Kei Zaitsu (Presenter)
Institute for Advanced Research, Nagoya University
Authorship: Kei Zaitsu (1, 2), Yumi Hayashi (1, 2), Hiroaki Kanayama (3), Ryosuke Takagi (4), Saki Noda (2), Maiko Kusano (2), Toshikazu Minohata (5), Jun-ichi Azuma (3), Nobuhiro Kanayama (3), Tetsuya Ishikawa (2), Hitoshi Tsuchihashi (2), Akira Ishii (2)
(1) Institute for Advanced Research, Nagoya University, Nagoya, Japan (2) Nagoya University Graduate School of Medicine, Nagoya, Japan (3) Sunactis Co., Ltd., Osaka, Japan (4) Kobe University, Kobe, Japan (5) Shimadzu Corporation, Kyoto, Japan
| Short Abstract This study demonstrated the real-time monitoring of Glutamic acid in mouse corpus striatum by direct coupling of microdialysis to tandem mass spectrometer with a newly-developed on-line desalting system. The on-line desalting device showed sufficient desalting efficiency for microdialysate, enabling direct coupling of microdialysis to mass spectrometer. Although there was a small time lag due to flow path length, real-time monitoring of glutamic acid in the mouse striatum was achieved. In conclusion, the present method showed future possibility not only for behavioral study of living animals, but also for in vivo real-time multiple-monitoring such as metabolome analysis. |
Long Abstract
Introduction
Real-time monitoring of endogenous compounds in living animals is one of the most exciting challenges in analytical chemistry, especially in mass spectrometry, not only for understanding the dynamics of organisms but also for various future application possibilities. In fact, research groups have been applying mass spectrometry to monitoring the dynamics of biogenic compounds such as metabolites, nucleic acids and neurotransmitters (1)-(3). Our group has additionally achieved the real-time monitoring of tricarboxylic acid (TCA)-cycle intermediates in a living mouse liver by a probe electrospray/tandem mass spectrometry technique (4). To achieve real-time monitoring of endogenous compounds by mass spectrometry, direct sampling technique and its direct coupling to a mass spectrometer are crucial. Microdialysis is a well-known, classical sampling technique in brain research (5), and it is highly useful in monitoring neurotransmitters and other compounds in extracellular brain regions. However, it is impossible to directly connect the microdialysis system to mass spectrometers unless an on-line desalting system for removing salts from microdialysates is developed.
Therefore, in this study, we have developed a novel on-line desalting system based on a new concept, and investigated its usefulness upon direct connection to a high-spec tandem mass spectrometer (MS) for real-time monitoring of L-glutamic acid (Glu) in mouse brain.
Materials and methods
Microdialysis: Brain microdialysis surgery was carried out according to the report by Fuwa et al. (6) with minor modifications. Briefly, a 15-week male ICR mouse (41 g) was anesthetized under isoflurane (Forane, Abbie GK, Tokyo, Japan) and moved to a stereotaxic frame (SR-5M, Narishige, Tokyo, Japan). A microdialysis probe with 2 mm active membrane length (A-I-4-02, Eicom, Kyoto, Japan) was stereotaxically implanted into the corpus striatum (0.7 mm anterior and 2.2 mm lateral from the bregma, and 3.4 mm depth from brain surface). The probe was fixed to a cranial bone according to the procedure described by Fuwa et al. One day after the implantation surgery, the mouse, confirmed to have recovered back to its original state, was subjected to real-time Glu monitoring experiment. Microdialysis was performed in a freely-moving condition. Other analytical conditions used in microdialysis were as follows: a saline solution was perfused by a syringe pump (KDS100 ifusion pump, KD Scientific, Holliston, MA, USA) at 3 ƒÊl/min perfusion rate. Equilibration time for the permeation of endogenous compounds into the probe was set at 2 hours before microdialysis.
Analytical conditions for mass spectrometry: An LCMS-8060 (Shimadzu Corporation, Kyoto, Japan) was used for real-time analysis. Operating parameters of MS were optimized using an authentic standard of Glu (Tokyo Chemical Industry Corporation, Tokyo, Japan). ESI negative mode was used for analysis. Selected reaction monitoring (SRM) transitions and collision energy (CE) for Glu were set at m/z 146 to 102 at 15 eV, m/z 146 to 128 at 15 ev, and m/z 146 to 100 at 16 eV, respectively. Dwell time of each transition was set at 200 msec.
On-line desalting system: An on-line desalting system was constructed by Sunactis Co., Ltd. (Osaka, Japan), of which system details cannot be disclosed because of a patent pending at present. To optimize parameters and estimate the desalting efficiency of the system, a saline solution treated under several conditions was measured for its electrical conductivity. Also, Glu (1 ƒÊg/ml) in saline solution was desalted by the system, and its recovery rate was investigated.
Real-time monitoring system: Microdialysates from the mouse corpus striatum was directly introduced into the above-mentioned on-line desalting system at 3 ƒÊl/min. Immediately after desalting by the system, the solution was sent to a T-joint plumbing, where methanol was also supplied by another syringe pump (KDS100) at the same flow rate as used in microdialysis, and the mixture of the desalted solution and methanol was sent to the MS ion source. An animal experiment including microdialysis surgery was approved by the animal experiment committee of Nagoya University Graduate School of Medicine.
Results and discussion
Evaluation of implantation surgery in microdialysis: The implantation surgery was completed within 1 hour after anesthesia to prevent excess physical burden of the mouse. In the pre-experiment, microdialysate from the mouse was pooled for 2 hours before direct connection to MS. After protein precipitation of the pooled sample by threefold volume of methanol, the supernatant was analyzed by flow-injection/tandem mass spectrometry, resulting in the detection of Glu: it provided adequacy of the microdialysis procedure in this experiment.
Estimation of the developed desalting system: Under the optimized conditions, electrical conductivity of the treated saline solution showed very low values, proving high efficiency of the desalting system. Sufficient recovery rate for Glu by the system was also confirmed. To prevent carry over of the target compound, interior of the apparatus was cleaned after every analysis. There was no severe contamination or carryover of the system.
MS: To assess the sensitivity of the instrument, a low-flow (3 ƒÊl/min) injection analysis using a syringe pump (KDS100) was executed. Under the optimized MS conditions, Glu at 100 ng/ml solution was detected in spite of the extremely low flow rate, demonstrating its sufficient sensitivity.
Real-time monitoring of Glu in the mouse corpus striatum: About 1.5 minutes after introducing the desalted microdialysates to MS, Glu peak was observed in the SRM chromatogram. Although there was a small time lag, mainly due to the flow path length from the out port of the desalting system to the MS ion source, real-time change in the Glu level in mouse corpus striatum was able to be monitored every 200 msec. In the beginning of the monitoring time, the mouse was sleeping and the detected Glu levels were very low. When the mouse was intentionally stimulated, higher Glu response signal was observed.
Glu is a well-known excitatory transmitter in the brain, and the extracellular Glu level in the corpus striatum is correlated to behavioral regulation. Therefore, the responses of Glu observed in this experiment were convincing, and it strongly supports that the present real-time monitoring system captured real-time changes in Glu levels in the corpus striatum.
Future perspectives and limitation: Although real-time monitoring of Glu in the mouse corpus striatum was achieved in this study, the brain concentration of Glu is known to be higher than those of other amino acids. Thus, to monitor other endogenous compounds in real-time by the present method, experimental conditions, especially in microdialysis, need to be further optimized. Also, time lag due to the flow path length must be minimized. However, the present study demonstrates the possibility of in vivo real-time monitoring of multiple biogenic compounds such as the metabolome, and in vivo real-time metabolome analysis is currently underway.
Conclusion
This study demonstrated the achievement of real-time monitoring of Glu in mouse corpus striatum by direct coupling of microdialysis to mass spectrometer with a newly-developed on-line desalting system. The on-line desalting device showed sufficient desalting efficiency for microdialysate, and it enabled direct coupling of microdialysis to mass spectrometer. Although there was a small time lag due to flow path length, real-time monitoring of Glu in the mouse corpus striatum was achieved by the present method. Correlation between mouse behavior and Glu response tendencies were also observed, suggesting future possibility of applying the present method to behavioral study of living animals. The present method shows high potential in in vivo real-time multiple-monitoring such as metabolome analysis.
References & Acknowledgements:
References
(1) C. Liu et al. Anal. Chem. 1996, 68: 3295-3299.
(2) R.A. Saylor et al. J. Chromatogr. A 2015, 1382: 48-64.
(3) C.C. Hsu et al. Anal. Chem. 2013, 85: 7014-7018.
(4) K. Zaitsu et al. Anal. Chem. 2016, 88: 3556-3561.
(5) P. Nandi et al. Anal. Chim. Acta. 2009, 651: 1-14.
(6) T. Fuwa et al. J. Toxicol, Sci. 2016, 41: 329-337.
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