Determining plasma and cerebrospinal fluid concentrations of EGFR-TKI in lung cancer patients.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are commonly used to treat advanced non-small cell lung cancer (NSCLC). A rapid and reliable method for measuring plasma and cerebrospinal fluid (CSF) concentrations of EGFR-TKIs is needed for therapeutic drug monitoring. By using UHPLC‒MS/MS with multiple reaction monitoring mode, we developed a method for rapidly determining the plasma and CSF concentrations of gefitinib, erlotinib, afatinib, and osimertinib. Protein precipitation was employed to remove protein interference for plasma and CSF matrix. The LC‒MS/MS assay was validated to be satisfactory in terms of linearity, precision, and accuracy. This method was successfully applied to measure plasma (n = 44) and CSF (n = 6) concentrations of EGFR-TKIs in NSCLC patients. The chromatographic separation was achieved by a Hypersil Gold aQ column within 3 min. The median plasma concentrations were 325.76, 1981.50, 42.62, 40.27, and 340.92 ng/ml for gefitinib erlotinib, afatinib 30 mg/day, afatinib 40 mg/day, and osimertinib, respectively. The CSF penetration rates were 2.15% for the patients receiving erlotinib therapy, 0.59% for afatinib, 0.08-1.12% for osimertinib 80 mg/day, and 2.18% for those receiving osimertinib 160 mg/day. This assay helps to predict the effectiveness and toxicities of EGFR-TKIs in the pursuit of precision medicine for lung cancer patients.

Low-dose tributyltin triggers human chondrocyte senescence and mouse articular cartilage aging.

Tributyltin (TBT) is known as an endocrine-disrupting chemical. This study investigated the effects and possible mechanisms of TBT exposure on inducing human articular chondrocyte senescence in vitro at the human-relevant concentrations of 0.01-0.5 µM and mouse articular cartilage aging in vivo at the doses of 5 and 25 µg/kg/day, which were 5 times lower than the established no observed adverse effect level (NOAEL) and equal to NOAEL, respectively. TBT significantly increased the senescence-associated ß-galactosidase activity and the protein expression levels of senescence markers p16, p53, and p21 in chondrocytes. TBT induced the protein phosphorylation of both p38 and JNK mitogen-activated protein kinases in which the JNK signaling was a main pathway to be involved in TBT-induced chondrocyte senescence. The phosphorylation of both ataxia-telangiectasia mutated (ATM) and histone protein H2AX (termed γH2AX) was also significantly increased in TBT-treated chondrocytes. ATM inhibitor significantly inhibited the protein expression levels of γH2AX, phosphorylated p38, phosphorylated JNK, p16, p53, and p21. TBT significantly stimulated the mRNA expression of senescence-associated secretory phenotype (SASP)-related factors, including IL-1ß, TGF-ß, TNF-α, ICAM-1, CCL2, and MMP13, and the protein expression of GATA4 and phosphorylated NF-κB-p65 in chondrocytes. Furthermore, TBT by oral gavage for 4 weeks in mice significantly enhanced the articular cartilage aging and abrasion. The protein expression of phosphorylated p38, phosphorylated JNK, GATA4, and phosphorylated NF-κB-p65, and the mRNA expression of SASP-related factors were enhanced in the mouse cartilages. These results suggest that TBT exposure can trigger human chondrocyte senescence in vitro and accelerating mouse articular cartilage aging in vivo.

Comparison of fatal traumatic medico-legal cases with postmortem computed tomography and autopsy: A pilot study in Taiwan.

In order to determine the performance of postmortem computed tomography (PMCT) in identifying traumatic-relevant macroscopic findings in medico-legal cases, this retrospective observational pilot study involving nine trauma casualties who had received PMCT prior to autopsy. The comparison of these findings in six anatomical regions as dictated in Injury Severity Score (ISS) were performed. 104 traumatic-relevant findings were identified with achievement of 51% congruent findings. PMCT and autopsy had additionally found 22 and 29 findings respectively. PMCT had highest sensitivity for extremity injury (81.82%), followed by chest (73.91%), head, neck and face (71.43%), and abdomino-pelvic area (50%). It had excellent detection rate in abnormal air collection, fracture, foreign body localization, internal ballistic and intracranial pathology. However, the solid organ and vascular injuries as well as integumentary lesions were the major drawback. In conclusion, incorporation of PMCT to autopsy in medico-legal investigation helps to preserve the most abundant traumatic-relevant injuries compared to either modality.

Gender differences in clinical characteristics of emergency department patients involving illicit drugs use with analytical confirmation.

BACKGROUND: To compare gender differences in socio-demographics, clinical manifestations, and laboratory test results of individuals who visited emergency departments (EDs) involving drug use. METHODS: We retrospectively collected the data from 10 hospitals in Taiwan on drug-related ED visits from May 2017 to December 2020. We then examined the gender differences in their socio-demographics, clinical manifestations, urine toxicological results, and other laboratory tests results using chi-square or multivariable logistic regression. RESULTS: Among individuals with drug-related ED visits, there were 546 (73.7%) men and 195 (26.3%) women. The most commonly used drugs were meth/amphetamine, followed by synthetic cathinones, and ketamine and its analogs. Compared to men, women were younger (32.03 ± 10.86 vs. 36.51 ± 10.84 years, p < 0.001) and more likely to use new psychoactive substances (NPS) (p = 0.011). Men were more likely to have human immunodeficiency virus infection (p < 0.001), whereas women were more likely to report psychiatric comorbidities (p = 0.003). Women were less likely to have aggressive behaviors (odds ratio (OR): 0.59, 95% CI: 0.39-0.88). After adjusting for socio-demographics and drug types, women were still less likely to have aggressive behaviors than men (adjusted OR: 0.59, 95% CI: 0.38-0.93). The likelihood of rhabdomyolysis and intensive care unit admission was higher in men (p < 0.001). CONCLUSION: We found considerable gender differences in clinical characteristics among ED-visiting drug users, which could offer valuable information for the future development of more tailored gender-specific drug prevention and treatment strategies.

Therapeutic Ultrasound Halts Progression of Chronic Kidney Disease In Vivo via the Regulation of Markers Associated with Renal Epithelial-Mesenchymal Transition and Senescence.

Low-intensity pulsed ultrasound (LIPUS), a therapeutic type of ultrasound, is known to enhance bone fracture repair processes and help some tissues to heal. Here, we investigated the therapeutic potential of LIPUS for the treatment of chronic kidney disease (CKD) in two CKD mouse models. CKD mice were induced using both unilateral renal ischemia/reperfusion injury (IRI) with nephrectomy and adenine administration. The left kidneys of the CKD mice were treated using LIPUS with the parameters of 3 MHz, 100 mW/cm2, and 20 min/day, based on the preliminary experiments. The mice were euthanized 14 days after IRI or 28 days after the end of adenine administration. LIPUS treatment effectively alleviated the decreases in the body weight and albumin/globulin ratio and the increases in the serum renal functional markers, fibroblast growth factor-23, renal pathological changes, and renal fibrosis in the CKD mice. The parameters for epithelial-mesenchymal transition (EMT), senescence-related signal induction, and the inhibition of α-Klotho and endogenous antioxidant enzyme protein expression in the kidneys of the CKD mice were also significantly alleviated by LIPUS. These results suggest that LIPUS treatment reduces CKD progression through the inhibition of EMT and senescence-related signals. The application of LIPUS may be an alternative non-invasive therapeutic intervention for CKD therapy.

Characteristics of patients with analytically confirmed γ-hydroxybutyric acid/γ-butyrolactone (GHB/GBL)-related emergency department visits in Taiwan.

The recreational drug γ-hydroxybutyric acid (GHB) is a central nervous system depressant, and can produce euphoria at low doses. GHB is a controlled substance in Taiwan. However, the organic solvents γ-butyrolactone (GBL) and 1,4-butanediol (BD), which are unregulated, may be used as an alternative source of GHB. There is no clinical report of analytically confirmed GHB use in Taiwan. We retrospective reviewed the clinical characteristics from the medical charts between May 2017 and April 2020. The urine samples of patients presented to the emergency departments with drug-related complaints were sent for toxicological analysis. Patients with urine samples detected GHB >10 µg/mL by liquid chromatography/tandem mass spectrometry were included. Overall, 11 men and one woman with an average age of 35.3 ± 8.7 years were included. Most patients co-ingested amphetamine (n = 6) and initially presented with depressed consciousness levels (n = 7). One patient presented with out-of-hospital cardiac arrest and one with respiratory depression. All patients regained consciousness within 6 h of admission. All patients used GBL to evade conviction. Although patients recovered with supportive care, respiratory failure and cardiac arrest occurred after GHB/GBL use. It is important to legislate GBL and BD as controlled chemical substances in Taiwan.

Determination of synthetic cathinone α-pyrrolidinovalero-phenone and its metabolite in urine using solid-phase extraction and gas chromatography-mass spectrometry.

RATIONALE: The presence of α-pyrrolidinovalerophenone (α-PVP) and its metabolites in urine is evidence of the administration of α-PVP. A toxicological challenge is that the metabolites of α-PVP exhibit amphoteric properties, which make them unsuitable for detection using gas chromatography-mass spectrometry (GC/MS). In the study reported, proper derivatization and sample extraction were essential for improving the sensitivity for GC/MS analysis. METHODS: An automated solid-phase extraction (SPE) method has been developed and optimized. The derivatization efficiency was tested using longer reaction time and the addition of polar pyridine into a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane. Method validation, including linearity, limit of detection, precision, accuracy, and recovery, was evaluated using automatic SPE and GC/MS. RESULTS: The results suggested that adding pyridine to BSTFA (1:1, v/v) significantly improved derivatization efficiency and precision. After optimization, the linear range was from 25 to 1000 ng mL-1 with R2 > 0.9950. The limit of detection was 5 ng mL-1 for α-PVP and 25 ng mL-1 for OH-α-PVP. The recovery for SPE was over 88%. The inter-day and intra-day precisions were less than 15%. A forensic sample has been found containing α-PVP (67.3 ng mL-1 ) and OH-α-PVP (560.2 ng mL-1 ). CONCLUSIONS: This study is the first to validate an auto-SPE-GC/MS method for the quantification and qualification of α-PVP and OH-α-PVP in urine. We have successfully improved the derivatization efficiency and developed a sensitive and semi-automatic approach. This approach is desirable for the detection of synthetic cathinone at trace levels in biological samples.

Arsenic induces human chondrocyte senescence and accelerates rat articular cartilage aging.

Arsenic-contaminated drinking water is known to be a serious human health problem. A previous epidemiological study has indicated that arsenic levels in blood were higher in arthritis patients compared to age-matched control subjects. Bone is known as an important arsenic store compartment in the body. Arsenic exposure has been suggested to promote senescence in human mesenchymal stem cells that may affect the balance of adipogenic and osteogenic differentiation. The toxicological effect and mechanism of arsenic exposure on articular chondrocytes still remain unclear. Here, we investigated the arsenic-induced senescence in cultured human articular chondrocytes and long-term arsenic-exposed rat articular cartilage. Arsenic trioxide (As2O3; 1-5 µM) significantly induced senescence in human articular chondrocytes by increasing senescence-associated ß-galactosidase (SA-ß-Gal) activity and protein expression of p16, p53, and p21. Arsenic induced the phosphorylation of p38 and c-Jun N-terminal kinase (JNK) proteins. The inhibitors of p38 and JNK significantly reversed the arsenic-induced chondrocyte senescence. Arsenic could also trigger the induction of GATA4-NF-κB signaling and senescence-associated secretory phenotype (SASP) by increasing IL-1α, IL-1ß, TGF-ß, TNF-α, CCL2, PAI-1, and MMP13 mRNA expression. The increased cartilage senescence and abrasion were also observed in a rat model long-term treatment with arsenic (0.05 and 0.5 ppm) in drinking water for 36 weeks as compared to age-matched control rats. The phosphorylation of p38 and JNK and the induction of GATA4-NF-κB signaling and SASP were enhanced in the rat cartilages. Taken together, these findings suggest that arsenic exposure is capable of inducing chondrocyte senescence and accelerating rat articular cartilage aging and abrasion.

Characteristics of analytically confirmed illicit substance-using patients in the Emergency Department.

BACKGROUND/PURPOSE: Although illicit substance use-induced toxicity or complication is a frequent cause of visit to the emergency department (ED), there are limited data on cases confirmed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis. This study aimed to describe clinical presentations of patients who visited the ED because of acute illicit substance-related complications. METHODS: We performed a retrospective study between May 2017 and August 2018 on patients presenting to the ED with positive urine illicit substance analysis by LC-MS/MS. RESULTS: Of 203 patients with at least one illicit substance detected in their urine, 162 (79.8%) showed traditional illicit substances, and 56 (32.0%) showed new psychoactive substances (NPS). Methamphetamine was the most common illicit substance (67.9%). The most common NPS was ketamine (21.7%), followed by synthetic cathinones (14.8%). We divided patients into traditional, NPS and combined (both traditional illicit substance and NPS) groups. Polysubstance use was more common in the NPS group than in the traditional group (P < 0.001). Most patients were men (78.3%), and the average age was lower in the NPS group compared to the traditional group (P < 0.001). Although the chemical structures of cathinones are similar to that of amphetamine, 92.0% of the cathinone use cases without combination with methamphetamine use showed negative immunoassay results. CONCLUSION: Our study provided the acute illicit substance complications at ED by LC-MS/MS analysis in Taiwan. Our study showed that more than one-third cases studied were NPS users. Young adults and polysubstance users were more common among NPS users.

In vivo imaging of insulin-secreting human pancreatic ductal cells using MRI reporter gene technique: A feasibility study.

PURPOSE: The purpose of this study was to investigate the feasibility of in vivo imaging of human pancreatic ductal cells by OATP1B3 reporter gene under MRI. METHODS: A human cell line (PANC-1) derived from the pancreatic ductal epithelium was used in this study. After transduction of OATP1B3, the cellular physiological functions and the ability of intracellular uptake of the MRI contrast medium (Gd-EOB-DTPA) were examined. Induced differentiation of the PANC-1 cells into hormone-secreting cells were performed to simulate pancreatic ß-like cells. The hormone-secreting cells were implanted into rats and in vivo MRI was evaluated. RESULTS: The mRNA and proteins of OATP1B3 were highly expressed. No significant change of cellular physiological functions was found after the expression. After induced differentiation, the hormone secretion capacities of the OATP1B3-expressing PANC-1 cells were confirmed. Intra-cellular uptake of Gd-EOB-DTPA was determined in vitro by inductively coupled plasma mass spectrometry and MRI. In vivo MRI of the OATP1B3-expressing xenograft revealed an increased signal intensity after contrast enhancement. CONCLUSION: OATP1B3 can be used as a safe and feasible in vivo MRI gene reporter for human pancreatic ductal cells.

Tributyltin exposure at noncytotoxic doses dysregulates pancreatic ß-cell function in vitro and in vivo.

Tributyltin (TBT) is an endocrine disruptor. TBT can be found in food and in human tissues and blood. Several animal studies revealed that organotins induced diabetes with decreased insulin secretion. The detailed effect and mechanism of TBT on pancreatic ß-cell function still remain unclear. We investigated the effect and mechanism of TBT exposure at noncytotoxic doses relevant to human exposure on ß-cell function in vitro and in vivo. The ß-cell-derived RIN-m5F cells and pancreatic islets from mouse and human were treated with TBT (0.05-0.2 µM) for 0.5-4 h. Adult male mice were orally exposed to TBT (25 µg/kg/day) with or without antioxidant N-acetylcysteine (NAC) for 1-3 weeks. Assays for insulin secretion and glucose metabolism were carried out. Unlike previous studies, TBT at noncytotoxic concentrations significantly increased glucose-stimulated insulin secretion and intracellular Ca2+ ([Ca2+]i) in ß-cells. The reactive oxygen species (ROS) production and phosphorylation of protein kinase C (PKC-pan) and extracellular signal-regulated kinase (ERK)1/2 were also increased. These TBT-triggered effects could be reversed by antiestrogen ICI182780 and inhibitors of ROS, [Ca2+]i, and PKC, but not ERK. Similarly, islets treated with TBT significantly increased glucose-stimulated insulin secretion, which could be reversed by ICI182780, NAC, and PKC inhibitor. Mice exposed to TBT for 3 weeks significantly increased blood glucose and plasma insulin and induced glucose intolerance and insulin resistance, which could be reversed by NAC. These findings suggest that low/noncytotoxic doses of TBT induce insulin dysregulation and disturb glucose homeostasis, which may be mediated through the estrogen receptor-regulated and/or oxidative stress-related signaling pathways.

Hemorrhagic cystitis in children treated with alkylating agent cyclophosphamide: The experience of a medical center in Taiwan.

BACKGROUND/PURPOSE: Hemorrhagic cystitis is a common complication with chemotherapeutic alkylating agents. We investigated the possible prognostic factors of cyclophosphamide-induced hemorrhagic cystitis in children. METHODS: Medical records of children (< 18 years old) with cyclophosphamide-related hemorrhagic cystitis were collected retrospectively from January 2000 to December 2010 in a tertiary care center. We also prospectively enrolled children (< 18 years old) with cyclophosphamide treatment. RESULTS: The retrospective study consisted of 23 patients whose median age was 11 years. The median day of onset time was 1 day after cyclophosphamide usage. The hemato-oncological diseases included acute leukemia (39.1%), lymphoma (13%), blastoma (13%), sarcoma (13%), aplastic anemia (13%), and others (8.7%). Patients who received bone marrow transplantation (BMT) had significantly longer duration of hemorrhagic cystitis than those who did not receive BMT (p < 0.05). Serum uric acid, checked prior to and after the onset of hemorrhage cystitis, was significantly lower after the development of hemorrhagic cystitis (p < 0.05). In the prospective study, 11 children were enrolled with a median age of 5 years. The urinary nitrite/nitrate and 8-iso-prostaglandin F2α levels increased significantly after cyclophosphamide usage (p < 0.05). CONCLUSION: Alteration serum uric acid level and BMT could be indicators for severe hemorrhagic cystitis. The elevated levels of urinary nitrite/nitrate and 8-iso-prostaglandin F2α may indicate the essential roles played by nitric oxide syntheses and reactive oxidative stress in cyclophosphamide-induced hemorrhagic cystitis. These findings may help clinicians formulate a better strategy for treating cyclophosphamide-induced hemorrhagic cystitis.

Impurities in over-the-counter pseudoephedrine leading to methcathinone detection in urine.

Methcathinone, a psychoactive substance with stimulant properties, has raised concerns in recent years due to its presence in urine screenings, even among individuals with no history of drug abuse. To prevent misjudgment, this work aims to explore the source of methcathinone in urine. A total of 58 urine samples tested positive for methcathinone in the National Taiwan University Hospital cohort, with 27 linked to illicit drug use and 31 from individuals with no drug use history. Co-occurrence analysis revealed a strong association between methcathinone and over-the-counter cold medications containing pseudoephedrine or ephedrine. In an in vivo experiment, participants who consumed pseudoephedrine-containing drugs showed the presence of methcathinone in their urine, suggesting a connection between these substances. Additionally, tests on pharmaceutical products containing pseudoephedrine detected small amounts of methcathinone as impurities. The findings suggest that the presence of methcathinone in nonillicit drug users may be attributed to impurities in over-the-counter pseudoephedrine-containing medications. This raises concerns about potential misinterpretations of drug screening results and underscores the need for more comprehensive criteria for assessing drug use. This study contributes to our understanding of the origin of methcathinone in urine, which has implications for legal justice and drug screening practices.

Rapid Screening of New Psychoactive Substances Using pDART-QqQ-MS.

Drug abuse is a severe social problem worldwide. Particularly, the issue of new psychoactive substances (NPSs) have increasingly emerged. NPSs are structural or functional analogs of traditional illicit drugs, such as cocaine, cannabis, and amphetamine; these molecules provide the same or more severe neurological effects. Usually, immunoassays are utilized in the preliminary screening method. However, NPSs have poor detectability in commercially available immunoassay kits. Meanwhile, various chromatography combined with the mass spectrometry platform have been developed to quantify NPSs. Still, a significant amount of time and resources are required during these procedures. Therefore, we established a rapid analytical platform for NPSs employing paper-loaded direct analysis in real time triple quadrupole mass spectrometry (pDART-QqQ-MS). We implemented this platform for the semiquantitative analysis of forensic drug tests in urine. This platform significantly shrinks the analytical time of a single sample within 30 s and requires a low volume of the specimen. The platform can detect 21 NPSs in urine mixtures at a lower limit of qualification of concentration ranging from 20 to 75 nanograms per milliliter (ng mL-1) and is lower than the cutoff value of currently available immune-based devices for detecting multiple drugs (1000 ng mL-1). Urine samples from drug addicts have been collected to verify the platform's effectiveness. By combining efficiency and accuracy, our platform offers a promising solution for addressing the challenges posed by NPSs in drug abuse detection.

Involvement of caspase-12-dependent apoptotic pathway in ionic radiocontrast urografin-induced renal tubular cell injury.

Contrast medium (CM) induces a direct toxic effect on renal tubular cells. This toxic effect subjects in the disorder of CM-induced nephropathy. Our previous work has demonstrated that CM shows to activate the endoplasmic reticulum (ER)-related adaptive unfolding protein response (UPR) activators. Glucose-regulated protein 78 (GRP78)/eukaryotic initiation factor 2α (eIF2α)-related pathways play a protective role during the urografin (an ionic CM)-induced renal tubular injury. However, the involvement of ER stress-related apoptotic signals in the urografin-induced renal tubular cell injury remains unclear. Here, we examined by the in vivo and in vitro experiments to explore whether ER stress-regulated pro-apoptotic activators participate in urografin-induced renal injury. Urografin induced renal tubular dilation, tubular cells detachment, and necrosis in the kidneys of rats. The tubular apoptosis, ER stress-related pro-apoptotic transcriptional factors, and kidney injury marker-1 (kim-1) were also conspicuously up-regulated in urografin-treated rats. Furthermore, treatment of normal rat kidney (NRK)-52E tubular cells with urografin augmented the expressions of activating transcription factor-6 (ATF-6), C/EBP homologous protein (CHOP), Bax, caspase-12, JNK, and inositol-requiring enzyme (IRE) 1 signals. Urografin-induced renal tubular cell apoptosis was not reversed by the inhibitors of ATF-6, JNK signals or CHOP siRNA transfection, but it could be partially reversed by the inhibitor of caspase-12. Taken together, the present results and our previous findings suggest that exposure of CM/urografin activates the ER stress-regulated survival- and apoptosis-related signaling pathways in renal tubular cells. Caspase-12-dependent apoptotic pathway may be partially involved in the urografin-induced nephropathy.

Defective determination of synthetic cathinones in blood for forensic investigation.

Antemortem specimens are sometimes the sole sources available for forensic investigation, and samples collected in nonideal ways are inevitably employed to achieve toxicological analysis. It is essential to assess the effects of blood collection tubes on the recoveries of emerging synthetic cathinones (SC) to estimate actual drug concentrations, and no such systematic investigations have been previously carried out. Seventy-one SC with various LogP values were employed to examine commonly used blood collection tubes, including plasma tubes, serum tubes and gel-containing tubes in recoveries which determined by a reliable LC-MS/MS method. Significantly poor recoveries for hydrophobic SC were obtained using serum separating tubes (SST). Notably, the suppressed recoveries in SST can be reversed by adding anticoagulants. Adding a procoagulant to a plasma separating tube (PST) considerably reduced recoveries, which indicated that clotting processes in the presence of polymeric gels contributed to poor recoveries of these hydrophobic drugs. In this study, we find that clotting formation in the presence of polymeric gels could significantly affect the determination of hydrophobic drugs. However, in real-world scenarios, nonideal collection methods are inevitably employed for antemortem specimens. Thus, it is important to rigorously interpret forensic toxicological results, especially for susceptible species.

Development of the dried blood spot preparation protocol for comprehensive evaluation of the hematocrit effect.

The application of dried blood spots (DBS) has gradually increased in different fields because of its several advantages. The hematocrit (Hct) effect is one major analytical challenge that may affect the quantification accuracy of DBS samples and should be investigated when developing a novel DBS method. However, previous studies usually overlooked the Hct-related distribution bias when evaluating the Hct effect. This study aimed to propose an effective DBS preparation protocol for the comprehensive evaluation of the Hct effect. We selected voriconazole and posaconazole as the demonstration drugs. Fifteen microliters of the blood samples were spotted on DBS cards followed by whole spot extraction. An LC-MS/MS method was first developed to quantify voriconazole and posaconazole in DBS samples. The quantitation accuracy for both azole drugs was within 93.5%-111.7%, except for the accuracies of posaconazole at the LLOQ, which were less than 119%. The intra- and interday precision were below 11%. The validated LC-MS/MS method was used to develop the DBS preparation protocol for evaluating the Hct effect. Three critical parameters that may affect the observed Hct effect were investigated. The results showed that using the solid-state of the target analytes, spiking the target analytes before preparing different Hct levels, and allowing enough equilibrium time after spiking target analytes can provide a more holistic Hct effect evaluation. The validity of the proposed new protocol was verified by conversion factors obtained from 71 paired DBS and plasma samples. Conversion factors calculated by clinical samples were consistent with the Hct effect evaluated by manually prepared DBS samples. This new DBS preparation protocol eliminated the common pitfalls in studying the Hct effect and offered a comprehensive strategy to assess the Hct effect for further DBS studies.

A ubiquitous endocrine disruptor tributyltin induces muscle wasting and retards muscle regeneration.

BACKGROUND: Organotin pollutant tributyltin (TBT) is an environmental endocrine disrupting chemical and is a known obesogen and diabetogen. TBT can be detected in human following consumption of contaminated seafood or water. The decrease in muscle strength and quality has been shown to be associated with type 2 diabetes in older adults. However, the adverse effects of TBT on the muscle mass and function still remain unclear. Here, we investigated the effects and molecule mechanisms of low-dose TBT on skeletal muscle regeneration and atrophy/wasting using the cultured skeletal muscle cell and adult mouse models. METHODS: The mouse myoblasts (C2C12) and differentiated myotubes were used to assess the in vitro effects of low-dose tributyltin (0.01-0.5 µM). The in vivo effects of TBT at the doses of 5 and 25 µg/kg/day (n = 6/group), which were five times lower than the established no observed adverse effect level (NOAEL) and equal to NOAEL, respectively, by oral administration for 4 weeks on muscle wasting and muscle regeneration were evaluated in a mouse model with or without glycerol-induced muscle injury/regeneration. RESULTS: TBT reduced myogenic differentiation in myoblasts (myotube with 6-10 nuclei: 53.9 and 35.8% control for 0.05 and 0.1 µM, respectively, n = 4, P < 0.05). TBT also decreased myotube diameter, upregulated protein expression levels of muscle-specific ubiquitin ligases (Atrogin-1 and MuRF1), myostatin, phosphorylated AMPKα, and phosphorylated NFκB-p65, and downregulated protein expression levels of phosphorylated AKT and phosphorylated FoxO1 in myotubes (0.2 and 0.5 µM, n = 6, P < 0.05). Exposure of TBT in mice elevated body weight, decreased muscle mass, and induced muscular dysfunction (5 and 25 µg/kg, P > 0.05 and P < 0.05, respectively, n = 6). TBT inhibited soleus muscle regeneration in mice with glycerol-induced muscle injury (5 and 25 µg/kg, P > 0.05 and P < 0.05, respectively, n = 6). TBT upregulated protein expression levels of Atrogin-1, MuRF1, myostatin, and phosphorylated AMPKα and downregulated protein expression level of phosphorylated FoxO1 in the mouse soleus muscles (5 and 25 µg/kg, P > 0.05 and P < 0.05, respectively, n = 6). CONCLUSIONS: This study demonstrates for the first time that low-dose TBT significantly inhibits myogenic differentiation and triggers myotube atrophy in a cell model and significantly decreases muscle regeneration and muscle mass and function in a mouse model. These findings suggest that low-dose TBT exposure may be an environmental risk factor for muscle regeneration inhibition, atrophy/wasting, and disease-related myopathy.

Therapeutic ultrasound treatment for the prevention of chronic kidney disease-associated muscle wasting in mice.

Low-intensity pulsed ultrasound (LIPUS) is a kind of therapeutic ultrasound. It can help improve bone fracture repair and soft tissue healing. Our previous study found that LIPUS treatment could halt the chronic kidney disease (CKD) progression in mice; unexpectedly, we observed the improvement of CKD-reduced muscle weights by LIPUS treatment. Here, we further tested the protective potential of LIPUS on CKD-associated muscle wasting/sarcopenia using the CKD mouse models. Mouse models of both unilateral renal ischemia/reperfusion injury (IRI) with nephrectomy and adenine administration were used to induce CKD. LIPUS with condition of 3 MHz, 100 mW/cm2, 20 min/day was applied to the kidney of CKD mice. LIPUS treatment significantly reversed the increased serum BUN/creatinine levels in CKD mice. LIPUS effectively prevented the decrease in grip strength, muscle weight (soleus, tibialis anterior, and gastrocnemius muscles), cross-section areas of muscle fibres, and muscular phosphorylated Akt protein expression by immunohistochemistry, and the increase in muscular atrogenes Atrogin1 and MuRF1 protein expression by immunohistochemistry in CKD mice. These results indicated that LIPUS could help improve weak muscle strength, muscle mass loss, muscle atrophy-related protein expression, and Akt inactivation. LIPUS application may be an alternative non-invasive therapeutic intervention on the management of CKD-associated muscle wasting.