Distribution of Zeptomole-Abundant Doxorubicin Metabolites in Subcellular Fractions by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Abstract

Doxorubicin (DOX) treatment of NS-1 mouse hybridoma cells results in the formation of zeptomole amounts of metabolites per cell that are difficult to determine by confocal microscopy or HPLC. The native fluorescence of DOX and its metabolites together with laser-induced fluorescence detection (HF) has previously been used to detect a maximum of four components. In this study, we use capillary electrophoresis with postcolumn LIF (CE-LIF) to separate and detect 12 components attributed to DOX metabolism, resulting from treatment of NS-1 cells with 25 microM DOX for 8 h. The so-called metabolites 8 and 10 have been identified as doxorubicinone (DOXone) and 7-deoxydoxorubicinone (7-deoxyDOXone), respectively, by comigration with the corresponding synthetic standard. Due to comigration of DOX with doxorubicinol (DOXone), the presence of DOXone had to be determined separately by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The rest of the metabolites remain unidentified and are referred to by their number assignment. In comparison with the whole cell lysate, fractionation by differential centrifugation results in a better separation resolution of metabolites due to reduced amounts of metabolites in each fraction. This approach was chosen to compare the distribution of 13 metabolites in three subcellular fractions that form a pellet at< 1,400 g, 1,400-14,000 g, and > 14, 000 g and that generically are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosolic components, respectively. The most abundant metabolite, DOXone, was estimated to be 90 +/- 15, 18 +/- 2, and 60 +/- 12 amol/cell (n = 5) in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively. In contrast, the total amount of other metabolites in a given fraction varied from 0 to 1,300 zmol. 7-DeoxyDOXone is the only metabolite that was present at similar levels in the three fractions. Other salient observations are metabolites 3, 7, and 11 are not detectable in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively; metabolite 9 and DOXone are more abundant in the nuclear-enriched fraction than in the other two fractions. The observations presented here suggest that subcellular fractionation followed by CE-LIF could be a powerful diagnostic for monitoring drug distribution, which is highly relevant to DOX cytoxicity studies.

Capillary electrophoresis of single mammalian cells

“…In these studies, CE with LIF detection was used to monitor different mitochondria preparations by differentiating between intact and fragmented organelles and determining the number of mitochondria in a sample. Subcellular fractions containing nuclei, oraganelles, and cytosolic components prepared from mammalian cells also have been examined using CE with LIF detection [35].…”

Recent developments in the determination of urinary cancer biomarkers by capillary electrophoresis

“…Researchers have downscaled the sample volume for CE analysis to less than picoliters, but the mass detection limits remain in the zeptomole range [31,32]. Recently, CE has been reportedly used in single cell analysis [33][34][35] and even subcellular-level analysis [36,37]. (ii) CE's waste is safer for the environment than that from HPLC, which produces large amounts of organic waste, or than that from GC, which produces volatile air pollutants [28].…”

Separation of doxorubicin and doxorubicinol by cyclodextrin‐modified micellar electrokinetic capillary chromatography

“…Detection of DOX and DOXol was accomplished using a post-column detection system previously described [20]. A 635 6 27.5-nm bandpass filter (model XF3015, Omega Optical, Brattleboro, VT) was used to select DOX fluorescence [11], and a 535 6 17.5-nm bandpass filter (model XF3007, Omega Optical) was used to select fluorescein fluorescence. Sheath flow buffer for all analyses consisted of BS10 buffer.…”

“…While MEKC is capable of separating many DOX metabolites with low LOD and without preparative steps for the analysis of biological matrices [11], this technique is not capable of separating DOX from DOXol or separating DOXol stereoisomers. Cyclodextrin-modified MEKC (CD-MEKC) has previously been demonstrated to separate analytes with closely related structures such as pesticides and drugs [12,13], including their stereoisomers [14,15].…”

“…CE with LIF detection (CE-LIF) is the best technique available for DOX analysis due to low LOD (60 zmol for DOX) and nanoliter injection volumes [11]. By incorporating micelles into the separation buffer, direct analysis of biological samples (e.g.…”