Determination of coenzyme [Q.sub.10] status in blood mononuclear cells, skeletal muscle, and plasma by HPLC with di-propoxy-coenzyme [Q.sub.10] as an internal standard.
When assessing tissue Co[Q.sub.10] 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 micro-organisms (9,10). There is a need, therefore, for an alternative IS that is not influenced by exogenous/endogenous ubiquinones. Di-ethoxy-Co[Q.sub.10] has been suggested as a nonphysiologic IS to determine Co[Q.sub.10] (11). In this study we evaluated this IS along with di-propoxy-Co[Q.sub.10] for their suitability to determine tissue Co[Q.sub.10]. Reference intervals were established for the Co[Q.sub.10] concentration of skeletal muscle, blood mononuclear cells (NINCs), and plasma. A patient with a suspected Co[Q.sub.10] 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 Co[Q.sub.10] 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 Co[Q.sub.10] status and between skeletal muscle and plasma Co[Q.sub.10] status were determined with samples from group 1; Correlations between MNC and plasma Co[Q.sub.10] status were determined with samples from groups 1 and 2.
The patient with a suspected Co[Q.sub.10] 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 ACCUSPIN[TM] system-Histopaque[R]-1077 (Sigma-Aldrich). The MNCs were suspended in phosphate-buffered saline (150 mmol/L NaCl, 150 mmol/L sodium phosphate), pH 7.2 (200 [micro]L per 5 mL of blood), and stored at -70[degrees]C until analysis. During this procedure, plasma was separated from the sodium EDTA-anticoagulated blood and stored at -70[degrees]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-Co[Q.sub.10] was undertaken as described by Edlund (11). The synthesis of di-propoxy-Co[Q.sub.10] was based on the method of Edlund (11), substituting propan-1-ol for ethanol. The concentration of di-propoxy-Co[Q.sub.10] was estimated based on the molar absorptivity for Co[Q.sub.10] at 275 nm (14.6 x [10.sup.3]), and the di-propoxy-Co[Q.sub.10] was diluted in ethanol to give a final concentration of 1.5 [micro]mol/L.
Samples were prepared for HPLC analysis of total Co[Q.sub.10] concentration by the addition of IS (30 [micro]L) to skeletal muscle (50 [micro]L), to MNCs (150 [micro]L), and to plasma (200 [micro]L) to give a final concentration of 150 nmol/L in the reconstituted extract. The ubiquinones (Co[Q.sub.10] and IS) were extracted by the method of Bonier et al. (14). The extracts were evaporated under NZ and reconstituted in ethanol (300 [micro]L). HPLC analysis was performed according to the method of Bonier et al. (14).
Co[Q.sub.10] and di-propoxy-Co[Q.sub.10] were analyzed at concentrations of 50 [micro]mol/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-Co[Q.sub.10] and skeletal muscle, MNC, and plasma Co[Q.sub.10], and between age and the MNC, skeletal muscle, and plasma Co[Q.sub.10] concentration. The relationship between sex and tissue Co[Q.sub.10] concentration was assessed by the Mann-Whitney U-test. Spearman rank correlation coefficients were calculated to assess the association between the Co[Q.sub.10] 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-Co[Q.sub.10] 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-Co[Q.sub.10], [M+Na]. An observed increase in molecular mass of 56 Da in di-propoxy-Co[Q.sub.10] relative to Co[Q.sub.10] (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-Co[Q.sub.10] (see Fig. 1B in the online Data Supplement). Production analysis of both Co[Q.sub.10] and dipropoxy-Co[Q.sub.10] (Fig. 2, A and B, in the online Data Supplement) demonstrated clearly that these impurities were not Co[Q.sub.10] analogs, but we were unable to confirm their identities. At the concentration of di-propoxy-Co[Q.sub.10] used in tissue determinations (150 nmol/L), these impurities would be undetected on reversed-phase HPLC. Di-propoxy-Co[Q.sub.10] is stable during the tissue extraction procedure and can be stored for up to 1 year at -70[degrees]C with no evidence of degradation. Di-ethoxy-Co[Q.sub.10] was poorly resolved from Co[Q.sub.10] on reversed-phase HPLC (see Fig. 3 in the online Data Supplement), and no further evaluation of this IS was undertaken. In contrast, dipropoxy-Co[Q.sub.10] was clearly separated from Co[Q.sub.9] and Co[Q.sub.10] (Fig. 1). The ultraviolet absorbance (275 nm) of di-propoxy-Co[Q.sub.10] showed linearity ([r.sup.2] = 0.999) over the concentration range 0-1000 nmol/L. Use of this IS (500 nmol/L Co[Q.sub.10] added to skeletal muscle homogenate with an endogenous Co[Q.sub.10] concentration of 350 nmol/L) gave a mean (SE) recovery of 99.8 (2.9)% (n = 5) of Co[Q.sub.10] in the assay. The intraassay CVs for the assessment of Co[Q.sub.10] in skeletal muscle, plasma, and NINC 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 Co[Q.sub.10] 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-Co[Q.sub.10] was used as IS. Detection of Co[Q.sub.10] was linear between 10 and 1000 nmol/L in skeletal muscle ([r.sup.2] = 0.997), NINCs ([r.sup.2] = 0.995), and plasma ([r.sup.2] = 0.991). The limit of detection of Co[Q.sub.10] was 6 nmol/L for all tissues.
[FIGURE 1 OMITTED]
Reference intervals for skeletal muscle, MNCs, and plasma were established from the observed range of Co[Q.sub.10] 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 Co[Q.sub.10] reported by other laboratories. Age and sex had no significant influence on tissue Co[Q.sub.10] 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 Co[Q.sub.10] 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.
The decreased Co[Q.sub.10] status of MNCs and skeletal muscle from this patient suggested that a relationship might exist between the Co[Q.sub.10] status of these tissues, and this prompted us to assess the relationship between skeletal muscle, MNC, and plasma Co[Q.sub.10]. We found a close association between skeletal muscle and MNC Co[Q.sub.10] concentrations in the 12 disease control patients and in the Co[Q.sub.10] deficient patient (r = 0.89; P <0.02; n = 13). Exclusion of the Co[Q.sub.10] 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 Co[Q.sub.10] concentrations (r = 0.015; n = 10) or between MNC and plasma Co[Q.sub.10] concentrations (r = 0.21; n = 24).
In conclusion, we have synthesized a di-propoxy-Co[Q.sub.10] IS that can be used in Co[Q.sub.10] assessment in MNCs, skeletal muscle, and plasma, allowing precision and a good recovery. This IS enabled the establishment of reference intervals for the Co[Q.sub.10] concentrations of skeletal muscle, NINCs, and plasma, which has facilitated the identification of a patient with a Co[Q.sub.10] deficiency.
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DOI : 10.1373/clinchem.2005.054643
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.
Andrew J. Duncan, [1,3] Simon J.R. Heales, [1,2] Kevin Mills,  Simon Eaton,  John M. Land, [1,2] and Iain P. Hargreavesn [1,2] * ( Division of Neurochemistry, Institute of Neurology, and  Neurometabolic Unit, National Hospital for Neurology and Neurosurgery, London, United Kingdom;  Biochemistry Unit, Institute of Child Heath, London, United Kingdom; * address 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 email@example.com)
Table 1. Reference intervals for skeletal muscle, MNC, and plasma Co[Q.sub.10] concentrations. Co[Q.sub.10] Units concentration Skeletal muscle Observed range 140-580 pmol/mg of protein Mean (SD) 241 (95) pmol/mg of protein MNCs Observed range 37-133 pmol/mg of protein Mean (SD) 65 (24) pmol/mg of protein Plasma Observed range 227-1432 nmol/L Mean (SD) 675 (315) nmol/L
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|Title Annotation:||Technical Briefs|
|Author:||Duncan, Andrew J.; Heales, Simon J.R.; Mills, Kevin; Eaton, Simon; Land, John M.; Hargreaves, Iain P|
|Date:||Dec 1, 2005|
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