I posted a comment at The Autism Crisis which referred to a recent talk given by Dr Matthew Belmonte. Ms Dawson felt the comments were off topic and so I have moved the discussion to my blog.
I am still in the process of trying to understand the comment that Dr Belmonte left, in particular the concept of the veridical percept. When I do understand it I will post a reply.
List of citations for Dr Belmonte. They are listed in approximate order of relevance. Also posted at
http://daedalus2u.blogspot.com/index.html
I have added some commentary. I have tried to use as few citations as possible. This is more to show that NO physiology is sufficiently complex to support the many complex physiological effects observed in autism and ASDs.
Thomas DD, Ridnour LA, Isenberg JS, Flores-Santana W, Switzer CH, Donzelli S, Hussain P, Vecoli C, Paolocci N, Ambs S, Colton CA, Harris CC, Roberts DD, Wink DA. The chemical biology of nitric oxide: implications in cellular signaling. Free Radic Biol Med. 2008 Jul 1;45(1):18-31. PubMed link
This is quite a good review paper on signaling by NO. Dave Wink is a senior researcher in the field, he just chaired the Gordon Conference on NO last week. Most of the higher concentration effects are relevant for metabolic physiology (and his specialty cancer), not so much neurological effects. The levels that are most relevant for neuronal effects are in the 1-30 nM/L range (0.03 to 0.9 ppb by weight). That is the basal level is around 1 nM/L up to a few times the EC50 of sGC in cells (~10 nM/L). There are no techniques to measure those levels in vivo on the time and length scales that are known to be important. We know that levels can't be that high (10 nM/L) long term in the endothelium because if they were, there would be systemic hypotension (as in septic shock). This is the first review that really puts together all the different concentration scales that NO is important at.
Because NO sources and sinks are small, and NO is highly diffusible, there are large gradients in NO concentration. Because NO is ~10x more soluble in isotropic lipid, lipid membranes have a large effect on the diffusion. Many lipid membranes are not isotropic. What effect that has on NO diffusion has not been investigated.
Sources are important and sinks are important. Superoxide is an important sink (depending on its concentration). I have a slight quibble (actually very slight). They mention that SOD levels are high enough that superoxide shouldn't affect NO levels in the nM/L level. If everything were uniform and isotropic I would agree, but SOD levels are not isotropic, and superoxide is usually generated vectorally to the inside of vesicles, mitochondria and microsomes. Superoxide is confined by lipid membranes so levels can (in theory) get high inside of those vesicles. Superoxide is an anion, so it will be affected by local electric fields and be held up against the inside of the lipid membrane that is confining it. The SOD would be floating around in the bulk. NO is ~10x more soluble in the lipid membrane, so depending on the precise locations of the NO source, the superoxide source, the SOD, the local electric field and the lipid volumes in the vicinity the effects could be complex.
The major take home message is that because NO can diffuse everywhere, and each NO sensor only senses the sum of NO from all sources, all sources and all sinks matter, including the basal level. The basal level is more important the lower the levels are, the lowest levels mediate things acutely through sGC, as in the brain. There can be effects from lower NO levels which don't activate sGC, for example when NO combines with superoxide and forms peroxynitrite and nitrates proteins. These proteins can accumulate NOx over long periods and integrate a NO/ROS signal over that time. I think this is important in regulating mitochondria number in neurons where mitochondria biogenesis (regulated by NO) must be matched to mitochondria need for the entire neuron (which can vary by 3 or more orders of magnitude depending on axon length). My hypothesis is that mitochondria accumulate nitrated proteins over their metabolic lifetime and that integrates the metabolic load on those mitochondria. When those mitochondria are recycled via autophagy, the NOx is released and produces a NO signal that triggers the biogenesis of the appropriate number of mitochondria to support the needed metabolic load.
Garthwaite J. Concepts of neural nitric oxide-mediated transmission. Eur J Neurosci. 2008 Jun;27(11):2783-802. PubMed link
This is a good review on neuronal signaling via NO, pointing out that NO does cause both LTP and LTD and that the quantities needed for activity are very small in the nM/L range. That is the same level that activates sGC and causes vasodilation.
Many neuropeptides and growth factors, when they are endocytosed by their receptor, activate nitric oxide synthase, and the receptor/NOS complex is conveyed back to the cell body while it is producing NO. I suspect that the NO that the endocytosed receptor complex produces is important in the LTP produced by many of these growth factors as that NO from inside the axon adds to the local NO on the outside. Myelin is transparent to NO, but the alternating layers of lipid and aqueous would certainly modulate its diffusion in complex ways.
NOS binds to the serotonin transporter which couples NO release to serotonin uptake.
Wang S, Paton JF, Kasparov S. Differential sensitivity of excitatory and inhibitory synaptic transmission to modulation by nitric oxide in rat nucleus tractus solitarii. Exp Physiol. 2007 Mar;92(2):371-82. PubMed link
This paper shows changes in membrane potential following exposure to ~ nM/L levels of NO. My conceptualization is that the NO release that causes the vasodilation observed in fMRI BOLD also changes the sensitivity of neurons in that affected volume element to be triggered.
It has been shown (Goense & Logothetis, 2008) that there can be vasodilation without neuronal activation. But we already knew that because even in a volume element that is observed to be highly activated, not every neuron is in a state of discharge. The NO primes the neurons to discharge, but which ones do discharge depends on which ones the action potentials propagate into.
Krause DN, Duckles SP, Pelligrino DA. Influence of sex steroid hormones on cerebrovascular function. J Appl Physiol. 2006 Oct;101(4):1252-61. PubMed link
This paper is quite interesting because it relates to the "extreme male brain" hypothesis of ASDs. Testosterone decreases NO levels and estrogen increases NO levels. This paper relates that primarily to blood flow in adults, I think the effects on fetal neurodevelopment in utero would be important. NO inhibits Leydig cell production of testosterone, so low NO causes high testosterone levels. High testosterone levels cause a more hirsute phenotype, which expands the niche for the bacteria I am studying, causing increased NO/NOx production by them, exerting feedback control on androgen synthesis. My hypothesis is that the association of in utero testosterone levels with ASDs and ASD-like behaviors is real, but that both of them are secondary to low NO status. Many conditions associated with hyperandrogenic effects in adults are also associated with low NO, as in polycystic ovarian syndrome. Stress causes low NO and stress tends to cause high androgen levels.
I think a better characterization of the autism would be as the "extremely stressed brain" because most of the differences are not gender specific.
In utero levels of testosterone and pathways by which that can affect neurodevelopment lead into the next papers, on epigenetic programming of adult physiology.
Racasan S, Braam B, Koomans HA, Joles JA. Programming blood pressure in adult SHR by shifting perinatal balance of NO and reactive oxygen species toward NO: the inverted Barker phenomenon. Am J Physiol Renal Physiol. 2005 Apr;288(4):F626-36. Epub 2004 Nov 16. Review. PubMed link
This paper relates to the epigenetic programming of adult physiology by NO/ROS balance in utero. The mechanism(s) for epigenetic programming by NO/ROS balance are not fully understood. The mechanisms for epigenetic modification (changes to DNA methylation for example) are mechanisms that could pertain in essentially every cell type and in essentially every tissue compartment. If NO/ROS mediated epigenetic programming modified adult function in some organs it would be surprising if that same mechanism(s) did not pertain in many organs in that the substrate (genomic DNA) is the same.
Stress in utero in animals is known to change adult brain size (some types of stress increase it) and adult behaviors. Stress in utero is also known to increase the incidence of ASDs. People with ASDs are exquisitely sensitive to stress. Chronic stress does increase acute sensitivity to stress even after the chronic stress is removed, even in adults.
MeCP2 deletion is known to cause autism-like symptoms (Rett Syndrome). MeCP2 codes for the gene that allows for differential expression of methylated DNA, DNA that is methylated as a consequence of the epigenetic programming of those cells. Presumably if aberrant readout of methylated DNA can "cause" autism-like symptoms, then sufficient changes to methylation of DNA at any step along the way could conceivably do similar things.
I don't think of Rett Syndrome as "autism", rather I see it as "autism-like". I see "autism" and the ASDs as being due to neuroanatomy and epigenetic programming of that neuroanatomy most of which occurs in utero (due to low NO (my hypothesis)). I see "autism-like" as being due to acute decreases in NO brought about by a variety of mechanisms. An important mechanism is epigenetic programming of the brain in utero to be in a lower NO state. Social isolation does this also, so can metabolic stress as in mitochondrial disorders (which generate lots of superoxide) and things like Rett Syndrome where there is metabolic stress due to tissue compartments being mosaic (with different MeCP2 status) resulting in cells not working "in sync". I think that raising NO levels can help "autism-like" symptoms, changing the neuroanatomy is (I think) not possible. I think that raising NO levels soon enough in neurodevelopment can switch an individual from development on an ASD trajectory to an NT trajectory (to some extent).
I think the acute resolution of autism symptoms with fever (Zimmerman 2007) is due to increased NO from iNOS. I have an extensive blog about what I see as the physiology behind it.
Godfrey KM, Barker DJ. Fetal nutrition and adult disease. Am J Clin Nutr. 2000 May;71(5 Suppl):1344S-52S. Review. PubMed link
See table 1, a list of 10 tissue compartments where there is evidence of in utero programming in humans. The physiology of many adult human organs is known to be epigenetically programmed in utero. It would be beyond surprising if the most important organ, the brain, was not.
Showing posts with label low NO hypothesis. Show all posts
Showing posts with label low NO hypothesis. Show all posts
Tuesday, March 10, 2009
Sunday, May 25, 2008
Is the secret to good health in a bowl of cherries?
In a word, no. As we all know the secret to good health is NO.
I received a question regarding a product that mentions specific antioxidant activity against peroxynitrite. Rather than leave the answer as a comment I am posting it as a blog. I looked at the CherryActive website and while their product may have abundant antioxidants of certain (and uncharacterized) types there is no compelling evidence on their website or in the literature that consuming supplemental antioxidants (of any type) has any positive health value and some modest evidence that modest levels supplemental antioxidants are actually harmful.
Cherries as a unique neutraceutical? Unlikely at best.
Even supposing that supplemental antioxidants had good effects, the only place those good effects could occur would be inside cells at the sites of free radical formation. Ingested antioxidants must survive digestion in the stomach and gut, be absorbed through the gut into the blood stream, be transported through the portal system, be metabolized by the liver (most likely) and then distributed by the blood stream throughout the body in exactly the correct amounts of each type of antioxidant in each different tissue compartment simultaneously. Is there any evidence that such things might occur with intact cherry antioxidants from this specific type of cherry? (I say intact because that is what the in vitro tests of cherry juice measured, antioxidant capacity before digestion and absorption, not afterward.) Not really. Perhaps it could happen with antioxidants that human physiology evolved to work best with, but such evolution would take a long time, many generations. For specific antioxidants to be a continuous and integral part of the human diet for many generations, those antioxidants could not be unique to a single food type which would only be available in the "wild" for a short period while in season (perhaps 1 month out of the year for cherries).
The only way humans could evolve physiology adapted to cherries as the ideal source of specific and unique antioxidants would be if the lack of the ability to utilize cherry antioxidants produces such a detriment to survival and reproduction that humans who could utilize those antioxidants had a substantial advantage. There certainly has been no report of the kinds of adverse effects from not consuming cherries that would be required to provide sufficient evolutionary pressure for humans to evolve a physiology that matched the phytochemical profile of cherries, particularly not a particular type of cherries. In any case, cherries were domesticated pretty recently, in Europe, Asia and North America (not Africa where most of human evolution occurred).
Free radicals, peroxynitrite, and superoxide: They say it like it is a bad thing.
They cite an article on NO and peroxynitrite (with the author's name misspelled). The article is a pretty good review; however the authors don't seem to appreciate the (extreme) importance of the location of the formation of superoxide to the location of the formation of peroxynitrite. Superoxide is a charged anion (as is peroxynitrite). Lipid membranes block anions very well. Superoxide can't pass through a lipid membrane except through an anion channel. Virtually all superoxide is generated inside of vesicles and is isolated from the cytoplasm by a lipid membrane. Mitochondria do generate a lot of superoxide, but it is vectorally produced to the inside of the inner matrix. There are 2 lipid membranes confining the superoxide, and the mitochondrial potential (~140 mV) keeps anions inside too. Superoxide (as superoxide) can't get out of mitochondria while the potential is intact and/or while the membranes are intact. Superoxide is dismutated to H2O2 and that being uncharged can diffuse out. Acute effects of superoxide or peroxynitrite in mitochondria would be expected to be confined to the inner matrix. Peroxynitrite does decompose producing NO2 which is uncharged and so can diffuse through the lipid membranes. NO2 could diffuse out, but NO2 is quite reactive and so can't diffuse very far. Nitration of mitochondrial proteins not in the inner matrix might be mediated through NO2. There might be a number of important control mechanisms utilizing nitration of mitochondrial proteins with NO2. Nitration of mitochondrial proteins is known to be important. The detailed mechanisms of that are still mostly unknown.
The other main sites of superoxide production are microsomes where a lot of metabolism of xenobiotic (and non-xenobiotic) chemicals takes place utilizing the cytochrome P450 enzymes. In microsomes too, the superoxide is vectorally produced to the inside of the vesicle. Cytochrome P450 enzymes are involved in a great many synthetic operations in the body. That is how cholesterol is synthesized, also steroids, and also how many toxins are rendered less toxic (and sometimes more toxic). Virtually all of the P450 enzymes are highly uncoupled, that is they make a lot of superoxide. Mitochondria only make a few percent superoxide from the O2 they consume, the P450 enzymes make a lot more, in some cases as much as 50% of the O2 consumed ends up as superoxide. This superoxide is important in the regulation of the activity of the P450 enzymes.
Normally the P450 enzymes are inhibited by NO. NO binds to the active site where O2 binds and prevents O2 from binding and beginning the reaction. Normally there is a little activity, so that electrons trickle through and generate enough superoxide to keep the NO level in balance. When the P450 enzyme becomes activated, the superoxide level goes up, the NO level goes down, and the activity of the P450 enzyme goes up, metabolizing what ever normal or xenobiotic chemical needs metabolizing. The pulse of superoxide and other free radicals produced by that the pulse of metabolic activity has important regulatory effects.
The oxidation of xenobiotic chemicals is called Phase I of the drug metabolizing process. This is followed by Phase II of the drug metabolizing process where the oxidation products are tagged with molecules that make them excretable. This includes glutathione, sulfate and even glucose. Glutathione and glucose tag the chemical for excretion in the bile, sulfate tags it for excretion in the urine.
The free radicals and electrophiles produced during the Phase I process cause the expression of the Phase II genes which then detoxify those oxidation products by conjugating them so they can be excreted. There is considerable thought that a significant part of the observed health effects of different phytochemicals in food is due to their metabolism by the P450 system, the normal generation of superoxide and other free radicals, and the stimulation of the Phase II system by those free radicals. It may be the production of free radicals rather than the destruction of free radicals that is mechanism by which fruits and vegetables are protective (if they actually are protective which is not clear). There have actually been no blinded studies of food intake on health.
In vitro, peroxynitrite is only observed when there is near stoichiometric production of superoxide and NO. When either is in several fold excess, peroxynitrite and effects attributed to peroxynitrite are not observed. In other words the problem of peroxynitrite (if is actually is a problem) is likely due to either not enough superoxide or not enough NO, rather than from too much of either. The association of nitrated proteins with adverse health states may be an effect not a cause. Peroxynitrite only occurs when the quantities are essentially equal and equal production occurs for extended periods of time. To me that sounds more like a signaling problem, that the "signal" of peroxynitrite wasn't high enough to switch physiology hard enough to get it back into the "good" state of either excess NO or excess superoxide. If that is the case, blocking peroxynitrite signaling would be a bad thing and would exacerbate peroxynitrite problems.
There is extremely good evidence that free radicals are extremely important signaling molecules that are absolutely essential in the right amounts at the right time in the right place and for the right duration. Free radicals tend to be small, are extremely reactive toward many different molecules, and have a very low background (because they are so reactive). Those properties make free radicals excellent signaling molecules capable of achieving a high signal to noise ratio even at very low levels and with very fast kinetics on a large variety of biological molecules simultaneously over multiple different time scales. The pathways controlled by free radical reactions include just about everything, energy production, gene transcription, the cell cycle, DNA replication, ischemic preconditioning. There are just about no physiological pathways that are not closely coupled to pathways utilizing free radicals as signaling molecules. Those ideal properties of free radicals as signaling molecules also make experiments to understand the signaling pathways those free radicals are involved in very challenging. It is not at all surprising that over evolutionary time organisms would evolve physiology to use such ideal signaling molecules many times, at many places, in many tissue compartments, for many reasons to do many things by many different pathways over many different time scales by many different mechanisms.
Signaling by free radicals as signaling molecules are only going to work well in the media they evolved to work in. That media is quite complex (multiple different phases, i.e. lipid and aqueous of different sizes and compositions), and is different in each tissue compartment (blood, plasma, CFS, cytoplasm, mitochondrial matrix, depot fat, liver, kidney, etc, etc.). Adding different compounds that bind strongly to free radicals (that would be supplemental antioxidants) is virtually certain to perturb that media and interfere with that signaling in complex ways.
All the large, long term double-blind placebo controlled trials of supplemental antioxidants have shown either no effect or a slight negative effect. A good recent meta-analysis summarizing many of those antioxidant trials is in the NEJM. A more recent Cochrane review on the subject (which I have only seen the abstract of) found essentially no benefit from supplemental antioxidants.
To me, that is not at all a surprise. My own feeling is that free radical signaling is too important to organisms for them to allow the background environment that signaling occurs in to be set "at random" by arbitrary (and in the wild highly variable) dietary levels of antioxidants. Humans (and every other organism) didn't evolve for their physiology to be optimized by consuming a certain number of a certain type of cherries (or other specific foods) per day. There simply has to be feedback control(s) that regulate the internal environment(s) the free radical signal(s) propagate in. Oxidative stress is such an important parameter to regulate that organisms "learned" how to regulate their own state of oxidative stress billions of year ago. Bacteria regulate their level of oxidative stress (as does every other organism). Stress response genes, including genes for responding to oxidative stress are among the most highly conserved genes in organisms. Why would we think that humans somehow lost that ability? Especially when there is no evidence that humans have lost it. If a particular human did lose the ability to regulate oxidative stress, death would occur in a few minutes (or less).
There are many compounds in vivo that have antioxidant properties in vitro. The natural modulation of the concentrations of a few dozen such compounds in vivo would be difficult to measure, particularly since the antioxidant properties of these molecules can be different by many orders of magnitude. For example NO is an excellent antioxidant. It has a free electron and reacts with free radicals at near diffusion limited kinetics. It is a better antioxidant than is Vitamin E by about a factor of 5,000.
NO rapidly reacts with peroxidized lipids, quenching the free radical mediated chain reaction. NO reacts with peroxidized lipid radicals considerably faster than does vitamin E (~2E9 M-1 s-1 vs. 5E5), and considerably faster than the propagation reaction (1.3E3). In vitro, 7 nM/min NO was as effective as 50 µM/L α- tocopherol in free radical catalyzed linoleic acid oxidation. Oxidative consumption of α- tocopherol did not occur until NO concentrations fell below ~10 nM/L. The combination of NO and α- tocopherol prevented lipid oxidation better than either alone. A slight increase in the NO level has a much larger effect on antioxidant activity than a much larger increase in Vitamin E levels.
10 nM/L NO is somewhat higher than the normal background (which is around 1 nM/L). 1 nM/L is about 30 parts per trillion by weight. There are no techniques to measure that in vivo on the length and time scales that are important. The only reason we know that the basal level is about that is because at higher levels NO activates sGC and causes local vasodilation which brings in oxyhemoglobin which removes the NO.
Summary antioxidant effects
There is no compelling evidence that supplemental antioxidants of any type have any positive health value. Everything important in physiology is regulated by feedback regulation. Oxidative stress is no exception. Attempting to perturb physiology artificially will simply force it to work harder to maintain the setpoint it is trying to achieve. I think this is the reason why all the large double blind placebo controlled studies show slight negative effects of supplemental antioxidants. Diet choice is a major compensatory pathway by which an organism regulates its state of oxidative stress. When people have high levels of oxidative stress they choose to eat foods with lower levels of antioxidants so the body doesn't need to destroy them metabolically (by generating even more superoxide). Diet choice is an effect of ill health, not the cause.
Gout
They mention that cherries seem to have pharmacological effects at reducing urate levels, and imply (or actually state) that lowering urate levels would be a good thing. They cite a paper that does show an acute lowering of plasma urate levels (funded in part by the California Cherry Advisory Board). The reductions reported in plasma urate are quite small and (in my opinion) not physiologically important. The "normal range" for plasma urate (in females) is 139-393 micromoles/L (serum). The levels measured in this study were all within the normal range, the reduction was small and it was an extremely short study (5 hours). Urinary levels of urate did increase in the cherry group. It isn't clear what is the mechanism for the very modest plasma urate reduction. If it is due to an acute inhibition due to cherry flavonoids, in the longer term (days, weeks, months), there may be compensatory increase in the metabolic systems that produce urate such that there would be no chronic effect, or the chronic effect could be to increase urate levels. If it is increased urinary excretion, levels in the urine are what determine the formation of sodium urate kidney stones. Physiology is inherently non-linear with feedback and hysteresis. The only way to determine if a treatment has a long term effect is to measure the effects of that treatment long term.
Gout is caused by the formation and deposition of crystals of sodium urate. Urate is the terminal metabolite of purines which are heterocyclic nitrogen containing bases used in RNA, DNA, ATP and some other fundamental biochemical pathways. Normally they are ultimately degraded to xanthine and then oxidized by xanthine oxidoreductase into urate. For gout to occur, the solubility of sodium urate crystals has to be exceeded in the relevant tissue compartment and nucleation of sodium urate has to occur. If the sodium urate level is below the solubility limit, further reductions in urate levels will have no preventative effect.
The usual treatment for gout is administration of allopurinol which inhibits xanthine oxidoreductase. A great many natural flavonoids also inhibit xanthine oxidase in vitro. Many natural flavonoids have urate lowering activity in vivo. Hypersensitivity to allopurinol is a significant triggering mechanism for Stevens Johnson Syndrome. The mechanism by which that happens is not understood. I would be concerned that any pharmacologically active compound that inhibits xanthine oxidase might have side effect profiles similar to allopurinol. That includes the unidentified compounds in cherries.
It is not clear that a lower plasma urate level is actually a benefit. There is pretty good evidence of an association of higher plasma urate with a reduced incidence of Parkinson's disease. There may also be a protective effect of urate in a number of diseases (I have only seen the abstract of this), but which is cause and which is effect is not clear. Urate is a pretty good antioxidant itself. Trading an insignificant reduction of the possibility of getting gout for a significant increase in the possibility of getting Parkinson's or Alzheimer's disease isn't a choice that I would make.
Without knowing one's urate status, taking pharmacologically active agents to lower it does not seem prudent to me. If one did need to lower one's urate level, using a compound of known composition, purity, efficacy and side-effect profile produced under controlled conditions seems more prudent to me than using some fruit juice. Allopurinol is generic so it is cheap, most insurance would pay for it with the proper medical indication (which is the only reason you want to take anything pharmacologically active), and you need to have your urate status actually measured by your MD in case that joint pain is actually something more serious.
Muscle damage
The CherryActive website claims
"Research study from University of Vermont and published in the British Journal of Sports Medicine shows that drinking cherry juice can help repair damaged muscle leading to an increased recovery rate from strenuous exercise"
The actual report (funded by Cherrypharm Inc and done by researchers who each own 2.5% equity in Cherrypharm Inc) only talks about symptoms and says:
"Although the results of this study indicate a protective effect of cherry juice, it is not possible to conclude that cherry juice supplementation prevented muscle damage, because only two of four indirect markers of damage showed an effect."
They used a proprietary blend of cherry juice and apple juice.
"The cherry juice blend was prepared by mixing freshly prepared tart cherry juice with commercially available apple juice in a proprietary ratio (Cherrypharm Inc, West Hartford, Connecticut, USA). Frozen tart cultivar Montmorency cherries were used to prepare the cherry juice following standard procedures that simulate industrial processing."
They only used subjective measures of muscle function, how the subjects felt about how their muscles were functioning. They specifically chose to not use objective measures of muscle damage, myoglobin and/or creatine kinase. They rationalize their decision in terms of potential confounding due to unauthorized muscle activity not within the protocol they were testing. Why they considered that objective instrumental measures of muscle injury would be more subject to confounding than subjective measures is not explained. I suspect it has to do with each of them owning 2.5% of Cherrypharm Inc. They suggest that future tests might be run using actual measurements but no future papers have been forthcoming. Either they never did the tests, or they did the tests and did not publish them.
They suggest no plausible mechanism by which consuming a cherry juice apple juice blend would have any effects on muscle damage or healing.
The statements on the CherryActive website are clearly not supported by the research they cite. They are being dishonest to pretend that it does.
Conclusion.
Cherries are a fine and tasty food. I have nothing against cherries, I like cherries and I eat cherries when in season. Moderate consumption of cherries as part of a varied diet is likely no more and no less healthful than moderate consumption of any other fruits and vegetables. Cherries do tend to be somewhat pricy for me even when in season. The cherry products sold by CherryActive are extremely pricy. I see no reason to consume any of CherryActive's products other than as simple foods, and virtually any other source of cherries would be more economical as are many other fruits and vegetables.
I didn't know very much about cherries when I started looking into CherryActive. I suspect that I will keep my cherry intake more moderate primarily because of the xanthine oxidoreductase inhibition effects. An extremely important property of xanthine oxidoreductase is the reduction of nitrate to nitrite and the reduction of nitrite to NO. NO from nitrite is extremely important in many emergency and extreme stress situations. Nitrite is substantially protective against infarcts due to acute ischemia in heart muscle, liver, kidney, brain. Messing with the xanthine oxidoreductase system by inhibiting it with cherries is not something one should do without good reasons. The reasons that CherryActive provide are not good enough for me.
I received a question regarding a product that mentions specific antioxidant activity against peroxynitrite. Rather than leave the answer as a comment I am posting it as a blog. I looked at the CherryActive website and while their product may have abundant antioxidants of certain (and uncharacterized) types there is no compelling evidence on their website or in the literature that consuming supplemental antioxidants (of any type) has any positive health value and some modest evidence that modest levels supplemental antioxidants are actually harmful.
Cherries as a unique neutraceutical? Unlikely at best.
Even supposing that supplemental antioxidants had good effects, the only place those good effects could occur would be inside cells at the sites of free radical formation. Ingested antioxidants must survive digestion in the stomach and gut, be absorbed through the gut into the blood stream, be transported through the portal system, be metabolized by the liver (most likely) and then distributed by the blood stream throughout the body in exactly the correct amounts of each type of antioxidant in each different tissue compartment simultaneously. Is there any evidence that such things might occur with intact cherry antioxidants from this specific type of cherry? (I say intact because that is what the in vitro tests of cherry juice measured, antioxidant capacity before digestion and absorption, not afterward.) Not really. Perhaps it could happen with antioxidants that human physiology evolved to work best with, but such evolution would take a long time, many generations. For specific antioxidants to be a continuous and integral part of the human diet for many generations, those antioxidants could not be unique to a single food type which would only be available in the "wild" for a short period while in season (perhaps 1 month out of the year for cherries).
The only way humans could evolve physiology adapted to cherries as the ideal source of specific and unique antioxidants would be if the lack of the ability to utilize cherry antioxidants produces such a detriment to survival and reproduction that humans who could utilize those antioxidants had a substantial advantage. There certainly has been no report of the kinds of adverse effects from not consuming cherries that would be required to provide sufficient evolutionary pressure for humans to evolve a physiology that matched the phytochemical profile of cherries, particularly not a particular type of cherries. In any case, cherries were domesticated pretty recently, in Europe, Asia and North America (not Africa where most of human evolution occurred).
Free radicals, peroxynitrite, and superoxide: They say it like it is a bad thing.
They cite an article on NO and peroxynitrite (with the author's name misspelled). The article is a pretty good review; however the authors don't seem to appreciate the (extreme) importance of the location of the formation of superoxide to the location of the formation of peroxynitrite. Superoxide is a charged anion (as is peroxynitrite). Lipid membranes block anions very well. Superoxide can't pass through a lipid membrane except through an anion channel. Virtually all superoxide is generated inside of vesicles and is isolated from the cytoplasm by a lipid membrane. Mitochondria do generate a lot of superoxide, but it is vectorally produced to the inside of the inner matrix. There are 2 lipid membranes confining the superoxide, and the mitochondrial potential (~140 mV) keeps anions inside too. Superoxide (as superoxide) can't get out of mitochondria while the potential is intact and/or while the membranes are intact. Superoxide is dismutated to H2O2 and that being uncharged can diffuse out. Acute effects of superoxide or peroxynitrite in mitochondria would be expected to be confined to the inner matrix. Peroxynitrite does decompose producing NO2 which is uncharged and so can diffuse through the lipid membranes. NO2 could diffuse out, but NO2 is quite reactive and so can't diffuse very far. Nitration of mitochondrial proteins not in the inner matrix might be mediated through NO2. There might be a number of important control mechanisms utilizing nitration of mitochondrial proteins with NO2. Nitration of mitochondrial proteins is known to be important. The detailed mechanisms of that are still mostly unknown.
The other main sites of superoxide production are microsomes where a lot of metabolism of xenobiotic (and non-xenobiotic) chemicals takes place utilizing the cytochrome P450 enzymes. In microsomes too, the superoxide is vectorally produced to the inside of the vesicle. Cytochrome P450 enzymes are involved in a great many synthetic operations in the body. That is how cholesterol is synthesized, also steroids, and also how many toxins are rendered less toxic (and sometimes more toxic). Virtually all of the P450 enzymes are highly uncoupled, that is they make a lot of superoxide. Mitochondria only make a few percent superoxide from the O2 they consume, the P450 enzymes make a lot more, in some cases as much as 50% of the O2 consumed ends up as superoxide. This superoxide is important in the regulation of the activity of the P450 enzymes.
Normally the P450 enzymes are inhibited by NO. NO binds to the active site where O2 binds and prevents O2 from binding and beginning the reaction. Normally there is a little activity, so that electrons trickle through and generate enough superoxide to keep the NO level in balance. When the P450 enzyme becomes activated, the superoxide level goes up, the NO level goes down, and the activity of the P450 enzyme goes up, metabolizing what ever normal or xenobiotic chemical needs metabolizing. The pulse of superoxide and other free radicals produced by that the pulse of metabolic activity has important regulatory effects.
The oxidation of xenobiotic chemicals is called Phase I of the drug metabolizing process. This is followed by Phase II of the drug metabolizing process where the oxidation products are tagged with molecules that make them excretable. This includes glutathione, sulfate and even glucose. Glutathione and glucose tag the chemical for excretion in the bile, sulfate tags it for excretion in the urine.
The free radicals and electrophiles produced during the Phase I process cause the expression of the Phase II genes which then detoxify those oxidation products by conjugating them so they can be excreted. There is considerable thought that a significant part of the observed health effects of different phytochemicals in food is due to their metabolism by the P450 system, the normal generation of superoxide and other free radicals, and the stimulation of the Phase II system by those free radicals. It may be the production of free radicals rather than the destruction of free radicals that is mechanism by which fruits and vegetables are protective (if they actually are protective which is not clear). There have actually been no blinded studies of food intake on health.
In vitro, peroxynitrite is only observed when there is near stoichiometric production of superoxide and NO. When either is in several fold excess, peroxynitrite and effects attributed to peroxynitrite are not observed. In other words the problem of peroxynitrite (if is actually is a problem) is likely due to either not enough superoxide or not enough NO, rather than from too much of either. The association of nitrated proteins with adverse health states may be an effect not a cause. Peroxynitrite only occurs when the quantities are essentially equal and equal production occurs for extended periods of time. To me that sounds more like a signaling problem, that the "signal" of peroxynitrite wasn't high enough to switch physiology hard enough to get it back into the "good" state of either excess NO or excess superoxide. If that is the case, blocking peroxynitrite signaling would be a bad thing and would exacerbate peroxynitrite problems.
There is extremely good evidence that free radicals are extremely important signaling molecules that are absolutely essential in the right amounts at the right time in the right place and for the right duration. Free radicals tend to be small, are extremely reactive toward many different molecules, and have a very low background (because they are so reactive). Those properties make free radicals excellent signaling molecules capable of achieving a high signal to noise ratio even at very low levels and with very fast kinetics on a large variety of biological molecules simultaneously over multiple different time scales. The pathways controlled by free radical reactions include just about everything, energy production, gene transcription, the cell cycle, DNA replication, ischemic preconditioning. There are just about no physiological pathways that are not closely coupled to pathways utilizing free radicals as signaling molecules. Those ideal properties of free radicals as signaling molecules also make experiments to understand the signaling pathways those free radicals are involved in very challenging. It is not at all surprising that over evolutionary time organisms would evolve physiology to use such ideal signaling molecules many times, at many places, in many tissue compartments, for many reasons to do many things by many different pathways over many different time scales by many different mechanisms.
Signaling by free radicals as signaling molecules are only going to work well in the media they evolved to work in. That media is quite complex (multiple different phases, i.e. lipid and aqueous of different sizes and compositions), and is different in each tissue compartment (blood, plasma, CFS, cytoplasm, mitochondrial matrix, depot fat, liver, kidney, etc, etc.). Adding different compounds that bind strongly to free radicals (that would be supplemental antioxidants) is virtually certain to perturb that media and interfere with that signaling in complex ways.
All the large, long term double-blind placebo controlled trials of supplemental antioxidants have shown either no effect or a slight negative effect. A good recent meta-analysis summarizing many of those antioxidant trials is in the NEJM. A more recent Cochrane review on the subject (which I have only seen the abstract of) found essentially no benefit from supplemental antioxidants.
To me, that is not at all a surprise. My own feeling is that free radical signaling is too important to organisms for them to allow the background environment that signaling occurs in to be set "at random" by arbitrary (and in the wild highly variable) dietary levels of antioxidants. Humans (and every other organism) didn't evolve for their physiology to be optimized by consuming a certain number of a certain type of cherries (or other specific foods) per day. There simply has to be feedback control(s) that regulate the internal environment(s) the free radical signal(s) propagate in. Oxidative stress is such an important parameter to regulate that organisms "learned" how to regulate their own state of oxidative stress billions of year ago. Bacteria regulate their level of oxidative stress (as does every other organism). Stress response genes, including genes for responding to oxidative stress are among the most highly conserved genes in organisms. Why would we think that humans somehow lost that ability? Especially when there is no evidence that humans have lost it. If a particular human did lose the ability to regulate oxidative stress, death would occur in a few minutes (or less).
There are many compounds in vivo that have antioxidant properties in vitro. The natural modulation of the concentrations of a few dozen such compounds in vivo would be difficult to measure, particularly since the antioxidant properties of these molecules can be different by many orders of magnitude. For example NO is an excellent antioxidant. It has a free electron and reacts with free radicals at near diffusion limited kinetics. It is a better antioxidant than is Vitamin E by about a factor of 5,000.
NO rapidly reacts with peroxidized lipids, quenching the free radical mediated chain reaction. NO reacts with peroxidized lipid radicals considerably faster than does vitamin E (~2E9 M-1 s-1 vs. 5E5), and considerably faster than the propagation reaction (1.3E3). In vitro, 7 nM/min NO was as effective as 50 µM/L α- tocopherol in free radical catalyzed linoleic acid oxidation. Oxidative consumption of α- tocopherol did not occur until NO concentrations fell below ~10 nM/L. The combination of NO and α- tocopherol prevented lipid oxidation better than either alone. A slight increase in the NO level has a much larger effect on antioxidant activity than a much larger increase in Vitamin E levels.
10 nM/L NO is somewhat higher than the normal background (which is around 1 nM/L). 1 nM/L is about 30 parts per trillion by weight. There are no techniques to measure that in vivo on the length and time scales that are important. The only reason we know that the basal level is about that is because at higher levels NO activates sGC and causes local vasodilation which brings in oxyhemoglobin which removes the NO.
Summary antioxidant effects
There is no compelling evidence that supplemental antioxidants of any type have any positive health value. Everything important in physiology is regulated by feedback regulation. Oxidative stress is no exception. Attempting to perturb physiology artificially will simply force it to work harder to maintain the setpoint it is trying to achieve. I think this is the reason why all the large double blind placebo controlled studies show slight negative effects of supplemental antioxidants. Diet choice is a major compensatory pathway by which an organism regulates its state of oxidative stress. When people have high levels of oxidative stress they choose to eat foods with lower levels of antioxidants so the body doesn't need to destroy them metabolically (by generating even more superoxide). Diet choice is an effect of ill health, not the cause.
Gout
They mention that cherries seem to have pharmacological effects at reducing urate levels, and imply (or actually state) that lowering urate levels would be a good thing. They cite a paper that does show an acute lowering of plasma urate levels (funded in part by the California Cherry Advisory Board). The reductions reported in plasma urate are quite small and (in my opinion) not physiologically important. The "normal range" for plasma urate (in females) is 139-393 micromoles/L (serum). The levels measured in this study were all within the normal range, the reduction was small and it was an extremely short study (5 hours). Urinary levels of urate did increase in the cherry group. It isn't clear what is the mechanism for the very modest plasma urate reduction. If it is due to an acute inhibition due to cherry flavonoids, in the longer term (days, weeks, months), there may be compensatory increase in the metabolic systems that produce urate such that there would be no chronic effect, or the chronic effect could be to increase urate levels. If it is increased urinary excretion, levels in the urine are what determine the formation of sodium urate kidney stones. Physiology is inherently non-linear with feedback and hysteresis. The only way to determine if a treatment has a long term effect is to measure the effects of that treatment long term.
Gout is caused by the formation and deposition of crystals of sodium urate. Urate is the terminal metabolite of purines which are heterocyclic nitrogen containing bases used in RNA, DNA, ATP and some other fundamental biochemical pathways. Normally they are ultimately degraded to xanthine and then oxidized by xanthine oxidoreductase into urate. For gout to occur, the solubility of sodium urate crystals has to be exceeded in the relevant tissue compartment and nucleation of sodium urate has to occur. If the sodium urate level is below the solubility limit, further reductions in urate levels will have no preventative effect.
The usual treatment for gout is administration of allopurinol which inhibits xanthine oxidoreductase. A great many natural flavonoids also inhibit xanthine oxidase in vitro. Many natural flavonoids have urate lowering activity in vivo. Hypersensitivity to allopurinol is a significant triggering mechanism for Stevens Johnson Syndrome. The mechanism by which that happens is not understood. I would be concerned that any pharmacologically active compound that inhibits xanthine oxidase might have side effect profiles similar to allopurinol. That includes the unidentified compounds in cherries.
It is not clear that a lower plasma urate level is actually a benefit. There is pretty good evidence of an association of higher plasma urate with a reduced incidence of Parkinson's disease. There may also be a protective effect of urate in a number of diseases (I have only seen the abstract of this), but which is cause and which is effect is not clear. Urate is a pretty good antioxidant itself. Trading an insignificant reduction of the possibility of getting gout for a significant increase in the possibility of getting Parkinson's or Alzheimer's disease isn't a choice that I would make.
Without knowing one's urate status, taking pharmacologically active agents to lower it does not seem prudent to me. If one did need to lower one's urate level, using a compound of known composition, purity, efficacy and side-effect profile produced under controlled conditions seems more prudent to me than using some fruit juice. Allopurinol is generic so it is cheap, most insurance would pay for it with the proper medical indication (which is the only reason you want to take anything pharmacologically active), and you need to have your urate status actually measured by your MD in case that joint pain is actually something more serious.
Muscle damage
The CherryActive website claims
"Research study from University of Vermont and published in the British Journal of Sports Medicine shows that drinking cherry juice can help repair damaged muscle leading to an increased recovery rate from strenuous exercise"
The actual report (funded by Cherrypharm Inc and done by researchers who each own 2.5% equity in Cherrypharm Inc) only talks about symptoms and says:
"Although the results of this study indicate a protective effect of cherry juice, it is not possible to conclude that cherry juice supplementation prevented muscle damage, because only two of four indirect markers of damage showed an effect."
They used a proprietary blend of cherry juice and apple juice.
"The cherry juice blend was prepared by mixing freshly prepared tart cherry juice with commercially available apple juice in a proprietary ratio (Cherrypharm Inc, West Hartford, Connecticut, USA). Frozen tart cultivar Montmorency cherries were used to prepare the cherry juice following standard procedures that simulate industrial processing."
They only used subjective measures of muscle function, how the subjects felt about how their muscles were functioning. They specifically chose to not use objective measures of muscle damage, myoglobin and/or creatine kinase. They rationalize their decision in terms of potential confounding due to unauthorized muscle activity not within the protocol they were testing. Why they considered that objective instrumental measures of muscle injury would be more subject to confounding than subjective measures is not explained. I suspect it has to do with each of them owning 2.5% of Cherrypharm Inc. They suggest that future tests might be run using actual measurements but no future papers have been forthcoming. Either they never did the tests, or they did the tests and did not publish them.
They suggest no plausible mechanism by which consuming a cherry juice apple juice blend would have any effects on muscle damage or healing.
The statements on the CherryActive website are clearly not supported by the research they cite. They are being dishonest to pretend that it does.
Conclusion.
Cherries are a fine and tasty food. I have nothing against cherries, I like cherries and I eat cherries when in season. Moderate consumption of cherries as part of a varied diet is likely no more and no less healthful than moderate consumption of any other fruits and vegetables. Cherries do tend to be somewhat pricy for me even when in season. The cherry products sold by CherryActive are extremely pricy. I see no reason to consume any of CherryActive's products other than as simple foods, and virtually any other source of cherries would be more economical as are many other fruits and vegetables.
I didn't know very much about cherries when I started looking into CherryActive. I suspect that I will keep my cherry intake more moderate primarily because of the xanthine oxidoreductase inhibition effects. An extremely important property of xanthine oxidoreductase is the reduction of nitrate to nitrite and the reduction of nitrite to NO. NO from nitrite is extremely important in many emergency and extreme stress situations. Nitrite is substantially protective against infarcts due to acute ischemia in heart muscle, liver, kidney, brain. Messing with the xanthine oxidoreductase system by inhibiting it with cherries is not something one should do without good reasons. The reasons that CherryActive provide are not good enough for me.
Wednesday, May 2, 2007
Can the low NO hypothesis of ASDs be proven false?
The answer is yes, it can. If nitric oxide is raised in an ASD individual and that individual does not experience an improvement in symptoms, then low NO is not the problem for that individual.
NO is involved in a lot of pathways, and the pathways that seem to be producing symptoms characteristic of ASDs seem to be affected in the direction that low basal NO would produce.
My perspective on ASDs is that they are a natural consequence of low NO in utero. They are a "feature", which programs the brain to produce the "tool-making and tool-using" phenotype, most precisely characterized by the person with Asperger's. But like all evolved features, if you push it too hard, it becomes dysfunctional. Just like anaphylaxis.
That is why I am extremely confident that there will never be a genetic test for ASDs. If your genotype is such that development in utero under conditions of low NO would not lead to an ASD phenotype, then you are not human. Tool making and tool using is such a fundamental part of being human, that you can't remove it and remain human.
NO will not be a "cure" along the lines of what the curebies want. It won't change the characteristic brain structures that lead to ASD behaviors and neurological characteristics. An ASD individual with high NO will still have an ASD. They won't melt down as much, or as badly, they won't be as anxious or as easy to bully. They will be better able to lead happier and more productive lives as they perceive them to be. A lot of things will get "better". It won't turn them into NTs.
If ASDs were actually a "disorder", then it would be too complicated to be treated. If it is a disorder, it is obviously a disorder that affects neural development on many levels, and has ongoing effects in physiology. A simple treatment will not be able to have complicated effects unless the "simple treatment" ties into the already existing control systems used in phsyiology. The physiological pathways that seem to be "dysfunctional" in ASDs are too coupled and to complex to regulate artifically, that is with anything other than the innate regulatory systems that the body and physiology already has.
If you look at what some of the DAN! practicioners are trying to do, they are trying to substitute external pharmacological interventions via supplements, drugs, vitamins, glutathione, ect, etc, etc, for things that physiology regulates on its own. That approach is not going to work. Physiology is too complicated.
So far, only one person with an ASD has tried my stuff, that would be me. It has not made me NT, but it has greatly improved my ability to be in social situations. I can now notice that there is a lot of body language and non-verbal communication that I am missing. I am getting a lot more than I did before. I can't "focus" like I used to be able to do, that is, I can't work while listening to NPR. My health is a lot better. A lot of things are better. Has my personality changed? I don't think so. Certainly I am different, but not in any ways that I find objectionable. I would not revert to the way I was before I raised my NO level.
Is my "treatment" permanant? I don't think so, not like a course of treatment that resolves a problem and makes it go away, like an antibiotic. It is more like a nutrient, one that you absorb through the skin.
NO is involved in a lot of pathways, and the pathways that seem to be producing symptoms characteristic of ASDs seem to be affected in the direction that low basal NO would produce.
My perspective on ASDs is that they are a natural consequence of low NO in utero. They are a "feature", which programs the brain to produce the "tool-making and tool-using" phenotype, most precisely characterized by the person with Asperger's. But like all evolved features, if you push it too hard, it becomes dysfunctional. Just like anaphylaxis.
That is why I am extremely confident that there will never be a genetic test for ASDs. If your genotype is such that development in utero under conditions of low NO would not lead to an ASD phenotype, then you are not human. Tool making and tool using is such a fundamental part of being human, that you can't remove it and remain human.
NO will not be a "cure" along the lines of what the curebies want. It won't change the characteristic brain structures that lead to ASD behaviors and neurological characteristics. An ASD individual with high NO will still have an ASD. They won't melt down as much, or as badly, they won't be as anxious or as easy to bully. They will be better able to lead happier and more productive lives as they perceive them to be. A lot of things will get "better". It won't turn them into NTs.
If ASDs were actually a "disorder", then it would be too complicated to be treated. If it is a disorder, it is obviously a disorder that affects neural development on many levels, and has ongoing effects in physiology. A simple treatment will not be able to have complicated effects unless the "simple treatment" ties into the already existing control systems used in phsyiology. The physiological pathways that seem to be "dysfunctional" in ASDs are too coupled and to complex to regulate artifically, that is with anything other than the innate regulatory systems that the body and physiology already has.
If you look at what some of the DAN! practicioners are trying to do, they are trying to substitute external pharmacological interventions via supplements, drugs, vitamins, glutathione, ect, etc, etc, for things that physiology regulates on its own. That approach is not going to work. Physiology is too complicated.
So far, only one person with an ASD has tried my stuff, that would be me. It has not made me NT, but it has greatly improved my ability to be in social situations. I can now notice that there is a lot of body language and non-verbal communication that I am missing. I am getting a lot more than I did before. I can't "focus" like I used to be able to do, that is, I can't work while listening to NPR. My health is a lot better. A lot of things are better. Has my personality changed? I don't think so. Certainly I am different, but not in any ways that I find objectionable. I would not revert to the way I was before I raised my NO level.
Is my "treatment" permanant? I don't think so, not like a course of treatment that resolves a problem and makes it go away, like an antibiotic. It is more like a nutrient, one that you absorb through the skin.
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