Screening anabolic androgenic steroids in human urine: an application of the state-of-the-art gas chromatography-Orbitrap high-resolution mass spectrometry.

Over the past few decades, anabolic androgenic steroids (AASs) have been abused in and out of competition for their performance-enhancing and muscle-building properties. Traditionally, AASs were commonly detected using gas chromatography-mass spectrometry in the initial testing procedure for doping control purposes. Gas chromatography-Orbitrap high-resolution mass spectrometry (GC-Orbitrap-HRMS) is a new technology that has many advantages in comparison with GC-MS (e.g., a maximum resolving power of 240,000 (FWHM at m/z 200), excellent sub-ppm mass accuracy, and retrospective data analysis after data acquisition). Anti-doping practitioners are encouraged to take full advantage of the updated techniques of chromatography-mass spectrometry to develop sensitive, specific, and rapid screening methods for AASs. A new method for screening a wide range of AASs in human urine using GC-Orbitrap-HRMS was developed and validated. The method can qualitatively determine 70 anabolic androgenic steroids according to the minimum required performance limit of the World Anti-Doping Agency. Moreover, the validated method was successfully applied to detect six metabolites in urine after the oral administration of metandienone, and their excretion curves in vivo were studied. Metandienone M6 (17ß-hydroxymethyl-17α-methyl-18-nor-androst-1,4,13-trien-3-one) has been identified as a long-term urinary metabolite which can be detected up to 7 weeks, thus providing a longer detection window compared with previous studies. This study provides a rationale for GC-Orbitrap-HRMS in drug metabolism and non-targeted screening.

Dietary carbon loaded with nano-ZnO alters the gut microbiota community to mediate bile acid metabolism and potentiate intestinal immune function in fattening beef cattle.

BACKGROUND: To our knowledge, carbon loaded with nano-ZnO (NZnOC) represents a new nutritional additive for the animal husbandry industry. However, the mechanism by which NZnOC mediates beef cattle growth and intestinal health is not fully understood. This study aimed to investigate the effects of carbon loaded with nano-ZnO (NZnOC) supplementation on growth performance, gut microbiota, bile acid (BAs) metabolism and intestinal immunity in fattening cattle. Twenty cattle (16 ± 0.95 months) were randomly assigned to two dietary groups: CON (control, without feed additive) and NZnOC (diet supplemented with 80 mg NZnOC/kg diet dry matter basic) for 60 d. The colon digesta microbiota composition and BAs concentration were determined by microbiota metagenomics and gas chromatography methods, respectively. RESULTS: The results showed that the NZnOC-supplemented cattle had greater final weight, average daily gain and gain-to-feed ratio than those in the CON group. Cattle fed the NZnOC diet had a higher relative abundance of the secondary BAs synthesizing phyla Firmicutes, Tenericutes and Actinobacteria than those fed the CON diet. Dietary supplementation with NZnOC increased the relative abundance of the secondary BAs synthesis microbiota genera Clostridium, Ruminococcus, Eubacterium, and Brevibacillus in colon digesta. Cattle fed the NZnOC diet had increased activities of 3α-hydroxysteroid dehydrogenase (EC: 1.1.1.52) and bile acid-CoA ligase BaiB (EC: 6.2.1.7) in the colon digesta compared with those fed the CON diet. The primary BAs taurocholic acid, taurochenodeoxycholic acid and taurodeoxycholate acid were significantly decreased by dietary NZnOC supplementation, while the secondary BAs deoxycholic acid, taurolithocholic acid, beta-muricholic acid, 12-ketolithocholic acid and ursodeoxycholic acid were significantly increased. Dietary supplementation with NZnOC increased the mRNA abundance of G protein-coupled bile acid receptor 1, protein kinase cAMP-activated catalytic subunit alpha, cyclic-AMP response element binding protein 1 and interleukin (IL)-10 in the colon mucosa of cattle, while the mRNA abundance of tumor necrosis factor and IL-1ß were significantly decreased. CONCLUSIONS: In summary, dietary supplementation with NZnOC can facilitate the growth performance and intestinal immune function of cattle by improving BAs metabolism. NZnOC can be supplemented in the diet as a safe regulator of gut microbiota and as a feed additive in the ruminants industry.

[Interaction between ATM and radiation-activated phosphorylation of P53 and P21].

BACKGROUND & OBJECTIVE: ATM gene is a member of PI-3K kinase family. ATM protein is capable of controlling DNA repair process and cell cycle checkpoint. In AT cells from ataxia-telangiectasia (AT) patients, ATM gene mutation leads to the deficiency of ionizing radiation-activated phosphorylation of P53 and P21. It shows ATM gene could mediate the phosphorylation of P53 and P21. This study was to explore the interaction between ATM and P53, and to observe whether ATM directly medicates the phosphorylation of P21 in a P53-independent way. METHODS: pEBS7-YZ5 vector containing ATM cDNA was transfected into AT cells by electroperforation. The cells expressing ATM protein stably were screened with hygromycin, and identified by reverse transcription-polymerase chain reaction (RT-PCR). The interaction between ATM and P53 in pEBS7-YZ5-AT cells was assessed by co-immunoprecipitation and Western blot. K562 cells served as a P53 mutation cell model to study whether ATM could interact with the phosphorylation of P21. RESULTS: pEBS7-YZ5 was transfected into AT cells successfully. RT-PCR detected fragment of ATM cDNA. After exposed to ionizing radiation, P53 of pEBS7-YZ5-AT cells was phosphorylated, and immunoprecipitation showed interaction between ATM and P53; P21 of K562 cells was phosphorylated, P21 protein was detected in the immunoprecipitation of ATM antibody-complex. CONCLUSION: Ionizing radiation-activated ATM kinase could interact with the phosphorylation of P53 and P21 in both P53 wild type and mutant type cells.