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Technical Briefs |
1 Division of Neurochemistry, Institute of Neurology, and2 Neurometabolic Unit, National Hospital for Neurology and Neurosurgery, London, United Kingdom;3 Biochemistry Unit, Institute of Child Heath, London, United Kingdom
aaddress correspondence to this author at: Neurometabolic Unit, National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, United Kingdom; fax 44-0-20-7829-1016, e-mail ihargrea{at}ion.ucl.ac.uk
Coenzyme Q10 (CoQ10), the predominant ubiquinone species in humans, functions as an electron carrier in the mitochondrial electron transport chain (ETC) and as an intracellular antioxidant (1). Although primary CoQ10 deficiency is rare, a profound deficiency in skeletal muscle CoQ10 has been reported in patients with multisystem mitochondrial encephalomyopathies (2)(3). Cardiovascular disease has been associated with a CoQ10 deficiency (4)(5), and it is becoming increasingly apparent that other groups of patients may become CoQ10 deficient, particularly individuals with ataxia (6) and some patients receiving statins (7).
When assessing tissue CoQ10 status, we have found that the lack of a commercially available nonphysiologic internal standard (IS) is a major difficulty. Although naturally occurring ubiquinones have been used as ISs in this determination, they are not free from the influence of ubiquinones that might be present in human tissue as the result of dietary contamination (8) or synthesis by microorganisms (9)(10). There is a need, therefore, for an alternative IS that is not influenced by exogenous/endogenous ubiquinones. Di-ethoxy-CoQ10 has been suggested as a nonphysiologic IS to determine CoQ10 (11). In this study we evaluated this IS along with di-propoxy-CoQ10 for their suitability to determine tissue CoQ10. Reference intervals were established for the CoQ10 concentration of skeletal muscle, blood mononuclear cells (MNCs), and plasma. A patient with a suspected CoQ10 deficiency was subsequently identified.
Reference intervals were established for the following: (a), skeletal muscle from 26 patients [mean (SE) age, 24.5 (3.9) years; range, 0.5–59 years; ratio of males to females, 7:6] with no evidence of an ETC deficiency detected in their skeletal muscle biopsies; (b), MNCs from 17 healthy volunteers and 13 disease controls with no clinical evidence of an ETC deficiency [mean (SE) age, 32.6 (2.6) years; range, 1–61 years; ratio of males to females, 7:8]; and (c), plasma from 24 patients [mean (SE) age, 14.3 (2.9) years; range, 1–57 years; ratio of males to females, 2:1] with no clinical evidence of a ETC deficiency.
The correlation between skeletal muscle, MNC, and plasma CoQ10 status was assessed in 2 groups of patients with no clinical or biochemical evidence of an ETC deficiency: Group 1 consisted of 12 patients [mean (SE) age, 13.21 (4.03) years; range, 1–43 years; male/female, 2:1]; plasma was obtained from 10 patients in this group. Group 2 consisted of 14 patients [mean (SE) age, 14.3 (3.7) years; range, 1–57 years; male/female, 4:3]. Correlations between skeletal muscle and MNC CoQ10 status and between skeletal muscle and plasma CoQ10 status were determined with samples from group 1; correlations between MNC and plasma CoQ10 status were determined with samples from groups 1 and 2.
The patient with a suspected CoQ10 deficiency was a 47-year-old female, mentally retarded since birth, ataxic, and with poor vision and hypertrophic cardiomyopathy, in whom evidence of an ETC complex II-III (succinate cytochrome c reductase) deficiency [0.015; reference interval, 0.040–0.204 (activity expressed as a ratio to citrate synthase activity to allow for mitochondrial enrichment) (12)] had been detected in skeletal muscle.
MNCs were isolated from 5–10 mL of sodium EDTA–anticoagulated blood within 24 h of venesection by use of the ACCUSPINTM system–Histopaque®-1077 (SigmaAldrich). The MNCs were suspended in phosphatebuffered saline (150 mmol/L NaCl, 150 mmol/L sodium phosphate), pH 7.2 (200 µL per 5 mL of blood), and stored at –70 °C until analysis. During this procedure, plasma was separated from the sodium EDTA–anticoagulated blood and stored at –70 °C until analysis.
Skeletal muscle biopsy homogenates were prepared as described by Heales et al. (12). Protein concentration was determined by the method of Lowry et al. (13). The synthesis of di-ethoxy-CoQ10 was undertaken as described by Edlund (11). The synthesis of di-propoxy-CoQ10 was based on the method of Edlund (11), substituting propan-1-ol for ethanol. The concentration of di-propoxy-CoQ10 was estimated based on the molar absorptivity for CoQ10 at 275 nm (14.6 x 103), and the di-propoxy-CoQ10 was diluted in ethanol to give a final concentration of 1.5 µmol/L.
Samples were prepared for HPLC analysis of total CoQ10 concentration by the addition of IS (30 µL) to skeletal muscle (50 µL), to MNCs (150 µL), and to plasma (200 µL) to give a final concentration of 150 nmol/L in the reconstituted extract. The ubiquinones (CoQ10 and IS) were extracted by the method of Boitier et al. (14). The extracts were evaporated under N2 and reconstituted in ethanol (300 µL). HPLC analysis was performed according to the method of Boitier et al. (14).
CoQ10 and di-propoxy-CoQ10 were analyzed at concentrations of 50 µmol/L by mass spectrometry using a Quattro micro triple-quadrupole tandem mass spectrometer operating in both the scan and parent ion modes (15).
We used regression analysis to assess the correlation between ultraviolet absorbance (275 nm) and the concentrations of di-propoxy-CoQ10 and skeletal muscle, MNC, and plasma CoQ10, and between age and the MNC, skeletal muscle, and plasma CoQ10 concentration. The relationship between sex and tissue CoQ10 concentration was assessed by the Mann–Whitney U-test. Spearman rank correlation coefficients were calculated to assess the association between the CoQ10 concentrations in skeletal muscle, MNCs, and plasma. A P value <0.05 was considered significant.
Analysis of the mass spectrum obtained in scan mode for the di-propoxy-CoQ10 IS demonstrated 1 predominant ion of m/z 942 (see Fig. 1B in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue12). This corresponded with the theoretical mass calculated for the sodium adduct of di-propoxy-CoQ10, [M+Na]. An observed increase in molecular mass of 56 Da in di-propoxy-CoQ10 relative to CoQ10 (see Fig. 1A in the online Data Supplement) would correspond to the formation of the di-propoxy derivative. A small amount of impurities (<5%) was observed in the straight-scan analysis of di-propoxy-CoQ10 (see Fig. 1B in the online Data Supplement). Production analysis of both CoQ10 and di-propoxy-CoQ10 (Fig. 2, A and B, in the online Data Supplement) demonstrated clearly that these impurities were not CoQ10 analogs, but we were unable to confirm their identities. At the concentration of di-propoxy-CoQ10 used in tissue determinations (150 nmol/L), these impurities would be undetected on reversed-phase HPLC. Di-propoxy-CoQ10 is stable during the tissue extraction procedure and can be stored for up to 1 year at –70 °C with no evidence of degradation. Di-ethoxy-CoQ10 was poorly resolved from CoQ10 on reversed-phase HPLC (see Fig. 3 in the online Data Supplement), and no further evaluation of this IS was undertaken. In contrast, di-propoxy-CoQ10 was clearly separated from CoQ9 and CoQ10 (Fig. 1
). The ultraviolet absorbance (275 nm) of di-propoxy-CoQ10 showed linearity (r2 = 0.999) over the concentration range 0–1000 nmol/L. Use of this IS (500 nmol/L CoQ10 added to skeletal muscle homogenate with an endogenous CoQ10 concentration of 350 nmol/L) gave a mean (SE) recovery of 99.8 (2.9)% (n = 5) of CoQ10 in the assay. The intraassay CVs for the assessment of CoQ10 in skeletal muscle, plasma, and MNC samples were 3.4% (mean concentration, 791 nmol/L; n = 6), 4.4% (201 nmol/L; n = 6), and 2.6% (331 nmol/L; n = 5), respectively. The interassay CVs for CoQ10 determination in skeletal muscle, MNCs, and plasma were 3.1% (861 nmol/L; n = 4), 3.5% (471 nmol/L; n = 5), and 4.5% (760 nmol/L; n = 4), respectively, when the di-propoxy-CoQ10 was used as IS. Detection of CoQ10 was linear between 10 and 1000 nmol/L in skeletal muscle (r2 = 0.997), MNCs (r2 = 0.995), and plasma (r2 = 0.991). The limit of detection of CoQ10 was 6 nmol/L for all tissues.
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Reference intervals for skeletal muscle, MNCs, and plasma were established from the observed range of CoQ10 concentrations for these tissues (Table 1
). The reference intervals for skeletal muscle and plasma were comparable to those reported by Artuch et al. (16) and Miles et al. (17) for skeletal muscle and plasma, respectively. To our knowledge, there have been no reference intervals for MNC CoQ10 reported by other laboratories. Age and sex had no significant influence on tissue CoQ10 concentrations in the reference population, allowing the effect of these variables to be excluded from the study (results not shown). By comparing the reference intervals, we found evidence of a CoQ10 deficiency in skeletal muscle (33 pmol/mg of protein) and MNCs (20 pmol/mg of protein) in the 47-year-old female patient with low skeletal muscle complex II-III activity.
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The decreased CoQ10 status of MNCs and skeletal muscle from this patient suggested that a relationship might exist between the CoQ10 status of these tissues, and this prompted us to assess the relationship between skeletal muscle, MNC, and plasma CoQ10. We found a close association between skeletal muscle and MNC CoQ10 concentrations in the 12 disease control patients and in the CoQ10-deficient patient (r = 0.89; P <0.02; n = 13). Exclusion of the CoQ10-deficient patient from this correlation did not significantly alter this relationship (r = 0.86; P <0.02; n = 12). We found no correlation between skeletal muscle and plasma CoQ10 concentrations (r = 0.015; n = 10) or between MNC and plasma CoQ10 concentrations (r = 0.21; n = 24).
In conclusion, we have synthesized a di-propoxy-CoQ10 IS that can be used in CoQ10 assessment in MNCs, skeletal muscle, and plasma, allowing precision and a good recovery. This IS enabled the establishment of reference intervals for the CoQ10 concentrations of skeletal muscle, MNCs, and plasma, which has facilitated the identification of a patient with a CoQ10 deficiency.
Acknowledgments
A.J. Duncan was supported by a grant from the Brain Research Trust (UK) awarded to Dr. S.J.R. Heales. Dr. I.P. Hargreaves is the recipient of an Association of Clinical Biochemists (UK) scholarship award, which also funded part of this work.
References
The following articles in journals at HighWire Press have cited this article:
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  S Rahman and M G Hanna Diagnosis and therapy in neuromuscular disorders: diagnosis and new treatments in mitochondrial diseases J. Neurol. Neurosurg. Psychiatry, September 1, 2009; 80(9): 943 - 953. [Abstract] [Full Text] [PDF]
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 P. Shelley, M. S. Martin-Gronert, A. Rowlerson, L. Poston, S. J. R. Heales, I. P. Hargreaves, J. M. McConnell, S. E. Ozanne, and D. S. Fernandez-Twinn Altered skeletal muscle insulin signaling and mitochondrial complex II-III linked activity in adult offspring of obese mice Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2009; 297(3): R675 - R681. [Abstract] [Full Text] [PDF]
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 S. Rahman, I. P. Hargreaves, M. Hirano, R. A. Marti, S. DiMauro, P. F. Chinnery, and J. Vissing ALLOGENEIC STEM CELL TRANSPLANTATION CORRECTS BIOCHEMICAL DERANGEMENTS IN MNGIE Neurology, May 22, 2007; 68(21): 1872 - 1873. [Full Text] [PDF]
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