1. First observation of the complex of BPA with blood component in whole blood by 10 B-NMR K. Yoshino, F. Chino, K. Saito, A.Ishikawa, H. Ohki Shinshu Univ. JAPAN

2. 10 B－NMR Spectrum for Aqueous Mixed Solution of 10 B-BPA and Fructose

3. Introduction

4. Blood used was Red Cell Concentrates including Mannitol, Adenine, Phosphate (RCC-MAP).

6. Y.Mori, A.Suzuki K.Yoshino, H.Kakihana, Pigment Cell Research 2:273-277(1989) 11 B-NMR Spectra 90MHz 1 H-NMR Apparatus BPA complex

7. 10 B-NMR original Spectrum of 4ppM 10 B (BPA:Fr=1:2) in Blood (RCC-MAP) 10 B-NMR Spectrum 500MHz 1 H-NMR Apparatus

8. 10 B-NMR Spectrum after base-line correction 10 B 10ppm BPA:Fr=1:2 RCC-MAP BPA BPA-Sugar Complex

9. Calibration Curves for total 10 B in Blood (RCC-MAP) and aq. Soln.) (total 10 B-NMR Signal Area vs. total 10 B ） green line : Blood (RCC- MAP) Black Line : Aq. Solution

10. 100 μg 10 B/mL BPA Mother Solution (NaCl 0.16M pH7.4) Whole Blood (include. EDTA) K.Yoshino 50 μg 10 B/mL BPA in whole Blood 10 B-NMR Measurement Experimental

11. Result BPA only in whole Blood

12. Discussion What is the Shoulder Peak? Chemical Shift and Half Width of NMR Signal BPA-Monosaccharide Complex BPA-Glucose Complex

13. Simulation Time Course of BPA, BPA-Fructose and BPA-Glucose Concentrations in Patient Blood

15. Simulation Conditions 1.Diffusion and discharge speeds of 3 Compounds (BPA, Fructose,BPA-Fr) in whole Body are same (1min). 2.The complex formation constants in blood are the same as in aqueous solution. 3.Prepared concentrations of BPA and Fructose are used following the reference*. (Mishima group original data) BPA 100mg and Fructose 220mg in 3mL water with 2N NaOH 0.28mL 4.Glucose concentration is 100mg/dL Blood *H. Kakihana, K. Yoashino, Y.Mori, Advaxes in Neutron Caoture Therapy, Edited by A.H.Soloway et al., Plenum Press, New York, 1993.

16. BPA BPA-Fructose (BPA-Fr) D -Fructose BPA-Fructose Complex formation Equilibrium K*

17. Y.Mori, A.Suzuki K.Yoshino, H.Kakihana, Pigment Cell Research 2:273- 277(1989)

18. Free BPA, BPA-Fructose and BPA- Glucose Complexes in Blood % Uncomplexed BPA Total BPA conc. ( 10 Bppm) in Blood

19. Conclusion 1.First observation of the complex of BPA with glucose in whole blood by 10 B-NMR 2. The simulated percent of the BPA-Glucose complex is less than 5 %. 3.The calculated percent of BPA-Fructose complex in whole blood is increased up to 40 percent ( 10 B ppm 10 B conc.) by drop intravenous infusion into a patient.

21. ☮ Materials and methods Reagents 10B-BPA…Stera Pharma Co. Ltd. (95 ％ 10 B enriched) NMR parameters NMR:JEOL ECA-500 10 B Resonance Freauency:53.375 MHz Receiver Gain:60 Relaxation delay:30 ms Scans:100000 Esternal Ref.:BF 3 ­·OEt 2

22. Strahlenther. Onkol. 165 (1989)

23. Article Title H. Kakihana, K.Yoshino, Y. Mori

24. Conclusion Since the discovered complex was anionic compound, the solubility of BPA would be increased by becoming BPA-blood compound complex. This means the amount of fructose can be decreased when one prepares the BPA- fructose solution in actual BNCT.