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.