Category Archives: Cardiovascular Disease

Disease Begins in the Mucus

Posted by on June 23, 2015 53 comments

Hippocrates famously said, “All disease begins in the gut.” I think we can narrow it down further: much modern disease begins in the mucus.

In a recent post, I put up this picture:

Antimicrobial peptides (AMPs), including defensins and cathelicidins, constitute an arsenal of innate regulators of paramount importance in the gut.

A depleted mucosal layer leads to inflammation and gut permeability.

It’s from a paper on the role of antimicrobial peptides in maintaining gut health. [1] The point of the left panel is that a healthy gut is characterized by a thick mucosal layer that shields our intestinal and immune cells from direct contact with bacteria. The inner mucus layer is infused with antimicrobial peptides to minimize its bacterial content. The outer mucus layer contains a population of friendly mucin-degrading bacteria – symbionts like Akkermansia who evolved to feed on our mucus. These friendly bacteria provide another layer of defense against infectious pathogens; bacteria tend to be quite good at keeping out competitors. Akkermansia has been found to prevent obesity.

In an unhealthy gut, on the other hand, the mucosal layer often gets stripped away. The image (right panel) attributes this to infection, which is one possibility, but nutritional factors also matter. For example:

  • Deficiencies of vitamins A or D will reduce production of antimicrobial peptides, making it easier for pathogens to reach our gut cells;
  • On very low-carb diets, production of mucin-2, the primary constituent of gut mucus, may be limited in order to preserve glucose for the brain (see “Dangers of Zero-Carb Diets, II: Mucus Deficiency and Gastrointestinal Cancers,” Nov 15, 2010);
  • Deficiencies of dietary fiber, vinegar, choline, and other nutrients may impair gut motility, leading to concentrations of partially digested food and bacteria at specific points in the intestine.

Regardless of why it happens, once bacteria come into direct contact with our gut and immune cells, they trigger inflammation and tend to loosen the gut barrier. This allows live bacteria and cell wall components from dead bacteria to enter the body from the gut.

Endotoxins – toxic compounds released when bacteria die, such as lipopolysaccharides from the cell walls of gram-negative bacteria – are immunogenic and inflammatory. A large influx of endotoxins into the body is “endotoxemia” – poisoning by endotoxins. As little as 2 nanograms LPS per kilogram body weight will induce fever, and 1 microgram LPS per kilogram of body weight will induce shock. [2]

Diseases caused by endotoxemia include:

  • Hepatitis [3]
  • Diabetes [4]
  • Heart disease [5]
  • Obesity [6]
  • Pulmonary hypertension [7]
  • Dyslipidemia [8]
  • Chronic kidney disease [9]

Many of today’s most prevalent diseases are caused by chronic endotoxemia.

So it behooves us to avoid it. If endotoxemia is fundamentally caused by the loss of a protective mucosal layer in the gut, how do we assure healthy production of mucus?

A recent paper sheds valuable light on that question.

Natural Whole Foods, High-Fat Diets, and Gut Health

It’s well known that in mice, “high-fat diets” induce endotoxemia. But these diets aren’t necessarily high in fat – any pelleted rodent food in which fat provides more than 20% of calories may be called “high-fat.” The critical difference of “high-fat diets” from chow is that they are composed of purified nutrients – starch, sugar, oil, vitamins, and minerals – whereas chow is composed of natural whole foods such as wheat, corn, and seeds.

A recent study tried to distinguish whether the cause of endotoxemia is the fat, or the purified starch, sugar, and oil. It made up three diets of varying fat content (8%, 48%, and 74% of energy respectively), but composed of natural whole foods. [10]

The result was remarkable:

[U]sing complex [i.e. natural whole foods] HFD, no associations were observed between dietary lipid amounts and the magnitude of endotoxemia, inflammation, and physiological alterations developed.

It turns out the endotoxemia that typically develops on high-fat diets is due to getting the calories from purified sources – starch, sugar, oil – rather than from whole foods. On a whole foods diet, the amount of endotoxin entering the body is more or less independent of the amount of fat consumed.

This is surprising in one respect. Lipopolysaccharide is fat-loving and enters the body along with dietary fat. So it stands to reason that a higher-fat diet would carry more endotoxin into the body.

But it turns out the body has mechanisms to regulate how much endotoxin enters the body. It wants a small amount so that the immune system can sample the gut microbiome, but not so much as to cause inflammation.

The primary mechanism for controlling endotoxin influx? Mucus production. The study noted that the mice eating higher-fat “had an increased number of goblet cells … [and] an increased MUC2 production.” MUC2 is mucin-2, the primary component of mucus in the gut.

Here is a picture with mucin-2 in the mucin-producing goblet cells stained red:

disease begins in mucus 02

It’s obvious that mucin production goes up dramatically as the fat content of the diet increases.

The study concludes,

“We show that, in complex HFDs based on chow ingredients and milk fat, there was no association between dietary lipid amounts and the magnitude of metabolic endotoxemia or low-grade inflammation.”

If high-fat diets are healthy, we can thank our mucin-producing goblet cells.

One last note: the fact that mice can produce healthy amounts of mucus on a 74% fat diet does not necessarily mean that humans can do the same. Humans have much larger brains than mice, and as a result our carbohydrate needs are larger. It’s possible that mice can maintain mucus production on a low-carb diet better than humans can.

References

[1] Muniz LR, Knosp C, Yeretssian G. Intestinal antimicrobial peptides during homeostasis, infection, and disease. Front Immunol. 2012 Oct 9;3:310. http://pmid.us/23087688.

[2] Warren HS et al. Resilience to bacterial infection: difference between species could be due to proteins in serum. J Infect Dis. 2010 Jan 15;201(2):223-32. http://pmid.us/20001600.

[3] Parlesak A, Schäfer C, Schütz T, Bode JC, Bode C. Increased intestinal permeability to macromolecules and endotoxemia in patients with chronic alcohol abuse in different stages of alcohol-induced liver disease. J Hepatol. 2000 May;32(5):742-7. http://pmid.us/10845660.

[4] Moreno-Navarrete JM et al. Circulating lipopolysaccharide-binding protein (LBP) as a marker of obesity-related insulin resistance. Int J Obes (Lond). 2012 Nov;36(11):1442-9. http://pmid.us/22184060.

[5] Lepper PM et al. Association of lipopolysaccharide-binding protein and coronary artery disease in men. J Am Coll Cardiol. 2007 Jul 3;50(1):25-31. http://pmid.us/17601541.

[6] Cani PD et al. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes. 2007 Jul;56(7):1761-72. http://pmid.us/17456850.

[7] Dschietzig T, Alexiou K, Richter C, Bobzin M, Baumann G, Stangl K, Brunner F. Endotoxin causes pulmonary hypertension by upregulating smooth muscle endothelin type-B receptors: role of aldose reductase. Shock. 2008 Aug;30(2):189-96. http://pmid.us/18091567.

[8] Lassenius MI et al. Bacterial endotoxin activity in human serum is associated with dyslipidemia, insulin resistance, obesity, and chronic inflammation. Diabetes Care. 2011 Aug;34(8):1809-15. http://pmid.us/21636801.

[9] McIntyre CW et al. Circulating endotoxemia: a novel factor in systemic inflammation and cardiovascular disease in chronic kidney disease. Clin J Am Soc Nephrol. 2011 Jan;6(1):133-41. http://pmid.us/20876680.

[10] Benoit B et al. Increasing fat content from 20 to 45 wt% in a complex diet induces lower endotoxemia in parallel with an increased number of intestinal goblet cells in mice. Nutr Res. 2015 Apr;35(4):346-56. http://pmid.us/25687164.

 

Nitric Oxide and AO+Mist Skin Probiotic at the Perfect Health Retreat

Posted by on April 30, 2015 40 comments

Our May Perfect Health Retreat begins on Saturday, and we’re thrilled to announce a new partner: AOBiome.

In January I blogged (“UBiome and the May 2015 Perfect Health Retreat”) about our partnership with uBiome.com. UBiome has contributed two gut microbiome sequencing kits for each guest, and we’re sequencing gut microbiomes pre-retreat and at the end of the retreat to see if a week together in a PHD-optimized environment causes microbiomes to converge to a “Perfect Health Diet” pattern.

Now, AOBiome is donating a four week supply of their AO+Mist skin probiotic to each guest. To see why I’m excited about this, I have to say a little about nitric oxide.

Nitric Oxide and Health

Nitric oxide is a gaseous signaling molecule with powerful effects on blood vessels, nerves, the immune system, gut and skin. It has proven to be such an important molecule that the three pharmacologists who established its role in relaxing blood vessels were awarded the Nobel Prize for Medicine in 1998.

Nitroglycerin, the explosive which turned out to be an effective remedy for heart failure, works by increasing nitric oxide levels.

A list of health conditions that may be improved through better nitric oxide status would be too long to attempt, but here is a brief sampling of Pubmed links. Nitric oxide may be helpful for high blood pressure and cardiovascular disease, gut conditions like constipation and impaired gut barrier integrity, obesity and diabetes, immunity against infections, dementia, lung dysfunction / COPD, and kidney disease. Nitric oxide has been reported to improve reaction time and exercise performance. It’s been proposed that nitric oxide may slow aging.

How do we obtain nitric oxide? It is a gas so you can’t eat it. Rather, we eat green leafy vegetables and beets to obtain nitrates; nitrates and bacteria-generated derivatives like nitrites are stored in the body, especially the skin; and then sunshine on the skin, among other processes, generates NO.

Here is dermatologist Richard Weller explaining why nitric oxide may be the reason sunshine is so good for our hearts, and why Scotsmen die so young despite the benefits of malt whisky:

Ammonia-Oxidizing Bacteria

Unfortunately, most of us don’t eat enough spinach to optimize our nitrate status. It would be wonderful if there were another way to obtain nitric oxide precursors.

David Whitlock, the scientist-founder of AOBiome, realized that there is. He was wondering why horses and other animals wallow in the mud, and realized that they might be obtaining probiotic bacteria for the skin from the soil.

Investigation revealed that a class of bacteria called ammonia-oxidizing bacteria are present in soil and, when they colonize the skin, can transform ammonia excreted in sweat to nitrite which can be re-absorbed by the body. These “AOBs” not only improve nitric oxide status, they improve body odor by eliminating ammonia. But they are destroyed by soaps and chlorinated water. Since most people use soap, take chlorinated showers, and rarely wallow naked in the mud, we lack these AOBs.

So AOBiome came up with the idea of a skin probiotic – some AOBs dissolved in water that you spritz on your skin after a morning shower, to re-colonize your skin each morning.

AOBiome has a variety of information on their web site if you’d like to read more: information about the skin microbiome in general, how modern lifestyle has changed it, why having a healthy skin microbiome is an important part of health, and the basics about the bacteria in the AO+ Mist.

Thank You, AOBiome

The Perfect Health Retreats represent our best effort to develop natural ancestral healing methods based on diet, lifestyle, and a healthful environment. Microbes are an important part of our environment, and managing our microbial environment has the potential to significantly improve health.

AO+Mist has not been tested in clinical trials, and there is no direct evidence that it will be beneficial to health. But the mechanisms seem logical, and I’m delighted that our guests will have a way to obtain ammonia-oxidizing skin bacteria without nakedness or mud. Thank you, AOBiome!

You don’t have to attend the retreat to benefit from AOBiome’s generosity. For readers of our blog, AOBiome is offering a 25% discount. Go here to purchase AO+Mist and use the coupon code phd25 for your discount.

More Evidence for Low-Carb Diets

Posted by on November 30, 2012 49 comments

In our book we point out a number of dietary tactics that appear to substantially decrease risk of cardiovascular disease. They include:

  • Optimizing tissue omega-6 to omega-3 balance by minimizing intake of omega-6 fats and eating an oily marine fish like salmon or sardines once a week.
  • Optimizing various micronutrients including vitamins D and K2, choline, magnesium, iodine, and selenium.
  • Reducing carbohydrate intake to the body’s natural level of glucose utilization, about 30% of total calories.

We cited two main sources for the claim that reducing carbohydrate intake reduces risk of cardiovascular disease:

–          The Nurses Health Study found that risk of coronary heart disease went down steadily as dietary carbohydrates were reduced and replaced by fat. Those eating a 59% carb diet were 42% more likely to have heart attacks than those eating a 37% carb diet. [1]

–          Replacing dietary carbohydrate with saturated or monounsaturated fat raises HDL and lowers triglycerides, changes that are associated with low rates of cardiovascular disease. Blood lipids are optimized when carb intake drops to 30% of energy or less. [2]

I think this is pretty strong evidence. It is not completely bulletproof, because associations don’t prove causation and improving risk factors doesn’t necessarily improve disease risk; but, combined with supportive evidence from cellular biology and clear evidence that evolutionary selection favors a carbohydrate intake around 30%, I consider it convincing.

However, it’s always good to have more evidence; and two new studies provide some. One directly relates utilization of carbohydrates for energy to atherosclerosis, and the other conducted a 12-month clinical trial of a carbohydrate restricted diet.

Carbohydrate Utilization is Associated With Atherosclerosis

Via Stephan Guyenet comes a study that directly links carbohydrate metabolism to atherosclerosis: “Metabolic fuel utilization and subclinical atherosclerosis in overweight/obese subjects.” [3]

The study used intima-media thickness in the carotid artery, which serves the head and neck, as a measure of atherosclerosis. As Wikipedia notes,

Since the 1990s, both small clinical and several larger scale pharmaceutical trials have used carotid artery IMT as a surrogate endpoint for evaluating the regression and/or progression of atherosclerotic cardiovascular disease. Many studies have documented the relation between the carotid IMT and the presence and severity of atherosclerosis.

To assess metabolism it measured the “respiratory quotient” or RQ. RQ is the ratio of carbon dioxide (CO2) generated in the body to oxygen (O2) consumed in the body.

RQ indicates which fuels are being burned for energy in the body. When carbohydrates are burned, the reaction involves carbon exclusively, so for every O2 molecule consumed there is a CO2 molecule created. This makes the RQ 1.0 when carbohydrates are burned.

Fats, however, donate both carbon and hydrogen, and the hydrogens react with oxygen to make water (H2O). So some of the oxygen consumed when fats are burned goes into water, not carbon dioxide, and the RQ when fats are burned is about 0.7. Ketones also have an RQ around 0.7.

Amino acids from protein have variable amounts of hydrogen and carbon, some amino acids are ketogenic and some are glucogenic, and so the RQ of protein depends on its amino acid mix. Typically RQ from different types of food protein is between 0.8 and 0.9.

However, most people eat a fairly consistent amount of protein, around 15% of energy, so the variable that generally determines RQ in practice is the ratio of carbs to fat in the diet. Higher RQ indicates a higher-carb diet.

Another study had previously shown that calorie restriction, which also reduces RQ by replacing dietary carbohydrate with fat released from adipose tissue, reduces the thickness of the carotid intima-media. [4] This study was the first testing whether the RQ-CIMT relationship holds also in subjects not known to be restricting calories.

The study found that indeed it does: the lower RQ, the less atherosclerosis the subjects had. Unfortunately they don’t present data in a visually useful way (a scatter plot of RQ vs CIMT would have been helpful); here is what they do show:

RQ was better than waist circumference or BMI at predicting degree of atherosclerosis. Only age was a stronger predictor of atherosclerosis than RQ.

RQ predicted atherosclerosis equally well in subjects with and without obesity. This tells us two things:

  1. It supports the idea that it was habitual diet rather than recent calorie restriction (which decreases RQ by replacing food-sourced calories with fat from adipose tissue) that generated low RQ and low CIMT.
  2. As the authors say, it indicates “the main role of metabolic factors rather than BMI” in generating atherosclerosis – metabolic factors meaning burning glucose for energy rather than fat.

It is also supporting evidence for one of the more controversial lines of our book, that “mitochondria prefer fat.”

One caution: Most of the subjects in this study were eating diets that were around 50% to 55% carbohydrate, so the study was testing whether it’s better to eat a little above or below this carb intake. It tells us, I think, that a 45% carb diet is healthier than a diet with more than 50% carbs. It doesn’t tell us what carb intake is optimal.

The Clinical Trial

In a trial lasting 12 months, restricting carbohydrates to 600 to 850 calories per day – that is, about the 30% of energy that we recommend – in the context of a slightly hypocaloric diet improved cardiovascular risk factors. [5]

Overweight and obese subjects in the trial lost 2.8 kg (6 pounds) over the year-long trial, so it couldn’t have been severely calorie restricted. Changes in other risk factors:

–          Blood pressure dropped from 121/79 to 112/72;

–          Fasting blood glucose dropped from prediabetic 106 mg/dl to normal 96 mg/dl;

–          Lipids improved, with triglycerides decreasing from 217 to 155 mg/dl and HDL increasing from 39 to 45 mg/dl.

They conclude:

The results of this study indicate that a moderately restricted calorie and carbohydrate diet has a positive effect on body weight loss and improves the elements of metabolic syndrome in patients with overweight or obesity and prediabetes. These results underscore the need to provide dietary recommendations focusing on calorie and carbohydrate restrictions … Our results are in agreement with reports produced by other authors who also assessed a carbohydrate-reduced diet …

Conclusion

A number of simple dietary and nutritional changes appear to reduce the risk of atherosclerosis and cardiovascular disease generally. One of them is reducing carbohydrate intake.

I believe the optimum carbohydrate intake is around 30% of energy. Many studies generate clear evidence of benefits as carbs are brought down into the range of 20% to 30% of energy, especially in metabolic disorders like metabolic syndrome, diabetes, and obesity. It’s good to see that evidence from other diseases, such as CVD, also supports the same carb intake.

Because most people’s diets are flawed in so many different ways, and fixing an individual factor is often associated with a reduction in CVD risk of 40% to 70%, it’s possible that we could reduce CVD risk by 90% or more by implementing all of the dietary optimizations described in our book.

It’s well worth pursuing all these little optimizations!

References

[1] Halton TL et al. Low-carbohydrate-diet score and the risk of coronary heart disease in women.  N Engl J Med. 2006 Nov 9;355(19):1991-2002. http://pmid.us/17093250.

[2] Krauss RM. Atherogenic lipoprotein phenotype and diet-gene interactions. J Nutr. 2001 Feb;131(2):340S-3S. http://pmid.us/11160558.

[3] Montalcini T et al. Metabolic fuel utilization and subclinical atherosclerosis in overweight/obese subjects. Endocrine. 2012 Nov 28. [Epub ahead of print] http://pmid.us/23188694.

[4] Iannuzzi A et al. Comparison of two diets of varying glycemic index on carotid subclinical atherosclerosis in obese children. Heart Vessels. 2009 Nov;24(6):419-24. http://pmid.us/20108073.

[5] Velázquez-López L et al. Low calorie and carbohydrate diet: to improve the cardiovascular risk indicators in overweight or obese adults with prediabetes. Endocrine. 2012 Sep 1. [Epub ahead of print] http://pmid.us/22941424.

Chocolate: What is the Optimal Dose?

Posted by on November 3, 2012 146 comments

Bret asked us how much chocolate is needed for good health:

I have a question about having dark chocolate daily. Does it need to be every day or what is the mininum grams per day. I have been having around 35g a day of 70% but I wondered if less would be ok or not having it at all.

This is a great time for this question, since Halloween candy will be running out soon, and those on tight budgets may be tempted to skimp on their chocolate. Should they?

Chocolate Is Not Considered Essential … Yet

Chocolate has not yet been recognized by the Food and Nutrition Board of the National Academies as an essential nutrient. We haven’t either: Our food plate lists it among “pleasure foods,” which are healthful but optional.

However, we are becoming ever-more chocolate friendly. In the new edition of our book, we list chocolate among our “supplemental foods” which we recommend consuming regularly. But our suggested dose is “as desired.” Perhaps we should narrow that down a little.

Chocolate Against Cardiovascular Disease

We’ve previously warned of the danger of chocolate deficiency, based on a systematic review that found: “The highest levels of chocolate consumption were associated with a 37% reduction in cardiovascular disease and a 29% reduction in stroke.” [1]

Here’s a visual summary of their findings:

The review authors report that every study accounted for chocolate intake in a different way, so they could only compare the groups with highest and lowest chocolate consumption in each study, not specific doses of chocolate.

Chocolate Against Diabetes

Bret was concerned about the sugar in chocolate, but if this is a problem, it’s outweighed by the benefits of chocolate. A Japanese study found that the rate of diabetes was reduced by 30% in those who consume the most chocolate. [2]

Chocolate Against Dementia and In Support of Cognitive Function

Several studies [3, 4] have found that chocolate consumption reduces risk of dementia and enhances performance on tests of cognitive function.

One of them found that cognitive function was optimized with a relatively low dose of chocolate – ten grams per day:

The associations between intake of these foodstuffs and cognition were dose dependent, with maximum effect at intakes of approximately 10 g/d for chocolate and approximately 75-100 mL/d for wine, but approximately linear for tea. [3]

The other found that cognition improved with intake of cocoa flavanols up to quite high doses – elderly given 1 g/day cocoa flavanols performed significantly better on cognitive tests than those given lower doses. [4]

Unfortunately I don’t know what fraction of chocolate is made of flavanols. I’m guessing it’s not more than a few percent, in which case this research suggests the optimal dose of chocolate may be 50 g/day or more.

Chocolate in Support of Circadian Rhythms

Most authors attribute the benefits of chocolate to their flavanols, which are thought to improve endothelial function and increase blood flow to the brain, among other effects.

However, there are other active compounds in chocolate, include peptides that interact with the opioid receptor. The opioid receptor has a role in circadian rhythms, which is one reason low-dose naltrexone (which blocks opioid function at night) works. It’s possible that eating chocolate during the day may support circadian rhythms via opioid receptor stimulation, especially if the peptides can reach the systemic circulation.

Indirect evidence that this may be beneficial comes from a Russian study in which exorphins (opioid receptor ligands) were injected into rats:

The chronic intraperitoneal administration of the peptide at the same dose of 5 mg/kg significantly increased exploratory activity, decreased anxiety, and improved learning. [5]

I don’t know how much chocolate would have to be eaten to achieve a similar exorphin dose in humans, but I imagine it’s large.

Chocolate in Support of Nobel Prizes

So how shall we resolve the issue of optimal chocolate dose? For me, the decisive evidence comes from a recent study by Franz Messerli published in the New England Journal of Medicine.

Based on chocolate’s support for cognitive function, he decided to see if chocolate consumption was related to another measure of cognition – Nobel Prize awards per capita. He counted Nobel Prizes and compared them to the recipient’s country’s chocolate consumption. These were his findings [6]:

There is clearly a strong correlation. The correlation coefficient is .79; p < 0.0001.

The correlation coefficient if Sweden is removed increases to .86 – which is suspicious:

Given its per capita chocolate consumption of 6.4 kg per year, we would predict that Sweden should have produced a total of about 14 Nobel laureates, yet we observe 32. Considering that in this instance the observed number exceeds the expected number by a factor of more than 2, one cannot quite escape the notion that either the Nobel Committee in Stockholm has some inherent patriotic bias when assessing the candidates for these awards or, perhaps, that the Swedes are particularly sensitive to chocolate, and even minuscule amounts greatly enhance their cognition. [6]

Those dastardly Swedes! Giving themselves more Nobel Prizes than their chocolate consumption warrants!

But I apologize, I’ve been diverted. The key point is, is there an optimum chocolate consumption?

the dose–response curve reveals no apparent ceiling on the number of Nobel laureates at the highest chocolate-dose level of 11 kg per year. [6]

11 kg/yr is an average of 30 g/day. So benefits are still increasing at that dose.

Of course, this was only a population level study. We still need to measure the doses in individual laureates to gain confidence. But anecdotally, there appears to be a correlation:

“I attribute essentially all my success to the very large amount of chocolate that I consume,” said Eric Cornell, an American physicist who received the Nobel Prize in 2001. “Personally I feel that milk chocolate makes you stupid. Now dark chocolate is the way to go. It’s one thing if you want like a medicine or chemistry Nobel Prize…but if you want a physics Nobel Prize it pretty much has got to be dark chocolate.”

Dark chocolate is, indeed, the PHD-approved form of this highly beneficial food.

Conclusion

This dose-response data might not be strong enough to define an RDA, but I’m going to take a stand: Bret’s intake of 35 g/day is healthy. Indeed, it’s right in line with the Nobel Prize-maximizing chocolate intake of the Swiss.

In regard to your last question, Bret – can you eat less chocolate, or none at all – the answer is clear. Yes, you can. But you must accept the consequences. You probably won’t be winning the next Nobel Prize for Physics.

References

[1] Buitrago-Lopez A et al. Chocolate consumption and cardiometabolic disorders: systematic review and meta-analysis. BMJ. 2011 Aug 26;343:d4488. doi: 10.1136/bmj.d4488. http://pmid.us/21875885.

[2] Oba S et al. Consumption of coffee, green tea, oolong tea, black tea, chocolate snacks and the caffeine content in relation to risk of diabetes in Japanese men and women. Br J Nutr. 2010 Feb;103(3):453-9. http://pmid.us/19818197.

[3] Nurk E et al. Intake of flavonoid-rich wine, tea, and chocolate by elderly men and women is associated with better cognitive test performance. J Nutr. 2009 Jan;139(1):120-7. http://pmid.us/19056649.

[4] Desideri G et al. Benefits in cognitive function, blood pressure, and insulin resistance through cocoa flavanol consumption in elderly subjects with mild cognitive impairment: the Cocoa, Cognition, and Aging (CoCoA) study. Hypertension. 2012 Sep;60(3):794-801. http://pmid.us/22892813.

[5] Belyaeva YA et al. Effects of acute and chronic administration of exorphin C on behavior and learning in white rat pups. Moscow University Biological Sciences Bulletin Volume 64, Number 2 (2009), 66-70, DOI: 10.3103/S0096392509020035. http://www.springerlink.com/content/qt537481061656gt/?MUD=MP.

[6] Messerli FH. Chocolate consumption, cognitive function, and Nobel laureates. N Engl J Med. 2012 Oct 18;367(16):1562-4. doi: 10.1056/NEJMon1211064. Epub 2012 Oct 10. http://pmid.us/23050509.