U.S. patent application number 12/910457 was filed with the patent office on 2011-02-10 for method for measuring hypochlorite ion.
This patent application is currently assigned to Tetsuo NAGANO. Invention is credited to Tetsuo NAGANO, Ken-ichi SETSUKINAI, Yasuteru URANO.
Application Number | 20110033944 12/910457 |
Document ID | / |
Family ID | 33417374 |
Filed Date | 2011-02-10 |
United States Patent
Application |
20110033944 |
Kind Code |
A1 |
SETSUKINAI; Ken-ichi ; et
al. |
February 10, 2011 |
METHOD FOR MEASURING HYPOCHLORITE ION
Abstract
A method for measuring hypochlorite ion, which comprises the
steps of: (A) reacting, with hypochlorite ion, a compound
represented by the following general formula (I): ##STR00001##
wherein R.sup.1 represents a 2-carboxyphenyl group which may be
substituted; R.sup.2 represents a phenyl group which is substituted
with a substituted or unsubstituted amino group; X.sup.1 and
X.sup.2 each independently represents either 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.
Inventors: |
SETSUKINAI; Ken-ichi;
(Tokyo, JP) ; URANO; Yasuteru; (Kanagawa, JP)
; NAGANO; Tetsuo; (Tokyo, JP) |
Correspondence
Address: |
GREENBLUM & BERNSTEIN, P.L.C.
1950 ROLAND CLARKE PLACE
RESTON
VA
20191
US
|
Assignee: |
NAGANO; Tetsuo
Tokyo
JP
SEKISUI MEDICAL CO., LTD.
Tokyo
JP
|
Family ID: |
33417374 |
Appl. No.: |
12/910457 |
Filed: |
October 22, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12493995 |
Jun 29, 2009 |
|
|
|
12910457 |
|
|
|
|
12099979 |
Apr 9, 2008 |
|
|
|
12493995 |
|
|
|
|
10437437 |
May 14, 2003 |
7378282 |
|
|
12099979 |
|
|
|
|
Current U.S.
Class: |
436/124 |
Current CPC
Class: |
Y10T 436/14 20150115;
G01N 2021/7786 20130101; Y10T 436/196666 20150115; Y10T 436/19
20150115; G01N 21/6428 20130101; Y10T 436/200833 20150115 |
Class at
Publication: |
436/124 |
International
Class: |
G01N 21/76 20060101
G01N021/76 |
Claims
1. A kit for measuring hypochlorite ion, which comprises
individually packaged a compound represented by the general formula
(I) or a salt thereof: ##STR00004## wherein R.sup.1 represents a
2-carboxyphenyl group which may be substituted; R.sup.2 represents
a phenyl group which is substituted with a substituted or
unsubstituted amino group; X.sup.1 and X.sup.2 each independently
represents hydrogen atom or a halogen atom; and a compound
represented by the general formula (II) or a salt thereof:
##STR00005## wherein R.sup.3 represents a 2-carboxyphenyl group
which may be substituted; R.sup.4 represents a hydroxyphenyl group;
X.sup.3 and X.sup.4 each independently represents hydrogen atom or
a halogen atom.
2. The kit according to claim 1 wherein R.sup.2 is 4-aminophenyl
group.
3. The kit according to claim 1 wherein R.sup.4 is 4-hydroxyphenyl
group.
4. The kit according to claim 2 wherein R.sup.4 is 4-hydroxyphenyl
group.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
12/493,995, filed Jun. 29, 2009, which is a continuation of
application Ser. No. 12/099,979, filed Apr. 9, 2008, now abandoned
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.
TECHNICAL FIELD
[0002] 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.
BACKGROUND ART
[0003] 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.
[0004] 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.
DISCLOSURE OF THE INVENTION
[0005] 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.
[0006] 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).
[0007] 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.
[0008] 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):
##STR00002##
(wherein R.sup.1 represents a 2-carboxyphenyl group which may be
substituted; R.sup.2 represents a phenyl group which is substituted
with a substituted or unsubstituted amino group; X.sup.1 and
X.sup.2 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 X.sup.1 and X.sup.2 are hydrogen atoms or a salt
thereof.
[0009] From another aspect, the present invention provides a
measuring method for hypochlorite ion which comprises the following
steps of
(C) reacting a compound represented by the aforementioned general
formula (I) or a salt thereof with a reactive species containing
oxygen; (D) reacting a compound represented by the following
general formula (II):
##STR00003##
(wherein R.sup.3 represents a 2-carboxyphenyl group which may be
substituted; R.sup.4 represents a hydroxyphenyl group; X.sup.3 and
X.sup.4 each independently represents either hydrogen atom or a
halogen atom) or a salt thereof with said reactive species
containing oxygen; (E) measuring fluorescence of a dearylated
compound generated in each of the aforementioned processes (C) and
(D) or a salt thereof; and (F) judging that said reactive species
containing oxygen is hypochlorite ion when a dearylated compound is
generated in the aforementioned process (C) and a dearylated
compound is not substantially generated in the aforementioned
process (D).
[0010] From further aspect, the present invention provides a kit
for measuring hypochlorite ion which comprises a compound
represented by the aforementioned general formula (I) or a salt
thereof and a compound represented by the aforementioned general
formula (II) or a salt thereof.
BRIEF EXPLANATION OF THE DRAWING
[0011] FIG. 1 shows a result of a measurement of hypochlorite ion
using HPF and APF.
BEST MODE FOR CARRYING OUT THE INVENTION
[0012] The compounds represented by the general formula (I) and the
compounds represented by the general formula (II) which are used in
the measuring method of the present invention are both disclosed in
WO 01/64664, and easily prepared by a method disclosed in this
publication.
[0013] R.sup.1 and R.sup.3 each represents a 2-carboxyphenyl group
which may be substituted, and is preferred to be an unsubstituted
2-carboxyphenyl group.
[0014] As R.sup.2, a phenyl group substituted with substituted or
unsubstituted amino groups can be used. A phenyl group substituted
with one substituted or unsubstituted amino group is preferred and
a phenyl group substituted with one unsubstituted amino group is
more preferred. When the amino group has a substituent, the types
of the substituent are not particularly limited. For example, an
alkyl group, an alkenyl group, an alkanoyl group, and arylcarbonyl
group can be used as a substituent. When the amino group has two or
more substituents, they may be the same or different. 4-Aminophenyl
group is preferred as R.sup.2.
[0015] R.sup.4 represents a hydroxyphenyl group, and
4-hydroxyphenyl group is preferred.
[0016] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 each represents
either hydrogen atom or a halogen atom. In a compound represented
by the general formula (I), X.sup.1 and X.sup.2 are preferred to be
hydrogen atoms, and in a compound represented by the general
formula (II), X.sup.3 and X.sup.4 are preferred to be hydrogen
atoms. When X.sup.1 and X.sup.2 are halogen atoms in a compound
represented by the general formula (I), they may be the same or
different, and for example, fluorine atom and chlorine atom are
preferably used. The same is true when X.sup.3 and X.sup.4 are
halogen atoms in a compound represented by the general formula
(II).
[0017] Examples of the salt of a compound represented by the
general formula (I) or the general formula (II) include a base
addition salt, an acid addition salt, and an amino acid addition
salt. Examples of a base addition salt include metal salts such as
sodium, potassium, calcium, and magnesium; a salt of ammonia; and
salts of organic amines such as trimethylamine, piperidine, and
morpholine. Examples of an acid addition salt include salts with
mineral acids such as hydrochloric acid, sulfuric acid, and nitric
acid; salts with organic acids such as methanesulfonic acid,
p-toluenesulfonic acid, citric acid, and oxalic acid. An example of
an amino acid addition salt includes glycine salt. However, the
salts are not limited to the above examples.
[0018] Among them, a physiologically acceptable water soluble salt
is preferably used for the agent and the measuring method of the
present invention. A compound represented by the general formula
(I) and the general formula (II) or a salt thereof in a free form,
may exist as a hydrate or a solvate, which may be used for the
method of the present invention. Types of the solvent which forms
the solvate are not limited. Such solvents can be exemplified by
ethanol, acetone, and isopropanol.
[0019] A compound represented by the general formula (I) or (II)
may have one or more of asymmetric carbon atoms depending on types
of their substituents and stereoisomers such as optical isomers, or
diastereoisomers may exist. For the method of the present
invention, any of the followings may be used: stereoisomers in a
pure form, any mixtures of stereoisomers, racemates and the like.
The compound represented by the general formula (I) or (II) may
form a lactone ring in the molecule. The compound in which a
lactone ring is formed may also be used for the method of the
present invention.
[0020] The term "measurement" used in the present specification
shall be construed in its broadest sense, including quantification,
qualification, measurements performed for the purpose of diagnosis,
tests, detections and the like.
[0021] The first embodiment of the measuring method of the present
invention comprises the steps of (A) reacting a compound
represented by the general formula (I) or a salt thereof with
hypochlorite ion; and (B) measuring fluorescence of a dearylated
compound generated in the aforementioned process (A) or a salt
thereof. A compound represented by the general formula (I) is
featured to have high reactivity to hypochlorite ion, and
substantially no reactivity to singlet oxygen, superoxide anion,
hydrogen peroxide, or nitric oxide. Therefore, hypochlorite ion
contained in samples can be measured by the method of the
aforementioned embodiment.
[0022] The second embodiment of the measuring method of the present
invention comprises the steps of (C) reacting a compound
represented by the general formula (I) or a salt thereof with a
reactive species containing oxygen; (D) reacting a compound
represented by the general formula (II) or a salt thereof with the
reactive species containing oxygen; and (E) measuring fluorescence
of a dearylated compound generated in the aforementioned processes
(C) and (D) or a salt thereof. The term "reactive species
containing oxygen" used in the present specification includes
hypochlorite ion as well as reactive oxygen species such as
hydroxyl radical, singlet oxygen, superoxide anion, hydrogen
peroxide, and nitric oxide.
[0023] As explained above, a compound represented by the general
formula (I) has high reactivity to hypochlorite ion, and
substantially no reactivity to singlet oxygen, superoxide anion,
hydrogen peroxide, or nitric oxide. However, such a compound may
have reactivity to hydroxyl radical. In contrast, the compound
represented by the general formula (II) has substantially no
reactivity to singlet oxygen, superoxide anion, hydrogen peroxide,
nitric oxide, and hypochlorite ion, and has reactivity only to
hydroxyl radical. Therefore, in the aforementioned second
embodiment, it can be judged that the reactive species containing
oxygen subjected to the measurement is hypochlorite ion when a
dearylated compound is generated in the aforementioned step (C) and
a dearylated compound is not substantially generated in the
aforementioned step (D). When dearylated compounds are generated in
both of the aforementioned processes (C) and (D), the reactive
species containing oxygen subjected to the measurement can be
judged hydroxyl radical.
[0024] Fluorescence of the dearylated compounds or salts thereof
can be measured by an ordinary method. For example, a method of
measuring fluorescence spectra in vitro, or a method of measuring
fluorescence spectra in vivo by a bioimaging technique can be
employed. A compound represented by the general formula (I) or (II)
used in the present invention has a feature to be taken up into a
cell. Therefore, hypochlorite ion localized in individual cells can
be measured with high sensitivity by a bioimaging technique. Using
the method of the present invention, hypochlorite ion localized in
individual cells and specific tissues can be precisely and easily
measured, which enables elucidation of the role of hypochlorite ion
in organisms. In addition, diagnoses of specific diseases can be
achieved by using the method of the present invention. For example,
hypochlorite ion is known to be generated by neutrophils in the
presence of Helicobacter pylori. Further, diseases wherein
neutrophils are involved in an immune reaction are exemplified by
bacterial infectious diseases including gastritis, gastroduodenal
ulcer, and gastric cancer. The method of the present invention is
also useful for convenient and rapid diagnoses of these
diseases.
[0025] As the agent for measuring hypochlorite ion provided by the
present invention, a compound of the aforementioned general formula
(I) or a salt thereat per se, may be used. The agent may also be
used as a composition formulated with additives ordinarily used for
preparation of reagents, if desired. For example, as additives for
a use of the agent under a physiological condition, additives such
as dissolving aids, pH adjusters, buffers, isotonic agents and the
like can be used, and amounts of these additives can suitably be
chosen by those skilled in the art. The compositions may be
provided as those in appropriate forms, for example, powdery
mixtures, lyophilized products, granules, tablets, solutions and
the like.
[0026] The kit provided by the present invention, which can be used
as a kit to conduct the measuring method according to the
aforementioned second embodiment, comprises a compound represented
by the aforementioned general formula (I) or a salt thereof and the
compound represented by the aforementioned general formula (II) or
a salt thereof. Each of the compounds represented by the
aforementioned general formula (I) or a salt thereof and the
compounds represented by the aforementioned general formula (II) or
a salt thereof is provided as an individually packaged form. Each
of the packaged forms can be provided as a composition formulated
with additives ordinarily used for preparation of reagents, if
desired. The additives and compositions are the same as those
explained above.
EXAMPLES
[0027] The present invention will be explained more specifically by
referring to the following examples. However, the scope of the
present invention is not limited to the examples.
[0028] In the following example, a compound wherein R.sup.1 is
2-carboxyphenyl group, and R.sup.2 is 4-aminophenyl group in the
general formula (I) (hereinafter referred to as "APF") and a
compound wherein R.sup.3 is 2-carboxyphenyl group, and R.sup.4 is
4-hydroxyphenyl group in the general formula (II) (hereinafter
referred to as "HPF") were used. Each of HPF and APF is dissolved
in dimethylformamide (DMF) to obtain a 10 mM solution, and then
added with a 100 mM sodium phosphate buffer (pH 7.4) to obtain a
final concentration of 10 .mu.M, unless otherwise specifically
mentioned.
[0029] Excitation spectra and fluorescence spectra were analyzed
with Hitachi F4500. The slit width was 2.5 nm in both of the
excitation spectra and fluorescence spectra. The photomultiplier
voltage was 950 V. Unless otherwise specifically mentioned,
excitation wavelength was 490 nm, fluorescence wavelength was 515
nm, and the measurement was performed at 37.degree. C.
Example 1
Comparison of Reactivity of APF and HPF Between Reactive Species
Containing Oxygen
[0030] Reactivities of APF and HPF to various reactive species
containing oxygen were studied. Reactivity of
2',7'-dichlorodihydrofluorescein (DCFH), used as a reference, was
studied in a similar manner. For a preparation of DCFH, DCFH-DA
(diacetate) was dissolved in DMF to obtain a 10 mM solution, which
is further added to an aqueous sodium hydroxide solution (0.01 M)
to obtain a 10-fold diluted solution, and the mixture was incubated
at 37.degree. C. for 30 minutes under dark condition for
deacetylation. The resulting solution was then added to a sodium
phosphate buffer (pH 7.4; 0.1M) to obtain a 10 .mu.M solution. The
measurement was performed with excitation wavelength at 500 nm and
with fluorescence wavelength at 520 nm for DCFH.
(1) Hydroxyl Radical (.sup.-OH)
[0031] A 10 .mu.M fluorescence probe solution was added with an
aqueous hydrogen peroxide solution (a final concentration of 1 mM),
and further slowly added with an aqueous ferrous perchlorate (a
final concentration of 100 .mu.M). Fluorescence intensities before
and after the addition of the hydrogen peroxide solution and the
aqueous ferrous perchlorate were measured. The measurement of the
fluorescence intensities was performed at room temperature.
(2) Singlet Oxygen (.sup.1O.sub.2)
[0032] A 10 .mu.M fluorescence probe solution was put in a
fluorescence cell, added with
3-(1,4-dihydro-1,4-epidioxy-1-naphthyl)propionic acid (EP-1; a
final concentration of 100 .mu.M), and was reacted for 30 minutes.
Fluorescence intensities before the addition of EP-1 and 30 minutes
after the addition were measured.
(3) Superoxide (O.sub.2.sub.-)
[0033] A 10 .mu.M fluorescence probe solution was put in a
fluorescence cell, added with KO.sub.2 (a final concentration of
100 .mu.M), and was reacted for 30 minutes. Fluorescence
intensities before the addition of KO.sub.2 and 30 minutes after
the addition were measured.
(4) Hydrogen Peroxide (H.sub.2O.sub.2)
[0034] A 10 .mu.M fluorescence probe solution was put in a
fluorescence cell, added with H.sub.2O.sub.2 (a final concentration
of 100 .mu.M), and was reacted for 30 minutes. Fluorescence
intensities before the addition of H.sub.2O.sub.2 and 30 minutes
after the addition were measured.
(5) Nitric Oxide (NO)
[0035] A 10 .mu.M fluorescence probe solution was put in a
fluorescence cell, added with
1-hydroxy-2-oxo-3-(3-aminopropyl)-3-ethyl-1-triazine (NOC.sub.13; a
final concentration of 100 .mu.M), and was reacted for 30 minutes.
Fluorescence intensities before the addition of NOC.sub.13 and 30
minutes after the addition were measured.
(6) Hypochlorite Ion (.sup.-OCl)
[0036] A 10 .mu.M fluorescence probe solution was put in a
fluorescence cell, added with an aqueous sodium hypochlorite
solution (a final concentration of 1.0 .mu.M). Fluorescence
intensities before and after the addition of an aqueous sodium
hypochlorite solution were measured.
(7) Autoxidation
[0037] A 10 .mu.M fluorescence probe solution was put in a
fluorescence cell, and was left under a fluorescent lamp for 2.5
hours. Fluorescence intensities before and after being left were
measured.
[0038] Results are shown in Table 1. DCFH well reacted with each of
the reactive oxygen species, and no specificity was observed. In
addition, DCFH was susceptible to autoxidation. In contrast,
absolutely no autoxidation of APF and HPF was observed. Both of APF
and HPF showed high reactivity to hydroxyl radical. APF also showed
high reactivity to hypochlorite ion, while HPF had no reactivity to
hypochlorite ion.
TABLE-US-00001 TABLE 1 Reactive species containing oxygen HPF APF
DCFH .sup.-OH 730 1200 7400 .sup.-OCl 6 3600 86 .sup.1O.sub.2 5 9
26 O.sub.2.sup.- 8 6 67 H.sub.2O.sub.2 2 <1 190 NO 6 <1 150
Autoxidation <1 <1 2000
Example 2
Application to Myeloperoxidase/H.sub.2O.sub.2 System
[0039] (A) By using a 100 mM sodium phosphate buffer (pH 7.4)
containing 150 mM sodium chloride, a 10 .mu.M HPF solution and a 10
.mu.M APF solution were prepared. Myeloperoxidase (human) were
added to these solutions to obtain a final concentration of 11.2
nM. Each of the solution was put in each fluorescence cell, and a
change in the fluorescence intensity with passage of time was
measured. The temperature of the solutions were set at 37.degree.
C. About 5 minutes after the start of the measurement, hydrogen
peroxide was added to obtain a final concentration of 1 .mu.M. As
clearly shown in FIG. 1(A), by the addition of hydrogen peroxide,
an increase in the fluorescence intensity was not observed in HPF,
while a significant increase in the fluorescence intensity was
observed in APF.
[0040] (B) By using a 100 mM sodium phosphate buffer (pH 7.4)
containing 150 mM sodium chloride, a 10 .mu.M HPF solution and a 10
.mu.M APF solution were prepared. Myeloperoxidase (human) were
added to these solutions to obtain a final concentration of 11.2
nM. Each of the solutions was put in each fluorescence cell and a
fluorescence intensity was measured at 37.degree. C. Then, hydrogen
peroxide was added to each solution to obtain final concentrations
of 0.1 .mu.M, 0.2 .mu.M, 0.5 .mu.M, and 1.0 .mu.M. The solutions
were left with stirring for 5 minutes, and the fluorescence
intensity was measured again after the 5 minutes. A correlation
between a concentration of hydrogen peroxide and an increase in the
fluorescence intensity before and after the addition of hydrogen
peroxide is shown in FIG. 1(B). No increase in the fluorescence
intensity was observed for HPF. Whilst, for APF; fluorescence
intensity was increased in a manner dependent on the concentration
of the added hydrogen peroxide.
[0041] Since an increase in the fluorescence intensity by APF was
observed, while no increase in the fluorescence intensity by HPF
was observed, the target reactive species containing oxygen was
identified as hypochlorite ion. It can be concluded that
hypochlorite ions, generated by the oxidation of chloride ions with
the addition of hydrogen peroxide in the presence of
Myeloperoxidase, were detected by APF. Since the measurement of
hypochlorite ion by APF is highly sensitive, the measurement was
free from the effect of a slightly detected fluorescence increase
caused by an addition of hydrogen peroxide. In addition, the
measured increase of fluorescence was dependent on the
concentration of the added hydrogen peroxide.
Example 3
Fluorescence Imaging of Neutrophils
[0042] According to the method described in Wakeyama, H.,
Takeshige, K., Takayanagi, R., and Minakami S. (1982) Biochem. J.
205, 593-601 and Kakinuma, K., Kaneda, M., Chiba, T., and Ohnishi,
T. (1986) J. Biol. Chem. 261, 9426-9432, neutrophils were obtained
from 1.8 liters of porcine blood. Erythrocytes in the buffy coat
collected from the blood were hemolyzed with a large volume of an
ice-cooled 0.2% aqueous sodium chloride solution for 30 minutes and
then the preparation was promptly mixed with an equal volume of a
1.6% aqueous sodium chloride solution to restore the isotonic
condition. Neutrophils were separated from platelets and
mononuclear cells by the Conrey-Ficoll method. The neutrophils were
suspended in Krebs Ringer phosphate buffer (114 mM NaCl, 4.6 mM
KCl, 2.4 mM MgSO.sub.4, 1.0 mM, CaCl.sub.2, 15 mM
NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4, pH7.4).
[0043] The separated neutrophils were seeded onto a glass-bottomed
dish. Then the cells were loaded with HPF or APF (10 .mu.M) after
incubation for 30 minutes at room temperature. These neutrophils
were stimulated with 4.beta.-phorbol-12-myristate-13-acetate
(PMA)(2 ng/mL). Fluorescence imaging was measured twice in each
experiment: one is before and the other is 10 minutes after the
stimulation with PMA. For the measurement, LSM510 cofocal laser
scanning unit (Carl Zeiss Co., Ltd.) coupled with an Axiovert 100M
inverted microscope with a Plan-Neofluar 100.times./1.3 objective
lens (Carl Zeiss Co., Ltd.) was used. The excitation wavelength was
488 nm, and the emission was filtered using a 505-550 nm barrier
filter. In HPF-loaded neutrophils, no difference was observed in
fluorescence intensity before and after the stimulation with PMA,
while in APF-loaded neutrophils, a significant increase was
observed in fluorescence intensity after stimulation with PMA. From
these results, the reactive species containing oxygen generated by
the stimulation with PMA was shown to be hypochlorite ion in
vivo.
INDUSTRIAL APPLICABILITY
[0044] The method for measuring hypochlorite ion of the present
invention is useful, in particular, as a method for precisely and
conveniently measuring hypochlorite ion localized in specific cells
and tissues in organisms by a bioimaging technique. In addition, a
sole participation of hypochlorite ion, among various reactive
species containing oxygen, can be clearly verified by using the
method of the present invention.
* * * * *