Thursday, August 11, 2016

Protein catabolism should generate an RQ of around 0.8

Anyone who has read Martha's story and put her narrative together with the folks in Phinney's 1980 study will have immediately wondered: How many of Phinney's subjects were lactating? Even just a little bit?

I think we can say, pretty categorically, that none of them were lactating. Gluconeogenesis from lipid is very likely to have been occurring but obviously (now) this can only drop the RQ when the glucose produced is not being oxidised. Clearly my initial idea expressed in the Phinney post is wrong.

Martha is easy, her child took the sugars hence the spectacularly low RQ. Trying to explain why a protein supplemented fast should drop the RQ below 0.69 needs a little more thought.

This is what Phinney thought might be happening under moderate exercise:

"The low RQ value of 0.66 observed during the final exercise test was surprising, as long chain fatty acid oxidation occurs at an RQ of 0.69. (The only common fuel oxidized at a lower ratio is ethanol at 0.67). The answer to this disparity may lie, at least in part, in the rise in serum ketone concentration observed during exercise. As the hepatic production of ketones from long-chain fatty acids occurs at an RQ of zero, a net retention of ketones in body fluids will result in a reduction in observed RQ due to non steady-state conditions. By calculating the increase in the whole body ketone pool associated with exercise, one can account for approximately half of the decrement of CO2 production that would be necessary to explain the decrease in RQ below 0.69. Other factors that could contribute to this low RQ include losses of ketones in the urine and loss of acetone in the breath after decarboxylation of acetoacetate in the blood, as well as CO2 utilized in urea genesis".

However, the non steady state accumulation of ketones does not apply to the at-rest readings from the Eskimo in Heinbecker's study.

I'd like to have a guess at the more "steady state" condition.

Full oxidation of a "typical" protein such as albumin produces a value of around 0.8 for the RQ. So I've invented a single mythical amino acid which gets close to the average RQ of protein. It looks like this:

NH2 - CH - COOH
            CH2
            CH3

Two of these amino acids oxidise using nine molecules of oxygen to give one molecule of urea and seven molecules of CO2, giving an RQ of 0.78. If this was replaced with a dietary equivalent the RQ would stay around 0.8 and the RQ of 0.69 from saturated fat would be increased somewhat. If the oxidised amino acid was not replaced the RQ change would be exactly the same but muscle wastage would occur.

What if, as a ketosis induced protein sparing effect, certain non-essential amino acids, were synthesised from urea plus carbon from fats plus a little oxygen. I'm not suggesting for a moment that this is exactly what happens, but the equation must balance whatever pathways might be used.

I've spent quite some time with scraps of paper working out how much oxygen has to be added to a couple of -CH2-CH2- moieties from saturated fat, along with a urea molecule, to reassemble the above pair of amino acids. "Mythical" protein turnover...

It takes 3O2 and liberates one CO2.

Combining this with the 9 O2 and 7 CO2 from oxidation, the whole repalcement of this amino acid would use:

12 O2 and generate 8 CO2 giving and an RQ of 0.67.

So the replacement of one "typical" amino acid using part of the acyl-chain of a saturated fatty acid generates an RQ of 0.67.

That's getting us somewhere below 0.69, what then matters is how general this effect might be which obviously depends on protein turnover, protein intake, protein quality and anything else anyone can think of. The value is pushed further down by the loss of oxygen rich ketone molecules through the breath and urine.

I'm very aware that minor errors in logic or arithmetic might alter the above calculations.

What an RQ well below 0.69 speaks very clearly against is gross muscle catabolism (which pushes the RQ upwards towards 0.80). Clearly, muscle loss does occur but I can see no reason why muscle loss should be an essential pre requisite for fat oxidation during fasting. The ability to minimise muscle loss under fasting (or ketogenic eating) might just provide some advantage on an evolutionary basis.

Marking out amino acid oxidation (ie loss of protein) as an essential pre requisite to fatty acid oxidation (in the absence of carbohydrate) suggests a rather odd view of reality. If it were correct it should show as elevated RQ's above 0.69 in proportion to the amount of amino acid oxidation which might be going on.

Which is not the case.

Peter

Edit for raphi: the arithmetic:










Tuesday, August 09, 2016

Glucose from fatty acids: RQ of 0.454


This is a section from Table V of Heinbecker's 1928 paper:

Studies on the Metabolism of Eskimos








This tells us certain very, very interesting things.  The subject is a young Eskimo woman. Column 6 gives her RQ and, by day 3.5 of fasting, it is 0.454. Which is clearly impossible. Maybe. It took me a few minutes to realise that the result is probably correct, certainly within the limits of measuring RQ in 1928 in a tent in the Arctic. Let's assume it's ballpark correct.

I've been through this too many times. An RQ below 0.69 suggests the generation of oxygen rich molecules from fatty acids. An RQ of 0.454 suggests a huge amount of (probable) gluconeogenesis from fat is going on.

The other thing which becomes obvious from simple logic is that any oxygen rich molecule generated from fat must NOT be oxidised for it to drop the RQ. If you oxidise stearic acid to CO2 and water you will get the same amount of CO2 per unit O2 consumed whether that process goes via acetyl-CoA (as it usually does) or via ketones, oxaloacetate or glucose.

The girl, Martha, was breast feeding a baby throughout the study:

"Subject II. Nursing female".

She has eaten nothing for 3.5 days, she is excreting both glucose and galactose in her milk. She has used up her glycogen stores. Where is the glucose/galactose for the milk coming from?

The RQ is 0.454, the milk sugars are coming from fat.

Sooooooo. Question:

How much gluconeogenesis is possible from fatty acids?

Answer:

A lot.

How much is a lot? It's not really practical to put a number to this, but enough to drop the RQ to 0.454 or, equally, enough to make a continuous supply of human breast milk. Both seem to be reasonable answers.

Unless you have an agenda.

Peter

Sunday, August 07, 2016

Just a heads-up

Protons (44) has been markedly updated. Just a heads up in case the update doesn't come through as a "New Post" to anyone who follows. Perhaps best not leave comments here on this little notification post.

Peter

Thursday, August 04, 2016

Protons (44) Does fatty acid oxidation really drive reverse electron transport and superoxide generation at complex I?

This post has been extended and adjusted quite considerably in the light of further information. The first five comments in the comments section are from pre update.

I suppose I should say now that I am particularly interested in data which trash the Protons hypothesis. I am so deeply biased in its favour that contradictory evidence has to be taken very seriously. Hence the initial post (preserved and embedded below) and the current extension of it based on another paper, also via Mike. It just goes to show how deeply selective people can be with the information which they pass on and how limited they are in coming forward with what they really think is happening. Personally, I'm interested in how stuff works. That's what I write about. Any agenda comes from the biases I have about how well the Protons hypothesis, largely self generated, fits most of the data.

Needless to say, other papers (Back in Protons 3) using mitochondrial preparations show they generate significant amounts of superoxide using palmitoyl carnitine. Anyway, here we go with the edited post:


The original post:

Well, should I develop any leisure time not taken up with the beach, crabbing, canoeing or any one of the hundreds of school holiday activities which are on-going, I have some serious reading to do!

From Mike Eades:

Mitochondrial fatty acid oxidation and oxidative stress: lack of reverse electron transfer-associated production of reactive oxygen species

The group seem pretty good and are supportive of succinate and mtG3Pdh driven RET, but not of ETFdh driven RET. You can imaging how much that gives me to think about! Needless to say, in view of the age of the paper, the group has interesting stuff published more recently which may have something to say about FFA oxidation and ROS generation.

Life is never as simple as you might like it to be!!!!

More to come, will take time.

Peter

End of original post.


It's worth adding this quote from the results to make things absolutely clear:


"The rate of ROS release from heart mitochondria oxidizing carnitine esters of long- and medium-chain fatty acids was much lower than that in the presence of succinate (Fig. 1A, B, C and D) and comparable to that with NAD-linked substrates, pyruvate or glutamate (not shown). An increase of acylcarnitine concentration from 0.5 mM up to 5 mM (examined with butyryl- and octanoylcarnitine) did not enhance ROS production (not shown)".

You can get ROS to be produced in this preparation, but only by using an ETC inhibitor. That's not physiological. Okay.



Now here is some more current (2013) thinking from Schönfeld and Reiser. This is the Schönfeld, as in the first author of the above (2010) paper. Here is what he says in this review:


Why does brain metabolism not favor burning of fatty acids to provide energy? - Reflections on disadvantages of the use of free fatty acids as fuel for brain


"This should be substantiated by the following quantitative analysis: during complete degradation of one glucose molecule, two molecules FADH2 and 10 molecules of NADH are formed, which corresponds to a FADH2/NADH ratio of 0.2. In contrast, b-oxidation of palmitic acid generates 15 molecules of FADH2 and 31 molecules of NADH, with an FADH2/NADH ratio of approx 0.5. Consequently, during b-oxidation there is competition of NADH and FADH2 electrons for oxidized ubiquinone as electron acceptor. This situation would most likely enhance oxidative stress in neurons for two reasons. Thus, slow NADH oxidation maintained the redox state of the electron carriers upstream of complex III in a highly reduced state, a situation similar to rotenone inhibition of complex I. Such situation enhances the superoxide generations by ETC. Moreover, at a high FADH2/NADH ratio, more FADH2 becomes oxidized by the electron transfer flavoprotein-ubiquinone oxidoreductase, a reaction known to be a potent source for superoxide generation".

Let's zoom in:

"Consequently, during b-oxidation there is competition of NADH and FADH2 electrons for oxidized ubiquinone as electron acceptor".

The whole quote and most especially the crucial snippet could have been lifted almost directly from the Protons thread. This is exactly the argument I made for the use of lactate rather than palmitate in neurons. This is simply one facet of the overall Protons concept, which is largely based on the FADH2/NADH ratio.

NB In the 2010 paper there was no difference in total ROS generated between feeding the mitochondria on pyruvate or palmitoyl canitine. Go figure!

Bear in mind that in his 2010 paper Schönfeld found very low generation of superoxide from any fatty acid source (using heart and liver mitochondria) and, although the group have some info since then from brown adipose tissue mitochondrial ROS, they don't appear to have generic data to support Schönfeld's (roughly correct) Protons-like hypothesis above. You can read their quote as well as I can. FADH2 via ETFdh is accepted as driving ROS generation via CoQ reduction. i.e. ROS are generated in proportion to FADH2 which is generated in proportion to the length and saturation of beta oxidised FFAs. They don't specify RET, the ROS may come from ETFdh directly, but I can live with that (should it turn out to be correct). It's the CoQ reduction and FADH2 input that speak to me.

They didn't find anything like this in their 2010 paper comparing ROS from butyric acid to octanoic acid to palmitic acid! All three substrates generated ROS comparable to pyruvate despite the FADH2/NADH ratio being very different.

My presumption is that Schönfeld considers his version of the Protons FADH2/NADH concept to be correct and I'd be willing to bet he even knows exactly why the 2010 model doesn't show this.

But he ain't sayin' nuffing. There's a lot of it about.

Summary: I don't think I'm about to discard my pet hypothesis quite yet!

Peter

Tuesday, August 02, 2016

Acetone to oxaloacetate

Some time back in May this year I posted on what I considered to be gluconeogenesis from acetoacetate via acetone (You don't have to read that post, the next link is far more important. OK, read the post then, I enjoyed writing it). It's a simple four or five step conversion from ketone to oxaloacetate, which can enter the Krebs Cycle allowing the regeneration of citric acid. This is my illustration:






















Notice the oxaloacetate (and the citrate off to the bottom right). Now, back in 2012 Chris Masterjohn posted on gluconeogenesis from acetoacetate via acetone and oxaloacetate (Follow the link and this time READ the post). This is his illustration:


















Perhaps not as pretty or as comprehensive as my pic but pretty well saying the same thing.

I think it is reasonable to say that both Dr Masterjohn and myself are both fully aware, on record, that acetone from acetoacetate is a substrate for the generation of oxaloacetate.

As Dr M says, fat burns in the flame of oxaloacetate. This does not have to come from glucose. It does not have to come from amino acids. He seems to have forgotten his own post from 2012. Fat provides oxaloacetate. Can anyone imagine that a period of food deprivation would not supply the necessary metabolites to utilise ketone bodies? Well duh.

I know this. He knows this. Now, try this video from this post, starting at about 3 minutes in. I gave up at around 8 minutes, so apologies if he returns to acetone as a source of oxaloacetate later on. It doesn't seem likely from the bits I listened to.

So, the big question is: What happened to Dr Masterjohn's knowledge about ketones and oxaloacetate between July 2012 and August 2016?

This is beyond me. I find it incomprehensible. In 2012 he knew... In 2016??????????

Peter

Thanks to Karl for the heads-up.

Sunday, July 31, 2016

A load of crap in Gartnavel

This came to me via Facebook from Wally Courie:

Poop Pill’s Surprise Failure Shows That the Microbiome is Still a Mystery

The original faecal transplant work was done in Glasgow and got written up here:

Faecal transplant for recurrent Clostridium difficile-associated diarrhoea: a UK case series.


Many years ago, probably around 2006 when the above paper was being written/published, I attended a presentation at a probiotics meeting in central London by a medic from Gartnavel General Hospital in Glasgow. Might have been by MacConnachie himself. He was quite deadpan, described their very simple procedure for obtaining, filtering and administering the faecal transplant by NG tube. You can read the abstract of the paper to get some idea of the success rate of the procedure. Hint; very high.

What really stuck in my memory was his comment was that, as they removed the NG tube, the patient cured. Many of these these folks hadn't left the house, often never left the loo, for years before referral. Their next bowel movement was going to be normal.

With a success rate as high as documented there has never been a double blind placebo controlled trial. I rather like that.

So I can imagine him watching the development (as I did) of the multiple probiotic capsule which was (or wasn't!) going to cure C. difficile gut-rot without all of the "ick-factor" of the trip to Gartnavel. Then quietly going in to work to cure some more people, assisted by a pooh sample.

Peter

Wednesday, July 06, 2016

Arteriosclerosis (6) and Subbotin

It seems a very long time ago that I wrote a series of posts on the mechanism of the development of the arterial tunica intima from a single layer of cells through to a thickened structure of similar or greater thickness than the muscularis layer. I've gone back and re-labelled them under Arteriosclerosis (1) through to (5).

Very briefly: Arteriosclerosis (1) covered the development of diffuse intima thickening (DIT) from birth through to about three years of age. DIT occurs over areas of disrupted elastin, is on the lumenal side of the elastin and is largely composted of glycosaminoglycans (GAG) and a few cells.

Arteriosclerosis (2) covered the role of platelets in supplying ILGF-1 which generates GAG at the site of platelet adhesion. That is where the platelets stick to damaged endothelium and strengthen an area at which the elastic tissue has been ruptured. It is a physiological process of tissue reinforcement.

Arteriosclerosis (3) covered the condition of von Willebrand's Disease, where platelets fail to adhere to damaged tissue. In this condition the intima stays thin and there is no DIT with age, assuming the vWD is not fatal at a young age. The elastin layer gets completely trashed as there no ability to generate reinforcement.

Arteriosclerosis (4) illustrated this process by showing the formation of surface microthombi to deliver platelets and generate GAG, it covered mucoplysaccaride disorders with massive intimal thickening when GAG cannot be degraded/remodelled and introduced the idea of intimal thickening in the arterial supply to the atrioventricular node as a precursor to sudden cardiac death.

Arteriosclerosis (5) is just the pictures to complete part (4).

To summarise: Arteriosclerosis begins as non pathological DIT which is a reinforcement system to maximise the correct development of the arterial tree to best withstand the loads to which it is subjected. It gives a tailor-made cardiovascular system for a given individual.

That's normal.

Subbotin has recently published a review article documenting his view that pathological arteriosclerosis, with lipid infiltration, commences when the tunica intima becomes too thickened to receive adequate oxygenation without developing a vascular supply of its own. It is normally avascular, getting oxygen and nutrients by diffusion from either the arterial lumen or the arterioles embedded in the muscularis layer. Once the thickened tunica intima develops a vascular supply all hell breaks loose with apoB labelled lipids attaching themselves to the GAG on the mistaken basis that this the extra cellular matrix of damaged tissue.

This is a highly plausible scenario and fits with the microscopy much better than any fairytale about apoB lipoproteins sneaking through the arterial endothelium and burrowing deep, deep in to the intima before depositing themselves on the border with the muscularis layer, leaving the surface layer completely unaffected. Which is what the histology shows. Shrug. Never forget: Only purple spotted cholesterol causes CVD, anything else is bollocks.

There are several things which spring to mind about DIT, insulin and oxygenation. If, as I think likely, a general thickening occurs under the effect of chronic hyperinsulinaemia acting on the ILGF-1 receptors which are on the lookout for platelets, we have a reason why insulin, not glucose, drives CVD. Anything which hastens thickening of the tunica intima hastens CVD. Insulin.

Second is that not only do VLC diets drop insulin, but they also reduce tissue oxygen extraction while maintaining normal ATP production. If Subbotin is correct that localised tissue hypoxia is what converts a benign reinforcement area to a fragile pool of lipid, then acetoacetate and beta-hydroxybutyrate (and stearic acid) are your first obvious port of call. AcAc/BHB looks like an excellent tool to blunt the impact of hypoxia on the need for blood vessel invasion of a given tissue.

Third: What happens if you keep insulin high with a diet of complete crap while supplementing ketone esters to reduce tissue oxygen needs? My guess, and it's only a guess, is that intimal thickening will progress apace but will only develop vascularisation at a later stage of the process, when the slack provided by the exogenous ketones has been taken up. Equally, the worry might be that you take ketone esters for a year, living on crap, and then run out of funds while you have thickened avascular DIT which was quite happy under ketones but is then going to need more oxygen per unit ATP under glucose oxidation. At least by going the ketogenic diet route you should have had basal insulin to minimise DIT progression in combination with your AcAc/BHB, up until the time you feel that you'd rather die than continue to live without eating pizza.......

Peter