Ciarán F. Duffy

Determination of the cardiolipin content of individual mitochondria by capillary electrophoresis with laser-induced fluorescence detection

We report the application of capillary electrophoresis (CE) with postcolumn laser‐induced fluorescence (LIF) detection to measure the cardiolipin content of individual mitochondria from cultured NS1 cells. Mitochondria were isolated by differential centrifugation and stained with the fluorescent dye 10‐N‐nonyl acridine orange which stoichiometrically binds to cardiolipin in a 1:1 or 2:1 ratio depending on the dye concentration. The green fluorescence resulting from the 1:1 complex was chosen for analysis because it is substantially more intense than the red fluorescence resulting from the 2:1 complex. Two dye concentrations that resulted in maximal and submaximal formation of the 1:1 10‐N‐nonyl acridine orange – cardiolipin complex were identified by spectrofluorometry. Individual mitochondria stained with both dye concentrations were separated and detected by CE with LIF detection. The data from mitochondria dosed with the lower dye concentration, where it is assumed that all the dye added to the mitochondrial sample was bound to cardiolipin, were used to derive a sensitivity factor relating fluorescence intensity of a mitochondrial event to its cardiolipin content. Using this factor, the cardiolipin contents of individual mitochondria stained with the higher dye concentration were determined, and ranged from 1.2 to 920 amol, with a median value of 4 amol. These results suggest a new strategy for estimating the organellar content of compounds that can be fluorescently tagged.

Determination of individual microsphere properties by capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis with postcolumn laser‐induced fluorescence detection was used to individually detect 6.0, 1.0, 0.5, and 0.2 νm diameter polystyrene microspheres and individually measure their electrophoretic mobility. The analysis of a nanoliter‐size volume from a microsphere suspension results in an electropherogram characterized by several narrow spikes in a well‐defined migration time window. Each spike is associated with one microsphere because, when one single microsphere is introduced into the capillary by micromanipulation, the electropherogram has only one spike in the same migration time window. The distributions of individual measurements resulting from an electropherogram were used to evaluate the reproducibility from run to run, observe the effect of sodium dodecyl sulfate (SDS) added to the running buffer, and to investigate the origin of electrophoretic dispersion. As expected from the interactions between microspheres and SDS, the addition of this surfactant to the running buffer narrowed the range and shifted the average electrophoretic mobility to more negative values. After evaluating common sources of broadening in capillary electrophoresis, electrophoretic dispersion was attributed to microsphere heterogeneity. Unlike electropherograms displaying Gaussian‐like profiles, the two‐dimensional representations of the individual measurements provide a new alternative to evaluate and study electrophoretic‐related properties of microspheres.

Detection of heteroplasmy in individual mitochondrial particles

AbstractMitochondrial DNA (mtDNA) mutations have been associated with disease and aging. Since each cell has thousands of mtDNA copies, clustered into nucleoids of five to ten mtDNA molecules each, determining the effects of a given mtDNA mutation and their connection with disease phenotype is not straightforward. It has been postulated that heteroplasmy (coexistence of mutated and wild-type DNA) follows simple probability rules dictated by the random distribution of mtDNA molecules at the nucleoid level. This model has been used to explain how mutation levels correlate with the onset of disease phenotype and loss of cellular function. Nonetheless, experimental evidence of heteroplasmy at the nucleoid level is scarce. Here, we report a new method to determine heteroplasmy of individual mitochondrial particles containing one or more nucleoids. The method uses capillary cytometry with laser-induced fluorescence detection to detect individual mitochondrial particles stained with PicoGreen, which makes it possible to quantify the mtDNA copy number of each particle. After detection, one or more particles are collected into polymerase chain reaction (PCR) wells and then subjected to real-time multiplexed PCR amplification. This PCR strategy is suitable to obtain the relative abundance of mutated and wild-type mtDNA. The results obtained here indicate that individual mitochondrial particles and nucleoids contained within these particles are not heteroplasmic. The results presented here suggest that current models of mtDNA segregation and distribution (i.e., heteroplasmic nucleoids) need further consideration. FigureSetup for collection of individual mitochondrial particles into PCR vials after their laser-induced fluorescence detection (image in the background). Laser-induced fluorescence detection of individual mitochondria particles (upper right). Multiplex real time PCR of the collected sample reveals the presence of wild type and deleted mtDNA (i.e., heteroplasmy) when the traces cross the upper and lower thresholds, respectively (lower right).

Determination of 7-hydroxycoumarin and its glucuronide and sulphate conjugates in liver slice incubates by capillary zone electrophoresis

The simultaneous detection of the phase I metabolite of coumarin, 7-hydroxycoumarin, and the two phase II metabolites, 7-hydroxycoumarin-glucuronide and 7-hydroxycoumarin-sulphate by capillary electrophoresis with UV detection using liver slice incubations was investigated. Separation was carried out on an untreated fused silica capillary with detection at 320 nm. Separation was achieved in under 6 min with a total run time of 8 min. Both phase two metabolites were produced following an in vitro incubation of liver slices in Krebs-Hanseleit buffer with 100 microM 7-hydroxycoumarin. Limits of detection were 5.52 microM (2 micrograms ml-1) for the glucuronide, 2.21 microM (0.5 microgram ml-1) for the sulphate and 6.17 microM (1 microgram ml)-1 for 7-hydroxycoumarin. Mean inter- and intra-assay results are presented for all three analytes, respectively.

Determining under- and oversampling of individual particle distributions in microfluidic electrophoresis with orthogonal laser-induced fluorescence detection.

This report investigates the effects of sample size on the separation and analysis of individual biological particles using microfluidic devices equipped with an orthogonal LIF detector. A detection limit of 17 ± 1 molecules of fluorophore is obtained using this orthogonal LIF detector under a constant flow of fluorescein, which is a significant improvement over epifluorescence, the most common LIF detection scheme used with microfluidic devices. Mitochondria from rat liver tissue and cultured 143B osteosarcoma cells are used as model biological particles. Quantile–quantile (q–q) plots were used to investigate changes in the distributions. When the number of detected mitochondrial events became too large (>72 for rat liver and >98 for 143B mitochondria), oversampling occurs. Statistical overlap theory is used to suggest that the cause of oversampling is that separation power of the microfluidic device presented is not enough to adequately separate large numbers of individual mitochondrial events. Fortunately, q–q plots make it possible to identify and exclude these distributions from data analysis. Additionally, when the number of detected events became too small (<55 for rat liver and <81 for 143B mitochondria) there were not enough events to obtain a statistically relevant mobility distribution, but these distributions can be combined to obtain a statistically relevant electrophoretic mobility distribution.

In vitro glucuronidation of 7-hydroxycoumarin and determination of 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide by capillary electrophoresis

Abstract A method was developed for the determination of the in vitro production of 7-hydroxycoumarin glucuronide and β-glucuronidase activity in rabbit tissue homogenates (liver, kidney, heart, lung, spleen, large intestine and fat). Separation of 7-hydroxycoumarin (7-HC) and 7-hydroxycoumarin glucuronide (7-HCG) by capillary electrophoresis was carried out on a 27 cm untreated fused silica capillary using a 100 m M phosphate buffer, pH 7.0 at 17.5 kV, with detection at 320 nm. 7-HC and 7-HCG were separated within 4 min and could be determined in the same run. Rabbit tissues, containing uridine diphosphate (UDP) glucuronyl transferase (UDPGT), were homogenised in Tris–HCl, pH 7.4, and used for the production of 7-HCG by the reaction of 7-HC and UDP-glucuronic acid (UDPGA). The conversion of 7-HC to 7-HCG is catalysed by UDPGT and the reverse reaction by β-glucuronidase. A sample of the reaction mixture was removed and added to acetonitrile, centrifuged and analysed by capillary electrophoresis. For the reverse reaction (β-glucuronidase reaction), the rabbit tissue samples were homogenised in 100 m M acetate buffer, pH 4.3. To this 7-HCG was added and its metabolism to 7-HC and the decrease in 7-HCG content was determined after stopping the reaction with a β-glucuronidase inhibitor, protein precipitation and centrifugation. β-Glucuronidase activity was observed in all tissue types, but not all tissues displayed UDPGT activity. The highest UDPGT activity was detected in the liver, followed by the kidney. The limit of detection was 1 μg/ml for 7-HC, and 2 μg/ml for the glucuronide, with a linear detection range for both analytes from 0–100 μg/ml.

Determination by HPLC of the UDP-Glucuronyl Transferase Activity For 7-Hydroxycoumarin in Rabbit Tissue Samples

Abstract HPLC was used to determine the uridine diphosphate (UDP) glucuronyl transferase activity in rabbit tissues. The crude tissue samples prepared were from the liver, kidney, bladder, large intestine, lung, spleen, heart and fat. A metabolic reaction mixture was prepared that included the rabbit tissue sample, 7-hydroxycoumarin and UDP-glucuronic acid (UDPGA). The method was used to determine the level of production of 7-hydroxycoumarin-glucuronide from 7-hydroxycoumarin. Seperation of the analyte was carried out under gradient elution by reverse phase chromatography on a C18 column, with UV detection at 320 nm. It was possible to determine 7-hydroxycoumarin-glucuronide (7-OHCG) produced over time as well as the decrease in 7-hydroxycoumarin concentration as the reaction progressed. The rate of the reaction was calculated from a plot of the concentration of 7-OHCG produced versus time for each organ, and the rate calculated from the slope of the linear part of the curve. The liver showed the highest ...