Summary of Study ST004037

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 PR002529. The data can be accessed directly via it's Project DOI: 10.21228/M8R840 This work is supported by NIH grant, U2C- DK119886.

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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 IDST004037
Study TitleUntargeted Metabolomics Reveal Long-Term Metabolic Changes in the Oral Cavity Following Disease Stress and Recovery
Study TypeLongitudinal
Study SummaryBackground The human oral microbiome, comprising over 700 microbial species, plays a critical role in both oral and systemic health. While next-generation sequencing (NGS) has linked oral dysbiosis to diseases such as cardiovascular disease, inflammatory bowel disease, and diabetes, the role of microbial metabolites in oral disease progression remains underexplored. This study aimed to characterize salivary metabolite dynamics during gingivitis initiation, progression, and recovery, and assess their diagnostic potential. Methods This longitudinal human study investigates salivary metabolomic changes during experimentally induced gingivitis. Healthy adult participants were asked to refrain from oral hygiene for 21 days to induce gingival inflammation, followed by a recovery period. We conducted untargeted metabolomics and 16S rRNA profiling on saliva samples from 20 healthy participants across seven time points during a 35-day experimental gingivitis model. Subjects discontinued oral hygiene for 21 days, followed by professional prophylaxis and a 14-day recovery period. Partial least squares-discriminant analysis (PLS-DA) identified temporal metabolite shifts. In vitro culture and biofilm assays linked key metabolites to microbial taxa. Machine learning models, Random Forest classification and regression, were trained with 5-fold cross-validation to classify inflammation and predict gingival index scores. Results We identified 765 salivary metabolites, of which 143 exhibited significant temporal variation. Gingivitis progression was marked by increases in amino acids, peptides, and carbohydrates, while protective alkaloids declined. Metabolites such as putrescine and eicosanoic acid were linked to inflammation and tissue degradation. Although microbiome composition returned to baseline by Day 35, metabolic profiles remained altered, indicating a persistent “metabolic lag.” Notably, diketopiperazines (DKPs) including cyclo(L-Val-L-Pro) and cyclo(L-Tyr-L-Pro) were detected in the oral cavity for the first time. Cyclo(L-Val-L-Pro), isolated from Streptococcus mitis, selectively inhibited Aggregatibacter actinomycetemcomitans biofilms. As a preliminary analysis, we applied Random Forest classifiers to assess whether metabolite profiles could distinguish inflammation states. While not intended as a diagnostic tool, the model revealed strong predictive potential of lipid-derivatives metabolites (R² = 0.96 discovery, 0.72 validation). Conclusion Our study reveals functional metabolic shifts that persist beyond microbial recovery, highlighting the utility of salivary metabolomics for non-invasive monitoring of oral inflammation. The identification of bioactive DKPs and inflammation-linked metabolites suggests new therapeutic targets. These findings support a paradigm shift toward function-based, personalized diagnostics and recovery timelines in oral health management and support the use of metabolomics-informed algorithms for oral inflammation monitoring while providing a pilot framework for future, larger-scale studies.
Institute
University of Michigan
DepartmentLife Science Institute
LaboratorySherman Laboratory
Last NameOkiye
First NameMaribel
Address201 Washtenaw Ave
Emailmaribelo@umich.edu
Phonemaribelo@umich.edu
Submit Date2025-06-26
Num Groups1
Total Subjects20
Num Males6
Num Females14
Study CommentsMetabolomics analysis focused on a subset of 14 subjects based on complete sample availability across all time points. 6 additional subjects were recruited and samples stored as a reserve to support potential resampling or further validation. Metabolite filtering retained features detected in ≥14 subjects.
PublicationsIn review at Microbiome journal (Springer Nature); bioRxiv doi: https://doi.org/10.1101/2023.06.26.546600
Raw Data AvailableYes
Raw Data File Type(s)mzML, mzXML
Analysis Type DetailLC-MS
Release Date2025-07-17
Release Version1
Maribel Okiye Maribel Okiye
https://dx.doi.org/10.21228/M8R840
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002529
Project DOI:doi: 10.21228/M8R840
Project Title:Untargeted Metabolomics Reveal Long-Term Metabolic Changes in the Oral Cavity Following Disease Stress and Recovery (University of Michigan)
Project Type:Human longitudinal study
Project Summary:This study investigates the temporal metabolomic dynamics of saliva during experimentally induced gingivitis in healthy individuals. Using a no-brushing model over 21 days followed by recovery, we collected saliva from 20 subjects across seven time points and analyzed extracted metabolites with n=14 via LC-MS/MS. The study highlights persistent metabolic signatures even after clinical recovery, suggesting a metabolic lag behind microbial resolution.
Institute:University of Michigan
Department:Life Science Institute
Laboratory:Sherman Laboratory
Last Name:Okiye
First Name:Maribel
Address:201 Washtenaw Ave
Email:maribelo@umich.edu
Phone:maribelo@umich.edu
Project Comments:Metabolomics study includes two extraction protocols, multiple time points, and matched microbial data.
Publications:doi: https://doi.org/10.1101/2023.06.26.546600
Contributors:Maribel E. Okiye, Michelle A. Velez, Victoria Voung, Janet Kinney, James Sugai, William V. Giannobile, Ashootosh Tripathi, and David H Sherman

Subject:

Subject ID:SU004183
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Genotype Strain:N/A
Age Or Age Range:20-36
Weight Or Weight Range:Not recorded
Height Or Height Range:Not recorded
Gender:Male and female
Human Race:13 Caucasian, 4 Asian, 1 African American, 1 Bi-racial, 1 Tri-racial
Human Ethnicity:N/A
Human Trial Type:Longitudinal observational study
Human Lifestyle Factors:Healthy, non-smokers, not on medications, no previous cavities, nonpregnant
Human Medications:None
Human Prescription Otc:None
Human Smoking Status:Non-smokers
Human Alcohol Drug Use:No regular use reported
Human Nutrition:Standard American diet
Human Inclusion Criteria:Healthy adults aged 20–36 with no cavities, no active oral health issues, not pregnant, non-smokers, not on antibiotics in the past 3 months, and able to comply with oral hygiene restriction protocol.
Human Exclusion Criteria:Chronic illness, recent antibiotic use (within 3 months), pregnancy, current or past tobacco use, active dental disease (e.g., gingivitis, periodontitis), or inability to comply with protocol.
Species Group:N/A

Factors:

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

mb_sample_id local_sample_id Sample source time.point gender race.ethnicity
SA466178Subject 5_X0.8saliva 0 0 African American
SA466179Subject 5_X0saliva 0 0 African American
SA466180Subject 5_X0.1saliva 0 0 African American
SA466181Subject 5_X0.3saliva 0 0 African American
SA466182Subject 5_X0.4saliva 0 0 African American
SA466183Subject 5_X0.5saliva 0 0 African American
SA466184Subject 5_X0.6saliva 0 0 African American
SA466185Subject 5_X0.7saliva 0 0 African American
SA466186Subject 5_X0.2saliva 0 0 African American
SA466187Subject 9_X0.1saliva 0 0 Asian
SA466188Subject 7_X0.4saliva 0 0 Asian
SA466189Subject 9_X0.3saliva 0 0 Asian
SA466190Subject 9_X0.4saliva 0 0 Asian
SA466191Subject 9_X0.5saliva 0 0 Asian
SA466192Subject 9_X0.6saliva 0 0 Asian
SA466193Subject 9_X0.7saliva 0 0 Asian
SA466194Subject 7_X0saliva 0 0 Asian
SA466195Subject 7_X0.1saliva 0 0 Asian
SA466196Subject 7_X0.2saliva 0 0 Asian
SA466197Subject 7_X0.3saliva 0 0 Asian
SA466198Subject 7_X0.5saliva 0 0 Asian
SA466199Subject 9_X0.8saliva 0 0 Asian
SA466200Subject 9_X0.9saliva 0 0 Asian
SA466201Subject 9_X0saliva 0 0 Asian
SA466202Subject 9_X0.2saliva 0 0 Asian
SA466203Subject 18_X0.5saliva 0 0 Caucasian
SA466204Subject 18_X0.7saliva 0 0 Caucasian
SA466205Subject 18_X0.8saliva 0 0 Caucasian
SA466206Subject 18_X0.6saliva 0 0 Caucasian
SA466207Subject 18_X0.9saliva 0 0 Caucasian
SA466208Subject 18_X0.10saliva 0 0 Caucasian
SA466209Subject 18_X0.11saliva 0 0 Caucasian
SA466210Subject 3_X0saliva 0 0 Caucasian
SA466211Subject 3_X0.1saliva 0 0 Caucasian
SA466212Subject 18_X0.1saliva 0 0 Caucasian
SA466213Subject 18_X0.4saliva 0 0 Caucasian
SA466214Subject 15_X0.5saliva 0 0 Caucasian
SA466215Subject 14_X0.2saliva 0 0 Caucasian
SA466216Subject 14_X0.3saliva 0 0 Caucasian
SA466217Subject 15_X0saliva 0 0 Caucasian
SA466218Subject 15_X0.1saliva 0 0 Caucasian
SA466219Subject 15_X0.2saliva 0 0 Caucasian
SA466220Subject 15_X0.3saliva 0 0 Caucasian
SA466221Subject 15_X0.4saliva 0 0 Caucasian
SA466222Subject 15_X0.6saliva 0 0 Caucasian
SA466223Subject 18_X0.3saliva 0 0 Caucasian
SA466224Subject 15_X0.7saliva 0 0 Caucasian
SA466225Subject 15_X0.8saliva 0 0 Caucasian
SA466226Subject 15_X0.9saliva 0 0 Caucasian
SA466227Subject 15_X0.10saliva 0 0 Caucasian
SA466228Subject 15_X0.11saliva 0 0 Caucasian
SA466229Subject 18_X0saliva 0 0 Caucasian
SA466230Subject 3_X0.3saliva 0 0 Caucasian
SA466231Subject 18_X0.2saliva 0 0 Caucasian
SA466232Subject 3_X0.2saliva 0 0 Caucasian
SA466233Subject 4_X0saliva 0 0 Caucasian
SA466234Subject 3_X0.4saliva 0 0 Caucasian
SA466235Subject 6_X0.6saliva 0 0 Caucasian
SA466236Subject 8_X0.9saliva 0 0 Caucasian
SA466237Subject 8_X0.8saliva 0 0 Caucasian
SA466238Subject 8_X0.7saliva 0 0 Caucasian
SA466239Subject 8_X0.6saliva 0 0 Caucasian
SA466240Subject 8_X0.5saliva 0 0 Caucasian
SA466241Subject 8_X0.4saliva 0 0 Caucasian
SA466242Subject 8_X0.3saliva 0 0 Caucasian
SA466243Subject 8_X0.2saliva 0 0 Caucasian
SA466244Subject 8_X0.1saliva 0 0 Caucasian
SA466245Subject 8_X0saliva 0 0 Caucasian
SA466246Subject 6_X0.9saliva 0 0 Caucasian
SA466247Subject 6_X0.8saliva 0 0 Caucasian
SA466248Subject 6_X0.7saliva 0 0 Caucasian
SA466249Subject 6_X0.5saliva 0 0 Caucasian
SA466250Subject 3_X0.5saliva 0 0 Caucasian
SA466251Subject 6_X0.4saliva 0 0 Caucasian
SA466252Subject 6_X0.3saliva 0 0 Caucasian
SA466253Subject 6_X0.2saliva 0 0 Caucasian
SA466254Subject 6_X0.1saliva 0 0 Caucasian
SA466255Subject 6_X0saliva 0 0 Caucasian
SA466256Subject 14_X0saliva 0 0 Caucasian
SA466257Subject 4_X0.2saliva 0 0 Caucasian
SA466258Subject 4_X0.1saliva 0 0 Caucasian
SA466259Subject 3_X0.9saliva 0 0 Caucasian
SA466260Subject 3_X0.8saliva 0 0 Caucasian
SA466261Subject 3_X0.7saliva 0 0 Caucasian
SA466262Subject 3_X0.6saliva 0 0 Caucasian
SA466263Subject 14_X0.1saliva 0 0 Caucasian
SA466264Subject 1_X0.1saliva 0 0 Tri-racial
SA466265Subject 1_X0saliva 0 0 Tri-racial
SA466266Subject 1_X0.6saliva 0 0 Tri-racial
SA466267Subject 1_X0.4saliva 0 0 Tri-racial
SA466268Subject 1_X0.2saliva 0 0 Tri-racial
SA466269Subject 1_X0.3saliva 0 0 Tri-racial
SA466270Subject 1_X0.8saliva 0 0 Tri-racial
SA466271Subject 1_X0.7saliva 0 0 Tri-racial
SA466272Subject 1_X0.9saliva 0 0 Tri-racial
SA466273Subject 1_X0.5saliva 0 0 Tri-racial
SA466274Subject 16_X0.2saliva 0 1 Asian
SA466275Subject 16_X0saliva 0 1 Asian
SA466276Subject 16_X0.1saliva 0 1 Asian
SA466277Subject 16_X0.4saliva 0 1 Asian
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Collection:

Collection ID:CO004176
Collection Summary:Longitudinal saliva collection over 35 days. Seven time points per subject (Days 0–35). Gender 0 = male, 1 = female.
Collection Protocol Filename:collection_protocol.txt
Collection Protocol Comments:Unstimulated whole saliva was collected at each visit. Subjects refrained from brushing for 21 days.
Sample Type:Saliva
Collection Method:Subjects rinsed their mouths vigorously with water for 30 seconds to remove debris, then expectorated. After a two-minute waiting period to establish baseline levels of saliva, the subjects tipped their heads toward the graduated test tubes and expectorated 5mL of whole saliva in the plastic funnel inserted in the plastic, sterile tubes, labeled with the subject's initials, sample name, and date of harvest. The sample was immediately placed on wet ice and transported to the lab. Aprotinin (1mg/ml) and Phenylmethylsulphonyl fluoride (100 mM in MeOH) were added to the whole saliva at a dilution of 1:100 and 1:200, respectively. The whole saliva was then stored at -80°C until analysis.
Collection Location:University of Michigan School of Dentistry, Ann Arbor, MI
Collection Frequency:7 time points per subject: Days 0, 4, 7, 14, 21, 28, 35
Collection Duration:35 days
Volumeoramount Collected:5 mL per time point
Storage Conditions:Described in summary
Collection Vials:Sterile 15 mL graduated polypropylene conical tubes with funnel
Storage Vials:10 mL Eppendorf tubes
Collection Tube Temp:Room temperature for collection (~22°C), then immediately chilled on wet ice
Additives:Aprotinin (1 mg/mL), PMSF (100 mM in MeOH), added 1:100 and 1:200 dilutions respectively

Treatment:

Treatment ID:TR004192
Treatment Summary:Subjects ceased all oral hygiene practices (brushing/flossing) for 21 days to induce gingivitis. Oral hygiene resumed after Day 21.
Treatment Protocol Comments:No chemical compounds administered. Gingivitis was induced by hygiene cessation.
Treatment:Behavioral (Hygiene cessation)
Treatment Compound:None
Treatment Route:N/A
Treatment Dose:N/A
Treatment Dosevolume:N/A
Treatment Doseduration:21 days (hygiene cessation period)
Treatment Vehicle:N/A
Human Fasting:No fasting required
Human Endp Clinical Signs:Gingival inflammation assessed via gingival and plaque index

Sample Preparation:

Sampleprep ID:SP004189
Sampleprep Summary:Whole saliva was centrifuged and separated into supernatant and pellet. Supernatant was extracted using three organic solvents and dried under vacuum.
Sampleprep Protocol Filename:sample_prep_protocol.txt
Sampleprep Protocol Comments:Supernatants from centrifuged whole saliva were extracted in parallel with Hexanes, EtOAc, and Butanol. Dried extracts were stored at −80°C until analysis.
Processing Method:Centrifugation at 10,000 rpm for 10 minutes to separate pellet and supernatant.
Processing Storage Conditions:4℃
Extraction Method:Liquid-liquid extraction using Hexanes, Ethyl Acetate, and Butanol, sequentially.
Extract Enrichment:None
Extract Cleanup:No additional cleanup
Extract Storage:4℃
Sample Resuspension:MeOH (BtOH, EtOAc, AQ layers); MeCN (Hexanes layer)
Sample Derivatization:None
Sample Spiking:None
Subcellular Location:N/A

Chromatography:

Chromatography ID:CH005072
Chromatography Summary:Metabolic extracts were analyzed by UPLC-QTOF-MS using a Phenyl-Hexyl column and a water–acetonitrile gradient. A divert valve was set to waste during the first minute.
Methods ID:EGM-DHS-WVG-004
Methods Filename:sherman001.txt
Chromatography Comments:ESI-MS detection was performed in positive ion mode; UV spectra were also acquired (195–400 nm).
Instrument Name:Agilent 1290 Infinity II
Column Name:Phenomenex Kinetex Phenyl-Hexyl (50 × 4.6mm, 5um, 100 Å)
Column Temperature:35
Flow Gradient:0–1 min: 90% A (isocratic) 1–8 min: Linear gradient to 100% B 8–10 min: 100% B (isocratic plateau)
Flow Rate:0.4 mL/min
Sample Injection:5 μL of 100 μg/mL extract
Solvent A:100% water
Solvent B:100% acetonitrile
Randomization Order:N/A
Chromatography Type:Reversed phase

Analysis:

Analysis ID:AN006675
Laboratory Name:Sherman and Tripathi Lab, Natural Products Discovery Core
Analysis Type:MS
Analysis Protocol File:sherman001.txt
Software Version:Time-of-Flight
Data Format:.d
Chromatography ID:CH005072
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST004037_AN006675_Results.txt
Units:Nomalized Peak Intensities
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