Reagents and Materials
Harmine, harmane, and harmaline (purity > 98%) were isolated by HPLC from seeds of P. harmala in our laboratory. Nor-harmane was purchased from Sigma Aldrich Co. (St. Louis, MO, United States). Scopolamine hydrobromide was purchased from TCI (Shanghai) Development, Co., Ltd. (Shanghai, China). L-tryptophan (L-Trp), 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), acetylcholine chloride (ACh), choline chloride (Ch), L-glutamic acid monosodium salt monohydrate (L-Glu), L-phenylalanine (L-Phe), L-tyrosine(L-Tyr), theophylline, tacrine (internal standard), and heparin sodium were purchased from Sigma Aldrich Co. (St. Louis, MO, United States). Perchloric acid and sodium hydroxide were purchased from Meilunbio® Biotech, Co. Ltd. (Dalian, China). Bovine serum albumin was purchased from YEASEN Biotechnology, Co. Ltd. (Shanghai, China). Acetonitrile, methanol, and formic acid of HPLC grade were purchased from Fisher Scientific, Co. (Santa Clara, CA, United States). Deionized water (> 18 mΩ) was purified by Milli-Q Academic System (Millipore, Corp, Billerica, MA, United States).
Animals
Twenty adult and 11 pregnant Sprague-Dawley rats and 10 C75BL/6 mice were obtained from the Drug Safety Evaluation and Research Center of Shanghai University of Traditional Chinese Medicine. Ten APP/PS1 double transgenic mice in C57BL/6 background aged 5–6 months with their age-matched littermates were obtained from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). Animals were introduced to the experimental holding rooms at least two days prior to commencement of the study. Animals were housed in a well-lighted air-conditioned room under standard environmental conditions (room temperature and relative humidity were kept at 25°C ± 1 ℃ and 60–65%, respectively) and given free access to rodent chow and tap water prior to the study. All animal-use procedures were in accordance with the regulations for animal experimentation issued by the State Committee of Science and Technology of the People’s Republic of China on 14 November 1988 and the protocol was approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (NO. PZSHUTCM190912018; Approval date: 12 September, 2019).
Voluntary subjects
Participants were recruited from two cohorts: Shanghai University of Traditional Chinese Medicine (i.e., the SH cohort) and Kashi Prefecture First People’s Hospital, Xinjiang China (i.e., the XJ cohort). All of participants were free from any neurodegeneration. A total of 131 participants were recruited from the SH cohort. Most of them were students aged from 24 to 30, and 15 volunteers were staffs aged from 31 to 55. The other 419 participants were enrolled from the XJ cohort. All of them went to Kashi Prefecture First People’s Hospital for physical examination. The 419 healthy volunteers were in the age range of 19 to 81. All subjects were given informed consent and asked no intervention before and throughout the study period. Exclusion criteria were not available. The study was approved by the Research Ethics Committee of Kashi Prefecture First People’s Hospital, and all participants provided informed consent (NO.2017-03; Approval date: February 28, 2017).
Analysis of target βCBs in plasma and tissues of mammals by UPLC-ESI-MS/MS
The concentrations of alkaloids were quantified with SHIMADZU LC-30AD UPLC system (Shimadzu, Kyoto, Japan) connected to an AB Sciex QTRAP® 6500 triple quadrupole mass spectrometer (SCIEX, United Sates) equipped with an ESI source using positive ion detection mode for multiple reaction monitoring. According to a previously validated method, chromatographic separation was conducted using a UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 µm, Waters, USA) [41]. Given the difference of linearity range, the sample pretreatment was slightly adjusted, and the methods and results are shown in supplementary materials Fig. S1 and Table S1–S3. The mobile phase consisted of an aqueous solution of 0.1% formic acid (solvent A) and an acetonitrile (solvent B) with a flow rate of 0.4 mL/min. The gradient elution was established as follows: 0–2.5 min, 9–13% B; 2.51–3 min, 14–14.5% B; 3–4 min, 14.5–15.5% B; 4–5 min, 15.5–90% B; 5–6 min, 90–90% B; 6–7 min, 90–9% B; 7–8 min, 9% B. All other instrumental parameters were set according to previous studies, and the method was well validated and successfully applied to determine the concentrations of alkaloids [41].
Analysis of neurotransmitters in plasma of mammals by UPLC-ESI-MS/MS
The concentrations of neurotransmitters were determined with SHIMADZU LC-30AD UPLC system (Shimadzu, Kyoto, Japan) connected to an AB Sciex QTRAP® 6500 triple quadrupole mass spectrometer (SCIEX, US) equipped with an ESI source. Chromatographic separation was conducted using a ZIC-cHILIC column (150 mm × 2.1 mm, 3 µm) with a SeQuant ZIC-cHILIC guard column (20 mm × 2.1 mm, 5 µm, Merck-Sequant, Germany). The mobile phase was an acetonitrile (A)–water mixture containing 0.1% of formic acid (B), and the gradient elution was established as follows: 0–8 min, 65% A. All other instrumental parameters and sample pretreatment were set according to previous studies, and the method was well validated and successfully applied to determine the concentrations of neurotransmitters [42].
Exposure levels of target alkaloids in the developmental phase of newborn rats within 29 days
Eleven Sprague-Dawley pregnant rats were used in this study. The pregnant rats were raised under environmentally controlled room and with free access to food and water all throughout the experiment. After parturition, pup rats were grouped into 11 on the basis of mother rats. The individual group indicated one sampling point and contained 10 pup rats obtained from 10 different mother rats (n = 10). The pup rats were sacrificed at postnatal days 1 (P1), 3 (P3), 5 (P5), 7 (P7), 9 (P9), 12 (P12), 15 (P15), 18 (P18), 21 (P21), 25 (P25), and 29 (P29) for sample collection, in which 11 sampling points were collected. The specific time is shown in Fig. 1A. Each pregnant rat could give birth to around ten pup rats, and every pup rat was sacrificed at certain time to ensure each point in time contained 10 samples (half female and half male). The sex of pup rats was determined by anogenital distance. Pup rats were anesthetized at specified time for sample collection, and the grouping and sampling time is shown in supplementary materials Table S4. Prior to blood sampling, the pups were anesthetized to unconsciousness with 4% isoflurane via isoflurane vaporizers. After blood collection, pup rats were sacrificed by inhaling excessive isoflurane, and the tissues were collected. Approximately 0.5 mL of blood sample was collected from the angular vein of each rat and transferred into a 1.5 mL heparinized tube. The supernatant plasma (100 µL) was transferred into another 1.5 mL centrifuge tube after centrifugation of blood at 3000 × g at 4 ℃ for 10 min. The tissues of the brain, heart, liver, spleen, lung, kidney, genital organ, muscle, white fat, and brown fat of each pup rat were also collected. Various tissues were weighted and stored at a suitable tube. All plasma and tissue samples were stored at − 80 ℃ until analysis.
Exposure levels of alkaloids in fodder and bedding
The concentrations of harmine, harmaline, harmane, and nor-harmane in the fodder and bedding were detected. The fodder and bedding were obtained from cages of above 11 pregnant rats and their pup rats. Detection method was referred to quality specification established by Yang et al. [43]. The fodder and bedding were accurately powdered and weighted. The powders were added to methanol, which was 25 times its volume, and ultrasound treated for 25 min. In addition, power and frequency were kept at 250 W and 30 kHz, respectively. Afterward, supernatant (5 mL) was transferred to another tube and evaporated to dry at 37 ℃ under a slight stream of nitrogen. The dried residue was reconstituted with 100 µL of 9% acetonitrile and vortexed for 2 min. After centrifugation at 13000 × g at 4 ℃ for 10 min, 20 µL of supernatant was injected into the UPLC-ESI-MS/MS system for alkaloid analysis.
Exposure levels of alkaloids and neurotransmitters in the growing process of 18-month-old rats (from youth to old stage)
Twenty Sprague-Dawley rats (200–220 g) with 10 males and 10 females were used in this study. The rats were raised under environmentally controlled room and with free access to food and water all throughout the experiment. The experimentation lasted for 16 months. The blood samples were collected once a month, and blood was drawn at around 9:00 am to 11:00 am. The schematic diagram is shown in Fig. 1B. Blood samples were collected from the angular vein for hematological analyses every month after rats were anesthetized to unconsciousness with 4% isoflurane via isoflurane vaporizers. The blood samples were promptly centrifuged at 3000 × g at 4 ℃ for 10 min, and all of the supernatant plasma was transferred into a new 1.5 mL centrifuge tube. Plasmas were stored at − 80 ℃ until analysis. After sample collection, the plasmas were used for the analyses of some alkaloids and neurotransmitters after pretreatment.
Alkaloid exposure levels in different physiological states of mice
Two AD mouse models were used, including 10 male APP/PS1 double transgenic mice and 10 male scopolamine molding mice. All mice were housed under environmentally controlled room and with free access to food and water before experiment. The APP/PS1 double transgenic mice were proven to have impaired spatial learning and memory compared with C57BL/6 through the Morris Water Maze test [44]. Mice were anesthetized to unconsciousness with 4% isoflurane, and the blood were collected from the right orbital vein [44]. The blood samples were centrifuged at 3000 × g at 4 ℃ for 10 min. Then, the supernatant plasmas were collected and stored at − 80 ℃ until analysis. Another AD mouse model was male C57BL/6 mice molded through intraperitoneal injection of scopolamine (1 mg/kg) for 7 days [13]. Compared with C57BL/6, the scopolamine-molded mice were proven to have impaired memory through the Morris Water Maze test [13]. After molding, the model mice and control were anesthetized with isoflurane, and the blood was collected from the right orbital vein. The blood samples were centrifuged at 3000 × g at 4 ℃ for 10 min. Then, the supernatant plasmas were collected and stored at − 80 ℃ until analysis.
Exposure levels of alkaloids in different developmental and health conditions in human
All 550 individuals were given written informed consents before drawing blood. In addition, the participants and their relatives were informed that anonymized data might be used in clinical research studies. Moreover, health examination and questionnaires were necessary. The Guardian was requested to do the questionnaires if participant could not answer. The survey involved various aspects, including name, age, nationality, gender, living habits, and contact information. Living habits included drinking and smoking. Detail information is shown in Table 1. Health examination about physiological states included neurodegeneration and any other disease. This experiment aimed to detect harmine, harmaline, harmane, and nor-harmane exposure levels in different developmental and physiological states. Furthermore, the difference of alkaloid exposure levels by gender, race, and lifestyle was detected. Blood sampling and numbering were directed by nurses in Kashi Prefecture First People’s Hospital and Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine. Serial numbers corresponded to detail information about every participant. The numbers were randomly allocated by sequence of arrival at hospital. Analysis about alkaloids in plasma was developed without further information except for serial numbers. Approximately 1 mL of venous blood was collected from participants who have fasted for at least 12 h. The blood samples were centrifuged at 3000 × g at 4 ℃ for 10 min to obtain plasma. Then, plasma samples were stored at − 80 ℃ until analysis.
Table 1
Characteristics of healthy participants and AD patients
Characteristics of subjects
|
SH cohort
|
XJ cohort
|
Number
|
131
|
419
|
Gender (Women/men)
|
79/52
|
101/318
|
Age (mean ± SD)
|
28 ± 4
|
41 ± 12
|
Race (Han/Uyghur)
|
124/7
|
274/145
|
Drinking (Yes/No)
|
13/118
|
199/220
|
Smoking (Yes/No)
|
9/122
|
162/257
|
-: no relevant statistics. |
Statistical analysis
Statistical evaluation was performed with SPPS version 18.0 software, and the data were presented as mean ± SD. Unadjusted P values were reported as this study is an exploratory rather than a confirmatory analysis. This study aimed to confirm whether harmine, harmaline, harmane, and nor-harmane were endogenous and compare their exposure levels in different gender, nationality, and lifestyle. Thus, four separate stepwise logistic regression analyses were conducted, that is, one analysis each for harmine, harmaline, harmane, and nor-harmane as the dependent variable. Data distribution was evaluated graphically using histograms and Q–Q plots. Nonparametric and parametric tests were used in the study. T test was used to compare the concentrations of alkaloids among the different groups. A nonparametric test was applied as part of statistical analyses for non-normal distribution. Participants were divided into two groups, and 60 years old was selected as the division point. Kruskal–Wallis H nonparametric test was used to compare the two groups. The threshold for statistical significance was set at P < 0.05 (2-tailed).