Quantitative Metabolome Profiling Reveals Novel Potential Biomarkers

in Human Herpesvirus 6 EncephalopathyYoshihiko Kawano1, MD, Yuka Torii1, MD, Hajime Sato2, Tamaki Fujimori2, PhD, Kazunori Sasaki2,

Jun-ichi Kawada1, MD, PhD, Yoshiaki Ohashi2, PhD, and Yoshinori Ito1, MD, PhD1Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya; and 2Human Metabolome Technologies, Inc., Tsuruoka, Japan

Contact Information

Address: 65 Tsurumai-cho,

Showa-ku, Nagoya

466-8550,

Japan

Tel: +81-52-741-2111

Fax: +81-52-744-2974

e-mail: y-kawano@med.nagoya-

u.ac.jp

Hoshino et al. Brain Dev. 2012

Pathogens of Acute Encephalopathy

during 2007-2010 in Japan Primary infection with human herpesvirus (HHV)-6

causes roseola (exanthema subitum), occasionally

with accompanying neurological complications such

as febrile seizures and acute encephalopathy, for

which the pathology remains unclear

Metabolomics is a remarkable method that searches

for disease-specific biomarkers by comprehensive

analysis of the kinetics of metabolites, resulting in an

assessment of disease pathophysiology

Metabolome profiling was used to identify novel

biomarkers in HHV-6 encephalopathy

Introduction What are Metabolites…? CE-MS-based Metabolomics

Results

Genome Transcriptome Proteome Metabolome

Human: 22,000 <1,000,000<100,0001122 (serum)

458 (urine), 309 (CSF)

The Human Metabolome Database

GENOMICS

Gene-omics

TRANSCRIPTOMICS

Transcript-omics

PROTEOMICS

Protein-omicsMETABOLOMICS

Metabolite-omics

mRNA Protein PhenotypeDNA

The technologies of genomics, transcriptomics, and

proteomics have been widely used in many studies.

The target of metabolomics is the metabolome, consisting

of metabolites, which are low-molecular weight

intermediates in metabolism.

Migration time (min)

Mass

/ c

harg

e (

m/z)

Target

compounds

MS range: m/z 50-1,000

Run time: 30 min

Capillary Electrophoresis-Time of Flight MS

(CE-TOFMS)

Features of Capillary Electrophoresis

Target Compounds of MS-based Metabolomics

HHV-6

Encephalopathy*

(n=9)

Febrile

Seizure†

(n=20)

Control#

(n=7)

Sex (Male/Female) 3/6 12/8 2/5

Age, mean years SD

(median)

1.1 0.4

(1.0)

2.0 1.4

(1.5)

2.6 2.0

(2.3)

Sample preparation

CE-TOFMS analysis

Metabolite peak information based on m/z and migration time

Hierarchical Cluster Analysis (HCA), Principal Component Analysis (PCA)

Metabolome-wide pathway

Extraction

Inactivation of enzymes

50 μL serum from a patient

HHV-6 infection was defined by either the detection of HHV-6 DNA from the CSF or serum in the early stage of

the disease. Encephalopathy was defined as showing altered consciousness or loss of consciousness that

persisted for more than 24 hours.

Febrile seizure included HHV-6 infection and non-HHV-6 infection.

Control was non-HHV-6 infection without febrile seizure.

*

†

#

Introduction, Materials & Methods

Metabolic Profile in a K yn u renine Pathway

159 metabolites were identified in the serum

samples, and of those, 56 metabolites were

absolutely quantified.

Results of Metabolite Measurements

Rel. Area Mann-Whitney U test Kruskal-Wallis test

Compound name

HHV-6

EncephalopathyFebrile Seizure Control

HHV-6

Encephalopathy

vs.

Febrile Seizure

HHV-6

Encephalopathy

vs.

Control

Febrile Seizure

vs.

Control

Mean S.D. Mean S.D. Mean S.D. Ratio ¶ p-value Ratio ¶ p-value Ratio ¶ p-value p-value

HHV-6 Encephalopathy > Febrile Seizure

Kynurenine 1.8 x 10-3 5.7 x 10-4 1.1 x 10-3 3.0 x 10-4 9.8 x 10-4 4.1 x 10-4 1.6 0.001 1.8 0.013 1.2 0.304 0.003

Quinolinic acid 1.9 x 10-4 8.9 x 10-5 1.0 x 10-4 3.8 x 10-5 8.0 x 10-5 3.7 x 10-5 1.9 0.002 2.4 0.003 1.3 0.255 0.002

3-Aminobutyric acid 1.7 x 10-3 1.4 x 10-3 7.4 x 10-4 4.0 x 10-4 1.2 x 10-3 8.6 x 10-4 2.3 0.001 1.4 0.580 0.6 0.255 0.013

Pipecolic acid 5.9 x 10-4 2.6 x 10-4 3.3 x 10-4 1.1 x 10-4 4.4 x 10-4 2.1 x 10-4 1.8 0.006 1.3 0.338 0.7 0.232 0.024

HHV-6 Encephalopathy < Febrile Seizure

Tryptophan 1.3 x 10-2 5.5 x 10-3 1.9 x 10-2 3.7 x 10-3 1.5 x 10-2 4.9 x 10-3 0.7 0.008 0.9 0.392 1.3 0.069 0.016

cis-Aconitic acid 7.4 x 10-4 1.1 x 10-4 1.0 x 10-3 2.7 x 10-4 8.0 x 10-4 2.6 x 10-4 0.7 0.000 0.9 0.580 1.3 0.126 0.004

Isocitric acid 5.8 x 10-4 8.4 x 10-5 7.2 x 10-4 1.5 x 10-4 6.4 x 10-4 3.0 x 10-4 0.8 0.009 0.9 0.392 1.1 0.112 0.030

Butyric acid 1.3 x 10-3 3.5 x 10-4 1.9 x 10-3 4.3 x 10-4 1.5 x 10-3 5.1 x 10-4 0.7 0.001 0.8 0.244 1.2 0.100 0.005

Hexanoic acid 1.0 x 10-3 2.8 x 10-4 1.3 x 10-3 2.2 x 10-4 1.3 x 10-3 1.6 x 10-4 0.8 0.011 0.8 0.033 1.0 0.707 0.026

Aspartic acid 4.7 x 10-3 1.2 x 10-3 6.9 x 10-3 2.3 x 10-3 6.6 x 10-3 2.3 x 10-3 0.7 0.006 0.7 0.044 1.0 0.788 0.020

Taurine 1.9 x 10-3 6.0 x 10-4 2.9 x 10-3 8.9 x 10-4 2.9 x 10-3 1.4 x 10-3 0.7 0.003 0.7 0.338 1.0 0.518 0.025

m-Toluic acid

o-Toluic acid4.9 x 10-3 1.5 x 10-3 6.2 x 10-3 1.2 x 10-3 7.0 x 10-3 1.5 x 10-3 0.8 0.013 0.7 0.033 0.9 0.255 0.019

Glu-Gly

Gly-Glu2.5 x 10-4 8.8 x 10-5 4.6 x 10-4 3.0 x 10-4 5.3 x 10-4 4.3 x 10-4 0.5 0.002 0.5 0.226 0.9 0.933 0.023

¶ The latter was calculated as denominator

Metabolites Up-regulated/Down-regulated, Comparing HHV-6 Encephalopathy and Febrile Seizure

HHV-6 encephalopathy > Febrile seizure

4 metabolites

Kynurenine, Quinolinic acid,

3-Aminobutyric acid, Pipecolic acid

HHV-6 encephalopathy < Febrile seizure

9 metabolites

Trytophan, cis-Aconitic acid, Isocitric acid,

Butyric acid, Hexanic acid, Aspartic Acid, Taurine,

m-Toluic acid/o-Toluic acid, Glu-Gly/Gly-Glu

0

2

3

4

5

6

7

Co

nc

en

tra

tio

n (

μM

)

1

HHV-6

Encephalopathy

Febrile

SeizureControl

P=0.001

P=0.013

Kynurenine Quinolinic acid

0

0.5

1

1.5

2

2.5

3

4.5

Co

nc

en

tra

tio

n (

μM

)

4

3.5

HHV-6

Encephalopathy

Febrile

SeizureControl

P=0.003

P=0.002

0

0.005

0.01

0.015

0.02

0.025

0.03

Tryptophan

Re

l. A

rea

HHV-6

Encephalopathy

Febrile

SeizureControl

P=0.008

Kynurenine showed

higher levels in the HHV-

6 encephalopathy group

than in both the febrile

seizure group and the

control group.

Quinolinic acid showed

higher levels in the HHV-

6 encephalopathy group

than in both the febrile

seizure group and the

control group.

Tryptophan showed lower

levels in the HHV-6

encephalopathy group

than in the febrile seizure

group.

Discussion

HHV-6 Infection and Kynurenine PathwayTRYPTOPHAN

Kynurenine

3-hydroxykynurenine

3-hydroxyanthranilic acid

Quinolinic acid

Nicotinamide adenosine dinucleotide

(NAD+)

Indoleamine or Tryptophan

2,3-dioxygenase

(IDO,TDO)

neurotoxicityNMDA

receptor

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Serotonin

Rel. A

rea

x 10-4

HHV-6Encephalopathy

FebrileSeizure

Control

0

0.5

1

1.5

2

2.5

3

3.5x 10-4

Rel. A

rea

Indole-3-acetic acid

HHV-6Encephalopathy

FebrileSeizure

Control

0

0.5

1

1.5

2

2.5

3

3.5

Quinolinic acid

Rel. A

rea

x 10-4

HHV-6Encephalopathy

FebrileSeizure

Control

P=0.003

P=0.002

0

0.5

1

1.5

2

2.5

3

Kynurenine

Rel. A

rea

x 10-3

HHV-6

Encephalopathy

Febrile

SeizureControl

P=0.001

P=0.013

0

0.005

0.01

0.015

0.02

0.025

0.03

Tryptophan

Rel. A

rea

HHV-6

Encephalopathy

Febrile

SeizureControl

P=0.008

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D. N.D. N.D.

N.D.

N.D.

N.D.N.D.

N.D.N.D.

Heyes CNS bacterial infection CSF Quinolinic acid

1995 Intraventricular hemorrhage

J Neurol Sci. Brain tumor

Valle HIV-associated neurocognitive disorder CSF Quinolinic acid

2004

Brain.

Valle Amyotrophic lateral sclerosis (ALS) Serum Quinolinic acid

2004 CSF Kynurenine

Brain.

Torii Influenza-associated encephalopathy Serum Quinolinic acid

IDWeek 2013 Kynurenine

Presentation Number 1200

The Kynurenine Pathwayin Neurological Disorders

Conclusion

Metabolite profiles of serum from patients with

HHV-6 encephalopathy were investigated

using CE-TOFMS

Metabolome profiling revealed 13 metabolites

including kynurenine and quinolinic acid as

potential biomarkers for HHV-6

encephalopathy

The tryptophan-kynurenine metabolic process

could be associated with the pathophysiology

of HHV-6 encephalopathy

Principal Component Analysis

Control

HHV-6 Encephalopathy

Febrile Seizure

-6 -4 -2 0 2 4 6 8 10-8

-6

-4

-2

0

2

4

6

8

10

12

PC1 (12.2%)

PC

2 (

9.7

%)

Principal component analysis

was performed. The score plots

for each group showed partial

discriminative separation of the

subjects, with groups showing

some clustering although they

were not clearly divided.

Hierarchical Cluster Analysis

HHV-6

Encephalopathy

Febrile

Seizure

Control

The profiles of the levels of metabolites appeared to be

different among the groups.

0 1 2 3 4 5 6 7

0

1

2

3

4

5

Kynurenine [μM]

Qu

ino

lin

ic a

cid

[μ

M]

§R=0.793 (P<0.01)

Control

HHV-6 Encephalopathy

Febrile Seizure

Correlation between Kynurenine and Quinolinic acid

§Pearson’s correlation coefficient

analysis

Kynurenine and quinolinic acid

were significantly correlated.

Encephalopathy

HHV-6 infection Cytokine

Metabolic Profiles

in Kynurenine

Pathway