Background
Methods
Subject recruitment
No | Age (years) | Gender | ADOS score | CARS total score | CARS activity level item score (hyperactivity) | CARS body use item score (stereotypes) | CARS total number of items with score ≥ 3 | Autism severity level | Cognitive/developmental impairment | Onset pattern (1, early; 2, regressive; 3, mixed)† | Probability of autism‡ from algorithm no. | |||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | |||||||||||
1 | 6.0 | m | 22 | 41.0 | 2.0 | 3.0 | 9 | Severe | Severe | 1 | 0.93 | 0.79 | 0.82 | 0.80 |
2 | 5.6 | m | 14 | 34.0 | 2.0 | 2.0 | 2 | Mild | Mild | 1 | 1.00 | 0.71 | 0.99 | 0.77 |
3 | 5.5 | m | 18 | 40.5 | 2.5 | 3.5 | 9 | Severe | Moderate | 1 | 1.00 | 0.86 | 1.00 | 0.29 |
4 | 5.4 | f | 17 | 31.5 | 2.0 | 2.0 | 1 | Mild | Mild | 3 | 0.88 | 0.69 | 0.55 | 0.27 |
5 | 8.5 | m | 22 | 36.5 | 2.0 | 2.5 | 6 | Mild | Mild | 3 | 0.98 | 0.64 | 1.00 | 0.88 |
6 | 8.7 | m | 19 | 44.5 | 3.0 | 3.0 | 11 | Severe | Moderate | 2 | 1.00 | 0.69 | 1.00 | 0.74 |
7 | 6.8 | m | 19 | 36.5 | 2.5 | 2.0 | 7 | Mild | Normal IQ | 1 | 1.00 | 0.63 | 1.00 | 0.91 |
8 | 5.5 | f | 15 | 41.5 | 2.5 | 2.0 | 9 | Severe | Borderline IQ | 1 | 0.73 | 0.77 | 0.72 | 0.92 |
9 | 11.9 | m | 22 | 44.5 | 2.5 | 3.0 | 11 | Severe | Severe | 2 | 0.20 | 0.91 | 0.34 | 0.45 |
10 | 9.2 | f | 15 | 47.5 | 3.5 | 3.0 | 13 | Severe | Moderate | 1 | 0.84 | 0.88 | 0.94 | 0.36 |
11 | 12.0 | m | 20 | 39.0 | 3.0 | 3.5 | 8 | Severe | Severe | 3 | 0.94 | 0.68 | 0.82 | 0.63 |
12 | 6.2 | m | 22 | 42.5 | 3.0 | 2.5 | 10 | Severe | Severe | 1 | 1.00 | 0.69 | 1.00 | 0.98 |
13 | 6.7 | m | 21 | 40.5 | 2.5 | 3.0 | 9 | Severe | Severe | 2 | 0.83 | 0.51 | 0.85 | 0.56 |
14 | 6.6 | m | 19 | 37.0 | 2.5 | 3.0 | 7 | Severe | Moderate | 1 | 0.83 | 0.63 | 0.74 | 0.97 |
15 | 5.5 | m | 22 | 40.5 | 3.0 | 3.0 | 9 | Severe | Moderate | 3 | 0.68 | 0.57 | 0.82 | 0.98 |
16 | 5.7 | m | 20 | 41.5 | 2.5 | 3.0 | 11 | severe | severe | 1 | 0.98 | 0.81 | 0.98 | 0.81 |
17 | 7.8 | m | 21 | 46.0 | 3.5 | 4.0 | 11 | Severe | Severe | 1 | 0.83 | 0.35 | 0.52 | 0.79 |
18 | 5.7 | f | 20 | 43.5 | 2.5 | 3.0 | 10 | Severe | Normal IQ | 3 | 0.85 | 0.39 | 0.58 | 0.68 |
19 | 7.8 | m | 20 | 48.5 | 3.0 | 4.0 | 12 | Severe | Severe | 2 | 0.95 | 0.58 | 0.93 | 0.57 |
20 | 6.8 | m | 19 | 42.0 | 3.0 | 3.0 | 9 | Severe | Moderate | 2 | 0.67 | 0.61 | 0.54 | 0.77 |
21 | 9.6 | m | 19 | 39.5 | 3.0 | 3.0 | 7 | Severe | Severe | 1 | 0.55 | 0.44 | 0.62 | 0.51 |
22 | 6.2 | m | 19 | 41.0 | 2.5 | 3.0 | 9 | Severe | Severe | 3 | 0.81 | 0.32 | 0.78 | 0.73 |
23 | 8.3 | m | 16 | 35.0 | 2.5 | 2.0 | 3 | Moderate | Moderate | 1 | 0.70 | 0.35 | 0.63 | 0.56 |
24 | 7.1 | m | 22 | 41.0 | 2.5 | 2.0 | 7 | Severe | Moderate | 1 | 0.83 | 1.00 | 0.99 | 1.00 |
25 | 6.2 | f | 17 | 38.0 | 2.0 | 2.5 | 7 | Severe | Severe | 1 | 0.28 | 0.82 | 0.15 | 0.79 |
26 | 10.3 | f | 21 | 33.5 | 2.0 | 2.0 | 4 | Moderate | Normal IQ | 3 | 0.89 | 0.55 | 0.82 | 0.86 |
27 | 10.7 | m | 19 | 39.0 | 2.0 | 2.5 | 7 | Moderate | Normal IQ | 1 | 0.89 | 0.60 | 0.97 | 0.52 |
28 | 6.5 | m | 17 | 39.0 | 3.0 | 2.5 | 6 | Moderate | Normal IQ | 3 | ND | ND | ND | 0.76 |
29 | 8.0 | m | 22 | 44.0 | 2.5 | 3.0 | 11 | Severe | Moderate | 1 | ND | ND | ND | 0.54 |
30 | 7.0 | m | 18 | 39.5 | 2.5 | 3.0 | 8 | Moderate | Normal IQ | 1 | ND | ND | ND | 0.96 |
31 | 9.5 | m | 21 | 47.0 | 3.0 | 3.5 | 13 | Severe | Severe | 1 | ND | ND | ND | 0.29 |
32 | 8.6 | m | 16 | 41.5 | 4.0 | 2.0 | 10 | Severe | Moderate | 3 | ND | ND | ND | 0.33 |
33 | 10.9 | m | 18 | 35.0 | 2.0 | 3.0 | 3 | Mild | Normal IQ | 1 | ND | ND | ND | 0.28 |
34 | 5.8 | f | 18 | 43.0 | 2.5 | 3.0 | 11 | Severe | Moderate | 2 | ND | ND | ND | 0.30 |
35 | 5.3 | f | 17 | 39.5 | 2.5 | 2.5 | 6 | Moderate | Normal IQ | 1 | ND | ND | ND | 0.62 |
36 | 5.3 | f | 17 | 41.0 | 2.5 | 2.5 | 7 | Severe | Normal IQ | 1 | ND | ND | ND | 0.77 |
37 | 8.2 | m | 20 | 41.0 | 2.0 | 3.5 | 9 | Severe | Moderate | 1 | ND | ND | ND | 0.58 |
38 | 11.1 | m | 13 | 34.0 | 2.0 | 1.5 | 2 | Mild | Normal IQ | 3 | ND | ND | ND | 0.87 |
Blood and urine sampling
Assay of markers of protein glycation, oxidation, and nitration
Machine learning analysis
Statistical analysis
Results
Children with autistic spectrum disorder recruited for this study
Plasma protein glycation, oxidation, and nitration
Glycation markers | Healthy controls | ASD | P value |
---|---|---|---|
FL (mmol/mol lys) | 1.27 ± 0.39 | 1.41 ± 0.53 | NS |
CML (mmol/mol lys) | 0.158 ± 0.026 | 0.190 ± 0.038 | 0.0018* |
CEL (mmol/mol lys) | 0.117 ± 0.044 | 0.092 ± 0.054 | NS |
G-H1 (mmol/mol arg) | 0.012 ± 0.005 | 0.016 ± 0.011 | NS |
MG-H1 (mmol/mol arg) | 0.473 ± 0.074 | 0.535 ± 0.100 | 0.021 |
3DG-H (mmol/mol arg) | 0.165 ± 0.037 | 0.138 ± 0.027 | 0.0052 |
CMA (mmol/mol arg) | 0.054 (0.043–0.067) | 0.077 (0.066–0.101) | 0.000082** |
MOLD (mmol/mol lys) | 0.027 ± 0.011 | 0.025 ± 0.016 | NS |
GSP (mmol/mol lys) | 0.514 ± 0.111 | 0.571 ± 0.206 | NS |
DT (mmol/mol tyr) | 0.025 (0.019–0.031) | 0.047 (0.035–0.094) | 0.000012*** |
NFK (mmol/mol trp) | 15.6 ± 1.7 | 15.0 ± 1.5 | NS |
AASA (mmol/mol lys) | 0.154 ± 0.048 | 0.152 ± 0.081 | NS |
GSA (mmol/mol arg) | 0.639 ± 0.327 | 0.713 ± 0.350 | NS |
3-NT (mmol/mol tyr) | 0.0056 (0.0045–0.0069) | 0.0053 (0.0045–0.0064) | NS |
Plasma glycated, oxidized, and nitrated amino acids and amino acid metabolome
Amino acid | Plasma (nM) | Urine (nmol/mg creatinine) | ||||
---|---|---|---|---|---|---|
Healthy controls | ASD | P value | Healthy controls | ASD | P value | |
FL | 1489 (987–1863) | 751 (361–1.570) | 0.047 | 56.7 (33.8–128.7) | 90.8 (42.4–167.9) | |
CML | 807 (587–1051) | 853 (219–1222) | 26.2 (19.7–34.7) | 33.1 (26.6–42.6) | 0.016 | |
CEL | 402 (298–477) | 420 (310–599) | 0.435 (0.202–0.848) | 0.472 (0.180–0.940) | ||
G-H1 | 0.819 (0.553–1.26) | 0.527 (0.366–0.959) | 0.037 | 1.88 (1.11–3.04) | 2.57 (1.53–3.75) | 0.024 |
MG-H1 | 271 (176–475) | 335 (213–500) | 18.6 (8.00–27.4) | 24.5 (9.14–37.6) | ||
3DG-H | 413 (303–637) | 360 (280–434) | 2.06 (0.587–4.38) | 2.92 (1.09–6.26) | ||
CMA | 9.18 (6.67–12.5) | 17.7 (13.2–24.8) | 0.00052** | 1.46 (0.636–1.97) | 1.78 (1.14–2.91) | 0.037 |
GSP | 12.8 (7.4–17.1) | 12.9 (9.3–22.0) | 1.58 (1.15–1.98) | 1.53 (1.20–1.98) | ||
MOLD | 1.79 (0.800–3.42) | 1.10 (0.503–0.2.21) | 0.025 (0.013–0.050) | 0.040 (0.017–0.068) | 0.027 | |
Pyrraline | 22.0 (12.2–30.4) | 24.2 (19.4–40.6) | 20.6 (14.9–44.2) | 34.2 (22.7–72.5) | 0.047 | |
DT | 0.501 (0.286–0.771) | 0.676 (0.500–0.847) | 0.070 (0.058–0.085) | 0.086 (0.075–0.109) | 0.0022* | |
NFK | 15.2 (12.5–18.1) | 11.3 (6.23–14.3) | 0.030 | 0.117 (0.084–0.231) | 0.179 (0.107–0.238) | 0.037 |
AASA | 19.7 (16.9–29.1) | 30.6 (21.1–46.4) | 0.0063 | 1.08 (0.805–2.76) | 1.80 (1.13–2.89) | 0.040 |
GSA | 73.9 (53.2–129) | 109 (80.1–203) | 0.039 | 17.3 (13.2–22.7) | 34.5 (12.7–48.0) | 0.0018* |
3-NT | 1.10 (0.90–1.26) | 1.17 (0.79–1.58) | 0.0044 (0.001–0.010) | 0.0077 (0.003–0.014) |
Amino acid | Plasma (μM) | Urine (nmol/mg creatinine) | ||||
---|---|---|---|---|---|---|
Healthy controls | ASD | P value | Healthy controls | ASD | P value | |
Ala | 294 ± 71.5 | 330 ± 98.3 | 301 (224–432) | 392 (305–533) | 0.030 | |
Arg | 39.4 ± 10.3 | 48.4 ± 16.6 | 0.016 | 52.1 ± 17.1 | 64.0 ± 18.0 | 0.014 |
Asn | 33.2 ± 4.57 | 34.3 ± 10.6 | 92.4 (72.1–132) | 169 (123–236) | 0.0012* | |
Asp | 35.5 ± 7.37 | 37.0 ± 10.0 | 142 (112–179) | 160 (117–241) | ||
Cys (total) | 41.7 ± 10.4 | 46.1 ± 15.0 | 25.3 (20.4–35.9) | 28.3 (21.1–43.3) | ||
Gln | 463 ± 38.6 | 496 ± 61.2 | 0.024 | 529 (452–704) | 741 (609–876) | 0.0048 |
Glu | 1672 ± 324 | 1999 ± 619 | 0.034 | 373 (254–495) | 475 (367–690) | 0.010 |
Gly | 272 (187–1061) | 241 (121–515) | 1.12 (1.19–2.78) | 2.58 (1.84–3.45) | 0.019 | |
His | 68.0 ± 8.51 | 72.8 ± 9.96 | 2.19 ± 0.88 | 2.90 ± 1.21 | 0.027 | |
IIe | 50.7 ± 15.3 | 51.6 ± 12.4 | 13.0 ± 4.37 | 18.1 ± 6.50 | 0.0059 | |
Leu | 126 ± 34.4 | 132 ± 25.6 | 43.1 ± 13.6 | 52.0 ± 13.5 | 0.028 | |
Lys | 132 ± 20.3 | 146 ± 36.7 | 136 (83.3–264) | 171 (117–352) | ||
Met | 23.1 ± 6.41 | 26.2 ± 5.84 | 17.9 ± 7.98 | 23.1 ± 7.20 | 0.0037 | |
Phe | 68.9 ± 11.3 | 68.8 ± 16.8 | 101 ± 38.9 | 110 ± 32.9 | ||
Pro | 195 ± 65.4 | 223 ± 94.7 | 14.5 ± 6.21 | 20.7 ± 5.37 | 0.00073** | |
Ser | 128 ± 23.5 | 132 ± 18.7 | 461 (413–621) | 677 (519–910) | 0.0021* | |
Thr | 116 ± 36.0 | 141 ± 46.8 | 0.041 | 216 (170–265) | 283 (221–405) | 0.0039 |
Trp | 5.22 ± 1.26 | 3.92 ± 2.40 | 0.0055 | 114 ± 73.6 | 164 ± 48.6 | 0.0043 |
Tyr | 84.4 ± 21.3 | 87.9 ± 26.8 | 204 (133–255) | 240 (173–317) | ||
Val | 125 ± 31.3 | 120 ± 26.0 | 25.6 ± 9.52 | 34.8 ± 10.9 | 0.00073** |
Urinary glycated, oxidized, and nitrated amino acids and amino acid metabolome and renal clearance
Amino acid | Renal clearance (μL/mg creatinine) | ||
---|---|---|---|
Healthy controls | ASD | P value | |
FL
#
| 0.696 (0.375–1.16) | 0.866 (0.384–2.31) | |
CML | 0.297 (0.249–0.387) | 0.259 (0.171–0.782) | |
CEL | 0.0068 (0.003–0.019) | 0.0042 (0.002–0.011) | |
G-H1 | 21.2 (13.1–34.7) | 23.9 (15.0–57.7) | |
MG-H1
#
| 0.718 (0.496–1.04) | 0.628 (0.363–0.912) | |
3DG-H | 0.067 (0.033–0.163) | 0.087 (0.042–0.131) | |
CMA | 1.57 (0.997–2.08) | 0.791 (0.465–1.36) | 0.0011* |
GSP | 0.121 (0.083–0.274) | 0.112 (0.067–0.195) | |
MOLD | 0.214 (0.075–0.487) | 0.269 (0.153–0.656) | |
Pyrraline | 0.81 (0.58–1.17) | 1.07 (0.64–1.96) | |
DT | 0.119 (0.657–2.02) | 0.747 (0.545–0.107) | 0.0025 |
NFK | 0.062 (0.038–0.109) | 0.096 (0.049–0.139) | 0.030 |
GSA | 29.9 (11.1–45.8) | 17.8 (1.96–26.1) | |
3-NT | 0.068 (0.034–0.100) | 0.042 (0.036–0.129) |
Amino acid | Renal clearance (mL/mg creatinine) | ||
---|---|---|---|
Healthy controls | ASD | P value | |
Ala | 1.03 (0.746–1.712) | 1.27 (0.890–1.65) | |
Arg | 0.011 (0.009–0.015) | 0.008 (0.006–0.010) | 0.0019* |
Asn | 2.80 (2.22–4.76) | 4.30 (3.05–7.14) | 0.0055 |
Asp | 3.81 (3.04–4.96) | 4.73 (2.92–7.82) | |
Cys (total) | 0.646 (0.518–0.812) | 0.629 (0.452–1.133) | |
Gln | 1.18 (0.913–1.53) | 1.54 (1.14–1.96) | 0.025 |
Glu | 0.210 (0.158–0.304) | 0.243 (0.179–0.357 | 0.049 |
Gly | 0.0050 (0.002–0.009) | 0.0067 (0.004–0.029) | 0.018 |
His | 0.029 (0.024–0.040) | 0.040 (0.027–0.053) | |
IIe | 0.262 (0.192–0.347) | 0.354 (0.256–0.441) | 0.0116 |
Leu | 0.358 ± 0.135 | 0.402 ± 0.105 | |
Lys | 0.011 (0.005–0.018) | 0.0071 (0.005–0.011) | |
Met | 0.0066 ± 0.0015 | 0.0056 ± 0.0023 | |
Phe | 1.32 (0.959–2.02) | 1.65 (1.33–1.95) | |
Pro | 0.077 ± 0.031 | 0.104 ± 0.039 | 0.0138 |
Ser | 3.66 (2.95–4.79) | 5.22 (3.99–6.78) | 0.0081 |
Thr | 2.023 ± 0.765 | 2.44 ± 1.05 | |
Trp | 0.209 (0.155–0.242) | 0.287 (0.166–0.497) | 0.010 |
Tyr | 0.0209 (0.016–0.025) | 0.015 (0.012–0.025) | |
Val | 0.0017 (0.0014–0.0022) | 0.0016 (0.0013–0.0026) |
Development of diagnostic algorithms for ASD
Algorithm no | 1 | 2 | 3 | 4 |
---|---|---|---|---|
Compartment and analyte | Plasma protein adduct residues | Plasma amino acids | Plasma protein adduct residues and amino acids | Urinary amino acids |
Features | CML, 3DG-H, CMA, and DT | CML and CMA | CML, 3DG-H, CMA, and DT residues with G-H1 and GSA free adducts | GSA and pyrraline free adducts |
Accuracy (%) | 88.3 (85.5–91.2) | 74.8 (71.7–77.9) | 89.0 (87.0–91.0) | 76.8 (74.6–79.0) |
Sensitivity (%) | 91.9 (89.1–94.6) | 80.5 (75.1–86.0) | 90.4 (87.7–93.1) | 77.1 (73.4–80.8) |
Specificity (%) | 83.9 (79.3–88.4) | 67.1 (58.9–75.4) | 87.3 (84.1–90.5) | 76.4 (72.0–80.8) |
AUROC | 0.94 (0.91–0.96) | 0.80 (0.77–0.83) | 0.95 (0.94–0.96) | 0.79 (0.76–0.81) |
Positive likelihood ratio | 5.69 (4.49–6.89) | 2.85 (2.16–3.55) | 7.23 (6.09–8.38) | 4.16 (2.88–5.44) |
Negative likelihood ratio | 0.10 (0.07–0.13) | 0.28 (0.21–0.35) | 0.11 (0.08–0.14) | 0.30 (0.25–0.34) |
Positive predictive value (%) | 88.2 (85.0–91.4) | 77.1 (72.9–81.4) | 90.2 (87.9–92.5) | 80.6 (77.6–83.5) |
Negative predictive value (%) | 89.1 (85.5–92.6) | 75.0 (70.6–79.4) | 88.0 (85.1–91.0) | 73.7 (71.0–76.5) |
F score | 0.90 (0.87–0.92) | 0.78 (0.75–0.81) | 0.90 (0.88–0.92) | 0.78 (0.76–0.81) |