Method for measuring hypochlorite ion

This application is a continuation of application Ser. No. 12/099,979, filed Apr. 9, 2008, which is a continuation of application Ser. No. 10/437,437 filed May 14, 2003, now U.S. Pat. No. 7,378,282, issued May 27, 2008, the entire disclosures of which are incorporated by reference herein in their entireties.

The present invention relates to a method for measuring hypochlorite ion. The present invention also relates to an agent for measuring hypochlorite ion and a kit used for said measuring method.

Hypochlorite ion is one of reactive oxygen species whose function in organisms has recently been focused. It is considered that bactericidal action of neutrophils is mainly derived from hypochlorite ion. Hypochlorite ion has been shown to be generated from hydrogen peroxide and chloride ions by myeloperoxidase in azurophilic granules in vitro (Klebanoff, S. J., and Clark, R. A. (1978) The Neutrophils: Function and Clinical Disorders, North-Holland Publishing Company, Amsterdam, Netherlands). In addition, hypochlorite ion is considered to play an important role in injury to the vascular endothelial surface in platelet-activating factor-induced microvascular damage (Suematsu, M., Kurose, I., Asako, H., Miura, S., and Tsuchiya, M. (1989) J. Biochem. 106, 355-360). However, it has been difficult to conclude that hypochlorite ion participates directly in the aforementioned mechanism in organisms, because a completely selective measuring method for hypochlorite ion, especially a measuring method in vivo had not been established.

Ten and several methods such as chemiluminescence, electron spin resonance (ESR), and luminescence are known as methods for measuring reactive oxygen species. Among them, the fluorescence detection method is superior from viewpoints of sensitivity and experimental convenience. In the fluorescence detection method, DCFH (2′,7′-dichlorodihydrofluorescein) or the like is used as a fluorescence probe for measuring reactive oxygen species. However, DCFH cannot successfully distinguish types of reactive oxygen species, and as a consequence, fails to selectively measure hypochlorite ion.

An object of the present invention is to provide a method for selectively measuring hypochlorite ion. Another object of the present invention is to provide an agent for measuring hypochlorite ion. Further object of the present invention is to provide a kit used for the aforementioned measuring method. Specifically, an object of the present invention is to provide a means that enables measurement of hypochlorite ion localized in specific cells and tissues in organisms.

The inventors of the present invention previously provided non-fluorescent compounds which effectively react with reactive oxygen under physiological conditions to generate dearylated fluorescent compounds, and by using said compounds, they succeeded in a selective and highly sensitive measurement of reactive oxygen localized in living cells and living tissues by fluorescence detection method (WO 01/64664).

On the basis of these compounds, the inventors of the present invention conducted further researches to achieve the foregoing objects, and as a result, they found that hypochlorite ion can be measured by using a compound represented by the following general formula (I) alone, or in a combination with a compound represented by the following general formula (II). The present invention was achieved on the basis of these findings.

The present invention thus provides a measuring method for hypochlorite ion which comprises the following steps:

(A) reacting, with hypochlorite ion, a compound represented by the following general formula (I):

(wherein R¹ represents a 2-carboxyphenyl group which may be substituted; R² represents a phenyl group which is substituted with a substituted or unsubstituted amino group; X¹ and X² each independently represents a hydrogen atom or a halogen atom) or a salt thereof, and
(B) measuring fluorescence of a dearylated compound generated in the aforementioned step (A) or a salt thereof. The present invention also provides an agent for measuring hypochlorite ion which comprises a compound represented by the aforementioned formula (I) or a salt thereof. According to a preferred embodiment, there is provided the agent for measuring hypochlorite ion which comprises the aforementioned compound wherein both of X¹ and X² are hydrogen atoms or a salt thereof.