LDL Cholesterol Particle Size and Number What Gives ?

ApoB_LDL_2_Jeffrey Dach MDLDL Particle Size and Particle Number, What Gives?

Superiority of Calcium Score

Ron is a 72 year old retired engineer, and has a total cholesterol of 174 which hasn’t changed over the seven years we have been following him. This is quite low. Yet, Ron is concerned because his LDL particle number and LDL particle size are “outside of the lab range”.  He is very worried about this and is concerned about his risk for future heart attack.  I explained to Ron the lab range doesn’t apply to him.  Ron’s Calcium Score is low, and his total cholesterol is 174, and he does not have metabolic syndrome or diabetes, so he doesn’t need to worry about the LDL particle size or particle number.

What does the mainstream cardiology say about the value of LDL particle size and number?

The Quebec Study – Small Dense LDL Associated with Increased Mortality from Coronary Artery Disease

Small Dense LDL associated with Increased Risk St Pierre QuebecYou might say “wait just a minute here”, the Quebec study followed 2072 males over 13 years and found that small dense LDL was associated with increased mortality from cardiovascular disease above chart).(6)  The above chart is very convincing, and the three lines for small dense LDL are nicely separated with greater numbers of small dense LDL associated with increased mortality.. (6) However, as pretty as the above chart looks, Correlation is not necessarily causation.  If increased small dense LDL particle number causes coronary artery disease, then intervention to reduce small dense LDL particle number should be preventive.  A new study shows this is false.  Reducing small particle LDL with a new drug FAILS to reduce heart attack rate compared to placebo.!!  Above image courtesy of Medscape.

Houston, We Have a Problem,  New Drug Reduces Small LDL,

However, No Benefit in Preventing Heart Disease

Treatment with the new cholesterol lowering drug, Evacetrapib, resulted in significant decreases in “total LDL particle number (LDL-P) (up to -54%), and small LDL particle (sLDL) (up to -95%) concentrations”.(5) Yet, according to Dr Lincoff in NEJM 2017,

“treatment with evacetrapib did not result in a lower rate of cardiovascular events than placebo among patients with high-risk vascular disease.”(4)

As a matter of fact, Eli Lilly abandoned drug development after this failed study.(4)  So we see that reducing total LDL particle number, or increasing LDL particle size had no benefit for preventing death from heart disease.  The benefit was same as a placebo.

Anacetrapib, another CETP inhibitor similar to evacetrapib, under development by Merck had a similar outcome.  The drug reduced LDL cholesterol, and increased HDL cholesterol, yet did not prevent heart attacks. In October 2017,  Merck announced they would not seek FDA approval and abandoned the drug.(29)

Dr Allaire  agrees that LDL particle size is not very useful.  Dr Allaire writes in 2017 Current Opinion in Lipidology:(1)

“LDL particle size….has not been independently associated with CVD risk after adjustment for other risk factors such as LDL cholesterol, triglycerides, and HDL-C and that routine use of information pertaining to particle size to determine and manage patients’ risk is not yet justified.”(1)

In other words, according to Dr Allaire,  the LDL particle size is not a good predictor of cardiovascular risk.(1)

Dyslipidemia of Metabolic Syndrome and AODM (Adult Onset Diabetes)

We still have yet to explain the association without causation of small dense LDL with increased mortality from cardiovascular disease.   The answer is fairly obvious.  Increased small dense LDL particles is a marker for metabolic syndrome and diabetes milletus (AODM), both of which are associated with accelerated cardiovascular disease.  The pattern of “diabetic dyslipidemia” is described nicely by Dr Rivas-Urbina.(9)

“Diabetic or atherogenic dyslipidemia, is characterized by high levels of triglycerides and apoB, low concentration of high density lipoprotein (HDL) cholesterol, and increased postprandial lipidemia. This abnormal lipid profile is typical of diabetes but it is also present in pre-diabetic situations such as insulin resistance and metabolic syndrome….This means that at a given LDL cholesterol concentration, diabetic patients have a greater number of LDL particles”

Dyslipidemia with small particle LDL is only a marker for metabolic syndrome.   The prediabetic state called metabolic syndrome and diabetes itself are associated with high risk for accelerated cardiovascular disease.(7-10)     As we have seen above, the failed drug study with Evacetrapib reveals the futility of modifying Lipoprotein subfractions with a drug, while ignoring metabolic syndrome.

Reduction in cardiovascular risk is best acheived by addressing underlying metabolic syndrome, the pattern is overweight, insulin resistance, elevated blood sugar, and elevated Hgb A1c.   This is best achieved by diet and lifestyle modification to reduce fasting blood sugar, reduce hemoglobin A1c, reduce weight, and reduce blood pressure.   Other interventions such as Metformin and Berberine are also useful for blood sugar control.

Leaky Gut and Low Level Endotoxemia

Studies show that patients with metabolic syndrome and diabetes have leaky gut with  low level endotoxemia, implicated in the pathogenesis of many diseases. (13)  Dr Robert Munford in 2016 states:(11)

“Gram-negative bacterial endotoxin (LPS) been invoked in the pathogenesis of so many diseases—not only as a trigger for septic shock, once its most cited role, but also as a contributor to atherosclerosis, obesity, chronic fatigue, metabolic syndrome, and many other conditions.”(11)

Low level endotoxemia from “leaky gut” triggers release of inflammatory cytokines such as IL-6.(13)  This inflammatory state is a direct cause of coronary artery disease.(12)  Low Level endotoxemia is associated not only with inflammation, but also infection of the arterial wall.  Indeed, atheromatous plaque has been shown infected with polymicrobial biofilm by  modern 16s Ribosome testing techniques.(17-21) See my article on this topic.

Addressing leaky gut with the well known protocol of gluten free diet, probiotics, glutamine, colostrum, berberine, aged garlic etc. is necessary to prevent progression of calcium score and plaque.  See my previous article on Leaky Gut and Low level Endotoxemia and its association with coronary artery disease.

Predicting Risk: LDL Subfraction Vs. Calcium Score

Below chart shows calcium score (CCS) correlates closely with Mortality from Cardiovascular Disease and Myocardial Infarction. CCS=0 (Zero score, red line) has best survival, while over CCS>400 (blue line) has worst survival. Note: Authors recommended adding CAT coronary angiography in symptomatic individuals suspected of having CAD to aid in discrimination.(22)

Coronary Calcium Score Risks CONFIRM Registry. Eur Heart J Cardiovasc Imaging 2014 Jeffrey Dach Above Chart is Fig 2 from Eur Heart J Cardiovasc Imaging. 2014 Mar;15(3):267-74 (22).  Note CCS = Calcium Score.

The next question you might ask: “If the cholesterol lipoprotein panel is useful only as a marker for metabolic syndrome, and not useful as a predictor of risk for coronary artery disease,  then what is a better test we should use to predict risk for cardiovascular disease?” 

ore Jeffrey dach Hecht 2015The answer is the Calcium Score which is an inexpensive test which uses a CAT scan to measure the amount of calcium in the coronary arteries.(23)  (Left Image)

Studies show that the higher the calcium score number the greater the risk, the lower the number the smaller the risk.(22)  None of the cholesterol subfractions can provide this type of information, and in my opinion should be relegated to the medical museum, a relic of the past. Left image shows CAT scan of coronary artery calcification, white color on arteries within  yellow circle. Fig 1 from Hecht 2015  (25)

Serial Calcium Scores Reveal Response to Treatment.

Progression of calcium score less that 15% per year implies Benign Prognosis.  However Progression of calcium score greater than 15% a year implies poor prognosis. See chart below.

CAC= Calcium Score.  Above image is  Figure 5 from Hecht 2015 (25). originally from Raggi 2004(28). Progression of CAC and Risk of First MI in 495 Asymptomatic Patients Receiving Cholesterol-Lowering Therapy.
(Left Chart) CAC progression of <15% per year is associated with a benign prognosis irrespective of the baseline CAC, implying stabilization of the atherosclerotic process.
(Right Chart) CAC progression of >15% per year is associated with a poor prognosis directly related to the baseline CAC, implying new plaque formation and inadequacy of treatment. CAC = coronary artery calcium; MI = myocardial infarction.  JACC: Cardiovascular Imaging Volume 8, Issue 5, May 2015  Coronary Artery Calcium Scanning Past, Present, and Future Harvey S. Hecht (25). Original data from Raggi, 2004(28)

Paradigm Shift in One Chart

Fifteen Per Cent Annual Change in Calcium score

Raggi 2004 calcium Score 15 per cent change Fig.2This left chart shows the MAJOR FINDING: Less than 15% annual increase in calcium score is associated with a good prognosis.(upper line)

Greater than 15% progression show a declining mortality curve indicating a poor prognosis.(lower line).  When you see the above chart for calcium score, let me remind you, there are no similar data charts for LDL cholesterol.  As a matter of fact, there was no difference in LDL levels for the 41 heart attack patients compared to 450 others free of heart attack.(28)  There was no difference !!!

Dr Raggi says:

Mean LDL level did not differ between groups (118 mg/dL versus 122 mg/dL, MI versus no MI).(Dr Raggi 2004 (28)  (Note: MI = Myocardial Infarction)

Dr Raggi found that LDL cholesterol is a useless marker for predicting future heart attack. Above Left Chart Fig2 Courtesy of Dr Raggi 2004 (28)

Why is Calcium Score a Superior Predictor Over Cholesterol ?

Cholesterol and subfractions are substances we measure in the blood stream, distant from the wall of the artery where the pathology is located. With calcium score, we are measuring the pathology directly in the wall of the artery.   Progression of calcium score indicates progression of pathological change in the wall of the artery.

Conclusion: Dr Raggi’s study in 2004 showed superiority of calcium score over LDL cholesterol in predicting future heart attack.  More than a decade later, mainstream cardiology is still in denial, having buried and ignored Dr Raggi’s study.  This is nothing less than a paradigm shift in how heart disease should be managed.  When it comes down to a contest between LDL Cholesterol and Calcium Score, there is no contest.  The Calcium Score wins every time.

Jeffrey Dach MD
7450 Griffin Road Suite 190
Davie Florida 33314
954 792-4663

Articles with Related Interest

Low Level Endotoxemia LPS Theory of Coronary Artery Disease

Leaky Gut and Low level Endotoxemia

Aged Garlic Benefits for Calcium Score

Vitamin K , Is There Anything It Cant Do? Benefits for Calcium Score

Autopsy Studies Show No Correlation

Statin Denialism Internet cult

Links and References

Header Image LDL particle courtesy of Drs Wolfson

1) Curr Opin Lipidol. 2017 Jun;28(3):261-266. LDL particle number and size and cardiovascular risk: anything new under the sun? Allaire J1, Vors C, Couture P, Lamarche B.

LDL particle size, on the other hand, has not been independently associated with CVD risk after adjustment for other risk factors such as LDL cholesterol, triglycerides, and HDL-C and that routine use of information pertaining to particle size to determine and manage patients’ risk is not yet justified.

We provide here an up-to-date perspective on the potential use of LDL particle number and size as complementary risk factors to predict and manage cardiovascular disease (CVD) risk in the clinical realm.
RECENT FINDINGS:  Studies show that a significant proportion of the population has discordant LDL particle number and cholesterol indices [non-HDL cholesterol (HDL-C)]. Data also show that risk prediction may be improved when using information on LDL particle number in patients with discordant particle number and cholesterol data. Yet, most of the current CVD guidelines conclude that LDL particle number is not superior to cholesterol indices, including non-HDL-C concentrations, in predicting CVD risk. LDL particle size, on the other hand, has not been independently associated with CVD risk after adjustment for other risk factors such as LDL cholesterol, triglycerides, and HDL-C and that routine use of information pertaining to particle size to determine and manage patients’ risk is not yet justified.
SUMMARY:  Additional studies are required to settle the debate on which of cholesterol indices and LDL particle number is the best predictor of CVD risk, and if such measures should be integrated in clinical practice.

Women with discordant high particle concentration were more likely to have metabolic syndrome (MetS) and diabetes

2) Clin Chem. 2017 Apr;63(4):870-879. doi: 10.1373/clinchem.2016.264515. Epub 2017 Feb 7.  Discordance between Circulating Atherogenic Cholesterol Mass and Lipoprotein Particle Concentration in Relation to Future Coronary Events in Women.
Lawler PR1,2,3,4, Akinkuolie AO1,3, Ridker PM2,3, Sniderman AD5, Buring JE3,4, Glynn RJ3,4, Chasman DI3, Mora S6,2,3.
It is uncertain whether measurement of circulating total atherogenic lipoprotein particle cholesterol mass [non-HDL cholesterol (nonHDLc)] or particle concentration [apolipoprotein B (apo B) and LDL particle concentration (LDLp)] more accurately reflects risk of incident coronary heart disease (CHD). We evaluated CHD risk among women in whom these markers where discordant.
METHODS:Among 27533 initially healthy women in the Women’s Health Study (NCT00000479), using residuals from linear regression models, we compared risk among women with higher or lower observed particle concentration relative to nonHDLc (highest and lowest residual quartiles, respectively) to individuals with agreement between markers (middle quartiles) using Cox proportional hazards models.
RESULTS:Although all 3 biomarkers were correlated (r ≥ 0.77), discordance occurred in up to 20.2% of women. Women with discordant high particle concentration were more likely to have metabolic syndrome (MetS) and diabetes (both P < 0.001). Over a median follow-up of 20.4 years, 1246 CHD events occurred (514725 person-years). Women with high particle concentration relative to nonHDLc had increased CHD risk: age-adjusted hazard ratio (95% CI) = 1.77 (1.56-2.00) for apo B and 1.70 (1.50-1.92) for LDLp. After adjustment for clinical risk factors including MetS, these risks attenuated to 1.22 (1.07-1.39) for apo B and 1.13 (0.99-1.29) for LDLp. Discordant low apo B or LDLp relative to nonHDLc was not associated with lower risk.
CONCLUSIONS:Discordance between atherogenic particle cholesterol mass and particle concentration occurs in a sizeable proportion of apparently healthy women and should be suspected clinically among women with cardiometabolic traits. In such women, direct measurement of lipoprotein particle concentration might better inform CHD risk assessment.

3)   Curr Opin Endocrinol Diabetes Obes. 2018 Jan 10. Discordance between lipoprotein particle number and cholesterol content: an update.
Cantey EP1, Wilkins JT2.
The cholesterol content within atherogenic apolipoprotein-B (apoB) containing lipid particles is the center of consensus guidelines and clinicians’ focus whenever evaluating a patient’s risk for atherosclerotic cardiovascular disease. The pathobiology of atherosclerosis requires the retention of lipoprotein particles within the vascular intima over time followed by maladaptive inflammation resulting in plaque formation and rupture in some. The cholesterol content is widely variable within each particle creating either cholesterol-deplete or cholesterol-enriched particles. This variance in particle cholesterol content varies within and between individuals. Discordance analysis exploits this difference in cholesterol content of particles to demonstrate the differential significance of LDL-cholesterol (LDL-C) and non-HDL-C from measures of lipoprotein particle number in terms of assessing atherosclerotic cardiovascular disease risks.
RECENT FINDINGS:Three studies have added to the growing body of literature of discordance analysis. Despite wide variability of discordance cutoffs, baseline risk of atherosclerotic disease, and populations sampled, the conclusion remains the same: risk of atherosclerotic disease follows apoB lipid particle concentration rather than cholesterol content of lipid particles.
SUMMARY:In addition to traditional lipid fractions, assessments of atherogenic particle number should be strongly considered whenever assessing CVD risk in nontreated and treated individuals. There is a need for clinical trials that focus not only on the reduction in LDL-C but apoB, as well.

4)  Lincoff, A. Michael, et al. “Evacetrapib and cardiovascular outcomes in high-risk vascular disease.” New England Journal of Medicine 376.20 (2017): 1933-1942.

Although treatment with evacetrapib has resulted in reductions of 60 to 70% in the levels of small dense LDL particles,29 effects on the total number of LDL particles and on apolipoprotein B levels (reductions of 22% and 20%, respectively) are considerably less pronounced.

CONCLUSIONS:Although the cholesteryl ester transfer protein inhibitor evacetrapib had favorable effects on established lipid biomarkers, treatment with evacetrapib did not result in a lower rate of cardiovascular events than placebo among patients with high-risk vascular disease.

5)  Nicholls SJ, Ruotolo G, Brewer HB, et al. Evacetrapib alone or in combination with statins lowers lipoprotein(a) and total and small LDL particle concentrations in mildly hypercholesterolemic patients. J Clin Lipidol 2016;10:519-527.e4

Potent CETP inhibitors reduce plasma concentrations of atherogenic lipoprotein biomarkers of cardiovascular risk.
OBJECTIVES:To evaluate the effects of the cholesteryl ester transfer protein (CETP) inhibitor evacetrapib, as monotherapy or with statins, on atherogenic apolipoprotein B (apoB)-containing lipoproteins in mildly hypercholesterolemic patients.
METHODS:VLDL and LDL particle concentrations and sizes (using nuclear magnetic resonance spectroscopy) and lipoprotein(a) concentration (using nephelometry) were measured at baseline and week 12 in a placebo-controlled trial of 393 patients treated with evacetrapib as monotherapy (30 mg/d, 100 mg/d, or 500 mg/d) or in combination with statins (100 mg plus simvastatin 40 mg/d, atorvastatin 20 mg/d, or rosuvastatin 10 mg/d; Clinicaltrials.gov Identifier: NCT01105975).
RESULTS:Evacetrapib monotherapy resulted in significant placebo-adjusted dose-dependent decreases from baseline in Lp(a) (up to -40% with evacetrapib 500 mg), total LDL particle (LDL-P) (up to -54%), and small LDL particle (sLDL) (up to -95%) concentrations. Compared to statin alone, coadministration of evacetrapib and statins also resulted in significant reduction from baseline in Lp(a) (-31%), LDL-P (-22%), and sLDL (-60%) concentrations. The percentage of patients with concentrations above optimal concentrations for LDL-P (>1000 nmol/L) and sLDL (>600 nmol/L) decreased from 88% and 55% at baseline, respectively, to 20% and 12% at week 12, for patients treated with evacetrapib plus statins. Evacetrapib, alone or with statins, significantly increased LDL-P size.

CONCLUSIONS:Evacetrapib, as monotherapy or with statins, significantly reduces the concentrations of atherogenic apoB-containing lipoproteins, including Lp(a), LDL-P, and sLDL.

6) St-Pierre, Annie C., et al. “Low-density lipoprotein subfractions and the long-term risk of ischemic heart disease in men: 13-year follow-up data from the Quebec Cardiovascular Study.” Arteriosclerosis, thrombosis, and vascular biology 25.3 (2005): 553-559.Low density lipoprotein Risk of ischemic heart disease Quebec St Pierre Annie Arterio thrombo vasc bio 2005

The objective of the present study was to investigate the association between large and small low-density lipoprotein (LDL) and long-term ischemic heart disease (IHD) risk in men of the Quebec Cardiovascular Study.
METHODS AND RESULTS:Cholesterol levels in the large and small LDL subfractions (termed LDL-C> or =260A and LDL-C<255A, respectively) were estimated from polyacrylamide gradient gel electrophoresis of whole plasma in the cohort of 2072 men of the population-based Quebec Cardiovascular Study. All men were free of IHD at the baseline examination and followed-up for a period of 13 years, during which 262 first IHD events (coronary death, nonfatal myocardial infarction, and unstable angina pectoris) were recorded. Our study confirmed the strong and independent association between LDL-C<255A levels as a proxy of the small dense LDL phenotype and the risk of IHD in men, particularly over the first 7 years of follow-up. However, elevated LDL-C> or =260A levels (third versus first tertile) were not associated with an increased risk of IHD over the 13-year follow-up (RR=0.76; P=0.07).

CONCLUSIONS:These results indicated that estimated cholesterol levels in the large LDL subfraction were not associated with an increased risk of IHD in men and that the cardiovascular risk attributable to variations in the LDL size phenotype was largely related to markers of a preferential accumulation of small dense LDL particles.

Diabetic Dyslipidemia

7) Nat Clin Pract Endocrinol Metab. 2009 Mar;5(3):150-9. doi: 10.1038/ncpendmet1066.
Dyslipidemia in type 2 diabetes mellitus.Mooradian AD1. Department of Medicine, University of Florida College of Medicine, Jacksonville, FL 32209, USA.

Dyslipidemia is one of the major risk factors for cardiovascular disease in diabetes mellitus. The characteristic features of diabetic dyslipidemia are a high plasma triglyceride concentration, low HDL cholesterol concentration and increased concentration of small dense LDL-cholesterol particles.

8)  Schofield, Jonathan D., et al. “Diabetes dyslipidemia.” Diabetes Therapy 7.2 (2016): 203-219.  The dyslipidemia of type 2 diabetes is characterized by high triglyceride levels and decreased high-density lipoprotein (HDL) cholesterol, changes observed many years before the onset of clinically relevant hyperglycemia [9, 30]. Recent evidence suggests that low HDL cholesterol is an independent factor not only for cardiovascular disease but also for the development of diabetes itself [31]. These changes, and the presence of small dense LDL particles, probably contribute to accelerated atherosclerosis even before diabetes is formally diagnosed

9) Rivas-Urbina, Andrea, et al. “Modified low-density lipoproteins as biomarkers in diabetes and metabolic syndrome.” Frontiers in bioscience (Landmark edition) 23 (2018): 1220-1240.
anomalous lipid profile, known as diabetic or atherogenic dyslipidemia, is characterized by high levels of triglycerides and apoB, low concentration of high density lipoprotein (HDL) cholesterol, and increased postprandial lipidemia. This abnormal lipid profile is typical of diabetes but it is also present in pre-diabetic situations such as insulin resistance and metabolic syndrome

This means that at a given LDL cholesterol concentration, diabetic patients have a greater number of LDL particles

10) Sánchez-Quesada, José Luis, and Antonio Pérez. “Modified lipoproteins as biomarkers of cardiovascular risk in diabetes mellitus.” Endocrinología y Nutrición (English Edition) 60.9 (2013): 518-528.  Lipoproteins as biomarkers of cardiovascular risk diabetes mellitus Sánchez Quesada Endocrinología Nutrición 2013

Metabolic syndrome (MS), defined as abdominal obesity,insulin resistance, hypertension, and an abnormal lipid profile, 5 is frequently associated with type 2 diabetes. Although all four factors are associated with the early development of atherosclerosis, an abnormal lipid profile is probably the factor most directly related to atherogenesis. This lipid profile, known as diabetic or atherogenic dyslipidemia,is characterized by hypertriglyceridemia, decreased high density lipoprotein (HDL) cholesterol levels, increased apolipoprotein B (apoB) levels, 6 and increased postprandial lipidemia

metabolic endotoxemia

11)  Munford, Robert S. “Endotoxemia—menace, marker, or mistake?.” Journal of leukocyte biology 100.4 (2016): 687-698.
Endotoxemia is in its scientific ascendancy. Never has blood-borne, Gram-negative bacterial endotoxin (LPS) been invoked in the pathogenesis of so many diseases—not only as a trigger for septic shock, once its most cited role, but also as a contributor to atherosclerosis, obesity, chronic fatigue, metabolic syndrome, and many other conditions.

12) Curr Cardiol Rev. 2016 Sep 1. Endotoxin, Toll-like Receptor-4, and Atherosclerotic Heart Disease. Horseman MA1, Surani S, Bowman JD.

Endotoxin is a lipopolysaccharide (LPS) constituent of the outer membrane of most gram negative bacteria. Ubiquitous in the environment, it has been implicated as a cause or contributing factor in several disparate disorders from sepsis to heatstroke and Type II diabetes mellitus. Starting at birth, the innate immune system develops cellular defense mechanisms against environmental microbes that are in part modulated through a series of receptors known as toll-like receptors. Endotoxin, often referred to as LPS, binds to toll-like receptor 4 (TLR4)/ myeloid differentiation protein 2 (MD2) complexes on various tissues including cells of the innate immune system, smooth muscle and endothelial cells of blood vessels including coronary arteries, and adipose tissue. Entry of LPS into the systemic circulation ultimately leads to intracellular transcription of several inflammatory mediators. The subsequent inflammation has been implicated in the development and progression atherosclerosis and subsequent coronary artery disease and heart failure.
OBJECTIVE:The potential roles of endotoxin and TLR4 are reviewed regarding their role in the pathogenesis of atherosclerotic heart disease.
CONCLUSION:Atherosclerosis is initiated by inflammation in arterial endothelial and subendothelial cells, and inflammatory processes are implicated in its progression to clinical heart disease. Endotoxin and TLR4 play a central role in the inflammatory process, and represent potential targets for therapeutic intervention. Therapy with HMG-CoA inhibitors may reduce the expression of TLR4 on monocytes. Other therapeutic interventions targeting TLR4 expression or function may prove beneficial in atherosclerotic disease prevention and treatment.

13)  Hawkesworth, S., et al. “Evidence for metabolic endotoxemia in obese and diabetic Gambian women.” Nutrition & diabetes 3.8 (2013): e83.
Emerging evidence from animal models suggests that translocation of bacterial debris across a leaky gut may trigger low-grade inflammation, which in turn drives insulin resistance. The current study set out to investigate this phenomenon, termed ‘metabolic endotoxemia’, in Gambian women.
Methods: In a cross-sectional study, we recruited 93 age-matched middle-aged urban Gambian women into three groups: lean (body mass index (BMI): 18.5–22.9 kg m−2), obese non-diabetic (BMI: ⩾30.0 kg m−2) and obese diabetic (BMI: ⩾30.0 kg m−2 and attending a diabetic clinic). We measured serum bacterial lipopolysaccharide (LPS) and endotoxin-core IgM and IgG antibodies (EndoCAb) as measures of endotoxin exposure and interleukin-6 (IL-6) as a marker of inflammation.
Results: Inflammation (IL-6) was independently and positively associated with both obesity and diabetes (F=12.7, P<0.001). LPS levels were highest in the obese-diabetic group compared with the other two groups (F=4.4, P<0.02). IgM EndoCAb (but not total IgM) was highly significantly reduced in the obese (55% of lean value) and obese diabetic women (30% of lean; F=21.7, P<0.0001 for trend) compared with lean women.
Conclusion: These data support the hypothesis that gut-derived inflammatory products are associated with obesity and diabetes. Confirmation of these findings and elucidation of the role of the microbiota, gut damage and the pathways for translocation of bacterial debris, could open new avenues for prevention and treatment of type 2 diabetes.

14)  Gomes, Júnia Maria Geraldo, Jorge de Assis Costa, and Rita de Cássia Gonçalves Alfenas. “Metabolic endotoxemia and diabetes mellitus: a systematic review.” Metabolism-Clinical and Experimental 68 (2017): 133-144.
In this systematic review we analyzed studies that assessed serum concentrations of lipopolysaccharide (LPS) and/or lipopolysacharide-binding protein (LBP) in diabetic patients compared with healthy people. Articles were selected using PubMed and Scopus. Search terms used were endotoxemia, endotoxins, LPS, LBP, diabetes mellitus (DM), type 1 (T1DM), type 2 (T2DM), insulin resistance, humans, epidemiologic studies, population-based, survey, representative, cross-sectional, case-control studies, observational, and clinical trials. Two authors independently extracted articles using predefined data fields, including study quality indicators. There was a great variability in the estimates of metabolic endotoxemia among the studies. Most of the studies observed higher LPS or LBP concentrations in diabetic subjects than in healthy controls. T1DM and T2DM subjects presented higher mean fasting LPS of 235.7% and 66.4% compared with non-diabetic subjects, respectively. Advanced complications (e.g. macroalbuminuria) and disease onset exacerbate endotoxemia. Antidiabetic medications decrease fasting LPS concentrations. Among these medications, rosiglitazone and insulin present higher and lower effects, respectively, compared with other treatments. T1DM and T2DM seem to increase metabolic endotoxemia. However, some confounders such as diet, age, medication, smoking and obesity influence both diabetes and endotoxemia manifestation. A better understanding of the interaction of these factors is still needed.

15)  Tremellen, Kelton, Natalie McPhee, and Karma Pearce. “Metabolic endotoxaemia related inflammation is associated with hypogonadism in overweight men.” Basic and clinical andrology 27.1 (2017): 5.

Obesity is associated with both impaired testosterone production and a chronic state of low grade inflammation. Previously it was believed that this inflammation was mediated by a decline in the immunosuppressive action of testosterone. However, more recently an alternative hypothesis (GELDING theory) has suggested that inflammation originating from the passage of intestinal bacteria into the circulation (metabolic endotoxaemia) may actually be the cause of impaired testicular function in obese men. The aim of this study is to investigate if metabolic endotoxaemia, as quantified by serum Lipopolysaccharide Binding Protein (LBP), is associated with impaired testicular endocrine function.
Methods A total of 50 men aged between 21 and 50 years (mean 35.1 ± 6.8 years) were assessed for adiposity (BMI, waist circumference and % body fat using bio-impedance), inflammatory status (serum CRP, IL-1β, IL-6, TNFα and LBP) and testicular endocrine function (serum testosterone, estradiol, AMH, LH and FSH). Statistical analysis was performed using Pearson correlation analysis, with log transformation of data where appropriate, and multi-variate regression.
Results Overall increasing adiposity (% body fat) was positively associated with metabolic endotoxaemia (LBP, r = 0.366, p = 0.009) and inflammation (CRP r = 0.531, p < 0.001; IL-6 r = 0.463, p = 0.001), while also being negatively correlated with serum testosterone (r = −0.403, p = 0.004). Serum testosterone levels were significantly negatively correlated with inflammation (CRP r = −0.471, p = 0.001; IL-6 r = −0.516, p < 0.001) and endotoxaemia (LBP) after adjusting for serum LH levels (p = −0.317, p = 0.03). Furthermore, serum IL-6 was negatively associated with AMH levels (r = −0.324, p = 0.023), with a negative trend between LBP and AMH also approaching significance (r = −0.267, p = 0.064).
Conclusions Obesity and its associated metabolic endotoxaemia helps initiate a pro-inflammatory state characterised by raised serum IL-6 levels, which in turn is correlated with impairment of both Leydig (testosterone) and Sertoli cell function (AMH). These results open up the potential for new treatments of obesity related male hypogonadism that focus on preventing the endotoxaemia associated chronic inflammatory state.

16) Tremellen, Kelton. “Gut Endotoxin Leading to a Decline IN Gonadal function (GELDING)-a novel theory for the development of late onset hypogonadism in obese men.” Basic and clinical andrology 26.1 (2016): 7.


Bacterial profile in human atherosclerotic plaques.

17)   Hansen, Gorm Mørk, et al. “Pseudomonas aeruginosa Microcolonies in Coronary Thrombi from Patients with ST-Segment Elevation Myocardial Infarction.” PloS one 11.12 (2016): e0168771.
Chronic infection is associated with an increased risk of atherothrombotic disease and direct bacterial infection of arteries has been suggested to contribute to the development of unstable atherosclerotic plaques. In this study, we examined coronary thrombi obtained in vivo from patients with ST-segment elevation myocardial infarction (STEMI) for the presence of bacterial DNA and bacteria. Aspirated coronary thrombi from 22 patients with STEMI were collected during primary percutaneous coronary intervention and arterial blood control samples were drawn from radial or femoral artery sheaths. Analyses were performed using 16S polymerase chain reaction and with next-generation sequencing to determine bacterial taxonomic classification. In selected thrombi with the highest relative abundance of Pseudomonas aeruginosa DNA, peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) with universal and species specific probes was performed to visualize bacteria within thrombi. From the taxonomic analysis we identified a total of 55 different bacterial species. DNA from Pseudomonas aeruginosa represented the only species that was significantly associated with either thrombi or blood and was >30 times more abundant in thrombi than in arterial blood (p<0.0001). Whole and intact bacteria present as biofilm microcolonies were detected in selected thrombi using universal and P. aeruginosa-specific PNA-FISH probes. P. aeruginosa and vascular biofilm infection in culprit lesions may play a role in STEMI, but causal relationships remain to be determined.

18) Ziganshina, Elvira E., et al. “Bacterial communities associated with atherosclerotic plaques from Russian individuals with atherosclerosis.” PloS one 11.10 (2016): e0164836.
Atherosclerosis is considered a chronic disease of the arterial wall and is the major cause of severe disease and death among individuals all over the world. Some recent studies have established the presence of bacteria in atherosclerotic plaque samples and suggested their possible contribution to the development of cardiovascular disease. The main objective of this preliminary pilot study was to better understand the bacterial diversity and abundance in human atherosclerotic plaques derived from common carotid arteries of individuals with atherosclerosis (Russian nationwide group) and contribute towards the further identification of a main group of atherosclerotic plaque bacteria by 454 pyrosequencing their 16S ribosomal RNA (16S rRNA) genes. The applied approach enabled the detection of bacterial DNA in all atherosclerotic plaques. We found that distinct members of the order Burkholderiales were present at high levels in all atherosclerotic plaques obtained from patients with atherosclerosis with the genus Curvibacter being predominant in all plaque samples. Moreover, unclassified Burkholderiales as well as members of the genera Propionibacterium and Ralstonia were typically the most significant taxa for all atherosclerotic plaques. Other genera such as Burkholderia, Corynebacterium and Sediminibacterium as well as unclassified Comamonadaceae, Oxalobacteraceae, Rhodospirillaceae, Bradyrhizobiaceae and Burkholderiaceae were always found but at low relative abundances of the total 16S rRNA gene population derived from all samples. Also, we found that some bacteria found in plaque samples correlated with some clinical parameters, including total cholesterol, alanine aminotransferase and fibrinogen levels. Finally, our study indicates that some bacterial agents at least partially may be involved in affecting the development of cardiovascular disease through different mechanisms.

19)  Atherosclerosis. 2017 Aug;263:177-183. Bacterial profile in human atherosclerotic plaques.Lindskog Jonsson A1, Hållenius FF1, Akrami R1, Johansson E2, Wester P3, Arnerlöv C4, Bäckhed F5, Bergström G6.

Several studies have confirmed the presence of bacterial DNA in atherosclerotic plaques, but its contribution to plaque stability and vulnerability is unclear. In this study, we investigated whether the bacterial plaque-profile differed between patients that were asymptomatic or symptomatic and whether there were local differences in the microbial composition within the plaque.
METHODS:Plaques were removed by endarterectomy from asymptomatic and symptomatic patients and divided into three different regions known to show different histological vulnerability: A, upstream of the maximum stenosis; B, site for maximum stenosis; C, downstream of the maximum stenosis. Bacterial DNA composition in the plaques was determined by performing 454 pyrosequencing of the 16S rRNA genes, and total bacterial load was determined by qPCR.
RESULTS:We confirmed the presence of bacterial DNA in the atherosclerotic plaque by qPCR analysis of the 16S rRNA gene but observed no difference (n.s.) in the amount between either asymptomatic and symptomatic patients or different plaque regions A, B and C. Unweighted UniFrac distance metric analysis revealed no distinct clustering of samples by patient group or plaque region. Operational taxonomic units (OTUs) from 5 different phyla were identified, with the majority of the OTUs belonging to Proteobacteria (48.3%) and Actinobacteria (40.2%). There was no difference between asymptomatic and symptomatic patients, or plaque regions, when analyzing the origin of DNA at phylum, family or OTU level (n.s.).
CONCLUSIONS:There were no major differences in bacterial DNA amount or microbial composition between plaques from asymptomatic and symptomatic patients or between different plaque regions, suggesting that other factors are more important in determining plaque vulnerability.

20)  Lanter, Bernard B., Karin Sauer, and David G. Davies. “Bacteria present in carotid arterial plaques are found as biofilm deposits which may contribute to enhanced risk of plaque rupture.” MBio 5.3 (2014): e01206-14.

Atherosclerosis, a disease condition resulting from the buildup of fatty plaque deposits within arterial walls, is the major underlying cause of ischemia (restriction of the blood), leading to obstruction of peripheral arteries, congestive heart failure, heart attack, and stroke in humans. Emerging research indicates that factors including inflammation and infection may play a key role in the progression of atherosclerosis. In the current work, atherosclerotic carotid artery explants from 15 patients were all shown to test positive for the presence of eubacterial 16S rRNA genes. Density gradient gel electrophoresis of 5 of these samples revealed that each contained 10 or more distinct 16S rRNA gene sequences. Direct microscopic observation of transverse sections from 5 diseased carotid arteries analyzed with a eubacterium-specific peptide nucleic acid probe revealed these to have formed biofilm deposits, with from 1 to 6 deposits per thin section of plaque analyzed. A majority, 93%, of deposits was located proximal to the internal elastic lamina and associated with fibrous tissue. In 6 of the 15 plaques analyzed, 16S rRNA genes from Pseudomonas spp. were detected. Pseudomonas aeruginosa biofilms have been shown in our lab to undergo a dispersion response when challenged with free iron in vitro. Iron is known to be released into the blood by transferrin following interaction with catecholamine hormones, such as norepinephrine. Experiments performed in vitro showed that addition of physiologically relevant levels of norepinephrine induced dispersion of P. aeruginosa biofilms when grown under low iron conditions in the presence but not in the absence of physiological levels of transferrin.

21) Allen, H. B., et al. “Arteriosclerosis: the novel finding of biofilms and innate immune system activity within the plaques.” J Med Surg Pathol 1.135 (2016): 2. Arteriosclerosis novel finding of biofilms within the plaques Allen HB J Med Surg Pathol 2016

Calcium Score

22) Eur Heart J Cardiovasc Imaging. 2014 Mar;15(3):267-74. doi: 10.1093/ehjci/jet148. Epub 2013 Aug 21.  Does coronary CT angiography improve risk stratification over coronary calcium scoring in symptomatic patients with suspected coronary artery disease? Results from the prospective multicenter international CONFIRM registry.
Al-Mallah MH1, Qureshi W, Lin FY, Achenbach S, Berman DS, Budoff MJ, Callister TQ, Chang HJ, Cademartiri F, Chinnaiyan K, Chow BJ, Cheng VY, Delago A, Gomez M, Hadamitzky M, Hausleiter J, Kaufmann PA, Leipsic J, Maffei E, Raff G, Shaw LJ, Villines TC, Cury RC, Feuchtner G, Plank F, Kim YJ, Dunning AM, Min JK.
The prognostic value of coronary artery calcium (CAC) scoring is well established and has been suggested for use to exclude significant coronary artery disease (CAD) for symptomatic individuals with CAD. Contrast-enhanced coronary computed tomographic angiography (CCTA) is an alternative modality that enables direct visualization of coronary stenosis severity, extent, and distribution. Whether CCTA findings of CAD add an incremental prognostic value over CAC in symptomatic individuals has not been extensively studied.
METHODS AND RESULTS:we prospectively identified symptomatic patients with suspected but without known CAD who underwent both CAC and CCTA. Symptoms were defined by the presence of chest pain or dyspnoea, and pre-test likelihood of obstructive CAD was assessed by the method of Diamond and Forrester (D-F). CAC was measured by the method of Agatston. CCTAs were graded for obstructive CAD (>70% stenosis); and CAD plaque burden, distribution, and location. Plaque burden was determined by a segment stenosis score (SSS), which reflects the number of coronary segments with plaque, weighted for stenosis severity. Plaque distribution was established by a segment-involvement score (SIS), which reflects the number of segments with plaque irrespective of stenosis severity. Finally, a modified Duke prognostic index-accounting for stenosis severity, plaque distribution, and plaque location-was calculated. Nested Cox proportional hazard models for a composite endpoint of all-cause mortality and non-fatal myocardial infarction (D/MI) were employed to assess the incremental prognostic value of CCTA over CAC. A total of 8627 symptomatic patients (50% men, age 56 ± 12 years) followed for 25 months (interquartile range 17-40 months) comprised the study cohort. By CAC, 4860 (56%) and 713 (8.3%) patients had no evident calcium or a score of >400, respectively. By CCTA, 4294 (49.8%) and 749 (8.7%) had normal coronary arteries or obstructive CAD, respectively. At follow-up, 150 patients experienced D/MI. CAC improved discrimination beyond D-F and clinical variables (area under the receiver-operator characteristic curve 0.781 vs. 0.788, P = 0.004). When added sequentially to D-F, clinical variables, and CAC, all CCTA measures of CAD improved discrimination of patients at risk for D/MI: obstructive CAD (0.82, P < 0.001), SSS (0.81, P < 0.001), SIS (0.81, P = 0.003), and Duke CAD prognostic index (0.82, P < 0.0001).
CONCLUSION: In symptomatic patients with suspected CAD, CCTA adds incremental discriminatory power over CAC for discrimination of individuals at risk of death or MI.

23) CAC_Coronary Calcium Score Position_Statement_2017_New Zealand
New Zealand Society of Cardiology Position Statement on Coronary calcium Score 2017

24) William Davis MD How to reduce Coronary calcium Score Nov 2017 Wheat Belly Blog

25)   Hecht, Harvey S. “Coronary artery calcium scanning: past, present, and future.” JACC: Cardiovascular Imaging 8.5 (2015): 579-596.

Figure 5.   Progression of CAC and Risk of First MI in 495 Asymptomatic Patients Receiving Cholesterol-Lowering Therapy
(Left) CAC progression of <15% per year is associated with a benign prognosis irrespective of the baseline CAC, implying stabilization of the atherosclerotic process. (Right) CAC progression of >15% per year is associated with a poor prognosis directly related to the baseline CAC, implying new plaque formation and inadequacy of treatment. CAC = coronary artery calcium; MI = myocardial infarction.

Coronary artery calcium scanning (CAC) has emerged as the most robust predictor of coronary events in the asymptomatic primary prevention population, particularly in the intermediate-risk cohort. Every study has demonstrated its superiority to risk factor–based paradigms, e.g., the Framingham Risk Score, with outcome-based net reclassification indexes ranging from 52.0% to 65.6% in the intermediate-risk, 34.0% to 35.8% in the high-risk, and 11.6% to 15.0% in the low-risk cohorts. CAC improves medication and lifestyle adherence and is cost-effective in specified populations, with the ability to effectively stratify the number needed to treat and scan for different therapeutic strategies and patient cohorts. Data have emerged clearly demonstrating the worse prognosis associated with increasing CAC on serial scans, suggesting a potential role for evaluating residual risk and treatment success or failure. CAC is also strongly associated with the development of stroke and congestive heart failure.

26)  The cholesterol and calorie hypotheses are both dead — it is time to focus on the real culprit: insulin resistance Clinical Pharmacist 14 JUL 2017 By Maryanne Demasi, Robert H Lustig, Aseem Malhotra

27) Calcium Score Study Linus Pauling Protocol Vitamin C HALTS PROGRESSION OF CORONARY ATHEROSCLEROSIS COMPUTED TOMOGRAPHY Matthias Rath MD Aleksandra Niedzwiecki Ph.D.  

28)  Raggi, Paolo, Tracy Q. Callister, and Leslee J. Shaw. “Progression of coronary artery calcium and risk of first myocardial infarction in patients receiving cholesterol-lowering therapy.” Arteriosclerosis, thrombosis, and vascular biology 24.7 (2004): 1272-1277.

Objective— Statins reduce cardiovascular risk and slow progression of coronary artery calcium (CAC). We investigated whether CAC progression and low-density lipoprotein (LDL) reduction have a complementary prognostic impact.

Methods and Results— We measured the change in CAC in 495 asymptomatic subjects submitted to sequential electron-beam tomography (EBT) scanning. Statins were started after the initial EBT scan. Myocardial infarction (MI) was recorded in 41 subjects during a follow-up of 3.2±0.7 years. Mean LDL level did not differ between groups (118±25 mg/dL versus 122±30 mg/dL, MI versus no MI). On average, MI subjects demonstrated a CAC change of 42%±23% yearly; event-free subjects showed a 17%±25% yearly change (P=0.0001). Relative risk of having an MI in the presence of CAC progression was 17.2-fold (95% CI: 4.1 to 71.2) higher than without CAC progression (P<0.0001). In a Cox proportional hazard model, the follow-up score (P=0.034) as well as a score change >15% per year (P<0.001) were independent predictors of time to MI.

Conclusions— Progression of CAC was significantly greater in patients receiving statins who had an MI compared with event-free subjects despite similar LDL control. Continued expansion of CAC may indicate failure of some patients to benefit from statin therapy and an increased risk of having cardiovascular events.

29) 17 October 2017 Anacetrapib failure marks the death of the CETP class of cholesterol-lowering therapies By GlobalData Healthcare
Merck & Co recently decided not to apply for regulatory approval of anacetrapib, one of the last-standing drugs from the doomed cholesteryl ester transfer protein (CETP) class of cholesterol-lowering therapies.CETP transfers cholesterol from high-density lipoprotein C (HDL-C) particles, more widely known as ‘good cholesterol’, to the atherogenic low and very low-density lipoprotein (LDL and VLDL) particles, sometimes referred to as ‘bad cholesterol’. The rationale for inhibiting CETP is that it would result in increased levels of good versus bad cholesterol.

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LDL Cholesterol Particle Size and Number What Gives
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What is the predictive value of LDL Cholesterol Particle Size and Number ?
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