CNS: central nervous system
DNA: deoxyribonucleic acid
DNMTs: DNA methyltranferases
EAAT: excitatory amino acid transporter
EAAT3: excitatory amino acid transporter 3
ENS: enteric nervous system
FODMAP: Fermentable Oligo-, Di- and Mono-saccharides and Polyols
GERD: gastroesophageal reflux disease
IBS: irritable bowel syndrome
SIFBO: small intestinal fungal and bacterial overgrowth
VAS: visual analog scale
As those of you who have read this blog for any length of time know, I’m not a big fan of foods that form opioid peptides upon digestion. As I explained in part two of my GERD series, all exogenous opioids, whether derived from prescription medications, illicit drugs or food slow, and in severe cases stop, both gastric and intestinal movement.
In the stomach, reducing the frequency by which food is released into the small intestine can result in acid reflux. The longer food sits in the stomach the more prone it is to work its way back into the esophagus, especially if swollen by insoluble fiber.
In the intestines, inhibition of the wave-like muscle movements that propel remnants of digested food to its final destination is a major predisposing factor for the development of small intestinal fungal and bacterial overgrowth (SIFBO). It is these peristaltic movements that are mainly responsible for preventing bacteria from the colon, especially the gram-negative variety, from working their way into the small intestine. It is for this same reason that thyroid disorders that depress metabolic rate like Hashimoto’s thyroiditis and euthyroid sick syndrome predispose to developing SIFBO. (1)
But opioids are best known for their pain-suppressing, and not gut-paralyzing, effects. It is this very quality that makes them so effective as analgesics.
There has been some debate about how pronounced these actions really are when it comes to opioid peptides (exorphins) formed from digesting A1 casein dairy and gluten grains. Given the studies I cited in my GERD post, I believe the evidence is strong enough to warrant caution.
Further evidence for these effects was inadvertently provided by a dietary study published earlier this year. Many of you know that the originators of the FODMAP diet found no evidence that adding gluten to this diet led to a worsening of symptoms in those reporting non-celiac gluten sensitivity or in those with confirmed irritable bowel syndrome (IBS). (2)
There were many words spilled in the blogosphere about this study with some arguing that gluten was not a factor in GI distress outside of celiac disease, and that there was no benefit in eliminating these grains from the diet of IBS sufferers. Those saying this, however, failed to account for the gastric delaying and SIFBO promoting effects of gluten opioid peptides, not to mention other extra-intestinal symptoms that clearly plague many, including those afflicted with silent celiac disease; silent only in the fact that these people experience no subjective gastrointestinal discomfort.
But for me this wasn’t the most interesting finding. No, what I found really fascinating was that those on the high-gluten supplement reported the fewest overall IBS symptoms. This couldn’t be ascribed to mere chance as the reduction in symptoms met statistical significance:
This graph shows what happened to GI symptoms over seven days as assessed by the visual analog scale (VAS). The VAS is a series of questions about gastrointestinal symptoms typically used to diagnose IBS. The lower the VAS value, the better.
All participants experienced a worsening of symptoms over seven days that the researchers ascribed to the nocebo effect. This effect is caused by a purely psychological expectation that the blinded treatment you are about to get may cause you harm.
Note how the fewest symptoms were reported in the high-gluten group, while the highest scores were in the placebo or whey-protein group. Those participants given the low-gluten supplement fell somewhere in the middle.
Why would this be? Why would those people given the most gluten report the fewest symptoms? I believe the answer is gluten opioid peptides.
The ability to feel sensation from your gut ultimately depends on the enteric nervous system (ENS). This system, second only to your central nervous system (CNS) in the number of neurons it contains (estimated at between 200 to 600 million), relays sensory information from your gut to your brain via the vagus nerve.
The vagus also works bi-directionally to convey information from your brain to your gut. It explains the butterflies you feel in your stomach when engaging in activities outside your comfort zone.
80% to 90% of the neurons in the vagus are composed of afferent nerves. These nerves are designed to convey information from the gut to the brain. Exciting research is focusing on how gut bacteria, both beneficial and pathogenic, affect these neurons leading to changes in emotion and behavior in both animals and humans. (3) (4)
Perception of pain or other signals from the gut is therefore reliant on communication between the ENS and the CNS. It has long been known that people who for whatever reason have higher visceral (gut) sensitivity are also more likely to be diagnosed with IBS.
This makes perfect sense. If those signals are not reaching the brain, chances are low that anyone would be visiting a doctor complaining of gastrointestinal distress, any more than a diabetic with peripheral neuropathy is likely to complain of burning sensations when an appendage is exposed to an open flame.
In this study, addition of gluten to the low FODMAP diet did what we would expect all opioids to do: reduce or numb perception of pain and other uncomfortable symptoms when these peptides bind to Mu opioid receptors.
This would suggest that people with dysbiotic guts who suddenly stop eating foods that form these opioids may be more prone to experience a surge in uncomfortable feelings that had been largely masked up to that point. This may explain the common onset of gut discomfort in some who go on either a Paleo or low-carb diet where reduction or elimination of wheat, and perhaps dairy, is common. It may also explain why many go back to eating these foods as a means of deadening these sensations.
Of course doing so is ultimately self-defeating. Symptoms of dysbiosis are a sign that gut flora is seriously out-of-whack and needs to be corrected. Dousing intestinal opioid receptors with exorphins is akin to turning up the radio to avoid hearing the clanking sound emanating from a car engine. It does work, but only until something more serious stops the car dead in its tracks.
It is this cycle of opioid use to treat GI symptoms that leads to the harmful merry-go-round I first blogged about in part two of my GERD series:
However, opioids have other biological effects besides slowing gastrointestinal motility or numbing pain and discomfort, which brings me to the research paper that is the subject of today’s post. This was an in vitro study on the effect that opioid peptides have, both from morphine and dietary sources, on antioxidant status and a very important biological process known as DNA methylation. (6)
Now as is true with any in vitro experiment, these results may not translate to animals or humans. After all, living organisms are far more complex than what occurs in a Petri dish. Nevertheless, there is clearly biological plausibility for what was seen in this study. And as you’ll soon read, these results may help explain what is witnessed in those either taking opiates or experiencing relief from diets that eliminate them.
The impetus for this study was to find the biological mechanism(s) for why gluten and/or dairy elimination diets often cause an improvement in a number of conditions including autoimmune disease, schizophrenia, psychosis, autism spectrum disorders, fibromyalgia, etc. (7) (8) (9) (10) (11) (12) (13)
Now in the two years that I’ve written this blog (Good God! I’m shocked at how fast that time has flown!), I’ve never reprinted an entire abstract from a research paper. Mainly because I feel it my duty to interpret these studies in a way that is readily understandable to readers unfamiliar with medical nomenclature. However, today I’m making an exception because this study is, in my opinion, groundbreaking.
Before I begin, I wish to thank Dr. Richard Deth, a co-author of this paper, for taking the time to answer my questions via email. I truly appreciate your correspondence.
The abstract, and by extension the study, will be unintelligible to most of you without explaining some biological processes. As always, I’ll try to simplify this as best I can.
The first concept to understand is the role of cysteine in human health. Cysteine is a sulfur-containing amino acid (amino acids are the building blocks of proteins and their shorter chain-length equivalents, peptides), and serves as the rate-limiting amino acid in the formation of the body’s major antioxidant, glutathione. What this means is that cellular antioxidant or glutathione status is ultimately dependent on the precursor levels of this amino acid in the body.
As I explained in my post Are Probiotics the new Antioxidants:
“Glutathione is the body’s most significant non-enzymatic antioxidant and exists in relatively large amounts if not depleted by combating high levels of oxidation. Not only does it neutralize free radicals, it preserves the antioxidant status of both vitamins C and E. It is also a necessary part of biological processes responsible for DNA synthesis and repair.”
Glutathione is also vitally important for the detoxifying activity of the liver. The reason an overdose of acetaminophen (paracetomal) is toxic is because its metabolite, N-acetyl-p-benzoquinoneimine, rapidly depletes the liver of glutathione leading to cell injury and death that can result in liver failure if not treated in time.
Glutathione in the liver, along with nitric oxide, is also necessary for the sensitization of that organ to insulin. (14) Without this, there is little to prevent the liver from continually producing and raising blood glucose via gluconeogenesis even in the presence of adequate insulin.
Glutathione is also needed for the removal of toxic formaldehyde from the body. Formaldehyde is produced as a necessary byproduct of metabolizing methionine, choline, methanol (alcohol dehydrogenase) sarcosine and other endogenous substances or xenobiotics. (15)
Glutathione also plays a major role in regulating proliferation of cells, including those lining the digestive tract. (16) Proper control of the rate intestinal cells divide is not only important when it comes to preventing digestive tract tumors, but also vital for maintaining gut-wall integrity.
Glutathione plays a role in the production and viability of sperm. (17) It is also essential for proper immune function. Both in vitro and animal experiments have shown that good glutathione status inhibits infection by influenza. (18)
Now these are only a handful of things glutathione does to keep us healthy. But one thing is certain: anything that causes it to be continually depleted will lead to deterioration of health.
Let’s now return to cysteine for a moment. It’s classified as a non-essential amino acid because the body can synthesize it as long as there are adequate quantities of another sulfur-containing amino acid by the name of methionine. However, in those who are most vulnerable to nutritional malabsorption disorders like infants, the elderly, people with uncontrolled celiac disease, those with small-gut tumors, persons with shortened bowels, anyone with SIFBO, etc., both cysteine and methionine status can readily become compromised. This will result in less intracellular glutathione and increased oxidative stress from free radicals that are no longer neutralized by this antioxidant.
Now that I’ve explained the importance of cysteine and glutathione in human health, I need to cover another biological system mentioned in today’s study by the name of DNA methylation.
This biological process obviously involves deoxyribonucleic acid or DNA, so let’s start there. DNA is defined by Merriam-Webster’s as:
“…any of various nucleic acids that are usually the molecular basis of heredity, are constructed of a double helix held together by hydrogen bonds between purine and pyrimidine bases which project inward from two chains containing alternate links of deoxyribose and phosphate, and that in eukaryotes are localized chiefly in cell nuclei.”
DNA is what mostly makes you you and me me. It’s a major reason the Kardashians are the way they are, and please feel free to interpret that however you like!
DNA is composed of four organic molecules known as nucleotides. These nucleotides are cytosine, guanine, thymine and adenine. They, along with a sugar known as deoxyribose and a phosphate group, are the building blocks that DNA is made of.
DNA methylation is the process of adding a methyl group to cytosine nucleotides. I realize this is all Greek to most of you, but stick with me here.
When a cell divides, DNA methylation ensures that these cells carry forward the correct genetic information. It prevents a normal cell, for instance, from reverting to a stem cell or becoming a different cell type.
Stem cells are the types of cells that make up an embryo, and they can morph into all the different cell types that form a human body. Stem cells are obviously essential for proper fetal development, but not desirable when a cell has already been differentiated into a particular type. For example, you want liver cells to produce other liver cells, and not a cell characteristic of your pancreas or kidney, otherwise you’ll end up with a very dysfunctional organ.
DNA methylation is also vital for DNA repair. It can turn genes on or off in a process known as epigenetics. Many of us are born with less than stellar genes that may predispose us to certain nasty diseases, and it is DNA methylation that makes sure those genes stay off.
This biological process is also necessary for the proper functioning of neurons, both in the central and enteric nervous systems. When DNA methylation is not functioning properly, faulty or incomplete transmission between neurons will lead to all kinds of issues affecting sensation, behavior, mood and memory.
Moreover, this process is involved in regulation of neurotransmitters. These are chemicals that nerves rely on to properly communicate with each other and other systems in the body like the immune system. It’s also involved in hormone, cholesterol and histamine regulation, as well as repair of protein structures.
In sum, DNA methylation is a very important reason a healthy person stays that way. Dysfunction here can lead to serious diseases like birth defects, cancer, autism, asthma, autoimmune disorders, etc.
DNA methylation is dependent on a group of enzymes known as DNA methyltranferases (DNMTs). These enzymes are in turn reliant on intracellular levels of S-adenosylmethionine (SAM).
Recall that methyl must be combined with the DNA nucleotide cytosine for DNA methylation reactions to happen. SAM provides these methyl groups for over 200 of these reactions making SAM status very, very important.
Like glutathione, SAM is dependent on availability of cysteine. So anything that inhibits cysteine absorption or synthesis also affects SAM and downstream DNA methylation reactions.
OK, now that you understand all that, it’s time to reprint the study abstract:
“Dietary interventions like gluten-free and casein-free diets have been reported to improve intestinal, autoimmune and neurological symptoms in patients with a variety of conditions; however, the underlying mechanism of benefit for such diets remains unclear. Epigenetic programming, including CpG methylation and histone modifications, occurring during early postnatal development can influence the risk of disease in later life, and such programming may be modulated by nutritional factors such as milk and wheat, especially during the transition from a solely milk-based diet to one that includes other forms of nutrition. The hydrolytic digestion of casein (a major milk protein), and gliadin (a wheat-derived protein) releases peptides with opioid activity, and in the present study, we demonstrate that these food-derived proline-rich opioid peptides modulate cysteine uptake in cultured human neuronal and gastrointestinal (GI) epithelial cells via activation of opioid receptors. Decreases in cysteine uptake were associated with changes in the intracellular antioxidant glutathione and the methyl donor S-adenosylmethione [SAM]. Bovine and human casein-derived opioid peptides increased genome-wide DNA methylation in the transcription start site region with a potency order similar to their inhibition of cysteine uptake. Altered expression of genes involved in redox and methylation homeostasis was also observed. These results illustrate the potential of milk- and wheat-derived peptides to exert antioxidant and epigenetic changes that may be particularly important during the postnatal transition from placental to GI nutrition. Differences between peptides derived from human and bovine milk may contribute to developmental differences between breastfed and formula-fed infants. Restricted antioxidant capacity, caused by wheat- and milk-derived opioid peptides, may predispose susceptible individuals to inflammation and systemic oxidation, partly explaining the benefits of gluten-free or casein-free diets. [Emphasis mine]”
Courtesy: Food-derived opioid peptides inhibit cysteine uptake with redox and epigenetic consequences. (6)
Here we see a representation of how cysteine uptake is inhibited in two different cell lines. The chart on the left represents neuronal cells, and the one on the right gut cells. Morphine suppressed the uptake of cysteine the most as illustrated by the black line at the bottom of each chart. Next came beta-casomorphin 7 derived form A1 casein dairy depicted by the red line.
In neuronal cells, alpha gliadin 7 (shown in green) derived from gluten, came next with the least inhibition seen from human breast milk. In gut epithelial cells, this gluten opioid showed about the same inhibitory effect on cysteine uptake as human breast milk.
I cautioned earlier that experiments like these can never replicate real-world conditions because our nervous and digestive systems are far more complex than this. So let’s explore why the results seen for A1 casein opioids may be different when consumed in whole food.
None of us, including children, are just consuming casein protein when we drink milk. Milk also contains whey protein, which is rich in both cysteine and methionine amino acids, as is true for most animal-based foods. This fact alone will likely offset some of what was seen in this study.
As I wrote in this post, there is compelling evidence that both casein and whey proteins improve gut-barrier function, and are therefore more likely to seal a leaky gut than cause one. And whole bovine milk, especially from grass-fed cows, is a nutrient-dense food rich in fat soluble vitamins, minerals and conjugated linoleic acid–substances known to be healthful to humans.
As in human breast milk, but to a greater extent in A1 dairy, these opioids serve a purpose by slowing gastrointestinal peristalsis to allow the nutrients in these liquids enough time to be absorbed by the infants ingesting them. And in their natural state, both come with oligosaccharides to feed beneficial gut bacteria, although the prebiotics in cow’s milk are found in highest concentrations in its colustrum. (19) (20)
Nevertheless, as the evidence continues to mount that the state of human gut flora both in the small and large bowel is intimately involved in central nervous system function, as well as regulation of the immune system, including a food that clearly depresses gastric and intestinal motility is highly problematic in those with dysbiotic guts.
Why? Because it:
1) allows more time for noxious metabolites and components from pathogenic gut organisms to cross the gut wall and enter systemic circulation increasing inflammatory immune responses, and
2) makes normalization of intestinal flora next to impossible as both proper gastric and intestinal motility is essential for long-term gut health.
In other words, while inhibition of cysteine uptake is clearly a concern, in my mind the bigger issue is the effect these opioids have on gut motility.
I need to emphasize that these dairy opioids are only formed from digestion of A1 casein dairy, not from A2 casein sources like goat’s milk, milk from Jersey cows or milk from Asian and African breeds. So while I stand by my belief that dairy does an intestinal tract good (at least in those without lactose intolerance), it’s mainly true in regards to A2 casein milk.
However, when it comes to gluten grains there are no compensatory components of these foods to counteract their gliadin-derived opioid peptides. Whereas A1 dairy forms just one opioid type upon digestion, gluten has been shown to form many. These include gluten exorphins A5, A4, B5, B4, and C5. (21) (22)
Addition of alpha gliadin 7 to this list gives us at least six different opioid peptides formed upon digestion of gluten grains, all with the potential to negatively affect cysteine uptake, antioxidant status and DNA methylation. This no doubt explains why the women I wrote about in this post regained their health on a gluten-free diet, but didn’t experience any relief eliminating dairy.
Combining A-1 dairy and gluten opioids, as would normally occur when eating a wheat-based breakfast cereal for example, would suggest that in the real-world the effect on cysteine uptake would be cumulatively worse.
But this is far from the only issue with gluten grains. As I wrote in this post, these foods, especially in their whole-grain form, are rife with anti-nutrients and natural pesticides chief among the latter being wheat germ agglutinin. They also contain phytic acid which prevents the proper absorption of the minerals they contain unless first soaked in an acidic medium for a specific length of time.
These grains also contain amylase and trypsin inhibitors. Amylase is an enzyme necessary for the proper digestion of starches, while trypsin is needed to properly digest protein. These inhibitors have been shown to cause the release of pro-inflammatory cytokines via the same receptors lipopolysaccharides (LPSs) trigger to activate the immune system. (23)
All these factors explain why there is not a lick of scientific evidence, outside of confounder-prone nutritional epidemiology, that consuming these grains are healthy. That is made abundantly clear in Anthony Colpo’s book: Whole Grains, Empty Promises: The Surprising Truth about the World’s Most Overrated ‘Health’ Food.
Those who believe that these issues are confined to modern gluten grains and can therefore be avoided by eating more ancient wheat varieties are sadly mistaken. These ancient grains have an even higher prolamine-rich content than modern wheat.
A research paper that examined two ancient wheat strains–Graziella Ra and Kamut–found no evidence that they were any less toxic to celiacs than modern wheat. (24) And the reason is because we DO NOT produce either the pancreatic or brush-border enzymes necessary to break these prolamine bonds.
The 33-mer peptide that is the trigger for celiac disease in the genetically susceptible remains intact after fifteen hours of exposure to human digestive enzymes. (25) If that particular peptide is so resistant to digestion, what hope do any of us have when it comes to gliadin opioid peptides?
That said, there is some evidence to suggest that in an intestine with healthy gut flora the pro-inflammatory effect of these grains may be somewhat mitigated. Bifidobacterium bifidus (B. bifidus), for example, has been shown to counter the inflammatory effects of gluten on cells lining the gut wall. (26) And as I wrote in this post, addition of prebiotics to pasta countered the negative effects of gluten on intestinal, tight-junction proteins.
Nonetheless, given the wide prevalence of autoimmune disorders, allergies, metabolic syndrome, anxiety, depression, chronic fatigue syndrome, fibromyalgia, IBS, ulcerative colitis, Crohn’s disease, SIFBO, etc., etc., etc., I think we can safely dispense with the notion that most people are sporting healthy gut flora, especially in the age of antibiotic overuse. On the contrary, all signs point to increasing levels of gut dysbiosis.
Earlier, I alluded to the fact that while this study was in vitro, there was more than enough biological plausibility to believe it holds true in everyday life. Morphine and other opioid derivatives have been shown to increase oxidation reactions and negatively affect immune function. (27) They have also been shown to cause brain damage in rodents, and increase oxidative stress in those receiving morphine analgesics to control cancer pain. (28) (29)
Inhibition of cysteine absorption by opioids appears to be mediated by excitatory amino acid transporters (EAAT), and in particular, excitatory amino acid transporter 3 (EAAT3). EAATs, also known as glutamate transporters, are involved in stopping excitatory signalling to neurons by their uptake of glutamic acid, also known as glutamate. Failure to do so leads to neuronal cell death resulting from over-stimulation. EAAT3 had earlier been shown by this same research group to be involved in the proper uptake of cysteine. (30)
Following is a diagram showing how activation of Mu opioid receptors affects the cellular absorption of cysteine via EAAT3 transport, and the downside consequences from doing so:
I advise all my readers to steer clear of these opioid-producing foods when trying to resolve gastrointestinal issues or chronic disease states. I have no doubt that beneficial gut flora mitigates the damage, but there are limits to everything, especially in an age of caloric excess.
At the heart of chronic disease is immune activation, an inherently pro-oxidative condition that calls forth compensatory cortisol secretion and generation. Eating foods that enhance that state–and that also includes excessive amounts of refined fructose, omega 6 polyunsaturated vegetable oils and alcohol–is not doing you any favors.
Don’t say I didn’t warn you.