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Endotoxin Antagonists - Part 1
 
This is the first in a series on things that might lower endotoxin transport or the inflammatory response to endotoxin. I have about 100 references and I'm pretty sure that's only scratching the surface. 

If you’ve been listening to the previous podcasts you will know that one of the ways to induce metabolic endotoxaemia in rodents and humans is through a high-fat diet or high-fat meals. We can definitely see increases in endotoxin in the plasma from more than 40 g of fat in one sitting. Looking at some of the experiments we seem to see about 1.4 fold increase in endotoxin from 40 g and closer to 2.5 fold increase from 70 g. It’s hard to say what amount of fat is safe, certainly something that is needed for absorption of the fat-soluble vitamins. Alcohol then is another factor that is very potent at initiating metabolic endotoxaemia. I’m not gonna go to deep into those two factors but just imagine them here at the beginning.

Okay so the first thing we will look at is citrus fruits. Reference number two looks at orange juice in the context of a high-fat meal induced increase in endotoxin and toll -like receptor 4. The experiment took three groups of 10 healthy people, and after an overnight fast they had either glucose, orange juice, or a water along with a 900 cal meal.(2)

The meal consisted of egg muffin and sausage muffin sandwiches with potato hash browns. The macronutrient breakdown was about 80 g carbohydrates, 50 g of fat, and 32 g of protein. They then had 300 cal worth of orange juice, or a glucose drink, or water. The orange juice was packaged not from concentrate Florida orange juice. From the figures unable to get on the amount of sugar or calories in orange juice this was probably quite a large amount of orange juice, maybe close to a litre.

There are some studies that are often passed around looking at the effects of sugar or high fructose corn syrup and claiming to compare the levels of inflammation from ingesting both. I’ve read two similar studies recently, neither showed much control over the dietary factors and both showed similar levels of inflammation from the diet when including either sucrose, glucose, or high fructose corn syrup. So the question I had reading them was whether the infflammation had anything to do with the sugars at all, as there are plenty of studies showing inflammation from high-fat diets.

So this study was interesting because it tested water along with sugary drinks. In general the results show that the glucose and water only groups had increased inflammation, some of the markers were higher in the water group and some higher in the glucose group, So that might be pretty specific differences in various inflammatory markers with or without glucose, but the trend with both was the same.

The study found that the orange juice prevented the increase in inflammatory markers and reactive oxygen species generation which occurred in the other two groups. Orange juice prevented the increase in plasma endotoxin concentrations. Notably orange juice also suppressed the increase in toll -like receptor four and two, these are involved in sensing endotoxin and creating inflammation in response to the endotoxin.

The paper indicates that these effects are probably due to the flavonoids, namely hesperidin and naringenin and references studies on those.

The next paper is similar (3). This study took four groups of 12 normal weight people and gave them a drink with 300 cal of glucose, orange juice, whipping cream, or its 300 mL of water after an overnight fast. One of the things they measured was toll like receptor four. This was increased by whipping cream only. TNF alpha was significantly increased by both glucose and cream. NFKB was also significantly increased by glucose or cream, but not water or orange juice. And then the plasma endotoxin concentrations increased significantly after cream only. Then in the conclusions they note that orange juice does not increase oxidative stress, inflammation or plasma endotoxin unlike cream or to some degree glucose.

The next paper looks at the effect of one of the citrus flavonoids, namely naringenin on hyperglycaemic mediated inflammation in the liver and pancreas tissue of rats (4). They created a drug induced model of type II diabetes in rats, and then looked at the effect of oral naringenin. They looked at a lot of different markers of inflammation and concluded that naringenin protects against inflammation by blocking the activation of NFKB, thereby lowering pro-inflammatory cytokines and inducible nitric oxide synthase expression, this in turn prevented hyperglycaemic inflammation and damage to the liver and pancreas.

The dose of naringenin was 50 mg per kilogram, which would be an enormous dose to get from food, I’ve linked up a site that analyses some of the content of polyphenols in foods, so for example you need about 3 L of grapefruit juice to get 50 mg of naringenin, however citrus fruit will contain multiple polyphenols so it’s pretty hard to make a one-to-one comparison anyway. (10)

Though grapefruit juice has higher levels of naringenin than orange juice, the latter is probably a better source due to grapefruit interactions with liver enzymes.

The next paper (5) looks at naringenin’s effects on experimental colitis, toll -like receptor four, NFKB. The paper tells us that along with grapefruit and citrus naringenin is also present in tomatoes and has been used as an anti-inflammatory for centuries. Colitis, which is inflammation of the colon, was induced in rodents using dextran sulphate sodium. Some of the rodents were given naringenin prior to the drug and this led to reduced colitis, lower inducible nitric oxide synthase, Cox-2 , TNF alpha and toll -like receptor four among others. The paper concludes that the mechanism is inhibition of the TLR4 and nfKB pathways. The dosage of naringenin was the same 50 mg per kilogram. There are a number of drugs that can ameliorate inflammatory bowel diseases that are shown to limit TLR4, tianeptine and low dose naltrexone come to mind.

The next paper (6) looks at the effect of naringenin and another similar polyphenol naringin on endotoxin induced uveitis in rats. Uveitis Is a group of inflammatory diseases that affect the eye. Rodents were injected in the foot with endotoxin and given various doses of the polyphenols intravenously. The doses were .4, four and 40 µg per kilogram of either polyphenol. In each case the polyphenol was administered three times, 30 minutes before and after a long with during the injection of endotoxin. They found that the polyphenols limited the development of eye inflammation and reduced prostaglandin E2 and nitric oxide in a dose-dependent manner, the paper suggested the anti-inflammatory effect may be due to suppression of prostaglandin E2 and nitric oxide.

The review paper Citrus flavonoids and lipid metabolism (7) Looks at citrus flavonoids and their effect on metabolic dysregulation. So this paper infers a lot of protective effects from citrus fruit on the effect of endotoxin. It looks at the treatment of diabetes, liver problems, obesity, dyslipidaemia, and atherosclerosis.

The paper talks about a number of studies using refined flavonoids. For Example 500 mg a day of hesperidin was seen to reduce triglycerides and apolipoprotein B (apoB). 400 mg of naringin could reduce LDL-cholesterol by more than 14% in people with high cholesterol. That study was eight weeks long, another study which was only four weeks long using 800 mg hesperidin and 500 mg naringin had no effect on moderately hypercholesterolemic individuals. 

They mentioned a study in genetically obese mice where 2% of the diet by weight was the peel of citrus fruit called unshiu, which is high in citrus flavonoids led to significantly lower plasma triglyceride and hepatic steatosis after six weeks. There are many other references in that paper giving doses in some form or other of citrus flavonoids in human and animal experiments. Some of these flavonoids are easily available now in supplement form and relatively cheap.

The next paper looks at the effect of orange juice on cognitive function. This was a study in middle-aged healthy males. The paper notes epidemiological evidence suggesting that consumption of fruit-based flavonoids is associated with cognitive benefits. They gave the subjects 240 mL of a flavonoid rich orange juice, there was measured to be 272 mg of total flavonoids in this dose. The results showed significantly improved executive function and psychomotor speed correlated with subjective alertness following the flavonoid rich orange drink relative to placebo. 

We’ll switch now from citrus flavonoids to cocoa flavanols. (11)

The first paper I selected on Coco looks at the effect of cocoa powder on metabolic endotoxaemia and adipose tissue inflammation in mice fed a high-fat diet. The paper goes through a lot of the mechanisms are to be involved in high-fat diet induced obesity and diabetes, it tells us that arachidonic acid is the source of one of the most potent classes of endogenous inflammatory mediators, the ecosanoids, which when released from membrane phospholipids can be synthesised into the ecosanoids via either cyclooxygenase or lipoxygenase. Mice are deficient in either of these enzymes or the polyunsaturated fatty acids are resistant to diet induced obesity.

So the study used a 10% fat diet which was low-fat, and a high-fat diet which was 60% from fat. Another group was put on the high-fat diet and was supplemented cocoa powder at 80 mg per gram. It’s not made clear whether there is 80 mg per gram of rodent or per gram of rodent food. I’m guessing it programme of rodent. 

The study showed that long-term supplementation with dietary cocoa lowers the high-fat diet induced adipose tissue inflammation, down regulates NFKB and ecosanoid metabolism, suggesting improved gut barrier function and lowered metabolic endotoxemia.

The next study looks at similar markers in rodents. (12) This study used an 8% cocoa powder intervention. They found that cocoa powder supplementation decreased TNF alpha, interleukin-6, inducible nitric oxide synthase, and arachidonic acid in the adipose tissue. Cocoa also reduce the levels of ecosanoid generating enzymes, phospholipase A2 and Cox-2 buy over 50%. Plasma Endotoxin was 40% lower in the cocoa treated animals.

The next paper is a review of randomly controlled trials on the efficacy of cocoa flavonols. (12a)This found that cocoa flavonols intake significantly improved insulin sensitivity and lipid profile, the paper stated the need for large long-term trials on the effects of cocoa flavonol looking at diabetes and cardiovascular events. 

The next reference contains polyphenol analysis of commercially available cocoa and chocolate products. (12b) The paper looked at antioxidants and procyanadins also. It found the general trend for all three categories and that was that the higher the amount of non-fat cocoa solids the more polyphenols, procyanadins and antioxidants. So that is cocoa powder came out on top with 2 to 3 times more than dark chocolate and 10 to 12 times more than milk chocolate. 


The next substance were going to look at is coffee.

The first paper is on decaffeinated coffee and the expression of tight junction proteins in rats with non-alcoholic fatty liver disease. (13)

So the study took three groups of rats, the first group got high-fat diet for five months, the second group got the same but with added decaffeinated coffee from the fourth month, the third group got a standard diet with no coffee. The high-fat diet was 58% fat, the standard or low-fat diet was 5% fat. The study measured markers of tight junction integrity, namely zonulin and occludin, along with toll -like receptor four.

There is an interesting passage on the background to the study regarding coffee, that it's made up of a mixture of about 1000 compounds including polyphenols with numerous antioxidant, anti-inflammatory, and prebiotic properties. It tells us that caffeine is responsible for many of the psychoactive and invigorating effects of coffee, there are other papers looking at the effects of caffeine on liver disease with good outcome, my assumption is that they used decaf in order to separate the effects of coffee in general from caffeine.

The experimental animals were free feeding and their food intake was recorded, the energy intake was higher in the high-fat diet groups than the standard diet, which is similar to other studies that have seen. While the energy intake in high-fat diet with and without coffee was similar the high-fat diet without coffee induced significantly higher body weight than the high-fat diet with coffee, both were significantly higher than the standard diet.

The most significant differences in weight gain really start to become apparent only after week eight, which is similar to some of the data on citrus polyphenols in rodents. As the coffee intervention group only received coffee from month four too month five I should point out that there were significant differences in body weight between the two high-fat diet groups prior to coffee intervention. So I have to wonder about that outcome on its own.

The effects on markers of tight junction permeability were that the coffee intervention mostly ameliorated the damage from the high-fat diet. The coffee interventions significantly lowered toll -like receptor four in the tissue sampled but the reduction here was not as great and the coffee intervention is closer to the high-fat diet than to the standard diet in this measure.

The next reference I have used is not a paper but it's a letter to the Journal of alimentary pharmacology and therapeutics. (14) The latter is regarding a review article on the experimental epidemiological, and clinical studies on coffee intake and non-alcoholic fatty liver disease, non-alcoholic fatty liver disease as I have described in the previous podcasts can be created by increased endotoxin detected in the portal vein, that is going from the digestive system to the liver.

The letter writer wishes to propose another mechanism, or set of mechanisms for the effect, and that is the modulation of gut microbiota by coffee. Presented firstly is evidence for the link between gut microbiota and chronic liver disease, next the writer reports the differences in microbiota from high-fat diets and the resulting increase in endotoxin.

Then the author references human studies in which a daily dose of 3 cups of coffee for three weeks resulted in an increase in bifido bacterium population, this type of bacteria is something that I've seen consistently associated with lower plasma endotoxin. Another reference given shows that coffee exhibits a regulatory effect on the microbiota, as we've seen in previous podcasts a dysregulation, or a lowering of diversity along with a decrease of bifido tend to occur with increased metabolic endotoxaemia.

The writer finishes up by saying that while it plausible that the effect of coffee and gut microbiota might contribute to reducing the risk of liver disease, it's not yet known what the mechanisms of action are.

There is a huge amount of positive data out there on coffee and it’s something that will probably look at in more detail in future. For this podcast I’ll include just one more reference and coffee, one that backs up my own experience with it and seems to be common to a lot of people. So the study looked at inflammatory markers in different people (14a). I think the title gives us a good overview of what it’s about the title is “Coffee consumption modulates inflammatory processes in an individual fashion.”

So the paper looked at blood markers of inflammation in eight people. So the markers were IL6, IL8, PGA2, PGD2, and prostaglandin E2 (PGE2). The paper found significant effects on inflammatory markers in individuals. In some individuals the markers went up, while in others they went down. I have experienced what seemed like both effects from coffee at different time periods.

The next category a look at, dairy is another food that has pretty variable effects on different people at different times. Some of the factors are metabolic rate, I only tolerated dairy after eating a lot of carbohydrate for a few months, another factor might be the casomorphins and in that case A2 cow's milk or standard goat milk might be more tolerable, if you haven't eaten dairy while it might take a few days for the lactase enzyme to increase to be able to handle certain amount of lactose. 

So the first thing we need to look at is IAP which is an acronym for intestinal alkaline phosphotase. This is in the category of brush border enzymes, it controls endotoxin information by dephosphorylating endotoxin. So for the same amount of endotoxin in the system higher IAP is better(15a).

This next paper looks at calcium and IAP, dairy of course being a very high source of calcium in general (16). The study was looking at various different elements in rodents but the interesting thing in the context of this discussion was that it found that calcium in increased IAP. Another study in rodents (17) found evidence of increased IAP with supplementation of the milk sugar lactose.

Milk products fermented with some strains of lactobacillus can increase IAP(20), at least in rodents, though the overall effect might not be great if there is an increase in lactate either from the fermented product or by increased population in the gut from the bacteria. A well strained yogurt might remove a lot of the lactate. 

Milk also contains casein glycomacropeptide, though it’s much higher in some cheeses (20a,20b). This is seen to lower the inflammatory response to endotoxin by binding it.

The last substance look at in this podcast is olive. I released a short podcast little while ago on findings that low adrenomedullin was associated with longevity. One of the things that came to mind when I saw that story was the prevalence of olive oil this part of the world, the article was a town in Italy and this was most people have heard about the potential benefits of the Mediterranean diet. In rodents endotoxin increases adrenomedullin which increases nitric oxide(21a).

So the first paper on olive products looks at the molecular mechanisms of the olive oil phenolic compound olectanthal.(21)

This an in-depth paper looks at a lot of different information on virgin olive oil as well as specifics on the polyphenol in question. Some of the things that it points out is that olecanthal inhibits Cox one and Cox two similar to ibuprofen. Cox two is particularly involved in endotoxin induced inflammation, so suppression of this will lower the damage from endotoxin. Cox two deficient mice are much less susceptible to endotoxin induced organ damage and death(21b).

The paper notes the suppression of endotoxin induced nitric oxide synthase along with an inhibitory effect on tau fibrilisation which is related to dementias like Alzheimer's and Parkinson's. There are a number of mechanisms by which endotoxin can be causal in dementia's which I hope to get to a future podcast. The paper notes that olive oil phenols also inhibit other inflammatory factors including NFKB and TNFa, both central to endotoxin damage. The COX-2 inhibition leads to lower Thromboxane A2 which seems central to some major cardiovascular diseases. 

The next paper (22) looks at these survival rates of rodents injected with endotoxin on differing diets. The mice groups were fed a standard lab mouse diet supplemented with 7% from either canola, sesame, soybean, or virgin olive oil. All of the supplemented groups had similar increases in body weight and energy intake. The mice were dosed with endotoxin at week six. Inflammation and increased lipid body formation was seen in all groups but was significantly diminished only in the virgin olive oil group. All of the mice died between 48 and 72 hours after endotoxin injection except for the olive oil group which had a survival rate of 60% one week later. Plasma TNF alpha prostaglandin E2 were two of the markers that were ameliorated by the olive oil diet.

The next paper looks at some of the markers of inflammation and the effect of phenolics from extra-virgin olive oil(23). So this paper took macrophages from rodents. Macrophages are inflammatory and immune effector cells central in the development of chronic inflammatory diseases and they are known to be activated by endotoxin where they then produce inflammatory mediators like nitric oxide, prostaglandin E2 and TNF alpha. The paper found that extra-virgin olive oil polyphenols reduced oxidative stress and inflammatory responses, decreased nitric oxide, reactive oxygen species generation, COX-2 and gave an indirect indicator of lower NFKB.

The next paper (24) looked looked at NFKB directly in a rodent model of rheumatoid arthritis. the orally administered polyphenol extract from extra-virgin olive oil showed decreased joint oedema, cell migration, cartilage degradation and bone erosion. It significantly reduced inflammatory cytokines and PGE2 along with decreasing NFKB and down regulating the arthritic process.

Another olive product with active anti-inflammatory polyphenols is olive leaf extract(25). The extract will contain more polyphenols than even high quality extra virgin olive oil which can be difficult in itself to get and the paper tells us that many of these polyphenols have a structural difference that may increase their beneficial effects. It provides analysis for some of the polyphenol content in olive oil and olive leaf extract.

The subject of the paper is cancer, it mentions that the Mediterranean diet is thought to have a protective role against cancer and that this mechanism may be related to reduced inflammatory response and ecosanoid pathways. See 25a “Unsaturated fatty acids: Nutritionally essential, or toxic?” for more on ecosanoids and free fatty acids in cancer and inflammatory disease.

I think there are at least two factors involved with the Mediterranean diet and inflammation from ecosanoids, as firstly the polyphenols limit the inflammatory damage and secondly eating a diet were the supplementary fat is primarily monounsaturated rather than polyunsaturated will lead to potentially lower ecosanoids in the first place as they are metabolites of polyunsaturated fats. Olive oil itself does contain around 10% polyunsaturated fatty acids however.

There are flavonoids in the olive leaf extract that are not detected in olive oil that are known to have anti-cancer properties. So olive oil polyphenols can inhibit NFKB, down regulate COX-2, inhibit TLR4 which is involved in detecting endotoxin and starting the inflammatory cascade, the OLE polyphenols down regulate nitric oxide, interleukin-6, interleukin beta, and TNF alpha.The paper goes into a lot of detail on potential effects of olive leaf extract or Mediterranean diet on a number of different cancers and discusses the potential mechanisms of action so it’s well worth the read but that is where I’m going to leave the podcast off for today. And about a quarter of the way through the references that I have for lowering endotoxin or the inflammation from endotoxin so I expect there will be probably three more shows in this topic. 


1

High fat diets


Citrus

2

http://www.ncbi.nlm.nih.gov/m/pubmed/20200256/ 

Orange juice neutralizes the proinflammatory effect of a high-fat, high-carbohydrate meal and prevents endotoxin increase and Toll-like receptor expression.


3

http://care.diabetesjournals.org/content/33/5/991

Differential Effects of Cream, Glucose, and Orange Juice on Inflammation, Endotoxin, and the Expression of Toll-Like Receptor-4 and Suppressor of Cytokine Signaling-3


4

https://www.ncbi.nlm.nih.gov/pubmed/23841752 

Ameliorative effect of naringenin on hyperglycemia-mediated inflammation in hepatic and pancreatic tissues of Wistar rats with streptozotocin- nicotinamide-induced experimental diabetes mellitus.


5

https://www.ncbi.nlm.nih.gov/pubmed/23506745 

Protective effect of naringenin against experimental colitis via suppression of Toll-like receptor 4/NF-κB signalling.


6

https://www.ncbi.nlm.nih.gov/pubmed/16117693 

The effects of naringin and naringenin on endotoxin-induced uveitis in rats.


7

https://www.ncbi.nlm.nih.gov/pubmed/23254473 

Citrus flavonoids and lipid metabolism.


8

http://link.springer.com/article/10.1007/s00394-015-1016-9 

Flavonoid-rich orange juice is associated with acute improvements in cognitive function in healthy middle-aged male


9

https://www.ncbi.nlm.nih.gov/pubmed/17384340/ 

Orange juice or fructose intake does not induce oxidative and inflammatory response.


10

http://nopr.niscair.res.in/bitstream/123456789/9379/1/NPR%203(1)%2012-15.pdf 

Quantitative Distribution of Hesperedin in Citrus Species, ...

http://phenol-explorer.eu 



COCOA


11

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4034375/ 

Dietary Cocoa Reduces Metabolic Endotoxemia and Adipose Tissue Inflammation in High-Fat Fed Mice


12

https://www.ncbi.nlm.nih.gov/pubmed/23494741 

Dietary cocoa ameliorates obesity-related inflammation in high fat-fed mice.


12a

http://sci-hub.cc/10.3945/jn.116.237644

Cocoa Flavanol Intake and Biomarkers for Cardiometabolic Health: A Systematic Review and Meta-Analysis of Randomized Controlled Trials


12b

http://sci-hub.cc/10.1021/jf060290o

Antioxidant Activity and Polyphenol and Procyanidin Contents of Selected Commercially Available Cocoa-Containing and Chocolate Products in the United States


COFFEE

13

http://www.ffhdj.com/index.php/ffhd/article/view/268 

Decaffeinated coffee induces expression of tight junction proteins in rats with NAFLD.


14

http://onlinelibrary.wiley.com/doi/10.1111/apt.12781/full 

Letter: gut microbiota modulation contributes to coffee's benefits for non-alcoholic fatty liver disease


14a

https://www.ncbi.nlm.nih.gov/pubmed/27425673

Coffee consumption modulates inflammatory processes in an individual fashion.


Milk and IAP

15

https://www.ncbi.nlm.nih.gov/pubmed/20536777


16

https://www.ncbi.nlm.nih.gov/pubmed/22018098 

Luminal calcium concentration controls intestinal calcium absorption by modification of intestinal alkaline phosphatase activity.


17

https://www.ncbi.nlm.nih.gov/pubmed/15207765

Enhancement by lactose of intestinal alkaline phosphatase expression in rats.


20

https://www.ncbi.nlm.nih.gov/pubmed/9693207

Diet supplemented with yoghurt or milk fermented by Lactobacillus casei DN-114 001 stimulates growth and brush-border enzyme activities in mouse small intestine.


20a

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567326/ 

Chemical and functional properties of glycomacropeptide (GMP) and its role in the detection of cheese whey adulteration in milk: a review

20b

https://www.ncbi.nlm.nih.gov/pubmed/25923657

Endotoxin-Binding Peptides Derived from Casein Glycomacropeptide Inhibit Lipopolysaccharide-Stimulated Inflammatory Responses via Blockade of NF-κB activation in macrophages.


Olive


21

http://sci-hub.cc/10.2174/138161211795428911 

http://www.ingentaconnect.com/content/ben/cpd/2011/00000017/00000008/art00002 

Molecular Mechanisms of Inflammation. Anti-Inflammatory Benefits of Virgin Olive 

Oil and the Phenolic Compound Oleocanthal 


21a

https://www.ncbi.nlm.nih.gov/pubmed/8632695

Up-regulation of rat adrenomedullin gene expression by endotoxin: relation to nitric oxide synthesis.


21b

http://www.fasebj.org/content/17/10/1325.short

Cyclooxygenase-2-deficient mice are resistant to endotoxin-induced inflammation and death1


22

http://journals.lww.com/shockjournal/Abstract/2005/02000/Mechanisms_of_Increased_Survival_After.12.aspx 

MECHANISMS OF INCREASED SURVIVAL AFTER LIPOPOLYSACCHARIDE-INDUCED ENDOTOXIC SHOCK IN MICE CONSUMING OLIVE OIL-ENRICHED DIET


23

https://www.ncbi.nlm.nih.gov/pubmed/24740524 

Extra virgin olive oil polyphenolic extracts downregulate inflammatory responses in LPS-activated murine peritoneal macrophages suppressing NFκB and MAPK signalling pathways.


24

https://www.ncbi.nlm.nih.gov/pubmed/25294776 

Anti-inflammatory and joint protective effects of extra-virgin olive-oil polyphenol extract in experimental arthritis.


25

http://www.mdpi.com/2072-6643/8/8/513/htm 

Evidence to Support the Anti-Cancer Effect of Olive Leaf Extract and Future Directions


25a

http://raypeat.com/articles/articles/unsaturatedfats.shtml 

Unsaturated fatty acids: Nutritionally essential, or toxic?


Seems unshiu is a type of mandarin orange probably quite easy to get.

Quince looks interesting too.

http://real.mtak.hu/17425/1/Hegedusetal2013IJHSreview.pdf