Attack of the Hydrogen Eaters
From 2 BILLION B.C.


By Durk Pearson & Sandy Shaw

Hypothesis: Methanogen Bacteria Are Responsible for Increased Risk of Cardiovascular Disease Associated With Phosphatidylcholine and Its Metabolites (Choline, TMAO)

T he recent publication of papers indicating increased cardiovascular risk in human consuming high levels of phosphatidylcholine and its metabolites (such as choline)1 and in mouse models of atherosclerosis fed phosphatidylcholine containing supplements2 has resulted in a lot of concern. These are results that require explanation.

Our hypothesis for a major mechanism for these results is that methanogen bacteria in the gut, using hydrogen produced by other gut bacteria, convert methyl groups obtained from methyl donors such as phosphatidylcholine and choline into methane with the release of energy that is then used by the methanogens. In the process, the methanogens deplete hydrogen produced in the gut, thus reducing the hydrogen available for diffusion into body tissues to provide protection against oxidative stress. Hydrogen is a highly selective antioxidant that scavenges hydroxyl radicals as well as the powerful oxidant peroxynitrite and, unlike most antioxidants, can reach virtually all body tissues including mitochondria. Interestingly, scientists have been working on mitochondrial-targeted antioxidants for many years to overcome the inability of most antioxidants to enter mitochondria,2A where most of the ROS (reactive oxygen species) are created.

THE METHANOGENS

The methanogens (Archaea) are a type of anaerobic bacteria that emerged around 2 BILLION years ago when the Earth had a reducing atmosphere containing considerable methane and hydrogen, but very little oxygen. But the Earth’s gravity is not strong enough to retain hydrogen, which eventually largely escapes the atmosphere. However, ultraviolet energy is able to split water into hydrogen and oxygen, so over a long period of time, Earth’s atmosphere released oxygen and retained it, releasing hydrogen into space and became oxidizing, which is toxic to the methanogens. Today, therefore, the methanogens are living (and making a very good living at that) in protected anaerobic environments such as the cooler areas around hydrothermal vents and in the colons of animals such as ourselves. (The hydrogen emerging from the hydrothermal vents is created by a chemical reaction between ferrous iron basalt and water.)

TEST YOUR MEMORY: Joe the methanogen goes to the store. Is he shopping for A) hydrogen, B) oxygen, or C) methane?

An earlier hypothesis paper2D suggested that glucosidase inhibitors, which prevent digestion of carbohydrates in the upper gastrointestinal tract thus allowing it to reach the gut microbiota in the lower gastrointestinal tract, could be related to the 49% risk reduction of cardiovascular events as reported in the STOP-NIDDM trial. They suggested that the increased hydrogen gas in the lower gastrointestinal tract from undigested fermentable carbohydrates might explain some of these beneficial effects. These researchers2D studied 11 healthy volunteers who were given the alpha-glucosidase inhibitor acarbose (100 mg three times a day) for four days under free-feeding conditions. The result was an increase in exhaled hydrogen at every time point examined, whereas there was only modest effects on methane production. (Acarbose treatment had no effect on hydrogen or methane in 2 of the 11 subjects.) The participation of methanogens in the process was not identified in this study. This same group of researchers had previously published work on the protective effect of hydrogen gas against damage resulting from myocardial ischemia/reperfusion injury in the rat.2E

One plausible way to combat this hydrogen depletion could be to increase consumption of prebiotic soluble fibers (such as the long chain fructooligosaccharides) that enable hydrogen producing gut bacteria to make more hydrogen. The data on hydrogen and methane as measured in human stool vary as some studies report higher levels of methanogens in the stool of obese humans,2B while others have found the opposite.2B A recent paper2BB with a larger sample size (792) found that the presence of both methane and hydrogen on breath testing is associated with increased BMI (body mass index) and percent body fat. The authors of this paper2BB suggest that the hydrogen-using methanogen Methanobrevibacter smithii increases nutrient availability for the host and may, therefore, contribute to weight gain. More elderly humans (over 65) have been reported to have higher methanogenic and archaea diversities.2B Methane breath excretion has also been reported to be high among patients with malignant or pre-malignant colonic pathology.X Methane producers are reported to have lower breath hydrogen levels than nonproducers,XX possibly as a result of the methanogens consuming hydrogen to create methane and energy. It has been noted that fermentable fibers may produce small increases in breath methane in methane-producing subjects.XX

One paper2C reports that the Archaea in the human gut are resistant to many antibiotics but were generally sensitive to metronidazole, which inhibited all strains against which it was tested, with a minimum inhibitory concentration (MIC) of 0.5–64 mg./ml. A further study2CC showed that prophylactic oral metronidazole in bone marrow transplant recipients suppressed fecal methanogenic Archaea.

TEST YOUR MEMORY: Reduced supplies of hydrogen are associated with what source of health risks? A) oxidative stress, B) loss of iron, or C) dehydration

Could Methanogens Be Found in Other Anaerobic Microenvironments in the Human Body? Meth­an­o­gens Depleting Hydrogen in Periodontal Plaques and Atherosclerotic Plaques

A new paper2DD reports on the plausible theory that bacteria in the form of biofilms, that are resistant to antibacterial therapy, may be part of the pathogenesis of atherosclerosis. The authors of the new paper, noting that persistent inflammation is a causative factor in atherosclerosis and also recognizing that the source of the persistent inflammation involved has not been identified, hypothesized that bacterial biofilms that thrive in a hyperinflammatory host niche might fit the requirements for promoting atherosclerosis. In this study, the researchers examined plaque tissue from 10 patients (removed during a surgical procedure called atherectomy) and found bacterial DNA in amounts far exceeding what would be there as a result of contamination, “suggesting the bacteria may be propagating in the plaque.”2DD As the bacteria would be in the form of biofilm, the authors hypothesized, it would “produce a hyperinflammatory response in host environments, and therefore is a candidate for being the “engine” for the persistent inflammation necessary for the pathogenesis of atherosclerosis.”2DD

There is general agreement, the authors explain, that whatever spreads the inflammatory cytokines and other factors derived from an oxidative environment, this oxidative stress generator is the putative cause for the production of atherosclerotic plaques.

The authors reasoned that, although bacteria have been found in many atherosclerotic plaques, it has never been determined whether they are causative factors driving the construction of plaques or merely bacteria taking advantage of a damaged environmental niche within which they can grow. The possibility of bacteria in the form of biofilms being part of atherosclerotic plaques is plausible because biofilms are complex constructs highly resistant to immune attack as compared to solitary circulating bacteria. The authors2DD note that, “when a biofilm community is fully established, it exhibits powerful tolerance to antibiotics up to 1,000 times MIC [minimum inhibitory concentration].” When the bacteria reach a high enough concentration, they have their own efficient communication network in which special quorum-sensing chemicals are released to communicate to other bacteria how many of them their are, which regulates whether they transform into the biofilm community. (Keep in mind that diverting resources so as to support the needs of a biofilm is an energy-requiring process and, hence, must be tightly regulated to prevent energy waste and subsequent weakening of the biofilm.

The authors paint a picture of the developing biofilm: “the bacteria growing in biofilm phenotype produces host lesions characterized by increased proinflammatory cytokines, matrix metalloproteases, reactive oxygen species, elastase, myeloperoxidase, and excess neutrophils and macrophages.” In other words, suggest the authors, you find the same type of inflammatory biochemistry and cellular architecture consistent with those found in atherosclerosis.

There is some knowledge already available on biofilms as developed by researchers in fields such as implanted medical devices, pipes and fixtures in plumbing, artificial organs, surfaces where food comes into contact, and other areas. For example, researchers investigating bacterial biofilms forming on dental surfaces found that essential oil of turmeric inhibited the formation of biofilms by Streptococcus mutans, a bacterium known to play a significant role in the formation of dental plaques and caries in humans.2EE

Another source2FF reports that “Of products commonly found in households, effectiveness against three pathogens (Salmonella, E. coli 0157:H7 and L. monocytogenes) increased in the order: household bleach (0.0314%)>hydrogen peroxide(3%)>undiluted vinegar>baking soda (50% sodium bicarbonate), while pathogen sensitivity followed the order: Salmonella>E. coli 0157:H7>L. monocytogenes. So watch out! The Centers for Disease Control and Prevention estimated (as reported in the June 11, 2008 Journal of the American Medical Association) warned that more than 65% of infections are caused by bacteria growing in biofilms.

TEST YOUR MEMORY: Atherosclerotic plaques are good homes for methanogens because A) they are sites that are low in oxygen, B) they have ample supplies of oxygen or C) they are not good niches for methanogens.

In Case You Wondered....

Why Have We Added Memory Tests to Our Article?

The reason is that a new paperXYZ reports that interpolating memory tests reduced mind wandering and improved learning of online lectures. This simple method has been reported before, as in studies showing that interpolating the study of lists of words, face-name pairs, and prose passages with memory tests substantially improves the ability to learn toward the end of a period of prolonged study. In experiments described by the authors of the paper,XYZ a group of students were tested after each list of words they were studying, while another group were tested only after the fifth and final list. The group that was tested after each list learned the final list twice as well as those who were only tested after the final list. A remarkable improvement! So, pop memory quizzes can add to the ability of your written or spoken material to inform others. Thinking up silly questions is fun, too!

In a final encore, the authorsXYZ conducted another two experiments and found they could reduce the tendency of the students to “daydream” (what they called mind wandering) during a study period learning the contents of a 21 minute video lecture (introduction to statistics) by including a 2 minute period of test questions following each section of the video to half of the students but only after the fourth lecture segment to the other half. The students that were tested more frequently reported less mind wandering. Surprisingly, however, those tested more frequently actually experienced less anxiety toward the final cumulative test than those not so tested.

TEST YOUR MEMORY: Having pop quizzes as part of a lecture may help improve your A) ability to learn the material, B) ability to play SCRABBLE, or C) ability to experience refreshing naps during the lecture.

Reference

  1. XYZ. Szpunar et al. Interpolated memeory tests reduce mind wandering and improve learning of online lectures. Proc Natl Acad Sci USA. 110(16):6313-7 (2013).

Tooth Decay With the Help of Methanogens

Mounting evidence links bacterial biofilms even more tightly to periodontal disease than before. One paper2GG reports that three hydrogenotrophic (hydrogen producing) microbial groups, along with methanogenic archaea (an ancient form of hydrogen consuming bacteria that produce methane), and acetogenic bacteria (hydrogen consuming bacteria that produce acetate) are associated with plaque biofilms of periodontal disease. The three groups of hydrogen consuming bacteria compete with each other for access to hydrogen. Thus, “interspecies hydrogen transfer” was described by the authors2DD as a “possible driving force to promote proliferation of fermenting pathogens.” Interestingly, the gut methanogens and the oral methanogens have close phylogenetic affinity. Oral methanogens are also found to be similar to those found in atherosclerotic plaques.

Indeed, a paper on methanogensX from 2009 noted that “[s]ignificant associations were observed … between levels of methanogens in periodontal pockets and severity of periodontitis and between quantities of methanogens in the large intestine and diseases such as colon cancer and diverticulosis.”

TEST YOUR MEMORY: Methanogens residing in periodontal plaques could be responsible for A) bad breath (because of the methane they excrete), B) tooth decay, C) establishing colonies in atherosclerotic plaques, or D) all of the above.

One final curiosity is found in a paper published in 2007 on the association between choline and the risk of colorectal adenoma in women. See next section below.

Increased Dietary Choline Does Not Reduce the Risk of Colorectal Adenoma

In a study with uncanny parallels to the recent studies of increased cardiovascular risk factors in association with phosphatidylcholine supplementation,1,2 an epidemiological study of dietary choline found in 2408 cases of colorectal adenoma in women that increasing dietary choline intake was dose-dependently associated with an elevated risk of colorectal adenoma.XXXX Though this might be thought to be because of the fat contained in foods most likely to contain high choline levels (e.g., eggs, meats), nevertheless there is a similarity to the recent papers on methyl donors such as choline and cardiovascular risk that it seemed worth mentioning. Sometimes what appears to be coincidence is, in biology, a useful link to a relationship. (Of course, it may also be a coincidence.)

The subjects were the 2408 cases of colorectal adenoma occurring in 39,246 women enrolled in the Nurses’ Health Study.

The authors had hypothesized, based on the fact that lower folate levels are associated with an increased risk of colorectal adenoma, that other dietary sources supporting one-carbon metabolism (sources of methyl groups) might also reduce the risk. In this large epidemiological study, choline has the opposite effect. Betaine, on the other hand, did have a nonlinear inverse association with colorectal adenoma.

Addendum:

We add here a new discovery concerning how gut microbiota use choline that provide additional insights into how methanogens, as proposed above, may play a role in atherosclerosis.

The Conversion of Choline to Trimethylamine by Gut Microbes Requires a Glycyl Radical Enzyme



Trimethylaminuria (Fish Odor Syndrome) is a rare metabolic disorder that causes people’s breath, urine and sweat to smell like fish.
It was recently discovered that anaerobic gut microbes convert choline to trimethylamine (TMA) via a glycyl radical enzyme.1 The enzyme, called choline TMA-lyase, is said to be a member of an enzyme family that uses highly reactive protein-based radical intermediates to promote a diverse set of chemical transformations including C-C bond formation and C-C bond cleavage. “This finding strengthens earlier hypotheses that anaerobic choline degradation is a major source of TMA [trimethylamine] formation within this environment [gastrointestinal tract] …”1 The authors suggest that the choline conversion to TMA via this enzyme may offer further insight into the link to atherosclerosis. For example, if the enzyme, with its “highly reactive protein-based radical intermediates” is active in atherosclerotic plaques, it may promote the development of the plaques, such as by oxidizing LDL.

TMA is then converted to TMAO in the liver and kidney. We do not consider TMA or TMAO themselves to be toxic. Note, for example, that the amounts of TMA and TMAO naturally found in halibut (the fish makes them to act as an anti-freeze), is very high at 8230.2 ± 564.8 μμmol/8 hr in urine following ingestion of 227 g of halibut.2 For reasons explained in the article above, we think that depletion of hydrogen by methanogens in the conversion of methyl donors such as choline to methane and energy is the source of cardiovascular risks. A small fraction of individuals do not have the liver or kidney enzyme required to convert TMA to TMAO and end up with trimethyl­aminuria, which causes them to smell fishy.

TEST YOUR MEMORY: People who lack the liver and kidney enzymes needed to convert TMA to TMAO can end up smelling A) like cat litter, B) like garlic, or C) fishy.

References

  1. Craciun and Balskus. Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme. Proc Natl Acad Sci USA. 109(52):21307-12 (2012).
  2. Zhang et al. Dietary precursors of trimethylamine in man: a pilot study. Food Chem Toxicol. 37:515-20 (1999).

Our working hypothesis, as explained above, is that the cardiovascular risk factors are a result of methanogen bacteria that convert dietary sources of methyl groups into methane, using hydrogen in the lower digestive tract emitted by hydrogen producing bacteria for the conversion. CH3 + H = CH4 We propose that the depletion of hydrogen from the gut by the methanogens leads to an increased risk of atherosclerosis by reducing the availability of hydrogen in the bloodstream and in atherosclerotic plaques, where it would otherwise protect against oxidative stress (as in the oxidation of LDL).

In a similar way, methanogens could be a factor in the increased risk of colorectal adenoma with choline used as a source of methyl groups for methanogen synthesis of methane associated with depletion of hydrogen.

What Are We Doing About Methanogens?

After reading many papers on methanogens, we have increased our intake of our prebiotic formulation designed to provide food for hydrogen producers in the human gut to three servings a day. While it is not certain that increased hydrogen won’t also increase the growth of methanogens in any particular individual, the extant data suggests that methane increases less than the increase in hydrogen. The hydrogen eaters have done well, surviving from around 2 BILLION BC to present. Their presence in our very bodies as a threat to our viability hasn’t even been recognized until very recently. Well done, hydrogen eaters, but now that we have noticed you, we don’t plan to sit still while you deprive us of our hydrogen.

As one recent review of the microbial hydrogen economyV explained, “Perhaps the most exciting link between H2 and human disease is emerging evidence that this microbial-derived gas has potent antioxidative, antiapoptotic and anti-inflammatory activities in a wide range of disease models.” “… decreases in the net production of H2 might increase the risk of inflammatory or metabolic diseases. It is intriguing to consider whether manipulation of microbial H2 metabolism, either through enhanced production or diminished utilization, might provide a novel means of regulating colonic homeostasis.”

We suggest that one way to do this is to ingest prebiotics that stimulate hydrogen producers to increase their hydrogen output. We are participants in a great experiment in cooperation in which we join together with an alien species to improve life on Earth (well, in our guts anyway, which is a good start or perhaps just a good fart).

TEST YOUR MEMORY: One way you might be able to increase the ratio of hydrogen producing bacteria to methanogens in your mouth is: A) chase the methanogens down with very tiny fly swatters, B) eat prebiotics that encourage hydrogen producers to excrete more hydrogen, or C) move to another planet that doesn’t issue visas to methanogens.

Hypothesis: Taurine Deficiency May Be Another Mechanism for Cardiovascular Risk From Consumption of Phosphatidylcholine and Its Metabolites

Taurine regulates the conversion of phosphatidyl­ethanolamine to phosphatidylcholine, inhibiting the enzyme that catalyzes this conversion.XXX When taurine supplies are low, the ratio of phosphatidylethanolamine to phosphatidylcholine is lower because more of the former is converted into the latter. This ratio change can affect calcium signaling in the heart that fosters heart failureXXX as occurs in dogs, cats, and foxes on low taurine diets.

TEST YOUR MEMORY: If you are a dog, cat, or fox, you should be very careful to get enough of what in your diet: A) chlorine, B) antidepressants or C) taurine.

References

1. Tang et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med. 368:1575-84 (2013).
2. Wang et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature. 472:57-63 (2011).
2A. Coulter et al. Mitochondrially targeted antioxidants and thiol reagants. Free Radic Biol Med. 28(10):1547-54 (2000).
2B. Saengkerdsub and Ricke. Ecology and characteristics of methanogenic archaea in animals and humans. Crit Rev Microbiol. Early online 1-20 DOI: 10.3109/1040841X.2013.763220
2BB. Mathur et al. Methane and hydrogen positivity on breath test is associated with greater body mass index and body fat. J Clin Endocrinol Metab. 98(4):E698-E702 (2013).
2C. Dridi et al. Archaea as emerging organisms in complex human microbiomes. Anaerobe. 17:56-63 (2011).
2CC. Ansorg et al. Influence of intestinal decontamination using metronidazole on the detection of methanogenic Archaea in bone marrow transplant recipients. Bone Marrow Transplant. 31:117-9 (2003).
2D. Suzuki et al. Are the effects of alpha-glucosidase inhibitors on cardiovascular events related to elevated levels of hydrogen gas in the gastrointestinal tract? FEBS Lett. 583:2157-2159 (2009).
2DD. Wolcott et al. A possible role of bacterial biofilm in the pathogenesis of atherosclerosis. J Bacteriol Parasitol. 3(127):(2012) doi:10.4172/2155-9597.1000127
2EE. Lee et al. Essential oil of Curcuma longa inhibits Streptococcus mutans biofilm formation. J Food Sci. 76(9):H226 (2011).
2FF. Sofos. Biofilms: Our Constant Enemies. Food Safety Magazine, Feb./March 2009.
2GG. Vianna et al. Quantitative analysis of three hydrogenotrophic microbial groups, methanogenic archaea, sulfate-reducing bacteria, and acetogenic bacteria, within plaque biofilms associated with human periodontal disease. J Bacteriol. 190(10):3779-85 (2008).
2E. Hayashida et al. Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia-reperfusion injury. Biochem Biophys Res Commun. 373:30-5 (2008).
X. de Macario and Macario. Methanogenic archaea in health and disease: a novel paradigm of microbial pathogenesis. Int J Med Microbiol. 299:99-108 (2009).
XX. Wolever et al. Effect of guar, pectin, psyllium, soy polysaccharide, and cellulose on breath hydrogen and methane in healthy subjects. Am J Gastroenterol. 87(3):305-310 (1992).
XXX. Hamaguchi et al. Interaction of taurine with methionine: inhibition of myocardial phospholipid methyltransferase. J Cardiovasc Pharmacol. 18:224-30 (1991).
XXXX. Cho et al. Dietary choline and betaine and the risk of distal colorectal adenoma in women. J Natl Cancer Inst. 99(16):1224-31 (2007).
V. Carbonero et al. Contributions of the microbial hydrogen economy to colonic homeostasis. Nat Rev Gastroenterol Hepatol. 9:504-18 (2012).


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