The science-free cult of homeopathy has struck again. Steve Novella at Science Based Medicine has the scoop. Apparently a blogger in Italy wrote about the homeopathic treatment Oscillococcinum which is used to treat flu and the homeopathic manufacturer threatened him with legal action, simply for stating the truth about Oscillococcinum, that it is a bogus treatment based on nonsensical ideas compounded together.
A good place to start is always QuackWatch. Which has a nice discussion of the “theory” behind homeopathy and how it is only a placebo. Another good site is What's the harm, a site that documents the real harms that can come from using placebos like homeopathy instead of real treatments.
The Main Stream Media has picked up on this, the BMJ has an article on how Boiron is threatening the blogger. There are some blogs about quack veterinarians being upset that real veterinarians only want science based treatments.
It is a pretty sad state of affairs when a big corporation uses hired lawyers as shills to try and keep people from speaking the truth, and when governmental laws and regulations allow them to do so. I am not the only one who thinks so, those of us in the reality based community are opposed to magical thinking fake treatments such as homeopathy.
Things are looking up for victims of homeopathy in the US, there are a couple of lawsuits for fraud via homeopathy that are proceeding.
Showing posts with label placebo. Show all posts
Showing posts with label placebo. Show all posts
Wednesday, August 17, 2011
Wednesday, January 30, 2008
Physiology behind the Magic Light Helmet for Alzheimer's
There have been a number of different blogs about the Magic Light Helmet for Alzheimer’s. Respectful Insolence and again! (Must be Orac's self-identification with blinking LED based intelligence boosting schemes ;), Science Based Medicine.
This is a more elaborate explanation of the quick comment I made at Science Based Medicine. My explanation is based on my understanding of the cause of Alzheimer's (which is not one of the common explanations (all of which I am quite skeptical of (in other words I think they are wrong)). I see Alzheimer's as being strictly due to low NO which then causes low ATP (via sGC), and invokes ischemic preconditioning (IP). The low perfusion is secondary to the low NO. Ischemic preconditioning can be mediated by oxidative stress in multiple ways. Normally IP is thought of as a good thing because it does reduce damage during ischemia. IP is invoked by brief periods of ischemia and after it is invoked organisms can survive periods of ischemia that would have been fatal. IP is a low ATP state, and it is also a low ATP consuming state. If organisms could be in the low ATP consuming status of the IP state long term, they would have evolved to do so. Spending less ATP on basal metabolism means more ATP for reproduction. Organisms haven't evolved to do so, which means there must be something incompatible with life and/or reproduction from being in the IP state long term. Neurons don't divide, so there must be something incompatible with long term survival for neurons to be on the IP state long term. I discuss more details of why accumulation of amyloid is a major outcome in Alzheimer’s below.
I am assuming that this Magic Light Helmet actually has physiological effects beyond the placebo effect. It might not. The placebo effect is mediated through NO, and all disorders associated with low NO are very susceptible to being improved by the placebo effect. That would include Alzheimer's. I will use the analogy of an internal combustion engine a lot. Not because it is an especially good analogy, but because it is easy to understand how bypassing the very simple controls of an engine to get “better performance” could easily shorten its lifetime by orders of magnitude. What does bypassing the extremely complex control system of the brain do? Most of which is completely unknown (but is likely already disrupted for that brain to have Alzheimer’s in the first place)? It is extremely unlikely (virtually inconceivable) that bypassing the normal control system of the brain would only have benign effects.
We know that Alzheimer's is not caused by a "deficiency" of light at 1072 nM. This Magic Light Helmet is not replacing some physiological need that is missing in Alzheimer's. The fundamental problem of Alzheimer's is low ATP supply (discussed below). Since the brain does not have photosynthetic centers to capture the energy of light at 1072 nM and convert that energy into ATP, any change in ATP supply can occur only via changing the regulation of existing ATP production systems via mechanisms susceptible to light at 1072 nM. Since physiology didn't evolve to be physiologically regulated by an external source of 1072 nM light these lights must be doing something else, something that is non-physiologic. Even if there are physiological receptors of 1072 nM light, they would be for sensory purposes (maybe setting day/night biorhythms?) and the levels used in the magic light helmet are many orders of magnitude greater, and narrow band rather than broad band natural sources (sunlight or fire).
If this NIR light does do "something", it pretty much has to be via modulation of kinetics of either free radical reactions, decay of "excited transition states" in activated molecules, or in photodissociation of things from other things (such as NO from cytochrome c oxidase). There could easily be hundreds of things that are being affected, none of which are well understood. Or it might just be placebo.
There are at least 4 main regions which might be important and which I will discuss, by far the most important is ATP production in mitochondria and I spend most of my time on that. Synthesis of compounds by the cytochrome P450 enzymes, catecholamine metabolism and acetylcholinesterase activity might be important too. There might be others, but these are 4 important ones, changes in any of them could conceivably be consistent with the "data" that a handful of non-blinded observers noticed improvement in 6 weeks of use. Although I will be discussing these somewhat separately, they are not separate. Physiology doesn't divide what it does into neat little orthogonal and independent boxes. In physiology everything is coupled to everything else. There is no evolutionary driving force to keep things separate or modular. There are only evolutionary driving forces for survival, and for economy (so more resources are available for reproduction).
ATP production
In neurons virtually all ATP is made by mitochondria through oxidation of substrates, lactate, ketone bodies, small acids such as acetate, aspartate. Mitochondria are small organelles that have a few thousand proteins, 13 of which are coded for by mitochondrial DNA, all the others are coded by nuclear DNA. Mitochondria have 2 lipid membranes, inside the inner one is where the DNA is and the protein manufacture stuff (the mitochondria matrix) and the enzymes of the citrate cycle and most everything that mitochondria do. Mitochondria work by generating an electrical potential and a pH gradient across that inner membrane. The different respiration complexes take electrons and protons from chemical compounds and extract energy from the chemical reactions as those electrons and protons are moved across the membrane and store that energy in the electrical and pH gradient. In the terminal enzyme, cytochrome c oxidase those electrons are gathered and 4 of them are simultaneously put onto O2 along with 4 protons making two molecules of water. Doing so many things all at once is a “tricky” and complicated thing for an enzyme to do. That energy gradient is then used to make ATP.
The 13 proteins are all parts of the respiration chain, usually the part containing the active site. All animals (except for a few invertebrates) have these same 13 proteins coded in their mitochondria. Plants have a few extra. These proteins are all large and quite hydrophobic. Why these (and only these) proteins are coded in mitochondria is not understood. I think it has to do with regulation of mitochondria, some of which has to be local to each mitochondrion and sometimes that regulation means turning off part of the respiration chain and then turning it back on. One mechanism might be destroying the protein and then making the protein again. In most cells mitochondria are close to the nucleus so that proteins can be made from DNA in the nucleus and then transported to mitochondria (in principle, whether this happens or not is unknown). In neurons that can't happen because the distance between the cell body (where the nuclear DNA is and the protein synthesis capacity) and mitochondria can be inches or even a meter in motor neurons. There simply isn't time for a signal to propagate from mitochondria to the cell body, trigger protein synthesis and then transport proteins out to mitochondria in need of them. If protein synthesis is needed for control of mitochondria, that synthesis must occur locally using locally available DNA.
If neurons are deprived of substrates, O2 and organic compounds, disruption of neuronal function (unconsciousness) occurs in seconds.
NIR does cause the photodissociation of “poisons” from cytochrome c oxidase. This is discussed in terms of NO in a recent article in Nature. They get the basic chemistry right, however they get the implications of how NO fits in physiology wrong. The problem is not too much NO, the problem is too little. One of the papers cited by the Nature article epitomizes much of what is misunderstood in the NO field (but the author is quite senior, so I won't link to the article). NO and superoxide can form peroxynitrite, however superoxide from mitochondria is confined to the mitochondria matrix, there are 2 lipid membranes the superoxide has to go through before it can get to the cytosol. Both of those lipid membranes have ~10x higher NO levels than the cytoplasm (NO partitions into lipid ~10x over aqueous). NO reacts with superoxide at near diffusion limited kinetics. It is completely implausible to me that superoxide could get out of the mitochondria matrix. Particularly when the mitochondrial potential (~140 mV) is going to keep the negatively charged superoxide inside. When peroxynitrite forms in the mitochondria inner matrix, it is consumed by cytochrome c oxidase, or decomposes generating NO2 (which is also a signaling molecule).
NO and superoxide only form peroxynitrite at near stoichiometric ratios. When NO or superoxide is in excess that doesn't happen. A much better conceptualization of what happens when NO and superoxide are generated in cells is by Wink et al. They make a very important (and correct) statement
"We demonstrate that the primary consequence of [superoxide] O2- generation concomitant with NO production is not the toxicity associated with the formation of higher nitrogen oxides, but rather the resultant phenotypic cellular changes that occur because of limiting the bioavailability of NO and H2O2."
This is precisely correct. The problem of oxidative stress is not the presence of nitrating NOx species, the problem is not enough NO. The problem of nitrated proteins in neurodegenerative diseases is from not enough NO, not from too much.
Of course what is a "poison" in one circumstance may be an absolutely necessary regulatory pathway in another. Normally cytochrome c oxidase is tonally inhibited by NO, which blocks O2 from binding and is the major regulatory pathway by which mitochondria regulate their O2 consumption. If you look at figure 1, you can see that in the absence of NO, mitochondria consume O2 down to ~5 microMolar. The solubility of O2 in plasma (assume same as in cytoplasm) at 1 atmosphere is 23 cm3/L (at 38 C) or about 1.1 mM/L. 5 micromolar O2 is then a partial pressure of about 0.0045 atm or 3.42 Torr.
The only reason mitochondria can regulate their O2 consumption is because NO "poisons" cytochrome c oxidase and inhibits O2 consumption. Remove that inhibition and mitochondria consume O2 to very low partial pressure, well below what is the "normal" basal O2 level at the location of the mitochondria. At "rest", the O2 flux to a mitochondrion in the heart is 1. The O2 consumption by that mitochondrion can increase by 10x. The flux of O2 from the blood vessel to the mitochondrion is purely passive down a concentration gradient. For the flux to go up 10x, either the gradient has to go up 10x, or the distance has to go down by 10x because the concentration at the blood vessel stays the same. For the gradient to go up by 10x, the concentration at the mitochondrion has to go down, and go down a lot, by a factor of 10x. It has to go down while the mitochondrion is increasing its O2 consumption by 10x. The specific O2 consumption by that mitochondrion, moles O2/mg protein/Torr O2 has to go up by a factor of ~100. This is only achieved by removing the "poisoning" of cytochrome c oxidase by NO during the “at rest” condition.
With NO blocking cytochrome c oxidase, the electron respiration chain becomes fully reduced and O2 can pick up electrons from complex III, forming superoxide. This superoxide is vectorally produced in the inner matrix, where MnSOD dismutates it at near diffusion controlled kinetics. NO also reacts with superoxide at near diffusion controlled kinetics and it is the destruction of NO by superoxide generated by too great a reduction of the respiration chain that lowers the NO level and disinhibits cytochrome c oxidase so that O2 can consume electrons (and be reduced to water).
If this light does dissociate NO from cytochrome c oxidase it would cause the respiration chain to become more oxidized and would reduce the quantity of superoxide formed. This would increase NO levels and upregulate ATP levels via sGC.
An analogy by looking at the regulation of an engine would be to consider that the “throttle” “poisons” the engine so that the engine does not run at 100% full throttle 100% of the time. What the Magic Light Helmet might be doing is bypassing the normal mitochondria regulation, an effect analogous to jury rigging a separate wire to the carburetor to bypass the normal controls. If you had only one mitochondrion to control, perhaps external control might work. Each cell has many thousands, if not millions of mitochondria. If they are not working together and sharing the load, some are working harder than others, consuming O2 that others could consume, generating non-physiological O2 gradients perhaps generating non-physiological gradients in ATP, or in other substrates consumed by mitochondria. A better analogy would be to think of it as a power grid, with millions of engines connected together. Removing the throttles on some of them might improve the total power production of the grid, until those engines running at 100% full power 100% of the time start to fail, or until the idle engines (those that are not doing anything) get shut down. Of course if the engines running at 100% full throttle were uniformly distributed it might work out. If they were only the ones within 50 miles of the ocean (analogous to limited light transmission in the brain), the non-uniform distribution might set up instabilities in power flow leading to roving brownouts or blackouts or even grid failures. Maybe not too bad for a carbureted engine, but what if they are fuel injected? What if your jury-rigged control bypass only delivered extra fuel to some cylinders and not others? Do you start getting bad vibration? Do you start getting bad fuel-air mixtures? Too rich in some and too lean in others? If you were as ignorant about engines as we are about physiology it would be easy to do serious damage due to ignorance. Of course the more ignorant you are, the more difficult it is to tell just how ignorant you are.
A figure that shows the mitochondria respiration chain is here. There is control mediated all along the respiration chain. Exactly how the respiration chain is regulated under what conditions in which tissue compartments under what normal and abnormal circumstances is a good question that all of the senior researchers working in the field would like to understand. This particular paper is about nitration of proteins in mitochondria under conditions of hypoxia and denitration when that hypoxia is removed. This nitration and denitration occurs rapidly and reproducibly. It is likely some sort of control mechanism for controlling each of the proteins that get nitrated and denitrated. The nitration is likely mediated via NO reacting with superoxide and forming peroxynitrite, or forming NO2, both of which can nitrate proteins. Precisely how that happens is unknown. Virtually all the nitration is on tyrosines which are in specific parts of the enzymes that are so regulated. Tyrosine gets nitrated more than other things because it is aromatic (has a benzene ring in it) and the pendant groups on that benzene ring direct agents that do nitration to specific carbons on that benzene ring. Tyrosine also tends to form tyrosyl radicals, that has an unpaired electron distributed on the aromatic ring (which stabilizes it somewhat). Tyrosyl radicals are much more reactive than tyrosine. It may be (actually is quite likely) that formation of tyrosyl radicals and then quenching of that radical by something else is likely part of a regulatory system.
Low ATP caused by low NO hypothesis of Alzheimer's
One of the most consistent symptoms of Alzheimer's is a reduction in brain metabolism. Amyloid (and all the other protein aggregates of the neurodegenerative disorders) is normally cleared by ATP powered proteases in the proteasome, or during autophagy (which requires an ATP powered pH gradient). My hypothesis of Alzheimer's is that the buildup of amyloid is a natural compensatory mechanism to cope with there being not enough ATP. There are simply more important things to use ATP for (when there is not enough) than to use it to get rid of amyloid (or to make new mitochondria). If the ATP setpoint is too low, my hypothesis is that ATP conservation pathways are invoked (which include not getting rid of amyloid) as in ischemic preconditioning. I discuss this in my blog on fevers and autism . Getting rid of garbage is something that can always be put off "a little bit longer" if there is something better to do. I think that Alzheimer's happens when that heuristic is taken a bit too far.
Just about every other physiological function inside neurons is more important than getting rid of amyloid. Nerve conduction is a lot more important. If your nerves are not conducting properly, a bear could catch and eat you. Keeping nerves alive is more important too. Amyloid buildup causes essentially no problems for quite a while, months, even years. My mother and both her parents died with advanced Alzheimer's, so I am not being flip, I know what it can do. But that the brain can function so well and for so long with such a serious decline in metabolism is quite remarkable to me. One of the things that has helped me in my research is that I inherited my mother's low nitric oxide physiology, so I experienced quite dramatic changes when I corrected it (I appreciate that my experiences are anecdotes).
The Magic Light Helmet might work in the short term for people who already have low ATP. I would be very concerned how it modifies the normal regulatory pathway(s) by which the number of mitochondria per neuron is regulated. My research indicates that regulation involves formation of long lived NOx species which accumulate in mitochondria and which release NO during autophagy and then trigger appropriate mitochondria biogenesis. I think those long lived NOx species include nitrated proteins, which derive only from the combination of NO and superoxide. The formation of superoxide by mitochondria under metabolic “stress” is then the signal by which the cell “measures” the metabolic stress of the mitochondria it has, and then infers how many it needs to make. In a neuron that number can be different by 3 or more orders of magnitude depending on the length of the axons.
In the rat CNS, mitochondria lifetime is on the order of a month. How long it is in humans is unknown. It might be somewhat longer, but likely not more than an order of magnitude. The turnover time for mitochondria might be inferable from the progression rates of some neurodegenerative diseases. If we assume that a neurodegenerative disease affects mitochondria biogenesis, and is not acutely toxic to mitochondria, then the minimum course of that disease might reflect mitochondria turnover. ALS tends to have a course longer than a certain minimum length. A more stringent criteria might be the woman who received an acute toxic dose of dimethyl mercury but had no symptoms for 5 months and was dead at 7 months post exposure. Her symptoms were consistent with neurodegeneration. If the mercury had acutely poisoned mitochondria her death would have been much sooner. If it only poisoned mitochondria biogenesis, then her survival to 5 and 7 months might reflect how long her inventory of mitochondria at exposure could sustain neuronal activity. In any case, a treatment for Alzheimer’s has to extend beyond several mitochondria lifetimes to be considered an effective treatment.
The Magic Light Helmet might slow the progression of Alzheimer’s for a while by operating existing mitochondria in a way that generates less superoxide. But that would likely compromise the regulation of mitochondria by superoxide. Mitochondria regulation by superoxide includes regulation of O2 consumption under normoxia and hypoxia, and very likely includes regulation of mitochondria number. The progression of Alzheimer’s could greatly accelerate at some future time when mitochondria wear out faster than they are replaced.
A (poor) analogy would be to suppose that one had a car with 100,000 miles on it, and it is starting to run not as well as it used to. A salesman says he can "fix" the car by installing nitrous oxide injection and by adding nitromethane to the fuel. Sure enough when you do that, the car "runs" better than it ever has. But if the car had been nursed along gently, it could have easily gone 150,000 miles, but with nitromethane it only goes 100,500. That is why long term trials with the proper endpoints (i.e. death) are most appropriate for terminal conditions like Alzheimer's. Four weeks is too short. I imagine that the right dose of cocaine or amphetamine might produce "improvement" in Alzheimer's for 4 weeks but would accelerate decline and hasten death.
On the Science Based Medicine blog, neurocritic made a comment about the result reported on the very rapid improvement of Alzheimer's on enterocept injection (into the spine). I see this as consistent with the low NO hypothesis of Alzheimer's with NO being what regulates the acute functional connectivity in the brain. I replied to neurocritic in this comment.
Dysregulation of cytochrome P450 metabolism
Cytochrome c oxidase is not the only heme enzyme inhibited by NO. All the cytochrome P450 enzyme are too. Many of them are active in the brain. How is changing the operating point of all of those enzymes going to change things? Very complexly, and very likely not in only benign ways.
There are about 60 cytochrome P450 enzymes, which synthesize such things as steroids, cholesterol via a reaction cycle that generates superoxide. Normally the P450 enzymes generate significant superoxide which is vectorally produced to the inside of the microsomes the enzyme is active in. Microsomes normally have superoxide dismutase and catalase inside them. Testosterone synthesis is known to be inhibited by NO.
Dysregulation of catecholamine metabolism
I bring up catecholamine metabolism because many aspects of it are also quite involved with free radical chemistry and so would likely be susceptible to disruption by NIR. Parkinson’s disease has some similarities to Alzheimer’s (it is characterized by buildup of protein inclusions too, but of a different composition). Dopamine pathways are highly involved in feelings of wellbeing. Parkinson’s disease-like symptoms can be reliably induced by killing certain nerves associated with the dopamine pathways. The usual experimental mechanism is with a compound that damages mitochondria (MPTP) in those neurons, causing their death via oxidative stress and ATP depletion.
Dysregulation of acetylcholinesterase metabolism
NIR does affect the activity of acetylcholinesterase enzymes in red blood cells (abstract only). Not surprisingly, the effect is complex with low levels reducing it and higher levels increasing it. Which is the Magic Light Helmet doing? Neither or some of both?
Acetylcholinesterase inhibitors are used to treat Alzheimer’s. There is a treatment effect, and the treatment effect is dose dependant, but the treatment effect is small. There does seem to be an association of increased executive function with increased inhibition of acetylcholinesterase.
If the Magic Light Helmet is inhibiting acetylcholinesterase, it is doing so in a spatially non-uniform manner. It might even be increasing activity in some parts of the brain and decreasing it in others. Because some nerve cells are larger than the region where the light has a single flux (and hence a single dose-response), some cells might experience both increased and decreased acetylcholinesterase activity but in different regions.
Expression of acetylcholinesterase is ultimately regulated in the cell body because that is where the DNA is that codes for it. If one axon of a nerve has it inhibited and one has it enhanced, how does the cell decide how much to make? What does that do to the actual operation and long term regulation of those two different axons? It would be extremely unlikely that the nerve cells most in need of having their acetylcholinesterase enzyme activity modified by the Magic Light Helmet were located in the regions of the brain where that could actually happen.
Safety
With no understanding of the mechanism there is no basis for saying if it is safe or not.
They claim it works, but offer no physiological explanation. They have a 6 week open label trial, but with no controls and no blinding of patients or investigators. All of physiology is non-linear. You can’t extrapolate from known conditions to unknown conditions when the underlying phenomena are non-linear (and unknown). The lifetime of mitochondria is likely longer than 6 weeks. Adverse effects might not show up during that time.
What concerns me most about this device is that the investigators don't seem to be asking the right questions about what it is actually doing, if it is doing anything. Rushing to human testing is (in my opinion) quite premature with something with little to no understanding behind it. This could easily be a situation where anecdotes were used instead of actual clinical trials and people become seriously injured.
Summary:
In summary, I have presented several plausible (but speculative) physiological mechanisms by which the Magic Light Helmet could have actual physiological effects. In none of these mechanisms does the Magic Light Helmet produce an effect that is representative of actual physiological needs that the brain has. The Magic Light Helmet doesn't regulate anything by any mechanism that is known or understood.
If it does any of those things it is likely to do so indiscriminately. There is nothing "magic" about 1072 nM, these investigators looked at a handful of wavelengths (where LEDs are available for cheap). Something like that might have a short term positive effect but is very likely to be very bad in the longer term. Physiology is too complex and too coupled to simply whack away at it indiscriminately and make it "better".
This is a more elaborate explanation of the quick comment I made at Science Based Medicine. My explanation is based on my understanding of the cause of Alzheimer's (which is not one of the common explanations (all of which I am quite skeptical of (in other words I think they are wrong)). I see Alzheimer's as being strictly due to low NO which then causes low ATP (via sGC), and invokes ischemic preconditioning (IP). The low perfusion is secondary to the low NO. Ischemic preconditioning can be mediated by oxidative stress in multiple ways. Normally IP is thought of as a good thing because it does reduce damage during ischemia. IP is invoked by brief periods of ischemia and after it is invoked organisms can survive periods of ischemia that would have been fatal. IP is a low ATP state, and it is also a low ATP consuming state. If organisms could be in the low ATP consuming status of the IP state long term, they would have evolved to do so. Spending less ATP on basal metabolism means more ATP for reproduction. Organisms haven't evolved to do so, which means there must be something incompatible with life and/or reproduction from being in the IP state long term. Neurons don't divide, so there must be something incompatible with long term survival for neurons to be on the IP state long term. I discuss more details of why accumulation of amyloid is a major outcome in Alzheimer’s below.
I am assuming that this Magic Light Helmet actually has physiological effects beyond the placebo effect. It might not. The placebo effect is mediated through NO, and all disorders associated with low NO are very susceptible to being improved by the placebo effect. That would include Alzheimer's. I will use the analogy of an internal combustion engine a lot. Not because it is an especially good analogy, but because it is easy to understand how bypassing the very simple controls of an engine to get “better performance” could easily shorten its lifetime by orders of magnitude. What does bypassing the extremely complex control system of the brain do? Most of which is completely unknown (but is likely already disrupted for that brain to have Alzheimer’s in the first place)? It is extremely unlikely (virtually inconceivable) that bypassing the normal control system of the brain would only have benign effects.
We know that Alzheimer's is not caused by a "deficiency" of light at 1072 nM. This Magic Light Helmet is not replacing some physiological need that is missing in Alzheimer's. The fundamental problem of Alzheimer's is low ATP supply (discussed below). Since the brain does not have photosynthetic centers to capture the energy of light at 1072 nM and convert that energy into ATP, any change in ATP supply can occur only via changing the regulation of existing ATP production systems via mechanisms susceptible to light at 1072 nM. Since physiology didn't evolve to be physiologically regulated by an external source of 1072 nM light these lights must be doing something else, something that is non-physiologic. Even if there are physiological receptors of 1072 nM light, they would be for sensory purposes (maybe setting day/night biorhythms?) and the levels used in the magic light helmet are many orders of magnitude greater, and narrow band rather than broad band natural sources (sunlight or fire).
If this NIR light does do "something", it pretty much has to be via modulation of kinetics of either free radical reactions, decay of "excited transition states" in activated molecules, or in photodissociation of things from other things (such as NO from cytochrome c oxidase). There could easily be hundreds of things that are being affected, none of which are well understood. Or it might just be placebo.
There are at least 4 main regions which might be important and which I will discuss, by far the most important is ATP production in mitochondria and I spend most of my time on that. Synthesis of compounds by the cytochrome P450 enzymes, catecholamine metabolism and acetylcholinesterase activity might be important too. There might be others, but these are 4 important ones, changes in any of them could conceivably be consistent with the "data" that a handful of non-blinded observers noticed improvement in 6 weeks of use. Although I will be discussing these somewhat separately, they are not separate. Physiology doesn't divide what it does into neat little orthogonal and independent boxes. In physiology everything is coupled to everything else. There is no evolutionary driving force to keep things separate or modular. There are only evolutionary driving forces for survival, and for economy (so more resources are available for reproduction).
ATP production
In neurons virtually all ATP is made by mitochondria through oxidation of substrates, lactate, ketone bodies, small acids such as acetate, aspartate. Mitochondria are small organelles that have a few thousand proteins, 13 of which are coded for by mitochondrial DNA, all the others are coded by nuclear DNA. Mitochondria have 2 lipid membranes, inside the inner one is where the DNA is and the protein manufacture stuff (the mitochondria matrix) and the enzymes of the citrate cycle and most everything that mitochondria do. Mitochondria work by generating an electrical potential and a pH gradient across that inner membrane. The different respiration complexes take electrons and protons from chemical compounds and extract energy from the chemical reactions as those electrons and protons are moved across the membrane and store that energy in the electrical and pH gradient. In the terminal enzyme, cytochrome c oxidase those electrons are gathered and 4 of them are simultaneously put onto O2 along with 4 protons making two molecules of water. Doing so many things all at once is a “tricky” and complicated thing for an enzyme to do. That energy gradient is then used to make ATP.
The 13 proteins are all parts of the respiration chain, usually the part containing the active site. All animals (except for a few invertebrates) have these same 13 proteins coded in their mitochondria. Plants have a few extra. These proteins are all large and quite hydrophobic. Why these (and only these) proteins are coded in mitochondria is not understood. I think it has to do with regulation of mitochondria, some of which has to be local to each mitochondrion and sometimes that regulation means turning off part of the respiration chain and then turning it back on. One mechanism might be destroying the protein and then making the protein again. In most cells mitochondria are close to the nucleus so that proteins can be made from DNA in the nucleus and then transported to mitochondria (in principle, whether this happens or not is unknown). In neurons that can't happen because the distance between the cell body (where the nuclear DNA is and the protein synthesis capacity) and mitochondria can be inches or even a meter in motor neurons. There simply isn't time for a signal to propagate from mitochondria to the cell body, trigger protein synthesis and then transport proteins out to mitochondria in need of them. If protein synthesis is needed for control of mitochondria, that synthesis must occur locally using locally available DNA.
If neurons are deprived of substrates, O2 and organic compounds, disruption of neuronal function (unconsciousness) occurs in seconds.
NIR does cause the photodissociation of “poisons” from cytochrome c oxidase. This is discussed in terms of NO in a recent article in Nature. They get the basic chemistry right, however they get the implications of how NO fits in physiology wrong. The problem is not too much NO, the problem is too little. One of the papers cited by the Nature article epitomizes much of what is misunderstood in the NO field (but the author is quite senior, so I won't link to the article). NO and superoxide can form peroxynitrite, however superoxide from mitochondria is confined to the mitochondria matrix, there are 2 lipid membranes the superoxide has to go through before it can get to the cytosol. Both of those lipid membranes have ~10x higher NO levels than the cytoplasm (NO partitions into lipid ~10x over aqueous). NO reacts with superoxide at near diffusion limited kinetics. It is completely implausible to me that superoxide could get out of the mitochondria matrix. Particularly when the mitochondrial potential (~140 mV) is going to keep the negatively charged superoxide inside. When peroxynitrite forms in the mitochondria inner matrix, it is consumed by cytochrome c oxidase, or decomposes generating NO2 (which is also a signaling molecule).
NO and superoxide only form peroxynitrite at near stoichiometric ratios. When NO or superoxide is in excess that doesn't happen. A much better conceptualization of what happens when NO and superoxide are generated in cells is by Wink et al. They make a very important (and correct) statement
"We demonstrate that the primary consequence of [superoxide] O2- generation concomitant with NO production is not the toxicity associated with the formation of higher nitrogen oxides, but rather the resultant phenotypic cellular changes that occur because of limiting the bioavailability of NO and H2O2."
This is precisely correct. The problem of oxidative stress is not the presence of nitrating NOx species, the problem is not enough NO. The problem of nitrated proteins in neurodegenerative diseases is from not enough NO, not from too much.
Of course what is a "poison" in one circumstance may be an absolutely necessary regulatory pathway in another. Normally cytochrome c oxidase is tonally inhibited by NO, which blocks O2 from binding and is the major regulatory pathway by which mitochondria regulate their O2 consumption. If you look at figure 1, you can see that in the absence of NO, mitochondria consume O2 down to ~5 microMolar. The solubility of O2 in plasma (assume same as in cytoplasm) at 1 atmosphere is 23 cm3/L (at 38 C) or about 1.1 mM/L. 5 micromolar O2 is then a partial pressure of about 0.0045 atm or 3.42 Torr.
The only reason mitochondria can regulate their O2 consumption is because NO "poisons" cytochrome c oxidase and inhibits O2 consumption. Remove that inhibition and mitochondria consume O2 to very low partial pressure, well below what is the "normal" basal O2 level at the location of the mitochondria. At "rest", the O2 flux to a mitochondrion in the heart is 1. The O2 consumption by that mitochondrion can increase by 10x. The flux of O2 from the blood vessel to the mitochondrion is purely passive down a concentration gradient. For the flux to go up 10x, either the gradient has to go up 10x, or the distance has to go down by 10x because the concentration at the blood vessel stays the same. For the gradient to go up by 10x, the concentration at the mitochondrion has to go down, and go down a lot, by a factor of 10x. It has to go down while the mitochondrion is increasing its O2 consumption by 10x. The specific O2 consumption by that mitochondrion, moles O2/mg protein/Torr O2 has to go up by a factor of ~100. This is only achieved by removing the "poisoning" of cytochrome c oxidase by NO during the “at rest” condition.
With NO blocking cytochrome c oxidase, the electron respiration chain becomes fully reduced and O2 can pick up electrons from complex III, forming superoxide. This superoxide is vectorally produced in the inner matrix, where MnSOD dismutates it at near diffusion controlled kinetics. NO also reacts with superoxide at near diffusion controlled kinetics and it is the destruction of NO by superoxide generated by too great a reduction of the respiration chain that lowers the NO level and disinhibits cytochrome c oxidase so that O2 can consume electrons (and be reduced to water).
If this light does dissociate NO from cytochrome c oxidase it would cause the respiration chain to become more oxidized and would reduce the quantity of superoxide formed. This would increase NO levels and upregulate ATP levels via sGC.
An analogy by looking at the regulation of an engine would be to consider that the “throttle” “poisons” the engine so that the engine does not run at 100% full throttle 100% of the time. What the Magic Light Helmet might be doing is bypassing the normal mitochondria regulation, an effect analogous to jury rigging a separate wire to the carburetor to bypass the normal controls. If you had only one mitochondrion to control, perhaps external control might work. Each cell has many thousands, if not millions of mitochondria. If they are not working together and sharing the load, some are working harder than others, consuming O2 that others could consume, generating non-physiological O2 gradients perhaps generating non-physiological gradients in ATP, or in other substrates consumed by mitochondria. A better analogy would be to think of it as a power grid, with millions of engines connected together. Removing the throttles on some of them might improve the total power production of the grid, until those engines running at 100% full power 100% of the time start to fail, or until the idle engines (those that are not doing anything) get shut down. Of course if the engines running at 100% full throttle were uniformly distributed it might work out. If they were only the ones within 50 miles of the ocean (analogous to limited light transmission in the brain), the non-uniform distribution might set up instabilities in power flow leading to roving brownouts or blackouts or even grid failures. Maybe not too bad for a carbureted engine, but what if they are fuel injected? What if your jury-rigged control bypass only delivered extra fuel to some cylinders and not others? Do you start getting bad vibration? Do you start getting bad fuel-air mixtures? Too rich in some and too lean in others? If you were as ignorant about engines as we are about physiology it would be easy to do serious damage due to ignorance. Of course the more ignorant you are, the more difficult it is to tell just how ignorant you are.
A figure that shows the mitochondria respiration chain is here. There is control mediated all along the respiration chain. Exactly how the respiration chain is regulated under what conditions in which tissue compartments under what normal and abnormal circumstances is a good question that all of the senior researchers working in the field would like to understand. This particular paper is about nitration of proteins in mitochondria under conditions of hypoxia and denitration when that hypoxia is removed. This nitration and denitration occurs rapidly and reproducibly. It is likely some sort of control mechanism for controlling each of the proteins that get nitrated and denitrated. The nitration is likely mediated via NO reacting with superoxide and forming peroxynitrite, or forming NO2, both of which can nitrate proteins. Precisely how that happens is unknown. Virtually all the nitration is on tyrosines which are in specific parts of the enzymes that are so regulated. Tyrosine gets nitrated more than other things because it is aromatic (has a benzene ring in it) and the pendant groups on that benzene ring direct agents that do nitration to specific carbons on that benzene ring. Tyrosine also tends to form tyrosyl radicals, that has an unpaired electron distributed on the aromatic ring (which stabilizes it somewhat). Tyrosyl radicals are much more reactive than tyrosine. It may be (actually is quite likely) that formation of tyrosyl radicals and then quenching of that radical by something else is likely part of a regulatory system.
Low ATP caused by low NO hypothesis of Alzheimer's
One of the most consistent symptoms of Alzheimer's is a reduction in brain metabolism. Amyloid (and all the other protein aggregates of the neurodegenerative disorders) is normally cleared by ATP powered proteases in the proteasome, or during autophagy (which requires an ATP powered pH gradient). My hypothesis of Alzheimer's is that the buildup of amyloid is a natural compensatory mechanism to cope with there being not enough ATP. There are simply more important things to use ATP for (when there is not enough) than to use it to get rid of amyloid (or to make new mitochondria). If the ATP setpoint is too low, my hypothesis is that ATP conservation pathways are invoked (which include not getting rid of amyloid) as in ischemic preconditioning. I discuss this in my blog on fevers and autism . Getting rid of garbage is something that can always be put off "a little bit longer" if there is something better to do. I think that Alzheimer's happens when that heuristic is taken a bit too far.
Just about every other physiological function inside neurons is more important than getting rid of amyloid. Nerve conduction is a lot more important. If your nerves are not conducting properly, a bear could catch and eat you. Keeping nerves alive is more important too. Amyloid buildup causes essentially no problems for quite a while, months, even years. My mother and both her parents died with advanced Alzheimer's, so I am not being flip, I know what it can do. But that the brain can function so well and for so long with such a serious decline in metabolism is quite remarkable to me. One of the things that has helped me in my research is that I inherited my mother's low nitric oxide physiology, so I experienced quite dramatic changes when I corrected it (I appreciate that my experiences are anecdotes).
The Magic Light Helmet might work in the short term for people who already have low ATP. I would be very concerned how it modifies the normal regulatory pathway(s) by which the number of mitochondria per neuron is regulated. My research indicates that regulation involves formation of long lived NOx species which accumulate in mitochondria and which release NO during autophagy and then trigger appropriate mitochondria biogenesis. I think those long lived NOx species include nitrated proteins, which derive only from the combination of NO and superoxide. The formation of superoxide by mitochondria under metabolic “stress” is then the signal by which the cell “measures” the metabolic stress of the mitochondria it has, and then infers how many it needs to make. In a neuron that number can be different by 3 or more orders of magnitude depending on the length of the axons.
In the rat CNS, mitochondria lifetime is on the order of a month. How long it is in humans is unknown. It might be somewhat longer, but likely not more than an order of magnitude. The turnover time for mitochondria might be inferable from the progression rates of some neurodegenerative diseases. If we assume that a neurodegenerative disease affects mitochondria biogenesis, and is not acutely toxic to mitochondria, then the minimum course of that disease might reflect mitochondria turnover. ALS tends to have a course longer than a certain minimum length. A more stringent criteria might be the woman who received an acute toxic dose of dimethyl mercury but had no symptoms for 5 months and was dead at 7 months post exposure. Her symptoms were consistent with neurodegeneration. If the mercury had acutely poisoned mitochondria her death would have been much sooner. If it only poisoned mitochondria biogenesis, then her survival to 5 and 7 months might reflect how long her inventory of mitochondria at exposure could sustain neuronal activity. In any case, a treatment for Alzheimer’s has to extend beyond several mitochondria lifetimes to be considered an effective treatment.
The Magic Light Helmet might slow the progression of Alzheimer’s for a while by operating existing mitochondria in a way that generates less superoxide. But that would likely compromise the regulation of mitochondria by superoxide. Mitochondria regulation by superoxide includes regulation of O2 consumption under normoxia and hypoxia, and very likely includes regulation of mitochondria number. The progression of Alzheimer’s could greatly accelerate at some future time when mitochondria wear out faster than they are replaced.
A (poor) analogy would be to suppose that one had a car with 100,000 miles on it, and it is starting to run not as well as it used to. A salesman says he can "fix" the car by installing nitrous oxide injection and by adding nitromethane to the fuel. Sure enough when you do that, the car "runs" better than it ever has. But if the car had been nursed along gently, it could have easily gone 150,000 miles, but with nitromethane it only goes 100,500. That is why long term trials with the proper endpoints (i.e. death) are most appropriate for terminal conditions like Alzheimer's. Four weeks is too short. I imagine that the right dose of cocaine or amphetamine might produce "improvement" in Alzheimer's for 4 weeks but would accelerate decline and hasten death.
On the Science Based Medicine blog, neurocritic made a comment about the result reported on the very rapid improvement of Alzheimer's on enterocept injection (into the spine). I see this as consistent with the low NO hypothesis of Alzheimer's with NO being what regulates the acute functional connectivity in the brain. I replied to neurocritic in this comment.
Dysregulation of cytochrome P450 metabolism
Cytochrome c oxidase is not the only heme enzyme inhibited by NO. All the cytochrome P450 enzyme are too. Many of them are active in the brain. How is changing the operating point of all of those enzymes going to change things? Very complexly, and very likely not in only benign ways.
There are about 60 cytochrome P450 enzymes, which synthesize such things as steroids, cholesterol via a reaction cycle that generates superoxide. Normally the P450 enzymes generate significant superoxide which is vectorally produced to the inside of the microsomes the enzyme is active in. Microsomes normally have superoxide dismutase and catalase inside them. Testosterone synthesis is known to be inhibited by NO.
Dysregulation of catecholamine metabolism
I bring up catecholamine metabolism because many aspects of it are also quite involved with free radical chemistry and so would likely be susceptible to disruption by NIR. Parkinson’s disease has some similarities to Alzheimer’s (it is characterized by buildup of protein inclusions too, but of a different composition). Dopamine pathways are highly involved in feelings of wellbeing. Parkinson’s disease-like symptoms can be reliably induced by killing certain nerves associated with the dopamine pathways. The usual experimental mechanism is with a compound that damages mitochondria (MPTP) in those neurons, causing their death via oxidative stress and ATP depletion.
Dysregulation of acetylcholinesterase metabolism
NIR does affect the activity of acetylcholinesterase enzymes in red blood cells (abstract only). Not surprisingly, the effect is complex with low levels reducing it and higher levels increasing it. Which is the Magic Light Helmet doing? Neither or some of both?
Acetylcholinesterase inhibitors are used to treat Alzheimer’s. There is a treatment effect, and the treatment effect is dose dependant, but the treatment effect is small. There does seem to be an association of increased executive function with increased inhibition of acetylcholinesterase.
If the Magic Light Helmet is inhibiting acetylcholinesterase, it is doing so in a spatially non-uniform manner. It might even be increasing activity in some parts of the brain and decreasing it in others. Because some nerve cells are larger than the region where the light has a single flux (and hence a single dose-response), some cells might experience both increased and decreased acetylcholinesterase activity but in different regions.
Expression of acetylcholinesterase is ultimately regulated in the cell body because that is where the DNA is that codes for it. If one axon of a nerve has it inhibited and one has it enhanced, how does the cell decide how much to make? What does that do to the actual operation and long term regulation of those two different axons? It would be extremely unlikely that the nerve cells most in need of having their acetylcholinesterase enzyme activity modified by the Magic Light Helmet were located in the regions of the brain where that could actually happen.
Safety
With no understanding of the mechanism there is no basis for saying if it is safe or not.
They claim it works, but offer no physiological explanation. They have a 6 week open label trial, but with no controls and no blinding of patients or investigators. All of physiology is non-linear. You can’t extrapolate from known conditions to unknown conditions when the underlying phenomena are non-linear (and unknown). The lifetime of mitochondria is likely longer than 6 weeks. Adverse effects might not show up during that time.
What concerns me most about this device is that the investigators don't seem to be asking the right questions about what it is actually doing, if it is doing anything. Rushing to human testing is (in my opinion) quite premature with something with little to no understanding behind it. This could easily be a situation where anecdotes were used instead of actual clinical trials and people become seriously injured.
Summary:
In summary, I have presented several plausible (but speculative) physiological mechanisms by which the Magic Light Helmet could have actual physiological effects. In none of these mechanisms does the Magic Light Helmet produce an effect that is representative of actual physiological needs that the brain has. The Magic Light Helmet doesn't regulate anything by any mechanism that is known or understood.
If it does any of those things it is likely to do so indiscriminately. There is nothing "magic" about 1072 nM, these investigators looked at a handful of wavelengths (where LEDs are available for cheap). Something like that might have a short term positive effect but is very likely to be very bad in the longer term. Physiology is too complex and too coupled to simply whack away at it indiscriminately and make it "better".
Sunday, April 15, 2007
Placebo and nocebo effects
Involovement of nitric oxide
Some of this derives from a couple posters I presented at a scientific conference. Send me your email address (via posting a message with email and then deleting it) and I will send you a copy. I get quite a bit into ATP physiology, but that is necessary to understand why the placebo effect actually works to promote healing.
One of the strongest effects in medicine is the placebo (I will please) effect, yet some of the known physiological mechanisms behind this effect are not well known or appreciated, and the details are still not fully understood. The nocebo (I will harm) effect is similar, and is usually considered to be the opposite of the placebo effect, but actually is somewhat different.
Contrary to popular belief, both of these effects are misnamed, in that harmful effects can occur via placebos, and beneficial effects via nocebos. Contrary to popular belief, these placebo and nocebo effects are quite real and can be completely indistinguishable from effects due to efficacious treatments, including improved healing, decreased pain, and these effects can often be detected instrumentally.
Virtually any treatment can have some effect, including those that have no conceivable physical mechanism for working, including homeopathy, Chi manipulation, prayer, sacrificing animals, treatment of surrogates, sham devices and pharmacologically inert pills. An interesting rapid response to this article is from a Dr. Jane Woo (who I presume is perhaps "the expert" on placebos), saying "One of my residents once said that he advocated morphine injections, as opposed to tablets, because, "There's something about steel hitting skin and having a doctor say, 'This is going to make you feel better.' Injections simply work better than pills.""
One of the earliest "treatments" that children receive from their mothers is known as "kiss it and make it better". Any parent anecdotally knows that this is an "effective" treatment. While saliva does have nitrite from the reduction of salivary nitrate by commensal bacteria on the tongue, usually a motherly healing kiss is insufficiently slobbery to transfer sufficient nitrite, and the therapeutic effect is faster than nitrite or NO transfer from the treated boo-boo would allow.
Most placebo research has been associated with pain relief, and increased analgesia from placebo effects is well documented. I will focus this discussion more on non-pain effects of placebos and nocebos, and specifically on how placebos actually do improve healing.
It is well known that physiology is extremely complicated, and the regulation achieved by normal physiology is exquisite. So exquisite, that it has been endowed with the imaginary and mythical property of "homeostasis". In reality, nothing in physiology is static, rather our inability to measure the changes that we know must be present simply leaves us ignorant of those changes. While the default assumption of stasis is simple, it is clearly an assumption based on ignorance, and is clearly wrong. But this blog is about placebos, not homeostasis.
One of the best regulated physiological parameters is the ATP concentration. Not surprising because ATP is used by just about every physiological process, hundreds of thousands, if not millions of different pathways, in each cell, regulated simultaneously.
So how is ATP regulated? The answer is, extremely well!
ATP is considered to be one of the mythic "homeostatic" parameters, that is regulated to be constant. But that cannot be correct. The only way a parameter can be regulated is via feedback which necessitates a deviation from a setpoint followed by a compensatory response. Our inability to measure that deviation does not mean it does not exist. It only shows that our instruments are insufficiently sensitive and precise.
ATP cannot be measured in individual cells on the length and time scales where it matters, at least not non-destructively. The usual way is to freeze the tissue by clamping it with liquid nitrogen cooled copper tongs, then taking a small piece and assaying it. Depending on technique, the assay might be the average of only 100 cells or so. More likely a few thousand.
We know that ATP is regulated within individual cells because ATP doesn't diffuse through lipid membranes. It is difficult to get any information on the dynamics of ATP production and consumption by destructively measuring the average of a few hundred cells. Most any parameter would look pretty "constant" if the only measurements were averages of a few thousand values. If the only measurements of heart rate were averages over an hour (3600 seconds) wouldn't it look pretty constant too? You could measure the increase due to a marathon (2-3 data points), but not from a 4 minute mile.
ATP is not stored; the instantaneous production always equals the instantaneous consumption. If this were not the case, then there would be rapid accumulation or depletion of ATP. Each mole of glucose that is oxidized produces about 38 moles of ATP. A 2000 calorie diet then produces some 55 kg of ATP per day. Obviously, the ADP and P are recycled, and ATP production and consumption is very precisely matched.
In all control systems, the sooner you can start pulling levers to change things, and the more levers you have to pull, the better control you can exert. Physiology is no exception. Invoking physiological pathways in anticipation of a need for that pathway improves performance of the system. Neurological control of some aspects of physiology is well known. It would not be surprising if other aspects were controlled as well.
The key to understanding physiology is to understand that everything is a compromise, just like engineering. Every task that physiology needs to do has a cost in terms of ATP production to do the task, manufacture of molecules to carry out the task, DNA to code for the information to make the molecules to do the task, machinery to turn the DNA into what ever molecules are needed when they are needed, and the control system to do all of these things at the right time and in the right place, and time for all of this to happen. All of these things take up space, and have a "cost" in terms of maintenance and a "cost in forgone reproduction. Cells, and ultimately organisms that did these things most "efficiently" had more resources to use on reproduction, and so had more descendents, and so are the extant organisms we observe.
I will consider two extreme metabolic states, the "fight or flight" state (FoF), and the resting and relaxed state (RnR). It is well known that organisms can invoke such states and FoF results in the characteristic physiological effects of "stress", and if prolonged can cause physical disorders associated with "stress". RnR reverses the effects of stress, but to do so, has to occur within a certain time period.
I suggest that placebos invoke the RnR response, and that nocebos invoke the FoF response. Usually then, a placebo will promote healing and other effects associated with rest, and a nocebo will promote effects associated with stress, which usually are detrimental, but can be beneficial, as in the reduction in nausea, also in this example. The expectation of low nausea via a placebo produced greater nausea than the expectation of high nausea via the identical substance as a nocebo. In the last example, gastric tachyarrhythmia was instrumentally measured to be greater with placebo than nocebo, thus the increased gastric symptoms were not merely subjective. An explanation for this is discussed later.
We know that ATP production and consumption can vary by large amounts. Muscle for example can increase its oxygen consumption by a factor of 10. An interesting property of muscle is that it can be worked to exhaustion that is until ATP levels fall enough that the cells necrose and die. A useful "feature", when you are running from a bear. Death from exhaustion is balanced by death from being eaten by a bear. Physiological systems evolved to minimize death due to the sum of both events. Presumably, organisms do this "efficiently". That is they allocate ATP in an optimum manner to maximize organism survival. That is, ATP consumption is prioritized. Since ATP not used by a low priority pathway is as good as ATP produced, the "optimum" control system for ATP consumption will allocate ATP to the most vital pathways first, and when demand exceeds supply, turn off low priority pathways. What are "low priority" pathways? Well, anything that takes a long time (longer than the ATP crisis) must be low priority.
In the running from a bear example, anything that takes longer than the escape time can be put off. If what ever that pathway is going to produce won't happen until after you have either escaped or been eaten, it can't contribute to your escape, and so can be shut off to supply a few more molecules of ATP. In the limit of a perfectly regulated ATP system, the longest term pathways would be shut off first, then shorter term and later, still shorter term pathways as ATP demand exceeds supply. When the time horizon of the pathways being shut down reaches the present is when you drop dead from exhaustion.
One thing that can be put off is cell maintenance. Maintaining cells consumes ATP. Damaged proteins are ligated to ubiquitin, carried to the proteasome for disassembly, first unfolded by ATP powered unfoldases, and then broken into little bits by ATP powered proteases, and then replacement proteins are manufactured. All of these steps require ATP. No doubt some damaged proteins don't need to be removed immediately, but can be stored until later, until after the ATP crisis is over.
Accumulation of damaged proteins, as in amyloidosis, is common in virtually all of the disorders that are exacerbated by "stress" including obesity, diabetes, end stage kidney failure, dilative cardiomyopathy, neurodegenerative diseases, and so on. Accumulation of damaged proteins is harmful, but cells can tolerate quite large quantities and still remain viable, and there is some suggestion that such aggregation is actually protective. From my own experience (as a bachelor living alone), greater quantities of garbage can be tolerated if they are aggregated in a few places, rather than when distributed uniformly. I suggest that the "problem" is not so much increased production of these damaged proteins, but rather reduced removal. In "steady state", removal must equal production. If there is accumulation, then production exceeds removal. Presumably aggregate removal is regulated, and the "problem" of accumulation is either dysregulation, or precise regulation about a bad setpoint. The dysregulation hypothesis requires multiple cells and cell types to simultaneously develop the same dysregulation, an implausible coincidence. Since removal requires ATP, and some accumulation can be tolerated, I suggest that chronically low ATP will cause accumulation due to a bad setpoint. The "setpoint" is a function of energy status, and if there isn't enough ATP, clearing bad proteins gets put off until later. How much later? Until ATP is back up where it "should" be. What if that never happens? Then you are SOL (shit out of luck). Remember, physiological pathways can't "compensate" because it is precisely those pathways that are affected.
Reduction in metabolic rate is well known in degenerative diseases, for example in Alzheimer's there is a very well documented reduction in brain metabolism that precedes pathology. For a very striking image of this look here. As well as a reduction in metabolic activity, there is a reduction in blood flow. Blood flow is regulated by the vasodilatation produced by NO, and the vascular changes observed in Alzheimer's are consistent with low NO.
What form does the reduction in metabolism observed take? Is it a failure of 1, 10, or 100 or more pathways (of the millions the cell regulates) each pathologically consuming a little less ATP? I would presume that if only a few pathways were involved, they would necessarily represent a large fraction of normal brain metabolism, and disruption of such presumably important pathways would likely have effects more prompt than slow degradation over years. If many pathways are involved, how can many pathways simultaneously "go bad" in diverse areas of the brain? On the other hand, if it is a "bad setpoint", that is if ATP is low because of low NO (discussed later), then the brain would "gracefully" consume less ATP via the extremely robust ATP consumption hierarchies by turning off the least important pathways first, the long term maintenance pathways. This could go on for years, and may even reverse itself at times as the cells go down the low NO death spiral.
The proteasome disassembles proteins one at a time. Larger damaged assemblies including damaged mitochondria can only be disposed of via autophagy. Mitochondria have a finite life. In the rat, CNS mitochondria turnover in about a month. In other organs the turnover is faster. Mitochondria are tricky to recycle, particularly damaged mitochondria because they can be sources of superoxide and hydrogen peroxide, and because they contain abundant Fenton reactive metals which turn hydrogen peroxide into hydroxyl radicals which will damage anything they touch. I will discuss the mechanisms for putting off of autophagy during times of low ATP in a future blog. This is also directly mediated by low NO and low ATP.
Mitochondria are unique, in that they have their own DNA and ribosomes, and manufacture some of their own proteins. The vast majority of mitochondrial proteins (perhaps a couple thousand) are coded in the nucleus, synthesized in the cell's ribosome, and ported into mitochondria during mitochondria biogenesis. Only 13 proteins are coded for by mitochondrial DNA, the active sites of the respiration chain complexes. The vast majority of the complexes are coded for in the nucleus, but these are regulatory subunits, not the active sites.
The number of pathways that consume ATP is not small. For the purposes of this analysis, we need to look at each pathway separately. Rather than look at generic "protein synthesis", we need to consider synthesis of proteins (protein aaaa, protein aaab, protein, aaac… protein zzzz) all separately because that is how they are regulated. Under conditions of ATP depletion, expression of some proteins is upregulated, heat shock proteins and others. I have denoted each protein by 4 letters because that is about how many different proteins are expressed, 26^4 ~ 10^5. Each protein has on the order of a few hundred amino acids, so the number of individual steps that are involved is many millions. We know that the expression of each protein is controlled "just right" because if it wasn't, either there would be not enough, or the cell would explode from too much.
So, how does a cell control a few million pathways and prioritize them based on ATP level? What can it use as a "signal"? I suggest that it must use ATP itself. There are not enough other molecules for it to use a different molecule for each one; some must be controlled by the same molecule, but by different concentrations. For this discussion I am not particularly concerned with the mechanism(s) involved. No doubt there are many.
In a cell, there are 3 ATP parameters, ATP concentration, ATP production rate, and ATP consumption rate. These 3 parameters are independent, and can (and are) controlled independently. Since muscle can consume ATP to the point of death, low ATP will necessarily stimulate maximum ATP production. Just short of death, the cell will "want" to turn off all non-essential systems to stave off ATP depletion for as long as possible. So low ATP turns off the "housekeeping" pathways. So what sets the ATP concentration? In part, that is set by NO via soluble guanylyl cyclase and cGMP.
When physiology calls for maximum ATP production, one of the first things it does is lower NO levels, to disinhibit cytochrome c oxidase. Under basal conditions, cytochrome oxidase is mostly inhibited by NO, which blocks O2 from binding and being reduced to water, the ultimate sink for electrons. O2 consumption can go up an order of magnitude. That means an order of magnitude more O2 must diffuse to the mitochondria and be reduced to water. O2 is only transported by passive diffusion. In the lungs, O2 diffuses into the blood and is absorbed by hemoglobin forming oxyhemoglobin. The blood carries the O2Hb to tissues where the O2 comes off and diffuses to the mitochondria down a concentration gradient. The lowest O2 concentration in the body is at the mitochondria where the O2 is consumed. For the flux of O2 to increase by an order of magnitude, the O2 concentration gradient must increase an order of magnitude. How does this happen? The concentration in the blood doesn't change, the spacing between vessels and mitochondria doesn't change much, so to increase the flux, the concentration at the mitochondria must drop by an order of magnitude. Then with the higher gradient, more O2 can diffuse to the more active mitochondria and more ATP can be produced. The O2 consumption by cytochrome c oxidase increases an order of magnitude while the O2 concentration drops an order of magnitude. The specific O2 consumption (moles O2/Torr O2/mg protein) must go up 2 orders of magnitude. This is accomplished by lowering the NO level local to the mitochondria.
So the low NO necessary for disinhibition of cytochrome c oxidase also serves to lower the ATP setpoint. This lowers the ATP concentration, which turns off non-essential systems. The lower ATP concentration upregulates ATP production by the mitochondria. When mitochondria don't have enough O2, the respiration chain becomes reduced, what little O2 is present becomes reduced by single electrons, not on cytochrome c oxidase, and superoxide is formed. This superoxide destroys NO at diffusion limited kinetics, pulls down the NO level, disinhibits cytochrome c oxidase which then pulls down the O2 level allowing more O2 to diffuse to the mitochondria.
So, under conditions of FoF, the NO level is lowered. The more severe the FoF, the lower the NO level is taken. NO is a small uncharged molecule that diffuses readily through lipid membranes. The only barrier to NO in the body is crystalline bone. A state of low NO, is then propagated to all cells, so that the metabolic status of all cells can be regulated in sync. This is important because to maximize the ability to run from a bear, O2 and glucose consumption by non-essential systems must be curtailed as well as ATP consumption by muscle repair systems.
Is the hypothesis of ATP hierarchies plausible? Well, we know that physiology does behave this way. There is an effect called ischemic preconditioning, where a brief ischemic event induces a transient state where a prolonged ischemic event will produce less damage. This is well observed in a number of different organs. Transient ischemia reduces ATP demand and so cells can survive ischemia that would otherwise kill them. This behavior is what the ATP hierarchies hypothesis would predict. The mechanisms behind ischemic preconditioning are mostly unknown. No doubt as a stress response from deep evolutionary time there are many pathways involved in very complex and redundant ways, which may (is likely to) be different for different organs. Oxidative stress is known to be involved in some aspects of ischemic preconditioning.
Presumably ischemic preconditioning has some detrimental long term effects, otherwise cells would evolve to be in that state continuously. They don't, therefore there must be long term negative consequences. Those negative consequences might not show up for some time, but they must be present. This is a danger of short term endpoints in clinical trials. A treatment may prevent short term damage but if continued may cause increased long term damage.
This is one of the dangers of pain relief. If it merely masks the pain symptoms, and people then behave as if they are in the RnR state when they are actually still in the FoF state, then running themselves to death is much easier. Similarly, what do "stimulants" actually do? Do they increase the ability of cells to make ATP? Doubtful that a drug could improve on a few billion years of evolution. They do increase ATP availability (otherwise they wouldn't be stimulants), most likely by invoking the FoF state and turning off non-voluntary pathways like long term maintenance, but without the pain that normally warns of degraded repair systems.
So what happens under conditions of RnR? Well, to activate all the repair pathways, ATP needs to be high, so via sGC, NO levels have to be high too. What triggers mitochondria biogenesis is NO, so to make more mitochondria NO levels need to be high too. So RnR is a state of high NO.
How is this state of high NO produced? One mechanism is by a reduction in mitochondrial potential. To generate high ATP flux, mitochondria increase their potential to increase the driving force for ATP production. This does increase the rate, but it also increases superoxide production, a valuable feature, which pulls down the NO level to increase O2 diffusion. When the demand for ATP drops, the potential drops, the superoxide formation rate drops, the NO destruction rate drops, and the NO concentration rises provided there is sufficient basal NO production to begin with. If the basal NO production rate is too low, then the reduction in the NO destruction rate doesn't raise the NO level.
This presents a problem, if the state of FoF is prolonged sufficiently that mitochondria biogenesis suffers. The only reason that organisms have the ability to increase their metabolic activity over basal levels is because there are "excess" mitochondria. That is mitochondria in excess of the minimum necessary to supply basal ATP requirements.
Fewer mitochondria can supply the same ATP by increasing mitochondrial potential. This results in greater superoxide production, and also greater "slip", that is a reduction in the number of ATP molecules produced per mole of O2 reduced. A hallmark of many of degenerative diseases is weight loss, often inappropriately termed malnutrition, where the actual problem is increased basal metabolism. Elevated basal metabolism is observed in dilative cardiomyopathy, chronic renal failure, HIV infection, liver cirrhosis, chronic obstructive pulmonary disease, Does an increased basal metabolism mean the body is doing "more stuff"? Likely not, rather it is doing the same "basal metabolism stuff" but using ATP generated less efficiently with fewer mitochondria as observed in heart failure. It might even be doing less, because low ATP has turned off the repair pathways which is why the liver, kidneys, heart are failing in the first place. In HIV, a standard treatment is via highly active anti-retroviral therapy (HAART). A side effect of this treatment is reductions in mitochondria biogenesis. This can result in hyperlactatemia because of increased glycolysis to supply ATP. But if the liver and kidneys don't have sufficient mitochondria to recycle the lactate via the Cori cycle, where does it go? Perhaps into ectopic fat. I suspect that this is one of the problems of obesity. NO selectively partitions into lipid, and adipose tissue is a source of inflammation and oxidative stress. If NO drops sufficiently to impact mitochondria biogenesis, there may be no internal mechanism to raise it sufficiently for a long enough time to reverse the mitochondria depletion.
So how does all of this relate to the placebo effect? Well, if healing and cellular repair is accomplished most effectively during periods of RnR, then invoking that state will promote healing, well being, and long life. One of the things that does invoke feelings of rest and relaxation is love. It is well known that married people live longer lives (and it isn't just that it seems longer). The well known maternal "kiss it and make it better" treatment does relieve pain and presumably resets the RnR state. Presumably regular episodes of love and affection from a romantic partner can reset the RnR state too.
At the heart of energy metabolism is nitric oxide. A major determinant of whether an organism is in the FoF state, or the RnR state is the level of NO. Because NO is freely diffusible, and is created and destroyed at many sites in the body, the basal level has an impact on the signaling effects of NO. Low basal NO will affect every NO mediated signal with no threshold. This is an extremely important point. Anything that increases basal NO will shift physiology to the RnR state and away from the FoF state. There are many things that will do this, placebos are one of them. The relaxation response causes the production of NO. My own favorite method is via commensal ammonia oxidizing bacteria on the skin. No matter what the basal NO level is, physiology can always destroy that NO very rapidly with superoxide. Mitochondria have an essentially unlimited capacity to make superoxide, limited only by the supply of O2 and reducing equivalents. What ever the NO level is, mitochondria can pull it down to zero. This has important implications in acute respiratory distress syndrome, and is what is responsible for the multiple organ failure which sometimes occurs.
Long term meditation does result in reduced age-associated loss of cortical white matter. I presume by increased repair, improved energy status, reduced apoptosis, better clearing of damaged proteins, and perhaps increased axonogenesis. Many neurotrophic factors have effects mediated through NO.
Meditation modulates the immune system and increases antibody titers due to vaccination. Meditation reduces the symptoms of the metabolic syndrome and improves a number of heart health parameters.
If placebos increase NO levels and invoke the RnR state, then nocebos likely reduce NO levels and induce the FoF state. What conditions might be improved by the FoF state? In the earlier example, nausea was reduced by a nocebo. Much of the enteric nervous system is nitrergic that is the nerves generate NO. If the basal level of NO is reduced by a nocebo, then the response of the enteric nervous system to CNS generated nausea signals mediated by NO will be reduced by a nocebo and enhanced by a placebo. When running from a bear, it is a "feature" to delay vomiting.
When coaches try to motivate athletes, usually it is via negative and violent symbolism, not by restful and peaceful symbolism. Invoking FoF is good when going into combat, even the ritualized combat of athletic events. However, the FoF state has costs associated with forgone cell repair and maintenance. It is a state used when necessary, but not a state that can be sustained long term. It would therefore be desirable to have a mechanism to terminate the FoF state, and to invoke the RnR state. This is the "relaxation response". Young children haven't yet learned to invoke this state, so it can be invoked for them by a parent by the "kiss and make it better" treatment.
So how does this all relate to pain? In this context, pain is a signal from your body telling you that your ATP consumption is exceeding what physiology can provide without shutting important stuff down. Your body will let you run yourself to death, because escaping from a bear is more important than any other damage short of death.
Implications of the placebo effect being mediated by NO. Every disease and disorder that is characterized by low NO will be helped by increasing NO, and so will be helped by placebos. This is not an imagined improvement, but an actual improvement. ASDs are caused by low NO, so they are helped by placebos and made worse by nocebos. This is why bullying is particularly bad for people with ASDs. They already have low NO, so bullying which invokes the FoF state makes that worse. What ASDs need is love and affection. As do children, and as do adults. As does everyone.
Some of this derives from a couple posters I presented at a scientific conference. Send me your email address (via posting a message with email and then deleting it) and I will send you a copy. I get quite a bit into ATP physiology, but that is necessary to understand why the placebo effect actually works to promote healing.
One of the strongest effects in medicine is the placebo (I will please) effect, yet some of the known physiological mechanisms behind this effect are not well known or appreciated, and the details are still not fully understood. The nocebo (I will harm) effect is similar, and is usually considered to be the opposite of the placebo effect, but actually is somewhat different.
Contrary to popular belief, both of these effects are misnamed, in that harmful effects can occur via placebos, and beneficial effects via nocebos. Contrary to popular belief, these placebo and nocebo effects are quite real and can be completely indistinguishable from effects due to efficacious treatments, including improved healing, decreased pain, and these effects can often be detected instrumentally.
Virtually any treatment can have some effect, including those that have no conceivable physical mechanism for working, including homeopathy, Chi manipulation, prayer, sacrificing animals, treatment of surrogates, sham devices and pharmacologically inert pills. An interesting rapid response to this article is from a Dr. Jane Woo (who I presume is perhaps "the expert" on placebos), saying "One of my residents once said that he advocated morphine injections, as opposed to tablets, because, "There's something about steel hitting skin and having a doctor say, 'This is going to make you feel better.' Injections simply work better than pills.""
One of the earliest "treatments" that children receive from their mothers is known as "kiss it and make it better". Any parent anecdotally knows that this is an "effective" treatment. While saliva does have nitrite from the reduction of salivary nitrate by commensal bacteria on the tongue, usually a motherly healing kiss is insufficiently slobbery to transfer sufficient nitrite, and the therapeutic effect is faster than nitrite or NO transfer from the treated boo-boo would allow.
Most placebo research has been associated with pain relief, and increased analgesia from placebo effects is well documented. I will focus this discussion more on non-pain effects of placebos and nocebos, and specifically on how placebos actually do improve healing.
It is well known that physiology is extremely complicated, and the regulation achieved by normal physiology is exquisite. So exquisite, that it has been endowed with the imaginary and mythical property of "homeostasis". In reality, nothing in physiology is static, rather our inability to measure the changes that we know must be present simply leaves us ignorant of those changes. While the default assumption of stasis is simple, it is clearly an assumption based on ignorance, and is clearly wrong. But this blog is about placebos, not homeostasis.
One of the best regulated physiological parameters is the ATP concentration. Not surprising because ATP is used by just about every physiological process, hundreds of thousands, if not millions of different pathways, in each cell, regulated simultaneously.
So how is ATP regulated? The answer is, extremely well!
ATP is considered to be one of the mythic "homeostatic" parameters, that is regulated to be constant. But that cannot be correct. The only way a parameter can be regulated is via feedback which necessitates a deviation from a setpoint followed by a compensatory response. Our inability to measure that deviation does not mean it does not exist. It only shows that our instruments are insufficiently sensitive and precise.
ATP cannot be measured in individual cells on the length and time scales where it matters, at least not non-destructively. The usual way is to freeze the tissue by clamping it with liquid nitrogen cooled copper tongs, then taking a small piece and assaying it. Depending on technique, the assay might be the average of only 100 cells or so. More likely a few thousand.
We know that ATP is regulated within individual cells because ATP doesn't diffuse through lipid membranes. It is difficult to get any information on the dynamics of ATP production and consumption by destructively measuring the average of a few hundred cells. Most any parameter would look pretty "constant" if the only measurements were averages of a few thousand values. If the only measurements of heart rate were averages over an hour (3600 seconds) wouldn't it look pretty constant too? You could measure the increase due to a marathon (2-3 data points), but not from a 4 minute mile.
ATP is not stored; the instantaneous production always equals the instantaneous consumption. If this were not the case, then there would be rapid accumulation or depletion of ATP. Each mole of glucose that is oxidized produces about 38 moles of ATP. A 2000 calorie diet then produces some 55 kg of ATP per day. Obviously, the ADP and P are recycled, and ATP production and consumption is very precisely matched.
In all control systems, the sooner you can start pulling levers to change things, and the more levers you have to pull, the better control you can exert. Physiology is no exception. Invoking physiological pathways in anticipation of a need for that pathway improves performance of the system. Neurological control of some aspects of physiology is well known. It would not be surprising if other aspects were controlled as well.
The key to understanding physiology is to understand that everything is a compromise, just like engineering. Every task that physiology needs to do has a cost in terms of ATP production to do the task, manufacture of molecules to carry out the task, DNA to code for the information to make the molecules to do the task, machinery to turn the DNA into what ever molecules are needed when they are needed, and the control system to do all of these things at the right time and in the right place, and time for all of this to happen. All of these things take up space, and have a "cost" in terms of maintenance and a "cost in forgone reproduction. Cells, and ultimately organisms that did these things most "efficiently" had more resources to use on reproduction, and so had more descendents, and so are the extant organisms we observe.
I will consider two extreme metabolic states, the "fight or flight" state (FoF), and the resting and relaxed state (RnR). It is well known that organisms can invoke such states and FoF results in the characteristic physiological effects of "stress", and if prolonged can cause physical disorders associated with "stress". RnR reverses the effects of stress, but to do so, has to occur within a certain time period.
I suggest that placebos invoke the RnR response, and that nocebos invoke the FoF response. Usually then, a placebo will promote healing and other effects associated with rest, and a nocebo will promote effects associated with stress, which usually are detrimental, but can be beneficial, as in the reduction in nausea, also in this example. The expectation of low nausea via a placebo produced greater nausea than the expectation of high nausea via the identical substance as a nocebo. In the last example, gastric tachyarrhythmia was instrumentally measured to be greater with placebo than nocebo, thus the increased gastric symptoms were not merely subjective. An explanation for this is discussed later.
We know that ATP production and consumption can vary by large amounts. Muscle for example can increase its oxygen consumption by a factor of 10. An interesting property of muscle is that it can be worked to exhaustion that is until ATP levels fall enough that the cells necrose and die. A useful "feature", when you are running from a bear. Death from exhaustion is balanced by death from being eaten by a bear. Physiological systems evolved to minimize death due to the sum of both events. Presumably, organisms do this "efficiently". That is they allocate ATP in an optimum manner to maximize organism survival. That is, ATP consumption is prioritized. Since ATP not used by a low priority pathway is as good as ATP produced, the "optimum" control system for ATP consumption will allocate ATP to the most vital pathways first, and when demand exceeds supply, turn off low priority pathways. What are "low priority" pathways? Well, anything that takes a long time (longer than the ATP crisis) must be low priority.
In the running from a bear example, anything that takes longer than the escape time can be put off. If what ever that pathway is going to produce won't happen until after you have either escaped or been eaten, it can't contribute to your escape, and so can be shut off to supply a few more molecules of ATP. In the limit of a perfectly regulated ATP system, the longest term pathways would be shut off first, then shorter term and later, still shorter term pathways as ATP demand exceeds supply. When the time horizon of the pathways being shut down reaches the present is when you drop dead from exhaustion.
One thing that can be put off is cell maintenance. Maintaining cells consumes ATP. Damaged proteins are ligated to ubiquitin, carried to the proteasome for disassembly, first unfolded by ATP powered unfoldases, and then broken into little bits by ATP powered proteases, and then replacement proteins are manufactured. All of these steps require ATP. No doubt some damaged proteins don't need to be removed immediately, but can be stored until later, until after the ATP crisis is over.
Accumulation of damaged proteins, as in amyloidosis, is common in virtually all of the disorders that are exacerbated by "stress" including obesity, diabetes, end stage kidney failure, dilative cardiomyopathy, neurodegenerative diseases, and so on. Accumulation of damaged proteins is harmful, but cells can tolerate quite large quantities and still remain viable, and there is some suggestion that such aggregation is actually protective. From my own experience (as a bachelor living alone), greater quantities of garbage can be tolerated if they are aggregated in a few places, rather than when distributed uniformly. I suggest that the "problem" is not so much increased production of these damaged proteins, but rather reduced removal. In "steady state", removal must equal production. If there is accumulation, then production exceeds removal. Presumably aggregate removal is regulated, and the "problem" of accumulation is either dysregulation, or precise regulation about a bad setpoint. The dysregulation hypothesis requires multiple cells and cell types to simultaneously develop the same dysregulation, an implausible coincidence. Since removal requires ATP, and some accumulation can be tolerated, I suggest that chronically low ATP will cause accumulation due to a bad setpoint. The "setpoint" is a function of energy status, and if there isn't enough ATP, clearing bad proteins gets put off until later. How much later? Until ATP is back up where it "should" be. What if that never happens? Then you are SOL (shit out of luck). Remember, physiological pathways can't "compensate" because it is precisely those pathways that are affected.
Reduction in metabolic rate is well known in degenerative diseases, for example in Alzheimer's there is a very well documented reduction in brain metabolism that precedes pathology. For a very striking image of this look here. As well as a reduction in metabolic activity, there is a reduction in blood flow. Blood flow is regulated by the vasodilatation produced by NO, and the vascular changes observed in Alzheimer's are consistent with low NO.
What form does the reduction in metabolism observed take? Is it a failure of 1, 10, or 100 or more pathways (of the millions the cell regulates) each pathologically consuming a little less ATP? I would presume that if only a few pathways were involved, they would necessarily represent a large fraction of normal brain metabolism, and disruption of such presumably important pathways would likely have effects more prompt than slow degradation over years. If many pathways are involved, how can many pathways simultaneously "go bad" in diverse areas of the brain? On the other hand, if it is a "bad setpoint", that is if ATP is low because of low NO (discussed later), then the brain would "gracefully" consume less ATP via the extremely robust ATP consumption hierarchies by turning off the least important pathways first, the long term maintenance pathways. This could go on for years, and may even reverse itself at times as the cells go down the low NO death spiral.
The proteasome disassembles proteins one at a time. Larger damaged assemblies including damaged mitochondria can only be disposed of via autophagy. Mitochondria have a finite life. In the rat, CNS mitochondria turnover in about a month. In other organs the turnover is faster. Mitochondria are tricky to recycle, particularly damaged mitochondria because they can be sources of superoxide and hydrogen peroxide, and because they contain abundant Fenton reactive metals which turn hydrogen peroxide into hydroxyl radicals which will damage anything they touch. I will discuss the mechanisms for putting off of autophagy during times of low ATP in a future blog. This is also directly mediated by low NO and low ATP.
Mitochondria are unique, in that they have their own DNA and ribosomes, and manufacture some of their own proteins. The vast majority of mitochondrial proteins (perhaps a couple thousand) are coded in the nucleus, synthesized in the cell's ribosome, and ported into mitochondria during mitochondria biogenesis. Only 13 proteins are coded for by mitochondrial DNA, the active sites of the respiration chain complexes. The vast majority of the complexes are coded for in the nucleus, but these are regulatory subunits, not the active sites.
The number of pathways that consume ATP is not small. For the purposes of this analysis, we need to look at each pathway separately. Rather than look at generic "protein synthesis", we need to consider synthesis of proteins (protein aaaa, protein aaab, protein, aaac… protein zzzz) all separately because that is how they are regulated. Under conditions of ATP depletion, expression of some proteins is upregulated, heat shock proteins and others. I have denoted each protein by 4 letters because that is about how many different proteins are expressed, 26^4 ~ 10^5. Each protein has on the order of a few hundred amino acids, so the number of individual steps that are involved is many millions. We know that the expression of each protein is controlled "just right" because if it wasn't, either there would be not enough, or the cell would explode from too much.
So, how does a cell control a few million pathways and prioritize them based on ATP level? What can it use as a "signal"? I suggest that it must use ATP itself. There are not enough other molecules for it to use a different molecule for each one; some must be controlled by the same molecule, but by different concentrations. For this discussion I am not particularly concerned with the mechanism(s) involved. No doubt there are many.
In a cell, there are 3 ATP parameters, ATP concentration, ATP production rate, and ATP consumption rate. These 3 parameters are independent, and can (and are) controlled independently. Since muscle can consume ATP to the point of death, low ATP will necessarily stimulate maximum ATP production. Just short of death, the cell will "want" to turn off all non-essential systems to stave off ATP depletion for as long as possible. So low ATP turns off the "housekeeping" pathways. So what sets the ATP concentration? In part, that is set by NO via soluble guanylyl cyclase and cGMP.
When physiology calls for maximum ATP production, one of the first things it does is lower NO levels, to disinhibit cytochrome c oxidase. Under basal conditions, cytochrome oxidase is mostly inhibited by NO, which blocks O2 from binding and being reduced to water, the ultimate sink for electrons. O2 consumption can go up an order of magnitude. That means an order of magnitude more O2 must diffuse to the mitochondria and be reduced to water. O2 is only transported by passive diffusion. In the lungs, O2 diffuses into the blood and is absorbed by hemoglobin forming oxyhemoglobin. The blood carries the O2Hb to tissues where the O2 comes off and diffuses to the mitochondria down a concentration gradient. The lowest O2 concentration in the body is at the mitochondria where the O2 is consumed. For the flux of O2 to increase by an order of magnitude, the O2 concentration gradient must increase an order of magnitude. How does this happen? The concentration in the blood doesn't change, the spacing between vessels and mitochondria doesn't change much, so to increase the flux, the concentration at the mitochondria must drop by an order of magnitude. Then with the higher gradient, more O2 can diffuse to the more active mitochondria and more ATP can be produced. The O2 consumption by cytochrome c oxidase increases an order of magnitude while the O2 concentration drops an order of magnitude. The specific O2 consumption (moles O2/Torr O2/mg protein) must go up 2 orders of magnitude. This is accomplished by lowering the NO level local to the mitochondria.
So the low NO necessary for disinhibition of cytochrome c oxidase also serves to lower the ATP setpoint. This lowers the ATP concentration, which turns off non-essential systems. The lower ATP concentration upregulates ATP production by the mitochondria. When mitochondria don't have enough O2, the respiration chain becomes reduced, what little O2 is present becomes reduced by single electrons, not on cytochrome c oxidase, and superoxide is formed. This superoxide destroys NO at diffusion limited kinetics, pulls down the NO level, disinhibits cytochrome c oxidase which then pulls down the O2 level allowing more O2 to diffuse to the mitochondria.
So, under conditions of FoF, the NO level is lowered. The more severe the FoF, the lower the NO level is taken. NO is a small uncharged molecule that diffuses readily through lipid membranes. The only barrier to NO in the body is crystalline bone. A state of low NO, is then propagated to all cells, so that the metabolic status of all cells can be regulated in sync. This is important because to maximize the ability to run from a bear, O2 and glucose consumption by non-essential systems must be curtailed as well as ATP consumption by muscle repair systems.
Is the hypothesis of ATP hierarchies plausible? Well, we know that physiology does behave this way. There is an effect called ischemic preconditioning, where a brief ischemic event induces a transient state where a prolonged ischemic event will produce less damage. This is well observed in a number of different organs. Transient ischemia reduces ATP demand and so cells can survive ischemia that would otherwise kill them. This behavior is what the ATP hierarchies hypothesis would predict. The mechanisms behind ischemic preconditioning are mostly unknown. No doubt as a stress response from deep evolutionary time there are many pathways involved in very complex and redundant ways, which may (is likely to) be different for different organs. Oxidative stress is known to be involved in some aspects of ischemic preconditioning.
Presumably ischemic preconditioning has some detrimental long term effects, otherwise cells would evolve to be in that state continuously. They don't, therefore there must be long term negative consequences. Those negative consequences might not show up for some time, but they must be present. This is a danger of short term endpoints in clinical trials. A treatment may prevent short term damage but if continued may cause increased long term damage.
This is one of the dangers of pain relief. If it merely masks the pain symptoms, and people then behave as if they are in the RnR state when they are actually still in the FoF state, then running themselves to death is much easier. Similarly, what do "stimulants" actually do? Do they increase the ability of cells to make ATP? Doubtful that a drug could improve on a few billion years of evolution. They do increase ATP availability (otherwise they wouldn't be stimulants), most likely by invoking the FoF state and turning off non-voluntary pathways like long term maintenance, but without the pain that normally warns of degraded repair systems.
So what happens under conditions of RnR? Well, to activate all the repair pathways, ATP needs to be high, so via sGC, NO levels have to be high too. What triggers mitochondria biogenesis is NO, so to make more mitochondria NO levels need to be high too. So RnR is a state of high NO.
How is this state of high NO produced? One mechanism is by a reduction in mitochondrial potential. To generate high ATP flux, mitochondria increase their potential to increase the driving force for ATP production. This does increase the rate, but it also increases superoxide production, a valuable feature, which pulls down the NO level to increase O2 diffusion. When the demand for ATP drops, the potential drops, the superoxide formation rate drops, the NO destruction rate drops, and the NO concentration rises provided there is sufficient basal NO production to begin with. If the basal NO production rate is too low, then the reduction in the NO destruction rate doesn't raise the NO level.
This presents a problem, if the state of FoF is prolonged sufficiently that mitochondria biogenesis suffers. The only reason that organisms have the ability to increase their metabolic activity over basal levels is because there are "excess" mitochondria. That is mitochondria in excess of the minimum necessary to supply basal ATP requirements.
Fewer mitochondria can supply the same ATP by increasing mitochondrial potential. This results in greater superoxide production, and also greater "slip", that is a reduction in the number of ATP molecules produced per mole of O2 reduced. A hallmark of many of degenerative diseases is weight loss, often inappropriately termed malnutrition, where the actual problem is increased basal metabolism. Elevated basal metabolism is observed in dilative cardiomyopathy, chronic renal failure, HIV infection, liver cirrhosis, chronic obstructive pulmonary disease, Does an increased basal metabolism mean the body is doing "more stuff"? Likely not, rather it is doing the same "basal metabolism stuff" but using ATP generated less efficiently with fewer mitochondria as observed in heart failure. It might even be doing less, because low ATP has turned off the repair pathways which is why the liver, kidneys, heart are failing in the first place. In HIV, a standard treatment is via highly active anti-retroviral therapy (HAART). A side effect of this treatment is reductions in mitochondria biogenesis. This can result in hyperlactatemia because of increased glycolysis to supply ATP. But if the liver and kidneys don't have sufficient mitochondria to recycle the lactate via the Cori cycle, where does it go? Perhaps into ectopic fat. I suspect that this is one of the problems of obesity. NO selectively partitions into lipid, and adipose tissue is a source of inflammation and oxidative stress. If NO drops sufficiently to impact mitochondria biogenesis, there may be no internal mechanism to raise it sufficiently for a long enough time to reverse the mitochondria depletion.
So how does all of this relate to the placebo effect? Well, if healing and cellular repair is accomplished most effectively during periods of RnR, then invoking that state will promote healing, well being, and long life. One of the things that does invoke feelings of rest and relaxation is love. It is well known that married people live longer lives (and it isn't just that it seems longer). The well known maternal "kiss it and make it better" treatment does relieve pain and presumably resets the RnR state. Presumably regular episodes of love and affection from a romantic partner can reset the RnR state too.
At the heart of energy metabolism is nitric oxide. A major determinant of whether an organism is in the FoF state, or the RnR state is the level of NO. Because NO is freely diffusible, and is created and destroyed at many sites in the body, the basal level has an impact on the signaling effects of NO. Low basal NO will affect every NO mediated signal with no threshold. This is an extremely important point. Anything that increases basal NO will shift physiology to the RnR state and away from the FoF state. There are many things that will do this, placebos are one of them. The relaxation response causes the production of NO. My own favorite method is via commensal ammonia oxidizing bacteria on the skin. No matter what the basal NO level is, physiology can always destroy that NO very rapidly with superoxide. Mitochondria have an essentially unlimited capacity to make superoxide, limited only by the supply of O2 and reducing equivalents. What ever the NO level is, mitochondria can pull it down to zero. This has important implications in acute respiratory distress syndrome, and is what is responsible for the multiple organ failure which sometimes occurs.
Long term meditation does result in reduced age-associated loss of cortical white matter. I presume by increased repair, improved energy status, reduced apoptosis, better clearing of damaged proteins, and perhaps increased axonogenesis. Many neurotrophic factors have effects mediated through NO.
Meditation modulates the immune system and increases antibody titers due to vaccination. Meditation reduces the symptoms of the metabolic syndrome and improves a number of heart health parameters.
If placebos increase NO levels and invoke the RnR state, then nocebos likely reduce NO levels and induce the FoF state. What conditions might be improved by the FoF state? In the earlier example, nausea was reduced by a nocebo. Much of the enteric nervous system is nitrergic that is the nerves generate NO. If the basal level of NO is reduced by a nocebo, then the response of the enteric nervous system to CNS generated nausea signals mediated by NO will be reduced by a nocebo and enhanced by a placebo. When running from a bear, it is a "feature" to delay vomiting.
When coaches try to motivate athletes, usually it is via negative and violent symbolism, not by restful and peaceful symbolism. Invoking FoF is good when going into combat, even the ritualized combat of athletic events. However, the FoF state has costs associated with forgone cell repair and maintenance. It is a state used when necessary, but not a state that can be sustained long term. It would therefore be desirable to have a mechanism to terminate the FoF state, and to invoke the RnR state. This is the "relaxation response". Young children haven't yet learned to invoke this state, so it can be invoked for them by a parent by the "kiss and make it better" treatment.
So how does this all relate to pain? In this context, pain is a signal from your body telling you that your ATP consumption is exceeding what physiology can provide without shutting important stuff down. Your body will let you run yourself to death, because escaping from a bear is more important than any other damage short of death.
Implications of the placebo effect being mediated by NO. Every disease and disorder that is characterized by low NO will be helped by increasing NO, and so will be helped by placebos. This is not an imagined improvement, but an actual improvement. ASDs are caused by low NO, so they are helped by placebos and made worse by nocebos. This is why bullying is particularly bad for people with ASDs. They already have low NO, so bullying which invokes the FoF state makes that worse. What ASDs need is love and affection. As do children, and as do adults. As does everyone.
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