Quantification of normetanephrine in canine urine using ELISA: evaluation of factors affecting results

Catecholamine release increases in dogs with pheochromocytomas and in situations of stress. Although plasma catecholamines degrade rapidly, their metabolites, normetanephrine (NME) and metanephrine (ME), are stable in acidified urine. Our aim was to verify a human urine ELISA kit for the quantification of NME and ME in canine urine and to determine the effects on metabolite stability of sampling time (morning or midday) and day (ordinary or day spent in a clinic). We analyzed 179 urine samples from 17 healthy dogs. For NME, the mean intra-assay CV was 6.0% for all samples and 4.3% for the canine control; inter-assay CVs were 3.3, 3.8, and 12% for high and low concentration human urine positive controls supplied in the ELISA kit and a positive canine control, respectively; spike-recovery was 90–101%. For ME, mean intra-assay CV was 6.5% for samples and 9.0% for the canine control; inter-assay CVs were 12.7, 7.2, and 22.5% for high and low concentration human urine positive controls supplied in the ELISA kit and a positive canine control, respectively; spike-recovery was 85–89%. Dilution recovery was unsatisfactory for both metabolites. Based on our verification results, NME was selected for remaining analyses. We found no effect on NME concentrations of acidification or room temperature storage for up to 24 h. The NME:creatinine ratio was higher after the first of 3 clinic days compared to the same morning (111.2 ± 5.5 vs. 82.9 ± 5.3; p < 0.0001), but not on the other days. NME verification results were generally superior to ME. Dilution studies were unsatisfactory for both metabolites. Given that NME was stable without acidification at room temperature, urine samples can be collected at home. The clinic environment can cause higher NME:creatinine ratios, especially in unaccustomed dogs.

Lysine demethylase KDM6B regulates HIF-1α mediated systemic and cellular responses to intermittent hypoxia

Intermittent hypoxia (IH) is a hallmark manifestation of Obstructive Sleep Apnea (OSA). Rodents treated with IH exhibit hypertension. Hypoxia-inducible factor (HIF)-1-dependent transcriptional activation of NADPH oxidases (Nox) and the resulting increase in reactive oxygen species (ROS) levels is a major molecular mechanism underlying IH/OSA-induced hypertension. Jumanji C (JmjC)-containing histone lysine demethylases (JmjC-KDMs) are coactivators of HIF-1-dependent transcriptional activation. In the present study, we tested the hypothesis that JmjC-KDMs are required for IH-evoked HIF-1 transcriptional activation of Nox4 and the ensuing hypertension. Studies were performed on pheochromocytoma (PC)12 cells and rats. IH increased KDM6B protein and enzyme activity in PC12 cells in a HIF-1-independent manner as evidenced by unaltered KDM6B activation by IH in HIF-1α shRNA treated cells. Cells treated with IH showed increased HIF-1-dependent Nox4 transcription as indicated by increased HIF-1α binding to hypoxia responsive element (HRE) sequence of the Nox4 gene promoter demonstrated by chromatin immunoprecipitation (ChiP) assay. Pharmacological blockade of KDM6B with GSKJ4, a specific KDM6 inhibitor, or genetic silencing of KDM6B with shRNA abolished IH-induced Nox4 transcriptional activation by blocking HIF-1α binding to the promoter of the NOX4 gene. Treating IH exposed rats with GSKJ4 showed: a) absence of KDM6B activation and HIF-1-dependent Nox4 transcription in the adrenal medullae, as well as b) absence of elevated plasma catecholamines and hypertension. Collectively, these findings indicate that KDM6B functions as a coactivator of HIF-1-mediated Nox4 transactivation and demonstrate a hitherto uncharacterized role for KDM's in IH-induced hypertension by HIF-1.

Effects of Slaughter Positions on Catecholamine, Blood Biochemical and Electroencephalogram Changes in Cattle Restrained Using a Modified Mark IV Box

The proper slaughter positioning of animals is among the most crucial factors in animal welfare. The lateral position in Halal slaughter is a technique used around the world by Muslims, with a few practicing the upright position. The literature on the effects of slaughter in upright versus lateral positions on pain and stress is scarce. Thus, this study was designed to evaluate the effects of slaughter positions on blood biochemical parameters, plasma catecholamines, and electroencephalographic (EEG) responses. Twenty Brahman crossbred steers were subjected to slaughter in either lateral recumbency (LP) (n = 10) or an upright position (UP) (n = 10). There was a significant increase in adrenaline (p < 0.0001) and noradrenaline (p < 0.05) at T2 compared to T1 in the animals of both groups. A significant difference (p < 0.0001) was observed in the median frequency (MF) and total power (Ptot) of EEG, parameters for pain and stress, between the animals slaughtered in the upright and the lateral position. However, MF and delta waves were significantly higher (p < 0.05) after slaughter in the UP group than in the LP group. The results demonstrate a lesser amount of stress and pain responses among the LP group.

LianXia Formula Granule Attenuates Cardiac Sympathetic Remodeling in Rats with Myocardial Infarction via the NGF/TrKA/PI3K/AKT Signaling Pathway

Sympathetic remodeling may cause severe arrhythmia after myocardial infarction (MI). Thus, targeting this process may be an effective strategy for clinical prevention of arrhythmias. LianXia Formula Granule (LXFG) can effectively improve the symptoms of patients with arrhythmia after MI, and modern pharmacological studies have shown that Coptidis Rhizoma and Rhizoma Pinelliae Preparata, the components of LXFG, have antiarrhythmia effects. Here, we investigated whether LXFG can mitigate sympathetic remodeling and suppress arrhythmia and then elucidated its underlying mechanism of action in rats after MI. Sprague-Dawley (SD) rats that had undergone a myocardial infarction model were randomly divided into 6 groups, namely, sham, model, metoprolol, and LXFG groups, with high, medium, and low dosages. We exposed the animals to 30 days of treatment and then evaluated incidence of arrhythmia and arrhythmia scores in vivo using programmed electrical stimulation. Moreover, we determined plasma catecholamines contents via enzyme-linked immunosorbent assay and detected expression of tyrosine hydroxylase (TH) at infarcted border zones via western blot, real-time PCR, and immunohistochemical analyses to assess sympathetic remodeling. Finally, we measured key molecules involved in the NGF/TrKA/PI3K/AKT pathways via western blot and real-time PCR. Compared with the model group, treatment with high dose of LXFG suppressed arrhythmia incidence and arrhythmia scores. In addition, all the LXFG groups significantly decreased protein and mRNA levels of TH, improved the average optical density of TH-positive nerve fibers, and reduced the levels of plasma catecholamines relative to the model group. Meanwhile, expression analysis revealed that key molecules in the NGF/TrKA/PI3K/AKT pathways were downregulated in the LXFG group when compared with model group. Overall, these findings indicate that LXFG suppresses arrhythmia and attenuates sympathetic remodeling in rats after MI. The mechanism is probably regulated by suppression of the NGF/TrKA/PI3K/AKT signaling pathway.

Correlation between plasma catecholamines, weight, and diabetes in pheochromocytoma and paraganglioma

Context Pheochromocytomas and paragangliomas (PCC/PGL) are neuroendocrine tumors with discrete catecholamine profiles that cause incompletely understood metabolic and physiologic changes. Objective The objective was to evaluate relationships between plasma catecholamines, body weight, and hemoglobin A1C (HA1C). We hypothesized that individual catecholamines would correlate negatively with weight and glucose control. Design A retrospective cohort study was performed (1999–2020). Wilcoxon rank-sum tests compared non-parametric, continuous variables; mixed-effect linear modeling (MEM) evaluated relationships between catecholamines and weight or HA1C. The median study duration was 54.2 months (IQR: 19.0–95.1). Setting Tertiary academic hospital. Patients 360 patients were identified prospectively by referral to our center for management or surveillance of PCC/PGL. The median age was 59 years (IQR: 45–67) and 56.4% (N=203) were female. Main outcome measures The primary and secondary outcomes were weight and HA1C, respectively. Results On multivariable MEM, norepinephrine (p&lt;0.0005) negatively correlated with weight when all catecholamines and their derivatives were tried in the model, and normetanephrine (p&lt;0.0005) correlated when only metanephrines were included. In the surgical cohort (N=272), normetanephrine decreased postoperatively and was inversely associated with weight (p&lt;0.0005). Elevated norepinephrine or normetanephrine at the study termination, indicative of metastatic and/or recurrent disease (MRD), correlated with weight loss. Norepinephrine and normetanephrine (p&lt;0.0005) directly correlated with HA1C. Conclusion Plasma norepinephrine and its metabolite directly correlate with HA1C and inversely correlate with weight in PCC/PGL. After resection, declining normetanephrine levels correlate with improving HA1C despite an increase in patient body weight. Persistently elevated catecholamines and decreasing weight are observed in MRD.

Case of pheochromocytoma mimicking MINOCA

We present a 52-year-old woman who was admitted to the emergency department with a short history of palpitations, sweating and nausea. An electrocardiogram (ECG) that was performed suggested inferolateral ischaemia with a significant troponin rise. The patient underwent an invasive coronary angiogram that showed mild non-obstructive coronary disease. She was thus given a provisional diagnosis of myocardial infarction with non-obstructive arteries (MINOCA), treated as an acute coronary syndrome (ACS) and subsequently discharged home. The patient represented within 72 hours with a recurrence of symptoms and a further troponin rise. While on the ward severe recurrent orthostatic hypertensive episodes were noted. Further investigations revealed increased urinary and plasma metanephrines, increased plasma catecholamines and imaging revealed a left adrenal 5.7 cm mass, demonstrating probable pheochromocytoma. The patient was treated with curative surgery. This case highlights the importance of thorough history-taking in patients with atypical symptoms for acute coronary syndrome and diagnosed with MINOCA.

Active hexose correlated compound restores the gene expression and protein secretion of protective cytokines of immune cells in a murine stress model during Chlamydia muridarum genital infection

Chlamydia trachomatis genital infection is the most common bacterial sexually transmitted disease worldwide. Previously we reported that cold-induced stress results in immune suppression of mice that subsequently leads to increased intensity of Chlamydia muridarum genital infection. Furthermore, we demonstrated that stressed mice orally fed with active hexose correlated compound (AHCC) have reduced shedding of C. muridarum from the genital tract. However, the mechanism of AHCC on reducing the organ load and changes in the immune response in the stress model is not well known. This study evaluated infection and changes in immunological parameters of stressed AHCC-fed mice with or without C. muridarum genital infection. We hypothesized that AHCC feeding to stressed mice restores the protective immune function and reduces susceptibility to C. muridarum genital infection. Results show that oral feeding of stressed mice with AHCC resulted in decreased shedding of C. muridarum from the genital tract, reduced production of plasma catecholamines, increased expression of T-bet and reduced GATA-3 in CD4+ T cells, increased production of IL-12 and IFN-γ, and reduced production of IL-4 in CD4+ T cells, and enhanced expression of surface markers and co-stimulatory molecules of CD4+ T cells, bone marrow-derived dendritic cells (BMDCs), and natural killer cells. Co-culturing of mature BMDCs with splenic CD4+ T cells led to the increased and decreased production of T-helper 1 and T-helper2 cytokines, respectively. Overall, our results show that AHCC fosters the restoration of Th1 cytokine production while reducing Th2 cytokine production, which would promote C. muridarum clearance in the murine stress model.