The Pathophysiological Rationale for Personalized Metabolic Therapy of ASD. Promising Treatments

A metabolic disorder is a serious problem. Changes that occur at the cellular level and are associated with biochemical processes lead to malfunctioning of the cell, and further, respectively, of tissue, organ, of the whole organism. Metabolic care is the basis of metabolic therapy. For almost every metabolic regimen of autism spectrum disorder, there is a counter-argument. For this reason, a unified approach to therapy is impossible, since, apart from the “autistic triad”, each person has his own peculiarities. At the present stage, in addition to a gluten-free and casein-free diet, vitamins of groups B and D, polyunsaturated fatty acids, various methods of microbiome correction are used in therapy, but there is no serious evidence base for the effectiveness of therapy for autism spectrum disorders. The article proposes options for the study of metabolic changes in the body, which are the rationale for the development of a scheme of metabolic therapy in the framework of a personalized medical approach to the treatment of autism spectrum disorders.

A metabolic disorder is a serious problem. Changes that occur at the cellular level and are
associated with bio- chemical processes lead to malfunctioning of the cell, and further,
respectively, of tissue, organ, of the whole organism. Metabolic care is the basis of metabolic
therapy. For almost every metabolic regimen of autism spectrum disorder, there is a
counter-argument. For this reason, a unified approach to therapy is impossible, since, apart from
the “autistic triad”, each person has his own peculiarities. At the present stage, in addition to a
gluten-free and casein-free diet, vitamins of groups B and D, polyunsaturated fatty acids, various methods of microbiome correction are used in therapy, but there is no serious evidence base for the effectiveness of therapy for autism spectrum disorders. The article proposes options for the study of metabolic changes in the body, which are the rationale for the development of a scheme of metabolic therapy in the framework of a personal- ized medical approach to the treatment of autism spectrum disorders.

Keywords: autism spectrum disorders, autism, metabolic therapy, personalized therapy.
he purpose of this article is to acquaint with the complex biochemical processes occur- ring in the brain, their dependence on the dietary intake, metabolic features, and the gut flora con- dition.
It seems that the detailed description of some biochemical processes, given in the article, may be of interest to young researchers and ex- perienced professionals working with children
with autism spectrum disorders.
People with ASD have varying degrees of brain function deficiency. This is due to the violation of neural pathways, the imbalance of inhibiting and activating biologically active
substances, which leads to a decrease in communication skills, the impossibility of social-
ization, and to some people — to cognitive impairment. Neurotransmitters are included in a wide variety of chemical processes at the level of neurons and synapses. The roles of gamma-
aminobutyric acid (GABA), glutamate/gluta- minergic mechanism, transport and synthesis of creatine, cholesterol, pyridoxine — which are cofactors of many reactions of neurotrans- mitter synthesis (serotonin, norepinephrine), biotin, carnitine were studied. Autism, as a symptom,  is described in violation of the diseases of the urea cycle, partly due to receptor deficiency in oxytocin and other mechanisms [4; 8; 11: 19; 20]. In a small number of studies on an autopsy of the brain of patients with autism, glial cells (neuroglia) predominate over neurons, whereas normally glial cells make up about 40% of all brain cells. The brain sizes of children under the age of one with autism exceed the brain sizes of neurotypical children. But with age, the brain composition changes: there is an increase of white matter, the cerebellum, precisely due to glia [4—6; 8; 11; 14,17; 19; 22; 24]. Gliosis is the result of the replacement of neurons by glia, and the causes of neuron destruction in cases of autism are variable. There are various theories: autoimmune lesions, post-vaccination reactions with immune damage, infectious, trophic (including hypoxic), metabolic. It is important to determine the causes of gliosis and its location, which significantly affects the clinical manifestations, aphasia, emotional sphere, motor symptoms of people with ASD. In 3/4 of the results of magnetic resonance spectroscopy of patients with autistic traits, there was a decrease in the content of glutamic acid and glutamate in the brain [8].
An individual approach to the treatment of each patient is primarily the identification  of
personal features of the metabolism. Patients with ASD are  known  to  have  unusual  eating
behavior, hypersensitivity to certain foods, which can manifest themselves as aerophagia, abdominal pain, bowel disorders (both diarrhea and constipation), bloating, which ultimately leads to unwillingness to try new food and the formation of fear of eating [2]. The symptoms of metabolic disorders that appear after a year are described. That is, precisely when children ex- pand their diets and try to introduce new foods into their diets. And, first of all, it concerns glu-
ten-containing products [14; 15].

The essence of gluten intolerance Cereal grains, especially wheat, are high in high molecular weight protein, gluten. The following  are  the  current  ideas about the mechanisms of gluten intolerance with a description of specific clinical entities and manifestations:
— Allergic: wheat allergy (respiratory and food allergies, gluten-dependent anaphylaxis, contact
urticaria);
— autoimmune (celiac disease, gluten ataxia, Duhring’s disease);
— and non-autoimmune non-allergic  gluten intolerance (or non-coeliac gluten intolerance), see
fig. 1.
Coeliac disease is a disease that often occurs not only in children, but also in 1-3% of the
western population, including the United States, which corresponds to 5 million people living in
Europe. Celiac disease is particularly common in Sweden.
Wheat allergy is a type of allergy that is most common in children in their early infan- cy. In
adolescents and adults, it is much less common. In most cases, children “overgrow” this type of
allergy by the age of 12.
Non-coeliac gluten intolerance is a disease whose symptoms are triggered by the use of gluten by a patient without coeliac disease or food allergies. For gluten intolerance, not associated with coeliac disease, there are no biological markers.
The most important step in the diagnosis of this disease is the exclusion of coeliac disease and 
wheat allergy.
Gluten has two fractions — gluten and gliadin. It is the amino acid composition of these proteins
that determine the properties and characteristics of hydrolyzed wheat gluten. The essence of the
biochemical transforma- tions of gluten in the process of digestion is the overproduction of
glutamic acid.

Sources and biological significance of glutamic acid glutamic acid is a part of proteins and means a lot in their metabolism, being an amino acid with a neurally mediated effect [16]. glutamic acid belongs to the group of replaceable amino acids and means a lot in the body. In the body, it is up to 25% of all amino acids.

Glutamate (a salt of glutamic acid) pro- vides, in particular, the functioning of NMDA receptors
and activates them, ensuring the conduction of nervous impulses, organizing such cognitive
functions as learning and mem- ory. According to some authors, the dysfunc- tion of NMDA receptors leads to ASD [16].
The key amino acid in the hydrolysis  of gluten, gliadin, and glutelin is glutamine acid. Another
important point: glutamic acid itself and its salts are flavor enhancers, creating a socalled
“umami” taste that the baby already feels with breast milk, although in essence, it is the taste of
meat that was extracted during long-term cooking. For all substances, toxicity is a
dose-dependent concept. For adults, it is considered safe to consume up to 9 g of glutamate per
day.
The list of products excluded from the diet of patients with ASD is expanded to the big four
harmful: gluten, casein, soy, and corn.

These foods, rich in glutamic acid, contribute to atrophy of the villi in the small intestine and
have neurotoxicity, which is a hot theme for discussion by Neuro Dietitians [21].
The mechanism of molecular mimicry explains the clinical intolerance of not only these four
(gluten (including millet and oats), casein, soy, and corn) but already six products: rice and
yeast are added to the list.
Contrary to popular belief, the corn-grain protein consists of zein (a partial protein that does 
not contain lysine) and glutelin (native protein), i.e., 40% per one of each. Corn glutelin
contains a large (10—40%) amount of glutamine in its composition; moreover, breeders grow
varieties enriched with protein for use in the food industry and to create animal feed [1]. Soy
protein contains 21.6% of glutamine — it is more than whey (16.9%), casein (19.5%), egg (13.5%), beef (14.5%) [20]. Despite the combative advertising of soybeans and products

Fig. 1. Gluten intolerance scheme, diagnostic markers. A fragment of non-coeliac intolerance,
characteristic of children with ASD, is highlighted.

from it, soy is a stodgy product, that has a large number of “insertions” in the molecule,
does not swell and, therefore, has low biological value, referring to partial proteins, because
does not contain methionine. There is a lot of information about the useful and not very useful
properties of soy, including products from genetically modified soy.
The lack of gluten in the product is not a guarantee of intolerance. It is not about allergy and
celiac disease, it is a hydrolysis product — glutamic acid and peptides  of  gliadin with opiate
activity [12].
Glutamic acid is found in all foods of both vegetable and animal origin. In grains— from 1500
mg/100 g of protein in product (corn), to  3400  mg/100  g  of  protein  in  Poltavskaya grain
(wheat groats), in whole chicken eggs — 1773, in milk from 509 (cow’s milk protein) to 1164 mg/100 g of sheep protein milk, cheese — from 4000 to 6300, fish 1700—3000 mg/100 g of protein. The higher the fat content of the product, the smaller the proportion of protein and, accordingly, amino acids. Therefore, the transition to a ketogenic, more fat-laden diet is often useful not only due to a shift in metabolism from  carbohydrates  to  fats.  So,  soy contains more than 6000 mg/100 g of glutamic acid. Then a little less — in green peas and parmesan cheese, and only then follow the meat varieties (beef — 2800), etc.
An important role in the hydrolysis of proteins is assigned to the enzyme dipeptidyl-peptidase-4 (DPP-IV). It is a nonspecific enzyme of many protein substrates, and in particular, gluten
and casein. The activity of DPP-IV reduces the content of polypeptides with an opioid effect on the CNS — gluten, and casein. If necessary, on the Internet you can find the trade names of enzymes containing DPP-IV, to use them to improve the digestion of gluten. Glutamine is an integral part of the most important antioxidant complex — glutathi- one, which protects cells from oxidative damage. In addition, glutamine is an energy source for  rapidly dividing cells, including cells of the immune system. About 1/3 of the energy of these cells is obtained by the oxidation  of glutamine.

Ammonia as a neurotoxin High levels of ammonia and glutamate in the brain are neurotoxic [7; ten]. Their effect on specific brain receptors (NMDA receptors) causes anxiety states. The “circulation” of glu- tamate is a complex mechanism that ensures the operation of NMDA receptors [7; 10; 23]. Deamination of glutamine to glutamate leads to the formation of ammonia, which, in turn, is associated with a free proton and excreted into the renal tubule lumen, leading to a de- crease in  acidosis.  The  conversion  of  gluta- mate to α-ketoglutarate also occurs with the formation of ammonia. But the reverse process of ammonia binding leads to an increase of glutamine (“time bomb”) with a potential risk of a hyperammonaemia crise, which, in a severe case, leads to coma, and in a lighter one — to headache, vomiting, impaired  consciousness and behavior, other signs of intoxication. In the central nervous system is about 106 glutamatergic neurons. Elevated level of glutamate in the synapses between neurons can destroy them, but astrocyte glial cells  absorb  excess glutamate.  It is  transported  into  these  cells using  the  GLT1 transport protein, which is present in the astrocyte cell membrane. Being absorbed by astroglia cells, glutamate no longer causes damage to neurons.

The role of nutrition in hyperammonaemia Food  selectivity,  the  rejection  of  certain foods, which is often peculiar to people with ASD, can lead to a lack of such amino acids as ornithine, arginine, citrulline, which are involved in  ammonia  detoxification.  Refusal of  protein  foods,  in  particular,  of  meat,  fish, eggs, cottage cheese, etc., can be associated with excessive formation of ammonia during the hydrolysis of dietary protein, as a result of which ammonia is not sufficiently neutralized by the liver, or too much of it is formed. This once again underlines the importance of the principle  of  balanced  nutrition  (a little protein — bad, a lot — it can be even worse).

And yet —  the  autoregulation  of  metabolic processes  consists  in  the  rejection  of  protein
foods, meat, cottage cheese, that is, in the met- abolically grounded selectivity of appetite. It
should be considerate towards this. Ammonia (and its ion — ammonium) is the primary nitrogenous slag, which must be converted into less toxic urea in the liver and excreted with urine. With an excess of formation, or with its insufficient neutralization, ammonia easily penetrates the blood-brain barrier and has a neurotoxic effect. The pathogenetic effect of ammonia on the central nervous system is associated with a violation of the Krebs cycle and a decrease in ATP synthesis — adenosine triphosphoric acid [10]. Ammonium causes alkalization of blood (metabolic alkalosis), inhibits gaseous exchange by increasing the affinity of hemoglobin for oxygen, which causes oxygen deprivation in tissues. A vicious circle is formed — hypoxia leads to increased respiration, loss of carbon dioxide, alkalization of blood (respiratory alkalosis); a combination of metabolic and respiratory alkalosis exacerbates impaired gas exchange and damages the cellular Na+/K+ pump.
Glutamine is formed from ammonia with glutamic acid, has a high osmolarity, accumulates in the cell, and, in excess, causes cerebral edema, especially astrocytes. The concentration of ammonium in tissues and in the brain, in particular, is 10 times higher than in the blood, but it is the brain that is most sensitive to hyperammonaemia. In the cell, glutamine and asparagine are
deaminated, respectively, by glutaminase and asparaginase to form ammonium ion [10]. When
collapsing, glutamine in high  concentrations  leads  to  hyperam- monaemia — this cycle can be
repeated many times, supporting intoxication, in a crisis situation — cerebral edema, and in
milder cases — behavioral disturbance, vomiting, headache.
There are too little works aimed at studying the level of ammonium in the blood of people with ASD.
Some  clinical  cases  are  combined in  reviews  of  studies  of  adult  patients  with
psychiatric diseases [7; 9; 28].
Hyperammonaemia as a symptom may be due to various reasons: hypercatabolism ofthe protein (due to trauma, excessive physical activity), protein overeating, starvation (and as a result — autophagy, rhabdomyolysis — processes in which the own tissues are used to maintain
homeostasis), disruption of micro- biota functioning both in terms of production and utilization of ammonia, lack of enzymes of the urea cycle.
Thus, the ammonia toxicity does not cause any doubts, the level of ammonium more than 60 μmol/l (110 μg/dl) is considered patholog- ical; the normal level of ammonium ions does not exceed 35 μMol/l (or 60 μg/dl), intermediate questionable  results  fall  into  the  “gray zone” and require repeated research.
The determination of the level of ammonia is carried out at any time, not only on an empty
stomach. Physiological amount of protein (1 g/kg) challenge is more informative.
Ammonia is also formed from other amino acids by deamination to form ammonium ion, for example, cysteine into pyruvate + ammonia, histidine is converted into urocaic acid and ammonia, glycine into glyoxalic acid and ammonia, glucosamine-6 phosphate into glucose-6-phosphate and ammonia, glutamine to glutamic acid and ammonia. It is important to know for the correct assessment of the spectra of the amino acids, the prescription of a low protein diet and amino acid mixtures and drugs.

The role of microflora in ammonia homeostasis The growth of yeast and bacteria in the intestine is accompanied by the formation of ammonia, which is easily soluble and overcomes the hematoencephalic barrier [14; 17; 18].   A diet rich in animal protein can also increase the level of ammonia in the body. Mag- nesium, zinc and taurine are agonists that prevent the activation of NMDA receptors and, accordingly, reduce anxiety, including during hyperammonaemia [16; 25].
It is important to emphasize that the study of the intestinal microbiota by the bacteriological
method makes it possible to identify only 10% of the intestinal microbiota, since already dead  microorganisms  are  taken  for analysis. In addition, the microbial community of the small intestine is represented not only by the luminal, but also by the parietal flora, located under the protective layer of the epepithelial mucus.
Alternative methods for the study  of  microbiota  include  the  sequencing  of  its representatives,  but  this  is  a  costly  and  in- accessible  study  to  date.  Also  used  is 
registered more than 20 years  ago,  the  diagnostic method G.A. Osipov “Mass spectrometry of
microbial markers” (MSMM). This method is based on the reconstruction of microbiota by microbial markers: components of microorganism membranes, serum lipopolysaccharides, punctates, drainage fluid, coprofiltrates, urine, saliva. The advantage of this method allows to evaluate the functional activity and the qualitative diversity of the representatives of the microbiota (including the intestine, and it is more powerful). This promising method is being actively developed.
Deviations in the intestinal microbiota in patients with ASD lead to an imbalance of repre-
sentatives: decrease in the number of bifidobacteria and lactobacilli (parietal flora), increase
in clostridia, fungi, some anaerobes (translucent flora). Symbiont digestion, complementary to its own, is accompanied by bloating, pain, increased intestinal permeability. This important point in conjunction with sensitization makes the intestine a very weak barrier to the metabolites of both the flora itself and the products of protein hydrolysis with opioid action.
A personalized approach to the prescription of a gluten-free and casein-free diet should be based primarily on clinical efficacy, but laboratory diagnosis of gluten and casein sensitization can be an argument in favor of this restrictive diet. Prescription of the DPP-IV enzyme does not solve the problems of coeliac disease, but it has proven itself very well for the occasional use of gluten for allergies and is especially effective for non-coeliac gluten intolerance.
The study of amino acids and the determination of ammonium must be included in the diagnostic program of children with ASD. Moreover, in the arsenal of specialists there are medications for the correction of hyperammonaemia and various dietary supplements.
As a result of two years of work on the study of metabolic features, eating behavior, diet
peculiarities and microbiota in children with ASD, the diagnostic program presented below is
proposed.

Table
Diagnostic program for the study of potentially significant metabolic abnormalities in children
with autism spectrum disorders
Research methods                                Therapeutic approach                              
Advanced research and
prescriptions

Exclusion of antiglyadin antibodies (to tissue transglutaminase, deamidated gliadin peptide) and
genetic susceptibility to coeliac of HLA-DQ2/ HLA-DQ8

Individual approach to a gluten-free and casein-free diet
— Test diet for at least 3 months. Prescriptions:
— alpha-galactosidase
— DPP-IV
— Synthesis of continuity of the intestinal barrier
— Mucous reparants
— Antisecretory drugs to reduce the aggressive prop- erties of luminal fluids
— Production of physiological rest (enzymes)
— Correction of defecation (including in the absence of hygienic skills)
— FODMAP1 diet with the administration of alpha- galactosidase (allowed from 5 years to off-lable)

— Determination of exorphins of gluten and casein in urine
— Magnetic resonance spectroscopy of the brain
— LCHF2 diet in patients with epilepsy

1 Fermentable Oligosaccharides, Disaccharides, Monosaccharides and Polyols.
2 Low Carb High Fat

In conclusion, it is important to emphasize that there are no uniquely bad or good molecules in
the metabolism, to ensure  homeostasis  (constancy  of  the  internal  environment of the body) there must be a mobile biochemical  equilibrium.  As  a  doctor  with more than thirty years of
experience, I can recommend a reasonable and gradual nutritional intervention of new products, diets, and medicines.
And, of course, any medical recommendations should be understood and accepted by parents,
especially in the case of restrictive  and prohibitive measures.

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