GB2215044A - Detection of diamines in biological fluids - Google Patents

Abstract

A fast-acting and portable diagnostic kit for detecting diamines in biological fluids is in the form of a diamine oxidase and a chromogenic system to detect the presence of hydrogen peroxide resulting from oxidation of such diamines. The kit may especially be used to detect cadaverine or putrescine or a mixture thereof, for example in a vaginal secretion, in order to detect Gardnerella-related bacterial vaginosis. The chromogenic system may be composed of peroxidase, 4-aminoantipyirine and a phenolic compound (e.g. 3, 5-dichloro-2-hydroxybenzenesulphonic acid).

Description

DETECTION OF DIAMINES IN BIOLOGICAL FLUIDS This invention relates to a method for the identification of diamines, particularly putrescine and cadaverine in vaginal secretions, and the use of this Ir. diagnosis of a certain clinical condition, ad to a diagnostic kit to enable the method to be applied routinely.

Vaginitis is a widespread problem in general practice. One common form of vaginitis was characterised by Gardner and Dukes (H.L.Gardner and C.D.Dukes, Am.J.Obstet. Gynecol., 1955, 69, 962- 976) who dexeribed a condition associated with a grey homogeneous discharge having a pH of 5.0-5.5 and accompanied by ninimal inflammation The condItion appears to be assoclated with the organism Gardnerella vasinalts in the presence of other organisms, notably anaerobic bacteria. This for of vaginitis is now often called bacterial vaginosis to acknowledge the complexity of its mierobilogical orign.

Clinical diagnosis of this condition is aided by identification of "clue cells (vaginal epithelial cells with adherent surface bacteria), a raised pH, and a "fishy" odour generated on adding alkall to the secretion (the "amine" test) (H.L.Gardner and C.D.Dukes, loc.cit.; T.Pheifer et al., New Eng.J.Med., 1978, 29@, 1429-1434; R.Amsel et al., Am.J.Med., 1983, 74, 14-22; Report of The Working Group on the Diagnosis of Bacteria Vaginosis", Scand.J.Urol.Nephrol. (Suppl.), 1984, 86, 260-261).

Mi@robiological confirmation of G.vaginalis is not straightforward and culture results take 2-3 days. There is no simple, rapid, diagnostic procedure. It is known that the diamines putrescine and cadaverine @@@@ at significantly elevated levels in Gardnerelle-related vaginit (K.C.S.Chen et al., J.Clin. Invest., 1979, 63, 828-635); these diamines are probably produced by the symbiotic growth of G.vaginalis in the presence of the anaerobes. Earlier work showed that the presence of the diamines correlated with clinical symptoms in 96b of patients (K.C.S.Chen et al., J.Infect.Dis. 1982, 145, 337-345) Previously the detection of the diamines has depended on electrophoresis or thin-layer chromatography (K.C.S.Chen et al., loc.cit.) requiring specialist equipment and personnel. The aim of this invention i to provide a method to establish the presence of the diamines in a way that can be carried out in a laboratory, clinic, surgery, or in the home, by persons nct skilled in chemical analysis or microbiological procedures.

The invention uses a selected diamine oxidase which reacts with the diamines, putrescine and cadaverine, to give hydrogen peroxide. This : detected by a colour reaction produced by peroxidase and a suitable chromogenic system. A suitable diamine oxidase (E.C. 1.4.3.6) can h isolated from pea seedlings (R.E.McGowan and R.M.Muir, Plant Physiol., 1971, 47, 644-649); this oxidises putrescine and cadaverine with a high specificity. Other diamine oxidases, for example that from pig kidney(Sigma Chemical Co.), can be used in a similar way. Peroxidase is a well-known enzyme that is commercially available. For the chromogenic system, a number of different reagents ny be used.A mixture of 4-aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulphonic acid, introduced by Barhan and Trinder (D.Barham and P.Trinder, Analyst, 1972, 97, 142-145) to detect hydrogen peroxide, proves very satisfactory, but other phenolic components, for example chromotropic acid or 2,4,6-tribromo-3-hydroxybenzoic acid, may also be used s@tisf@@@@@ other chromogenio systeme which also work successfully in@@@de 3,3',5,5'-tetramethylbenzidine and 2,2'-azino-bis-(3-ethylbenzthiazoline6-sulphonate).

The diagnostic test in its first form involves taking a vaginal swab and agitating it in a solution containing the diamine oxidase, peroxidase, and the chromogenic compounds. When a mixture of 4-aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulphonic acid is used as the chromogenic system, the positive magenta colour develops in under two minutes and is stable for more than one hour. Quantitatlve measurement can be obtained from the optical denslty of the solution, but this is not necessary for the clinical diagnostic test. Negative samples give no colour, or at most a very pale colour. The solution of reagents for the test in this form can be made by simple addition of water to a freeze-dried mixture of all the ingredients prepared in a suitable vessel. This freeze-dried mixture can be stored without deterioration of its efficiency.Alternatively the reagents can be constituted in other ways, for example in a tablet form or adsorbed on to paper or other surface layer.

Cllnical samples of 183 vaginal secretions have been tested and the results correlated with existing diagnostic procedures - the "amine" test, "clue cells and culture of G.vaginalis. These correlations, and the sensitivities and speclficlties, are presented in Example 7 (see below).The diamine test results correlate closely with the microbiological culture and with the two sideroom techniques; clue cello and "amine" tests.The agreements in the comparisons with "clue cells" and the "amine" test are marginally better than those with the microbiological results from the Public Heaith Laboratory Service. n a tree comparisone the diamine test gave slightly poere@ appa@ent spetifi@@@, than sensitivity. Thus, sensitivity for miorobiological, "cl@@ cell" wamlner tests was 86%, 96% and 93% respectively. The new test thus works well as a diagnostic procedure.

EXAMPLES Example 1 Linearity of response of the diamlne oxidase/dye system to increasing concentrations of putrescine.

Procedure.

Each incubation contained the following reagents: 4-aminoantipyrine (5mg/ml; 0.1ml); 3,5-dichloro-2-hydroxy-benzenesulphonic acid (35mg/ml; 0.1ml); peroxidase (1mg/ml; 0.1ml); diamine oxidase (5-15 units/ml; 0.02ml); putrescine (0-1.25mM; 0.1ml); buffer (0.1M tris-HCl, pH 7.5; 2ml).

The reagents were mixed and the absorbance at 515nm was recorded after a 10 minute incubation. The absorbance was proportional to the amount or putrescine added, so establishing that the relationship between absorbance and the quantity of putrescine in the assay was linear over the range studied (0 - 125 nmol putrescine per assay).

Table nmol putrescine A 515nm nmol putrescine A 515nm 0 0 25 0.292 1 1 0.012 50 0.584 2.5 0.026 75 0.885 5 0.058 100 1.160 7.5 0.088 125 1.446 10 0.113 Example 2 Addltivity of the response of the diamine oxidase/dye system to the two diamines, putrescine and cadaverine.

The procedure was as descrlbed in Example 1, but using either putrescine or cadaverine, or mixtures -of both putrescine and cadaverine. Putrescine and cadaverine gave similar absorbances on a mclar basis and when present together in the incubation the absorbance produced was that predicted by a simple addition of the absorbances given by the two diamines separately.

Table Absorbance 515nm column 1 column 2 column 3 column 4 Substrate mixture of concentration putrescine cadaverlne putrescine column 1 of each diamine alone alone + + cadaverine column 2 1 nmol 0.011 0.009 0.018 0.020 10 nmoi 0.113 0.096 0.225 0.209 100 nmol 1.105 1.104 2.163 2.209 (Column 4 is calculated as the addition of the absorbances in columns 1 and 2.) Example 3 Response of the diamine oxidase/dye system to other amines or diamines.

The procedure was as described in Example 1, but the putrescine was replaced by other biologically important amines. The other amines included: alanine, asparagine, aspartic acid, arginine, cysteine, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, valine, 4-aminobutyric acid, histamine, phenethylamine, spermine and tyramine. Colour producti@@ was not detectable, or was elight or very slow to develop with these other a@@@es Thue, the response of the diamine oxidase/dye system to these biologically important amines that may be present in secretions or fluids could be readily distinguished from the colour production by putrescine and cadaverine.

Example 4 Alternatlve dye systems used in conjunction with the diamine oxidase.

The procedure was as described in Example 1, except that the incubetion contained 2,2'-azino-bis-(3-ethylbenthiazoline-6-sulphonate)(14mg/ml; 0.1ml) instead of 4-aminoantipyrine and 3,5-dichloro-2-hydroxybenzene sulphonic acid. The absorbance was measured at 646nm after an incubation at room terperature for 60 minutes Table nmol putrescine A 646nm nmol putrescine A 646nm 0 0 25 C.172 1 0.008 -50 0.336 2.5 0.018 75 0.490 5 0.035 100 0.6 7.5 0.054 125 10 0.070 This alternative dye system gave absorbances proportional to the putrescine concentration and thus demonstrated that the procedure was not dependent on the dye system quoted in Example 1 but would operate with other hydrogen peroxide detection systems.

Example 5 Stability of the reagents in a dried form.

Transparent polystyrene tubes were prepared containing in freeze-dried form the following components: 4-aminoantipyrine (0.5mg); 3,5-dichloro-2- hydroxybenzenesulphonic acid (3.5mg); peroxidase (0.1mg); diamine oxidase (0.2 units); tris-HCl, pF 7.5 (0.2mmol).

This freeze-dried mixture contained the same amounts of the reagents a in Example 1, with the omission of putrescine.

Each tube was reconstituted with 2.3ml water after 6 weeks storage at 4 C and the reactivity compared with a fresh solution of the same composition.

Putrescine (50 nmol) was added to each test (in triplicate) and the absorbances compared.

Table Absorbances 515nm mean Fresh 0.588 0.587 0.612 0.596 Reconstituted 0.608 0.607 0.609 0.608 Thus, when the componente were stored in a dried form there was no loss of reactivity by the diamine oxidase/dye system. Similarly, when the same components were dried on to a strip of Whatman No.1 chromatography paper and the paper dipped into a solution containing putrescine, a magenta colour developed.

Example 6 Appllcatlon of the diamine oxidase/dye system to the analysls of diamines in vaginal secretlons.

Vaginal swabs were taken from: (i) patients consulting for vaginltis, (ii) patients consulting for cervical smears or family planning advice, (iii) women invited to health checks.

Each swab was washed twice with lml 0.1M-tris/HCl buffer, pH7 5. Any particulate material in the combined washings was removed by brief centrifugatlon and/or filtration. An aliquot (0.5ml) was added to a mlxture containing: tris/HCl buffer (pH7.5, 0.1M, 1.5ml); 3,5-dichloro-2-hydroxybenzenesulphonic acid (35mg/ml, 0.1ml); 4-aminoantipyrine (5mg/ml, 0.1ml); peroxidase (lmg/ml; O.lml); diamine oxidasc(l0units/ml, 0.02ml).

The absorbance at 515nm was measured after a 10 minute incubation at roon temperature and the amount of diamine calculated from a standard calibration. From a survey of 183 swabs the amount of diamine in eac swab was estimated. The range of values was found to be 0-540 nmol diamines/swab.

Example? 183 vaginal swabs were assayed quantitatively by the procedure described in Example 6 and the results compared with the microbiological culture by the Public Health Laboratory Service and with current sideroom methods of diagnosing Gardnerella-related vaginitis. Details of the "amine" test, identification of "clue" cells and of the taking, storing, transporting and cultures of swabs at the Public Health Laboratory Service have been described (T.C.O'Dowd et al., J.Roy.Coll. Gen.Pract., 1987, 37, 59-61).

Microbiological comparison The Public Health Laboratory Service identified 57 swabs as Gardnerella vaginal is positive and the remaining 126 swabs as Gardnerella vaginal negative. The rear quantity of diamine measured per swab fc Gardnerella pesitive group was 16@@@@ diamines(+146nm@@ s.d.). and for the Gardnerella negative group was 37nmol diamines(+86nm@l s.d.). The dlfference was statistically hlghly significant.

If a cut-off at 25nmol diamines/swab is chosen as the dividing line between a positive and a negative result in the diamine oxidase/dye system assay, the sensitivity of the diamine test was 49/57 = 86t, i.e. 8 apparent false negatives", and the specificity of the diamine test was 1001126 = 79S, i.e. 26 apparent "false positives".

"Clue cell" comparison 171 swabs were evaluated for "clue cells". 52 swab were described as "clue cell positive", 17 swabs as "clue cell uncertain" and 102 swabs as clue cell negative". The results of the diamine test were 200nmol diamines (+141nmol s.d.)/swab, 54nmol diamines(+103nmol s.d.)/swab and 22nmol diamines (+60nmol s.d.)/swab for the three groupe respectively. The statistical evaluation of the three groups revealed that the samples fron the "clue cell uncertain" group were statistlcally different from those of the "clue cell positive" group but not from those of the "clue cell negative" group. Thus, the "clue cell uncertain" and "clue cell negative" groups were considered as constituting a single group.

If a cut-off at 25nmol diamines/swab is chosen as the dividing line between a positive and a negative result in the diamine oxidase/dye systen assay, the aensitlvlty of the diamine test was 50/52 = 96%, i.e. 2 apparent false negatives", and the specificity was 97/119 = 82%, i.e.

22 apparent "false positives".

"Amine" test comparison Using the same cut-off value of 25nmol diamines/swab in the dlamine oxldase/dye system assay, 40 of 42 swabs judged positive in the "amine" test were also positive by the diamine test. Similarly, 13 of 15 swabs judged possibly posltlve in the "amine" test and 14 of 87 swabs judged negative for wamlnesw gave a positive result in the diamine test. If those swabs considered possibly positive are grouped with the positive, the relative sensitivity was 53/57 = 93%, i.e. 4 apparent "false negatives", and the relative specificlty 73/87 = 84%, i.e. 14 apparent "false positives".

The diamine oxidase/dye system results agreed well with the microbiological culture and with the two sideroom techniques; "clue cell" and "amine" tests. It is apparent that agreements in the comparisons with clue cells* and the "amine" test are marginally better than those with the microbiological results from the Public Health Laboratory Service.

In all three comparisons the diamine test gave slightly poorer apparent specificity, an indication of "false positives", than sensitivity, an indication of "false negatives". Thus, sensitivity for microbiological, "clue cell" and "amine" tests was 86%, 96t and 93% respectively, whereas the incidence of "false positives" was 21t, 18% and 16t for the microbiological, "clue cell" and "amine" tests respectively. The close agreement with the "amine" test might be expected because the diamine test measures the diamines that create the characteristic odour associated with this sideroom test.

In each of these comparisone the efficiency of the diamine assay is being assessed on the basis that the true answer is given by either the microbiological culture or a sideroom test. However, no single test is currently recognised as a definitive test and diagnosis resides on a collective evaluation of the several tests employed in the comparisons presented here. Thus, the evaluation of the diamine oxidase/dye system assay indicates that it is as accurate as other recognised procedures, but has the advantages of portability and speed, and may be performed by unskilled, untrained personnel or by the patient.

Example 8 Application of the diamlne oxidase/dye system to the analysls of diamines in the presence of human serum.

Procedure.

Each incubation contained the following reagents: 4-aminoantipyrine (5mg/ml; 0.1ml); 3,5-dichloro-2-hydroxy-benzenesulphonic acid (35mg/ml; O.lml); peroxidase (lmg/ml; O.lml); diamine oxidase (5-15 units/ml; 0.02m1); putrescine or cadaverine (G, 10, 50 or 100 nmol; O.lml) human serum or buffer (0.1M tris-HCl, pH 7.5) (0.5ml) buffer (0.1e tris-Hol, pH 7.5) to make a final volume of 2.1ml.

The reagents were mixed and the absorbance at 515nm was recorded after a 10 minute incubation. The absorbance was propertional to the amount cf putrescine or cadeverine added, so establishing that the relationship between absorbance and the quantity of either ptresine or cadaverine in the assay was linear in the presence of human serum. over the range studied (0 - 100 nmol diamine per assay).

Table (undialysed serum) putrescine A 515nm cadaverine A 515nm nmol -serum +serum nmol -serum +serum 10 0.111 0.091 10 0.127 0.096 50 0.56' 0.425 50 0.616 0.503 100 1.118 0.948 100 1.265 1.078 The presence of serum in the assay reduced the absorbance by 15-20z; this decrease in colour formation was the same for both putrescine and cadaverine.

Two similar series of incubations were established, one using human serum that had been dialysed at 40 C for 24 hours against physiological saline (0.154M NaCl), and the other using putrescine and cadaverine in the same incubation.

Table (dialysed serum) putrescine A 515nm cadaverine A 515nr.

nmol -serum +serum nmol -serum +serum 10 0.111 0.089 10 0.127 0.104 50 0.562 0.480 50 0.616 0.544 100 1.118 0.947 100 1.265 1.096 The presence of dialysed serur in the assay ha a sirilar effect to that exhibited by undialysed serum and the decrease in colour formation was the same for both putrescine and cadaverine.

In a similar manner of the findings reported in, Example 2, colour formation from putrescine and cadaverine was additive in the presence of either dialysed or undialysed serum.

Claims (13)

1. A method for the detection of at least one diamine in a biological fluid, by adding to the biological fluid in vitro a diamine oxidase and a chromogenic system to detect the presence of hyrogen peroxide resulting from the oxidation of such diamine.

2. A method according to claim 1, wherein the diamine is putrescine or cadaverine, or a mixture thereof.

3. A method according to claim 1 or 2, wherein the chromogenic system is composed of peroxidase, 4-aminoantipyrine, and a phenolic compound.

4. A method according to claim 3, wherein the phenolic compound is 3, 5-dichloro-2-hydroxybenzenesulphonic acid.

5. A method according to any preceding claim, wherein the diamine oxidase is derived from pea seedlings.

6. A method according to any preceding claim for the detection of Gardnerella-related bacterial vaginosis and wherein the biological fluid is a vaginal secretion.

7. A diagnostic kit for detecting the presence of one or more diamines, comprising a diamine oxidase and a chromogenic system to detect the presence of hydrogen peroxide resulting from oxidation of such diamines.

8. A diagnostic kit according to claim 7 for the detection of cadaverine or putrescine or a mixture thereof.

9. A diagnostic kit according to claim 7 or claim 8, wherein the chromogenic system is composed of peroxidase, 4-aminoantipyrine, and a phenolic compound.

1C. A diagnostic kit according to claim 9, wherein the phenolic compound is 3,5-dichloro-2-hydroxybenzenesulphonic acid.

11. A diagnostic kit according to any of claims 7 to 10, wherein the diamine oxidase is derived from pea seedlings.

12. A diagnostic kit according to any of claims 7 to 11, wherein the reagents are in freeze-dried form in sealed containers, or on a portable surface, for example paper.

13. A diagnostic kit according to any of claims 7 to 12, for the detection of Gardnerella-related bacterial vaginosis and wherein the biological fluid is a vaginal secretion.