Molecular Study of Oral Dysbacteriosis in People With Prediabetes and Diabetes
Conditions: PreDiabetes, Diabetes Mellitus, Type 2, Obesity, Oral Dysbiosis, Mouth Disease
Sponsor: Fundación Pública Andaluza para la Investigación de Málaga en Biomedicina y Salud

 
ENDOCANNABINOIDS IN ORAL DYSBACTERIOSIS
 
 
 
29 JUNE 2023 
HOSPITAL REGIONAL UNIVERSITARIO DE MALAGA (MALAGA, SPAIN). 
Unidad Endocrinología y Nutrición, Hospital Civil, Pabellón 7, planta 2 
VERSION 3 

SMILE ORAL DYSBACTERIOSIS IN PEOPLE WITH OBESITY AND PREDIABETES OR DIABETES 
1. SUMMARY 
1.1. STUDY TITLE 
MOLECULAR STUDY OF ORAL DYSBACTERIOSIS IN PEOPLE WITH OBESITY 
AND PREDIABETES OR DIABETES ( SMILE ) 
1.2. PRINCIPAL INVESTIGATOR OF THE TRIAL 
Dr. Rodolfo Matias Ortiz Flores 
Dr. Francisco Javier Bermudez Silva 
1.3. CENTER WHERE THE TEST IS PLANNED TO BE CARRIED OUT 
Hospital Regional Universitario de Malaga (Malaga). 
1.4. RESEARCH ETHICS COMMITTEE THAT EVALUATED THE TRIAL 
Investigación Provincial de Málaga Ethics Committee 
1.5. MEDICATION/SUBSTANCE WITH WHICH THE TEST IS GOING TO BE 
CARRIED OUT 
No medication or substance will be tested on patients, it is a descriptive observational study.   
1.6. MAIN OBJECTIVE OF THE TRIAL 
Investigate the possible differences in endocannabinoid levels and their correlation with 
inflammatory cytokines and microbial load in the oral cavity depending on the presence or 
absence of oral cavity disease in people with obesity and prediabetes or diabetes. 
1.7. TRIAL DESIGN 
Descriptive observational study. 
1.8. ILLNESS OR DISORDER UNDER STUDY 
• 
Prediabetes 
• 
Diabetes mellitus type 2 
• 
Obesity 
• 
Oral dysbiosis 
• 
Oral cavity disease (OCD) 
1.9. MAIN AND SECONDARY VARIABLES 
As MAIN VARIABLE, endocannabinoids in saliva and plasma (2-AG, AEA, PEA, OEA, 
DHEA, 2-LG, 2-OG; pmol/ml) will be studied . 
And as SECONDARY VARIABLES are going to be studied: 
• 
The bacteriological profile of the oral cavity (the following bacterial strains: 
Aggregatibacter actinomycetemcomitans , Porphyromonas gingivalis , Tannerella 
forsythia , Treponema denticola , Prevotella intermedia, Fusobacterium nucleatum, 
Parvimonas 
micra, 
Campylobacter 
rectus, 
Eikenella 
corroe, 
Veillonella 
kindergarten and Actinomyces naeslundii for periodontal disease; and streptococci 
mutans , S. sanguis, S. mitior, S. salivarius and S. milleri for dental caries) , 
• 
The concentration of inflammatory cytokines in saliva and blood (interleukin [IL]-
1β, IL-6, IL-8, IL-10, IL-17, leptin, adiponectin, vascular endothelial growth factor 
[VEGF], Interferon [IFN] -γ and Tumor Necrosis Factor [TNF]-α), 
• 
Glucose management (fasting blood glucose (mg/ dL), insulin (mUI/mL), HbA1c 
(%), HOMA-IR, HOMA2-IR, QUICKY, HOMA2%S, HOMA2%B) , 
• 
Adiposity and atherogenic profile [BMI (kg/m²)], waist circumference (cm), 
waist/hip ratio, waist/height ratio, blood pressure ( mmHg ), triglycerides (mg/dL), 

SMILE ORAL DYSBACTERIOSIS IN PEOPLE WITH OBESITY AND PREDIABETES OR DIABETES 
total cholesterol (mg/dL), HDL cholesterol (mg/dL), LDL cholesterol (mg/dL), total 
cholesterol/HDL, LDL/HDL and Triglycerides/HDL ratios) , 
• 
Salivary parameters through sialometry (salivary flow), salivary viscosity, salivary 
pH and the impact profile on oral health (OHIP-14sp; WHO questionnaire) . 
1.10. 
STUDY POPULATION AND TOTAL NUMBER OF PATIENTS 
A total of 60 adult patients are expected to be included: 15 diagnosed with obesity and 
prediabetes with OCD, 15 diagnosed with obesity and prediabetes without OCD, 15 diagnosed 
with obesity and diabetes with OCD, and 15 diagnosed with obesity and diabetes without OCD. 
1.11. 
TEST DURATION 
Twelve months. 
1.12. 
SCHEDULE AND EXPECTED COMPLETION DATE 
• 
Study authorization date: Second quarter of 2023. 
• 
Date of inclusion of the first patient: Fourth quarter of 2023. 
• 
Date of inclusion of the last patient: Second quarter of 2024. 
• 
Study closing date: Fourth quarter of 2024. 
2. GENERAL INFORMATION 
2.1. TEST IDENTIFICATION 
Title and acronym : 
MOLECULAR STUDY OF ORAL DYSBACTERIOSIS IN PEOPLE WITH OBESITY AND 
PREDIABETES OR DIABETES - SMILE 
2.2. DESCRIPTION OF THE MEDICATION/SUBSTANCE UNDER STUDY 
No medication or substance will be tested on patients, this is a descriptive observational 
study 
2.3. DATA RELATING TO THE PROMOTER 
Fundación Pública Andaluza para la Investigación de Málaga en Biomedicina y Salud 
(FIMABIS) 
2.4. DATA RELATING TO THE PRINCIPAL INVESTIGATOR 
• 
Dr. Rodolfo Matias Ortiz Flores 
• 
Dr. Francisco Javier Bermudez Silva 
UGC Endocrinología y Nutrición, Hospital Regional de Málaga - Instituto de 
Investigación Biomédica de Málaga (IBIMA) 
Hospital Civil, Pabellón 2, sótano, Laboratorio de Investigación 
2.5. CENTER WHERE THE TEST IS PLANNED TO BE CARRIED OUT 
Hospital Regional Universitario de Málaga 
Pabellón C (Hospital Civil) 
Plaza del Hospital Civil, s/n 
29009 Málaga. 
 
2.6. TEST DURATION 
Twelve months. 
3. RATIONAL BASIS OF THE ESSAY 
3.1. INTRODUCTION 
According to the International Diabetes Federation, 537 million adults (20-79 years) are 
living with diabetes, 1 in 10 people worldwide. This number is projected to rise to 643 million 

SMILE ORAL DYSBACTERIOSIS IN PEOPLE WITH OBESITY AND PREDIABETES OR DIABETES 
by 2030 and 783 million by 2045 1. Type 2 diabetes (T2D) is the most common type of diabetes, 
accounting for more than 90% of all diabetes worldwide 2,3. It has been estimated that, in 2021, 
541 million adults worldwide (10.6% of the total) had glucose intolerance and some 319 million 
adults (6.2% of the world's adult population), had altered basal glucose (ABG) 1, and it is 
estimated that by the year 2045, these figures will increase to 730 million adults (11.4%) and 
441 million adults (6.9%), respectively 1. 
Both glucose intolerance and ABG are conditions of elevated blood glucose levels 
above the normal range and below the diagnostic threshold for T2D, both situations being 
collectively called prediabetes. Prediabetes implies a greater risk of future development of T2D 
and the appearance of cardiovascular disease (CVD) 3. It is estimated that the cumulative 
incidence of progression of T2D at five years after the diagnosis of glucose intolerance or GBA 
is 26% and 50%, respectively. T2D, in the absence of proper control, can cause damage to 
multiple organs and systems, leading to disabling and life-threatening health complications, 
such as CVD, nerve damage (neuropathy), kidney damage (nephropathy), amputation of lower 
limbs, vision loss and even blindness. Prediabetes and T2D are also important risk factors for 
oral cavity diseases (OCD), with this risk being three times higher than among people without 
diabetes 4,5. Oral health depends on the maintenance of stable microbial communities, appearing 
oral disease occurs when pathogenic species exceed the normal flora 6. 
In the oral cavity, the presence of representative pathogenic bacteria is usually 
associated with two main diseases; streptococcus mutans and Lactobacilli with dental caries, 
and Porphyromonas gingivalis , Fusobacterium nucleatum and Treponema denticola with 
periodontal disease 7. Gingivitis is a frequent form of inflammation of the gums, a step prior to 
periodontitis. ECHO are complications associated with T2D, being generally underestimated 
and underdiagnosed. Interestingly, there is evidence of a bidirectional relationship between the 
T2D condition and said oral conditions, with salivary levels of different pathogens correlated 
with blood glucose levels 8–10. However, although inflammation seems to play a central role, the 
molecular mechanisms that link these pathological conditions are currently poorly understood. 
The endocannabinoid system (ECS) is a lipid signaling machinery that regulates 
various biological processes within the body 11,12. Originally, it was composed of the lipid 
signaling molecules anandamide (AEA) and 2-arachidonoylglycerol (2-AG), two receptors, 
CB1 and CB2, and five enzymes responsible for the biosynthesis and degradation of these 
ligands: N - acyl phosphatidylethanolamine phospholipase D (NAPE-PD), diacylglycerol lipase 
alpha and beta (DAGL) for the synthesis of endocannabinoids, and fatty acid amide hydrolase 
(FAAH) and monoacylglycerol lipase (MAGL) for the catabolism of AEA and 2-AG, 
respectively 13. This original conception of the SEC, however, has turned out to be too 
simplistic. Receptor heterodimers, non-canonical cannabinoid-sensitive receptors, and the 
ability of some ligands to act as receptor allosteric modulators have amplified this signaling 
system.14,15 constituting what is now called an endocannabinoidome 16,17. It is now well known 
that the endocannabinoidome plays a key role in energy homeostasis and related diseases such 
as obesity, prediabetes, and T2D by acting on different organs, including the gut microbiota 
18,19, and modulating key processes such as inflammation and oxidative stress. 
For all these reasons, the present study aims to unravel the possible role of 
endocannabinoids and related lipids as mediators of OCD, which could help to develop new 
therapies based on this signaling system aimed at preventing and treating OCD in the context of 
prediabetes/T2D. 
The association between T2D and periodontitis is bidirectional. Results from 
mechanistic studies indicate that T2D leads to a hyperinflammatory response in the periodontal 
microbiota and also affects inflammation resolution and repair, leading to accelerated 
periodontal destruction. On the other hand, it has been reported that periodontitis negatively 
affects glycemic control in patients with diabetes mellitus and contributes to the development of 
complications 20. Mechanistic links between periodontitis and T2D involve elevations in 
interleukin (IL)-1-β, tumor necrosis factor-α, IL-6, activator of nuclear factor receptor-kappa B 
ligand/ osteoprotegerin, oxidative stress, and expression Toll -Like Receptor (TLR)-2/4 21. 
Therefore, inflammation is a central feature of the pathogenesis of diabetes and periodontitis. 

SMILE ORAL DYSBACTERIOSIS IN PEOPLE WITH OBESITY AND PREDIABETES OR DIABETES 
However, the relationship between the oral microbiota and T2D is less explored. 
Several studies indicate that there are probably subtle differences in the microbial composition 
of the subgingival biofilm between people with and without T2D 22, but the clinical relevance of 
this is not fully clear. Such differences may arise from the effect of T2D by altering the local 
environment within the periodontal pocket, in such a way that the growth of certain bacterial 
species is favored 23. Unfortunately, the specific mechanisms that exacerbate inflammation, oral 
microbiota dysbiosis, and periodontal disease in people with prediabetes and T2D are still 
unknown. 
3.2. JUSTIFICATION 
Diseases of the oral cavity are more frequent in people with prediabetes and diabetes, 
implying a decrease in the quality of life of these patients, adding to the multiple complications 
that these people develop during the progression of the disease. Precisely, one of the possible 
mechanisms that mediate the relationship between inflammation and oral dysbiosis in T2D is 
the endocannabinoidome. The novelty of this proposal consists precisely in studying the 
possible correlation between endocannabinoids and related lipids, inflammatory markers and 
oral dysbiosis in people with prediabetes and T2D, determining the levels of these molecules in 
saliva and blood samples. To date, there are no studies that have addressed the analysis of these 
relationships, which may open the door to new ways to prevent and treat OCD in people with 
prediabetes and T2D. 
3.3. HYPOTHESIS 
For all of the above, our hypothesis is the following: the endocannabinoidome has a 
similar role in the oral microbiota as in the intestinal one, so that its deregulation produces 
dysbiosis and an inflammatory state in the oral cavity, which may be linked to the OCD 
appearance. 
In fact, elevated levels of the endocannabinoids AEA and 2-AG have been found in the 
saliva of obese people 24. Furthermore, ECS has been described in the oral cavity 25–27and 
several studies have demonstrated the presence and physiological relevance of the 
endocannabinoidome in the salivary glands 25,28. 
3.4. AIMS 
Based on this hypothesis, we propose the following main objective: to study the possible 
differences in the levels of endocannabinoids (2-AG, AEA, PEA, OEA, DHEA, 2-LG, 2-
OG; pmol/ml), both in saliva and plasma, in a sample of patients with/without oral cavity 
disease, and with obesity and prediabetes, or with obesity and T2D. 
As secondary objectives will be studied: 
- The bacteriological profile of the oral cavity (increase in the bacterial 16S rRNA amplicon 
, compared to the reference genes, for the following bacterial strains: Aggregatibacter 
actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola 
, Prevotella intermedia, Fusobacterium nucleatum , Parvimonas micra, Campylobacter rectus , 
Eikenella corroe, Veillonella kindergarten and Actinomyces naeslundii for periodontal disease; 
and streptococci mutans , S. sanguis, S. mitior , S. salivarius and S. milleri for dental caries). 
- Inflammatory cytokines in saliva and plasma (interleukin [IL]-1β, IL-6, IL-8, IL-10, IL-
17, leptin, adiponectin, vascular endothelial growth factor [VEGF], Interferon [IFN]-γ and 
Tumor Necrosis Factor [TNF]-α). 
- Glucose management through fasting blood glucose levels (mg/dL), insulin (mUI/mL), 
HbA1c (%), HOMA-IR, HOMA2-IR, QUICKY, HOMA2%S, HOMA2%B. 
- Adiposity and atherogenic profile, through BMI (kg/m²), waist circumference (cm), 
waist/hip ratio, waist/height ratio, blood pressure (mmHg), triglycerides (mg/dL), total 
cholesterol 
(mg/dL), 
HDL 
cholesterol 
(mg/dL), 
LDL 
cholesterol 
(mg/dL), 
total 
cholesterol/HDL, LDL/HDL, and Triglycerides /HDL ratios. 
- Salivary parameters (sialometry (salivary flow), salivary viscosity, salivary pH and the 
impact profile on oral health (OHIP-14sp; WHO questionnaire)). 
 

SMILE ORAL DYSBACTERIOSIS IN PEOPLE WITH OBESITY AND PREDIABETES OR DIABETES 
3.5. BIBLIOGRAPHIC REFERENCES 
1.  
IDF Diabetes Atlas 10th edition. Accessed September 29, 2022. www.diabetesatlas.org 
2.  
Aleixandre MA, Miguel M. Syndrome metabolic . Endocrinology and Nutrition . 2007 ;54 (9). doi: 
10.1016/S1575-0922(07)71487-0 
3.Reaven  
GM . Role of insulin resistance in human disease. diabetes . 1988 ;37 (12). 
doi:10.2337/diab.37.12.1595 
4.  
Bissett SM, Stone KM, Rapley T, Preshaw PM. An exploratory qualitative interview study about 
collaboration between medicine and dentistry in relation to diabetes management. BMJOpen . 2013 ;3 
(2):e002192. doi: 10.1136/BMJOPEN-2012-002192 
5.  
Tavares R da CR, Ortigara GB, Tatsch KF, Ferreira CM, Boligon J, Moreira CHC. Association between 
periodontitis and glycated Hemoglobin levels in individuals living in rural Southern Brazil. Clin Oral 
Investig . 2021 ;25 (12). doi: 10.1007/s00784-021-03980-y 
6.  
Kriebel K, Hieke C, Müller-Hilke B, Nakata M, Kreikemeyer B. Oral biofilms from symbiotic to pathogenic 
interactions and associated disease - Connection of periodontitis and rheumatic arthritis by peptidylarginine 
deiminate . Front Microbiol . 2018 ;9 (JAN). doi:10.3389/fmicb.2018.00053 
7.  
Canepari P, Zerman N, Cavalleri G. Lack of correlation between salivary Streptococcus mutans and 
lactobacilli counts and caries in IDDM children. Minerva Stomatol . 1994 ;43 (11):501-505. Accessed 
September 29, 2022. https://pubmed.ncbi.nlm.nih.gov/7739481/ 
8.  
Estrich CG, Araujo MWB, Lipman RD. Prediabetes and Diabetes Screening in Dental Care Settings: 
NHANES 2013 to 2016. JDR Clin Trans Res . 2019 ;4 (1):76-85. doi: 10.1177/2380084418798818 
9.  
Kocher T, Holtfreter B, Petersmann A, et al. Effect of Periodontal Treatment on HbA1c among Patients with 
Prediabetes. J Dent Res . 2019 ;98 (2). doi: 10.1177/0022034518804185 
10.  
Buczkowska CAEO. Oral Health Problems among Diabetic Patients – Part of Dental Professionals in 
Diagnostic and Therapy. J Oral Hyg Health . 2014 ;02 (06). doi:10.4172/2332-0702.1000167 
11.  
say Marzo V, Silvestri C. Lifestyle and metabolic syndrome: Contribution of the endocannabinoidome . 
Nutrients . 2019 ;11 (8). doi: 10.3390/nu11081956 
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Gatta-Cherifi B, Cota D. New insights on the role of the endocannabinoid system in the regulation of energy 
balance. Int J Obes . 2016 ;40 (2). doi:10.1038/ijo.2015.179 
13.  
Bermudez-Silva FJ, Cardinal P, Cota D. The role of the endocannabinoid system in the neuroendocrine 
regulation 
of 
energy 
balance. 
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of 
Psychopharmacology 
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2012 
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(1). 
doi: 
10.1177/0269881111408458 
14.  
say March V. New approaches and challenges to targeting the endocannabinoid system. Nat Rev Drug 
Discov . 2018 ;17 (9). doi:10.1038/nrd.2018.115 
15.Hillard  
CJ. Circulating Endocannabinoids: From Whence Do They Come and Where are They Going? 
Neuropsychopharmacology 2018 43:1 . 2017 ;43 (1):155-172. doi:10.1038/npp.2017.130 
16.  
Arturo IF, Fabiana P. Endocannabinoidome . eLS . Published online November 16, 2018:1-10. 
doi:10.1002/9780470015902.A0028301 
17.  
Davis MP. Overview of the Endocannabinoid System and Endocannabinoidome . In: Cannabis and 
Cannabinoid-Based Medicines in Cancer Care . ; 2022. doi: 10.1007/978-3-030-89918-9_1 
18.  
Cani PD, Plovier H, van Hul M, et al. Endocannabinoids-at the crossroads between the gut microbiota and 
host metabolism. Nat Rev Endocrinol . 2016 ;12 (3). doi:10.1038/nrendo.2015.211 
19.  
Romero- Zerbo SY, Bermúdez-Silva FJ. Cannabinoids, eating behavior, and energy homeostasis. Drug Test 
Anal . 2014 ;6 (1-2). doi:10.1002/dta.1594 
20.  
Lalla E, Papapanou PN. Diabetes mellitus and periodontitis: a tale of two common interrelated diseases. Nat 
Rev Endocrinol . 2011 ;7 (12):738-748. doi:10.1038/NRENDO.2011.106 
21.  
Sanz M, Ceriello A, Buysschaert M, et al. Scientific evidence on the links between periodontal diseases and 
diabetes: Consensus report and guidelines of the joint workshop on periodontal diseases and diabetes by the 
International Diabetes Federation and the European Federation of Periodontology. j clin Periodontol . 2018 
;45 (2). doi:10.1111/jcpe.12808 
22.  
Rohani B. Oral manifestations in patients with diabetes mellitus. World J Diabetes . 2019 ;10 (9). 
doi:10.4239/wjd.v10.i9.485 
23.  
Thorstensson H, Dahlén G, Hugoson A. Some suspected periodontopathogens and serum antibody response 
in adult long‐duration insulin‐dependent diabetics. j clin Periodontol . 1995 ;22 (6). doi:10.1111/j.1600-
051X.1995.tb00176.x 
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Takahashi K, Nishimura F, Kurihara M, et al. Subgingival microflora and antibody responses against 
periodontal bacteria of young Japanese patients with type 1 diabetes mellitus. J Int acad Periodontol . 2001 
;3 
(4):104-111. 
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29, 
2022. 

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https://neuro.unboundmedicine.com/medline/citation/12666949/Subgingival_microflora_and_antibody_resp
onses_against_periodontal_bacteria_of_young_Japanese_patients_with_type_1_diabetes_mellitus_ 
25.  
Prestifilippo JP, Fernández-Solari J, de La Cal C, et al. Inhibition of Salivary Secretion by Activation of 
Cannabinoid Receptors: https://doi.org/101177/153537020623100816 . 2016 ;231 (8):1421-1429. doi: 
10.1177/153537020623100816 
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Matias I, Gatta-Cherifi B, Tabarin A, et al. Endocannabinoids Measurement in Human Saliva as Potential 
Biomarker of Obesity. PLoS One . 2012 ;7 (7):e42399. doi:10.1371/JOURNAL.PONE.0042399 
27.  
Piscitelli F, di March V. “Redundancy” of endocannabinoid inactivation: New challenges and opportunities 
for pain control. In: ACS Chemical Neuroscience . Vol 3. ; 2012. doi: 10.1021/cn300015x 
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Andreis K, Billingsley J, Naimi Shirazi K, et al. Cannabinoid CB1 receptors regulate salivation. Sci Rep . 
2022 ;12 (1). doi: 10.1038/S41598-022-17987-2 
29.  
Oral health. Global Burden of Disease Study 2019. Published 2019. Accessed September 30, 2022. 
https://www.who.int/news-room/fact-sheets/detail/oral-health 
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4. STUDY DESIGN 
4.1. TYPE OF STUDY 
Descriptive observational study. 
A study with four groups is proposed: a group of obese diabetic subjects with a healthy 
mouth, a group of obese diabetic subjects with a diseased mouth, a group of obese pre-diabetic 
subjects with a healthy mouth, and a group of obese pre-diabetic subjects with sick mouth. All 
patients will be recruited on a voluntary and unpaid basis. After a meeting with each participant, 
where a detailed explanation about the study will be given, written informed consent will be 
obtained, as well as the necessary information to complete the medical evaluation questionnaire, 
which will allow determining their state of health and the criteria of inclusion and exclusion. 
Once the patient's eligibility is confirmed, they will be scheduled to fast for a saliva and blood 
extraction and an oral health questionnaire and/or an oral clinical examination. 
The samples of the patients included in the study will be processed and frozen at -80ºC until 
their subsequent analysis following standardized protocols. The samples will be stored in the 
biobank of the Hospital Regional de Málaga-IBIMA, which is part of the biobank of the public 
health system of Andalucía (BSSPA). 
5. SUBJECT SELECTION 
5.1. STUDY POPULATION 
A population of patients of both sexes aged 40-65 years, with obesity and diagnosed with 
pre-diabetes, and with obesity and diabetes, and who meet the rest of the inclusion criteria, as 
well as who do not present any of the criteria, will be studied. of exclusion, and who agree to 
participate voluntarily in the study. For this, three studies led by our unit will be based on: The 
Pizarra study, an epidemiological study of the UGC of Endocrinología y Nutrición del Hospital 
Regional de Málaga that has provided detailed information on anthropometric, biochemical, 
eating habits and style parameters. of life in a cohort of about 1,700 subjects, the di@bet.es 
study, a national epidemiological study carried out on a sample of more than 5,000 people, 
coordinated by this Unit, and the April study, a nutritional intervention study on people with 
obesity and prediabetes. In addition, the databases of the obesity consultations of the UGC of 
Endocrinología y Nutrición del Hospital Regional de Málaga will be available. Based on these 
databases, we will proceed to contact the people who could meet the inclusion criteria, in order 
to arrange a recruitment visit for the study, following the guidelines of the Declaration of 
Helsinki (Fortaleza 2013) and the Standards of Good Clinical Practices. Personal data will be 
processed in accordance with REGULATION (EU) 2016/679 OF THE EUROPEAN 
PARLIAMENT AND OF THE COUNCIL of April 27, 2016 on the protection of natural 
persons with regard to the processing of personal data and the free movement of these dates; and 
Organic Law 3/2018, of December 5, Protection of Personal Data and guarantee of digital rights 

SMILE ORAL DYSBACTERIOSIS IN PEOPLE WITH OBESITY AND PREDIABETES OR DIABETES 
5.2. SAMPLE SIZE 
Based on previous publications that have reported the levels of endocannabinoids in saliva 
(20), at least 12 subjects are needed to detect as significant differences of 0.5pmol/ml of AEA 
and 25pmol/ml of 2-AG, which are the differences found between healthy people and obese 
people. It is intended to recruit 15 people with obesity, prediabetes and a healthy mouth, 15 
people with obesity, prediabetes and a diseased mouth, 15 people with obesity, diabetes and a 
healthy mouth, and 15 people with obesity, diabetes and diseased mouth. To differentiate a 
healthy mouth from a diseased mouth, a complete oral evaluation will be carried out on all 
participants, which includes a review of the mucous membranes, an evaluation of the bacterial 
plaque and the presence of dental caries, and an evaluation of the health of the teeth. gums, 
including the depth of probing and the presence of gingival bleeding (see point 5.6). 
5.3. INCLUSION CRITERIA 
• 
Adults (40-65 years) 
• 
With obesity and prediabetes: BMI 30-40 and HbA1c 5.7-6.4 
• 
With obesity and diabetes: BMI 30-40 and previous diagnosis of diabetes 
 
5.4. EXCLUSION CRITERIA 
• 
Pregnant women 
• 
Diagnosis of some type of neoplasia or treatment with radiotherapy and/or 
chemotherapy in the last year. 
• 
Ongoing inflammatory diseases (Crohn's disease, ulcerative colitis, arthritis, etc.) and/or 
anti-inflammatory treatments 
• 
Presence of systemic diseases of vital organs 
• 
Participants in treatment with  drugs that could alter salivary flow 
• 
Smokers. 
• 
Participants who have not followed the specifications prior to sampling 
• 
Participants who did not sign the informed consent 
 
5.5. ELIMINATION CRITERIA 
• 
Inadequate amount of saliva to perform the tests 
 
5.6. HEALTHY MOUTH vs DISEASE MOUTH CRITERIA 
The differentiation between a healthy mouth and a diseased mouth will be made through a 
detailed observation and a clinical examination carried out by a dentist. This will be done in 
accordance 
with 
the 
guidelines 
of 
the 
World 
Health 
Organization 
(WHO; 
https://www.who.int/publications/m/item/draft-global-oral-health-action-plan-[2023-2030]). 
The signs that indicate a healthy mouth are: 
• 
Firm, pink gums that do not bleed during brushing or flossing. 
• 
Clean teeth without stains, without cavities or fractures. 
• 
Absence of pain or dental sensitivity. 
• 
No presence of bad breath or halitosis. 
• 
No signs of inflammation or infection in the gums or around the teeth. 
On the other hand, the signs that indicate a sick mouth are: 
• 
Red, swollen or bleeding gums. 
• 
Teeth with stains, cavities or fractures. 
• 
Tooth pain or sensitivity, especially when biting or chewing food. 
• 
Bad breath or chronic halitosis. 
• 
Presence of abscesses or infections in the teeth or gums. 
 

SMILE ORAL DYSBACTERIOSIS IN PEOPLE WITH OBESITY AND PREDIABETES OR DIABETES 
In the present study, based on these criteria, the dentist will make the diagnosis of "without oral 
cavity disease" or "with oral cavity disease" for each subject. 
 
5.7. SUBJECT IDENTIFICATION 
Patients will be identified with a sequential numerical identification code, which will be 
assigned to each patient according to the correlative order of inclusion when they give their 
informed consent. In the event that a patient is included in the study and is considered unfit to 
continue in the study or the patient withdraws their consent, the code assigned to that patient 
cannot be reused for the next consecutively assigned patient. 
The promoter will only be able to identify the subjects by the code that has been assigned to 
them, their date of birth, sex and date of signing the informed consent. The principal 
investigator will keep a record, protected by password, with the names of the patients and the 
assigned identification code. 
 
6. DEVELOPMENT OF THE TRIAL AND EVALUATION OF THE 
RESPONSE 
6.1. ASSESSMENT OF EFFICACY 
Not applicable. 
6.2. SAFETY ASSESSMENT 
Not applicable. 
6.3. DEVELOPMENT OF THE TEST 
Selection of patients and survey. Participants will attend the recruitment appointment after 
fasting for at least 8 hours. After verifying that they meet the inclusion and exclusion criteria 
and sign the informed consent, they will be given a survey that collects data on affiliation and 
lifestyle habits related to health and daily oral hygiene, including smoking and alcohol 
consumption; medical history that includes the medication taken, last visit to the dentist and risk 
factors and symptoms of oral disease, through questions that are easy to answer when self-
identifying general symptoms compatible with gingivitis, periodontitis and dental caries (eg 
localized tooth sensitivity and in gums, bleeding gums, loose teeth, hypersensitivity in teeth to 
cold and/or heat, persistent bad breath, elongated teeth with receding and/or separated gums, 
modified bite, dry mouth, change of color in teeth, etc.). 
Collection of salivary and blood samples. After completing the medical history, all 
patients will have their anthropometric data such as height, weight, waist circumference, hip 
circumference, systolic blood pressure, and diastolic blood pressure recorded. The research team 
dentist will perform a complete oral health diagnosis on the study subjects, using the World 
Health Organization (WHO) guidelines for diagnosing oral cavity disease 29 ,30. Likewise, the 
oral health questionnaire will be passed. After the oral examination, saliva samples will be 
collected under resting conditions using the passive drooling method, in 15 mL tubes , every 1 
min, for 5 min in the same tube. Together with the saliva samples, trained personnel will collect 
9 ml of blood samples , under resting conditions, in EDTA tubes. The tubes will be centrifuged 
and the supernatant will be collected and stored at -80ºC in the Biobank. 
Analysis and detection of eCBs levels and profile of inflammatory biomarkers. In 
saliva and plasma, the analysis and detection of endocannabinoids and related lipids (2-
arachidonoyl-glycerol [2-AG], N- arachidonoylethanolamine [AEA], N- palmitoylethanolamine 
[PEA], N- oleoylethanolamine [OEA], N- docosahexanoylethanolamine [DHEA], 2-
linoleoylglycerol, [2-LG] and 2-oleoylglycerol [2-OG]). The analysis and detection of these 
factors will be carried out by HPLC chromatography. Inflammatory markers will also be 
quantified in saliva and plasma (interleukin [IL]-1β, IL-6, IL-8, IL-10, IL-17, leptin, 
adiponectin, vascular endothelial growth factor [ VEGF ], Interferon [IFN]-γ and Tumor 
Necrosis Factor [TNF]-α), using Procarta multiplex assay (TermoFisher Scientific) following 
the manufacturer's instructions. In addition, biochemical parameters such as blood glucose, 
insulin, HbA1c, triglycerides , total cholesterol, HDL cholesterol, LDL cholesterol will be 
analyzed in the hospital laboratory in the blood samples . 

SMILE ORAL DYSBACTERIOSIS IN PEOPLE WITH OBESITY AND PREDIABETES OR DIABETES 
Analysis of oral bacteriological profiles. The bacteriological profiles in the saliva 
samples will be analyzed using massive sequencing techniques and amplification of the 
bacterial 16S rRNA amplicon of the following microbial strains: Aggregatibacter 
actinomycetemcomitans, 
Porphyromonas 
gingivalis, 
Tannerella 
forsythia, 
Treponema 
denticola, Prevotella intermedia, Fusobacterium nucleatum, Parvimonas micra, Campylobacter 
rectus , Eikenella corroe, Veillonella parvula and Actinomyces naeslundii for periodontal 
diseases ; and streptococci mutans , S. sanguis, S. mitior, S. salivarius and S. milleri for tooth 
decay. In addition, saliva will be analyzed for sialometry (salivary flow), salivary viscosity, 
salivary pH and the impact profile on oral health (OHIP-14sp; WHO questionnaire). 
7. STATISTIC ANALYSIS 
7.1. COLLECTED VARIABLES 
The following groups of variables are included in this essay: 
• 
Anthropometric data: weight, height, body mass index, waist circumference, hip 
circumference, blood pressure. 
• 
Clinical data: Previous or ongoing illnesses, current medication. 
• 
Blood test data: Standard biochemistry (plasma glucose, insulin, plasma HbA1c, lipid 
profile, renal function, liver function, lymphocytes, hs-CRP, etc.), inflammatory 
markers (panel of inflammatory cytokines, including IL-6 , IL- 1beta, and TNF-alpha), 
endocannabinoids (2-AG, AEA, PEA, OEA, DHEA, 2-LG, 2-OG). 
• 
Saliva data: Salivary flow (to calculate unstimulated oral saliva flow), salivary 
viscosity, salivary pH, salivary protein concentration. Inflammatory markers (panel of 
inflammatory cytokines , including IL-6, IL-1beta and TNF-alpha), endocannabinoids 
(2-AG, AEA, PEA, OEA, DHEA, 2-LG, 2-OG), Bacteriological profiles. In addition, 
the oral health impact profile (OHIP-14sp) will be evaluated, both through the dental 
examination and through questions. 
7.2. STATISTICAL ANALYSIS 
For the statistical study, the statistical software package SPSS 25.0 will be used. A database 
equivalent to the data collection notebook will be created with all the variables. First, the sample 
will be studied (descriptive statistics) using absolute and relative frequencies and then 
inferential statistics will be performed. The parametricity of the variables will be studied using a 
normality test. The parametric analysis will be carried out using ANOVA, followed by the 
corresponding post-test analysis. If the distribution of the variable is not normal, it will be 
converted to log or a non-parametric test will be used to contrast the differences. All hypothesis 
contrasts will be made with a significance level of 5%. 
8. ADVERSE EVENTS 
Given the nature of this trial, no type of adverse event or adverse reaction is anticipated. 
9. ETHICAL ASPECTS 
The collection and processing of human saliva and peripheral blood samples and personal 
and anthropometric data, as well as the data obtained in the aforementioned tests will be carried 
out in accordance with current legal regulations on the use of human samples, preserving and 
codifying adequately the identity of the subjects involved in the study. A copy of the informed 
consent to be signed by each subject included in the study is attached. This study will not begin 
until it is informed and approved by the Provincial CEI of Malaga. Once the trial is finished, the 
rest of the human samples obtained in this study will be stored in the Biobank of the Hospital 
Regional Universitario de Málaga - IBIMA (which is part of the Biobank of the SSPA), so that 
they can be used by any researcher who considers them useful. 
 

CONSENTIMIENTO INFORMADO - INFORMACIÓN AL PACIENTE 
Antes de proceder a la firma de este consentimiento informado, lea atentamente la información 
que a continuación se le facilita y realice las preguntas que considere oportunas. 
Nombre del Estudio: 
ESTUDIO MOLECULAR DE LA DISBACTERIOSIS ORAL EN PERSONAS CON 
PREDIABETES Y DIABETES - SMILE 
Naturaleza: 
Las enfermedades de la cavidad oral son más frecuentes en personas con prediabetes y diabetes, 
suponiendo una disminución de la calidad de vida de estos pacientes, uniéndose a las múltiples 
complicaciones que desarrollan estas personas durante la progresión de la enfermedad. Por tanto, 
el Hospital Regional de Málaga está realizando un estudio que permitan aclarar esta relación y 
descubrir nuevas dianas moleculares que permitan prevenir y/o tratar estas afecciones. Para ello, 
los participantes acudirán a la cita de reclutamiento tras un ayuno de al menos 10 horas y se les 
entregará una encuesta a rellenar, que recoge datos de filiación y hábitos de estilo de vida 
relacionados con la salud y la higiene oral diaria. Luego se procede a la recogida de una muestra 
de sangre venosa, y de repetidas muestras (una por cada minuto) de saliva total (en reposo) 
utilizando el método de babeo pasivo, durante 5 min. 
Importancia: 
Toda la información recogida servirá para determinar el vínculo causal entre la disglicemia y las 
enfermedades de la cavidad oral. El sistema endocannabinoide podría ser el mediador de estas 
alteraciones, siendo asi diana terapéutica potencial. Este estudio persigue dilucidar si el sistema 
de señalización intercelular por endocannabinoides está alterado en la cavidad oral de personas 
con prediabetes y diabetes que tienen enfermedades de la cavidad oral. 
Implicaciones para el donante/paciente: 
La donación/participación es totalmente voluntaria. 
El donante/paciente puede retirarse del estudio cuando así lo manifieste, sin dar explicaciones y 
sin que esto repercuta en sus cuidados médicos. 
• Todos los datos carácter personal, obtenidos en este estudio son confidenciales y se tratarán
conforme a la Ley Orgánica de Protección de Datos de Carácter Personal 15/99.
• La donación/información obtenida se utilizará exclusivamente para los fines específicos de este
estudio.
Riesgos de la investigación para el donante/paciente: 
La donación de sangre apenas tiene efectos secundarios; lo más frecuente es la aparición de 
pequeños hematomas en la zona de punción que desaparecen transcurridos 1 ó 2 días. 
Si requiere información adicional se puede poner en contacto con nuestro personal 
de Investigación en el teléfono: 951 29 02 26 o en el correo electrónico: smile@ibima.eu. 

 
CONSENTIMIENTO INFORMADO - INFORMACIÓN AL PACIENTE 
 
Estudio molecular de la disbacteriosis oral en personas con prediabetes y diabetes - 
SMILE 
 
Yo (Nombre y Apellidos):_______________________________________ 
 
• 
He leído el documento informativo que acompaña a este consentimiento (Información al 
Paciente).  
• 
He podido hacer preguntas sobre el Estudio molecular de la disbacteriosis oral en 
personas con prediabetes y diabetes - SMILE. 
• 
He recibido suficiente información sobre el Estudio molecular de la disbacteriosis oral 
en personas con prediabetes y diabetes - SMILE. 
• 
He hablado con el profesional sanitario informador:_____________________________. 
• 
Comprendo que mi participación es voluntaria y soy libre de participar o no en el estudio. 
• 
Se me ha informado que todos los datos obtenidos en este estudio serán confidenciales y 
se tratarán conforme establece la Ley Orgánica de Protección de Datos de Carácter 
Personal 15/99. 
• 
Se me ha informado de que la donación/información obtenida sólo se utilizará para los 
fines específicos del estudio. 
• 
Deseo ser informado/a de los datos que se obtengan en el curso de la investigación, 
incluidos los descubrimientos inesperados que se puedan producir, siempre que esta 
información sea necesaria para evitar un grave perjuicio para mi salud o la de mis 
familiares biológicos. 
 
 Si   
 No 
 
Comprendo que puedo retirarme del estudio: 
• 
Cuando quiera 
• 
Sin tener que dar explicaciones 
• 
Sin que esto repercuta en mis cuidados médicos 
 
Presto libremente mi conformidad para participar en el proyecto titulado 
Estudio molecular de la disbacteriosis oral en personas con prediabetes 
y diabetes - SMILE. 
 
 
 
 
 
Firma del paciente 
(o representante legal en su caso) 
 
 
 
 
 
 
Nombre y apellidos:___________________________ 
Fecha:  _____________________________________  
 
Firma del profesional 
sanitario informador 
 
 
 
 
 
 
Nombre y apellidos:___________________________ 
Fecha:  _____________________________________