RU2329509C2 - Method of parodentium tissues inflammatory diseases diagnostics - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns stomatology, and can be used for diagnostics of parodentium tissues inflammatory diseases. Mixed non-stimulated saliva is biochemically tested for calcium, phosphorus content, alkaline phosphatase, common and tartrate-resistive phosphatase activity. Value system indicates gingivitis or periodontitis of slight or medium-severe level.

EFFECT: method application provides increased accuracy of gingivitis and/or periodontitis severity levels, and forecast of disease course by means of a simple and noninvasive test method.

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Description

The invention relates to medicine, namely to dentistry, and can be used to diagnose inflammatory diseases of periodontal tissues.

In the diagnosis and treatment of inflammatory periodontal diseases, an objective assessment of the severity of the disease is very important. No less relevant is the problem of accurate biochemical control of the effectiveness of treatment and prognosis of the course of the disease.

A known method for the diagnosis of periodontitis using the determination in the gingival blood of a patient the presence of R-protein [RF Patent No. 2061961, G01N 33/68, 1996].

This method is based on the fact that it uses as a diagnostic criterion increased concentrations of R-protein in the capillary blood of the gum, a specific marker of inflammation.

This method allows you to determine the presence of inflammation in the periodontium. However, it is not accurate enough and sensitive in the early stages and in the chronic course of the inflammatory process. In addition, it is invasive and involves a painful and time-consuming procedure for taking blood from the gums.

Closest to the proposed method is a method for diagnosing inflammatory periodontal disease (periodontitis) by biochemical study of the intensity of formation of lipid peroxidation products and saliva antioxidant system in mixed unstimulated saliva by the intensity of induced chemiluminescence on a BHL-06 biochemiluminometer [Bezrukova I.V. Rapidly progressive periodontitis. Etiology. Clinic. Treatment. // Abstract. diss .... doct. honey. sciences. M. 2001].

A feature of this method is that it uses, as a diagnostic criterion, excessive accumulation of lipid peroxidation products, which are one of the links in the pathogenesis of the inflammatory process, as well as a decrease in the activity of antioxidant systems, which accompanies the exhaustion of natural protective anti-inflammatory mechanisms.

However, its sensitivity is insufficient to detect the inflammatory process in the early stages, especially when tissue destruction processes are distributed mainly in the soft tissues of the gums, without affecting significantly the periodontal structure, in particular with gingivitis, as well as with chronic periodontal inflammation. In addition, this method requires the use of sophisticated equipment and expensive reagents, not always available to ordinary biochemical laboratories, which increases the cost of analysis and does not allow recommending it for widespread use.

To solve the problem of timely treatment and prevention of periodontal diseases, early diagnosis and treatment of the beginning periodontal pathology is necessary. The development of simple, affordable for any biochemical laboratory and preferably non-invasive methods for studying the state of periodontal and gum tissue remains urgent.

The technical result of this invention is to improve the accuracy of the diagnosis of inflammatory diseases of periodontal tissues using a simple and non-invasive research method.

The technical result is achieved by the fact that in the method for the diagnosis of inflammatory diseases of periodontal tissues by biochemical study of mixed unstimulated saliva, a distinctive feature is that in the biochemical study of mixed unstimulated saliva, the content of calcium, phosphorus, alkaline phosphatase activity, total and tartrate-resistant acid phosphatase are determined and diagnosed:

Gingivitis in the content:

Calcium 1-3 mmol / L

Phosphorus no more than 15 mmol / l

Alkaline phosphatase is not less than 0.7 μkat / l or total acid phosphatase is not less than 700 nkat / l

Tartrate-resistant acid phosphatase not more than 200 ncat / l and not more than 20% of total acid phosphatase

Mild severity of periodontitis with the content:

Calcium 1.5-2 mmol / L

Phosphorus more than 10 mmol / l

Alkaline phosphatase 0.7-1.2 mkkat / l

Total acid phosphatase 700-1500 nkat / l

Tartrate-resistant acid phosphatase greater than 200 ncat / l or more than 20% of total acid phosphatase

Moderate severity of periodontitis with content:

Calcium 1.5-3.0 mmol / L

Phosphorus more than 10 mmol / l

Alkaline phosphatase is greater than 1.2 μkat / l or total acid phosphatase is greater than 1500 nkat / l

Tartrate-resistant acid phosphatase greater than 200 ncat / l and more than 20% of total acid phosphatase

Indicators are considered consistent with the norm (no signs of inflammation) when kept in saliva:

Calcium 1-4 mmol / L

Phosphorus 2.5-10 mmol / L

Alkaline phosphatase 0.1-0.7 mkcat / l and total acid phosphatase not more than 700 nkat / l

Tartrate-resistant acid phosphatase not more than 200 ncat / l and not more than 20% of total acid phosphatase

If the indicators do not meet any combination of diagnostic criteria, this indicates the presence of a concomitant pathology of a non-inflammatory nature, for example, a violation of mineral metabolism, endocrine diseases. In this case, the diagnosis can be carried out after eliminating the concomitant disease.

It is advisable to take mixed unstimulated saliva from the mouth at least 2 hours after a meal. After medical manipulations, accompanied by trauma to the soft tissues of the oral cavity (curettage, patchwork, etc.), the study can be carried out no earlier than 10-14 days later. Diagnosis cannot be carried out in the presence of purulent processes in the tissues of the oral cavity and inflammatory diseases of the salivary glands.

Determination of total and tartrate-resistant acid phosphatases must be carried out on the day of selection. Alkaline phosphatase can also be determined in saliva, which remained frozen for up to 2-3 months, but only after a single thaw. Calcium and phosphorus can be determined in saliva, which has remained frozen for several months in a hermetically sealed container, even after repeated freezing and thawing.

Determination of calcium and phosphorus can be carried out by any unified method, in particular calcium - with glyoxal bis-2-hydroxyaniline, with arsenazo III; phosphorus - by the method with molybdate-vanadate, with molybdate.

The activity of alkaline phosphatase is recommended to be determined by the rate of hydrolysis of p-nitrophenyl phosphate in N-methyl-D-glucamine buffer (for example, reagent kits manufactured by LAHEMA, Czechoslovakia).

The activity of total and tartrate-resistant acid phosphatases is recommended to be determined by the rate of hydrolysis of p-nitrophenyl phosphate in citrate buffer (for example, reagent kits made by LAHEMA, Czechoslovakia).

The proposed method is non-invasive, simple, does not require expensive reagents, sophisticated equipment and is available for any biochemical laboratory.

Example 1

Patient B., born in 1979 I applied to the TsNIIS prevention department for teeth whitening. Clinical examination revealed the absence of pathology of the gum and periodontal tissues. Since periodontal pathology is a contraindication for teeth whitening, the patient was asked to conduct an additional biochemical study of saliva.

Calcium is determined by the method with arsenazo III, reagent kits of the company BioSystems (Spain).

The composition of the reagent: arsenazo III 0.2 mmol / l, imidazole 75 mmol / l.

Reference solution: calcium 2.5 mol / l.

Measure (ml) Try Comparison solution Control solution Try 0.015 - - Reference solution - 0.015 - Water - - 0.015 Reagent 1.00 1.00 1.00

After 2-15 minutes after mixing the sample with the reagent, the optical density of the sample solution is determined at 650 nm against the control solution (A _pr ). At the same time, the optical density of the comparison solution is determined against the control solution (A _cf. ).

The concentration of calcium in the sample, mmol / l:

X = 2.5 × A _pr / A _cf

A _np = 0.2357; And _avg = 0.4572.

X = 2.5 × 0.2357 / 0.4572 = 1.289 mmol / L.

Phosphorus is determined by the method with molybdate-vanadate, reagent kits manufactured by LAHEMA.

Reagents:

1. Reference solution: an aqueous solution of potassium dihydrogen phosphate, 5 mmol / l;

2. Solution 1: an aqueous solution of trichloroacetic acid, 0.62 mol / l;

3. Solution 2: ammonium vanadate 1 mmol / L, ammonium molybdate 15 mmol / L.

Measure (ml) Try Comparison solution Control solution Try 0.20 - - Reference solution - 0.10 - Water 0.60 0.70 0.80 Solution 1 0.80 0.80 0.80 Stirred and after 5 min centrifuged for 10 min at 3000 rpm Supernatant 0.80 0.80 0.80 Solution 2 1.00 1.00 1.00 Stirred and after 20 minutes measure the optical density of the sample (A _CR ) and the comparison solution (A _CP ) against the control solution at a wavelength of 405 nm in a cuvette of 1.00 cm

The concentration of phosphorus in the sample, mmol / l:

X = 2.5 × A _pr / A _cf.

A _np = 0.3558; And _avg = 0.1508.

X = 2.5 × 0.3558 / 0.1508 = 5.90 mmol / L.

Alkaline phosphatase is determined by the rate of hydrolysis of p-nitrophenyl phosphate in N-methyl-D-glucamine buffer.

Reagents:

1. Substrate: an aqueous solution of p-nitrophenyl phosphate disodium salt 92 mmol / l;

2. Buffer: 0.426 mol / L N-methyl-D-glucamine buffer pH 10.1 (37 ° C).

To 1.00 ml of buffer add 0.040 ml of sample, thermostat at 37 ° C for 2 min, add 0.20 ml of substrate and measure the slew rate of absorbance ΔA / min at 37 ° C in the range of 60-240 s after adding the substrate at a wavelength 405 nm in a cell 1.00 cm.

The activity of alkaline phosphatase, mkkat / l:

X = 15.5432 (ΔA / min)

ΔA / min = 0.00938;

X = 15.5432 × 0.00938 = 0.1458 μkat / L.

Total and tartrate resistant acid phosphatase is determined by the rate of hydrolysis of p-nitrophenyl phosphate in citrate buffer

Reagents:

1. Solution 1: nitrate buffer, pH 4.95, 0.05 mol / l; p-nitrophenyl phosphate disodium salt, 5.5 mmol / l

2. Solution 2: potassium sodium tartrate, 0.5 mmol / l

3. Solution 3: p-nitrophenol, 0.54 mol / L in ethanol

4. Inhibitor: Trilon B (complex III) 0.03 mol / L, sodium hydroxide 0.1 mol / L

Measure (ml) Sample 1 Sample 2 Control solution Standard Solution 1 0.50 0.50 0.50 - Solution 2 - 0.02 - - Stir, incubate for 5 min at 37 ° C and add Try 0.05 0.05 - - Incubate for exactly 30 minutes at 37 ° C and add Inhibitor 2.00 2.00 2.00 2.00 Try - - 0.05 - Solution 3 - - - 0.05 Mix and measure the optical density of sample 1 (A ₁ ), sample 2 (A ₂ ), control solution (A ₃ ) and standard (A ₄ ) against water at a wavelength of 405 nm in a cuvette of 1.00 cm

The activity of acid phosphatase total in the sample, ncat / l:

The activity of acid phosphatase tartrate-resistant in the sample, ncat / l:

A ₁ = 0.281; A ₂ = 0.184; A ₃ = 0.169; A _{4 =} 0.188.

The results of the study of saliva:

Calcium: 1.289 mmol / L

Phosphorus: 5.90 mmol / L

Alkaline phosphatase: 0.1458 μkat / L

Acidic phosphatase total: 179 nkat / l

Tartrate-resistant acid phosphatase: 24 nkat / l (14% of the total)

Conclusion: The indicators are normal, signs of inflammation of the periodontal and gum tissue are absent. The clinical diagnosis is biochemical.

Example 2

Patient K., born in 1980 appealed to the TsNIIS prevention department for teeth whitening.

Somatic status: almost healthy (according to the patient).

Clinical examination results: The presence of soft plaque in the frontal area of the lower jaw on the lingual side, soft plaque in the neck of all groups of teeth. The mucous gums are hyperemic, swollen, when probing the gingival sulcus, bleeding of the gums is observed.

There are no changes on the orthopantomogram.

Clinical diagnosis: chronic gingivitis.

Since periodontal pathology is a contraindication for teeth whitening, the patient was asked to conduct an additional biochemical study of saliva according to the method described in example 1.

The results of a biochemical study of saliva:

Calcium: 1.302 mmol / L

Phosphorus: 2.84 mmol / L

Alkaline phosphatase: 0.7504 μkat / L

Acid phosphatase total: 1301 nkat / l

Acidic tartrate-resistant phosphatase: 192 nkat / l (15% of the total)

Conclusion: inflammation without the destruction of mineralized tissues, which is a sign of gingivitis.

The clinical diagnosis is biochemical.

Example 3

Patient E., born in 1969 He appealed to the periodontology department of the Central Scientific Research Institute of Hygiene with complaints of recurrent bleeding of the gums when brushing your teeth, bad breath. Somatic status: almost healthy (according to the patient).

Clinical examination results: The presence of soft plaque and hard dental deposits in the frontal area of the lower jaw, pigmented plaque in the neck of all groups of teeth.

Depth of periodontal pockets 1-2 mm. Bleeding when probing periodontal pockets.

On the orthopantomogram: the absence of a compact plate of the interdental partitions of the anterior teeth, the resorption of the vertices of the interdental partitions by 1/4 of the height.

Clinical diagnosis: mild chronic generalized periodontitis.

To clarify the diagnosis, a biochemical study of saliva was carried out according to the method described in example 1.

The results of a biochemical study of saliva:

Calcium: 1.825 mmol / L

Phosphorus: 11.03 mmol / L

Alkaline phosphatase: 0.8055 μkat / L

Acid phosphatase total: 918 nkat / l

Acidic phosphatazatartrate-resistant: 192 nkat / l (21% of the total)

Conclusion: inflammation with the destruction of mineralized tissues, which is a sign of mild periodontitis.

The clinical diagnosis is biochemical.

Example 4

Patient K., born in 1980 He appealed to the periodontology department of the Central Scientific Research Institute of Hygiene with complaints of recurrent bleeding of the gums when brushing your teeth, the presence of dental deposits, and soreness when chewing solid food. Somatic status: almost healthy (according to the patient).

Clinical examination results: The presence of soft plaque and hard dental deposits in the frontal area of the lower jaw on the lingual side, pigmented plaque in the neck of all groups of teeth.

Depth of periodontal pockets 3-6 mm. Bleeding when probing periodontal pockets.

On the orthopantomogram: destruction of the cortical plate of the interdental septum, resorption of the vertices of the interdental septum by 1/2 height.

Clinical diagnosis: chronic generalized periodontitis of moderate severity.

To clarify the diagnosis, a biochemical study of saliva was carried out according to the method described in example 1.

The results of a biochemical study of saliva:

Calcium: 1.51 mmol / L

Phosphorus: 10.16 mmol / L

Alkaline phosphatase: 1.4397 μkat / L

Acid phosphatase total: 1547 nkat / l

Tartrate-resistant acid phosphatase: 322 nkat / L (21% of the total)

Conclusion: inflammation with the destruction of mineralized tissues, which is a sign of periodontitis of moderate to severe severity.

The clinical diagnosis is biochemical.

Example 5

Patient Ch., Born in 1954 appealed to the periodontology department of the Central Scientific Research Institute of Hygiene with complaints of recurrent bleeding of the gums when brushing teeth, the mobility of individual teeth. Somatic status: almost healthy (according to the patient).

Clinical examination results: The presence of soft plaque and hard dental deposits in the frontal area of the lower jaw on the lingual side, pigmented plaque in the neck of all groups of teeth.

Tremas:

Diastema:

The depth of periodontal pockets is 5-8 mm. Bleeding when probing periodontal pockets.

Mobility I degree:

On the orthopantomogram: destruction of the cortical plate of the interdental septum, resorption of the vertices of the interdental septum at 1 / 2-2 / 3 of the height.

Clinical diagnosis: chronic generalized periodontitis of moderate severity.

To clarify the diagnosis, a biochemical study of saliva was carried out according to the method described in example 1.

The results of a biochemical study of saliva:

Calcium: 2.175 mmol / L

Phosphorus: 11.41 mmol / L

Alkaline phosphatase: 1.5106 μkat / L

Acid phosphatase total: 1589 nkat / l

Acidic tartrate-resistant phosphatase: 852 ncat / l (54% of the total)

Conclusion: inflammation with the destruction of mineralized tissues, which is a sign of periodontitis of moderate severity.

The clinical diagnosis is biochemical.

The proposed method for the diagnosis of inflammatory diseases of periodontal tissues allows to identify:

- the presence of an inflammatory process in the tissues of the oral cavity and evaluate its severity and

- the presence of periodontal tissue damage and evaluate its severity.

The proposed method is simple, does not require special and expensive equipment, gives reproducible results.

Claims (1)

A method for the diagnosis of inflammatory diseases of periodontal tissues by biochemical research of mixed unstimulated saliva, characterized in that during a biochemical study of mixed unstimulated saliva, calcium, phosphorus, alkaline phosphatase, total and tartrate-resistant acid phosphatase are determined and diagnosed: gingivitis in the content: calcium 1-3 mmol / l; phosphorus no more than 15 mmol / l; alkaline phosphatase not less than 0.7 mccat / l or total acid phosphatase not less than 700 ncat / l; tartrate-resistant acid phosphatase not more than 200 ncat / l and not more than 20% of total acid phosphatase, mild severity of periodontitis with content: calcium 1.5-2 mmol / l; phosphorus greater than 10 mmol / l; alkaline phosphatase 0.7-1.2 mkkat / l; total acid phosphatase 700-1500 nkat / l; tartrate-resistant acid phosphatase greater than 200 ncat / l or more than 20% of total acid phosphatase, moderate severity of periodontitis with content: calcium 1.5-3.0 mmol / l; phosphorus greater than 10 mmol / l; alkaline phosphatase greater than 1.2 mccat / l or total acid phosphatase greater than 1500 ncat / l; tartrate-resistant acid phosphatase more than 200 ncat / l and more than 20% of total acid phosphatase.