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

Monday, May 23, 2016

The degradation of mitochondrial research


Does everyone remember this?



















Most especially this bit:























Control is using pyruvate at 5 mmol/l and the ATP synthesis shut down is generated using palmitoyl carnitine at 10 micromol/l. Got that?

Try this one too, plots of pyruvate against palmitoyl carnitine:


















And how about this one, increasing doses of palmitoyl carnitine alone, showing self destruction of ATP synthesis with rising doses of palmitoyl carnitine:
















Is everyone convinced that allowing anything over, say 5 micromolar palmitoyl carnitine anywhere near a mitochondrion is going to crash ATP synthesis? Lots of experiments, lots of evidence.

Now, this is pretty basic science. What happens when a lab takes their basic science and goes clinical? This is the same lab:

Chronic Reduction of Plasma Free Fatty Acid Improves Mitochondrial Function and Whole-Body Insulin Sensitivity in Obese and Type 2 Diabetic Individuals

Here is the key statement, from the methods:

"Mitochondrial ATP synthesis rate was measured ex vivo with a chemiluminescence technique as previously described (16)".

Reference 16 is the one from which all of the above graphs have been taken. The isolation, washing and feeding of the mitochondria have not been changed. Yet now, in a clinical study showing the wonders of free fatty acid reduction, we get this:












We can ignore the acipimox groups and use the pre treatment open columns. Look at ATP yield from Pyr, this is pyruvate 2.5 mmol/l. Now look at PMC 0.5 and PMC 1. Here we have palmitoyl carnitine being added at either 0.5 mmol/l, ie 500 micromol/l or even 1000 micromol/l, giving comparable rates of ATP synthesis to pyruvate 2.5 mmol/l.  That 1000 micromol/l is one hundred times the concentration used in their first paper to shut down electron flow and collapse delta psi.

Where did the inhibition of electron transfer from reduced CoQ to complex III by palmitoyl carnitine go to? What changed?

They went from basic science to a clinical application. Was the basic science correct? Is the clinical paper correct? An interesting set of changes. Makes me thing of the degradation we see so commonly in research, from something which looks sound to something which looks incomprehensible.

Peter

Will fasting destroy your mitochondria? (No).

I started with this paper:

Prolonged Fasting Identifies Skeletal Muscle Mitochondrial Dysfunction as Consequence Rather Than Cause of Human Insulin Resistance

"Prolonged" here means 60 hours. I had to look up "dysfunction" in a dictionary as I feel it carries negative connotations. It does.

"abnormality or impairment in the operation of a specified bodily organ or system"

Not eating for a couple of days renders your mitochondrial either abnormal or impaired. That's a big assertion to put in to a title.

So I don't like this group. They are fully aware that fasting requires insulin resistance and that this insulin resistance is physiological. Under such conditions there are, undoubtedly, changes in mitochondrial function. What sort of a label you apply to the changes says rather more about the mindset of the authors than it does about the mitochondria. These folks are deep lipophobes.

There are two respiration states under which the mitochondria from fed people outperform those from the same people after a 60 hour fast:

State 3 respiration is when you supply so much ADP that the ATP synthase complex can run at its absolute maximum rate. It's a measure of the ability of the ETC to pass a very high electron flow while using the membrane voltage. Oxygen consumption (a surrogate for electron flow) is reduced in preparations from fasted people.

The second condition where mitochondrial oxygen consumption is reduced by fasting is under pharmacological uncoupling.  FCCP, just such an uncoupler, allows the absolute maximum flow of electrons down the ETC, completely unfettered by any need to drive ATP synthase at all and probably with no delta psi to work against when pumping protons. Under fasting conditions there is measurably less oxygen consumed under FCCP than when tissue is isolated from fed subjects.

Out of interest they also treated a set of mitochondria with oligomycin (which blocks ATP synthase) and checked for physiological uncoupling (state 4o respiration). There is no difference in oxygen consumption under either nutritional state.

How badly are the humans crippled by this level of mitochondrial "dysfunction" under fasting?

"Twenty-four hour energy expenditure during the last 24 h of the 60 h intervention was slightly but significantly reduced upon prolonged fasting (10.88+/- 0.33 vs. 10.30+/- 0.30 MJ/day, in fed versus fasted, respectively, P less than 0.02). The difference was mainly caused by a reduction in diet-induced thermogenesis, and not caused by a decrease in resting metabolic rate (data not shown)".

They are normal.

These subjects went from a free fatty acid concentration of just over 200 micromol/l after an overnight fast to just under 2000 micromol/l (no typo) after a 60 hour fast. Fatty acid oxidation is supposed to be a supply controlled system. With a ten fold rise in a metabolic substrate perhaps we might expect a ten fold rise in ATP production driven by an attempted ten fold rise in membrane potential? Reductio ad absurdum. Do you think we might need some sort of control system to be applied? Like a brake?

The brake looks interesting to me. The next layer to dig in to is this group:

Deleterious action of FA metabolites on ATP synthesis: possible link between lipotoxicity, mitochondrial dysfunction, and insulin resistance.

OK, you've read the title. Are they lipophobes? No Brownie points there I'm afraid!

Now, you have to be careful. The 60h starvation folks used permeablised muscle tissue. What every had happened to the mitochondria under starvation is (pretty much) still in place and didn't budge. This next group of lipophobes washed and isolated their mitochondria. We are now dealing with squeaky clean mitochondria with all of the cytoplasm washed off. And all of the fatty acids and their derivatives, along with any cytoplasmic enzymes to interconvert them, also washed off.

In the following diagram they added palmitoyl carnitine to mitochondria being fed on pyruvate and ATP production went through the floor. They could do something similar using palmitoyl-CoA and oleoyl-CoA, even without added carnitine. They could also simply wash those mitochondria to restore pre-lipotoxicity ATP synthesis rates:


















Treating mitochondria with palmitoyl carnitine reduces ATP synthesis by 90%. Washing those mitochondria restores normal function. It looks very much like the site of action of the fatty acid derivatives is a) on the outside of the mitochondria and b) a non-covalently bound effect.

This is what the authors say:

"We therefore postulate that a rise in intramyocellar fatty acyl-CoA interferes with mitochondrial ATP synthesis by inhibiting the electron transport chain and decreasing the inner mitochondrial membrane potential. As a result, fatty acyl-CoA oxidation is reduced, leading to a further rise in intracellular FACoA concentration and exacerbation of the mitochondrial dysfunction. This sequence of events leads to a self-perpetuating negative feedback cycle whereby a small rise in intramyocellar FACoA impairs mitochondrial function and further increases the intramyocellar fatty acyl-CoA concentration".

NB I think they mean a positive feedback loop, to be producing runaway mitochondrial dysfunction and an ATP supply crisis. You know, skip a meal and "phut" your mitochondria snuff out.

My impression is slightly different. I look at the lethal effects of my favourite fatty acid's carnitine derivative and ask, scratching my head: How do people function with FFAs of 2000 micromol/l if 5 micromol/l of palmitoyl carnitine is going to kill them?

For a reality check we can just look at those 60 hour fasted people in the respiratory chamber. Are they wanting to die or wanting to go out for a steak?

Mmmmmmmm.... Steak......


The mitochondrial-washers are suspicious (but have no evidence) that fatty acid derivatives are dropping in to the binding pocket on complex III where reduced CoQ should be docking and so blocking the ability for electrons to pass from reduced CoQ to complex III. If this is true, and yes the docking site really is very close to the outer surface of the inner mitochondrial membrane, we have a system where fatty acid derivatives can limit the flow of electrons down the ETC to the levels needed for perfect ATP production while maintaining a physiological insulin resistant state to keep the brain supplied with glucose.

We know this system works because people can go without eating for 60 hours without looking anything other than slightly slimmer than they did when they walked in to the respiratory chamber.

We also know that people can go for six weeks on a protein supplemented fast and actually improve their exercise ability, based purely on fatty acid catabolism.

Of course, we must ask what happens to the redox state of the CoQ couple if you point blank refuse to allow reduced CoQ to hand on its electrons on to complex III. All of the CoQ will end up fully reduced. Reverse electron transport through Complex I is inevitable, given a decent membrane voltage. That means targeted superoxide production and the Good Things that flow from this. That means physiological insulin resistance. I view this as one layer up in control systems from the NADH:FADH2 ratio within the mitochondria. Note that the this particular effect of fatty acid derivatives does NOT require active oxidation of those derivatives. There are a number of papers out there where fatty acids induce insulin resistance even when beta oxidation is pharmacologically blocked. The data in this paper are the explanation, they make complete sense (more than you can say for the authors).

Peter

Sunday, May 15, 2016

Fruit Flies and NDI1

The Protons thread originated when I asked myself: What is the difference between fat oxidation and glucose oxidation? This rapidly led to the redox state of the CoQ couple as a driver for reverse electron transport (RET) through complex I, superoxide generation and the benefits of ROS signalling. It was a period of deep insight, especially about electron transporting flavoprotein and its dehydrogenase. Core to the Protons thread is the redox state of the CoQ couple and the generation of reverse electron transport.

In amongst the recent flurry of blogging about calorimetry and associated physiology Mike Eades forwarded this text to me:

Mitochondrial ROS Produced via Reverse Electron Transport Extend Animal Lifespan

Mostly on the basis of its title, I think. It's a very complex paper, drosophila based, couched in terminology which is probably completely routine if you work with flies but as clear as mud if you don't. I think we can reduce the paper to its title, discussion headings and three diagrams, one of which I'm going to butcher, the way you do. It's free full text if anyone wants to bend their brain.

Those titles:

ROS Production Increases with Age and Correlates with a Decrease in Complex I-Linked Respiration

Over-Reduction of the CoQ Pool Increases ROS Production and Extends Lifespan

CoQ-Mediated ROS Signalling Can Rescue Pathology Induced by Oxidative Stress

Loss of CoQ-Mediated ROS Signalling Accelerates Ageing

I mean, to a superoxidophile, what more could you ask? Just so long as the superoxide is RET derived from complex I...

OK, now the images. This one is core. It's taken from the Graphical Abstract and looks to be hand drawn in pencil. Note the NDI1 super-fly logo on the chest. This research group is crazy. I like that:























We've met NDI1 before. Its a small NADH oxidase from yeasts which reacts the reducing equivalents from NADH with oxygen to give water, reducing the CoQ couple in the process. It drives, given an adequate delta psi, RET through complex I. Inserting the gene for this protein in to a fly does this to life span:













Obviously, the line off to the right represents the NDI1 positive flies. NDI1>daGAL4, as "fly people" might say. They then went on to use an almost infinite supply of other tricks, in other flies, to show that it really is RET through complex I via CoQ reduction that extends life via site specific ROS generation. I won't slog through the arguments, you now know where the paper is!

And here is the lamb to the slaughter picture, presented as part of Figure 2:















There's NDI1 in blue, using NADH to reduce the CoQ couple and generate ROS. The main thing I dislike about this image is that the ROS seem to be popping out of the CoQ couple. They actually come from RET through complex I, so let's change it so it really looks that way:












That's better. Now I, personally, don't have and don't really want to have an NDI1 sitting on the matrix side of my inner mitochondrial membrane. Perhaps there is some similar enzyme available? Ah yes, let's mentally substitute ETFdh:












And of course, if we want to drive this process, we need FADH2, transported to ETFdh via electron transporting flavoprotein, generated by the first step of beta oxidation of saturated fats. Any double bonds mean this step gets skipped and all we supply is NADH. If we want FADH2 it's palmitate and stearate all the way:
















I have absolutely no idea whether using FADH2 from beta oxidation will do, in mammals, what NDI1 does in fruit flies. But I like the paper, and I like that idea.

And the fly doodle of course!

Peter

The addendum; because this post is not totally irrelevant to my recent blogging ideas:

Veech doesn't like fatty acid oxidation. He has little time for acetoacetate but loves beta hydroxybutyrate because it, specifically, reduces the NAD+/NADH couple while oxidising the CoQ couple, increasing the redox span. ie BHB oxidises the CoQ couple.

Kwasniewski is very pro saturated fat and rather anti ketosis. He wants FADH2 driven metabolism which enters the ETC by reducing the CoQ couple. He has a disturbing habit of being correct without providing any science.

The flies suggest going with Kwasniewski rather than buying bulk BHB by the tanker-load when it eventually hits the market as an affordable ketone ester. But they're only flies...

Friday, May 13, 2016

Uncoupling in a can?

In response the suggestion from E-S, at top of comments to the last post, that caloric output should be measured using a calorimeter:

Direct calorimetry identifies deficiencies in respirometry for the determination of resting metabolic rate in C57Bl/6 and FVB mice

"In a carefully controlled study, Walsberg and Hoffman (10) examined the accuracy of respirometry in multiple species, including the kangaroo rat (Dipodomys merriama Mearns), dove (Columbina inca Lesson), and quail (Coturnix communis Linnaeus), by comparing simultaneous outputs from animals studied with both direct and respirometry methods. Those authors concluded that when disparate species were studied under various conditions that estimations of heat production by RER-based respirometry calculations led to errors averaging 21% for kangaroo rats, 15% for doves, and 17% for quail".

"Here, we demonstrate that the rate of inaccuracy of respirometry [for the CL57Bl/6 mouse] is roughly 10–12% and posit that this magnitude of inaccuracy, given the target range, is unacceptably large. We conclude that the challenges faced by the obesity therapeutics research community in identifying or validating novel therapeutic targets in mice (and likely other species as well) may be compounded by the inappropriate yet almost universal and sole reliance upon respirometry".


RER underestimates calorie output, compared to a calorimeter. But you can't buy a calorimeter off the shelf.

So folks use RER and that table. "Everyone does it". Yeah. Well.

Peter

Wednesday, May 11, 2016

Uncoupling and weight loss

I've spent the last three posts making the point that fatty acid oxidation (supplemented by ketosis) increases the amount of ATP (and energy yield of ATP hydrolysis) available per unit oxygen consumed. This is particularly clear under the conditions of extended, intensely hypocaloric eating described by Phinney, where exercise can be sustained for longer, at a lower VO2, than on a mixed diet.

Now, oxygen consumption is a surrogate for caloric output. How many calories you "spend" per unit oxygen consumption is a complex calculation and depends on your fat to carb ratio.

But we don't run on calories. We run on ATP (mostly), or rather we run on the energy yielded from ATP hydrolysis.

To make that absolutely clear: We know, from Phinney, that under pure fat oxidation, we can generate enough ATP energy (physical treadmill load) to sustain moderate exercise by using less calories (ie lower VO2) on fat oxidation than on mixed diet oxidation. The increase in ability shows as a 25% drop in VO2, ie a 25% drop in calories needed to get enough ATP energy to move at 70% VO2 max.

That, to me, is pure survival adaptation. It's elegant, neat, cool etc.

It's not providing a metabolic advantage for weight loss.



Now, once again, I must wander off in to rodent studies.

If you take a rat or a mouse and feed it a genuine ketogenic diet you get some interesting effects. Let's look at this small study in rats. Here's heat output. Red is chow fed, grey is ketogenic:








Day or night, energy output is lower for the ketogenic rats compared to the chow fed rats. Phinney got a 25% drop in VO2 on his treadmill, the rats have calorie output down by an average of 11%, at a similar RQ. Running on fat (+/-ketones) requires less calories to generate adequate ATP levels.

Note, these are not real heat outputs in the rats. No one measured heat flux in any way. They're calculated from the VO2. They're done using the software built in to a CLAMS device around well accepted values of calories used per litre of oxygen consumed. This drop in calculated heat output, in itself, is not a surprise in view of Phinney's work.

What is surprising is that VO2 actually increased to generate this reduced heat output:









The rats should be using less oxygen per minute to produce their whole-body ATP energy requirement running on fat, according to Phinney. And me. And the chart. They're not, they're using more, in absolute terms.

The conclusion here is that the VO2 has gone the wrong way. So we have to ask: What is the difference between a fasting, exercising human on an RQ of 0.66 and a ketogenic rat slumming around its cage with a very similar RQ of 0.7?


The rats are uncoupled. They pump protons through complexes I, III and IV but a significant number of those protons drop straight back in to the mitochondria through open uncoupling proteins. Calories and oxygen are used (at the same RQ as any other more productive oxidation) but no ATP is produced from any protons which do not use ATP synthase. VO2 moves in two directions. It goes down (and so do calories used) due to switch from glucose oxidation to fat oxidation. It goes up due to uncoupling. The overall effect, up or down, on VO2 depends on the relative effects of RQ change, uncoupling, gluconeogenesis, NEAT and actual exercise.

Phinney's treadmill walkers had high FFAs and high ketones but absolutely no suggestion of any sort of uncoupling. Why?

To get any further we have to go to the Protons thread back in 2014.

Uncoupling proteins are kept closed by cytoplasmic ATP. And there is always enough cytoplasmic ATP in a functional cell to keep UCPs closed. There is one particular way (of several) to open them. The inhibition from cytoplasmic ATP can be overcome by an excess of mitochondrial ATP. Mitochondrial ATP, obviously, enters the UCP from the opposite end to cytoplasmic ATP and gets in the way of the latter's binding. Mitochondrial ATP cannot reach far enough into the UCP to induce the closed conformation itself, so the pore opens. The blog post has nice images and a more thorough description. Here's my fave picture:























How do we keep mitochondrial ATP levels low?

Phinney had six week starved humans on a treadmill showing every probability of low mitochondrial ATP and UCPs closed tighter than the proverbial monkey's @rsehole.

On the opposite front we have rats in a cage whose biggest effort is to move over to the hopper of ketogenic pellets and have a munch. These animals uncouple like mad while eating to satiety. They also either maintain low fat reserves or lose fat reserves if previously made obese from fat/sucrose feeding. We've all read this mouse study even if today's rat epic is very inaccessible (thanks Mike).


It seems to me that it is possible to maximise the efficiency of energy usage to ensure survival under near starvation conditions. However fat based your metabolism, you are not going to uncouple your oxidative metabolism unless you have adequate ATP within the mitochondrial matrix.

It's very clear that an ad libitum ketogenic diet allows uncoupling and metabolic inefficiency down to a lean bodyweight, certainly in rodents. This is not arguable. Here's the graph. No mouse was forcibly calorie restricted:



Days 1-4 after switch to ketosis they ate less, by day eight after the switch to ketogenic eating they were eating more calories (ns) than other groups but staying weight stable.

The question to me is: By how much do you have to deliberately restrict the calories of a ketogenic diet fed human to eliminate the uncoupling effect? Or, more simply, turn the question round: How do you get a human to lose weight most effectively on a ketogenic diet? This is easier to answer.

As Amber O'Hearn suggests:

Eat meat.
Not too little.
Mostly fat.

Perhaps someone should tell Dr Hall this. Better still, make it his epitaph as science progresses.

Peter

Monday, May 02, 2016

On Stephen Phinney and an RQ of 0.62

Now, oxidising long chain saturated fat gives you an RQ of 0.69. Lower than this needs a supplementary process of some sort. In the last post I had Table II from Stephen Phinney's 1980 paper. There are RQs below 0.69 all over the place and even the mean RQ of the 6 week fasting exercise test was 0.66,  with some individuals down at 0.62.
















So how can we manipulate RQ values?

This is a graph taken from that nice paper on ketogenic diets for rats. The black line is the RQ of the chow fed rats. They are on 17% or so calories from fat, 64% of calories from starch and the rest is protein. Grey zones are night, white zones are daytime. Ratties are nocturnal, they eat their high carbohydrate diet at night. While they are eating they run their metabolism on glucose. This should give an RQ of 1.0 but we can see the RQ is greater than 1.0 during the times at which the rats are feeding:











We've seen this before during an OGTT in massively weight reduced people. Show them some glucose and they will immediately convert it to lipid and store it. After a mere 75g of glucose during an OGTT, these post obese ladies will develop an RQ over 1.0, see the top dashed line:











This is de novo lipogenesis, either routine in the rats on a 64% carbohydrate or pathological in the post obese ladies. Glucose arrives as an oxygen rich molecule. During the reorganisation to a very oxygen poor molecule oxygen is provided without it needing to be taken up through the lungs. Smaller oxygen flux per unit CO2 produced gives an RQ greater than 1.0.

So it's pretty easy to get a RQ above 1.0. How easy is it to get an RQ below 0.69?

As we all know, acetoacetate is unstable, spontaneously decarboxylating to acetone and CO2. On its own this isn't fast enough to be useful so we have acetoacetate decarboxylase to speed the process up. You find it in the liver and in the brain, mostly. The sorts of places where glucose might be useful.

Apart from being exhaled, what is the fate of acetone in the body? I can't imagine that we are deliberately forming the stuff enzymatically just to breathe it out... Well, here's a pathway I cribbed earlier, can't remember from which paper but one on basic acetone metabolism:























Soooo theoretically ketone bodies, via acetone and oxaloacetate , are glucose precursors. If you radio label acetone with (14)C, where does it end up?

"Radioactivity from (14)C acetone was not detected in plasma free fatty acids, acetoacetate, beta-hydroxybutyrate, or other anionic compounds, but was present in plasma glucose, lipids, and proteins".

Ketones to glucose. How much?

“On the basis of our specific activity data, we have calculated that 4-11% of plasma glucose production could theoretically be derived from acetone”.

The 11% was calculated for 21 day starved humans.

The most logical explanation for an RQ of 0.62 is that the person is performing a significant conversion of fat to glucose. This is completely plausible via acetoacetate, acetone and oxaloacetate. The exact steps are unimportant. What matters is that there will be an increased consumption of oxygen per unit CO2 produced. The RQ is just a ratio so increasing oxygen use will make it drop, possibly below that 0.69 of saturated fat oxidation.

Summary: We already know that total O2 consumption must and did drop on fat adaptation. We know from simple arithmetic that CO2 production drops even more that O2 usage when fat (vs glucose) is oxidised, to give us that normal RQ of 0.69.

If there is a further usage of O2 in the process of converting ketones derived from fat in to glucose, this would explain an RQ of 0.62.

Despite this "waste" of oxygen you still use less O2 per ATP from fat oxidation, even if doing some gluconeogenesis. We know this from the absolute VO2 measurements combined with the RQ values in Phinney's Table II and my back of envelope calculations.

I sit in awe of fat oxidation. We carry fat as long term energy storage for use in times of need. Under those conditions of privation this long term energy store allows very efficient ATP generation per unit oxygen, at the same time as reducing CO2 production, at the same time as generating a significant amount of glucose. Fatty acids and beta oxidation, with ketones thrown in, are just awesome.

I'm also hugely impressed by how far ahead of its time Stephen Phinney's paper was and how well it still stacks up against modern papers.

Peter

Friday, April 29, 2016

Endurance and oxygen flux during fatty acid oxidation

I was browsing Pubmed looking for other things when I came across this ancient publication by Stephen Phinney, from back in 1980. Those were the days. That was the Summer I contracted mycoplasmal arthritis as a final year vet student seeing practice in North Devon. Lots of NSAIDs then six weeks on tetracyclines once the serology came back positive. Yum. Anyway:

Capacity for Moderate Exercise in Obese Subjects after Adaptation to a Hypocaloric, Ketogenic Diet

Free full text, nice read. Replicable, if you have the equipment.

This is table II. For the first endurance test the treadmill was set at 3 mph with a personalised incline. The same test was then repeated after one and six weeks of a protein supplemented fast. For the six week treadmill run the settings were identical to the first sessions but the subjects wore backpacks containing weights equivalent to the bodyweight that they had lost during the 6 weeks of their fast. There was no exercise training in the interim.

















The column to look at is the one in the middle labelled VO2 and expressed as ml/min. On a mixed diet the treadmill settings required a flux of oxygen of 1875ml/min to maintain this significant effort. After 6 weeks on a protein supplemented fast, wearing weights in a backpack to offset the bodyweight loss, with the same treadmill settings, subjects required 1497 ml/min of oxygen. That's a big drop.

Some of this is from the magic of ketones, BHB increasing the free energy of hydrolysis of ATP. A large chunk of this component should have been in place by the one week treadmill test as everyone was well in to ketosis at this time. There was some drop in VO2 needed by this time point, but nowhere like the drop by six weeks.

We can tell by the RQ values that at baseline (mean RQ 0.76) the subjects were oxidising a mixture of fat and carbohydrate (and some protein I guess). At one week there was more fat oxidation occurring (RQ 0.72). By six weeks we have the rather strange RQ of 0.66, lower than is produced even by the oxidation of pure saturated fat.

Oxidation of a fully saturated fat produces an RQ of 0.69. Hmmmmm...

I worked out that oxidising fat, on a simple mathematical basis, should give you a 5% improvement in ATP production per unit oxygen consumed compared with glucose. With Veech's increased Gibbs free energy of ATP hydrolysis while oxidising ketones we gain a combined 5% improvement in ATP supply alongside a decreased total ATP requirement. These are components of the decreased VO2 which didn't make it in to the discussion as they were either unknown (free energy of ATP hydrolysis) or hadn't been worked out (ATP per O2). Not a bad gain for six weeks of fasting. I can see, very clearly, that anyone with a chronic lung disease would be crazy to eat any way other than for a metabolism based on lipid oxidation with ketosis. It just makes sense.

I'll leave it now for this post and put up some ideas about the subnormal RQ figure as the next post.

Peter