Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Serum Levels of Coenzyme Q10 in Patients with Multiple System Atrophy

  • Takashi Kasai ,

    kasaita@koto.kpu-m.ac.jp

    Affiliations Department of Neurology, Research Institute for Geriatrics, Kyoto Prefectural University of Medicine, Kyoto 602–0841, Japan, AMED-CREST, Japan Agency for Medical Research and Development Kyoto 602–0841, Japan

  • Takahiko Tokuda,

    Affiliations Department of Neurology, Research Institute for Geriatrics, Kyoto Prefectural University of Medicine, Kyoto 602–0841, Japan, AMED-CREST, Japan Agency for Medical Research and Development Kyoto 602–0841, Japan, Department of Molecular Pathobiology of Brain Diseases, Research Institute for Geriatrics, Kyoto Prefectural University of Medicine, Kyoto 602–0841, Japan

  • Takuma Ohmichi,

    Affiliation Department of Neurology, Research Institute for Geriatrics, Kyoto Prefectural University of Medicine, Kyoto 602–0841, Japan

  • Ryotaro Ishii,

    Affiliation Department of Neurology, Research Institute for Geriatrics, Kyoto Prefectural University of Medicine, Kyoto 602–0841, Japan

  • Harutsugu Tatebe,

    Affiliations Department of Neurology, Research Institute for Geriatrics, Kyoto Prefectural University of Medicine, Kyoto 602–0841, Japan, Department of Zaitaku (Homecare) Medicine, Kyoto Prefectural University of Medicine, Kyoto 602–0841, Japan

  • Masanori Nakagawa,

    Affiliation North Medical Center, Kyoto Prefectural University of Medicine, Kyoto 629–2261, Japan

  • Toshiki Mizuno

    Affiliation Department of Neurology, Research Institute for Geriatrics, Kyoto Prefectural University of Medicine, Kyoto 602–0841, Japan

Serum Levels of Coenzyme Q10 in Patients with Multiple System Atrophy

  • Takashi Kasai, 
  • Takahiko Tokuda, 
  • Takuma Ohmichi, 
  • Ryotaro Ishii, 
  • Harutsugu Tatebe, 
  • Masanori Nakagawa, 
  • Toshiki Mizuno
PLOS
x

Abstract

The COQ2 gene encodes an essential enzyme for biogenesis, coenzyme Q10 (CoQ10). Recessive mutations in this gene have recently been identified in families with multiple system atrophy (MSA). Moreover, specific heterozygous variants in the COQ2 gene have also been reported to confer susceptibility to sporadic MSA in Japanese cohorts. These findings have suggested the potential usefulness of CoQ10 as a blood-based biomarker for diagnosing MSA. This study measured serum levels of CoQ10 in 18 patients with MSA, 20 patients with Parkinson’s disease and 18 control participants. Although differences in total CoQ10 (i.e., total levels of serum CoQ10 and its reduced form) among the three groups were not significant, total CoQ10 level corrected by serum cholesterol was significantly lower in the MSA group than in the Control group. Our findings suggest that serum CoQ10 can be used as a biomarker in the diagnosis of MSA and to provide supportive evidence for the hypothesis that decreased levels of CoQ10 in brain tissue lead to an increased risk of MSA.

Introduction

Multiple system atrophy (MSA) is a progressive neurodegenerative disease, clinically characterized by autonomic failure in addition to various combinations of parkinsonism, cerebellar ataxia, and pyramidal dysfunction. The distribution of pathologies classically encompass three functional systems in the central nervous system (CNS): the striatonigral system; the olivopontocerebellar system; and autonomic nuclei of the brainstem and spinal cord in which cytoplasmic aggregates of alpha-synuclein are primarily observed in oligodendroglia [1,2]. However, the pathogenic mechanisms underlying this disease remain unclear, making it difficult to develop effective therapies and diagnostic biomarkers.

The COQ2 gene encodes an enzyme essential for biogenesis of coenzyme Q10 (CoQ10). Mutations in COQ2 have recently been found in autosomal-recessive MSA families from Japan [3]. Moreover, screening for COQ2 polymorphisms in sporadic MSA cases has revealed variants conferring an increased disease risk for MSA in Japanese cohorts [3]. CoQ10, or ubiquinone, is a lipophilic molecule present in cell membranes that functions as an essential cofactor for electron transport in the mitochondrial respiratory chain and as an endogenous antioxidant [4] That discovery prompted a reconsideration of the roles of mitochondrial function and oxidative stress in the pathogenesis of this neurodegenerative disease, and also suggested the potential usefulness of CoQ10 as a blood-based diagnostic biomarker in patients with MSA.

The aim of this study was to assess and compare serum levels of CoQ10 in patients with MSA, patients with Parkinson’s disease (PD) and a control population, and to evaluate whether serum levels of this antioxidant can be used to diagnose MSA.

Material and Methods

Ethics statement, subject recruitment, and sample collection

All study subjects provided written informed consent to participate, and the study protocols were approved by the University Ethics Committee (ERB-G-12-1, Kyoto Prefectural University, Kyoto, Japan). Study procedures were designed and performed in accordance with the Declaration of Helsinki. Patients were registered in this institute from April 2008 to August 2014. We enrolled 20 patients with MSA (MSA group) according to the current consensus criteria [5]. We also enrolled 20 patients with PD (PD group) according to the UK Parkinson’s Disease Society Brain Bank criteria [6] and 20 participants with non-neurodegenerative diseases (Control group) as age-matched controls in the same registration. Of note, some participants in the MSA group were diagnosed with “possible MSA” according to the consensus criteria when clinical information and serum samples were obtained. However, we confirmed that all converted to “probable MSA” within a 3-year follow-up period. The modified Rankin Scale (mRS), which is a commonly used scale for measuring dependence [7], was used to grade all participants based on their medical records, because daily activities may affect levels of CoQ10 [2]. Since statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) reduce the biosynthesis of CoQ10 in addition to the synthesis of cholesterol [8], we excluded two subjects with MSA and two subjects with non-neurodegenerative diseases who were receiving statin therapy. We thus ultimately analyzed 18 samples from patients with MSA (MSA group), 20 samples from patients with PD (PD group), and 18 samples from participants with non-neurodegenerative disease (Control group). No participants were using medicines or food supplements containing CoQ10. The Control group comprised neurologically normal individuals (n = 1) or patients with various neurological disorders, including demyelinating diseases of the CNS (n = 5), epilepsy (n = 2), brain infarction (n = 3), subdural hematoma (n = 1), myositis (n = 1), normal-pressure hydrocephalus (n = 1), subarachnoid hemorrhage (n = 1), herpes zoster (n = 1), cervical spondylosis (n = 1), and neuropathy (n = 1). We avoided collecting samples for research use alone where possible. Most samples were taken when the participants were required to give blood for routine clinical diagnosis or treatment.

Serum samples were taken through venous puncture and a total of 10 ml of blood was collected in blood collection tubes with clot activator and gel separator (Terumo, Tokyo, Japan). After being allowed to clot for 15 min at room temperature, serum was separated by centrifugation for 10 min at 3000 rpm and distributed in polypropylene vials. Fresh samples obtained from the enrolled subjects were immediately stored at -80°C until used for analysis.

Measurement of CoQ10

Levels of CoQ10 and its reduced form (CoQ10H2) in serum were measured by SRL Inc. (Tokyo, Japan) according to the previously established method using high-performance liquid chromatography [9]. CoQH2 is easily oxidized to CoQ10 from the moment of sample extraction. Since precautions were not taken to prevent oxidation of CoQH2 at sample collection, we evaluated the total CoQ10 (sum total of CoQ10 and CoQ10H2) as an index of CoQ10 deficiency [10]. Corrected CoQ10 levels were further defined by dividing serum CoQ10 levels by total cholesterol levels (T-Cho) (i.e., total CoQ10/T-Chol, CoQH2/T-Cho), because cholesterol levels influence CoQ10 levels by forming a conjugated form in blood [11]. Levels of serum cholesterol were enzymatically determined in our clinical laboratory.

Statistics

The level of significance was set at P<0.05. When data were on a continuous or ordinal scale, the homogeneity of groups was analyzed using the Mann-Whitney U test for comparing two groups and the Kruskal-Wallis for three groups. If the Kruskal-Wallis test yielded significant results, Dunn’s post-hoc test was performed. The Chi-square test was used to evaluate the statistical significance of differences in categorical variables. All analyses were performed using SPSS for Windows version 23 software (IBM Japan Ltd, Tokyo, Japan).

Results

The demographic characteristics of participants in the MSA, PD and Control groups are summarized in Table 1. We found no significant difference in age or gender among groups: mean ages were 62.3 years in the MSA group, 63.9 years in the PD group, and 61.6 years in the Control group. Female ratios in these groups were 33.3% in the MSA group, 70% in the PD group, and 44.4% in the Control group. Mean disease duration in the MSA group (30.2 months) tended to be slightly, but not significantly, shorter than that in the PD group (67.0 months). The mRS scores did not differ significantly among the three groups: median values were 3 in the MSA group, 2 in the PD group, and 2 in the Control group. The MSA group included no familial cases of MSA, 16 patients (88.9%) with MSA-C and 2 patients (11.1%) with MSA-P (Table 2).

thumbnail
Table 1. Characteristics of participants in the MSA, PD and Control groups.

Data for continuous variables are expressed as mean ±standard deviation or median (maximum-minimum).

https://doi.org/10.1371/journal.pone.0147574.t001

Levels of total CoQ10 are shown in Fig 1A. Mean levels of total CoQ10 were lowest in the MSA group and highest in the Control group. However, comparison of the three groups with the Kruskal-Wallis test showed no significant differences. When we compared corrected levels of total CoQ10 using the ratio of total CoQ10 to T-Cho (total CoQ10/T-Cho) among groups, the ratio was significantly lower in the MSA group than in the Control group (Fig 1B). Mean total CoQ10/T-Cho was higher in the PD group than in the MSA group, although statistical significance was not maintained after post-hoc analysis. An outlier was seen in the Control group, for which we could find no specific factor contributing to the extreme elevation of CoQ10 (e.g., unbalanced diet). To exclude the possibility that the statistical results were unduly influenced by this outlier, we reanalyzed the groups after removing the outlier from the data-set. Exclusion of the outlier made the mean ±standard deviation (SD) values for total CoQ10 and CoQ10/T-cho in the Control group changed from those shown in the Table 2C to 771.1±244.9 and 4.58±1.67, respectively. The significant difference in total CoQ10/T-Cho between the MSA and Control groups was robustly confirmed even after excluding the outlier (Fig 1C). We also examined levels of CoQ10H2, which comprised the major part of total CoQ10, although these values were “only advisory”, due to the possibility of sample oxidation. CoQ10H2/T-Cho was significantly lower in the MSA group than in the Control group, even when the outlier was excluded, similar to the findings for total CoQ10 (data not shown). Raw data for each participant are summarized in Tables 24.

thumbnail
Fig 1. (A) Scatter plot for total CoQ10 level in serum in the MSA group (n = 18), PD group (n = 20), and control group (n = 18). Mean levels of total CoQ10 were lowest in the MSA group (593.2 nmol/l) and highest in the Control group (985.3 nmol/l), although no significant difference was seen between groups. (B) Scatter plot for total CoQ10/T-Cho ratio in serum in the three groups. A significant difference was seen between groups. Post-hoc analysis showed that mean level was significantly lower in the MSA group (3.04) than in the Control group (5.92). (C) The scatter plot for total CoQ10/T-Cho ratio in serum in the three groups after excluding an outlier from the Control group.

Similar statistical results to those shown in Graph B were also found. Bars indicate mean values. One participant in the Control group showed an extremely high total CoQ10/T-Cho ratio (Graph B). Graph C therefore shows participants after excluding this outlier. P values over the columns were obtained using the Kruskal-Wallis test. When the Kruskal-Wallis test yielded significant findings, post-hoc analysis was conducted; the results of which are shown on the right side of the graph.

https://doi.org/10.1371/journal.pone.0147574.g001

Discussion

The present study showed decreased total CoQ10/T-Cho ratios in MSA compared with controls, although differences in total CoQ10 levels between groups were not significant. Since CoQ10 is distributed into lipoproteins in the liver and released into the circulation because of its hydrophobicity [12], serum CoQ10 concentrations are highly dependent on lipoprotein levels. Levels of CoQ10 normalized to T-Cho levels have thus been accepted as a better biomarker of CoQ10 deficiency than non-corrected CoQ10 levels [9]. To the best of our knowledge, this is the first study to report an association between decreased levels of serum CoQ10 and risk of MSA. Our findings suggest that serum CoQ10 has potential as a biomarker in the diagnosis of MSA. The results correspond to the facts that functionally impaired variants of the COQ2 gene are associated with decreased levels of CoQ10 in brain tissue and an increased risk of MSA [3].

Leaving aside the obvious limitation of the small sample size that may have undermined the reliability of the results, we would like to note the bias caused by the predominance of the MSA-C subtype as a possible confounder. Phenotype distributions for MSA differ between populations. The MSA-P subtype predominates among Caucasian MSA patients, while MSA-C is more common in the Japanese population [13,14]. The percentage of patients exhibiting the MSA-C subtype in our study (88.9%) was similar to that in a previous study of Japanese patients with clinically diagnosed MSA (83.8%) [15]. According to a study of primary CoQ10 deficiency, concentrations of CoQ10 in the human brain were lowest in the cerebellum, which may thus be selectively vulnerable to CoQ10 deficiency [16]. Such findings suggest that compromised COQ2 function and/or decreased CoQ10 concentrations may contribute to cerebellar degeneration in MSA. This idea is also supported by the fact that the ratio of patients with MSA-C to those with MSA-P was higher among carriers of deleterious COQ2 variants than among non-carriers [3]. Considering such observations, our findings might hold true only for patients with MSA-C, and not for those with MSA-P, although the two participants with MSA-P in this study did not show marked differences in CoQ10/T-Cho ratio from the rest of the MSA group (Table 2).

In future, large-scale case-control studies including adequate numbers of MSA-P patients are needed to confirm our findings and to elucidate whether decreased ratios of serum total CoQ10/T-Cho ratio are also found in patients with MSA-P.

Author Contributions

Conceived and designed the experiments: TK TT. Performed the experiments: TK. Analyzed the data: TK TT TO RI HT. Wrote the paper: TK TT MN TM.

References

  1. 1. Coppadoro A, Berra L, Kumar A, Pinciroli R, Yamada M, Schmidt UH, et al. Critical illness is associated with decreased plasma levels of coenzyme Q10: a cross-sectional study. J Crit Care 2013; 28: 571–576, pmid:23618779
  2. 2. Del Pozo Cruz J, Rodriguez Bies E, Navas Enamorado I, Del Pozo Cruz B, Navas P, Lopez Lluch G. Relationship between functional capacity and body mass index with plasma coenzyme Q10 and oxidative damage in community-dwelling elderly-people. Exp Gerontol 2014; 52: 46–54, pmid:24512763
  3. 3. Multiple-System Atrophy Research Collaboration. Mutations in COQ2 in familial and sporadic multiple-system atrophy. N Engl J Med 2013; 369: 233–244, pmid:23758206
  4. 4. Faust JR, Goldstein JL, Brown MS. Synthesis of ubiquinone and cholesterol in human fibroblasts: regulation of a branched pathway. Arch Biochem Biophys 1979; 192: 86–99. pmid:219777
  5. 5. Montero R, Sanchez Alcazar JA, Briones P, Hernandez AR, Cordero MD, Trevisson E et al. Analysis of coenzyme Q10 in muscle and fibroblasts for the diagnosis of CoQ10 deficiency syndromes. Clin Biochem 2008; 41: 697–700, pmid:18387363
  6. 6. Hughes AJ, Daniel SE, Kilford L, Lees AJ. Accuracy of clinical diagnosis of idiopathic Parkinson's disease: a clinico-pathological study of 100 cases. J Neurol Neurosurg Psychiatry 1992; 55: 181–184. pmid:1564476
  7. 7. van Swieten JC, Koudstaal PJ, Visser MC, Schouten HJ, van Gijn J. Interobserver agreement for the assessment of handicap in stroke patients. Stroke 1988; 19: 604–607. pmid:3363593
  8. 8. Laaksonen R, Riihimaki A, Laitila J, Martensson K, Tikkanen MJ, Himberg JJ. Serum and muscle tissue ubiquinone levels in healthy subjects. J Lab Clin Med 1995; 125: 517–521. pmid:7706908
  9. 9. Steele PE, Tang PH, DeGrauw AJ, Miles MV. Clinical laboratory monitoring of coenzyme Q10 use in neurologic and muscular diseases. Am J Clin Pathol 121 2004; Suppl: S113–120. pmid:15298157
  10. 10. Molina JA, de Bustos F, Jimenez Jimenez FJ, Gomez Escalonilla C, Garcia Redondo A, Esteban J et al. Serum levels of coenzyme Q10 in patients with amyotrophic lateral sclerosis. J Neural Transm 2000; 107: 1021–1026. pmid:11041280
  11. 11. Kaikkonen J, Nyyssonen K, Tuomainen TP, Ristonmaa U, Salonen JT. Determinants of plasma coenzyme Q10 in humans. FEBS Lett 1999; 443: 163–166. pmid:9989597
  12. 12. Bhagavan HN, Chopra RK. Coenzyme Q10: absorption, tissue uptake, metabolism and pharmacokinetics. Free Radic Res 2006; 40: 445–453, pmid:16551570
  13. 13. Ozawa T, Paviour D, Quinn NP, Josephs KA, Sangha H, Kilford L et al. The spectrum of pathological involvement of the striatonigral and olivopontocerebellar systems in multiple system atrophy: clinicopathological correlations. Brain 2004; 127: 2657–2671. pmid:15509623
  14. 14. Ozawa T, Tada M, Kakita A, Onodera O, Tada M, Ishihara T et al. The phenotype spectrum of Japanese multiple system atrophy. J Neurol Neurosurg Psychiatry 2010; 81: 1253–1255, pmid:20571046
  15. 15. Yabe I, Soma H, Takei A, Fujiki N, Yanagihara T, Sasaki H. MSA-C is the predominant clinical phenotype of MSA in Japan: analysis of 142 patients with probable MSA. J Neurol Sci 2006; 249: 115–121. pmid:16828805
  16. 16. Naini A, Lewis VJ, Hirano M, DiMauro S. Primary coenzyme Q10 deficiency and the brain. Biofactors 2003; 18: 145–152. pmid:14695930