Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1990-10-02
1992-06-23
Nucker, Christine
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 25, 435 29, 435 34, C12Q 128, C12Q 126, C12Q 102, C12Q 116
Patent
active
051242540
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a method for the identification of diamines, particularly putrescine and cadaverine in vaginal secretions, and the use of this in diagnosis of a certain clinical condition, and 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 described a condition associated with a grey homogeneous discharge having a pH of 5.0-5.5 and accompanied by minimal inflammation. The condition appears to be associated with the organism Gardnerella vaginalis in the presence of other organisms, notably anaerobic bacteria. This form of vaginitis is now often called bacterial vaginosis to acknowledge the complexity of its microbiological origin.
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 alkali to the secretion (the "amine" test) (H. L. Gardner and C. D. Dukes, loc. cit.; T. Pheifer et al., New Eng. J. Med., 1978, 298, 1429-1434; R. Amsel et al., Am. J. Med., 1983, 74, 14-22; Report of The Working Group on "The Diagnosis of Bacterial Vaginosis", Scand. J. Urol. Nephrol. (Suppl.), 1984, 86, 260-261).
Microbiological 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 occur at significantly elevated levels in Gardnerella-related vaginitis (K. C. S. Chen et al., J. Clin. Invest., 1979, 63, 828-835); 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 96% 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 is 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 not 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 is detected by a colour reaction produced by peroxidase and a suitable chromogenic system. A suitable diamine oxidase (E. C. 1.4.3.6) can be 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 may be used. A mixture of 4-aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulphonic acid, introduced by Barham 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 satisfactorily. Other chromogenic systems which also work successfully include 3,3',5,5'-tetramethylbenzidine and 2,2'-azino-bis-(3-ethylbenzthiazoline-6-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. Quantitative measurement can be obtained from the optical density of
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patent: 4550078 (1985-10-01), Yamada et al.
patent: 4617263 (1986-10-01), Yamada et al.
Chen et al. (1979) Amine Content of Vaginal Fluid . . . J. Clin. Invest. 63: 828-835.
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"A Sensitive, Rapid, Chemiluminescence-Based Method for the Determination of Diamines and Polyamines", Uriel Bachrach et al., Analytical Biochemistry, 152, 423-431 (1986).
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Davis Robert H.
Hewlins Michael J. E.
O'Dowd Thomas C.
Winterburn Peter J.
Nucker Christine
Preston David R.
University College Cardiff Consultants Limited
Welsh Medical School Enterprises Limited
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