JPH08271602A - Selective observation method of phosphorus 31 nuclear magnetic resonance signal, and method for utilizing the selective observation method - Google Patents

Abstract

PURPOSE: To selectively observe the phosphorus 31 nuclear magnetic resonance signal of a specific phosphoric acid compound observing oxygen 17 lobeld phosphoric acid group derived phosphorus 31 nuelear magnetic resonance signal by using a probe which can excite oxygen 17 and phosphorus 31 simultaneously and a spectoscope. CONSTITUTION: A probe 15 is a double resonance NMR probe which can simultaneously excite oxygen 17 and phosphorus 31 in a sample. A sample 2, an oxygen 17 irradiation coil 10 and a phosphorus 31 irradiation and observation coil 11 are built in the probe. Then, a phosphorus 31 nuclear magnetic resonance signal which is derived from a spin-spin coupling operation to the oxygen 17 is observed by oxygen 17 irradiation pulses and by the phase cycling of a phosphorus 31 receiver, and a phosphorus 31 unclear magnetic resonance signal derivad from the phosphorus 31 which has no spin- spin coupling operation to the oxygen 17 is eliminated. In addition, when a phosphorus 31 unclear magnetic resonance signal from the sample 2 is received, the phosphorus 31 irradiation and detection coil 11 is connected to a receiver 7, and a phosphorus 31 irradiation circuit 6 is cut off.

Description

Detailed Description of the Invention

[0001]

The present invention relates to phosphorus 31 nuclear magnetic resonance (N
MR) signal observation method.

[0002]

2. Description of the Related Art Phosphorus 31 (atomic number 15, mass number 31) is a nuclide having a natural abundance ratio of 100% and capable of observing an NMR phenomenon.

As shown in FIG. 2, the phosphate group has one oxygen atom having a double bond with phosphorus and three oxygen atoms having a single bond.
A single-bonded oxygen atom is further composed of R ₁ ,
It is bonded to the other atomic groups represented by R ₂ and R ₃ . For example,
When R ₁ , R ₂ and R ₃ are all hydrogen atoms, phosphoric acid,
When all of R ₁ , R ₂ and R ₃ are phenyl groups, it becomes triphenyl phosphate. Further, as shown in FIG. 14, there is also a compound having a polyphosphoric acid structure in which a plurality of phosphoric acid groups are dehydrated and condensed. For example, a phosphoric acid compound containing adenosine in the atomic group indicated by R in FIG. 14 and having two or three phosphoric acid groups bonded linearly is adenosine diphosphate (ADP) or adenosine triphosphorus, respectively. It is called acid (ATP). The phosphoric acid group in polyphosphoric acid is referred to as the alpha position, beta position, or gamma position from the side closer to R.

Oxygen 17 (atomic number 8 and mass number 17) is a stable isotope of oxygen with a natural abundance of 0.037% and capable of observing the NMR phenomenon. The natural abundance of isotopes of oxygen is
It is known, for example, from the Encyclopedia of Chemistry (Kyoritsu Shuppan, 1963) that 99.759% and oxygen 18 are 0.204%. Oxygen 1
Neither 6 nor oxygen 18 shows the NMR phenomenon.

A phosphoric acid compound labeled with oxygen 17 is disclosed in Japanese Patent Application No. 6-2390.
As suggested by 9 and the structural formula shown in FIG.
A compound having a chemical structure in which one oxygen atom is bonded to a phosphorus atom, and at least one of these oxygen atoms is labeled with oxygen 17 at a ratio higher than the natural abundance.

Polarization Transfer is
This is a phenomenon in which the energy level occupancy of observed nuclei changes due to transient magnetic excitation of non-observed nuclei in NMR in which a coupled spin system in which heteronuclear spin-spin coupling exists is observed. Examples of pulse sequences that use polarization transfer include APT (APT), SMT (SEMUT), INP (INEPT), DPT (DEPT), etc. Literature
Earl Earl Ernst, The Bodenhausen, Erbogan, The Principles Of New Clear Magnetic Resonance In One And
Two Dimensions, known by Oxford Science Publications (1987).

HMC (HMQC,
Heteronuclear Multiple Quantum Coherence) is published in the literature Avax, RL Griffey, BL Hawkins, Journal of Magnetic Resonance, 55, 301-
315 (1983) is a pulse sequence using polarization transfer.

FIG. 5 shows a timing chart of the HMQC pulse sequence for observing hydrogen by irradiating with nitrogen 15. 42 in the figure,
43 is a waiting time, 44 is a detection period, and 45 is a non-observing nuclear first
The pulse, 46 is the second pulse of the non-observed nucleus, and 47 is the development period. Since the pulse sequence described in the literature is intended to observe the chemical shift correlation two-dimensional NMR spectrum of hydrogen and nitrogen 15, the development period 47 and the detection period 44 in the pulse sequence are
There are two independent time domains. In the literature, the first pulse 45 of the nitrogen 15 channel was used to obtain quadrature quadrupole detection with respect to the chemical shift axis of the nitrogen 15 corresponding to the development period 47.
Perform phase cycling in 90 degree units with both receiver phases. It is shown in the document that the choice of phase cycling allows one to choose between zero quantum coherence and two quantum coherence, and that phase cycling eliminates the hydrogen signal without the bond to nitrogen 15. ing.

DPT (DEPT,
Distortionless Enhancement by Polarization Transfe
r, distortion-free enhancement using polarization transfer) is a pulse sequence using polarization transfer described in the documents DM Dodrel, D. Peg, MR Bendall, Journal of Magnetic Resonance, 48, 323-324 (1982). Is. In FIG. 6, irradiating with hydrogen,
3 shows a timing diagram of a DEPT pulse sequence for observing carbon 13. In the figure, 52, 53, 55 are waiting times, 54 is a detection period, and 57 is a non-observed nuclear third pulse. The pulse sequence described in the literature is the multiplicity of the carbon 13 spectrum, namely carbon.
For the purpose of distinguishing the number of hydrogen atoms bonded to 13 atoms, in this document, both the hydrogen channel third pulse 57 in FIG. 6 and the carbon 13 receiver phase in the detection period 54 are described.
Perform 180 degree phase cycling. This phase cycling eliminates signals for quaternary carbons, such as carbonyl carbons, to which no hydrogen bonds.

In both HMQC and DEPT, signals derived from nuclear spins in which heteronuclear spin-spin coupling exists are observed, and signals derived from isolated spins without coupling are erased.

When the HMQC pulse sequence is used, the maximum value of the signal intensity of phosphorus 31 passing through polarization transfer is expressed by the following equation.

[0012]

[Equation 1]

Where M (PT) is the signal intensity of the phosphorus 31 nucleus passing through polarization transfer, M (FID) is the signal intensity when the phosphorus 31 resonance signal is taken in immediately after the first pulse excitation of phosphorus 31. η is a polarization transfer enhancement factor, f is a periodic function having the waiting time of the observation pulse sequence and the oxygen 17-phosphorus 31 spin-spin coupling constant of the sample, and d is the attenuation of the signal intensity due to lateral relaxation.

The enhancement factor η corresponds to the limit of signal intensity passing through polarization transfer, and is expressed by the following equation.

[0015]

[Equation 2]

Here, γ (17O) and γ (31P) are oxygen 1
7. Phosphorus 31 gyromagnetic ratio, N (17O), N (31P) are oxygen 17 in the coupled spin system, phosphorus 31 is the number of atoms, and I and S are phosphorus 3.
1, the nuclear spin quantum number of oxygen 17. The sign of the right side of the equation 2 is negative because the gyromagnetic ratio of oxygen 17 is negative and the gyromagnetic ratio of phosphorus 31 is positive. That is, the phase of the signal of phosphorus 31 that has passed through polarization transfer is inverted with reference to the signal that has not passed.

For simplicity, of the 12 energy levels of the oxygen 17-phosphorus 31 bond spin system, focusing on only the four energy levels involved in the central transition of oxygen 17, the periodic function f is expressed.

[0018]

(Equation 3)

Where π is the circular constant, J is oxygen 17-phosphorus 31
The spin-spin coupling constant, τ, is the waiting time in the HMQC pulse sequence.

Attenuation of signal strength due to lateral relaxation is expressed by the following equation.

[0021]

[Equation 4]

Here, T ₂ is the transverse relaxation time of phosphorus 31.

As the observation conditions, for example, the waiting time τ shown by 32 and 33 in FIG. 4 is set as in the following equation.

[0024]

(Equation 5)

At this time, the sign of the periodic function represented by (Equation 3) is positive, and the sign of the signal strength of (Equation 1) is negative. Further, in the case of the condition of (Equation 5), the value of (Equation 3) becomes the maximum value, but the observed signal includes the signal attenuation due to the lateral relaxation represented by (Equation 4). Lateral relaxation time T
_{When 2} and the waiting time τ are almost equal to each other, the waiting time τ shown by 32 and 33 in FIG.

[0026]

(Equation 6)

Equation 6 is a convenient observation condition, and the optimum value of the waiting time τ is influenced by the lateral relaxation time T ₂ .

Double-resonance NMR probes can be found in literature buoys.
It is an NMR probe that can excite two nuclei with different resonance frequencies at the same time, as is known from R. Cross, R. K. Hester, JS Wow, Review of Scientific Instruments, p1486 (1976).

The tracking of intracellular phosphate metabolism in microbial cultures using phosphorus 31 nuclear magnetic resonance signals is described in, for example, the literature AJ Mihan, SJ Eski, E.
P. Koretsuki, M. Edomak, Biotechnology and Bioengineering, Vol.40, p1
359 (1992). Phosphorus 31 nuclear magnetic resonance signal chemical shifts of in-vivo phosphate compounds are described in, for example, J. Earl van Waza, Earl Ditchfield, Phosphorus Compounds and There 31P Chemical Shifts, Titibert, Ed., Phosphorus NMR in Biology. , CRC
Known by Press (1987). These references include
Phosphate compounds in vivo, for example, creatine phosphate, phosphate monoester, phosphate diester, inorganic phosphate, nicotinamide adenine dinucleotide (NAD), AT
It has been shown that P alpha-phosphate group, beta beta-phosphate group, gamma-phosphate group, ADP alpha-phosphate group, beta beta-phosphate group, etc. can be observed as phosphorus 31 nuclear magnetic resonance signal spectra. There is.

ATP can be obtained, for example, from the literature B Alberts, D. Bragg, J. Lewis, Emraf,
11-13 kc / mole when hydrolyzed to ADP and inorganic phosphoric acid as shown by K. Roberts, J. R. Watson, Molecular Biology of the Cell, Garland Publishing Inc. (1983).
releases al (kilocalorie) chemical energy to the reaction system,
It is known that this chemical energy advances biological reactions in nerve cells, muscle cells, and the like.

[0031]

In the conventional phosphor 31 nuclear magnetic resonance signal observation method, the NMR signal of the excited phosphor 31 nucleus is non-selectively observed. That is, there is no particular relation between the chemical structure of the phosphoric acid compound contained in the measurement sample and the signal observation, and in the mixed sample in which the phosphoric acid compounds having different chemical structures are present, the phosphorous compounds derived from the respective chemical species are used. Superposition of nuclear magnetic resonance signals is observed.

For this reason, when a plurality of phosphoric acid compounds having different resonance frequencies and having resonance frequency differences close to or less than the spectral line width are present in the sample, the spectra overlap, Analysis becomes difficult.

This problem is encountered when observing a wide-range phosphorus 31 nuclear magnetic resonance signal of phospholipids forming a biological membrane or when measuring an in-vivo phosphate compound existing in a space such as a cell where the magnetic susceptibility is not uniform. , Especially becomes an obstacle to analysis. For example, phosphoric acid monoester, phosphoric acid diester, and inorganic phosphoric acid in the living body have a partial overlap of the phosphor 31 nuclear magnetic resonance signal spectrum,
Part of the spectra of NAD, ATP alpha-phosphate group and ADP alpha-phosphate group overlap, and part of the spectra of ATP beta-phosphate group and ADP beta-phosphate group overlap.

Further, the phosphoric acid diester and phosphoric acid monoester have a plurality of chemical species having different fatty acid chain structures.

That is, phosphorus 31 nuclear magnetic resonance signal spectra derived from ATP, ADP and NAD are separately measured, and phospholipids containing phosphodiester and phosphate monoester are identified from conventional phosphorus 31 nuclear magnetic resonance signal spectra. It can be said that it is extremely difficult to do.

The present invention has been made in view of the above points, and in a mixture sample containing a plurality of compounds such as living tissues such as living tissues, microbial cells, and animal and plant cells, phosphorus 31 nucleus of a specific phosphate compound is used. The subject is to selectively observe magnetic resonance signals.

[0037]

In order to solve the above-mentioned problems, the phosphorus 31 nuclear magnetic resonance signal selective observation method of the present invention uses a compound containing a phosphate group labeled with oxygen 17 at a ratio higher than the natural abundance ratio. A sample prepared by mixing multiple compounds containing it was measured, and phosphorus 31 nuclear magnetic resonance derived from the oxygen 17-labeled phosphate group was measured using a probe and a spectrometer capable of simultaneously exciting oxygen 17 and phosphorus 31. Observe the signal.

[0038]

In the selective observation method of the phosphorus 31 nuclear magnetic resonance signal of the present invention, the phosphorus 31 nucleus derived from phosphorus 31 having spin-spin coupling with oxygen 17 is obtained by the oxygen 17 irradiation pulse and the phase cycling of the phosphorus 31 receiver. Observe the magnetic resonance signal, oxygen
Phosphorus 31 derived from phosphorus 31 without spin-spin coupling with 17
Erase the nuclear magnetic resonance signal.

[0039]

【Example】

(Structure of Observing Device and Sample) FIG. 1 is a structural diagram showing a relationship among a mixed sample, an NMR probe, an NMR spectrometer, and a magnet, which are measurement targets of the phosphorus 31 nuclear magnetic resonance signal selective observation method according to the present invention.

The magnets 1 and 1'are magnets attached to the NMR apparatus. For example, superconducting magnets having a magnetic flux density of 11.7 T (tesla) at the center of the magnetic field are used. At this magnetic flux density, the resonance frequency of phosphorus 31 is about 202 MHz (megahertz), and the resonance frequency of oxygen 17 is about 67.8 MHz.

In Sample 2, both a phosphoric acid compound containing an oxygen 17-labeled phosphate group (referred to as compound A) and a phosphoric acid compound containing oxygen 17 in a natural abundance ratio (referred to as compound B) were used in a glass tube or the like. It is arranged so that both Compound A and Compound B are magnetically excited by the oxygen 17 irradiation coil 10 and the phosphorus 31 irradiation and observation coil 11.

The probe 15 is composed of oxygen 17 and phosphorus 31 in the sample.
It is a double resonance NMR probe capable of simultaneously exciting a sample 2, a sample 2, an oxygen 17 irradiation coil 10, a phosphorus 31 irradiation and an observation coil 11 are incorporated. This probe 15 is used for the sample 2
Are installed so that they are located at the magnetic field centers of the magnets 1 and 1 '.
The oxygen 17 irradiation coil 10 is connected to the oxygen 17 irradiation circuit 5 of the NMR spectrometer 8. Phosphorus 31 irradiation and detection coil 11
Is connected to the switch 19 and this switch 19
Is connected to the phosphorus irradiation circuit 6 and the receiver 7 of the NMR spectrometer 8.

When magnetically exciting the sample 2, the switcher 19 connects the phosphorus 31 irradiation circuit 6 and the phosphorus 31 irradiation and detection coil 11 and disconnects the receiver 7. When the phosphorus 31 nuclear magnetic resonance signal from the sample 2 is received, the phosphorus 31 irradiation / detection coil 11 and the receiver 7 are connected and the phosphorus 31 irradiation circuit 6 is disconnected.

The NMR probe 15 contains oxygen 17 and phosphorus 31.
In addition to the irradiation function of H.sub.2, a function of irradiation of hydrogen nuclei for erasing hydrogen-phosphorus 31 spin-spin coupling, and a function of irradiation of deuterium nuclei for carrying out magnetic field lock may be attached. The oxygen 17 irradiation coil 10 and the phosphorus 31 irradiation and observation coil 11 may be independent as shown in FIG. 1, and a double resonance circuit is connected to a single coil to form a single oxygen 17 and phosphorus 31 unit. It may be a coil double resonance probe. The type of resonant circuit connected to each coil does not matter.

Compounds A and B may be mixed so that the phosphorus 31 nuclei of the phosphate groups contained therein are magnetically excited, and they may be mixed so as to come into contact with each other, or separated so as not to come into contact with each other. It may have been done. As a support for the sample, a glass tube having one end sealed can be arbitrarily used.
Alternatively, a solution containing a mixed phosphate compound, such as a culture solution of a microorganism, may be prepared in a container installed outside the probe 15 and introduced into the position of the sample 2 at the center of the magnetic field with a silicon tube or the like for measurement. Good.

(Observation of phosphorus 31 nuclear magnetic resonance signal via polarization transfer with oxygen 17) An example of observation of phosphorus 31 nuclear magnetic resonance signal via polarization transfer with oxygen 17 using a one-dimensional HMQC pulse sequence. 4 will be described with reference to FIG. FIG. 4 is a pulse sequence in which the development period 47 of the HMQC pulse sequence described in the literature shown in FIG. 5 is changed to a fixed waiting time and only the detection period is treated as the time domain of the input of the Fourier transform.
In the same way as the DEPT pulse sequence described in the literature, the phase cycling is performed in units of 180 degrees.

In the first observation, the high frequency phases of the phosphorus 31 channel first pulse 29 and the second pulse 30 are matched, and the high frequency phases of the oxygen 17 channel first pulse 35 and the second pulse 36 are matched. During the detection period 34, phosphorus
Capture the 31 signal. In the second observation, the high frequency phase of either the oxygen 17 channel first pulse 35 or the second pulse 36 is inverted 180 degrees, and the phase of the receiver 7 is inverted 180 degrees. Capture the signal of. The signals of the first and second times are added.

With respect to the signal derived from phosphorus 31 to which oxygen 17 is not bound, the phase of the phosphorus 31 resonance signal observed at the first and second times is also inverted due to the inversion of the phase of the receiver. Offset. However, the signal derived from phosphorus 31 to which oxygen 17 was bound was found to be oxygen 17 in the second observation.
Since the high frequency phase of the signal is inverted, the resonance signal of phosphorus 31 passing through the polarization transfer is also inverted, and the phase of the receiver is also inverted, so that the signal is observed in the same phase as the first time. Therefore, the strength is increased by the addition. In this way, the NMR signal of phosphorus 31 bound with oxygen 17 was observed,
The signal of phosphorus 31 without coupling can be eliminated.

In FIGS. 7 and 8, as a measurement example, 25.7 atomic%
The oxygen 17-phosphorus 31 HMQC spectrum obtained by integrating 256 times the phosphoric acid labeled with oxygen 17 as a sample is shown. The pulse width of oxygen 17 irradiation is 1 μs (microsecond) in FIG. 7 and 100 in FIG.
μs. The HMQC spectrum intensity at the same number of times of integration varies depending on the observation device, but in the observation device of this measurement example, the intensity is maximum when the oxygen 17 irradiation pulse width is 220 μs. In FIG. 7, the spectrum is not observed because the amount of oxygen 17 which is magnetically excited is smaller than that in the condition of FIG. 8, but the spectrum is observed in FIG.
That is, in the spectrum observed in FIG. 8 but not observed in FIG. 7, the transient magnetic excitation of oxygen 17 is reflected in the signal intensity, and the phosphorus 31 nuclear magnetic field via polarization transfer with oxygen 17 is observed. It is a resonance signal spectrum. Further, the fact that FIG. 7 does not include the spectrum above the noise level indicates that the phosphorus 31 nuclear magnetic resonance signal derived from phosphoric acid having no bond with oxygen 17 is erased by phase cycling.

In the examples of FIGS. 7 and 8, the HMQC pulse sequence for performing phase cycling in units of 180 degrees is used.
You may observe using a DEPT pulse sequence.

(Selective Observation in Mixed Sample) FIG. 9 shows a mixed sample of oxygen 17-labeled phosphoric acid and unlabeled pyrophosphoric acid.
Inorganic phosphoric acid and pyrophosphoric acid encapsulated in a glass capillary are put in a glass tube 63 having an outer diameter of 5 mm (millimeter) and sealed with a fluororubber cap 60. This mixed sample was mixed with oxygen 17
The spectra observed using the -phosphorus 31HMQC pulse sequence are shown in FIGS.

FIG. 10 shows the HMQC pulse sequence
FIG. 11 shows the spectrum when only the second pulse of oxygen 17 is phase-inverted and the receiver phase is held constant.
It is a spectrum at the time of carrying out phase cycling of the 2nd pulse and a receiver phase in a unit of 180 degrees.

In FIG. 10, phosphoric acid is observed at a phosphorus 31 nuclear magnetic resonance signal chemical shift value of 0 ppm, and pyrophosphoric acid is observed at -13 ppm. However, in FIG. 11, only an inverted spectrum of phosphoric acid is observed. The phosphoric acid spectrum is erased.

In FIG. 11, the phosphoric acid spectrum is inverted because the sign of the enhancement factor of (Equation 2) described in the prior art is negative.

(Selective Observation Pulse Sequence) The pulse sequence used in the selective observation method of the present invention is an HMQC pulse sequence,
Not limited to DEPT pulse sequence, oxygen 17
Anything is acceptable as long as it can observe the signal of phosphorus 31 via the polarization transfer with.

The selective observation method of the present invention is signal selection during the detection period of the phosphorus 31 nuclear magnetic resonance signal observation method, and can be applied to measurement other than one-dimensional chemical shift. For example, two-dimensional N
It can be applied to the detection period of multidimensional NMR observation methods such as MR and three-dimensional NMR. That is, by replacing the detection period of the multidimensional NMR pulse sequence with the selective observation method of the present invention,
A multi-dimensional NMR observation pulse sequence for selectively observing an oxygen-17-labeled phosphate compound with a mixed sample as an observation target may be created. Similarly, by replacing the detection period of the relaxation measurement pulse sequence with the selective observation method of the present invention, a relaxation measurement pulse sequence for selectively observing the oxygen-17-labeled phosphate compound with the mixed sample as the observation target may be created. Similarly, by replacing the detection period of the pulse sequence of the diffusion constant measurement with the selective observation method of the present invention, a mixed sample can be used as an observation target for oxygen.
A pulse sequence for selectively observing the diffusion constant of the 17-labeled phosphate compound may be created.

The pulse sequence used in the selective observation method of the present invention is a phosphorus derived from an unlabeled phosphate compound during the detection period.
It includes all pulse sequences in which the 31 nuclear magnetic resonance signal is eliminated and the phosphorus 31 nuclear magnetic resonance signal derived from the oxygen-17-labeled phosphate compound can be detected.

(Observation of Phosphate Metabolism System in Living Body) With reference to FIGS. 12 and 13, application of the selective observation method of the present invention to a living body's phosphate metabolism system accompanied by ATP synthesis is shown.

FIG. 12 is an explanatory view showing that oxygen-17-labeled phosphate 70 administered from the outside of the cell is taken up into the cell and bound to ADP72 through the phosphate metabolism pathway to synthesize ATP73. . These phosphate compounds are
By using the selective observation method of the present invention, as shown schematically in FIG. 13, extracellular phosphoric acid 70 has a spectrum 80, intracellular phosphoric acid 71 has a spectrum 81, and AT
The gamma-phosphate group of P73 can be identified spectroscopically as spectrum 82.

In the conventional method, since all the phosphate compounds inside and outside the cell are observed, the spectrum 83 which does not particularly reflect the process of phosphate metabolism can be obtained.

Similarly, referring to FIG. 15 and FIG.
The application of the selective observation method of the present invention to the phosphoric acid metabolism system of the living body accompanied with decomposition is shown.

FIG. 15 is an explanatory diagram showing that oxygen-17-labeled ATP90 administered from the outside is taken up into the cell and decomposed into oxygen-17-labeled phosphate 92 and ADP93 through the phosphate metabolism pathway. . By using the selective observation method of the present invention, these phosphoric acid compounds can be detected in the gamma position oxygen of extracellular ATP90, as schematically shown in FIG.
17 labeled phosphate group is spectrum 100, intracellular AT
The gamma position oxygen 17-labeled phosphate group of P91 has a spectrum 10
1, the oxygen 17 labeled phosphate 92 can be spectroscopically identified as spectrum 102.

In the conventional method, since all the phosphate compounds inside and outside the cell are observed, the spectrum 103 which does not particularly reflect the process of phosphate metabolism can be obtained.

(Control of culture conditions, application to imaging)
For the phosphate metabolism system of living body, living tissue, microbial culture liquid,
To administer an oxygen-17-labeled phosphate compound such as oxygen-17-labeled phosphate, observe the phosphorus-31 nuclear magnetic resonance signal of the oxygen-17-labeled phosphate compound by the selective observation method of the present invention, and trace the time-dependent change in signal intensity. Therefore, it can be applied to a method for measuring phosphate metabolism in a living body. In addition, since the change in signal intensity over time reflects the state of phosphate metabolism by cells, cell growth and substance metabolism can be estimated. Therefore, based on the temporal change in signal strength,
By controlling the supply amount of nutrients and oxygen to the culture solution, it becomes possible to efficiently control the culture conditions of microbial cells and animal and plant cells.

The selective observation method of the present invention was applied during the detection period of phosphorus 31-MRI, a reagent containing oxygen-17-labeled phosphate compound was administered to a subject, and phosphorus 31-MR derived from labeled phosphate was administered.
By selectively observing the I signal or the phosphorus 31-MRSI signal, it is possible to image the phosphate metabolism of the living body.

[0066]

As described above in detail, when the phosphorus 31 nuclear magnetic resonance signal selective observation method of the present invention is used, the phosphorus 31 nuclear magnetic resonance signal is observed through the polarization transfer with oxygen 17, and the phosphorus 31 nuclear magnetic resonance signal is transferred through the polarization transfer. Signal can be eliminated, so that oxygen 17 contained in a mixed sample containing multiple phosphate compounds with different chemical structures can be eliminated.
Phosphorus 31 nuclear magnetic resonance signal spectra of labeled phosphate compounds can be selectively observed.

[Brief description of drawings]

FIG. 1 is a mixed sample to be measured, an NMR probe, an NM in the phosphorus 31 nuclear magnetic resonance signal selective observation method according to the present invention.
The block diagram which shows the relationship of R spectrometer and a magnet.

FIG. 2 is a structural formula showing a phosphoric acid compound according to the present invention.

FIG. 3 is a structural formula showing an oxygen-17-labeled phosphate compound according to the present invention.

FIG. 4 is a diagram showing an example of timing for explaining an HMQC pulse sequence used in the selective observation method according to the present invention.

FIG. 5 is a diagram showing an example of timing for explaining a nitrogen 15-hydrogen two-dimensional chemical shift correlation HMQC pulse sequence described in the literature.

FIG. 6 is a diagram showing an example of timing for explaining a carbon 13 DEPT pulse sequence described in a document.

FIG. 7 is a diagram showing an example of a spectrum of oxygen-17-labeled phosphoric acid (oxygen-17 pulse width 1 μs) according to the selective observation method of the present invention.

FIG. 8 is a spectrum of oxygen-17-labeled phosphoric acid according to the selective observation method of the present invention (a diagram showing an example of oxygen-17 pulse width 100 μs).

FIG. 9 is a view showing an example of a sample tube containing both labeled phosphoric acid and unlabeled pyrophosphoric acid arranged in a phosphor 31 nuclear magnetic resonance signal detection space.

FIG. 10 is a diagram showing an example of spectra of a mixed sample of labeled phosphoric acid and unlabeled pyrophosphoric acid (without receiver phase cycling) according to the selective observation method of the present invention.

FIG. 11 is a diagram showing an example of spectra (with receiver phase cycling) of a labeled phosphoric acid-unlabeled pyrophosphoric acid mixed sample by the selective observation method of the present invention.

FIG. 12: Oxygen 17-labeled inorganic phosphate administered from the outside of the cell is taken up into the cell and AD is transmitted through the phosphate metabolism pathway.
Explanatory drawing which shows that ATP is synthesize | combined by couple | bonding with P.

FIG. 13 is an explanatory view of selective observation of a phosphate metabolism system including an ATP synthesis reaction by the selective observation method of the present invention and observation by a conventional method.

FIG. 14 is a structural formula showing an oxygen-17-labeled polyphosphate compound according to the present invention.

FIG. 15: Oxygen 17-labeled ATP administered extracellularly
Explanatory drawing showing that it is taken up into cells and is decomposed into ADP and oxygen-17-labeled phosphate through the phosphate metabolism pathway.

FIG. 16 is an explanatory view of selective observation of a phosphate metabolism system including an ATP decomposition reaction by the selective observation method of the present invention and observation by a conventional method.

[Explanation of symbols]

1, 1 '... Magnet, 2 ... Sample, 5 ... Oxygen 17 irradiation circuit, 6 ...
Phosphorus 31 irradiation circuit, 7 ... Receiver, 8 ... NMR spectrometer, 10
... oxygen 17 irradiation coil, 11 ... phosphorus 31 irradiation and detection coil, 15 ... probe, 19 ... switcher, 29 ... phosphorus 31
First pulse, 30 ... Phosphorus 31 Second pulse, 32, 33, 4
2, 43, 52, 53, 55 ... Waiting time, 34, 44,
54 ... Detection period, 35 ... Oxygen 17 first pulse, 36 ... Oxygen
17 Second pulse, 45 ... Non-observed nuclear first pulse, 46 ... Non-observed nuclear second pulse, 47 ... Expansion period, 57 ... Non-observed nuclear third
Pulse, 60 ... Rubber cap, 61 ... Unlabeled pyrophosphoric acid-encapsulated glass capillary tube, 62 ... Oxygen 17-labeled phosphoric acid aqueous solution, 63
… Glass tube, 70… Extracellular phosphate, 71… Intracellular phosphate, 72… ADP, 73… ATP, 75, 95… Cell,
80 ... Example of phosphorus 31 selective observation NMR spectrum of extracellular phosphate, 81 ... Example of phosphorus 31 selective observation NMR spectrum of intracellular phosphate, 82 ... Example of phosphorus 31 selective observation NMR spectrum of ATP gamma-phosphate , 103 ... Example of phosphorus 31 nuclear magnetic resonance signal spectrum of living body observed by conventional method, 9
0 ... Extracellular ATP, 91 ... Intracellular ATP, 92 ... Phosphate, 93 ... ADP, 100 ... Extracellular ATP Phosphorus group of gamma phosphate group 31 Selective observation NMR spectrum example, 101 ...
Phosphorus 31 selective observation NMR of intracellular ATP gamma phosphate group
Spectrum example, 102 ... Phosphoric acid Phosphorus 31 Selective observation NM
Example of R spectrum.

Claims (5)

[Claims] 1. An oxygen 17-labeled phosphate compound in which at least one oxygen atom of a phosphate group is labeled with oxygen 17 at a ratio higher than the natural abundance in observing phosphorus 31 nuclear magnetic resonance signals, and a natural abundance of oxygen. Oxygen-17-labeled by using a pulse sequence capable of observing phosphorus-31 nuclear magnetic resonance signal via polarization transfer based on spin-spin coupling of oxygen-17 and phosphorus-31, using a substance containing a phosphate compound having 17- Phosphorus 31 nuclear magnetic resonance signal derived from a non-phosphoric acid compound is erased, and a phosphorus 31 nuclear magnetic resonance signal derived from a compound containing a phosphate group labeled with oxygen 17 is observed. Selective observation method. 2. The phosphorus according to claim 1, wherein a phosphoric acid 31-labeled phosphate compound derived from the labeled phosphate group is observed by administering an oxygen-17-labeled phosphate compound to a living body that metabolizes phosphate over time. 31 Phosphate metabolism measurement in vivo using selective observation method of nuclear magnetic resonance signals. 3. The method for observing phosphate metabolism according to claim 2, wherein the metabolic rate of microbial cells, animal and plant cells, etc. is calculated based on the amount of oxygen-17-labeled phosphate compound consumed in a certain period of time, A method for controlling cell culture, which comprises controlling a supply rate of a nutrient to be supplied to the cells. 4. A phosphorus 31 nuclear magnetic resonance signal derived from an oxygen 17-labeled phosphate group is observed by using magnetic resonance imaging or magnetic resonance spectroscopy. Phosphate metabolism imaging method. 5. A measurement comprising an oxygen-17-labeled phosphate compound in which at least one oxygen atom of a phosphate group is labeled with oxygen-17 at a ratio higher than the natural abundance, and a phosphate compound having a natural abundance of oxygen-17. A container for storing a sample, an irradiation coil for magnetically exciting oxygen 17, an irradiation coil and an observation coil for magnetically exciting phosphorus 31, for applying a voltage of a predetermined frequency to each irradiation coil. Detects the nuclear magnetic resonance signal of the sample obtained through the irradiation circuit and the observation coil, and performs the specified processing on the detected nuclear magnetic resonance signal, and the phosphorus 31 nucleus derived from the oxygen-containing 17-phosphate-containing compound A selective observation apparatus for phosphorus 31 nuclear magnetic resonance signals comprising a receiver for observing magnetic resonance signals and means for applying a predetermined magnetic field to the sample and the coil.