Coenzyme Q10 changes are associated with metabolic syndrome

Clinica Chimica Acta 344 (2004) 173 – 179 www.elsevier.com/locate/clinchim

Coenzyme Q10 changes are associated with metabolic syndrome Michael V. Miles a,b,*, John A. Morrison c, Paul S. Horn d, Peter H. Tang a,b, Amadeo J. Pesce e a Division of Pathology and Laboratory Medicine, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center and The University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA b Division of Pediatric Neurology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center and The University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA c Division of Cardiology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center and The University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA d Department of Mathematical Sciences, The University of Cincinnati, Cincinnati, Ohio 45221-0025, Psychiatry Service, Veteran’s Affairs Medical Center, Cincinnati, OH 45220, USA e Department of Pathology and Laboratory Medicine, The University of Cincinnati Medical Center, Cincinnati, OH 45267-0559, USA

Received 10 January 2004; received in revised form 13 February 2004; accepted 23 February 2004

Abstract Background: The purpose of this study was to determine whether coenzyme Q10 (CoQ) concentrations and redox status are associated with components of the metabolic syndrome. Methods: This is a cross-sectional survey of 223 adults (28 – 78 years), who were drawn from the ongoing Princeton Follow-up Study in greater Cincinnati. Individuals were assessed for measures of fatness, blood pressure, glucose, lipid profiles, C-reactive protein (CRP), reduced CoQ (ubiquinol), oxidized CoQ (ubiquinone), total CoQ and CoQ redox ratio (ubiquinol/ubiquinone). Results: After adjusting for age, sex and race, we found that total CoQ, ubiquinol and CRP levels are significantly increased in metabolic syndrome. Comparison of minimal risk and high-risk metabolic syndrome groups indicates an increased CoQ redox ratio in the high risk group ( p < 0.05). Step-wise logistic regression analysis, using age, sex, race, (ln)CRP, total cholesterol, LDL, ubiquinol, ubiquinone and total CoQ as predictors, shows that only age ( p = 0.001), total CoQ adjusted for plasma lipids ( p < 0.0001) and (ln)CRP ( p < 0.005) were significant predictors of metabolic syndrome. Conclusions: The presence of metabolic syndrome components are associated with increased plasma total CoQ and ubiquinol concentrations after adjusting for age, sex and race. An increase in CoQ redox ratio may indicate a gender-specific adaptive response to oxidative stress in females, but not males. D 2004 Elsevier B.V. All rights reserved. Keywords: Coenzyme Q10; Ubiquinone; Ubiquinol; Metabolic syndrome; Antioxidant; Oxidative stress

Abbreviations: CRP, C-reactive protein; CoQ, coenzyme Q10; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoproteins. * Corresponding author. Cincinnati Children’s Hospital Medical Center, OSB-5449, 3333 Burnet Ave., Cincinnati, OH 45229, USA, Tel.: +1-513-636-7871; fax: +1-513-636-1888. E-mail address: [email protected] (M.V. Miles). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.02.016

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1. Introduction The clustering of insulin resistance, hyperinsulinemia, glucose intolerance, hypertension, dyslipidemia and central obesity constitutes a metabolic syndrome, which is now recognized as a powerful determinant of cardiovascular disease and type 2 diabetes [1,2]. The Third Report of the National Cholesterol Education Program Adult Treatment Panel (ATP III) emphasized the need for identification and treatment of the metabolic syndrome because of its strong association with cardiovascular disease [3]. Specific criteria for the metabolic syndrome defined by ATP III include abdominal obesity (waist circumference >102 cm in men and >88 cm in women), impaired fasting plasma glucose ( z 6.1 mmol/l), hypertriglyceridemia ( z 1.7 mmol/l), low HDL ( < 1.0 mmol/l in men and < 1.3 mmol/l in women) and increased blood pressure ( z 130/85 mm Hg) [3]. A recent report using data from the Third National Health and Nutrition Examination Survey (NHANES III), a representative sample of the U.S. population, found that the metabolic syndrome is present in >20% of adults, and varies according to ethnicity even after correction for BMI, age, socioeconomic status and other predictors of cardiovascular disease [4]. Coenzyme Q10 (CoQ) may have potential relevance to the metabolic syndrome because it is required for mitochondrial electron transport chain function, and has tissue-protective and antioxidant properties in the biochemically reduced state (ubiquinol) [5]. CoQ is the only lipid-soluble antioxidant synthesized in living organisms [6]. Its strong hydrophobicity allows insertion of the molecule into the membrane phospholipid bilayer [6]. CoQ is also a regulatory factor in adenosine triphosphate production and may be essential for the function of uncoupling proteins like UCP3 [7]. CoQ has a unique function in mitochondria, as it transfers electrons from primary substrates to the oxidase system and at the same time it transports protons from the mitochondrial matrix to the intermembrane space [6]. Because of the presence of a high percentage of ubiquinol in plasma CoQ, there has been interest in the function of CoQ both as an endogenous antioxidant and as a therapeutic agent. Previous reports have suggested that the redox state of CoQ, characterized by the ratio of ubiquinol to the oxidized

form (ubiquinone), might be a useful marker of oxidative stress [8,9]. This study investigated the relationship between components of metabolic syndrome and ubiquinol, ubiquinone and total CoQ concentrations in adults.

2. Materials and methods This study was approved by the Institutional Review Board of the Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. Written informed consent was obtained from all participants. It is a cross-sectional epidemiological survey of a subset of individuals drawn from the ongoing Princeton Follow-up Study (PFS, HL62394), a 28-year follow-up of former students and their parents from the Lipid Research Clinics (LRC) Princeton Study [10 – 12]. Participants were excluded from the study if they reported taking CoQ or lipid-lowering medications, or had a history of medically diagnosed diabetes mellitus. A medical history/health status questionnaire, anthropometric and blood pressure measurements, and phlebotomy were completed during their study visit. Participants were seen between September 1, 2000 and August 31, 2001. Fasting blood specimens were collected from the antecubital vein into glass vacuum tubes containing sodium heparin or EDTA. Blood was immediately refrigerated, then tubes were centrifuged within 3 h of collection at 2000  g for 10 min at + 5jC. Plasma specimens were immediately transferred to a pre-labeled 1.5-ml screw-capped polypropylene tubes, then stored at 80jC until analysis. Heparinized plasma specimens were analyzed for ubiquinone, ubiquinol and total The HPLC method for the determination of ubiquinol and ubiquinone concentrations was described previously [13]. Lipid profiles were measured in the Clinical Laboratory of the Cincinnati Children’s Hospital Medical Center in accordance with the manufacturers’ guidelines. All computations and statistical analyses were conducted using SAS software (ver. 8, SAS Institute, Cary, NC). C-reactive protein (CRP) results were transformed using the natural logarithm because of the skewed distribution of these tests. The Mann – Whitney test was used to compare CoQ redox ratios in sub-group analyses. Spearman correlation coefficients

M.V. Miles et al. / Clinica Chimica Acta 344 (2004) 173–179 Table 1 Characteristics of 223 study participants Metabolic syndrome ( ) (n = 133) Age (years) 43.0 [10.8] Female (%) 84 (63%) Black/White 44 (33%)/89 (67%) race (%) Smokers (%) 19% Alcoholic drink consumption of respondents V 1 per month 35% >1 per month 34% < 2 per week z 3 per week 22% BMI (kg/m2) 26.5 [4.9] Waist (cm) 91.2 [12.3] Systolic BP 121.1 [14.2] (mm Hg) Diastolic BP 76.9 [9.6] (mm Hg) Fasting glucose 4.7 [0.7] (mmol/l) C-reactive protein 0.34 [0.9] (mg/l) Total cholesterol 5.1 [0.8] (mmol/l) HDL (mmol/l) 1.3 [0.3] LDL (mmol/l) 3.2 [0.8] Triglycerides 1.1 [0.6] (mmol/l)

Metabolic syndrome (+) (n = 90) 49.9 [13.3]a,b 41 (46%)c,d 20 (22%)/70 (78%)c 28%c (%) 41% 41% 9%d,e 33.2 [5.4]a,b 109.4 [12.5]a,b 139.8 [17.4]a,b 87.0 [10.8]a,b 5.5 [1.5]a,b 0.60 [0.67]a,b 5.5 [1.0]a,b 1.0 [0.3]a,b 3.5 [0.9]a,b 2.3 [1.2]a,b

a

Wilcoxon rank-sum test. p < 0.001. c Fisher’s exact test. d p < 0.05. Mean [S.D.]. e Chi-square test. b

were computed for various pairs of continuous variables. Each correlation was tested and the significance of the results (vs. 0) was reported. Linear models were fit to examine the differences in lipids, CoQ measurements and (ln)CRP between the two study groups. The mean differences were adjusted for the demographic variables age, sex and race. Parameters defined as criteria for the metabolic syndrome were not included in the analysis. BMI was also omitted because of its close relationship with waist circumference. Step-wise logistic regression was conducted where the dichotomous response was the presence or absence of metabolic syndrome. The regressor variables were the demographic variables (age, sex and race), ubiquinol, ubiquinone, total CoQ, ubiquinol/ubiquinone ratio, total CoQ/total cholesterol index, total CoQ/LDL index, CoQ/triglyceride index,

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total CoQ/total cholesterol + triglyceride index, (ln)CRP, LDL and total cholesterol. Results are expressed as mean [S.D.]. The level of significance was set at p < 0.05.

3. Results A total of 323 individuals were screened for this study; however, 11 were excluded because they failed to provide sufficient data to determine their metabolic syndrome status. A total of 134 individuals were identified as metabolic syndrome (+) based upon the presence of 3 or more of the ATP III criteria [3]. Forty-four members of this group were excluded because they were taking lipid-lowering medication or had medically diagnosed diabetes mellitus. A total of 178 individuals were identified as metabolic syndrome ( ) ( < 3 ATP III criteria present) and 45 were excluded because of concurrent lipid-lowering medication therapy or medically diagnosed heart disease or diabetes mellitus. Characteristics of the final study populations are summarized in Table 1. Table 2 Comparison of mean C-reactive protein and CoQ measures in metabolic syndrome individuals after adjusting for age, sex and race Measure

C-reactive protein (mg/l)a Total CoQ (Amol/l) Ubiquinol (Amol/l) Ubiquinone (Amol/l) Total CoQ/total cholesterol index (Amol/mmol) Total CoQ/LDL index (Amol/mmol) Total CoQ/triglyceride index (Amol/mmol) Total CoQ/total cholesterol + triglyceride index (Amol/mmol) Ubiquinol/ubiquinone ratio

Metabolic syndrome ( ) (n = 133)

Metabolic syndrome (+) (n = 90)

p-value

0.13

0.37

< 0.0001

1.13 1.08 0.04 0.22

1.33 1.28 0.05 0.25

0.0001 0.0001 NS < 0.001

0.35

0.40

0.005

1.18

0.79

< 0.0001

0.18

0.18

NS

27.0

p-value based on Wilcoxon sum test. a Expressed as the geometric mean.

29.1

NS

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The percentage of smokers is not significantly different between the metabolic syndrome (+) and ( ) groups (Table 1). The rates of alcoholic drink consumption among respondents are significantly different (Table 1). Although ethanol intake is not quantified, individuals consuming 3 or more drinks/ week are more common in the metabolic syndrome ( ) group (Table 1). Comparisons of total plasma CRP and CoQ measures in the metabolic syndrome (+) and ( ) groups are presented in Table 2. After adjustment for age, sex and, race significant differences exist for (ln)CRP, total CoQ, ubiquinol, CoQ/total cholesterol index, CoQ/LDL index and CoQ/triglyceride index (Table 2). The total CoQ index to the sum of total cholesterol + triglyceride is not significantly different

(Table 2). Adjustment of CoQ concentrations for plasma lipids has been recommended, because virtually all CoQ is incorporated into lipoprotein particles. LDL cholesterol is purported to be associated with f 65% of CoQ, followed by f 25% in HDL, and f 10% in VLDL [14]. Bivariate correlations between the metabolic syndrome components and study measurements are presented in Table 3. Total CoQ and ubiquinol have the strongest correlations with triglyceride concentration (Table 3). Ubiquinone, the oxidation product of ubiquinol, is positively correlated with blood pressure and triglyceride concentration. CoQ redox ratio and log-transformed CRP are positively correlated with waist circumference (Table 3), which is important because of the strong relation-

Table 3 Comparison of Spearman correlations between biochemical parameters and metabolic syndrome (ATP III) components in the study population

C-reactive protein

Ubiquinol

Ubiquinone

Total coenzyme Q

Total coenzyme Q/total cholesterol index Total coenzyme Q/LDL index Total coenzyme Q/triglyceride index Total coenzyme Q/total cholesterol + triglyceride index Ubiquinol/ubiquinone ratio

Data are presented as: a Spearman, r. b Prob> | r| under H0: Rh. c n.

Waist girth

Systolic BP

Diastolic BP

Fasting glucose

Triglyceride

HDL

0.425a < 0.0001b 214c 0.332 < 0.0001 218 0.107 NS 218 0.327 < 0.0001 218 0.289 < 0.0001 218 0.184 < 0.01 218 0.219 0.001 218 0.134 < 0.05 218 0.192 < 0.005 218

0.304 < 0.0001 219 0.312 < 0.0001 223 0.164 < 0.05 223 0.310 < 0.0001 223 0.235 < 0.0005 223 0.257 0.0001 223 0.290 < 0.0001 223 0.070 NS 223 0.095 NS 223

0.210 < 0.005 219 0.328 < 0.0001 223 0.197 < 0.005 223 0.325 < 0.0001 223 0.271 < 0.0001 223 0.245 < 0.0005 223 0.212 < 0.005 223 0.120 NS 223 0.096 NS 223

0.249 < .0005 203 0.079 NS 206 0.041 NS 206 0.073 0.297 206 0.034 NS 206 0.037 NS 206 0.207 < 0.005 206 0.068 NS 206 0.118 NS 206

0.357 < 0.0001 219 0.410 < 0.0001 223 0.272 < 0.0001 223 0.411 < 0.0001 223 0.219 0.001 223 0.272 < 0.0001 223 0.816 < 0.0001 223 0.186 < 0.01 223 0.047 NS 223

0.307 < 0.0001 219 0.199 < 0.005 223 0.106 NS 223 0.199 < 0.005 223 0.198 < 0.005 223 0.034 NS 223 0.463 < 0.0001 223 0.062 NS 223 0.050 NS 223

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ship between intra-abdominal obesity and metabolic syndrome [3]. Comparison of population extremes, i.e., individuals having low risk of metabolic syndrome ( < two components) vs. individuals having high risk (>three components), shows that the mean CoQ redox ratio is increased in the presence of metabolic syndrome ( p < 0.05) (Fig. 1). It is interesting to note that females with high risk have a significantly increased mean CoQ redox ratio compared to females with low risk ( p < 0.05); however, no significant difference exists in males (Fig. 1). Step-wise logistic regression analysis of age, sex, race, (ln)CRP, total cholesterol, LDL, ubiquinol, ubiquinone, total CoQ, CoQ/total cholesterol index, CoQ/LDL index, CoQ/triglycerides index and CoQ/ total cholesterol + triglycerides index shows that only age ( p = 0.001), CoQ/LDL index ( p < 0.0001), CoQ/ total cholesterol index ( p < 0.0001), CoQ/total cholesterol + triglycerides index ( p < 0.0001) and (ln)CRP ( p < 0.005) are significant predictors of the presence of the metabolic syndrome. This analysis shows that the odds for developing the metabolic syndrome increases by approximately 5.5% per year if all other factors are held fixed. Similarly, for each 0.01-unit increase in the CoQ/total cholesterol index the odds of having the metabolic syndrome increases by a factor of 3.3 if all other factors are held fixed.

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4. Discussion The metabolic syndrome is comprised of a constellation of metabolic abnormalities, which are associated with major risk factors for cardiovascular diseases and type 2 diabetes mellitus. Dyslipidemia, intra-abdominal obesity, high blood pressure, insulin resistance and thrombotic states are considered to be important components in the pathogenesis of diseases associated with the metabolic syndrome [3]. Lowgrade inflammation associating elevation of CRP with metabolic syndrome has also been reported in a report taken from NHANES III [15]. The current study results indicate a relationship between CRP and specific metabolic syndrome components similar to the findings of previous reports [16,17]. An important strength of the current study is the analytical validity, which was made possible by the improved HPLC method for ubiquinol and ubiquinone determination [13]. Also by excluding individuals who reported taking lipid-lowering medications the potential confounding effect of ‘‘statin’’ drugs is avoided. Statins tend to lower CoQ plasma concentrations along with cholesterol according to some sources [5,6]. The main limitations are study design and sample size. The cross-sectional design limits the ability to make conclusive statements about the causeeffect relationship between CoQ and the metabolic syndrome. Therefore, it is not possible to evaluate the

Fig. 1. Comparison of mean (S.D.) ubiquinol/ubiquinone ratios in individuals with minimal risk ( < 2 ATP III components; shaded bars) vs. high risk (>3 ATP III components; open bars) of metabolic syndrome ( p-values based on Mann – Whitney test).

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effect of the difference in the rate of alcoholic drink consumption in the study populations (Table 1). This observation does raise an interesting question, however, because a recent study reported that the prevalence of metabolic syndrome is lower in women who were moderate wine drinkers (1– 30 g/day) than in women who were low alcohol drinkers [18]. To our knowledge, the current study is the first to provide evidence that individual components of the metabolic syndrome are correlated with increased ubiquinol concentrations and redox ratio (Table 2). Ubiquinol and total CoQ concentrations are positively correlated with several components of the metabolic syndrome, although the significance of these correlations disappears after adjusting for total cholesterol plus triglyceride (Table 3). The effect of triglyceride concentration on CoQ is somewhat surprising because, as mentioned previously, VLDL contains only a minor portion of plasma CoQ [14]. The possibility that ubiquinol increases in triglyceride-rich lipoproteins, e.g. VLDL, with the presence of metabolic syndrome may be important because of evidence that small quantities of ubiquinol in VLDL provide highly efficient antioxidant protection [19]. The relatively strong correlations between triglyceride and ubiquinol, ubiquinone and total CoQ (Table 3) suggest that triglyceride content may be more important in CoQ transport than previously thought. This question deserves further investigation. The current study results indicate that ubiquinol concentration is increased with the presence of metabolic syndrome, while ubiquinone is unchanged (Table 2). This seems contrary to what some have suggested [20], because it has been generally assumed that systemic antioxidant deficiency precedes and predisposes to the deleterious effects of oxidative stress [6]. However, Lenaz et al. [21] proposed that antioxidant defenses should actually increase in response to oxidative stress. Evidence of an increase in CoQ is provided in several animal studies where coenzyme Q9, the predominant CoQ form in rodents, is significantly increased in response to the prooxidant effects of diabetes mellitus [22 – 24]. None of these studies evaluated changes in CoQ redox state, however. A potentially important finding is the increased CoQ redox ratio observed in women with high risk of metabolic syndrome, which is not apparent in highrisk men (Fig. 1). Sex-related differences in absolute

CoQ levels have been described in healthy adults [25], but to our knowledge the current study is the first to provide evidence of gender-related difference in CoQ redox status. Others have also observed increased oxidative stress biomarkers in older females which may be related to CoQ redox state [26], but further studies are obviously required to understand these relationships. We propose that the increase in ubiquinol and CoQ redox ratio may be a part of the natural antioxidant defense response to certain components of the metabolic syndrome. Relationships between CoQ and factors, such as gender, inflammatory mediators and other antioxidants, need to be considered in future investigations.

Acknowledgements This study was supported in part by NIH grant HL62394.

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