MERC Proteins in Saliva and GCF in Periodontal Disease (ELISA Study) (EKNMERC-GCF-SL)

Expression of Mitochondria-Endoplasmic Reticulum Contact Proteins in Saliva and Gingival Crevicular Fluid in Patients With Periodontal Disease: ELISA and Calcium Analysis

This observational cross-sectional study investigates the levels of mitochondria-endoplasmic reticulum contact site (MERC) proteins in saliva and gingival crevicular fluid (GCF) of individuals with different periodontal conditions. MERCs are specialized regions where mitochondria and the endoplasmic reticulum physically connect, and they play important roles in calcium signaling, oxidative stress regulation, and cell death (apoptosis). Disruption of these contact sites has been linked to inflammatory diseases, including periodontal disease.

The study includes 48 systemically healthy, non-smoking adults divided into three groups: periodontally healthy (n=16), gingivitis (n=16), and Stage III Grade B periodontitis (n=16). Five MERC-associated proteins (VAPB, PTPIP51, IP3R, GRP75, and VDAC) are measured in GCF and saliva samples using ELISA. Additionally, caspase-3 (a marker of apoptosis), reactive oxygen species (a marker of oxidative stress), and calcium levels are measured to explore relationships between MERC proteins and key cellular processes involved in periodontal tissue destruction.

The purpose of this study is to determine whether MERC protein levels differ across periodontal conditions and to evaluate their associations with clinical periodontal parameters, oxidative stress, calcium metabolism, and apoptosis.

Study Overview

• Status: Completed
• Conditions: Periodontitis; Gingivitis and Periodontal Diseases; Pathways; Biomarker Discovery and Validation
• Intervention / Treatment: Diagnostic test: ELISA Biomarker Analysis; Diagnostic test: Calcium Measurement

Detailed Description

Mitochondria-endoplasmic reticulum contact sites (MERCs) are specialized structural domains that physically link mitochondria and the endoplasmic reticulum. These contact sites are enriched with functional proteins that coordinate calcium transfer between the two organelles, regulate lipid metabolism, modulate oxidative stress responses, and control apoptotic signaling pathways. Key MERC-associated proteins include VAPB (vesicle-associated membrane protein-associated protein B), PTPIP51 (protein tyrosine phosphatase-interacting protein 51), IP3R (inositol 1,4,5-trisphosphate receptor), GRP75 (glucose-regulated protein 75), and VDAC (voltage-dependent anion channel). The IP3R-GRP75-VDAC complex facilitates calcium transfer from the ER to mitochondria, while the VAPB-PTPIP51 complex modulates this process and influences ATP production and autophagy. Disruption of MERC homeostasis has been implicated in neurodegenerative diseases, metabolic disorders, and periodontal disease. Periodontal disease is a chronic inflammatory condition driven by microbial dental plaque, leading to destruction of periodontal tissues and alveolar bone. Previous studies demonstrated dysregulation of MERC-related gene expression (MFN1, IP3R, GRP75, PINK1, SIGMAR1) in human gingival fibroblasts exposed to Porphyromonas gingivalis and Fusobacterium nucleatum.

The present study measures the concentrations and total amounts of five MERC proteins-VAPB, PTPIP51, IP3R, GRP75, and VDAC-together with caspase-3 (an apoptosis marker), reactive oxygen species (ROS, an oxidative stress marker), and calcium levels in gingival crevicular fluid (GCF) and unstimulated whole saliva obtained from systemically healthy, non-smoking participants. Participants are classified into three groups according to the 2017 World Workshop classification: periodontally healthy (n=16), gingivitis (n=16), and Stage III Grade B periodontitis (n=16), with equal sex distribution (8 males and 8 females per group). GCF samples are collected from three single-rooted teeth per participant using standardized paper strips (Periopaper), with absorbed volume measured using a Periotron 8000. Saliva samples are collected using the passive drooling method. Biomarker levels are quantified using commercially available ELISA kits. Clinical periodontal parameters recorded include Plaque Index (PI), Gingival Index (GI), Probing Depth (PD), Bleeding on Probing (BOP%), and Clinical Attachment Level (CAL). Correlations between biomarker levels and clinical parameters are analyzed. Receiver operating characteristic (ROC) curve analysis is performed to evaluate the diagnostic potential of the biomarkers.

• Study Type: Observational
• Enrollment (Actual): 48

Contacts and Locations

Study Locations

• Turkey (Türkiye)
  • Malatya
    • Malatya, Malatya, Turkey (Türkiye), 44210
      • Inönü University Faculty of Dentistry, Department of Periodontology

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

Systemically healthy, non-smoking individuals aged 18-65 years who presented to the Department of Periodontology, Faculty of Dentistry, Inonu University, Malatya, Turkiye for examination and/or treatment between January 2024 and May 2024. Participants were classified into three groups based on the 2017 World Workshop criteria: periodontally healthy (n=16), gingivitis (n=16), and Stage III Grade B periodontitis (n=16), with equal sex distribution (8 males and 8 females per group).

Description

Inclusion Criteria:

• Systemically healthy individuals aged 18-65 years
• Non-smokers or smoking cessation more than 5 years ago
• Healthy group: No history of periodontal disease, ≥20 teeth, probing depth (PD) ≤3 mm at all sites, bleeding on probing (BOP) <10%, radiographic CEJ-to-alveolar bone crest distance ≤3 mm in ≥95% of teeth
• Gingivitis group: No history of periodontal disease, ≥20 teeth, PD ≤3 mm at all sites, BOP ≥30%, radiographic CEJ-to-alveolar bone crest distance ≤3 mm in ≥95% of teeth
• Periodontitis group: ≥15 teeth, >30% of teeth affected by periodontal disease, PD ≥6 mm, clinical attachment loss (CAL) ≥5 mm, vertical bone loss ≥3 mm, Class II or III furcation involvement, radiographic alveolar bone loss extending to the middle third or beyond (≥33%), bone loss-to-age ratio between 0.25 and 1.00

Exclusion Criteria:

• Periodontal treatment or antibiotic use within the past 6 months
• Fewer than 20 teeth (excluding third molars)
• Presence of any systemic disease
• Current smoking or cessation within the past 5 years
• Use of immunosuppressive medication
• Alcohol consumption
• Regular medication use
• Pregnancy or lactation
• Need for antibiotic prophylaxis prior to dental procedures
• Prosthetic restorations on the teeth selected for sampling

Study Plan

How is the study designed?

Number of groups / cohorts

3

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Periodontally Healthy; Systemically healthy, non-smoking individuals with no history of periodontal disease. Inclusion criteria: ≥20 teeth, probing depth (PD) ≤3 mm at all sites, bleeding on probing (BOP) <10%, and radiographic CEJ-to-alveolar bone crest distance ≤3 mm in ≥95% of teeth. n=16 (8 females, 8 males).	Diagnostic test: ELISA Biomarker Analysis; Levels of MERC-associated proteins (VAPB, PTPIP51, IP3R, GRP75, VDAC), caspase-3, reactive oxygen species, and calcium were measured in gingival crevicular fluid and unstimulated whole saliva samples using commercially available ELISA kits. Absorbance was read at 450 nm. No therapeutic intervention was applied; this is an observational biomarker measurement study.; Diagnostic test: Calcium Measurement; Calcium levels were measured in gingival crevicular fluid and unstimulated whole saliva samples using a commercially available ELISA kit (ELK Biotechnology, China). Concentrations were expressed as μmol/mL and total amounts as μmol/30s. Analytical range: 0.15-40 μmol/mL. No therapeutic intervention was applied.
Gingivitis; Systemically healthy, non-smoking individuals with gingival inflammation but no attachment loss. Inclusion criteria: ≥20 teeth, PD ≤3 mm at all sites, BOP ≥30%, and radiographic CEJ-to-alveolar bone crest distance ≤3 mm in ≥95% of teeth. n=16 (8 females, 8 males).	Diagnostic test: ELISA Biomarker Analysis; Levels of MERC-associated proteins (VAPB, PTPIP51, IP3R, GRP75, VDAC), caspase-3, reactive oxygen species, and calcium were measured in gingival crevicular fluid and unstimulated whole saliva samples using commercially available ELISA kits. Absorbance was read at 450 nm. No therapeutic intervention was applied; this is an observational biomarker measurement study.; Diagnostic test: Calcium Measurement; Calcium levels were measured in gingival crevicular fluid and unstimulated whole saliva samples using a commercially available ELISA kit (ELK Biotechnology, China). Concentrations were expressed as μmol/mL and total amounts as μmol/30s. Analytical range: 0.15-40 μmol/mL. No therapeutic intervention was applied.
Stage III Grade B Periodontitis; Systemically healthy, non-smoking individuals with Stage III Grade B periodontitis. Inclusion criteria: ≥15 teeth, >30% of teeth affected, PD ≥6 mm, CAL ≥5 mm, vertical bone loss ≥3 mm, Class II/III furcation involvement, radiographic bone loss extending to the middle third or beyond (≥33%), and bone loss-to-age ratio between 0.25 and 1.00. n=16 (8 females, 8 males).	Diagnostic test: ELISA Biomarker Analysis; Levels of MERC-associated proteins (VAPB, PTPIP51, IP3R, GRP75, VDAC), caspase-3, reactive oxygen species, and calcium were measured in gingival crevicular fluid and unstimulated whole saliva samples using commercially available ELISA kits. Absorbance was read at 450 nm. No therapeutic intervention was applied; this is an observational biomarker measurement study.; Diagnostic test: Calcium Measurement; Calcium levels were measured in gingival crevicular fluid and unstimulated whole saliva samples using a commercially available ELISA kit (ELK Biotechnology, China). Concentrations were expressed as μmol/mL and total amounts as μmol/30s. Analytical range: 0.15-40 μmol/mL. No therapeutic intervention was applied.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
GCF VAPB Levels	Concentrations and total amounts of VAPB in gingival crevicular fluid measured by ELISA.	At baseline (single visit)
Salivary VAPB Levels	Concentrations of VAPB (pg/mL) in unstimulated whole saliva measured by ELISA.	At baseline (single visit)
GCF PTPIP51 Levels	Concentrations and total amounts of PTPIP51 in gingival crevicular fluid measured by ELISA.	At baseline (single visit)
Salivary PTPIP51 Levels	Concentrations of PTPIP51 (ng/L) in unstimulated whole saliva measured by ELISA.	At baseline (single visit)
GCF IP3R Levels	Concentrations and total amounts of IP3R in gingival crevicular fluid measured by ELISA.	At baseline (single visit)
Salivary IP3R Levels	Concentrations of IP3R (ng/mL) in unstimulated whole saliva measured by ELISA.	At baseline (single visit)
GCF GRP75 Levels	Concentrations and total amounts of GRP75 in gingival crevicular fluid measured by ELISA.	At baseline (single visit)
Salivary GRP75 Levels	Concentrations of GRP75 (ng/mL) in unstimulated whole saliva measured by ELISA.	At baseline (single visit)
GCF VDAC Levels	Concentrations and total amounts of VDAC in gingival crevicular fluid measured by ELISA.	At baseline (single visit)
Salivary VDAC Levels	Concentrations of VDAC (ng/mL) in unstimulated whole saliva measured by ELISA.	At baseline (single visit)
GCF Caspase-3 Levels	Concentrations and total amounts of caspase-3 (ng/mL) in gingival crevicular fluid measured by ELISA.	At baseline (single visit)
Salivary Caspase-3 Levels	Concentrations of caspase-3 (ng/mL) in unstimulated whole saliva measured by ELISA.	At baseline (single visit)
GCF Reactive Oxygen Species Levels	Concentrations and total amounts of reactive oxygen species (ROS) in gingival crevicular fluid measured by ELISA.	At baseline (single visit)
Salivary Reactive Oxygen Species Levels	Concentrations of reactive oxygen species (ROS) in unstimulated whole saliva measured by ELISA.	At baseline (single visit)
GCF Calcium Levels	Concentrations and total amounts of calcium (µmol/mL) in gingival crevicular fluid measured by ELISA.	At baseline (single visit)
Salivary Calcium Levels	Concentrations of calcium (µmol/mL) in unstimulated whole saliva measured by ELISA.	At baseline (single visit)
Plaque Index	Plaque Index (PI) recorded at sampling sites.	At baseline (single visit)
Gingival Index	Gingival Index (GI) recorded at sampling sites.	At baseline (single visit)
Probing Depth	Probing Depth (PD) recorded at sampling sites.	At baseline (single visit)
Bleeding on Probing	Bleeding on Probing (BOP%) recorded at sampling sites.	At baseline (single visit)
Clinical Attachment Level	Clinical Attachment Level (CAL) recorded at sampling sites.	At baseline (single visit)
Gingival Crevicular Fluid Volume	Gingival crevicular fluid (GCF) volume measured at sampling sites.	At baseline (single visit)

Collaborators and Investigators

• Sponsor: Inonu University
• Collaborators: University of Minnesota; Harvard University
• Investigators: Study Chair: Cüneyt A Aral, Professor, DDS, PhD, Inonu University

Publications and helpful links

General Publications

• Papapanou PN, Sanz M, Buduneli N, Dietrich T, Feres M, Fine DH, Flemmig TF, Garcia R, Giannobile WV, Graziani F, Greenwell H, Herrera D, Kao RT, Kebschull M, Kinane DF, Kirkwood KL, Kocher T, Kornman KS, Kumar PS, Loos BG, Machtei E, Meng H, Mombelli A, Needleman I, Offenbacher S, Seymour GJ, Teles R, Tonetti MS. Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J Clin Periodontol. 2018 Jun;45 Suppl 20:S162-S170. doi: 10.1111/jcpe.12946.
• Chapple ILC, Mealey BL, Van Dyke TE, Bartold PM, Dommisch H, Eickholz P, Geisinger ML, Genco RJ, Glogauer M, Goldstein M, Griffin TJ, Holmstrup P, Johnson GK, Kapila Y, Lang NP, Meyle J, Murakami S, Plemons J, Romito GA, Shapira L, Tatakis DN, Teughels W, Trombelli L, Walter C, Wimmer G, Xenoudi P, Yoshie H. Periodontal health and gingival diseases and conditions on an intact and a reduced periodontium: Consensus report of workgroup 1 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J Periodontol. 2018 Jun;89 Suppl 1:S74-S84. doi: 10.1002/JPER.17-0719.
• Lim D, Dematteis G, Tapella L, Genazzani AA, Cali T, Brini M, Verkhratsky A. Ca2+ handling at the mitochondria-ER contact sites in neurodegeneration. Cell Calcium. 2021 Sep;98:102453. doi: 10.1016/j.ceca.2021.102453. Epub 2021 Aug 5.
• Lee S, Min KT. The Interface Between ER and Mitochondria: Molecular Compositions and Functions. Mol Cells. 2018 Dec 31;41(12):1000-1007. doi: 10.14348/molcells.2018.0438. Epub 2018 Dec 12.
• Kirmizigul OA, Sabanci A, Disli F, Yildiz S, Milward MR, Aral K. Evaluation of the role of mitofusin-1 and mitofusin-2 in periodontal disease. J Periodontol. 2024 Jan;95(1):64-73. doi: 10.1002/JPER.23-0072. Epub 2023 Jul 24.
• Aral K, Milward MR, Cooper PR. Gene expression profiles of mitochondria-endoplasmic reticulum tethering in human gingival fibroblasts in response to periodontal pathogens. Arch Oral Biol. 2021 Aug;128:105173. doi: 10.1016/j.archoralbio.2021.105173. Epub 2021 May 27.

Study record dates

Study Major Dates

• Study Start (Actual): January 10, 2024
• Primary Completion (Actual): January 1, 2025
• Study Completion (Actual): April 1, 2025

Study Registration Dates

• First Submitted: May 13, 2026
• First Submitted That Met QC Criteria: May 20, 2026
• First Posted (Actual): May 22, 2026

Study Record Updates

• Last Update Posted (Actual): May 22, 2026
• Last Update Submitted That Met QC Criteria: May 20, 2026
• Last Verified: May 1, 2026

More Information

Terms related to this study

• Keywords: Oxidative Stress; Saliva; Calcium; Reactive Oxygen Species; Apoptosis; Caspase-3; ELISA; Gingival Crevicular Fluid; MERC; Mitochondria-Endoplasmic Reticulum Contact; VAPB; PTPIP51; IP3R; GRP75; VDAC
• Additional Relevant MeSH Terms: Neurologic Manifestations; Mouth Diseases; Stomatognathic Diseases; Brain Diseases; Central Nervous System Diseases; Nervous System Diseases; Genetic Diseases, Inborn; Infections; Neurodegenerative Diseases; Gingival Diseases; Heredodegenerative Disorders, Nervous System; Spinal Cord Diseases; Dyskinesias; Cerebellar Diseases; Cerebellar Ataxia; Spinocerebellar Degenerations; Ataxia; Congenital, Hereditary, and Neonatal Diseases and Abnormalities; Periodontitis; Periodontal Diseases; Spinocerebellar Ataxias; Gingivitis
• Other Study ID Numbers: IUCTEC.2024/06; TDH-2024-3464 (Other Grant/Funding Number: Inönü University Scientific Research Projects Coordination Office)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: UNDECIDED

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No