Wednesday, January 16, 2013

Easy Whey to Prevent LPS Induced Inflammation? Whey Protein Prevents LPS Binding to TRL-4 and IL-8 Production. Surprise: Pressurized Denatured Whey Works Best!

Unlikely that this or whatever whey protein you have bought as of late was pressurized with an Avure High Pressure Processing System at more than 500mPa in order to denature it (yeah you're reading right) and produce a bunch of fancy new peptides which appear to have even more potent anti-LPS effects than those in regular whey protein (hydrolysate).
Saturday's post on saturated fatty acids and their negative effects on post-prandial endotoxemia has turned out to be (un-)surprisingly popular. I am still not sure if this will be same for the post at hand; and that despite the fact that it revolves around the exact same topic, namely the inflammatory reaction to lipopolysaccharides (LPS). In the case of endotoxins such as LPS, the inflammation, which is, as you all should by now be aware of, an endogenous "alert, defense and repair" reaction of our bodies, is actually triggered by their interaction with the so-called toll-like-receptor. Now scientists from the McGill University in Montreal have found that there is a substance all of you are familiar with and some of you may even be consuming on daily or at least regular basis that can block this interaction, as well as the ensuing overproduction of cytokines and/or other well-meant, but in the end potentially hazardous immune responses.

There is al-wheys something new about whey ;-)

As the researchers point out the beneficial effects of whey proteins don't just go way beyond their muscle building effects and are mediated by several and not just one of it's amino acid, protein and peptide ingredients:
"Whey proteins (WP), a by-product of the cheese-making industry, possess nutritional benefits as a source of protein of high biological value. Whey products and whey-derived peptides have demonstrated a number of anti-inflammatory effects. These anti-inflammatory effects include decreased cytokine release in rodent models of ischaemia– reperfusion and exposure to LPS. In addition, individual whey constituents, such as lactoferrin or glycomacropeptide, and peptides released from these by pepsin– pancreatin hydrolysis exhibit anti-inflammatory effects, such as suppression of tissue neutrophilia or inhibition of inflammatory cytokine release." (Iskandar. 2013)
In that, whey proteins have been shown to be particularly useful for the treatment and/or management of chronic inflammatory diseases such as cystic fibrosis, a disease passed down through families that causes thick, sticky mucus to build up in the lungs, digestive tract, and other areas of the body and a disease that will probably make you - just like me - think about the high cysteine content of whey, immediately.

"Under pressure..."

For the study at hand the researchers used two different epithelial cell types and added lipopsaccharides (LPS), as well as
  • regular whey (Inpro 90 Whey Protein Isolate from Vitalus Nutrition) that had been enzymatically hydrolysated (=predigested) to yield a product that would be similar to what many supplement are now selling you as either intra- or post-workout "super whey" (their not my claim ;-), or
  • pressurized whey, which was based on the same raw material, but was pressurized before being hydrolysated
to their petri dishes and observed the effect the additional whey proteins had on the LPS-induced interleukin-8 (IL-8) production and the binding of the Escherichia coli LPS to the TRL4s (toll-like receptor 4) on the surface of the epithelial cells.
Figure 1: Differential effects of different doses (in µg/ml) regular and pressurized whey protein hydrolysate on LPS-induced IL-8 secretion in 1HAEo- cells (left) and corresponding LPS-binding to toll-like receptors on the surface of the cells (right); data expressed relative to LPS only (Iskandar. 2013)
As the data in figure 1 clearly shows, both whey protein hydrolysate, the regular, as well as the pressurized one had similar effects on the binding of LPS. The ensuing decrease in cytokine production (IL-8) was yet statistically significant only in the dish with the pressurized whey protein hydrolysate. Moreover at the highest dosage of the normal whey protein hydrolysate, there is what you could call a "rebound effect", if this reached statistical significance - which it obviously didn't.

Figure 2: Effect of 500 or 1000mg/ml of pressurized whey protein and native whey protein hydrolysates on 1HAEo- cell culture medium ferric-reducing antioxidant power  (FRAP); data expressed relative to basal levels (Iskandar. 2013)
That said, the overall effect size dependent not just on the type of whey protein hydrolysate, but also on the cell type: While the 1HAEOo- cells (shown in figure 1) needed the highest tested dose to show statistically significant reductions in IL-8 production, the effect reached significance at 500 µg/ml for the CFTE29o- cells using the pressurized whey protein hydrolysate and at 1,000µg/ml for the regular WPH (obviously no rebound here).

A similar difference was observed in the results FRAP essay (FRAB stands for ferric reducing ability of plasma and the results provide information about the general antioxidant defenses of the cells). While the CFTE29o- cells (shown in figure 2) were happy with both whey protein hydrolysate, the pressurized WPH had a minimal, but statistically non-significant edge in the 1HAEOo- cells.

Is there anything special about pressurized hydrolysates?

In previous studies the researchers had already established that pressurization of WP improves its
in vitro digestibility, promotes the release of novel peptides by gastrointestinal digestive enzymes and enhances the antiinflammatory effect (Vilela. 2006).
"These in vitro findings were also confirmed in clinical studies. Thus, a 2-week supplementation with pressurised whey increased the levels of glutathione, a crucial low-molecular anantioxidant, in peripheral blood mononuclear cel. Further, we have reported that a 1-month dietary supplementation with pressurised whey improved nutritional status and markers of systemic inflammation in patients with CF [cystic fibrosis]." (Iskandar. 2012)
In the study at hand, the research team from Canada did now want to (a) investigate the potential anti-inflammatory and antioxidant effects of pressurized and regular whey protein hydrolysates in the context of cystic fibrosis and non- CF respiratory epithelial cells and (b) explore the mechanisms by which pressurised and native whey exert their beneficial anti-inflammatory effects. Their research hypothesis was that it is the difference in peptide (=complex bond of amino acids that has different effects from the same amino acids in isolation) availability that is enhanced by the pressurisation of whey that's responsible for it's superiority compared to regular whey proteins.

Establishing the (leaky) gut, bacteria, non-alcoholic fatty liver disease connection

With the researchers focus being on cystic fibrosis, the results of this in-vitro trial are still highly meaningful for all of us. In particular the more potent increase in overall anti-oxidant capacity upon exposure of the epithelial cell lines would suggest that the ingestion of pressurized whey proteins could exert similar benficial effects in other parts of the body, specifically the digestive tract, as well.

Suggested read: "Plus: 20+ Things to Protect and Restore the Integrity of Your Intestinal Wall" (read more)
On the other hand, the more important blockage of the toll-like receptors, was virtually identical with both the regular and the pressurized whey protein and the general implications of these findings are actually pretty far-reaching. After all, TRL4 has only recently been implicated in the development of fibrosis in non-alcoholic fatty liver disease subsequent to alteration of gut microbiota, increased intestinal permeability and the ensuing increase in exposure of the liver to gut-derived bacterial products (Frainarius. 2012). The exact same horror-scenario many of you will probably have had on mind, when they read about the effects the high saturated fat content had in the study by Mani et al. from Saturday.

Bottom line: So will just having your daily whey protect your gut from all assaults? Probably not, but is may be just another one of the many small things which may not render your intestinal wall and overall immune system bullet-proof but will at least help them to come with the omnipresent and 24/7 assault they are exposed to. Whether it's really got to be pressurized whey, on the other hand, remains questionable. Personally, I don't think so - if you take a look at the dose-dependently reduced expression of IL-8 in response to LPS exposure upon co-administration with pressurized and normal whey, it seems as if the pressurized variety did not only have the edge, but was also lacking the rebound effect that occurred at very high doses in the 1HAEo-cells.

If you want to live out your OCD tendencies on your whey intake, start with rule #1 "Never Sip Your Whey" (read why).
Since, previous have shown that pressurization does impart significant changes not just to the digestibility of whey protein hydrolysates, but also to their peptide structure (Vilela. 2006), it could thus well be that the observed differences are of real world importance, as well, and cannot be compensated for by just taking more of the "regular" whey protein hydrolysates.  I hope there will soon be comparative studies in human subjects available, until then I would not worry too much about not getting the "optimal" whey protein to combat TRL-4 binding of LPS and increase anti-oxidant defenses. As a regular SuppVersity reader, you are probably apart of the privileged part of the Western society which has to care least about LPS and overall (bad) inflammation, anyways. Plus, as the study goes to show "regular" whey has similar, albeit not "optimal" effects in this regard.

  • Iskandar MM, Dauletbaev N, Kubow S, Mawji N, Lands LC. Whey protein hydrolysates decrease IL-8 secretion in lipopolysaccharide (LPS)-stimulated respiratory epithelial cells by affecting LPS binding to Toll-like receptor 4. Br J Nutr. 2013 Jan 3:1-11.
  • Frasinariu OE, Ceccarelli S, Alisi A, Moraru E, Nobili V. Gut-liver axis and fibrosis in nonalcoholic fatty liver disease: An input for novel therapies. Dig Liver Dis. 2012 Dec 29.
  • Vilela RM, Lands LC, Chan HM, Azadi B, Kubow S. High hydrostatic pressure enhances whey protein digestibility to generate whey peptides that improve glutathione status in CFTR-deficient lung epithelial cells. Mol Nutr Food Res. 2006 Nov;50(11):1013-29.