**4. GI abnormalities in ASD**

Autism is associated with gastrointestinal pathology from the esophagus to the colon [87–91]. The literature suggests that GI pathophysiology is an intrinsic component of autism in many patients and may be a central component to the etiology of the disease. GI problems have been reported in 42% of children with ASD and 12% of controls, with chronic diarrhea and constipation being the most prevalent problems. The severity of these problems correlates with the severity of ASD [92]. It is noteworthy that in both GI dysfunction and ASD imaging reveals abnormalities in brain regions associated with emotional and sensory functions [93, 94], and GI problems contribute to behavioral problems, attentional deficits, and self injury [95]. Gut bacteria influence intestinal permeability, mucosal immunity, the enteric nervous system, pituitary functions, and the modulation of pain (cited [96]).

There is increasing reason to believe that the interaction between gastric microbiota and the brain are contributory to the symptoms seen in ASD. This is mediated via the autonomic innervation of the intestine and the hypothalamic– pituitary axis which is innervated by catecholamines and which generates GI signaling molecules affecting enteroendocrine and mucosal immune cells. The "Gut Brain Axis" is comprised of central and peripheral nervous systems as well as the neuroendocrine and immune systems, and communication is bidirectional, with vagal inputs to the brain as well as endocrine and neuroendocrine signaling [97]. Catecholamines are associated with stress reactions and, interestingly, GI microbiota respond to stress with changes in their efferent and afferent catecholamine responses (reviewed in [98]).

A trial of 36 autistic children found pain, chronic diarrhea, bloating, GI irritability, chronic gastritis, esophagitis, chronic duodenitis, diminished carbohydrate digestive enzymes and reduced pancreatic exocrine secretion in response to secretin challenge [88]. Secretin has not been found to be an effective treatment for autism. In a survey of parents of 500 autistic children, half responded that their children had loose stools or chronic diarrhea, and intolerance for wheat and cow's milk [99]. A number of reports mention improvements in autistic symptoms when reduced gluten and casein diets are implemented and the return of symptoms when these diets are terminated (cited in [100]).

Lucarelli et al. [101] observed an improvement in social skills and the ability to communicate in a trial of 36 autistics who were given diets with diminished

gluten and/or cow's milk, with improvements observed in 5 of 7 objective behavioral scales. Similar findings have been reported by others [102–105]. Following one year on this diet symptoms returned upon termination of the dietary restrictions [104]. Intestinal permeability was found increased to lactose in a number of high functioning autistic children compared to age matched controls, with no increased permeability to mannitol, which was interpreted to mean a diminution in the tight junctions of gut epithelium [106] and the subsequent release of incomplete gluten and casein digestive products. Autistic patents reportedly manifest significantly higher levels of IgA for casein, gluten, lactalbumin and β-lactoglobulin [101, 107].

These observations give rise to the Leaky Gut Hypothesis of Autism, which states that various digestion products can enter the blood through leaky tight junctions in the gut and interact with the immune and central nervous systems in ways that facilitate the onset of autism. Gut peptidases release short chain peptides called exorphins that have structural similarity to endorphins. Gliadomorphins and casomorphins are stable examples of these peptides that are known to induce psychosis [108]. β-Casomorphin-7 is elevated in the urine of autistic patients [104], and when in infused into the blood stream of rats has been shown to activate the transcription of the gene *c-Fos* in the brain [109]. However, dietary restrictions do not cure autism.

While controversial, elevated short chain fatty acids (SCFA) have been associated with autism [110], and both central and peripheral administration of propionic acid (PPA) to rats induces ASD-like impairments that include aberrant motor movements, stereotyped repetition, EEG changes, cognitive deficits, perseveration and social impairment, as well as increased oxidative stress, glutathione depletion, neuro-inflammation, altered lipid profiles and more [111]. SCFA are digestive products derived from fiber and protein. The most common SCFA include propionic PPA and butyric acid (BA) [112]. BA and PPA are metabolized in the liver via the portal circulation, however areas of the distal colon are outside of the portal circulatory bed, and the systemic effects of BA and PPA are believed to be significantly underestimated [110, 112]. SCFA, including PPA, activate G protein coupled neural, effect neurotransmitter synthesis and release, and mediate such diverse events in the nervous system as Ca++ gating, mitochondrial function, lipid metabolism, immune function, gene expression and the role of tight junctions [110]. SCFA are believed to modify the activity of TH, and there are 3 ways in which the SCFA BA modifies TH activity: (1) modulation of transcription via chromaffin remodeling, (2) activation of various transcription mediators, and (3) by interfering with TH mRNA [113–116]. Subsequent work by Nankova, et al. [117] have shown that PPA elevates TH mRNA levels and that SCFA increase TH and subsequent catecholamine synthesis.

This dietary model allows for the elevation of cortical and striatal dopamine activity via elevated TH synthesis and activation, and which has been invoked as a potential mechanism for the actions of risperidone [118, 119], one of the two drugs approved for the treatment of the irritability associated with autism. It is worth noting in this context that PPA is structurally similar to valproic acid (VA), and has similar effects to VA, which is a treatment known to induce autistic symptoms, and used as a model for this purpose [120–122]. As cited above, VA appears to induce ASD- like symptoms by stimulating TH transcription in a manner similar to butyrate [86].

It is worth noting that relative to the participation of gastrointestinal events which may underlie autism, recent developments in the study of the human biome and investigations into GI function have revealed that the gut is the source of a number of neurotransmitters and neurotrophic factors, thus opening a previously

**105**

*L1-79 and the Role of Catecholamines in Autism DOI: http://dx.doi.org/10.5772/intechopen.95052*

properties of luminal bacteria [127].

metabolic and neurologic pathologies [129].

consistent with findings in swine [140].

predominant neurosteroid [145].

understudied source of pharmacologic agents which may regulate CNS function. *E. coli* and Clostridium sp. have been shown to elevate free GI catecholamines and dopamine increases colonic water absorption [123]. GI microbiota produce catecholamines and recognize them in the environment [124–126]. Epinephrine and norepinephrine are implicated in the virulence, ability to adhere, and chemotaxic

In the work discussed above, it is important to note that PPA activates peroxisome proliferator-activated receptor gamma (PPAR-γ), and that this orphan receptor has been shown to have independent effects on the mediation of catecholamine and opioid pathways by SCFA [128], and that, as discussed below, PPAR is considered to be a "master regulator" of lipid homeostasis both centrally and peripherally. This later finding plays into the growing literature of lipid metabolism dysregulation in autism. PPAR also has immunologic functions that have been found to be related to

**5. Dopamine underlies autistic symptoms in the gut and the CNS**

the amygdala and prefrontal cortex in children with ASD [137, 138].

Any comprehensive approach to the treatment of autism must accommodate many different organ systems, certainly the gut and the CNS. As discussed above, the neurotransmitter function of dopamine is well known, including its modulatory effects on motor function, mood, emotion, irritability, reward, and other systems which are affected by autism. However, there also exists in the mesentery a paracrine dopaminergic system that regulates the secretion of bicarbonate [130], the secretion of digestive enzymes by the exocrine pancreas [131], and which controls sodium transport in the lower intestine [132]. Dopamine also has documented effects on gut motility and mucosal blood flow [133–135]. As early as 1994 elevated blood levels of levels of dopamine have been associated with autism [136]. It is known that ASD is associated with elevated levels of dopamine in the tracts linking

What is less known is that approximately 42–46% of the dopamine in the body is produced in the gut. Eisenhower and colleagues at NIH [139] studied 8 patients undergoing elective abdominal surgery and 47 patients who underwent cardiac catheterization. Tissue samples from the stomach and duodenum were obtained and compared, as were arteriovenous concentration differences and rates of renal clearance of dopamine and its metabolites in conditions of different sympathetic nervous backgrounds for dopamine not converted to norepinephrine. They found considerable dopamine synthesis in the stomach, pancreas, and duodenum, with renal elimination of dopamine and its metabolites. Dopamine has a naturetic function in the kidney; however, there was significant overflow of dopamine into the renal venous circulation that allows for systemic effects. As expected, cells in the stomach, pancreas, and duodenum stained positive for TH. The authors could not account for the amount of dopamine added to the mesenteric venous circulation, as it cannot be explained by sympathetic activity or diet, and their results were

In keeping with the concepts presented herein, it is relevant that in the liver, bile salt production and release are also under the control of dopamine [141, 142]. Bile salts are known to occur not only in the periphery, but in the CNS as well, where they appear to contribute to neurologic decline and blood brain barrier permeability [143, 144]. Bile acids are the predominant steroid in the brain, with levels that are 10x greater than those found in the blood indicating local synthesis, and with higher titers than that of pregnanolone, which was once considered the

*L1-79 and the Role of Catecholamines in Autism DOI: http://dx.doi.org/10.5772/intechopen.95052*

*Autism Spectrum Disorder - Profile, Heterogeneity, Neurobiology and Intervention*

β-lactoglobulin [101, 107].

not cure autism.

gluten and/or cow's milk, with improvements observed in 5 of 7 objective behavioral scales. Similar findings have been reported by others [102–105]. Following one year on this diet symptoms returned upon termination of the dietary restrictions [104]. Intestinal permeability was found increased to lactose in a number of high functioning autistic children compared to age matched controls, with no increased permeability to mannitol, which was interpreted to mean a diminution in the tight junctions of gut epithelium [106] and the subsequent release of incomplete gluten and casein digestive products. Autistic patents reportedly manifest significantly higher levels of IgA for casein, gluten, lactalbumin and

These observations give rise to the Leaky Gut Hypothesis of Autism, which states that various digestion products can enter the blood through leaky tight junctions in the gut and interact with the immune and central nervous systems in ways that facilitate the onset of autism. Gut peptidases release short chain peptides called exorphins that have structural similarity to endorphins. Gliadomorphins and casomorphins are stable examples of these peptides that are known to induce psychosis [108]. β-Casomorphin-7 is elevated in the urine of autistic patients [104], and when in infused into the blood stream of rats has been shown to activate the transcription of the gene *c-Fos* in the brain [109]. However, dietary restrictions do

While controversial, elevated short chain fatty acids (SCFA) have been associated with autism [110], and both central and peripheral administration of propionic acid (PPA) to rats induces ASD-like impairments that include aberrant motor movements, stereotyped repetition, EEG changes, cognitive deficits, perseveration and social impairment, as well as increased oxidative stress, glutathione depletion, neuro-inflammation, altered lipid profiles and more [111]. SCFA are digestive products derived from fiber and protein. The most common SCFA include propionic PPA and butyric acid (BA) [112]. BA and PPA are metabolized in the liver via the portal circulation, however areas of the distal colon are outside of the portal circulatory bed, and the systemic effects of BA and PPA are believed to be significantly underestimated [110, 112]. SCFA, including PPA, activate G protein coupled neural, effect neurotransmitter synthesis and release, and mediate such diverse events in the nervous system as Ca++ gating, mitochondrial function, lipid metabolism, immune function, gene expression and the role of tight junctions [110]. SCFA are believed to modify the activity of TH, and there are 3 ways in which the SCFA BA modifies TH activity: (1) modulation of transcription via chromaffin remodeling, (2) activation of various transcription mediators, and (3) by interfering with TH mRNA [113–116]. Subsequent work by Nankova, et al. [117] have shown that PPA elevates TH mRNA levels and that SCFA increase TH and subsequent catecholamine

This dietary model allows for the elevation of cortical and striatal dopamine activity via elevated TH synthesis and activation, and which has been invoked as a potential mechanism for the actions of risperidone [118, 119], one of the two drugs approved for the treatment of the irritability associated with autism. It is worth noting in this context that PPA is structurally similar to valproic acid (VA), and has similar effects to VA, which is a treatment known to induce autistic symptoms, and used as a model for this purpose [120–122]. As cited above, VA appears to induce ASD- like symptoms by stimulating TH transcription in a manner similar to

It is worth noting that relative to the participation of gastrointestinal events which may underlie autism, recent developments in the study of the human biome and investigations into GI function have revealed that the gut is the source of a number of neurotransmitters and neurotrophic factors, thus opening a previously

**104**

synthesis.

butyrate [86].

understudied source of pharmacologic agents which may regulate CNS function. *E. coli* and Clostridium sp. have been shown to elevate free GI catecholamines and dopamine increases colonic water absorption [123]. GI microbiota produce catecholamines and recognize them in the environment [124–126]. Epinephrine and norepinephrine are implicated in the virulence, ability to adhere, and chemotaxic properties of luminal bacteria [127].

In the work discussed above, it is important to note that PPA activates peroxisome proliferator-activated receptor gamma (PPAR-γ), and that this orphan receptor has been shown to have independent effects on the mediation of catecholamine and opioid pathways by SCFA [128], and that, as discussed below, PPAR is considered to be a "master regulator" of lipid homeostasis both centrally and peripherally. This later finding plays into the growing literature of lipid metabolism dysregulation in autism. PPAR also has immunologic functions that have been found to be related to metabolic and neurologic pathologies [129].

## **5. Dopamine underlies autistic symptoms in the gut and the CNS**

Any comprehensive approach to the treatment of autism must accommodate many different organ systems, certainly the gut and the CNS. As discussed above, the neurotransmitter function of dopamine is well known, including its modulatory effects on motor function, mood, emotion, irritability, reward, and other systems which are affected by autism. However, there also exists in the mesentery a paracrine dopaminergic system that regulates the secretion of bicarbonate [130], the secretion of digestive enzymes by the exocrine pancreas [131], and which controls sodium transport in the lower intestine [132]. Dopamine also has documented effects on gut motility and mucosal blood flow [133–135]. As early as 1994 elevated blood levels of levels of dopamine have been associated with autism [136]. It is known that ASD is associated with elevated levels of dopamine in the tracts linking the amygdala and prefrontal cortex in children with ASD [137, 138].

What is less known is that approximately 42–46% of the dopamine in the body is produced in the gut. Eisenhower and colleagues at NIH [139] studied 8 patients undergoing elective abdominal surgery and 47 patients who underwent cardiac catheterization. Tissue samples from the stomach and duodenum were obtained and compared, as were arteriovenous concentration differences and rates of renal clearance of dopamine and its metabolites in conditions of different sympathetic nervous backgrounds for dopamine not converted to norepinephrine. They found considerable dopamine synthesis in the stomach, pancreas, and duodenum, with renal elimination of dopamine and its metabolites. Dopamine has a naturetic function in the kidney; however, there was significant overflow of dopamine into the renal venous circulation that allows for systemic effects. As expected, cells in the stomach, pancreas, and duodenum stained positive for TH. The authors could not account for the amount of dopamine added to the mesenteric venous circulation, as it cannot be explained by sympathetic activity or diet, and their results were consistent with findings in swine [140].

In keeping with the concepts presented herein, it is relevant that in the liver, bile salt production and release are also under the control of dopamine [141, 142]. Bile salts are known to occur not only in the periphery, but in the CNS as well, where they appear to contribute to neurologic decline and blood brain barrier permeability [143, 144]. Bile acids are the predominant steroid in the brain, with levels that are 10x greater than those found in the blood indicating local synthesis, and with higher titers than that of pregnanolone, which was once considered the predominant neurosteroid [145].

In the brain, it has been observed that chenoxydecholic acid or deoxycholic acid induce the phosphorylation of occludin and increase the permeability of tight junctions via an Rac-1 dependent mechanism [146], making it conceivable that tight junctions in the gut are similarly effected by bile salts under the control of dopamine. It is well known that bile salts upregulate the orphan X receptors Farnesoid X Receptor (FXR), Liver X Receptor (LXR), Retinoid X Receptor (RXR) and PPAR. These nuclear receptors regulate the metabolism and homeostasis of glucose and lipids in numerous ways, including the transcription of the genes that regulate energy metabolism. Beyond the role of lipids in cell membranes and myelin sheaths, there is a growing body of literature to support the concept that lipids play a crucial signaling and regulatory role in cognition and other CNS events. This would appear to be significant as the brain comprised fundamentally of lipid and has the highest rate of glucose utilization in the body.

While it is commonly stated that autism occurs more frequently in males, at a rate of 4 boys for each girl [147], it is less commonly known that in severe autism this ratio increases to 11 to 1 [148]. Numerous sexual dimorphisms in the brain have been described (reviewed in [149]), such as brain size, hemispheric communications, differential gene expression, and more. It is worth noting that there is a growing body of literature implicating dopamine modulation of behavior as part of these sexual dimorphisms. One mechanism which may underlie the sexual dimorphism seen in the expression of ASD may relate to *SRY*, the sex-determining region on the Y chromosome, which is responsible for many male traits, including the differentiation of bipotential embryonic gonads to become testes. *SRY* is an intronless gene that co-localizes with dopaminergic neurons in the hypothalamus, frontal and temporal cortex, striatum, ventral tegmental area (VTA), locus coeruleus and substantia nigral. In humans, *SRY* expression is found in a population of TH positive neurons in the VTA, which is the origin of the dopaminergic cell bodies of the mesocorticolimbic dopamine system which is widely implicated in the drug and natural reward circuitry of the brain. It is important in cognition, motivation, orgasm, drug addiction, intense emotions relating to love, and several psychiatric disorders. SRY has been found to regulate the transcription of TH via the AP-1 binding site on the TH promotor. The synthesis of MAO-A, an enzyme which inactivates DA, and which has polymorphisms associated with the severity of ASD, is also mediated by *SRY* in a manner that elevates extracellular dopamine. Thus, *SRY* appears to be expressed in regions of the brain, and have pharmacologic activity on dopaminergic function, in a manner that is consistent with the preponderance of ASD in males that is pathognomic for this syndrome (reviewed in [149]).

Consistent with the increased prevalence of ASD in males, work in a mouse model has shown that a 16p11.2 gene deletion, which is associated with autism, affects the striatal reward system. While both sexes had 50% reductions in mRNA associated with ERK1, an important signaling kinase, in males there was an increase in ERK1 activation at baseline and in response to sugar in a manner associated with reduced striatal plasticity not shown in females. These changes were associated with an overexpression of dopamine D2 receptors in the striatum [150].

A mechanism by which sleep disturbances associated with ASD may be mediated involves the striatum, an area known to coordinate reward, learning and cognitive behaviors [151, 152] as well as to modulate circadian locomotor and retinal responses [153, 154]. This locus has been shown to be responsible for the maintenance of normal circadian rhythm functionality, and this system appears to be controlled by dopamine. Activation of D2 receptors has been found to regulate clock genes in the striatum, controlling circadian events. Hood et al. [155] found that depletion of striatal dopamine by various methods, including the use of

**107**

*L1-79 and the Role of Catecholamines in Autism DOI: http://dx.doi.org/10.5772/intechopen.95052*

**6. The role of energy metabolism**

(for reviews see: [158–165]).

the brain occurs *de novo* within the CNS.

required to maintain normal circadian rhythmicity.

as changes in secretory, digestive and excretory functions.

AMPT, blunts normal circadian functions and that daily dopaminergic activation is

ASD and energy metabolism are associated in several ways beyond the gut with glucose and lipid metabolism affected. Key among them is the role of bile salts under the control of catecholamines. Bile regulates FXR, LXR, and PPAR which are involved in the regulation of glucose metabolism, insulin sensitivity, lipid signaling and homeostasis. As a class, these ligand-inducible receptors are upregulated in the presence of their ligand, such as bile salts, and after binding they migrate to the nucleus where they exert genomic and epigenomic effects upon transcription and translation of the genes that mediate glucose and lipid utilization

Diabetes and metabolic syndrome are recognized comorbidities of ASD [154]. Catecholamines regulate bile acid release and the FXR upregulates the synthesis and secretion of bile salts from the gall bladder by stimulating the bile salt efflux pump in order to provide bile to solubilize fat soluble nutrients and vitamins from the gut and may have a similarly stimulate FXR in the brain. In mice, FXR deficiency leads to insulin resistance and reduced glucose tolerance [163, 166, 167], and the finding of FXR in pancreatic islet cells that affects insulin release allows for a regulatory role of local bile acid concentrations in insulin release and glucose tolerance [168, 169]. In the CNS, it is commonly known that the influence glucose receptors in the hypothalamus, carotid bodies, and other sites summate to mediate the central nervous control of glucose metabolism. Eating, satiety and similar energy mediated events in the brain are known to be modulated by the sympathetic nervous system, predominantly by dopaminergic systems [170]. Sympathetic afferents from the hypothalamus and other central site, under the control of various agents such as catecholamines and leptin are known to regulate glucose synthesis, insulin sensitivity and similar events (reviewed in [171–174]). Severing the autonomic projections to the islets of Langerhans resulted in a 75–90% impairment in the ability to regulate serum glucose in response to insulin induced hypoglycemia [175]. Although the effects of catecholamines on lipid homeostasis have been defined in the gut, these mechanism can also serve as a model in brain tissue, since this organ contains 25% of the body's cholesterol but only 2% of its mass [176], and most of the lipid synthesis and metabolism in

There exists a growing body of work that lipid metabolism underlies cognitive function. Accumulating evidence supports the idea that HDL and the mechanisms that regulate lipid metabolism also influence neurodegenerative diseases including autism, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and others [177]. Just as HDL have a demonstrably cardio-protective role, they

In the context of a dopamine mediated model of autism, it is interesting to note that bile acids under catecholamine control inhibit the GABAA receptor [156, 157] in a manner that that diminished GABA related inhibitory post synaptic potentials. Inhibition was observed to occur in a stereospecific receptor-ligated, ion channel dependent manner, independent of lipophilicity, and consistent with the behavior of other known GABA receptor blockers. Interestingly, the inhibitory potencies of various bile salts corresponded best with their binding constants with albumin. Taken together there is evidence for a dopaminergic system which might underlay and unite the symptoms of autism which manifest as central nervous system changes in mood, attention, cognition, socialization, etc., and those seen in the gut

#### *L1-79 and the Role of Catecholamines in Autism DOI: http://dx.doi.org/10.5772/intechopen.95052*

*Autism Spectrum Disorder - Profile, Heterogeneity, Neurobiology and Intervention*

rate of glucose utilization in the body.

In the brain, it has been observed that chenoxydecholic acid or deoxycholic acid induce the phosphorylation of occludin and increase the permeability of tight junctions via an Rac-1 dependent mechanism [146], making it conceivable that tight junctions in the gut are similarly effected by bile salts under the control of dopamine. It is well known that bile salts upregulate the orphan X receptors Farnesoid X Receptor (FXR), Liver X Receptor (LXR), Retinoid X Receptor (RXR) and PPAR. These nuclear receptors regulate the metabolism and homeostasis of glucose and lipids in numerous ways, including the transcription of the genes that regulate energy metabolism. Beyond the role of lipids in cell membranes and myelin sheaths, there is a growing body of literature to support the concept that lipids play a crucial signaling and regulatory role in cognition and other CNS events. This would appear to be significant as the brain comprised fundamentally of lipid and has the highest

While it is commonly stated that autism occurs more frequently in males, at a rate of 4 boys for each girl [147], it is less commonly known that in severe autism this ratio increases to 11 to 1 [148]. Numerous sexual dimorphisms in the brain have been described (reviewed in [149]), such as brain size, hemispheric communications, differential gene expression, and more. It is worth noting that there is a growing body of literature implicating dopamine modulation of behavior as part of these sexual dimorphisms. One mechanism which may underlie the sexual dimorphism seen in the expression of ASD may relate to *SRY*, the sex-determining region on the Y chromosome, which is responsible for many male traits, including the differentiation of bipotential embryonic gonads to become testes. *SRY* is an intronless gene that co-localizes with dopaminergic neurons in the hypothalamus, frontal and temporal cortex, striatum, ventral tegmental area (VTA), locus coeruleus and substantia nigral. In humans, *SRY* expression is found in a population of TH positive neurons in the VTA, which is the origin of the dopaminergic cell bodies of the mesocorticolimbic dopamine system which is widely implicated in the drug and natural reward circuitry of the brain. It is important in cognition, motivation, orgasm, drug addiction, intense emotions relating to love, and several psychiatric disorders. SRY has been found to regulate the transcription of TH via the AP-1 binding site on the TH promotor. The synthesis of MAO-A, an enzyme which inactivates DA, and which has polymorphisms associated with the severity of ASD, is also mediated by *SRY* in a manner that elevates extracellular dopamine. Thus, *SRY* appears to be expressed in regions of the brain, and have pharmacologic activity on dopaminergic function, in a manner that is consistent with the preponderance of ASD in males that is pathognomic for this syndrome

Consistent with the increased prevalence of ASD in males, work in a mouse model has shown that a 16p11.2 gene deletion, which is associated with autism, affects the striatal reward system. While both sexes had 50% reductions in mRNA associated with ERK1, an important signaling kinase, in males there was an increase in ERK1 activation at baseline and in response to sugar in a manner associated with reduced striatal plasticity not shown in females. These changes were associated with

A mechanism by which sleep disturbances associated with ASD may be mediated involves the striatum, an area known to coordinate reward, learning and cognitive behaviors [151, 152] as well as to modulate circadian locomotor and retinal responses [153, 154]. This locus has been shown to be responsible for the maintenance of normal circadian rhythm functionality, and this system appears to be controlled by dopamine. Activation of D2 receptors has been found to regulate clock genes in the striatum, controlling circadian events. Hood et al. [155] found that depletion of striatal dopamine by various methods, including the use of

an overexpression of dopamine D2 receptors in the striatum [150].

**106**

(reviewed in [149]).

AMPT, blunts normal circadian functions and that daily dopaminergic activation is required to maintain normal circadian rhythmicity.

In the context of a dopamine mediated model of autism, it is interesting to note that bile acids under catecholamine control inhibit the GABAA receptor [156, 157] in a manner that that diminished GABA related inhibitory post synaptic potentials. Inhibition was observed to occur in a stereospecific receptor-ligated, ion channel dependent manner, independent of lipophilicity, and consistent with the behavior of other known GABA receptor blockers. Interestingly, the inhibitory potencies of various bile salts corresponded best with their binding constants with albumin.

Taken together there is evidence for a dopaminergic system which might underlay and unite the symptoms of autism which manifest as central nervous system changes in mood, attention, cognition, socialization, etc., and those seen in the gut as changes in secretory, digestive and excretory functions.

## **6. The role of energy metabolism**

ASD and energy metabolism are associated in several ways beyond the gut with glucose and lipid metabolism affected. Key among them is the role of bile salts under the control of catecholamines. Bile regulates FXR, LXR, and PPAR which are involved in the regulation of glucose metabolism, insulin sensitivity, lipid signaling and homeostasis. As a class, these ligand-inducible receptors are upregulated in the presence of their ligand, such as bile salts, and after binding they migrate to the nucleus where they exert genomic and epigenomic effects upon transcription and translation of the genes that mediate glucose and lipid utilization (for reviews see: [158–165]).

Diabetes and metabolic syndrome are recognized comorbidities of ASD [154]. Catecholamines regulate bile acid release and the FXR upregulates the synthesis and secretion of bile salts from the gall bladder by stimulating the bile salt efflux pump in order to provide bile to solubilize fat soluble nutrients and vitamins from the gut and may have a similarly stimulate FXR in the brain. In mice, FXR deficiency leads to insulin resistance and reduced glucose tolerance [163, 166, 167], and the finding of FXR in pancreatic islet cells that affects insulin release allows for a regulatory role of local bile acid concentrations in insulin release and glucose tolerance [168, 169]. In the CNS, it is commonly known that the influence glucose receptors in the hypothalamus, carotid bodies, and other sites summate to mediate the central nervous control of glucose metabolism. Eating, satiety and similar energy mediated events in the brain are known to be modulated by the sympathetic nervous system, predominantly by dopaminergic systems [170]. Sympathetic afferents from the hypothalamus and other central site, under the control of various agents such as catecholamines and leptin are known to regulate glucose synthesis, insulin sensitivity and similar events (reviewed in [171–174]). Severing the autonomic projections to the islets of Langerhans resulted in a 75–90% impairment in the ability to regulate serum glucose in response to insulin induced hypoglycemia [175]. Although the effects of catecholamines on lipid homeostasis have been defined in the gut, these mechanism can also serve as a model in brain tissue, since this organ contains 25% of the body's cholesterol but only 2% of its mass [176], and most of the lipid synthesis and metabolism in the brain occurs *de novo* within the CNS.

There exists a growing body of work that lipid metabolism underlies cognitive function. Accumulating evidence supports the idea that HDL and the mechanisms that regulate lipid metabolism also influence neurodegenerative diseases including autism, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and others [177]. Just as HDL have a demonstrably cardio-protective role, they

also appear to have a neuro-protective role. HDL are made throughout the body and serve to remove excess cholesterol from peripheral tissues for excretion in the bile and for steroidogenesis. In a study of 139 centenarians it was found that plasma HDL correlated with mental acuity in age [178]. This was confirmed in another study of 159 centenarians [179], again in a longitudinal population study in Amsterdam [180], and supported by the finding that low HDL was associated with intellectual impairment in age [181–183]. Effectors like cholesteryl ester transfer protein (CETP), which increase HDL are similarly associated with durable cognitive function in later age [179, 184, 185].

Bile salts can be released inappropriately via a "leaky gut" syndrome that ASD or they can be made locally in the brain under the control of catecholamines. Their synthesis and biologic functions have been described in a variety of non-gastric tissues, including the brain. As reviewed by Quinn and DE Marrow [186], bile acids and their salts are now viewed as steroid hormones, and not merely as detergents that solubilize lipids. Consistent with their role as the predominant brain steroid [145], in the rat that the primary bile acid chenoxydecholic acid composed 95% of brains bile acid. Further, the most abundant oxysterols found in the CNS are the C22 and C26 intermediates of bile acid synthesis.

One of the agents that regulates HDL homeostasis is the LXR, which is upregulated in the presence of the bile salts that solubilize and accompany plasma and tissue lipids. LXR is a cholesterol sensing and regulating molecule and cholesterol functionality is necessary for heathy cell membrane function, which is crucial to synaptic function. LXR has been demonstrated to improve cognitive performance in animal models of Alzheimer's disease presumably via the induction of HDL [cited: [177]].

Once believed to be the master regulator of glucose and lipid metabolism, PPAR-γ is associated with the maturation and development of adipocytes, the deposition of lipids, glucose metabolism, insulin sensitivity and other related events [187]. PPAR-γ has been shown to be mediated by bile salts and dopamine via phospholipase C in a calcium dependent manner, with elevations in dopamine resulting in increased PPAR-γ in cardiac myocytes [188]. PPAR-α is abundantly expressed in skeletal muscle, liver and brain [189, 190], and is associated with dyslipidemia, a condition often seen in autistic patients [190–193]. PPAR-α has been associated in the literature with central dopaminergic function as it appears to influence the activity of antipsychotic agents known to interact with dopaminergic neurologic systems [194, 195]. It has also been implicated in reduced GABAergic interneuron firing in pyramidal neurons resulting in cortical excitation [196–199]. D'Agastino et al. [200] have shown that central nervous system reduction in this "Master Regulator of Lipid Homeostasis" is associated with autistic like behaviors that include; repetitive and perseverative behaviors, loss of cognitive flexibility and reduced spatial information processing. They documented PPAR-α deprivation resulted in resistance to central glutamate stimulation via NMDA receptors, reduced GABAergic interneurons in the frontal cortex and hippocampus with dystrophic neurons in these structures, and increased gamma waves with decreased theta wave frequency.

Historically, there is a well-defined relationship between stress, catecholamines, and plasma lipids (reviewed in [25]). Stress, which is characterized by elevated levels of circulating catecholamines, is associated with increased plasma lipids, reduced glycemic control, diminished insulin secretion and insulin insensitivity, all of which can be associated with ASD. This is consistent with the aggressive fighting responses associated with catecholamines significantly elevating NGF in sympathetic ganglia and in the absence of ACTH or corticoids [201]. Various central mechanisms have been implicated in these events, including, the ventromedial

**109**

*L1-79 and the Role of Catecholamines in Autism DOI: http://dx.doi.org/10.5772/intechopen.95052*

exemplified in autism.

**7. Nerve growth factors**

nucleus of the hypothalamus and hippocampal efferents to the hypothalamus. These central nervous system events can be translated into hyperlipidemia in three ways: via adrenal epinephrine release, via elevated pancreatic glucagon secretion, and via the regulation of hepatic glycolysis and gluconeogenesis. All three of these

These findings fit with an emerging metabolic model of autism in which CNS control of energy metabolism and the autonomic nervous system as an integrating modality that senses and regulates those changes in the periphery and modifies these effects centrally. Integration of reward, satiety, insulin release and sensitivity, related endocrine events, as well as circadian clock mechanisms and similar systems which are impaired in autism appear to be mediated largely in the hypothalamus and brain stem via various nutrient sensing mechanisms which reticulate throughout the CNS to the cortex, basal ganglia, pyramids and so forth. (for a review see [202]). Cholesterol, LXR, PPARs and other agents which are known to regulate energy metabolism in the periphery appear to do so in the CNS as well and these mechanisms map well to the deficiencies seen in autism. It is particularly noteworthy that many of the events mediated by the nuclear receptors LXR and PPAR are cell and ligand specific, and that changes in cholesterol metabolism can have profound changes on membranes and their functions. That these changes can vary as a function of cell type provides a mechanism by which metabolic impairments in discrete brain regions may occur in ways that compromise specific nuclei and tracts. Taken together, there appears to be linkage between catecholamine metabolism both centrally and peripherally, the regulation of energy homeostasis, and central nervous system function in a variety of pathologic states. There is a growing body of evidence to indicate a relationship between central and peripheral nervous system regulation of glucose and energy homeostasis, and abnormal cognitive function, as

Nerve growth factor (NGF) in the brain is stimulated by catecholamine synthesis [203] and regulates the morphology of the catecholamine synapse. Neurotropic NGF is required for catecholaminergic neuron survival and differentiation. It is released into the synapse with catecholamines and it determines the synaptic architecture with elevated levels of NGF resulting in elevated levels of TH, catecholamine synthesis and synaptic neurotransmission [204–206] since NGF concentrations have a direct effect on the budding and arborization of catecholamine dendrites [207, 208] as well as the density of target tissue innervation [209, 210] Similarly, elevated catecholaminergic transmission is associated in a dose dependent manner with brain derived nerve growth factor (BDNF) in a pre-synaptic manner [211]. This is consistent with the finding that the loss of a Brain Derived Nerve Growth Factor (BDNF) allele in a mouse knockout model.prevented the loss of

sympathetic islet innervation in an immune based diabetic model [212].

neurons, and innervation density [207, 217].

NGF has a hyperplastic, hypertrophic effect on catecholaminergic neurons characterized by elevated TH [213–215] that results from binding to its tyrosine kinase receptor TrkA expressed on the axons of catecholaminergic neurons [216]. In this way pre-synaptic release of neurotransmitters exerts a differentiating effect post-synaptically to mediate catecholamine synaptic architecture, the number of

NGF is known to increase with increased catecholaminergic nerve traffic and with stress [218], and results in the sprouting of new nerve fibers in the stellate ganglion and elsewhere in the sympathetic nervous system [219, 220]. NGF and

pathways are regulated by the sympathetic nervous system.

#### *L1-79 and the Role of Catecholamines in Autism DOI: http://dx.doi.org/10.5772/intechopen.95052*

*Autism Spectrum Disorder - Profile, Heterogeneity, Neurobiology and Intervention*

function in later age [179, 184, 185].

and C26 intermediates of bile acid synthesis.

[cited: [177]].

also appear to have a neuro-protective role. HDL are made throughout the body and serve to remove excess cholesterol from peripheral tissues for excretion in the bile and for steroidogenesis. In a study of 139 centenarians it was found that plasma HDL correlated with mental acuity in age [178]. This was confirmed in another study of 159 centenarians [179], again in a longitudinal population study in Amsterdam [180], and supported by the finding that low HDL was associated with intellectual impairment in age [181–183]. Effectors like cholesteryl ester transfer protein (CETP), which increase HDL are similarly associated with durable cognitive

Bile salts can be released inappropriately via a "leaky gut" syndrome that ASD or they can be made locally in the brain under the control of catecholamines. Their synthesis and biologic functions have been described in a variety of non-gastric tissues, including the brain. As reviewed by Quinn and DE Marrow [186], bile acids and their salts are now viewed as steroid hormones, and not merely as detergents that solubilize lipids. Consistent with their role as the predominant brain steroid [145], in the rat that the primary bile acid chenoxydecholic acid composed 95% of brains bile acid. Further, the most abundant oxysterols found in the CNS are the C22

One of the agents that regulates HDL homeostasis is the LXR, which is upregulated in the presence of the bile salts that solubilize and accompany plasma and tissue lipids. LXR is a cholesterol sensing and regulating molecule and cholesterol functionality is necessary for heathy cell membrane function, which is crucial to synaptic function. LXR has been demonstrated to improve cognitive performance in animal models of Alzheimer's disease presumably via the induction of HDL

Once believed to be the master regulator of glucose and lipid metabolism, PPAR-γ is associated with the maturation and development of adipocytes, the deposition of lipids, glucose metabolism, insulin sensitivity and other related events [187]. PPAR-γ has been shown to be mediated by bile salts and dopamine via phospholipase C in a calcium dependent manner, with elevations in dopamine resulting in increased PPAR-γ in cardiac myocytes [188]. PPAR-α is abundantly expressed in skeletal muscle, liver and brain [189, 190], and is associated with dyslipidemia, a condition often seen in autistic patients [190–193]. PPAR-α has been associated in the literature with central dopaminergic function as it appears to influence the activity of antipsychotic agents known to interact with dopaminergic neurologic systems [194, 195]. It has also been implicated in reduced GABAergic interneuron firing in pyramidal neurons resulting in cortical excitation [196–199]. D'Agastino et al. [200] have shown that central nervous system reduction in this "Master Regulator of Lipid Homeostasis" is associated with autistic like behaviors that include; repetitive and perseverative behaviors, loss of cognitive flexibility and reduced spatial information processing. They documented PPAR-α deprivation resulted in resistance to central glutamate stimulation via NMDA receptors, reduced GABAergic interneurons in the frontal cortex and hippocampus with dystrophic neurons in these structures, and increased gamma waves with decreased theta wave

Historically, there is a well-defined relationship between stress, catecholamines,

and plasma lipids (reviewed in [25]). Stress, which is characterized by elevated levels of circulating catecholamines, is associated with increased plasma lipids, reduced glycemic control, diminished insulin secretion and insulin insensitivity, all of which can be associated with ASD. This is consistent with the aggressive fighting responses associated with catecholamines significantly elevating NGF in sympathetic ganglia and in the absence of ACTH or corticoids [201]. Various central mechanisms have been implicated in these events, including, the ventromedial

**108**

frequency.

nucleus of the hypothalamus and hippocampal efferents to the hypothalamus. These central nervous system events can be translated into hyperlipidemia in three ways: via adrenal epinephrine release, via elevated pancreatic glucagon secretion, and via the regulation of hepatic glycolysis and gluconeogenesis. All three of these pathways are regulated by the sympathetic nervous system.

These findings fit with an emerging metabolic model of autism in which CNS control of energy metabolism and the autonomic nervous system as an integrating modality that senses and regulates those changes in the periphery and modifies these effects centrally. Integration of reward, satiety, insulin release and sensitivity, related endocrine events, as well as circadian clock mechanisms and similar systems which are impaired in autism appear to be mediated largely in the hypothalamus and brain stem via various nutrient sensing mechanisms which reticulate throughout the CNS to the cortex, basal ganglia, pyramids and so forth. (for a review see [202]). Cholesterol, LXR, PPARs and other agents which are known to regulate energy metabolism in the periphery appear to do so in the CNS as well and these mechanisms map well to the deficiencies seen in autism. It is particularly noteworthy that many of the events mediated by the nuclear receptors LXR and PPAR are cell and ligand specific, and that changes in cholesterol metabolism can have profound changes on membranes and their functions. That these changes can vary as a function of cell type provides a mechanism by which metabolic impairments in discrete brain regions may occur in ways that compromise specific nuclei and tracts.

Taken together, there appears to be linkage between catecholamine metabolism both centrally and peripherally, the regulation of energy homeostasis, and central nervous system function in a variety of pathologic states. There is a growing body of evidence to indicate a relationship between central and peripheral nervous system regulation of glucose and energy homeostasis, and abnormal cognitive function, as exemplified in autism.

#### **7. Nerve growth factors**

Nerve growth factor (NGF) in the brain is stimulated by catecholamine synthesis [203] and regulates the morphology of the catecholamine synapse. Neurotropic NGF is required for catecholaminergic neuron survival and differentiation. It is released into the synapse with catecholamines and it determines the synaptic architecture with elevated levels of NGF resulting in elevated levels of TH, catecholamine synthesis and synaptic neurotransmission [204–206] since NGF concentrations have a direct effect on the budding and arborization of catecholamine dendrites [207, 208] as well as the density of target tissue innervation [209, 210] Similarly, elevated catecholaminergic transmission is associated in a dose dependent manner with brain derived nerve growth factor (BDNF) in a pre-synaptic manner [211]. This is consistent with the finding that the loss of a Brain Derived Nerve Growth Factor (BDNF) allele in a mouse knockout model.prevented the loss of sympathetic islet innervation in an immune based diabetic model [212].

NGF has a hyperplastic, hypertrophic effect on catecholaminergic neurons characterized by elevated TH [213–215] that results from binding to its tyrosine kinase receptor TrkA expressed on the axons of catecholaminergic neurons [216]. In this way pre-synaptic release of neurotransmitters exerts a differentiating effect post-synaptically to mediate catecholamine synaptic architecture, the number of neurons, and innervation density [207, 217].

NGF is known to increase with increased catecholaminergic nerve traffic and with stress [218], and results in the sprouting of new nerve fibers in the stellate ganglion and elsewhere in the sympathetic nervous system [219, 220]. NGF and

BDNF are important mediators of neurologic function in the brain with the ability to mediate short and long term neurologic function in areas associated with ASD like the cortex and hippocampus [221, 222]. NGF has been shown to promote sympathetic neural growth, differentiation and to enhance target innervation [205, 208–210, 223, 224] and NGF is known to be elevated in PTSD [225–227], a disease with a similar constellation of symptoms to autism. NGF leads to sympathetic sprouting and supports dendritic geometry of the newly sprouted nerve terminals for the life of the sympathetic neural substrate [228, 229]. NGF is known to effect memory directly [230], indirectly [220, 231, 232], and through its actions on NE, as well as indirectly via hypothalamically mediated release of cortisol [233].

#### **8. L1-79**

LI:79 is D,L α-methyl-para-tyrosine, abbreviated AMPT. It inhibits the activity of TH, which catalyzes the first transformation in catecholamine biosynthesis, i.e., the conversion of tyrosine to dihydroxyphenylalanine (DOPA) which is the rate limiting step in catecholamine synthesis. L α-methyl-para-tyrosine was approved by the FDA in 1979, is marketed under the name Demser®, and is typically called metyrosine and abbreviated AMT.

α-methyl-para-tyrosine is a tyrosine analog that competes competitively for TH and is excreted mostly unchanged in the urine. Demser was approved for presurgical use in the treatment of pheochromocytoma, a catecholamine producing tumor which when manipulated surgically releases pathologic levels of catecholamines into the circulation that can result in serious AE. Demser minimizes this potentially serious complication and can treat pheochromocytoma patients who were not qualified for surgery. It is approved for use in doses between 1 and 4 g/day in divided doses. The doses of L1-79 used in autism clinical trials was 90 mg tid to 400 mg tid of which only 50% is the L-isomer.

While Demser is intended to deplete adrenal medullary catecholamines as fully as possible L1-79 is intended to reduce catecholaminergic tone slightly, a use for which Demser is inappropriate. The published half-life for Demser is 3.53 hours [234], whereas the half-life for L1-79 has been found to be between 10.3–14.3 hours [235]. This is presumed to result from a competitive inhibition between the dextro and levo forms of the molecule for the L-amino acid transport mechanisms in the body resulting in more time on target for the racemate. Since only 50% of L1-79 is the active L-isomer, and as it persists at the receptor for a longer duration, L1-79 is suitable for bid dosing and is much better tolerated at the lower doses used to get a therapeutic effect in ASD.

Adverse events associated with Demser include sedation that typically habituates but might persist at doses >2 g/d, temporary changes in sleep, extrapyramidal signs including tremor at high doses, trismus and parkinsonism at high doses, dose dependent confusion that resolves with dose reduction, dose related diarrhea, and infrequent AE that include crystalluria, nausea and vomiting, and impotence. None of these have been observed in autism except for 2 patients who manifest crystalluria without clinical consequence at the 200 mg tid dose.

It should be noted that D,L α-methyl-para-tyrosine as described herein for the treatment of autism is also used in a polytherapeutic regimen for the treatment of patients with late stage cancer (SM-88) under the Tyme Technologies Inc. at doses that are a fraction of the lowest approved dose for Demser, and has been well tolerated.

**111**

**Figure 1.**

*visits for all participants.*

*L1-79 and the Role of Catecholamines in Autism DOI: http://dx.doi.org/10.5772/intechopen.95052*

**9. Preliminary clinical observations**

In a proof of concept trial in 8 patients of both sexes between the ages of 2.75 to 24 years of age and without Rett or Fragile X syndrome. Doses began at doses of 90 mg tid and were escalated to 200 mg tid for most patients with two patients receiving a brief course at 400 mg tid, which was not found to increase efficacy. All doses were well tolerated. Patients were washed out of their legacy medications and 6 patients were maintained on L1-79 alone. Two patients were restarted on one of their legacy medications at lower than their pre-study dose. L1-79 in this study had a therapeutic effect on the core symptoms of autism as defined by the ABC-C (**Figure 1**), the CPRS (**Figure 2**), ADOS (**Figure 3**), and the CGI (**Figure 4**). This includes improvements in socialization, communication, repetitive movements, sleep disturbances, and other symptoms of ASD. Interestingly, the Autism Diagnostic Observation Schedule 2 (ADOS), which is the "gold standard" for quantifying the lifetime severity of ASD was profoundly influenced by L1-79 treatment. In the 6 patients in whom the ADOS was measured a mean decrease of 30% was observed with one patient experiencing a reduction of 47% (**Figure 3**) which took him below the threshold for a diagnosis of autism following 10 weeks of treatment, although

*Proof of concept study: Aberrant behavior checklist-community (ABC-C) scores. Domain scores for each participant during weeks 1 to 8. Because of participant-specific factors, ABC-C scores were not recorded at all*  *Autism Spectrum Disorder - Profile, Heterogeneity, Neurobiology and Intervention*

BDNF are important mediators of neurologic function in the brain with the ability to mediate short and long term neurologic function in areas associated with ASD like the cortex and hippocampus [221, 222]. NGF has been shown to promote sympathetic neural growth, differentiation and to enhance target innervation [205, 208–210, 223, 224] and NGF is known to be elevated in PTSD [225–227], a disease with a similar constellation of symptoms to autism. NGF leads to sympathetic sprouting and supports dendritic geometry of the newly sprouted nerve terminals for the life of the sympathetic neural substrate [228, 229]. NGF is known to effect memory directly [230], indirectly [220, 231, 232], and through its actions on NE, as well as indirectly via hypothalamically mediated release of

LI:79 is D,L α-methyl-para-tyrosine, abbreviated AMPT. It inhibits the activity of TH, which catalyzes the first transformation in catecholamine biosynthesis, i.e., the conversion of tyrosine to dihydroxyphenylalanine (DOPA) which is the rate limiting step in catecholamine synthesis. L α-methyl-para-tyrosine was approved by the FDA in 1979, is marketed under the name Demser®, and is typically called

α-methyl-para-tyrosine is a tyrosine analog that competes competitively for TH and is excreted mostly unchanged in the urine. Demser was approved for presurgical use in the treatment of pheochromocytoma, a catecholamine producing tumor which when manipulated surgically releases pathologic levels of catecholamines into the circulation that can result in serious AE. Demser minimizes this potentially serious complication and can treat pheochromocytoma patients who were not qualified for surgery. It is approved for use in doses between 1 and 4 g/day in divided doses. The doses of L1-79 used in autism clinical trials was 90 mg tid to 400 mg tid

While Demser is intended to deplete adrenal medullary catecholamines as fully as possible L1-79 is intended to reduce catecholaminergic tone slightly, a use for which Demser is inappropriate. The published half-life for Demser is 3.53 hours [234], whereas the half-life for L1-79 has been found to be between 10.3–14.3 hours [235]. This is presumed to result from a competitive inhibition between the dextro and levo forms of the molecule for the L-amino acid transport mechanisms in the body resulting in more time on target for the racemate. Since only 50% of L1-79 is the active L-isomer, and as it persists at the receptor for a longer duration, L1-79 is suitable for bid dosing and is much better tolerated at the lower doses used to get a

Adverse events associated with Demser include sedation that typically habituates but might persist at doses >2 g/d, temporary changes in sleep, extrapyramidal signs including tremor at high doses, trismus and parkinsonism at high doses, dose dependent confusion that resolves with dose reduction, dose related diarrhea, and infrequent AE that include crystalluria, nausea and vomiting, and impotence. None of these have been observed in autism except for 2 patients who manifest crystal-

It should be noted that D,L α-methyl-para-tyrosine as described herein for the treatment of autism is also used in a polytherapeutic regimen for the treatment of patients with late stage cancer (SM-88) under the Tyme Technologies Inc. at doses that are a fraction of the lowest approved dose for Demser, and has been well

luria without clinical consequence at the 200 mg tid dose.

**110**

tolerated.

cortisol [233].

metyrosine and abbreviated AMT.

of which only 50% is the L-isomer.

therapeutic effect in ASD.

**8. L1-79**
