Salivary and serum enzymes as diagnostic biomarkers at patients with periodontal disease
Daniela Miricescu1, PhD, Alexandra Totan1, PhD, Bogdan Calenic1*, PhD, Brânduşa Mocanu2, PhD and Maria Greabu1, PhD
1Department of Biochemistry, Faculty of Dental Medicine, Carol Davila, University of Medicine and Pharmacy, Bucharest, Romania
2Department of Periodontology, Faculty of Dental Medicine, Carol Davila, University of Medicine and Pharmacy, Bucharest, Romania
1Teaching Assistant, Department of Biochemistry, Faculty of Dental Medicine, “Carol Davila”, University, 8 Blvd Eroii Sanitari st, Sector 5, Bucharest,RO-050474, Romania
1Lecturer, Department of Biochemistry, Faculty of Dental Medicine, “Carol Davila”, University, 8 Blvd Eroii Sanitari st, Sector 5, Bucharest, RO-050474, Romania
2Teaching Assistant, Department of Periodontology, Faculty of Dental Medicine, “Carol Davila”, University, 19 Calea Plevnei st, Sector 1, Bucharest, RO- 010221, Romania
1Professor, Head of Department, Department of Biochemistry, Faculty of Dental Medicine, “Carol Davila”, University, 8 Blvd Eroii Sanitari st, Sector 5, Bucharest,RO-050474, Romania
* Corresponding author: Calenic Bogdan, DDS, PhD; Address: 8 Blvd Eroii Sanitari, Sector 5, RO-050474 Bucharest, Romania; Telephone: +40755044047; Fax:021 3110984, E-mail: firstname.lastname@example.org
Introduction: Periodontal disease is one of the most widespread oral disease.
Aim: The general aim of the present study was to detect salivary and serum enzyme activities in response to periodontal infection, involving the release of enzymes from stromal, epithelial, inflammatory and bacterial cells.
Methodology: We included 20 patients with chronic periodontitis and 20 controls. Unstimulated whole saliva and serum was used to detect the following enzymes: aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and gamaglutamil transferase (GGT). Periodontal status was established using classic clinical parameters, plaque index, bleeding index and probing depth.
Results: In patient’s saliva with periodontal disease we detected significantly decreased levels of LDH and ALP. Salivary level for GGT was decreased and for AST were increased but no statistical significance was found when compared to controls. Significantly increased levels for serum ALP and GGT were observed in patients with periodontal disease. At the same time no statistical difference was found between controls and periodontitis patients for AST and LDH.
Discussions: Our results reflect changes of intracellular enzymes both in saliva and serum, at patients with periodontal disease group versus healthy subjects.
Conclusions: The activity of this enzymes in saliva and serum of patients with periodontal disease can be useful in diagnosis, monitorization and treatement of this inflammatory disease.
Keywords: saliva, intracellular enzymes, periodontal infection
Introducere: Boala parodontală este una dintre cele mai răspândite afecţiuni orale.
Scop: Principalul obiectiv al acestui studiu, a fost detectarea activităţilor enzimatice serice şi salivare, ca răspuns la boala parodontală, afectiune care implică eliberarea de enzime din celulele stromale, epiteliale, inflamatorii și bacteriene.
Metodologie: În cadrul acestui studiu, am inclus 20 de pacienţi cu parodontopatie cronică şi 20 de subiecţi sănătoşi. Am recolatat salivă totală nestimulată și ser pentru a detecta următoarele enzime: aspartat aminotransferaza (AST), lactat dehidrogenaza (LDH), fosfataza alcalina (ALP) și gamaglutamil transferaza (GGT).
Statusul parodontal a fost stabilit, utilizându-se următorii parametrii clinici: indicele de placă, indicele de sângerare si adâncimea pungilor parodontale.
Rezultate: În saliva pacienţilor cu boală parodontală am detectat un nivel statistic scăzut de LDH și ALP, comparativ cu lotul subiecţilor sănătoşi. Nivel salivar pentru GGT a fost scăzut, iar pentru AST crescut, dar nesemnificative statistic, la pacienţii cu boală parodontală, versus control. Nivele serice statistic crescute au fost observate pentru ALP și GGT la pacienţii cu parodontopatii. În același timp, nu am fost detectat nici o diferenţă semnificativ statistică între subiecţi sănătoşi si pacienţi cu boală parodontală în cazul valorilor obţinute în ser pentru AST și LDH.
Discuții: Rezultatele studiului nostru indică modificări ale enzimelor intracelulare atât în saliva cât şi în serul pacienţilor cu boală parodontală versus lotul subiecţilor sănătoşi.
Concluzii: Activitatea acestor enzime în saliva şi serul pacienţilor cu boală parodontală poate fi utilă în diagnosticul, monitorizarea și tratarea acestei boli inflamatorii.
Cuvinte cheie: saliva, enzime intracelulare, infectie parodontală
Whole saliva is secreted by three major and many minor glands. It contains locally produced biomolecules as well as other molecules derived from systemic circulation. Whole saliva can contain proteins, serum products, electrolytes immune and epithelial cells and gingival crevicular fluid (GCF) [1, 2].
Periodontal disease is one of the most widespread oral disease. It is a chronic bacterial infection characterized by inflammation, connective tissue breakdown and finally alveolar bone loss . An response of an organism to inflammation such as periodontal disease, includes the release of several enzymes from stromal, bacterial, inflammatory and epithelial cells .
The intracellular enzymes are released from damaged cells of periodontal tissues into the GCF, saliva and surrounding fluids .
The most important enzymes are : aspartate and alanine aminotransferases (AST and ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), gamaglutamil transferase (GGT), creatine kinase (CK) and acidic phosphatase (ALP) [4-6].
The main aim of our study is to detect the activities of AST, LDH, ALP and GGT in saliva and serum of patients with periodontal disease.
This study was reviewed and approved by the ethics board of University of Medicine and Pharmacy Carol Davila, Faculty of Dental Medicine. Informed consent was obtained from each participant who agreed to participate voluntarily in this study. The study was carried in 20 patients with chronic periodontitis (5 males and 15 females, with a mean age of 51.26±7.4).
Twenty healthy subjects with no gingival inflammation, good oral hygiene and no history of periodontal disease were grouped as controls. All patients included in the study fulfilled the following criteria: absence of any systemic disease; no use of systemic medications like antibiotics within the preceding three months, anti-inflammatory drugs or anti-oxidant drugs, no history of tobacco usage or alcoholism. Periodontal status was determined by measuring PD (probing depth), gingival index (GI) and plaque index (PI).
The subjects included in this study, were told not to eat or drink anything in the morning before collection the samples. Unstimulated whole saliva was collected into sterile tubes between 9 and 10 a.m. after a single mouth rise with 10 mL of distilled water to wash out exfoliated cells. About 2 mL of unstimulated whole saliva collected was immediately centrifuged at 3000 rpm for 10 min to remove cell debris. The supernatant was kept and stored in small aliquots at -80ºC until further analysis.
Serum sampling At the same time 5 ml of blood were collected and the serum obtained was used for our determinations.
All biomarkers were performed using the kinetic method at an automatic analyzer A15 Biosystems, Spain.
Serum and salivary AST: AST catalyzes the transfer of amino groups from aspartate molecule to 2-oxoglutaratului, forming oxaloacetate and glutamate. Enzyme activity is determined by measuring the decrease in NADH concentration at 340nm by the reaction catalyzed by malate dehydrogenase (MDH).
Principle: Aspartam + 2-oxoglutarat → glutamate + oxaloacetate
Oxaloacetate + NADH + H + → L-malate + NAD +
Salivary and serum assay for ALP.Under ALP action of p-nitrophenilphosphate (colorless) is converted to p-nitrophenol, the yellow colored compound. The color intensity is proportional to the activity of ALP in the sample.
Principle: p-Nitro-phenylphosphate + H20 → p-Nitrophenol + Phosphate
Salivary and serum assay for LDH
L-lactate + pyruvate + NAD + ← → NADH, H +
Salivary and serum assay for GGT. GGT- catalyzes the transfer of γ-glutamate from L-γ-glutamyl-3-carboxy-4-nitroanilida in glycylglycine to form L-γ-glutamilglicilglicina and 5-amino-2-nitrobenzene yellow. The absorbance is read at 405 nm
Principle: L-γ-glutamyl-3-carboxy-4-nitroanilida + glycylglycine – → L-γ-glutamilglicilglicina + 5 amino2-nitrobenzene
Data distributions were expressed as means, standard deviations (SD), ranges, and percentages, as appropriate. The Pearson’s correlation coefficient and ANOVA test were used. The data were analysed statistically on the computer using StataIC 11 (StataCorp. 2009. Stata: Release 11. StatisticalSoftware. College Station, TX, USA). A p-value < 0.05 was considered statistically significant.
Patients and healthy controls were investigated for plaque index (PI), bleeding index (GI), probing depth (PD) ( p<0.05). Results are detailed in Table 1.
In patient’s saliva with chronic periodontitis we detect an significantly decreased levels for LDH and ALP. Salivary level for GGT was decreased and for AST was increased but both were statistically insignificant, compared with controls (table 2).
Significantly increased levels for serum ALP and GGT were obtained from patients with periodontal disease. Increased serum levels for AST and LDH were also found (Table 3).
During the past few decades, many biomarkers have been proposed for the diagnosis of periodontal disease, such as intracellular enzymes. Their activity can be detected in unstimulated saliva and in GCF. If the periodontal tissue is damaged or if the cells are affected due to edema or cellular damage, intracellular enzymes are released into the GCF and saliva [4, 7].
LDH, AST, GGT are intracellular enzymes also present in oral soft tissues. Their activity is increased in saliva as a consequence of release from damaged cells in periodontal soft tissue and inflamed gums. In our study these enzymes have been detected in unstimulated whole saliva .
ALP is an intracellular enzyme present predominantly in bone, being the first enzyme determined in periodontal disease [7, 8] . Dabra et al. detected an increased level of this enzyme in stimulated saliva of patients with periodontal disease (6). Todorovic et al. also reported an increased activity of ALP in unstimulated saliva of 187 patients with periodontal disease .
In our study we obtained a statistically significant decrease of salivary of ALP in patients with periodontal disease compared to healthy patients group. Studies have shown a remarkable increase of this enzyme in the acute phase of periodontal disease [7-10]. Increased activity in saliva is probably a consequence of destructive processes in the alveolar bone in advanced stages of periodontal disease.
Yan F et al. considered that ALP was increased in acute phase of periodontal disease, suggesting that periodontal disease is well advanced  .
Salivary enzymatic activity of ALP significantly decreases during periodontal ligament attachment loss and bone resorption. Release of cytokines (IL-1β) during bone resorption may inhibit proliferation periodontal ligament cells such as osteoblasts . LDH level was also significantly decreased in saliva of patients with periodontal disease compared to healthy patients group.
Zappacosta B et al, detected an increased salivary level of LDH at patients with periodontal probing pocket depth > 5mm . Atici et al measured LDH in GCF and observed that the progression of periodontal disease is associated with this enzyme .
GGT is a key enzyme , which can be regarded as a new important oxidative stress biomarker and a indicator of cellular damage [15,8]. Dabra et al. detected an increased level of GGT in stimulate saliva of patients with periodontal disease . Todorovic et al. obtained and increased level of GGT in unstimulated saliva. In our study we detected decreased levels of GGT but without significant difference . The decreased level of GGT, may reflect the presence of oral oxidative stress in patients with periodontitis.
The only enzyme that showed increased levels in unstimulated whole saliva of patients with periodontitis was AST. Increased levels of AST were also reported by Todorovic et al. in unstimulated saliva of patients with periodontal disease .
The results obtained in our study may have several explanations:
After determining these intracellular enzymes patients included in the study may or may not be in an advanced stage of periodontal disease.
At the time of collection of biological samples, it is possible that patients were in different stages of periodontal disease. Our study used unstimulated whole saliva vs GCF because saliva collection has several clear avantages: no need for specialized equipment or techniques, much faster and more convenient for both the patient and medical staff.
Because whole saliva contains numerous biomarkers derived from all structures of the oral cavity, analyzing biomarkers in saliva may provide a thorough overview of the periodontal status compared with GCF. Studies show that different results are due primarily to different processing methods saliva. Therefore the study design requires careful standardization in the collection and processing of saliva.
Numerous studies show that there is a direct relationship between periodontal complications and many systemic diseases such as cardiovasculat disease, metabolic syndrome or diabetes [16-20]. Another aim of the present study was to test the hypothesis that periodontal disease can influence general health by analyzing enzymatic levels.
In serum of patients with periodontal disease we have obtained a series of changes in enzyme activity of ALP, LDH, GGT and AST in patients with periodontal disease when compared with the control group. ALP is present specially in bones and liver, duodenum and kidney. Increased level of this enzyme has been recorded in skeletal damage associated with osteoblastic reaction, cholestasis [21, 22]. Overall our results show that enzyme serum levels in patients with periodontal disease are statistically increased when compared to healthy patients group. Previous studies show an association between periodontal disease and osteoporosis, especially in postmenopausal women [23, 24]. Our group of patients with periodontal disease has 15 females, with the average age over 50 years, so they present higher risk of osteoporosis.
LDH is present especially in muscle, liver, myocardium, kidney and erythrocytes. Marked increase of LDH enzyme activity is found in myocardial infarction, toxic liver damage or testicular cancer. Moderate increments of LDH were also found in muscle disease, hemolysis, malignant lymphoma [22, 25].
In our experiments LDH was increased but not statistically significant. This increase in LDH may be a warning sign even it was not a statistically significant value. Beck and colleagues have postulated a connection between periodontal disease and atherosclerosis, so people suffering from periodontal disease may be at increased risk of atherosclerosis .
GGT is present in kidney, pancreas, liver. Significant increases of GGT activity has been recorded in cholestasis, alcoholism, hepatic tumors. Moderate increases were observed for chronic hepatitis, pancreatitis . In our experiments GGT levels were statistically increased in serum from patients with periodontal disease versus healthy subjects. AST is a widespread enzyme, mainly localized in the liver, myocardium or muscle, but also being present in small amounts in the lungs, kidneys, pancreas and erythrocytes. Marked increments of AST are present in myocardial infarction, acute hepatitis or toxic liver damage. Moderate increases are observed in patients with chronic hepatitis, infectious mononucleosis [22, 27]. The enzymatic activity of AST was increased but without statistical significance in serum of patients with periodontal disease versus healthy group.
In conclusion the salivary and serum enzymes detected in our study can be useful in monitorization of patients with periodontal disease.
This study was supported by the Sectorial Operational Programme Human Programme Human Resources Development (SOP HRD), financed from the European Social Fund and by the Romanian Government under the contract number POSDRU/6/1.5/S/S17.
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