Distribution of platinum between nuclear and cytosolic fractions – Can subcellular fractionation be performed quantitatively?
[Display omitted] •Cisplatin-exposed cells were fractionated into cytosol and nuclei.•Obtained nuclei were not contaminated with cytosol and vice versa.•18% and 66% of Pt taken up by cells were located in nuclei and cytosol respectively.•Accumulated uncertainty and fraction purity had a negative eff...
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Veröffentlicht in: | Journal of pharmaceutical and biomedical analysis 2019-02, Vol.165, p.82-89 |
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Format: | Artikel |
Sprache: | eng |
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•Cisplatin-exposed cells were fractionated into cytosol and nuclei.•Obtained nuclei were not contaminated with cytosol and vice versa.•18% and 66% of Pt taken up by cells were located in nuclei and cytosol respectively.•Accumulated uncertainty and fraction purity had a negative effect on Pt recovery.•Quantitative fractionation is feasible with an 81% recovery.
The feasibility of quantitatively tracking platinum, derived from platinum-based compounds, during subcellular fractionation was studied. Cisplatin-exposed murine Ehrlich Lettré Ascites cells were fractionated into cytosolic and crude nuclear fractions. The latter was subsequently purified. Every residue and fraction produced during the fractionation procedure were collected and the platinum content determined by inductively coupled plasma mass spectrometry. Western blotting verified that the nuclear and cytosolic fractions were pure. It was found that 18% of platinum taken up by the cells was located in the nuclear fraction while 66% was located in the cytosolic fraction. Accumulated uncertainty originating from invariable sample characteristics and giving fraction purity priority had a negative effect on platinum recovery. Thus, overall 81% (n = 3, RSD = 3.4%) of the platinum taken up by the cells was recovered in the residues and final fractions. In conclusion, a reliable intracellular localization and quantitation of platinum following administration of Cisplatin can be determined by application of the method. |
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ISSN: | 0731-7085 1873-264X |
DOI: | 10.1016/j.jpba.2018.11.046 |