JP3069298B2 - Polyamine analysis method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for analyzing polyamines.

[0002]

2. Description of the Related Art Biological polyamines (putrescine, cadaverine, spermidine, spermine) are known to exhibit high urine levels in various cancer patients. The measurement sensitivity required in this case is as high as about pmol. Conventionally used analysis methods include labeling with a fluorescent dye and HPL
This is a method combining separation by C.

[0003] As a labeling reagent, those reacting with an amino group are often used. At this time, other amino group-containing components mixed with the polyamine, such as amino acids and oligopeptides, are similarly labeled. Accordingly, there is a problem that the fluorescent-labeled polyamine must be separated from other components.

[0004]

SUMMARY OF THE INVENTION In view of the above-mentioned problems, the present invention provides a method for easily and selectively preparing polyamines without separating the monoamino group-containing components even if the monoamino group-containing components are present in a large amount. And a method for analyzing the mixture thereof, thereby completing the present invention.

[0005]

Means for Solving the Problems The present inventor has made intensive studies to solve the above-mentioned problems, and when two or more amino groups of a polyamine are labeled with a pyrene fluorescent molecule, when the pyrene fluorescent molecule is photoexcited. The inventors have found that excimer fluorescence is efficiently generated, and have completed the present invention.

That is, the present invention labels a polyamine (or mixture) having two or more amino groups with a labeling reagent having a pyrene group, and has a longer wavelength region (450 to 520 nm) than monomer fluorescence based on the pyrene group. A method for analyzing the amount of the polyamine (or a mixture thereof) based on the measurement of the fluorescence intensity and / or the fluorescence intensity decay curve of the fluorescence generated in the step (a).

[0007] The present invention also relates to a method for producing a polyamine, wherein the fluorescence generated in a longer wavelength region than the fluorescence based on the pyrene group is obtained by labeling at least two amino groups of the polyamine with the reagent, A method for analyzing the polyamine, characterized in that it is excimer fluorescence formed in the molecule of the polyamine.

Further, the present invention provides a method for analyzing a polyamine, wherein the polyamine mixture contains at least one member selected from the group consisting of lysine, ornithine, putrescine, cadaverine, spermidine and spermine. To provide.

Hereinafter, the present invention will be described in detail with reference to embodiments.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION

(Polyamine and Sample Containing Polyamine) The polyamine, the polyamine mixture, and the sample containing them that can be analyzed by the method of the present invention are not particularly limited. A sample in a solution state, a sample in a semi-solid state, or a sample in a solid state may be used, as long as it is diluted with an appropriate solvent to a concentration at which a labeling reaction described below can be performed.

[0011] Specifically, samples from living bodies include polyamines contained in urine samples, blood samples and the like. Further, there is no particular limitation on the pretreatment of the sample when using the method according to the present invention, but as described below, the method according to the present invention labels an amino group of a polyamine, In addition, since a detection method based on fluorescence measurement is used, it is preferable to remove various coexisting components in advance. Specific pretreatment includes removal of mixed acidic components or removal of mixed protein components. A common ion-exchange column chromatography can easily remove mixed acidic components, and a normal acid, organic solvent, or a method of heating can be used to remove mixed protein components. Can be easily performed. In particular, some protein components exhibit strong fluorescence, which causes background fluorescence in the method of the present invention. Therefore, this is a preferable treatment for using the method of the present invention for high-sensitivity analysis.

Further, in the method according to the present invention, a large amount of a monoamine component may be mixed in the polyamine. Here, specific examples of the monoamine component include various amino acid components in the biological sample described above. In the method according to the present invention, these monoamine components are also non-selectively labeled by a labeling reaction with a labeling reagent having a pyrene group. On the other hand, these labeled monoamine components generate fluorescence of the pyrene group itself upon photoexcitation, but do not generate fluorescence in a longer wavelength region than the fluorescence of the pyrene group itself according to the present invention, or excimer fluorescence due to excimer formation. Therefore, the method according to the present invention is not affected by the presence of these mixed monoamine components.

Further, when the polyamine component has a special structure in the sample and does not exist in a free form, the polyamine may be decomposed by a treatment including various chemical reactions to release the polyamine. This is a preferred pretreatment. Specifically, when the polyamine is present in a conjugated form, it is preferable to hydrolyze the sample containing the polyamine in advance to make the polyamine in a free form.

(Analyzable Polyamine) The polyamine to be analyzed by the method of the present invention is not particularly limited, and may be any component having two or more amino groups in the molecule. Here, the amino group means a primary amino group, a secondary amino group, or a tertiary amino group. In the present invention, a primary amino group or a secondary amino group is preferable. Components having the above are preferred.

The polyamine analyzed by the method according to the present invention may have a structure in which various other substituents are present in the molecule. Specifically, examples of the structure having another substituent include an amino acid structure, a nucleic acid structure, a sugar structure, a protein structure, and a lipid structure. As the protein structure, more specifically, an amino acid having two or more amino groups, or an oligopeptide containing the amino acid (including a protein)
Is mentioned. Examples of amino acids having two or more amino groups include lysine and ornithine. Furthermore,
Polyamines having an alkyl skeleton or cyclic alkyl skeleton, polyamines containing an aromatic ring, and other polyamine compounds are to be analyzed in the present invention. Among them, the method according to the present invention is particularly known as a biological polyamine,
It is preferred to analyze polyamines such as putrescine, cadaverine, spermidine, spermine, or mixtures thereof.

The method according to the present invention can be preferably used even in a state where each polyamine component is present alone. In this case, the amount of the polyamine can be analyzed.

Further, the method according to the present invention can be preferably used even in a state where a plurality of polyamine components are mixed. In this case, the total amount of polyamine present can be analyzed. At this time, as described below, the total amount of the polyamines present can be analyzed without separating the plurality of polyamine components. The amount of each component of the mixture can be analyzed by separating the mixture by means such as HPLC.

(Labeling Reagent) The labeling reagent usable in the method of the present invention has a pyrene group. Here, the pyrene group includes an unsubstituted pyrene ring and a substituted pyrene ring. The substituent on the substituted pyrene ring is not particularly limited, but a substituent that does not inhibit excimer formation or a substituent that promotes excimer formation can be preferably used. In the method according to the present invention, a labeling reagent having an unsubstituted pyrene ring is particularly preferable.

The labeling reagent usable in the method of the present invention is not particularly limited to other structures as long as it contains a pyrene group. Accordingly, various commonly known or commercially available pyrene-containing labeling reagents for amino group labeling can be preferably used. In the present invention, succinimidyl pyrene butyrate is particularly preferably used.

In the present invention, the reaction selectivity of the labeling reagent is not limited to a reagent that preferentially reacts with a primary amino group, but may be one that reacts with a secondary amino group. Also, primary and secondary amino group labeling reactions may coexist. Therefore, the polyamine may be labeled with three or more pyrene groups. Even in this case, as described below, an excimer is formed by two pyrene groups in the molecule, and excimer fluorescence can be observed. Therefore, polyamine analysis can be performed using the method according to the present invention.

In the method according to the present invention, the amino group in the component having a monoamino group mixed in the sample is also non-selectively labeled by the labeling reagent containing a pyrene group. In such a case, excimer formation in the molecule is impossible, and no excimer fluorescence is generated.

Further, even if an excess of the labeling reagent is unreacted or undergoes hydrolysis and is mixed in the post-solution, excimer fluorescence does not occur similarly to the above, so that there is no need for separation.

(Excimer Fluorescence) As is generally known, when one molecule has two aromatic molecules such as pyrene (eg, 1,3-bis- (1-pyrenyl) -propane), it is photoexcited. Pyrene associates in the excited state,
Emit intramolecular excimer fluorescence. Here, the excimer fluorescence is generated by the association of two molecules of the excited molecule, and when this association occurs within one molecule, the fluorescence intensity does not depend on the concentration of the molecule. Furthermore, according to conventional knowledge, in order for the above-mentioned excimer to occur, it is necessary that two pyrene groups take a preferable spatial position for excimer formation, and therefore, due to a specific structure between the two pyrene groups, There is a need to maintain the preferred spatial position described above.

On the other hand, in the method according to the present invention, the fluorescence generated when the labeled polyamine is photoexcited is measured. However, the method according to the present invention is not limited to the fluorescence from the pyrene group itself. This is to observe new fluorescence appearing in a longer wavelength region than the fluorescence. This new fluorescence is considered to be an excimer-based fluorescence (excimer fluorescence) formed by photoexcitation of a pyrene group in a polyamine molecule, similarly to the above-described excimer fluorescence. That is, the present inventor considers not only fluorescence from the pyrene group itself but also excimer fluorescence by labeling with a labeling reagent having a pyrene group at two or more amino groups of various polyamines found in the living body. It was found that new fluorescence was observed in a longer wavelength region than the obtained fluorescence, and that the fluorescence intensity hardly depended on the type (chemical structure) of the polyamine. Further, in the case of a mixture of various polyamines, it was found that the intensity of the obtained fluorescence was proportional to the total amount of the polyamines present. In addition, the above results show that as a polyamine mixture, in vivo polyamines putrescine, cadaverine,
It has been found that spermidine, spermine or the diamino amino acids lysine, ornithine or mixtures thereof are also applicable. FIG. 1 shows a fluorescence spectrum when spermidine is labeled with a pyrene group labeling reagent. In the case of using only the labeling reagent, only the fluorescence spectrum of pyrene itself is observed (excitation wavelength 34
At 5 nm, the emission maximum at 375 nm and 395 nm), and as a result of the reaction with sperimidine, the emission of the pyrene group itself decreases, and emission with a maximum at 475 nm is newly observed.
This fluorescence is considered to be the excimer fluorescence described above, and is particularly strongly observed in a polar solvent containing water.
In a water-containing polar solvent, for example, it is considered that pyrene aromatic rings due to hydrophobic interaction are located close to each other. The excimer fluorescence intensity depends on the composition of the solvent (water content, type of polar solvent, and their abundance).

These polyamines are amines having the following chemical structures. The spatial distance between these primary amino groups is evaluated by the number of CC (and CN) bonds,
5 (putrescine) to 13 (spermine), and the spatial distance between the primary amino group and the secondary amino group is 4 to
There are five ranges.

[0026]

Embedded image

After labeling a mixture of these various polyamine standards, the fluorescence intensity obtained by directly measuring the fluorescence and measuring the excimer fluorescence (475 to 550 nm) intensity, and further, this mixture is analyzed by HPLC. Separate and measure the excimer fluorescence intensity (after correcting the excimer fluorescence intensity with the gradient solvent) based on the peak of each labeled polyamine, and measure the same value (variation coefficient 5%) as the sum of the integrated fluorescence intensity values of each component. ) (FIG. 1).

These results indicate that, in the analysis method according to the present invention, the excimer fluorescence intensities based on the pyrene groups of these polyamine compounds having widely different structures are substantially the same.

Therefore, by using the method according to the present invention, the total amount of polyamine present in the sample can be easily analyzed regardless of the kind of polyamine without being inhibited by the mixed monoamine component. It is shown that it is.

(Fluorescence Measurement) There is no particular limitation on the fluorescence measurement device that can be used in the method according to the present invention. Excitation light (preferably at a wavelength of 345 nm) for exciting a pyrene group,
And a detector that observes excimer fluorescence (wavelength 475 nm)
Is preferable). The sample concentration, measurement cell, and measurement conditions suitable for the measurement are not particularly limited, and generally known conditions and instruments can be preferably used. If necessary, measurement conditions can be optimized by a generally known method.

As an example of a specific measuring means, a polyamine mixture (putrescine, cadaverine, spermidine,
Equivalent mixture of spermine) in the solvent tetrahydrofuran-
A solution having a sample concentration of 1 × 10 ⁻⁵ to 10 ⁻⁸ M (in terms of polyamine) labeled with succinimidylpyrene butyrate using DMSO-water (1: 2: 1 volume / volume) was added to 1 solution.
Measure in a ~ 4 ml quartz cell. In this case, the excitation wavelength is 3
45 nm and the measurement wavelength is 475 nm. Regarding the obtained excimer fluorescence intensity, the intensity in the range of a wavelength of 475 nm to 550 nm is integrated. A sample without a polyamine or a sample without labeling is measured under the same background (or as a control).

(Method of calculating total polyamine amount) The method of calculating (quantifying) total polyamine in a sample based on the obtained excimer fluorescence intensity is not particularly limited. For example, a standard curve of a single polyamine of a known concentration (available from Wako Pure Chemical Industries, Ltd.) is used to perform the labeling reaction according to the present invention, and a calibration curve is created from excimer fluorescence intensity obtained by fluorescence measurement. By doing so, it is possible to preferably calculate the amount of the polyamine.

Alternatively, a standard addition method in which a standard polyamine having a known concentration is added can be preferably used. In this case, it is preferable to use one of the polyamines presumed to be contained in the sample as described above. Specifically, for example, the excimer fluorescence intensity obtained by adding a known concentration of lysine or spermidine to a sample and then treating with a labeling reagent is measured, and the measured value and the excimer fluorescence intensity obtained without adding the above-mentioned standard product By comparing the measured values of the excimer fluorescence intensity, it is possible to analyze the amount of polyamine in the sample.

(Fluorescence Decay Curve of Excimer Fluorescence) In the present invention, the amount of polyamine can be analyzed based on the fluorescence decay curve of the excimer fluorescence. In this case, it is possible to selectively measure only the fluorescence based on the excimer fluorescence, and it is possible to perform measurement with higher sensitivity.

At this time, the time-resolved fluorescence spectrum measuring apparatus is not particularly limited, and it is only necessary to have a means for exciting the pyrene group and a means for time-resolved measurement of the obtained excimer fluorescence.

[0036]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist.

Example 1 As polyamines, ornithine, lysine, putrescine, cadaverine, spermidine,
And spermine, respectively, and an equal mixture thereof (100 nmol each) were combined with succinimidylpyrene butyrate (1 nmol, manufactured by Molecular Probes) and 200 μl of tetrahydrofuran-DMSO-water (1:
(2: 1 volume / volume) and allowed to react for about 15 hours to perform labeling.

The separation of the labeled polyamine mixture is performed by high performance liquid chromatography (hereinafter referred to as HPLC) (conditions: column; TSKgel SuperOctyl (4.6 mm × 5 cm, Tosoh), mobile phase), gradient elution of water-acetonitrile (acetonitrile concentration 50- 80% / 20 min, linear) The separated peaks are the normal fluorescence wavelength of pyrene group (excitation wavelength 3).
45 nm, observation wavelength 375 nm) and pyrene excimer fluorescence wavelength (excitation wavelength 345 nm, observation wavelength 475 nm) (FIG. 2).

Further, the results of mass spectrometry (TOF-MS, MALDI IV, manufactured by Shimadzu Corporation) of each of the above labeled polyamines separated by HPLC show that all of these polyamines are labeled at two places. Was shown. This result indicates that the primary amino group is mainly labeled by succinimidyl pyrene butyrate.

FIG. 3 shows the fluorescence spectra of the reaction mixture of lysine, ornithine, alanine, glycine and a blank sample similarly labeled with succinimidylpyrene butyrate. In this case, in the measurement solution,
An unreacted reagent and its hydrolyzate (succinimidylpyrene butyrate and pyrene butyrate, respectively) are also present in excess at the same time, and the fluorescence derived from these is strongly observed in all samples (fluorescence peak: 375 nm).

With respect to the blank and the monoamino compound, no excimer fluorescence (maximum wavelength: 475 nm), which is fluorescence having a longer wavelength than that of pyrene itself, is not observed at all. In contrast, new excimer fluorescence is clearly observed around 470-600 nm for the polyamines lysine and ornithine.

Further, FIG. 4 shows a time decay curve of the e-fluorescence due to lysine and ornithine in the excimer fluorescence region of this measurement sample. The delay in the emission of the new fluorescence compared to the excitation light indicates that this emission is excimer fluorescence based on excimer formation.

(Example 2) The results of comparing the fluorescence intensity of 470-600 nm, which is the excimer fluorescence region after the labeling reaction of each amine component, with each sample are shown in the table below. Lysine and ornithine were measured as polyamines, and alanine and glycine were measured as monoamines.

=============================== Amine Fluorescence Intensity (Any unit after blank correction) −−−−−−−−−−−−−−−−−−−−−−−−−−− Lysine 0.9 Ornithine 0.8 Glycine 0 Alanine 00 ================ ================ Polyamines (lysine, ornithine) show a marked increase in strength, monoamines (glycine, alanine) show no increase in strength at all. Lysine and ornithine also showed substantially the same strength.

Example 3 Influence of polyamine on excimer fluorescence under the condition that monoamines coexist in an excessive amount.

A mixture of lysine in which monoamine compounds (alanine and glycine) were co-existing in an amount of about 100 times each was labeled under the same conditions as in Example 1, and the fluorescence of the resulting reaction solution was measured. The intensity of excimer fluorescence measured under these conditions was 0.9 in relative intensity. As a comparison, the fluorescence of a similarly labeled reaction solution was measured under the condition that no monoamine compound was present. In this case, the measured intensity of excimer fluorescence was 0.9 in relative intensity. That is, there is no significant difference between these fluorescence intensities, and it is clear that there is no influence from the coexistence of a large amount of monoamines. This result indicates that the polyamine analysis method according to the present invention is not affected by the coexistence of a large amount of a monoamine compound in a sample.

(Example 4) Preparation of a calibration curve for polyamine analysis.

Various polyamines (lysine, ornithine,
Putrescine, cadaverine, spermidine, spermine)
Fig. 5 is a plot of the concentration of
10 to 10. These calibration curves are substantially the same and show little dependence on the type of polyamine. This result indicates that the polyamine analysis method according to the present invention can easily analyze all polyamines comprising a mixture of various polyamines in a sample. Furthermore, using separation means such as HPLC,
The use of a single component calibration curve also enables more sensitive analysis.

Further, based on these calibration curves, it is shown that the detection limit of the method according to the present invention is about 50 nM (the concentration giving a signal three times the standard deviation of the blank value).

[Brief description of the drawings]

FIG. 1: Succinimidyl pyrene butyrate (2.5 m
FIG. 4 is a diagram showing fluorescence spectra after reaction with only M) and sperimidine (1 mM).

FIG. 2 shows a mixture of polyamine compounds (putrescine, cadaverine, spermidine, spermine, 100 pmol each)
It is a figure which shows the chromatogram label | marked with the pyrene group, isolate | separated by HPLC, and detected by the excimer fluorescence measurement, (A) excitation wavelength 345nm, fluorescence observation wavelength 475.
(B) is an excitation wavelength of 34 nm.
It is a chromatogram at 5 nm and a fluorescence observation wavelength of 375 nm.

FIG. 3 shows a fluorescence spectrum (5 × 10 ⁻⁶ M of polyamine in water-DMSO (1: 1 volume / volume)) of various amino acids (ornithine, lysine, alanine, glycine) labeled with pyrene. FIG. 4 is a diagram showing ^-4 M succinimidyl pyrene butyrate (a solution obtained by diluting a solution obtained by labeling with 100 μl of DMSO-THF (1: 1 volume / volume) with acetonitrile 10-fold). .

FIG. 4 shows excimer fluorescence decay curves of ornithine and lysine labeled with pyrene.

FIG. 5 is a diagram showing a calibration curve of lysine.

FIG. 6 is a diagram showing a calibration curve of ornithine.

FIG. 7 is a diagram showing a calibration curve of spermine.

FIG. 8 is a diagram showing a calibration curve of spermidine.

FIG. 9 is a graph showing a calibration curve of putrescine.

FIG. 10 is a diagram showing a calibration curve of cadaverine.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-60-142250 (JP, A) JP-A-64-24884 (JP, A) JP-A-60-131457 (JP, A) JP-A 55-142 66542 (JP, A) JP-A-56-164795 (JP, A) JP-A-55-141199 (JP, A) JP-A-60-188096 (JP, A) Japanese Translation of PCT Application No. Hei 3-502333 (JP, A) (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/68 G01N 21/76 CA (STN) JICST file (JOIS)

Claims (3)

(57) [Claims] 1. A polyamine having two or more amino groups is labeled with a labeling reagent having a pyrene group, and
A method for analyzing the amount of the polyamine based on measurement of a fluorescence intensity and / or a fluorescence intensity decay curve of fluorescence generated in a wavelength region of 0 to 520 nm. 2. The fluorescence generated in the wavelength region, wherein two or more amino groups of the polyamine are labeled with the reagent,
2. The method according to claim 1, wherein the excimer fluorescence is formed in a molecule of the polyamine based on photoexcitation of the pyrene group. 3. The method according to claim 1, wherein said polyamine mixture further comprises at least lysine, ornithine, putrescine, cadaverine,
A method comprising any one of the group consisting of spermidine and spermine.