Serum Ubiquinone-10 in a Pediatric Population

To the Editor:

Ubiquinone-10 is a lipid implicated in several biological functions. In tissues, it has an important role in electron transport and ATP synthesis related to the mitochondrial respiratory chain. The reduced form of ubiquinone protects cells from peroxidative damage (1). In blood, ubiquinone-10 is transported by lipoproteins, is one of the antioxidants within LDL, and prevents free radical damage caused by neutrophils and oxidative injury by endothelial cells in ischemia-reperfusion (1). Adequate serum concentrations of ubiquinone seem necessary to prevent peroxidative damage.

Various procedures to measure total ubiquinone-10 have been described, with HPLC with ultraviolet (UV) detection being the most common (2). Reference intervals for adults (3) and newborns (4) have been reported, but to our knowledge, no data are available for children.

Our aim was to establish serum reference values for a pediatric population in our geographical area with our working conditions.

Specimens were collected from apparently healthy children (by history and analytical data) before minor surgical intervention (n = 102; 38 females and 64 males; ages, 1 month–18 years), in accordance with the Helsinki Declaration of 1975, as revised in 1983. We collected 1.5 mL of 12-h fasting blood samples in Venoject silicone-coated gel tubes (Terumo Corp.), protected the samples from light and centrifuged them (2000g for 10 min at 4 °C). The serum was separated, frozen at −40 °C, and analyzed within 2 weeks. The total ubiquinone-10 (reduced plus oxidized) concentration was measured by HPLC with UV detection (275 nm) according to Zierz et al. (2), with slight modifications (ubiquinone-7 was used instead of ubiquinone-9 as the internal calibrator). Briefly, we added ubiquinone-7 (Sigma Chemical Co.) to 400 μL of serum or calibrator [375 μL of 154 mmol/L sodium chloride + 25 μL of ubiquinone-10 (Sigma Chemical Co.)]. The final concentration of both calibrators was 1.25 μmol/L. They were dissolved in 200 mL/L n-hexane–800 mL/L ethanol. Samples and calibrators were added to 1 mL of methanol containing 10 g/L pyrogallol and were saponified with 50 μL of 500 g/L potassium hydroxide for 10 min at 56 °C. The ubiquinone was extracted with 1 mL of n-hexane, mixed thoroughly for 1 min, desiccated under nitrogen, and dissolved in 200 μL of ethanol.

Chromatographic conditions were as follows: a Perkin-Elmer Integral 4000 HPLC system with a Perkin-Elmer Turbochrom Data Analysis module, a Nucleosil C₁₈ column (150 × 4 mm, 5 μm particle size), and a C₁₈ precolumn. The eluting solvent was 7 g of NaClO₄·H₂O in 1000 mL of 700 mL/L ethanol–300 mL/L methanol–1 mL/L HClO₄; the flow rate was 1.3 mL/min. The equilibration time was 3 min, and the total chromatographic time was 8 min.

The results for ubiquinone-10 were expressed as molar concentration (μmol/L serum) and related to serum cholesterol concentrations (μmol/mol cholesterol). Cholesterol was measured by standard procedures (Cobas Integra Analyzer, Roche Diagnostic Systems).

The within-run imprecision (CV; n = 20) was 4.4% (0.79 ± 0.035 μmol/L) and 4.7% (1.1 ± 0.051 μmol/L); the between-day CV (n = 18) was 6.7% (0.87 ± 0.058 μmol/L) and 7.5% (1.25 ± 0.093 μmol/L). Linearity was apparent between 0.15 and 10 μmol/L (r = 0.99). The recovery was 101% ± 10%.

Because serum ubiquinone-10 values in our sample pediatric population did not follow a gaussian distribution (assessed by the Kolmogorov–Smirnov test), we calculated the median and the 2.5 and 97.5 percentiles for reference values. The ubiquinone concentration was independent of sex (P >0.05, Mann–Whitney test) but decreased significantly with age (r = −0.383, n = 102, P <0.0001 for ubiquinone in μmol/L; r = −0.384, n = 102, P <0.0005 for ubiquinone in μmol/mol cholesterol), whereas cholesterol values did not change with age. Ubiquinone concentrations correlated with cholesterol concentrations (r = 0.485, P <0.00001, Spearman test). After applying statistical analysis to all age groups (Kruskal–Wallis), we established two groups whose ubiquinone concentrations (in μmol/L and μmol/mol cholesterol) were the most significantly different from one another (Mann–Whitney, P <0.005): 1 month–7 years [n = 62; median, 0.8 μmol/L (interval, 0.46–1.38 μmol/L) and 203 μmol/mol cholesterol (interval, 137–341 μmol/mol cholesterol)], and 8–18 years [n = 40; median, 0.57 μmol/L (interval, 0.34–1.03 μmol/L) and 169 μmol/mol cholesterol (interval, 111–248 μmol/mol cholesterol)].

HPLC with UV detection is a rapid and useful procedure for the quantification of total ubiquinone and correlates well with other procedures (5). We established reference intervals for serum ubiquinone in a Mediterranean pediatric population, showing a decrease of ubiquinone related to age, as was also reported by other authors in several tissues (1). Dietetic habits may influence serum ubiquinone-10 concentrations (6). Therefore, little difference in serum ubiquinone-10 values would be expected between several populations. In our experience, both ubiquinone-10 and ubiquinone-9 are present in substantial amounts in serum (data not shown). Ubiquinone-9 is not synthesized by humans; however, small amounts have been found in serum by some authors (7) but not by others (2). Interestingly, vegetable oils are the richest sources of ubiquinone-9 (6), an important food in Mediterranean area. Consequently, ubiquinone-9 is not suitable for use as internal calibrator to assay serum ubiquinone-10, at least in our population; we, therefore, chose ubiquinone-7.

Serum ubiquinone results are usually reported as molar concentrations but rarely are related to cholesterol (8). The reference intervals thus obtained are different, and the ubiquinone-to-cholesterol ratio yields a more adjusted reference interval. Moreover, ubiquinone-10 in serum protects lipoproteins from peroxidation. Therefore, the relationship between ubiquinone and cholesterol concentrations seems the best means of assessing its biological function in blood.

This work was supported in part by the Ministerio de Sanidad of Spain, grant FIS (95/0040).

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