Coenzyme Q10 (CoQ10) is present in cellular membranes of lipophilic cell organelles and in serum lipoproteins (1,2). Because it is part of a redox couple, an equilibrium exists between oxidized CoQ10 (CoQ10ox; ubiquinone) and reduced CoQ10 (CoQ10red; CoQ10H2; ubiquinol) (Fig. 1) (3). Approximately 60% of plasma CoQ10 is associated with low-density lipoproteins (LDLs), 25% with high-density lipoproteins (HDLs) and 15% with other lipoproteins (2,4). Hence, a strong correlation is found between plasma CoQ10 and LDL, and thus cholesterol concentration (4,5). Additionally, other antioxidants, such as α-tocopherol and β-carotene, are also predominantly transported by LDL (6,7).
Coenzyme Q10 originates both from endogenous synthesis and dietary intake (4,8,9). Although plasma levels are significantly influenced by dietary uptake, the tissue levels of CoQ10 depend mainly on de novo synthesis (10). Coenzyme Q10red acts as a potent antioxidant in mitochondria and lipid membranes by scavenging free oxygen radicals, thus efficiently protecting membrane phospholipids, serum polyunsaturated fatty acids and lipoproteins as LDL from free radical–induced oxidative damage (2–4,11). Coenzyme Q10red also recycles oxidized α-tocopheryl back to α-tocopherol (vitamin E; Fig. 2), thus potentiating its antioxidative effect (3,12,13). In addition, CoQ10ox acts as an essential electron carrier in the mitochondrial adenosine triphosphate synthesis of all body cells (4).
Cystic fibrosis (CF) is an autosomal recessive inherited disease predominantly characterized by chronic pulmonary inflammation and infection in combination with maldigestion and malabsorption due to pancreatic insufficiency (14). These symptoms are the result of a defective cystic fibrosis transmembrane conductance regulator (CFTR) protein, which has an essential role in chloride transport (15). In the lungs, the defective CFTR protein leads to poor hydration of the airway surface lining fluid, resulting in thick mucus, defective ciliary clearance and airway plugging, the combination of which promotes recurrent bacterial infection and chronic inflammation, and thereby induces oxidative stress. These reactive oxygen species, combined with the impaired absorption of fat-soluble antioxidant nutrients, such as α-tocopherol and β-carotene, result in an oxidant-antioxidant imbalance (16–18).
The surplus of free oxygen radicals causes peroxidation of polyunsaturated fatty acids in cellular biomembranes and of lipoproteins in the circulation, and contribute to the progressive deterioration of pulmonary function (19–21).
We hypothesized that the equilibrium between CoQ10ox and CoQ10red in patients with cystic fibrosis might be disturbed. In addition, we assumed that the fat malabsorption characteristic for cystic fibrosis may adversely influence the intestinal uptake of CoQ10, resulting in reduced plasma levels.
PATIENTS AND METHODS
Pediatric patients ages 8 to 18 years were enrolled in the study. Patients with CF in clinical stable condition were recruited from the CF center of the Wilhelmina Children's Hospital. The control patients consisted of children from the pediatric gastroenterology outpatient clinic and children who underwent ENT (ear, nose and throat) or orthopedic surgery. The exclusion criteria were clinical signs of infection and diseases associated with oxidative stress (eg, cardiac, metabolic, cerebral, neuromuscular, or mitochondrial diseases). All patients with CF routinely received α-tocopherol supplementation (mean ± SD = 120 ± 75 mg). The α-tocopherol levels were monitored annually to ascertain the necessary amount of supplementation. This study was approved by the UMC Medical Ethics Committee, and informed consent was obtained from all patients and/or their parents.
Peripheral blood samples were collected in Venoject 2-mL capacity heparinized tubes (cholesterol) and 7-mL capacity EDTA tubes (CoQ10 and α-tocopherol). The EDTA tubes were immediately protected from light, briefly stored on ice, and subsequently centrifuged for 10 minutes at 2000g at 4°C. The plasma specimens were transferred to prelabeled capped polypropylene tubes and stored at −80°C until analysis.
Total serum cholesterol level was measured by routine clinical dry chemistry and colorimetric methods using cholesterol ester hydrolase and cholesterol oxidase (Dry Chemistry Vitros 950; OrthosClinical Diagnostics).
Before extraction and analysis of CoQ10red, CoQ10ox and α-tocopherol concentrations, the frozen plasma samples were thawed in a water bath of ambient temperature. All sample handling was conducted under subdued light to avoid photochemical decomposition. Subsequently, 1-mL plasma was transferred into a glass tube and 100 μL of a glutathione solution was added to a final concentration of 0.01 mol/L. This mixture was slowly added to 2 mL of 2-propanol containing Q9 (concentration, 1.5 mg/L) under continuous agitation, exposed for 15 minutes at a temperature of approximately 4°C and centrifuged at 2000g for 15 minutes at 4°C. Then, 750 μL of the supernatant was injected directly into a high-performance liquid chromatography system (quaternary Waters 2690 pump with automated injector and 2 reversed-phase suplex pkb100 columns [dimension = 250 × 4.6 mm; particle size = 5 μm]), preceded by a short precolumn (suplex pkb100; dimension = 20 × 4.0 mm; particle size = 5 μm); Supelco, Bellefonte, PA). Detection was performed by a photodiode array detector (Waters Corporation, Milford, MA). Column oven and sample tray temperatures were 20°C and 4°C, respectively. The system was controlled by Millennium software version 3.05 (Waters Corporation). The mobile phase consisted of ammonium acetate (concentration = 7.5 g/L) in (1) methanol, (2) acetonitril, (3) 2-propanol and (4) a solution consisting of 50% acetonitril and 50% water with a linear gradient. The flow rate was 1.5 mL/min and the run time was 60 minutes.
Quantification of Metabolite Concentrations
Coenzyme Q10red and α-tocopherol levels were measured simultaneously by means of a high-performance liquid chromatography system with diode-array detection at 292 nm, whereas CoQ10ox and Q9 levels were measured at 275 nm. Concentrations of CoQ10ox and CoQ10red were calculated on the basis of peak areas using internal standardization with Q9. The total CoQ10 was calculated as the sum of CoQ10ox and CoQ10red. Coenzyme Q10 is not commercially available as reference standard; therefore, a relative response factor (RRF) for CoQ10red (at 292 nm) compared with CoQ10ox (at 275 nm) was determined. The RRF was established in 3-fold by reducing a known amount of CoQ10ox using sodium tetrahydroborate and comparing the response area of the formed CoQ10red (at 292 nm) with the response area of the original solution containing only CoQ10ox (at 275 nm). The RRF was 4.0 ± 0.4 (mean ± SD). α-Tocopherol was quantified by external standardization. The results for CoQ10 were expressed as molar concentrations (μmol/L). Because lipophilic antioxidants, such as CoQ10, are carried by the circulating lipoproteins in plasma, CoQ10 was also expressed as μmol/mol cholesterol. The CoQ10 redox status was expressed as the percentage of CoQ10red in the total CoQ10.
Coenzyme Q10 plasma levels, CoQ10red/CoQ10 ratio (redox status), cholesterol level and anthropometric parameters (height, weight and body mass index [BMI]) were measured or calculated in both patients and controls. In the CF group, the data for the most recent forced expiratory volume in 1 second, expressed as percentage of the predicted value (FEV1 % predicted), and the fat resorption quotient were also collected.
Results are presented as means ± SD. Data were entered in an SPSS database and the differences were tested for significance using an independent Student t test. The Spearman rank correlation was used for calculating the correlation between the CoQ10 levels and FEV1, plasma cholesterol and α-tocopherol levels, and fat resorption quotient.
The cases of 30 preadolescent and adolescent children with stable CF and 30 age-matched clinically healthy control subjects were studied. There was no significant difference in age or sex distribution between the children with CF and the controls. Although a considerable difference in SD for height (P < 0.01) was observed, the SD for weight-for-height and BMI were not different (P = 0.42 and P = 0.57, respectively). The characteristics of patients are summarized in Table 1. Anthropometric data are shown as SD scores, according to the Dutch growth charts, generated by the 4th Nationwide Dutch Growth Study 1997 (22).
Plasma Levels of CoQ10ox and CoQ10red and Cholesterol
The total CoQ10 levels in patients with cystic fibrosis were significantly lower than those in the control group (0.87 ± 0.42 μmol/L vs 1.35 ± 0.39 μmol/L; P < 0.001). No significant difference was found in the redox status (CoQ10red to total CoQ10 ratio). The cholesterol levels (P < 0.001) and the ratio of CoQ10 to cholesterol (P < 0.05) were significantly lower in the CF group. The α-Tocopherol values were similar in both the CF and the control groups (18.4 ± 6.9 and 21.0 ± 3.3 μmol/L, respectively). The values are presented in Table 2.
The CoQ10 levels between the male and the female subjects within the study group and the control group were not significantly different. Likewise, between 2 age groups (7–11 and 12–18 years), no significant difference in CoQ10 level was found.
The Spearman rank correlation showed positive correlations between CoQ10 and cholesterol plasma levels (r = 0.6, P = 0.003) in the study group, in contrast with the control group in which no correlation was found (r = 0.3, P = 0.16). The CoQ10 plasma levels did not correlate with the FEV1 % predicted (r = 0.2, P = 0.2) nor with the severity of the cystic fibrosis when categorized into mild CF (FEV1 % predicted ≥ 80%) and moderate CF (FEV1 % predicted < 80%) (23). In addition, the Spearman rank correlation showed some correlation between CoQ10 and α-tocopherol (r = 0.4, P = 0.02), but no correlation between CoQ10 and the fat resorption quotient (r = 0.1, P = 0.6).
This study and an earlier abstract (24) clearly showed lowered CoQ10 plasma levels in children with CF, even when corrected for serum cholesterol levels. Low plasma CoQ10 levels, like low α-tocopherol and low β-carotene plasma levels, could contribute to impaired resistance to oxidation of LDL and, thus, to increased lipid peroxide formation, which, in turn, could result in enhanced propagation of disease processes, for example, in the lungs and liver of these patients (6,7). Interestingly, in our patients with CF, the percentage of reduced Q10 and oxidized Q10 was not different from controls. This is in contrast with other diseases associated with increased oxidative stress, such as coronary heart disease (25), hyperlipidemia (26), neurodegenerative disorders (27,28) and liver diseases (29), in which this equilibrium is disturbed. However, in pediatric patients with hyperthyroidism, Menke et al. (30) also demonstrated a normal CoQ10 redox status in combination with significantly reduced plasma CoQ10 levels. Their suggestion that the CoQ10 redox status in plasma does not reflect the intracellular redox status might also be applicable to patients with CF. Indeed, in another study, no clear correlation was found between the plasma CoQ10 concentration and the levels in platelets, representing mitochondria containing tissues (9). However, the normal percentage of CoQ10ox, as found in our group of patients with CF with mild to moderate disease, could also be due to a still adequate regeneration of CoQ10red by nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (11).
Although the low overall CoQ10 plasma levels may be due to the hypermetabolic state in patients with hyperthyroidism (30), a CoQ10red uptake from the gut is likely to be the primary problem in patients with CF. The correlation we found in this study between low vitamin E level and CoQ10 plasma levels supports this conclusion. Therefore, in a way, CoQ10 deficiency in CF resembles the low CoQ10 levels found in phenylketonuria and total parenteral nutrition, where a diminished influx from the intestinal compartment, albeit for dietary reasons, results in low plasma CoQ10 levels (31,32).
Malabsorption of lipid soluble vitamins, including the antioxidants beta carotene and α-tocopherol, is well known in CF; therefore, routine supplementation is advised (33,34). Obviously, the low plasma Q10 levels we now found in patients with CF could also be boosted with adequate supplementation (9,35). Because the CoQ10 redox equilibrium was not disturbed in the current group of patients with mild to moderate disease and no active infection at the time of sampling was observed, we speculate that the patients, especially those with more advanced disease and those with CF during pulmonary exacerbation, might benefit. However, because the results of CoQ10 supplementation in other patient groups with chronic lung disease had inconsistent outcomes (36), a further trial with clearly defined outcomes, including measurements of tissue CoQ10 levels (eg, platelets) and biomarkers of oxidative stress, is necessary before routine supplementation of Q10 is implemented.
In summary, we found significantly lower levels of CoQ10 in pediatric patients with cystic fibrosis as compared with age-matched controls, but a normal redox status in these patients with mild to moderate pulmonary disease.
The authors thank their colleagues at the Department of Metabolic and Endocrine Disorders for technical support.
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