Mitochondrial Coenzyme Q10 Determination by Isotope ... - CiteSeerX

Clinical Chemistry 59:8 1260–1267 (2013)

Other Areas of Clinical Chemistry

Mitochondrial Coenzyme Q10 Determination by Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry Outi Itkonen,1* Anu Suomalainen,1,2,3 and Ursula Turpeinen1

BACKGROUND: Coenzyme Q10 (CoQ10) is an essential part of the mitochondrial respiratory chain. Unlike most other respiratory chain disorders, CoQ10 deficiency is potentially treatable. We aimed to develop and validate an accurate liquid chromatography– tandem mass spectrometry (LC-MS/MS) method for the determination of mitochondrial CoQ10 in clinical samples. METHODS: We used mitochondria isolated from muscle biopsies of patients (n ⫽ 166) suspected to have oxidative phosphorylation deficiency. We also used fibroblast mitochondria from 1 patient with CoQ10 deficiency and 3 healthy individuals. Samples were spiked with nonphysiologic CoQ10-[2H6] internal standard, extracted with 1-propanol and with ethanol and hexane (2 mL/5 mL), and CoQ10 quantified by LCMS/MS. The method and sample stability were validated. A reference interval was established from the patient data. RESULTS: The method had a limit of quantification of 0.5 nmol/L. The assay range was 0.5–1000 nmol/L and the CVs were 7.5%– 8.2%. CoQ10 was stable in concentrated mitochondrial suspensions. In isolated mitochondria, the mean ratio of CoQ10 to citrate synthase (CS) activity (CoQ10/CS) was 1.7 nmol/U (95% CI, 1.6 –1.7 nmol/U). We suggest a CoQ10/CS reference interval of 1.1–2.8 nmol/U for both sexes and all ages. The CoQ10/CS ratio was 5-fold decreased in fibroblast mitochondria from a patient with known CoQ10 deficiency due to recessive prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2) mutations. CONCLUSIONS: Normalization of mitochondrial CoQ10 concentration against citrate synthase activity is likely to reflect most accurately the CoQ10 content available for the respiratory chain. Our assay and the established

1

HUSLAB, Helsinki University Central Hospital, 2 Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, and 3 Department of Neurology, Helsinki University Central Hospital, Helsinki, Finland. * Address correspondence to this author at: HUSLAB, Helsinki University Central Hospital, P.O. Box 140, FIN-00029 HUS, Helsinki, Finland. Fax ⫹358-9-47174806; e-mail [email protected]. Part of this manuscript has been published as a poster at the Nordic Congress in

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reference range should facilitate the diagnosis of respiratory chain disorders and treatment of patients with CoQ10 deficiency. © 2013 American Association for Clinical Chemistry

Coenzyme Q10 (CoQ10),4 also called ubiquinone, is an essential part of the mitochondrial respiratory chain that transforms energy in the diet to ATP for cellular functions (1 ). In this process of oxidative phosphorylation (OXPHOS), CoQ10 transfers reducing equivalents from respiratory complexes I and II to complex III (2 ). In addition, CoQ10 also allows protons to be extruded from the mitochondrial matrix to the intermembrane space (3 ), acts as a pro- or antioxidant (4 ), and has a role in pyrimidine biosynthesis (5 ) and apoptosis modulation (6 ). CoQ10 is synthesized in the mitochondrial inner membrane from tyrosine or phenylalanine and acetyl coenzyme A by the enzyme decaprenyl diphosphate synthase followed by condensation, decarboxylation, hydroxylation, and methylation steps (7 ). Disorders in the OXPHOS chain cause lack of energy in the cell and tissue, resulting in heterogeneous symptoms in the patient. Patients with primary and secondary CoQ10 deficiency have been reported (4, 8 – 12 ). There are no curative treatments for OXPHOS disorders. However, some patients with CoQ10 deficiency benefit from CoQ10 substitution (10, 13 ), and the treatment is relatively inexpensive and lacks severe side effects. CoQ10 quantification may be used to aid in the diagnosis of mitochondrial CoQ10 deficiency. Thus far, few mass-spectrometry (MS)-based assays for CoQ10 have been reported (14 –19 ). However, in these assays CoQ11 (14 ), CoQ9 (15–17,19 ), or dipropoxy-CoQ10 (18 ) is used as an internal standard (IS). Here, we describe the development and validation

Clinical Chemistry LabMed 2012, Reykjavik, Iceland. Received December 4, 2012; accepted April 11, 2013. Previously published online at DOI: 10.1373/clinchem.2012.200196 4 Nonstandard abbreviations: CoQ10, ubiquinone or coenzyme Q10; OXPHOS, oxidative phosphorylation; MS, mass spectrometry; LC-MS/MS, liquid chromatography–tandem MS; IS, internal standard; ESI, electrospray ionization; LOD, limit of detection; LOQ, limit of quantification; CS, citrate synthase.

Mitochondrial Coenzyme Q10 Determination

of a liquid chromatography–tandem MS (LC-MS/MS) method for the measurement of tissue mitochondria CoQ10 using isotopically labeled CoQ10 as internal standard. We also established a reference interval for CoQ10 in muscle mitochondria. Materials and Methods PATIENT SAMPLES

We used enriched mitochondrial fractions from quadriceps (vastus lateralis) muscle samples of 166 patients. The analysis was done as part of their diagnostic workup for suspicion of mitochondrial disorder (i.e., clinical symptoms, laboratory findings, and/or histopathological findings suggesting mitochondrial disease). The samples were collected between May 2008 and September 2012. The median age of the patients was 27.5 years (range 4 days to 80 years); 80 patients were men (median age 27.5 years, range 21 days to 80 years) and 86 were women (median age 26.5 years, range 4 days to 70 years). Mitochondria were enriched from fresh specimens within 2 h as previously described (20 ). Briefly, the muscle sample (mean 115 mg, range 19 –204 mg) was homogenized for 4 min in a Potter-Elvehjelm homogenizer fitted with a Teflon pestle. The volume (␮L) of homogenizing buffer (100 mmol/L KCl, 50 mmol/L Tris, 5 mmol/L MgCl2, 1.8 mmol/L ATP, 1 mmol/L EDTA, pH 7.2) corresponded to muscle weight (mg) ⫻ 20 (min 700 ␮L). The sample was first centrifuged for 3 min at 650g and then the supernatant was centrifuged for 3 min at 15 000g. The pelleted mitochondria were washed with 300 ␮L of homogenizing buffer and suspended in resuspension solution [250 mmol/L sucrose (BDH Chemicals), 15 mmol/L K2HPO4, 2 mmol/L MgAc2, 0.5 mmol/L EDTA, and 0.5 g/L albumin (human, essentially fatty acid free, Sigma), pH 7.2], in a volume (␮L) corresponding to muscle weight (mg) ⫻ 4 (min 200 ␮L). All reagents and materials were cooled and kept ice cold during the entire isolation procedure (20 ). Enriched mitochondrial fractions were then forwarded for immediate diagnostic biochemical analysis (see below), and when stated, for CoQ10 quantification. The rest of isolated mitochondria were stored aliquoted at ⫺80 °C (1 day to 4.4 years) or at ⫺20 °C (1–28 days) until analyzed. The study was approved by the ethics committee of Helsinki University Central Hospital, Finland. REAGENTS AND CALIBRATORS

We used MS-grade methanol (Fluka, Sigma-Aldrich) and HPLC grade 1-propanol (Sigma-Aldrich). All other reagents were of the highest analytical quality. As calibrator we used CoQ10 (Sigma-Aldrich) and as sta-

ble isotope-labeled IS [2H6]-CoQ10 (IsoSciences http://isosciences.com/). A 700 ␮mol/L (600 mg/L) calibrator stock solution of CoQ10 was prepared in chloroform and stored aliquoted at ⫺20 °C. We found it important to dissolve the stock calibrator into chloroform because concentrated CoQ10 showed poor solubility in 1-propanol. A 1000 nmol/L calibrator was diluted from the stock solution with eluent A (see below) and stored aliquoted at ⫺20 °C. A 1.2 mmol/L (1 g/L) stock solution of IS was dissolved in 1-propanol and stored aliquoted at ⫺80 °C. A working solution of 0.5 ␮mol/L was prepared in 1-propanol and stored at ⫹4 °C. LC-MS/MS ANALYSIS

The LC-MS/MS analysis was performed using a SunFire C18 analytical column (2.1 ⫻ 50 mm, 3.5-␮m particle size; Waters Corporation) kept at 40 °C and coupled to an Agilent Technologies 1200 HPLC (Agilent Technologies) and an AB Sciex 4000 triple quadrupole MS (AB Sciex) with a Turbo-V electrospray ionization (ESI) ion source. During the HPLC run, the system was kept at 100% eluent A [methanol and 1-propanol (205 mL/45 mL) containing 500 ␮mol/L ammonium acetate], ramped to 100% eluent B [methanol and 1-propanol (125 mL/125 mL) containing 500 ␮mol/L ammonium acetate] at 0.2– 0.21 min, kept at 100% B until 2 min, and then ramped back to 100% eluent A at 2–2.1 min. The total run time was 10 min and the flow rate was 300 ␮L/min. The MS was operated in positive ion mode with the ion source spray voltage at ⫹5500 V, declustering potential at 70 V, temperature at 275 °C, and collision energy at 27 V. The curtain gas was 20 L/min, gas 1 45 L/min, gas 2 30 L/min, and collision gas setting 4. For MS/MS detection we followed the transitions of m/z 880.8 3 197.2 (dwell time 500 ms) corresponding to the ammonium adduct of CoQ10 and m/z 886.8 3 203.2 (250 ms) for the ammonium adduct of [2H6]-CoQ10. SAMPLE AND CALIBRATOR PREPARATION

The mitochondrial suspension (0.25 mg tissue wet weight/␮L) was diluted with water to 0.01 mg wet weight/␮L. We added 20 ␮L of IS working solution to 100 ␮L of diluted mitochondrial suspension and vortex mixed the resulting solution. Then we added 300 ␮L of 1-propanol and mixed as above. After centrifugation at 10 000g for 2 min the organic layer was extracted with 3 mL ethanol and hexane (2 mL/5 mL) and mixed for 3 min in a multitube vortex-type mixer (Labtek International).Then 500 ␮L of water was added and the solution was mixed for 3 min. The organic layer was removed and evaporated to dryness under nitrogen. Clinical Chemistry 59:8 (2013) 1261

The residue was dissolved in 100 ␮L of eluent A and 5 ␮L of the sample was injected into the HPLC. The highest 1000-nmol/L calibrator was produced batch-wise as described above. Other calibrators (500, 250, 100, 10, and 1 nmol/L) were prepared by dilution of the highest calibrator in eluent A. The calibrators were not extracted, but 20 ␮L of IS working solution was added to 100 ␮L of calibrator before LC-MS/MS analysis. EFFECT OF OXIDATION OF MITOCHONDRIAL CoQ10

We compared CoQ10 results with and without oxidation in 5 samples containing 4.8 –25 nmol CoQ10/g wet weight stored at ⫺80 °C for 17–28 months, in freshly isolated mitochondria (0.9 –5.9 nmol CoQ10/g wet weight, n ⫽ 3) and with the same samples after 1 week of storage at ⫺20 °C. The oxidation was performed by adding 10 ␮L of 10 mmol/L K3Fe(CN)6 to the mitochondrial suspension containing the IS, followed by sample extraction as above. ANALYTICAL VALIDATION OF THE CoQ10 ASSAY

The limit of detection (LOD) was based on a signal-tonoise ratio of 3 (n ⫽ 10). Linearity and limit of quantification (LOQ) was determined by preparing 9 calibrators by serial dilution to concentrations of 0.125–1000 nmol/L performed on 4 separate days. The calibration curves were derived using 1/x2 weighted linear least-squares regression by the Analyst 1.5 software (AB Sciex). The LOQ and linear range were defined as the lowest concentration and range, respectively, that could be measured with an inaccuracy (percentage relative error) and imprecision (CV%) ⬍20% (n ⫽ 4). Intraassay and interassay imprecision values were calculated from 15 replicates of 3 mitochondrial CoQ10 concentrations, respectively. The matrix effect was studied with 5 mitochondrial suspensions containing 1.6 –12.2 nmol CoQ10/g wet weight. After extraction, the samples were supplemented with 76 nmol/L of CoQ10 and 167 nmol/L IS. The same concentrations of CoQ10 and IS were added to eluent A. Analyte recovery was determined using 5 mitochondrial suspensions containing 4.1–13 nmol CoQ10/g wet weight supplemented with 11.4 nmol/L, 45.4 nmol/L, or 114 nmol/L of CoQ10. SAMPLE STABILITY

We studied the stability of CoQ10 in isolated mitochondria using 3 patient samples in suspensions of 0.25 mg wet weight/␮L resuspension solution and 0.01 mg wet weight/␮L water. CoQ10 was assayed immediately after the isolation of mitochondria and after 1, 2, 3, and 4 weeks at ⫺20 °C. Other aliquots of the same samples were stored at ⫺20 °C and then 1262 Clinical Chemistry 59:8 (2013)

thawed and assayed at the same time points and put back to ⫺20 °C for repeated analysis. We considered mitochondrial CoQ10 stable if the concentration changed by ⬍20%. To estimate the effect of prolonged storage of mitochondrial suspension (0.25 mg wet weight/␮L) at ⫺80 °C, we compared the CoQ10 content in the samples against storage time by Pearson correlation. Furthermore, we compared the median concentration of CoQ10 in samples that had been stored for 0 –1.5 years (n ⫽ 83) and 1.5– 4.4 years (n ⫽ 83). ESTABLISHMENT OF REFERENCE INTERVAL

For calculation of the reference interval, we included results from patient samples for which biochemical respiratory chain analysis showed no evidence for the OXPHOS defect (n ⫽ 115). Partitioning by age and sex was considered by the Mann–Whitney U-test. The reference interval was calculated as the central 95% reference interval according to the IFCC guidelines (CLSI C28-A3c). CoQ10 IN CULTURED SKIN FIBROBLAST MITOCHONDRIA

We compared CoQ10 content in mitochondria isolated from cultured skin fibroblasts from 3 healthy individuals and 1 CoQ10-deficient patient who had a CoQ10 synthetic pathway defect caused by prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2) mutations (21 ). Informed consent was obtained from all individuals. The cells were grown under standard cell culture conditions with uridine supplementation (50 g/L). Mitochondria were isolated from 1.5–12.4 ⫻ 106 cells and suspended in 100 ␮L water. Citrate synthase (CS) (EC 2.3.3.1) activity (22 ) and CoQ10 in the suspensions were measured and the results calculated as nmol/(min ⫻ 106 cells) and nmol/106 cells, respectively. DIAGNOSTIC METHODS

Analysis of mitochondrial ATP production rate (20 ) and respiratory chain enzyme complex I (23 ), II, I⫹III, II⫹III, IV, and CS (22, 24 ) activity were performed as previously described. CS activity was used as a matrix marker enzyme and determined by following the reduction of 5,5⬘-dithiobis(2-nitrobenzoic acid) at 412 nm coupled to the reduction of coenzyme A in the presence of oxaloacetate (22 ) in a solution containing 0.12% n-dodecyl-␤-D-maltopyranoside (Anatrace; Affymetrix). STATISTICAL METHODS

The Pearson correlation test, Mann–Whitney U-test, and paired t-test were performed by Analyse-it for Microsoft® Excel 2003 (version 2.04) (Analyse-it Software


Fig. 1. Ion chromatograms of (A), 0.2 nmol/L CoQ10 calibrator and (B), extracted mitochondria from a patient muscle containing 15 nmol CoQ10/ g wet weight. The signal-to-noise ratio of the 0.2 nmol/L solution was 7.3. IS refers to [2H6]-CoQ10.

http://www.analyse-it.com/; 2009). A P value of ⬍0.05 was considered statistically significant.

weight in fresh samples after 1 week at ⫺20 °C, respectively (paired t-test P ⬎ 0.22 for all experiments).

Results

SAMPLE STABILITY

ANALYTICAL VALIDATION OF CoQ10 ASSAY

CoQ10 and IS eluted at a retention time of 4.7 min (Fig. 1). The method was linear over the concentration range of 0.5–1000 nmol/L (0.432– 863 ng/mL) with an LOD of 0.06 nmol/L (52 pg/mL) and LOQ of 0.5 nmol/L (432 pg/mL). We routinely used 1 nmol/L as the lowest calibrator. The intraassay CVs were 6.0%, 4.2%, and 5.4% at concentrations of 3.5, 11.3, and 28.2 nmol/g wet weight, and interassay CVs were 8.2%, 8.0%, and 7.5% at concentrations of 1.7, 6.1, and 16.3 nmol/g wet weight, respectively. The mean recovery of CoQ10 in 5 mitochondrial samples containing 4.4 –13 nmol/g wet weight endogenous CoQ10 was 78% (range 72%– 82%), 100% (range 97%–107%), and 94% (range 92%–96%) when supplemented with 11.4 nmol/L, 45.4 nmol/L, and 114 nmol/L of CoQ10, respectively. The ion suppression reduced the measured peak signal on average to 90% (range 83%–95%) for CoQ10 and to 99% (range 88%–106%) for [2H6]CoQ10, respectively. The mean CoQ10 concentration with and without oxidation was 12.9 and 12.5 nmol/g wet weight in frozen samples (n ⫽ 5), 3.4 and 3.4 nmol/g wet weight in fresh samples (n ⫽ 3), and 3.4 and 3.9 nmol/g wet

In mitochondrial suspensions (0.25 mg wet weight/␮L, n ⫽ 3), the mean CoQ10 on day 0 was 13.4 nmol/g wet weight and after 1– 4 weeks at ⫺20 °C it was 16.7–17.7 nmol/g wet weight, i.e., the measured CoQ10 concentration increased by 25% during 1 week at ⫺20 °C and then after 2, 3, and 4 weeks decreased by 6%, 5%, and 3%, respectively (Fig. 2B). In suspensions (n ⫽ 3) corresponding to 0.01 mg wet weight/␮L the mean CoQ10 was 13.1 nmol/g wet weight on day 0. After 1– 4 weeks at ⫺20 °C it was 13.2–15.2 nmol/g wet weight. The increase in measured CoQ10 concentration in 1 week was 14%, after which it changed by 1%–12% (Fig. 2A). In mitochondrial suspensions (n ⫽ 3) corresponding to 0.25 mg wet weight/␮L the mean increase in CoQ10 concentrations after 1 freeze-thaw cycle was 30%. After the second, third, and fourth cycle the decrease was 11%, 7%, and 16%, respectively (Fig. 2B). The respective decreases in mitochondrial suspension corresponding to 0.01 mg wet weight/␮L after 1– 4 freeze–thaw cycles were 3%, 15%, 30%, and 33%, respectively (Fig. 2A). There was no correlation between storage time (3 days to 4.4 years) at ⫺80 °C and the measured CoQ10 concentration (nmol/g wet weight; P ⫽ 0.29). The meClinical Chemistry 59:8 (2013) 1263

A

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CoQ10 (nmol/g wet weight)


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20 15 10 5 0 0

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40 35 30 25

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60 years

3 2.5

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CoQ10/CS (nmol/U)


P = 0.0324

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Time (weeks) or freeze–thaw cycles

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25 20 15 10 5

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Fig. 2. Stability of CoQ10 in mitochondrial suspension at ⴚ20 °C [⽧, mean (SD), n ⴝ 3] and after repeated freezing and thawing (䡺, mean). The mitochondrial suspension was either (A), 0.01 mg wet weight/␮L or (B), 0.25 mg wet weight/␮L.

dian CoQ10 concentration in mitochondrial suspensions stored for 1.5– 4.4 and for 0 –1.5 years was 8.6 (95% CI, 7.0 –10.7) nmol/g wet weight and 8.6 (95% CI, 6.8 –10.2) nmol/g wet weight, respectively (P ⫽ 0.85; n ⫽ 166). MITOCHONDRIAL CoQ10 CONTENT AND THE REFERENCE INTERVAL

The mitochondrial CoQ10 content in muscle from men [median 10.8 (95% CI, 8.4 –12.7) nmol/g wet weight (n ⫽ 80)] was higher (P ⫽ 0.0168) than that in women [median 7.2 (95% CI, 5.9 –9.1) nmol/g wet weight (n ⫽ 86)]. Furthermore, there were significant differences in mitochondrial CoQ10 with age (Fig. 3A). However, when the CoQ10 concentration (nmol/g wet weight) was normalized against CS activity [nmol/ (min ⫻ g wet weight)] in the sample, there was no difference in the mitochondrial CoQ10/CS ratio between the sexes [median 1.7 (95% CI, 1.5–1.8) nmol/U for men and 1.6 (95% CI, 1.5–1.7) nmol/U for women; P ⫽ 0.34] or between various age groups (P ⬎ 0.08) (Fig. 3B). For establishing the reference interval, 115 1264 Clinical Chemistry 59:8 (2013)

0

Age group

Fig. 3. Interquartile box plot of mitochondrial CoQ10 concentrations in muscle samples expressed as (A), nanomoles per gram wet weight or as (B), CoQ10/CS (nmol/U). Median is shown in the box. Values outside the 1.5⫻ interquartile range are marked as outliers (⫹). The P value is shown when the difference between the adjacent groups is significant (P ⬍ 0.05).

reference samples with no biochemical evidence for an OXPHOS defect were included. The median CoQ10/CS of the reference values [1.7 nmol/U (95% CI, 1.6 –1.8) nmol/U] was higher (P ⫽ 0.0020) than that in the samples for which the biochemical outcomes were abnormal [1.5 nmol/U (90% CI, 1.3–1.7) nmol/U]. We suggest a CoQ10/CS reference interval of 1.1 (90% CI, 1.0 – 1.1) to 2.8 (90% CI, 2.6 –3.0) nmol/U. COMPARISON OF CoQ10 DEFICIENT AND NORMAL MITOCHONDRIA

The CoQ10/CS ratio values were 0.43, 0.45, and 0.52 nmol/U in mitochondria isolated from cultured skin fibroblasts from 3 healthy individuals. In mitochondria isolated from fibroblasts from a CoQ10deficient patient (23 ) the CoQ10/CS ratio was 0.09 nmol/U.


Discussion We have validated an LC-MS/MS method for mitochondrial CoQ10 that, to our knowledge, employs for the first time isotopically labeled CoQ10 as the IS. In previously reported LC-MS– based assays for CoQ10 in biological samples, either CoQ11 (14 ), CoQ9 (15– 17, 19 ), or dipropoxy-CoQ10 (18 ) has been used as the IS. A potential source of error for MS assays is ionization suppression that varies during a chromatographic run and between the samples. Therefore, it is ideal that the IS coelutes with the analyte for minimization of matrix effects and for sample recovery. Our method has an LOQ of 0.5 nmol/L. Other groups have reported LOQs of 2.9 nmol/L (2.5 ng/mL) to 58 nmol/L (20 ng/mL) (14 –17, 19 ) for LC-MS CoQ10 assays. Most previously reported assays for CoQ10 in biological samples are based on HPLC with ultraviolet (8, 21, 25–28 ) or electrochemical (29 –32 ) detection. The reported LOD of the HPLC assays is at best 1.6 nmol/L (30 ). The low LOD of our assay is an advantage both for diagnostic and research purposes. In tissues, CoQ10 exists as oxidized ubiquinone and reduced ubiquinol. Approximately 54%–59% of CoQ10 was reported to be in the reduced form in human muscle homogenates (32 ). Ubiquinol readily oxidizes to ubiquinone during sample procession (33 ). We found that practically all mitochondrial CoQ10 is oxidized during sample pretreatment and no ubiquinol oxidation is needed for quantification of total CoQ10 in isolated mitochondria. Decreased activities of CoQ10-dependent enzymes (complexes I⫹III and II⫹III) are usually associated with CoQ10 deficiency. However, in cases of mild CoQ10 deficiency activities of complexes I⫹III or II⫹III, or both, may be normal (13 ). Therefore, direct measurement of CoQ10 concentration in muscle is considered the most reliable test for diagnosis of a CoQ10 defect (34 ). For determination of muscle CoQ10 most studies have used muscle homogenates (10, 11, 28, 30, 35, 36 ). We and others (8, 9 ) used isolated muscle mitochondria. CoQ10 content has been shown to differ in different muscle types (27 ). Therefore, quantification of mitochondrial CoQ10 enables direct analysis of CoQ10 available for the respiratory chain. The activity of CS is generally accepted as a matrix enzyme and a marker for mitochondrial abundance in a sample. When we calculated CoQ10 concentration as nanomoles per gram wet weight, we found that muscle CoQ10 concentration was higher in men than in women. This result agrees with the findings of Päivä et al. (33 men and 15 women) in quadriceps homogenates (28 ). In our study, mitochondrial CoQ10 (nmol/g wet weight) was the lowest during the first year of life,

reaching the highest values between 11 and 26 years of age. However, when we normalized CoQ10 concentrations against CS activity of the sample, there was no difference between the sexes or correlation with age. In other studies using total protein content or tissue wet weight for normalization of muscle homogenate CoQ10, no dependence with sex or age was found (11, 27 ). Clinically, the most important application of CoQ10 assay is detection of CoQ10 deficiency of the respiratory chain. For this purpose, normalization of the CoQ10 concentration in relation to CS activity, i.e., mitochondrial abundance, is likely to reflect CoQ10 content available most accurately. Previously, provisional reference ranges for muscle mitochondria CoQ10 determined by using a limited number of reference individuals have been published. Ogasahara et al. established CoQ10 “control values” for quadriceps (vastus lateralis) muscle mitochondria of 2.1 (0.1) nmol/mg protein [mean (SD), 1811 (99) ng/mg protein; n ⫽ 10] (8 ) and Boitier et al. for quadriceps muscle mitochondria of 2.1 (0.1) ␮mol/mg protein [1856 (112) ␮g/mg protein; n ⫽ 5] (9 ). The vast discrepancy between the concentrations may partly be explained by the very small number of samples, and differences in assay calibration, IS, and the muscle sample (8, 9 ). The mean mitochondrial CoQ10 concentration of all samples in our study was 9.6 nmol/g wet weight (95% CI, 8.6 –10.5 nmol/g wet weight) or CoQ10/CS 1.7 nmol/U (95% CI, 1.6 –1.7 nmol/U). In our study, storage of mitochondrial suspensions (n ⫽ 5) at ⫺20 °C for 1 week resulted in increased CoQ10 concentrations by 15%–30% or, when repeated (n ⫽ 3), in a slight but nonsignificant change in CoQ10 concentrations. The reason for the increased CoQ10 concentrations in our initial experiment remains unclear. After a further 2– 4 weeks, the concentration remained practically unchanged. CoQ10 was stable during 4 repeated freeze–thaw cycles in concentrated (0.25 mg wet weight/␮L) but not in dilute suspensions (0.01 mg wet weight/␮L). Therefore, we conclude that CoQ10 is stable for at least 4 weeks at ⫺20 °C in mitochondrial suspensions, and storage of concentrated rather than dilute suspensions is suggested. Others have reported that CoQ10 is stable in frozen muscle after prolonged storage at and below ⫺70 °C (37 ). This finding is in line with our finding that there was no correlation between storage time at ⫺80 °C and CoQ10 concentrations in isolated mitochondria (0.25 mg wet weight/␮L) over 4.4 years storage time, and no difference between CoQ10 concentrations in samples that had been stored for 0 –1.5 years and for 1.5– 4.4 years. We studied the performance of our CoQ10 assay in tissue mitochondria as a diagnostic test using culClinical Chemistry 59:8 (2013) 1265

tured skin fibroblasts from a patient with confirmed CoQ10 deficiency (21 ). The CoQ10/CS ratio in fibroblast mitochondria from this patient was 5-fold lower than that in mitochondria from normal fibroblasts. This result and the finding that the decrease in CoQ10 concentrations may be less marked in cultured fibroblasts than muscle samples (8 –10 ) suggest that our method can detect CoQ10 deficiency in clinical samples. However, further studies are needed to establish the diagnostic sensitivity and specificity of our assay. In conclusion, we have validated an LC-MS/MS method for quantification of mitochondrial CoQ10 with special attention to preanalytical issues and diagnostic usefulness. CoQ10 is stable in isolated mitochondria for at least 4 weeks at ⫺20 °C and during 4 freeze–thaw cycles in concentrated suspensions. The CoQ10/CS ratio was 5-fold decreased in fibroblast mitochondria from a patient with confirmed CoQ10 deficiency. No differences in the CoQ10/CS ratio were found

according to sex or age. Our assay and the established CoQ10/CS reference interval are likely to improve diagnosis and treatment of patients with CoQ10 deficiency.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest. Role of Sponsor: No sponsor was declared. Acknowledgments: The authors thank Prof. Agne`s Rötig for the CoQ10 deficient fibroblast cell line, the Mito-team at HUSLAB Department of Obstetrics and Gynecology, and Anu Harju for expert technical assistance.

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