Detection of hypoglycin A and MCPA-carnitine in equine serum and muscle tissue: Optimisation and validation of a LC-MS-based method without derivatisation.
- 作者列表："González-Medina S","Hyde C","Lovera I","Piercy RJ
BACKGROUND:Measurement of hypoglycin A (HGA) and its toxic metabolite, methylenecyclopropylacetic acid (MCPA), in equine serum confirms a diagnosis of atypical myopathy (AM), a pasture-associated toxic rhabdomyolysis with high mortality linked to the ingestion of Acer trees plant material. Supportive diagnostic tests include plasma acyl-carnitine profiling and urine organic acid testing, but these are not specific for AM. Previously reported HGA and MCPA analytical techniques used liquid chromatography-mass spectrometry (LC-MS) with a derivatising step, but the latter prolongs testing and increases costs. OBJECTIVES:To develop a rapid LCMS method for detection of serum and tissue HGA and MCPA that enables expedited diagnosis for horses with AM. STUDY DESIGN:Analytical test validation. METHODS:Validation parameters to industry standards using as criteria precision, accuracy, linearity, reproducibility and stability in analyte-spiked samples were calculated on 9-calibration points and 3 different validation concentrations in both serum and muscle tissue. RESULTS:The test was successfully validated for the detection of HGA and MCPA-carnitine in equine serum and muscle. Test linearity was excellent (r2 = .999), accuracy was very good for both analytes (93%-108%), precision did not exceed 10% coefficient of variation and reproducibility met the requirements of the Horwitz equation. Stability was unaffected by storage at a range of temperatures. MAIN LIMITATIONS:The spectrum of the tested analytes was limited to only two relevant analytes in favour of a quick and easy analysis. Linearity of the muscle method was not evaluated as calibration curves were not produced in this matrix. CONCLUSION:We report an optimised, simplified and validated method for detection of HGA and MCPA-carnitine in equine serum and muscle suitable for rapid diagnosis of suspected AM cases. The serum-based test should also enable risk assessment of toxin exposure in cograzing horses and assessment of horses with undiagnosed myopathies, while the tissue detection test should help to confirm cases post-mortem and to determine toxin distribution, metabolism and clearance across different tissues.
背景: 在马血清中测定低甘氨酸A (HGA) 及其毒性代谢产物亚甲基环丙基乙酸 (MCPA)，证实了非典型肌病 (AM) 的诊断，AM是一种牧场相关的毒性横纹肌溶解症，高死亡率与摄入枫树植物材料有关。支持性诊断测试包括血浆酰基肉碱分析和尿液有机酸测试，但这些不是AM特异性的。先前报道的HGA和MCPA分析技术使用具有衍生化步骤的液相色谱-质谱 (lc-ms)，但后者延长了测试并增加了成本。 目的: 开发一种快速LCMS方法来检测血清和组织HGA和MCPA，从而能够快速诊断患有AM的马。 研究设计: 分析测试验证。 方法: 使用作为标准的工业标准的验证参数在9个校准点和血清和肌肉组织中的3种不同的验证浓度上计算掺入分析物的样品中的精密度、准确度、线性、再现性和稳定性。 结果: 本试验成功用于马血清和肌肉中HGA和MCPA-肉碱的检测。测试线性良好 (r2 = .999)，两种分析物的准确度都非常好 (93%-108%)，精密度不超过10%，变异系数和再现性符合Horwitz方程的要求。稳定性不受在一定温度范围内储存的影响。 主要限制: 测试的分析物的光谱仅限于两种相关分析物，有利于快速和容易的分析。没有评价肌肉方法的线性，因为在该矩阵中没有产生校准曲线。 结论: 我们报告了一种优化、简化和验证的方法，用于检测马血清和肌肉中HGA和MCPA-肉碱，适用于疑似AM病例的快速诊断。以血清为基础的试验还应能够对共放牧马的毒素暴露进行风险评估，并对患有未确诊肌病的马进行评估，而组织检测试验应有助于在死后确认病例，并确定毒素在不同组织中的分布、代谢和清除情况。
METHODS::The ATP binding-cassette superfamily corresponds the mostly transmembrane transporters family found in humans. These proteins actively transport endogenous and exogenous substrates through biological membranes in body tissues, so they have an important role in the regulation of many physiological functions necessary for human homeostasis, as well as in response regulation to several pharmacological substrates. The development of multidrug resistance has become one of the main troubles in conventional chemotherapy in different illnesses including cancer, being the increased efflux of antineoplastic drugs the main reason for this multidrug resistance, with a key role of the ABC superfamily. Likely, the interindividual variability in the pharmacological response among patients is well known, and may be due to intrinsically factors of the disease, genetic and environmental ones. Thus, the understanding of this variability, especially the genetic variability associated with the efficacy and toxicity of drugs, can provide a safer and more effective pharmacological treatment, so ABC genes are considered as important regulators due to their relationship with the reduction in pharmacological response. In this review, updated information about transporters belonging to this superfamily was collected, the possible role of these transporters in cancer, the role of genetic variability in their genes, as well as some therapeutic tools that have been tried to raise against main transporters associated with chemoresistance in cancer.
METHODS:BACKGROUND:Cholinergic neurotransmission regulates neuroinflammation in Parkinson disease (PD). RESEARCH DESIGN AND METHODS:The authors conducted a delayed-start study of donepezil for cognitive decline in non-demented PD patients. The study consisted of a 96-week randomized placebo-controlled double-blind phase 1, followed by a 24-week donepezil extension phase 2. The primary outcome measure was a change in the Mini-Mental State Examination (MMSE) at week 120. RESULTS:A total of 98 patients were randomly allocated to the early-start (donepezil-to-donepezil) and delayed-start (placebo-to-donepezil) groups. Mean (SD) of the baseline MMSE was 27.6 (2.0) and 28.0 (2.1), respectively. MMSE change at week 120 was better in the early-start group than in the delayed-start group, but the difference was not significant. The MMSE declined in apolipoprotein ε4 carriers, but not in non-carriers, and the factor interaction (intervention × ε4 genotype) was highly significant (P < 0.001). Analyzed with the interaction, the difference was significant (group difference 1.95 [0.33 to 3.57], P = 0.018). The MMSE decline slope in phase 1 was significantly better in the early-start group than in the delayed-start group (P = 0.048). CONCLUSIONS:Cognitive function deteriorated in ε4 carriers, but not in non-carriers, and early-start donepezil may postpone cognitive decline in the former.
METHODS::Since the discovery of dental pulp stem cells, a lot of teams have expressed an interest in dental pulp regeneration. Many approaches, experimental models and biological explorations have been developed, each including the use of stem cells and scaffolds with the final goal being clinical application in humans. In this review, the authors' objective was to compare the experimental models and strategies used for the development of biomaterials for tissue engineering of dental pulp with stem cells. Electronic queries were conducted on PubMed using the following terms: pulp regeneration, scaffold, stem cells, tissue engineering and biomaterial. The extracted data included the following information: the strategy envisaged, the type of stem cells, the experimental models, the exploration or analysis methods, the cytotoxicity or viability or proliferation cellular tests, the tests of scaffold antibacterial properties and take into account the vascularization of the regenerated dental pulp. From the 71 selected articles, 59% focused on the "cell-transplantation" strategy, 82% used in vitro experimentation, 58% in vivo animal models and only one described an in vivo in situ human clinical study. 87% used dental pulp stem cells. A majority of the studies reported histology (75%) and immunohistochemistry explorations (66%). 73% mentioned the use of cytotoxicity, proliferation or viability tests. 48% took vascularization into account but only 6% studied the antibacterial properties of the scaffolds. This article gives an overview of the methods used to regenerate dental pulp from stem cells and should help researchers create the best development strategies for research in this field.