METHOD FOR MONITORING PERIODONTAL DISEASE BY MONITORING ENDOTOXINS AND INFLAMMATORY AGENTS

Abstract: A method of quantitatively determining the amount of bacterial endotoxin which includes the steps of: (a) obtaining a sample from the periodontal pocket of the subject; (b) contacting the sample with an amebocyte lysate under conditions so as to activate an enzyme capable of cleaving a bond between an arginyl group and a nitrogen-containing group; (c) contacting the activated enzyme with a substrate comprising an arginyl group and a suitable nitrogen-containing group bound to the arginyl group so as to form an amine; (d) treating the resulting amine to produce a detectable product; and, (e) quantitatively determining the amount of product formed and thereby the amount of bacterial endotoxins present in the periodontal pocket. The invention also provides a method of diagnosing a periodontal disease which comprises quantitatively determining the amount of bacterial endotoxins present and correlating the amount so determined with known periodontal disease-causing amounts of endotoxin.

Description: -1- METHOD FOR MONITORING PERIODONTAL DISEASE BY MONITORING ENDOTOXINS AND INFLAMMATORY AGENTS Background of the Invention Bacterial endotoxins are produced by Gram negative bacteria, many of which are very dangerous or deadly in human beings and animals. In periodontal pockets. Gram-negative bacteria, their endotoxins and other inflammatory agents causes gingivitis and other periodontal diseases. It is important to identify the existence of endotoxins and, if possible, the concentration of the endotoxins in the periodontal pockets in order to promptly initiate the proper medical and dental treatment. The monitoring of endotoxins in the periodontal pockets of a subject may also be used to diagnose periodontal diseases and the effect of treatment programs. It has been known for many years that bacterial endotoxins activate the blood clotting enzyme occurring in amebocyte lysates of horseshoe crabs. This property has been described in many publications, e.g. J. Levin and F. B. Bangs, 1968, Thomb. Diath. Hae onth, 19:186 and S. Nakamura, 1976, Biochemical and Biophysical Research Communication, 72:902. These investigations demonstrated that the coagulation of Limulus amebocyte lysate (LAL) requires activation of the proclotting enzyme by endotoxin in the presence of divalent cations. The active enzyme thus formed cleaves coagulogen at the C-carboxyl terminal between the glycine and arginine subunits. The cleaved coaculogen molecules polymerized and thus bring about coagulation. The first endotoxin tests were based on observation of the gel thus formed. The gelation reaction test method is deficient for use in monitoring periodontal disease because of the considerable length of time necessary to carry it out, because it does not accurately measure endotoxin concentration, because it is difficult to standardize and because it requires highly skilled experienced personnel to perform it. Fluorogenic or chromogenic substrates have been used for the assay of many enzymes, e.g. trypsin, thrombin, thro bobiastin, serine protease, plasmin and plas inogen. It was also realized that fluorogens or chromogens can be used as LAL-substrates. See e.g. Iwanaga, et al., 1978; Homeostatis, 2 :183 and patent publications U.S. Patent No. 4,188,265, U.S. Patent No. 4,406,832 and U.S. Patent No. 4,510,241. The use of chromogenic substrates has become a means to both study and clinically monitor various enzymes and inhibitors in the complex coagulation processes of man. An extensive list of enzyme specific substrates are commercially available for measuring enzymes such as trypsin, thrombin, thromboplastin, plasmin, plasmin Kallikrein, Urokinase, and plasminogen. Iwanaga, et al., Hemostasis 7:183-188 (1978) discloses that synthetic substrates can be used to measure the level of endotoxin activated pro-clot enzyme in LAL prepared from the blood of both the Japanese (Tachypleus tridentatus) and the American (Limulus polyphemus) horseshoe crabs. Chromogenic substrate methods for assaying bacterial endotoxins using Limulus amebocyte lysate are also described in pages 209-220 of "Bio edical Applications of the Horseshoe Crab" (1979) Allen R. Lis, Inc. and U.S. Patent No. 4,301,245. However, these disclosures are directed to the testing of blood which may containing endotoxins or the testing of food for endotoxin contamination. There are three methods commonly used for monitoring bacteria in a periodontal pocket: 1. Dark field or phase microscopy; 2. Cultural microbiological evaluation; and 3. Fluorescent antibody identification. The first method is inaccurate, time consuming, and requires knowledge of a specialist. The second method is expensive and too cumbersome to be undertaken by the dentist in his office. The third method is also difficult for the dentist, and too expensive to be performed at each individual periodontal pocket site. With the present method, it is possible to conduct the desired endotoxin test within a short period of time in a dental office and without the need for a specialist or expensive e-quipment. The time period for the method of the present invention is much shorter than that required for previous test methods for bacterial endotoxins. Moreover, the present method simply and accurately measures the concentration of the endotoxin to indicate the extent of the Gram negative bacterial infection in the periodontal pocket. -4- Summary of the Invention The present invention concerns a method of quantitatively determining the amount of bacterial endotoxin present in a periodontal pocket of a subject. The method comprises: a) obtaining a sample from the periodontal pocket of the subject; 10 b) contacting the sample with an amebocyte lysate under conditions so as to activate an enzyme capable of cleaving a bond between an arginyl group and a nitrogen-containing 15 group; c) contacting the activated enzyme with substrate comprising an arginyl group and a suitable nitrogen-containing group bound t 20 the arginyl group so as to form an amine; d) treating the resulting amine to produce detectable product; and 25 e) quantitatively determining the amount o product formed and thereby the amount o bacterial endotoxins present in th periodontal pocket. 30 In the preferred embodiments, the method comprise treating the sample prior to contacting with th amebocyte lysate so as to inactivate enzymes present i the sample which would interfere with amebocyte lysate endotoxin interaction. 35 The invention also provides a method of diagnosing a periodontal disease which comprises quantitatively determining the amount of bacterial endotoxins present in a periodontal pocket of a subject by the method of the present invention and correlating the amount so determined with known periodontal disease-causing amounts of endotoxin. The invention also provides kits for carrying the methods. Brief Description of the Fig-ures Figure 1 shows the optical densities of the final volumes of p-nitroaniline measured at various endotoxin concentrations of the reaction mixture. Figure 2 shows relative values of optical densities for p-nitroaniline as such and its azo dyes with H-Acid and N-na. Reaction mixture volumes equal 5.0 cc. Figure 3 shows endotoxin standard curve for p- nitroaniline coupled with H-Acid. Fi»gure 4 shows optical densities of endotoxins eluted from a periodontal strip at various dilutions for p- nitroaniline coupled with H-Acid. Figure 5 shows optical densities of p-nitroaniline coupled with N-na at various concentrations of endotoxins in the reaction mixture. Detailed Description of the Invention The present invention concerns a method of quantitatively determining the amount of bacterial endotoxin present in a periodontal pocket of a subject. The method comprises: a) obtaining a sample from the periodontal pocket of the subject; b) contacting the sample with an amebocyte lysate under conditions so as to activate an enzyme capable of cleaving a bond between an arginyl group and a nitrogen-containing group; c) contacting the activated enzyme with a substrate comprising an arginyl group and a suitable nitrogen-containing group bound to the arginyl group so as to form an amine; d) treating the resulting amine to produce a detectable product; and e) quantitatively determining the amount of product formed and thereby the amount of bacterial endotoxins present in the periodontal pocket. In the preferred embodiments, the method comprises treating the sample prior to contacting with the amebocyte lysate so as to inactivate enzymes present in the sample which would interfere with amebocyte lysate- endotoxin interaction, such as trypsin or trypsin-like enzymes. The sample may also be diluted in order to reduce the effect of gram positive bacteria on the amebocyte lysate-endotoxin interaction. On the average the gram negative bacteria are approximately 1 x 10 times more active in the limulus assay than any of the gram positive bacteria. Accordingly, the distinction between gram negative and gram positive bacteria can be readily ascertained and maximized by dilutions of plaque organisms in the sample. In this manner all limulus provoking activity of gram positive bacteria can be minimized by the dilution effect and thus only gram negative bacteria will be capable of provoking a positive limulus response. Since ver 98% of the suspected periodontal pathogens are gram negative, the dilution of plaque organisms in the sample increases the effectiveness of the present method as a clinical tool for determining levels of gram negative bacteria in a pocket site and thereby the effectiveness of the method in diagnosing periodontal diseases. The sample may be obtained by placing a small filte paper strip into a periodontal pocket of the subjec and collecting the sample on the paper strip. In th preferred embodiments, the strip is then subjected t heating, e.g. hot air from a hair dryer set on hig temperature for 1 minute, in order to inactivat endogenous enzymes present in the sample which ma interfere with the amebocyte lysate-endotoxin reaction. In carrying out the present method, the sample is mixe with a king crab amebocyte lysate, preferably Limulu amebocyte lysate. However, lysate from other forms o horseshoe crabs such as Tachypleus tridentatus, th Japanese horseshoe crab, can be used. The lysate ca be prepared in accordance with conventional procedures. Procedures such as are disclosed in Journal of Clinical Investigation, Volume 51, July 1972, Bulletin of Johns Hopkins Hospital, Volume 115, pages 265-274 (1964) and Proceedings of the Society for Experimental Biochemical Medicine, Volume 137, pages 334-342, can be used to recover the lysate. Briefly, the conventional procedure usually involves withdrawing crab blood by sterile needle from the crab heart, placing the blood into a mixture which prevents aggregation of the blood cells and premature lysis os the cells, separating the amebocytes from the remainder of the blood by centrifugation, lysing the amebocytes by mechanical breaking, freeze-thawing or by osmotic lysis in pyrogen-free distilled water in a volume ratio of water to cells of about 3-6:1 or the like. The lysate thus obtained is centrifuged free of the broken cells and usually freeze dried, that is, lyphophilized to preserve it. When it is ready for use, the lysate powder is diluted with approximately 50 volumes of sterile pyrogen-free distilled water to bring it to a solids concentration per ml. of about 0.02 gm. In the present method, lysate is used which may either be one freshly prepared or one which has been preserved, usually in lyophilized powder form, and which has been reconstituted or which during the preparation of the reaction mixture is directly added as a powder to the liquid mixture. In any event, the lysate is present in a solids concentration in the reaction mixture of about 1.50 to about 2.50 gm/100 ml. The substrate utilized in the present method contains a selected colorimetric indicator capable of being split off from the substrate by lysate enzyme produced by conversion of proenzyme by endotoxin in the sample. -10- For such purposes, any suitable substrate generally of the type described in, for example, U.S. Pat. No. 4,028,318 can be utilized. However, certain of such substrates have been found to be more advantageous then others. In this regard, a substrates characterized by the general formula R.-Ile-Glu-Gly-Arg-pNA are preferred. The R 1 blocking group in the general formula above may be N-tert butoxycarbonyl, alkanoyl of 1 to 12 carbon atoms, cyclohexylcarbonyl, and N-benzoyl, acetyl and benzoyl substituted with one or more halogens, lower alkyl, e.g., methyl and ethyl, amino or phenyl groups, or may be H when the N-terminal L-amino acid of th peptide structure is replaced with a N-terminal D-amin acid. Suitable chromogenic or fluorogenic groups include nitrophenyl, methylnitrophenyl, dinitrophenyl, naphthyl, nitronapththyl, methoxyhaphththyl, indoxyl, methylindoxyl, (4-methyl-)umbelliferyl and resorfin. The most preferred substrates comprise benzoyl (o acetyl)-isoleucine-glutamic acid-glycine-arginine-p nitroanalide benzoylarginine-β-naphthylamide, benzoylarginine-p-nitroanilide. Further suitabl substrates can easily be determined by minima experimental testing. Such substrates may contain, fo example, 2-naphthyl-amide as the color indicator or p nitroanilide. Also contemplated by the present invention are the aci addition salts of the above peptide-type compounds Suitable acid salts include those from mineral acid such as hydrochloric, hydrobromic, hydrosulf ric an hydrophosphoric or from organic acids such as formic cetic, oxalic, tartaric, methane sulfonic and benzen sulfonic. In accordance with the present method, the desired test reaction is effected by mixing together the sample, the amebocyte lysate and the selected substrate in suitable relative concentrations. In carrying out the method the sample can, for example, be added to a reaction zone such as a sterile test tube, can then be diluted with about five to ten volumes of pyrogen-free sterile water and powdered Limulus amebocyte lysate or other king crab amebocyte lysate in a suitable concentration can then be added to each such test tube. The temperature of each test tube is preferably kept within a suitable range, for example, 35"-40 , C, most preferably about 37*C. This mixture is thoroughly mixed together and incubated for about 7-12 minutes, preferably about 10 minutes. The substrate is then added to each test tube and the resulting mixture is then mixed together and incubated for about 2-4 minutes, preferably about 3 minutes, at the desired previously indicated temperature of about 37 * C. for a total reaction time (initial incubation plus final incubation) of approximately 10-12 minutes, preferably about 11 minutes, whereupon acetic acid, for example, I N, is added to each test tube in a volume sufficient to stop the reaction from continuing, usually 100 μl. The mixture is then left in each test tube and read on a colorimeter at 405 nm. The concentration of the endotoxin is calculated from a standard curve previously prepared from known dilutions of a standard endotoxin. The method may also be performed by using a reagent sheet as described in the Experimental Detail section which follows: Alternatively, the contacting of the sample with amebocyte lysate may be preformed simultaneously with the contacting of the trypsin-like enzyme with the substrate. In one embodiment the sample is collected on a filtered paper strip and contacted with a reagent sheet which contains the amebocyte lysate and the substrate. Preferably the sheet also contains a buffer, such as Tris. The sample on the filter paper stripe may also be immersed in a solution containing the amebocyte lysate and the substrate or it may be immersed with the above described reagent sheet in a solution containing the buffer and/or pyrogen-free water. The treating of the enzyme to produce a protectable product preferably consists of diazotization of the amine to produce a diazonium salt and reacting the diazonium salt with a suitable coupling agent to produce the detectable product. Suitable coupling agents are well known to those skilled in the art and include histidine, 8-amino-l-hydroxynapthalene-3,6- disulfonic acid, or N-(l-naphthyl)-ethylenediamine dihydrochloride or a salt thereof. The invention also provides a method of diagnosing a periodontal disease which comprises quantitatively determining the amount of bacterial endotoxins present in a periodontal pocket of a subject by the method of the present invention and correlating the amount so determined with known periodontal disease-causing amounts of endotoxin. In the preferred embodiments the periodontal disease is gingivitis or advanced periodontitis. Lastly, the invention provides a diagnostic kit for diagnosing periodontal diseases in a patient. The invention is further illustrated in the experimental detailed section and the examples which follow. These sections are set forth to aid in an understanding of the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter. Experimental Details Experiments performed in the laboratories of the inventors demonstrated a positive, strong correlation between stages of gingivitis and levels of endotoxin as measured by the limulus lysate assay. In such experiments it was possible to quantitatively evaluate endotoxins obtained by placing a small filter paper strip into a single, minimally inflamed gingival pocket. Material eluted from the strip was quantitated by the limulus lysate assay, and recorded as nanograms of endotoxin in a sample from the contents of one pocket site. A correlation value of r=0.95 was observed between levels of gingivitis and limulus lysate activity. Based on the results obtained i these initial experiments, work on a simple application of the limulus lysate assay to make it convenientl applicable within the practical limits of the dental practitioner was performed. For this purpose chromogenic substrate was utilized for developing color following an endotoxin-limulus lysat interaction. The present invention involves the use of an extremel sensitive colorimetric or fluorometric assay for th determination of minute amounts (picograms, 10 -1 grams) of specific biochemical constituents of Gram negative bacteria in periodontal pockets. The metho reguires that the dental practitioner obtain a sampl of the patient's periodontal pocket contents by standardized method using small filter paper strips. paper strip acts as a wick to collect bacteria an their free biochemical consituents contained in th pocket. The strip can be treated according to a "Dry assay" or "Wet-assay" method. In the former case th -15- strip is placed on a "Reagent sheet" containing buffering components and limulus lysate ingredients. The site of the strip placed on the "Reagent sheet" is wetted with pyrogen-free water, and following an endotoxing-limulus lysate interaction the color developed is compared with color intensities of a color chart. If the substrate is fluorogenic, the fluorescence developed is similarly evaluated by illuminating the reaction spot in a dark room. Color or fluorescence charts are made on the basis of amounts of endotoxin required under the standard conditions. Alternatively, in the "Wet-assay" method the sample strip and the "Reagent sheet" are immersed in 0.5 ml of pyrogen-free water for interaction and development of color or fluorescence measured with a colorimeter or fluorometer. Values obtained are examined with a standard curve of color or fluorescence versus amount of endotoxin. The reaction-developing color or fluorescence proceeds as the appropriate chromophore is freed by action of a trypsin-like enzyme generated from a zymogen form present in the limulus lysate. Activation of the zymogen is the result of zymogen- endotoxin interaction. The intensity of color or fluorescence is a direct measure of any inflammatory substances present in the periodontal pocket, mostly bacterial endotoxins contributed by Gram-negative bacteria. In this manner the dental practitioner can monitor Gram-negative bacteria and/or inflammatory agents contained in the periodontal pockets of a patient, and, therefore, have a quantitative criterion to follow-up the success of the therapy applied. Examples TYPICAL LIMULOS AMEOBOCYTE LYSATE (LAL) TEST using a chromogenic substrate according to the kit QCL-1000 ® by Whittaker M.A. Bioproducts. Materials: Lyophilized LAL to be dissolved in 1.4 cc of pyrogen- free water. E. Coii Endotoxin Standard (about 20 endotoxin units (EU)) ' , to be dissolved in 1.0 cc of pyrogen-free water. Acetyl-Ile-Glu-Gly-Arg-p-Nitroanalide Subs--rate to be dissolved in 6.0 cc of pyrogen-free water (2.2 mM solution) . Tris Buffer, 0.05 M, pH 9.0, 0.2 ionic strength adjusted with NaCl, in the presence of Ca and Mg ions. Pyrogen-Free Water. Method: Solution Incubation Time and Temp. 50 μl LAL 50 " Buffer 10 min, 37 * 50 " Endotoxin Standard or unknown 50 " Substrate 3 min, 37 * 100" 25 % Acetic acid Total Volume: 0.3 cc. Measure O.D. of p-Nitroaniline at 405 nm. MODIFIED, CHROMOGENIC LIMULUS AMEOBOCYTE LYSATE (LAL) TEST specifically developed for the evaluation of periodontal disease in dental office. The main characteristics of the Modified Chromogenic LAL Test are: 1. An increase of the reaction mixture volume from 0.3 to 5.0 cc. Volume increase is necessary to permit the use of an inexpensive colorimeter instead of a special, expensive spectrophotometer used in a typical LAL test. 2. Possibility of visual comparison of strong red colorations instead of relatively weak yellow colorations due to p-nitroaniline. Red colorations are obtained by coupling p-nitroaniline with H-Acid (cherry-red) or with N-na (purplish red) . 3. Minimal chance of error in a procedure performed by dental office personnel. Materials: All materials are commercially available. LAL, Endotoxin Standard, Chromogenic Substrate, Buffer and Pyrogen-Free Water, as provided in the QCL-1000 Kit by Whittaker M.A. Bioproducts. For p-nitroaniline - H-Acid Coupling: 1 % Sodium Nitrite 3 " Urea 6 " Sodium Bicarbonate 0.75 % H-Acid (8-amino-l-hydroxynaphthalene-3,6- disulfonic acid,Na salt.1.5 HOH) 0.05 N HC1 For p-nitroaniline - N-an Coupling: 1 % Sodium Nitrite 3 " Urea 0.75 % N-na (S-(l-naphthyl)-ethylenediamine . 2 HCl) 0.35 N HCl H-Acid and N-na obtained from Aldrich Chemical Co. Methods: The Modified LAL Test was developed in stages starting with an evaluation of the nature and conditions of the typical LAL test. In addition to the disadvantage of a 0.3 cc reaction mixture volume and the yellow coloration that is not an optimal criterion for visual comparisons in the typical LAL test, there are two undesirable conditions in this test: 1. The practice of a 10-min. incubation for the zymogen-enzy e conversion followed by the 3-min. enzyme-substrate interaction re-quires strict timing that may result in stress and errors on the part of those performing the tests especially when several samples are handled in one run. There is no reason for the substrate not to be present from the beginning, and acted upon as the enzyme is being generated. Therefore, it was decided to allow the total mixture o LAL, endotoxin, substrate and buffer to be held at 37* for 15 min. The modification of the one-stage 15-min. tota interaction is considerably safer with regard to timin -19- errors than the typical LAL test characterized by a first stage 10-min. incubation followed by a second stage 3-min. enzymatic reaction. Furthermore, the 15- min. total interaction results in a higher concentration of p-nitroaniline generated. 2. There is no particular advantage in using 25 % acetic acid in the typical LAL test. Thus, in the Modified LAL Test it was decided to use a solution of HCl with a dual purpose: first, to terminate the enzyme-substrate interaction, and second, to convert the generated p-nitroaniline into its diazonium salt to be coupled with the appropriate agent. The p- nitroaniline - H-Acid coupling was performed under alkaline conditions. Thus, 0.05 N HCl was sufficient to terminate enzymatic activity and liberate the nitrous acid to interact with p-nitroaniline. For the p-nitroaniline - N-na coupling method 0.35 N HCl was used to terminate enzymatic activity, form the diazonium salt of p-nitroaniline and provide the required acidity for coupling. Experimental work began by maintaining the mixture of LAL,endotoxin, substrate and buffer at 37° for 15 min., and terminating enzymic activity with 0.5 cc of 0.05 N (or 0.35 N) HCl: 50 ul LAL 50 " Buffer 50 " Endotoxin Standard or Unknown 50 " Substrate 15 min., 37* One-stage, 15 min. total interaction 500 ul 0.05 N (or 0.35 N) HCl The resulting final volume of 0.7 cc permitted p- nitroaniline O.D. measurements at 405 nm using an ordinary spectrophotometer and 1-cc cuvettes. In subsequent work, 1.0 cc a/ acid was added giving a final volume of 1.2 cc. The 1.2 cc volume was appropriate for the formation of the p-nitroaniline diazonium salt followed by coupling to form an azo-dye. After coupling with H-Acid or N-na, neither the reaction mixture was diluted to 5.0 cc and its O.D. was measured at 540 n or sample and standard colorations were visually compared. Modified LAL Test p-nitro.milin* coupled with H-Acid: 50 ul LAL 50 " Buffer 50 " Endotoxin Standard or Unknown Control, with 50μl Pyrogen-Free Water 50 " Substrate 15 min., 37" 1.0 cc 0.05 N HCl 100 ul 1 % Sodium Nitrite 3 % Urea 6 % Sodium Bicarbonate 0.75 % H-Acid Water to 5.0 cc (3.1 cc) O.D. measured at 540 nm Comments: Addition of sodium nitrite in the acid medium results in liberation of nitrous acid which in turn reacts with p-nitroaniline to form its diazonium salt. Urea is added to destroy any excess nitrous acid, thus preventing a reaction of this acid with the amino group of the H-Acid. Sodium bicarbonate provides the alkalinity for the H- Acid coupling. The cherry-red color obtained was stable for weeks. S T R U C T U R A L F O R M U L A E P-nitroaniline oupled NO. Modified LAL Test p-nitroaniline coupled with N-na: 50 ul LAL 50 " Buffer 50 " Endotoxin Standard or Unknown Control, with 50 μl Pyrogen Free Water 50 " Substrate 15 min. , 37* 1.0 cc 0.35 N HCl 100 ul 1 % Sodium Nitrite loo " 3 % Urea 100 " 0.75 % H-na Water to 5.0 cc (3.5 cc) O.D. measured at 540 nm Comments: Under the conditions of acidity and 1.4 cc volume prior to addition of N-na, introduction of this coupler results in instantaneous formation of a purplish color. The color is practically unchanged for 24 hours, but fades in about two weeks to an extent visually observed in a comparison of fresh and old reaction mixtures. The O.D. of N-na coupled reaction mixtures is about 1.4 times higher than the O.D. associated with H-Acid coupling and about 6 times higher than the O.D. of the p-nitroaniline not subjected to coupling. Modified LAL Test for endotoxins obtained from the fluid of a periodontal pocket: Fluid of periodontal pockets was sampled by inserting the professionally employed paper strips for 30 sec. 1.0 cc of Buffer was added to a strip and the mixture was gently warmed and vortexed for one min. This master solution of endotoxins was diluted with pyrogen- free water 50, 100, 500 and 1000 times. The Modified LAL Test was performed only with diluted solutions because master solutions of endotoxins exhibited a concentration that was over-saturating the LAL material. It is noted that a certain amount of LAL material contains a certain amount of zymogen that is converted to an enzyme by a proportional amount of endotoxin. Endotoxin supplied in excess of the requirement for the total zymogen present is useless and no more enzyme can be generated. Accordingly, excess endotoxin is manifested as a plateau effect fo the p-nitroaniline liberated. Example Measuring the Endotoxins of a Periodontal Pocket: A strip was inserted for 30 sec. in a 6-mm periodonta pocket of a patient with advanced periodontitis. Th strip was eluted in 1.0 cc of Buffer to form a maste solution of endotoxins. A Modified LAL Test with H Acid coupling was performed using 100 ul of the 5 times diluted master solution of endotoxins. An O.D of 0.234 was measured at 540 nm. According to the Standard Endotoxin Curve (linea function) prepared under identical conditions, an O.D of 0.234 at 540 nm corresponds to an endotoxi concentration of 0.93 EU/cc (Endotoxin Units/cc) . Therefore, the 50 times diluted solution of endotoxin had an endotoxin concentration of 0.93 EU/cc. Thus the endotoxin concentration in 1.0 cc of strip eluat (master solution) exhibited an endotoxin content o 46.5 EU/cc (0.93 X 50) . The Endotoxin Standard Curve: As mentioned in the Materials section for the typical LAL test, Standard E. Coli Endotoxin is supplied with a specification of X Endotoxin Units/vial (EU/vial) . Addition of 1.0 cc of Pyrogen-Free Water yields a Standard Endotoxin Solution of X EU/cc. One Endotoxin Unit represents 0.2 nanograms of LPS (lipopolysaccharide) . It is convenient to dilute an original Standard Endotoxin solution of X EU/cc X times in order to obtain a Standard Endotoxin solution of 1.0 EU/cc, an aliquot of which can be further diluted (e.g., 2, 4, 5 and 10 times) . The 1.0 EU/cc solution and its further dilutions are used to perform a LAL test and plot O.D. measured versus EU/cc of endotoxin solution employed. General Considerations concerning the Modified Chromogenic LAL Test: The spectrophotometric determinations of aniline and many of its derivatives based on procedures of diazotization-coupling were reported by George Norwitz and Peter Kellher Anal. Chem. 53_, 56-60 (1981); Anal. Chem. 5_3, 1238-1240 (1981); Anal. Chem. 54_, 807-809 (1982); Anal. Chem. 55, 1226-1229 (1983)). Norwitz and Kellher selected H-Acid and N-na for diazotization- coupling with aniline and its derivatives and investigated the conditions of the reactions for the production of azo dyes that are reliable and useful in spectrophoto etry. However, in this work certain of Norwitz's and Kellher 7 s conditions were modified for practical purposes. For instance, the coupling agents were added to a considerably smaller volume of solutions containing the diazonium salt of p-nitroaniline to achieve higher concentrations and faster reactivity. Also, urea, a substance less costly than ammonium sulfamate, was employed to destroy excess nitrous acid. H-Acid and N-na, like all aromatic hydrocarbons and their derivatives, are substances that must be handled with care. For reasons of safety and also because H- Acid (not recrystallized material) solutions darken upon standing even without exposure to light, it was necessary to seek an alternative to the use of coupling agent solutions. Strips cut from thick glass-fiber filtration disks (Whatman) were wetted with coupling agent solutions and allowed to dry. Such dry coupling reagent-strips were successfully used for the addition of the coupling agents. There was an immediate development of red color of the same optical density as in the case of addition of fresh coupling reagent solution. The Modified LAL Tests were also performed as "spot tests" on thick glass-fiber filtration disks. However, the above-described methods are more appropriate for a visual comparison of colorations in solutions held in the path of light. Optical densities of low value observed in control experiments (Pyrogen-Free Water added instead of Endotoxin Solution) are due to the spontaneous hydrolysis of the substrate exposed to pH 9, at 37*C. For this reason the Substrate solution is prepared with Pyrogen-Free Water, and can be kept refrigerated for about six months without a significant release of p- nitroaniline. The Substrate is exposed to the Buffer only during the performance of a LAL test. To date the Limulus Activity of the following 9 oral isolates have been analysed: (1)- gram (+) bacteria including- S. mutans, S. sanguis, A. israelii, A. viscosus, A. neaslundi; (2)- gram (-) bacteria including-C. gingivalis, C. ochracea, A. actinomycetemcomitans, and V. alcalescens. On the average the gram (-) bacteria are approximately 1 x 10 times more active in the Limulus Assay than any of the gram (+) bacteria tested using the clot and tube dilution assay. Negligible differences were found when whole cell suspensions were compared to sonic extracts derived from analagous whole cell suspensions. Thus it can be assumed that either whole cells or cell fragments derived from the same cell source can provoke limulus activity with equal facility. These results suggest that distinctions between gram (-) and gram (+) bacteria can be readily ascertained and maximized by dilutions of plaque organisms. In this manner all limulus-provoking activity of gram (+) bacteria can be obliterated by the dilution effect and thus only gram (-) bacteria will be capable of provoking a positive limulus response. Since over 98% of the suspected periodontal pathogens are gram (-) this could be a valuable clinical tool for determining levels of gram (-) bacteria in a pocket site. A collection procedure that can be used at chairside by dentists in an effort to determine levels of endotoxin in specific pocket areas has been developed (see Example below) . PROTOCOL FOR LIMULUS COLLECTION (1) - Isolate area with cotton rolls (2) - Wipe area dry with cotton pledget (3) - Place one #3 sterile absorbent endodontic paper point into pocket of concern for ten seconds (4) - Holding point in pliers dry point with air from hair dryer set on high temperature for one minute (5) - Place paper point in sterile tube, label tube and place in freezer (6) - Try to collect from 2 diseased sites and one "normal" site per patient (7) - Perform LAL test or modified LAL on paper point and determine endotoxin concentration by comparison with color charts.

Claims: What is claimed is: 1. A method of quantitatively determining the amount of bacterial endotoxins present in a periodontal pockets of a subject which comprises: a) obtaining a sample from the periodontal pocket of the subject; b) contacting the sample with an amebocyte lysate under conditions so as to activate an enzyme capable of cleaving a bond between an arginyl group and a nitrogen-containing group; c) contacting the activated enzyme with a substrate comprising an arginyl group and a suitable nitrogen-containing group bound to the arginyl group so as to form an amine; d) treating the resulting amine to produce a detectable product; and e) quantitatively determining the amount of product formed and thereby the amount of bacterial endotoxins present in the periodontal pocket. 2. A method of claim 1 further comprising treating the sample prior to contacting with the amebocyte lysate so as to inactivate endogenous enzymes present in the sample which would interfere with amebocyte lysate-endotoxin interaction. 3. A method of claim 2, wherein the treating of the sample comprises heating of the sample. 4. A method of claim 1 further comprising dilution of the sample prior to contacting with amebocyte lysate. 5. A method of claim 1, wherein the amebocyte lysate is Limulus amebocyte lysate. 6. A method of claim 1, wherein the sample is obtained by placing a filter paper strip into the periodontal pocket of the subject and collecting the sample on the paper strip. 7. A method of claim 6, wherein the sample obtained on the filter paper strip is heated prior to contacting with the amebocyte lysate. 8. A method of claim 1, wherein the contacting of the sample with amebocyte lysate is performed in the presence of a buffer. 9. A method of claim 1, wherein the contacting of the sample with amebocyte lysate and the contacting of the activated enzyme with the substrate is performed simultaneously. 10. A method of claim 9, wherein the contacting of th sample with amebocyte lysate and the contacting o the activated enzyme with the substrate i effected by obtaining the sample on a filter pape strip and contacting the strip with a reagen sheet containing the amebocyte lysate and th substrate. -Si¬ 11. A method of claim 9, wherein the contacting of the sample with amebocyte lysate and the contacting of the enzyme with the substrate is effect by obtaining the sample on a filter paper strip and immersing th»_ strip in a solution comprising the amebocyte lysate and the substrate. 12. A method of claim 1, wherein the activated enzyme is trypsin or a trypsin-like enzyme. 13. A method of claim 1, wherein the nitrogen- containing group is naphthylamide or p- nitroanilide and the amine formed is napthylamine or p-nitroaniline. 14. A method of claim 13, wherein the substrate is benzoylarginine-β-naphthylamide , benzoylarginine- p-nitroanilide, or acetyl-Ileu-Glu-Gyl-Arg-(p- nitroanilide) . 15. A method of claim 14, wherein the substrate is acetyl-Ileu-Glu-Gyl-Arg-(p-nitroanilide) . 16. A method of claim 1, wherein the treating of the amine comprises diazotization of the amine to produce a diazonium salt and reacting the diazonium salt with a suitable coupling agent to produce the detectable product. 17. A method of claim 16, wherein the coupling agent is histidine, 8-amino-l-hydroxynapthalene-3,6- disulfonic acid, or N-(l-naphthyl)-ethylenediamine dihydrochloride or a salt thereof. 18. A method of claim 17, wherein the detectable product is a colored product. 19. A method of claim 1, wherein quantitatively determining the product formed is effected by comparing the detectable product with standards of known endotoxin concentrations. 20. A method of claim 18, wherein quantitatively determining the colored product is effected by comparing the colored product to standard curves of color versus endotoxin concentration. 21. A method of claim 20, wherein quantitatively determining the colored product is effected by comparing the optical density of the colored product to optical densities of solutions of known endotoxin concentrations. 22. A method of quantitatively determining the amount of bacterial endotoxins or other inflammatory agents present in a periodontal pocket of a subject which comprises: a) collecting a sample from the periodontal pocket of the subject on a filter paper strip; b) treating the strip containing the sample so as to inactivate endogenous enzymes which would interfere with limulus amebocyte lysate-endotoxin interaction; c) contacting the treated strip with limulus amebocyte lysate and a substrate comprising -33- an arginyl group and a naphthyl mide or p- nitroanilide group bound to the arginyl group so as to form an aromatic amine; d) treating the aromatic amine to form a diazonium salt; e) reacting the diazonium salt with a suitable coupling agent to produce a colored product; f) quantitatively determining the amount of colored product produced and thereby the amount of bacterial endotoxins present in the periodontal pocket by comparing the color intensity of the colored product to standards of known endotoxins concentrations. 23. A method of claim 22, wherein the treating of the strip containing the sample is effected by heating the strip. 24. A method of diagnosing a periodontal disease which comprises quantitatively determining the amount of endotoxin present in the periodontal pockets of a subject by the method of claim 1 and correlating the amount so determined with known periodontal disease-causing amounts of endotoxin. 25. A method of claim 24, wherein the periodontal disease is gingivitis or advanced periodontitis. 26. A periodontal disease diagnostic kit comprising a support means on which the test sample is to be place, a container of lyophilized limulus amebocyte lysate, a container of E. Coli endotoxin standard of about 20 endotoxin units, a container of a substrate comprising an arginyl group and a suitable nitrogen-containing group bound to the arginyl group, a container of a buffer, a container of pyrogen-free water, and a container of acetic acid. 27. A kit of claim 26, wherein the substrate is acetyl-Ile-Glu-Gly-Arg-p-nitroanalide. 28. A kit of claim 26, wherein the buffer is Tris buffer at 0.05M, pH 9.0, 0.2 ionic strength adjusted with NaCl in the presence of Ca and Mg ions. 29. A kit of claim 26 further comprising containers of sodium nitrite, urea, sodium bicarbonate, sodium salt of 8-amino-l-hydroxynaphthalene-3,6- disulfσnic acid, and HCl. 30. A kit of claim 26 further comprising container of sodium nitrite, urea, N-(l-naphthyl)- ethylenediamine, and HCl.

Inventors: FINE DANIEL H, VRATSANOS SPYROS M, FINE, DANIEL, H., VRATSANOS, SPYROS, M.
Applicants: UNIV COLUMBIA, THE TRUSTEES OF COLUMBIA UNIVERSITY
Application year: 1989
Main classification: C12Q1
IPC codes: C12Q1, G01N33