Investigation on the endogenous metabolicchange by intestinal gut microflora usingmass spectrometry based metabolomics 한국과학기술연구원 생체분자기능연구 센터 정병화 Relative concentrations of bacteria at various locations within the gut. Arq Bras Endocrinol Metab. 2009
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)Because1) 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.
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 andResearch Gene mapping Identification of Quantitation Area protein function of metabolite
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
Strategies for metabolomics investigation Mass Spectrometry Reviews 2006
Gut is one of the most metabolically active tissues in the body Alteration of diet Immune systemDrug metabolism Gut Microbiome Antibiotic use Allergy obesity Formation of gall stone
? What endogenous metabolic pathway is affected by gut microflora ? Preparation of pseudo germ free rat Metabolomic approach with urinary metabolites
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 0.56Antibiotic treatment(200 mg/kg for each (mg/㎗) 18.9 ± 4.2 19.5 ± 6.2 0.85 BUNantibiotics, twice a dayfor 6 days) (mg/㎗) 0.4 ± 0.0 0.4 ± 0.0 0.21 CRE • neomycin sulfate • Streptomycin • Bacitracin
Retention time Positive ionization UPLC/TOF/MS Retention time Negative ionization MS2 pattern m/z comparison of metabolic profiling Identification of metabolites
Metabolomic ApproachFlow diagram of a typical MS-based metabolite profiling workflow.
Acceptance Criteria Suggested by the Validation Process
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
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 ……… QCConditioning Run order sequence
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 data set
PCA plotspositive ionization mode negative ionization mode control (●) pseudo-germ free group (▲) QC samples (■)
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
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
Instrumental ConditionsAnalytical Instruments Oven Temp. Profile 320ºC 2 min6890 Series Gas Chromatography5975 Series Mass Selective Detector 1ºC/minGC Parameter 290ºC 2 minColumn: Ultra-I (25m x 0.2mm x 0.33μm)Injection volume: 2.0 μL 20ºC/minInlet mode: SplitSplit Ratio 10:1 240ºCInlet Temp: 280ºCMode: Constant FlowFlow Rate: 0.9 mL/min, HeMS ParameterMS Source: 230ºCMS Quad. 150ºCAuxiliary temperature: 300°CSolvent delay: 5 minAcq. Mode: SIM
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 hrsLiquid-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
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
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 HHO 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
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 50Ursodeoxycholic 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
160 **Cholic acid/chenodeoxycholic acid 140 120 100 80 60 40 20 0 Normal control Pseudo germ free Treated groups Figure 4.
CONCLUSION1) 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.
Acknowledgement Dr. Oh Seong Kwon Dr. Bong Chul Chung Dr. Soo Hyun Lee Ji Hye Ahn Salil Bhowmik KumarSupported by KFDA KIST MEST