RU2706238C1 - Method for determining intensity of inflammatory-destructive changes of periodontal tissues in periodontitis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to dentistry, and can be used for diagnosing the intensity of inflammatory-destructive changes (IIDC) periodontal tissues in periodontitis. Determination method IIDC periodontal tissues in periodontitis consists in examination of oral liquid by spectrophotometric method, as an indicator IIDC in periodontium tissues concentration of free hydroxyproline is determined, and as an index of reparative restoration of periodontium tissues, the concentration of protein-bound hydroxyproline is determined, and if the values are <1.0 mcg/ml and >20.5 mcg/ml, the absence of a pathology of oral tissues is diagnosed; at values 1.1–1.8 mcg/ml and 15.5–20.4 mcg/ml respectively diagnosed low IIDC; with values of 1.81–2.5 mcg/ml and 11.0–17.4 mcg/ml respectively diagnosing the average IIDC; at values >2.5 mcg/ml and <10.9 mcg/ml respectively diagnosed high IIDC periodontal tissues.

EFFECT: disclosed is a method of determining IIDC periodontal tissues in periodontitis.

1 cl, 6 ex

Description

The invention relates to medicine, namely to dentistry, and can be used to diagnose the intensity of inflammatory and destructive changes in periodontal tissues with periodontitis.

Today, innovative methods of treatment based on the latest scientific achievements of medicine are increasingly being introduced into dental practice, using equipment and treatment methods developed taking into account the pathogenesis and etiology of inflammatory periodontal diseases. However, metabolic processes occurring in the periodontium are normal and, moreover, with inflammation, are still not well understood.

Periodontal diseases have a pronounced inflammatory and destructive nature. Most often, people get sick with generalized periodontitis, which differs in the speed, aggressiveness and depth of the course of destructive processes in periodontal tissues, involving the bone tissue of the alveolar process in advanced stages of the disease. Periodontitis can rapidly progress and in a short period of time lead to loss of all or part of the teeth (Borovsky E.V., Groshikov M.I., Patrikeev V.K., Barysheva Yu.D., Lemetskaya T.I. Therapeutic dentistry. Textbook for students of medical universities. Moscow, Publishing House "Medicine", 1982. S. 329-331)

In the course of recent studies, it was proved that protein and mineral metabolism are one of the most important processes that determine the activity, level and prognosis of the development of inflammatory and dystrophic phenomena in periodontal disease. Violation of this type of metabolic reactions significantly affects the clinical picture of the disease and determines the specifics of drug treatment in the complex treatment of patients with generalized periodontitis (Handbook of Dentistry. Edited by Academician of the USSR Academy of Medical Sciences AI Rybakov. Moscow, Publishing House "Medicine", 1993. S.93-96).

Inflammation of the gums and periodontal tissues is characterized by the destruction of collagen, the main protein of connective tissue, which, without a doubt, leads to an increase in saliva levels of such an amino acid as 4-hydroxyproline, which is characteristic exclusively of collagen (T. Vavilova, Biochemistry of tissues and oral fluids. M. : GEOTAR-Media, 2008, p. 215).

Proline and its metabolite, hydroxyproline, are part of collagen, the main protein of connective tissue, and therefore clinical analysis is important in the diagnosis of diseases associated with collagen pathology, which includes periodontitis.

However, in the special dental literature on the diagnosis of periodontal inflammatory diseases, we were able to find only a limited number of data on the use of the concentration of hydroxyproline in saliva as a marker of these diseases, which are mainly of a theoretical nature (Dutov A.A. et al. HPLC analysis of proline and 4-hydroxyproline in biological fluids. Sorption and chromatographic processes. 2013. V.13. Issue 2. - S.229-237).

It is known that the diagnosis of periodontal diseases is carried out based on a subjective assessment of the periodontal condition by the patient himself, for example, bleeding gums during brushing or pain during eating.

The final diagnosis is based on an analysis of a number of objective indicators - the depth of periodontal pockets, the presence of bleeding, suppuration, plaque and tartar. An X-ray examination is also important, in which the resorption of the alveolar interdental septa, the decrease in part of the bone tissue of the alveolar processes of the jaws, are determined.

However, when diagnosing periodontitis, clarifying its severity, such as mild, moderate, or severe, based on an analysis of the above data, is not enough for adequate therapy.

The physician needs to understand the dynamics of the inflammatory process in order to proportionately evaluate the effectiveness of the therapy.

For this purpose, low, medium and high intensity of inflammatory and destructive changes in periodontal tissues (IVDI) are distinguished. This indicator at a qualitative level shows in which direction the intensity of the pathological process shifts, helping the doctor determine the most rational and correct type of dental care. The most common methods for diagnosing IVDI using chemical reagents and specific biochemical markers.

A known method for the diagnosis of periodontal disease by determining the glycogen content on the surface of the gums (Schiller-Pisarev test). For this, the mucous membrane of the gums is stained with Lugol's solution. The color change in various areas after staining is used to judge the presence of an inflammatory process (Diagnosis, treatment and prevention of dental diseases. Yakovleva V.I., Trofimova E.K., Davidovich T.P., Prosviryak G.P. - 2nd ed. ., rev. and add. - Mn .: Vysh.shk., 1994. - P.28).

There is also a method for evaluating collagen metabolism (patent RU 2214596), related to clinical diagnostic studies, based on determining the content in saliva of free and protein-bound hydroxyproline in the dynamics of healing of gastroduodenal ulcers.

A method for evaluating collagen metabolism involves collecting biological fluid, isolating free and protein-bound hydroxyproline from it by extraction with ethyl alcohol and centrifugation, where free hydroxyproline is isolated from the supernatant. To isolate protein-bound hydroxyproline, barium hydroxide hydrolyzate is used, followed by ion-exchange chromatography. The concentration of oxyproline is determined colorimetrically at a wavelength of 540 nm in a 0.5 cm cuvette by adding Erlich reagent. As biological fluid, saliva of the examined person in the amount of 4 ml is used. Add 16 ml of ethyl alcohol to it, mix, centrifuged for 15 min at a speed of 3000 rpm. The isolation of protein-bound hydroxyproline is carried out on a chromatographic column with a diameter of 3 mm, a filling height of KRS-8p 6 mm ion exchange resin, with a rate of neutralization of 0.2 mm per minute. Oxyproline is eluted with 1 ml of 1N. hydrochloric acid solution, after which the hydrochloric eluate is neutralized with 5n. sodium hydroxide solution, while collagen decomposition and its biosynthesis are evaluated by increasing the concentration of free or protein-bound hydroxyproline, respectively.

A known method for the diagnosis of inflammatory diseases of periodontal tissues (patent RU 2329509), the essence of which is in determining the content of calcium, phosphorus, alkaline phosphatase activity, total and tetra-resistant acid phosphatase in mixed unstimulated saliva. However, the method is more time-consuming and expensive, requiring special equipment.

The technical result of the proposed method is to increase the accuracy of diagnosis with minimal time by determining the concentration of free oxyproline in saliva, which is an objective assessment of the stage of the inflammatory process in the oral cavity, and this is done on equipment that is available in any clinical laboratory.

The closest in essence and selected as a prototype was the "Method for the diagnosis of periodontal tissue diseases at different stages" (patent for invention RU 2581921; Aleksakhina E.L. Tomilova I.K.).

In the known method, the concentration of free oxyproline in the saliva of patients who went to the dentist, including complaints of bleeding gums and tooth mobility, the authors determined spectrophotometrically using a modified method C.S. Tetyanets, which is used to determine free oxyproline in serum (S. S. Tetyanets. Method for the determination of free oxyproline in serum. Laboratory case, 1985, N 1, p. 61-62).

To analyze free 4-hydroxyproline, 0.25 ml of oral fluid was poured into test tubes, 1 ml of absolute ethanol was added, centrifuged for 10 minutes at 3000 rpm. All supernatant was transferred to volumetric centrifuge tubes. With stirring, a 0.25 ml solution of chloramine T in acetate buffer (1: 4) was added. 0.25 ml of isopropyl alcohol was added. After 4 min, 0.75 ml of Erlich reagent was poured. The total volume of the solution was adjusted with ethyl alcohol to 2.5 ml. After stirring, the tubes were closed with stoppers and thermostated at 60 ° C for 30 minutes. Then cooled to 20 ° C.

The authors emphasize that in the claimed method they use the value of an objective indicator of the concentration of free oxyproline in saliva, which allows you to diagnose the inflammatory process on an objective basis. The research results are shown in the table, where it is shown that the concentration of free oxyproline in the saliva of patients varies within the limits defined by the authors. For patients with healthy periodontal tissues, the corresponding concentration range of free hydroxyproline is <0.80 mg / l; patients with acute catarrhal gingivitis - 0.80-1.00 mg / l; patients with chronic gingivitis - 1.01-1.50 mg / l; patients with periodontitis> 1.50 mg / l.

Advantages of the prototype: the method improves the accuracy of diagnosis with minimal time by determining the concentration of free oxyproline in saliva, which is an objective assessment of the stage of the inflammatory process in the oral cavity, and is performed on equipment that is available in any clinical laboratory.

The prototype disadvantages: the method does not allow to determine the stage of the pathological process and, most importantly, its direction, since the biochemical markers used by the authors (the concentration of free hydroxyproline in saliva) reflect the final changes in resorption and bone formation, which can be directed in one direction or another, but do not reflect their orientation - the disease intensifies or the patient recovers.

The task: to increase the accuracy of diagnosing the intensity of inflammatory and destructive changes in periodontal tissues (IVDI) by determining the concentration of protein-bound and free hydroxyproline in saliva for an objective assessment of the severity of the pathological process.

To solve this problem, a bone formation marker (protein-bound hydroxyproline in the oral fluid) and a bone destruction marker (free hydroxyproline in the oral fluid) were used.

The content of hydroxyproline in biological fluids is an important indicator of collagen metabolism. In some inflammatory and inflammatory-destructive diseases accompanied by impaired collagen synthesis, the transverse bonds in collagen fibrils are destroyed, which increases the content of readily soluble forms of collagen. In this regard, in patients with chronic periodontitis amid impaired metabolism of the connective tissue, the content of the free fraction of hydroxyproline in the oral fluid increases and the content of its protein-bound fraction decreases. Thus, free hydroxyproline is a marker of destruction, protein-bound - a marker of repair (recovery).

The use of these biochemical markers makes it possible to assess the intensity of inflammatory and destructive changes in periodontal tissues (IVDI) in patients depending on the diagnosis: periodontitis of mild, moderate or severe clinical manifestations, evaluating either pathological changes or the effect of the anti-inflammatory therapy in the treatment dynamics.

The method was carried out as follows.

The collection of oral fluid was performed on an empty stomach. 10 minutes before the intake of oral fluid, patients were advised to thoroughly rinse their mouth with warm water, special mechanical means for cleaning the oral cavity before the intake of oral fluid were not used.

The analysis used the principle based on the determination of the optical density of the chromogen obtained as a result of the condensation of the hydroxyproline oxidation products with paradimethylaminobenzaldehyde). The collected oral fluid of each patient was centrifuged. 0.5 ml of 5% trichloroacetic acid and 0.5 ml of 5% perchloric acid were added to the centrifuge tube. Then centrifuged at 3000 rpm for 6 minutes. The supernatant was distributed in 0.75 ml into two tubes to determine the free (tube No. 1) and protein-bound fraction of hydroxyproline (tube No. 2).

The contents of test tube No. 1 were neutralized with a 24% NaOH solution in the presence of an indicator solution (until a stable slightly purple color appeared). To the contents of test tube No. 2 was added 0.5 ml of distilled water, 0.5 ml of 5% trichloroacetic acid and 0.5 ml of 57% perchloric acid, hydrolyzed in a water bath for 6 hours at a temperature of 60 ° C. Then, 0.25 g of activated carbon was added and centrifuged at 3000 rpm for 6 minutes. The transparent part of the hydrolyzate was neutralized with a 24% NaOH solution in the presence of an indicator solution (until a stable slightly purple color appeared).

Then, 0.5 ml of a solution of chloramine B, 0.5 ml of 57% perchloric acid and 0.5 ml of para-dimethylbenzaldehyde were added to all tubes. The mixture was placed in a water bath for 20 min at 60 ° C. Then measured the optical density of the contents of tubes No. 1-2 at 557 nm. The concentration of hydroxyproline fractions was calculated using the calibration curve on a Shimadzu SPD-M20 spectrophotometric detector (Shimadzu, Japan). The data obtained were processed using the statistical program SPSS for Windows 17.0, the differences between the comparison groups were determined by the Mann-Whitney test in the confidence interval at p <0.05.

The research results showed that the concentrations of free hydroxyproline and protein-bound hydroxyproline in the oral fluid of patients change in different directions.

For patients with healthy periodontal tissues, the range of free hydroxyproline concentrations is <1.0 μg / ml; patients with low IVDI - 1.1-1.8 μg / ml; patients with an average IVDI - 1.81-2.5 mcg / ml; patients with high IVDI> 2.5 mcg / ml.

Patients with healthy periodontal tissues correspond to a concentration range of protein-bound hydroxyproline <20.5 μg / ml, patients with low IVDI - 15.5-20.4 μg / ml; patients with an average IVDI - 11.0-17.4 mcg / ml; patients with high IVDI> 10.9 mcg / ml.

Experimentally identified changes in free hydroxyproline indicate activation of catabolism and depression of anabolism of periodontal tissues (for example, due to disease progression and the absence of adequate anti-inflammatory therapy).

The increase in the concentration of protein-bound hydroxyproline, on the contrary, indicates the activation of collagen synthesis and intensive regeneration of periodontal tissues (for example, due to effective anti-inflammatory therapy).

Thus, a clear relationship was established between the degree of deviation from the normal levels of the content of free and bound hydroxyproline fractions in the oral fluid and the presence of changes in the metabolism of periodontal tissues. The claimed quantitative and qualitative determination of hydroxyproline can be used in the diagnosis of the degree of intensity of inflammatory and destructive changes in periodontal tissues.

The results of the study are confirmed in a clinical setting. Clinical examples.

1. Patient Zn, born 2001 turned to the dentist with the goal of sanitation of the oral cavity and professional hygiene.

Complaints: for the presence of smoker's teeth.

Objectively in the oral cavity: the mucous membrane of the attached gums is pale pink, moderately moist.

An instrumental examination of the oral cavity revealed a small amount of plaque on the lingual surface of the frontal and chewing teeth. The gingival connection is not broken. Pathological changes in the survey radiograph were not detected.

Using clinical indices (KPU = 2.35, GAME = 0.3, PMA = 0.1) an objective assessment of the condition of the teeth and gums was carried out.

The results of the study of saliva:

Free hydroxyproline - 1.35 μg / ml.

Protein-bound hydroxyproline - 22.8 μg / ml.

Summary: there are no signs of inflammation and destruction of bone tissue, healthy.

Diagnosis: K 03.66 Plaque.

2. Patient F., born in 1991 turned to the dentist with the goal of sanitation of the oral cavity and professional oral hygiene.

Complaints: bad breath, bleeding gums when brushing your teeth and biting hard foods, plaque.

Objectively in the oral cavity: the mucous membrane of the attached gums is slightly hyperemic, pale pink, in places slightly hyperemic, moderately moist.

An instrumental examination of the oral cavity revealed soft and hard supragingival and subgingival plaque in all groups of teeth of the upper and lower jaw. When probing a gingival sulcus with a button probe, slight bleeding is noted in the chewing and frontal groups of teeth. Depth of periodontal pockets 1-3 mm. The gingival connection is broken in the frontal area of the teeth. An overview radiograph revealed a decrease in the height of the interdental alveolar septa in the frontal section of the lower jaw by 1 / 4-1 / 3 of their height.

Using clinical indices (KPU = 4.8, GAME = 1.75, PMA = 4.4), an objective assessment of the condition of teeth and gums was carried out.

The results of the study of saliva:

Free hydroxyproline - 1.58 μg / ml.

Protein-bound hydroxyproline - 16.5 μg / ml.

Conclusion: signs of inflammation with phenomena of destruction of the interdental alveolar septa were detected.

Diagnosis: Chronic generalized periodontitis of mild severity, with a low intensity of inflammatory and destructive changes in periodontal tissues.

3. Patient S. a, born in 1977 appealed to the dentist.

Complaints: an aesthetic defect in the frontal area, plaque, bad breath.

Objectively: the mucous membrane of the attached gums is cyanotic, edematous, moderately moist. Clinical examination of the oral cavity shows supragingival and subgingival tartar in the region of 34, 33, 32, 31, 41, 42, 43, 44, 45 teeth, pigmented plaque on all groups of teeth. When probing with a button probe, periodontal pockets up to 5 mm deep were found, gum recession 2-3 mm. Mobility of teeth of 1 degree. On the survey x-ray revealed the destruction of the cortical plate of the interdental septa at 1 / 2-1 / 3 of their height.

Using clinical indices (KPU = 12.8, GAME = 2.3, PMA = 4.4), an objective assessment of the condition of teeth and gums was carried out.

The results of the study of saliva:

Free hydroxyproline - 2.3 μg / ml.

Protein-bound hydroxyproline - 12.8 μg / ml.

Conclusion: signs of inflammation with pronounced destruction of the interdental alveolar septa were detected.

Diagnosis: Chronic generalized periodontitis of moderate severity, with an average intensity of inflammatory and destructive changes in periodontal tissues.

4. Patient B-in, 1979 p. He went to the dentist for an orthopedic treatment.

Complaints: the absence of a group of teeth, tooth mobility, plaque, bad breath.

Objectively: the mucous membrane of the attached gum is hyperemic, edematous, moderately moist.

Clinical examination revealed supragingival and subgingival tartar on all teeth of the lower jaw, pigmented plaque on all groups of teeth of the upper jaw, when probing with a button probe, bleeding periodontal pockets with a depth of more than 5 mm were revealed. In the area of periodontal pockets 35, 34, 33, 32, 31, 41, 42, 43, 44, when probing along with blood, pus is released. Mobility of the teeth of the frontal group of 2 degrees. On the survey roentgenogram, complete destruction of the cortical plate of the interdental septa, a decrease in the height of the alveolar part of the lower jaw in the area of missing teeth of the chewing group by 5-7 mm are observed.

Using clinical indices (KPU = 18.9, GAME = 4.8, PMA = 5.5), an objective assessment of the condition of teeth and gums was carried out.

The results of the study of saliva:

Free hydroxyproline - 2.8 μg / ml.

Protein-bound hydroxyproline - 9.5 μg / ml.

Conclusion: an inflammatory-dystrophic process with pronounced destruction of the interdental alveolar septa.

Diagnosis: Chronic generalized periodontitis of a severe degree, with a high intensity of inflammatory and destructive changes in periodontal tissues.

5. Patient A-ko, b. 1982 turned to the dentist for the purpose of professional oral hygiene.

Complaints: bleeding gums during brushing and biting off solid foods.

Objectively: the mucous membrane of the attached gums is slightly hyperemic, moderately moist.

Clinical examination of the oral cavity revealed supragingival and subgingival tartar on teeth 33, 32, 31, 41 42, 43 teeth. When probing a periodontal sulcus with a button probe, bleeding is noted. The gingival connection is broken in the area of the teeth of the frontal group of the lower jaw. On the survey radiograph, there are no visible pathological changes in the area of the tops of the interdental bone septa.

Using clinical indices (KPU = 11.3, GAME = 1.6, PMA = 3.1) an objective assessment of the condition of the teeth and gums was carried out.

The results of the study of saliva:

Free hydroxyproline - 2.6 μg / ml.

Protein-bound hydroxyproline - 12.8 μg / ml.

Conclusion: signs of inflammation without pronounced destruction of the interdental alveolar septa were detected.

Diagnosis: Chronic generalized periodontitis of mild severity, with an average intensity of inflammatory and destructive changes in periodontal tissues. The prognosis is unfavorable.

6. Patient I., b. 1968 turned to the dentist for the purpose of oral hygiene. Complaints: for the presence of dental plaque, halitosis.

Objectively: the mucous membrane of the attached gum is hyperemic, edematous, moderately moist.

Clinical examination of the oral cavity revealed supragingival and subgingival tartar in the area of chewing teeth of the lower jaw, pigmented plaque on all groups of teeth. When probing the periodontal sulcus, bleeding, tooth mobility of 1 degree are noted. Periodontal pockets 4-5 mm deep. On the survey roentgenogram, partial destruction of the cortical plate, resorption of the vertices of the interdental septa by 1 / 3-1 / 2 of the height is observed.

Using clinical indices (KPU = 8.5, IGRU = 1.3, PMA = 3.9), an objective assessment of the condition of teeth and gums was carried out.

The results of the study of saliva:

Free hydroxyproline - 1.65 μg / ml.

Protein-bound hydroxyproline - 20.8 μg / ml.

Conclusion: signs of inflammation with phenomena of destruction of the interdental alveolar septa were detected.

Diagnosis: Chronic generalized periodontitis of moderate severity, with a low intensity of inflammatory and destructive changes in periodontal tissues. The forecast is favorable.

Thus, the results obtained are of paramount importance for determining the appropriateness of starting / continuing / discontinuing drug treatment: the lower the values of the activity of bone metabolism and the lower the density of bone tissue compared with the criteria for a healthy periodontium (determined, for example, radiologically, by densitometry method), the greater the need for the appointment of special agents that enhance bone metabolism.

As a result of using the proposed method, it was possible to increase the efficiency of diagnosing the intensity of inflammatory and destructive changes in periodontal tissues in all clinical cases.

Claims (1)

A method for determining the intensity of inflammatory and destructive changes in periodontal tissues in periodontitis, including the study of the oral fluid by spectrophotometric method, characterized in that the concentration of free hydroxyproline is determined as an indicator of the intensity of inflammatory and destructive changes (IVDI) in the periodontal tissues, and as an indicator of reparative restoration of periodontal tissues determine the concentration of protein-bound hydroxyproline and at values <1.0 μg / ml and> 20.5 μg / ml, respectively they essentially diagnose the absence of pathology of the tissues of the oral cavity; at values of 1.1-1.8 μg / ml and 15.5-20.4 μg / ml, respectively, low IVDI is diagnosed; at values of 1.81-2.5 μg / ml and 11.0-17.4 μg / ml, respectively, the average IVDI is diagnosed; at values> 2.5 μg / ml and <10.9 μg / ml, respectively, high IVDI of periodontal tissues is diagnosed.