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

26 comments:

Passthecream said...

Perhaps relevant, a computational model of mt ros, free text.

http://www.ncbi.nlm.nih.gov/pubmed/23972856

Peter said...

Thanks Pass. The problem is largely how you design your model, what you base it on. And the mitochondrial work gives very different results in the presence of downstream inhibitors cf free flow. And whether you use FFAs, carnitine derivatives etc. There is a lot to pick through. The model will be interesting to look at...

Peter

Passthecream said...

and I think this is the paper which, if you read them both at the same time I thought your head might explode. Now I have three to reconcile.

http://www.hindawi.com/journals/omcl/2014/457154/

oh boy, rotenone plus metformin plus hyperglycaemia all at once. I saw some 'jicama beans' at a market recently which are a Mexican crispy snack vegetable. I was appalled when I looked them up to find that the plant is a source of rotenone. Plants will kill you every time.

Peter said...

Ugh.

Tucker Goodrich said...

"...which may have something to say about FFA oxidation and ROS generation."

There are other sources of pathological ROS generation. I wonder if oxidation is even the major source? I tend to think not.

Rattus said...

Hey Peter,

What do you think about the differing ratios of MUFA to SFA in beef tallow and butter? Butter is close to 2.5:1 SFA to MUFA, whereas beef tallow is closer to 1.25:1. If thinking about things in terms of the "Fat Hunter" paper you cited me a while back, primitive man would have relied more heavily on animal fat than dairy fat, and the difference in the ratios seems substantial enough to exert some kind of effect on health in the longer term if you are sourcing your diet primarily from fat. Obviously there are pastoral societies that relied on dairy as well, but even they seem to have gotten a lot of their fat from the animal itself rather than the milk [mongols putting sheep tail fat in their milk tea].

Peter said...

Hi Rattus,

And much of the SFA in butter is shorter chain SFAs, so will be significantly more ketogenic than beef tallow, via the MCT route. My main problem with non dairy SFA based diets is that I tried to eat non dairy VLC and getting the fat content high enough was very difficult without protein overload. I decided dairy was an acceptable fudge... (not literally!).

Peter

raphi said...

This is great...and timely for me.

Sorry to deviate from the core of this thread, but it brings up a question I'm wrestling with.

In mitochondrially defective (aka cancer) cells, glutamine may enter TCA via enzymatic magic as alpha-KG, eventually replenishing the succinate pool thanks to oxidative and reductive metabolism (aka bidirectional metabolism).

Any thoughts as to what this the lactate-to-pyruvate and NAD+/NADH ratio would look like in such a (mis)behaving cell?
Shouldn't these ratios be different than seen in a typical Warburg Effect cancer cell gorging on straight glucose rather than amino acids for its ATP needs? Seems like the pseudo-respiration of the amino acid gorging one would have a smaller NAD+/NADH ratio than the glucose gorging one, no?

You may notice my confusion.

PS: this paper may be useful for those trying to grasp the details of your threads - nice illustrations "Sites of reactive oxygen species generation by mitochondria oxidizing different substrates" by Quinlan et al. 2013 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3757699&tool=pmcentrez&rendertype=abstract

Tucker Goodrich said...

@raphi:

Fascinating paper, thanks for posting. Those authors are entirely missing the primary source of oxidative stress in the mitochondria, however. It's not due to oxidation of normal fuel sources: while those do generate ROS, the mitochondria are entirely capable of handling that load: there are highly effective anti-oxidants for just that job.

The primary source of pathological oxidative damage is auto-oxidation of cardiolipin, also in the mitochondria, but not necessarily dependent on the fuel source (although as cardiolipin is located near complex I, it should be more susceptible to ROS there). Cardiolipin not only produces ROS indepedent of oxidation for energy production, but unstable lipin peroxidation products that are several orders of magnitude more damaging, and can leave the mitochondria.

Oh, and they're mutagenic, to your cancer point.

I read a paper recently (which, sadly, I can't find at the moment) that observed that since NAPDH is a strong anti-oxidant, the mitochondrial dysfunction typical of cancer may be an attempt by the cell to use NAPDH to fight excessive oxidative damage coming from the mitochondria. Interesting idea.

Peter said...

raphi, just got to look at the paper. Nice. I've looked at some of Brand's papers before and he does seem to have pretty good insight. They make nice reads. I always think I should go and read more recent work from authors I've cited 5-6 years ago and see where they are now. FeS N1-a, what do foks think about it and superoxide generation now?

Tucker, agree that non physiological ROS are high on the list re pathology. I keep meaning to go and work through your cardiolipin ideas but, until this stupidity from Chris Masterjohn, I keep getting lured down Na+ energetics in modern hyperthermophilic archaea, Ech and Rfn. Sometimes my brain's a pain!

Peter

raphi said...

Peter,

About FeS N1-a, this is 11 years old but they say:

"No consensus has yet been reached on the site of superoxide production in mitochondrial complex I. Proposals have included the flavin (11–13, 35), bound reduced nucleotide (14), FeS clusters N2 (15) and N1a (16), and a semiquinone radical (17, 18)."
and
" In complex I, the flavin radical is thermodynamically unstable (30), supporting redistribution, but it is not possible to identify a single FeS cluster to oxidize (or re-reduce) it. It is tempting to propose a specific role for cluster N1a, because it is close to the flavin yet has no obvious role in energy transduction (21). However, N1a is unlikely to be important during catalysis, when electrons are flowing from flavin to ubiquinone, and its potential is too low [−0.45 V (28)] for it to compete effectively with the flavin [−0.42 V (30)] for the extra electron."
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1472492/ (The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria)

Tucker,

You said "since NAPDH is a strong anti-oxidant, the mitochondrial dysfunction typical of cancer may be an attempt by the cell to use NAPDH to fight excessive oxidative damage coming from the mitochondria" ==> ha, that's an interesting angle...need to think about it some more. As to your paint about where mitochondrial oxidative damage comes from, there's (surprisingly?) still a lot that's not known about ROS origins. Seyfried certainly points to cardiolipin as a fragile entity worth looking after.

Tucker Goodrich said...

@Raphi: "As to your paint about where mitochondrial oxidative damage comes from, there's (surprisingly?) still a lot that's not known about ROS origins. Seyfried certainly points to cardiolipin as a fragile entity worth looking after."

The mechanisms of how lipid peroxidation produces superoxide and other mediators of oxidative damage is well-known, well-studied, and the evidence for it is, IMHO, overwhelming. The research has all been done, it's not needed to look after it...

"Currently, lipid peroxidation is considered as the main molecular mechanisms involved in the oxidative damage to cell structures and in the toxicity process that lead to cell death."

http://www.intechopen.com/books/lipid-peroxidation/lipid-peroxidation-chemical-mechanism-biological-implications-and-analytical-determination

The primary markers are all dependent on dietary intake, and can be reduced by altering dietary intake.

This mechanism explains rather a lot:

"Mutagenic effects of 4-hydroxynonenal triacetate, a chemically protected form of the lipid peroxidation product 4-hydroxynonenal, as assayed in L5178Y/Tk+/- mouse lymphoma cells."

"The results indicate that, in the presence of serum that approximates physiological conditions, 4-HNE generated intracellularly but not extracellularly is a strong mutagen via a clastogenic action at concentrations that may occur during oxidative stress."

http://www.ncbi.nlm.nih.gov/pubmed/15701709

HNE is produced in the mitochondria by the peroxidation of polyunsaturated-fat-altered cardiolipin by cytochrome-c, a process that also generates singlet oxygen.

Seyfried's been focused on the wrong mechanism, I think.

Tucker Goodrich said...

"... I keep getting lured down Na+ energetics in modern hyperthermophilic archaea, Ech and Rfn. Sometimes my brain's a pain!"

LOL. Well that's certainly a pretty tempting topic!

raphi said...

@Tucker Goodrich,

I agree that the chemistry of lipid peroxidation is well understood.

However I wasn't talking about that but about the exact origins of ROSs and how their balance shifts according to the mix of substrates &/or inhibitors in play. In other words from Quinlan et al. (from only 3 years ago) "Mitochondrial radical production is important in redox signaling, aging and disease, but the relative contributions of different production sites are poorly understood [...] at least ten different sites of superoxide/H2O2 production in the electron transport chain and associated enzymes (Krebs cycle, β oxidation etc.) have been identified in mammalian mitochondria". The title of Peter's post "Does fatty acid oxidation really drive reverse electron transport and superoxide generation at complex I?" also implies that answers are building up, slowly.

About HNE. It's not the first time I've seen you mention it, making me want to look into it more. What do you mean regarding Seyfried's misdirected focus? In 2008 he collaborated on a cardiolipin peroxidation paper (Kiebish MA, Han X, Cheng H, Chuang JH, Seyfried TN. Cardiolipin and electron transport chain abnormalities in mouse brain tumor mitochondria). In Chapter 5 of his book he dedicates a large section to cardiolipin peroxidation.

Tucker Goodrich said...

@raphi:

From what I've read in many of the papers about lipid peroxidation the assumption has been that LP gets started from ROS generation in the mitochondrial complexes. I figured this was the case, and that carbs were involved. So I was surprised by many of Peter's posts showing that ROS generation is higher with fats.

But MetSym improves on a low-carb diet, including those parts associated with lipid peroxidation. How can that be?

I think the answer is simple: carbs aren't consumed in isolation in the SAD, but with omega-6 fats. One could accurately define junk food as refined carbohydrates in combination with refined seed oils, which are the primary source of omega-6 fats in the SAD, of course.

The nail in the coffin for me for the high-carb-intake-causes MetSym hypothesis was the recent study that demonstrated 100% cure of non-alcoholic fatty liver disease through reducing omega-6 intake, on a high-carb diet.

Many have observed that people can be healthy on higher-carb traditional diets: they don't get MetSym. Well, the thing missing in traditional diets like the Kitavans are refined seed oils.

Linoleic-Acid-saturated cardiolipin in the mitochondria doesn't need ROS to oxidize. It will auto-oxidize when it comes into contact with iron, such as that in cytochrome-c. Cardiolipin is in constant contact with cyt-c. And they've demonstrated that this will cause a death spiral, where the lipid peroxidation products thus generated created further peroxidation, which continues (in vitro) until all the LA-saturated cardiolipin is consumed.

Non LA-saturated cardiolipin does not auto-oxidize...

So I think focusing on the relative ROS load from traditional foods is missing the main avenue. We're well-adapted to traditional foods, by definition. We're not well-adapted to novel foods like refined seed oils. We're even adapted to a moderate omega-6 load, which is what you'd see in a traditional diet. But pass that point, and the oxidative load on the body overwhelms our adaptive mechanisms, and you get the MetSym.

I do still think that a high-carb diet is suboptimal for a variety of reasons, btw, but I no longer think it's causative in MetSym.

Oh, and the interesting thing about HNE is that it's present in every part of the MetSym, in a dose-dependent fashion, and can only be produced from LA.

So in a nutshell, the low-carb movement has been tricked by the correlation between high carb and high seed oil intake. They (we) picked the wrong culprit.

Tucker Goodrich said...

@raphi:

Oh, and thanks for pointing that out about Seyfried. I've seen him speak, but haven't read his book. But his focus is pretty clearly on carbs, not LA.

However, I just read the 2008 paper, which I was not aware of. Thank you, as it confirms one of my hypotheses about CL and cancer. It contains a number of inaccuracies about the nature of CL, I think. For one, they describe "immature" and "mature" CL, yet others have shown that CL fatty-acid composition is directly and linearly related to short-term dietary consumption. I suspect that the "mature" CL they describe is pathological, as CL seems to be created in one form, and then modified based on the fatty-acid compositions of the diet.

LA does not cross the blood-brain barrier very effectively, but AA (arachidonic acid) does, so the observation about a complete lack of longer-chain n-6 CL is very interesting to me. Perhaps these cells have undergone the auto-oxidation death spiral, leaving none but those CL species not susceptible to it? That would mean the Warburg effect is the cell's attempt to recover from the failure of the mitochondria. An interesting factoid for you: it's not possible to induce cancer in laboratory animals if they have an LA-free diet. It's also not possible to induce alcoholic fatty liver disease on an LA-free diet.

I've not really yet looked into AA and cardiolipin, as most AA in the body is made from LA, and there's precious little research about LA and cardiolipin as it is! But, as that paper notes, the toxic n-6 metabolites are present in many of the neurological defects currently widespread, including stroke and traumatic brain injury. So there could well be a role for for AA-laden CL as well as LA-saturated CL. I've suspected as much, but not found any evidence to support that view until this paper.

Thanks again!

Tucker Goodrich said...

OK, just came across this. Not sure if it's correct, but it would tie cardiolipin into the proton series quite nicely!

"Cardiolipin is a special phospholipid found only in the inner mitochondrial membrane, and it is closely tied to the efficiency with which we produce energy, or ATP: the higher the cardiolipin saturation index, the lower the proton conductance, and the more ATP we produce. What is more, a higher saturation index renders mitochondria highly resistant to damage by free radicals and reactive oxygen species (ROS), including those produced by fish oil, and is associated with lower levels of ROS in vivo."

http://www.andrewkimblog.com/2013/04/pufa-lipid-peroxidation-processes-and_9.html

raphi said...

@Tucker Goodrich,

"it's not possible to induce cancer in laboratory animals if they have an LA-free diet." ==> really?? I have to verify that...

You bring up quite a few interesting points, so I'll get back to you when I've had the chance to mull them over.

Thanks!

Tucker Goodrich said...

Indeed see here:

http://health120years.com/cn/pdf/hd_Cancer_LA_Summary.pdf

Jack Kruse said...

You guys all need to real a lot more Dr. Doug Wallace and a lot less Seyfried and the LCHF guys. They just do not understand how mitochondria work. For more data on ROS production from mitochondrial look at Roeland van Wijk's work.

Tucker Goodrich said...

@Jack Kruse:

I presume you're referring to this recent post of yours:

"TIME # 19: IS TIME TOLD BY BIOPHOTON EMISSION?"
https://www.jackkruse.com/time-for-biophotons/

To judge from your post, you're confusing cause and effect here. Your post mentions cytochrome c, but not cardiolipin (as we've been discussing above). The cause of the light release which is indicative of the pathological state your describing is simply an effect of cardiolipin oxidation by cyt-c, as detailed in this paper:

"Cytochrome c-promoted cardiolipin oxidation generates singlet molecular oxygen" (PDF)
http://www.producao.usp.br/bitstream/handle/BDPI/36322/wos2012-1518.pdf?sequence=1

The root cause of cardiolipin oxidation by cyt-c is well understood, as is how to prevent it.

Again, scroll up for my previous comments on this topic.

Bob said...

Hi, Tucker,

From your link (http://health120years.com/cn/pdf/hd_Cancer_LA_Summary.pdf):

"Nevertheless, there appears
to be a minimum required intake of linoleic acid for tumor devel-
opment in rodents. However, this requirement is about as low or
lower than intakes recommended to prevent deficiency of essen-
tial fatty acids, and reducing linoleic acid intake to below these
amounts is neither realistic nor desirable."

And,

"The available evidence does not suggest that a high intake of
linoleic acid substantially raises the risk of breast, colorectal, or
prostate cancer. Nevertheless, a small increase in risk cannot be
excluded"

This hardly seems like support for the idea that LA is carcinogenic. Am I missing something?

Bob said...

Hi, Tucker,

From your link:

"Nevertheless, there appears
to be a minimum required intake of linoleic acid for tumor devel-
opment in rodents. However, this requirement is about as low or
lower than intakes recommended to prevent deficiency of essen-
tial fatty acids, and reducing linoleic acid intake to below these
amounts is neither realistic nor desirable."

And,

"The available evidence does not suggest that a high intake of
linoleic acid substantially raises the risk of breast, colorectal, or
prostate cancer. Nevertheless, a small increase in risk cannot be
excluded"

This hardly seems like support for the idea that LA is carcinogenic. Am I missing something?

Tucker Goodrich said...

@Bob

That paper is from 1998. I included it for the comment about LA being required for inducing cancer in lab animals. A lot's happened since then...

See here for a paper from 2013:

"In summary, the results from pre-clinical studies provide compelling evidence that PUFAs can mediate cancer progression in vitro and in vivo in models of several different types of cancer. Mediation may occur through several mechanisms including regulation of gene expression, angiogenesis, cell migration, and apoptosis...."

http://journal.frontiersin.org/article/10.3389/fonc.2013.00224/full

Moreover, the metabolites of LA are well-recognized as mutagens and carcinogens.

Keep in mind that most of the studies described in that paper are short-term. You need to smoke for 20 to 30 years (on a population basis) to get lung cancer, for instance. LA isn't going to give you cancer tomorrow, anymore than one cigarette will.

Bob said...

Hi, Tucker,

Thanks for the response. Very interesting review paper (and fairly readable for this layman despite the abundant acronyms).

An idea jumped out at me I suspect Jack Kruse might like. Many of us have gone low-carb, because the damage from many years of higher sugar consumption has exacted a toll on our ability to process non-sugar carbohydrate.

By analogy, I wonder if many years of higher LA consumption has exacted a toll on our ability to handle small amounts of AA as found in meat, egg yolk, and dairy, typically the foods found in the low-carb diet. This implies more fish would be appropriate for a post-LA diet.

I know, I know. More saturated fat means less PUFA in cardiolipin and thus changes in mitochondrial "resistance". And of course the damage from years of excess AA may not be analagous to the damage from years of excess sugar. Besides, the entire subject is littered with complexity and uncertainty. Still, I'd be curious on your thoughts.

Thanks again for the link.

Tucker Goodrich said...

@Bob:

Short answer: It depends. LOL.

Fish have arachidonic acid too, for starters, especially if they're fed seeds or grains, as farmed fish are.

A major pathway in the body is LA to AA, and reducing LA intake reduces AA conversion and lowers concentration in the body (with lots of qualifiers).

Since omega-6 fats concentrate up the food chain, you want to reduce eating animals that are fed seeds (chicken, pork, farmed fish). Instead eat those that don't concentrate it (ruminants like cattle, sheep, goats); or just avoid grain-fed animals (pastured chicken or pork, wild-caught fish).

Yes, I do find it fascinating that the animals that spend the most time eating grass and therefore seeds (ruminants) don't concentrate LA and convert it into conjugated LA, which seems to block some of the harmful effects of LA. It suggests to me there is some evolutionary pressure, which other experiments have confirmed (poor breeding performance on high-LA diets).

See here:
"Popular Fish, Tilapia, Contains Potentially Dangerous Fatty Acid Combination"
https://www.sciencedaily.com/releases/2008/07/080708092228.htm

and here:
"Anti-inflammatory effects of a low arachidonic acid diet and fish oil in patients with rheumatoid arthritis."
http://www.ncbi.nlm.nih.gov/pubmed/12548439