Wednesday, February 14, 2018

Saturated fat and fatty liver. Payday in Colorado.

Dophamn supplied the link to another interesting study:

The role of visceral and subcutaneous adipose tissue fatty acid composition in liver pathophysiology associated with NAFLD

Here is the money shot that supports the religion of saturated fat as the devil incarnate:

"Overall, these data suggest that diets enriched in saturated fatty acids are associated with liver inflammation, ER stress and injury".

Meanwhile, in the study detail:

I would agree that the stearic acid rats stayed comparable in weight to others despite eating more calories than either Crapinabag or PUFA fed rats, as in Table 1. There is NO evidence that they developed inflammatory changes in their liver! They had a statistically significant increase in messenger RNA expression for seven genes associated with inflammatory liver disease. The question is whether these mRNA changes actually result in detectable inflammatory changes in the liver, or are they markers of the normal response to reverse electron transport though complex I derived superoxide which might also trigger life extending increases in SOD and/or catalase gene expression? ROS generation is essential to mitochondrial biogenesis. It MUST affect signalling molecules. At what level does physiological signalling degenerate in to pathology? Easy to find this out, just look at the liver histology.

What we need to know is whether there is histological evidence of NASH development. After all, we know from the methods that they took terminal samples of liver and snap froze them in liquid nitrogen. Either sticking some in formalin at the same time or getting histology done on the frozen samples (not ideal from the histologist point of view but quite possible) would allow them to correlate their mRNAs with actual damage in the liver. They didn’t do this.

So why did they freeze liver samples at all? As they say in the methods:

“Liver tissue was homogenized in buffer (100mM Tris, pH 7.8) and alanine aminotransferase (ALT) concentration was determined from supernatant via manufacture instructions (Cayman Chemical, Ann Arbor, MI)”.

[Not my typo in the copy/paste. I can do enough of my own when I feel that way!!!]

In the results section in Figure 4 this is converted to:

Plasma alanine aminotransferase concentration was higher in SAT compared with CON and PUFA”.

[My shouting emphasis on "supernatant" and "plasma"]

Plasma???? No. The methods clearly state that it was liver homogenate supernatant! Plasma ALT is an absolutely routine, standard, everyday marker of liver damage. It is a surrogate for hepatocellular damage, i.e. a normal component of liver cytoplasm which has leaked in to plasma in response to liver injury. It’s measured every day in any patient undergoing any sort of health/illness monitoring blood work. It is a COMPLETELY normal cytoplasm component while it is contained within the liver hepatocytes. It is LEAKAGE  to the blood stream that we are interested in as a surrogate for hepatic damage. The rats all had terminal blood samples taken. The group could have measured ALT for a few pence in real plasma from this blood. They didn’t. They homogenised liver and measured ALT in the supernatant. They described this as “plasma”. All we can say from Fig 4 is that the liver of stearic acid fed rats has more of ALT within its hepatocytes. ALT is a normal enzyme used for interconverting certain components of the TCA/amino acid metabolism. Who knows why it is increased under stearate feeding, but it's not a marker of hepatocellular damage unless it is being released in to the blood stream... I think we can assume plasma ALT was completely normal. I'd be willing to bet it was measured in a pilot study and failed to pass the pay-dirt test.

The related studies cited in this paper are equally interesting and say nothing about much other than the ingenuity of the researchers. As always, my fascination is about the mindset involved.

Who decided to homogenise liver to get “plasma” ALT? Who decided to bin the histology friendly liver samples?

Why?

Peter

11 comments:

jesrad said...

Nice find again !

"I think we can assume plasma ALT was completely normal. I'd be willing to bet it was measured in a pilot study and failed to pass the pay-dirt test."

Yup I bet someone had a Bright(tm) idea to save the day after the "Uh results say it is the reverse" moment. I doubt they realized how bad the idea really was. More likely they went "oh so that was what we were doing wrong", and no one was enlightened. I've seen it happen in my work so often...

cavenewt said...

What are the chances someone will point this out in the letters section of the publication?

Unknown said...
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Edward Edmonds said...
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Edward Edmonds said...

Hilarious. Snap frozen liver sections and no histology. Maybe the cryostat was broken. Ha. The the supernatant/plasma error is unforgivable. The ideal way to do this would be to take the liver sections and put them straight into glyoxal or formaldehyde and post fix (i.e. post osmication) in osmium tetroxide or osmium imidazole.... or dichromate is an option if you have the time. H&E's on a frozen sections only if you want to peak at morphology and conserve tissue for other molecular methods and pick and choose what you want fixed. Osmium tetroxide/imidazole will show unsaturated lipids and triglycerides in a black/brown color. The benefit of this method verses other lipid stains is that you can then stain with other special stains other then routine H&E's e.g. such as Perl's Prussian Blue for demonstration of Iron (which I would be curious about), Trichome, Reticulin, PAS, etc,... it would prove to be a wonderful and insightful display of color and useful structural information. This would allow you to have nice consecutive serial sections all morphologically similar assuming the microtomist isn't a ditz.

Cheers,
Edward

Peter said...

Yes Ed, you can certainly get some really pretty (and informative) stains on liver. I think my wife likes the aesthetics of Trichrome best! I agree iron would be interesting. I've long had this suspicion that iron retention in liver is an evolutionary conserved event to assist the generation of ROS when there is inadequate metabolic ROS to generate insulin resistance and the hepatocytes really, really don't one any more calories. Under PUFA there will be too few normal metabolic ROS to induce insulin resistance so the cells retain Fe (or Cu) to generate enough ROS to limit caloric ingress, or rather to limit caloric retention in the case of hepatocytes. Even hepatocytes can need to say enough is enough on the Standard American Diet, PUFA won't let them signal this, those hepatocytes which retain transition metals manage to limit their caloric retention and do OK except the ROS from transition metals are poorly targeted/timed and then the cell ends up with 13HODE, 4HNE etc etc and everything then falls to pieces......

Just a background thought. One of the banes of my wife's life as a histopathologist is the request from specialists for hepatic Cu stains looking for genetic copper storage disease. By the time the dog is terminally cirrhotic for any reason there is usually a ton of copper there anyway, secondary to the cirrhosis.

Peter

cavenewt said...

Your mention of iron allows me to get tangential.

Tucker pointed out in a blog post that more and more members of the health community (some that I consider good sources of information) are warning of iron overload, to the point of recommending regular blood donation as a means of keeping it under control. To my great relief, Tucker concluded it was not a real cause for concern. http://yelling-stop.blogspot.com/2018/01/iron-overload-myth-in-healthy-people.html

Preferring to always look at things from an evolutionary perspective, I've found this iron-overload warning troubling, unless Paleo folks had regular blood loss from injuries, which is not an unlikely possibility. Important substances in the body tend to be closely regulated.

Peter said...

The paper I have on iron being released from storage to cytoplasm is somewhere on my hard drive but was in yeasts. Not exactly hepatocytes. But I have my eyes open for a similar process in liver cells under PUFA.... Avoid PUFA and hang on to essential iron!

Peter

Edward Edmonds said...

I'm homozygous for hemochromatosis. Shrug. I've always considered elevated iron as a surrogate for something is wrong much like cholesterol. An innocent bystander. Secondary. There was something I saw years ago about a protein discovered to regulate iron storage and have totally lost the link. I think non-heme is the one to worry about. But then again my Siberian mitochondrial heritage might predispose me to think that.

Aside to iron ROS insulin resistence: DHA (Dehydroascorbic acid): Always thought the Pauling idea of AA mutation interesting, came across an old paper where "they" (not Pauling) talk about DHA having a diabetogenic effect at 500mg, it seems odd to me that any reasonable person would think a 63 million year old conserved genetic mutation irrational and use it to justify multi-gram doses of AA w/ the accompanying rise in mutagenic lesions. It would seem a useful mutation for animals that live off of palmitic acid and need to sleep for the winter or store a bit for hard times (eat a bit of fructose store a bit of iron live to see tomorrow, the ability of fructose to shut down palmitic acid OXPHOS is interesting, makes me think that de novo is re-diverted palmitic acid verses legit de novo but I haven't looked closely at tracer studies). I come into mysterious contact with a lot of people trying the gallon of orange juice and skim milk a day developing diabetic like symptoms. Skinny diabetics. Makes me wonder, but so many ways to go with that train of thought.

Peter said...

I doubt any of your orange juice/slim milk friends are about to volunteer a liver biopsy for iron staining in the immediate future!