DIAGNOSTIC METHOD FOR DETECTING PERIODONTAL DISEASE

Abstract: Method for detecting the presence of periodontal dis­ ease by contacting saliva from a patient suspected of having said disease condition with hydrogen peroxide and a buf­ fered aqueous solution of 4-aminoantipyrine and a phenolic component and observing the color produced as an indica­ tion of the presence of periodontal disease, said phenolic component being (i) sodium 2-hydroxy-3,5-dichlorobenzene sulfonate; (ii) p-hydroxybenzoic acid; (iii) p-methoxyphenol; or (iv) 2,6-dichlorophenol.

Description: DIAGNOSTIC METHOD FOR DETECTING PERIODONTAL DISEASE Background of the Invention Periodontal diseases such as, for example, periodontitis, stomatitis, gingivitis and the like, are inflammatory conditions of the mouth characterized by inflammatory oral tissue changes usually due to local irritation. The destructive inflammatory process involves the interaction between bacteria, food debris, oral leukocytes and the epithelial attachment around the tooth and periodontal membrane, the result of which causes bone resorption (the supportive bone around the roots of the teeth) and inflammation of the tissues (gingiva) immediately surrounding human teeth involving the portion upward from the epithelial attachment (between the surface of gingiva and epithelial attachments only). The early detection of such inflammatory conditions is very important to proper dental treatment. Such detection is herein accomplished by taking advantage of the known fact that, at the site of inflammation, the body produces an increased number of leukocytes such as polymorphonuclear (PMN) leukocytes, which contain a peroxidase enzyme which is a good marker for detecting the presence of the inflammatory periodontal disease condition. When the inflammatory condition is present, the PMN leukocyte count will increase and, concommitantly, so will the peroxidase enzyme activity. Detailed Description of Invention The instant invention provides a simple saliva test for detecting the presence of inflammation due to periodontal disease. It takes advantage of the known chemical ability of peroxidase to catalyze the oxidation of a colorless hydrogen donor (i.e., a chromogen) to a colored product which can be determined colorimetrically. The subject peroxidatic test method is rapid and reliable with a strong positive correlation between the presence of peroxidase in the saliva sample and the existence of'such disease. The determination of peroxidase activity is herein achieved with a reagent mixture of hydrogen peroxide as the substrate and 4-aminoantipyrine as the chromogen in a buffered aqueous solution also containing a phenolic component as a coupling aaent, said phenolic component being a member selected from the group consisting of (i) sodium 2-hydroxy-3,5-dichlorobenzene sulfonate; (ii) p-hydroxybenzoic acid; (iii) p-methoxyphenol; or (iv) 2,6-dichlorophenol The 4-aminoantipyrine is oxidized by the hydrogen peroxide in the presence of peroxidase from the saliva sample and the intensity of the resultant color formation is proportional to the enzyme concentration and, accordingly, to the severity of the periodontal inflammation. In our prior copending European Patent Application EP-A2 0 158 796 (Application No. 85 10 2113.9), a peroxidatic saliva test is described wherein phenol itself constitutes the coupling agent in the chromogen solution. Due to the physical properties of said phenol, however, such chromogen solution cannot be lyophilized as can the chromogen solutions of the present invention. The present invention, therefore, constitutes an improvement over said prior application. For purposes of convenience, the following symbols will be used for each phenolic component, respectively: (i) sodium 2-hydroxy-3,5 dichlorobenzene sulfonate (preferred): HDCBS (ii) p-hydroxybenzoic acid: pHBA (iii) p-methoxyphenol: pMP (iv) 2,6-dichlorophenol: dCP (outside the human) The reaction is carried out by mixing/measured amounts of the chromoqen and substrate solutions with a measured amount of saliva. The reaction is initiated upon contact of the hydrogen peroxide substrate with the saliva so that the order of mixing may first proceed by mixinq the chromoen and substrate solutions to which mixture the saliva is then added, or, alternatively, by mixing-the chromogen solution and saliva together and then adding the substrate solution. The end point of the assay will obviously depend on the particular assay conditions employed. For example, increasing the concentration of chromogen and/or substrate, up to an optimum, will enhance the color formation, that is, the time required for observing the color produced by the reaction will be shortened. Thus, one can advantaqeously adjust the concentrations of chromogen and substrate within the given parameters such that the end point of the assay will be observable within a convenient period of time. Any such adiustment will be readily apparent and easily accomplished by the practitioner of the assay with some familiarization therewith. Although minimal concentrations of chromogen and substrate may require longer assay times, for example, in excess of 1 hour, it has been generally found that use of the hereinafter indicated preferred concentrations to the maximum range concentrations will provide -suitable assays in about 2 to about 60 minutes and, preferably, in about 3 to about 30 minutes, at room temperature (20-26µC), at which time the color intensity in the reaction mixture is observed. Sliahtly elevated temperatures up to about 45°C (i.e., below the inactivation temperature of the peroxidase enzyme) may also be employed to enhance the rate of reaction. The lack of color formation indicates a normal condition, that is, an absence of PMN leukocytes in the saliva which, in turn, indicates the absence of periodontal disease. The formation of color, however, correlates to the presence of periodontal disease and the color intensity correlates to the severity of the disease. A convenient scoring system for each of the phenolic components, from 1 to 4, is as follows: HDCBS pHBA pMB dCP 1 = normal: ------------no no color developed 2 = mild: -- light pink 3 = moderate: purplish pinkish pinkish purplish pink red red pink 4 = severe: dark dark dark dark purple red red purple The chromogen solution consists of the 4-aminoantipyrine chromogen, the phenolic coupling agent and appropriate buffers, preferably conventional alkali metal (sodium or potassium) phosphate buffers, to obtain a desired pH of about 4-9, preferably 6.8 +0.05, for the reaction conditions. Chromogen Solution % w/v (pH - 6.8) Components Range Preferred 4-Aminoantipyrine 0.001-0.15 q 0.017 g Phenolic Component: if HDCBS 0.07-1.0 g 0.42 g if pHBA 0.10-15.0 g 1.5 g if pMP 4.1-130.0 m 25.0 mg if dCP 6.5-1000.0 m 65.0 mg Potassium phosphate (monobasic) 0.15-12.0 g 1.5 q Potassium phosphate (dibasic) 0.15-13.0 q 1.6 g Water, q.s. 100 ml 100 ml The hydroperoxide substrate solution is a diluted aqueous solution of hydrogen peroxide. Substrate Solution % v/v Components Ranae Preferred Hydrogen peroxide (30%) 0.001-10 ml 0.55 ml Water, q.s. 100 ml 100.00 ml In general, the method of this invention will utilize about 2-15 unit volumes of the chromogen solution and about 0.1-2 unit volumes of the substrate solution per unit volume of saliva. Optimum efficient amounts may vary, however, depending upon the concentrations of chromogen and/or substrate and the particular assay technique employed in the test. Such techniques, as more fully detailed hereinafter, include test tubes, filter pad sections, porcelain wells, spectrophotometers and the like. With theaforementioned preferred chromagen and substrate solutions we have found, for example, that about 2 unit volumes each of the substrate and chromogen solutions per unit volume of saliva is preferred when using test tubes or porcelain wells; that about 2 unit volumes of the chromogen solution and 1 unit volume of the substrate solution per unit volume of saliva is preferred when using filter pad sections (e.g. discs); and that about 15 unit volumes of the chromogen solution and 0.1 unit volume of the substrate solution per unit volume of saliva is preferred when using spectrophotometric instruments. To obtain consistently reliable color formation, the pH and peroxide concentration must be controlled. As buffers, there can be used, for example, phosphate (preferred), phthalate, citrate, Tris, borate, succinate and the like buffers, the pH value and capacity thereof being selected in such a manner that the reaction mixture has a pH value of from about 4.0 to about 9.0, and preferably about 6.8. When mixing the chromogen solution and the peroxide substrate solution, it is advantaaeous to use from about 1.1 x 10-4 mole to about 189 moles of 4-aminoantipyrine and from about 2.5 X 10'3 mole to about 8.57 x 102 moles of HDCBS, or from about 5.93 x 10'3 mole to about 2.14 x 104 moles of pHBA, or from about 3.13 x 10'4 mole to about 2.39 x 102 moles of pMB, or from about 3.77 x 10'4 mole to about 1.39 x 103 moles of dCP, respectively, in the chromogen solution for each mole of hydrogen peroxide in the substrate solution. Preferably, the equivalent ratio for each of these reactants is from about 7.5 x 10-3 to about 1.9 x 10-2 mole of 4-aminoantipyrine and from about 0.15 mole to about 0.36 mole of HDCBS, or from about 0.88 mole to about 2.14 moles of pHBA, or from about 0.019 mole to about 0.045 mole of pMB, or from about 0.038 mole to about 0.091 mole of dCP, respectively, in the chromogen solution for each mole of hydrogen peroxide in the substrate solution. Preferably, the chromogen solution is lyophilized to a dry powdery form which provides easier storage, increased shelf life and handling convenience than does the solution itself. The lyophilization step is performed in accordance with well-known and conventional methodologies. The method of the invention may be employed in the form of a convenient testinq kit providing a stoppered test tube for the chromogen, either in solution or in lyophilized form, an air-tiht container such as a pliable sealed vial or another stopper test tube for the peroxide solution and a calibrated dropper for the saliva sample. The testing kit may contain, for example, a sufficient amount of the buffered chromogen and the peroxide solution for one or more tests. In conducting the test, the peroxide solution is simply added to the chromogen in the test tube followed by a measured sample of saliva, that is, an amount sufficient for the test, previously collected in the dropper to the calibrated measurement. The test tube may then be re-stoppered, shaken and then allowed to stand for the indicated time interval. Means can be provided in the kit for positioninq the test tube such that, after the color reaction is completed, the resultant color formation may he immediately compared to a color indicator chart also provided with the kit and from which the absence or presence and the severity of the inflam- matory periodontal disease can be determined. The instant invention thus provides a kit for detecting the presence of inflammatory periodontal disease comprising the following separately contained components: (a) a hydrogen peroxide solution; and (b) a lyophilized reagent consisting of 4-aminoantipyrine; a phenolic component selected from the group consisting of sodium 2-hydroxy-3,5-dichloroben zene sulfonate, p-hydroxybenzoic acid, p-methoxyphenol and 2,6-dichlorophenol; and a buffer to provide a pH of about 4-9 upon admixture of said lyophilized reaqent with said hydrogen peroxide solution. In place of said lyophilized reagent, the kit may contain the heretofore described aqueous buffered chromogen solution. Alternate methods include the use of porcelain wells which provide excellent receptacles for performing the assay with small volumes of reactants and a white background for observing the assay color result; as does the use of a suitable absorbent material, for example, commercially available filter support pads cut into convenient shapes for unit testing, e.g. discs, which maintain all the reactants thereon without leakaqe and from which the assay color result is readily discernible; and the use of conventional spectrophotometric instruments to automatically "color read" the test assay solutions. The following example is given for the purpose of illustrating the invention. Example 1 An independent clinical trial was conducted by a research dentist in order to compare and correlate the clinical results thus obtained with the in-vitro results obtained from the subject assay. Each patient was clinically examined to determine the absence or presence of inflammatory periodontal disease and the severity thereof. The result of each clinical diagnosis was scored from 1 (normal) to 4 (severe). A total. of 22 male and female sub jects participated in the trial. Each participant was asked to refrain from any oral hygiene or food intake before collecting approximately 5 mls of saliva after awakening in the morning. Each patient was asked to spit once into a spital collectinq tube. Each of the saliva samples were analyzed to detect the presence of the peroxidase enzyme according to the following test tube method. Test Tube Method: The test reaction was carried out at room temperature in the following manner: a. 50 microliters of the Preferred Chromoaen Solution was lyophilized in a test tube; b. 110 microliters of the Preferred Substrate Solution was added to the lyophilized chro mogen; c. 30 microliters of the saliva test sample was added to initiate the reaction; d. 30 minutes after the saliva addition, the presence or absence and intensity of the developed color due to the presence of per oxidase enzyme in the saliva was scored on the aforementioned scale of 1 to 4. The results of the clinical diagnosis in the clinical trial and the results of the in-vitro saliva assay for each of the 22 subjects are shown in Table I. TABLE I In-Vivo Patient In Vitro Score Clinical Number HDCBS pHBA pMP dCP Score 1 2 2 2 2 1 2 1 1 1 1 2 3 2 2 1 2 1-2 4 1 1 1 1 1-2 5 3 3 2 2 1 6 1 1 1 1 2 7 3 3 3 2 1 8 3 3 3 2 1-2 9 4 4 4 4 4 10 1 1 1 1 2 11 3 3 2 3 1 12 1 1 1 1 2 13 2 3 3 2 1 14 3 3 3 3 1 15 1 1 1 1 1 16 1 1 1 1 2-3 17 1 1 1 1 1 18 1 1 2 1 2-3 19 3 3 2 3 1 20 1 1 1 1 1 21 1 1 1 1 1 22 2 2 2 2 2-3

Claims: CLAIMS 1. A method for detecting the presence of inflammatory periodontal disease in a human suspected of being afflicted with same which comprises admixing a saliva sample from such human with an aqueous solution of 4-aminoantipyrine and a phenolic component selected from the group consisting of sodium 2-hy droxy-3 , 5-dichlorobenzene sul fonate, p-hydroxybenzoic acid, p-methoxyphenol and 2,6-dichlorophenol buffered to a pH about 4-9 and with hydrogen peroxide solution and observing the color produced as an indicator of such presence. 2. The method of Claim 1 wherein said phenolic component is 2-hydroxy-3,5-dichlorobenzene sulfonate. 3. The method of Claim 1 wherein said phenolic component is p-hydroxybenzoic acid. 4. The method of Claim 1 wherein said phenolic component is p-methoxyphenol. 5. The method of Claim t wherein said phenolic component is 2,6-dichlorophenol. 6. A method for detecting the presence of in inflammatory periodontal disease in a human suspected of being afflicted with same which comprises admixing a unit volume of saliva sample from such human with about 2-15 unit volumes of a chromogen solution consisting essentially of: % w/v 4-aminoantipyrine 0.001 - 0.15 g phenolic component: if HDCBS 0.07 - 1.0 g if pHBA 0.10 - 15.0 g if pMP 4.1 - 130.0 mg if dCP 6.5 - 1000.0 mg KH2PO4 0.15 - 12.0 g K2HPO4 0.15 - 13.0 g Water 100 ml and with about 0.1-2 unit volumes of a substrate solution consisting essentially of: % v/v hydrogen peroxide (30%) 0.001 - 10 ml water, q.s. 100 ml and observing the color produced as an indicator of such presence. 7. The method of Claim 6 wherein said chromogen solution consists essentially of: % w/v 4-aminoantipyrine 0.01v7 g sodium 2-hydroxy-3,5-dichloro benzene sulfonate 0.42 g KH2P04 (monobasic) 5.98 g K2HPO4 (dibasic) 6.27 g water, Q.s. 100 ml and said substrate solution consists essentially of: % v/v hydrogen peroxide (30%) 0.55 ml water, q.s. 100 ml 8. The method of Claim 6 wherein said chromogen solution consists essentially of: % w/v 4-aminoantipyrine 0.017 q p-hydroxybenzoic acid 1.5 mg KH2P04 (monobasic) 5.98 g K2HPO4 (dibasic) 6.27 g water, q.s. 100 ml and said substrate solution consists essentially of: % v/v hydrogen peroxide (30%) 0.55 ml water, q.s. 100 ml 9. The method of Claim 6 wherein said chromogen solution consists essentially of: % w/v 4-aminoant ipyrine 0.017 o p-methoxyphenol 25.0 g KH2P04 (monobasic) 5.98 g K2HPO (dibasic) 6.27 g water, q.s 100 ml and said substrate solution consists essentially of: % v/v hydrogen peroxide (30%) 0.55 ml water, q.s. 100 ml 10. The method of Claim 6 wherein said chromogen solution consists essentiaaly of: % w/v 4-aminoant ipyrine 0.017 g 2, 6-dichlorophenol 25.0 mg KH2PO4 (monobasic) 5.98 g K2HPO4 (dibasic) 6.27 g water, q.s. 100 ml and said substrate solution consists essentially of: % v/v hydrogen peroxide (30%) 0.55 ml water, q.s. 100 ml 11. A kit for detecting the presence of inflammatory periodontal disease comprising the following separately outlined components: (a) a hydrogen peroxide solution; and (b) a lyophilized reagent consisting essentially of 4-aminoantipyrine; a phenolic component selected from the group consisting of sodium 2-hydroxy-3,5-dichlorobenzene sulfonate, p-hydroxybenzoic acid, p-methoxyphenol and 2,6-dichlorophenol; and a buffer to provide a pH of about 4-9 upon admixture of said lyophilized reagent with said hydrogen peroxide solution. 12. The kit of Claim 11 wherein said phenolic component is 2-hydroxy-3,5-dichlorobenzene sulfonate. 13. The kit of Claim 11 wherein said phenolic component is p-hydroxybenzoic acid. 14. The kit of Claim 11 wherein said phenolic component is p-methoxyphenol. 15. The kit of Claim 11 wherein said phenolic component is 2,6-dichlorophenol.

Inventors: CHAUDHARI, PANNA R., SHAH, NUTAN B.
Applicants: RICHARDSON-VICKS, INC.
Application year: 1986
Main classification: C12Q1
IPC codes: C12Q1, A61C19, G01N33