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Methods in Molecular Biology (Clifton,... 2024We hereby present a fast, high-throughput, and clinical LC-MS/MS assay for the simultaneous analysis of barbiturates in human urine. It is deployed as a quantitative...
We hereby present a fast, high-throughput, and clinical LC-MS/MS assay for the simultaneous analysis of barbiturates in human urine. It is deployed as a quantitative assay for phenobarbital, butalbital, pentobarbital/amobarbital, and secobarbital, as well as for confirmations following positive immunoassay drug screens in patient urine. Briefly, urine specimens are processed via dilute and shoot, i.e., by mixing the sample with 20 times volume of internal standard reagent and injecting 50 μL of that mixture into the analytical instrument. Chromatographic separation is performed using a reversed-phase C18 column in a mobile-phase system doped with <1 mM ammonium fluoride. Mass spectrometric detection occurs via negative-mode electrospray ionization multiple reaction monitoring in the TSQ Quantiva triple-quadrupole instrument. All the analytes in the mixture are detected and quantified simultaneously with respect to internal calibration in the range 20-2500 ng/mL. However, the assay cannot distinguish pentobarbital from amobarbital, which are isobaric analytes. Nonetheless, the assay is sensitive, robust, and amenable to harmonization with other assays that employ barbiturate cutoffs in the range of 20-150 ng/mL.
Topics: Humans; Pentobarbital; Chromatography, Liquid; Amobarbital; Tandem Mass Spectrometry; Barbiturates; Reproducibility of Results; Chromatography, High Pressure Liquid
PubMed: 38036813
DOI: 10.1007/978-1-0716-3541-4_9 -
Methods in Molecular Biology (Clifton,... 2024In the method described here, an aliquot of a urine sample is analyzed to detect barbiturates through dilution and ultra-high-performance chromatography-tandem mass...
In the method described here, an aliquot of a urine sample is analyzed to detect barbiturates through dilution and ultra-high-performance chromatography-tandem mass spectrometry (UPLC-MS/MS) using deuterated internal standards. This assay detects the presence of nine barbiturate drugs-amobarbital, barbital, butalbital, butabarbital, mephobarbital, secobarbital, pentobarbital, phenobarbital, and thiopental. This protocol describes two LC separation methods-first LC method (2.2 min/sample) is intended to be used as a first step of the analysis that does not separate amobarbital and pentobarbital, and a second, longer (2.7 min/sample) LC method is intended to be used only for samples which have a peak in the amobarbital/pentobarbital retention time on the shorter LC method. Since the frequency at which amobarbital and pentobarbital are observed in clinical populations is low, the shorter LC method helps gain efficiency in a high-volume laboratory environment. Additional features of this protocol that help in efficiency gain are automated extraction using Hamilton™ liquid handling system and algorithmic data review using Ascent™ software.
Topics: Pentobarbital; Amobarbital; Chromatography, Liquid; Tandem Mass Spectrometry; Barbiturates
PubMed: 38036812
DOI: 10.1007/978-1-0716-3541-4_8 -
Methods in Molecular Biology (Clifton,... 2024Antiepileptic drugs (AEDs) are a chemically diverse group of medications that are used to control seizures and different clinical forms of epilepsy. AEDs can be used as...
Antiepileptic drugs (AEDs) are a chemically diverse group of medications that are used to control seizures and different clinical forms of epilepsy. AEDs can be used as single agents but are commonly administered in combination, as a multi-drug regimen. AEDs have narrow therapeutic windows. Therapeutic ranges may not be properly defined, and symptoms of toxic serum concentrations may include increased frequency of seizures, as seen when AED concentrations are subtherapeutic. Pentobarbital, a barbiturate, is a potent anti-seizure medication, but it is also used in the treatment of head injury. Therapeutic drug monitoring (TDM) is required for optimal treatment of epilepsy. The method presented here is designed to measure serum concentrations of six commonly administered antiepileptic drugs (levetiracetam (Keppra), lamotrigine, lacosamide, 10-hydroxycarbazepine (oxcarbazepine metabolite), topiramate, zonisamide) and that of pentobarbital by LC-MS/MS. Liquid-liquid sample extraction is followed by reversed-phase chromatography using biphenyl HPLC column and gradient elution. Two MRM transitions are monitored for each drug, and their heavy isotope labeled internal standards. Six-point calibration curve is generated with each batch of analysis for quantitation of AEDs. The method's AMR covers the clinically relevant concentration range for each AED. The method has <10% CV throughout the AMR, is free of matrix effect commonly found in clinical samples, and is free from cross reactivity by other AEDs.
Topics: Humans; Anticonvulsants; Pentobarbital; Chromatography, Liquid; Tandem Mass Spectrometry; Levetiracetam; Epilepsy; Drug Monitoring
PubMed: 38036809
DOI: 10.1007/978-1-0716-3541-4_5 -
Heliyon Nov 2023High fructose diet has been linked with impaired body metabolism and cardiovascular diseases. Sodium butyrate (NaB) was documented to improve glucoregulation and...
BACKGROUND
High fructose diet has been linked with impaired body metabolism and cardiovascular diseases. Sodium butyrate (NaB) was documented to improve glucoregulation and cardiometabolic problems associated with high fructose diet (HFrD) but the mechanisms behind it are unclear. As a result, the purpose of this study was to look into the effects of NaB on VEGF and cardiac lactate in HFrD-induced dysmetabolism.
METHODS
Twenty male Wistar rats of weight 130-140 g were assigned randomly after a week of acclimation into four groups: Control diet (CTR), High fructose drink (HFrD); 10 % (w/v), NaB (200 mg/kg bw), and HFrD + NaB (200 mg/kg bw). The animals were induced to be unconscious with 50 mg/kg of pentobarbital sodium intraperitoneally, blood samples were taken via cardiac puncture and cardiac tissue homogenates were obtained for Fasting Blood Sugar (FBS) and plasma insulin, cardiac glycogen, plasma and cardiac glycogen synthase, plasma and cardiac nitric oxide as well as vascular endothelial growth factor (VEGF).
RESULT
HFrD resulted in statistical elevation body and cardiac weight, plasma glucose, plasma insulin, cardiac lactate, glycogen and decreased nitric oxide level (NO) when compared with the control group. Administration of NaB reduced cardiac weight, blood glucose, plasma insulin, cardiac lactate while nitric oxide and glycogen increased (P < 0.05). NaB increased plasma glycogen synthase in normal rats, plasma and cardiac circulating VEGF in HFrD administered rats (P < 0.05) while no change was produced in plasma and cardiac glycogen synthase level of HFrD treated rats.
CONCLUSION
Sodium butyrate improves glucoregulation by reducing cardiac lactate and increasing circulating VEGF in HFrD-treated rats.
PubMed: 38034766
DOI: 10.1016/j.heliyon.2023.e22008 -
Studies in Health Technology and... Nov 2023Insomnia is one of the most common sleep-related diseases. In traditional Chinese medicine, Flos daturae has been used as a traditional herbal totreatment of sizens of...
Insomnia is one of the most common sleep-related diseases. In traditional Chinese medicine, Flos daturae has been used as a traditional herbal totreatment of sizens of diseases. The research objective was to investigate the sedative and hypnotic effects of Flos Daturae. Kunming mice were divided into control group, Estazolam (positive drug, 0.0005 g/kg) group and Flos Daturae groups (0.01, 0.02, 0.04g/kg) with random, ig once a day for 7 days. The central sedative effect of flos Daturae on the spontaneous activity of mice was observed using the locomotive activity test, and the hypnotic effect of Flos Daturae was observed in mice using the direct sleep test and the sleep latency with synergistic supra-and sub-threshold doses of pentobarbital sodium. Flos Daturae (0.04g/kg) significantly inhibited mice locomotive activity (P<0.05) and had no direct sleeping effect (P>0.05), increased the number rate of sleep (P<0.05), and significantly shortening sleep latency (P<0.05), enhanced pentobarbital sodium-induced sleep. Flos Daturae possesses have sedative-hypnotic properties.
Topics: Mice; Animals; Hypnotics and Sedatives; Pentobarbital; Sleep Initiation and Maintenance Disorders; Sleep
PubMed: 38007723
DOI: 10.3233/SHTI230822 -
Current Molecular Pharmacology 2024With the increasing risk of infections and other serious complications, the underlying molecular mechanism of wound healing impairment in diabetes deserves attention....
BACKGROUND
With the increasing risk of infections and other serious complications, the underlying molecular mechanism of wound healing impairment in diabetes deserves attention. Cold shock proteins (CSPs), including CIRP and RBM3 are highly expressed in the skin; however, it is unknown whether CSPs are involved in the wound-healing impairment of diabetic skin.
OBJECTIVES
The objective of this study is to investigate the effects of RBM3 on skin wound healing in diabetes.
METHODS
In vitro experiments, western blot assay was used to test the levels of proteins in HaCaT cells treated with different concentrations of glucose. RBM3 was over-expressed in HaCaT cells using lentivirus particles. Cell viability was analyzed by Cell-Counting Kit-8 assay and colony formation assay. The migration of HaCaT cells at different concentrations of glucose was evaluated by wound healing assay. In vivo experiments, the mouse model of diabetes was established by intraperitoneal injection of streptozotocin. Four weeks later, the mice were anesthetized by intraperitoneal injection of pentobarbital sodium for skin tissue collection or wound healing experiments. RBM3 knockout mice were established by removing exons 2-6 using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technique and then used in skin wound healing experiments with or without diabetic stress.
RESULTS
In this study, the expression of RBM3, rather than CIRP, was altered in the skin of diabetic specimens, and the RBM3's overexpression accelerated the cell viability and proliferation of HaCaT cells under high glucose conditions. RBM3 deficiency caused delayed wound healing in RBM3 knockout in diabetic conditions. Moreover. RBM3 enhanced the ERK1/2 signaling pathway, and its inhibitor FR180204 blocked the beneficial effect of RBM3 overexpression on skin wound healing in diabetes.
CONCLUSION
RBM3 activated the ERK1/2 signal to facilitate skin wound healing in diabetes, offering a novel therapeutic target for its treatment.
Topics: Animals; Mice; MAP Kinase Signaling System; Wound Healing; Signal Transduction; Diabetes Mellitus; Glucose
PubMed: 37982286
DOI: 10.2174/0118761429260980231005105929 -
Clinical Hemorheology and... 2024Pentobarbital and isoflurane are commonly used veterinary anesthetics. Due to the dangers of overdose by repeat-bolus regimen of pentobarbital, isoflurane has been...
BACKGROUND
Pentobarbital and isoflurane are commonly used veterinary anesthetics. Due to the dangers of overdose by repeat-bolus regimen of pentobarbital, isoflurane has been recommended. However, literature suggests isoflurane-induced inhibition of cytokine and adhesion molecule release, impacting leukocyte adhesion.
OBJECTIVE
This study aims to characterize the impacts of pentobarbital versus isoflurane on leukocyte interactions within the intestinal microcirculation with and without endotoxin challenge.
METHODS
Female BALB/c mice were subjected to pentobarbital or isoflurane (N = 20) and challenged with endotoxin or saline by intraperitoneal injection. The mice were kept under anesthesia for 2 hours. Fluorochromes, rhodamine-6 G and fluorescein isothiocyanate, were injected intravenously. To visualize leukocyte adhesion within the intestinal microcirculation, laparotomy and intravital microscopy was performed. Leukocyte rolling and adhesion was quantified offline in a blinded fashion.
RESULTS
Within collecting venules, leukocyte rolling and adhesion showed no significant differences between pentobarbital and isoflurane anesthesia under basal conditions. Endotoxin challenge caused a similar response in both anesthetic groups. Within postcapillary venules, no statistical differences between the two anesthetics were found for adhering leukocytes under basal conditions or following endotoxin challenge either. However, leukocyte rolling after LPS-challenge was significantly decreased in postcapillary venules during isoflurane anesthesia compared to pentobarbital anesthesia.
CONCLUSIONS
Isoflurane anesthesia showed only minor differences in the immune response to endotoxin within the intestinal microcirculation compared to pentobarbital anesthesia. Due to the superior safety profile of volatile anesthetics, immunological studies may choose isoflurane over pentobarbital as the veterinary anesthetic of choice.
Topics: Animals; Isoflurane; Mice; Microcirculation; Female; Endotoxins; Pentobarbital; Mice, Inbred BALB C; Intestines; Leukocytes; Anesthetics, Inhalation; Leukocyte Rolling; Cell Adhesion; Anesthesia
PubMed: 37980655
DOI: 10.3233/CH-231989 -
Journal of Analytical Toxicology Jan 2024The barbiturate drug pentobarbital is commonly used by veterinarians for the euthanasia of domestic animals. During the veterinary forensic autopsy, it is sometimes...
The barbiturate drug pentobarbital is commonly used by veterinarians for the euthanasia of domestic animals. During the veterinary forensic autopsy, it is sometimes necessary to determine whether the animal was chemically euthanized with pentobarbital. The use of a human immunochromatographic test for barbiturate screening utilizing dog or cat urine has been previously validated; however, the use of alternative matrices for this purpose is yet to be explored when urine is not available. Postmortem heart, liver, spleen, skeletal muscle, blood and/or urine samples from 20 dogs and 26 cats with a reported chemical euthanasia status were processed using two different methods, bead homogenization and sonication, and screened for barbiturates using a human immunochromatographic test. There was 100% agreement of the immunochromatographic test results using the sonication method with the reported euthanasia status of both dogs and cats. Using the bead homogenization method, agreement with the reported euthanasia status was 93.3% and 96.7% for dogs and cats, respectively, due to invalid test results from four dog and two cat samples. A subset of liver samples (10 canine and 10 feline) was analyzed via gas chromatography-mass spectrometry, and there was 100% agreement between the immunochromatographic test results and gas chromatography-mass spectrometry results for both cats and dogs. Overall, our results support the use of a variety of alternative matrices for barbiturate screening in cats and dogs.
Topics: Humans; Cats; Dogs; Animals; Pentobarbital; Cat Diseases; Dog Diseases; Barbiturates; Immunoassay; Animals, Domestic
PubMed: 37978839
DOI: 10.1093/jat/bkad087 -
MMW Fortschritte Der Medizin Nov 2023
Topics: Humans; Pentobarbital; Physicians; Sodium
PubMed: 37973746
DOI: 10.1007/s15006-023-3155-x -
Psychopharmacology Apr 2024Cabergoline (CAB) is an ergot derivative typically prescribed for the treatment of hyperprolactinemia. It suppresses the release of prolactin through agonist actions on...
RATIONALE
Cabergoline (CAB) is an ergot derivative typically prescribed for the treatment of hyperprolactinemia. It suppresses the release of prolactin through agonist actions on dopamine (DA) D2 receptors; however, it possesses binding affinity for other DA and 5-HT receptors. Side effects that exacerbate valvular heart disease can occur with high doses.
OBJECTIVE
The present study examined the acute, subchronic, and chronic dose-response effects of CAB and a derivative dimethylcabergoline (DMC) which acts as an antagonist instead of agonist at 5-HT 2B receptors, on appetitive and consummatory sexual behaviors of male rats.
METHODS
CAB (0, 0.03, 0.15, or 0.3 mg/kg/ml) was administered daily to sexually experienced male rats (N = 10/dose) by oral gavage for a total of 68 days. Sexual behavior was tested every 4 days during this period for a total of 16 trials. On the 17 trial, rats were administered their dose of CAB, and 4 h after were overdosed with sodium pentobarbital, perfused intracardially, and their brains processed for Fos immunohistochemistry. DMC (0, 0.03, 0.15, 0.3 mg/kg/ml) was administered daily to sexually experienced male rats (N = 10/dose) by oral gavage for a total of 36 days. Sexual behavior was tested every 4 days for a total of 9 trials.
RESULTS
CAB increased anticipatory level changes, intromissions, and ejaculations significantly across all timepoints, with the medium and high doses being most potent. The medium and high doses also increased Fos protein significantly within the medial preoptic area, whereas in the nucleus accumbens shell, the low and medium doses decreased Fos protein but the high dose increased it significantly from control. Similar to CAB, the medium and high doses of DMC increased the number of ejaculations significantly. Rats in all drug dose groups appeared healthy for the duration of the experiments.
CONCLUSIONS
Both CAB and DMC facilitate ejaculations, and CAB further facilitates measures of anticipatory sexual motivation and intromissions. These data suggest that both could be used as treatments for sexual arousal disorders and ejaculation/orgasm disorders with little or no untoward side effects at low doses.
Topics: Rats; Male; Animals; Cabergoline; Sexual Behavior, Animal; Copulation; Motivation; Brain; Gonadal Steroid Hormones; Receptors, Dopamine D2
PubMed: 37968530
DOI: 10.1007/s00213-023-06501-9