Yumi Hayashi (Presenter)
Institute for Advanced Research, Nagoya University
Authorship: Yumi Hayashi(1.2), Kei Zaitsu(1,2), Tasuku Murata(3), Hiroki Nakajima(3), Maiko Kusano(2), Hitoshi Tsuchihashi(2), Akira Ishii(2), Tetsuya Ishikawa(2)
(1) Institute for Advanced Research, Nagoya University, Nagoya, Japan (2) Nagoya University Graduate School of Medicine, Nagoya, Japan (3) Shimadzu Corporation, Kyoto, Japan
| Short Abstract Probe electrospray ionization (PESI), which is one of the ambient ionization techniques, enables direct analysis of endogenous compounds in biological tissues without sample preparation. In this study, we applied PESI/tandem mass spectrometry (MS/MS) to intact metabolome analysis of biological tissues and investigated the possibility of applying the present method to local distribution analysis of metabolites and real-time monitoring in a living animal. In conclusion, the present method achieved intact endogenous metabolite analysis without sample pretreatment. PESI/MS/MS also demonstrated its applicability not only to local distribution analysis in brain but also to in vivo real-time analysis of a living mouse liver. |
Long Abstract
Introduction
Intact analysis of biological tissues is one of the ultimate goals for metabolome analysis to obtain solid information on metabolome alteration because it can achieve rapid data acquisition while excluding bias arising from sample preparation. Probe electrospray ionization (PESI), which is one of the ambient ionization techniques, enables direct analysis of endogenous compounds in biological tissues without sample preparation. PESI utilizes an extremely thin needle (700 nm tip diameter) both as a sampling and ionization probe, expecting not only noninvasive analysis of living animals but also local distribution analysis based on narrow sampling area. In this presentation, we will show the application of PESI/tandem mass spectrometry (MS/MS) to intact metabolome analysis of mice liver and frontal cortex (1). We will also demonstrate a real-time monitoring of metabolites in a live mouse liver and the possibility of the present method to be applied to regional distribution analysis of metabolites in brain.
Materials and Methods
Analytical conditions
An LCMS-8040 tandem mass spectrometer with probe electrospray ionization source (Shimadzu Corporation, Kyoto, Japan) was used. A solid needle (700 nm tip diameter, Shimadzu Corporation) was used as a sampling and ionization probe. The probe was exchanged between sample analyses to prevent contamination. Both positive and negative modes were used for ionization polarity. Product ion scan and selected reaction monitoring (SRM) modes were used for qualitative and quantitative analyses, respectively.
Experimental conditions
All animal experiments described below were approved by the animal experiment committee of Nagoya University Graduate School of Medicine.
Experiment 1: Intact analysis of mice liver
CCl4 olive oil solution (0.5 ml/kg body weight) or olive oil solution were intraperitoneally administrated to 6-week male C57BL/6J mice (n=5 in each group). After 24-hours starvation, liver and serum were collected by dissection and quickly frozen on dry ice. To estimate the extent of hepatic injury, serum concentration of deviation enzymes, aspartate transaminase (AST) and alanine transaminase (ALT), were measured by BBx type1 kit (Nittobo Medical Co., Ltd., Japan). Ca. 3 mm-square frozen liver sample was set on a sample plate with 30 microliter of 50% ethanol solution, and the sample was directly analyzed by PESI/MS/MS. The cycle time of probe movement was set at 4.55 Hz and the data acquisition time was set at 0.25 min to obtain sufficient data points. Triplicate analyses were executed for each sample and the obtained data were averaged. Multivariate analysis using SIMCA-P+ software (Umetrics, Umea, Sweden) was carried out for the normalized data by unit variance and the metabolites that primarily contributed to group separation on principal component analysis (PCA) score plots were extracted by PCA loading plots. Finally, statistical analyses were performed by Welch's test for the extracted metabolites (significant level: p<0.05). The results obtained by PESI/MS/MS were further validated comparing with the established metabolome analysis method by gas chromatography-tandem mass spectrometry.
Experiment 2: Intact analysis of mice brain and local distribution analysis
A cannabinoid receptor (CB) agonist (AM-2201), which is known to disrupt energy metabolism in rodent's brain, was used for preparing brain disorder model mouse. AM-2201 DMSO solution (15 mg/kg body weight) or DMSO were intraperitoneally administered to 12-week male ICR mice (n=5 in each group). One hour after administration, frontal cortexes and hippocampi were collected by dissection and quickly frozen with liquid nitrogen, and the frozen samples were directly analyzed by PESI/MS/MS. The cycle time of probe movement was set at 4.5 Hz, and the data acquisition time was set at 0.2 min. To profile the metabolome in the frontal cortexes, multivariate and statistical analyses were executed in a similar manner as described above. We also investigated the potential of PESI/MS/MS for local distribution analysis by comparing the metabolic profiles between frontal cortex and hippocampus in each mouse.
Experiment 3: Real-time monitoring of a living mouse liver
In this experiment, we tested the real-time monitoring of tricarboxylic acid (TCA) cycle intermediates, fumaric acid and alpha-ketoglutaric acid, in a live mouse liver. However, unlike the above-mentioned two off-line experiments, ethanol, which enhances ionization efficiency of PESI, needed to be supplied without damaging the liver. Thus, we designed an appropriate plastic cup which can supply ethanol to the probe needle tip while preventing ethanol from contacting the liver surface. The cup was made by shaping a polypropylene tube, and thin plastic films were attached to the bottom of the cup.
A 25 gauge infusion needle was cannulated into the hepatic portal vein in a male 10-week 129/sv mouse under isoflurane anesthesia. The above-mentioned plastic cup was then mounted directly on the surface of the liver and fixed with an adhesive. After pouring 150 microliter of 50% ethanol solution with 0.5 mg/mL heparin to the cup, fumaric acid and alpha-ketoglutaric acid in a live mouse liver were analyzed by PESI/MS/MS, where the cycle time of probe movement was set at 0.1 Hz. To intentionally enhance TCA cycle activity in the liver, pyruvic acid (400 mg/kg body weight) saline solution was injected via the cannulated tube 4.5 min after the start of analysis, and dynamic changes of the intermediate levels were monitored in real time.
Results and discussion
Experiment 1: Intact analysis of mice liver
Serum levels of the deviation enzymes in CCl4-administrated mice were significantly higher than those in control mice (p<0.05 by Welch's test), confirming expression of acute hepatic injury by CCl4 administration under the current condition. Twenty-six metabolites were detected in mice liver samples by PESI/MS/MS. Results from multivariate analysis showed clear separation of the control and hepatic injury model groups on PCA score plots. Taurine was extracted by loading plots as the primary contributor to group separation, and its levels between control and hepatic injury model groups were significantly different, demonstrating that PESI/MS/MS was able to detect the metabolome alteration directly. Furthermore, the result for taurine obtained by PESI/MS/MS was well correlated with that obtained by the established GC/MS/MS metabolome analysis method (Pearsonfs r = −0.943), proving the present methodfs validity.
Experiment 2: Intact analysis of mice frontal cortexes and local distribution analysis
Twenty-five metabolites were directly detected in mice frontal cortexes by PESI/MS/MS. However, some metabolites showed somewhat low repeatability of the quantitative results. To improve it, a pre-homogenization step, which utilizes a rapid up and down movement of the needle, was added before the analysis, resulting in decreasing the RSD values (37% to 4% for N-acetyl-L-aspartate, 26% to 6% for L-phenylalanine, and 12% to 3% for GABA, before and after adding homogenization step, respectively). Multivariate analysis showed clear separation of the control and model groups, demonstrating PESI/MS/MS was able to capture metabolic profiles in the frontal cortex. Glutamic acid, succinic acid and D-glucose-6-phosphate were extracted by projection to latent structures-discriminant analysis (PLS-DA) loading plots as the primary contributors to group separation, suggesting energy metabolism and glutamatergic system disruption induced by the present CB receptor agonist. These results are in good agreement with our previous results obtained by GC/MS/MS metabolome analysis (2). Also, the metabolite levels between the frontal cortex and the hippocampus of each mouse were able to be monitored by PESI/MS/MS, suggesting PESI/MS/MS has the potential for regional distribution analysis of biological specimens.
Experiment 3: Real-time monitoring of living mice liver
Unlike off-line analysis of dissected samples, blood coagulation on the surface of the probe needle can be problematic in real-time analysis of a living mouse. Therefore, heparin was added to ethanol solution for anticoagulation, and the effect of heparin on ionization efficiency of metabolites was negligible, achieving in real-time analysis of a live mouse liver. At the beginning of monitoring, peak intensities of fumaric acid and alpha-ketoglutaric acid were low in SRM chromatograms. However, their peak intensities dramatically increased after pyruvic acid injection via the cannula: PESI/MS/MS succeeded in capturing the phenomena of the enhancement of TCA cycle activity in real time. As it is well known, pyruvic acid can permeate the mitochondrial outer membrane and enhance TCA cycle activity. Thus, the phenomena captured by PESI/MS/MS is understandable regarding TCA cycle activation caused by pyruvic acid permeation to mitochondria in the mouse liver.
Conclusion
Intact endogenous metabolite analysis of mice liver and brain without sample pretreatment was achieved by PESI/MS/MS. In addition, PESI/MS/MS was able to capture real-time phenomena in a live mouse liver, suggesting future expansion of the present method to various scientific fields.
References & Acknowledgements:
(1) K. Zaitsu et al. Anal Chem. 2016, 88: 3556-3561.
(2) K. Zaitsu et al. Life Sciences. 2015, 137: 49-55.
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |