Plasma free metanephrine and normethanephrine levels correlated to plasma catecholamine after acute running in amateur runner.

BACKGROUND: Catecholamine is a typical index of exercise intensity, but it is difficult to detect. Plasma metanephrine (MN) and normethanephrine (NMN) levels are more stable than those of catecholamines. This study aimed to investigate plasma MN and NMN levels during acute exercise running in amateur runners. METHODS: Samples were collected from eight healthy male participants. They were either sedentary or running at low or high intensity for 30 min. Blood samples were collected under these conditions. Measurements taken included plasma adrenaline, noradrenaline, MN, and NMN. RESULTS: Plasma adrenaline levels increased after high-intensity exercise compared with sedentary subjects. Plasma noradrenaline, MN, and NMN levels increased after both low- and high-intensity exercise compared with sedentary subjects. In addition, these levels were also significantly higher at high intensity than at low intensity. Plasma adrenaline and noradrenaline levels were positively correlated with plasma free MN and NMN levels after acute running, respectively. CONCLUSION: This study revealed that plasma MN and NMN levels transiently increased depending on exercise intensity in amateur runners. In addition, plasma NMN levels are better markers than plasma MN levels because of their stronger correlation with plasma catecholamine levels.

Effects of renalase deficiency on liver fibrosis markers in a nonalcoholic steatohepatitis mouse model.

Progression of nonalcoholic steatohepatitis (NASH) is attributed to several factors, including inflammation and oxidative stress. In recent years, renalase has been reported to suppress oxidative stress, apoptosis and inflammation. A number of studies have suggested that renalase may be associated with protecting the liver from injury. The present study aimed to clarify the effects of renalase knockout (KO) in mice with NASH that were induced with a choline­deficient high­fat diet (CDAHFD) supplemented with 0.1% methionine. Wild type (WT) and KO mice (6­week­old) were fed a normal diet (ND) or CDAHFD for 6 weeks, followed by analysis of the blood liver function markers and liver tissues. CDAHFD intake was revealed to increase blood hepatic function markers, lipid accumulation and oxidative stress compared with ND, but no significant differences were observed between the WT and KO mice. However, in the KO­CDAHFD group, the Adgre1 and Tgfb1 mRNA levels were significantly higher, and α­SMA expression was significantly lower compared with the WT­CDAHFD group. Furthermore, the Gclc mRNA and phosphorylated protein kinase B (Akt) levels were significantly lower in the KO­ND group compared with the WT­ND group. The results of the current study indicated that as NASH progressed in the absence of renalase, oxidative stress, macrophage infiltration and TGF­ß expression were enhanced, while α­SMA expression in NASH may be partly suppressed due to the decreased phosphorylation of Akt level.

The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay.

BACKGROUND: With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. METHODS: Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 µL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. RESULTS: In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. CONCLUSIONS: These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR.

With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.