- Email: [email protected]
Whole-body spatially-resolved metabolomics method for profiling the metabolic differences of epimer drug candidates using ambient mass spectrometry imaging
Talanta 202 (2019) 198–206
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Whole-body spatially-resolved metabolomics method for profiling the metabolic differences of epimer drug candidates using ambient mass spectrometry imaging
T
Zhigang Luoa, Dan Liua, Xuechao Panga, Wanqi Yanga, Jiuming Hea, Ruiping Zhanga, Chenggen Zhua, Yanhua Chena, Xin Lia, Jianjun Zhanga, Jiangong Shia, Zeper Abliza,b,∗ a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, PR China Center for Imaging and Systems Biology, School of Pharmacy, Minzu University of China, Beijing, 100081, PR China
b
ARTICLE INFO
ABSTRACT
Keywords: Epimers Air flow-assisted ionisation Sedative-hypnotic Whole-body mass spectrometry imaging analysis Neurotransmitters
Investigation of the in vivo drug action and metabolic differences of epimer drugs is challenging. Whole-body MSI analysis can visually present the stereoscopic distribution of molecules related to the interaction of drugs and organisms, and can provide more comprehensive organ-specific profiling information. Herein, we developed a whole-body spatially-resolved imaging metabolomics method based on an air flow-assisted ionisation desorption electrospray ionisation (AFADESI)-MSI system coupled with a high-resolution mass spectrometer and highly discriminating imaging software. The epimeric sedative-hypnotic drug candidates YZG-331 and YZG-330 were selected as examples, and rats administered normal or high oral doses were used. By performing multivariate statistical data-mining on the combined MSI data, organ-specific differential ions were screened. By comparing the variations in the relative contents of the drugs, their metabolites, and endogenous neurotransmitters throughout whole-body tissue sections of the rats, rich information that could potentially explain the more significant sedative-hypnotic effects of YZG-330 compared to YZG-331 was obtained. Such as the increased ratio of gamma-aminobutyric acid in the brain and stomach of the rats (0.25, 0.47, 0.68, 0.30, and 0.89 for the control and YZG-331-H, YZG-330-H, YZG-331-L, and YZG-330-L, respectively) were interesting. This study provided a convenient and visual method to investigate in vivo molecular metabolic differences and provide insight towards a better understanding of the pharmacodynamic mechanisms of these sedative-hypnotic drug-candidates.
1. Introduction The different stereoisomers of chiral drugs often exert different pharmacodynamic actions [1–3]. Investigating the drug action and comparing the in vivo molecular metabolic differences of chiral drugs has important practical value, and can provide a strategic basis for chiral drug development. Epimers are defined as a pair of stereoisomers that contain multiple chiral centres but differ in configuration at only one stereocentre [4]. The separation and evaluation of the stereoselective pharmacokinetic or pharmacodynamic properties of epimer drugs is a long-standing research topic, in which HPLC analysis has been the most widely used method [5–7]; however, cell viability assays, maximal electroshock seizure tests, dual energy x-ray absorptiometry, differential mobility spectrometry tandem mass spectrometry, and
LC–MS/MS methods have also been used [8–11]. Due to their excellent separation ability, LC–MS/MS methods can be used for the accurate quantitative evaluation of relative differences in molecular metabolism in vivo, regardless of the spatial distribution of the molecule throughout the body. Whole-body autoradiography (WBA) uses radioactive labelling to analyse the drug distribution in whole-body tissue sections; but at the cost of reduced chemical specificity and the inability to detect multiple molecules simultaneously [12,13]. Mass spectrometry imaging (MSI) [14–20] is an effective tool that can distinguish the spatial distributions of drugs, metabolites, and thousands of endogenous molecules by their corresponding spatial locations. In particular, the air flow-assisted ionisation desorption electrospray ionisation (AFADESI)-MSI method [21,22], which does not require complex pre-treatment and labelling, is particularly suitable for
∗ Corresponding author. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, PR China. E-mail address: [email protected] (Z. Abliz).
https://doi.org/10.1016/j.talanta.2019.04.068 Received 5 February 2019; Received in revised form 4 April 2019; Accepted 27 April 2019 Available online 30 April 2019 0039-9140/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Talanta 202 (2019) 198–206
Z. Luo, et al.
ambient whole-body tissue section analysis. Other than the in vitro methods, whole-body MSI analysis can visually present the stereoscopic distribution of molecules related to the interaction of drugs with organisms, and can provide more comprehensive organ-specific profiling information that is rarely analysed in general research with LC–MS/MS method. This information is useful in evaluating the in vivo spatial differences in the molecular metabolism of epimer drug candidates at the whole-body level. However, performing whole-body MSI analysis involves many challenges. First, it is critical to obtain the same whole-body tissue layer from different animal individuals in order to allow comparability between samples; the preparation of large-size tissue sections can present technical limitations. Secondly, whole-body MSI analysis requires a long acquisition time and produces very large datasets, which can complicate analysis of the metabolomic data, especially when a high resolution mass spectrometer is used. The third, but not the last, issue is that different organic tissues in the whole-body section have different matrix effects, which makes the accurate quantitative comparison of the spatial distribution of metabolites more difficult. Investigation of metabolites is very important for understanding the complex biochemical processes, and metabolomics complements other omics technologies by revealing the contributions of nongenetic factors [22–24]. Herein, we developed a whole-body spatially resolved imaging metabolomics method based on an AFADESI-MSI system coupled with a high-resolution mass spectrometer by integrating highly discriminating imaging software. AFADESI coupled with high-resolution mass spectrometer has been demonstrated to provide a stable mass axis, high m/z resolution and identification [23,25], which allows ion distributions from different samples or different organs in a whole-body tissue section to be compared. The custom-developed imaging software (Δm/z = 0.001) [26] was further developed to allow the combination of two sets of whole-body MSI data into one dataset. However, based on the organ profile in the optical images of whole-body tissue section, metabolomic analysis between the same organs in different samples can be performed based on image pixel points. In this way, region of interest (ROI) background subtraction and multivariate statistical datamining can be performed conveniently in order to identify the exogeneous and endogeneous discriminating ions, simplifying the data processing and hardware requirements compared to our previous research [22]. It is especially suitable for researchers who do not have professional data processing knowledge. Furthermore, by calculating the average ion intensity of the delineated ROI of the same organs, a quantitative relative comparison can be directly obtained, regardless of matrix effects. Scheme 1 shows the strategy by which this is accomplished. In this study, we investigated the in vivo drug-body interactions and differences in metabolic profiles of a pair of epimeric sedative-hypnotic drug candidates YZG-331 and YZG-330 using the integrated wholebody AFADESI-MSI method. This pair of epimers is a derivative of N6(4-hydroxybenzyl) adenine riboside (NHBA), one of the bioactive components of Gastrodia elata [27]. Previous studies have demonstrated that both YZG-331 and YZG-330 have excellent sedative and hypnotic effects [28,29], and that YZG-330 is more potent than YZG-331. However, whether these pharmacodynamic differences are caused by the drug itself, its metabolites, or associated endogenous neurotransmitters is still unclear. Herein, MSI metabolic analysis on wholebody tissue sections of rats administered YZG-331 and YZG-330 was performed, and a number of organ-specific differential ions were explored. According to these differential ions, the differences between the spatial distribution of the metabolites of the epimers were further investigated. Factors that may potentially be associated with the differential sedative-hypnotic effects of YZG-331 and YZG-330 were discovered. These results demonstrated that the method can be used for evaluating in vivo differences in the molecular metabolism of epimer drug candidates.
2. Materials and methods Details of the experimental parameters of the AFADESI-MSI system, comparison between ionisation characters of YZG-331 and YZG-330, and error between measured and theoretical values of characteristic ions can be found in the Supplementary materials. 2.1. Chemicals HPLC-grade methanol and formic acid were purchased from Merck (Darmstadt, Germany). Pure water was obtained from a local market (Wahaha, Hangzhou, China). 95% alcohol of analytical grade was purchased from Beijing Chemical Works. Epimeric sedative–hypnotic drug-candidates YZG-331 and YZG-330 (purity > 99%, HPLC) were synthesized by Professor Jiangong Shi, Department of Phytochemistry of the Institute of Materia Medica (Beijing, China). The corresponding structures are shown in Fig. S2. YZGs (abbreviation for “YZG-331 and YZG-330”) were dissolved in CMC-Na solution to prepare normal dose (4.5 mg/ml) and high dose (13.5 mg/ml) solutions. 2.2. Animals The animal study was approved by the Animal Care and Welfare Committee of the Institute of Materia Medica, CAMS, and Peking Union Medical College (Beijing, China; approval no. 11401300054060). Six adult male rats (SD) weighing approximately 170–180 g were housed under normal conditions (See Supplementary material). Water and food were given ad libitum. One rat (weight 176 g) was randomly selected as a control. Two rats (weight 174 g and 180 g) were orally administered high-dose YZG-331 and YZG-330 solutions (150 mg/kg), another two rats (weight 171 g and 185 g) were orally administered with a normal dose of YZGs solutions (50 mg/kg). Each of the five rats was administered approximately 2 ml of solution. When the rats began to fall asleep (about 20 min after administration), they were euthanized under CO2 gas and snap-frozen in a liquid nitrogen bath. The control rat was euthanized according to the same procedure, and all of the rats were embedded/blocked in 3.5% aqueous carboxymethylcellulose, and frozen at −80 °C until sectioning. 2.3. Preparation of whole-body tissue sections Sagittal whole-body cryosections (40 μm thick) were prepared using a Leica CM3600 cryomacrotome (Leica Microsystems Ltd., Wetzlar, Germany), a fully computerized cryomacrotome for whole-body sectioning. To obtain tissue sections from a comparable layer in different animals, a layer located at a depth of 2 mm below the layer with the first exposure of brain tissue was selected for sectioning. Most of the major organs were exposed in the selected layer and three consecutive tissue sections were prepared for each rat. The tissue area in the slide was approximately 140 mm × 45 mm. All sections were stored at −80 °C, and then removed and immediately transferred to a vacuum desiccator for 40 min prior to MSI analysis. 2.4. MS and MS/MS analysis of the YZGs The optical rotation of the materials (1.02 g/100 ml in methanol) was determined using an automatic optical rotator (SGW-5). The YZGs were prepared in the solvent (2 mg/mL in 95% alcohol), and then diluted 10,000-fold with HPLC-grade methanol for ESI analysis using a quadrupole time-of-flight mass spectrometer (QSTARTM Elite, Applied Biosystems, Foster, CA/MDS Sciex, Concord, Canada) coupled with an Agilent 1200 Series rapid resolution liquid chromatography system (Agilent Technologies, Waldbronn, Germany). The mobile phase was composed of 0.1% formic acid in water (A, 20%) and methanol (B, 80%). The flow rate was 100 μL/min. The data was acquired using an 199
Talanta 202 (2019) 198–206
Z. Luo, et al.
Scheme 1. Strategy of whole-body spatially-resolved imaging metabolomics method.
the MS spectra from .raw to .cdf.
ESI source in positive ion mode. Each sample was injected six times with an injection volume of 1 μL. The total ion chromatograms, base peak intensity, and peak areas of the extracted ion chromatograms of the ESI-MS spectra were compared. Data acquisition and processing were performed using Analyst software version QS2.0. High-resolution MS/MS experiments were performed using the AFADESI-MSI system coupled with a Q-Orbitrap mass spectrometer (Q Exactive, Thermo Scientific, Bremen, Germany) to further confirm the identity of the YZGs and the important differential metabolites detected in the MSI analysis. A known amount of a methanol solution of the standard materials was dropped onto a clean glass slide, and the sample region was then scanned in AFADESI-MSI mode. The MS/MS spectra were acquired in positive ion mode; along with moving the glass slide manually.
2.6. Data processing The custom-developed imaging software MassImager 1.0 was used to perform image analysis. The collected profile data of each line was saved as a raw data file and then converted to .cdf format. Using MassImager software (minimum bin width: Δm/z = 0.001), all of the consecutive data files in .cdf format were converted to a cache file database and a loading file. For differential ion discovery, the wholebody MSI data of the control rat and the rats administered YZG-331 were loaded into the imaging software and saved as one cache file database and the corresponding loading file. Then, combined imaging could be obtained. Generally, for exogenous ions, the mass spectrum extracted from one organ region of the control rat image (based on the optical image of the tissue section) was designed as the background, and was subtracted from the profile of the same organ of the drugadministered rat. For endogenous ions, orthogonal partial least squares discriminant analysis (OPLS-DA) based on image pixel points were used to explore the differences in the molecules between the same organ regions in the combined imaging. Furthermore, using a function of the software, the average ion intensity of the delineated region of interest (ROI) was calculated, and the high resolution data information of each peak was exported into an Excel spreadsheet. The average ion intensity of the selected ion was extracted from the file. Each ROI corresponding to an organ profile was delineated and calculated three times to reduce error. A comparison of the ion intensity was performed between the same organs. For different characteristic ions within the same organ region, the corresponding ion intensity ratio was calculated.
2.5. AFADESI-MSI analysis All MSI experiments were performed using an AFADESI-MSI system coupled with a Q-Orbitrap mass spectrometer (Q Exactive, Thermo Scientific, Bremen, Germany). The key parameters of the custom-built AFAI ion source were similar those used in the previous work [21,22]. The parameters of the mass spectrometer were optimized accordingly, and are summarized in Table S1. The spray voltage was set at 7000 V. A mixture of methanol and water (v/v 4:1) with 0.1% formic acid was used as the electrospray desorption solvent, and was delivered to the sprayer by an injection pump (LSP01-2A, Longer Precision Pump Co., Ltd. Baoding, China) at a flow rate of 5 μL/min. Prior to the imaging experiments, optical images of whole-body tissue sections were acquired using a Microtek scanner (MRS-2400A48U, Shanghai Microtek Technology Ltd., Shanghai, China). A unidirectional scanning mode was achieved by continuously moving the sagittal section surface in the y-direction at a velocity of 400 μm/s. The step size in the x-direction was 500 μm. For one section sample, the scanning time was approximately 7–8 h. Data in the 100–1000 m/z range were acquired in positive full scan mode along with target-selected ion monitoring (t-SIM) for the YZGs (m/z 386.1823). Xcalibur Qual browser software version 3.0 (ThermoFisher Scientific) was used to convert the data file format of
3. Results and discussion 3.1. Evaluation of the ionisation and MS/MS character of the YZGs The optical rotations of YZG-331 and YZG-330 were determined to be −35.522 and −59.096, respectively (22 °C, 589 nm). These results 200
Talanta 202 (2019) 198–206
Z. Luo, et al.
indicated that YZG-331 and YZG-330 were epimers, but not enantiomers. Before comparing the differences between the distributions of YZG-331 and YZG-330 in different organs, the ionisation characteristics of the epimers at the same concentrations were determined in ESI mode. The difference in their ionisation behaviour was determined by comparing the total ion chromatograms, base peak intensities, and peak areas of the extracted ion chromatograms of the ESI-MS spectra (see Fig. S1). By calculating the average peak areas of YZG-331 and YZG-330 across six separate acquisition events, a ratio of 1.31 was obtained (see Fig. S1c). MS/MS experiments were performed on the [M+H]+ ion of the YZGs at m/z 386.1823, and the results showed that YZG-331 and YZG-330 shared the same fragment ions, and the product ions at m/z 254.1400 and m/z 136.0620 were observed dominantly in the AFADESI-MS/MS spectra (Figs. S2a and Fig. S2b).
consistent with the results in previous studies using the LC–MS/MS method [28]. The error between the measured value and theoretical value of these ions were also calculated and the mass accuracy was less than 5 ppm in all cases (see Tables S2). To further confirm that the ions detected at m/z 386.1815 in the MSI analysis were the [M+H]+ ions of the YZGs, MS/MS experiments of the ions at m/z 386.1815 in the tissues were performed for comparison with the materials of YZGs. The results showed that the ions at m/z 386.1815 in the MSI analysis had the same fragment ions as the YZGs (Fig. S2c). MS/MS analysis of the abovementioned metabolic ions was also performed by scanning the organ regions in the tissue directly. The characteristic product ions were obtained, and provided information regarding the metabolic pathway of the YZGs (see Table S3). However, that of ion at m/z 270.1349 was not detected. In order to explore the changes in the endogenous molecular metabolism induced by the YZGs, OPLS-DA analysis was used to screen organ-specific discriminating molecules using the combined wholebody MSI data. An illustration of the result of OPLS-DA analysis in the brain is shown in Fig. 1d. There was an obvious distinction between the brains of the control and YZGs-administered rats. The ion at m/z 104.0707 was not only explored as a discriminating ion in the brain, but also was found in the stomach region using the whole-body MSI method. Many of the screened endogenous molecules showed changes in response to YZGs administration, including molecules that are important in neurohumoral transmission, such as gamma-aminobutyric acid (GABA, m/z 104.0707), glutamic acid (Glu, m/z 148.0600), glutamine (Gln, m/z 147.0762), adenosine (m/z 268.1027), and others. These extracted adducted ions were compared with data from the free databases HMDB (http://hmdb.ca/) and Metlin (http://metlin.scripps. edu). The error between the measured values and theoretical values of these ions were also calculated, and the mass accuracy within 5 ppm in all cases (see Tables S2). The MS/MS spectra of GABA, Glu, Gln, and
3.2. Data-mining of the characteristic metabolic ions In order to explore the characteristic ions that could be used for the comparative MSI analysis of the epimers, the MSI data from a control rat and a rat administered a high dose of YZGs were combined into one dataset. The combined dataset allowed the exploration of the discriminating molecules and multivariate statistical analysis to be performed conveniently, although the size of the dataset reached about 12 GB. Using background subtraction, many exogenous ions were discovered based on the comparison of organ profiles in the combined whole-body imaging. An illustration of background subtraction in the stomach region is shown in Fig. 1c. In addition to the protonated ion of the YZGs at m/z 386.1815 and the speculated metabolic product ions at m/z 254.1400 based on phase I drug metabolism, other speculated metabolic product ions at m/z 372.1666, m/z 402.1772, and m/z 270.1349 were also found in the stomach, kidney and intestine regions (imaging of some discriminating ions see Fig. S3). These ions were
Fig. 1. Mass spectra extracted from the stomach region of the combined whole-body MSI data of a rat administered YZGs (a) and the control rat (b). Mass spectrum extracted after background subtraction (c). The mass loading plot (d) and score plot (e) obtained from OPLS-DA analysis. 201
Talanta 202 (2019) 198–206
Z. Luo, et al.
Fig. 2. Optical images and mass images of the [M+H]+ ions at m/z 386.1823, m/z 254.1400, and m/z 372.1666, corresponding to the YZGs and their metabolites in rats (a). Relative intensity of the YZGs ions and the ions at m/z 254.1400 and m/z 372.1666 (b). The ratio of the intensity of the m/z 254.1400 to that of the YZGs, and the ratio of the intensity of the m/z 372.1666 to that of the YZGs (c).
adenosine obtained from the tissue imaging analysis were acquired and compared with the MS/MS spectra of standard samples to further confirm the identity of these endogenous molecules. (Figs. S5–S7). The discriminating ions of the YZGs, their metabolites, and important neurohumoral transmitters were explored using the integrated whole-body AFDESI-MSI method, and were used to compare the spatial differences in the metabolism of the epimers.
quantify drug content in the gastric region using our previous method [30]. Since the analytes were desorbed and ionized in the same organ, the MS response rate corresponded to the same matrix, and could be considered the same for both samples. Furthermore, the data was integrated and normalised prior to extraction. Thus, the average ion intensity of a particular ion in a delineated ROI in the images could be calculated and averaged, and was used to compare the differences in the distribution of the drug and its metabolites (see Tables S4). From the results, the relative intensity of the ion at m/z 386.1823 in the YZG-330H section was 1.42 times that of YZG-331-H. In contrast, the intensity of this ion in YZG-330-L was 10.6-fold greater than in YZG-331-L (Fig. 2b). By calculating the ratio of the intensity of the hydrolysis products (m/z 254.1400) to that of the drug, the hydrolysis rate was determined. The results in Fig. 2c show that YZG-330 was more easily hydrolysed than YZG-331. Similarly, YZG-330 was also more easily demethylated than YZG-331, particularly at high doses. These results may be correlated with their pharmacodynamic differences. For the ion at m/z 270.1349, the maps of the YZG-331-dosed rats showed a relatively higher intensity than those of the YZG-330-dosed rats; however, for YZG-331-H this ion was mainly localised in the intestine, while for YZG-331-L it was distributed throughout almost the entire body. None of the maps showed a particularly high intensity for this ion (Fig. 3a). The ion at m/z 402.1772 was observed mainly in the rats that received a high YZGs dose. YZG-331-H was found in the liver, kidney, and intestinal tissue. However, YZG-330-H was distributed in the stomach and intestine (Fig. 3b). The ion at m/z 402.1772 was extracted in the kidney, liver, and stomach regions, and its intensity in the different maps was compared. Almost none of this ion was extracted for the normal dose-treated rats, except for a small amount in the stomach region of YZG-330-L (Fig. 3c). No obvious pattern correlated with the pharmacodynamic differences was obtained.
3.3. Comparison of the distribution of epimers and their metabolites All of the rats that were administered a YZGs drug candidate entered a sedative-hypnotic state, regardless of whether YZG-331 or YZG-330 was administered and regardless of the dose. However, the rats administered YZG-330 exhibited more obvious sedative-hypnotic and muscle relaxation effects than the rats administered YZG-331. To clearly delineate the results, the images of rats that received high YZGs doses were marked as YZG-331-H and YZG-330-H, and the images of the rats that received the normal dose were marked as YZG-331-L and YZG-330-L. Images of the designated ions based on their exact molecular weights were quickly obtained. Fig. 2a shows the mass images acquired from the tissue sections of the drug-administered rats using AFADESI-MSI, which were produced by extracting the ion corresponding to the drugs at m/z 386.1823 and those of their tentative metabolite ions at m/z 254.1400 and 372.1666. The ions at m/z 254.1400 were the hydrolysis product, which was the main metabolite of the YZGs. The ions at m/z 372.1666 were speculated to be the demethylation product according to phase I drug metabolism (Fig. 2a), and the characteristic product ions at m/z 240.1245 was detected in the MS/MS analysis (see Tables S3). The drugs were mainly distributed in the stomach and gastric contents, rather than in solid organs (brain, lung, liver, etc.). As such, it was difficult to 202
Talanta 202 (2019) 198–206
Z. Luo, et al.
Fig. 3. Images of the [M+H]+ ions at m/z 270.1349 (a) and m/z 402.1772 (b) in the treated rats. Relative intensity of the hydroxylation product (m/z 402.1772) in the kidney, liver, and stomach regions (c).
3.4. Comparison of the distribution of key neurotransmitters
stomach to the brain following YZG-330 treatment was clearly higher than that of YZG-331, regardless of the dose (Fig. 4b). Thus, such relationships can be observed and analysed using the developed integrated whole-body MSI analysis method. According to the classical metabolic route, Glu is the main substrate for GABA synthesis [31], and Gln is a major substrate for Glu synthesis. Extraction of the ions associated with Glu (m/z 148.0604) and Gln (m/z 147.0764) was used to produce the images shown in Fig. 5a. Glu and Gln were found to be distributed in the brain and throughout the whole body. Compared to the control, high doses of YZG-331 and YZG-330 increased Glu and Gln in the brain. YZG-330 increased the Glu content in the stomach regardless of the dose (Fig. 5b). The ratio of Glu to Gln reflects a dynamic balance, such that calculating the value in the same region across treatments may provide insight into the pharmacodynamic differences. In the stomach, the ratios of Glu to Gln following YZG-330-H and YZG-330-L treatment were 54.05 and 16.28, respectively (Fig. 5c), which were far higher than the values following treatment with YZG-331. It is possible that the increased Glu production in the stomach resulted in the entry of additional Glu into the Krebs cycle, resulting in energy restoration during sleep. However, in the brain, although the differences were not as obvious (the Glu/Gln ratios were 1.69, 1.24, 1.18, 0.92, and 0.76 for the control and YZG-331-H, YZG-330-H, YZG-331-L, and YZG-330-L, respectively), the ratio generally decreased for the rats administered YZGs compared to the controls. In particular, the ratio for the rats administered YZG-330 decreased more than that for the rats administered YZG-331. Glu is an
Although treatment with both epimers induced a sedative-hypnotic effect, imaging did not show the presence of either epimer in the brain. We surmised that some neurotransmitters may have been responsible for the sedative-hypnotic effects; thus, each step involved in neurohumoral transmission may be a potential point of drug action. Therefore, the relative content and distribution of some key neurotransmitters was compared based on the whole-body MSI analysis. Gamma-aminobutyric acid (GABA) is the most well-known neurotransmitter in the induction of sedative-hypnotic effects. GABA is an inhibitory transmitter mainly found in the CNS system. Increases in GABA concentration can result in central nervous system depression and sleep. Here, GABA (m/z 104.0706) was found in both the brain and stomach regions (Fig. 4a). Compared with that of the control rat, the GABA content in the brain decreased for the YZGs-administered rats, except in response to YZG-330-H administration. The presence of GABA in the stomach was an interesting finding; it may originate from the stomach contents or from stomach secretions. Furthermore, the levels of GABA in the stomach were increased by drug treatment, except in the case of YZG-331-L. Glutamic acid (Glu) can be converted directly to GABA in the brain; however, other sources also exist, such as pyruvic acid, glucose, and glutamine (Gln). Since a relationship may exist between the brain and the stomach, the ratio of the ion intensity of GABA in the stomach to that in the brain was calculated. Although the ratio could not explain the observed drug action, the ratio of GABA in the
Fig. 4. Optical images and mass images of the [M+H]+ ions at m/z 104.0706 for GABA in the control and YZGs-administered rats (a). The relative intensity of GABA in the stomach and the brain with the corresponding intensity ratios (b). 203
Talanta 202 (2019) 198–206
Z. Luo, et al.
Fig. 5. Optical images and mass images of the [M+H]+ ions at m/z 148.0604 and m/z 147.0764 for Glu and Gln in the control and treated rats (a). Relative intensity of Glu and Gln in the stomach and brain (b). Relative intensity ratio of Glu to Gln in the stomach and brain (c).
excitatory neurotransmitter, and reductions in this neurotransmitter favour sedative-hypnotic effects. The trend in the Glu/Gln ratio was consistent with this effect, as well as with the observed pharmacodynamic differences. Whole-body imaging analysis allows for the evaluation of organs that often receive less attention. 5-Hydroxytryptophan (5-HT) is a widely studied inhibitory neurotransmitter. The ion corresponding to 5HT was not found in any of the images, but its tentative direct metabolite 5-hydroxyindoleacetic acid (5-HIAA, m/z 192.0655) was distributed in the subcutaneous tissue of the back and neck (Fig. 6a). By extracting the ion representing 5-HIAA at m/z 192.0655 and calculating the average ion intensity in the selected region, an increase in 5-HIAA was observed in the YZGs-treated rats relative to the control (Fig. 6b).
Each treatment resulted in increased intensity of the m/z 192.0655 signal, but the effect was more pronounced for YZG-330 than for YZG331. 5-HT released into the synaptic cleft is reabsorbed into the presynaptic nerve endings and degraded into 5-HIAA [32,33]. The treatments in this study may promote the degradation of 5-HT, but the significance of the accumulation of the m/z 192.0655 ion in the back and neck requires further study. Tryptophan (Trp, m/z 205.0972) is the precursor to 5-HT, and an increased level of Trp in the blood is associated with a higher level of 5-HT in the brain. Although only YZG-330H showed an increased m/z 205.0972 intensity in the stomach compared with the control, the content of the ions at m/z 205.0972 in rats administered YZG-330 was several times higher than that in the rats given either dose of YZG-331. These results were also consistent with
Fig. 6. Optical images and mass images of the [M+H]+ ions at m/z 192.0655 and m/z 205.0972 in the control and treated rats (a). Relative intensities of the m/z 192.0655 (b) and m/z 205.0972 (c) signals in the back, neck, and stomach. 204
Talanta 202 (2019) 198–206
Z. Luo, et al.
Fig. 7. Images of the [M+H]+ ions at m/z 268.1040 (a) and m/z 132.0768 (b) in the control and treated rats.
Fig. 8. Optical images and mass images of the [M+H]+ ions at m/z 112.0869 and m/z 126.1026 in the control and treated rats.
study focused on evaluating selected neurotransmitters and modulators involved in the sedative-hypnotic response, and potential factors responsible for the greater sedative-hypnotic effects of YZG-330 compared to those of YZG-331 were discovered. In particular, the distribution of GABA, glutamic acid, and glutamine in the brain and stomach regions is worthy of further study. Although these high-resolution ions were suitable for comparison, further tandem MS/MS analysis is needed to analyse additional characteristic ions. This study provided a simplified method to investigate the in vivo molecular metabolic differences of the epimeric sedative-hypnotic drug candidates, and provided clues to clarify their pharmacodynamic mechanisms.
the observed pharmacodynamic differences (Fig. 6c). Obvious changes in some regions can be observed by simple visual comparison of the whole-body images. Adenosine (m/z 268.1040) plays a role in regulating the sleep-wake cycle. Creatine (m/z 132.0768) functions in energy metabolism, and increased creatine levels help to overcome brain fatigue. In Fig. 7a, adenosine is observed only in the image of the control rat, and almost no adenosine signal is seen in the brains of the treated rats. This was consistent with our study of NHBA [22]. In Fig. 7b, the intensity of the ion at m/z 132.0768 increased in the treated rats. The high doses induced a more significant effect than the normal dose, and YZG-330 had a more considerable effect than YZG-331. Histamine (m/z 112.0869) is an important modulator that acts as a “waking” substance. The histamine content in the brain correlates positively with wakefulness. However, no apparent signal at m/z 112.0869 was observed in the brains of the control or treated rats. Curiously, YZGs treatment resulted in an increase in the ions at m/z 112.0869 in the peripheral tissues, particularly YZG-330-H (Fig. 8a). Since methylation is the principal inactivation mechanism of histamine, the tentative methylated metabolite t-methylhistamine (m/z 126.1026) was evaluated. As shown in Fig. 8b, the ion at m/z 126.1026 was mainly distributed in the stomach, intestine, and kidney. The ion intensity following treatment with YZG-330-H and YZG-331-H in the treated rats was increased compared with that of the control group. However, no signal corresponding to this ion was observed in the kidneys of the YZG331-L and YZG-330-L-treated rats, indicating the potential renal toxicity of high dose treatment. In addition to the above-mentioned characteristic ions, there are many other discriminating ions whose correlations could be evaluated using the integrated whole-body MSI method to investigate the potent multi-target drug action of the epimers.
Acknowledgement The authors are grateful for financial support from the National Scientific and Technological Major Project for New Drugs (Grant No. 2018ZX09711001-002-004); the CAMS Innovation Fund for Medical Science (Grant No. 2017-I2M-3-010, 2017-I2M-1-012); and the Fundamental Research Funds for the Central Universities (PUMC Youth Fund, Grant No. 3332016055). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.04.068. References [1] W.C. Cheng, J.H. Wang, H.Y. Li, S.J. Lu, J.M. Hu, W.Y. Yun, C.H. Chiu, W.B. Yang, Y.H. Chien, W.L. Hwu, Bioevaluation of sixteen ADMDP stereoisomers toward alpha-galactosidase A: development of a new pharmacological chaperone for the treatment of Fabry disease and potential enhancement of enzyme replacement therapy efficiency, Eur. J. Med. Chem. 123 (2016) 14–20, https://doi.org/10.1016/ j.ejmech.2016.07.025. [2] C. Bianchini, P. Lavery, S. Agellon, H.A. Weiler, The generation of C-3alpha epimer of 25-hydroxyvitamin D and its biological effects on bone mineral density in adult rodents, Calcif. Tissue Int. 96 (2015) 453–464, https://doi.org/10.1007/s00223015-9973-9. [3] H. Alkadi, R. Jbeily, Role of chirality in drugs: an overview, Infect. Disord. - Drug Targets 18 (2018) 88–95, https://doi.org/10.2174/ 1871526517666170329123845. [4] L.A. Nguyen, H. He, C. Pham-Huy, Chiral drugs: an overview, Int. J. Biomed. Sci. : IJBS 2 (2006) 85–100. [5] A. Solyomvary, A. Alberti, A. Darcsi, R. Konye, G. Toth, B. Noszal, I. Molnar-Perl, L. Lorantfy, J. Dobos, L. Orfi, S. Beni, I. Boldizsar, Optimized conversion of antiproliferative lignans pinoresinol and epipinoresinol: their simultaneous isolation and identification by centrifugal partition chromatography and high performance liquid chromatography, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1052
4. Conclusions Our study developed a whole-body spatially-resolved imaging metabolomics method that can be used to conveniently screen discrimination metabolites in vivo, and to evaluate the distribution and relative content differences induced by the administration of epimeric drug candidates. Here, the epimeric sedative-hypnotic drug-candidates YZG-331 and YZG-330 were used as examples. By comparing the differences in the distribution and relative content of the drugs, their metabolites, and important neurotransmitters, information regarding the pharmacodynamic differences of the epimers was obtained. This 205
Talanta 202 (2019) 198–206
Z. Luo, et al. (2017) 142–149, https://doi.org/10.1016/j.jchromb.2017.03.036. [6] M. Gumustas, S.A. Ozkan, B. Chankvetadze, Analytical and preparative scale separation of enantiomers of Chiral drugs by chromatography and related methods, Curr. Med. Chem. 25 (2018) 4152–4188, https://doi.org/10.2174/ 0929867325666180129094955. [7] L. Wan, X. Sun, Y. Li, Q. Yu, C. Guo, X. Wang, A stereospecific HPLC method and its application in determination of pharmacokinetics profile of two enantiomers of naringenin in rats, J. Chromatogr. Sci. 49 (2011) 316–320, https://doi.org/10. 1093/chrsci/49.4.316. [8] S. Zhang, Y. Tang, J. Cao, C. Zhao, Y. Zhao, Crystallization-induced dynamic resolution R-epimer from 25-OCH3-PPD epimeric mixture, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1005 (2015) 39–46, https://doi.org/10.1016/j.jchromb. 2015.09.011. [9] D. Zolkowska, A. Dhir, K. Krishnan, D.F. Covey, M.A. Rogawski, Anticonvulsant potencies of the enantiomers of the neurosteroids androsterone and etiocholanolone exceed those of the natural forms, Psychopharmacology (Berl) 231 (2014) 3325–3332, https://doi.org/10.1007/s00213-014-3546-x. [10] C. Liang, H. Sun, X. Meng, L. Yin, J.P. Fawcett, H. Yu, T. Liu, J. Gu, Separation and simultaneous quantitation of PGF2alpha and its epimer 8-iso-PGF2alpha using modifier-assisted differential mobility spectrometry tandem mass spectrometry, Acta Pharm. Sin. B 8 (2018) 228–234, https://doi.org/10.1016/j.apsb.2018.01. 011. [11] S.W. Blue, A.J. Winchell, A.V. Kaucher, R.A. Lieberman, C.T. Gilles, M.N. Pyra, R. Heffron, X.L. Hou, R.W. Coombs, K. Nanda, N.L. Davis, A.P. Kourtis, J.T. Herbeck, J.M. Baeten, J.R. Lingappa, D.W. Erikson, Simultaneous quantitation of multiple contraceptive hormones in human serum by LC-MS/MS, Contraception 97 (2018) 363–369, https://doi.org/10.1016/j.contraception.2018.01.015. [12] V. Kertesz, G.J. Van Berkel, M. Vavrek, K.A. Koeplinger, B.B. Schneider, T.R. Covey, Comparison of drug distribution images from whole-body thin tissue sections obtained using desorption electrospray ionization tandem mass spectrometry and autoradiography, Anal. Chem. 80 (2008) 5168–5177. [13] Y. Xue, H. Gao, Q.C. Ji, Z. Lam, X. Fang, Z. Lin, M. Hoffman, D. Schulz-Jander, N. Weng, Bioanalysis of drug in tissue: current status and challenges, Bioanalysis 4 (2012) 2637–2653. [14] R.M. Caprioli, Imaging mass spectrometry: molecular microscopy for the new age of biology and medicine, Proteomics 16 (2016) 1607–1612, https://doi.org/10.1002/ pmic.201600133. [15] P. Chaurand, Imaging mass spectrometry of thin tissue sections: a decade of collective efforts, J. Proteom. 75 (2012) 4883–4892, https://doi.org/10.1016/j.jprot. 2012.04.005. [16] E.G. Solon, A. Schweitzer, M. Stoeckli, B. Prideaux, Autoradiography, MALDI-MS, and SIMS-MS imaging in pharmaceutical discovery and development, AAPS J. 12 (2010) 11–26. [17] J.M. Wiseman, D.R. Ifa, Y. Zhu, C.B. Kissinger, N.E. Manicke, P.T. Kissinger, R.G. Cooks, Desorption electrospray ionization mass spectrometry: imaging drugs and metabolites in tissues, Proc. Natl. Acad. Sci. Unit. States Am. 105 (2008) 18120–18125. [18] J.C. Vickerman, Molecular imaging and depth profiling by mass spectrometry-SIMS, MALDI or DESI? Analyst 136 (2011) 2199–2217, https://doi.org/10.1039/ c1an00008j. [19] E. Moreno-Gordaliza, D. Esteban-Fernandez, A. Lazaro, B. Humanes, S. Aboulmagd, A. Tejedor, M.W. Linscheid, M.M. Gomez-Gomez, MALDI-LTQ-Orbitrap mass spectrometry imaging for lipidomic analysis in kidney under cisplatin chemotherapy, Talanta 164 (2017) 16–26, https://doi.org/10.1016/j.talanta.2016.11.
026. [20] B. Feng, J. Zhang, C. Chang, L. Li, M. Li, X. Xiong, C. Guo, F. Tang, Y. Bai, H. Liu, Ambient mass spectrometry imaging: plasma assisted laser desorption ionization mass spectrometry imaging and its applications, Anal. Chem. 86 (2014) 4164–4169, https://doi.org/10.1021/ac403310k. [21] Z. Luo, J. He, Y. Chen, J. He, T. Gong, F. Tang, X. Wang, R. Zhang, L. Huang, L. Zhang, H. Lv, S. Ma, Z. Fu, X. Chen, S. Yu, Z. Abliz, Air flow-assisted ionization imaging mass spectrometry method for easy whole-body molecular imaging under ambient conditions, Anal. Chem. 85 (2013) 2977–2982, https://doi.org/10.1021/ ac400009s. [22] J. He, Z. Luo, L. Huang, J. He, Y. Chen, X. Rong, S. Jia, F. Tang, X. Wang, R. Zhang, J. Zhang, J. Shi, Z. Abliz, Ambient mass spectrometry imaging metabolomics method provides novel insights into the action mechanism of drug candidates, Anal. Chem. 87 (2015) 5372–5379, https://doi.org/10.1021/acs.analchem.5b00680. [23] A. Palmer, P. Phapale, I. Chernyavsky, R. Lavigne, D. Fay, A. Tarasov, V. Kovalev, J. Fuchser, S. Nikolenko, C. Pineau, M. Becker, T. Alexandrov, FDR-controlled metabolite annotation for high-resolution imaging mass spectrometry, Nat. Methods 14 (2017) 57–60, https://doi.org/10.1038/nmeth.4072. [24] A. Sepehri, M.-H. Sarrafzadeh, Effect of nitrifiers community on fouling mitigation and nitrification efficiency in a membrane bioreactor, Chem. Eng. Process. - Process Intens. 128 (2018) 10–18, https://doi.org/10.1016/j.cep.2018.04.006. [25] J. He, C. Sun, T. Li, Z. Luo, L. Huang, X. Song, X. Li, Z. Abliz, A sensitive and wide coverage ambient mass spectrometry imaging method for functional metabolites based molecular histology, Adv. Sci. (Weinh) 5 (2018) 1800250, , https://doi.org/ 10.1002/advs.201800250. [26] J. He, L. Huang, R. Tian, T. Li, C. Sun, X. Song, Y. Lv, Z. Luo, X. Li, Z. Abliz, MassImager: a software for interactive and in-depth analysis of mass spectrometry imaging data, Anal. Chim. Acta 1015 (2018) 50–57, https://doi.org/10.1016/j.aca. 2018.02.030. [27] Y. Zhang, M. Li, R.X. Kang, J.G. Shi, G.T. Liu, J.J. Zhang, NHBA isolated from Gastrodia elata exerts sedative and hypnotic effects in sodium pentobarbital-treated mice, Pharmacol. Biochem. Behav. 102 (2012) 450–457, https://doi.org/10.1016/ j.pbb.2012.06.002. [28] Z. Liu, Y. Yang, L. Sheng, Y. Li, Interspecies variation of in vitro stability and metabolic diversity of YZG-331, a promising sedative-hypnotic compound, Front. Pharmacol. 8 (2017) 527, https://doi.org/10.3389/fphar.2017.00527. [29] L. Liu, S. Jia, J. Dong, Y. Zhang, R. Xu, J. Zhang, Sedative–hypnotic effect of YZG330 and its effect on chloride influx in mouse brain cortical cells, Acta Pharm. Sin. B 3 (2013) 234–238, https://doi.org/10.1016/j.apsb.2013.05.005. [30] Z. Luo, J. He, J. He, L. Huang, X. Song, X. Li, Z. Abliz, Quantitative analysis of drug distribution by ambient mass spectrometry imaging method with signal extinction normalization strategy and inkjet-printing technology, Talanta 179 (2018) 230–237, https://doi.org/10.1016/j.talanta.2017.11.005. [31] Z.Y. Hong, Z.L. Huang, W.M. Qu, N. Eguchi, Y. Urade, O. Hayaishi, An adenosine A receptor agonist induces sleep by increasing GABA release in the tuberomammillary nucleus to inhibit histaminergic systems in rats, J. Neurochem. 92 (2005) 1542–1549, https://doi.org/10.1111/j.1471-4159.2004.02991.x. [32] L. Imeri, M.G. Desimoni, R. Giglio, A. Clavenna, M. Mancia, Changes in the serotonergic system during the sleep-wake cycle - simultaneous polygraphic and voltammetric recordings in hypothalamus using a telemetry system, Neuroscience 58 (1994) 353–358, https://doi.org/10.1016/0306-4522(94)90042-6. [33] M. Asikainen, J. Toppila, L. Alanko, D.J. Ward, D. Stenberg, T. Porkka-Heiskanen, Sleep deprivation increases brain serotonin turnover in the rat, Neuroreport 8 (1997) 1577–1582, https://doi.org/10.1097/00001756-199705060-00006.
206
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Whole-body spatially-resolved metabolomics method for profiling the metabolic differences of epimer drug candidates using ambient mass spectrometry imaging
T
Zhigang Luoa, Dan Liua, Xuechao Panga, Wanqi Yanga, Jiuming Hea, Ruiping Zhanga, Chenggen Zhua, Yanhua Chena, Xin Lia, Jianjun Zhanga, Jiangong Shia, Zeper Abliza,b,∗ a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, PR China Center for Imaging and Systems Biology, School of Pharmacy, Minzu University of China, Beijing, 100081, PR China
b
ARTICLE INFO
ABSTRACT
Keywords: Epimers Air flow-assisted ionisation Sedative-hypnotic Whole-body mass spectrometry imaging analysis Neurotransmitters
Investigation of the in vivo drug action and metabolic differences of epimer drugs is challenging. Whole-body MSI analysis can visually present the stereoscopic distribution of molecules related to the interaction of drugs and organisms, and can provide more comprehensive organ-specific profiling information. Herein, we developed a whole-body spatially-resolved imaging metabolomics method based on an air flow-assisted ionisation desorption electrospray ionisation (AFADESI)-MSI system coupled with a high-resolution mass spectrometer and highly discriminating imaging software. The epimeric sedative-hypnotic drug candidates YZG-331 and YZG-330 were selected as examples, and rats administered normal or high oral doses were used. By performing multivariate statistical data-mining on the combined MSI data, organ-specific differential ions were screened. By comparing the variations in the relative contents of the drugs, their metabolites, and endogenous neurotransmitters throughout whole-body tissue sections of the rats, rich information that could potentially explain the more significant sedative-hypnotic effects of YZG-330 compared to YZG-331 was obtained. Such as the increased ratio of gamma-aminobutyric acid in the brain and stomach of the rats (0.25, 0.47, 0.68, 0.30, and 0.89 for the control and YZG-331-H, YZG-330-H, YZG-331-L, and YZG-330-L, respectively) were interesting. This study provided a convenient and visual method to investigate in vivo molecular metabolic differences and provide insight towards a better understanding of the pharmacodynamic mechanisms of these sedative-hypnotic drug-candidates.
1. Introduction The different stereoisomers of chiral drugs often exert different pharmacodynamic actions [1–3]. Investigating the drug action and comparing the in vivo molecular metabolic differences of chiral drugs has important practical value, and can provide a strategic basis for chiral drug development. Epimers are defined as a pair of stereoisomers that contain multiple chiral centres but differ in configuration at only one stereocentre [4]. The separation and evaluation of the stereoselective pharmacokinetic or pharmacodynamic properties of epimer drugs is a long-standing research topic, in which HPLC analysis has been the most widely used method [5–7]; however, cell viability assays, maximal electroshock seizure tests, dual energy x-ray absorptiometry, differential mobility spectrometry tandem mass spectrometry, and
LC–MS/MS methods have also been used [8–11]. Due to their excellent separation ability, LC–MS/MS methods can be used for the accurate quantitative evaluation of relative differences in molecular metabolism in vivo, regardless of the spatial distribution of the molecule throughout the body. Whole-body autoradiography (WBA) uses radioactive labelling to analyse the drug distribution in whole-body tissue sections; but at the cost of reduced chemical specificity and the inability to detect multiple molecules simultaneously [12,13]. Mass spectrometry imaging (MSI) [14–20] is an effective tool that can distinguish the spatial distributions of drugs, metabolites, and thousands of endogenous molecules by their corresponding spatial locations. In particular, the air flow-assisted ionisation desorption electrospray ionisation (AFADESI)-MSI method [21,22], which does not require complex pre-treatment and labelling, is particularly suitable for
∗ Corresponding author. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, PR China. E-mail address: [email protected] (Z. Abliz).
https://doi.org/10.1016/j.talanta.2019.04.068 Received 5 February 2019; Received in revised form 4 April 2019; Accepted 27 April 2019 Available online 30 April 2019 0039-9140/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Talanta 202 (2019) 198–206
Z. Luo, et al.
ambient whole-body tissue section analysis. Other than the in vitro methods, whole-body MSI analysis can visually present the stereoscopic distribution of molecules related to the interaction of drugs with organisms, and can provide more comprehensive organ-specific profiling information that is rarely analysed in general research with LC–MS/MS method. This information is useful in evaluating the in vivo spatial differences in the molecular metabolism of epimer drug candidates at the whole-body level. However, performing whole-body MSI analysis involves many challenges. First, it is critical to obtain the same whole-body tissue layer from different animal individuals in order to allow comparability between samples; the preparation of large-size tissue sections can present technical limitations. Secondly, whole-body MSI analysis requires a long acquisition time and produces very large datasets, which can complicate analysis of the metabolomic data, especially when a high resolution mass spectrometer is used. The third, but not the last, issue is that different organic tissues in the whole-body section have different matrix effects, which makes the accurate quantitative comparison of the spatial distribution of metabolites more difficult. Investigation of metabolites is very important for understanding the complex biochemical processes, and metabolomics complements other omics technologies by revealing the contributions of nongenetic factors [22–24]. Herein, we developed a whole-body spatially resolved imaging metabolomics method based on an AFADESI-MSI system coupled with a high-resolution mass spectrometer by integrating highly discriminating imaging software. AFADESI coupled with high-resolution mass spectrometer has been demonstrated to provide a stable mass axis, high m/z resolution and identification [23,25], which allows ion distributions from different samples or different organs in a whole-body tissue section to be compared. The custom-developed imaging software (Δm/z = 0.001) [26] was further developed to allow the combination of two sets of whole-body MSI data into one dataset. However, based on the organ profile in the optical images of whole-body tissue section, metabolomic analysis between the same organs in different samples can be performed based on image pixel points. In this way, region of interest (ROI) background subtraction and multivariate statistical datamining can be performed conveniently in order to identify the exogeneous and endogeneous discriminating ions, simplifying the data processing and hardware requirements compared to our previous research [22]. It is especially suitable for researchers who do not have professional data processing knowledge. Furthermore, by calculating the average ion intensity of the delineated ROI of the same organs, a quantitative relative comparison can be directly obtained, regardless of matrix effects. Scheme 1 shows the strategy by which this is accomplished. In this study, we investigated the in vivo drug-body interactions and differences in metabolic profiles of a pair of epimeric sedative-hypnotic drug candidates YZG-331 and YZG-330 using the integrated wholebody AFADESI-MSI method. This pair of epimers is a derivative of N6(4-hydroxybenzyl) adenine riboside (NHBA), one of the bioactive components of Gastrodia elata [27]. Previous studies have demonstrated that both YZG-331 and YZG-330 have excellent sedative and hypnotic effects [28,29], and that YZG-330 is more potent than YZG-331. However, whether these pharmacodynamic differences are caused by the drug itself, its metabolites, or associated endogenous neurotransmitters is still unclear. Herein, MSI metabolic analysis on wholebody tissue sections of rats administered YZG-331 and YZG-330 was performed, and a number of organ-specific differential ions were explored. According to these differential ions, the differences between the spatial distribution of the metabolites of the epimers were further investigated. Factors that may potentially be associated with the differential sedative-hypnotic effects of YZG-331 and YZG-330 were discovered. These results demonstrated that the method can be used for evaluating in vivo differences in the molecular metabolism of epimer drug candidates.
2. Materials and methods Details of the experimental parameters of the AFADESI-MSI system, comparison between ionisation characters of YZG-331 and YZG-330, and error between measured and theoretical values of characteristic ions can be found in the Supplementary materials. 2.1. Chemicals HPLC-grade methanol and formic acid were purchased from Merck (Darmstadt, Germany). Pure water was obtained from a local market (Wahaha, Hangzhou, China). 95% alcohol of analytical grade was purchased from Beijing Chemical Works. Epimeric sedative–hypnotic drug-candidates YZG-331 and YZG-330 (purity > 99%, HPLC) were synthesized by Professor Jiangong Shi, Department of Phytochemistry of the Institute of Materia Medica (Beijing, China). The corresponding structures are shown in Fig. S2. YZGs (abbreviation for “YZG-331 and YZG-330”) were dissolved in CMC-Na solution to prepare normal dose (4.5 mg/ml) and high dose (13.5 mg/ml) solutions. 2.2. Animals The animal study was approved by the Animal Care and Welfare Committee of the Institute of Materia Medica, CAMS, and Peking Union Medical College (Beijing, China; approval no. 11401300054060). Six adult male rats (SD) weighing approximately 170–180 g were housed under normal conditions (See Supplementary material). Water and food were given ad libitum. One rat (weight 176 g) was randomly selected as a control. Two rats (weight 174 g and 180 g) were orally administered high-dose YZG-331 and YZG-330 solutions (150 mg/kg), another two rats (weight 171 g and 185 g) were orally administered with a normal dose of YZGs solutions (50 mg/kg). Each of the five rats was administered approximately 2 ml of solution. When the rats began to fall asleep (about 20 min after administration), they were euthanized under CO2 gas and snap-frozen in a liquid nitrogen bath. The control rat was euthanized according to the same procedure, and all of the rats were embedded/blocked in 3.5% aqueous carboxymethylcellulose, and frozen at −80 °C until sectioning. 2.3. Preparation of whole-body tissue sections Sagittal whole-body cryosections (40 μm thick) were prepared using a Leica CM3600 cryomacrotome (Leica Microsystems Ltd., Wetzlar, Germany), a fully computerized cryomacrotome for whole-body sectioning. To obtain tissue sections from a comparable layer in different animals, a layer located at a depth of 2 mm below the layer with the first exposure of brain tissue was selected for sectioning. Most of the major organs were exposed in the selected layer and three consecutive tissue sections were prepared for each rat. The tissue area in the slide was approximately 140 mm × 45 mm. All sections were stored at −80 °C, and then removed and immediately transferred to a vacuum desiccator for 40 min prior to MSI analysis. 2.4. MS and MS/MS analysis of the YZGs The optical rotation of the materials (1.02 g/100 ml in methanol) was determined using an automatic optical rotator (SGW-5). The YZGs were prepared in the solvent (2 mg/mL in 95% alcohol), and then diluted 10,000-fold with HPLC-grade methanol for ESI analysis using a quadrupole time-of-flight mass spectrometer (QSTARTM Elite, Applied Biosystems, Foster, CA/MDS Sciex, Concord, Canada) coupled with an Agilent 1200 Series rapid resolution liquid chromatography system (Agilent Technologies, Waldbronn, Germany). The mobile phase was composed of 0.1% formic acid in water (A, 20%) and methanol (B, 80%). The flow rate was 100 μL/min. The data was acquired using an 199
Talanta 202 (2019) 198–206
Z. Luo, et al.
Scheme 1. Strategy of whole-body spatially-resolved imaging metabolomics method.
the MS spectra from .raw to .cdf.
ESI source in positive ion mode. Each sample was injected six times with an injection volume of 1 μL. The total ion chromatograms, base peak intensity, and peak areas of the extracted ion chromatograms of the ESI-MS spectra were compared. Data acquisition and processing were performed using Analyst software version QS2.0. High-resolution MS/MS experiments were performed using the AFADESI-MSI system coupled with a Q-Orbitrap mass spectrometer (Q Exactive, Thermo Scientific, Bremen, Germany) to further confirm the identity of the YZGs and the important differential metabolites detected in the MSI analysis. A known amount of a methanol solution of the standard materials was dropped onto a clean glass slide, and the sample region was then scanned in AFADESI-MSI mode. The MS/MS spectra were acquired in positive ion mode; along with moving the glass slide manually.
2.6. Data processing The custom-developed imaging software MassImager 1.0 was used to perform image analysis. The collected profile data of each line was saved as a raw data file and then converted to .cdf format. Using MassImager software (minimum bin width: Δm/z = 0.001), all of the consecutive data files in .cdf format were converted to a cache file database and a loading file. For differential ion discovery, the wholebody MSI data of the control rat and the rats administered YZG-331 were loaded into the imaging software and saved as one cache file database and the corresponding loading file. Then, combined imaging could be obtained. Generally, for exogenous ions, the mass spectrum extracted from one organ region of the control rat image (based on the optical image of the tissue section) was designed as the background, and was subtracted from the profile of the same organ of the drugadministered rat. For endogenous ions, orthogonal partial least squares discriminant analysis (OPLS-DA) based on image pixel points were used to explore the differences in the molecules between the same organ regions in the combined imaging. Furthermore, using a function of the software, the average ion intensity of the delineated region of interest (ROI) was calculated, and the high resolution data information of each peak was exported into an Excel spreadsheet. The average ion intensity of the selected ion was extracted from the file. Each ROI corresponding to an organ profile was delineated and calculated three times to reduce error. A comparison of the ion intensity was performed between the same organs. For different characteristic ions within the same organ region, the corresponding ion intensity ratio was calculated.
2.5. AFADESI-MSI analysis All MSI experiments were performed using an AFADESI-MSI system coupled with a Q-Orbitrap mass spectrometer (Q Exactive, Thermo Scientific, Bremen, Germany). The key parameters of the custom-built AFAI ion source were similar those used in the previous work [21,22]. The parameters of the mass spectrometer were optimized accordingly, and are summarized in Table S1. The spray voltage was set at 7000 V. A mixture of methanol and water (v/v 4:1) with 0.1% formic acid was used as the electrospray desorption solvent, and was delivered to the sprayer by an injection pump (LSP01-2A, Longer Precision Pump Co., Ltd. Baoding, China) at a flow rate of 5 μL/min. Prior to the imaging experiments, optical images of whole-body tissue sections were acquired using a Microtek scanner (MRS-2400A48U, Shanghai Microtek Technology Ltd., Shanghai, China). A unidirectional scanning mode was achieved by continuously moving the sagittal section surface in the y-direction at a velocity of 400 μm/s. The step size in the x-direction was 500 μm. For one section sample, the scanning time was approximately 7–8 h. Data in the 100–1000 m/z range were acquired in positive full scan mode along with target-selected ion monitoring (t-SIM) for the YZGs (m/z 386.1823). Xcalibur Qual browser software version 3.0 (ThermoFisher Scientific) was used to convert the data file format of
3. Results and discussion 3.1. Evaluation of the ionisation and MS/MS character of the YZGs The optical rotations of YZG-331 and YZG-330 were determined to be −35.522 and −59.096, respectively (22 °C, 589 nm). These results 200
Talanta 202 (2019) 198–206
Z. Luo, et al.
indicated that YZG-331 and YZG-330 were epimers, but not enantiomers. Before comparing the differences between the distributions of YZG-331 and YZG-330 in different organs, the ionisation characteristics of the epimers at the same concentrations were determined in ESI mode. The difference in their ionisation behaviour was determined by comparing the total ion chromatograms, base peak intensities, and peak areas of the extracted ion chromatograms of the ESI-MS spectra (see Fig. S1). By calculating the average peak areas of YZG-331 and YZG-330 across six separate acquisition events, a ratio of 1.31 was obtained (see Fig. S1c). MS/MS experiments were performed on the [M+H]+ ion of the YZGs at m/z 386.1823, and the results showed that YZG-331 and YZG-330 shared the same fragment ions, and the product ions at m/z 254.1400 and m/z 136.0620 were observed dominantly in the AFADESI-MS/MS spectra (Figs. S2a and Fig. S2b).
consistent with the results in previous studies using the LC–MS/MS method [28]. The error between the measured value and theoretical value of these ions were also calculated and the mass accuracy was less than 5 ppm in all cases (see Tables S2). To further confirm that the ions detected at m/z 386.1815 in the MSI analysis were the [M+H]+ ions of the YZGs, MS/MS experiments of the ions at m/z 386.1815 in the tissues were performed for comparison with the materials of YZGs. The results showed that the ions at m/z 386.1815 in the MSI analysis had the same fragment ions as the YZGs (Fig. S2c). MS/MS analysis of the abovementioned metabolic ions was also performed by scanning the organ regions in the tissue directly. The characteristic product ions were obtained, and provided information regarding the metabolic pathway of the YZGs (see Table S3). However, that of ion at m/z 270.1349 was not detected. In order to explore the changes in the endogenous molecular metabolism induced by the YZGs, OPLS-DA analysis was used to screen organ-specific discriminating molecules using the combined wholebody MSI data. An illustration of the result of OPLS-DA analysis in the brain is shown in Fig. 1d. There was an obvious distinction between the brains of the control and YZGs-administered rats. The ion at m/z 104.0707 was not only explored as a discriminating ion in the brain, but also was found in the stomach region using the whole-body MSI method. Many of the screened endogenous molecules showed changes in response to YZGs administration, including molecules that are important in neurohumoral transmission, such as gamma-aminobutyric acid (GABA, m/z 104.0707), glutamic acid (Glu, m/z 148.0600), glutamine (Gln, m/z 147.0762), adenosine (m/z 268.1027), and others. These extracted adducted ions were compared with data from the free databases HMDB (http://hmdb.ca/) and Metlin (http://metlin.scripps. edu). The error between the measured values and theoretical values of these ions were also calculated, and the mass accuracy within 5 ppm in all cases (see Tables S2). The MS/MS spectra of GABA, Glu, Gln, and
3.2. Data-mining of the characteristic metabolic ions In order to explore the characteristic ions that could be used for the comparative MSI analysis of the epimers, the MSI data from a control rat and a rat administered a high dose of YZGs were combined into one dataset. The combined dataset allowed the exploration of the discriminating molecules and multivariate statistical analysis to be performed conveniently, although the size of the dataset reached about 12 GB. Using background subtraction, many exogenous ions were discovered based on the comparison of organ profiles in the combined whole-body imaging. An illustration of background subtraction in the stomach region is shown in Fig. 1c. In addition to the protonated ion of the YZGs at m/z 386.1815 and the speculated metabolic product ions at m/z 254.1400 based on phase I drug metabolism, other speculated metabolic product ions at m/z 372.1666, m/z 402.1772, and m/z 270.1349 were also found in the stomach, kidney and intestine regions (imaging of some discriminating ions see Fig. S3). These ions were
Fig. 1. Mass spectra extracted from the stomach region of the combined whole-body MSI data of a rat administered YZGs (a) and the control rat (b). Mass spectrum extracted after background subtraction (c). The mass loading plot (d) and score plot (e) obtained from OPLS-DA analysis. 201
Talanta 202 (2019) 198–206
Z. Luo, et al.
Fig. 2. Optical images and mass images of the [M+H]+ ions at m/z 386.1823, m/z 254.1400, and m/z 372.1666, corresponding to the YZGs and their metabolites in rats (a). Relative intensity of the YZGs ions and the ions at m/z 254.1400 and m/z 372.1666 (b). The ratio of the intensity of the m/z 254.1400 to that of the YZGs, and the ratio of the intensity of the m/z 372.1666 to that of the YZGs (c).
adenosine obtained from the tissue imaging analysis were acquired and compared with the MS/MS spectra of standard samples to further confirm the identity of these endogenous molecules. (Figs. S5–S7). The discriminating ions of the YZGs, their metabolites, and important neurohumoral transmitters were explored using the integrated whole-body AFDESI-MSI method, and were used to compare the spatial differences in the metabolism of the epimers.
quantify drug content in the gastric region using our previous method [30]. Since the analytes were desorbed and ionized in the same organ, the MS response rate corresponded to the same matrix, and could be considered the same for both samples. Furthermore, the data was integrated and normalised prior to extraction. Thus, the average ion intensity of a particular ion in a delineated ROI in the images could be calculated and averaged, and was used to compare the differences in the distribution of the drug and its metabolites (see Tables S4). From the results, the relative intensity of the ion at m/z 386.1823 in the YZG-330H section was 1.42 times that of YZG-331-H. In contrast, the intensity of this ion in YZG-330-L was 10.6-fold greater than in YZG-331-L (Fig. 2b). By calculating the ratio of the intensity of the hydrolysis products (m/z 254.1400) to that of the drug, the hydrolysis rate was determined. The results in Fig. 2c show that YZG-330 was more easily hydrolysed than YZG-331. Similarly, YZG-330 was also more easily demethylated than YZG-331, particularly at high doses. These results may be correlated with their pharmacodynamic differences. For the ion at m/z 270.1349, the maps of the YZG-331-dosed rats showed a relatively higher intensity than those of the YZG-330-dosed rats; however, for YZG-331-H this ion was mainly localised in the intestine, while for YZG-331-L it was distributed throughout almost the entire body. None of the maps showed a particularly high intensity for this ion (Fig. 3a). The ion at m/z 402.1772 was observed mainly in the rats that received a high YZGs dose. YZG-331-H was found in the liver, kidney, and intestinal tissue. However, YZG-330-H was distributed in the stomach and intestine (Fig. 3b). The ion at m/z 402.1772 was extracted in the kidney, liver, and stomach regions, and its intensity in the different maps was compared. Almost none of this ion was extracted for the normal dose-treated rats, except for a small amount in the stomach region of YZG-330-L (Fig. 3c). No obvious pattern correlated with the pharmacodynamic differences was obtained.
3.3. Comparison of the distribution of epimers and their metabolites All of the rats that were administered a YZGs drug candidate entered a sedative-hypnotic state, regardless of whether YZG-331 or YZG-330 was administered and regardless of the dose. However, the rats administered YZG-330 exhibited more obvious sedative-hypnotic and muscle relaxation effects than the rats administered YZG-331. To clearly delineate the results, the images of rats that received high YZGs doses were marked as YZG-331-H and YZG-330-H, and the images of the rats that received the normal dose were marked as YZG-331-L and YZG-330-L. Images of the designated ions based on their exact molecular weights were quickly obtained. Fig. 2a shows the mass images acquired from the tissue sections of the drug-administered rats using AFADESI-MSI, which were produced by extracting the ion corresponding to the drugs at m/z 386.1823 and those of their tentative metabolite ions at m/z 254.1400 and 372.1666. The ions at m/z 254.1400 were the hydrolysis product, which was the main metabolite of the YZGs. The ions at m/z 372.1666 were speculated to be the demethylation product according to phase I drug metabolism (Fig. 2a), and the characteristic product ions at m/z 240.1245 was detected in the MS/MS analysis (see Tables S3). The drugs were mainly distributed in the stomach and gastric contents, rather than in solid organs (brain, lung, liver, etc.). As such, it was difficult to 202
Talanta 202 (2019) 198–206
Z. Luo, et al.
Fig. 3. Images of the [M+H]+ ions at m/z 270.1349 (a) and m/z 402.1772 (b) in the treated rats. Relative intensity of the hydroxylation product (m/z 402.1772) in the kidney, liver, and stomach regions (c).
3.4. Comparison of the distribution of key neurotransmitters
stomach to the brain following YZG-330 treatment was clearly higher than that of YZG-331, regardless of the dose (Fig. 4b). Thus, such relationships can be observed and analysed using the developed integrated whole-body MSI analysis method. According to the classical metabolic route, Glu is the main substrate for GABA synthesis [31], and Gln is a major substrate for Glu synthesis. Extraction of the ions associated with Glu (m/z 148.0604) and Gln (m/z 147.0764) was used to produce the images shown in Fig. 5a. Glu and Gln were found to be distributed in the brain and throughout the whole body. Compared to the control, high doses of YZG-331 and YZG-330 increased Glu and Gln in the brain. YZG-330 increased the Glu content in the stomach regardless of the dose (Fig. 5b). The ratio of Glu to Gln reflects a dynamic balance, such that calculating the value in the same region across treatments may provide insight into the pharmacodynamic differences. In the stomach, the ratios of Glu to Gln following YZG-330-H and YZG-330-L treatment were 54.05 and 16.28, respectively (Fig. 5c), which were far higher than the values following treatment with YZG-331. It is possible that the increased Glu production in the stomach resulted in the entry of additional Glu into the Krebs cycle, resulting in energy restoration during sleep. However, in the brain, although the differences were not as obvious (the Glu/Gln ratios were 1.69, 1.24, 1.18, 0.92, and 0.76 for the control and YZG-331-H, YZG-330-H, YZG-331-L, and YZG-330-L, respectively), the ratio generally decreased for the rats administered YZGs compared to the controls. In particular, the ratio for the rats administered YZG-330 decreased more than that for the rats administered YZG-331. Glu is an
Although treatment with both epimers induced a sedative-hypnotic effect, imaging did not show the presence of either epimer in the brain. We surmised that some neurotransmitters may have been responsible for the sedative-hypnotic effects; thus, each step involved in neurohumoral transmission may be a potential point of drug action. Therefore, the relative content and distribution of some key neurotransmitters was compared based on the whole-body MSI analysis. Gamma-aminobutyric acid (GABA) is the most well-known neurotransmitter in the induction of sedative-hypnotic effects. GABA is an inhibitory transmitter mainly found in the CNS system. Increases in GABA concentration can result in central nervous system depression and sleep. Here, GABA (m/z 104.0706) was found in both the brain and stomach regions (Fig. 4a). Compared with that of the control rat, the GABA content in the brain decreased for the YZGs-administered rats, except in response to YZG-330-H administration. The presence of GABA in the stomach was an interesting finding; it may originate from the stomach contents or from stomach secretions. Furthermore, the levels of GABA in the stomach were increased by drug treatment, except in the case of YZG-331-L. Glutamic acid (Glu) can be converted directly to GABA in the brain; however, other sources also exist, such as pyruvic acid, glucose, and glutamine (Gln). Since a relationship may exist between the brain and the stomach, the ratio of the ion intensity of GABA in the stomach to that in the brain was calculated. Although the ratio could not explain the observed drug action, the ratio of GABA in the
Fig. 4. Optical images and mass images of the [M+H]+ ions at m/z 104.0706 for GABA in the control and YZGs-administered rats (a). The relative intensity of GABA in the stomach and the brain with the corresponding intensity ratios (b). 203
Talanta 202 (2019) 198–206
Z. Luo, et al.
Fig. 5. Optical images and mass images of the [M+H]+ ions at m/z 148.0604 and m/z 147.0764 for Glu and Gln in the control and treated rats (a). Relative intensity of Glu and Gln in the stomach and brain (b). Relative intensity ratio of Glu to Gln in the stomach and brain (c).
excitatory neurotransmitter, and reductions in this neurotransmitter favour sedative-hypnotic effects. The trend in the Glu/Gln ratio was consistent with this effect, as well as with the observed pharmacodynamic differences. Whole-body imaging analysis allows for the evaluation of organs that often receive less attention. 5-Hydroxytryptophan (5-HT) is a widely studied inhibitory neurotransmitter. The ion corresponding to 5HT was not found in any of the images, but its tentative direct metabolite 5-hydroxyindoleacetic acid (5-HIAA, m/z 192.0655) was distributed in the subcutaneous tissue of the back and neck (Fig. 6a). By extracting the ion representing 5-HIAA at m/z 192.0655 and calculating the average ion intensity in the selected region, an increase in 5-HIAA was observed in the YZGs-treated rats relative to the control (Fig. 6b).
Each treatment resulted in increased intensity of the m/z 192.0655 signal, but the effect was more pronounced for YZG-330 than for YZG331. 5-HT released into the synaptic cleft is reabsorbed into the presynaptic nerve endings and degraded into 5-HIAA [32,33]. The treatments in this study may promote the degradation of 5-HT, but the significance of the accumulation of the m/z 192.0655 ion in the back and neck requires further study. Tryptophan (Trp, m/z 205.0972) is the precursor to 5-HT, and an increased level of Trp in the blood is associated with a higher level of 5-HT in the brain. Although only YZG-330H showed an increased m/z 205.0972 intensity in the stomach compared with the control, the content of the ions at m/z 205.0972 in rats administered YZG-330 was several times higher than that in the rats given either dose of YZG-331. These results were also consistent with
Fig. 6. Optical images and mass images of the [M+H]+ ions at m/z 192.0655 and m/z 205.0972 in the control and treated rats (a). Relative intensities of the m/z 192.0655 (b) and m/z 205.0972 (c) signals in the back, neck, and stomach. 204
Talanta 202 (2019) 198–206
Z. Luo, et al.
Fig. 7. Images of the [M+H]+ ions at m/z 268.1040 (a) and m/z 132.0768 (b) in the control and treated rats.
Fig. 8. Optical images and mass images of the [M+H]+ ions at m/z 112.0869 and m/z 126.1026 in the control and treated rats.
study focused on evaluating selected neurotransmitters and modulators involved in the sedative-hypnotic response, and potential factors responsible for the greater sedative-hypnotic effects of YZG-330 compared to those of YZG-331 were discovered. In particular, the distribution of GABA, glutamic acid, and glutamine in the brain and stomach regions is worthy of further study. Although these high-resolution ions were suitable for comparison, further tandem MS/MS analysis is needed to analyse additional characteristic ions. This study provided a simplified method to investigate the in vivo molecular metabolic differences of the epimeric sedative-hypnotic drug candidates, and provided clues to clarify their pharmacodynamic mechanisms.
the observed pharmacodynamic differences (Fig. 6c). Obvious changes in some regions can be observed by simple visual comparison of the whole-body images. Adenosine (m/z 268.1040) plays a role in regulating the sleep-wake cycle. Creatine (m/z 132.0768) functions in energy metabolism, and increased creatine levels help to overcome brain fatigue. In Fig. 7a, adenosine is observed only in the image of the control rat, and almost no adenosine signal is seen in the brains of the treated rats. This was consistent with our study of NHBA [22]. In Fig. 7b, the intensity of the ion at m/z 132.0768 increased in the treated rats. The high doses induced a more significant effect than the normal dose, and YZG-330 had a more considerable effect than YZG-331. Histamine (m/z 112.0869) is an important modulator that acts as a “waking” substance. The histamine content in the brain correlates positively with wakefulness. However, no apparent signal at m/z 112.0869 was observed in the brains of the control or treated rats. Curiously, YZGs treatment resulted in an increase in the ions at m/z 112.0869 in the peripheral tissues, particularly YZG-330-H (Fig. 8a). Since methylation is the principal inactivation mechanism of histamine, the tentative methylated metabolite t-methylhistamine (m/z 126.1026) was evaluated. As shown in Fig. 8b, the ion at m/z 126.1026 was mainly distributed in the stomach, intestine, and kidney. The ion intensity following treatment with YZG-330-H and YZG-331-H in the treated rats was increased compared with that of the control group. However, no signal corresponding to this ion was observed in the kidneys of the YZG331-L and YZG-330-L-treated rats, indicating the potential renal toxicity of high dose treatment. In addition to the above-mentioned characteristic ions, there are many other discriminating ions whose correlations could be evaluated using the integrated whole-body MSI method to investigate the potent multi-target drug action of the epimers.
Acknowledgement The authors are grateful for financial support from the National Scientific and Technological Major Project for New Drugs (Grant No. 2018ZX09711001-002-004); the CAMS Innovation Fund for Medical Science (Grant No. 2017-I2M-3-010, 2017-I2M-1-012); and the Fundamental Research Funds for the Central Universities (PUMC Youth Fund, Grant No. 3332016055). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.04.068. References [1] W.C. Cheng, J.H. Wang, H.Y. Li, S.J. Lu, J.M. Hu, W.Y. Yun, C.H. Chiu, W.B. Yang, Y.H. Chien, W.L. Hwu, Bioevaluation of sixteen ADMDP stereoisomers toward alpha-galactosidase A: development of a new pharmacological chaperone for the treatment of Fabry disease and potential enhancement of enzyme replacement therapy efficiency, Eur. J. Med. Chem. 123 (2016) 14–20, https://doi.org/10.1016/ j.ejmech.2016.07.025. [2] C. Bianchini, P. Lavery, S. Agellon, H.A. Weiler, The generation of C-3alpha epimer of 25-hydroxyvitamin D and its biological effects on bone mineral density in adult rodents, Calcif. Tissue Int. 96 (2015) 453–464, https://doi.org/10.1007/s00223015-9973-9. [3] H. Alkadi, R. Jbeily, Role of chirality in drugs: an overview, Infect. Disord. - Drug Targets 18 (2018) 88–95, https://doi.org/10.2174/ 1871526517666170329123845. [4] L.A. Nguyen, H. He, C. Pham-Huy, Chiral drugs: an overview, Int. J. Biomed. Sci. : IJBS 2 (2006) 85–100. [5] A. Solyomvary, A. Alberti, A. Darcsi, R. Konye, G. Toth, B. Noszal, I. Molnar-Perl, L. Lorantfy, J. Dobos, L. Orfi, S. Beni, I. Boldizsar, Optimized conversion of antiproliferative lignans pinoresinol and epipinoresinol: their simultaneous isolation and identification by centrifugal partition chromatography and high performance liquid chromatography, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1052
4. Conclusions Our study developed a whole-body spatially-resolved imaging metabolomics method that can be used to conveniently screen discrimination metabolites in vivo, and to evaluate the distribution and relative content differences induced by the administration of epimeric drug candidates. Here, the epimeric sedative-hypnotic drug-candidates YZG-331 and YZG-330 were used as examples. By comparing the differences in the distribution and relative content of the drugs, their metabolites, and important neurotransmitters, information regarding the pharmacodynamic differences of the epimers was obtained. This 205
Talanta 202 (2019) 198–206
Z. Luo, et al. (2017) 142–149, https://doi.org/10.1016/j.jchromb.2017.03.036. [6] M. Gumustas, S.A. Ozkan, B. Chankvetadze, Analytical and preparative scale separation of enantiomers of Chiral drugs by chromatography and related methods, Curr. Med. Chem. 25 (2018) 4152–4188, https://doi.org/10.2174/ 0929867325666180129094955. [7] L. Wan, X. Sun, Y. Li, Q. Yu, C. Guo, X. Wang, A stereospecific HPLC method and its application in determination of pharmacokinetics profile of two enantiomers of naringenin in rats, J. Chromatogr. Sci. 49 (2011) 316–320, https://doi.org/10. 1093/chrsci/49.4.316. [8] S. Zhang, Y. Tang, J. Cao, C. Zhao, Y. Zhao, Crystallization-induced dynamic resolution R-epimer from 25-OCH3-PPD epimeric mixture, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1005 (2015) 39–46, https://doi.org/10.1016/j.jchromb. 2015.09.011. [9] D. Zolkowska, A. Dhir, K. Krishnan, D.F. Covey, M.A. Rogawski, Anticonvulsant potencies of the enantiomers of the neurosteroids androsterone and etiocholanolone exceed those of the natural forms, Psychopharmacology (Berl) 231 (2014) 3325–3332, https://doi.org/10.1007/s00213-014-3546-x. [10] C. Liang, H. Sun, X. Meng, L. Yin, J.P. Fawcett, H. Yu, T. Liu, J. Gu, Separation and simultaneous quantitation of PGF2alpha and its epimer 8-iso-PGF2alpha using modifier-assisted differential mobility spectrometry tandem mass spectrometry, Acta Pharm. Sin. B 8 (2018) 228–234, https://doi.org/10.1016/j.apsb.2018.01. 011. [11] S.W. Blue, A.J. Winchell, A.V. Kaucher, R.A. Lieberman, C.T. Gilles, M.N. Pyra, R. Heffron, X.L. Hou, R.W. Coombs, K. Nanda, N.L. Davis, A.P. Kourtis, J.T. Herbeck, J.M. Baeten, J.R. Lingappa, D.W. Erikson, Simultaneous quantitation of multiple contraceptive hormones in human serum by LC-MS/MS, Contraception 97 (2018) 363–369, https://doi.org/10.1016/j.contraception.2018.01.015. [12] V. Kertesz, G.J. Van Berkel, M. Vavrek, K.A. Koeplinger, B.B. Schneider, T.R. Covey, Comparison of drug distribution images from whole-body thin tissue sections obtained using desorption electrospray ionization tandem mass spectrometry and autoradiography, Anal. Chem. 80 (2008) 5168–5177. [13] Y. Xue, H. Gao, Q.C. Ji, Z. Lam, X. Fang, Z. Lin, M. Hoffman, D. Schulz-Jander, N. Weng, Bioanalysis of drug in tissue: current status and challenges, Bioanalysis 4 (2012) 2637–2653. [14] R.M. Caprioli, Imaging mass spectrometry: molecular microscopy for the new age of biology and medicine, Proteomics 16 (2016) 1607–1612, https://doi.org/10.1002/ pmic.201600133. [15] P. Chaurand, Imaging mass spectrometry of thin tissue sections: a decade of collective efforts, J. Proteom. 75 (2012) 4883–4892, https://doi.org/10.1016/j.jprot. 2012.04.005. [16] E.G. Solon, A. Schweitzer, M. Stoeckli, B. Prideaux, Autoradiography, MALDI-MS, and SIMS-MS imaging in pharmaceutical discovery and development, AAPS J. 12 (2010) 11–26. [17] J.M. Wiseman, D.R. Ifa, Y. Zhu, C.B. Kissinger, N.E. Manicke, P.T. Kissinger, R.G. Cooks, Desorption electrospray ionization mass spectrometry: imaging drugs and metabolites in tissues, Proc. Natl. Acad. Sci. Unit. States Am. 105 (2008) 18120–18125. [18] J.C. Vickerman, Molecular imaging and depth profiling by mass spectrometry-SIMS, MALDI or DESI? Analyst 136 (2011) 2199–2217, https://doi.org/10.1039/ c1an00008j. [19] E. Moreno-Gordaliza, D. Esteban-Fernandez, A. Lazaro, B. Humanes, S. Aboulmagd, A. Tejedor, M.W. Linscheid, M.M. Gomez-Gomez, MALDI-LTQ-Orbitrap mass spectrometry imaging for lipidomic analysis in kidney under cisplatin chemotherapy, Talanta 164 (2017) 16–26, https://doi.org/10.1016/j.talanta.2016.11.
026. [20] B. Feng, J. Zhang, C. Chang, L. Li, M. Li, X. Xiong, C. Guo, F. Tang, Y. Bai, H. Liu, Ambient mass spectrometry imaging: plasma assisted laser desorption ionization mass spectrometry imaging and its applications, Anal. Chem. 86 (2014) 4164–4169, https://doi.org/10.1021/ac403310k. [21] Z. Luo, J. He, Y. Chen, J. He, T. Gong, F. Tang, X. Wang, R. Zhang, L. Huang, L. Zhang, H. Lv, S. Ma, Z. Fu, X. Chen, S. Yu, Z. Abliz, Air flow-assisted ionization imaging mass spectrometry method for easy whole-body molecular imaging under ambient conditions, Anal. Chem. 85 (2013) 2977–2982, https://doi.org/10.1021/ ac400009s. [22] J. He, Z. Luo, L. Huang, J. He, Y. Chen, X. Rong, S. Jia, F. Tang, X. Wang, R. Zhang, J. Zhang, J. Shi, Z. Abliz, Ambient mass spectrometry imaging metabolomics method provides novel insights into the action mechanism of drug candidates, Anal. Chem. 87 (2015) 5372–5379, https://doi.org/10.1021/acs.analchem.5b00680. [23] A. Palmer, P. Phapale, I. Chernyavsky, R. Lavigne, D. Fay, A. Tarasov, V. Kovalev, J. Fuchser, S. Nikolenko, C. Pineau, M. Becker, T. Alexandrov, FDR-controlled metabolite annotation for high-resolution imaging mass spectrometry, Nat. Methods 14 (2017) 57–60, https://doi.org/10.1038/nmeth.4072. [24] A. Sepehri, M.-H. Sarrafzadeh, Effect of nitrifiers community on fouling mitigation and nitrification efficiency in a membrane bioreactor, Chem. Eng. Process. - Process Intens. 128 (2018) 10–18, https://doi.org/10.1016/j.cep.2018.04.006. [25] J. He, C. Sun, T. Li, Z. Luo, L. Huang, X. Song, X. Li, Z. Abliz, A sensitive and wide coverage ambient mass spectrometry imaging method for functional metabolites based molecular histology, Adv. Sci. (Weinh) 5 (2018) 1800250, , https://doi.org/ 10.1002/advs.201800250. [26] J. He, L. Huang, R. Tian, T. Li, C. Sun, X. Song, Y. Lv, Z. Luo, X. Li, Z. Abliz, MassImager: a software for interactive and in-depth analysis of mass spectrometry imaging data, Anal. Chim. Acta 1015 (2018) 50–57, https://doi.org/10.1016/j.aca. 2018.02.030. [27] Y. Zhang, M. Li, R.X. Kang, J.G. Shi, G.T. Liu, J.J. Zhang, NHBA isolated from Gastrodia elata exerts sedative and hypnotic effects in sodium pentobarbital-treated mice, Pharmacol. Biochem. Behav. 102 (2012) 450–457, https://doi.org/10.1016/ j.pbb.2012.06.002. [28] Z. Liu, Y. Yang, L. Sheng, Y. Li, Interspecies variation of in vitro stability and metabolic diversity of YZG-331, a promising sedative-hypnotic compound, Front. Pharmacol. 8 (2017) 527, https://doi.org/10.3389/fphar.2017.00527. [29] L. Liu, S. Jia, J. Dong, Y. Zhang, R. Xu, J. Zhang, Sedative–hypnotic effect of YZG330 and its effect on chloride influx in mouse brain cortical cells, Acta Pharm. Sin. B 3 (2013) 234–238, https://doi.org/10.1016/j.apsb.2013.05.005. [30] Z. Luo, J. He, J. He, L. Huang, X. Song, X. Li, Z. Abliz, Quantitative analysis of drug distribution by ambient mass spectrometry imaging method with signal extinction normalization strategy and inkjet-printing technology, Talanta 179 (2018) 230–237, https://doi.org/10.1016/j.talanta.2017.11.005. [31] Z.Y. Hong, Z.L. Huang, W.M. Qu, N. Eguchi, Y. Urade, O. Hayaishi, An adenosine A receptor agonist induces sleep by increasing GABA release in the tuberomammillary nucleus to inhibit histaminergic systems in rats, J. Neurochem. 92 (2005) 1542–1549, https://doi.org/10.1111/j.1471-4159.2004.02991.x. [32] L. Imeri, M.G. Desimoni, R. Giglio, A. Clavenna, M. Mancia, Changes in the serotonergic system during the sleep-wake cycle - simultaneous polygraphic and voltammetric recordings in hypothalamus using a telemetry system, Neuroscience 58 (1994) 353–358, https://doi.org/10.1016/0306-4522(94)90042-6. [33] M. Asikainen, J. Toppila, L. Alanko, D.J. Ward, D. Stenberg, T. Porkka-Heiskanen, Sleep deprivation increases brain serotonin turnover in the rat, Neuroreport 8 (1997) 1577–1582, https://doi.org/10.1097/00001756-199705060-00006.
206