Summary of Study ST003877

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 PR002431. The data can be accessed directly via it's Project DOI: 10.21228/M8DG0F 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.

Study ID	ST003877
Study Title	Intestinal permeability of N-acetylcysteine is driven by gut microbiota-dependent cysteine palmitoylation
Study Summary	Trillions of intestinal microbiota are essential to the permeability of orally administered drugs. However, identifying microbial-drug interactions remains challenging due to the highly variable composition of intestinal flora among individuals. Using single-pass intestinal perfusion (SPIP) platform, we establish the microbiota-based permeability screening framework involving germ-free (GF) and specific-pathogen-free (SPF) rats to compare in-situ Peff-values and metabolomic profiles of 32 orally administered drugs with disputable classifications of permeability, prior to the verifications of bioorthogonal chemistry and LC-MS/MS. In contrast with SPF controls, N-Acetylcysteine (NAC) exhibit significantly increased permeability in GF rats, which is inversely related to reduced cysteine-3-ketosphinganine by Bacteroides. To further validate these microbiome features, we integrate clinical descriptors from a prospective cohort of 319 participants to optimize a 15-feature eXtreme Gradient Boosting (XGB) model, which reveal that cysteine palmitoylation by intestinal microbiota has significantly affected NAC permeability. By comparison of net reclassification improvement (NRI) index, this machine learning (ML) model of clinical prediction model encompassing intestinal microbial features outperform other three commercial models in predicting NAC permeability. Here we have developed an intestinal microbiota-based strategy to evaluate uncharacterized NAC permeability, thus accounting for its discordant biopharmaceutics classification.
Institute	Peking University
Last Name	zhang
First Name	yuhang
Address	No. 8, Xi Shi Ku Street, Xicheng, Beijing, China
Email	13811376981@163.com
Phone	13811376981
Submit Date	2025-04-19
Raw Data Available	Yes
Raw Data File Type(s)	dat
Analysis Type Detail	LC-MS
Release Date	2025-05-14
Release Version	1

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Project:

Project ID:	PR002431
Project DOI:	doi: 10.21228/M8DG0F
Project Title:	Intestinal permeability of N-acetylcysteine is driven by gut microbiota-dependent cysteine palmitoylation
Project Summary:	Trillions of intestinal microbiota are essential to the permeability of orally administered drugs. However, identifying microbial-drug interactions remains challenging due to the highly variable composition of intestinal flora among individuals. Using single-pass intestinal perfusion (SPIP) platform, we establish the microbiota-based permeability screening framework involving germ-free (GF) and specific-pathogen-free (SPF) rats to compare in-situ Peff-values and metabolomic profiles of 32 orally administered drugs with disputable classifications of permeability, prior to the verifications of bioorthogonal chemistry and LC-MS/MS. In contrast with SPF controls, N-Acetylcysteine (NAC) exhibit significantly increased permeability in GF rats, which is inversely related to reduced cysteine-3-ketosphinganine by Bacteroides. To further validate these microbiome features, we integrate clinical descriptors from a prospective cohort of 319 participants to optimize a 15-feature eXtreme Gradient Boosting (XGB) model, which reveal that cysteine palmitoylation by intestinal microbiota has significantly affected NAC permeability. By comparison of net reclassification improvement (NRI) index, this machine learning (ML) model of clinical prediction model encompassing intestinal microbial features outperform other three commercial models in predicting NAC permeability. Here we have developed an intestinal microbiota-based strategy to evaluate uncharacterized NAC permeability, thus accounting for its discordant biopharmaceutics classification.
Institute:	Peking University
Last Name:	zhang
First Name:	yuhang
Address:	No. 8, Xi Shi Ku Street, Xicheng, Beijing, China
Email:	13811376981@163.com
Phone:	13811376981

Subject:

Subject ID:	SU004012
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116
Gender:	Male and female

Factors:

Subject type: Mammal; Subject species: Rattus norvegicus (Factor headings shown in green)

mb_sample_id	local_sample_id	Rat type
SA426585	W0_19_07	GF
SA426586	W0_26_28	GF
SA426587	W0_26_45	GF
SA426588	W0_38_11	GF
SA426589	W0_38_09	GF
SA426590	W0_28_35	GF
SA426591	W0_05_25	GF
SA426592	W0_34_11	GF
SA426593	W0_34_12	GF
SA426594	W0_26_43	GF
SA426595	W0_26_46	SPF
SA426596	W0_26_29	SPF
SA426597	W0_19_06	SPF
SA426598	W0_19_09	SPF
SA426599	W0_26_22	SPF
SA426600	W0_19_04	SPF
SA426601	W0_26_24	SPF
SA426602	W0_26_41	SPF
SA426603	W0_19_02	SPF
SA426604	W0_26_44	SPF

Showing results 1 to 20 of 20

Showing results 1 to 20 of 20

Collection:

Collection ID:	CO004005
Collection Summary:	Under anesthesia, the perfused and unperfused intestinal segments of rats were used to assess microbiomic changes. Each intestine was perfused with 37°C saline at a rate of 0.2 mL/min for 10 minutes. The intestinal fluids were then used for subsequent Metabolomics analysis.
Sample Type:	Intestinal fluid

Treatment:

Treatment ID:	TR004021
Treatment Summary:	For in-situ permeability experiments, the rats (n = 6 each group) were fasted for 12 h with free access to water before anesthetization by an intramuscular injection of ketamine-xylazine mixture (80 mg/kg and 20 mg/kg) and placed on the heated surface maintaining with 37 ± 1 °C. The laparotomy was implemented through a midline incision of 3-4 cm to expose the intestine and approximately 10 cm of the proximal jejunum portion was cannulated at both ends. The blank perfusion solution at 37 °C was pumped by peristaltic pump through the intestine at a flow rate of 0.5 mL/min for approximately 30 min. Then, a perfusion solution containing 30 mg/L NAC was administered at 37 °C through the intestinal lumen at a constant flow rate of 0.2 mL/min. To ascertain the steady state during the perfusion process, perfusate samples were collected from the distal portion of the jejunum at 15-minute intervals (at 15, 30, 45, 60, 75, 90, 105, and 120 minutes).

Treatment ID:

TR004021

Treatment Summary:

For in-situ permeability experiments, the rats (n = 6 each group) were fasted for 12 h with free access to water before anesthetization by an intramuscular injection of ketamine-xylazine mixture (80 mg/kg and 20 mg/kg) and placed on the heated surface maintaining with 37 ± 1 °C. The laparotomy was implemented through a midline incision of 3-4 cm to expose the intestine and approximately 10 cm of the proximal jejunum portion was cannulated at both ends. The blank perfusion solution at 37 °C was pumped by peristaltic pump through the intestine at a flow rate of 0.5 mL/min for approximately 30 min. Then, a perfusion solution containing 30 mg/L NAC was administered at 37 °C through the intestinal lumen at a constant flow rate of 0.2 mL/min. To ascertain the steady state during the perfusion process, perfusate samples were collected from the distal portion of the jejunum at 15-minute intervals (at 15, 30, 45, 60, 75, 90, 105, and 120 minutes).

Sample Preparation:

Sampleprep ID:	SP004018
Sampleprep Summary:	For intestinal microflora metabolites extraction, all samples were transferred into EP tubes three times with 1000 μL extract containing internal target (methanol acetonitrile volume ratio =1:1, internal standard concentration 20 mg/L), and vortex mixed for 30 seconds. Add steel ball for 45 Hz grinding instrument for 10 min and ultrasonic 10 min (ice bath). Then samples were centrifuged at 200 g at 4℃ for 15 min and remove 500 μL supernatant into EP tube. The extract is dried in a vacuum concentrator and 160 μL extract (acetonitrile-water ratio: 1:1) was added to the dried metabolites for resolution. Vortex 30 seconds, ice water bath ultrasonic 10 minutes and centrifuged at 200 g at 4 ℃ for 15 min. 120 μL supernatant of each sample was mixed into QC sample for LC/MS detection.

Sampleprep ID:

SP004018

Sampleprep Summary:

For intestinal microflora metabolites extraction, all samples were transferred into EP tubes three times with 1000 μL extract containing internal target (methanol acetonitrile volume ratio =1:1, internal standard concentration 20 mg/L), and vortex mixed for 30 seconds. Add steel ball for 45 Hz grinding instrument for 10 min and ultrasonic 10 min (ice bath). Then samples were centrifuged at 200 g at 4℃ for 15 min and remove 500 μL supernatant into EP tube. The extract is dried in a vacuum concentrator and 160 μL extract (acetonitrile-water ratio: 1:1) was added to the dried metabolites for resolution. Vortex 30 seconds, ice water bath ultrasonic 10 minutes and centrifuged at 200 g at 4 ℃ for 15 min. 120 μL supernatant of each sample was mixed into QC sample for LC/MS detection.

Chromatography:

Chromatography ID:	CH004832
Chromatography Summary:	Low pH polar (LC/MS Pos early)
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C18 (100 x 2mm, 1.7um)
Column Temperature:	45
Flow Gradient:	Linear gradient from 5% B to 80% B over 3.35 minutes
Flow Rate:	0.35 mL/min
Solvent A:	100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Solvent B:	100% methanol; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN006369
Analysis Type:	MS
Chromatography ID:	CH004832
Num Factors:	2
Num Metabolites:	245
Rt Units:	Minutes
Units:	Peak area