Metabolites as diagnostics for autism spectrum disorder in children with gastrointestinal symptoms

1 . A method of treating Autism Spectrum Disorder (ASD) in a subject diagnosed with a gastrointestinal (GI) problem, the method comprising:

(a) measuring or having measured a level of each metabolite in a selected combination of at least two metabolites in a biological sample obtained from the subject, wherein the at least two metabolites in combination are selected from the group consisting of tyramine O-sulfate and inosine 5′-monophosphate (IMP); IMP and iminodiacetate (IDA); IMP and methylsuccinate; nicotinamide riboside and sphingomyelin (d18:2/23:0, d18:1/23:1, d17:1/24:1); IMP and 1-stearoyl-GPS (18:0); nicotinamide riboside and bilirubin; IMP and sarcosine; IMP and leucylglycine; iminodiacetate (IDA) and 1-linoleoyl-2-arachidonoyl-GPC (18:2/20:4n6); galactonate and 10-undecenoate (11:1n1); nicotinamide riboside and tartarate; IMP and caprylate (8:0); nicotinamide riboside and inosine 5′-monophosphate (IMP); inosine 5′-monophosphate (IMP) and 1-(1-enyl-oleoyl)-GPE (P-18:1); 3-phosphoglycerate and glycerophosphoethanolamine; 3-phosphoglycerate and 1-oleoyl-GPS (18:1); nicotinamide riboside and biliverdin; IMP and 1-stearoyl-GPI (18:0); IMP and 1-arachidonoyl-GPI (20:4); carnitine and 2′-deoxyadenosine; 1-(1-enyl-oleoyl)-GPE (P-18:1) and hydroxyproline; adenine and N-stearoyl-sphingosine (d18:1/18:0);

(b) comparing the measured level of each metabolite in the selected combination against a database of control metabolite level for each metabolite, wherein the control level for each metabolite is determined by measuring a metabolite level of in control typically developing (TD) subjects with no history of bearing a child with ASD, wherein the database is stored on a computer system and wherein the comparing comprises calculating Type I (FPR; false positive rate) and Type II (FNR; false negative rate) errors for the selected combination of metabolites using FDA or logistic regression;

(c) indicating an ASD diagnosis when, for the selected combination, the Type I error is below 10%, and a Type II error is 10%; and

(d) administering a medical, behavioral, or nutritional treatment to the subject identified as having ASD, wherein the treatment comprises administering to the subject:

(i) a behavioral management therapy, a cognitive behavior therapy, an early intervention, an educational and school-based therapy, a joint attention therapy, an occupational therapy, a parent-mediated therapy, a physical therapy, social skills training, or a speech-language therapy;

(ii) a medication selected from an antipsychotic drug, a selective serotonin re-uptake inhibitor (SSRI), a tricyclic, a psychoactive or anti-psychotic medication, a stimulant, an anti-anxiety medication, or an anticonvulsant;

(iii) a nutritional supplementation with one or more of the metabolites of step (a) identified as having a Type I error of below 10%, and a Type II error of below 10%; or

(iv) a composition comprising fecal microbiota from a healthy neurotypical human donor.

2 . The method of claim 1 , further comprising preparing a sample extract from the biological sample using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) to obtain the levels of the at least two metabolites.

3 . The method of claim 2 , wherein the level of a metabolite is measured using:

i. reverse phase chromatography positive ionization methods optimized for hydrophilic compounds (LC/MS Pos Polar);

ii. reverse phase chromatography positive ionization methods optimized for hydrophobic compounds (LC/MS Pos Lipid);

iii. reverse phase chromatography with negative ionization conditions (LC/MS Neg); or

iv. a HILIC chromatography method coupled to negative (LC/MS Polar).

4 . The method of claim 2 , wherein the level of a metabolite is calculated from a peak area and standard calibration curve obtained for the metabolite using UPLC-MS/MS.

5 . The method of claim 4 , wherein measuring further comprises identifying each metabolite by automated comparison of ion features in the sample extract to a reference library of chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra.

6 . The method of claim 1 , wherein the database of metabolite control measured levels are measured in biological samples obtained from typically developing (TD) individuals with no GI problems.

7 . The method of claim 1 , wherein the biological sample is blood or blood plasma.

8 . The method of claim 7 , wherein the at least two metabolites in combination are selected from the group consisting of tyramine O-sulfate and inosine 5′-monophosphate; inosine 5′-monophosphate (IMP) and iminodiacetate (IDA); IMP and sarcosine; IMP and methylsuccinate; nicotinamide riboside and sphingomyelin (d18:2/23:0, d18:1/23:1, d17:1/24:1); IMP and caprylate (8:0); IMP and 1-stearoyl-GPS (18:0); nicotinamide riboside and bilirubin; IMP and sarcosine; IMP and leucylglycine; iminodiacetate (IDA) and 1-linoleoyl-2-arachidonoyl-GPC (18:2/20:4n6); galactonate and 10-undecenoate (11:1n1); nicotinamide riboside and tartarate; nicotinamide riboside and inosine 5′-monophosphate (IMP); inosine 5′-monophosphate (IMP) and 1-(1-enyl-oleoyl)-GPE (P-18:1); 3-phosphoglycerate and glycerophosphoethanolamine; 3-phosphoglycerate and 1-oleoyl-GPS (18:1); nicotinamide riboside and biliverdin; IMP and 1-stearoyl-GPI (18:0); and IMP and 1-arachidonoyl-GPI (20:4).

9 . The method of claim 1 , wherein the biological sample is a fecal sample.

10 . The method of claim 9 , wherein each two metabolite in a selected combination of at least two metabolites are selected from the group consisting of carnitine and 2′-deoxyadenosine; 2′-deoxyadenosine and 3-hydroxy-3-methylglutarate; 1-(1-enyl-oleoyl)-GPE (P-18:1) and hydroxyproline; and adenine and N-stearoyl-sphingosine (d18:1/18:0).

11 . A method of treating Autism Spectrum Disorder (ASD) in a subject diagnosed with a gastrointestinal (GI) problem, the method comprising:

(a) measuring or having measured a level of each metabolite in a selected combination of three metabolites in a biological sample obtained from the subject, wherein the three metabolites are selected from the group consisting of sarcosine, tyramine O-sulfate, and inosine 5′-monophosphate; IMP, iminodiacetate (IDA), and tyramine O-sulfate; IMP, caproate (6:0), and tyramine O-sulfate; heptanoate (7:0), biliverdin, and indolepropionate; IMP, heptanoate (7:0), and biliverdin; IMP, picolinate, and tyramine O-sulfate; IMP, heptanoate (7:0), and tyramine O-sulfate; IMP, 1-oleoyl-GPS (18:1), and creatine; caprylate (8:0), 1-palmitoyl-GPI (16:0), and 3-phosphoglycerate; and IMP, azelate (nonanedioate; C9), and biliverdin; indole, hydroxyproline, and 2′-deoxyadenosine; N-stearoyl-sphingosine (d18:1/18:0), delta-tocopherol, and diaminopimelate; galactonate, N-acetylsphingosine, and adenine; carnitine, N-stearoyl-sphingosine (d18:1/18:0), and adenine; imidazole propionate, N-acetylsphingosine, and adenine;

(b) administering a medical, behavioral, or nutritional treatment to the subject identified as a candidate for treatment of ASD with GI, wherein the treatment comprises administering to the subject:

(i) a behavioral management therapy, a cognitive behavior therapy, an early intervention, an educational and school-based therapy, a joint attention therapy, an occupational therapy, a parent-mediated therapy, a physical therapy, social skills training, or a speech-language therapy;

(ii) a medication selected from an antipsychotic drug, a selective serotonin re-uptake inhibitor (SSRI), a tricyclic, a psychoactive or anti-psychotic medication, a stimulant, an anti-anxiety medication, or an anticonvulsant;

(iii) a nutritional supplementation with one or more of the metabolites of step (a) identified as having a Type I error of below 10%, and a Type II error of below 10%; or

(iv) a composition comprising fecal microbiota from a healthy neurotypical human donor.

12 . A method of treating Autism Spectrum Disorder (ASD) in a subject diagnosed with a gastrointestinal (GI) problem, the method comprising:

(a) measuring or having measured a level of each metabolite in a selected combination of four metabolites in a biological sample obtained from the subject, wherein the four metabolites are selected from the group consisting of sarcosine, tyramine O-sulfate, inosine 5′-monophosphate, and arachidonate (20:4n6); IMP, picolinate, tyramine O-sulfate, and arachidonate (20:4n6); IMP, galactonate, picolinate, and tyramine O-sulfate; 3-phosphoglycerate, caproate (6:0), picolinate, and tyramine O-sulfate; IMP, iminodiacetate (IDA), tyramine O-sulfate, and arachidonate (20:4n6); IMP, iminodiacetate (IDA), leucylglycine, and tyramine O-sulfate; IMP, 2-aminophenol sulfate, picolinate, and tyramine O-sulfate; iminodiacetate (IDA), 3-phosphoglycerate, glycerophosphoethanolamine, and tyramine O-sulfate; iminodiacetate (IDA), 3-phosphoglycerate, tyramine O-sulfate, and arachidonate (20:4n6); IMP, sarcosine, sphingomyelin (d18:2/23:0, d18:1/23:1, d17:1/24:1), and tyramine O-sulfate; indole, 10-nonadecenoate (19:1n9), adenine, and theobromine; indole, adenine, biliverdin, and theobromine;

(b) administering a medical, behavioral, or nutritional treatment to the subject identified as a candidate for treatment of ASD with GI, wherein the treatment comprises administering to the subject:

(i) a behavioral management therapy, a cognitive behavior therapy, an early intervention, an educational and school-based therapy, a joint attention therapy, an occupational therapy, a parent-mediated therapy, a physical therapy, social skills training, or a speech-language therapy;

(ii) a medication selected from an antipsychotic drug, a selective serotonin re-uptake inhibitor (SSRI), a tricyclic, a psychoactive or anti-psychotic medication, a stimulant, an anti-anxiety medication, or an anticonvulsant;

(iii) a nutritional supplementation with one or more of the metabolites of step (a) identified as having a Type I error of below 10%, and a Type II error of below 10%; or

(iv) a composition comprising fecal microbiota from a healthy neurotypical human donor.

13 . A method of treating Autism Spectrum Disorder (ASD) in a subject diagnosed with a gastrointestinal (GI) problem, the method comprising:

(a) measuring or having measured a level of each metabolite in a selected combination of five metabolites in a biological sample obtained from the subject, wherein the five metabolites are selected from the group consisting of IMP, galactonate, picolinate, tyramine O-sulfate, and arachidonate (20:4n6); IMP, 1-stearoyl-GPS (18:0), picolinate, creatine, and tyramine O-sulfate; IMP, iminodiacetate (IDA), biliverdin, arachidonate (20:4n6), and 2-oxindole-3-acetate; IMP, picolinate, gamma-glutamylhistidine, tyramine O-sulfate, and arachidonate (20:4n6); IMP, iminodiacetate (IDA), leucylglycine, 1-palmitoyl-GPI (16:0), and tyramine O-sulfate; IMP, citrate, sphingomyelin (d18:2/23:0, d18:1/23:1, d17:1/24:1), fructose, and arachidonate (20:4n6); IMP, picolinate, 9,10-DiHOME, tyramine O-sulfate, and arachidonate (20:4n6); IMP, 2-methylserine, picolinate, tyramine O-sulfate, and arachidonate (20:4n6); IMP, caproate (6:0), tyramine O-sulfate, arachidonate (20:4n6), and 1-linoleoyl-2-arachidonoyl-GPC (18:2/20:4n6); IMP, bilirubin, sphingomyelin (d18:2/23:0, d18:1/23:1,d17:1/24:1), arachidonate (20:4n6), and tartarate; imidazole propionate, hydroxyproline, theobromine, 2-hydroxy-3-methylvalerate, and indole;

(b) administering a medical, behavioral, or nutritional treatment to the subject identified as a candidate for treatment of ASD with GI, wherein the treatment comprises administering to the subject:

(i) a behavioral management therapy, a cognitive behavior therapy, an early intervention, an educational and school-based therapy, a joint attention therapy, an occupational therapy, a parent-mediated therapy, a physical therapy, social skills training, or a speech-language therapy;

(ii) a medication selected from an antipsychotic drug, a selective serotonin re-uptake inhibitor (SSRI), a tricyclic, a psychoactive or anti-psychotic medication, a stimulant, an anti-anxiety medication, or an anticonvulsant;

(iii) a nutritional supplementation with one or more of the metabolites of step (a) identified as having a Type I error of below 10%, and a Type II error of below 10%; or

(iv) a composition comprising fecal microbiota from a healthy neurotypical human donor.

14 . A method of treating Autism Spectrum Disorder (ASD) in a subject diagnosed with a gastrointestinal (GI) problem, the method comprising: administering to the subject a medical, behavioral, nutritional treatment, or a composition comprising fecal microbiota from healthy neurotypical human, wherein the subject has been determined as having ASD with GI disorder by, for a selected combination of at least two metabolites, a Type I error of below 10%, and a Type II error of below 10% for a level of each two metabolite in a selected combination of at least two metabolites in a biological sample obtained from the subject, wherein two metabolites in combination are selected from the group consisting of tyramine O-sulfate and inosine 5′-monophosphate (IMP) IMP and iminodiacetate (IDA); IMP and sarcosine, IMP and methylsuccinate; nicotinamide riboside and sphingomyelin (d18:2/23:0, d18:1/23:1, d17:1/24:1); IMP and 1-stearoyl-GPS (18:0); nicotinamide riboside and bilirubin; IMP and sarcosine; IMP and leucylglycine; iminodiacetate (IDA) and 1-linoleoyl-2-arachidonoyl-GPC (18:2/20:4n6); galactonate and 10-undecenoate (11:1n1); nicotinamide riboside and tartarate; IMP and caprylate (8:0); nicotinamide riboside and inosine 5′-monophosphate (IMP); inosine 5′-monophosphate (IMP) and 1-(1-enyl-oleoyl)-GPE (P-18:1); 3-phosphoglycerate and glycerophosphoethanolamine; 3-phosphoglycerate and 1-oleoyl-GPS (18:1); nicotinamide riboside and biliverdin; IMP and 1-stearoyl-GPI (18:0), and IMP and 1-arachidonoyl-GPI (20:4), wherein the treatment comprises administering to the subject:

(a) a behavioral management therapy, a cognitive behavior therapy, an early intervention, an educational and school-based therapy, a joint attention therapy, an occupational therapy, a parent-mediated therapy, a physical therapy, social skills training, or a speech-language therapy;

(b) a medication selected from an antipsychotic drug, a selective serotonin re-uptake inhibitor (SSRI), a tricyclic, a psychoactive or anti-psychotic medication, a stimulant, an anti-anxiety medication, or an anticonvulsant;

(c) a nutritional supplementation with one or more of the metabolites identified as having a Type I error of below 10%, and a Type II error of below 10% for a level of each of the two metabolite in the combination; or

(d) a composition comprising fecal microbiota from a healthy neurotypical human donor.

15 . A method of classifying a subject as a candidate for treatment of Autism Spectrum Disorder (ASD) with a gastrointestinal (GI) disorder, the method comprising:

a) measuring a level of each of two metabolite in a selected combination of at least two metabolites in the biological sample using ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS), wherein the at least two metabolites in combination are selected from the group consisting of tyramine O-sulfate and inosine 5′-monophosphate (IMP) IMP and iminodiacetate (IDA); IMP and sarcosine, IMP and methylsuccinate; nicotinamide riboside and sphingomyelin (d18:2/23:0, d18:1/23:1, d17:1/24:1); IMP and 1-stearoyl-GPS (18:0); nicotinamide riboside and bilirubin; IMP and sarcosine; IMP and leucylglycine; iminodiacetate (IDA) and 1-linoleoyl-2-arachidonoyl-GPC (18:2/20:4n6); galactonate and 10-undecenoate (11:1n1); nicotinamide riboside and tartarate; IMP and caprylate (8:0); nicotinamide riboside and inosine 5′-monophosphate (IMP); inosine 5′-monophosphate (IMP) and 1-(1-enyl-oleoyl)-GPE (P-18:1); 3-phosphoglycerate and glycerophosphoethanolamine; 3-phosphoglycerate and 1-oleoyl-GPS (18:1); nicotinamide riboside and biliverdin; IMP and 1-stearoyl-GPI (18:0); IMP and 1-arachidonoyl-GPI (20:4); carnitine and 2′-deoxyadenosine; 1-(1-enyl-oleoyl)-GPE (P-18:1) and hydroxyproline; adenine and N-stearoyl-sphingosine (d18:1/18:0);

b) generating, by a processor, a Type I (FPR; false positive rate) and a Type II (FNR; false negative rate) error for the each of the metabolite in the selected combination of metabolites using Fisher discriminant analysis (FDA) or logistic regression by comparing the measured level of each metabolite in the selected combination against a model comprising a database of control metabolite level determined by measuring a metabolite level in control typically developing (TD) subjects with no history of bearing a child with ASD, wherein the model has a classification accuracy of at least 0.05 and misclassification error of less than 0.05;

c) classifying the subject as a candidate for treatment of ASD with GI when, for the selected combination, the Type I error is below 10%, and a Type II error is below 10%; and

d) administering a medical, behavioral, or nutritional treatment to the subject identified as a candidate for treatment of ASD with GI, wherein the treatment comprises administering to the subject:

(i) a behavioral management therapy, a cognitive behavior therapy, an early intervention, an educational and school-based therapy, a joint attention therapy, an occupational therapy, a parent-mediated therapy, a physical therapy, social skills training, or a speech-language therapy;

(ii) a medication selected from an antipsychotic drug, a selective serotonin re-uptake inhibitor (SSRI), a tricyclic, a psychoactive or anti-psychotic medication, a stimulant, an anti-anxiety medication, or an anticonvulsant;

(iii) a nutritional supplementation with one or more of the metabolites of step (a) identified as having a Type I error of below 10%, and a Type II error of below 10%; or

(iv) a composition comprising fecal microbiota from a healthy neurotypical human donor.