Abstract
Background: The percentage of reduced coenzyme Q10 (CoQ10H2) in total coenzyme Q10 (TQ10) is decreased in plasma of patients with prematurity, hyperlipidemia, and liver disease. CoQ10H2 is, however, easily oxidized and difficult to measure, and therefore reliable quantification of plasma CoQ10H2 is of clinical importance.
Methods: Venous blood was collected into evacuated tubes containing heparin, which were immediately placed on ice and promptly centrifuged at 4 °C. The plasma was harvested and stored in screw-top polypropylene tubes at −80 °C until analysis. After extraction with 1-propanol and centrifugation, the supernatant was injected directly into an HPLC system with coulometric detection.
Results: The in-line reduction procedure permitted transformation of CoQ10 into CoQ10H2 and avoided artifactual oxidation of CoQ10H2. The electrochemical reduction yielded 99% CoQ10H2. Only 100 μL of plasma was required to simultaneously measure CoQ10H2 and CoQ10 over an analytical range of 10 μg/L to 4 mg/L. Intra- and interassay CVs for CoQ10 in human plasma were 1.2–4.9% across this range. Analytical recoveries were 95.8–101.0%. The percentage of CoQ10H2 in TQ10 was ∼96% in apparently healthy individuals. The method allowed analysis of up to 40 samples within an 8-h period.
Conclusions: This optimized method for CoQ10H2 analysis provides rapid and precise results with the potential for high throughput. This method is specific and sufficiently sensitive for use in both clinical and research laboratories.
Coenzyme Q10 is an essential cofactor in the mitochondrial respiratory chain responsible for oxidative phosphorylation (1). Furthermore, coenzyme Q10 has a primary function as an antioxidant and is carried mainly by lipoproteins in the circulation (2). Approximately 60% of coenzyme Q10 is associated with LDL, 25% with HDL, and 15% with other lipoproteins (2). When LDL is subjected to oxidative stress in vivo (3), the reduced form of CoQ10 (CoQ10H2)1 functions as an antioxidant. It has been postulated that CoQ10H2 prevents lipid peroxidation in plasma lipoproteins and biological membranes (4). The antioxidative activity of CoQ10H2 depends not only on its concentration, but also on its redox status. Recent reports (5)(6)(7)(8)(9)(10)(11)(12)(13)(14) have suggested that the percentage of CoQ10H2 in total CoQ10 (CoQ10H2:TQ10) may be lower in patients with certain conditions, including Parkinson disease (5), prematurity (6), hemodialysis (7), chronic active hepatitis (8), liver cirrhosis (8), hepatocellular carcinoma (8), hyperlipidemia (9)(10), heart disease (11)(12), β-thalassemia (13), and DNA damage (14). Therefore, CoQ10H2 may be a useful marker of oxidative stress, and the measurement and function of CoQ10H2 are of considerable interest.
Several investigators have reported analytical techniques for measurement of CoQ10H2(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). In these publications, electrochemical (EC) detection was preferred for measurement of CoQ10H2 because of its high sensitivity. The EC reactions were measured at electrodes, which detected the current produced by the reduction of oxidized CoQ10 (CoQ10) or by the oxidation of CoQ10H2 (Fig. 1⇓ ).
Diagram depicting the EC reactivity of CoQ10H2 and CoQ10.
Investigation of CoQ10H2 in clinical studies has been hampered by instability during sample handling, storage, and processing (15)(16)(17)(18)(19)(20)(21). According to several investigators (22)(23)(24)(25), the concentration of CoQ10H2 decreases rapidly within 1 h after phlebotomy. At room temperature, it is oxidized at a rate of ∼3 nmol/L per min in the hexane extract of human plasma (23). Sample preparation may have a profound effect on the redox status of CoQ10, and the utmost care is required to ensure reliable quantification of CoQ10H2. Recently, investigators recommended that plasma samples be individually thawed, extracted, and analyzed immediately as a continuous process to minimize CoQ10H2 oxidation (22)(23)(24). This is very impractical for analyzing even small numbers of biological specimens. In earlier studies, biological fluid samples were converted into either CoQ10 by use of an oxidizing reagent such as ferric chloride, or into CoQ10H2 by a reducing agent such as sodium tetrahydroborate (NaBH4) or sodium dithionite (Na2S2O4; Table 1⇓ ). However, these methods are also inefficient, are susceptible to preanalytical degradation, and have increased potential for analytical error because of the lability of CoQ10H2.
We therefore developed a simple and rapid HPLC procedure with coulometric detection for simultaneous determination of CoQ10 and CoQ10H2 in human plasma.
Materials and Methods
materials
CoQ10 and coenzyme Q9 (CoQ9) were obtained from Sigma. Methanol, ethanol, 1-propanol, 2-propanol, hexane, sodium acetate, and glacial acetic acid were obtained from Fisher Scientific. All chemicals were HPLC grade and were used without further purification. Dade® immunoassay comprehensive tri-level controls were from Dade International.
apparatus
The HPLC-EC system configuration is depicted in Fig. 2⇓ . The system consists of an Model 582 Solvent Delivery Module (ESA) equipped with a double plunger reciprocating pump, an AS3000 variable-loop autosampler (Thermo Separation Products), an analytical column, an ESA CouloChem II Model 5200A EC detector, and a Dell Pentium II 350 Mz computer/controller with ChromQuest software (Thermo Separation Products). The system consisted of two cells (pre- and postcolumn) and an analytical cell (Fig. 2⇓ ). One carbon filter was placed before the precolumn cell and another between the analytical column and the postcolumn cell. Both pre- and postcolumn cells (E1 and E2) were coulometric electrodes (ESA Model 5020). The postcolumn cells were configured in series as described by Edlund (17). The analytical cell (ESA Model 5010) consisted of a series of two coulometric electrodes and was connected in series to the postcolumn cell; the first electrode (E3) was for reduction of CoQ10, and the second electrode (E4) was for detection of CoQ10H2.
Schematic diagram of the HPLC-EC system.
This system can be operated in three different modes, as described in the text.
The analytical column was a reversed-phase Microsorb-MV column (4.6 mm × 15 cm; 5 μm bead size; Rainin). A reversed-phase C18 guard column (4.6 × 10 mm; 5 μm bead size) was used to protect the analytical column. The AS3000 injector was set at a needle height of 1.5 mm, and the injection volume was set at 20 μL for each sample. The cooling temperature of the autosampler was set at 0 °C. The mobile phase for the isocratic elution of CoQ10 was prepared as follows: sodium acetate trihydrate (6.8 g), 15 mL of glacial acetic acid, and 15 mL of 2-propanol were added to 695 mL of methanol and 275 mL of hexane. The mobile phase was filtered through a 0.2 μm (47 mm diameter) nylon or analogous filter. The pH of the mobile phase was ∼6, and the flow rate was 1 mL/min.
preparation of calibrators
All sample preparation work was carried out under a dim light to avoid photochemical decomposition of CoQ10 and CoQ9. To prepare a 5 mg/L working solution of CoQ10, we dissolved 10 mg of CoQ10 in 10 mL of hexane and diluted this solution to 100 mL with 1-propanol. The solution was thoroughly vortex-mixed until complete dissolution. A working solution was then prepared by dilution with 1-propanol to 5 mg/L. The concentration of the working solution was then calculated by reading the absorbance on a spectrophotometer (275 nm wavelength; 1-cm quartz cuvette), using a molar absorptivity (ε) of 14 200. A series of calibration solutions was then prepared with the appropriate volume of 1-propanol to final CoQ10 concentrations of 10, 100, 500, 1000, 2000, and 4000 μg/L. The low control was prepared by diluting pooled plasma containing 0.45 mg/L CoQ10 with distilled water to a final concentration of 75 μg/L. The middle and high controls were prepared by adding working solutions containing 1.2 and 3.0 mg/L CoQ10 to pooled plasma samples to final concentrations of 1.65 and 3.45 mg/L, respectively. The calibrators and controls were stored in 1.8-mL polypropylene tubes (Sarstedt) without addition of argon or nitrogen at −80 °C and used throughout the study. CoQ9 was chosen as the internal standard. To prepare a CoQ9 solution, we dissolved 2 mg of CoQ9 in 100 mL of 1-propanol. The CoQ9 solution was thoroughly vortex-mixed until complete dissolution. A working solution of CoQ9 was then prepared by dilution with 1-propanol to 2 mg/L. All solutions were stored in 1.8-mL polypropylene tubes at −80 °C and used throughout the study.
preparation of plasma samples
Venous blood was collected into a Vacutainer® Tube (Becton Dickinson) containing heparin as anticoagulant and mixed by gentle inversion 5–6 times. The Vacutainer Tube was not opened to ambient air and was placed in ice or kept refrigerated before processing. Blood samples were 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 −80 °C until analysis.
liquid-liquid extraction
Under our experimental conditions, we optimized the extracting procedure of Edlund (17) and compared the efficiency of different mixtures of organic solvents for liquid-liquid extraction of CoQ10 and CoQ9 from human plasma. Quantitative recoveries (∼100%) of these compounds were obtained with two solvents: 1-propanol and a mixture of ethanol-hexane (2:5 by volume). The 1-propanol extraction procedure was used for subsequent studies.
coulometric detection
The hydrodynamic voltammograms were obtained by repeated injections into the HPLC system of a mixture of CoQ9 (1 mg/L) and CoQ10 (4 mg/L) in water-1-propanol (1:9 by volume). The detector potential was increased by 0.05 V in each subsequent run. Anodic currents for CoQ10H2 and cathodic currents for CoQ10 reached maximum response at applied voltages of +0.35 V and −0.65 V, respectively. On the basis of the assessed hydrodynamic voltammogram, the E2 cell potential was always set at +0.7 V to oxidize any electrochemically active eluates. The E3 and E4 cell potentials were set at −0.65 V and +0.45 V, respectively. When the E1 cell potential was set to −0.7 V for the precolumn reduction mode, all CoQ10 was reduced to CoQ10H2 before column separation. Total CoQ10H2 was then measured, and a calibration curve of CoQ10H2 was established. For the precolumn oxidation mode, the E1 cell potential was set at +0.7 V. All CoQ10H2 was oxidized to CoQ10, and TQ10 was measured. A calibration curve of CoQ10 was thus obtained. For simultaneous determination of CoQ10H2 and CoQ10, the E1 cell was turned off. Because no current flowed into the cell, all compounds remained at their original state.
assessment of possible interfering substances
To explore possible sources of interference, we processed several lyophilized products from human blood, highly purified chemicals, and biochemicals (included in the Dade high control) according to the developed method. Briefly, Dade high control, which contains 45 drugs and endogenous substances, was supplemented with 20 commonly prescribed drugs at concentrations exceeding clinically relevant values (Table 2⇓ ); 100-μL aliquots of the supplemented control were then placed in 1.8-mL capped polypropylene tubes, processed, and analyzed.
To assess the possible interference of endogenous CoQ9 or other substances in patient plasma samples, blood samples were collected from 25 patients (ages 1–18 years) in the neurology clinic at the Children’s Hospital Medical Center, Cincinnati, OH. These patients were diagnosed with a variety of neurological disorders. An additional 25 specimens were obtained from apparently healthy individuals (ages 0.2–65 years). Informed consent was obtained from all adults and from the parents (or guardians) of all minors.
sample analysis
We simultaneously processed samples in batches of 20, which is the capacity of our centrifugation instrument. Each frozen sample was thawed at room temperature, and then a 100-μL aliquot of the sample was placed in a 1.8-mL capped polypropylene tube containing 50 μL of internal standard solution. All tubes were kept in an ice bath. The sample was then mixed with 850 μL of cold 1-propanol. All tubes were vortex-mixed for 2 min on a mechanical vortex-type mixer and centrifuged for 10 min at 21 000 g and 0 °C. The resulting supernatant was separated from the precipitate and transferred to a glass autosampler vial. Sample vials were immediately placed in the autosampler tray at 0 °C. A batch of 20 samples was analyzed immediately in a single run sequence. A 20-μL aliquot of 1-propanol extract from a vial was injected immediately into an automated HPLC. Peak height and area measurements for each injection were obtained by the ChromQuest software. The CoQ10:CoQ9 peak-height ratios were used (peak area was optional) to obtain least-squares linear regression equations, which were used to calculate the CoQ10 concentrations of the frozen control samples and patient samples. If an error occurred in the system, the sample vials were resealed and immediately restored at −80 °C or below for further investigation. A single technician could complete the analysis of a 20-specimen batch routinely within 4 h.
preliminary reference interval data
To evaluate CoQ10H2:TQ10 reference intervals, blood samples were obtained from 25 apparently healthy individuals (5 males and 20 females; age range, 12–64 years) after obtaining their consent. Individuals were carefully screened and excluded if taking any medication chronically, had any history of acute or chronic illness, or were taking any form of coenzyme Q10 as a supplement.
Results
efficiency of reduction cell
CoQ10 was converted to CoQ10H2 electrochemically by the reduction cell. The efficiency of conversion was measured by comparing the peak heights of CoQ10H2 and CoQ10 per injected amount of CoQ10. A series of solutions containing 0.01, 0.1, 1.0, 1.5, 2.0, 3.0, and 4.0 μg/L of CoQ10 in water/1-propanol (1:9 by volume) were injected in duplicate into the HPLC system. Quantitative conversion rates of 99.4% ± 0.5% were obtained.
chromatographic analysis
As seen in Fig. 3⇓ , CoQ10 and CoQ10H2 could be measured in the same HPLC run. CoQ9 and CoQ10 eluted at ∼5.5 and ∼6.9 min, respectively (Fig. 3A⇓ ). Two peaks were observed for the reduced form (CoQ9H2) of CoQ9 (internal standard) and CoQ10H2 at ∼3.6 and ∼4.1 min, respectively (Fig. 3B⇓ ).
HPLC chromatograms of CoQ10 and CoQ9 calibrators.
(A), chromatogram showing two oxidation peaks for CoQ10 (8 ng on column) and CoQ9 (2 ng) calibrators at ∼5.5 and ∼6.9 min, respectively. (B), chromatogram showing two oxidation peaks for CoQ9H2 and CoQ10H2 calibrators at ∼3.6 and ∼4.1 min, respectively. Precolumn reduction was performed to transform CoQ10 and CoQ9 to CoQ10H2 and CoQ9H2, respectively.
calibration curves and linearity
Calibration curves for CoQ10H2 and CoQ10 are shown in Fig. 4⇓ . An excellent linear relationship was observed between the peak-height ratios of each compound vs CoQ9 over a wide concentration range from 10 μg/L to 4 mg/L. The regression equations were: y = 1.151x + 0.003 (r2 = 0.999) for CoQ10H2; and y = 0.846x + 0.001 (r2 = 0.998) for CoQ10. The detection limits of CoQ10H2 and CoQ10 were ∼5 μg/L (signal-to-noise ratio = 3).
Calibration curves for CoQ10 (○) and CoQ10H2 (□).
CoQ10 calibrators were dissolved in 1-propanol.
extraction efficiency
Under our experimental conditions, quantitative recoveries of CoQ10 and CoQ9 for the current method using 1-propanol were compared with previously published extraction methods (Table 1⇓ ). With the 1-propanol method, the mean recoveries were 99% ± 3% for CoQ10 and 100% ± 2% for CoQ9 (n = 6). Comparison with other extraction solvents (n = 6 replicates each) produced the following mean recoveries: 2-propanol, 89% ± 5%; ethanol, 88% ± 4%; n-butanol, 85% ± 5%; acetone, 71% ± 8%; methanol-hexane (0.2:2.5 by volume), 64% ± 10%; hexane, 52% ± 9%; acetonitrile, 19% ± 11%; and methanol, 19% ± 10%.
stability of CoQ10H2 in stored whole blood
Venous blood was collected from five healthy adults in tubes containing sodium heparinate. Blood samples, which were kept on ice or in refrigerated at 4 °C, were processed identically at hourly intervals up to 8 h after collection. Plasma from each blood specimen was separated and frozen at −80 °C until analysis. The results showed that CoQ10H2 in whole blood stored at 4 °C was stable for at least 8 h with a CV <5%. The mean (SD) ratio of CoQ10H2:TQ10 in 25 heparinized whole blood specimens was 95.3% (± 1.8%) 8 h after blood collection (4 °C). On the basis of these findings, we recommend that blood for CoQ10H2 analysis be refrigerated to ensure sample stability for up to 8 h after collection.
precision and accuracy
The analytical recoveries of CoQ10 in human plasma controls are shown in Table 3⇓ . The inter- and intraday assay CVs were <5% over four concentrations of CoQ10. Because CoQ10H2 is oxidized rapidly, no control tests for CoQ10H2 were performed. To verify the reproducibility of the CoQ10H2 analysis, human plasma samples from 10 healthy individuals were examined (5 replicates each; Table 4⇓ ). The reproducibility of the analysis is presented in Table 4⇓ . The CVs for the ratio of CoQ10H2:TQ10 were ≤1.0%, which shows the excellent reproducibility of analysis.
Reproducibility of the analysis of coenzyme Q10 in human plasma.
interference studies
Testing of the supplemented Dade control indicated that a few unidentified electroactive compounds and substances (Table 2⇓ ) eluted from the column within the first 3.5 min (data not shown). These compounds and substances may have been more hydrophilic than CoQ10H2 because an organic solvent-based mobile phase was used. Only one unknown compound eluted at ∼6.9 min, which corresponded exactly with the CoQ10 elution time. Because the Dade control is plasma-based, this peak most likely was residual CoQ10 in this control. None of the lyophilized products of human blood, highly purified chemicals, biochemicals, and medications added to the Dade High Control produced interference in the analysis.
Comparison between current and previous studies on CoQ10H2 analysis.
Lyophilized products from human blood, highly purified chemicals and biochemicals, and commonly prescribed drugs tested for potential interference with HPLC-EC.
To assess the possible interference of endogenous CoQ9 with that added as internal standard, 50 plasma samples, including 25 from apparently healthy individuals and 25 from patients, were extracted without adding the internal standard, CoQ9. Only one plasma sample, from a patient with a rare glycogen storage disease (type I), was found to have ∼25 μg/L CoQ9, which corresponded to ∼2.5% of the internal standard concentration. Measurable CoQ9 was not detected in the plasma samples from the remaining 25 healthy individuals or 24 patients. On the basis of these findings, we conclude that interference from endogenous CoQ9 is very unlikely to cause significant analytical error with our method, and thus is a very suitable internal standard (Fig. 5⇓ ).
Chromatograms of extracts of a patient’s plasma containing 1.693 mg/L CoQ10H2 and 43 μg/L CoQ10 with (B) or without (A) CoQ9 internal standard (2 ng on column).
preliminary reference interval results
To provide preliminary data for establishing the reference interval for the ratio of CoQ10H2:TQ10, plasma specimens were collected from 25 apparently healthy individuals. The CoQ10H2:TQ10 ratio was 96.3% ± 2.0% (mean ± SD). The mean plasma concentrations of CoQ10H2 and TQ10 were 803 (± 264) and 835 (± 276) μg/L, respectively.
Discussion
Various HPLC-EC methods have been described in the past that attempted to measure CoQ10 and CoQ10H2 (Table 1⇑ ). Because CoQ10 is somewhat insensitive to EC detection, in-line postcolumn reduction of CoQ10 to CoQ10H2 by a coulometric method (21)(22)(24) or a reduction column (23) has been reported recently, which allows for the sensitive detection of CoQ10 by an EC electrode. Historically, these methods used chemical reagents to obtain CoQ10H2 by reducing the commercially available CoQ10. This process typically involves the addition of an excess amount of the reducing reagent to convert CoQ10 into CoQ10H2 and to maintain the reduced form. Additional care is also required to avoid oxidation by ambient oxygen, usually by storage under argon or nitrogen. Preparation of calibrators and controls is particularly problematic to protect CoQ10H2 from oxidation. The current method uses in-line precolumn EC reduction to convert CoQ10 into CoQ10H2 and avoids the artifactual oxidation that frequently occurs during the chemical process to produce CoQ10H2. Coulometric detectors efficiently convert ∼99% of CoQ10 to CoQ10H2. This improvement is important because it dispenses with the need for a reducing agent and additional sample clean-up steps.
The first study to substantially improve earlier methods was reported by Grossi et al. (19), who introduced a precolumn oxidation cell for the CoQ10H2 study. However, their quantitative measurement of CoQ10H2 was unsuccessful (Table 1⇑ ).
Finckh et al. (21) developed a micromethod for simultaneous measurement of several lipophilic antioxidants using HPLC with coulometric EC detection (Table 1⇑ ). Postcolumn EC detectors in the reduction-reduction-oxidation mode, as described by Grossi et al. (19), were used. According to their procedure, 5 or 10 μL of sample was extracted with ethanol, β-hydroxytoluene, and hexane. After centrifugation, the hexane phase was evaporated to dryness under a stream of argon and redissolved in a mixture of ethanol and methanol. Poor recoveries of 54% ± 37% and 76% ± 36% were reported for CoQ10H2 and CoQ10, respectively, without internal standardization by CoQ9H2 and CoQ9. To correct this problem, the authors added internal standardization with CoQ9H2 and CoQ9. This improved the accuracy and precision, i.e., recoveries were 105% ± 21% and 97% ± 11%, respectively, for CoQ10H2 and CoQ10. However, the imprecision of the CoQ10H2 analysis was excessive. The instability of hexane-extracted CoQ10H2 after drying has been reported by other investigators (23) and may contribute to this problem. Again, it should be noted that this procedure is relatively tedious and complex.
Lagendijk et al. (22) also reported a rapid HPLC-EC procedure for the determination of CoQ10H2 and CoQ10 in 1-propanol extracts (Table 1⇑ ). They used the postcolumn EC electrodes in the oxidation-reduction-oxidation mode as described by Edlund (17). Their extraction and analysis procedures without internal standardization were also used to obtain the ratio between CoQ10H2 and CoQ10. To prevent a coulometric overload with an 80-μL injection of sample, a sophisticated switching valve was used to ensure that only the compounds of interest were channeled through the coulometric cells. Although they used a 1-propanol extraction similar to the one used in the current method, their sample and solvent volumes (300 μL and 1 mL, respectively) were much greater than the current method (100 and 900 μL, respectively), and their injection volume was fourfold increased (80 μL vs 20 μL). In addition, the current method uses in-line precolumn reduction, an autosampler, and cooling of the samples to 0 °C. Although the method used by Lagendijk et al. (22) may accurately measure the CoQ10H2:CoQ10 ratio (∼16.7:1), it may also be prone to analytical variation because it does not use internal standard for quantifying CoQ10H2 and CoQ10. According to the authors’ recommendations for reliable results, the time span from collection to analysis must be within 15 min. The instability of CoQ10H2 limits the practical application of their method because only 8–10 samples can be analyzed per day.
Yamashita and Yamamoto (23) reported a HPLC-EC procedure using single extraction with methanol-hexane (1:2 by volume; Table 1⇑ ). To prevent the air oxidation of CoQ10H2, they incorporated an immediate and direct injection step into their procedure. Their results clearly indicated that the hexane extract should be analyzed immediately after extraction, and the analysis of one sample at a time was emphasized. In addition, Finckh et al. (21) and Wang et al. (25) reported that hexane is not an efficient extraction solvent. Our data (unpublished) also indicate the poor recovery of CoQ10H2 (52–64%) with the use of hexane.
Kaikkonen et al. (24) reported a method similar to the one described by Finckh et al. (21) for measuring CoQ10H2, but their results indicated a lower mean (∼88%) and broader range for the CoQ10H2:TQ10 ratio (80.9–90.9%) than the current study (mean, 96.3% ± 2%; Table 1⇑ ). Their method also used a complex sample preparation procedure and evaporation under nitrogen, which may explain their decreased recovery of CoQ10H2: The long pretreatment, extraction, and evaporation procedures required for their method may have allowed the oxidation of a significant portion of CoQ10H2. Their method was very tedious and slow, and according to their own description was capable of analyzing only one sample at a time (24).
Wang et al. (25) recently reported a gradient HPLC method with automated precolumn reduction to assess CoQ10H2 and TQ10 concentrations in plasma. Their method uses chemical reduction of CoQ10H2. As a result, each clinical specimen requires duplicate injections to complete the analysis of CoQ10H2 and TQ10. In addition, CoQ10H2 and TQ10 determinations must be performed before and after each sample is mixed with reducing agent before HPLC analysis. Because the reducing reagent is very unstable, a fresh and adequate amount of reducing reagent must be prepared every three samples. Previous experience by the current investigators found that excess reducing agent may overload the EC electrode and shorten the life-span of the detector cells (unpublished data). Additionally, the method of Wang et al. (25) may be prone to analytical variation because they do not use an internal standard. According to the authors, their method is also limited to the analysis of a maximum of one sample per hour and requires continuous effort by a technician.
Although the current procedure requires 100 μL of plasma, the sample size could be further reduced to 25 or 50 μL depending on the detection of trace amounts of CoQ10. Because CoQ10H2 and CoQ10 are measured simultaneously, the total analysis time is substantially shorter than those for other methods. The method described herein makes it possible to analyze up to 40 samples within an 8-h period. Although other, longer methods have included additional analytes (16)(17)(18)(20)(21)(23), the current method has been optimized to measure CoQ10H2 and CoQ10 as rapidly, simply, accurately, and precisely as possible. Tables 3⇓ and 4⇑ summarize the excellent reproducibility of the analysis. The individual CVs for CoQ10H2, TQ10, and the CoQ10H2:TQ10 ratio were ≤3.8%, ≤3.7%, and ≤1.0%, respectively (Table 4⇑ ). The current method uses single extraction with 1-propanol as solvent to disrupt lipoproteins and efficiently solubilize CoQ10H2 and CoQ10. This eliminates the necessity of the repeated extraction procedures that frequently were required by earlier procedures in which mixtures of either methanol or ethanol and hexane were used. In contrast to earlier methods, no evaporation step and no additional cleanup of the 1-propanol extract are needed. Although >1000 samples have been injected into the current system over the previous 6 months, the in-line filters have been replaced only once. The current system also avoids the need for complex system configurations, such as coupled columns with column-switching valves and postcolumn two-way valves.
Precision results for CoQ10 analysis.
Some controversy exists concerning the use of CoQ9 as an internal standard for CoQ10H2 analysis. Evidence of endogenous CoQ9 in some individuals was cited by some investigators (26), but not others (27). Our current results and considerable experience indicate that CoQ9 is rarely found in measurable quantities in human plasma and thus is a suitable internal standard for this procedure.
The ranges for the percentage of CoQ10H2 in TQ10 from previous reports are quite variable (Table 1⇑ ). The percentages of CoQ10H2 in TQ10 reported recently, i.e., ∼95% (21), ∼94% (22), ∼96% (23), and ∼93% (25), agree well with the results of the current study (∼96%). Other methods may accurately measure CoQ10H2; however, they generally are more labor-intensive and more prone to error than the current method.
Accurate determination of CoQ10H2 makes it a possible marker for assessing the presence of oxidative stress in many pathologic states. Although significant differences in the plasma CoQ10H2:TQ10 ratio between controls and patients with atherosclerosis, coronary artery disease, and Alzheimer disease have not been observed by some investigators (10)(28), other researchers have reported decreased CoQ10H2 concentrations associated with certain disease processes. Hara et al. (6) suggested that the CoQ10H2:TQ10 ratio is a good marker of oxidative stress in infants with asphyxia (6). Hemodialysis patients have also been found to have significantly lower concentrations of plasma CoQ10H2 than healthy controls (7). According to one report (7), a single hemodialysis session causes a 30% decrease in mean plasma CoQ10H2 concentrations. Plasma CoQ10H2 was also found to be significantly lower in hyperlipidemic patients and in patients with liver disease (10). In 64 patients with chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma, significantly increased CoQ10 and decreased CoQ10H2 were observed (8). Palomäki et al. (12) observed that lovastatin treatment diminishes the CoQ10H2 concentration in the LDL of hypercholesterolemic patients with coronary heart disease. There are also concerns that patients could experience deleterious effects as a result of long-term therapy with hydroxymethylglutaryl-CoA reductase inhibitors or “statin” therapy. Monitoring of the effects of statin therapy on CoQ10H2 may be useful for diagnosing CoQ10H2 deficiency in many patient populations. These are but a few of a growing numbers of studies that suggest that CoQ10H2 deficiency may be related to pathophysiologic mechanisms.
Recent studies have reported new findings related to CoQ10H2 that may lead to a better understanding of the cellular function of CoQ10H2. One study in patients with β-thalassemia showed that severely depleted CoQ10H2 concentrations (−62.5%) are associated with increased plasma concentrations of lipoperoxidation byproducts and urinary concentrations of catecholamine metabolites and azelaic acid (13). These changes may indicate both neurological and lipoperoxidation stress (13). Gotz et al. (5) also reported that platelet CoQ10H2:CoQ10 ratios were significantly decreased in patients with Parkinson disease. An altered redox state of platelet coenzyme Q10 may reflect a change in membrane electron transport and the effectiveness of defense against toxic reactive oxygen species, such as hydrogen peroxide and superoxide (5). Another recent study suggested that CoQ10 enrichment may decrease oxidative DNA damage in human lymphocytes (14). Additional studies are needed to understand the function and protective role of CoQ10H2 in these and other diseases.
In conclusion, we developed a simple, rapid, and isocratic HPLC method for the determination of CoQ10H2 and CoQ10 in human plasma. An extraction process using 1-propanol as solvent allows rapid and simple sample extraction and minimizes oxidation of CoQ10H2 during sample processing. An in-line precolumn reduction cell is used to convert CoQ10 into CoQ10H2. The EC reduction yields 99% CoQ10H2 and avoids the artifactual oxidation that frequently occurs with CoQ10H2 produced through the chemical reduction process. This optimized method provides excellent sensitivity, precision, and accuracy for relatively high-throughput assessment of CoQ10H2 and CoQ10 in human plasma. This method is suitable for research and can be easily adapted for clinical testing purposes. Studies are in progress to establish reference intervals and to evaluate the clinical significance of plasma and cerebrospinal fluid concentrations of CoQ10H2 in several patient populations.
Acknowledgments
We thank Dr. Paul Steele for valuable suggestions, and Gail Chuck and Laura Schroer for assistance in collecting patient specimens and information for this study.
Footnotes
1 Values of CoQ10H2:TQ10 are mean ± SD.
2 Analytes in addition to CoQ10H2 and CoQ10 were included in this method.
3 NA, data not provided; UV, ultraviolet.
1 Drugs added to the commercial Dade® immunoassay control prior to extraction.
1 Diluted from pooled plasma containing 0.45 mg/L CoQ10.
2 Pooled plasma was fortified with 1.2 mg/L CoQ10 to a final concentration of 1.65 mg/L.
3 Pooled plasma was fortified with 3 mg/L CoQ10 to a final concentration of 3.45 mg/L.
↵1 Nonstandard abbreviations: CoQ10H2, reduced coenzyme Q10; CoQ10, oxidized coenzyme Q10; TQ10, total coenzyme Q10; EC, electrochemical; CoQ9, oxidized coenzyme Q9; and CoQ9H2, reduced coenzyme Q9.
- © 2001 The American Association for Clinical Chemistry
References
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