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Articles |
1
Division of Pediatric Neurology, The Children’s Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039.
2
Department of Pathology and Laboratory Medicine, College
of Medicine, University of Cincinnati, 231 Bethesda Ave., Cincinnati,
OH 45267-0559.
a Address correspondence to this author at: Clinical Neuropharmacology Laboratory, Division of Pediatric Neurology, The Children’s Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Fax 513-636-6359; e-mail Tangp0{at}chmcc.org.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
).
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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.
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We therefore developed a simple and rapid HPLC procedure with coulometric detection for simultaneous determination of CoQ10 and CoQ10H2 in human plasma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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The analytical column was a reversed-phase Microsorb-MV column (4.6
mm x 15 cm; 5 µm bead size; Rainin). A reversed-phase
C18 guard column (4.6 x 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 (
) 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.
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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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
).
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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).
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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.
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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.
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
).
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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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, ß-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.
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 ß-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.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-tocopherol. Proc Natl Acad Sci U S A 1991;88:1646-1650.-tocopherol during lovastatin treatment. FEBS Lett 1997;410:254-258.[Web of Science][Medline]
[Order article via Infotrieve]
-tocopherol and cholesterol in biological samples by coupled-column liquid chromatography with coulometric and ultraviolet detection. J Chromatogr 1988;425:87-97.[Web of Science][Medline]
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-tocopherol in biological samples by high performance liquid chromatography with platinum catalyst reduction and electrochemical detection. Biol Pharm Bull 1994;17:997-1002.[Web of Science][Medline]
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) and
CoQ10H2 (
).