3. 정병화식약청 사업단_심포지움_2011

Relative concentrations of bacteria at various lo-cations within the gut.

Arq Bras Endocrinol Metab. 2009

Micro gut flora.Investigation on the endogenous metabolic change by intestinal gut microflora using mass spectrometry based metabolomics

한국과학기술연구원생체분자기능연구 센터

정병화

The “OMICS” Cascade

What can happen

What appears to be happening

What makes it happen

What has happened and is happening(the most predictive of phenotype)

Phenotype

Metabolite(molecular weight:<1000Da)

Because

1)they are the end products of cellular regulatory processes.

2) their levels can be regarded as the ul-timate response of biological systems to genetic or environmental changes.

Research Area

Gene mappingSeparation of protein

Identification of protein function

Idetification and Quantitation

of metabolite

TechniqueSystemic DNA

sequencing

2D-gelpeptide mass fingerprinting

Hyphenated tech.NMR, MASS

Class Genomics Proteomics Metabolomics

Subject Gene Protein Metabolite

mw of subject

>100,000 5,000 – 200,000 100 – 1,000

Major technologies for Metabolomics

MS and NMR:

MS NMR

Identify and quantify metab-olite after separation by GC, HPLC, CE so on

Does not rely on the separa-tion of the analytes(all kind of metabolite can be measured simultaneously)

Very sensitive and selective Has significant limitations of sensitivity ( 10 mg/com-pound on column)

Identification of metabolite according to its fragmenta-tion pattern

Wikipedia

Strategies for metabolomics investigation

Mass Spectrometry Reviews 2006

GutMicrobiome

Immune system Alteration of diet

Antibiotic use

Al-lergy Formation

of gall stone

obesity

Gut is one of the most metabolically active tissues in the body

Drug metabolism

Micro gut flora.

Objective of study

? What endogenous metabolic path-way is affected by gut microflora ?

Preparation of pseudo germ free rat

Metabolomic approach with urinary metabolites

Plasma,Urine

Plasma,Urine

Pseudogerm-free

Control

Oral administration of wa-ter, twice a day for 6 days

• neomycin sulfate • Streptomycin• Bacitracin

After 2 day withdraw of drug

Antibiotic treatment(200 mg/kg for each an-tibiotics, twice a day for 6 days)

Preparation of psudo germ free rats

　 　 Control Pseudo-germ free p-value

AST (IU/ℓ) 95.7 ± 18.1 114.4 ± 19.7 0.17

ALT (IU/ℓ) 26.6 ± 5.0 33.6 ± 10.4 0.19

ALP (IU/ℓ) 126.9 ± 25.2 153.0 ± 25.1 0.15

CPK (IU/ℓ) 77.3 ± 15.2 108.3 ± 50.0 0.20

T-BIL

(mg/㎗ )

0.1 ± 0.0 0.1 ± 0.0 0.92

GLU

(mg/㎗ )

144.9 ± 27.6 154.7 ± 38.8 0.65

CHO

(mg/㎗ )

100.5 ± 6.4 103.8 ± 8.5 0.50

TG

(mg/㎗ )

42.8 ± 11.9 42.5 ± 13.6 0.98

PRO (g/ ㎗ ) 5.6 ± 0.1 5.6 ± 0.5 0.88

ALB (g/ ㎗ ) 3.0 ± 0.1 2.9 ± 0.2 0.64

A/G 1.1 ± 0.0 1.1 ± 0.1 0.56

BUN

(mg/㎗ )

18.9 ± 4.2 19.5 ± 6.2 0.85

CRE

(mg/㎗ )

0.4 ± 0.0 0.4 ± 0.0 0.21

m/z

Retention time

MS2 pattern

Retention time

Retention time

UPLC/TOF/MS

comparison of metabolic profiling Identification of metabolites

Positive ionization

Negative ionization

MS analysis

Metabolomic Approach

Flow diagram of a typical MS-based metabolite profiling workflow.

Acceptance Criteria Suggested by the Validation Process

Validation of analysis

QC sample(repeatability)

Test mix(repeatability, accuracy)

RT stability, peak shape, detector response

RT stability, peak shape, detector response, mass accuracy

Nat. Protocol. 2010

Method Validation Workflow

Preparation of QC samples and Test mix Test mix : commercially available standardsQC samples : pooled equal aliquots of each sample

UPLC-QTOF-MS

Nine QC QC Ten analytical samples QC QC

Test mix

Conditioning

Ten analytical samples QC………

Test mix Test mix

Run order sequence

XIC for selected ions from test mix and QC samples; RT, peak shape, intensity and mass accuracy

No trend with time of injection observed

Process data with MarkerLynx; Peak finding, alignment, first nine QC runs removed

Tight QC clustering prerequisite

Examine peak list table for QCsCalculate CV% of ion intensity

Check number of ions with CV<15%, <20%, <30%A percentage of ~70% of features with CV<30% denotes an acceptable data set

Summary of validation

positive ionization mode negative ionization mode

control (●)

pseudo-germ free group (▲) QC samples (■)

PCA plots

Repeatability of analysis

XIC from QC samples 　 RT a 　 Ion intensity b

RT m/z Average SD CV% Average SD CV%

Positive mode 　 　 　1.45 166.08 1.47 0.00 0.00 76.00 1.61 2.12

2.12 271.15 2.14 0.01 0.54 21.66 0.59 2.74

3.87 568.27 3.91 0.00 0.00 2.21 0.29 12.89

4.12 384.12 4.13 0.00 0.00 47.80 2.34 4.89

5.71 221.12 5.73 0.01 0.20 157.25 1.97 1.26

7.13 410.18 7.15 0.00 0.00 152.29 4.99 3.28

12.11 300.30 12.13 0.00 0.00 0.86 0.07 8.24

Negative mode

1.58 231.10 1.57 0.00 0.00 19.28 0.74 3.84

3.00 215.10 3.01 0.00 0.00 28.62 0.23 0.81

4.73 188.04 4.75 0.01 0.12 19.23 1.95 10.14

5.46 429.08 5.46 0.00 0.00 36.25 1.67 4.60

8.75 141.13 8.75 0.00 0.00 7.20 0.63 8.72

3.76 388.14 3.77 0.01 0.31 3.75 0.04 1.14

10.88 221.15 10.89 0.00 0.00 1.83 0.09 4.94

Repeatability of retention time and ion intensity in QC samples

QCs considered %CV < 15% %CV < 20% %CV < 30%

Positive mode 56.7 65.2 73.3

Negative mode 63.3 72.7 80.3%CV < 30%: 70 % 이상

Standard chemical

in test mixture

　 RT 　 Ion intensity

Average SD CV% Average SD CV%

Positive mode 　 　 　Acetaminophen 4.25 0.00 0.00 5959.00 732.50 12.29

Caffeine 5.00 0.01 0.23 28916.00 3289.07 11.37

Reserpnie 7.98 0.00 0.00 2835.00 367.02 12.95

Negative mode

Adipic acid 4.34 0.00 0.00 636.67 27.01 4.24

Hippuric acid 5.11 0.00 0.00 8511.00 305.18 3.59

Glycocholic acid 8.25 0.00 0.00 19612.00 985.29 5.02

Repeatability of retention time and ion intensity in test mixture.

Standard chemical in test mixture

exact mass(Da)

1st run 2nd run 3rd run

m/zMass difference

(mDa)m/z

Mass difference(mDa)

m/zMass difference

(mDa)

Positive mode 　 　 　Acetaminophen 151.0633 152.0699 1.4 152.0692 -2 152.0727 1.5

Caffeine 194.0803 195.0876 -0.6 195.0876 -0.6 195.0891 0.9

Reserpnie 608.2733 609.2799 -1.3 609.2807 -0.5 609.2823 1.1

Negative mode

Adipic acid 146.0579 145.0506 0.5 145.0509 0.8 145.0509 0.8

Hippuric acid 179.0582 178.0508 0.4 178.0504 0.7 178.0508 0.4

Glycocholic acid 465.3091 464.3014 0.2 464.3014 0.2 464.3 -1.2

Repeatability of mass accuracy in test mixture.

15-30 %

50 mDa

Typical chromatograms

control

Pseudo germ free

control

Pseudo germ free

Positive ionization mode.

Negative ionization mode.

(●) control group and(▲) pseudo-germ free group

OPLS-DA score plot

positive ionization mode negative ionization mode

Identification of metabolites

Identified metabolites that showed significant differences in urinary excretion between the control and pseudo-germ free rats

3.19_205.07 [M+H] C9H8N (-C2H5NO2) Tryptophane 1.40 0.0033

(C11H12N2O2) C11H13N2O (-O)

4.95_162.06 [M+H] C8H6N (-CH2O2) Indole-3-carboxylic acid 0.23 0.0001

(C9H7NO2) C9H6NO (-H2O)

C9H8NO (-O)

5.25_164.08 [M+H] C8H10O (-CNO) 3-Methyldixoyindole 0.00 b 0.0002

(C9H9NO2) C9H8NO (-H2O)

C6H7 (-C3H3NO2)

4.46_162.06 [M+H] C10H10N (-H2O) Tryptophanol 0.12 0.0051

(C10H11NO) C9H8N (-CH4O)

5.48_212.00 [M-H] C8H6NO (-O3S) Indoxyl sulfate 0.62 0.0434

(C8H7NO4S) O3S (-C8H6NO)

5.46_194.09 [M+H] C7H7 (-C3H5NO3) Phenylacetylglycine 0.27 0.0003

(C10H11NO3) C9H10NO (-CH2O2)

C9H12NO (-CO2)

5.65_283.08 [M-H] C6H10O5 (-C7H5O2) p-Cresol glucuronide 0.07 0.0006

(C13H16O7) C7H7O (-C6H8O6)

C5H8O4 (-C8H7O3)

5.10_180.07 [M+H] C7H5O (-C2H5NO2) Hippuric acid 0.28 0.0025

(C9H9NO3) C6H5 (-C3H5NO3)

RT_m/z (Formula)

Fragmentation Identified metabolite Fold change a p-value

1) Aromatic amino acid metabolism

Protein

Peptides

Tyrosine Phenylalanine Tryptophan

Cinnamic acidPhenylacetylglycine

P-cresol

Intestine

BodyP-cresol

P-cresol glucuronide

Benzoic acid

Hippuric acid

Indole sulfate 3-Methyldioxyindole

Tryptophanol

Indole pyruvate Tryptamine

Excreted in urine

Metabolism by gut microbiota

Aromatic amino acid metabolism

IsoflavonoidsRT_m/z (Formula)

FragmentationIdentified metabo-

liteFold change

ap-value

6.01_445.078 [M-H] C15H9O5 (-C7H12O5) Glycitin 5.87 0.0000 (C22H22O10) C5H5O3 (-C17H16O7)

5.54_285.08 [M+H] C15H10O5 (-CH3) Glycitein 5.92 0.0001 (C16H12O5) C6H6O3 (-C10H7O2)

3.65_433.17 [M+H] C6H6O2 (-C15H15O8) Genistin 4.24 0.0006

(C21H20O10) C8H9O2 (-C13H12O8)

C5H5O2 (-C16H16O8)

5.47_255.07 [M+H] C8H7O (-C7H4O3) Daidzein 3.15 0.0003 (C15H10O4) C7H7O2 (-C8H4O2) 2.96 0.0001

6.29_243.11 [M+H] C9H9O (-C6H6O2) Equol 0.01 0.0006 (C15H14O3) C7H7O2 (-C8H8O) 0.00 b 0.0003

OthersRT_m/z (Formula)

FragmentationIdentified metabo-

liteFold change

ap-value

5.21_447.10 [M+H] C17H17O2 (-C7H14O6) Estrone glucuronide (C24H30O8) C6H10O3 (-C18H21O5) 2.08 0.0048

C5H8O2 (-C19H23O6)

1.17_314.13 [M+H] C8H9O (-C6H11NO6

Tyramine glu-curonide

10.25 0.0023

(C14H19NO7) C5H4O2 (-C9H16NO5)

5.23_340.11 [M+H] C14H13NO3 (-CH5O5) 6-Hydroxy-5-methoxyindole glu-

curonide

0.01 0.0002

(C15H17NO8) C9H10NO2 (-C6H8O6)

　 C5H5O2 (-C10H13NO6) 　 　 　

2) Isoflavonoids and others

LXR (Liver X receptor), FXR (farnesoid X receptor) PXR (Pregnane X receptor)

The receptors closely related with liver toxicity

The ligand metabolite for LXR, FXR, PXR: cholesterol oxysterols and bile acids

Oxysterol and bile acids analysis by GC-MS/MS

Bile Acid Analysis

Metabolic pathway & related enzyme

Steroid

Cholesterol 7α-hydroxylase

Cholesterol 24-hydroxylase

Cholesterol 25-hydroxylase

Sterol 12α-hydroxylase

∆4-3-oxosteroid 5β-reductase

6β-hydroxylase7α-dehydroxylase

7α-dehydroxylase

7β-dehydroxylase

7α-hydroxysteroid dehydrogenase,

7β-hydroxysteroid dehydrogenase

CYP39A1, oxysterol 7α-hydroxylase

CYP7B1, oxysterol 7α-hydroxylase

3α-hydroxysteroid dehydrogenase3α-hydroxysteroid dehydrogenase

∆4-3-oxosteroid 5β-reductase

HOH

H

H

Cholesterol

OH

HOH

H

H

22 (R)- hydroxy cholesterolHO OH

H

H

H

7keto cholesterolHO OH

H

H

H

7 hydroxy cholesterol

OH

HOH

H

H

24(S)- hydroxy cholesterolHO

HO

H

H

H

25-hydroxy cholesterol

O OHH

H

H

7 hydroxy-4-cholesten-3-one

HO H OH

OHO

OH

H

H

H

Cholic acid

O

OH

HO

OH

H

H

H

H

Deoxycholic acid

O

OH

HOOH

OHH

H

H

H

-Muricholic acid

O

OH

HOOH

OHH

H

H

H

-Muricholic acidHO

O

OH

H

H

H

Lithocholic acid

HO OH

O

OH

H

H

H

Ursodeoxycholic acid

HO OH

O

OH

H

H

H

Chenodeoxycholic acid

3β-hydroxy-∆5-C27-steroid oxidoreductase

HO OH

OH

H

H

H

H

5-cholestane-3,7,12-triol

LXR ligand: Oxysterols

Cholesterol

PXR, FXR ligand: Bile acids

GC Parameter

Column: Ultra-I (25m x 0.2mm x 0.33μm)

Injection volume: 2.0 μL

Inlet mode: Split

Split Ratio 10:1

Inlet Temp: 280ºC

Mode: Constant FlowFlow Rate: 0.9 mL/min, He

MS Parameter

MS Source: 230ºC

MS Quad. 150ºC

Auxiliary temperature: 300°C

Solvent delay: 5 min

Acq. Mode: SIM

Analytical Instruments

6890 Series Gas Chromatography

5975 Series Mass Selective Detector

240ºC

290ºC

320ºC

20ºC/min

2 min

1ºC/min

2 minOven Temp. Profile

Instrumental Conditions

Sample preparation method

Urine 1ml

Add 10 μl of internal standard mixture(10 μg/ ml of 5α-cholestane and d4-cholic acid)

Add 1.5 ml of sodium acetate butter (0.2 M, pH 5.2)Add 50 μl of β-glucuronidase/aryl sulfataseStand at 550C at 3 hrs

Add 5 ml of t-butylmethyletherShaking for 10 minsCentrifuge 2500 rpm for 5 mins

EvaporateDry at P2O5/KOH

Add 40 μl of MSTFA/NH4I/dithioerythritol mixtureStand at 600C at 30 mins

Enzyme hyrolysis

Liquid-liquid extraction

Organic layer

ResidueDerivatization

GC-MSD

GC-MS chromatogram

Anal Biochem Submitted

Compounds Before treat-ment

After treat-ment

Mean± SD Mean± SD

7α-OH-cholesterol 53.56±22.18 63.40±36.47

7α-OH-4-cholesten-3one 120.5±67.83 166.7±140.4

7keto- cholesterol 13.76±2.94 18.70±9.73

22(R)-OH- cholesterol 7386±2360 5661±1606

24S-OH- cholesterol ND ND

25-OH- cholesterol 106.7±34.92 70.62±33.82*

Cholesterol 4008±1592 2866±1271*

Deoxycholic acid (DCA) 46.27±18.30 29.84±14.35*

α-Muricholic acid (α-MCA) 118.1±30.75 139.6±63.87

Chenodeoxycholic acid (CDCA)

94.96±20.28 158.9±111.3*

Cholic acid (CA) 55.10±17.04 78.58±53.64

Ursodeoxycholic acid (UDCA)

67.37±12.00 75.01±45.29

Lithocholic acid (LCA) 81.06±38.24 56.43±24.92

β-Muricholic acid (α-MCA) 53.91±13.58 74.43±43.61

5β-cholestane 3α,7α,12α-triol

47.58±10.16 77.73±51.49*

Urinary concentration (ng/mg of creatinine) of oxysterols and bile acids in before and after treatment of neomycin and streptomycin treated rats.

*: ≤ 0.05 between before treatment and after treatment; ND: not detected

HOH

H

H

Cholesterol

OH

HOH

H

H

22 (R)- hydroxy cholesterolHO OH

H

H

H

7keto cholesterol

HO OHH

H

H

7 hydroxy cholesterol

OH

HOH

H

H

24(S)- hydroxy cholesterolHO

HO

H

H

H

25-hydroxy cholesterol

O OHH

H

H

7 hydroxy-4-cholesten-3-one

HO H OH

OHO

OH

H

H

H

Cholic acid

O

OH

HO

OH

H

H

H

H

Deoxycholic acid

O

OH

HOOH

OHH

H

H

H

-Muricholic acid

O

OH

HOOH

OHH

H

H

H

-Muricholic acidHO

O

OH

H

H

H

Lithocholic acid

HO OH

O

OH

H

H

H

Ursodeoxycholic acid

HO OH

O

OH

H

H

H

Chenodeoxycholic acid

Cholesterol 7α-hydroxylase

3β-hydroxy-∆5-C27-steroid oxidoreductase

CYP39A1, oxysterol 7α-hy-droxylase

CYP7B1, oxysterol 7α-hy-droxylase

3α-hydroxysteroid dehydrogenase

∆4-3-oxosteroid 5β-reductase

Steroid

CYP8B1,sterol 12α-hydroxylase

∆4-3-oxosteroid 5β-reductase

3α-hydroxysteroid dehydrogenase

7α-dehydroxylase 6β-hydroxylase

24-hydroxylase25-hydroxylase

Cytochrome p450scc

7α-dehydroxylase

7α-hydroxysteroid dehy-drogenase,

7β-hydroxysteroid dehy-drogenase

HO OH

OH

H

H

H

H

5-cholestane-3,7,12-triol

7β-dehydroxylase

Cholesterol 7-hydroxylase

Treated groups

Normal control Pseudo germ free

7-O

H-c

hol

este

rol/c

hol

este

rol

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.01425-hydroxylase

Cholesterol

Normal control Pseudo germ free

25-O

H-c

hol

este

rol/c

hol

este

rol

0.000

0.005

0.010

0.015

0.020

0.025

3-OH-5-C27-steroid oxidoreductase

Treated groups

Normal control Pseudo germ free

7-O

H--

4-ch

otes

ten

-3-o

ne/

7-O

H-c

hol

este

rol

0

1

2

3

4

Treated groups

Normal control Pseudo germ free

5-c

hol

esta

ne-

3,7

,1

2-t

riol

/7

-OH

-4-c

hol

este

n-3

-on

e

0.0

0.2

0.4

0.6

0.8

1.0

Treated groups

Normal control Pseudo germ free

Col

ic a

cid

/5 -

chol

esta

ne-

3,7

,1

2-t

riol

0

100

200

300

400

500

4-3-oxosteroid 5-reductase3-hydroxysteroid dehydrogease

Treated groups

Normal control Pseudo germ freeCh

enod

eoxy

chol

ic a

cid

/7

-OH

-4-c

hol

este

n-3

-on

e

0

1

2

3

4

7-hydroxysteroid dehydrogenase

Treated groups

Normal control Pseudo germ free

Urs

odeo

xych

olic

aci

d/c

hen

odeo

xych

olic

aci

d

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Treated groups

Normal control Pseudo germ free

7ket

o-ch

oles

tero

l/ch

oles

tero

l

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.006

Cytochrome p450sce

Treated groups

Normal control Pseudo germ free

22(R

)-O

H-c

hol

este

rol/c

hol

este

rol

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0.008

CYP7B1, oxysterol 7-hydroxylase

Treated groups

Normal control Pseudo germ free

7-O

H-4

-ch

oles

ten

-3-o

ne/

25-O

H-c

hol

este

rol

0.0

0.5

1.0

1.5

2.0

2.5

0.05

7-dehydroxylase

Treated groups

Normal control Pseudo germ free

Deo

xych

olic

aci

d/c

hol

ic a

cid

0.0

0.1

0.2

0.3

0.4

0.5

0.0136-hydroxylase

Treated groups

Normal control Pseudo germ free

-m

uri

chol

ic a

cid

/ch

enod

eoxy

chol

ic a

cid

0

5

10

15

20

25

30

35

0.0116-hydroxylase

Treated groups

Normal control Pseudo germ free

-m

uri

chol

ic a

cid

/ch

enod

eoxy

chol

ic a

cid

0

20

40

60

80

100

0.032

7-dehydroxylase

Treated groups

Normal control Pseudo germ free

Lit

hoc

hol

ic a

cid

/ch

enod

eoxy

chol

ic a

cid

0

10

20

30

40

50

0.024

Treated groups

Normal control Pseudo germ free

Ch

olic

aci

d/c

hen

odeo

xych

olic

aci

d

0

20

40

60

80

100

120

140

160 **

Figure 4.

1) In the non targeted metabolic profiling, 20 metabolites were signi-ficantly related to the activities of gut microbiota.

2) They are in Aromatic amino acid, Isoflavonoid metabolism & phase II metabolism (glucuronide conjugation).

3) In the target approach on the bile acid and oxysterol, . Increase of hydroxylase and significant decrease of 7α-dehydroxylase were ob-served. The urinary concentration ratio of primary bile acids (cholic acid and chenodeoxycholic acid), marker for hepatotoxicity, in-creased in pseudo germ-free condition.

4) Those findings indicated that the gut microbiota could play a signifi-

cant role in the bile acid homeostasis and liver toxicity could be hap-pen in the absent of gut microbiota.

5) Therefore this study provided clear clue that the gut microbiota play important role in normal life directly and indirectly.

CONCLUSION

Acknowledgement

Dr. Oh Seong KwonDr. Bong Chul ChungDr. Soo Hyun LeeJi Hye AhnSalil Bhowmik Kumar

Supported by

KFDAKISTMEST