June 2017

Medical Mass Spectrometry Vol. 1 No. 1

Short Communication

A metabolome study on 90 autism spectrum disorder patientsby mass spectrometry
Qi Li, Chunhua Zhang
Department of Research & Development of MILS International,
3‒1‒1 Heiwa Machi, Kanazawa, Ishikawa 921‒8105, Japan
Abstract

Pathogenesis of autism spectrum disorder (ASD) remains unclear and there are still no proper clinical diagnosis

biomarkers or effective medical therapy methods for ASD. Previous studies have implicated physiological and metabolic
abnormalities in ASD. Here we performed a metabolome analysis of urine and dry blood spot samples from 90 ASD patients and 90 age-matched controls from China using mass spectrometry. To our certain knowledge, this is the ﬁrst study investigating ASD patients urinary and blood metabolites at the same time. Abnormalities in urinary and blood amino acids,organic acids and acylcarnitines showed a similar tendency to the reported physiological and metabolic abnormalities in
ASD patients. This thesis aimed at detailing the metabolome information of Chinese ASD patients, hoping to help the studyof ASD pathology, quantitative diagnosis or therapy methods.
Key words: metabolome, autism spectrum disorder, mass spectrometry, urine, dry blood spot

could contribute to an early and more accurate ASD diagno-Introduction

Autism spectrum disorder (ASD) represents a highly het-sis . Physiological and metabolic abnormalities have been

erogeneous collection of neurodevelopmental conditions
characterized by social and communication deﬁcits, stereo-

proved in ASD, particularly immune dysregulation or

typic and rigid patterns of behavior, restricted interests, andand environment toxicant exposure . Metabolome changes
can be the clue to explain the pathology of ASD; mean-

4)

inﬂammation, oxidative stress, mitochondrial dysfunction
5)

1)

unusual sensory processing with onset in early childhood .
In the US, about 1 in 68 or 1.5% of 8-year-old patients waswhile, it can point to potential quantitative diagnosis crite-identiﬁed with ASD in 2012, of which boys were 4.5 times

ria or treatment methods for ASD.

more likely to be identiﬁed with ASD than girls . There is

In this study, 90 ASD patients metabolomes were ana-

no ofﬁcial report on the prevalence of ASD in China. A
study in 2007 showed that the estimated incidence of 0‒4

lyzed compared to 90 healthy children s as controls. Target2)

metabolites including amino acids and organic acids in3)

years old Chinese patients with ASD was 5.49 per 10000 .

urine and amino acids and acylcarnitines in blood were

ASD diagnosis by now is relying on impaired social

detected based on our lab s inborn errors of metabolism

1)interaction skills combined with restrictive behaviors or

screening method using mass spectrometry.

ASD diagnostic interview. Many researchers have been

We aimed at investigating the urinary and blood metabo-

focusing on establishing quantitative diagnostic criteria thatlites changing trends in ASD patients to provide moredetails on metabolome information for clinical diagnosis of* Corresponding author

ASD patients, hoping to help the study of ASD pathology,

Chunhua Zhang

quantitative diagnosis or treatment methods.

Department of Research & Development of MILS International,
3‒1‒1 Heiwa Machi, Kanazawa, Ishikawa 921‒8105, JapanMaterials and Methods

Tel: 076‒243‒7711, Fax: 076‒243‒7751
E-mail: mils@yacht.ocn.ne.jp

Subjects

90 Chinese ASD patients diagnosed based on ASD diagnostic interview, and 90 age matched Chinese health con-Received December 26, 2016. Accepted April 10, 2017.
DOI: 10.24508/mms.2017.06.004
14

June 2017

Table 1.

Medical Mass Spectrometry Vol. 1 No. 1

85 m/z in positive mode (CE-25 V). Mass spectra for gly-Information of ASD patients and controlsASDcine, arginine, citrulline, ornithine and methionine wereacquired through multiple-reaction monitoring (MRM) inControlNumber9090

Age (year)
Range of age (year)

3.8±1.61.5‒8

2.9±1.7
0.1‒9Sex (male : female)

76 : 1465 : 25

positive mode.

Statistical analysis
Statistical analyses were conducted using Origin version

trols were analyzed as subjects. All the subjects urine and2016. Means and standard deviations were computed. Test
results were showed as mean±SD. All the statistical com-dry blood spot samples were sent to our lab for inborn
errors of metabolism screening. Details on age and genderparisons were made by means of independent studentt-Test. The signiﬁcance threshold was held at a nominalby groups are provided in Table 1. All experiments wereperformed in compliance with the relevant laws and institu-p＜0.05.

tional guidelines.

Results and Discussion

To our certain knowledge, this is the ﬁrst study investi-

Urine sample preparation and GC/MS analysis
For the urine samples analysis, 100 μL of urine was used.

gating ASD patients urinary and blood metabolites at theChemicals and the procedure for sample preparation havesame time. 175 kinds of urine metabolites including amino6)

been described previously . Treated samples were analyzedacids, organic acids, carbohydrates and nucleic acids, as

using gas chromatography coupled to mass spectrometry(GC/MS). GC/MS (JEOL JMS-Q1000GC) was equippedwell as 51 kinds of blood metabolites including aminowith Ultra Alloy Capillary Column (30 m×0.25 mm×0.25 μm). Metabolites peak identiﬁcation and analyses were per-focused on urine amino acids and organic acids combining
blood amino acids and acylcarnitines. Table 2 shows uri-formed using our lab s original routine metabolome datanary amino acids and organic acids, and blood amino acids

analysis software. Urine metabolites were quantiﬁed by

and acylcarnitines comparison data obtained in this study.

acids and acylcarnitines, were analyzed. This time we only

their ratio to creatinine.

Most urinary amino acids and organic acids showed nosigniﬁcant change between ASD patients and controls. Alanine, glycine, valine, leucine, pyroglutamic acid and N-ac-Dry blood spot sample preparation and LC/MS/MS
analysis

tylaspartate increased in ASD patients while histidine

Sample preparation for amino acids and acylcarnitines isdecreased. Lactate, succinate and malate, which are parts ofsummarized as below. Dried blood spot (DBS) was

the tricarboxylic acid cycle, showed lower levels in ASD
patients than controls. Pyroglutamic acid (PGA) as a gluta-

punched into 3 mm diameter and deposited in a microcell
plate. The sample was extracted by dispensing 300 μL of anmate (Glu) analog and a potential precursor and reserve of
Glu increased signiﬁcantly in ASD patients urine. Homova-extraction solution consisting of a mixture of methanol(200 μL) and an aqueous solution of 3 mmol/L hydratehy-

nillate and vanillylmandelate, which are metabolites of neu-drazine (100 μL). Internal standards (CIL free acid non-derivative NSK-B1 and NSK-A1), stable heavy isotope ana-

rotransmitters, decreased in ASD patients. Meanwhile,logsofseveralaminoacids,

freecarnitine

some other urinary organic acids, which are involved inandmany other metabolic pathways, also slightly changed

acylcarnitines were also present in the extract solution. Thesample was shaken for 25 min at room temperature, trans-

between groups. Whether these changes were meaningful

ferred to a new plate and dried under Nitrogen ﬂow. The

ASD patients data and research.

for ASD study still needs support from more diagnosed

sample was then eluted with 260 μL of acetonitrile/water

Different with urinary components, 19 of 21 blood amino(70/30; 0.1% formic acid). The extracted sample wasinjected into the Shimadzu LCMS-8040. Mass spectral dataacids were increased in ASD patients. Free carnitine, shortfor the amino acids were acquired through a neutral loss
scan of 46 Da in positive mode (CE-15 V), and for the acyl-and unsaturation ones have signiﬁcantly changed between

carnitines were acquired through a precursor ion scan ofThis study showed the metabolome analysis of 90 ASD

chain and medium chain acylcarnitines including saturation
ASD patients and control.

15

June 2017

Table 2.

Medical Mass Spectrometry Vol. 1 No. 1

Urinary amino acids and organic acids, and blood amino acids and acylcarnitines comparison by groups (̶, below detectable limit; *, signiﬁcantly changed component between groups; urine metabolites unit is ratio of metabolite quantitative ion peak area to creatinine quantitative ion m/z 329 peak area; blood amino acid and acylcarnitine unit is μM)
Component

Urinary amino acids*Alanine

Mean±SDpt statistic

1.09±1.35

0.010

2.60

ASD

Control

0.68±0.65*Glycine

1.54±1.22

2.39±3.27

0.022

2.32

Sarcosine*Valine0.051±0.058

0.045±0.047

0.42

−0.81

0.12±0.12

0.17±0.16

0.024

2.28

*Leucine

0.057±0.082

0.031±0.052

0.011

−2.55

Proline0.023±0.020

0.12±0.52

0.083

1.75

Isoleucine

0.031±0.0350.034±0.048

0.59

0.54

Serine0.64±0.71

0.64±0.97

0.98−0.023Threonine0.12±0.17

0.12±0.13

0.88

−0.15

0.0077±0017

0.0097±0.031

0.60

0.53

1.32±0.97̶

1.94±1.43
̶

8.89E-4
̶

3.39
̶

Phenylalanine*N-actylaspartate

0.070±0.088

0.054±0.073

0.19

−1.31

0.063±0.054

0.11±0.12

0.00103.34

Lysine0.0073±0.024

0.70

−0.38

Glutamine

0.0096±0.049

0.0057±0.031
̶

0.064

−1.87

Tyrosine*Histidine

0.36±0.40

0.42±0.36

0.31

1.01

0.48±1.47
̶0.14±0.52
̶

0.041
̶

−2.06
̶

Methionine*Pyroglutamic acid
Ornithine

N-actyltyrosine
Cysteine̶̶̶̶

Homocystine

̶̶

̶

̶

Homoserine*Phenylalanine/Tyrosine

0.0064±0.047

0.0067±0.030

0.96

0.046

0.20±0.12

0.16±0.12

0.029

−2.20

*Leucine/Alanine

0.098±0.13

0.052±0.086

0.0059−2.79

Urinary organic acids*Lactate0.15±0.19

0.39±0.70

0.0017

3.23

Glycolate

0.072±0.063

0.071±0.074

0.96−0.052Oxalate3-hydroxypropionate

0.0027±0.0040

0.0047±0.010

0.079

1.77

0.010±0.015

0.016±0.034

0.13

1.52

Cresol*3-hydroxyisobutyrate*2-hydroxyisovalerate

0.086±0.16

0.10±0.17

0.55

0.60

0.028±0.027
5.37E-4±0.0017

0.048±0.053

0.00173.20

0.0046±0.019

0.050

1.98

1.74±2.95

1.61±3.34

0.79

−0.27

3-aminoisobutyrate
3-hydroxyisovalerate*2-methyl-3-hydroxybutyrate

0.054±0.093

0.097±0.19

0.055

1.93

0.017±0.019

0.030±0.042

0.00942.64

2-ethyl-3-hydroxypropionate

0.0022±0.0048

0.0034±0.0060

0.15

1.45Phosphate*Succinate

6.00±15.5418.34±73.45

0.12

1.56

0.0021±0.006

0.0098±0.027

0.0093

2.65

Methylsuccinate

0.0020±0.0021

0.0030±0.0044

0.057

1.92

Fumarate*Glycerate

0.0014±0.011

0.0066±0.032

0.15

1.44

0.026±0.028

0.040±0.045

0.010

2.61

*2,3-dihydroxybutyrate

0.12±0.089

0.16±0.13

0.022

2.31

2,4-dihydroxybutyrate

0.089±0.082

0.12±0.13

0.058

1.91

16

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Medical Mass Spectrometry Vol. 1 No. 1

Table 2.
Urinary organic acids*3,4-dihydroxybutyrate

Continued

0.16±0.14

0.24±0.27

0.015

2.45Glutarate
3-methylglutarate*3-methylglutarconate

0.0028±0.0061
̶

0.0032±0.0046
̶

0.62
̶

0.50
̶

0.0012±0.0020

0.0031±0.0044

2.63E-4

3.75*Malate0.0013±0.0047

0.011±0.037

0.019

2.38

*Adipate

0.016±0.026
7.54E-4±0.0053

0.044±0.084

0.00283.06

0.10±0.62

0.12

1.58

1.10±0.82

1.66±1.45

0.00173.21

0.052±0.046
̶

0.09±0.11

0.00402.93

0.0011±0.0082

0.21

1.26

0.0073±0.013

0.0062±0.013

0.56

−0.58

4-hydroxyproline*Erythronate*2-hydroxyglutarate
Phenyllactate
3-hydroxyphenylacetate*4-hydroxyphenylacetate

0.14±0.15

0.20±0.22

0.038

2.09

0.017±0.040

0.011±0.028

0.20

−1.28

0.0031±0.0028

0.0059±0.0075

0.00133.30

Suberate*Cisaconate

0.0016±0.0031

0.0028±0.0057

0.082

1.75

0.13±0.16

0.26±0.32

9.93E-4

3.37*Glycerol-3-phosphate*Homovanillate

0.090±0.093

0.13±0.12
0.048±0.041

0.00894.50E-5

2.65

0.025±0.017*Citrate

0.036±0.068

0.20±0.54

0.0044

2.92

Methylcitrate

0.015±0.029

0.022±0.025

0.081

1.75

Hippurate*4-hydroxyphenyllactate

4.06±7.42

3.19±6.82

0.41

−0.82

0.014±0.023

0.036±0.073

0.0074

2.73

Vitamin C*Vanillylmandelate

0.44±1.32

0.93±2.55

1.62

0.11

0.15±0.092

0.25±0.19

5.96E-6

4.72

Galactonate*Gluconate

0.14±0.25

0.24±0.42

0.066

1.86

0.17±0.12
1.04E-4±7.49E-4

0.24±0.21
4.28E-4±0.0023

0.013

2.21

0.20

1.28

0.0011±0.0026

0.0060±0.018

0.015

2.48

0.17±0.29

0.15±0.35

0.69

−0.40

0.41±0.56

0.48±0.61

0.43

0.79

2-aminoadipate*3-hydroxy-3-methylglutarate

4-hydroxyphenylpyruvate*3-hydroxysebacate
3-hydroxyhippurate
4-hydroxyhippurate*Stearate

4.81

0.057±0.11

0.20±0.41

0.0023

3.13

2-ketoglutarate

2.96E-4±0.0021

0.0015±0.0077

0.14

1.48

Tartarate

0.0091±0.036

1.29

0.024±0.11

0.20

Pyruvate
2-ketoglutaraminate

0.16±0.20

0.23±0.35

0.10

1.64

0.088±0.11

0.072±0.093

0.30

−1.03

Orotate0.0080±0.027

0.0047±0.012

0.29−1.07Urate

2.01±2.81
̶

2.16±3.67̶0.76
̶

0.31
̶

1.47±1.32

2.15±2.01

0.00872.66

135.1±42.61

95.99±32.03*Arginine

11.75±5.44

4.28±3.81

7.38E-11
2.22E-20−10.66*Aspartate

46.80±15.89

36.41±15.66*Citrulline

17.38±4.79

11.61±3.16*Cysteine

0.95±0.21

0.77±0.20*Glutamine

40.03±6.15

Glutamate*Glycine

Sebacate*Lactate/Pyruvate
Blood amino acids*Alanine−6.97

1.72E-5
3.05E-17−9.54

27.88±6.85

2.82E-8
4.03E-26−12.5265.02±16.23

71.71±31.96

0.079

129.86±42.38

91.33±24.41

7.22E-12

17−4.42−5.81
1.77
−7.48

June 2017

Medical Mass Spectrometry Vol. 1 No. 1

Table 2.

Continued

Blood amino acids
Homocysteine*Histidine

9.65±4.99

8.99±4.08

0.33

−0.98

53.91±15.83

36.34±9.02−9.15*Leucine

85.96±21.58

61.86±17.65

5.70E-16
5.53E-14*Methionine

18.49±4.99

14.75±4.44−5.30*Ornithine

34.34±11.44

24.04±6.76

3.36E-7
1.37E-11*Phenylalanine

54.59±12.10

40.45±9.34

266.0±164.1

181.5±96.36

1.95E-15
4.41E-5−8.77*4-Piperidinepropionate−3.63−13.81*Proline

424.7±128.5

349.8±147.23.66E-4*Serine69.37±32.66

41.38±12.34*Tryptophan

8.74±1.65

5.45±1.54

8.72E-12
6.14E-30*Tyrosine

41.02±9.59

29.60±9.27*Threonine

39.71±12.72*Valine

−8.20
−7.35
−4.21
−7.61
−8.12

27.91±9.66

7.41E-14
5.85E-11

153.9±35.36

107.4±30.80

3.10E-17−9.40

Phenylalanine/Tyrosine

1.36±0.30

1.44±0.37

0.14

1.49

Leucine/Alanine

0.68±0.21

0.68±0.21

0.84

0.21

24.71±6.25

19.74±5.64
16.76±7.33

8.33E-8
1.21E-3−5.59

19.89±5.26−3.41

Blood acylcarnitines*Free carnitine (C0)*Acetyl- (C2)

−7.01

−3.33

*Propionyl- (C3)
*Isobutyryl-/butyryl- (C4)1.80±0.73

1.46±0.58

8.13E-40.16±0.05

0.13±0.06−4.35*Isovaleryl-/2-methylbutyryl- (C5)0.21±0.10

0.12±0.04

2.36E-5
6.34E-14*Hexanoyl- (C6)0.04±0.02

0.03±0.01

0.10±0.05

0.06±0.03

1.51E-5
1.46E-9−4.46*Octanoyl- (C8)
*Decanoyl- (C10)0.23±0.11

0.14±0.08−5.85*Dodecanoyl- (C12)0.08±0.03

0.06±0.03

2.70E-8
5.86E-3

Tetradecanoyl- (C14)
Hexadecanoyl- (C16)*Octadecanoyl- (C18)0.06±0.03

0.06±0.03

0.76

−0.30

1.11±0.38

1.01±0.41

0.07

−1.82

0.54±0.17

0.49±0.15

0.05

−2.01

*Tiglyl- (C5:1)

0.02±0.01

0.01±0.01−2.76*Octenoyl- (C8:1)

0.17±0.10

0.13±0.07

6.42E-3
3.53E-4*Decenoyl- (C10:1)

0.11±0.06

0.07±0.04

2.39E-7−5.39*Decadienoyl- (C10:2)*Dodecenoyl- (C12:1)

0.04±0.01

0.03±0.01

0.02

−2.40

0.04±0.02

0.03±0.02−3.99*Tetradecenoyl- (C14:1)

0.08±0.02

0.05±0.02

9.82E-5
1.04E-14

−3.08

−8.47
−6.41
−2.79

−3.65

−8.45

*Tetradecadienoyl- (C14:2)

0.02±0.01

0.01±0.01

2.38E-3Hexadecenoyl- (C16:1)*Octadecenoyl- (C18:1)

0.10±0.03

0.09±0.03

0.17

−1.37

0.40±0.12

0.36±0.11

0.05

−1.99

*Octadecadienoyl- (C18:2)

0.19±0.06

0.17±0.06

0.03±0.01

0.03±0.01

5.36E-3
9.03E-3−2.82*Glutaryl- (C5DC)
3-hydroxybutyryl- (C4OH)*3-hydroxyisovaleryl-/
2-methyl-3-hydroxybutyryl- (C5OH)

0.09±0.04

0.09±0.05

0.34

−0.96

0.16±0.06

0.14±0.05

0.05

−2.00

3-hydroxytetradecenoyl- (C14OH)*3-hydroxyhexadecenoyl- (C16:1OH)

0.01±0.01

0.01±0.01

0.10

−1.67

0.02±0.02

0.02±0.01−3.99*3-hydroxyhexadecanoyl- (C16OH)

0.04±0.05

0.01±0.01

1.10E-4
3.64E-73-hydroxyoctadecenoyl- (C18:1OH)*3-hydroxyoctadecanoyl- (C18OH)

0.02±0.01

0.01±0.01

0.11

−1.63

0.01±0.03

0.01±0.02

0.01

−2.56

18

−2.64

−5.48

June 2017

Medical Mass Spectrometry Vol. 1 No. 1patients and 90 age-matched controls from China. Urinaryspectrum disorders. Neuron 28: 355‒363, 2000.

amino acids and organic acids, as well as blood amino acids

2) Christensen DL, Baio J, Van Naarden BK, et al: Preva-and acylcarnitines, were compared between these twolence and characteristics of autism spectrum disordergroups. The abnormal levels of amino acids, organic acids

among children aged 8 years̶Autism and Developmen-and acylcarnitines in urine and blood of ASD patientstal Disabilities Monitoring Network, 11 sites, United

showed a similar tendency to reported physiological andmetabolic abnormalities in ASD patients. It s worth men-States, 2012. MMWR Surveill Summ 65(No. SS-3): 1‒23,

tioning that increased urinary PGA in ASD patients is3) Wong VC, Hui SL: Epidemiological study of autism spec-ﬁrstly reported this time. There are also issues need furthertrum disorder in China. J Child Neurol Jan 23(1): 67‒72,

studies like if the increased urinary PGA was meaningful or2008.

2016.

not in ASD patients and reason for different changing4) Zhang HF, Wang WQ, Li XM, Rauw G, Baker GB: Body

trends of amino acids in urine and blood.

ﬂuid levels of neuroactive amino acids in autism spectrum
disorders: A review of the literature. Amino Acids Sep 29,

Acknowledgements

2016.

We would like to thank all the ASD patients and their5) Rossignol DA, Frye RE: A review of research trends inparents for their kindly providing urine and dry blood spot

physiological abnormalities in autism spectrum disorders:samples for this study.

Immune dysregulation, inﬂammation, oxidative stress,
mitochondrial dysfunction and environmental toxicant ex-Conﬂict of Interest

posures. Mol Psychiatry 17: 389‒401, 2012.

The authors declare that they have no competing inter-6) Matsumoto I, Kuhara T: A new chemical diagnostic method for inborn errors of metabolism by mass spectrome-ests.

try-rapid, practical and simultaneous urinary metabolites

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