Duncan, AJ

Eaton, S

Hargreaves, IP

Heales, SJR

Land, JM

Mills, K

English

UCL Discovery

15. Gibbs DA, Watts RWE. The variation of urinary oxalate excretion with age.J Lab Clin Med 1969;736:901–8.16. Barratt TM, Kasidas GP, Murdoch I, Rose GA. Urinary oxalate and glycolateexcretion and plasma oxalate concentration. Arch Dis Child 1991;66:501–3.17. von Schnakenburg C, Byrd DJ, Latta K, Reusz GS, Graf D, Brodehl J.Determination of oxalate excretion in spot urines of healthy children by ionchromatography. Eur J Clin Chem Clin Biochem 1994;32:27–9.18. Detry O, Honore P, DeRoover A, Trimeche M, Demoulin JC, Beaujean M, etal. Reversal of oxalosis cardiomyopathy after combined liver and kidneytransplantation. Transplant Int 2002;15:50–2.19. Johnson JS, Short AK, Hutchison A, Parrott NR, Roberts IS. Small intestinalinfarction: a fatal complication of systemic oxalosis. J Clin Pathol 2000;53:720–1.20. Marconi V, Mofid MZ, McCall C, Eckman I, Nousari HC. Primary hyperoxalu-ria: report of a patient with livedo reticularis and digital infarcts. J Am AcadDermatol 2002;46:S16–8.21. Boer P, van Leersum L, Hene RJ, Mees EJ. Plasma oxalate concentration inchronic renal disease. Am J Kidney Dis 1984;4:118–22.22. Hylander E, Jarnum S, Nielsen K. Calcium treatment of enteric hyperoxaluriaafter jejunoileal bypass for morbid obesity. Scand J Gastroenterol 1980;15:349–52.23. Canos HJ, Hogg GA, Jeffery JR. Oxalate nephropathy due to gastrointestinaldisorders. Can Med Assoc J 1981;124:729–33.24. Drenick EJ, Stanley TM, Border WA, Zawada ET, Dornfeld LP, Upham T, et al.Renal damage with intestinal bypass. Ann Intern Med 1978;89:594–9.25. McLeod RS, Churchill DN. Urolithiasis complicating inflammatory boweldisease. J Urol 1992;148:974–8.26. Hassan I, Juncos LA, Milliner DS, Sarmiento JM, Sarr MG. Chronic renalfailure secondary to oxalate nephropathy: a preventable complication afterjejunoileal bypass. Mayo Clin Proc 2001;76:758–60.DOI: 10.1373/clinchem.2005.054353Determination of Coenzyme Q10 Status in Blood Mono-nuclear Cells, Skeletal Muscle, and Plasma by HPLCwith Di-Propoxy-Coenzyme Q10 as an Internal Standard,Andrew J. Duncan,1,3 Simon J.R. Heales,1,2 Kevin Mills,3Simon Eaton,3 John M. Land,1,2 and Iain P. Hargreaves1,2*(1 Division of Neurochemistry, Institute of Neurology,and2 Neurometabolic Unit, National Hospital for Neurol-ogy and Neurosurgery, London, United Kingdom;3 Bio-chemistry Unit, Institute of Child Heath, London, UnitedKingdom; * address correspondence to this author at:Neurometabolic Unit, National Hospital for Neurologyand Neurosurgery, Queen Square, London WC1N 3BG,United Kingdom; fax 44-0-20-7829-1016, e-mailihargrea@ion.ucl.ac.uk)Coenzyme Q10 (CoQ10), the predominant ubiquinone spe-cies in humans, functions as an electron carrier in themitochondrial electron transport chain (ETC) and as anintracellular antioxidant (1). Although primary CoQ10deficiency is rare, a profound deficiency in skeletal muscleCoQ10 has been reported in patients with multisystemmitochondrial encephalomyopathies (2, 3). Cardiovascu-lar disease has been associated with a CoQ10 deficiency(4, 5), and it is becoming increasingly apparent that othergroups of patients may become CoQ10 deficient, particu-larly individuals with ataxia (6) and some patients receiv-ing statins (7).When assessing tissue CoQ10 status, we have found thatthe lack of a commercially available nonphysiologic inter-nal standard (IS) is a major difficulty. Although naturallyoccurring ubiquinones have been used as ISs in thisdetermination, they are not free from the influence ofubiquinones that might be present in human tissue as theresult of dietary contamination (8) or synthesis by micro-organisms (9, 10). There is a need, therefore, for analternative IS that is not influenced by exogenous/endog-enous ubiquinones. Di-ethoxy-CoQ10 has been suggestedas a nonphysiologic IS to determine CoQ10 (11). In thisstudy we evaluated this IS along with di-propoxy-CoQ10for their suitability to determine tissue CoQ10. Referenceintervals were established for the CoQ10 concentration ofskeletal muscle, blood mononuclear cells (MNCs), andplasma. A patient with a suspected CoQ10 deficiency wassubsequently 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 tofemales, 7:6] with no evidence of an ETC deficiencydetected in their skeletal muscle biopsies; (b), MNCs from17 healthy volunteers and 13 disease controls with noclinical evidence of an ETC deficiency [mean (SE) age,32.6 (2.6) years; range, 1–61 years; ratio of males tofemales, 7:8]; and (c), plasma from 24 patients [mean (SE)age, 14.3 (2.9) years; range, 1–57 years; ratio of males tofemales, 2:1] with no clinical evidence of a ETC deficiency.The correlation between skeletal muscle, MNC, andplasma CoQ10 status was assessed in 2 groups of patientswith no clinical or biochemical evidence of an ETCdeficiency: 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]. Correlationsbetween skeletal muscle and MNC CoQ10 status andbetween skeletal muscle and plasma CoQ10 status weredetermined with samples from group 1; correlations be-tween MNC and plasma CoQ10 status were determinedwith samples from groups 1 and 2.The patient with a suspected CoQ10 deficiency was a47-year-old female, mentally retarded since birth, ataxic,and with poor vision and hypertrophic cardiomyopathy,in whom evidence of an ETC complex II-III (succinatecytochrome c reductase) deficiency [0.015; reference inter-val, 0.040–0.204 (activity expressed as a ratio to citratesynthase 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 ofthe ACCUSPINTM system–Histopaque®-1077 (Sigma-Aldrich). The MNCs were suspended in phosphate-buffered saline (150 mmol/L NaCl, 150 mmol/L sodiumphosphate), pH 7.2 (200 L per 5 mL of blood), and storedat 70 °C until analysis. During this procedure, plasmawas separated from the sodium EDTA–anticoagulatedblood and stored at 70 °C until analysis.Skeletal muscle biopsy homogenates were prepared asdescribed by Heales et al. (12). Protein concentration wasdetermined by the method of Lowry et al. (13). Thesynthesis of di-ethoxy-CoQ10 was undertaken as de-2380 Technical Briefsscribed by Edlund (11). The synthesis of di-propoxy-CoQ10 was based on the method of Edlund (11), sub-stituting propan-1-ol for ethanol. The concentration ofdi-propoxy-CoQ10 was estimated based on the molarabsorptivity for CoQ10 at 275 nm (14.6  103), and thedi-propoxy-CoQ10 was diluted in ethanol to give a finalconcentration of 1.5 mol/L.Samples were prepared for HPLC analysis of totalCoQ10 concentration by the addition of IS (30 L) toskeletal muscle (50 L), to MNCs (150 L), and to plasma(200 L) to give a final concentration of 150 nmol/L in thereconstituted extract. The ubiquinones (CoQ10 and IS)were extracted by the method of Boitier et al. (14). Theextracts were evaporated under N2 and reconstituted inethanol (300 L). HPLC analysis was performed accord-ing to the method of Boitier et al. (14).CoQ10 and di-propoxy-CoQ10 were analyzed at concen-trations of 50 mol/L by mass spectrometry using aQuattro micro triple-quadrupole tandem mass spectrom-eter operating in both the scan and parent ion modes (15).We used regression analysis to assess the correlationbetween ultraviolet absorbance (275 nm) and the concen-trations of di-propoxy-CoQ10 and skeletal muscle, MNC,and plasma CoQ10, and between age and the MNC,skeletal muscle, and plasma CoQ10 concentration. Therelationship between sex and tissue CoQ10 concentrationwas assessed by the Mann–Whitney U-test. Spearmanrank correlation coefficients were calculated to assess theassociation between the CoQ10 concentrations in skeletalmuscle, MNCs, and plasma. A P value 0.05 was consid-ered significant.Analysis of the mass spectrum obtained in scan modefor the di-propoxy-CoQ10 IS demonstrated 1 predominantion of m/z 942 (see Fig. 1B in the Data Supplement thataccompanies the online version of this Technical Brief athttp://www.clinchem.org/content/vol51/issue12). Thiscorresponded with the theoretical mass calculated for thesodium adduct of di-propoxy-CoQ10,[ M Na]. An ob-served increase in molecular mass of 56 Da in di-propoxy-CoQ10 relative to CoQ10 (see Fig. 1A in the online DataSupplement) would correspond to the formation of thedi-propoxy derivative. A small amount of impurities(5%) was observed in the straight-scan analysis ofdi-propoxy-CoQ10 (see Fig. 1B in the online Data Supple-ment). Production analysis of both CoQ10 and di-propoxy-CoQ10 (Fig. 2, A and B, in the online DataSupplement) demonstrated clearly that these impuritieswere not CoQ10 analogs, but we were unable to confirmtheir identities. At the concentration of di-propoxy-CoQ10used in tissue determinations (150 nmol/L), these impu-rities would be undetected on reversed-phase HPLC.Di-propoxy-CoQ10 is stable during the tissue extractionprocedure and can be stored for up to 1 year at 70 °Cwith no evidence of degradation. Di-ethoxy-CoQ10 waspoorly resolved from CoQ10 on reversed-phase HPLC (seeFig. 3 in the online Data Supplement), and no furtherevaluation of this IS was undertaken. In contrast, di-propoxy-CoQ10 was clearly separated from CoQ9 andCoQ10 (Fig. 1). The ultraviolet absorbance (275 nm) ofdi-propoxy-CoQ10 showed linearity (r2  0.999) over theconcentration range 0–1000 nmol/L. Use of this IS (500nmol/L CoQ10 added to skeletal muscle homogenate withan endogenous CoQ10 concentration of 350 nmol/L) gavea mean (SE) recovery of 99.8 (2.9)% (n  5) of CoQ10 inthe assay. The intraassay CVs for the assessment of CoQ10in skeletal muscle, plasma, and MNC samples were3.4% (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 determinationin 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 linearbetween 10 and 1000 nmol/L in skeletal muscle (r2 0.997), MNCs (r2  0.995), and plasma (r2  0.991). Thelimit of detection of CoQ10 was 6 nmol/L for all tissues.Fig. 1. Reversed-phase HPLC chromatogram of CoQ9, CoQ10, anddi-propoxy-CoQ10.Retention times of ubiquinones: 7.58 min (CoQ9), 9.36 min (CoQ10), and 12.33min (di-propoxy-CoQ10). UV, ultraviolet.Clinical Chemistry 51, No. 12, 2005 2381Reference intervals for skeletal muscle, MNCs, andplasma were established from the observed range ofCoQ10 concentrations for these tissues (Table 1). Thereference intervals for skeletal muscle and plasma werecomparable to those reported by Artuch et al. (16) andMiles et al. (17) for skeletal muscle and plasma, respec-tively. To our knowledge, there have been no referenceintervals for MNC CoQ10 reported by other laboratories.Age and sex had no significant influence on tissue CoQ10concentrations in the reference population, allowing theeffect 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 skeletalmuscle (33 pmol/mg of protein) and MNCs (20 pmol/mgof protein) in the 47-year-old female patient with lowskeletal muscle complex II-III activity.The decreased CoQ10 status of MNCs and skeletalmuscle from this patient suggested that a relationshipmight exist between the CoQ10 status of these tissues, andthis prompted us to assess the relationship betweenskeletal muscle, MNC, and plasma CoQ10. We found aclose association between skeletal muscle and MNCCoQ10 concentrations in the 12 disease control patientsand in the CoQ10-deficient patient (r  0.89; P 0.02; n 13). Exclusion of the CoQ10-deficient patient from thiscorrelation did not significantly alter this relationship (r 0.86; P 0.02; n  12). We found no correlation betweenskeletal muscle and plasma CoQ10 concentrations (r 0.015; n  10) or between MNC and plasma CoQ10concentrations (r  0.21; n  24).In conclusion, we have synthesized a di-propoxy-CoQ10IS that can be used in CoQ10 assessment in MNCs, skeletalmuscle, and plasma, allowing precision and a good recov-ery. This IS enabled the establishment of reference inter-vals for the CoQ10 concentrations of skeletal muscle,MNCs, and plasma, which has facilitated the identifica-tion of a patient with a CoQ10 deficiency.A.J. Duncan was supported by a grant from the BrainResearch Trust (UK) awarded to Dr. S.J.R. Heales. Dr. I.P.Hargreaves is the recipient of an Association of ClinicalBiochemists (UK) scholarship award, which also fundedpart of this work.References1. Ernster L, Dallner G. Biochemical, physiological and medical aspects ofubiquinone function. Biochim Biophys Acta 1995;1271:195–204.2. Ogasahara S, Engel AG, Frens D, Mack D. Muscle coenzyme Q10 deficiencyin familial mitochondrial encephalomyopathy. Proc Natl Acad Sci U S A1989;86:2379–82.3. Sobreira C, Hirano M, Shanske S, Keller RK, Haller RG, David E, et al.Mitochondrial encephalomyopathy with coenzyme Q10 deficiency. Neurology1997;48:1238–43.4. Mortensen SA, Vadhanavikit S, Muratsu K, Folkers K. Coenzyme Q10: clinicalbenefits with biochemical correlates suggesting a scientific breakthrough inthe management of chronic heart failure. Int J Tissue React 1990;12:155–62.5. Folkers K, Watanabe T. Bioenergetics in clinical medicine XIV. Studies on anapparent deficiency of coenzyme Q10 in patients with cardiovascular andrelated disease. J Med 1978;9:67–79.6. Musumeci O, Naini A, Slonim AE, Skavin N, Hadjigeorgiou GL, Krawiecki N,et al. Familial cerebellar ataxia with muscle coenzyme Q10 deficiency.Neurology 2001;56:849–55.7. Bleske BE, Willis RA, Anthony M, Casselberry N, Datwani M, Uhley VE. Theeffect of pravastatin and atorvastatin on coenzyme Q10. Am Heart J2001;142:E2.8. Weber C, Bysted A, Hllmer G. The coenzyme Q10 content of the averageDanish diet. Int J Vitam Nutr Res 1997;67:123–9.9. Daves GD, Muraca RF, Hitticks JS, Fris K, Siegel H. Discovery of ubiqui-none-1, -2, -3, and -4 and the nature of isoprenylation. Biochem J 1967;113:38P–9P.10. Sippel CJ, Goewert RR, Slachman FN, Olson RE. The regulation of ubiqui-none-6 biosynthesis by Saccharomyces cerevisiae. J Biol Chem 1983;258:1057–61.11. Edlund PE. Determination of coenzyme Q10, a-tocopherol and cholesterol inbiological samples by coupled-column liquid chromatography with coulumet-ric and ultraviolet detection. J Chromatogr 1988;425:87–97.12. Heales SJR, Hargreaves IP, Olpin SE, Guthrie P, Bonham JR, Morris AAM, etal. Diagnosis of mitochondrial electron transport chain defects in smallmuscle biopsies. J Inherit Metab Dis 1996;19(Suppl 1):151.13. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement withfolin phenol reagent. J Biol Chem 1951;193:265–75.14. Boitier E, Degoul F, Desguerre I, Christiane C, Francois D, Ponsot G, et al.A case of mitochondrial encephalomyopathy associated with a musclecoenzyme Q10 deficiency. J Neurol Sci 1998;156:41–6.15. Mills K, Eaton S, Ledger V, Young E, Winchester B. The synthesis of internalstandards for the quantification of sphingolipids by tandem mass spectrom-etry. Rapid Commun Mass Spectrom 2005;19:1739–48.16. Artuch R, Briones P, Aracil A, Ribes A, Pineda J, Galvan M. Familial cerebellarataxia with coenzyme Q10 deficiency. J Inherit Metab Dis 2004;27(Suppl1):118.17. Miles MV, Horn PS, Tang PH, Morrison JA, Miles L, Degrauw T. Age-relatedchanges in plasma coenzyme Q10 concentrations and redox state inapparently healthy children and adults. Clin Chim Acta 2004;347:139–44.DOI: 10.1373/clinchem.2005.054643Serum Tartrate-Resistant Acid Phosphatase 5b or Ami-no-Terminal Propeptide of Type I Procollagen for Mon-itoring Bisphosphonate Therapy in PostmenopausalOsteoporosis? Matti J. Va ¨lima ¨ki1* and Riitta Ta ¨htela ¨2 (1 Di-vision of Endocrinology, Department of Medicine, Hel-sinki University Central Hospital, Helsinki, Finland;2 Me-hila ¨inen Oy Laboratoriopalvelut, Helsinki, Finland;* address correspondence to this author at: Division ofEndocrinology, Department of Medicine, Helsinki Uni-versity Central Hospital, FIN-00290 Helsinki, Finland; fax358-9-47175798, e-mail matti.valimaki@hus.fi)Bone markers to monitor the efficacy of antiresorptivetherapy of osteoporosis are of great value to clinicians.Considerable decreases in markers can be seen within 3 to6 months after the start of an efficient treatment, withconsiderable increases in bone mineral density (BMD)Table 1. Reference intervals for skeletal muscle, MNC, andplasma CoQ10 concentrations.CoQ10 concentration UnitsSkeletal muscleObserved range 140–580 pmol/mg of proteinMean (SD) 241 (95) pmol/mg of proteinMNCsObserved range 37–133 pmol/mg of proteinMean (SD) 65 (24) pmol/mg of proteinPlasmaObserved range 227–1432 nmol/LMean (SD) 675 (315) nmol/L2382 Technical Briefs

A case of mitochondrial encephalomyopathy associated with a muscle coenzyme Q10 deficiency.

and the nature of isoprenylation.

Bioenergetics in clinical medicine XIV. Studies on an apparent deficiency of coenzyme Q10 in patients with cardiovascular and related disease.

Coenzyme Q10: clinical benefits with biochemical correlates suggesting a scientific breakthrough in the management of chronic heart failure.

Determination of coenzyme Q10, a-tocopherol and cholesterol in biological samples by coupled-column liquid chromatography with coulumetric and ultraviolet detection.

Diagnosis of mitochondrial electron transport chain defects in small muscle biopsies.

Familial cerebellar ataxia with coenzyme Q10 deficiency.

Familial cerebellar ataxia with muscle coenzyme Q10 deficiency.

Muscle coenzyme Q10 deficiency in familial mitochondrial encephalomyopathy.

physiological and medical aspects of ubiquinone function.

Protein measurement with folin phenol reagent.

The coenzyme Q10 content of the average Danish diet.

The effect of pravastatin and atorvastatin on coenzyme Q10. Am Heart J

The regulation of ubiquinone-6 biosynthesis by Saccharomyces cerevisiae.

The synthesis of internal standards for the quantification of sphingolipids by tandem mass spectrometry. Rapid Commun Mass Spectrom

Determination of coenzyme Q(10) status in blood mononuclear cells, skeletal muscle, and plasma by HPLC with Di-propoxy-coenzyme Q(10) as an internal standard

Determination of coenzyme Q(10) status in blood mononuclear cells, skeletal muscle, and plasma by HPLC with Di-propoxy-coenzyme Q(10) as an internal standard

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UCL Discovery