Phosphate radical identification method based on high field (> = 5.0 T) 31P CEST

Abstract

The invention discloses a phosphate radical identification method based on a high field (greater than or equal to 5.0 T) 31P CEST. According to the phosphate radical identification method, a high field MRI system greater than or equal to 5.0 T is adopted, the signal-to-noise ratio of the 31P CEST is effectively improved, and the problem of low saturation efficiency is solved; the lanthanide metal complex accelerates chemical exchange through a paramagnetic effect and is combined with spectral line separation under a high field, so that the contrast ratio of 31P CEST is remarkably improved, the problem of spectral peak overlapping is solved, and a phosphate radical signal is distinguished from adjacent phosphorus-containing molecules. In a word, through the synergistic effect of the high-field 31P CEST technology and the lanthanide series metal complex, phosphate radical recognition is achieved, and a new direction is provided for tumor metabolism research and drug development.

Description

Technical Field

[0001] The present invention belongs to the field of magnetic resonance imaging and detection analysis, and specifically relates to a method based on high field (≥5.0T) 31 Phosphate identification method of P CEST. Background Art

[0002] Phosphate ion (PO4 3- ) is a key participant in energy metabolism, genetic material and cell signal transduction in organisms. ATP (adenosine triphosphate), creatine phosphate, deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and cell membranes all contain phosphate. Abnormal concentrations of phosphate are closely related to various diseases such as tumors, Alzheimer's disease, and renal dysfunction.

[0003] Currently, the detection of phosphate mainly relies on in vitro chemical analysis or low-field nuclear magnetic resonance (NMR) technology. However, traditional methods have significant drawbacks. Chemical analysis is invasive and requires destructive sampling, which cannot achieve dynamic monitoring in vivo; low-field NMR is not sensitive enough to detect phosphate. 31 The detection of P signals is limited by the low gyromagnetic ratio (about 40% of 1H), and the signal-to-noise ratio is difficult to meet the needs of trace analysis. 1 The H CEST technique has low specificity, relies on proton chemical exchange, is easily interfered by water signals in the body, and has difficulty distinguishing phosphate from other phosphorus-containing metabolites such as creatine phosphate.

[0004] In recent years, chemical exchange saturation transfer (CEST) technology has provided a new approach for metabolite detection by selectively saturating exchangeable protons and transferring them to water signals. However, existing CEST research has mostly focused on 1 H nucleus, used for the detection of glucose, creatine and other substances, 31 The development of the CEST effect of P nuclei has lagged behind seriously. The main reasons include: 1. Field strength limitation. Under low-field MRI (<5T), 31 Chemical exchange rate of P (k ex ) and the Larmor frequency offset (Δω) are difficult to satisfy the CEST optimal condition (k ex ≈Δω), resulting in low saturation efficiency; Second, spectral peak overlap: 31 The chemical shift range of the P spectrum is relatively narrow, about 30 ppm, and the phosphate signal is easily confused with neighboring phosphorus-containing molecules. Summary of the Invention

[0005] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a high-field (≥5.0T) 31 The PCEST phosphate identification method has the following advantages: 1. High field-lanthanide collaborative design: using a ≥5.0T high field system, combined with slow electron relaxation lanthanide ions Pr3+ 、Yb 3+ etc. 31 PCR efficiency is improved; 2. Relaxation control strategy: by adjusting the concentration of lanthanide complexes (20-200 μM) and using strong field ligands such as DO3A, balance 31 The transverse relaxation time (T2) of P is shortened and the CEST signal gain is increased. 31 The high sensitivity of PCEST detection opens up new avenues for phosphate identification and quantitative analysis.

[0006] The technical solution adopted to achieve the above-mentioned purpose of the present invention is:

[0007] A high field (≥5.0T) 31 The phosphate identification method of P CEST includes the following steps:

[0008] S1, preparing an aqueous solution of a lanthanide metal complex;

[0009] S2. determining the concentration of the lanthanide metal complex aqueous solution;

[0010] S3. Prepare a neutral phosphate solution;

[0011] S4, respectively mixing the lanthanide metal complex aqueous solution with the phosphate solution to prepare a series of mixed solutions with gradient final concentrations of the lanthanide metal complex, and subjecting each mixed solution to high field heating. 31 P NMR, the phosphate in each mixed solution 31 The half-width and signal intensity of the P NMR signal were analyzed to determine the optimal final concentration of the lanthanide metal complex;

[0012] S5, mixing the lanthanide metal complex aqueous solution and the phosphate solution to prepare a test solution with the optimal final concentration of the lanthanide metal complex, 31 The corresponding NMR signal in the P NMR spectrum is set as the spectrum center and used as the large pool signal of CEST. The solution to be tested is subjected to high field 31 P CEST test, the results show that the solution to be tested 31 The chemical shift difference between the P CEST signal site and the spectrum center is ≥70 ppm, which realizes the identification of phosphate.

[0013] Further, in step S2, using 1 The concentration of lanthanide metal complexes in aqueous solution was determined by H NMR quantitative method.

[0014] Furthermore, the phosphate radical comes from a soluble phosphate or a phosphorus-containing biomolecule, the soluble phosphate is selected from at least one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate and disodium hydrogen phosphate, and the phosphorus-containing biomolecule is selected from at least one of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0015] Furthermore, the lanthanide metal complex is Pr-DO3A or Yb-DO3A.

[0016] The preparation method of lanthanide metal complexes Pr-DO3A or Yb-DO3A refers to the literature "Separation of Intra- and Extracellular Lactate NMR Signals Using a Lanthanide Shift Reagent, Silvio Aime,1*Mauro Botta,2Valentina Mainero,3and Enzo Terreno1".

[0017] Compared with the existing technology, the advantages and benefits of the present invention are: the present invention adopts a high-field MRI system of ≥5.0T, which effectively improves 31 The signal-to-noise ratio of PCEST solves the problem of low saturation efficiency; lanthanide metal complexes accelerate chemical exchange through paramagnetic effect, combined with spectral line separation under high field, significantly improves 31 The PCEST contrast solves the problem of peak overlap, thereby distinguishing the phosphate signal from the adjacent phosphorus-containing molecules. 31 The synergistic effect of P CEST technology and lanthanide metal complexes enables the identification of phosphate groups, providing a new direction for tumor metabolism research and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The quantitative analysis of Pr-DO3A prepared in Example 1 1 H NMR spectrum.

[0019] Figure 2 Yb-DO3A prepared in Example 1 is quantitatively 1 H NMR spectrum.

[0020] Figure 3 Eu-DO3A prepared in Example 1 was quantitatively 1 H NMR spectrum.

[0021] Figure 4 The quantitative Tb-DO3A prepared in Example 1 1 H NMR spectrum.

[0022] Figure 5 The phosphate concentrations of lanthanide metal complexes are shown in Table 1. 31 P NMR spectrum. Among them, Figure 5 a is the phosphate under different concentrations of Pr-DO3A 31 P NMR spectrum, Figure 5 b is the phosphate under different concentrations of Yb-DO3A 31 P NMR spectrum, Figure 5 c is the phosphate under different concentrations of Eu-DO3A 31 P NMR spectrum, Figure 5 d is the phosphate under different concentrations of Tb-DO3A 31 P NMR spectrum.

[0023] Figure 6 The effect of different concentrations of lanthanide metal complexes on phosphate 31 The influence diagram of the half-width height of the P NMR signal peak. Figure 6 a is the effect of different concentrations of Pr-DO3A on phosphate 31 P NMR signal peak half-width influence diagram, Figure 6 b is the effect of different concentrations of Yb-DO3A on phosphate 31 P NMR signal peak half-width influence diagram, Figure 6 c is the effect of Eu-DO3A on phosphate at different concentrations 31 P NMR signal peak half-width influence diagram, Figure 6 d is the effect of different concentrations of Tb-DO3A on phosphate 31 P NMR signal peak half-width effect diagram.

[0024] Figure 7 The effect of different concentrations of lanthanide metal complexes on phosphate 31 P NMR signal intensity effect diagram. Figure 7 a is the effect of different concentrations of Pr-DO3A on phosphate 31 P NMR signal intensity effect diagram, Figure 7 b is the effect of different concentrations of Yb-DO3A on phosphate 31 P NMR signal intensity effect diagram, Figure 7 c is the effect of Eu-DO3A on phosphate at different concentrations 31 P NMR signal intensity effect diagram, Figure 7 d is the effect of different concentrations of Tb-DO3A on phosphate 31 Effect diagram of P NMR signal intensity.

[0025] Figure 8 KH2PO4 in the presence of Pr-DO3A 31P CEST test results. Among them, 8(a) KH2PO4 at different saturation pulse powers 31 P CEST spectrum; 8(b) is the CEST exchange rate between Pr-DO3A-bound phosphate and free phosphate at different saturation pulse powers.

[0026] Figure 9 KH2PO4 in the presence of Yb-DO3A 31 P CEST test results. Among them, 9(a) KH2PO4 at different saturation pulse powers 31 P CEST spectrum; 9(b) is the CEST exchange rate between Yb-DO3A-bound phosphate and free phosphate at different saturation pulse powers.

[0027] Figure 10 KH2PO4 in the presence of Eu-DO3A 31 P CEST test results. Among them, 10 (a) KH2PO4 at different saturation pulse powers 31 P CEST spectrum; 10 (b) is the CEST exchange rate between Eu-DO3A-bound phosphate and free phosphate at different saturation pulse powers.

[0028] Figure 11 KH2PO4 in the presence of Yb-DO3A 31 P CEST test results. Among them, 11 (a) KH2PO4 at different saturation pulse powers 31 P CEST spectrum; 10(b) is the CEST exchange rate between Yb-DO3A-bound phosphate and free phosphate at different saturation pulse powers. DETAILED DESCRIPTION

[0029] In order to facilitate those skilled in the art to understand and implement the present invention, the present invention is further described in detail below in conjunction with embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0030] Example 1

[0031] 1. Preparation of lanthanide metal complex aqueous solution

[0032] 1.1. Prepare 10 mM aqueous solutions of praseodymium trifluoromethanesulfonate (Pr(CF3SO3)3), ytterbium trifluoromethanesulfonate (Yb(CF3SO3)3), europium trifluoromethanesulfonate (Eu(CF3SO3)3), and terbium trifluoromethanesulfonate (Tb(CF3SO3)3) as solutions A1-A4, respectively. Prepare 11.76 mM aqueous solution of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate (DO3A) as solution B.

[0033] 1.2. Take 5 mL of A1 solution and 5 mL of B solution and add them to 50 mL round-bottom centrifuge tubes respectively. Mix them evenly with magnetic stirring. At the same time, adjust the pH of the mixed solution to 5.5-6.0 with 1 M HCl and 1 M NaOH. Continue magnetic stirring. After the pH value stabilizes at 5.5-6.0, transfer the mixed solution to a 45°C constant temperature water bath and keep it for 2 hours. Then test the pH value of the reaction system. If the pH value of the reaction system exceeds the range of 5.5-6.0, continue to adjust the pH value of the reaction system with 1 M HCl and 1 M NaOH until the pH value of the reaction system remains stable in the range of 5.5-6.0, indicating that the reaction of metal Pr with DO3A is complete and Pr-DO3A solution is obtained.

[0034] 1.3. Treat solution A2 and solution B according to the method of step 1.2 to obtain a Yb-DO3A solution. Treat solution A3 and solution B according to the method of step 2 to obtain a Eu-DO3A solution. Treat solution A4 and solution B according to the method of step 2 to obtain a Tb-DO3A solution.

[0035] 2. Determination of concentration of lanthanide metal-DO3A complex aqueous solution

[0036] 2.1. Take 198 μL of each of the Pr-DO3A solution, Yb-DO3A solution, Eu-DO3A solution, and Tb-DO3A solution and mix them with 2 μL of isopropanol to obtain solutions C1-C4. Mix 495 μL of D2O with 5 μL of isopropanol to obtain solution D.

[0037] 2.2. The concentrations of Pr-DO3A, Yb-DO3A, Eu-DO3A and Tb-DO3A were quantified using magnetic resonance. The specific operation was as follows: 200 μL of C1, C2, C3 and C4 solutions were added to the inner tubes of four NMR tubes respectively, and 500 μL of D solution was added to the outer tubes of four NMR tubes respectively. The solutions in the inner and outer tubes of each NMR tube were measured using a 500 MHz magnetic resonance spectrometer. 1 H NMR test, the isopropanol in two environments was measured 1 H NMR signals and calculate the chemical shift difference (δx ), the concentrations c of Pr-DO3A, Yb-DO3A, Eu-DO3A, and Tb-DO3A were calculated using the following formula (1):

[0038]

[0039] Where, T is 295K, s=1 / 3, μ of Pr eff =3.620, μ of Yb eff =4.500, μ of Eu eff =3.455, μ of Tb eff =9.700.

[0040] 1 H NMR test results: δ x =0.09ppm, δ of Tb-DO3A x =0.80ppm, δ of Eu-DO3A x =0.090ppm, δ of Yb-DO3A x =0.15ppm (see Figure 1-4 ), δ x Substituting into the formula, the concentration c values ​​of Pr-DO3A, Yb-DO3A, Eu-DO3A, and Tb-DO3A are calculated to be 4.07 mM, 4.37 mM, 3.98 mM, and 4.83 mM, respectively.

[0041] 3. Preparation of phosphate solution

[0042] Mix 2.2 mL of D2O with 17.8 mL of deionized water to obtain 20 mL of an 11 v / v% D2O solution. Weigh 136.09 mg of KH2PO4 and dissolve it in the D2O solution. Adjust the pH to 7.0 to obtain a 50 mM KH2PO4 solution.

[0043] 4. Determination of the optimal final concentration of lanthanide metal complexes

[0044] 4.1. Pr-DO3A solution was mixed with 50mM KH2PO4 solution to prepare a series of mixed solutions with Pr-DO3A gradient final concentrations of 200, 100, 50 and 20μM. Each mixed solution was added to a 5mm NMR tube and collected on a 500MHz magnetic resonance spectrometer. 31 P NMR spectrum (accumulated 16 times), and analyze the phosphate 31 The PCEST test result signal changes with the added Pr-DO3A concentration.

[0045] 4.2. Treat the Yb-DO3A solution, Eu-DO3A solution, and Tb-DO3A solution according to the method in step 4.1.

[0046] 4.3、 31 P NMR test results:

[0047] Phosphate under different concentrations of Pr-DO3A, Yb-DO3A, Eu-DO3A, and Tb-DO3A 31 P NMR spectrum is as follows Figure 5 As shown in Figure 2, different concentrations of Pr-DO3A, Yb-DO3A, Eu-DO3A, and Tb-DO3A have an impact on the phosphate 31 The influence of the half-width of the P NMR signal peak is as follows Figure 6 As shown in Figure 2, different concentrations of Pr-DO3A, Yb-DO3A, Eu-DO3A, and Tb-DO3A have an impact on the phosphate 31 The influence of P NMR signal intensity is as follows Figure 7 As shown by Figure 5 It can be seen that as the concentration of lanthanide metal complexes increases, the 31 The half-width of the P NMR signal peak gradually increases, and the phosphate 31 The P NMR signal intensity gradually decreases, indicating that the lanthanide metal complexes can lead to 31 The transverse relaxation time (T2) of P is shortened.

[0048] Further analysis of phosphate 31 P NMR signal peak half-width and height 31 The P NMR signal intensities were quantified and the results showed that with the increase of the concentration of lanthanide metal complexes, the 31 The half-width of the P NMR signal peak gradually increases ( Figure 6 ), phosphate 31 The P NMR signal intensity gradually decreased ( Figure 7 ), further indicating that lanthanide metal complexes can lead to the 31 The transverse relaxation time (T2) of P NMR is shortened.

[0049] According to the above 31 P NMR results, taking into account 31 The T2 shortening and CEST signal gain of P were studied by selecting the concentration of Pr-DO3A, Yb-DO3A, Eu-DO3A, and Tb-DO3A at 50 μM. 31 P CEST experiment, identification of phosphate groups.

[0050] 5. Identification of phosphate

[0051] 5.1. Mix the Pr-DO3A solution with 50mM KH2PO4 solution to prepare a sample solution with a final concentration of 50μM Pr-DO3A. Take 500μL of the sample solution and add it to a 5mm NMR tube. Use a 500MHz magnetic resonance spectrometer to perform the spectrometry under different saturation pulse power conditions. 31 In the PCEST test, the saturation pulse power was 2, 4, 6, 8, and 10 μT, the saturation time was 2 seconds, and the exchange rate between the CEST site signal and the CEST large pool signal was calculated. 31 In the P CEST test, KH2PO4 31 The site (chemical shift) of the NMR signal in the PNMR spectrum is set to the spectrum center and used as the large pool signal of CEST. 31 PCEST test, KH2PO4 31 The PCEST saturation site was set to -200-200 ppm.

[0052] 5.2. Treat the Yb-DO3A solution, Eu-DO3A solution, and Tb-DO3A solution according to the method in step 5.1.

[0053] 5.3、 31 PCRest test results:

[0054] KH2PO4 in the presence of Pr-DO3A 31 P CEST test results are as follows Figure 8 As shown, Figure 8 It is shown that the observed 31 P CEST signal, and its signal intensity increases with the increase of saturation pulse power ( Figure 8 a), further calculation 31 The exchange rate of the phosphate coordinated with Pr-DO3A and the free phosphate was determined to be 2101 Hz by PCEST, and the residence time of the phosphate coordinated with Pr-DO3A was 476 μs ( Figure 8 b).

[0055] KH2PO4 in the presence of Yb-DO3A 31 P CEST test results are as follows Figure 9 As shown, Figure 9 It is shown that the observed 31 P CEST signal, and its signal intensity increases with the increase of saturation pulse power ( Figure 9 a), further calculation 31 The exchange rate of phosphate coordinated with Yb-DO3A and free phosphate was determined to be 5614 Hz by PCEST, and the residence time of phosphate coordinated with Yb-DO3A was 178 μs ( Figure 9 b).

[0056] KH2PO4 in the presence of Eu-DO3A 31 P CEST test results are as follows Figure 10 As shown, Figure 10 It is shown that the observed 31 P CEST signal, and its signal intensity increases with the increase of saturation pulse power ( Figure 10 a), further calculation 31 The exchange rate of phosphate coordinated with Eu-DO3A and free phosphate was determined to be 11760 Hz by PCEST, and the residence time of phosphate coordinated with Eu-DO3A was 85 μs ( Figure 10 b).

[0057] KH2PO4 in the presence of Tb-DO3A 31 P CEST test results are as follows Figure 11 As shown, Figure 11 It is shown that the observed 31 P CEST signal, and its signal intensity increases with the increase of saturation pulse power ( Figure 11 a), further calculation 31 The exchange rate of the phosphate coordinated with Tb-DO3A and the free phosphate was determined to be 21054 Hz by PCEST, and the residence time of the phosphate coordinated with Tb-DO3A was 47 μs ( Figure 11 b).

[0058] In the above phosphate 31 In the P CEST test, the lanthanide metal-DO3A complex was measured 31 The chemical shift difference between the P CEST signal site and the CEST large cell signal is ≥70ppm, which is sufficient to overcome the 31 The P NMR technique detects the signal stacking problem in phosphate (the chemical shift difference with phosphorus-containing metabolites in the body is less than 10 ppm), thus realizing the identification of phosphate.

[0059] exist 31 In the PCEST exchange rate test, the exchange rate refers to the frequency of conversion of molecules between different chemical environments, reflecting the speed of the chemical exchange process. 31P is an important parameter for CEST signal detection. A lower exchange rate is more conducive to experimental analysis. This is because when the exchange rate is lower than the chemical shift difference between chemical environments (i.e., in the "slow exchange" region), the signals of different chemical environments still maintain separate sharp peaks. By saturating the signal of a specific environment, the signal change of saturation transfer to another environment can be effectively observed, thereby producing a high-contrast CEST effect. If the exchange rate is too high, the rapid averaging of the signal will lead to spectral line broadening, decreased resolution, and reduced saturation transfer efficiency, making it difficult to accurately capture and analyze the chemical exchange process. Therefore, a lower exchange rate can ensure the specificity and detectability of the signal, facilitate quantitative research on phosphate, and is 31 The key factor in optimizing signal quality in PCEST experiments. From the above results, it can be seen that the exchange rates of phosphate coordinated with Pr-DO3A and Yb-DO3A and free phosphate are 2101Hz and 5614Hz, respectively, which are much lower than those of Eu-DO3A and Tb-DO3A (11760Hz and 21054Hz). 31 In the P CEST method for identifying phosphate, Pr-DO3A and Yb-DO3A are better choices than Eu-DO3A and Tb-DO3A.

Claims

1. A high field (≥ 5.0 T) 31 The phosphate identification method of P CEST is characterized by The steps include: S1, preparing an aqueous solution of a lanthanide metal complex; S2. determining the concentration of the lanthanide metal complex aqueous solution; S3. Prepare a neutral phosphate solution; S4, respectively mixing the lanthanide metal complex aqueous solution with the phosphate solution to prepare a series of mixed solutions with gradient final concentrations of the lanthanide metal complex, and subjecting each mixed solution to high field heating. 31 P NMR, the phosphate in each mixed solution 31 The half-width and signal intensity of the P NMR signal were analyzed to determine the optimal final concentration of the lanthanide metal complex; S5, mixing the lanthanide metal complex aqueous solution and the phosphate solution to prepare a test solution with the optimal final concentration of the lanthanide metal complex, 31 The chemical shift of the corresponding NMR signal in the P NMR spectrum is set as the spectrum center and used as the large cell signal of CEST. The solution to be tested is subjected to high field 31 P CEST test, the results show that the solution to be tested 31 The chemical shift difference between the PCEST signal site and the spectrum center is ≥ 70 ppm, which enables the identification of phosphate.

2. The high field according to claim 1 31 The phosphate identification method of P CEST is characterized by: In step S2, use 1 The concentration of lanthanide metal complexes in aqueous solution was determined by H NMR quantitative method.

3. The high field according to claim 1 31 The phosphate identification method of P CEST is characterized by: The phosphate radical comes from soluble phosphate or phosphorus-containing biomolecules, the soluble phosphate is selected from at least one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate and disodium hydrogen phosphate, and the phosphorus-containing biomolecule is selected from at least one of deoxyribonucleic acid and ribonucleic acid.

4. The high field according to claim 1 31 The phosphate identification method of P CEST is characterized by: The lanthanide metal complex is Pr-DO3A or Yb-DO3A.

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