JPH1183969A - Detection and imaging for phosphorus-31 nuclear magnetic resonance signal - Google Patents

Abstract

PROBLEM TO BE SOLVED: To observe and trace a change in an amount and a change with the lapse of time by receiving only a phosphorus-31 NMR signal derived from the beta phosphate radical of nucleoside triphosphate whose beta position is labeled with oxygen-17. SOLUTION: A transmitter 12 for a phosphorus-31 nucleus outputs radio waves at the resonance frequency of the beta phosphate radical of nucleoside triphosphate. In addition, when only a phosphate radical labeled with beta oxygen-17 in a sample is selected so as to be observed, its phsophorus-31 NMR signal is not observed even when phosphate compounds other than the nucleoside triphosphate radical labeled with the beta oxygen-17 exist in the sample, and only the phosphorus-31 NMR signal of the beta phosphate radical of the nucleoside triphosphate labeled with the beta oxygen-17 can be observed. This feature is obtained by connecting a frequency setting device 19 to a receiver 13 and to the transmitter 12. The frequency setting device 19 finds the resonance frequency of the beta phosphate radical of the nucleoside triphosphate labeled with the beta oxygen-17 on the basis of the output of the receiver 13, and it sets the output frequency of the transmitter 12.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting a phosphorus 31 nuclear magnetic resonance signal and a method for imaging a phosphorus 31 nuclear magnetic resonance signal.

[0002]

2. Description of the Related Art Phosphorus 31 and oxygen 17 are stable nuclides having a natural abundance of 100% and 0.037%, respectively, capable of observing NMR. The NMR resonance frequencies in an external magnetic field having a magnetic flux density of 11.74 Tesla are 202.4 MHz and 67.78, respectively. MHz is known, for example, from the literature, edited by The Chemical Society of Japan, “4th Edition, Experimental Chemistry Course 5 NMR”, Chapter 3.1, Table 3.1, page 348, Maruzen Co., Ltd. (1991).

The pulse NMR method is described, for example, in the literature TCFarra
r, EDBecker, "Pulse and Fourier Transform NMR",
This is a method of observing nuclear magnetic resonance signals known by Academic Press (1971) (Japanese translation, Akasaka, Imoto, "Pulse and Fourier Transform NMR", Yoshioka Shoten (1976)).

[0004] An oxygen 17-labeled phosphate compound is known, for example, from Japanese Patent Application No. 6-23909. By labeling at least one of four oxygen atoms of a phosphate group with oxygen 17, oxygen 17 and phosphorus 31 can be combined. Is a compound having a chemical bond. For this compound, using the “selective observation method of phosphorus 31 nuclear magnetic resonance signal and the method utilizing this” known from Japanese Patent Application No. 7-71137, the NMR of phosphorus 31 via polarization transfer with oxygen 17 A mixed phosphoric acid in which a signal is observable and a phosphate compound having a natural abundance of oxygen 17 and an oxygen 17-labeled phosphate compound having a higher content of oxygen 17 than the natural abundance are present in a mixed state. Phosphorus 3 derived from an oxygen 17-labeled phosphate compound in a compound sample
It is possible to selectively observe the 1 NMR signal. The selection of the observation signal is performed by phase cycling in which the high-frequency pulse phase for exciting the oxygen 17 in time and the reception phase of the receiver are repeatedly executed in a fixed combination.

[0005] Polarization transfer is
For example, references RRErnst, G. Bodenhausen, A. Wokaun, "Pr
inciples of Nuclear Magnetic Resonance in One and
Two Dimensions ", Oxford Scientific Publications (1
987) (Earl Ernst, J. Bodenhausen, E. Bogan, Principles of New Clare Magnetic Resonance in One
And Two Dimensions, Oxford
In NMR, which is known from Science Publication (1987) and uses a coupled spin system with heteronuclear spin-spin coupling, the energy levels of the observed nuclei are excited by transiently exciting the unobserved nuclei magnetically. This is a phenomenon in which the occupancy rate of the data changes. Examples of pulse sequences using polarization transfer are APT (APT),
EMU (SEMUT), INEP (INEPT), DEE
It is known from the literature, for example, that P.T.

[0006] HMCQ (HMQC,
Heteronuclear Multiple Quantum Coherence) is described in A. Bax, RH Griffy,
BLHawkins, J. Magn.Reson., 55, 301-315 (1983)
(Abax, RGH Griffey, Bee
El Hawkins, Journal of Magnetic Resonance, 55, 301-315 (1983)).
This is a pulse sequence that can observe polarization transfer between different nuclides.

Frequency selective excitation is described, for example, in the document H. Kessle.
r, H. Oschkinat, C. Griesinger, W. Bermel, Journal
Excitation frequency band artificially limited, known by H. Kessler, H. Oshkinato, H. Oshkinato, C. Grigginger, W. Baermel, Journal of Magnetic Resonance, Vol. 70, p. 106, 1986. It is a technique to do. As an example, the excitation pulse of the NMR observation method is not a general rectangular pulse but a reference C. Bauer, R. Freeman,
T. Frenkel, J. Keeler, and AJ Shaka, Journal of
Magnetic Resonance, volume 58, page 442-457 (198
4) (Sea Bauer, Earle Freeman, Ti Frenkel, J Keeler, AJ Shaka,
Journal of Magnetic Resonance, 58
Vol., Pp. 442-457, 1984). The excitation frequency band is obtained as a Fourier transform of an amplitude modulation function centered on the center frequency. By appropriately designing the function form, the excitation intensity in the side band can be suppressed as compared with the rectangular pulse. An example is a Gaussian function type selective excitation pulse. The frequency bandwidth of the Gaussian function type selective excitation pulse is described in, for example, the above-mentioned C. Bauer et al. Document, a Gaussian with a pulse width of 10 milliseconds and a flip angle of 90 °. It is shown that in the functional pulse, the intensity in the sidebands that are more than 300 Hz away from the center frequency can be ignored. Since the pulse width and the excitation band are inversely proportional,
From the numerical values in this document, it can be easily understood that when the pulse width is set to 1 millisecond, the excitation intensity at a frequency apart from the center frequency by about 3000 Hz can be ignored. Also, by adjusting the pulse width between 1 and 10 milliseconds, it is also possible to artificially adjust the spread of the sideband between 3000 and 300 Hz.

[0008] Nuclear magnetic resonance imaging (MRI) is described, for example, in the literature, PC Lauterbur, Nature, 242, 190 (1973).
(PC rotor bar, Nature, 242 volumes, 190
P. (1973)) and also described, for example, in the literature, CM Lai
and PC Lauterbur, J. Phys. E: Sci. Instrum., 1
3, 747 (1980) (CM Ray, PC Rotor Bar, Journal of Physics E Scientific Instruments, Vol. 13, p. 747 (198
0)), which uses the fact that the precession frequency of magnetic resonance of sample nuclear spins is distributed in a gradient magnetic field, and measures the spatial coordinates of observed spins from the frequency difference of free induction decay after pulse excitation. Is the law.

[0009] Magnetic resonance spectroscopy imaging (MRS or MRSI) is described, for example, in the literature, TR Brown, BM Kin.
caid, K. Ugurbil, Proc. Natl. Acad. Sci. USA, 79,
3523-3526 (1982) (T.E.R. Brown, BM Kincaid, Caughleville, Proceedings of National Academy of Sciences USA, 79, 3523-3526 (1982))
Is a spectroscopic means for obtaining an NMR spectrum in a specific space by utilizing an NMR phenomenon in a gradient magnetic field. Since the chemical shift value of the NMR spectrum is obtained with spatial resolution, it is also called chemical shift imaging (CSI). The MRS of phosphorus 31 can be found, for example, in the literature, R. McNamara, F. A-. Mendoza, and TR Brown,
NMR Biomed., 7, 237-242 (1994) (Earl McNamara, F. Arismendza, TE Earl Brown, NMR in Biomedicine, 7, 237-242
(1994)), and it is known that the localization of phospholipids inside a living human brain can be quantitatively analyzed using a 2.5 cm square cubic space as a unit of resolution.

It is known that the phosphate groups of nucleoside triphosphate and diphosphate are distinguished from each other by calling them alpha, beta and gamma from the side closer to the sugar phosphate ester bond. Biochemistry '' Hirokawa Shoten (1
990).

Adenosine triphosphate, adenosine monophosphate, and adenylate kinase enzyme are mixed at pH 7.5 and 25 ° C., and in a buffer solution which has sufficiently passed, adenosine triphosphate, adenosine diphosphate, The chemical equilibrium of the following equation 1 is established between adenosine monophosphate and
That the equilibrium constant of is 0.7, for example, in the literature, J. R.
einstein, IR Vetter, I. Schlichting, P. Roesch,
A. Wittinghofer, RS Goody, Biochemistry, 29, 7
440-7450 (1990) (J. Reinstein, IR Earl Better, Eye Slichting, P. Resh, A. Bittinhofer, Biochemistry,
29, 7440-7450 (1990)).

[0012]

(Equation 1)

[0013]

(Equation 2)

Here, [ATP], [AMP], [AD
P] are the concentrations of adenosine triphosphate, adenosine monophosphate, and adenosine diphosphate, respectively.

[0015]

In the conventional method of observing a phosphorus-31 nuclear magnetic resonance signal via polarization transfer with oxygen 17, a conventional method for observing all oxygen 17-labeled phosphate compounds existing in the observation space is used. Was. That is, when the administered phosphate compound changes into a metabolite over time in a living body or the like, since both the administered reagent and the product are observed, the received signal is identified in order to identify the respective resonance signals. It was necessary to split the spectrum as a frequency spectrum.

This has the advantage that the phosphate compound can be identified depending on the presence or absence of the label as compared with the conventional non-selective phosphorus 31-NMR observation and phosphorus 31-MRSI observation not using oxygen 17, but the frequency spectrum is always required. Must get. For this reason, a phosphorus 31-MR
When observing I, an observation method and a calculation process in which the spatial coordinates and the frequency spectrum are treated separately are indispensable.

The present invention has been made in view of the above points, and restricts a received signal to a phosphorus 31-NMR signal derived from a single chemical species oxygen-17-labeled phosphate group. By eliminating the originating resonance signal, phosphorus 31-
It is an object of the present invention to eliminate the necessity of NMR frequency spectrum observation and to know the presence or absence and amount of observed chemical species in a mixed phosphoric acid sample based on the intensity of a received signal.

[0018] The present invention also provides a phosphorous 3 with a gradient magnetic field.
In 1-MRI, it is an object to omit the treatment of the frequency spectrum of phosphorus 31 and to know the presence or absence and the amount of a single chemical species of oxygen 17-labeled phosphate group in a specific observation space.

Another object of the present invention is to measure a nucleoside phosphate in an observation sample in a non-invasive and non-destructive manner by observing a phosphorus 31-NMR signal.

[0020]

In order to solve the above-mentioned problems, the present invention provides a method for frequency-selectively exciting the beta phosphate group of nucleoside triphosphate and converting the beta phosphate group to oxygen 17.
With the labeled nucleoside phosphate compound as the measurement target,
A means is provided for selectively observing only the NMR signal of phosphorus 31 via polarization transfer with oxygen 17.

In the present invention, the beta phosphate group is replaced with oxygen 17
Observe the spatial distribution of the labeled nucleoside phosphate compound.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a block diagram showing an apparatus for observing a phosphorus 31 nuclear magnetic resonance signal.

The magnets 1 and 2 generate a predetermined static magnetic field required for the NMR phenomenon, and the observation sample is placed in the sample container 3 inside the detector 9. The shape of the sample container 3 can be determined according to the sample to be observed. The NMR spectrometer 6 is provided with a gradient magnetic field power supply 10, a transmitter 11, a transmitter 12, and a receiver 13, and the transmitter 11 transmits radio waves having a resonance frequency of oxygen 17,
The transmitter 12 outputs radio waves having the resonance frequency of the phosphorus 31. The high frequency filter 14 passes the resonance frequency of oxygen 17 and the high frequency filter 15 passes the resonance frequency of phosphorus 31. The radio wave having the resonance frequency of the oxygen 17 is applied to the sample container 3 through the irradiation coil 4 inside the detector 9.

To resonate the phosphorus 31 nucleus in the sample,
The switching device 16 includes the transmitter 12 and the irradiation and detection coil 5.
And the receiver 13 is shut off. The sample container 3 is irradiated with the radio wave having the resonance frequency of the phosphorus 31 through the irradiation inside the detector 9 and the detection coil 5. When receiving the resonance signal of the phosphorus 31 nucleus, the switching device 16 connects the irradiation and detection coil 5 to the receiver 13 and shuts off the transmitter 12. The resonance signal of phosphorus 31 is received by the irradiation and detection coil 5 and reaches the receiver 13.

When observing the spatial distribution of the oxygen-17-phosphate compound, a magnetic field gradient pulse synchronized with the observation pulse sequence is generated from the gradient magnetic field power supply 10 and the gradient magnetic field coils 7 and 8 are used.
Current flows through As the gradient magnetic field coils 7 and 8, coils of a type used in general NMR imaging apparatuses and MRI apparatuses can be used.

A feature of the present invention is that the transmitter 1 for the phosphorus 31 nucleus is used.
2 emits a radio frequency wave at the resonance frequency of the beta-phosphate group of the nucleoside triphosphate;
By selectively observing only the 7-labeled phosphate group, even if a phosphate compound other than beta oxygen 17-labeled nucleoside triphosphate is present in the sample, the phosphorus 31-NMR signal is not observed, and beta oxygen 17-labeled. This is where only the phosphorus 31-NMR signal of the beta phosphate group of nucleoside triphosphate can be observed. The feature is that the receiver 13 and the transmitter 1
2 is connected to the frequency setting device 19. The frequency setting device 19 obtains the resonance frequency of the beta phosphate group of the beta oxygen 17-labeled nucleoside triphosphate from the output of the receiver 13 and sets the output frequency of the transmitter 12.

In FIG. 1, gradient coils 7, 8 for generating a gradient magnetic field in a direction parallel to the static magnetic field generated by the magnets 1, 2
However, a gradient magnetic field coil for generating a gradient magnetic field perpendicular to the static magnetic field may be added as in the known MRI apparatus. The feature of the present invention is that the beta phosphate group of beta oxygen 17-labeled nucleoside phosphate in the observation space is targeted for observation, and phosphorus 3 via polarization transfer between oxygen 17 and phosphorus 31 is observed.
1—To observe the NMR signal, and if this signal can be observed, any arrangement of the gradient coil is possible.

Although the irradiation coil 4 and the irradiation and detection coil 5 are shown separately in FIG. 1, a single-coil double resonance type resonance element is formed, and the irradiation and detection coil for oxygen and phosphorus is one.
You may put them together. The coil may be of any shape, and may be a surface coil that is brought into contact with or left at the observation site of the sample.

FIG. 2 is a flowchart showing a procedure up to the determination of the frequency of the transmitter 12. A feature of the present invention resides in that the beta phosphate group of nucleoside triphosphates whose beta position is labeled with oxygen 17 is selectively excited, and polarization transfer between oxygen 17 and phosphorus 31 is observed. That is, it is necessary to first measure the phosphorus 31-NMR resonance frequency of the beta phosphate group of nucleoside triphosphate to determine the frequency of selective excitation.

However, if the resonance frequency of the beta phosphate group is known in advance, such as by repeatedly measuring the same sample or by observing another sample whose susceptibility can be regarded as being the same after performing selective observation on a certain sample, select The excitation frequency may use a known value.

FIG. 3 is a timing chart showing an observation pulse sequence according to the present invention. In this observation pulse sequence, the beta phosphate group of nucleoside triphosphate is frequency-selectively excited, and the phase cycling between the high-frequency phase of the second pulse 26 of the oxygen 17 channel and the receiver phase is performed via the polarization transfer with oxygen 17. Selectively observe only the NMR signal of phosphorus 31 and determine phosphorus 3 derived from other phosphate compounds.
1-Remove NMR signal.

The frequency selective excitation is performed as the phosphorus 31 excitation pulse 20 of FIG. The frequency difference between the resonance line of the nucleoside triphosphate beta-phosphate and the resonance line of the alpha-phosphate group is about 2000 Hz when a superconducting magnet having a static magnetic field strength of 11.7 T (tesla) is used, and the excitation frequency width is 2000 Hz.
This is achieved by using a selective excitation pulse within, for example, a Gaussian-type pulse with a pulse width of 1.5 ms.

In the present invention, the frequency-selective excitation of phosphorus 31 is provided by the phosphorus 31 excitation pulse 20, and the excitation frequency bands of the other high-frequency pulses 21, 25, and 26 do not matter. High frequency pulses 21, 25, 26
May be a rectangular pulse, or a frequency-selective excitation pulse such as a Gaussian function type.

FIG. 3 shows the HMQC pulse sequence.
The observation pulse sequence may be any pulse sequence capable of observing a phosphorus-31 nuclear magnetic resonance signal via polarization transfer between oxygen 17 and phosphorus 31, and may be, for example, an INEPT pulse sequence.

FIG. 4 is a timing chart showing an observation pulse sequence for the selective phosphorus 31 imaging according to the present invention. FIG. 4 shows only slice selection for limiting the observation space in the static magnetic field direction. The phosphorus 31 channel first pulse 20 is the same as that of FIG. 3, and the difference from the observation pulse sequence of FIG. 3 is that a gradient magnetic field pulse 30 synchronized with the phosphorus 31 channel first pulse 20 is used.

By using the gradient magnetic field pulse 30, the spatial distribution of phosphorus 31 where the resonance condition is satisfied in the gradient magnetic field is

[0037]

(Equation 3)

Is represented by Here, γ is the gyromagnetic ratio of the observed nucleus, B 'is the gradient magnetic field strength, l is the distance from the magnet center to the observation site on the coordinate axis in the gradient magnetic field direction, Δν ↓ A ↓ D ↓
C is the sampling frequency of the observation signal, Δν ↓ R ↓ F is the excitation band of the high-frequency pulse, and min is a function that selects one of the smaller ones. When frequency selective excitation is performed and experimental conditions are set such that Δν ↓ R ↓ F is smaller than the sampling period, Equation 3 can be expressed as the following equation.

[0039]

(Equation 4)

Equation 4 indicates that nuclear spins distributed in a narrower space can be observed as the frequency band of the selective excitation is narrowed or the gradient magnetic field strength is increased in the coordinate axis direction where the gradient magnetic field is applied. I have.

According to the present invention, by using the gradient magnetic field pulse 30 shown in the observation pulse sequence of FIG. 4, oxygen 17 labeled adenosine 3 existing in a specific space in the gradient magnetic field application direction is used.
The beta phosphate group of phosphoric acid can be selectively observed.

As shown in the description of FIG. 3, the frequency difference between the resonance line of the nucleoside triphosphate beta-phosphate group and the resonance line of the alpha-phosphate group is a superconducting magnet having a static magnetic field strength of 11.7 T (tesla). Since the frequency is about 2,000 Hz when using, the excitation band of the high-frequency pulse 20 is set to 2,000 Hz, and the gradient magnetic field intensity of the gradient magnetic field pulse 30 is set to 0.1 T / m (tesla per meter). The possible sample thickness can be calculated to be from about 4 to about 1.2 mm, and the beta phosphate group of beta oxygen 17-labeled nucleoside triphosphate existing in this range can be observed in the present invention.

FIG. 5 is a timing chart showing an observation pulse sequence of the selective phosphorus 31 imaging according to the present invention. In addition to FIG. 4, a gradient magnetic field pulse 31 for generating a gradient magnetic field in a plane perpendicular to the static magnetic field direction. , 32 are added. The operation of the gradient magnetic field pulses 31 and 32 is the same as that of generating a gradient magnetic field used in ordinary MRI, and the selective phosphorus 31 observation method of the present invention uses the generally used MRI.
It is possible to combine with the gradient magnetic field of the device.

FIG. 6 is a plan structural formula showing the chemical structure of beta oxygen 17-labeled adenosine triphosphate as an example of beta oxygen 17-labeled nucleoside triphosphate according to the present invention. The label for oxygen 17 is that at least one of the four oxygen atoms bonded to the beta-position phosphorus atom 40 in FIG.
Should be fine. Since two of the four oxygen atoms are located at the cross-linking portion of another phosphate group with the phosphorus atom, in the adenosine triphosphate in which the cross-linking portion is labeled with 17 oxygen atoms, the phosphate group at the alpha position Alternatively, also in the phosphate group at the gamma position, polarization transfer between oxygen 17 and phosphorus 31 can be observed. However, in the present invention, by using the frequency selective excitation shown in FIG. 3, even when the oxygen 17 atom is labeled on the cross-linking portion of polyphosphoric acid, only the polarization transfer with the beta-position phosphorus atom is observed. . Therefore, it is possible to observe a resonance signal derived from the phosphorus atom at the beta position without questioning whether or not the beta oxygen 17 label is located at the bridge portion.

FIG. 6 shows adenosine triphosphate whose base moiety is adenosine. According to the selective observation method of the present invention, the beta phosphate group is used as a measurement target for phosphorus 31 via polarization transfer between oxygen 17 and phosphorus 31. Is observed, the structure of the base portion of the nucleoside triphosphate may be anything, for example, guanosine triphosphate having a guanosine base.

FIG. 7 is a plan structural formula showing the chemical structure of beta oxygen 17-labeled adenosine diphosphate generated by hydrolysis of the terminal phosphate group of beta oxygen 17-labeled adenosine triphosphate in FIG. The beta-17 oxygen 17-labeled phosphate group was bonded to two adjacent phosphate groups in adenosine triphosphate, but in adenosine diphosphate, there was only one adjacent phosphate group. This difference in chemical structure is reflected in the phosphorus 31 chemical shift of the beta phosphate group, which is about -20 ppm for adenosine triphosphate and about -8 ppm for adenosine diphosphate. When this chemical shift difference is converted into frequency units, about 200 per 1 T (tesla) of the static magnetic field strength is obtained.
Hz, that is, about 2400 for a measured magnetic field of 11.7T.
Hz, and the frequency-selective excitation pulse shown in the description of FIG. 3 is used to selectively excite the beta phosphate group of adenosine triphosphate and to distinguish it from the beta phosphate group of adenosine diphosphate.

An example of an embodiment in which an enzyme reaction system is selected as a model experiment system of a bioenergetic system and phosphate metabolism can be selectively observed by using the selective observation method of the present invention will be described.

The beta oxygen 17 described with reference to FIGS.
As an example of selective phosphorus 31-NMR observation using a solution sample containing labeled adenosine triphosphate and diphosphate as an object of measurement, two molecules of adenosine diphosphate and one molecule of adenosine triphosphate and one molecule of adenosine monophosphate are used. Measurement experiment using adenylate kinase, an enzyme that produces one molecule, and dissolving the enzyme and adenosine diphosphate in a buffer solution at a constant temperature where adenylate kinase can maintain the enzyme activity and starting observation Is shown.

FIG. 8 is an explanatory diagram showing that the concentration of each nucleoside phosphate in the equilibrium system is kept constant. The horizontal axis represents time, and the vertical axis represents the concentration of nucleoside phosphate. The adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and adenylate kinase enzymes were adjusted to pH 7.6 so that the equilibrium constant of Equation 2 shown in the description of the prior art was 0.7.
When dissolved in the above buffer, the equilibrium constant of Formula 2 is maintained at the initial value under chemical equilibrium.

FIG. 9 shows that in the mixed nucleoside phosphate solution under the chemical equilibrium described with reference to FIG. 8, beta oxygen 17-labeled adenosine diphosphate was used as the nucleoside diphosphate constituting the equilibrium system. Unlabeled adenosine monophosphate as another nucleoside phosphate constituting
The concentration of oxygen-17-labeled adenosine diphosphate changes over time in a mixed solution prepared by using unlabeled adenosine triphosphate so that the equilibrium constant of each of the solutions in Equation 2 becomes 0.7. FIG. At the start time, all of the oxygen 17-labeled phosphate groups exist as beta phosphate groups of adenosine diphosphate, but the oxygen equilibrium 17-labeled phosphate groups that exist as beta phosphate groups of adenosine triphosphate with time due to the chemical equilibrium shown in Equation 1. The group increases.

The feature of the present invention is that, by selectively observing the oxygen-17-labeled phosphate group, the mixed nucleoside phosphate solution having a constant concentration under the chemical equilibrium described with reference to FIG. As described with reference to FIG. 9, it is possible to trace the transfer of the oxygen-17-labeled phosphate group from adenosine diphosphate to adenosine triphosphate.

By using the selective observation method of the present invention, a sample in which a nucleoside phosphate compound having a similar chemical structure is in a mixed state is measured, and an oxygen 17-labeled phosphate existing as a beta phosphate group of adenosine triphosphate is measured. By selectively observing only groups, it is possible to non-invasively measure changes over time in the energy metabolism system.

FIG. 10 is a schematic diagram showing a phosphorus 31-NMR spectrum observed using the mixed nucleoside phosphoric acid solution under the chemical equilibrium described with reference to FIG. 8 as an object to be measured. The resonance line 61 is derived from adenosine triphosphate gamma phosphate group, adenosine diphosphate beta phosphate group and adenosine monophosphate alpha phosphate group, and the resonance line 62 is derived from adenosine triphosphate alpha phosphate group and adenosine 2 phosphate group.
The resonance line 63 is derived from the adenosine triphosphate beta phosphate group due to the alpha phosphate group. In the mixed nucleoside phosphoric acid solution, the solution composition is maintained so that the equilibrium constant becomes 0.7, so that the intensity of the three resonance lines is constant over time in non-selective observation.

FIG. 11 is a schematic diagram showing a phosphorus-31 NMR spectrum through the polarization transfer of oxygen 17 and phosphorus 31 using the mixed nucleoside phosphoric acid solution described with reference to FIG. 10 as a measurement sample. The resonance line 71 is derived from the oxygen 17-labeled adenosine triphosphate beta phosphate group, and the resonance line 72 is derived from the oxygen 17-labeled adenosine triphosphate beta phosphate group. As described with reference to FIG. 9, even when the equilibrium constant is kept constant, by observing the oxygen 17-phosphorus 31 polarization transfer, the oxygen 17-labeled phosphate group contained in adenosine diphosphate can be determined by chemical equilibrium. It is possible to follow the decrease and increase of the oxygen 17 label contained in adenosine triphosphate.
However, only by observing the polarization transfer, the oxygen 17-labeled phosphate group contained in adenosine diphosphate can be converted to adenosine 3 phosphate.
Since the oxygen 17 label contained in the phosphoric acid is also observed together, it is necessary to identify the chemical species by spectroscopy of the observed signal.

FIG. 12 shows a phosphorus 31-NMR spectrum obtained by using the polarization transfer method using a frequency-selective excitation pulse according to the present invention as a measuring means and observing the mixed nucleoside phosphoric acid solution described with reference to FIG. 10 as a measuring sample. FIG. The resonance line 73 is derived from the oxygen 17-labeled adenosine triphosphate beta phosphate group. Selective excitation pulse 2 in FIG.
By making the frequency of 0 coincide with the resonance frequency of the beta phosphate group of adenosine triphosphate by the frequency setting machine 19 of FIG. 1, the signal of the oxygen 17-labeled phosphate group contained in adenosine diphosphate is not observed, Only the signal of the oxygen 17-labeled phosphate group contained in adenosine triphosphate is observed.

In the selective observation shown in FIG. 12, even if a plurality of nucleoside phosphate species containing an oxygen 17-labeled phosphate group exist in the measurement sample, the resonance line of the phosphorus 31-NMR spectrum shows the beta oxygen 17-labeled adenosine. Only one resonance line of the beta phosphate group of the triphosphate can be observed. In this regard, the present invention differs from the known selective observation method utilizing oxygen 17-phosphorus 31 polarization transfer.

In the selective observation shown in FIG. 12, the object to be observed is only the beta phosphate group of beta oxygen 17-labeled adenosine triphosphate, and the other phosphorus 31-NMR signals are erased regardless of the presence or absence of the label. The intensity itself corresponds to the average sample concentration in the observation space. Therefore, spectroscopy for distinguishing from other nucleoside phosphate chemical species is not particularly required, and only the intensity of the resonance signal need be known.

[0058]

As described in detail above, according to the present invention,
In a mixed sample in which nucleoside phosphate compounds of a plurality of chemical species are mixed and the concentrations of the phosphate compounds are in equilibrium with each other, nucleoside 3 labeled with oxygen 17 at the beta position is used.
Only the phosphorus 31-NMR signal derived from the beta phosphate group of the phosphoric acid is received, and the quantitative change and the change with time can be observed and tracked.

According to the present invention, even if a phosphate compound chemically equivalent to the oxygen 17-labeled phosphate compound is present in the sample in advance, the signal is not observed, and the time-dependent change of the administered oxygen 17-labeled phosphate compound is not observed. Only can be measured. The question is whether or not phosphate compounds other than the oxygen 17-labeled phosphate compound are present in the sample, and whether or not the phosphorus 31-NMR resonance frequency of those phosphate compounds overlaps with the beta oxygen 17-labeled nucleoside to be observed. Instead, only the phosphorus 31-NMR signal of the beta oxygen 17 labeled nucleoside can be tracked.

The method of observing the phosphorus 31 nuclear magnetic resonance signal of the present invention is similar to ordinary phosphorus 31-NMR observation, and non-invasively and non-destructively affects phosphorus 31 derived from nucleoside phosphate in the observed sample. -NMR signals can be observed.

[Brief description of the drawings]

FIG. 1 is a configuration diagram showing a phosphorus 31 nuclear magnetic resonance signal observation device.

FIG. 2 is a flowchart showing a procedure up to determination of a transmission frequency in the signal observation method according to the present invention.

FIG. 3 is a timing chart showing an oxygen 17-phosphorus 31 HMQC pulse sequence including a frequency selective excitation pulse according to the present invention.

FIG. 4 includes a frequency-selective excitation pulse according to the present invention.
FIG. 9 is a timing chart showing an observation pulse sequence of the dimensional phosphorus 31 imaging.

FIG. 5 includes a frequency selective excitation pulse 3 according to the invention.
FIG. 9 is a timing chart showing an observation pulse sequence of the dimensional phosphorus 31 imaging.

FIG. 6 is a diagram showing a planar structural formula representing a chemical structure of beta oxygen 17-labeled adenosine triphosphate.

FIG. 7 is a diagram showing a planar structural formula representing a chemical structure of beta oxygen 17-labeled adenosine diphosphate.

FIG. 8 is an explanatory diagram showing that each nucleoside phosphate concentration is kept constant in a mixed nucleoside phosphate solution constituting an equilibrium system.

FIG. 9 is an explanatory diagram showing that the concentrations of oxygen 17-labeled adenosine diphosphate and oxygen 17-labeled adenosine triphosphate change with time in a mixed nucleoside phosphate solution constituting an equilibrium system.

FIG. 10 is an explanatory diagram showing a phosphorus 31-NMR spectrum when a mixed nucleoside phosphoric acid solution in chemical equilibrium is observed by a known nonselective phosphorus 31-NMR observation method.

FIG. 11 is an explanatory diagram showing a phosphorus 31-NMR spectrum when a mixed nucleoside phosphoric acid solution in chemical equilibrium is observed by a known oxygen 17-phosphorus 31 polarization transfer observation method.

FIG. 12 is an explanatory diagram showing a phosphorus 31-NMR spectrum when a mixed nucleoside phosphoric acid solution at a chemical equilibrium is observed by an oxygen 17-phosphorus 31 polarization transfer observation method including a frequency-selective excitation pulse according to the present invention.

[Explanation of symbols]

1, 2, magnet; 3, sample container; 4, oxygen 17 irradiation coil; 5, phosphorus 31 irradiation and detection coil; 6, NMR spectrometer; 7, 8, gradient coil; 9, detector, 10: gradient magnetic field Power supply, 11 ... Oxygen 17 irradiation circuit, 12 ... Phosphorus 31 irradiation circuit, 13 ... Receiver, 14 ... High frequency filter, 15 ... High frequency filter, 16 ... Switcher, 19 ... Frequency setting device, 20 ... Phosphorus 31 channel first pulse 21: phosphorus 31 channel second pulse 22, 23: waiting time, 24: signal reception period, 25: oxygen 17 channel first pulse, 26 ... oxygen 17 channel second pulse, 27, 28 ... waiting time, 29 ... Oxygen 17 channel continuous irradiation pulse, 30 ... Z axis gradient magnetic field pulse, 35 ... X axis encode pulse, 36 ... Y axis gradient magnetic field pulse, 40 ... beta phosphate group, 41 Adenosine, 61 ... Phosphorus 31-NMR resonance line of adenosine triphosphate gamma phosphate group and adenosine diphosphate beta phosphate group observed by non-selective observation method, 62 ... Adenosine triphosphate alpha observed by non-selective observation method Phosphorus 31-NMR resonance line of phosphate group and adenosine diphosphate alpha phosphate group, 63 ... Phosphorus 31-NMR resonance line of adenosine triphosphate beta phosphate group observed by non-selective observation method, 71 ... Polarization transfer method 73-phosphorus 31-NMR resonance line of adenosine triphosphate gamma phosphate group and adenosine diphosphate beta phosphate group observed in step 72; phosphorus 31-NMR resonance line of adenosine triphosphate beta phosphate group observed by polarization transfer method; 73 ... Phosphorus 31-NMR resonance line of adenosine triphosphate beta phosphate group observed by the selective excitation polarization transfer method of the present invention.

Claims (6)

[Claims] 1. A nucleoside triphosphate containing a higher proportion of oxygen 17 in a beta phosphate group than the natural abundance is measured, and at the time of observation, the phosphoric acid 31 nucleus of the beta phosphate group is frequency-selectively excited to obtain oxygen 17 3 via polarization transfer with
1. A method for detecting a phosphorus-31 nuclear magnetic resonance signal, comprising: observing a nuclear magnetic resonance signal and observing a phosphorus-31 nuclear magnetic resonance signal derived from a beta phosphate group of beta oxygen 17 nucleoside triphosphate. 2. A gradient magnetic field is generated in the sample space to measure a nucleoside triphosphate containing a higher proportion of oxygen 17 in the beta phosphate group than the natural abundance ratio. 31 frequency-selective excitation of 31 nuclei, oxygen 1
And a phosphorous-31 nuclear magnetic resonance signal originating from the beta-phosphate group of beta-oxygen-17-labeled nucleoside triphosphate and passing through the polarization transfer with oxygen-17. 31 nuclear magnetic resonance imaging method characterized by obtaining a spatial distribution image of 3. The method for detecting a phosphorus-31 nuclear magnetic resonance signal according to claim 1, wherein the center frequency of the transmitter and that of the receiver are made to coincide with the resonance frequency of the beta phosphate group of beta oxygen 17 nucleoside triphosphate. Phosphorus 31 nuclear magnetic resonance signal detection method. 4. The phosphorus-31 nuclear magnetic resonance imaging method according to claim 2, wherein the center frequency of the transmitter and the receiver is matched with the resonance frequency of the beta phosphate group of beta oxygen 17 nucleoside triphosphate. 31 Nuclear magnetic resonance imaging method. 5. The method according to claim 1, wherein when setting the center frequency of the high-frequency pulse, the center necessary for frequency-selective excitation of the beta phosphate group of beta oxygen 17-labeled nucleoside triphosphate is set. A method for detecting a phosphorus-31 nuclear magnetic resonance signal, comprising using a known center frequency if the frequency is known. 6. The phosphorus 31 nuclear magnetic resonance imaging method according to claim 2, wherein the center frequency required for frequency-selective excitation of the beta phosphate group of beta oxygen 17-labeled nucleoside triphosphate when the center frequency of the high frequency pulse is set. Is used, the known center frequency is used, the method of imaging a phosphorus 31 nuclear magnetic resonance signal.