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ATIs: amylase and trypsin enzyme inhibitors

HFD: high-fat diet

HPA axis: hypothalamic-pituitary-adrenal axis

IAP: intestinal alkaline phosphatase (aka alkaline phosphatase)

IL1-β: interleukin 1 beta

LPSs: lipopolysaccharides

NF-kB: nuclear factor kappa B

PUFAs: polyunsaturated fatty acids

TLRs: toll-like receptors

TLR4: toll-like receptor 4

TNF-α: tumor necrosis factor alpha

WGA: wheat germ agglutinin

Today I want to write a post on the importance of a particular intestinal enzyme involved in gut-wall integrity and host defense. Intestinal alkaline phosphatase (IAP) is a brush-border enzyme expressed by absorptive cells (enterocytes) of the small intestine. It is secreted into both the lumen, or interior, of the digestive tract as well as the basolateral or systemic end.

Unlike gut flora, highest concentrations of IAP are found in the first section of the small intestine (duodenum) and decline the further down the digestive tract you go.

IAP secretion, just like any other small intestinal enzyme, is dependent on the health of enterocytes comprising what is known as the brush border. Intestinal cells that are chronically inflamed are by definition unhealthy, which is a major reason why those battling small intestinal dysbiosis are often deficient in these enzymes.

As you know, inflammation can be due to a number of factors: gluten, acetaldehyde, unsaturated fatty acids, enzyme inhibitors, gut infections, drugs, yeast overgrowth, thin to non-existent mucus layer, viruses, etc. Whatever the cause, inflammatory cytokines will affect not only the shape of these cells and the tight junction proteins that bind them together, but their ability to properly secrete enzymes.

IAP has some very important functions. First, it’s involved in regulating secretion of bicarbonate in the duodenum.

Bicarbonate helps to neutralize the very acidic semi-digested food (chyme) entering the small intestine after passing through the stomach. Failure to raise pH here can lead to acidified chyme injuring cells lining this part of the digestive tract. That can increase inflammation and intestinal permeability.

But IAP’s most important role is detoxifying lipopolysaccharides (LPSs) derived from the cell wall components of gram-negative gut bacteria. (1) It is therefore an important defense against endotoxemia.

LPSs initiate inflammatory immune responses by binding to proteins known as toll-like receptors (TLRs), and in particular toll-like receptor 4 (TLR4). This in turn induces two separate inflammatory pathways.

The first is nuclear factor kappa B (NF-kB). NF-kB is a protein that regulates inflammatory immune responses, and chronic activation of this pathway has been linked to cancer and autoimmune disorders.

The second pathway initiated by TLR4 activation is release of tumor necrosis factor alpha (TNF-α). As you recall from this post, TNF-α is a very powerful and potentially destructive inflammatory cytokine.

The binding of TLR4 is a necessary condition for immune responses to gram-negative gut bacteria when they come into direct contact with intestinal epithelial cells and the submucosa. This inflammatory cascade is always accompanied by an increase in cortisol generation and synthesis via activation of the hypothalamic-pituitary-adrenal (HPA) axis, and by increasing intracellular expression of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) via the cortisol-cortisone shunt.

Now, this immune complex causes the release of IAP as a defensive mechanism. IAP’s ability to detoxify LPSs prevents further immune stimulation. IAP also prevents NF-kB from migrating to the nucleus of enterocytes. So by both detoxifying LPSs and halting NF-kB, IAP serves as a potent anti-inflammatory intestinal enzyme.

Conversely, cytokines like TNF-α and interleukin 1 beta (IL1-β) depress the secretion of IAP from enterocytes. Why this is so no one really knows. However, as mentioned above, oxidative stress no doubt has something to do with it. Very little is more stressful to gut cells than being subjected to the chronic onslaught of inflammatory cytokines.

Therefore, as a general rule it’s safe to say that anything that chronically inhibits IAP intestinal expression will increase the risk of LPSs initiating inflammatory immune responses at the gut wall that can result in a leaky gut. In very severe cases sepsis can result should these bacterial components reach systemic circulation in appreciable amounts. At the very least, LPSs reaching the liver will provoke inflammation capable of damaging cells in that organ.

The protective role of IAP against endotoxemia has been demonstrated in animal studies. In mice genetically bred to be incapable of secreting this enzyme, high-fat feeding increases serum triglyceride levels, leads to visceral fat accumulation and causes these animals to develop a fatty liver. (2)

In another study published in 2013, researchers found that both endogenously produced IAP as well as oral supplementation with this enzyme reversed metabolic syndrome in rodents (3):

“The present study was undertaken based upon work in our laboratory and others that points to the role for IAP in protecting the host from bacterial toxins. The present data in mice provide a “proof of principle” that IAP could be an effective oral supplement against endotoxemia, thus protecting the host from metabolic syndrome. We have shown that IAP reduces corn-oil induced endotoxemia and prevents the inflammation and intestinal permeability changes that occur in response to an HFD [high fat diet].”

Dietary Fats and Intestinal Alkaline Phosphatase Expression

IAP expression is partly influenced by the type of fatty acids present in the small intestine after a meal. As I covered in this post, unlike short- and medium-chain fatty acids, long-chain fatty acids do not go directly to the liver absent a leaky gut:

“Longer-chain fatty acids…re-form into triacylglycerols within the enterocyte. Together with phospholipids, cholesterol and proteins, they form large particles within the absorptive cell. If there are lipopolysaccharides in the vicinity, these too hitch a ride on the newly formed lipoprotein vehicle or chylomicron.

As I wrote then, chylomicrons and all members of the cholesterol family bind to and inactivate LPSs with varying degrees of efficiency, which is a good thing as Homo Sapiens have been eating long-chain fatty acids for approximately 200,000 years, with most of these fats coming from animal sources.

However, cholesterol isn’t the only biological substance that defends us against endotoxins. In animal models, IAP secretion has been consistently shown to rise in the presence of saturated fatty acids.

If as a normal consequence of absorbing these fats, LPSs come into direct contact with brush border cells, it’s not at all surprising that this enzyme would be excreted as part of an innate immune defense against these potential troublemakers.

But while saturated fatty acids increase the secretion of this anti-inflammatory enzyme, polyunsaturated fatty acids (PUFAs)–both from omega-6 and omega-3 sources–have been shown in both pigs and rodents to either not provoke IAP release, or actively suppress it.

This is a decidedly undesirable state of affairs.

LPSs carried across the gut wall by long-chain PUFAs are also inactivated once incorporated into chylomicrons. However, the failure to increase IAP secretion when LPSs either reach enterocytes, or slip between them when tight-junction proteins are compromised, inevitably causes immune activation. Couple this with the propensity of these fatty acids to easily oxidize and generate lipid peroxidation byproducts, and the risk of inducing leaky gut and endotoxemia goes through the proverbial roof.

Is it any wonder then that rodents chronically fed alcohol while simultaneously fed PUFAs experienced the liver damage I showed you in this post, while saturated fats were protective under the same chronic alcohol feeding?

In pigs fed either corn oil or beef tallow for four weeks, corn oil feeding consistently decreased a number of brush-border intestinal enzymes (4):


Courtesy: Jejunal Brush Border Hydrolase Activity Is Higher in Tallow-Fed Pigs than in Corn Oil-Fed Pigs.

Courtesy: Jejunal Brush Border Hydrolase Activity Is Higher in Tallow-Fed Pigs than in Corn Oil-Fed Pigs.


Compare the quantity of enzymes secreted between the unsaturated high fat, corn-oil group shown in the middle column with the saturated high fat, beef-tallow group. In all cases, the expression of the five brush border enzymes listed on the left were lower in the pigs fed omega-6 PUFAs, even when measured against pigs fed a low corn oil diet.

IAP (shown as alkaline phosphatase) was significantly depressed in this group. This meant that any LPSs that reached intestinal cells had to provoke an inflammatory cascade that no doubt increased intestinal permeability and liver inflammation in these animals.

As an animal model, pigs have more in common with our digestive tract than rodents, so these results are sobering to say the least. What’s also surprising is how high corn oil feeding depressed other brush border enzymes like aminopeptidase, lactase, sucrase and maltase.

Aminopeptidases break down proteins for proper absorption. Lactase is essential for digesting lactose found in dairy while sucrase is needed to properly digest sucrose or sugar. Maltase is needed to digest maltose.

Dietary components that are not digested or hydrolyzed in the small intestine become substrates for bacteria once they reach the colon. Here, they will be set upon by specific species of colonic gut bacteria.

The more these substrates reach the colon, the larger the colonies of bacteria that are specialized to feed on them grow. I suspect that augmentation of these bacteria crowd out beneficial colonic gut flora like gram-positive Bifidobacterium that typically feed on fiber. So it shouldn’t be too surprising that depletion of bifidobacteria is highly associated with a great number of chronic disease states.

In another study comparing rats fed a control diet high in omega-6s with 16.3% saturated fat, an olive oil diet (mainly monounsaturated fat) containing 15.67% saturated fat, or a high oleic acid sunflower oil diet containing 9.96% saturated fat, the animals fed the sunflower oil diet also experienced lower IAP secretion. (5)

So why did rats fed the control or olive oil diets secrete higher amounts of IAP in contrast to those fed the high-oleic sunflower oil? While an explanation is missing from the study abstract, the discussion section lets the shy kitty peek its furry little head from out the bag:

“It has been reported that the specific activity of alkaline phosphatase is increased by saturated fatty acids. Since HOSO [high-oleic sunflower oil] diet contained a lesser amount of saturated fatty acids when compared to control and OO [olive oil] diet, it is possible that this HOSO diet induced a decrease in the activity of alkaline phosphatase.”

You think?

Canola oil, a current darling of those who earned their nutritional degrees while faithfully worshiping at the altar of nutritional epidemiology, contains just 6% saturated fat which is why it’s so beloved. It’s also relatively high in omega-3 PUFAs causing many dietitians to giddily shout Hosannas to the heavens when discussing the supposed health benefits of consuming these highly processed oils. Never mind that the intense heat, pressure and solvents used in their manufacture easily causes the PUFAs they contain to be oxidized long before anyone grabs them off the supermarket shelf.

Unfortunately for the public, omega-3 PUFAs also inhibit IAP expression. As I wrote in my post Ulcerative Colitis and Dietary PUFAs, omega-3s in conjunction with omega-6s have been shown to lower IAP expression in mice even when adjusted to levels that are typical for humans who take omega-3 supplements. (6) That was a major reason some of the mice died from sepsis during the study.

Let me reprint the relevant passage:

“While C. rodentium-induced inflammation promotes colonic damage in C57BL/6 mice…, these responses are also important for C. rodentium clearance… Our results suggest that ω-3 [omega 3] PUFA supplementation to an ω-6 [omega 6] PUFA rich diet results in a milder colitis but impairs infection-induced inflammatory responses important for preventing systemic C. rodentium infection. Similarly, another study demonstrated that mice fed high levels of ω-3 PUFAs had impaired immune function and could not produce a response against Helicobacter hepaticus-induced infection… Consumption of ω-3 PUFA rich diets may have anti-inflammatory properties however this may prevent the body from mounting appropriate immune responses critical for host defense.

In following with this, the mice fed ω-3 PUFA supplemented high-fat diets suffered increased mortality during C. rodentium infection. While the lack of colonic inflammatory responses likely played a role, a contributing factor was the inability to detoxify LPS through the lack of dephosphorylation activity in response to C. rodentium infection. In addition, there was a lack of IAP+ submucosal cells infiltrating into the colons of these mice. IAP prevents sepsis by dephosphorylating LPS. While the pathogen did not translocate across the intestinal barrier at an increased level in the mice fed diets supplemented with ω-3 PUFA, C. rodentium itself induces barrier dysfunction. Thus, some pathogen became systemic and this resulted in sepsis. In support of this, mice fed fish oil supplemented diets had elevated serum-associated LBP [lipopolysaccharide binding protein], a clinical biomarker of sepsis associated with mortality. Additionally, in these mice infection induced serum IL-15 and TNF-α expression. IL-15 induces sepsis as determined with IL-15 knockout mice which avoid sepsis through the lack of protease activation.”

Yessiree folks! Just keep cooking with “healthy” canola oil and dousing your insides with omega-3 flaxseed or fish oils and you too may one day be lucky enough to spend some quality time in your friendly neighborhood intensive care unit (ICU) should a pathogenic strain of gram-negative bacteria colonize your blood. You’re welcome!

According to a medical paper published in 2012:

“In the past decade we have realized that there have been significant longitudinal changes in the incidence of sepsis, most obviously in the USA. A two-decade study of US hospitalizations identified an increase in the incidence of sepsis among hospitalized patients by 8.7% per year. At present, it is estimated that there are more than 1,000,000 cases of sepsis among hospitalized patients each year in the USA. Numerous reports have shown the incidence of sepsis and severe sepsis increasing in excess of the growth of the population. Similar reports exist from the UK, Australia and from Croatia.”

Courtesy: Sepsis, severe sepsis and septic shock: changes in incidence, pathogens and outcomes. (7)

I wonder how many of these people ended up in the ICU because they foolishly believed that adding flaxseed oil or cod liver oil or krill oil or fish oil capsules to a diet already brimming with omega-6s was the royal road to spectacular health? How many were consuming foods sautéed or fried in PUFA-rich oils? How many were dressing their salads with expeller-pressed flaxseed, canola, soybean or walnut oils? In other words, how many were unwittingly depressing the very brush border enzyme necessary to detoxify LPSs?

I think these are good questions if I may say so myself. Unfortunately, it appears no one can answer them.

And how many of these same people were on statins to lower their total cholesterol? IAP and cholesterol are part of our innate immune defense against translocating endotoxins even if that fact seems to have escaped notice by the editors of Kuby Immunology.

A major reason I’m writing this post is that I have yet to talk or correspond with one person complaining of gut dysbiosis who was not taking omega-3 supplements. The belief in the curative powers of these fatty acids, at least in regards to gut health, seems to be ingrained in both conventional and alternative medicine.

However, the futility of supplementing with omega-3s to cure or prevent gut dysbiosis was illustrated in a randomized, placebo-controlled trial in breast-fed infants living in Gambia at high risk for intestinal diseases. (8)

In the hope that doing so might ward off gut dysbiosis in 172 of these infants and lead to other benefits including cognitive improvement, one group of infants was randomized to receive 500 mg of fish oil per day. The control, or placebo group, was given olive oil instead.

Supplementing began at three months of age and lasted for six. At both nine and twelve months, infants given fish oil were medically assessed and compared to their placebo controls.

Not only did fish oil fail to improve gut wall integrity via measures of intestinal permeability, it utterly failed to improve cognitive function, growth or resistance to disease, including intestinal ones. It did, however, reduce the incidence of diarrhea so there was that benefit.

If fish oil supplements were ineffective at preventing or curing gut dysbiosis in breast-fed infants, what’s the chance that anyone past the age of weaning will experience any curative effect?

In my case, one of the supplements I began taking before the onset of my gastrointestinal odyssey was cod liver oil. I was led to believe that because it was high in fat-soluble vitamins A and D, it would only do a body good. However, whatever benefit I thought I was deriving from taking it was soon eclipsed by the intestinal hell that followed. Mind you, I was quite careful in avoiding omega 6 PUFAs during this period.

Looking back at this experience and what I now know about how omega-3s depress IAP expression and immune function, it’s little wonder I had such difficulty overcoming a gut infection. I suspect many of you are doing the same if those who I’ve consulted with are any indication.

Now nothing I’ve just written is meant to dissuade any of you from eating seafood. On the contrary, getting your omega-3s from whole food is always recommended.

Seafood is a rich protein source. As protein is the most satiating macronutrient, the quantity of omega-3s you’re likely to ingest are within tolerable limits. It also contains selenium, which as a precursor to glutathione synthesis, increases anti-oxidant status.

But when it comes to omega-3 supplements, it’s quite easy to go overboard. I’ve known people who drench their salads in flaxseed oil or take ungodly amounts of fish oil because they’ve heard it’s good for them. The reasoning being, I suppose, that if a little is good, a lot must be better. Wrong!

So let me say it again. The best way to re-balance a warped omega-6/omega-3 ratio is not by supplementing with omega-3s, but by dramatically curtailing omega-6 intake.

And no, you can’t achieve this by consistently obtaining your breakfast, lunch and/or dinner from the drive-through window of your favorite corporate-owned restaurant. Restaurants are for-profit businesses and their first allegiance is to the bottom line, not your health. Many will use the cheapest food ingredients they can get away with, and when it comes to cooking oils that means PUFAs.

Nor can you achieve this by eating processed foods. PUFAs, both hydrogenated and non-hydrogenated, permeate many of these products.

If you refuse to cook your own food, or have it prepared by someone who knows what they’re doing, then there isn’t any advice I or anyone else can offer you that’s worth a damn when it comes to regaining your gut health.

Other factors affecting intestinal alkaline phosphatase secretion

Beneficial bacteria derived from eating fermented dairy protect the mucosal layer from being colonized by pathogenic organisms, including gram-negative ones. But what is becoming clear is that other components of dairy independently increase IAP activity.

For example, it has recently been discovered that calcium-rich foods increase IAP in rodents. (9) This no doubt partly accounts for the increase seen in IAP expression in animals fed yogurt or fermented dairy. (10)

Milk sugar or lactose has also been shown to increase IAP activity (11). So as I wrote here, the right type of dairy does indeed do a digestive tract good.

Whole-fat milk (the milk we drank back in the days before the obesity epidemic) coupled with its high calcium and lactose content would be particularly effective at increasing IAP excretion, especially if that milk comes from A2 dairy sources devoid of opioid peptides after digestion.

Now this isn’t going to work very well for you if you’re lactose intolerant. You can either supplement with lactase to overcome this issue, or eat fermented dairy like yogurt.

Simple and complex carbohydrates are also known to increase the production of IAP in rodents. That includes starches. (12) However, guar gum, a common food additive, was seen to lower IAP activity. (13) And speaking of starch, potato starch, a resistant starch currently in vogue among certain Paleo Diet advocates, has also shown a propensity to increase IAP. (14)

Both vitamins K1 and K2 also increase IAP levels in rats. (15) Both are found in meat, dairy and eggs.

K2 is also produced by lactobacillus in the small intestine, hinting that its production has more to do with increasing IAP activity against endotoxins instead of serving as a nutritional source for the host. Whatever benefits are currently ascribed to K2 supplementation may have more to do with this than anything else.

Other dietary components shown to increase IAP activity include black and red pepper, and ginger. (16) This suggests that consistently including these spices in your diet may also be of benefit.

Fiber also increases IAP activity in the colon, probably as a direct result of fermentation by beneficial colonic bacteria. (17)

Conversely, dietary factors shown to depress IAP in animals apart from PUFAs include soybeans and other phytoestrogens like coumestrol. (18) (19) Binding to estrogen receptors may be the reason for these results.

If so, it may offer a clue for why intake of phytoestrogens, birth control pills and estrogen replacement therapy are all associated with an increased risk for Crohn’s disease and ulcerative colitis in women. (20)

Fasting dramatically decreases IAP activity. (21) This is likely a major reason prolonged intravenous feeding (total parenteral nutrition) is highly associated with liver damage and failure. (22)

IAP is stimulated by feeding, so bypassing the small intestine during parenteral nutrition would naturally cause this important enzyme to decline, leading to increased translocation of gram-negative bacterial components to the liver where resident immune cells become chronically activated. As beneficial gut flora are also very important for maintaining gut wall integrity, the lack of the substrates they require to flourish (soluble prebiotic fibers) compounds the problem.

Nonetheless, I’m still a believer in intermittent fasting. When trying to resolve a chronic case of gut dysbiosis, there’s a lot to be said about giving your digestive tract a break to accelerate healing.

Fasting is also known to increase autophagy. This is an intracellular process where debris and pathogens hiding from immune detection are destroyed by cellular garbage collectors.

However, prolonged fasting carries downside risks like nutrient deprivation, thyroid and metabolic depression, and in the case of IAP, reduced secretion. My advice is to keep your fasts to no longer than twenty-four hours at a time, and to do them no more than once or twice a week.

Very low-fat diets also cause depression in IAP expression. As mentioned, IAP is increased with saturated fat intake, which is precisely the type of fat usually restricted in these diets.

Whatever fat is eaten is likely to be “heart healthy” omega-6 PUFAs, the absolutely worst fats you can eat if trying to avoid dysbiosis and a leaky gut. I suppose this helps explain why the Sidney Diet Heart Study found an increase in coronary artery disease in those eating the most dietary PUFAs.

Finally, few of my posts would be complete without mentioning gluten grains. There is no evidence that gluten has any effect on IAP expression. But since all opioids inhibit brush border enzyme secretion, I see no reason gluten opioid peptides wouldn’t do the same.

Other wheat factors affecting gut health are amylase and trypsin enzyme inhibitors (ATIs). (23) Both would inhibit the proper digestion of starch and proteins and increase these substrates in the colon as occurs with PUFA consumption.

But more importantly, these ATIs bind to and activate toll-like receptor 4:

“Here, we identified the nongluten ATIs CM3 and 0.19 and likely other homologous ATI variants as potent activators of TLR4. Based on our data, we believe that this interaction of ATI with TLR4 is a major signaling pathway in innate immune reactions to wheat. We did not find activity in gliadins, and reduction of disulfide bonds completely abolished TLR4-stimulating activity in ATI-enriched fractions. However, our findings do not completely rule out other, gliadin-dependent signaling pathways that still remain to be characterized. The highly disulfide-linked ATIs stimulate monocytes, macrophages, and DCs [dendritic cells] in vitro to produce IL-8 and other inflammatory cytokines such as IL-12, TNF, MCP-1, or RANTES. Notably, feeding ATIs to C57BL/6J mice resulted in up-regulation of proinflammatory cytokines in their duodenal mucosa, which confirms resistance of ATIs to enteric proteases and their stimulatory potential in vivo.

ATI, a “contaminant” in gluten preparations, rather than gluten itself might explain observations that in celiac patients in remission a duodenal and rectal gluten challenge increased proinflammatory chemokines and cytokines after only 1–4 h [hours], a time frame which is too short to induce adaptive immune responses. Of note, the local gluten doses applied in these studies (6–10 g) already come close to the doses that we used in mice in vivo. Moreover, we can assume that there is increased sensitivity toward ATI in patients with celiac disease or gluten sensitivity compared with healthy controls.”

In other words, separate and apart from issues concerning gliadin, zonulin, gluten opioid peptides or wheat germ agglutinin (WGA), these wheat-derived enzyme inhibitors activate the same toll-like receptors that lipopolysaccharides do. ATIs, like WGA, are natural pesticides produced by wheat to ward off predators. If you’re eating food containing this grain, you’re the predator! Congratulations.

This information reinforces what I wrote in PUFAs, Leaky Gut, Endotoxemia and the Liver. As intestinal inflammation always causes declines in beneficial gut flora populations, preventing unnecessary immune activation is one of the best ways to care for this vitally important, yet often overlooked microbial organ.


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