Background
Methods
Study design and sample collection and analysis
IRB approval and consent
Advertising
Participants
Diet and medical history
Biological sample collection
Laboratory tests
Statistical analysis
Univariate analysis
Multivariate analysis
Results
Univariate analysis
Participants
ASD (n = 30) | TD (n = 29) | p-Value of t-test (T) or Chi-Squared (C) | |
---|---|---|---|
Maternal age (years) | 35.4 (5.2) | 35.2 (5.8) | n.s. (T) |
Maternal BMI | 26.46 (5.10) | 25.50 (4.65) | n.s. (T) |
Maternal Ethnicitya | |||
Hispanic or Latino | 22% | 28% | n.s. (C) |
White | 86% | 93% | n.s. (C) |
Black or African American | 0% | 3% | n.s. (C) |
Asian | 4% | 0% | n.s. (C) |
American Indian or Alaska Native | 0% | 0% | N/A |
Native Hawaiian or Other Pacific Islander | 0% | 0% | N/A |
Do not wish to provide ethnic info | 7% | 0% | n.s. (C) |
ASD (n = 30) | TD (n = 29) | Chi-Squared | FDR | |
---|---|---|---|---|
Use of Any Medications | 43% (13) | 30% (9) | n.s. | |
Psychiatric | 17% (5) | 7% (2) | n.s. | |
Allergy | 10% (3) | 14% (4) | n.s. | |
Birth control | 33% (10) | 10% (3) | 0.03 | n.s. |
Inhaler | 0% (0) | 3% (1) | n.s. | |
Blood pressure | 7% (2) | 0% (0) | n.s. | |
Thyroid | 10% (3) | 7% (2) | n.s. | |
GI (Gastrointestinal) | 0% (0) | 3% (1) | n.s. | |
Pain | 0% (0) | 3% (1) | n.s. | |
Epi-pen as needed | 3% (1) | 0% (0) | n.s. | |
Use of Nutritional Supplements* | 7% (2) | 7% (2) | n.s. |
ASD (n = 30) | TD (n = 29) | Ratio of ASD/TD | t-test | FDR | |
---|---|---|---|---|---|
Fatigue | 1.15 (1.0) | 0.79 (0.8) | 1.46 | n.s. | |
Fibromyalgia | 0.11 (0.6) | 0 (0) | n.s. | ||
Depression | 0.67 (0.8) | 0.48 (0.7) | 1.38 | n.s. | |
Irritability | 0.78 (0.9) | 0.71 (0.8) | 1.09 | n.s. | |
Anxiety | 1.11 (0.9) | 0.75 (1.0) | 1.48 | 0.08 | n.s. |
Cognition | 0.56 (1.0) | 0.32 (0.6) | 1.73 | n.s. | |
Attention | 0.85 (1.0) | 0.32 (0.55) | 2.65 | 0.056 | n.s. |
Sensory Sensitivity | 0.44 (0.7) | 0.18 (0.4) | 2.49 | n.s. | |
Stool/GI Problems | 0.44 (0.8) | 0.71 (1.0) | 0.62 | n.s. | |
Sleep Problems | 0.74 (0.9) | 0.46 (0.74) | 1.60 | n.s. | |
Headaches | 0.56 (0.8) | 0.64 (0.7) | 0.86 | n.s. | |
Chemical Sensitivity | 0.19 (0.5) | 0.14 (0.5) | 1.30 | n.s. | |
Allergies | 0.78 (1.0) | 0.71 (0.7) | 1.09 | n.s. | |
Average | 0.60 (0.5) | 0.44 (0.4) | 1.37 | n.s. |
FOCM/TS metabolites
Metabolite | Test | ASD-M (mean ± std) N = 30 | TD-M (mean ± std) N = 29 | p-Values | FDR | AUC | Ratio (ASD-M/TD-M) |
---|---|---|---|---|---|---|---|
B12 Δ | MW | 355 ± 196 | 473 ± 173 | 2.40E-03 | 0.00 | 0.73 | 0.75 |
fCysteine Δ | t= | 23.8 ± 1.88 | 22.6 ± 1.94 | 0.01 | 0.00 | 0.70 | 1.06 |
Glu-Cys Δ | t= | 1.89 ± 0.22 | 1.72 ± 0.24 | 0.01 | 0.00 | 0.69 | 1.10 |
SAM/SAH Δ | t= | 1.94 ± 0.25 | 2.09 ± 0.20 | 0.01 | 0.00 | 0.67 | 0.93 |
fCystine Δ | t= | 24.1 ± 2.78 | 22.4 ± 2.43 | 0.02 | 0.01 | 0.67 | 1.07 |
tCysteine | t= | 248 ± 23.1 | 234 ± 28.3 | 0.04 | 0.40 | 0.64 | 1.06 |
tGSH | t= | 6.23 ± 0.98 | 5.85 ± 1.07 | 0.15 | 1.00 | 0.63 | 1.07 |
SAM | t= | 47.2 ± 5.37 | 49.3 ± 5.76 | 0.15 | 1.00 | 0.62 | 0.96 |
Methionine | t= | 19.9 ± 2.56 | 20.8 ± 2.98 | 0.19 | 1.00 | 0.61 | 0.95 |
MTHFR mut. (A1298C) | χ2 | 0.15 | 1.00 | 0.60 | |||
tGSH/GSSG | t= | 29.5 ± 6.21 | 27.3 ± 6.06 | 0.18 | 1.00 | 0.60 | 1.08 |
Folate | MW | 17.6 ± 6.19 | 21.1 ± 9.17 | 0.20 | 1.00 | 0.60 | 0.83 |
Homocysteine | MW | 8.63 ± 0.98 | 8.27 ± 1.18 | 0.21 | 1.00 | 0.60 | 1.04 |
tGSH/GSSG | t= | 29.5 ± 6.21 | 27.3 ± 6.06 | 0.18 | 1.00 | 0.60 | 1.08 |
SAH | t= | 24.5 ± 2.66 | 23.6 ± 2.04 | 0.15 | 1.00 | 0.59 | 1.04 |
Vitamin D3‡ | t= | 27.1 ± 9.10 | 24.9 ± 6.08 | 0.27 | 1.00 | 0.57 | 1.09 |
Ferritin | MW | 35.1 ± 31.0 | 29.5 ± 26.1 | 0.36 | 1.00 | 0.57 | 1.19 |
Cys-Gly | t= | 38.7 ± 5.06 | 37.6 ± 6.38 | 0.46 | 1.00 | 0.57 | 1.03 |
Adenosine | t= | 0.22 ± 0.03 | 0.21 ± 0.03 | 0.28 | 1.00 | 0.55 | 1.04 |
fGSH/GSSG | MW | 8.73 ± 2.08 | 8.81 ± 1.84 | 0.49 | 1.00 | 0.55 | 0.99 |
% Oxidized GSH | MW | 0.19 ± 0.03 | 0.19 ± 0.04 | 0.49 | 1.00 | 0.55 | 1.01 |
Chlorotyrosine | t= | 26.8 ± 4.27 | 27.6 ± 4.23 | 0.51 | 1.00 | 0.55 | 0.97 |
Nitrotyrosine | t ≠ | 32.9 ± 6.28 | 33.7 ± 4.67 | 0.59 | 1.00 | 0.55 | 0.98 |
fGSH | t= | 1.85 ± 0.32 | 1.89 ± 0.35 | 0.62 | 1.00 | 0.55 | 0.98 |
fCystine/fCysteine | t= | 1.01 ± 0.11 | 1.00 ± 0.10 | 0.59 | 1.00 | 0.54 | 1.02 |
Vitamin E | t= | 9.23 ± 2.53 | 9.82 ± 3.22 | 0.75 | 1.00 | 0.54 | 0.94 |
Isoprostane (U)‡ | MW | 0.15 ± 0.10 | 0.18 ± 0.14 | 0.70 | 1.00 | 0.53 | 0.83 |
MTHFR mut. (C677T) | χ2 | 0.90 | 1.00 | 0.53 | |||
GSSG | t= | 0.22 ± 0.04 | 0.22 ± 0.03 | 0.93 | 1.00 | 0.52 | 1.00 |
MMA | MW | 0.15 ± 0.06 | 0.15 ± 0.08 | 0.64 | 1.00 | 0.51 | 0.96 |
Global metabolic profile- Metabolon
Metabolite | Test | ASD-M (mean ± std) N = 30 | TD-M (mean ± std) N = 29 | p-Value | FDR | AUC | Ratio (ASD-M/TD-M) |
---|---|---|---|---|---|---|---|
Fructose Δ | t ≠ † | 2.08E6 ± 7.88E5 | 3.61E6 ± 2.07E6 | 6.88E-04 | 0.00 | 0.81 | 0.60 |
Histidylglutamate Δ | t ≠ | 1.05E5 ± 3.25E4 | 7.27E4 ± 2.05E4 | 2.67E-05 | 0.00 | 0.80 | 1.50 |
Decanoylcarnitine (C10) Δ b | t ≠ | 3.73E6 ± 1.48E6 | 5.85E6 ± 2.36E6 | 1.55E-04 | 0.00 | 0.78 | 0.66 |
S-1-pyrroline-5-carboxylate Δ | MW | 1.62E5 ± 1.06E5 | 2.65E5 ± 1.14E5 | 3.09E-04 | 0.00 | 0.77 | 0.63 |
Octanoylcarnitine (C8) Δ b | MW | 1.96E6 ± 7.67E5 | 3.00E6 ± 1.17E6 | 4.00E-04 | 0.00 | 0.77 | 0.67 |
4-vinylphenol sulfate Δ | t ≠ † | 6.52E5 ± 6.79E5 | 2.20E6 ± 2.27E6 | 1.30E-03 | 0.00 | 0.77 | 0.31 |
Cis-4-decenoylcarnitine (C10:1) Δ b | t= | 2.25E6 ± 7.42E5 | 3.12E6 ± 1.07E6 | 5.82E-04 | 0.00 | 0.74 | 0.75 |
N-formylanthranilic acid Δ | t= | 1.12E5 ± 5.20E4 | 1.67E5 ± 7.04E4 | 1.20E-03 | 0.00 | 0.74 | 0.69 |
N-acetylasparagine Δ | t ≠ † | 3.31E5 ± 6.33E4 | 4.37E5 ± 1.60E5 | 1.90E-03 | 0.00 | 0.73 | 0.78 |
Arachidoylcarnitine (C20)a Δ b | MW | 4.68E5 ± 2.09E5 | 5.80E5 ± 1.75E5 | 3.00E-03 | 0.00 | 0.73 | 0.85 |
N-palmitoylglycine Δ | t= | 7.60E4 ± 2.51E4 | 9.76E4 ± 2.65E4 | 2.20E-03 | 0.00 | 0.72 | 0.81 |
Citrulline Δ | t= | 3.36E7 ± 4.90E6 | 3.82E7 ± 6.37E6 | 2.60E-03 | 0.00 | 0.72 | 0.91 |
6-hydroxyindole sulfate Δ | t ≠ | 2.94E5 ± 1.56E5 | 5.16E5 ± 3.07E5 | 1.20E-03 | 0.00 | 0.72 | 0.59 |
N-palmitoylserine Δ | MW | 6.90E4 ± 3.61E4 | 9.33E4 ± 3.78E4 | 4.00E-03 | 0.00 | 0.72 | 0.77 |
Myristoylcarnitine (C14) Δ b | MW | 2.61E6 ± 1.21E6 | 3.30E6 ± 1.17E6 | 4.10E-03 | 0.00 | 0.72 | 0.82 |
Laurylcarnitine (C12) Δ b | MW | 1.41E6 ± 5.33E5 | 1.96E6 ± 8.25E5 | 4.30E-03 | 0.00 | 0.72 | 0.74 |
Stearoylcarnitine (C18) Δ b | MW | 2.15E7 ± 8.37E6 | 2.61E7 ± 6.68E6 | 0.01 | 0.00 | 0.71 | 0.85 |
Gamma-glutamylglycine Δ | t ≠ † | 2.91E4 ± 5.86E4 | 1.12E5 ± 1.24E5 | 2.20E-03 | 0.00 | 0.71 | 0.27 |
5-oxoproline Δ | t= | 9.59E6 ± 1.30E6 | 1.05E7 ± 1.29E6 | 0.01 | 0.00 | 0.70 | 0.94 |
Asparaginylalanine Δ | t= | 8.80E4 ± 2.68E4 | 6.89E4 ± 2.16E4 | 3.90E-03 | 0.00 | 0.70 | 1.32 |
Glutamine Δ | t= | 4.11E8 ± 5.43E7 | 4.46E8 ± 5.38E7 | 0.02 | 0.00 | 0.70 | 0.95 |
Catechol sulfate Δ | MW | 2.10E6 ± 1.54E6 | 3.22E6 ± 1.83E6 | 0.01 | 0.00 | 0.70 | 0.67 |
3-indoxyl sulfate Δ | t ≠ | 7.41E6 ± 3.56E6 | 1.15E7 ± 5.98E6 | 2.70E-03 | 0.00 | 0.70 | 0.67 |
7-methylxanthine Δ | MW | 1.56E5 ± 3.18E5 | 2.75E5 ± 2.48E5 | 0.01 | 0.00 | 0.70 | 0.58 |
Phenol sulfate Δ | MW | 1.22E7 ± 7.67E6 | 1.88E7 ± 1.21E7 | 0.01 | 0.00 | 0.70 | 0.67 |
Cinnamoylglycine Δ | t ≠ † | 1.73E5 ± 1.59E5 | 3.87E5 ± 4.08E5 | 0.01 | 0.00 | 0.70 | 0.46 |
Alpha-ketoglutaramatea Δ | t= | 2.53E6 ± 1.46E6 | 3.59E6 ± 1.77E6 | 0.02 | 0.00 | 0.70 | 0.73 |
Isovalerylglycine Δ | MW | 5.91E4 ± 2.88E4 | 7.81E4 ± 2.82E4 | 0.01 | 0.00 | 0.69 | 0.78 |
Propionylglycine Δ | MW | 9.15E4 ± 1.19E5 | 1.97E5 ± 1.89E5 | 0.01 | 0.00 | 0.69 | 0.48 |
Docosapentaenoylcarnitine (C22:5n3)a Δ b | MW | 1.61E5 ± 1.16E5 | 2.31E5 ± 1.39E5 | 0.01 | 0.00 | 0.69 | 0.72 |
N-acetyl-2-aminooctanoatea Δ | t ≠ | 7.20E4 ± 4.51E4 | 1.38E5 ± 1.05E5 | 3.20E-03 | 0.00 | 0.69 | 0.54 |
S-methylglutathione Δ | t= | 2.06E5 ± 5.87E4 | 2.47E5 ± 7.06E4 | 0.02 | 0.01 | 0.69 | 0.86 |
Gamma-glutamyltyrosine Δ | MW | 4.25E4 ± 3.40E4 | 6.83E4 ± 3.29E4 | 0.02 | 0.00 | 0.68 | 0.64 |
Succinylcarnitine (C4-DC) Δ b | t= | 2.80E6 ± 8.21E5 | 3.31E6 ± 9.04E5 | 0.03 | 0.07 | 0.68 | 0.87 |
Arachidonoylcarnitine (C20:4) Δ b | MW | 1.10E6 ± 5.59E5 | 1.47E6 ± 8.12E5 | 0.02 | 0.00 | 0.68 | 0.77 |
Glycine Δ | t= | 2.04E7 ± 4.44E6 | 2.42E7 ± 6.07E6 | 0.01 | 0.00 | 0.68 | 0.87 |
N-acetylvaline | t= | 7.51E4 ± 4.01E4 | 9.74E4 ± 3.92E4 | 0.04 | 0.28 | 0.68 | 0.80 |
Lignoceroylcarnitine (C24)a Δ b | t= | 6.12E5 ± 1.72E5 | 7.48E5 ± 2.61E5 | 0.02 | 0.01 | 0.68 | 0.84 |
Guaiacol sulfate Δ | MW | 4.23E5 ± 4.12E5 | 5.42E5 ± 3.12E5 | 0.02 | 0.01 | 0.68 | 0.81 |
5-methylthioadenosine (MTA) Δ | MW | 2.60E5 ± 1.28E5 | 3.12E5 ± 1.11E5 | 0.02 | 0.00 | 0.68 | 0.86 |
Proline Δ | MW | 2.44E8 ± 4.76E7 | 2.80E8 ± 5.83E7 | 0.02 | 0.00 | 0.68 | 0.90 |
Pyridoxate Δ | MW | 6.26E6 ± 2.78E5 | 8.61E5 ± 4.53E5 | 0.02 | 0.00 | 0.68 | 0.75 |
Palmitoylcarnitine (C16) Δ b | MW | 3.12E7 ± 8.25E6 | 3.89E7 ± 1.33E7 | 0.02 | 0.03 | 0.67 | 0.83 |
Eicosenoylcarnitine (C20:1)a Δ b | MW | 1.46E6 ± 4.30E5 | 1.82E6 ± 7.46E5 | 0.02 | 0.05 | 0.67 | 0.83 |
Nicotinamide adenine dinucleotide (NAD+) Δ | t ≠ † | 5.53E4 ± 5.46E4 | 1.39E5 ± 1.91E5 | 0.03 | 0.05 | 0.67 | 0.41 |
Dimethyl sulfone | MW | 6.06E5 ± 2.08E6 | 3.36E4 ± 1.04E5 | 0.01 | 0.23 | 0.67 | 18.7 |
Tiglylcarnitine (C5:1-DC) Δ b | MW | 3.18E4 ± 3.19E4 | 5.19E4 ± 3.40E4 | 0.02 | 0.02 | 0.67 | 0.63 |
Adrenoylcarnitine (C22:4)a b | MW | 2.67E5 ± 1.50E5 | 3.75E5 ± 2.38E5 | 0.03 | 0.11 | 0.67 | 0.74 |
3-methylxanthine | MW | 2.32E5 ± 3.76E5 | 3.22E5 ± 3.10E5 | 0.03 | 0.13 | 0.67 | 0.74 |
Mannose | MW | 1.49E7 ± 3.96E6 | 1.27E7 ± 2.19E6 | 0.03 | 0.19 | 0.67 | 1.21 |
Carnitine
Variable | Test | ASD-M (mean ± std) N = 28 | TD-M (mean ± std) N = 29 | p-Value | FDR | AUC | Ratio (ASD-M/TD-M) |
---|---|---|---|---|---|---|---|
Beef Frequency | MW | 2.50 ± 1.43 | 2.24 ± 0.91 | 0.73 | 1.00 | 0.53 | 1.12 |
Beef Quantity | MW | 1.46 ± 0.58 | 1.41 ± 0.50 | 0.83 | 1.00 | 0.51 | 1.41 |
Multivariate analysis
Subset | Combination | Type I Error | Type II Error |
---|---|---|---|
(i): FOCM/TS Metabolites | tCysteine, Glu-Cys, fCysteine, fCystine/fCystiene, Nitrotyrosine | 24% | 27% |
(ii): FOCM/TS metabolites plus nutritional information | SAM/SAH, Glu-Cys, GSSG, fCysteine, B12 | 24% | 27% |
(iii): FOCM/TS metabolites, nutritional information, and MTHFR gene information | SAM/SAH, tCysteine, Glu-Cys, B12, MTHFR mut. (A1298C) | 24% | 20% |
(iv): FOCM/TS metabolites, nutritional information, MTHFR gene information, and Metabolon metabolites | Glu-Cys, histidylglutamate, cinnamoylglycine, proline, adrenoylcarnitine (C22:4)a | 3% | 3% |
Metabolite | Correlation Coefficient | p-Value |
---|---|---|
Glu-Cys | ||
tGSH | 0.55 | 5.71E-06 |
tGSH/GSSG | 0.35 | 0.01 |
6-hydroxyindole sulfate | −0.25 | 0.05 |
SAM/SAH | −0.26 | 0.04 |
N-formylanthranilic acid | −0.28 | 0.03 |
5-methylthioadenosine (MTA) | −0.28 | 0.03 |
Pyridoxate | −0.31 | 0.02 |
Folate | −0.38 | 3.40E-03 |
Histidylglutamate | ||
Asparaginylalanine | 0.55 | 6.74E-06 |
Mannose | 0.40 | 1.70E-03 |
fCystine | 0.30 | 0.02 |
Succinylcarntine (C4-DC) | −0.27 | 0.04 |
Citrulline | −0.27 | 0.04 |
Fructose | −0.28 | 0.03 |
Octanoylcarnitine (C8) | −0.29 | 0.02 |
Gamma-glutamylglycine | −0.30 | 0.02 |
Isovaleryglycine | −0.32 | 0.01 |
Decanoylcarnitine (C10) | −0.33 | 0.01 |
Cinnamoylglycine | ||
N-acetyl-2-aminooctanoatea | 0.45 | 4.13E-04 |
N-formylanthranilic acid | 0.44 | 4.30E-04 |
3-indoxyl sulfate | 0.35 | 0.01 |
Citrulline | 0.33 | 0.01 |
6-hydroxyindole sulfate | 0.32 | 0.01 |
Chlorotyrosine | 0.29 | 0.02 |
Alpha-ketoglutaramatea | 0.28 | 0.03 |
Nicotinamide adenine dinucleotide (NAD+) | 0.28 | 0.03 |
Pyridoxate | 0.27 | 0.04 |
Guaiacol sulfate | 0.26 | 0.04 |
S-methylglutathione | 0.26 | 0.04 |
Methionine | −0.29 | 0.02 |
fCysteine | −0.33 | 0.01 |
Proline | ||
S-1-pyrroline-5-carboxylate | 0.59 | 1.22E-06 |
Gamma-glutamyltyrosine | 0.45 | 3.73E-04 |
3-indoxyl sulfate | 0.44 | 5.33E-04 |
6-hydroxyindole sulfate | 0.43 | 6.01E-04 |
Phenol sulfate | 0.41 | 1.20E-03 |
Glutamine | 0.36 | 0.01 |
Propionylglycine | 0.35 | 0.01 |
Glycine | 0.35 | 0.01 |
Gamma-glutamylglycine | 0.32 | 0.01 |
5-oxoproline | 0.30 | 0.02 |
Alpha-ketoglutaramatea | 0.28 | 0.03 |
Folate | 0.28 | 0.03 |
N-formylanthranilic acid | 0.27 | 0.04 |
Adenosine | −0.30 | 0.02 |
Adrenoylcarnitine (C22:4)a | ||
Arachidonoylcarnitine (C20:4) | 0.93 | 8.00E-26 |
Docosapentaenoylcarnitine (C22:5n3)a | 0.85 | 8.64E-18 |
Eicosenoylcarnitine (C20:1)a | 0.74 | 2.35E-11 |
Palmitoylcarnitine (C16) | 0.70 | 8.13E-10 |
Myristoylcarnitine (C14) | 0.69 | 2.17E-09 |
Laurylcarnitine (C12) | 0.49 | 8.88E-05 |
Fructose | 0.41 | 1.20E-03 |
N-acetylasparagine | 0.38 | 2.60E-03 |
Stearoylcarnitine (C18) | 0.31 | 0.02 |
Methionine | 0.26 | 0.04 |
Cys-Gly | 0.26 | 0.05 |
Arachidoylcarnitine (C20)a | 0.26 | 0.05 |
N-palmitoylserine | 0.25 | 0.05 |
fCysteine/fCystine | −0.29 | 0.03 |
fCystine | −0.30 | 0.02 |
Metabolites | Correlation Coefficient | P-value |
---|---|---|
Glu-Cys x Histidylglutamate | −0.01 | 0.92 |
Glu-Cys x Cinnamoylglycine | −0.06 | 0.63 |
Glu-Cys x Proline | −0.09 | 0.51 |
Glu-Cys x Adrenoylcarnitine (C22:4)a | 0.01 | 0.95 |
Histidylglutamate x Cinnamoylglycine | −0.03 | 0.81 |
Histidylglutamate x Proline | −0.07 | 0.59 |
Histidylglutamate x Adrenoylcarnitine (C22:4)a | −0.06 | 0.65 |
Cinnamoylglycine x Proline | 1.80E-03 | 0.99 |
Cinnamoylglycine x Adrenoylglycine (C22:4)a | −0.13 | 0.31 |
Proline x Adrenoylcarnitine (C22:4)a | 0.04 | 0.74 |