Inhibition of the Beta-oxidation Pathway of Fatty Acids and Dopamine- Beta-hydroxylase by Phenyl Derivatives of Short- Chain Fatty Acids from Gastrointestinal Clostridia Bacteria is a (the) Major Cause of Autism

Abstract

Background

A The author’s comprehensive evaluation of the biochemical metabolomic literature over more than 40 years discusses multiple studies documenting abnormal elevations of the neurotransmitter dopamine and its metabolites as well as inhibitors of dopamine beta hydroxylase (DBH) from Clostridia bacteria in urine samples and cerebrospinal fluid samples of children with autism.

Aims of Review

The evaluation intends to elucidate the reasons for the elevation of dopamine and its metabolites in urine and their relationship to increased Clostridia colonization of the gastrointestinal tract in children with autism. In addition, to the evaluation of Clostridia metabolism and its effects on abnormal dopamine metabolism in autism, a secondary aim intends to demonstrate as a hypothesis that one particular metabolite of Clostridia bacteria—3-hydroxy-(3-hydroxyphenyl)- 3-hydroxypropionic acid (HPHPA)—may cause even more severe effects on in autism than other metabolites by leading to depletion of free coenzyme A (CoASH). This depletion of free Coenzyme A leads to a deficiency of cholesterol and activated palmitic acid needed for activation of the key brain developmental protein sonic hedgehog, which has recently been research has shown to be severely abnormal in severe autism.

Key Scientific Concepts of Review

Laboratories throughout the world have consistently found high quantities of HPHPA and 4-cresol in high percentages of urine samples of children with autism. Those inhibitors, which intestinal Clostridia bacteria produce, cause an elevation in dopamine and its metabolites, which affect the brain’s and the sympathetic nervous system’s key enzyme dopamine-beta-hydroxylase (DBH). Excessive dopamine and its toxic metabolites due to these DBH inhibitors may cause brain damage due to excessive unstable dopamine quinones, toxic adducts of dopamine disrupting brain mitochondrial energy production, and oxygen superoxide. HPHPA, a short chain phenyl compound, may have additional biochemical effects on the brain in autism, causing a reduction in free CoASH needed to produce the CoA palmitic acid derivative necessary to activate the key brain developmental protein sonic hedgehog. The depletion of CoASH appears to be a new therapeutic target to reverse the adverse effects of the HPHPA metabolite on the beta oxidation of fatty acids and cholesterol synthesis that are prevalent in autism.

Conclusions

Variations in the severity of autism could be based on the types and concentrations of the Clostridia markers produced and the extent to which these markers, such as HPHPA, have depleted critical lipids, such as cholesterol and CoA palmitic acid derivative. Patients need those lipids for the activation of the developmental protein sonic hedgehog. In addition, the sequestration of coenzyme A by short chain adducts of Clostridia leads to the depletion of critical free CoASH, needed throughout intermediary metabolism, and creates a biochemical storm that especially affects brain function.

Introduction

Many studies of the urine samples from children with autism, some of which have focused on a small group of small molecules and others on as many as 154 metabolites, have begun to elucidate characteristic biochemical abnormalities related to neurotransmitters and gastrointestinal microbial metabolism in autism.^1-11

The author reviewed the results of these studies to determine if some of their abnormal results of these studies may be biochemically related and was especially impressed with the metabolomic study of a group of Italian investigators, Mussap et al,⁹ who found that “adipic acid, palmitic acid, and 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA) can be proposed as candidate biomarkers of autism severity.”

A somewhat surprising finding to the author from that study is that the single marker with the greatest correlation (0.65), to the severity of autism’s severity symptoms was palmitic acid, a very common saturated fatty acid. Those researchers based their findings of autism severity on the Autism Diagnostic Observation Schedule (ADOS) and found that palmitic acid was the marker that correlated the greatest with the ADOS severity score.

Another metabolomic research group, Khan et al in Pakistan,⁴ also identified palmitic acid as a key abnormal metabolite in the urine of children with autism. In that Pakastani study, 86% of the participants in the autism group had detectable palmitic acid in their urine versus none in the control group. However, Khan et al’s study did not measure 4-cresol or HPHPA. A short review of these three key biochemicals in autism includes palmitic acid (hexadecanoic acid), adipic acid, and HPHPA.

Biochemicals

Palmitic acid.

This acid (hexadecanoic acid) is a 16-carbon saturated fatty acid that is named after the most common source, palm oil. There is no published evidence that palmitic acid is present in excessive quantities in the diet of children with autism. If there is not excessive intake of palmitic acid occurs in their diets, the presence of excessive palmitic acid must be due to its reduced catabolism. What would decrease the catabolism of this common fatty acid?

Adipic acid.

This acid is a six-carbon dicarboxylic acid that is a product of the omega-oxidation pathway of fatty- acid oxidation, often considered to be a “back-up” system for the beta-oxidation of fatty acids when it’ is overloaded due to genetic mutations, such as medium chain acyl dehydrogenase deficiency (MCAD); deficiency of beta-oxidation cofactors, such as carnitine or coenzyme A (Co A), or excess substate, such as a high- fat diet.¹²

HPHPA.

This acid is a metabolic product of combined Clostridia bacteria and human metabolism, and was first structurally identified and reported to be elevated in autism, schizophrenia, and other disorders by Shaw.^3,13,14 One mechanism of its neurotoxicity appears to be severe inhibition of dopamine beta-hydroxylase (DBH), the enzyme that converts dopamine to norepinephrine.^13,14 The elevation of HPHPA in urine samples of children with autism has now been verified in multiple geographic regions, including Turkey,² China,⁵ Italy,^1,9 Latvia,¹¹ and the USA,^3,13,14 indicating that this abnormal compound appears to be prevalent in autism worldwide (Table 1).

Table 1.

Studies Implicating Clostridia Metabolites in Autism in Humans and Animals

Chemical	Reference	Geographic Location	Major Findings
HPHPA, 4-cresol	Shaw 2010,3 2015,13 202114	USA	Identified HPHPA in elevated amounts in urine samples from patients with autism and attributed that elevation to certain Clostridia species. Attributed DBH deficiency to HPHPA and 4-cresol.
HPHPA	Keşli et al, 20142	Turkey	HPHPA in 30 urine samples from an autism group was significantly higher than group of 30 controls.
HPHPA	Noto et al, 20141	Italy	Identified HPHPA in elevated amounts in urine samples from patients with autism.
HPHPA	Xiong et al, 20165	China	HPHPA in 62 urine samples from an autism group were significantly higher than those of a group of 62 controls. Autism symptoms improved after treatment of Clostridia. The high HPHPA diagnosed autism with a 96% accuracy.
HPHPA	Mussap et al, 20209	Italy	Three markers in an evaluation of 154 markers in urine samples, palmitic and adipic acids and HPHPA, were highly diagnostic of autism. Palmitic acid had the greatest correlation with autism severity.
HPHPA, 4-cresol	Daneberga et al, 202111	Latvia	Reported high incidence of both 4-cresol and HPHPA in 44 patients with autism.
4-cresol	Altieri et al, 201127	Italy	The 4-cresol was elevated in children with autism. The degree of elevation was higher in those with a greater severity of illness.
4-cresol	Persico, Napolioni, 2012	Italy	The 4-cresol was elevated in children with autism.
4-cresol	Gabriele et al, 201428	France	Total urinary 4-cresol, 4-cresylsulfate, and 4-cresylglucuronate were significantly elevated in 33 French patients with autism spectrum disorder (ASD) compared with 33 sex- and age-matched controls. High values were associated with stereotypic, compulsive, and repetitive behaviors.
4-cresol	Pascucci et al, 202032	Italy	The 4-cresol injections caused autistic behavior in mice.
4-cresol	Gevi et al, 20206	Italy	The elevated 4-cresol in 40 urine samples from patients with autism was directly correlated with high dopamine metabolites due to inhibition of dopamine beta hydroxylase.
4-cresol	Bermudez-Martin et al, 202131	France	Oral dosing with 4-cresol caused autistic behavior in mice.

Abbreviations: HPHPA, 3-(3-hydroxyphenyl)-3-hydroxypropionic acid.

Using HPHPA and two additional phenyl metabolites—3-hydroxyhippuric acid and 3-hydroxyphenylacetic acid—of Clostridia bacteria as diagnostic markers for autism, the Chinese researchers Xiong et al 5 found that an elevation of these 3 three markers had a 98.4% specificity for the diagnosis of an autism spectrum disorder (ASD), a remarkably high number that at least appears to be a significant diagnostic marker for a major subgroup of autism.

Effect of phenyl Clostridia compound HPHPA on fatty acid metabolism and its relation to early elucidation of fatty acid metabolism by Knoop

A possible interaction among the three major abnormalities that Mussap et al discovered in the urine metabolome of autism may be the pronounced effect that very large quantities of HPHPA may can have on the metabolism of fatty acids, especially the adipic and palmitic acids that stood out in that study.⁹ Shaw’s prior outline of the formation and catabolism of HPHPA³ relied heavily on the classic work of Knoop,¹⁵ (Figure 1) who used phenyl derivatives of fatty acids to elucidate their beta-oxidation pathway of fatty acids15 in the early 1900s (Figure 1).

Figure 1.

Knoop’s scheme (left) for the elucidation of beta-oxidation using 5-phenylvaleric acid and 4-phenylbutyric acid, contrasted with Shaw’s proposed pathway (right) for the metabolism of 3-phenylpropionic acid from a variety of Clostridia species which requires both bacterial and human metabolism.

Since HPHPA is a phenyl-containing fatty acid, it fits the same metabolic pathway for beta-oxidation of fatty acids of Knoop. The initial substrate for the pathway is based on the research of Elsden,¹⁶ who found that phenylpropionic acid to be the major product of phenylalanine fermentation by the Clostridia species botulinum, caloritolerans, and sporogenes, with lesser amounts produced by Clostridia species mangenoti, ghoni, bifermentans, difficile, and sordelii. Fourteen other Clostridia species were evaluated, including tetani, and did not produce that product.

Bhala et al also found that Clostridia species were the only species that produced phenylpropionic acid after they evaluated 67 different human stool isolates of microbes from nine different genera of bacteria and Candida albicans.¹⁷ Furthermore, they found that metronidazole, clindamycin, and a combined therapy of ticarcillin, clavulanate, and oxacillin (drugs that kill Clostridia) abolished gut flora that produce phenylpropionic acid. They also found that the antibiotics cefazolin, cefuroxime, ampicillin, chloramphenicol, and gentamicin did not abolish phenylpropionic production. Since one or more of these agents kill a large group of intestinal bacteria, including E. coli, Streptococci, Salmonella, Shigella, Proteus, Pseudomonas, and Klebsiella are killed by one or more of these agents, the persistence of phenylpropionic acid in the presence of these agents appears to eliminate those species as potential sources of phenylpropionic acid, the precursor of the HPHPA compound.

Clostridia or other bacteria may hydroxylate the phenyl ring of phenylpropionic acid to form 3-(3-hydroxyphenyl)-propionic acid (3-HPPA). Additional research is needed to confirm this reaction.

The 3-HPPA can then be converted to a thioester of Coenzyme A via the enzymatic action of fatty acyl Co A synthetase (Figure 2). The 3-HPA CoA thioester then enters the beta-oxidation pathway of human metabolism with its conversion to 3-(3-hydroxyphenyl) trans-2-propenyl- Co A coenzyme A thioester, mediated by the flavin-adenine dinucleotide- dependent acyl dehydrogenase.

Figure 2.

Catabolism of Clostridia Metabolite 3-(3-Hydroxyphenyl) Propionic Acid by Human Beta- oxidation Pathway of Fatty Acid Metabolism to HPHPA or 3-Hydroxyhippuric Acid. All the enzymes in the above pathway are of human origin.

Abbreviations: ATP, adenosine triphosphate; NAD+, nicotinamide adenine dinucleotide; CoASH, free Coenzyme A.

Next, the hydration of the double bond of this product by enoyl hydratase produces 3-(3-hydroxyphenyl)-3-hydroxypropionyl—Coenzyme Co A A thioester, which can then be metabolized by two alternate pathways: (1) conversion by a thiolase enzyme to produce 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA) and free Co A coenzyme A (CoASH) or (2) conversion by nicotinamide adenine dinucleotide (NAD+)- dependent 3-hydroxyacyl-dehydrogenase to form 3-(3-hydroxyphenyl)-3-oxopropionyl—Coenzyme Co A thioester. The thiolase enzyme producing 3-(3-hydroxyphenyl)-3-hydroxypropionic acid from its CoA thioester has not yet been elucidated.

Since 3-hydroxyacyldehydrogenase is a reversible enzyme reaction, a deficiency of free coenzyme A due to its sequestration by the Clostridia metabolite might shift the equilibrium to a predominance of 3-(3-hydroxyphenyl)-3-hydroxypropionyl—Co A thioester. Any 3-(3-hydroxyphenyl)-3-oxopropionyl—Co A thioester formed can then be converted to 3-hydroxyphenyl CoA and acetyl CoA by the catalysis of acyl-CoA betaketothiolase activity and an additional molecule of CoASH, which might not be available if it was tied up as 3-HPA thioesters of coenzyme A.

Any 3-hydroxyphenyl CoA formed would then be conjugated with glycine by the enzyme glycine N-acyltransferase to form 3-hydroxyhippuric acid, a compound that Xiong et al found in appreciable quantities in urine samples of patients with autism with elevated HPHPA in the same samples.⁵ Those researchers also found that the mean value of 3-hydroxyhippuric acid was 95 times greater in the urine samples of the group with autism compared to those of the normal controls. A diminished ratio of 3-hydroxyhippuric acid to HPHPA would seem to be an indicator of free coenzyme A availability.

Sequestration of Co A

Sequestration of Co A by excessive amounts of phenolic Clostridia compounds can have an impact on the beta-oxidation pathway and the formation of palmitic acid Co A thioester that is needed for sonic hedgehog activation.

The amount of HPHPA is sometimes so large that it is difficult to quantify the other organic acids in a chromatogram. Even when resolution is obtained, the amount of HPHPA sometimes exceeds the sum of all of the creatinine- corrected molar concentrations of all the other organic acids combined. (That observation has been the personal observation of the author in multiple cases).

The formation of Co A thioesters of 3-hydroxyphenylpropionic acid appears to be so great that it seems possible that there is an induced deficiency of free CoASH needed for all the other metabolic functions requiring CoASH.

Martines et al’s theoretical analysis¹² of the flux of intermediates through the beta-oxidation pathway revealed that the over-accumulation of Co A intermediates could lead to a logjam that shuts down the energy production necessary for maintaining adequate glucose.¹² It is logical to conclude that this deficiency of CoASH causes fatty acids to be metabolized by the omega-oxidation pathway, leading to the characteristic overabundance of adipic acid in urine samples of children with autism. The omega-oxidation pathway does not require CoASH as a cofactor.

The first enzymatic step, (fatty acid CoA ligase,) in the utilization of palmitic acid also requires its conversion to a coenzyme A thioester. That same enzyme will also likely be inhibited by high amounts of 3-HPPA the first reaction outlined in Figure 2. So, a deficiency of CoASH would impair this first step of palmitic acid utilization. If palmitic acid cannot be utilized in autism, free palmitic acid would be excreted in the urine in excessive amounts.

Sonic hedgehog is one of the most important proteins involved in the development of anatomic features. For example, children with mutations in the sonic hedgehog gene may have a single cyclops eye in the center of the head instead of 2 eyes. Sonic hedgehog protein requires activation by both cholesterol and palmitic acid Co A coenzyme A thioester.¹⁸ Activated sonic hedgehog has 30-fold greater activity than unactivated sonic hedgehog.

The sonic hedgehog concentrations are abnormal in serum samples of children with autism, with the highest values associated with the most severe autism.¹⁹ Presumably defective, unactivated sonic hedgehog cannot bind adequately to its receptor, leading to overproduction of unactivated sonic hedgehog because of a defective feedback regulation.

Total cholesterol has been reported to be significantly lower in autism^20,21 and extremely low in the genetic disease of cholesterol biosynthesis, Smith-Lemli-Opitz syndrome, in which autistic symptoms are very frequent but are markedly improved with cholesterol supplementation.²²

Co A is an essential coenzyme to produce biochemical intermediates, such as the 3-hydroxy-3-methylglutaric-acid- Co-A coenzyme A derivative, the substrate for 3-hydroxy-3-methylglutaryl (HMG) CoA reductase, which is the first committed intermediate for cholesterol production and the metabolic target for most of the cholesterol-lowering statin drugs.

Purkinje neurons secrete sonic hedgehog to sustain the division of granule- neuron precursors in the external granule layer in cerebral development. Xiao et al’s neuropathological studies found lower Purkinje-cell numbers, missed or ectopic neurons of deeper cerebellar nuclei (DCN), cortical-thickness alterations, foliation dysplasia, and migration impairments in the cerebellar cortex of individuals with autism spectrum disorders.²³

The fact that a key factor for sonic- hedgehog activation, palmitoyl-CoA, appears to be lacking in autism helps to explain the remarkably high correlation between autistic symptoms and palmitic acid concentration in urine. An additional abnormality in the Pakistani study of Khan et al⁴ is the finding that 3-hydroxyisovaleric acid, which requires adequate CoASH for its synthesis was 80% lower in the autistic participants than in the controls, with a P < .0001.

The production of this that marker as well as major cholesterol precursors from leucine are also greatly dependent on CoASH²⁴ for one of the first enzymes involved in the cholesterol synthesis metabolic pathway, branched-chain alpha-keto acid dehydrogenase.

4-Cresol, another common Clostridia metabolite detected in children with autism

Multiple studies have associated another phenolic substance in urine or fecal samples of children with autism, 4-cresol, has been associated with Clostridia overgrowth.^{6,11,13,14,25-29} of the gastrointestinal tract. Table 1 provides an overall summary of these studies. In addition, Bermudez-Martin et al³⁰ and Pascucci et al³¹ found that treatment of animals with 4-cresol causes autistic-like symptoms.

More important, behavioral abnormalities elicited by 4-cresol in Pascucci et al’s study in mice strikingly resemble core symptoms and co-morbid disorders clinically observed in human autistic individuals. The metabolite 4-cresol impairs dendritic development, synaptogenesis, and synapse function in hippocampal neurons in rat cell cultures.³²

It is interesting that only two of these studies reported both HPHPA and 4-cresol. (Table1).¹¹ The author suspects that one of the major issues has been a lack of a commercial analytical standard for HPHPA for identification and quantitation. In addition, HPHPA was likely not detected in the stool studies^25,26 since its production requires the combination of both human and microbial metabolism to convert phenylpropionic acid to HPHPA.

The 4-Cresol forms covalent bonds with two different tyrosine positions (amino acids 216 and 357)—of the DBH enzyme, which cause irreversible inhibition of the DBH enzyme activity.^34-36 Gevi et al demonstrated that the concentration of 4-cresol was directly related to the degree of inhibition of DBH, which is a critical enzyme that catalyzes the conversion of dopamine to norepinephrine.⁶

Shaw reported that both phenolic compounds, including HPHPA and 4-cresol, appear to be inhibitors of DBH (Figure 3).¹⁴ Goodhart et al reported that other phenols could be inhibitors of DBH, (Figure 4) including 3-cresol, 4-methylcatechol, 4-ethylphenol, and 3-hydroxybenzylalcohol.³³ It seems likely that other common phenols commonly found in urine, such as 3-hydroxyphenylacetic acid and 4-hydroxyphenylacetic acid, may also be in vivo inhibitors of DBH.

Figure 3.

Proven and suspected Inhibitors of Dopamine-beta-hydroxylase (DBH)

Figure 4.

Effects of Clostridia Metabolism on Altered Neurotransmitters by Inhibition of Dopamine Beta Hydroxylase (DBH) resulting in excessive dopamine and sequestration of free coenzyme A (CoASH) resulting in impaired Beta-oxidation of Fatty Acids, Impaired Cholesterol Synthesis, and Decreased Activated Sonic Hedgehog.

Hsiao et al³⁶ found that 4-ethylphenolsulfate, a chemical closely related to 4-cresol (a methyl phenol) has already been reported as a potent cause of autistic symptoms in a certain strain of mice and that the use of a strain of Bacillus probiotics could completely reverse the autistic symptoms of these mice can be completely reversed with the use of a strain of Bacillus probiotics³⁶; the non-sulfated metabolite is a strong inhibitor of DBH. The sulfated metabolite is also likely to be an inhibitor also but hasn’t yet been assessed.

Elevated Elevation of Dopamine and its Metabolites in autism, one of the most consistent metabolic abnormalities in autism

Elevated dopamine and its major metabolite homovanillic acid(HVA) has been a common finding in urine samples of children with autism for over 40 years.¹⁴ Both Lelord et al³⁷ and Garreau et al³⁸ reported that HVA levels were higher in autistic than in normal children. The former reported that the clinical improvement of these young autistic patients under vitamin B6 treatment was associated with a decrease of in their urinary HVA excretion. Abnormalities in dopamine metabolism included elevated concentrations of the dopamine metabolite homovanillic acid (HVA) in cerebrospinal fluid.³⁹

In addition, the degree of elevation of HVA in urine was highly correlated with the increased severity of autistic symptoms.³⁸ Several more recent studies^1,4,6,7 have confirmed the findings of the earlier studies of finding elevated dopamine metabolites in urine samples of children with autism, making this abnormality one of the most consistent replicated findings in autism research. What is the reason for the harmful effects of excess dopamine on the brain?

Toxicity of Excessive Dopamine and its metabolites to the brain

Excessive dopamine and its toxic metabolites in the brain due to these DBH inhibitors may cause brain damage due to excessive unstable dopamine quinones, toxic adducts of dopamine, and oxygen superoxide (Figure 4), leading: (1) to increasing oxidative stress, (2) to damage to neuronal mitochondria by covalent bonding of the dopamine metabolite aminochrome to the mitochondrial complexes I and III of the electron transport chain and to the Krebs cycle enzyme isocitrate dehydrogenase, (3) to neuron damage, and (3) to neuronal apoptosis.^40-42

The longer the production of DBH inhibitors continues in the gastrointestinal tract, the greater the damage to the brain may be and concomitantly the reduced possibility to use antimicrobial therapy to reverse the damage done to the brain.

The 4-cresol may be less harmful to human metabolism than phenylpropionic acid since it is detoxified in a different manner. 4-Cresol is either being excreted directly into the urine or converted to sulfate or glucuronate conjugates.⁴³ These detoxification methods mean that 4-cresol fails to sequester Co A as phenyl thioesters that cause inhibition of beta -oxidation of fatty acids, and reduction in cholesterol and activated palmitic acid.

Summary Discussion

Laboratories throughout the world have consistently found high quantities of HPHPA and 4-cresol in high percentages of urine samples of children with autism. A major reason for the elevation of dopamine and its metabolites in those children is the presence of those inhibitors, which intestinal Clostridia bacteria produce. They affect the brain’s and the sympathetic nervous system’s key enzyme dopamine-beta-hydroxylase (DBH).

The degree of DBH inhibition is proportional to the concentration of the microbial DBH inhibitor. Administration of 4-cresol to animals causes the production of autistic symptoms. Those symptoms also appear in animals with high amounts of a similar chemical, 4-ethylphenolsulfate, and probiotic bacteria can treat their autistic symptoms very effectively.

Excessive dopamine and its toxic metabolites in the brain due to these DBH inhibitors may cause brain damage due to excessive unstable dopamine quinones, toxic adducts of dopamine, and oxygen superoxide, leading: (1) to increasing oxidative stress; (2) damage to neuronal mitochondria by covalent bonding of the dopamine metabolite aminochrome to the mitochondrial complexes I and III of the electron transport chain and to the Krebs cycle enzyme isocitrate dehydrogenase; (3) neuron damage; and (4) neuronal apoptosis.

HPHPA, a short chain phenyl compound, may have additional biochemical effects on the brain in autism. The results of metabolomic studies provide evidence of fatty-acid metabolic abnormalities in autism, such as high palmitic acid in urine that correlates highly with autistic-symptom severity. These abnormalities appear to be related to a disturbance of fatty-acid metabolism due to a reduction in CoASH, which the initial step of palmitic-acid utilization requires.

This reduction appears to be caused by excessive sequestration of CoASH as phenyl Co A thioesters formed from Clostridia HPHPA. Elevated adipic acid in urine and low serum cholesterol in children with autism appears to be similarly caused by excessive omega oxidation due to inadequate CoASH for beta oxidation. The induced deficiency of CoASH and the low cholesterol and Co A-palmitic acid thioester due to high HPHPA in autism may fail to adequately activate the protein sonic hedgehog, which is critical for brain development. Abnormal metabolism of sonic hedgehog has been confirmed in autism and correlated with the severity of autism.

The knowledge of the role of these Clostridia metabolites on human metabolism may yield important new therapies for the prevention and treatment of autism.

The consistent finding of high quantities of HPHPA and 4-cresol, potent inhibitors of dopamine-beta-hydroxylase in high percentages of urine samples of children with autism in laboratories throughout the world correlated with high amounts of likely toxic dopamine metabolites indicates that these substances should be measured in urine samples of all cases of autism spectrum disorders. In addition, adipic acid and palmitic acid appear to be good indicators of free coenzyme A depletion by HPHPA. Furthermore, the likely biochemical effects of HPHPA, a short chain phenyl compound, on the depletion of free coenzyme A appears to be a new therapeutic target to reverse the adverse effects of this the HPHPA metabolite on impaired the beta-oxidation of fatty acids and cholesterol synthesis that are prevalent in autism. In addition, the induced deficiency of free coenzyme A and low cholesterol and coenzyme A-palmitic acid thioester due to high HPHPA likely causes a deficient activation of the critical brain development protein sonic hedgehog. HPHPA and 4-cresol should be measured in urine samples of all patients with ASDs. In addition, adipic acid and palmitic acid appear to be good indicators of CoASH depletion by HPHPA.

Supplementation with Co A coenzyme A or its precursors, such as pantothenic acid and/or pantothenic acid phosphate, may be an inexpensive and effective means to reverse the depletion of Co ASH by HPHPA. A recent study found that pantothenic acid is commonly low in urine samples of children with autism and is a major marker differentiating normal children from those with autism.⁴⁴

Conclusions

Variations in the severity of autism could be based on the types and concentrations of the Clostridia markers produced and the extent to which those markers, such as HPHPA, have depleted critical lipids, such as cholesterol and palmitic acid. Patients need those lipids for the activation of the developmental protein sonic hedgehog, and their lack leads to the depletion of critical CoASH, needed throughout intermediary metabolism, and creates a biochemical storm.