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Abstracts of Oak Ridge Posters |
1 Divisions of Neurology, Pathology, and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center, and Department of Pediatrics, School of Medicine, University of Cincinnati, 3333 Burnet Ave., Cincinnati, OH 45229-3039
aauthor for correspondence: fax 513-636-8850, e-mail peter.tang{at}cchmc.org
Reactive oxygen species are highly reactive molecules and have been implicated in the pathophysiology of many diseases, including diabetes mellitus, cancer, rheumatoid arthritis, and cardiovascular, renal, inflammatory, infectious, and neurologic diseases (1)(2). Cells and biological fluids have an array of protective antioxidant mechanisms, both for preventing the production of free radicals and for repairing oxidative damage (3). These antioxidant systems include enzymes, macromolecules, and small molecules, including ascorbic acid, 
-tocopherol, ß-carotene, ubiquinol-10, reduced lipoic acid (DHLA), reduced glutathione (GSH), methionine, uric acid, bilirubin, and some amino acids. Antioxidants within cells, cell membranes, and extracellular fluids can be up-regulated and mobilized to neutralize excessive and inappropriate formation of reactive oxygen species, but a deficiency of antioxidant defense may lead to a situation of increased oxidative stress.
Assays that measure the combined antioxidant effect of the nonenzymatic defenses in biological fluids may be useful in providing an index of ability to resist oxidative damage. Several methods (4)(5)(6) have been developed to assess the total antioxidant capacity of human serum or plasma because of the difficulty in measuring each antioxidant component separately and the interactions among different antioxidant components in the serum or plasma. However, the measured antioxidant capacity of a sample depends on which technology and which free radical generator or oxidant is used in the measurement. The ferric reducing ability of plasma (FRAP) assay uses an easily reduced oxidant in a redox-linked colorimetric method (6). Because the redox potential of the ferrous/ferric couple is 0.77 V, any substance or antioxidant with a redox potential <0.77 V will drive the ferric reduction, assuming stability of redox product. According to a study by Cao and Prior (7), the FRAP assay does not measure serum proteins and excludes the low-molecular-weight SH-group-containing antioxidants, such as GSH, DHLA, and some amino acids. We developed a novel method for measuring the ubiquinone-9-reducing ability of plasma (URAP). This new method estimates antioxidant systems with redox potentials 
0.1 V, which will drive the reduction of ubiquinone-9 (Fig. 1A
).
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HPLC analysis was performed with an ESA Model 582 Solvent Delivery Module, AS1000 autosampler (Thermo Separation Products), and a reversed-phase Microsorb-MV column [150 x 4.6 mm (i.d.); 5-µm bead size] from Rainin. The mobile phase consisted of a mixture of anhydrous sodium acetate (4.2 g; Sigma), 15 mL of glacial acetic acid (Mallinckrodt), 15 mL of 2-propanol (Mallinckrodt), 720 mL of methanol (Fisher), and 250 mL of hexane (Fisher). The flow rate was 1.1 mL/min. The electrochemical detector with ESA Model 5200A CouloChem II has been described previously (8). The electrochemical cells were a postcolumn guard cell and analytical cell containing dual electrodes in series. The potential of the postcolumn guard cell was set at +0.9 V to oxidize any electrochemically active compounds. The first electrode potential of the analytical cell was set at –0.9 V to transform ubiquinone-9 to ubiquinol-9, and the second electrode was set at +0.35 V with a sensitivity of 1 µA for monitoring ubiquinol-9. The concentrations of ubiquinol-9 and ubiquinone-9 were determined simultaneously. Analyses were performed at ambient temperature (
25 °C). Systat® software, Ver. 10 (SPSS), was used for statistical analyses. Results are expressed as the mean (SD) unless noted otherwise. P <0.05 was considered significant.
Coenzymes Q1, Q2, Q4, Q6, Q7, Q9, and Q10 were purchased from Sigma. Ubiquinol-9 was prepared from ubiquinone-9 by use of sodium borohydride, as described previously (9). Ubiquinol-9 and ubiquinone-9 calibrators in the range 0–20 µmol/L were used to prepare calibration curves in plasma. The linear regression equations for peak height (y; in arbitrary units) and concentration (µmol/L) of the injected calibrator (x) were as follows: y = 4.577 x 10–6x – 0.002 (r = 0.999) for ubiquinol-9 and y = 7.677 x 10–6x – 0.002 (r = 0.999) for ubiquinone-9. To estimate the limit of detection of the method for ubiquinol-9, we treated a plasma sample (100 µL) with a basal ubiquinol-9 concentration of 25 nmol/L with 1-propanol (700 µL). Injection of a 100-µL aliquot of this plasma extract, corresponding to 0.3 pmol of ubiquinol-9, yielded a ubiquinol-9 peak with a signal-to-noise ratio of 4:1. The experiment was performed in quadruplicate, and the impression (CV) was 14%.
Blood was collected into a VacutainerTM Tube (Becton Dickinson) containing heparin as anticoagulant and mixed by gentle inversion five to six times. The Vacutainer Tube was not opened to ambient air and was placed on ice or kept refrigerated until processing. Blood was processed within 4 h of collection and centrifuged at 2000g for 10 min at 4 °C. Plasma was collected, placed in a capped polypropylene tube, and immediately stored without addition of argon or nitrogen at or below –70 °C until analysis. Plasma samples were obtained from 30 healthy individuals [17 women and 13 men; mean (SD) age, 40 (10) years]. All plasma samples were stored at –70 °C, and analyzed within 1 week. Thawed plasma (100 µL) was vortex-mixed with 1-propanol (600 µL) and ubiquinone-9 solution (100 µL) at ambient temperature for 1 min and centrifuged for 10 min at 5600g. The supernatant layer was transferred to an autosampler vial, and the first portion of the extract (100 µL) was injected directly into the HPLC system. The second portion of extract in the same vial was injected after storage for a certain time interval. Sample preparation and HPLC analyses were performed at ambient temperature.
When the test was performed with plasma but with no ubiquinone-9 added to the reaction mixture, the HPLC chromatogram showed three peaks, including ubiquinol-9, ubiquinol-10, and ubiquinone-10. Ubiquinone-9 was not detectable, whereas the concentration of ubiquinol-9 was 2% of the total concentrations of ubiquinol-10 (1 µmol/L) and ubiquinone-10 (0.1 µmol/L). No change of ubiquinol-9 was observed over 4 h at room temperature.
To obtain the optimum concentration for plasma reducing power, we studied three concentrations of ubiquinone-9 (2.5, 5, and 7.5 µmol/L). Plasma (200 µL) was vortex-mixed with 1-propanol (1200 µL) and ubiquinone-9 solution (200 µL) at ambient temperature for 1 min and centrifuged for 10 min at 10 000 rpm. The supernatant layer was transferred to a vial, and the first portion of the extract (100 µL) was injected directly into the HPLC system. Portions of extract in the same vial were injected repeatedly after storage for certain time intervals. Linear responses over a time period up to 5 h were observed for three concentrations of ubiquinone-9 tested. Results indicated that the 0- to 240-min reaction time was suitable for the measurement of URAP. A reaction time of 35 min between two injections was used in the program.
The dose–response characteristics of ubiquinone-9 toward plasma showed that the activity is concentration dependent. A ubiquinone-9 concentration of 7.5-µmol/L is recommended for use. Coenzymes Q1, Q2, Q4, Q6, and Q7 have also shown activities similar to that of coenzyme Q9; they are therefore potential candidates for measuring plasma reducing power. Ubiquinone-9 was chosen for the current method because reduced and oxidized coenzymes Q9 and Q10 can be measured simultaneously in a single and short HPLC run (8 min). The representative chromatograms from the analysis of plasma in Fig. 1B
were obtained from the same plasma reaction mixture at time 1 (t1) and time 2 (t2), respectively. Reduced and oxidized coenzymes Q9 and Q10 were measured in the same HPLC run. The retention times for ubiquinol-9, ubiquinol-10, ubiquinone-9, and ubiquinone-10 were 3.6, 4.2, 5.5, and 6.8 min, respectively. The increase in the ubiquinol-9 peak after a 35-min reaction time was accompanied by a decrease in the ubiquinone-9 peak, indicating that the increased amount of ubiquinol-9 was attributable to the reduction of ubiquinone-9. Calculation of the ubiquinol-9 production rate (nmol · L–1 · h–1) was based on the increment of ubiquinol-9 concentration between t1 and t2.
The URAP assay gives reproducible results: within-run imprecision for a single sample was 6.5% (n = 6); between-run imprecision for 5 days was 8% (n = 10). The calculated activities of the various antioxidant added to plasma are given in Table 1
. Addition of -tocopherol (25 µmol/L), Trolox (25 µmol/L), ascorbic acid (100 µmol/L), and uric acid (250 µmol/L) decreased the plasma reducing power by 25–44%, whereas addition of DHLA (12.5 µmol/L) and ubiquinol-10 (1.25 µmol/L) enhanced the reducing power of plasma. The dose–responses of ubiquinol-10 and DHLA were linear, showing that activity is concentration-dependent. DHLA is an antioxidant the concentration of which is low in human plasma. However, the functional part of DHLA as an antioxidant is the SH group, which is also present in nonprotein antioxidants such as GSH and some amino acids. DHLA was therefore used here to represent the SH-group-containing nonprotein compounds. Dose–responses were linear for DHLA and ubiquinol-10 within the concentration range 0–50 µmol/L. The reducing activity did not exist for bilirubin and albumin, because their redox half-reaction potentials are more positive than 0.1 V.
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The mean (SD) ubiquinone-9 reduction rate in 30 healthy individuals was 94.8 (23.8) nmol · L–1 · h–1; after 28 days of storage at –70 °C, repeated analysis of some specimens showed that the URAP values (n = 12) were not significantly different from the values of fresh specimens. No correlation study between the current method and other assays, such as oxygen radical absorbance capacity, FRAP, and trolox equivalent antioxidant capacity, was attempted because these assays use different technologies. The current method is sensitive and specific and can be used to detect the first-line antioxidant system. Clinical studies comparing different population groups, measuring changes in URAP values associated with particular pathologic states, and monitoring URAP values during various treatment strategies are now needed.
References
The following articles in journals at HighWire Press have cited this article:
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  H. Shimada, D. Kodjabachian, and M. Ishida Specific and rapid analysis of ubiquinones using Craven's reaction and HPLC with postcolumn derivatization J. Lipid Res., September 1, 2007; 48(9): 2079 - 2085. [Abstract] [Full Text] [PDF]
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