Saturday, April 21, 2007

Autism Diva’s Curing Vegetables®

Autism Diva has written a very nice blog on her home manufacture of lactic acid bacteria fermented vegetables, a traditional food of Koreans, known as kimchi. While Autism Diva spoke of selling her Curing Vegetables® as a treatment for ASDs in jest (I presume), I think there is at least a plausible mechanism that could suggest that consuming Autism Diva’s Curing Vegetables® might be beneficial. Something that is completely lacking with every other so called “treatment” sold at a DAN!® conference (that I am aware of). At least it won't be harmful (as many of the DAN!® treatments are).

As is mentioned elsewhere on my blog, I subscribe to the “low NO hypothesis” of ASDs. NO is a pleiotropic signaling molecule that is involved in thousands of different physiological pathways, including many (if not all) known to be disrupted in ASDs. Because NO is used as a signaling molecule, and all “NO detectors” only detect the sum of all NO from all sources, the background level of NO will affect all NO pathways with no threshold.

There are a number of ways of increasing basal NO/NOx levels. As far as I know, there are no "pharmacological" ways. That is, there are no "drugs" per se, that will increase NO/NOx levels. Part of why is simple physiology. NO/NOx physiology is so well regulated that it is very difficult to perturb artificially. It is so coupled, and so complex, that it is virtually impossible to perturb artificially without adverse side effects. There is intense research in these areas, and compounds that are NO/NOx active are in use, but they don't strictly increase the basal NO/NOx level. Nitroglycerine, other organic nitrates have been used to treat angina for over a century, and they work, but precisely why remains unknown. They are not simple NO donors. Sodium nitroprusside is an NO donor, which is used to control refractory blood pressure, but it must be administered continuously. It has a very short lifetime in the blood, and the effect stops within a few minutes. Inhaled NO only has effects in the lung (pretty much). High doses of inhaled NO can have systemic effects, but that is likely do to the metabolite of inhaled NO, nitrite. Nitrite can be infused, and is as a treatment for cyanide poisoning. It works by oxidizing hemoglobin to methemoglobin, which binds cyanide, disinhibiting cytochrome oxidase while sulfuration pathways get rid of it. NO also reversed the inhibition of cytochrome oxidase by cyanide.

As Autism Diva mentioned, kimchi is produced by fermentation of various foods by lactic acid bacteria. Her favorite vegetables include cabbage. Green leafy vegetables are well known to have high nitrate content. A list of tables with nitrate contents of various vegetables. There is considerable thought that the nitrate in green leafy vegetables is part of what makes them so healthy to eat, and contributes to antimicrobial effects in the stomach. Lactic acid bacteria are responsible for the fermentation that turns milk into yogurt, and vegetables into kimchi. It is also responsible for turning chopped up maize stalks into silage.

Quite a number of different strains of lactobacilli are used and can be used to ferment vegetables, where they produce diverse metabolic products including lactic acid, acetic acid, and alcohol.
The reason that Daedalus considers that Autism Diva's Curing Vegetables® would be beneficial to individuals with ASDs is due to what lactic acid bacteria do to the nitrate that is abundantly present in most vegetables. Lactobacilli produce NO and nitrite from nitrate. This is a normal part of the curing process, which generates NO and nitrite and is what makes sausage such as salami bright red. The red color is from nitrosylmyoglobin (NOMb). That is myoglobin with NO attached to the heme where O2 is usually attached. Hemoglobin is red because O2Hb is red. Hb without O2 is blue. NOMb is red, but a slightly different shade (like bacon). COMb is a slightly different shade too. If you have a gas stove, sometimes you can see slightly different shades of red in meat as it is cooked due to the different myoglobin species.

Nitrite is unstable at low pH, and is also fairly easily reduced to NO. When nitrate is consumed, as in green leafy vegetables, the nitrate is well absorbed, concentrated in the saliva from the blood stream, then bacteria on the tongue reduce that nitrate to nitrite. When the nitrite is swallowed, it forms NO in the stomach, and adds considerable anti-microbial effects to the low pH of the stomach, killing the nasty bacteria that would like to live there.

Ingestion of nitrate has a "bad rap" because of the perception that it can cause methemoglobinemia in infants, otherwise known as blue baby. This is the reason that there are limits on nitrate in drinking water. However, the epidemiology of nitrate in drinking water causing methemoglobinemia is quite poor, essentially non-existent. Ingestion of nitrate can lead to methemoglobinemia, but the main dietary source is vegetables (which have ~1000-3000 ppm nitrate) not drinking water. Methemoglobinemia occurs when a large fraction of the blood's hemoglobin becomes oxidized to methemoglobin and so cannot carry O2. Usually this only presents problems if more than 50% of the blood is so converted. Usually this makes people look quite cyanotic, because metHb is dark colored.

With out analyzing Autism Diva's Curing Vegetables®, it would be difficult to predict what NOx species would be present. Nitrite is not stable at low pH, so the nitrite level would be low. But there are a great many other NOx species that might be important as I discuss in an earlier blog. I looked at some sauerkraut, and as expected, there was no nitrite, but there was substantial nitrate.

The method for preserving foliage for use as feed for ruminants by making silage is the same method as Autism Diva uses to make Autism Diva's Curing Vegetables®; that is fermentation with lactic acid bacteria. When silage is made, the bacteria reduce the nitrate that plants accumulate into nitrite, and that nitrite decomposes in the low pH forming NO which is then easily oxidized by the O2 in air to form NO2, nitrogen dioxide. NO2 is a brown gas, extremely toxic. When you look at the exhaust gases from a large power plant, if the exhaust stream is brown, that is NO2. NO2 can accumulate during silage production, and people have been killed by formation of "silo gas", which can kill with a single breath. NO2 is toxic like that, the threshold is a few ppm. NO isn't toxic at all, people can routinely breath hundreds of ppm, but only if provision is made to ensure there is no NO2 which can very rapidly form. Your nasal passages produce a few hundred ppb NO in the air you breathe to regulate the perfusion of the lung, and match it to air inhalation.

So what form are the NOx species in Autism Diva’s Curing Vegetables®? They could be S-nitrosothiols. When a thiol containing protein (which includes most every protein) is exposed to nitrosative conditions, (that is nitrite, low pH, and something oxidizing), then NO can attach to the thiol and form the S-nitrosothiol. S-nitrosoglutathione, S-nitrosoalbumin, S-nitrosocysteine are all well known S-nitrosothiols. Human blood has about 7 micromolar S-nitrosothiol, the most abundant S-nitrosothiol in the blood is S-nitrosoalbumin, which is present at variable levels, but in humans is about 5 micromolar in plasma. S-nitrosothiols are likely generated in the stomach from salivary nitrite, low stomach pH. Ingestion of nitrate does reportedly does have physiological effects most likely mediated through S-nitrosothiol formation in the stomach.

There is a higher incidence of stomach cancer among Koreans. Whether this is due to consumption of kimchi or to particular kinds of kimchi is unknown. Nitrosamines can be carcinogenic. This is the concern over nitrite in bacon and other cured meats, that under nitrosative conditions (low pH and nitrite and air), that nitrosamines can form. The reason they are carcinogenic is that they can attach to DNA and modify it. The risk associated with bacon and other nitrite-cured meats is purely theoretical. That is, there hasn't been good epidemiology to show that there is a connection to induction of cancer. Most studies have shown that when ascorbate is consumed with the nitrite containing food, that no nitrosamines can be detected.

If I had to guess, I would suppose that vegetable based kimchi was completely safe (due to the ascorbate), and that only meat-based kimchi was potentially problematic. Red meat is associated with increased gut cancers, and that is due to the heme content. Nitrosylating that heme might render it non-cancer forming. Heme is a reasonably good catalyst for oxidation reactions (most every oxidation enzyme contains heme for that reason). Nitrosylating heme does block its catalytic activity toward O2. Would nitrosylating heme prevent heme from being cancer forming? That isn't something that has been tested yet that I am aware of. Fish does have amines in it, that is what makes it smell "fishy". Amines plus nitrosating conditions does make nitrosamines. So I would consider it poor practice to put fish into kimchi while it is fermenting. If you do, you want to make sure that the container is completely anaerobic. That is, that the CO2 the lactobacilli are making has displaced all the air. Then you would want to consume the kimchi fairly soon, or keep it anaerobic until you do consume it. Putting it up in sealed jars is probably ok, but not in open containers. This mimics the action of the stomach, which is also anaerobic. The head space in the stomach can reach ~100 ppm NO due to the action of low pH on salivary nitrite. That level would probably kill you if you breathed it long term due to formation of NO2 from mixing with air. But in the stomach away from air, it is a natural and healthful source of NOx species. Mimicking those conditions while fermenting kimchi would probably be similarly healthful.

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.

Tuesday, April 3, 2007

dropped comment post from left/right brain blog

I accept that the burden of "proof" is on me, but until it is "proven", it is not wrong, but unproven.
I know that "proof" will require research. Research require funding and resources which I don't currently have. Obtaining such funding requires research results. It is a catch-22. The senior NO researcher I am working with is moving his lab to another continent (because of funding issues). That has set back our work together over a year already. Realistically he won't be able to do anything for another 6 months. The focus of his work is not ASDs, but NO/NOx systems biology. They are all related, so there is no stretch at all me working with him.

I believe in the scientific method too, that is what has made me a successful inventor and scientist, and what has gotten me this far. As someone with Asperger's, the science part is easy, a simple continuation of how I live my everyday life. The interpersonal stuff necessary to convince NTs that my ideas are worthwhile is much more difficult.

Unfortunately VCs and pharmo/biotech companies are not beating a path to my door. They are in business to make money on things they understand, things like viagra knock-offs, things that are "sexy" to other NTs. They are not in the business of funding research that they do not understand. No agency is. My approach is considered to be "high risk". What that "means", is not that it is has the potential to harm any patients, but that it is so "far out" of the mainstream that if it is funded, and fails, then the NTs who funded it will look foolish. NTs can't abide being thought of as foolish. So the peers who (don't quite) understand NO physiology look to ASD experts, who look to NO experts, who look to ASD clinicians, who look to neurophysiologists, who look to geneticists. None of them understand enough pieces of the puzzle to see the big picture, even when it is explained to them.

It is unfortunate that many people have (wrongly) marketed what they (falsely) believe to be a universal biologic panacea. It has made my efforts more difficult. The false idea that "mercury causes autism", has really poisoned the field in terms of considering the real biologic correlations in ASD physiology.
That was the reason I become knowledgable about mercury, not because there is any basis for effects in ASDs, but because the senior clinician I was talking too regarding ASDs was too enamoured with mercury to have the mental capacity to think of anything else.

"Curing" ASDs is a tiny market compared to the rest of what my stuff is good for. It is important to me because I have suffered from the effects of ASDs for my entire life. I know what it is like to be bullied until one is suicidal. I know that many NTs are simply unable to stop bullying. I think my stuff will greatly reduce the effects of that bullying on ASDs.

Barbara, did you get the stuff on ATP that I sent you? If not, I need your email address.

Monday, April 2, 2007

Discussion of financial interest

I have posted on my blog, a financial disclosure which states that I am actively researching and commercializing these bacteria to prevent and treat a number of disorders including ASDs, and that I have patents issued and applied for. I put the statement there in the interests of full disclosure. If any reader considers this bothersome or unacceptable, please don't read my blog.

There has been concern expressed that I am attempting to merely introduce "another" biological based "treatment" to "cure" ASDs, and that my motives and by implication my methods are no different than those of the quacks pushing their various "snake-oils".

My perception is that my motives and my methods are completely different than those of the quacks. I feel that I have nothing to hide, and that filing for patents is a normal, even a necessary part of commercialization of any product, including treatments for medical disorders. The only thing that patents provide is the ability to bar others from making, using or selling the patented thing. My control of this does prevent the quacks from using it once it becomes proven. Believe me, I will prevent them.

I have spent over 10,000 hours researching NO, ASDs, and other disorders associated with low NO. I have received no funding for this. I am starting a company to commercialize this (as I have to commercialize other products I have invented). I have invested my entire life-savings in this. If the company succeeds, so will I. If it doesn't, I will need to do something else. t should be noted, that on the AutismHub, there are "Autism Pro bloggers". These individuals make their living by providing services to ASD individuals. While I don't consider myself a "pro", no one begrudges them fair compensation for their labors.

I am extremely cognizant of the potential harm that unproven "treatments" or "experiments" can do. I am being extremely careful in my research, and in the claims I have made, and in how I am proceeding. I am on the spectrum myself. I started my NO research before I knew I was on the spectrum. The only reason that I started doing research on ASDs was because my Asperger's got better, a lot better.

I have been using this on myself for over 5 years now. I have been actively researching the connections to ASDs for 3 or 4 (since I realized I was on the spectrum). I haven't jumped to try and sell this, or lie to people about what I have, or what it will do for them. I am perfectly prepared to be wrong, but I don't think I am, because I have read an extremely large amount of the NO and ASD literature. I am not quite hyperlexic, but I can read (and understand) dozens of papers in an evening. As far as I can tell, everything in the literature is consistent with my hypothesis (after discarding the stuff that is wrong). My literature file has about 57,000 items in it, some of those are duplicates, and when a web page is stored there are multiple items from it. There are probably at least 20,000 papers in it that I have "read". No, I haven't read them all carefully, but because I read so fast, I don't need to, I can carry them with me on my laptop (it only takes up 16 GB) and refer to them when I need to. The senior researcher in the NO field thinks I am very likely the best read in the NO field. I think he is right. I read really fast, and I don't have to maintain a lab, a family, or a life like everyone else does.

This is why I am so convinced of my hypothesis. I am prepared to be wrong, but it will mean that much of what is in the literature is also wrong, and must be discarded. I consider the possibility that thousands of papers in the literature are all wrong in the direction that would erroneously suggest my hypothesis is correct to be very unlikely. It is somewhat "infuriating", when people suggest after listening to half an hour of explanation, that I am wrong because it "can't be that simple". Nothing about NO is simple; any system of thousands of coupled non-linear parameters cannot be "simple". Ferid Murad has stated that he thinks that we understand perhaps 15% of NO physiology. I think he is wrong by at least 3 or 4 orders of magnitude, perhaps more. If I am wrong, show me where.

I have been following the mercury/vaccine stuff very carefully, and have read virtually everything I could on it. There is no comparison between what I am doing and the fraud that is being committed by those quacks. My treatment is not "yet another “alternative” biomedical treatment to the appalling array already in existence." The "reason" there are so many "treatments" for ASDs, is because there is no effective treatment. I posted a blog about mercury, my mercury file has 450 items and is 93 MB. It is "enough" for something that has no relationship to ASDs. Things that are more important I have done more research on.

So far, the only ASD individual that has used this is me. I would like to get a clinical trial going, but I don't have a clinic or an IRB. I think the "risks" are near zero. But I only want a trial to go forward with everyone involved completely aware of every possible risk, even those that are extremely unlikely, or even impossible. I have done an extremely extensive safety review. There is not a single reported instance of these bacteria causing an "infection". It is likely than they cannot do so, even in immunocompromised individuals. These bacteria express no virulence factors, no toxins, no porters to excrete them; they are unable to grow on any media used to isolate pathogens. I understand that the word "bacteria" invokes fear and loathing. These bacteria are safer than the bacteria in yogurt, the daily consumption of many ounces of which is considered benign, even healthful. In contrast to the zero reported cases of infection from the type of bacteria I am using, there have been reports of liver abscesses from yogurt bacteria (in immunocompromised individuals (yes, plural)). The treatment modality I use is topical application to unbroken skin. What risk is there from the application of bacteria that are safer than yogurt to external unbroken skin? I think only risks that are quite small. What "therapeutic benefit" can justify the "risk" of applying bacteria that are safer than yogurt to unbroken skin? I think a relatively small benefit. I have experience benefits considerably greater than that.

I don't really consider this to be a medical "treatment" or "intervention" per se, any more than a nutritious diet, or moderate exercise, or sufficient rest is a medical "treatment" or "intervention". They are normal components of a healthy lifestyle. I think in time, it will be recognized that the proper surface biofilm of these bacteria is also a normal part of a healthy lifestyle.

What are the "risks" of a nutritious diet, moderate exercise and sufficient rest? There may be "side effects", but I don't think there are any "adverse side effects", or "risks". People may disagree on that. I would be happy to discuss that with an IRB.

I very much recognize that "cure" means different things to the ASD and to the NT communities. What I have has not made me "Asperger-free". I can still enjoy playing minesweeper for 10 hours straight. There is no way that I could "pass" for NT. But I am much less anxious, my mood is a lot better, I am more functional in many ways. I think that every ASD would "like" the changes that my stuff produces. I think that many parents would "like" the changes that my stuff would produce in their ASD children because it will make them happier, and lead richer, more fulfilling lives as the ASD individual sees it. I don't think that my "cure" should be imposed on anyone who doesn't "want" it.

I think that some NTs won't "like" what this does to ASDs, because it will make them less susceptible to bullying. I think that many NTs don't appreciate how important bullying is in their interactions with other NTs and with ASDs (think of John Best for example, his only way of interacting is via bullying. Were his bullying to be rendered ineffectual, he would be emasculated). I don't think the "curebie" parents will "like it" because it won't turn their children into the "Stepford children" that they want. What I think it will do is make ASDs more what the ASDs want to be.

As I understand it, having a biofilm of these bacteria was how our ancestors evolved, back 5, 10, 50, even 100 million years. Our physiology has come to need these bacteria, and it suffers when we don't have them. The loss of them mimics the effects of "stress", and invokes all the stress compensatory pathways, one of which I think is the ASD phenotype. I think all the other degenerative diseases of the developed world are also the consequences of overactive stress responses.

The mechanisms behind the "relaxation response" work because they cause the release of NO. Raising the level of NO with my bacteria rolls back the stress setpoint. It reduces the stress response to a given stressor. It does so without any "doping", or cognitive impairment.

One way to describe it is that it increases the effectiveness of rest. It does so by restoring the "natural" setpoint of the physiological processes that are activated during rest.

I am still working alone in ASDs. I have tried to interest clinicians, but they don't have the background to understand what I am saying, and in my ASD way, I can't explain it to them in the few minutes they have before they glaze over and think I am wacko and grandiose for thinking I can cure essentially every degenerative disease.

I have tried to publish, but keep being told I need a larger n. I can't get research grants because I don't have an institution that fits the profile, and I don't have any credentials and what I am proposing is too "high risk". What they "mean", is not that it is risky for patients, rather it is so "far out" that if it fails, the people who approved it will look foolish, something that NTs are extremely reluctant to even consider. Soil bacteria preventing disease? Who has heard of that? No one, because I discovered it.

I think it would stop meltdowns in a day or so. Depending on severity. How things proceed from there is hard to judge. I think that everyone can appreciate that a large reduction in meltdowns would have beneficial effects that would increase over time. I only have an n of 1 to work with, and my understanding of ASDs is limited.

Sunday, April 1, 2007

Background and summary NO and ASDs

Autism is a spectrum of sometimes debilitating development disorders. The "cause" remains unknown, but autism often becomes apparent in the first few years of life. It is during this time that the brain is growing rapidly and forming and reforming many new connections. There is some thought that autism occurs when these connections do not form properly. Among 3 to 4 year olds autistic children, brain volume was 10 to 13% greater than in normal children and in children with development delays that were not autistic.1 Improper brain growth, and in particular excessive brain volume, has been correlated with autism2.
Nitric oxide (NO) and related species are important in a large number of critical physiologic pathways, including regulation of O2 consumption by cytochrome oxidase, triggering of soluble guanylyl cyclase, initiation of mitochondria biogenesis, transcription via HIF-1α, inhibition of NFκB, regulation of Zn metallization of Zn finger proteins and transcription factors, as a neurotransmitter, as a mediator of neurotrophic and growth factor effects including BMPs, TGF-βs, IGF-I, BDNF, regulation of the cell cycle, regulation of steroid synthesis, initiation and inhibition of apoptosis, and for antimicrobial effects. NO and the major source of NO, nitric oxide synthase, are the subjects of intense interest and research.

Figure 1. some pathways involving NO species

As a labile diffusive signaling molecule, the background concentration of NO must be critically important in determining the effective range of a NO signal. Because many of the effects of NO are nonlinear, and are coupled to many other physiological processes, experimental determinations of the effects of NO are not simple, particularly when it is not easy to change or even measure NO levels at the length scales, concentrations or time scales known to be important.

Coupled non-linear effects are notoriously difficult to model (essentially impossible with more than a few), and observed effects are not always predictable, for example, inhibition of NOS with L-NAME can increase NO levels at particular sites.3 Nitric oxide is used in many regulatory pathways. One of the best understood is the activation of soluble guanylyl cyclase (sGC) which causes production of cGMP. cGMP then activates multiple kinases, cyclic nucleotide-gated channels and phosphodiesterases. The apparent threshold for activation (EC50) of sGC by NO is complex due to deactivation, but is ~20 nM/L for long activation times and ~45 nM/L for short.4 20 nM/L is 0.56 parts per billion by weight. The background NO concentration considered important in the context of this paper are somewhat less than that, perhaps 0.1 to 10 nM/L, or about 2.5 to 250 parts per trillion (ppt). By comparison, 10 ppt is less than 1 mm compared to the circumference of the Earth. These levels are difficult to measure in vitro on macroscopic samples; there are simply no techniques available to routinely measure such levels in sub-cellular compartments in vivo on the time (sub second) and length scales (sub micron) where they are obviously important.
Figure 2 some species which can deliver, store or produce NO or NO effects

It is not clear that the only component of "basal NO" is "free" NO. NO chemistry is quite complex, and there are many species that can release NO via numbers of different pathways (see Figure 2). Some of these, such as nitrite, are present at considerable concentrations. For example, nitrite levels in plasma, erythrocytes, and whole blood from 15 normal volunteers were 121 ± 9, 288 ± 47, and 176 ± 17 nM/L respectively.5 Nitrite is a metabolite of NO, so a nitrite pool used to generate NO would be partially regenerated. Nitrite is the major metabolite of these bacteria when oxidizing ammonia.

In Figure 2, it should be noted that once NO attaches to a molecule, that molecule becomes an NO species and can deliver an NO signal (or not) at another time or place depending on conditions at that time or place. It is important to recognize the extreme complexity of NO metabolism. NO is involved in hundreds of different pathways by numbers of quite different chemical reactions. NO directly couples to the ATP setpoint6 , and ATP couples to essentially everything. There are 3 NOS isoforms, and surprisingly knock-out animals have been produced for each isoform. It is surprising that these NOS knock-out animals do as well as
they do and can tolerate losing an entire NOS isoform, and necessarily all the regulatory pathways that feed into it and produce NO by activating that isoform. This demonstrates that NO metabolism is extremely robust.

No threshold for basal NO effects:
Because NO is used as a signaling molecule, the background concentration of NO is important in determining the onset time, the range and the duration of every NO signal. Because the NO level is already in the "active" range (that is it is actively being used as a regulatory signaling molecule), any change in the background NO level will change the onset time, the range and the duration of every NO signal with no threshold. This is an extremely important point and is illustrated in Figure 3. Because the NO signal is already in the active range, any change to the background level of NO will affect what ever pathway is being regulated via NO with no threshold. The diffusing signal of NO adds to the background NO concentration, and when the sum exceeds the action level, the action of the NO signal occurs. The focus of this paper is to discuss the rationale behind a low basal NO level (which I term nitropenia) causing, being associated with and exacerbating autism spectrum disorders.

Despite the extreme complexity and extreme robustness of NO physiology, a reduction in the basal level of NO will affect those pathways with no threshold. The effect may be

Figure 3. Operating point of NO feedback regulatory circuits shown to be affected by basal NO with no threshold

small, but it is non-zero. Because NO is inside the feedback loop of many pathways, those pathways cannot compensate for low basal NO levels because it is the compensation pathway itself that is affected. This is particularly important for something like the ATP setpoint. Low NO necessarily leads to low ATP, and that low ATP will invoke all the "low ATP" compensatory pathways.

It has been reported that there is an increased level of NO production in autistic individuals.7 Higher levels of NO were also reported in autistic individuals by others.8 It should be noted that actual NO concentrations were not measured in these studies, only the terminal metabolites nitrite and nitrate. NO does not accumulate, it is destroyed as rapidly as it is produced. Terminal metabolite levels only reflect production rate, they do not reflect NO concentration, any more than CO2 (the terminal metabolite of O2) reflects O2 concentration. Autism is well known to be a state of oxidative stress.9 A state of oxidative stress is characterized by increased levels of superoxide. NO and superoxide react with each with essentially diffusion limited kinetics. One therefore cannot have both superoxide and NO present simultaneously. Which ever one is in excess will destroy the one that isn’t.10 The nitration of tyrosine by fluxes of superoxide and NO occurs in vitro only at near equimolar fluxes.11 The actually nitrating species is thought to be NO2. NO2 rapidly reacts with NO to form N2O3 (k=1.1E9). When NO or superoxide is in excess, nitration does not occur.12 With a 2 to 3 fold excess of NO over superoxide, the oxidative effects of peroxynitrite are not observed.13 More relevant than the total quantity of NO produced (and measured as metabolites), is the quantity of NO relative to that of superoxide. Increased levels of NO metabolites and oxidative stress are consistent with an excess of superoxide. Such a state is actually a low NO state irrespective of NO production rates. The presence of nitrated proteins does not reflect "too much" NO, any more than the presence of ROS damages species reflects "too much" O2. Hypoxia can produce ROS and ROS damaged species, as can hyperbaric O2.

Lennart Gustafsson has suggested that autism might result from low NO due to inadequate levels of nitric oxide synthase14 and producing abnormal minicolumn architecture during development, which he suggests might also be produced by low levels of serotonin.15 He suggests that autism might be treated by increasing the activity of nitric oxide synthase in the brain, but offers no suggestions of how to do so. He notes that a nitric oxide explanation provides a rational for some of the seemingly disparate symptoms observed in autism spectrum disorders including comorbidity with epilepsy, motor impairment, sleep problems, aggression, and reduced nociception.

I agree with Gustafsson that low basal NO leads to autism via the mechanism that new connections in the brain are of insufficient number and are not "well formed", and that this malformation of connections is a consequence of insufficient basal nitric oxide, and in particular a lack of sufficient nitric oxide during sleep which is when I suggest that much refinement of neural connections occurs.

I suggest that additional symptoms noticed in autistic individuals also point to low NO as both a cause and as an ongoing problem, including the abnormalities in neuroanatomy including increased brain size and reduced minicolumn size, increased pitch discrimination, gut disturbances, immune system dysfunction, reduced cerebral blood flow, increased glucose metabolism, increased plasma lactate, attachment disorders, humming, joint hyperextensibility, oxidative stress, dietary selection, endocrine abnormalities .

It is my hypothesis that the reason for this deficiency in nitric oxide is the loss of the previously unrecognized (as commensal) autotrophic ammonia oxidizing bacteria (AAOB) that in the "wild" (under prehistoric conditions) would live on the scalp and external skin and generate nitric oxide from sweat derived urea. Modern bathing practices wash these bacteria off faster than they can proliferate and the loss of the nitric oxide they generate causes many of the chronic diseases of the modern world, including hypertension, heart disease, obesity, diabetes, and Alzheimer’s Disease16 . I have found that AAOB can live on external human skin for long periods (5 years now), subsisting solely on sweat residues and produce a NO flux significantly above that of uncolonized skin17 . A biofilm of these bacteria suppresses the growth of heterotrophic bacteria. In the presence of these bacteria, a scalp that had been unwashed for >2.5 years had a lower microbial diversity (showing only Staphylococci) than a scalp that had been unwashed for <1>

  1. Sparks BF, Friedman SD, Shaw DW, Aylward EH, Echelard D, Artru AA, Maravilla KR, Giedd JN, Munson J, Dawson G, Dager SR. Brain structural abnormalities in young children with autism spectrum disorder . Neurology 2002 Jul 23;59(2):184-92
  2. E.H. Aylward, PhD; N.J. Minshew, MD; K. Field, BA; B.F. Sparks, BS; and N. Singh, BS. Effects of age on brain volume and head circumference in autism. NEUROLOGY 2002;59:175–183.
  3. Ragnar Henningsson, Per Alm, Erik Lindstrom, and Ingmar Lundquist. Chronic blockade of NO synthase paradoxically increases islet NO production and modulates islet hormone release. Am J Physiol Endocrinol Metab 279: E95-E107, 2000.
  4. Tomas C. Bellamy and John Garthwaite. Sub-second Kinetics of the Nitric Oxide Receptor, Soluble Guanylyl Cyclase, in Intact Cerebellar Cells. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 6, Issue of February 9, pp. 4287–4292, 2001.
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  8. Sadik Sogut, S. Salih Zoroglu, Huseyin Ozyurt, H. Ramazan Yilmaz, Fikret Ozgurlu, Ercan Sivashi, Ozer Yetkin, Medaim Yanik, Hamdi Tutkun, Haluk A. Savas, Mehmet Tarakcioglu, Omer Akyol. Changes in nitric oxide levels and antioxidant enzyme activities may have a role in the pathophysiological mechanisms involved in autism. Clinica Chimica Acta 331 (2003) 111-117.
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  10. Andreas Daiber, Daniel Frein, Dmitry Namgaladze, and Volker Ullrich. Oxidation and Nitrosation in the Nitrogen Monoxide/Superoxide System. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 14, Issue of April 5, pp. 11882–11888, 2002.
  11. Michael Graham Espey, Sandhya Xavier, Douglas D. Thomas, Katrina M. Miranda, and David A. Wink. Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms. PNAS March 19, 2002 vol. 99 no. 6 3481–3486.
  12. Michael G. Espey, Douglas D. Thomas, Katrina M. Miranda, and David A. Wink Focusing of nitric oxide mediated nitrosation and oxidative nitrosylation as a consequence of reaction with superoxide. PNAS August 20, 2002 vol. 99 no. 17 11127–11132.
  13. Andreas Daiber, Daniel Frein, Dmitry Namgaladze, and Volker Ullrich. Oxidation and Nitrosation in the Nitrogen Monoxide/Superoxide System. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 14, Issue of April 5, pp. 11882–11888, 2002.
  14. Lennart Gustafsson. Neural network theory and recent neuroanatomical findings indicate that inadequate nitric oxide synthase will cause autism. In Pallade V, Howlett RJ, Jain L, editors. Lecture notes in artificial intelligence, Volume 2774, part II. New York: Springer-Verlag, P 1109-14.
  15. Lennart Gustafsson. Comment on "disruption in the inhibitory architecture of the cell minicolumns" Implications for autism". Neuroscientist 10 (3): 189-191, 2004. January 8, 2004.
  16. David R. Whitlock. NO production on human skin from sweat derived urea by commensal Autotrophic Ammonia Oxidizing Bacteria. Poster P208 Presented at: The 3rd International Conference on the Biology, Chemistry, and Therapeutic Applications of Nitric Oxide / The 4th annual Scientific meeting of the Nitric Oxide Society of Japan May 24-28, 2004.
  17. David R. Whitlock. Nitric oxide production on human skin from sweat derived ammonia by autotrophic ammonia oxidizing bacteria. (unpublished companion paper).
  18. Maria Gloria Dominguez-Bello, University of Puerto Rico, personal communication.