HMCS Max Bernays Pre-Deployment Brief (May 2024).pptx
3. 정병화식약청 사업단_심포지움_2011
1. Investigation on the endogenous metabolic
change by intestinal gut microflora using
mass spectrometry based metabolomics
한국과학기술연구원
생체분자기능연구 센터
정병화
Relative concentrations of bacteria at various locations
within the gut.
Arq Bras Endocrinol Metab. 2009
2. 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
3. Metabolite
(molecular weight:<1000Da)
Because
1) they are the end products of cellular
regulatory processes.
2) their levels can be regarded as the ultimate
response of biological systems to genetic or
environmental changes.
4. Class Genomics Proteomics Metabolomics
Subject Gene Protein Metabolite
mw of
>100,000 5,000 – 200,000 100 – 1,000
subject
Systemic DNA 2D-gel Hyphenated tech.
Technique peptide mass
sequencing fingerprinting NMR, MASS
Separation of protein Idetification and
Research
Gene mapping Identification of Quantitation
Area
protein function of metabolite
5. Major technologies for Metabolomics
MS and NMR:
MS NMR
Identify and quantify metabolite Does not rely on the separation
after separation by GC, HPLC, CE of the analytes
so on (all kind of metabolite can be
measured simultaneously)
Very sensitive and selective Has significant limitations of
sensitivity ( 10 mg/compound on
column)
Identification of metabolite
according to its fragmentation
pattern
Wikipedia
7. Gut is one of the most metabolically active
tissues in the body
Alteration of diet
Immune system
Drug metabolism Gut
Microbiome
Antibiotic use
Allergy obesity
Formation
of gall stone
8. ? What endogenous metabolic pathway is
affected by gut microflora ?
Preparation of pseudo germ free rat
Metabolomic approach with urinary metabolites
9. Oral administration of
water, twice a day for 6
days Control Pseudo-germ free p-value
AST (IU/ℓ) 95.7 ± 18.1 114.4 ± 19.7 0.17
Control 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
(mg/㎗) 0.1 ± 0.0 0.1 ± 0.0 0.92
Plasma, T-BIL
Urine GLU
(mg/㎗) 144.9 ± 27.6 154.7 ± 38.8 0.65
(mg/㎗) 100.5 ± 6.4 103.8 ± 8.5 0.50
CHO
Pseudogerm-free
(mg/㎗) 42.8 ± 11.9 42.5 ± 13.6 0.98
TG
After 2 day PRO (g/㎗) 5.6 ± 0.1 5.6 ± 0.5 0.88
withdraw of drug ALB (g/㎗) 3.0 ± 0.1 2.9 ± 0.2 0.64
A/G 1.1 ± 0.0 1.1 ± 0.1 0.56
Antibiotic treatment
(200 mg/kg for each (mg/㎗) 18.9 ± 4.2 19.5 ± 6.2 0.85
BUN
antibiotics, twice a day
for 6 days) (mg/㎗) 0.4 ± 0.0 0.4 ± 0.0 0.21
CRE
• neomycin sulfate
• Streptomycin
• Bacitracin
10. Retention time
Positive ionization
UPLC/TOF/MS
Retention time Negative ionization
MS2 pattern
m/z
comparison of metabolic profiling
Identification of metabolites
13. Validation of analysis
QC sample Test mix
(repeatability) (repeatability, accuracy)
RT stability, peak shape, RT stability, peak shape,
detector response detector response, mass accuracy
Nat. Protocol. 2010
14. Method Validation Workflow
Preparation of QC samples and Test mix
Test mix : commercially available standards
QC samples : pooled equal aliquots of each sample
UPLC-QTOF-MS
Test mix Test mix Test mix
Nine QC QC Ten analytical samples QC Ten analytical samples QC ……… QC
Conditioning
Run order sequence
15. Summary of validation
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 QCs
Calculate 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 d
ata set
25. Oxysterol and bile acids analysis by GC-MS/MS
The receptors closely related with liver toxicity
LXR (Liver X receptor),
FXR (farnesoid X receptor)
PXR (Pregnane X receptor)
The ligand metabolite for LXR, FXR, PXR: cholesterol oxysterols and bile acids
26. Metabolic pathway & related enzyme
H Cholesterol
H H
HO
Cholesterol
Cholesterol 7α-hydroxylase Cholesterol 25-hydroxylase
Cholesterol 24-hydroxylase
OH HO
OH
LXR ligand:
H H H H H Oxysterols
H H H H H H H H H H
HO HO OH HO OH HO HO
22 (R)- hydroxy cholesterol 7keto cholesterol 7hydroxy cholesterol 24(S)- hydroxy cholesterol 25-hydroxy cholesterol
CYP7B1, oxysterol 7α-hydroxylase
3β-hydroxy-∆5-C27-steroid oxidoreductase
H
Steroid H H
CYP39A1, oxysterol 7α-hydroxylase
O OH
7hydroxy-4-cholesten-3-one
Sterol 12α-hydroxylase
∆4-3-oxosteroid 5β-reductase
∆4-3-oxosteroid 5β-reductase
3α-hydroxysteroid dehydrogenase
3α-hydroxysteroid dehydrogenase
OH
O
PXR, FXR ligand: OH
OH Bile acids O
H OH
H
H H H H
H
HO OH HO OH
H
H H Chenodeoxycholic acid
7α-hydroxysteroid dehydrogenase,
Cholic acid HO OH
H 7β-hydroxysteroid dehydrogenase
7α-dehydroxylase 5-cholestane-3,7,12-triol 6β-hydroxylase
7α-dehydroxylase
O O O
OH OH OH
OH OH
OH O O
H H H
7β-dehydroxylase
H H
H H H H H H
HO OH H H H H
HO H HO OH
H OH H HO HO OH
OH
Deoxycholic acid -Muricholic acid -Muricholic acid Lithocholic acid Ursodeoxycholic acid
27. Instrumental Conditions
Analytical Instruments Oven Temp. Profile 320ºC 2 min
6890 Series Gas Chromatography
5975 Series Mass Selective Detector
1ºC/min
GC Parameter
290ºC 2 min
Column: Ultra-I (25m x 0.2mm x 0.33μm)
Injection volume: 2.0 μL
20ºC/min
Inlet mode: Split
Split Ratio 10:1 240ºC
Inlet Temp: 280ºC
Mode: Constant Flow
Flow 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
28. Sample preparation method
Urine 1ml
Add 10 μl of internal standard mixture
(10 μg/ ml of 5α-cholestane and d4-cholic acid)
Enzyme hyrolysis
Add 1.5 ml of sodium acetate butter (0.2 M, pH 5.2)
Add 50 μl of β-glucuronidase/aryl sulfatase
Stand at 550C at 3 hrs
Liquid-liquid extraction
Add 5 ml of t-butylmethylether
Shaking for 10 mins
Centrifuge 2500 rpm for 5 mins
Organic layer
Evaporate
Dry at P2O5/KOH
Residue
Derivatization
Add 40 μl of MSTFA/NH4I/dithioerythritol mixture GC-MS chromatogram
Stand at 600C at 30 mins
GC-MSD
Anal Biochem Submitted
29. Urinary concentration (ng/mg of creatinine) of oxysterols and bile acids in before and
after treatment of neomycin and streptomycin treated rats.
Compounds Before treatment After treatment
Mean± SD Mean± SD
63.40±36.47
7α-OH-cholesterol 53.56±22.18
120.5±67.83 166.7±140.4
7α-OH-4-cholesten-3one
13.76±2.94 18.70±9.73
7keto- cholesterol
7386±2360 5661±1606
22(R)-OH- cholesterol
ND ND
24S-OH- cholesterol
106.7±34.92 70.62±33.82*
25-OH- cholesterol
4008±1592 2866±1271*
Cholesterol
46.27±18.30 29.84±14.35*
Deoxycholic acid (DCA)
118.1±30.75 139.6±63.87
α-Muricholic acid (α-MCA)
94.96±20.28 158.9±111.3*
Chenodeoxycholic acid (CDCA)
55.10±17.04 78.58±53.64
Cholic acid (CA)
67.37±12.00 75.01±45.29
Ursodeoxycholic acid (UDCA)
81.06±38.24 56.43±24.92
Lithocholic acid (LCA)
53.91±13.58 74.43±43.61
β-Muricholic acid (α-MCA)
47.58±10.16 77.73±51.49*
5β-cholestane 3α,7α,12α-triol
*: ≤ 0.05 between before treatment and after treatment; ND: not detected
30. H 25-hydroxylase
Cytochrome p450scc H H 24-hydroxylase
HO
Cholesterol
OH HO
OH
H
Cholesterol 7α-hydroxylase H H
H H H H H H HH
HO OH HO HO
H H H H
HO HO OH 7hydroxy cholesterol 24(S)- hydroxy cholesterol 25-hydroxy cholesterol
22 (R)- hydroxy cholesterol 7keto cholesterol
CYP39A1, oxysterol 7α-
hydroxylase
3β-hydroxy-∆5-C27-steroid CYP7B1, oxysterol 7α-
oxidoreductase hydroxylase
Steroid
H
H H
O OH
7hydroxy-4-cholesten-3-one
CYP8B1,sterol 12α-hydroxylase ∆4-3-oxosteroid 5β-reductase
∆4-3-oxosteroid 5β-reductase
3α-hydroxysteroid dehydrogenase
OH 3α-hydroxysteroid dehydrogenase OH
O O
OH
H H
H H OH
H H
HO OH HO OH
H H
Cholic acid Chenodeoxycholic acid
H H 7α-hydroxysteroid
HO OH dehydrogenase,
H 6β-hydroxylase
7α-dehydroxylase 5-cholestane-3,7,12-triol 7α-dehydroxylase
O O 7β-hydroxysteroid
O
OH OH OH dehydrogenase OH
OH
OH O O
H H
H
H H
H H H H
H H
HO
HO
H
OH HO OH H H 7β-dehydroxylase H H
H OH H HO HO OH
OH
Deoxycholic acid -Muricholic acid -Muricholic acid Lithocholic acid Ursodeoxycholic acid
31. 0.014 1.8 0.025
Cholesterol 7-hydroxylase 25-hydroxylase
0.0012 Cytochrome p450sce
0.012 1.6
22(R)-OH-cholesterol/cholesterol
7-OH-cholesterol/cholesterol
25-OH-cholesterol/cholesterol
0.0010 1.4 0.008 0.020
0.006
7keto-cholesterol/cholesterol
0.010
1.2
0.008 0.0008 0.015
1.0
0.006 0.0006 0.8
0.010
0.6
0.004 0.0004
0.4 0.005
0.002
0.0002 0.2
0.000 0.0 0.000
Normal control Pseudo germ free 0.0000 Normal control Pseudo germ free Normal control Pseudo germ free
Treated groups Normal control Pseudo germ free
Treated groups Cholesterol
5-cholestane-3,7,12-triol/7-OH-4-cholesten-3-one
Treated groups
Chenodeoxycholic acid/7-OH-4-cholesten-3-one
7-OH--4-chotesten-3-one/7-OH-cholesterol
4 2.5
7-OH-4-cholesten-3-one/25-OH-cholesterol
3-OH-5-C27-steroid oxidoreductase 1.0 4
CYP7B1, oxysterol 7-hydroxylase 4-3-oxosteroid 5-reductase
3-hydroxysteroid dehydrogease
3
2.0 0.05 0.8
3
1.5 0.6
2
2
1.0 0.4
1
1
0.5 0.2
0 0.0 0.0 0
Normal control Pseudo germ free Normal control Pseudo germ free Normal control Pseudo germ free Normal control Pseudo germ free
Treated groups Treated groups Treated groups Treated groups
500 0.5 35 100
6-hydroxylase 6-hydroxylase
-muricholic acid/chenodeoxycholic acid
7-dehydroxylase
-muricholic acid/chenodeoxycholic acid
Colic acid/5-cholestane-3,7,12-triol
0.013 30 0.011 0.032
80
Deoxycholic acid/cholic acid
400 0.4
25
300 0.3 60
20
15 40
200 0.2
10
100 0.1 20
5
0 0.0 0 0
Normal control Pseudo germ free Normal control Pseudo germ free Normal control Pseudo germ free Normal control Pseudo germ free
Treated groups Treated groups Treated groups Treated groups
3.0 50
Ursodeoxycholic acid/chenodeoxycholic acid
7-hydroxysteroid dehydrogenase 7-dehydroxylase
Lithocholic acid/chenodeoxycholic acid
2.5
40
0.024
2.0
30
1.5
20
1.0
10
0.5
0.0 0
Normal control Pseudo germ free Normal control Pseudo germ free
Treated groups Treated groups
32. 160 **
Cholic acid/chenodeoxycholic acid
140
120
100
80
60
40
20
0
Normal control Pseudo germ free
Treated groups
Figure 4.
33. CONCLUSION
1) In the non targeted metabolic profiling, 20 metabolites were significantly
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 observed. The urinary
concentration ratio of primary bile acids (cholic acid and chenodeoxycholic
acid), marker for hepatotoxicity, increased in pseudo germ-free condition.
4) Those findings indicated that the gut microbiota could play a significant role in
the bile acid homeostasis and liver toxicity could be happen 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.
34. Acknowledgement
Dr. Oh Seong Kwon
Dr. Bong Chul Chung
Dr. Soo Hyun Lee
Ji Hye Ahn
Salil Bhowmik Kumar
Supported by
KFDA
KIST
MEST