2.1 Chemicals and reagents
OPD (F05975, purity: 98% by HPLC) and OPD' (F581298, purity: 98% by HPLC) were purchased from Shanghai EFE Biotechnology Co., Ltd. (Shanghai, China). Qualitative detection kit for urine hemosiderin test (Rous) was purchased from Shanghai Yuan Mu Biotechnology Co., Ltd. (Shanghai, China).
All other chemicals and solvents were analytical or HPLC grade. The ultra-pure water was prepared by the Milli-Q system (Millipore, Bedford, MA, USA). Methanol, acetonitrile, formic acid were purchased from CNW Technologies. GmbH (Düsseldorf, Germany). L-2-chlorophenylalanine was purchased from Shanghai Heng Chuang Bio-technology Co., Ltd. (Shanghai, China).
In vivo hemolytic study was conducted in the apparently healthy adult Wistar rats (180-200g, male and female). Animals were purchased from Beijing Charles River Animal Breeding Center (Production license: SCXK (Jing)-2018-0010). All animals were housed in an environmentally controlled breeding room (temperature: 22±2℃, humidity: 50±5%, dark/light cycle: 12/12 h). The animals were provided standard laboratory food and water. The experimental protocols were approved by the Animal Ethics Committee of Academy of Military Medical Sciences (No. IACUC-DWZX-2020-684) and were performed in accordance with the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals.
2.3 Parameters related to hemolysis
In this experiment, the groups were set as follows: normal control group (NC), OPD independent use group (OPD), OPD' independent use group (OPD'), OPD and OPD' combination group (OPD+OPD'), OPD and OPD' interval administration group (OPD→OPD'). 25 Wistar rats (5 rats in each group) were given by tail vein injection once a day for 30 days. The activities and physiological conditions of rats were observed and recorded every day, body weight were weighed every 7 days. At the end of experiments, carbon dioxide anesthesia, whole blood was collected, anticoagulation and perform hematology analysis. Plasma was separated by centrifugation for biochemical analysis. Urine were collected, one part was used to determine whether it contains hemosiderin by Rous method, another part was used for routine urine testing.
2.4 Metabolomic experiments
2.4.1 Sample preparation
22 rats were randomly assigned to 3 groups, they are normal control group (NC, n=6), OPD group (n=8) and OPD' group (n=8). All drugs were administrated by intravascular injection for 14 days. According to the limit of clinical hemolysis rate (5%) and the results obtained from in vitro hemolytic test, When the concentration of OPD' exceeds 5μg·ml-1, the hemolysis rate exceeds the clinical limit of 5%. Assuming an ideal situation, according to the blood volume of the experimental animals and this blood drug concentration (i.e. 5μg·ml-1), the doses of the OPD and OPD' of each group are defined as 0.25mg·kg-1. Plasma was prepared by centrifugation for metabolomics analysis.
100μL of plasma was added to a 1.5 mL Eppendorf tube with 10μL of 2-chloro-1-phenylalanine (0.3 mg·mL-1) dissolved in methanol as internal standard. Subsequently, 300μL of ice-cold mixture of methanol and acetonitrile (2/1, v/v) was added, and the mixtures were vortexed for 1 minute, ultrasonicated at ambient temperature (25℃ to 28℃) for 10 minutes, store at -20℃ for 30 minutes. The extract was centrifuged at 13000 rpm, 4℃ for 15 minutes, 300μL of supernatant in a brown and glass vial was dried in a freeze concentration centrifugal dryer, 400μL mixture of methanol and water (1/4, v/v) were added to each sample and then vortexed for 30 seconds, Repeat the centrifugation operation, 150μL supernatants from each tube were collected using crystal syringes, filtered through 0.22μm microfilters and transferred to LC vials for LC-MS analysis. QC samples were prepared by mixing aliquots of the all to be a pooled sample and the QCs were injected at regular intervals (every 10 samples) throughout the analytical run to provide a set of data from which repeatability can be assessed.
2.4.2 UPLC-MS/MS analysis
Instrument for this study is a LC-MS system composed of Dionex U3000 UPLC ultra-efficient liquid chromatography tandem a QE plus high-resolution mass spectrometer. Liquid phase separation was performed on an Acquity UPLC HSS T3 (100 mm×2.1 mm, 1.8 um) column. The flow rate was controlled at 0.35 mL·min-1, the temperature of the automatic injector was maintained at 4 ℃, and the column temperature was controlled at 50 ℃. The injection volume of all samples was 5μL. The mobile phase consisted of water and acetonitrile which both containing 0.1% formic acid. Gradient elution was performed as follows: (1) mobile phase A was at 95% at 0 min, (2) an isocratic elution was maintained at 95% A from 0 min to 1 min, (3) a linear gradient was decreased to 0% A from 1 min to 11 min, and (4) mobile phase A maintained at 0% from 11 min to 13 min.
The condition of mass spectrometry (MS) analysis was ESI source ionization in positive and negative ion mode respectively, and the scanning mode adopted centroid and continuum mode. The optimum conditions were shown in Table 1.
2.5 Lipidomic Experiments
2.5.1 Sample Preparation
After the same steps of metabolomic sample preparation. 100μL of plasma was added to a tube with 20μL of 2-chloro-1-phenylalanine (0.3 mg·mL-1) and Lyso PC (17:0) (0.01 mg·mL-1) dissolved in methanol as internal standard. Subsequently, 300μL Isopropanol was added, and vortex for 30s, and extract with ultrasound for 10min. Place at -20℃ for 30min, centrifuge for 10 min, take 300μL of supernatant and transfer to a new tube. Another 200μL of isopropanol was added to the original tube and extract again, take 200μL up layer and merge it into the new tube. After drying, the lipid residue in the centrifuge tube was reconstituted with 200μL isopropanol-methanol mixture (1:1, V/V ), vortex 30s, ultrasound 3min, centrifuged for 10 min (12000rpm, 4℃), take 150μL of the supernatant and put it into an LC-MS sample vial for LC-MS analysis. QC samples were prepared by mixing equal volumes of extracts from all samples, and each QC has the same volume as the sample.
2.5.2 UPLC-MS/MS analysis
The lipidomic analyses were performed on Q Exactive Mass Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) coupled to Nexera UPLC system (Shimadzu, Kyoto, Japan). An ACQUITY UPLC BEH C18 (2.1mm×100mm, 1.7μm, Waters, Milford, MA, USA) was used for separation of lipids. The mobile phase for HPLC was composed of solvent A (10 mM ammonium formate and 0.1% formic acid in acetonitrile/water (60:40)) and solvent B (10 mM ammonium formate and 0.1% formic acid in isopropanol/ acetonitrile (90:10)). LC gradient was as follows: 0.0–3.0min for 30% B, 3.0–5.0min to 62% B, 5.0–15.0min to 82% B, 15.0–16.5min to 99% B, 16.5–18.0min to maintain 99% B. The flow rate was 0.35 mL/min, and the injected sample amount was 5μL. Heated electrospray ionization (HESI) positive and negative ion modes were used for detection. Positive: Heater Temp 300 °C, Sheath Gas Flow rate 45 arb, Aux Gas Flow Rate15 arb, Sweep Gas Flow Rate 1arb, spray voltage 3.5KV, Capillary Temp 320 °C, S-Lens RF Level 50%. MS1 scan ranges: 120-1800. Negative: Heater Temp 300°C, Sheath Gas Flow rate 45arb, Aux Gas Flow Rate 15arb, Sweep Gas Flow Rate 1arb, spray voltage 3.1KV, Capillary Temp 320 °C, S-Lens RF Level 50%. MS1 scan ranges: 120-1800.
2.6 Data processing and statistical analysis
The acquired LC-MS raw data were analyzed by the progqenesis QI software (Waters Corporation, Milford, USA). The Excel file was obtained with three dimensions datasets including gm/z, peak RT and peak intensities, and RT–m/z pairs were used as the identifier for each ion. The resulting matrix was further reduced by removing any peaks with missing value (ion intensity=0) in more than 50% samples.
Metabolites were identified by progenesis QI (Waters Corporation, Milford, USA) Data Processing Software, based on public databases such as http://www.hmdb.ca/; http://www.lipidmaps.org/ and self-built databases. The software Lipid search (Thermo Fisher Scientific Inc., Waltham, MA, USA) was employed for peak picking and alignment of lipids.
The positive and negative data were combined to get a combine data which was imported into R package. Principle component analysis (PCA) and (orthogonal) partial least-squares-discriminant analysis (OPLS-DA) were carried out to visualize the metabolic alterations among experimental groups, after mean centering and Pareto variance scaling, respectively. The Hotelling’s T2 region, shown as an ellipse in score plots of the models, defines the 95% confidence interval of the modeled variation. Variable importance in the projection (VIP) ranks the overall contribution of each variable to the OPLS-DA model, and those variables with VIP>1 are considered relevant for group discrimination.