Sunday, October 28, 2012

Fats and LDL Oxidation

The fat composition of the diet highly influences the fat composition of adipose tissue, phospholipids and cholesterol esters.  Changes in fat composition of adipose tissue to changes in the diet are slow and take a few years to approximates the proportions of fats in the diet [1] 

The following studies fed humans a variety of diets rich in a particular type of fat for 5 weeks then sampled their blood and oxidised the lipids.  Malondialdehyde and conjugated dienes are products of lipid peroxidation and lag time refers to how long it takes for oxidisation to occur. 

Vitamin E (molecules/ LDL particle)
TBARS MDA (nmol/mg LDL protein)
Conjugated dienes (nmol/mg LDL protein)
Lag time (min)

Vitamin E (molecules/ LDL particle)
TBARS MDA (nmol/mg LDL protein)
Conjugated dienes (nmol/mg LDL protein)
Lag time (min)

(What’s interesting about the two studies above is the SFA group had higher linoleic acid in their phospholipids than the MUFA group despite a roughly equal amount in the diet.) 

PUFA (n-6)
Max rate of conjugated diene formation per minute
Lag Time (min)
TBARS before oxidation (ns)
(nmol MDA/mg protein)
TBARS after oxidation (ns)
(nmol MDA/mg protein)
Macrophage degradation before oxidation
(µg LDL x 5h-1 x mg cell protein)-1 (ns)
Macrophage degradation after oxidation
(µg LDL x 5h-1 x mg cell protein)-1

Vitamin E (nmol/mg)
Vitamin LDL bound (µM)
Inhibition time (min)
Peroxidation rate (nmol O2/min)

These studies suggest the linoleic acid to oleic acid ratio in phospholipids and the amount of vitamin E correlated very strongly with LDL oxidation rate.  And also that SFA and MUFA rich diets produce less lipid peroxidation products than PUFA rich diets.  As fat composition in adipose tissue takes a while to change, I expect the differences between SFA/MUFA and PUFA rich diets would be greater if these studies lasted longer than five weeks. 

However, the previous four studies used copper ions to oxidise LDL in vitro, which is artificial and would not happen in vivo.  To support their findings a study found a low fat and high PUFA diet increased LDL oxidation and Lp(a) compared to the participants normal diet [6] 

LDL Oxidation
Low Fat, High PUFA, High Vegetable
Low Fat, High PUFA, Low Vegetable

An interesting finding of this study is that the low fat, high PUFA diet decreased LDL-C, but increased Lp(a) – a marker of LDL-P [6] 

* Oxidised lipoproteins are rapidly cleared and plasma concentrations of oxidised LDL in healthy humans are extremely low [7] 

Further Reading:
(1) The Diet-Heart Hypothesis: Oxidized LDL, Part II

Sunday, October 21, 2012

The Diet Heart Hypothesis

The Diet Heart Hypothesis

The diet heart hypothesis is the most common application of the lipid hypothesis.  The version using total cholesterol goes like this:

  • A higher cholesterol level is associated with an increased risk of CVD
  • SFA (and dietary cholesterol) raises cholesterol and PUFA lowers cholesterol
  • Therefore SFA (and dietary cholesterol) increases the risk of CVD and PUFA decreases the risk of CVD. 

A possible new version of the lipid hypothesis using the Total:HDL-C ratio goes like this:

  • A higher Total:HDL-C ratio is associated with an increased risk of CVD
  • SFA raises the Total:HDL-C ratio and PUFA lowers the Total:HDL-C ratio
  • Therefore SFA increases the risk of CVD and PUFA decreases the risk of CVD. 

While SFA raises cholesterol and the Total:HDL-C ratio in the short term (see graphs below), it doesn’t seem to in the long term [1] [2]

 * If we go off the short term studies, replacing 10% carbohydrate with SFA will increase total cholesterol by about 0.4 mmol/l (15.2 mg/dl) and increase the Total:HDL-C ratio by 0.03.  For reference a good Total:HDL-C ratio might be between 3:1 to 4:1 a bad ratio around 5:1 or 6-1.  In other words replacing 10% carbohydrate with SFA increases the Total:HDL-C by less than 1%.  Then if we assume we can use the Total:HDL-C ratio as a risk factor we have increased our risk of CVD by less than 2.1%.  Then consider that no food in 100% SFA.  As we can see in the second graph all the fats are expected to reduce the Total:HDL-C ratio. 

Saturated Fat in Observational Studies

Cohort study: In women with CHD, the highest quartile of SFA intake had less atherosclerosis, and even slightly reversed it.  This initial result was not yet adjusted for confounding variables such as the higher rates of smoking, diabetes, trans fats, alcohol and less exercise, fibre and drug use.  Interestingly they had slightly lower LDL-C (ns).  They also had higher HDL-C, lower triglycerides, a lower total:HDL-C ratio, lower ApoB (ns) and less hypertension (ns).  Overall SFA and MUFA was associated with less progression of atherosclerosis, while PUFA and carbohydrates was associated with a greater progression of atherosclerosis [4]

SFA and CHD by European country [5]

And a meta-analysis found: 

“…insufficient evidence from prospective epidemiologic studies to conclude that dietary saturated fat is associated with an increased risk of CHD, stroke, or CVD” [6] 

Saturated Fat in Clinical Trials

Most of the clinical trials in this area are poorly designed.  Some aren’t randomised, most aren’t isolating one variable (SFA vs linoleic acid) and are instead replacing SFA and artificial TFA with omega 6 PUFA and omega 3 PUFA, and some change the whole diet along with SFA for the intervention group. 

“The American Heart Association (AHA) and individual scientists advise consumption of at least 5–10 % of energy as n-6 PUFA to reduce CHD risk. They note that randomised controlled trials (RCT) of CHD outcomes are considered to be the ‘gold-standard’ for guiding clinical practice decisions. Individual RCT, and two meta-analyses combining seven RCT, are cited as providing ‘the most convincing’ and ‘decisive’ evidence-base, with ‘immediate implications’ for ‘population and individual level recommendations’ to substitute n-6 PUFA-rich vegetable oils for SFA. However, the conclusions of these meta-analyses have been questioned due to their (1) omission of relevant trials with unfavourable outcomes; (2) inclusion of trials with weak design and dominant confounders; (3) failure to distinguish between trials that selectively increased n-6 PUFA, from trials that substantially increased n-3 PUFA; (4) failure to acknowledge that n-6 and n-3 PUFA replaced large quantities of trans-fatty acids (TFA), in addition to SFA, in several trials.” [7] 

Dietary recommendations to include linoleic acid have been based on data showing benefits when SFA and TFA have been replaced with both omega 3 and 6 PUFA, usually along with other inventions that improve food quality.  Replacing SFA and artificial TFA with both omega 6 and omega 3 PUFA decreased all-cause mortality by 22% [7] [8] 

However, replacing SFA and TFA with just linoleic acid increases CVD by 16% and all-cause mortality by 13% [7] [8].  Artificial TFA has been well established to promote CVD, so one would expect the effect of replacing SFA with omega 3 and 6 PUFA would be smaller than a 22% decrease in mortality and the effect of replacing SFA with linoleic acid would be greater than a 16% increase in CVD and a 13% increase in total mortality. 

“Unfortunately this potential confounding role of TFA was not appreciated. Similarly, the displacement of TFA, rather than the substitution of mixed n-3/n-6 PUFA for SFA, may account for some or all of the 22 % reduction in non-fatal MI+CHD death in our meta-analysis. By contrast, the increased CHD risks from n-6 specific PUFA diets in our meta-analysis may be underestimated as n-6 PUFA also replaced substantial quantities of TFA” [7] 

How is it that PUFA consumption is associated with lower CVD in observational studies, yet in clinical trials replacing SFA and TFA with LA increases CVD mortality?  There’s something called the healthy observer effect where those who observe officially endorsed health practices (such as replacing SFA with LA) have a lower risk of disease and death.  The healthy observer effect can explain why hormone replacement therapy was associated with lower CVD in observational studies but higher CVD in trials [8]

I’ll discuss in a later post a reason why replacing SFA and TFA with LA resulted in increased mortality.  If you would like to read more about the clinical trials comparing SFA with PUFA you can find that in most of the further reading and in ‘Perfect Health Diet’ by Paul and Shou-Ching Jaminet and ‘The Great Cholesterol Con’ by Anthony Coplo 

Further Reading:
(1) Diet-Heart Controlled Trials: a New Literature Review
(2) New Review of Controlled Trials Replacing Saturated fat with Industrial Seed Oils
(3) Good Fats, Bad Fats: Separating Fact from Fiction
(4) Precious Yet Perilous
(5) Myth: One High-Saturated Fat Meal Can Be Bad
(6) The Diet-Heart Hypothesis: Stuck at the Starting Gate
(7) Does Dietary Saturated Fat Increase Blood Cholesterol? An Informal Review of Observational Studies
(8) Saturated Fat Is Not Associated With CVD, Evidence of Publication Bias
(9) The Cholesterol Wars: Steinberg Strikes Back
(10) How Conflating the Lipid Hypothesis With the Diet-Heart Hypothesis Led to the Public Condemnation of Bacon, Butter, and Eggs

Sunday, October 14, 2012

Immune Related Mechanisms

Bacterial Infections and LPS 

Chronic infections and bacterial products such as LPS are associated with atherosclerosis and are found in atherosclerotic lesions [1].  Bacterial and viral infections can promote endothelial dysfunction, proliferation of endothelial cells, increase pro-inflammatory cytokines and lower HDL-C [2] [3].  LPS increases pro-inflammatory cytokines, platelet aggregation and induces endothelial cells to produce ROS that can oxidise LDL.  LPS also increases LDL-C and cholesterol synthesis perhaps because LDL-C is used by the innate immune system to bind to and neutralise LPS.  The LPS-LDL-C can be taken up by macrophages [2] 

Pro-inflammatory cytokines promote monocyte adhesion to endothelial cells [2], endothelial dysfunction, oxidative stress, apoptosis of endothelial cells are associated with worse outcomes [4].  The role of pro-inflammatory cytokines in heart disease is well illustrated by the association between RA and CVD (OR 3.17) [5].  Aspirin reduces MI by 55.7% in people with high CRP and 13.9% in people with low CRP [6] 

LPS induces mitochondrial dysfunction, which may be one of its major mechanisms of action.  People with periodontitis have 60% lower CoQ10, 78% lower citrate synthase and double the ROS.  These measures of mitochondrial function were almost completely returned to normal with CoQ10 supplementation [7] 

Chronic infections and bacterial products such as LPS are associated with CVD:

  • High levels of CD14, a receptor on monocytes for LPS, is associated with atherosclerosis and is increased by IL-1, TNF-a and glucocorticoids [8]
  • C. pneumoniae antibodies are associated with a 2-7 times increased risk of CHD [2]
  • An allele that reduces the expression of TLR4 (a receptor for LPS) has lower pro-inflammatory cytokines and an odds ratio for atherosclerosis of 0.54 [9]
  • 43% of people with antibodies to herpes virus developed restenosis (narrowing of blood vessel), compared to 8% without antibodies.  Restenosis correlated with chronic infections rather than acute infections [3]
  • High LPS is associated with atherosclerosis in smokers (OR 14.7) and ex-smokers (OR 9.5), but not non-smokers (OR 1.7 NS).  However non-smokers with a chronic infection and high LPS had an OR of 5.4 [10]
  • Either C. pneumoniae or H. pylori DNA was found in the artery wall of 50% of patients undergoing surgery for atherosclerosis, compared with 0% in the healthy controls [11]
  • LPS is associated with increased triglycerides and troponin and decreased HDL-C [12]
  • C. pneumoniae (OR 3.06) and H. pylori (OR 3.82) infections are associated with myocardial infarction [13] 

* Some of the heart problems in endurance athletes could be explained by LPS.  At high intensities of exercise blood flow to the intestines can drop to 20%, which disturbs normal functioning and may cause exercise-induced abdominal symptoms and bacterial translocation.  After a marathon 68% of athletes had mild endotoxemia and IL-6 levels were 27 times higher [14] 

** CVD is a common complication of AIDS, affecting 70-80% of people who have it [15] 


Myeloperoxidase (MPO) is found in neutrophils, monocytes and some macrophages, and is stored in an inactive form until these cells become active.  It produces hypochlorous acid (HOCl) from H2O2 and Cl- and oxidises tyrosine to tyrosyl radical by using H2O2.  HOCl and the tyrosyl radical are cytotoxic and are used by neutrophils to produce a respiratory burst [16]. 

Evidence to support the role of MPO in CVD:

  • MPO is active in atheroma, MPO oxidation products are found in atheroma and attract leukocytes [16]
  • People with CVD have more MPO products such as (nitrotyrosine and chlorotyrosine) [16]
  • MPO can modify HDL particles and ~50% of HDL particles in atherosclerotic lesions have some kind of MPO modification [16]
  • MPO promotes endothelial dysfunction by: reducing NO bioavailability, producing HOCl which inhibits NOS, chlorinating arginine and by producing ROS and RNS which uncouple NOS leading to SO production rather than NO [16]
  • MPO promotes endothelial cell apoptosis [16]
  • LDL/HDL from atherosclerotic lesions contains 100/6 times more 3-NO2Tyr compared with circulating LDL/HDL from healthy controls [17] 

MPO levels are very strongly associated with CVD:

  • Leukocyte and blood MPO is associated with CAD (OR of 11.9 and 20.4) [18]
  • MPO is associated with endothelial dysfunction (OR of 6.4) [19]
  • MPO modified HDL is associated with CVD (OR of 6 and 16 (depends on the MPO oxidation product) [20]
  • 60-66% of Americans have the GG genotype, which increases MPO and is associated with CV events (HR of 5.5) [21].
  • The AA genotype has an OR of 0.135 and the AG genotype has an OR of 0.639 [22]

Sunday, October 7, 2012

Endothelial Dysfunction

Endothelial Dysfunction

Endothelial cells secrete substances to maintain an appropriate degree of clotting, cell proliferation, blood pressure, etc.  Perhaps the most important of these substances is nitric oxide (NO), which inhibits LDL oxidation and is a vasodilator (relaxes the blood vessels to lower blood pressure).  A defect in the production or activity of NO leads to endothelial dysfunction (ED) [1] [2]. 

ED leads to an increase of endothelial permeability, LDL oxidation, platelet aggregation, leukocyte adhesion, pro-inflammatory cytokines, vasoconstriction and smooth muscle cell proliferation – all of which are factors that promote atherosclerosis to varying degrees [2].  Evidence to support the role of ED in atherosclerosis:

  • ED is a strong independent predictor of CVD [3].  In a sample of 157, those with mild ED and without ED had no cardiac events, while 6 people (14%) with severe ED had cardiac events after 28 months [4]
  • People with CVD tend to have ED, and atherosclerosis and hypertension strongly predict ED [5]
  • Correcting ED through several means (arginine (precursor to NO), statins and increasing NO availability/activity) improves cardiac outcomes [4]


ROS and reactive nitrogen species (RNS) are a cause of endothelial dysfunction [1].  In particular superoxide (O2-) can combine with nitric oxide (NO) to form peroxynitrite (ONO2).  This process generates a more powerful oxidant and displaces NO.  Peroxynitrite (PON) is a 2e oxidant and has a number of effects that promote CVD:

  • PON nitrates prostacyclin synthase, which decreases prostacyclin, an eicosanoid derived from arachidonic acid that is anti-inflammatory, a vasodilator and inhibits blood clotting [1]
  • PON can modify LDL and PON modified LDL has a high affinity for macrophage scavenger receptors [1]
  • PON inhibits mtSOD and the electron transport chain, which leads to more superoxide and more PON [1]
  • PON upregulates adhesion molecules in endothelial cells and neutrophil adhesion [1]
  • PON uncouples NO synthase which leads to the production of superoxide rather than NO [1]
  • PON triggers apoptosis in endothelial and smooth muscle cells [1]
  • PON promotes mitochondrial dysfunction [6]

Mitochondrial Dysfunction

Mitochondria are the energy factories of the cell, but in the process of processing energy mitochondria also produce reactive oxygen species (ROS).  If the ROS are not dealt with by antioxidant defenses, then they are free to cause oxidative stress.  Mitochondria and their DNA (mtDNA) are most vulnerable due to their close proximity.  ROS inhibit ATP production and can damage mtDNA, leading to mutations.  This initiates a positive feedback cycle whereby mutations in mtDNA compromise mitochondrial function, slow down the electron transport chain and lead to more ROS, more oxidative stress and more mutations. 

Mitochondrial dysfunction is a condition of low energy production, high oxidative stress, elevated pro-inflammatory cytokines, pro-apoptotic signaling, insulin resistance, etc.  Mitochondria produces 90% of the endogenous ROS [7], which begins as superoxide and can combine with NO to form PON and lead to ED.  Insulin resistance can lead to hyperglycemia and increased free fatty acids, which further increases superoxide generation in the mitochondria [8] [9].  Evidence supporting the role of mitochondrial dysfunction in CVD:

  • Hearts from patients with CAD had 8-2000 times more mtDNA deletions than controls [8]
  • Blood vessels often have inefficient ATP production before developing atherosclerosis [10]
  • Mitochondrial dysfunction precedes atherosclerosis in animal models of atherosclerosis [11]
  • There is more mtDNA damage in the blood vessels of people with atherosclerosis and mtDNA mutations correlate with atherosclerosis [11]
  • Age is one of the strongest risk factors for CVD and mitochondrial dysfunction is a major contributor to the aging process.  mtDNA mutations accumulate with age and are inversely correlated with maximal life span [8]
  • People with LDL >130mg/dl tend to have more mtDNA mutations and more oxidative stress [12]
  • Mitochondrial superoxide promotes atherosclerosis and mtSOD is protective against atherosclerosis [6]
  • Mitochondrial superoxide has been found to be required to oxidise LDL in vitro [13]
  • Atherosclerotic lesions in Alzheimer’s have increased mtDNA deletions [8]
  • Mitochondrial dysfunction is an important link between CVD with obesity [14] and the metabolic syndrome [15] 

* After 1 year, CoQ10 and statins reduced cardiac events in patients who previously had an MI  by 45.3% compared with B Vitamins and statins.  Also, the CoQ10 group had only one sixth of the side effects (fatigue) from statins [16]