Monday, July 24, 2017

An update

Hi All.

We've moved house. It has not been the simplest of moves. OK, it was awful. However it was also worth it as we live here now.


















While the house is in pretty good order the acre and a half of ground needs some TLC before we can get the chickens strip grazing and maybe some stock in, so I sort of doubt there will be a huge amount of free time to blog. Maybe a little musing on metformin might be possible...

Anyway, we're alive and busy and now live some distance from the nearest main road (in Norfolk terms).

Peter

Saturday, June 03, 2017

Why stop at formaldehyde?

If we consider the dissociation of hydrogen:





the right hand side of the equation can supply electrons to another reaction. The tendency for this to occur is in part dependent on the pH of the solution. If we consider alkaline hydrothermal vents we have a pH of around 11, this drives the reaction to the right because the protons avidly combine with hydroxyl ions to give water:
















Which means that there is a marked tendency to supply electrons for any electron-accepting reaction. The electrons can hop on to an FeS barrier (each changing the charge on an Fe from 3+ to 2+) which separates the vent fluid from CO2 rich, acidic oceanic water:













Deriving from fluid with a pH of 11 these electrons have a redox potential of -650mV, ie they are highly reducing.

If we now look at the situation on the oceanic side of the barrier we have:




and by adding on the factor of an acidic pH, with lots of protons driving the reaction to the right we have this:
















Under these conditions electrons supplied at -650mV are very able to allow the reaction to proceed to the right yielding CO. Repeating the process yields CH2O and metabolism is on its way.















OK. Nick Lane makes these points in his paper:

1. There is no contact between the H2 in the vent fluid and the CO2 in the ocean fluid. The two Hs in the formaldehyde come from oceanic protons combining with vent H2 derived electrons.

2. I've shown the reaction occurring once to CO and again to CH2O. Why stop at twice? Given a supply of -650mV electrons why not keep generating CO and inserting it, along with e- and H+, in to whatever hydrocarbon you have already got in the vent fluid? Nick Lane has reaction sketches for generating almost all of the Krebs cycle components on this basis.




Theoretically, if you wanted to make an origin of life reactor to test whether you can generate a multitude of the hydrocarbons at the core of metabolism you don't actually need a supply of alkaline hydrogen rich fluid. This only supplies electrons at -650mV. An alternative supply would be a 1.5 volt battery with some sort of voltage reduction to get from -1500mV to -650mv and you're away.

A microporous FeS electrode in Perrier water, energised by an AA battery via a couple of resistors and you might just be set up. Getting the apparatus anoxic and detecting the products might be more of a challenge!

Edit Finally followed Nick Lane's final reference. These folks have reached pyruvate via an energised FeS electrode. It's a lot more complex than Perrier water but it works. End edit

Peter

Thursday, June 01, 2017

Nick Lane on Proto-Ech

Nick Lane has a few more downloadable papers available on his website, two of which focus on ideas I've thought a lot about. Here are a few quotes:

Iron Catalysis at the Origin of Life

"Why does the reduction of ferredoxin via Ech depend on the proton-motive force? The answer is as yet unknown, but cannot relate to reverse electron flow [as originally proposed (49)] as these methanogens do not possess an electron-transport chain (37,38). A more pleasing possibility is that pH modulates reduction potential at the active site of the enzyme. The flux of protons through Ech from the relatively acidic exterior could lower the pH at the active site of the enzyme, which should facilitate reductions that depend on protons, including CO2 as well as some ferredoxins (50)".

My italics. Next:

Proton gradients at the origin of life

Aside: If you read the full text of Lane's paper you will take note of Jackson JB (2016) Natural pH gradients in hydrothermal alkali vents were unlikely to have played a role in the origin of life. And this passed scrutineering. Nick Lane does not seem impressed. End aside.

"One possibility is that prebiotic carbon and energy metabolism entailed the synthesis of reactive thioesters analogous to acetyl CoA, such as methyl thioacetate, coupled to substrate-level phosphorylation, generating acetyl phosphate and ultimately ATP [1, 17, 27, 60–63] as still happens in bacteria [14, 31]".

"Across the barrier, in acidic conditions, CO2 is more easily reduced, and so is more likely to be reduced by Fe2+ in the barrier. The semiconducting barrier should transfer electrons from Fe2+ on the alkaline side to Fe3+ on the acidic side. The thickness of the barrier does not matter, so long as it is semiconducting. The two phases do not come into direct contact - H2 and CO2 do not react directly (Fig. 3)".

This is really neat, it puts in to a published paper many of the logical concepts that went in to the Life series. I really like the pre biotic ideas of electron transfer across any-thickness FeS barriers. No need for membranes, indeed insulating "crud" membranes would hinder electron transfer from the FeS wall to the enzyme, necessitating the generation of a pore like structure (ancestor to NuoH) to get the voltage generating acidic pH to the active enzyme's site.

This ferredoxin reduction plus subsequent substrate-level phosphorylation is where it should all start. NuoH starts as a pH channel, not part of a nano machine. That comes later with reversal of proton flow and the development of complex I, a true advanced nano machine.

I still don't buy ATP synthase (another very complex nano machine) as running on the primordial vent proton gradient as Nick Lane holds to. Later developing Na+ energetics look much more likely, these following on from Proto-Ech's pore duplication to form a Na+/H+ antiporter, giving a usable Na+ gradient. That clearly post-dates some sort of membrane, which ferredoxin based metabolism must precede when using a geochemical proton gradient. NuoH becomes essential only after a crude membrane forms to impede this process of ferredoxin reduction.

Nice papers.

Peter

Tuesday, May 30, 2017

Adrian Ballinger on Everest

Back at the end of 2015 Mike Brampton and I had a conversation about climbing Everest.

Based on Graph A from Fig 3 in D'Agostino's rat paper

Therapeutic ketosis with ketone ester delays central nervous system oxygen toxicity seizures in rats

our conclusion was that summiting Everest might be best achieved using a ketogenic diet. I know nothing about extreme climbing or the culture which goes with it but it came as no surprise, via Mike, that they carb loaded and carb loaded and carb loaded. You know, sugar has its own partial oxygen supply built in to the molecule. No point trying to burn fat if there's no oxygen*. Understandable but, obviously, completely incorrect. I think Mike had been trying (frustratedly) to convert altitude folks to fat centred thinking for some years before this.

*It's true that there is no point trying to burn fat under anoxia. But given some oxygen ketosis pays dividends.

So it was interesting to pick up this link on Facebook:

How Adrian Ballinger Summited Everest Without Oxygen

This fits in with Veech's concept of increased metabolic efficiency per unit O2 consumed when burning ketones and D'Agostino's discovery of an "unexpected" rise in arterial PO2 in rats gavaged with a betahydroxybutyrate/acetoacetate combination precursor, while they were breathing room air (PaO2 from 100mmHg to 130mmHg, pardon the archaic units).

Very gratifying, even if completely different from the approach taken by Naked Mole Rats and their fructolysis.

Peter

Fructose and lactic acid in Naked Mole Rats

Naked Mole Rats appear to use fructose as their preferred metabolic substrate when exposed to both physiological hypoxia (which is common in their lifestyle) or complete anoxia under experimental conditions. It's irresistible to go and find out a little about why they might do this.

Fructose-driven glycolysis supports anoxia resistance in the naked mole-rat

I suppose the first thing to say is that the fact that fructose is protective against hypoxic cellular injury has been known for a long time, this paper coms from 1992:

Fructose protects rat hepatocytes from anoxic injury. Effect on intracellular ATP, Ca2+i, Mg2+i, Na+i, and pHi

There was a lot of work done in the 1980s and 90s looking at ways of preserving liver cells under anoxia. I'd guess this was looking to improve the survival of harvested livers within the transplant program.

If we look at ATP levels compared to an externally supplied control (MDPA) we have this graph, with hypoxia imposed at one hour and relieved at three hours:

















ATP falls faster within the first 30 minutes of anoxia with fructose. Although the trends are interesting, all else is ns after 45 minutes. So fructose causes a more severe ATP depletion than glucose. However a better marker is the ratio of ATP to Pi (phosphorylation potential), here plotted as the inverse for some reason, ie the lower the better in the graph:


















So under fructose there is less ATP in the cytoplasm than under glucose but the phosphate level is even lower, giving a similar or more favourable ratio of ATP to Pi except at the 30 minute mark. So the next question is: Where has the phosphate gone?

This might be related to the protective effect of cytoplasmic acidosis. It doesn't seem to matter how you acidify the cytoplasm (fructose is as good a way as any), it's the acidosis which appears to protect against mitochondrial failure. There's a nice paper here

Protection by acidotic pH and fructose against lethal injury to rat hepatocytes from mitochondrial inhibitors, ionophores and oxidant chemicals

and here

Intracellular acidosis protects cultured hepatocytes from the toxic consequences of a loss of mitochondrial energization

So if we go back to Gasbarrini's paper we can look at a surrogate for intracellular pH and how it differs between fructose and glucose:




















Fructose produces a much more profound acidosis. If we look at that basic ETC doodle I used in the rho zero cell post, but eliminate complexes I, II, III and IV we have this:









We have here two process which can be driven by an excess of protons in the cytoplasm over those in the mitochondrial matrix. Transport of Pi in to the mitochondria and synthesis of ATP. Which of these is most important to ensure cell survival is hard to say. It is even quite possible that it's neither and that maintaining an excess of protons outside the mitochondria maintains delta psi so defers the commitment to apoptosis or the occurrence of necrosis.

Later changes which confirm the commitment to cell death are an influx of extracellular calcium in to the cytoiplasm. This is marked under glucose and stays within tolerable limits with fructose. I strongly suspect the metabolic decision making is being controlled by the pH drop and the Ca2+ influx is consequent to a mitochondrial decision as to how badly damaged the cell might be. But it's hard to be sure with the data we have in these rather elderly papers.

About that acidosis:

Here are the reactions relevant to the pH change in lactic acidosis, all taken from the wiki entry on lactic acid. They are interesting. This is the situation down to pyruvate:



There are two protons generated to acidify the cytoplasm. Now look at this step where pyruvate is converted to lactate. The molecules in the red oval are needed to form the lactate.







So where did the two acidifying protons go to? They are consumed in converting pyruvate to lactate. Does lactic acid generation actually acidify the cytoplasm? It appears not to do so here but it must do because the overall reaction is:




So where are these two protons? They are in the two ATP molecules:




The conversion of ATP to ADP releases them. So lactate causes acidosis only when the ATP generated during glycolysis/fructolysis is consumed... Obviously ATP depletion is common in anaerobic exercise or hypoxia/anoxia. Hence lactic acidosis shows under these two conditions.

The Naked Mole Rat paper is very descriptive, with lots of experimental results but is light on insight as to hows and whys. I think the above scenario might well have explanatory power and might have been extended from the liver to the rest of the body in NMRs.

Peter

Thursday, May 18, 2017

Fructose and metabolic syndrome: Uric acid

Some weeks ago a friend sent me a full text copy of the Naked Mole Rats (NMR) paper

Fructose-driven glycolysis supports anoxia resistance in the naked mole-rat

which demonstrated that they (NMRs) appear to generate and use fructose as a coping stratagem for dealing with hypoxia or even anoxia. This is fascinating and leads back to research in the late 1980s, mostly looking at anoxia in liver or liver cells. I'm guessing that this liver work was funded to look at ways of improving the condition of transplant grafts. Fructose is significantly better than glucose for supporting anoxic liver cells, possibly something you might expect, possibly not. Perhaps in another post.

Anyway. So I've been looking at why fructose is different to glucose and to do this you end up asking rather difficult questions about the upper sections of both glycolysis and fructolysis.

Fructose enters the fructolytic pathway by being phosphorylated very rapidly to fructose-1-phosphate. Given a large enough supply of fructose this phosphorylation can deplete the ATP supply in a cell, most obviously in hepatocytes which bear the brunt of metabolising fructose. This takes place before aldolase generates the trioses which probably (or don't, in the case of fructose) control insulin signalling through mtG3Pdh and the glycerophosphate shuttle.

If this initial ATP depletion by fructokinase is profound it is perfectly possible to take two "waste" ADP molecules and transfer a phosphate from one to the other. This generates one ATP and one AMP. The ATP is useful to the cell and the excess AMP is degraded to uric acid.

This is all basic biochemistry.

In the Protons series I have worked on the (incorrect) basis that fructose should drive the glycerophosphate shuttle hard enough to generate RET (reverse electron transport) and so signal insulin resistance. The degree of insulin resistance should neatly reduce insulin mediated glucose supply by an appropriate amount to offset the fructose and so maintain a stable flux of ATP generation from the combined fructose and glucose. That's not quite how it appears to work. Even before the aldolase step in fructolysis, the body is starting to prepare the process of insulin resistance. This paper is not unique but shows general principles:

Uric acid induces hepatic steatosis by generation of mitochondrial oxidative stress: potential role in fructose-dependent and -independent fatty liver

The title of the paper is sneaky, it doesn't give away the answer! Nor does the abstract. If you don't want to read the paper, the missing link is NOX4.

NADPH oxidase 4 (NOX4), if exposed to uric acid (present here from fructolysis induced AMP degradation), translocates to the mitochondria and starts to generate enough hydrogen peroxide* to down regulate aconitase, abort the TCA and divert citrate out of the mitochondria through the citrate/malate shuttle for DNL. This will not just affect fructose metabolism, acetyl-CoA from glucose, entering the TCA as citrate, will also be diverted to DNL.

*The NOX family appear to be the only enzymes with no function other than to produce ROS, mostly superoxide. NOX4 is unique in that it always produces hydrogen peroxide. There is uncertainty if the "E loop" of the enzyme converts superoxide to hydrogen peroxide directly or if this is a docking site for superoxide dismutase, which does the conversion as an accessory module to NOX4.

I think it is a reasonable assumption that the hydrogen peroxide generated by NOX4 will be what signals the insulin resistance induced by fructose, rather than RET via mtG3Pdh. Quite why fructose doesn't drive the glycerophosphate shuttle is a difficult question to answer. Obviously the aldolase products of fructose-1-P (fructolysis) differ from those of fructose-1-6-bisphosphate (glycolysis) but these pathways are very difficult to get at experimentally and I've not found any papers looking at what controls why dihydroxyacetone phosphate from fructolysis doesn't drive mtG3Pdh, but that appears to be the case. There are hints that some activation of the glycerophosphate shuttle does occur but NOX4 seems to be the main player. It might relate to the consumption of NADH in the conversion of glyceradehyde to glycerol and so reducing the need to decrease it using the glycerophosphate shuttle. Hard to be sure.

So. Uric acid is the evil molecular link between fructose and metabolic syndrome via NOX4. And yes, yes, you can block metabolic syndrome using allopurinol to reduce uric acid production in rats but you have to give them a sh*tload of it. After that NOX4 might be considered evil or hydrogen peroxide is evil or aconitase is evil when it's on strike. Lots of drug targets available for molecular cleansing.


My own concept is that there is the necessity to developing insulin resistance when fructose is available so as to limit glucose ingress to offset the ATP from that fructose ingress. If that is done by NOX4, so be it. The facility to deal with fructose by the generation of hydrogen peroxide is not random, it's not some accidental mistake perpetrated by evolution on hapless humans who munched on a few Crab apples or found a little honey. It is an appropriate evolutionarily response to a relatively common occurrence. The fact that uric acid mediated insulin resistance is common to alcohol metabolism as well as to fructose metabolism suggests that this mechanism is a general approach to dealing with a calorie input which takes priority over metabolising glucose.

Developing a drug along the lines of allopurinol to block uric acid production, or an inhibitor of NOX4, or a hydrogen peroxide scavenger to avoid insulin resistance is simply trying to block a perfectly adaptive response to a reasonable dose of fructose.

All that's needed to avoid a pathological response to fructose is to avoid ingesting a pathological dose of the stuff. There is actually quite a lot of evidence to suggest that physiological levels of uric acid production might be beneficial...

Peter

Mulkidjanian: Na+ pump or Na+/H+ antiporter?

Mulkidjanian is a co-worker with Skulachev and extremely wedded to the primacy of Na+ bioenergetics, which is good. He has been looking at NuoH and NuoN subunits of complex I and their phylogenetics. In contrast to this, in the past I've discussed similarities between NuoH and NuoL. You just have to accept we're never going to be certain which component of complex I is most closely related to another... Anyway, I like this paper:

Phylogenomic Analysis of Type 1 NADH:Quinone Oxidoreductase

"Two recently published works independently noted the structural similarity between the NuoH and NuoN subunits and suggested their origin by some ancestral membrane protein duplication [13, 14]. Our analysis does not exclude the possibility that this duplication may have occurred even before the LUCA stage. In this case the initial NDH-1 form [proto-Ech in my terminology] had only one type of membrane subunit (the ancestor of NuoN and NuoH), which could function as a sodium transporter. The duplication of the gene would result in a different subunit, which improved the kinetic effectiveness of the redox-dependent sodium export pump (that participated in maintenance of [K+]/[Na+] greater than 1 in a primal cell) by facilitating proton translocation in the reverse direction".

Bear in mind that none of us can be certain exactly what a given protein might have been doing based on these family trees of genes.

I think there is general agreement that ancestor of NuoH and NuoN is a membrane pore and that it is primordial. In Mulkidjanian's scenario that pore is associated with a redox driven hydrogenase. His idea is that the hydrogenase is using preformed ferredoxin, or something similar, to extrude Na+ ions from the cell. This requires an external source of energy and his concept is for ZnS catalysed photosynthesis giving a localised organic "soup", ie heterotrophy. The refs are here and here. The source of K+ for the primordial cell cytoplasm is suggested here. I have to say, I'm not a convert to these aspects of his ideas, I'm staying more aligned with autotrophic thinking...

My own view is that the pore was a duct to localise oceanic acidic pH tightly to an NiFeS hydrogenase within alkaline vent "cytoplasm" to allow the hydrogenase to reduce ferredoxin, the primary energy currency of the proto-cell. The power source is the pH differential across an internal FeNiS moiety within the hydrogenase, combined with molecular hydrogen as the electron donor to reduce ferredoxin and so, eventually, CO2.


Given the almost certain ancestral gene duplication it is not difficult to make an antiporter out of NuoH/NuoN, whether you consider the ancestor to have been a proton pore or part of a Na+ pump. Even today, the membrane component of Complex I functions as an antiporter for Na+/H+ provided you separate it off from the hydrophilic matrix section:

The deactive form of respiratory complex I from mammalian mitochondria is a Na+/H+ antiporter

Given an antiporter sitting in a Na+ opaque membrane we can antiport a ton of Na+ out of the cell using a geological proton gradient to give us the result of a low intracellular Na+ concentration. Excess Na+ extrusion can be converted, by electrophoresis, to an elevated K+ inside giving the modern intracellular composition. In the early days the electrophoresis might not have been K+ specific, theoretically any positive ion other than Na+ would do. K+ is the long term preferred option.

As soon as we leave the vent there is no free antiporting so we need to have a system which provides energy to generate a Na+ potential (buffered by K+ electrophoresis). The power available to do this becomes very limited in the absence of a geothermal proton gradient, when all that is available is the reduction of CO2 using H2, the Wood–Ljungdahl pathway. The Na+ chemiosmotic circuit then comes in to it's own as a system for combining small amounts of free energy in to units large enough to generate one ATP molecule. Recall how the modern pyrophosphatase Na+ pump requires the hydrolysis of four PPi to give one ATP via chemiosmotic addition. Until the advent of photosynthesis and the possibility of heterotrophy, all free living prokaryotes would have been autotrophic and living on a meagre energy budget.

The switch from luxurious hydrothermal vent conditions to lean autotrophic conditions goes a long way to explaining the universality of chemiosmosis. Alkaline hydrothermal vents may be stable on geological time scales but not for 4 billion years of un-interrupted flow and if the Wood–Ljungdahl pathway is all there is to replace the vent power supply it's going to be chemiosmosis all the way...

Peter

Thursday, April 20, 2017

Skulachev addendum

This is the final paragraph in the discussion section of the paper by Skulachev, regarding the use of a Na+/K+ concentration gradient across a membrane to store potential energy, convertible to a Na+ or H+ gradient as needed, and why elevated K+ does not have to be a primordial feature of proto-cells:

"One might think that Na+ ions are incompatible with life and this is the reason why K+ is substituted for Na+ in the cell interior. Apparently, it is not the case as, e.g., in halophilic bacteria [Na+]int can reach 2 M [41]. The very fact that some enzyme systems work better in the presence of K+ than of Na+, may be considered as a secondary adaptation of enzymes to the K+-rich and Na+-poor conditions in the cytosol [40]. Besides, it would have been dangerous to couple any work performance with Na+ influx to the cytoplasm if Na+ were a cell poison".

That makes perfect sense to me.

Peter

Wednesday, April 19, 2017

From Skulachev to LUCA

TLDR: Cells become islands of raised K+ ion concentration when energy is supplied.


Okay, here come the doodles based on Skulachev's paper

Membrane-linked energy buffering as the biological function of Na+/K+ gradient

This is the scenario in ultra modern bacteria, the pinnacle of about 4 billion years of evolution. The membrane is tight to all significant ions at reasonable temperatures and concentration gradients. In this set of pictures the proton population represented within the red circle is holding a membrane voltage of 180mV, as per usual:






The trans-membrane potential from the pumped protons is stable while ever the pumping and the consumption of protons is balanced. The problem is that it doesn't need many protons to generate that 180mV. Pumping any more than basic needs generates too great a membrane voltage. The converse is that it doesn't take much excess proton consumption to collapse the potential. So you need a buffer which does not waste the energy used to pump.

If a bacterium suddenly increases proton pumping by eating some glucose we have this problem of a spike in membrane voltage:









We can get around this by allowing a positive ion to travel in the opposite direction. This will stop the rising membrane potential as the ion uses the membrane potential to enter the cell against a concentration gradient. It uses an ion-specific channel, in this case for potassium. This process is electrophoresis down the electrical gradient, against a concentration gradient, powered by the electrical component rather than the pH component of the rising proton gradient:










The number of K+ ions matches the excess protons pumped. The electrical potential is thus maintained at 180mV at the "cost" or "benefit" (semantics here!) of K+ entering the cell. But there is a problem in that the more protons pumped and the more K+ entering the cell, the higher the pH of the intracellular medium becomes. That K+ pool is actually tied to the OH- left behind by pumping out H+. Caustic potash...










This is not good for metabolic processes. But it is easily surmounted using a 1:1 ratio Na+/H+ (electro-neutral) antiporter to get some protons back in to the cell to offset the excess OH-












while still maintaining an electrical gradient of 180mV using H+, keeping an electro-neutral Na+/K+ gradient as an energy store:










Obviously the Na+/H+ antiporter is being driven by the pH component of the proton gradient. It's neat how evolution has separated out the pH and electrical components of a proton gradient!

The whole system is fully reversible so if there is a sudden drop in proton pumping the transmembrane Na+/K+ gradient can be reconverted to a proton gradient to "buffer" changes in proton translocation. This seems to be how modern, proton pumping bacteria with superbly proton tight membranes work. In E coli the ion channel and antiporter are ATP gated.

That's how Skulachev looked at modern bacteria in 1978.


I'm now going to wander off on my own and speculate about LUCA with a proton leaky but Na+/K+ tight membrane. This is just me from here onwards:

Let's have a think about LUCA, with a cell membrane which is tight to Na+, and probably K+ too, but highly leaky to both protons and hydroxyl ions. Metabolism is based on Na+ pumping and a Na+ specific ATP synthase. The initial Na+/H+ antiporter (from the Life series) is gone as a source of Na+ gradient as soon as LUCA leaves the alkaline hydrothermal vents.

I like the idea that LUCA used a pyrophosphatase to pump Na+ but with any Na+ pump we have the same problem as in modern bacteria: You can only store a small amount of energy as a 180mV Na+ gradient, as per H+ above:










But excess Na+ pumping can be easily be accommodated by K+ electrophoresis:










There is no need for the Na+/H+ antiporter in this scenario because there is no pH change associated with pumping Na+ ions, so all we need is the ion specific channel for K+.

This sets up a non-electrical energy store which is "accessible" to form an electrical gradient when primary Na+ pumping is low.

The buffer automatically implies the generation of a raised intracellular K+. We have here, based on a tiny step beyond Skulachev's ideas, a place within LUCA which is potassium rich. It's simply produced to buffer changes in ion pumping by the primary Na+ pump (or usage by ATP synthase) across relatively primitive membranes. And driving intracellular K+ higher is an indicator to the cell that there is excess of energy available, which should select for increased enzyme activity based on rising intracellular K+ concentration. Many of the "core" LUCA enzymes do indeed use K+ as a cofactor to function optimally.

Summary: Cells become islands of raised K+ ion concentration when more than basal a level of energy is supplied. Remember that for our later discussion about Mulkidjanian's ideas on the origin of life on Earth.

Peter