Sunday, October 12, 2014

Where has the superoxide gone?

This is the first section of Fig 1 section C from the paper using dihydroethidium (DHE) to view in vivo superoxide production in control and diabetic kidneys, though not in the figure below.

























It's a very important figure as it shows, very convincingly, that sudden onset hyperglycaemia has zero effect, none whatsoever, on superoxide production in their model of normal, non diabetic kidney tissue, that's the second column, identical to the first.

I have a lot of time for the failure to generate superoxide in diabetic kidneys, especially with pyruvate dehydrogenase complex down regulation limiting input to mitochondria from the end stage of glycolysis. But I have a concept that acute hyperglycaemia in normal, non Crabtree affected, tissues SHOULD generate superoxide, it should come from the respiratory chain and it should more particularly come from complex I in the region of the FAD moiety, preferably via the FeS cluster N1-a.

Now, if I had an in vivo tool for viewing superoxide generation, how would I do this? Well, I would use it in vivo. I would set up an iv glucose infusion, or perhaps a large intragastric glucose bolus, inject the DHE, wait a while, then look for superoxide/DHE derivative with my lovely optical scanner.

To keep the scrutineers happy I might have repeated the findings ex vivo, using the technique of paramagnetic detection of a superoxide/spin trap derivative, but the core finding, that superoxide generation on acute hyperglycaemia does NOT occur has to be shown in vivo. We already know it DOES occur ex vivo in multiple models, and the authors cite the studies to show this.

So, if hyperglycaemia triggers superoxide generation ex vivo in assorted non Crabtree adapted cells, why doesn't it do so in this study?

I don't know. There is a piece of core information which the scrutineers failed (miserably) to demand to be included in the study methods.

Figure 1C was not obtained in vivo. Column Ctrl was from a tissue homogenate of health kidney from non diabetic mice fed with pyruvate, malonate and ADP, subsequently flooded with 25mmol of glucose to produce the +HG column. That is not so bad. It's a model and it's clearly able to get GrantAid quality results.

But is it real?

Let's look at the equipment used. This is what they say:

"These studies were carried out in a MiniScope MS200 Benchtop EPR Spectrometer (Magnettech), which is designed to allow tight control of pO2 and temperature".

Why do they need tight control of pO2? You can obtain utterly rigid control control of pO2 by exposing your preparation to room air. Correct pressure to 760mmHG and pO2 is fixed at 21% of this.

To me the question is: What was the pO2 which failed to generate any superoxide when a mush of cytosol and mitochondria was exposed to 25mmol of glucose?

Was it 159.6mmHg, i.e. room air? Was it 40-50mmHg as other groups suspect mitochondria run at? Or was it 22mmHg?

This might matter. I got the 22mmHg value from the previous paper by the same authors which gave 3% oxygen as the likely conditions for normal mitochondrial function. This was a non referenced, throw away comment:

"Because the physiologic concentration of oxygen in mammals in vivo is less than 3% in most organs, we carried out a series of studies to determine whether ethidium or 2-hydroxyethidium was the specific oxidation product of DHE in vivo (i.e., in the intact animal, not cell culture/tissue slice) using several different validated animal models of increased or decreased superoxide".

Why it matters to me so much is that if an electron is thrown out of complex I due to hyperglycaemia triggered reverse electron flow through complex I, would it generate superoxide if the pO2 had been set to below physiological limits? Or if the guesstimate of 3% oxygen is correct and there is no superoxide generated, is there no reverse flow occurring? Or does the reverse flow occur, the electron is ejected, but it drops on to the surrounding protein structure rather than oxygen to be used as a distant signal via superoxide/H2O2/insulin receptor?

Using the in vivo technique would have told us exactly what was happening, at a true but non measured tissue pO2. I'm worried that the in vivo technique showed the anticipated (by me) hyperglycaemic superoxide and an ex vivo technique had to be developed and adjusted to maintain the fund generating core finding of no extra superoxide.

There was no reply to a simple polite email query as to the pO2 used.

Peter




13 comments:

Tucker Goodrich said...

Yes, this is OT to this post, but I thought you'd find it amusing:

"...a full-scale mitochondrial uncoupling drug would be a nasty proposition in humans (see, for example, dinitrophenol). DNP will indeed make you lose weight, while at the same time you ravenously try to eat your daily supply of ATP, but this is done at a significant risk of sudden death."

"Diabetes Progress"
http://pipeline.corante.com/archives/2014/10/13/diabetes_progress.php

Of course there's an obvious solution to burning more fat, if they really find that too much fat in the body is a cause of diabetes... :)

karl said...

@petro Where you wrote:
"clearly able to get GrantAid quality results."

Always important to separate what is science rather than grant-seeking-behavior'

There was a
recent paper in Nature (so it might end up retracted) where it appears they are confused about the effect of stopping the source of glucose in the diet.


That three unrelated AS chemicals would have identical effects is the first flag. I have no doubt that there is a change in intestinal flora on diets differing in carb or sugar content. Should this paper have made it through peer review?

raphi said...

i sent them an email reiterating your request (without mentioning you) and I've yet to receive any acknowledgment whatsoever.

Guillermo Fernandez said...

My expectation was exactly the opposite, increased glucose should increase superoxide in both cases (control and DM) but with a rather decreased amount in the DM case. My feeling is that stopping superoxide production as result of low PO2 would imply really low oxygen concentration, well below physiological limits and maybe, if that was the condition set up for this study, this could be something related with what would happen during hypoxia (upregulating glucose utilization for ATP production?)
The other option I see is that cells were already at maximum superoxide production (starvation maybe?) and that the glucose insult wouldn’t affect the total production for the control ones, which would be compatible with the Antimicyn A treated results because of the altered ETC (higher and maximum superoxide production under a blocked Complex III). In that case the conclusion would be that the DM cells would have a lower maximum superoxide production during starvation (ok) but would work poorly at superoxide production under onset hyperglycemia (???)

Jack Kruse said...

I just did a bio hack Guillermo and present it at the Bullet conference in Pasadena. I used induced hypoxia and two popular paleo books and the results were rather startling. I was able to keep my )2 levels between 15-18% for 9 months. Peter is on the right track. Superoxide production is tied to the electric current size and the resultant magnetic force generated from the current. You might want to remember some basic physics. Diatomic is the only gas that is naturally paramagnetic because of its two unpaired electrons. Your mitochondria uses this effect in a big way. Peter is pulling the veil back on how a low superoxide level is a proxy for ECT current changes and the polarity of magnetic flux at the 5th cytochrome: The ATPase. I would also remind you that the the ATPase has a Fo rotating head that also acts to the action of both ECT forces the the associate magnetic flux. Good stuff Peter yet again.

Jack Kruse said...

One other point I'd like to make regarding superoxide is how it is made by sunlight on the skin. When UVB sunlight is missing cholesterol can not be sulfated in the skin and superoxide also drops like a rock in the skin. When you look at most biologic cell membranes in skin, they normally give no intrinsic EPR signal because they normally have no unpaired electrons. However, when the skin is affected by UVB and IR sunlight that changes because eNOS is used to make cholesterol sulfate from cholesterol in the basal layers of the skin. The nitric oxide is the key to making cholesterol sulfate paramagnetic so that it is draw to tissues with strong magnetic flux. This means it is drawn to tissues with a lot of mitochondria. Unpaired electrons do show up because of the quantum action of UV sunlight on the nitrogen atom. This is why eNOS is the co factor in cholesterol sulfate synthesis. It become freely mobile in the skin lipid layers to change the structure of cholesterol to the sulfated version. Cholesterol goes from un-sulfated to sulfated and it become paramagnetic. This is how cholesterol works in a quantum fashion and how it is delivered to tissues. When this is not working LDL cholesterol rises and sulfated cholesterol drops and so does superoxide levels. When superoxide drops magnetic flux in the mitochondria are also destroyed leaving cholesterol in the blood too long allowing it to become oxidized. Sunlight makes cholesterol more water soluble in blood to go where the mitochondria need it to be.

Jack Kruse said...

When you understand 3 D molecular dynamics, superoxide is a fundamental signaling molecule where transition metals and sulfur exist. This happens in the skin and the cytochromes. Superoxide is actually needed to oxidize sulfur to make sulfated versions of proteins. This is how things are coupled in redox biochemistry. Complex dance......but this scale is where mother nature works.

Jack Kruse said...

1.http://www.ncbi.nlm.nih.gov/pubmed/21128718 2.http://www.sciencedirect.com/science/article/pii/S0009308411003070
3.Dermatotoxicology
edited by Klaus Peter Wilhelm, Hongbo Zhai, Howard I. Maibac
4.http://pubs.acs.org/doi/abs/10.1021/ja01046a038
5.http://www.researchgate.net/publication/256188810_An_Electron_Paramagnetic_Resonance_Method_for_Measuring_the_Affinity_of_a_Spin-Labeled_Analog_of_Cholesterol_for_Phospholipids
6.http://www.sciencedirect.com/science/article/pii/0005273687903385

karl said...

@Jack Kruse

Hmm - I've been looking for papers on photo-chemicals formed in skin for some time - not a lot of research - (there was one quote: "11 chemicals formed in skin via sunlight" never found the research or source).

What started this was all the Vit D research - lots of correlative stuff, lots of possibilities yet no controlled studies showing the use of D3 to maintain +50 levels doing any good. The problem is correlation vs causation. Sick people don't go for walks much - have lower vit D - cause or effect. And then it occured to me - how do we know it is vit D - not the reduction of the 7-dehydrocholesterol? (7-dehydrocholesterol is photolyzed by ultraviolet light in a 6-electron conrotatory ring-opening electrocyclic reaction; the product is previtamin D3.). Or the production of some other photochemical. Are the vit-D levels just an indicator of how much sun exposure someone has?

I have been amazed that as the Vit-D3 interest has spiked - instead of basic research to determine causation, we have piles of 'grant seeking activity' that can't possibly answer the important questions. Anyway, the S/N('signal to noise' ratio) in the journals is so low that I now think that reduced funding might actually help. Very few of the papers illuminate - most are 'muddying the water'.

(BTW - I don't think LDL is the cause of CAD, it is only the when the mis-identification of oxLDL causes an inappropriate immune response that we get the formation of foam-cells. I'm thinking that it is also less a factor of genetics than one of pathogen exposure - priming sensitivity to oxLDL that appears like a dying bacteria to a macrophage. Reducing oxLDL (not LDL) then becomes prudent, and easy if one keeps postprandial BG below 110. )

Jack Kruse said...

Peter you said, "Now, if I had an in vivo tool for viewing superoxide generation, how would I do this? Well, I would use it in vivo. I would set up an iv glucose infusion, or perhaps a large intragastric glucose bolus, inject the DHE, wait a while, then look for superoxide/DHE derivative with my lovely optical scanner."

My answer: We have the ability to do this already and maybe you don't see it yet.

As a free radical, superoxide has a strong EPR signal, and it is possible to detect superoxide directly using the EPR method when its abundance is high enough. In a mitochondria this is nearly impossible to see in vivo because we have no way of measuring the quantum effect yet, but we know this is happening because of our observations. For practical purposes, this can be achieved only in vitro under non-physiological conditions, when we use a high pH which slows the spontaneous dismutation with the enzyme xanthine oxidase. Peter, stop and think about how molybdenum and xanthine oxidase work in a cell and then think about your proton series and how this ties to pH. In vivo, xanthine oxidase does the same thing with the protons that flow through the cytochrome pores. Xanthine oxidase makes massive amounts of superoxide. But it requires molybdenum to work. Molybdenum no longer works to generate xanthine oxidase when non native EMF is present because of how microwaves affect transition metals. This means the wrong frequencies of light in the form of heat are the result. This effect is measurable in diabetics. None of the can control their thermal regulation or sweating. The FE-S core needs to be below 14 A to tunnel electrons. When things lose energy they swell. This is apoptosis is signaled in mitochondria. When a diabetic loses energy in a mitochondria Moly no longer is an electron sink and superoxide levels tank. The reason is because Mo has a very reactive amount of D shell electrons that cause specific and sensitive micro-molecular motions it does not maintain its proper size and shape relationships in the proteins, like xanthine oxidase where it is found in as a co factor in vivo. We have the ability........and it points out where MetSyn comes from fundamentally.

Jack Kruse said...

You said, " less than 3% in most organs, we carried out a series of studies to determine whether ethidium or 2-hydroxyethidium was the specific oxidation product of DHE in vivo (i.e., in the intact animal, not cell culture/tissue slice) using several different validated animal models of increased or decreased superoxide".

"Why it matters to me so much is that if an electron is thrown out of complex I due to hyperglycaemia triggered reverse electron flow through complex I, would it generate superoxide if the pO2 had been set to below physiological limits? Or if the guesstimate of 3% oxygen is correct and there is no superoxide generated, is there no reverse flow occurring? Or does the reverse flow occur, the electron is ejected, but it drops on to the surrounding protein structure rather than oxygen to be used as a distant signal via superoxide/H2O2/insulin receptor?"

My answer: Quantum biology is interested in how electrons are transferred in biochemical reactions using free radicals as their intermediaries. These are the mitochondrial signaling molecules that have lone electrons in their outermost electron shell. What makes free radicals different than other chemicals is that they have their outermost electrons paired up in atomic orbitals. This is a critical point because when you consider the queer quantum property of electron spin. Paired electrons cancel each other’s spins out to zero in quantum math, because paired electrons spin in opposite directions. Free radicals do not have a paired twin to spin cancel them out. All known free radical chemicals Peter have lone electrons in their outer shells giving them a quantum spin other than zero. When this happens it gives all free radicals a "net spin" that gives them a unique magnetic footprint. This is why EPR picks them up in experiment. It also means that free radicals can be aligned within a magnetic field. It should be clear to you now why mitochondria use free radicals to signal. Mitochondria are where EEG and MEG signals come from. They are the magnet that entangles the two unpaired electrons in free radicals. If ECT flow slows Peter what happens to the resultant magnetic field or to the reduction of oxygen? It lowers. The mitochondrial signal is sent to tissue distally by what? other free radicals such has CO, NO, and H2S. Also having unpaired electrons. Those signals tell tissue different things. H2S is the one that deals with hypoxia critically. Modern biology knows Jack shit about H2S because they think it is toxic. It is not. It is the key to cold Thermogenesis and magnetism in mitochondria. This is why the brain and heart have massive MEG signals when you sample them. What does H2S do Peter? Hydrogen sulfide is a critical mediator of multiple physiological processes in mammalian systems. The pathways involved in the production, consumption, and mechanism of action of H2S appear to be sensitive to alterations in the cellular redox state and O2 tensions. These were the focus of my recent bio-hack. And this is the answer to what perplexes you.

Jack Kruse said...

Now Peter H2S forms via photochemical change within plasma. So if you release the wrong frequency or the wrong amount of IR light from your mitochondria you wont get the right amount of free radical signaling distally in tissues. (Photochemical formation of H2S to make a free radical. http://pubs.acs.org/doi/abs/10.1021/j100865a037)

Jack Kruse said...

http://www.bbc.com/future/story/20140505-secrets-behind-the-big-sleep