Summary of Study ST001718

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001102. The data can be accessed directly via it's Project DOI: 10.21228/M8840R This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST001718
Study Title	Commensal intestinal microbiota regulates host luminal proteolytic activity and intestinal barrier integrity through β-glucuronidase activity
Study Type	Complex
Study Summary	Proteases constitute the largest enzyme gene family in vertebrates with intracellular and secreted proteases having critical roles in cellular and organ physiology. Intestinal tract contains diverse set of proteases mediating digestion, microbial responses, epithelial and immune signaling. Transit of chyme through the intestinal tract results in significant suppression of proteases. Although endogenous protease inhibitors have been identified, the broader mechanisms underlying protease regulation in the intestinal tract remains unclear. The objective of this study was to determine microbial regulation of proteolytic activity in intestinal tract using phenotype of post-infection irritable bowel syndrome, a condition characterized by high fecal proteolytic activity. Proteases of host pancreatic origin (chymotrypsin like pancreatic elastase 2A, 3B and trypsin 2) drove proteolytic activity. Of the 14 differentially abundant taxa, high proteolytic activity state was characterized by complete absence of the commensal Alistipes putredinis. Germ free mice had very high proteolytic activity (10-fold of specific-pathogen free mice) which dropped significantly upon humanization with microbiota from healthy volunteers. In contrast, high proteolytic activity microbiota failed to inhibit it, a defect that corrected with fecal microbiota transplant as well as addition of A. putredinis. These mice also had increased intestinal permeability similar to that seen in patients. Microbiota β-glucuronidases mediate bilirubin deconjugation and unconjugated bilirubin is an inhibitor of serine proteases. We found that high proteolytic activity patients had lower urobilinogen levels, a product of bilirubin deconjugation. Mice colonized with β-glucuronidase overexpressing E. coli demonstrated significant inhibition of proteolytic activity and treatment with β-glucuronidase inhibitors increased it. The findings establish that specific commensal microbiota mediates effective inhibition of host pancreatic proteases and maintains intestinal barrier function through the production of β-glucuronidases. This suggests an important homeostatic role for commensal intestinal microbiota.
Institute	Mayo Clinic
Department	Gastroenterology
Last Name	Grover
First Name	Madhu
Address	200 First Street SW, Rochester, MN
Email	dasari.surendra@mayo.edu
Phone	5072840513
Submit Date	2021-03-11
Num Groups	2
Total Subjects	16
Num Males	8
Num Females	8
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2022-03-09
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001102
Project DOI:	doi: 10.21228/M8840R
Project Title:	Fecal Metabolomics
Project Type:	Untargeted MS of mice fecal samples
Project Summary:	Proteases constitute the largest enzyme gene family in vertebrates with intracellular and secreted proteases having critical roles in cellular and organ physiology. Intestinal tract contains diverse set of proteases mediating digestion, microbial responses, epithelial and immune signaling. Transit of chyme through the intestinal tract results in significant suppression of proteases. Although endogenous protease inhibitors have been identified, the broader mechanisms underlying protease regulation in the intestinal tract remains unclear. The objective of this study was to determine microbial regulation of proteolytic activity in intestinal tract using phenotype of post-infection irritable bowel syndrome, a condition characterized by high fecal proteolytic activity. Proteases of host pancreatic origin (chymotrypsin like pancreatic elastase 2A, 3B and trypsin 2) drove proteolytic activity. Of the 14 differentially abundant taxa, high proteolytic activity state was characterized by complete absence of the commensal Alistipes putredinis. Germ free mice had very high proteolytic activity (10-fold of specific-pathogen free mice) which dropped significantly upon humanization with microbiota from healthy volunteers. In contrast, high proteolytic activity microbiota failed to inhibit it, a defect that corrected with fecal microbiota transplant as well as addition of A. putredinis. These mice also had increased intestinal permeability similar to that seen in patients. Microbiota β-glucuronidases mediate bilirubin deconjugation and unconjugated bilirubin is an inhibitor of serine proteases. We found that high proteolytic activity patients had lower urobilinogen levels, a product of bilirubin deconjugation. Mice colonized with β-glucuronidase overexpressing E. coli demonstrated significant inhibition of proteolytic activity and treatment with β-glucuronidase inhibitors increased it. The findings establish that specific commensal microbiota mediates effective inhibition of host pancreatic proteases and maintains intestinal barrier function through the production of β-glucuronidases. This suggests an important homeostatic role for commensal intestinal microbiota.
Institute:	Mayo Clinic
Department:	Biomedical Statistics and Informatics
Laboratory:	ENSP
Last Name:	Grover
First Name:	Madhu
Address:	200 First Street SW, Rochester, MN, 55905, USA
Email:	Dasari.Surendra@mayo.edu
Phone:	507-284-0513

Subject:

Subject ID:	SU001795
Subject Type:	Mammal
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	16-60
Weight Or Weight Range:	NA
Height Or Height Range:	NA
Gender:	Male and female

Factors:

Subject type: Mammal; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Factor
SA161897	ms5520-11	case
SA161898	ms5520-13	case
SA161899	ms5520-7	case
SA161900	ms5520-9	case
SA161901	ms5520-3	case
SA161902	ms5520-4	case
SA161903	ms5520-5	case
SA161904	ms5520-14	control
SA161905	ms5520-15	control
SA161906	ms5520-16	control
SA161907	ms5520-2	control
SA161908	ms5520-12	control
SA161909	ms5520-1	control
SA161910	ms5520-10	control
SA161911	ms5520-6	control
SA161912	ms5520-8	control

Showing results 1 to 16 of 16

Showing results 1 to 16 of 16

Collection:

Collection ID:	CO001788
Collection Summary:	Fecal supernatants (FSNs) were made fresh prior to each experiment. Feces from patients (0.1g) or mice (1 pellet) was added to 0.8mL of phosphate buffered saline (PBS) and subsequently homogenized with a pellet pestle for 5-10 seconds (Sigma-Aldrich, St. Louis, MO, USA). Homogenates were spun twice at 5,000 g for 10 min at 4°C and then added to a 0.22 µm Spin-X tube filter (Corning Life Sciences, Durham, NC, USA). Samples were filtered at 4°C, 10,000 g for 5 min and FSN was stored on ice until use.
Sample Type:	Feces

Treatment:

Treatment ID:	TR001808
Treatment Summary:	A total of 52 PI-IBS patients defined by Rome III criteria and 38 healthy volunteers were recruited. Those with a history of abdominal surgery (except hernia, C-section, hysterectomy, appendectomy or cholecystectomy), inflammatory bowel disease, microscopic colitis, or celiac disease were excluded. Additionally, recruited volunteers were not pregnant at the time of the study. Use of tobacco or alcohol for the duration of the study was prohibited. Following medications were prohibited 7 days prior to study participation: those affecting gastrointestinal transit, serotonergic agents, anti-cholinergic agents, antimuscarinics, narcotics, peppermint oil, antibiotics or new probiotics. Ingestion of artificial sweeteners such as SplendaTM (sucralose), Nutrasweet TM (aspartame), lactulose or mannitol was prohibited for 2 days before the start and during the study. All subjects taking part in the study were asked to complete the Hospital Anxiety and Depression Scale (HADS) and a 7-day bowel diary. All participants completed the Hospital anxiety and depression scale (HADS). PI-IBS patients also completed the Symptom Checklist-90 (SCL-90), IBS Symptom severity scale (IBS-SSS), IBS-quality of life (IBS-QoL) questionnaire as well as the Long Bowel Disease questionnaire (BDQ). Mayo Clinic Institutional Review Board approved human studies and all participants provided a written informed consent (IRB protocol: 12-006529; ClinicalTrials.gov identifier: NCT03266068).

Treatment ID:

TR001808

Treatment Summary:

A total of 52 PI-IBS patients defined by Rome III criteria and 38 healthy volunteers were recruited. Those with a history of abdominal surgery (except hernia, C-section, hysterectomy, appendectomy or cholecystectomy), inflammatory bowel disease, microscopic colitis, or celiac disease were excluded. Additionally, recruited volunteers were not pregnant at the time of the study. Use of tobacco or alcohol for the duration of the study was prohibited. Following medications were prohibited 7 days prior to study participation: those affecting gastrointestinal transit, serotonergic agents, anti-cholinergic agents, antimuscarinics, narcotics, peppermint oil, antibiotics or new probiotics. Ingestion of artificial sweeteners such as SplendaTM (sucralose), Nutrasweet TM (aspartame), lactulose or mannitol was prohibited for 2 days before the start and during the study. All subjects taking part in the study were asked to complete the Hospital Anxiety and Depression Scale (HADS) and a 7-day bowel diary. All participants completed the Hospital anxiety and depression scale (HADS). PI-IBS patients also completed the Symptom Checklist-90 (SCL-90), IBS Symptom severity scale (IBS-SSS), IBS-quality of life (IBS-QoL) questionnaire as well as the Long Bowel Disease questionnaire (BDQ). Mayo Clinic Institutional Review Board approved human studies and all participants provided a written informed consent (IRB protocol: 12-006529; ClinicalTrials.gov identifier: NCT03266068).

Sample Preparation:

Sampleprep ID:	SP001801
Sampleprep Summary:	Fecal samples were deproteinized with six times volume of cold acetonitrile:methanol (1:1 ratio), kept on ice with intermittent vortexing for 30 minutes at 4C, then centrifuged at 18000xg. 13C6-phenylalanine (3 µl at 250ng/µl) was added as internal standard to each sample prior to deproteinization. The supernatants were divided into 2 aliquots and dried down for analysis on a Quadrupole Time-of-Flight Mass Spectrometer (Agilent Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data were acquired under both positive and negative electrospray ionization conditions over a mass range of 100 - 1200 m/z at a resolution of 10,000-35,000 (separate runs). Metabolite separation was achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters). For each column, the run time is 20 min using a flow rate of 400 ul/min. A total of four runs per sample will be performed to give maximum coverage of metabolites. Samples were injected in duplicate or triplicate, and a quality control sample, made up of a subset of samples from the study was injected several times during a run. All raw data files obtained were converted to compound exchange file format using Masshunter DA reprocessor software (Agilent). Mass Profiler Professional (Agilent) was used for data alignment and to convert each metabolite feature (m/z x intensity x time) into a matrix of detected peaks for compound identification.

Sampleprep ID:

SP001801

Sampleprep Summary:

Fecal samples were deproteinized with six times volume of cold acetonitrile:methanol (1:1 ratio), kept on ice with intermittent vortexing for 30 minutes at 4C, then centrifuged at 18000xg. 13C6-phenylalanine (3 µl at 250ng/µl) was added as internal standard to each sample prior to deproteinization. The supernatants were divided into 2 aliquots and dried down for analysis on a Quadrupole Time-of-Flight Mass Spectrometer (Agilent Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data were acquired under both positive and negative electrospray ionization conditions over a mass range of 100 - 1200 m/z at a resolution of 10,000-35,000 (separate runs). Metabolite separation was achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters). For each column, the run time is 20 min using a flow rate of 400 ul/min. A total of four runs per sample will be performed to give maximum coverage of metabolites. Samples were injected in duplicate or triplicate, and a quality control sample, made up of a subset of samples from the study was injected several times during a run. All raw data files obtained were converted to compound exchange file format using Masshunter DA reprocessor software (Agilent). Mass Profiler Professional (Agilent) was used for data alignment and to convert each metabolite feature (m/z x intensity x time) into a matrix of detected peaks for compound identification.

Combined analysis:

Analysis ID	AN002799	AN002800	AN002801
Analysis type	MS	MS	MS
Chromatography type	Reversed phase	HILIC	HILIC
Chromatography system	Agilent 6550	Agilent 6550	Agilent 6550
Column	Agilent DB5-MS (15m x 0.25mm,0.25um)	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
MS Type	ESI	ESI	ESI
MS instrument type	QTOF	QTOF	QTOF
MS instrument name	Agilent 6550 QTOF	Agilent 6550 QTOF	Agilent 6550 QTOF
Ion Mode	POSITIVE	POSITIVE	NEGATIVE
Units	intensity	intensity	intensity

Chromatography:

Chromatography ID:	CH002068
Chromatography Summary:	C18 Reverse phase
Instrument Name:	Agilent 6550
Column Name:	Agilent DB5-MS (15m x 0.25mm,0.25um)
Flow Rate:	400 ul/min
Chromatography Type:	Reversed phase

Chromatography ID:	CH002069
Chromatography Summary:	HILIC
Instrument Name:	Agilent 6550
Column Name:	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Chromatography Type:	HILIC

MS:

MS ID:	MS002594
Analysis ID:	AN002799
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Fecal samples were deproteinized with six times volume of cold acetonitrile:methanol (1:1 ratio), kept on ice with intermittent vortexing for 30 minutes at 4C, then centrifuged at 18000xg. 13C6-phenylalanine (3 µl at 250ng/µl) was added as internal standard to each sample prior to deproteinization. The supernatants were divided into 2 aliquots and dried down for analysis on a Quadrupole Time-of-Flight Mass Spectrometer (Agilent Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data were acquired under both positive and negative electrospray ionization conditions over a mass range of 100 - 1200 m/z at a resolution of 10,000-35,000 (separate runs). Metabolite separation was achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters). For each column, the run time is 20 min using a flow rate of 400 ul/min. A total of four runs per sample will be performed to give maximum coverage of metabolites. Samples were injected in duplicate or triplicate, and a quality control sample, made up of a subset of samples from the study was injected several times during a run. All raw data files obtained were converted to compound exchange file format using Masshunter DA reprocessor software (Agilent). Mass Profiler Professional (Agilent) was used for data alignment and to convert each metabolite feature (m/z x intensity x time) into a matrix of detected peaks for compound identification.
Ion Mode:	POSITIVE

MS ID:	MS002595
Analysis ID:	AN002800
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Fecal samples were deproteinized with six times volume of cold acetonitrile:methanol (1:1 ratio), kept on ice with intermittent vortexing for 30 minutes at 4C, then centrifuged at 18000xg. 13C6-phenylalanine (3 µl at 250ng/µl) was added as internal standard to each sample prior to deproteinization. The supernatants were divided into 2 aliquots and dried down for analysis on a Quadrupole Time-of-Flight Mass Spectrometer (Agilent Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data were acquired under both positive and negative electrospray ionization conditions over a mass range of 100 - 1200 m/z at a resolution of 10,000-35,000 (separate runs). Metabolite separation was achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters). For each column, the run time is 20 min using a flow rate of 400 ul/min. A total of four runs per sample will be performed to give maximum coverage of metabolites. Samples were injected in duplicate or triplicate, and a quality control sample, made up of a subset of samples from the study was injected several times during a run. All raw data files obtained were converted to compound exchange file format using Masshunter DA reprocessor software (Agilent). Mass Profiler Professional (Agilent) was used for data alignment and to convert each metabolite feature (m/z x intensity x time) into a matrix of detected peaks for compound identification.
Ion Mode:	POSITIVE

MS ID:	MS002596
Analysis ID:	AN002801
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Fecal samples were deproteinized with six times volume of cold acetonitrile:methanol (1:1 ratio), kept on ice with intermittent vortexing for 30 minutes at 4C, then centrifuged at 18000xg. 13C6-phenylalanine (3 µl at 250ng/µl) was added as internal standard to each sample prior to deproteinization. The supernatants were divided into 2 aliquots and dried down for analysis on a Quadrupole Time-of-Flight Mass Spectrometer (Agilent Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data were acquired under both positive and negative electrospray ionization conditions over a mass range of 100 - 1200 m/z at a resolution of 10,000-35,000 (separate runs). Metabolite separation was achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters). For each column, the run time is 20 min using a flow rate of 400 ul/min. A total of four runs per sample will be performed to give maximum coverage of metabolites. Samples were injected in duplicate or triplicate, and a quality control sample, made up of a subset of samples from the study was injected several times during a run. All raw data files obtained were converted to compound exchange file format using Masshunter DA reprocessor software (Agilent). Mass Profiler Professional (Agilent) was used for data alignment and to convert each metabolite feature (m/z x intensity x time) into a matrix of detected peaks for compound identification.
Ion Mode:	NEGATIVE