Combination of micellar electrokinetic and high-performance liquid chromatographies to assess age-related changes in the in vitro metabolism of Fischer 344 rat liver

Abstract

The metabolism of doxorubicin, a widely used anticancer drug, is different in young adult and old cancer patients. In this study, we demonstrate that micellar electrokinetic chromatography with laser-induced fluorescence detection is highly suited to monitor the metabolism of doxorubicin in subcellular fractions isolated from young adult (11 months, 100% survival rate) and old (26 months, ~25% survival rate) Fischer 344 rat livers. The relative amounts of doxorubicin metabolized in both mitochondria-enriched and postmitochondria fractions of young adult were larger than the respective fractions of old rat liver. 7-Deoxydoxorubicinolone and 7-deoxydoxorubicinone were identified using internal standard addition and structural elucidation by high-performance liquid chromatography with combined laser-induced fluorescence and mass spectrometry detection. Although high-performance liquid chromatography with combined laser-induced fluorescence and mass spectrometry detection is more useful in the identification of compounds, micellar electrokinetic chromatography with laser-induced fluorescence detection has low-sample requirements, simplified sample processing procedures, short analysis times and low limit of detection. Therefore, the combination of these two techniques provides a powerful approach to investigate metabolism of fluorescent drugs in aging studies.

Figure 1.

Possible doxorubicin (DOX) metabolic pathways (13). Changes in the chemical structures upon reaction are indicated by boxes. (I) Carbonyl reduction by nicotinamide adenine dinucleotide phosphate-dependent carbonyl reductase, (II) reductase-type deglycosidation by NADPH-cytochrome c reductase, (III) hydrolase-type deglycosidation by glycosidase, and (IV) demethylation by cytochrome P450. The possible metabolic pathway of DOX in this study is shown in bold arrows.

Figure 2.

Identification metabolites with the aid of internal standard addition and HPLC-LIF-MS. (A) Electropherograms of 10 μM doxorubicin (DOX) incubated in mitochondria-enriched fraction (MF) for 1 h (Trace a) and with internal addition of DOX, DOXol, and DOXone (Trace b). Trace b is y-axis offset for clarity. M1 is 7-deoxyDOXone and M2 is 7-doxyDOXolone. Separations were performed in a 45.7-cm long, 50-μm i.d.–fused silica capillary at 400 V/cm in BS50-γ-CD20 buffer. Analytes were excited at 488 nm and fluorescence was detected at 635 ± 27.5 nm. (B) Mass chromatogram of DOX metabolites formed in a liver postmitochondria fraction (PMF). The PMF was treated with 50 μM doxorubicin and acid extracted after 10 min. Separation was conducted in a 150 × 0.3 mm C18 column under isocratic conditions (67% water, 0.1% formic acid: 33% acetonitrile). The ESI-MS used a Mass spectrometry was done with a Bruker MicrOTOFQ instrument. Peak 1: DOX, 2: 7-deoxyDOXolone, 3: Doxone, and 4: 7-deoxyDOXone. Doxone is formed during sample preparation and is not considered a natural metabolite (24).

Figure 3.

Effect of cofactors on the metabolic activity of rat liver. Electropherograms of 10 μM doxorubicin incubated in MF for 1 h without cofactors (Trace a) or with 1 mM β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate and 5 mM Mg²⁺ (Trace b); trace b is y-axis offset for clarity. Separations were performed in a 45.3-cm long, 50-μm i.d.–fused silica capillary. Other conditions were same as in Figure 2A.

Figure 4.

In vitro metabolism of doxorubicin (DOX) in postmitochondria fraction (PMF) and mitochondria-enriched fraction (MF) of young adult and old rat livers. Electropherograms of DOX metabolism in (A) PMF of young adult rat liver; (B) MF of young adult rat liver; (C) PMF of old rat liver, and (D) MF of old rat liver after 15 min (trace a), 30 min (trace b), 60 min (trace c), and 120 min (trace d). Traces b, c, and d are y-axis offset for clarity. M1 is 7-deoxyDOXone and M2 is 7-deoxyDOXolone. Separations were performed in a 45-cm (A and B) or 45.8-cm (C and D) long, 50-μm i.d.–fused silica capillary. Other conditions were same as in Figure 2A.

Figure 5.

Relative abundance of doxorubicin (DOX) and its metabolites in postmitochondria fraction (PMF) and mitochondria-enriched fraction (MF) as a function of incubation time. (A) Changes in DOX and 7-deoxyDOXolone in the young adult and old PMF. (B) Changes in DOX and 7-deoxyDOXolone in the young adult and old MF. (C) Changes in 7-deoxyDOXone, M3, and M4 in the young adult and old PMF. (D) Changes in 7-deoxyDOXone and M3 in the young adult and old MF. Percentages (molar) are calculated using equation 2. The young adult and old rat livers are represented in solid and dash lines, respectively. DOX and metabolites are labeled as DOX (×), 7-deoxyDOXone (◊), 7-deoxyDOXolone (o), M3 (Δ), and M4 (□).