MS enabled us to analyze intact metabolites in mouse brain without sample preparation. MS discriminated the metabolic profiles of energy metabolism disruption triple quadrupole mass spectrometry pdf mice from control mice. MS allowed us to perform local distribution analysis of mouse brain.
MS directly detected 25 metabolites in a mouse frontal cortex, and clearly discriminated the metabolic profiles of mice model with energy metabolism disruption from control mice. MS for direct metabolome profiling of mouse brain. Check if you have access through your login credentials or your institution. Yumi Hayashi and Kei Zaitsu contributed equally to this work. 7 230 156 230 154. 7 153 227 153 225.
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4 12 13 12 13. 5 14 11 14L1 14C0. After optimizing the cell reaction system, this method demonstrated a screening capability of 103 samples, including 78 single compounds and 25 extracts, in less than 12 h without manual sample preparation. DHT production in cultured DU145 cells.
In addition, naringenin boosted DHT production in DU145 cells. The method can facilitate the discovery of bioactive chemicals that modulate the DHT production, and four phytochemicals are potential candidates of nutraceuticals to adjust DHT levels in male hormonal dysfunction. Since DHT has a much higher binding affinity to the androgen receptor than testosterone, DHT is an active metabolite of testosterone. It is involved in several diseases in older males, including benign prostatic hyperplasia and androgenic alopecia.
Traditionally, two radio-label methods, which involve – and -testosterone, are used for inhibitory screening assays of the enzyme reaction. An in vitro biochemical assay using the recombinant enzyme is not applicable. Therefore, a cell-based high-throughput screening system with minimal sample preparation and maximal sensitivity should be developed to efficiently screen 5α-reductase inhibitors in complex biological matrices. However, this requires chemical derivatization of androgen metabolites or the radio-labeled substrates, which make them difficult for the high-throughput screening. In the present study, we develop a high-throughput screening system based on the direct and rapid determination of DHT produced from cultured prostate cells using TFC-LC-TQMS without labor-intensive manual sample preparation.
We have also discovered bioactive chemicals that modulate the DHT production for the potential candidates of nutraceuticals associated with male hormonal dysfunctions. Seoul National University, College of Pharmacy. LNCaP, DU145, and PC-3 human prostate cancer cells were purchased from ATCC. 96-well plate and incubated for 24 h. The cells were incubated with 300 μl fresh RPMI medium supplemented with the vehicle control, testosterone alone, or testosterone and tested chemicals for 48 h. C for further MS analysis.
We selected this chemical as the internal standard because it has similar retention time to DHT, which is a key metabolite for the screening. Solvent A and acetonitrile as Solvent B. Solvent B was used for 3. The column was maintained at room temperature. Positive-ion electrospray tandem mass spectra were recorded, and the obtained results were processed with the Analyst 1. GS1, and GS2 at 30, 35, and 40, respectively. The two SRM transitions for testosterone, DHT, and internal standard were used to enhance the selectivity of the detection signal in the culture medium.
MS method, whose detection limits of testosterone and DHT were 0. DU145 cells were treated with DMSO. MS method showed two prominent signals of testosterone and DHT at 2. MS method is suitable to discover bioactive chemicals that modulate the DHT production in cell reaction systems. To investigate the time-dependent conversion of testosterone to DHT in various human prostate cancer cells, 4 different prostate cancer cells were treated with testosterone for 6, 12, 24, and 48 h. Among four different prostate cell lines, DU145 had the highest enzyme activity, and there was an 8.