Development of a Liquid Chromatography/Mass Spectrometry-Based Inhibition Assay for the Screening of Steroid 5-α Reductase in Human and Fish Cell Lines
Abstract
:1. Introduction
2. Results
2.1. Method Validation
2.1.1. Linearity of the Calibration Curve and LLOQ
2.1.2. Accuracy and Precision
2.2. Assay Application in Human Cell Lines
2.3. Assay Application in SRD5A2-Overexpressing HEK-293 Cells
2.4. Assay Application in a Fish Cell Line
3. Discussion
4. Materials and Methods
4.1. Chemicals and Reagents
4.2. Cell Culture
4.3. Transient Overexpression
4.4. Cell Culture Assay Application
4.5. qRT PCR
4.6. Sample Preparation
4.7. Instrumental Conditions
4.8. Calibration Curve and LLOQ
4.9. Accuracy and Precision
4.10. Data Analysis
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
Sample Availability
References
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Low QC | Medium QC | High QC | |
---|---|---|---|
CV%—inter day a | 1.3 | 0.7 | 1.6 |
CV%—intra day b | 0.9 | 2.5 | 1.3 |
Accuracy%—inter day | 102.3 | 104.0 | 95.0 |
Accuracy%—intra day | 101.0 | 98.9 | 95.5 |
Vmax (nmol L−1 h−1) | KM (nM) | ||
---|---|---|---|
Human cell lines | LNCaP | 38.67 (34.36–45.16) * | 15.10 (12.35–19.25) |
DU-145 | 75.55 (66.61–90.45) | 9.15 (7.01–12.81) | |
SW-13 | 29.35 (27.94–30.90) | 19.42 (17.31–21.88) | |
Overexpression lines | Non vector | 4.473 (4.29–4.66) | 7.56 (6.74–8.46) |
SRD5A1 | 8.584 (7.86–9.35) | 2.29 (1.70–3.09) | |
SRD5A2 | 22.52 (20.01–25.42) | 0.36 (0.18–0.65) | |
Fish cell lines | ZFL | 116.60 (106.9–126.9) | 0.46 (0.30–0.68) |
RTG-2 | 13.09 (11.88–14.37) | 1.12 (0.78–1.61) |
IC50 Value | |||
---|---|---|---|
Finasteride (nM; 95% CI *) | Dutasteride (nM; 95% CI) | ||
Human cell lines | LNCaP | 241.0 (185.8–303.9) | 1.26 (1.02–1.57) |
DU-145 | 308.5 (217.0–415.5) | 3.83 (3.10–4.78) | |
SW-13 | 213.5 (180.2–250.7) | 4.75 (4.26–5.32) | |
Overexpression lines | SRD5A1 | 332.8 (260.9–424.8) | 1.27 (0.76–2.06) |
SRD5A2 | 69.83 (33.65–133.3) | 1.19 (0.96–1.47) | |
Fish cell lines | ZFL | 142.4 (121.5–165.7) | 7.33 (6.12–8.77) |
RTG-2 | 2667 (2394–2952) | 13.19 (10.73–16.54) |
Zebrafish | Rainbow Trout | ||
srd5a1 | srd5a1 | ||
Human srd5a1 | 51.7 | 50.2 | |
Zebrafish | Rainbow Trout | ||
srd5a2a | srd5a2b | srd5a2a | |
Human srd5a2 | 52.3 | 42.4 | 50.2 |
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Kim, D.; Cho, H.; Eggers, R.; Kim, S.K.; Ryu, C.S.; Kim, Y.J. Development of a Liquid Chromatography/Mass Spectrometry-Based Inhibition Assay for the Screening of Steroid 5-α Reductase in Human and Fish Cell Lines. Molecules 2021, 26, 893. https://doi.org/10.3390/molecules26040893
Kim D, Cho H, Eggers R, Kim SK, Ryu CS, Kim YJ. Development of a Liquid Chromatography/Mass Spectrometry-Based Inhibition Assay for the Screening of Steroid 5-α Reductase in Human and Fish Cell Lines. Molecules. 2021; 26(4):893. https://doi.org/10.3390/molecules26040893
Chicago/Turabian StyleKim, Dahye, Hyunki Cho, Ruth Eggers, Sang Kyum Kim, Chang Seon Ryu, and Young Jun Kim. 2021. "Development of a Liquid Chromatography/Mass Spectrometry-Based Inhibition Assay for the Screening of Steroid 5-α Reductase in Human and Fish Cell Lines" Molecules 26, no. 4: 893. https://doi.org/10.3390/molecules26040893