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.
5. André Dejam, Christian J. Hunter, Mildred M. Pelletier, Lewis L. Hsu1, Roberto F. Machado, Shruti Shiva, Gordon G. Power, Malte Kelm, Mark T. Gladwin, and Alan N. Schechter. Erythrocytes are the Major Intravascular Storage Sites of Nitrite in Human Blood. prepublished online March 17, 2005; DOI 10.1182/blood-2005-02-0567.
6. I. Ruiz-Stewart, S. R. Tiyyagura, J. E. Lin, S. Kazerounian, G. M. Pitari, S. Schulz, E. Martin, F. Murad, and S. A. Waldman. Guanylyl cyclase is an ATP sensor coupling nitric oxide signaling to cell metabolism. PNAS January 6, 2004, vol. 101 no. 1, 37–42. 5
7. Thayne L. Sweeten, David J. Posey, Sudha Shankar and Christopher J. McDougle High nitric oxide production in autistic disorder: a possible role for interferon-. Biological Psychiatry Volume 55, Issue 4, 15 February 2004, Pages 434-437.
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.
9. Woody R. McGinnis. OXIDATIVE STRESS IN AUTISM. ALTERNATIVE THERAPIES, nov/dec 2004, VOL. 10, NO. 6.
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.