead to lower levels of aT on the VLDL particles (6).

Deficiency in aT sets us up for oxidation of the LDL particles. Do we get more deficient when we have higher LDL levels? There is a possibility but it will depend on many factors. Are the high LDL levels caused by longer retention and thus the rate of production and rate of disappearance is slowed such as in our low carb hyperresponders? With a lower production rate the liver may have sufficient aT to load onto the particles. If there is an increase in the production rate then there is a possibility for insufficient aT.

But that is not all. aT gets redistributed. Extra-hepatic tissue exports aT which is picked up by HDL and transferred to the other lipid particles (7) so your level of HDL in the blood could be of importance as well.

HDL

Whether HDL numbers are important for the anti-oxidant effect of aT is difficult to know but HDL itself has similar functions. It can take away the lipid hydroperoxides from the LDL particle essentially functioning as an anti-oxidant (8).

It is hard to know the extend to which HDL levels are important but if they contribute to less oxLDL then it favors to have higher levels. We see this coming back in our low carb hyperresponders.

Resources

(2) “Vitamin E and Atherosclerosis: Beyond Prevention of LDL Oxidation” – Mohsen Meydani – 2001 – https://academic.oup.com/jn/article/131/2/366S/4686917

(3) “Alpha-Tocopherol Transfer Protein (α-TTP): Insights from Alpha-Tocopherol Transfer Protein Knockout Mice” – Yunsook Lim and Maret G. Traber – 2007 – https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2849030/

(4) “Expression of the Alpha Tocopherol Transfer Protein gene is regulated by Oxidative Stress and Common Single Nucleotide Polymorphisms” – Lynn Ulatowski, Cara Dreussi, Noa Noy, Jill Barnholtz-Sloan, Eric Klein, and Danny Manor – 2012 – https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3612136/

(5) “Differential Expression of Vitamin E and Selenium-Responsive Genes by Disease Severity in Chronic Obstructive Pulmonary Disease” – AH Aglera, RG Crystalb, JG Mezeyb, J Fullerb, C Gaoc, JG Hansena, and PA Cassano – 2013 – https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060420/

(6) “Secretion of α-Tocopherol in VLDL Is Decreased by Dietary Protein Insufficiency in Young Growing Rats” – Huey-Mei Shaw, Ching-jang Huang – 2000 – https://academic.oup.com/jn/article/130/12/3050/4686278

(7) “Complexity of vitamin E metabolism” – Lisa Schmölz, Marc Birringer, Stefan Lorkowski, Maria Wallert – 2016 – https://www.wjgnet.com/1949-8454/full/v7/i1/14.htm

(8) “Antioxidative activity of high-density lipoprotein (HDL): Mechanistic insights into potential clinical benefit” – Fernando Brites, Maximiliano Martina, Isabelle Guillas, Anatol Kontush – 2017 – https://www.sciencedirect.com/science/article/pii/S2214647417300326

(9) “Effects of alpha-tocopherol on superoxide production and plasma intercellular adhesion molecule-1 and antibodies to oxidized LDL in chronic smokers.” – van Tits LJ, de Waart F, Hak-Lemmers HL, van Heijst P, de Graaf J, Demacker PN, Stalenhoef AF – 2001 – https://www.ncbi.nlm.nih.gov/pubmed/11369502

Sleep/melatonin

There are indications that the quality of sleep can have an influence, suspected mainly through the action of melatonin. A hormone that is released when it gets dark. Melatonin has the ability to scavenge ROS which are part of the metabolites that oxidize LDL. A second paper looked at nocturnal levels of oxLDL and found it significantly associated.

“An assessment of oxidized LDL in the lipid profiles of patients with obstructive sleep apnea and its association with both hypertension and dyslipidemia, and the impact of treatment with CPAP” – Marcia C. Feresa, Francisco A.H. Fonseca, Fatima D. Cintra, Luciane Mello-Fujita, Altay Lino de Souza, Maria C. De Martino, Sergio Tufik, Dalva Poyares – 2015 – https://www.atherosclerosis-journal.com/article/S0021-9150(15)01309-X/abstract

“Elevated levels of oxidized low-density lipoprotein and impaired nocturnal synthesis of melatonin in patients with myocardial infarction” – A.Dominguez-Rodriguez P.Abreu-Gonzalez M.Garcia-Gonzalez J.Ferrer-Hita M.Vargas R.J.Reiter – 2005 – https://www.sciencedirect.com/science/article/abs/pii/S0021915004005830

Finally

What sets the risk for CAD apart is greatly defined by glucose control. Having large prolonged upswings due to insulin resistance are likely the biggest factor contributing to glycation… the inability to lower glycose quickly to baseline.

Our system HAS clearance capabilities for oxidized LDL and glycated LDL but it only works so well until it gets overwhelmed.

Going low carb and being active looks like it gives the best chance to avoid glycation and lower our risk of CAD. Not just because of reducing glycated LDL but because it also positively reduces the other risk factors such as hyperinsulinemia, hypertension etc.

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