Deficiency of leucine-rich repeat kinase 2 aggravates thioacetamide-induced acute liver failure and hepatic encephalopathy in mice.

BACKGROUND: Hepatic encephalopathy (HE) is closely associated with inflammatory responses. However, as a crucial regulator of the immune and inflammatory responses, the role of leucine-rich repeat kinase 2 (LRRK2) in the pathogenesis of HE remains unraveled. Herein, we investigated this issue in thioacetamide (TAA)-induced HE following acute liver failure (ALF). METHODS: TAA-induced HE mouse models of LRRK2 wild type (WT), LRRK2 G2019S mutation (Lrrk2G2019S) and LRRK2 knockout (Lrrk2-/-) were established. A battery of neurobehavioral experiments was conducted. The biochemical indexes and pro-inflammatory cytokines were detected. The prefrontal cortex (PFC), striatum (STR), hippocampus (HIP), and liver were examined by pathology and electron microscopy. The changes of autophagy-lysosomal pathway and activity of critical Rab GTPases were analyzed. RESULTS: The Lrrk2-/--HE model reported a significantly lower survival rate than the other two models (24% vs. 48%, respectively, p < 0.05), with no difference found between the WT-HE and Lrrk2G2019S-HE groups. Compared with the other groups, after the TAA injection, the Lrrk2-/- group displayed a significant increase in ammonium and pro-inflammatory cytokines, aggravated hepatic inflammation/necrosis, decreased autophagy, and abnormal phosphorylation of lysosomal Rab10. All three models reported microglial activation, neuronal loss, disordered vesicle transmission, and damaged myelin structure. The Lrrk2-/--HE mice presented no severer neuronal injury than the other genotypes. CONCLUSIONS: LRRK2 deficiency may exacerbate TAA-induced ALF and HE in mice, in which inflammatory response is evident in the brain and aggravated in the liver. These novel findings indicate a need of sufficient clinical awareness of the adverse effects of LRRK2 inhibitors on the liver.

Association of apolipoprotein E (APOE) polymorphisms with warfarin maintenance dose in a northern Han Chinese population.

BACKGROUND: Apolipoprotein E (apoE) induces the uptake of vitamin K-rich lipoproteins by the liver, which likely affects inter-individual variation of warfarin dosing requirements. Associations between APOE polymorphisms and warfarin dosing were previously reported inconsistently among different ethnic groups, so the present study investigated this association in northern Han Chinese patients with mechanical heart valve prosthesis. METHODS: A total of 186 patients who underwent mechanical heart valve replacement and attained a stable warfarin dose were included. APOE single nucleotide polymorphisms (SNPs) rs7412 and rs429358 were genotyped using Illumina SNP GoldenGate Assay. Genotyping results were confirmed by direct sequencing. PHASE v2.1 software was used to construct rs7412 and rs429358 haplotypes. The effects of different APOE genotypes on warfarin dose were analyzed statistically. RESULTS: The mean warfarin maintenance dose was 3.10 ± 0.96 mg/day, and the mean international normalized ratio (INR) was 2.09 ± 0.24. APOE E2, E3, and E4 allele frequencies were 11.6 %, 82.5 %, and 5.9 %, respectively. No E2/E2 or E4/E4 genotypes were detected in this population. E2/E3, E3/E3, E2/E4, and E3/E4 genotype frequencies were 21.0 %, 67.2 %, 2.2 %, and 9.7 %, respectively. Significant differences in warfarin dose requirements were observed among patients with E2/E3, E3/E3, and E3/E4 genotypes (p < 0.05). In post hoc comparison, daily warfarin maintenance doses were significantly higher in E2/E3 heterozygotes compared with E3/E3 homozygotes (p < 0.05), but no differences in dose requirements were found between E3/E4 and E3/E3, or E2/E3 and E3/E4 (p > 0.05). Patients were divided into low-intensity anticoagulant treatment group (1.6 ≤ INR <2.0) and relatively high-intensity anticoagulant treatment group (2.0 ≤ INR ≤ 2.5), and significantly higher warfarin dose requirements were observed in E2/E3 heterozygotes compared with E3/E3 homozygotes in both subgroups (p < 0.05). Multivariable analysis adjusting for other confounders showed that E2/E3 genotype was associated with a significantly higher warfarin dose compared with E3/E3 genotype (p < 0.05). CONCLUSIONS: APOE allele and genotype frequencies in the northern Han Chinese population appear to differ from other racial groups or populations living in other regions of China. The APOE E2 variant was associated with a significantly higher warfarin maintenance dose. Thus, APOE polymorphisms could be one of the predictors influencing warfarin doses in this population.

Tripchlorolide ameliorates experimental autoimmune encephalomyelitis by down-regulating ERK1/2-NF-κB and JAK/STAT signaling pathways.

Tripchlorolide (T4), an extract of the natural herb Tripterygium wilfordii Hook F, has been found to possess anti-inflammatory and immunosuppressive actions. In the current study, these actions were evaluated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis by scoring the clinical signs, observing the infiltration of inflammatory cells and myelin sheath in the lumbar spinal cord of EAE mice. The results demonstrated that T4 (at a dose of 40 µg/kg) significantly reduced the severity of EAE and slowed down the ongoing EAE. Further analysis showed that T4 suppressed the mRNA and protein levels of the transcription factors T-bet and RoRrt and mRNA levels of IFN-γ and IL-17 in the spinal cords. Furthermore, T4 down-regulated the ERK1/2-NF-κB and JAK/STAT signaling pathways. At 40 µg/kg, T4 did not induce side effects on hematological parameters. These findings suggest that T4 ameliorates EAE by immunosuppression, providing a new insight into T4 application in multiple sclerosis treatment.

Comparison of different calibration approaches for chloramphenicol quantification in chicken muscle by ultra-high pressure liquid chromatography tandem mass spectrometry.

Matrix-dependent signal suppression often occurs in quantitative analysis by ultra-high pressure liquid chromatography tandem mass spectrometry (UPLC-MS/MS). In this study, we investigated three calibration methods for compensation of signal suppression on chloramphenicol (CAP) quantification in chicken muscle. The data showed that the spiking recoveries by solvent standard calibration with a stable isotope labelled internal standard (SIL-IS) and matrix-matched standard calibration with a SIL-IS were significantly higher than by external matrix-matched standard calibration (P < 0.05). When the SIL-IS was used, standards prepared in the mobile phase solvent showed no significant difference as those prepared in the matrix (P > 0.05). The limit of detection (LOD) for external matrix matched standard calibration was 0.1 µg kg(-1), and that for SIL-IS calibration (including matrix matched and solvent dissolved standard) was 0.03 µg kg(-1).

[Establishment of a scoring system for predicting the positive rate of prostatic biopsy for prostate cancer].

OBJECTIVE: To identify the predictors of the positive results of transrectal ultrasound (TRUS)-guided biopsy for prostate cancer. METHODS: We performed univariate and multivariate logistic regression analyses on the relevant data on 385 male patients that underwent TRUS-guided biopsy for prostate cancer, including such potential predictors as age, body mass index (BMI), symptoms, results of digital rectal examination (DRE), tPSA, fPSA, free/total PSA ratio (f/tPSA), prostate volume (PV), and PSA density (PSAD) for identification of the risk factors related to the positive rate of biopsy. Then we constructed a scoring system as a tool for predicting prostate cancer in repeat biopsies and determined the sensitivity of the system by calculating the false positive rate using the receiver operating characteristic curve. RESULTS: Among the 385 patients, 139 (36.1%) were diagnosed with prostate cancer. On multivariate analysis, age (P < 0.01), DRE (P < 0.01), tPSA (P < 0.01), fPSA (P < 0.01), f/tPSA (P < 0.01), PV (P < 0.01), and PSAD (P < 0.01) were all significant predictors of prostate cancer. Multivariate logistic regression analysis showed age, tPSA, f/tPSA, PV, and PSAD to be independent predictors, with ORs and 95% CIs of 1.07 (1.05-1.16), 1.05 (1.02-1.15), 0.97 (0.86-0.99), 0.98 (0.87-0.96), and 1.79 (1.48-2.06), respectively. Moreover, patients with the risk score of 3-5 had a significantly higher rate of prostate cancer than those with 0-2 (64% vs 11%, P < 0.001). CONCLUSION: The scoring system on the key predictors of prostate cancer can help urologists to identify the men in need of prostatic biopsy.

Exposure assessment of heavy metals (Cd, Hg, and Pb) by the intake of local foods from Zhejiang, China.

Considering the environmental pollution, food safety is of great concern to the consumers. The present study was conducted to assess the health risk of cadmium (Cd), mercury (Hg), and lead (Pb) through the dietary intake in Zhejiang, China. Eight hundred and sixty two food samples including aquatic products, meat, vegetables, milk and dairy products, and cereal grains were analyzed. Only 2.44 % (Cd), 1.39 % (Hg), and 1.51 % (Pb) of the samples exceeded the maximum allowable concentration set by Chinese Ministry of Health. The average dietary intakes of Cd, Hg, and Pb were estimated to be 0.26, 0.14, and 0.55 µg/kg bw/day, respectively. Compared with the reference doses, the mean exposure of Cd, Hg, and Pb was all less than the tolerable intake value. Only at the 95th percentile level, Cd and Hg exposure exceeded the values of tolerable intakes by 40 and 277 %, respectively. It indicates that there is low health risk to the dietary exposure of Cd, Hg, and Pb for general people in Zhejiang province, China.

Antibiotic residues in commercial freshwater fish from southeast China: distribution and human health risk assessment.

We investigated 14 antibiotic residues in 8 marketed freshwater fish species from southeast China and estimated the associated health risks to local consumers. The antibiotic residues were determined by UPLC-MS/MS. Our findings revealed widespread distribution of quinolones (QNs), tetracyclines (TCs), and chloramphenicols (CAPs) in the freshwater fish. Notably, the average concentrations of enrofloxacin and ciprofloxacin reached levels as high as 62.5 µg/kg wet weight (ww) and 11.7 µg/kg ww, respectively, and detection frequencies were 68.7% for enrofloxacin and 31.6% for ciprofloxacin. Additionally, we detected chloramphenicol, a prohibited antibiotic, in samples with a detection frequency of 0.76%. Among the fish species, the mean concentration of total antibiotic residues was highest in bluntnose black bream (263.3 µg/kg), followed by English perch (52.4 µg/kg), crucian carp (46.3 µg/kg), black carp (28.6 µg/kg), yellowcheek carp (21.0 µg/kg), grass carp (15.3 µg/kg), bighead carp (3.78 µg/kg), and mandarin fish (3.69 µg/kg). We estimated the daily intake values of these antibiotic residues which were lower than the acceptable daily intake values and hazard indexes were much less than 1. It indicates that there is very low direct health risk to consumers. Despite that, investigation on the chronic impact, such as antibiotic-resistant bacteria, gut microbiota disruption, and allergic reactions, is urgently needed.

Distribution and Probabilistic Risk Assessment of Antibiotics, Illegal Drugs, and Toxic Elements in Gastropods from Southeast China.

We investigated fourteen antibiotics, three illegal drugs, and two toxic elements in commercially available gastropods from southeast China. The data revealed high detection frequencies (DFs) for florfenicol (61.32%), florfenicol amine (47.33%), and thiamphenicol (39.88%), with maximum concentrations of 1110, 2222, and 136 µg/kg wet weight (ww), respectively. The DFs of illegal drugs were 3.54% for leucomalachite green and 0.3% for chloramphenicol. The average levels of Cd and As were 1.17 and 6.12 mg/kg ww, respectively. All chemicals presented diverse DFs in different sampling months. The highest DFs of florfenicol, florfenicol amine, and thiamphenicol were in July. The health risk assessment showed that targeted hazard quotients (THQs) of antibiotics, Cd, and As for children, teens, and adults were all less than one. Notably, the toxic elements (Cd and As) were identified as the primary health risk in gastropods, contributing to over 90% of the total THQs.

Pterosin B improves cognitive dysfunction by promoting microglia M1/M2 polarization through inhibiting Klf5/Parp14 pathway.

BACKGROUND: Pterosin B (PB) exhibits strong neuroprotective effects in vitro, but its therapeutic effect and underlying mechanism on Alzheimer's disease (AD) remain elusive. PURPOSE: This study aimed to investigate the anti-AD effect and mechanism of PB. STUDY DESIGN: The therapeutic effect and mechanism of PB were investigated in APP/PS1 mice and lipopolysaccharide (LPS)-induced BV-2 cells. METHODS: After 8 weeks of oral administration of PB or donepezil, the cognitive function was assessed using behavioral tests. Pathological damage was evaluated using histological analysis and immunohistochemical staining. Flow cytometry was applied to detect M1/M2 polarization. The expression levels of glycolysis- and oxidative phosphorylation-related proteins as well as enzyme activities were determined using Western blot and biochemical kits, respectively. The levels of inflammatory cytokines and Kruppel-like factor 5 (Klf5) were measured using enzyme-linked immunosorbent assay. AD biomarkers in serum were analyzed using single-molecular array. RNA sequencing identified the downstream molecules of Klf5, and interaction was evaluated using dual-luciferase reporter assay. RESULTS: Our findings demonstrated that PB effectively ameliorated cognitive impairment and reduced pathological damage in APP/PS1 mice. Furthermore, PB facilitated the transition of the phenotype of LPS-induced BV-2 cells from M1 to M2 by modulating metabolic reprogramming. Additionally, Klf5 had high expression levels in the serum of patients with AD, which strongly correlated with cognitive performance and AD biomarkers. PB downregulated Klf5 expression both in vitro and in vivo. Subsequently, poly-ADP ribosyl polymerase 14 (Parp14) was identified as a downstream molecule of Klf5 involved in regulating metabolic reprogramming, and PB regulated microglia M1/M2 polarization by inhibiting the Klf5/Parp14 pathway. CONCLUSION: The findings suggested that PB ameliorated cognitive dysfunction in AD by modulating microglia M1/M2 polarization via inhibiting Klf5/Parp14 pathway.

[Profiles of reproduction-related mRNA expressions in hypothalamus of female mice with advancing age].

OBJECTIVE: To explore the effects of aging on the levels of reproduction-related mRNA genes including Gnrh, KISS1/KISS1r, estrogen receptor-alpha (ERα), estrogen receptor-beta (ERß) and progesterone receptor (PR) in hypothalamus. METHODS: Proestrus and metestrus in young (3-4 months) and middle-aged (10-11 months) female mice and diestrus in senile (18-19 months) female mice were observed. And the levels of related mRNA genes in preoptic area anterior hypothalamus (POA-AH) and medial basal hypothalamus (MBH) were determined by real-time polymerase chain reaction (RT-PCR). RESULTS: In middle-aged mice on proestrus, the level of Gnrh mRNA in POA-AH (0.896 ± 0.049) was significantly lower than that in young mice (1.228 ± 0.147, P = 0.049). The level of ERα mRNA in POA-AH decreased in young mice on proestrus whereas increased in middle-aged mice (0.432 ± 0.063 vs 0.603 ± 0.018, P = 0.016). The level of ERα mRNA of POA-AH, both in middle-aged mice (0.432 ± 0.063, P = 0.014) and senile mice (0.403 ± 0.145, P = 0.020) on diestrus, were significantly lower than that in young mice. The PR mRNA expression in middle-aged mice on proestrus (1.037 ± 0.037) was markedly lower than that in young mice (1.251 ± 0.081, P = 0.031) . In senile mice, the levels of Gnrh mRNA (1.520 ± 0.146, P = 0.004) and ERß mRNA (1.572 ± 0.184, P = 0.011) increased in POA-AH compared with that in young mice on metestrus. Aging had no effect upon KISS1 and KISS1r mRNA levels in POA-AH. In contrast, KISS1 mRNA level of MBH in middle-aged (1.663 ± 0.398, P = 0.037) and senile (2.622 ± 0.454, P = 0.014) mice obviously increased compared with the young mice group. CONCLUSION: Higher levels of ERα mRNA and decreases of PR and Gnrh mRNA in POA-AH in middle-aged mice on proestrus may play an important role in declining reproductive function.

[Impact and related mechanisms of stromal cell-derived factor-1α on serum deprivation-induced cardiac stem cells apoptosis].

OBJECTIVE: To explore the impact and related mechanisms of stromal cell-derived factor-1α (SDF-1α) on serum deprivation-induced apoptosis of cardiac stem cells (CSCs). METHODS: CSCs were isolated from adult mouse heart tissue and cultured in vitro. Obtained cells were purified using magnetic-activated cell sorting (MACS) with c-kit magnetic beads. C-kit(+)CSCs were divided into five groups: normal control group, serum deprivation group, serum deprivation+SDF-1α group, serum deprivation+SDF-1α+AMD3100 group, serum deprivation+SDF-1α+LY294002 group. Cell apoptosis was assessed using the DeadEnd Colorimetric TUNEL System and flow cytometry analyses with an Annexin V-FITC Apoptosis Detection Kit. The viability of CSCs was assessed by CCK-8. The protein expression of Bcl-2 and phosphorylated Akt were detected by Western blot. The caspase-3 activity was determined using caspase-3 Colorimetric Assay Kit. RESULTS: After magnetic separation, more than 85% of cardiosphere derived cells were positive for c-kit expression. Compared with the normal control group, the apoptosis rate of serum deprivation group was significantly increased[(27.03 ± 0.80)% vs. (1.51 ± 0.54)%, P < 0.01], which could be significantly reduced by SDF-1α in a concentration dependent manner and peak effect was seen with 100 ng/ml SDF-1α[(10.67 ± 1.06)% vs. (27.03 ± 0.80)%, P < 0.01]. The expressions of p-Akt and Bcl-2 were significantly increased and the activity of caspase-3 was significantly decreased in serum deprivation+SDF-1α group compared to serum deprivation group (P < 0.01). Further more, the expression of p-Akt and Bcl-2 were significantly decreased and the activity of caspase-3 was increased in both serum deprivation+SDF-1α+AMD3100 group and serum deprivation+SDF-1α+LY294002 group compared to serum deprivation+SDF-1α group (P < 0.01). CONCLUSIONS: SDF-1α reduces serum deprivation induced CSCs apoptosis via modulating PI3K/Akt signaling pathway.

Distribution of Cadmium in Fresh Vegetables Marketed in Southeast China and Its Dietary Exposure Assessment.

This study investigated concentrations of cadmium (Cd) in 2465 vegetable samples (52 species) from 2018 to 2022 and estimated the associated health risk for local consumers. The average concentration of Cd was 0.035 mg kg-1, and the percentage of samples exceeding the Chinese maximum allowed concentration was 3.89% (96/2465). The top five species with highest Cd levels were Lilium brownii F (0.182 mg kg-1), Allium chinense G (0.117 mg kg-1), Allium macrostemon Bunge (0.105 mg kg-1), Colocasia esculenta (0.064 mg kg-1), and Amaranthus tricolor L (0.054 mg kg-1). Bulb vegetables had a higher relative accumulation of Cd compared to other vegetables. The levels of Cd in vegetables varied significantly across sampling areas and years. The mean estimated daily intake (EDI) of cadmium through consumption of vegetables was 0.519 µg kg-1 bw per day for adults and 0.217 µg kg-1 bw per day for children. The target hazard quotients (THQs) were all less than the threshold of 1 for both adults and children. This indicates that there is low health risk for Cd through vegetable consumption. However, routine monitoring of Cd levels in food is still crucial to ensure food safety and protect public health.

Heavy metals accumulation in bivalve mollusks collected from coastal areas of southeast China.

The distribution of six heavy metal and metalloids (As, Cd, Cr, Hg, Ni and Pb) was analyzed in 597 bivalve mollusks (8 species) collected from coastal areas of southeast China. Target hazard quotient, total hazard index, and target cancer risk were calculated to evaluate potential human health risks from bivalve consumption. The mean concentrations of As, Cd, Cr, Hg, Ni and Pb were 1.83, 0.581, 0.111, 0.0117, 0.268 and 0.137 mg kg-1 wet weight in bivalves. The average estimated daily intakes for As, Cd, Cr, Hg, Ni and Pb were 1.156, 0.367, 0.07, 0.007, 0.167 and 0.087 µg kg-1 body weight/day. Health risk assessment showed that there was no non-carcinogenic health risk to general residents to these metals from consumption of bivalves. Cd intake through mollusks posed a potential cancer risk. Accordingly, regular monitoring for heavy metals, especially Cd is recommended with respect to potential contaminant on marine ecosystems.

Quantification of 37 glucocorticoids in chicken muscle by UHPLC-Q-Orbitrap-MS with parallel reaction monitoring.

The quantification capability of high-resolution mass spectrometry (HRMS) has received increasing interest from analysts. In this study, we present a method for analyzing 37 glucocorticoids in chicken muscle using UHPLC-Q-Orbitrap MS with parallel reaction monitoring (PRM). The analytes were extracted using acetonitrile (ACN) containing 0.1% formic acid and subjected to commercial PRiME HLB solid-phase extraction (SPE) cartridge clean-up. Under optimized conditions, the analytes were separated on an analytical column and subsequently detected using a high-resolution hybrid quadrupole/Orbitrap mass spectrometer coupled with PRM scan mode. The Q-Orbitrap with PRM exhibited remarkable sensitivity, with limits of quantification (LOQs) ranging from 0.08 µg kg-1 to 7.59 µg kg-1. To validate the method, we conducted intra- and inter-day tests using a blank matrix sample at different spiking levels. The achieved results demonstrated satisfactory recovery values (74.1-97.5%) and precise results (RSDs < 15%) for all the studied analytes. In application, we found dexamethasone with 6.5 µg kg-1 and fluorometholone with 3.9 µg kg-1 in two chicken samples. These findings suggest that the UHPLC-Q-Orbitrap system, in conjunction with the SPE sample preparation method, has great potential as a routine quantification approach for multiple glucocorticoid residues in chicken samples.

Toxic Elements in Beans from Zhejiang, Southeast China: Distribution and Probabilistic Health Risk Assessment.

This study described the distribution of As, Cd, Cr, Hg, and Pb in 692 bean samples from Zhejiang province, southeast China, and estimated the health risk using Monte Carlo simulation. The average levels of As, Cd, Cr, Hg, and Pb were 0.0349, 0.0379, 0.246, 0.0019, and 0.0246 mg kg-1. Correlation analyses showed very strong positive correlations for Cd-Pb in kidney beans and mung beans, Cd-As in black beans, and Pb-As in red beans. The target hazard quotients (THQs) were adopted for non-carcinogenic risk assessment, and THQs at the 50th percentile were all less than 1, indicating that there are no deleterious effects from rice exposure to these elements. When evaluating THQ for multiple elements, the certainty with a hazard index (HI) greater than 1 for children was 12.64%, for teens 11.54%, and for adults 1.01%. The sensitivity analysis reveals that the concentration of Cd in beans and ED (exposure duration) are the main principal factors that contributed to the total risk. The mean carcinogenic risks for children, teens, and adults were all less than 1 × 10-4, indicating no potential carcinogenic risk. Despite that, the routine monitoring of these elements, especially for Cd should be continued.

Dendrobine rescues cognitive dysfunction in diabetic encephalopathy by inhibiting ferroptosis via activating Nrf2/GPX4 axis.

BACKGROUND: Ferroptosis playsa crucial role in the development of dementia and dendrobine (Den)possesseshypoglycemic and neuroprotective effects. However, the character of ferroptosis in diabetic encephalopathy (DE) and Den's therapeutic effect remains unclear. PURPOSE: This study aimed to verify the effects of Den on ferroptosis in treating DE and underlying mechanisms. STUDY DESIGN: Den's therapeutic effect was assessed in db/db mice and advanced glycation end products (AGEs)-induced HT22 cells. METHODS: After oral administration with Den orMetformin for 8-week, behavioral tests were used to assess cognitive capacity. Then, biochemical analysis was preformed to detect glucose and lipid metabolism levels; histological analysis and transmission electron microscope were applied to evaluate pathological injuries. Meanwhile, EdU staining and flow cytometry were applied to test cell apoptosis. Furthermore, mitochondrial dynamics, iron transport, and Nrf2/GPX4 axis related proteins were detected by western blot or immunofluorescence. RESULTS: Our results demonstrated that Den remarkably alleviated glucose and lipid metabolism disorders, as well as ameliorated mnemonic deficits of db/db mice. Meanwhile, Den could protect AGEs-induced HT22 cells from death and apoptosis. In addition, we noted that Den inhibited lipid peroxidation by restoring mitochondrial function and reducing reactive oxygen species production. Furthermore, ferroptosis was proven to exist in db/db mice brain and Den could inhibit it via activating Nrf2/GPX4 axis. CONCLUSION: These findings indicated that Den could rescue cognitive dysfunction in DE by inhibiting ferroptosis via activating Nrf2/GPX4 axis.

Analytical Method Optimization of Tetrodotoxin and Its Contamination in Gastropods.

Tetrodotoxin (TTX) is an extremely potent marine biotoxin. An analytical method was developed for both trace contamination and extremely high levels of TTX in gastropods by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with clean-up of cation exchange solid phase extraction (SPE) in this study. The limit of detection (LOD) in the sample matrix was 0.5 µg/kg. With the calibration of a screened internal standard (validamycin, IS), the linear range was 0.1-100 ng/mL (1.5-1500 µg/kg in sample matrix) with a correlation coefficient of r2 > 0.999. The average recoveries at three spiking levels (1.5 µg/kg, 44 µg/kg, and 1500 µg/kg) were 82.6-94.4% with relative standard deviations (RSDs) less than 8.4%. TTX levels in seven gastropods (741 samples) were studied. The contamination and analogues in Neverita didyma (N. didyma, 565 samples collected in Zhejiang province, China, from 2016 to 2022) were first reported. The detection rate of TTX in N. didyma was 34.2%. The average concentration was 23.1 µg/kg, and the maximum value was 2327 µg/kg. The time distribution study indicated that high contaminations of TTX occurred from May to August for N. didyma.

Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels in cultured rat hippocampal neurons.

AIM: To investigate the effect of ginsenoside Rb1 on voltage-gated calcium currents in cultured rat hippocampal neurons and the modulatory mechanism. METHODS: Cultured hippocampal neurons were prepared from Sprague Dawley rat embryos. Whole-cell configuration of the patch-clamp technique was used to record the voltage-gated calcium currents (VGCCs) from the hippocampal neurons,and the effect of Rb1 was examined. RESULTS: Rb1 (2-100 µmol/L) inhibited VGCCs in a concentration-dependent manner, and the current was mostly recovered upon wash-out. The specific L-type Ca(2+) channel inhibitor nifedipine (10 µmol/L) occluded Rb1-induced inhibition on VGCCs. Neither the selective N-type Ca(2+) channel blocker ω-conotoxin-GVIA (1 µmol/L), nor the selective P/Q-type Ca(2+) channel blocker ω-agatoxin IVA (30 nmol/L) diminished Rb1-sensitive VGCCs. Rb1 induced a leftward shift of the steady-state inactivation curve of I(Ca) to a negative potential without affecting its activation kinetics or reversal potential in the I-V curve. The inhibitory effect of Rb1 was neither abolished by the adenylyl cyclase activator forskolin (10 µmol/L), nor by the PKA inhibitor H-89 (10 µmol/L). CONCLUSION: Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels, without affecting the N-type or P/Q-type Ca(2+) channels in hippocampal neurons. cAMP-PKA signaling pathway is not involved in this effect.

[The determination of phthalic acid esters in edible vegetable oils by gas chromatography-mass spectrometry].

OBJECTIVE: We aimed to establish a quantified method for the 17 phthalate acid esters (PAE) in edible vegetable oil by gas chromatography-mass spectrometry (GC-MS) with the pretreatment of acetonitrile extraction and silica/N-(n-propyl)ethylenediamine (silica/PSA) mixed solid phase extraction column and evaluated the PAE of 25 edible oil samples from supermarkets in Hangzhou city. METHODS: The internal standard solution (D4-DEHP) was added in edible vegetable oil sample. The analytes were extracted by acetonitrile with 1 min vortex, and centrifuged at 3050×g for 5 min. The supernatant was then cleaned with silica/PSA column, and eluted with acetonitrile. The elution was dried with N2 flow at 50°C and diluted to 1.0 ml with hexane. Then, 17 PAE were tested by GC-MS and quantified with internal standards. The repeatability and sensitivity of the assay were evaluated. PAE were then determined in 25 plastic buckets of edible vegetable oil from supermarkets in Hangzhou city. RESULTS: By the quantification of internal standard of D4-DEHP, a good linearity range of related 17 PAE was observed. The correlation coefficient was 0.994-1.000 and the standard lowest quantified level was 0.05-0.15 µg/ml. The spiking recoveries of 17 PAE were 78.3%-108.9% with the RSD of 4.3%-12.1% (n=6). The method detection limits were 0.1-0.2 mg/kg. In 25 plastic buckets of edible vegetable oil from Hangzhou, DMP, DEP, DIBP, DBP and DEHP were detected at the range of <0.1-1.8 mg/kg and the detection rates were 12% (3/25), 24% (6/25), 100% (25/25), 96% (24/25) and 100% (25/25), respectively. Other 12 PAE was not detected. For DBP with the level of <0.1 to 1.3 mg/kg, the results of 16% (4/25) samples exceeded the regular migrating limit of 0.3 mg/kg. For DEHP of <0.2-1.8 mg/kg, the data of 12% (3/25) samples were beyond the regular migrating limit of 1.5 mg/kg. CONCLUSION: The pretreatment by silica/PSA mixed solid phase extraction column can satisfy the PAE determination requirements in edible vegetable oils. The DMP, DIBP, DEP, DBP and DEHP were detected from the survey of 25 edible oil samples in Hangzhou city.

Determination of steroid hormone residues in farmed fish using high-resolution orbital ion trap mass spectrometry.

A method for the determination of 15 steroid hormones in farmed fish by liquid chromatography-orbital ion trap mass spectrometry has been developed and validated. The method involved sample preparation with acetonitrile extraction and clean-up, and separation in the LC using a C18 column. The orbital ion trap MS was operated at a resolution of 35 000 FWHM in selected ion monitoring mode. An ion source with heated electrospray ionization was used in positive ionization mode. The samples were prepared by solid-phase extraction. The limit of quantification of steroid hormones in fish samples was 2 µg kg-1. Good linearity was observed since correlation coefficients were more than 0.99 for all compounds. Recoveries of spiked fish samples (2 µg kg-1 and 20 µg kg-1) ranged from 80.8% to 112.6% with relative deviations less than 15%. The method was successfully applied to detect steroid hormones in real farmed fish samples at the µg kg-1 level.