THE EIGHTH CONFERENCE of the

INTERNATIONAL

COENZYME Q 10

ASSOCIATION

Organizing Committee

Placido Navas Giorgio Lenaz Anna Ida Falasca Maria Luisa Genova

BOLOGNA, ITALY October 8-11, 2015

PROGRAMME and ABSTRACTS

CONFERENCE SCHEDULE

Morning Afternoon October 8 Arrivals Registration, Opening Lecture & Welcome Party October 9 Scientific sessions Scientific sessions October 10 Scientific sessions Scientific sessions & Social Dinner October 11 Scientific sessions

Conference Venue

Centro San Domenico

Piazza San Domenico, 12 40124 Bologna (Italy) Tel: +39 051 581718

Scientific Committee

Flint Beal Gustav Dallner Frank Döring Michio Hirano Makoto Kawamukai Peter Langsjoen Gian Paolo Littarru Placido Navas Daniel Pella Franklin Rosenfeldt Roland Stocker Yorihiro Yamamoto

Scientific Secretariat

Giorgio Lenaz and Maria Luisa Genova University of Bologna, Department of Biomedical and Neuromotor Sciences, Via Irnerio 48, Bologna, Italy Tel: +39 051 20912-29/-14 Email: [email protected] [email protected] Monica Glebocki Tel: +39 ………. Email: [email protected] Programme of the 8th Conference of the International Coenzyme Q10 Association Bologna, Italy October 8-11, 2015

Thursday, October 8

9.00 – 12.00 Meeting of the Executive Committee

14.00 – 17.30 Registration

17.45 – 18.30 Introductory remarks P. Navas G. Lenaz R. Lodi

18.35 – 19.00  In Memory of Professor Svend Aage Mortensen  Plenary Lecture G.P. Littarru

19.15 Welcome reception (Palazzo Della Lana-Montagnani, Bologna)

I Friday, October 9

8.30 – 10.10 Mitochondrial Bioenergetics Chairpersons: M.L. Genova and R. Rossignol

8.30 R. Rossignol Metabolic remodeling in physiology and pathology 8.55 U. Brandt The central role of ubiquinone in the proton pumping mechanism of respiratory complex I 9.20 G. Paradies Role of cardiolipin in mitochondrial bioenergetics: molecular and physiopathological aspects 9.45 J.A. Enriquez Coenzyme Q redox status controls the dynamic configuration of the mitochondrial electron transport chain by reverse electron transport

Coffee break

10.50 – 12.45 Bioenergetics of Ageing Chairpersons: C. Franceschi and G. López-Lluch

10.50 G. López-Lluch Coenzyme Q is an important factor in aging 11.15 C. Franceschi The mitochondria beyond OXPHOS 11.40 A. Sanz Montero Redox state of mitochondrial Coenzyme Q regulates ROS production and determines lifespan in animals 12.05 K. Higuchi Reduced Coenzyme Q10 (Ubiquinol-10) activates mitochondrial functions and decelerates senescence by activating sirtuin pathways and inhibiting cAMP phosphodiesterases 12.30 F. Brugè Coenzyme Q10 content and oxidative stress during ageing in human dermal fibroblasts cultured under different oxygen tension

Lunch

II

13.45 – 16.15 Coenzyme Q Biosynthesis Chairpersons: C. Santos-Ocaña and M. Kawamukai

13.45 C. Santos-Ocaña The dual function of Coq4p in yeast: the nucleation of Coenzyme Q6 biosynthesis complex and the activation of complex III 14.10 M. Kawamukai Biosynthesis of CoQ10 in fission yeast 14.35 M. Santoro UBIAD1 is an antioxidant enzyme that regulates CoQ10 synthesis in endothelial cells 15.00 C.F. Clarke Characterization of proteins associated with the mitochondrial CoQ synthome in Saccharomyces cerevisiae 15.25 D.J. Pagliarini Elucidating the roles of COQ8 and COQ9 in Coenzyme Q biosynthesis 15.50 F. Pierrel About pABA and 4-hydroxybenzoic acid in yeast Coenzyme Q biosynthesis

Coffee break

16.45 – 18.50 Coenzyme Q redox activities and reactive oxygen species in cell signalling Chairpersons: R. Stocker and M.L. Bolognesi

16.45 M.P. Murphy The role of CoQ redox state in mitochondrial superoxide production 17.10 P. Bernardi An in vivo-generated metabolite of idebenone is a promising molecule for treatment of LHON 17.35 R. Fato Effect of CoQ10 supplementation on cellular bioenergetic status and oxidative stress: an in vivo and in vitro study 18.00 R. Ritchie Coenzyme Q10 targets mechanisms of diabetic cardiomyopathy in pre-clinical models 18.25 R. Stocker A role for Coenzyme Q in cellular metabolism

III Saturday, October 10

8.00 – 10.05 Mitochondrial myopathies Chairpersons: L. Salviati and M. Hirano

8.00 M. Hirano title t.b.c 8.25 R. Artuch Secondary Coenzyme Q10 deficiencies 8.50 D. Ghezzi COQ4, a novel disease-gene for mitochondrial disorders associated with CoQ10 deficiency 9.15 E. Trevisson Primary Coenzyme Q10 deficiency caused by COQ2 mutations: functional characterization of the human gene and genotype- phenotype correlations 9.40 P. Yeske The role of patient advocacy groups in facilitating the development of therapeutics for mitochondrial disease

Coffee break

10.40 – 12.20 Cardiovascular Diseases Chairpersons: F. Rosenfeldt and P. Langsjoen

10.40 F. Rosenfeldt The effect of Coenzyme Q10 on morbidity and mortality in chronic heart failure 11.05 J. Langsjoen Statin cardiomyopathy: the invisible pandemic 11.30 A. Molardi Ubiquinol supplementation in the elderly patients undergoing aortic valve replacement: biochemical and clinical effect 11.55 D. Pella Statin intolerance – definition, pathophysiology, risk factors and management - what is the role of Coenzyme Q10?

12.20 – 13.00 General assembly of the International Coenzyme Q10 Association

Lunch

IV

14.00 – 16.05 Coenzyme Q and cell homeostasis Chairpersons: J.M. Villalba and G. Dallner

14.00 J.M. Villalba Metabolic regulation by the antioxidant response axis: mitochondrial and Coenzyme Q alterations produced by genetic deletion of Nrf2 or NAD(P)H:quinone oxidoreductase 1 14.25 M. Bernier Cytochrome b5 reductase overexpression extends lifespan 14.50 D. Ross NQO1; Structure, function, polymorphisms and redox changes 15.15 R. de Cabo Novel metabolic roles of NQO1 in metabolic regulation 15.40 G. Dallner Effect of modified tocotrienols in diabetes

Coffee break

16.40 – 17.10 Poster session

17.10 – 18.25 Various clinical aspects Chairpersons: G. Lenaz and Y. Yamamoto

17.10 V. Parisi Effects of Coenzyme Q10 on retinal-evoked and cortical-evoked responses in patients with open-angle glaucoma 17.25 Y. Yamamoto Increased oxidative stress in patients with amyotrophic lateral sclerosis: the effect of edaravone administration and a possible need for Coenzyme Q10 supplementation 17.40 A. Knott Benefits of topical Q10 treatment on human skin 17.55 G. Balercia Protective effect of CoQ10 and aspartic acid on oxidative stress and DNA damage in subjects affected by idiopathic asthenozoospermia 18.10 Y. Watanabe Therapeutic effects of ubiquinol on fatigue and chronic fatigue syndrome

20.30 Social dinner (Palazzo Gnudi, Bologna)

V Sunday, October 11

8.30 – 9.45 Gene regulation and epigenetics Chairpersons: F. Döring and D.J.M. Fernández-Ayala

8.30 D.J.M. Fernández-Ayala Survival transcriptome in the Coenzyme Q deficiency syndrome is acquired by epigenetic modifications 8.55 F. Döring Physiological functions of ubiquinone- and ubiquinol- dependent gene expression 9.20 J. López-Miranda Effects of the mediterranean diet supplemented with Coenzyme Q10 on oxidative stress and inflammatory response in elderly men and women

9.45 – 10.30 Young Participant Award - In Memory of Professor Svend Aage Mortensen Chairperson: G. Lenaz

9.45 D.J. Fazakerley Loss of mitochondrial ubiquinone as a driver of insulin resistance 9.55 Z. Xu Ubiquinol-10 might suppress the aging process by inhibiting type 4 cAMP phosphodiesterases 10.05 M.A. Belousova Intravenous administration of solubilized Coenzyme Q10 provides neuroprotection in rat model of permanent cerebral ischemia 10.15 P. Ranadive Inverse metabolic engineering of Sporidiobolus johnsonii ATCC-20490 for improved production of Coenzyme Q10 and its process optimization

Coffee break

11.00 – 12.50 Multidisciplinary discussion: Ubiquinone vs. Ubiquinol Chairpersons: P. Navas and G.P. Littarru

11.00 G.P. Littarru Ubiquinol versus ubiquinone 11.10 L. Tiano Effect of Ubiquinol on mitochondrial potential and oxidative stress in relation to intensive physical exercise 11.35 L. Greci On the reactions of endogenous Coenzyme Q10 with nitric oxide and its metabolite nitrogen dioxide 12.00 Ch. López-Pedrera Potential impact of in vivo ubiquinol supplementation in the prevention of atherothrombosis in antiphospholipid syndrome patients. Preliminary results of a clinical trial 12.25 M. Failla Why is the bioavailability of ubiquinol greater than that of ubiquinone?

12.50 Concluding remarks

Lunch VI CONTENTS

ABSTRACTS of ORAL PRESENTATIONS

IN MEMORY OF SVEND AAGE MORTENSEN G.P. Littarru

METABOLIC REMODELING IN PHYSIOLOGY AND PATHOLOGY E. Obre, N. Bellance, P. Esteves, A. Brillac, C. Lalou, T. Imasawa, R. Rossignol

THE CENTRAL ROLE OF UBIQUINONE IN THE PROTON PUMPING MECHANISM OF RESPIRATORY COMPLEX I U. Brandt

ROLE OF CARDIOLIPIN IN MITOCHONDRIAL BIOENERGETICS: MOLECULAR AND PHYSIOPATHOLOGICAL ASPECTS G. Paradies, V. Paradies, F.M. Ruggiero, G. Petrosillo

COENZYME Q REDOX STATUS CONTROLS THE DYNAMIC CONFIGURATION OF THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN BY REVERSE ELECTRON TRANSPORT J.A. Enriquez

COENZYME Q IS AN IMPORTANT FACTOR IN AGING G. López-Lluch

THE MITOCHONDRIA BEYOND OXPHOS C. Franceschi

REDOX STATE OF MITOCHONDRIAL COENZYME Q REGULATES ROS PRODUCTION AND DETERMINES LIFESPAN IN ANIMALS A. Sanz Montero

REDUCED COENZYME Q10 (UBIQUINOL-10) ACTIVATES MITOCHONDRIAL FUNCTIONS AND DECELERATES SENESCENCE BY ACTIVATING SIRTUIN PATHWAYS AND INHIBITING CAMP PHOSPHODIESTERASES K. Higuchi, Z. Xu, G. Tian, J. Huo, H. Kubo K. Hosoe, J. Sawashita

COENZYME Q10 CONTENT AND OXIDATIVE STRESS DURING AGEING IN HUMAN DERMAL FIBROBLASTS CULTURED UNDER DIFFERENT OXYGEN TENSION F. Brugè, E. Damiani, P. Orlando, S. Silvestri, F. Marcheggiani, G.P. Littarru, L. Tiano

THE DUAL FUNCTION OF Coq4p IN YEAST: THE NUCLEATION OF COENZYME Q6 BIOSYNTHESIS COMPLEX AND THE ACTIVATION OF COMPLEX III E. García-Testón Páez, P. Navas Lloret, C. Santos-Ocaña

BIOSYNTHESIS OF CoQ10 IN FISSION YEAST M. Kawamukai

VII UBIAD1 IS AN ANTIOXIDANT ENZYME THAT REGULATES CoQ10 SYNTHESIS IN ENDOTHELIAL CELLS R. Sewduth, E. Panieri, C. Millia, M. Santoro

CHARACTERIZATION OF PROTEINS ASSOCIATED WITH THE MITOCHONDRIAL CoQ SYNTHOME IN SACCHAROMYCES CEREVISIAE C.F. Clarke, C.M. Allan, A. M. Awad, C. H. He, A. Hsu, H. Tong Lam, A. Nag, H. S. Tsui

ELUCIDATING THE ROLES OF COQ8 AND COQ9 IN COENZYME Q BIOSYNTHESIS D.J. Pagliarini

ABOUT PABA AND 4-HYDROXYBENZOIC ACID IN YEAST COENZYME Q BIOSYNTHESIS L. Payet, M. Leroux, M. Ozeir, U. Forsman, P. Sindelar, L. Pelosi, A. Ismail, C. Mellot-Drazniek, M. Fontecave, F. Pierrel

THE ROLE OF COQ REDOX STATE IN MITOCHONDRIAL SUPEROXIDE PRODUCTION M. Murphy

AN IN VIVO-GENERATED METABOLITE OF IDEBENONE IS A PROMISING MOLECULE FOR TREATMENT OF LHON V. Giorgio, M. Schiavone, M. Carini, T. Da Ros, M. Prato, F. Argenton, V. Petronilli, P. Bernardi

EFFECT OF CoQ10 SUPPLEMENTATION ON CELLULAR BIOENERGETIC STATUS AND OXIDATIVE STRESS: AN IN VIVO AND IN VITRO STUDY C. Bergamini, A. Fetoni, D. Troiani, G. Lenaz, R. Fato

COENZYME Q10 TARGETS MECHANISMS OF DIABETIC CARDIOMYOPATHY IN PRE-CLINICAL MODELS R. Ritchie

A ROLE FOR COENZYME Q IN CELLULAR METABOLISM A. Ayer, C. F. Clarke, I.W. Dawes, R. Stocker

SECONDARY COENZYME Q10 DEFICIENCIES R. Artuch

COQ4, A NOVEL DISEASE-GENE FOR MITOCHONDRIAL DISORDERS ASSOCIATED WITH CoQ10 DEFICIENCY D. Ghezzi

PRIMARY COENZYME Q10 DEFICIENCY CAUSED BY COQ2 MUTATIONS: FUNCTIONAL CHARACTERIZATION OF THE HUMAN GENE AND GENOTYPE- PHENOTYPE CORRELATIONS E. Trevisson, M. A. Desbats , M. Silic-Benussi, P. Navas, L. Salviati

THE ROLE OF PATIENT ADVOCACY GROUPS IN FACILITATING THE DEVELOPMENT OF THERAPEUTICS FOR MITOCHONDRIAL DISEASE P. Yeske VIII THE EFFECT OF COENZYME Q10 ON MORBIDITY AND MORTALITY IN CHRONIC HEART FAILURE S.A. Mortensen, F.L. Rosenfeldt, K. Filipiak, G.P.Littarru., K. Overvad, F. Skjøth, C. D. Sindberg, P. Dolliner, G. Steurer; V. Nagy, J. Feher, G. Paragh, P. Fülop, A. Kumar, H. Kaur, C.S. Ping, A.A.A. Rahim, M. Bronisz, M. Stopinski, M. Marchel, A. Kaplon-Cieslicka, W. Sinkiewicz, B. Wozakowsk-Kaplon, M. Bzymek, H. Wysocki, M. Krzciuk, D. Pella, I. Lazurova, U. Alehagen.

STATIN CARDIOMYOPATHY: THE INVISIBLE PANDEMIC P.H. Langsjoen, J.O. Langsjoen, A.M. Langsjoen.

UBIQUINOL SUPPLEMENTATION IN THE ELDERLYPATIENTS UNDERGOING AORTIC VALVE REPLACEMENT: BIOCHEMICAL AND CLINICAL EFFECTS F. Nicolini, A. Molardi, T. Gherli, F. Brugè, P. Orlando, S. Silvestri, G.P. Littarru and L. Tiano

STATIN INTOLERANCE- DEFINITION, PATHOPHYSIOLOGY, RISK FACTORS AND MANAGEMENT – WHAT IS THE EROLE OF COENZYLE Q10? D. Pella

METABOLIC REGULATION BY THE ANTIOXIDANT RESPONSE AXIS: MITOCHONDRIAL AND COENZYME Q ALTERATIONS PRODUCED BY GENETIC DELETION OF Nrf2 OR NAD(P)H: QUINONE OXIDOREDUCTASE 1 J.M. Villalba, J. Ariza, L. Fernández del Río, S.R. Levan, E. Mercken, H. Khraiwesh, J.A. González-Reyes, L. Jódar, M.I. Burón, M. Bernier, R. de Cabo

CYTOCHROME b5 REDUCTASE OVEREXPRESSION EXTENDS LIFESPAN M. Bernier

NQO1; STRUCTURE, FUNCTION, POLYMORPHISMS AND REDOX CHANGES D. Ross, D. Siegel

NOVEL METABOLIC ROLES OF NQO1 IN METABOLIC REGULATION R. de Cabo

EFFECT OF MODIFIED TOCOTRIENOLS IN DIABETES G. Dallner. K. Brismar

EFFECTS OF COENZYME Q10 ON RETINAL-EVOKED AND CORTICAL-EVOKED RESPONSES IN PATIENTS WITH OPEN-ANGLE GLAUCOMA V. Parisi

INCREASED OXIDATIVE STRESS IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS: THE EFFECT OF EDARAVONE ADMINISTRATION AND A POSSIBLE NEED FOR COENZYME Q10 SUPPLEMENTATION Y. Yamamoto, M. Nagase, Y. Miyazaki, H. Yoshino

BENEFITS OF TOPICAL Q10 TREATMENT ON HUMAN SKIN A. Knott

IX PROTECTIVE EFFECT OF COQ10 AND ASPARTIC ACID ON OXIDATIVE STRESS AND DNA DAMAGE IN SUBJECTS AFFECTED BY IDIOPATHIC ASTHENOZOOSPERMIA G. Tirabassi, A. Vignini, L. Tiano, E. Buldreghini, F. Brugè, S. Silvestri, P. Orlando, A. D’Aniello, L. Mazzanti, A. Lenzi and G. Balercia

THERAPEUTIC EFFECTS OF UBIQUINOL ON FATIGUE AND CHRONIC FATIGUE SYNDROME Y. Watanabe, S. Fukuda, K. Yamaguti, O. Kajimoto, K. Fujii, H. Kuratsune

SURVIVAL TRANSCRIPTOME IN THE COENZYME Q DEFICIENCY SYNDROME IS ACQUIRED BY EPIGENETIC MODIFICATIONS D.J.M. Fernández-Ayala, P. Navas Lloret

PHYSIOLOGICAL FUNCTIONS OF UBIQUINONE AND UBIQUINOL DEPENDENT GENE EXPRESSION F. Döring

EFFECTS OF THE MEDITERRANEAN DIET SUPPLEMENTED WITH COENZYME Q10 ON OXIDATIVE STRESS AND INFLAMMATORY RESPONSE IN ELDERLY MEN AND WOMEN J. López-Miranda

UBIQUINOL VERSUS UBIQUINONE G.P. Littarru

EFFECT OF UBIQUINOL ON MITOCHONDRIAL POTENTIAL AND OXIDATIVE STRESS IN RELATION TO INTENSIVE PHYSICAL EXERCISE L. Tiano, P. Orlando, S. Silvestri, F. Brugè, T. Bacchetti, G.P. Littarru

ON THE REACTIONS OF ENDOGENOUS COENZYME Q10 WITH NITRIC OXIDE AND ITS METABOLITE NITROGEN DIOXIDE L. Greci, G. P. Littarru

POTENTIAL IMPACT OF IN VIVO UBIQUINOL SUPPLEMENTATION IN THE PREVENTION OF ATHEROTHROMBOSIS IN ANTIPHOSPHOLIPID SYNDROME PATIENTS. PRELIMINARY RESULTS OF A CLINICAL TRIAL Ch. López-Pedrera, C. Pérez-Sánchez, M.A. Aguirre, F. Velasco, P. Ruiz-Limón, N. Barbarroja, Y. Jiménez-Gómez, P. Segui, E. Collantes-Estévez, L. Fernández del Río, J.A. González-Reyes, M.J. Cuadrado, J.M. Villalba

WHY IS THE BIOAVAILABILITY OF UBIQUINOL GREATER THAN THAT OF UBIQUINONE? M. Failla, C. Chitchumroonchokchai

X ABSTRACTS of POSTERS

INTRAVENOUS ADMINISTRATION OF SOLUBILIZED COENZYME Q10 PROVIDES NEUROPROTECTION IN RAT MODEL OF PERMANENT CEREBRAL ISCHEMIA. M.A. Belousova, O.S. Medvedev, E.A. Gorodetskaya, E.I Kalenikova

AGE-DEPENDENT CHANGES IN COENZYME Q-SYNTHESIS COMPLEX TRANSCRIPTOME C. Campos-Silva, I. Reyes-Torres, M. Rivera, C. Meza-Torres, E. Rodriguez-Bies, P. Navas, G. López-Lluch

RNA-BINDING PROTEINS REGULATE CELL RESPIRATION AND COENZYME Q BIOSYNTHESIS BY POST-TRANSCRIPTIONAL REGULATION OF COQ7. M. V. Cascajo, E. Siendones, J. M. Cuezva, D. M. Fernández-Ayala, P. Navas, M. Gorospe

VITAMIN K2 CANNOT SUBSTITUTE COENZYME Q10 AS ELECTRON CARRIER IN THE MITOCHONDRIAL RESPIRATORY CHAIN OF MAMMALIAN CELLS C. Cerqua, A. Casarin, L. Salviati, E. Trevisson.

MOLECULARLY IMPRINTED SOLID PHASE EXTRACTION BEFORE CoQ10 ANALYSIS FROM TWO COMPLEX MATRICES. M. Contin, C. Garcia Becerra, M. Moretton, S. Flor, D. Chiappetta, S. Lucangioli, V. Tripodi

UBIQUINOL SUPPLEMENTATION REDUCES OXIDATIVE DAMAGE ASSOCIATED TO STRENUOUS EXERCISE J. Diaz-Castro, A. Sarmiento, N. Kajarabille, M. Pulido-Morán, J. Moreno-Fernández, V.M. Osa, S. Hijano, J.J. Ochoa

PLASMATIC AND INTRACELLULAR MARKERS OF OXIDATIVE STRESS IN NORMAL WEIGHT AND OBESE PATIENTS WITH POLYCYSTIC OVARY SYNDROME C. Di Segni, S. Raimondo, A. Mancini, F. Guidi, D. Romualdi, R. Apa, A. Lanzone, C. Bergamini, F. Volta, R. Fato

LOSS OF MITOCHONDRIAL UBIQUINONE AS A DRIVER OF INSULIN RESISTANCE D. J. Fazakerley, R.Chaudhuri, G. J. Maghzal, C. Suarna, P.Yang, C. C. Meoli, S. J. Humphrey, K. H. Fisher-Wellman, J. R. Krycer, B. L. Parker, K. C. Thomas, J. R. Junutula, Z. Modrusan, G. Kolumam, J. Y. Yang, K. L. Hoehn, R. Stocker, D. E. James

UBIQUINONE AND UBIQUINOL DEPENDENT GENE EXPRESSION SIGNATURES UNDER AD LIBITUM AND DIETARY RESTRICTION CONDITION IN THE MODEL ORGANISM C. ELEGANS A. Fischer, P. Niklowitz, T. Menke, F. Döring

CONTROL OF MITOCHONDRIAL SUPEROXIDE ION GLIOBLASTOMA MULTIFORME: IMPACT ON TUMOR METABOLISM, NEOVASCULARIZATION AND INFLAMMATION J. Frontiñán-Rubio, R. Santiago-Mora, M. V. Gómez-Almagro, M. Villar-Rayo, G. Ferrín-Sánchez, J. R. Peinado, J. M. Villalba, F.J. Alcaín, M. Durán-Prado

XI NONLINEAR PHARMACOKINETIC OF SOLUBILIZED UBIDECARENONEAFTER INTRAVENOUS ADMINISTRATION E.A. Gorodetskaya, E.I. Kalenikova., O.S. Medvedev

THE COENZYME Q6 SYNTHESIS REGULATION BY THE COUPLE Coq7p/Ptc7p INTEGRATES THE CELL BIOENERGETICS STATE WITH THE MITOCHONDRIAL HOMEOSTASIS IN SACCHAROMYCES CEREVISIAE. P. Gutiérrez Ríos, I. González-Mariscal, A. Martín-Montalvo, P. Navas, C. Santos-Ocaña

COENZYME Q10 LEVELS ARE DECREASED IN THE CEREBELLUM OF MULTIPLE- SYSTEM ATROPHY PATIENTS I. Hargreaves, L. V. Schottlaender, C. Bettencourt, A. P. Kiely, A. Chalasani, V. Neergheen, J.L. Holton, H. Houlden

TISSUE DISTRIBUTION AND PHARMACOKINETIC PROFILE OF COENZYME Q 10 DURING 8 DAYS AFTER INTRAVENOUS ADMINISTRATION E.I. Kalenikova, E.A. Gorodetskaya, O.S. Medvedev

THE EFFECT OF LONG-TERM INTAKE OF UBIQUINOL ON IMPROVING QUALITY OF LIFE FOR COMMUNITY RESIDENTS T. Kinoshita, K. Maruyama, T. Tanigawa

EVALUATION OF PLASMA COENZYME Q10 IN ADULTS PATIENTS WITH GROWTH HORMONE DEFICIENCY A. Mancini, C. Di Segni, S. Raimondo, P. Orlando, S. Silvestri, L. Tiano, G.P. Littarru

COENZYME Q10 SUPPLEMENTATION IMPROVES ETHYNIL ESTRADIOL-INDUCED CHOLESTASIS IN RATS M. Martinefski, M. Rodriguez, S. Lucangioli, L. Bianciotti, V. Tripodi

EFFECT OF SHORT- AND LONG-TERM COMPLETE STARVATION AND REGENERATION DIET ON ANTIOXIDANTS, OXIDATIVE STRESS AND KIDNEY FUNCTION V. Mojto, A. Gvozdjáková, J. Kucharská, J. Mišianik, Z. Rausová, J. Valuch

PURIFICATION AND CHARACTERIZATION OF HUMAN RECOMBINANT COQ4 L. Morbiato, F. Tonello, P. Berto, G. Zanotti, M.A. Desbats, C. Montecucco, L. Salviati

UBIQUINOL SUPPLEMENTATION POSITIVELY CORRELATES WITH REDUCED SKELETAL MUSCLE DAMAGE IN PROFESSIONAL FOOTBALL PLAYERS. I. Navas-Enamorado, A. Sánchez-Cuesta, P. Bjork, J. del Rio, T. Viar, J. Lekue, F. Angulo, P. Navas, G. López-Lluch

IDENTIFICATION OF POPULATION SUBGROUPS WITH CRITICAL UBIQUINOL STATUS S. Onur, A. Fischer, P. Niklowitz, T. Menke, F. Döring

XII THE EFFECT OF DIETARY INTAKE OF COENZYME Q10 ON SKIN PARAMETERS AND CONDITION: RESULTS OF A PLACEBO-CONTROLLED HUMAN STUDY T. Pogačnik, J. Žmitek, K. Žmitek

INVERSE METABOLIC ENGINEERING OF SPORIDIOBOLUS JOHNSONII ATCC-20490 FOR IMPROVED PRODUCTION OF COENZYME Q10 AND ITS PROCESS OPTIMIZATION P. Ranadive, A. Mehta, S. George

MODELING COENZYME Q10 DEFICIENCY USING IPS CELLS D. Romero-Moya, C. Santos-Ocaña, P. González-Rodríguez, J. A. Rodríguez-Gómez, I Velasco, J. Castaño, A. Giorgetti, M. F Fraga, A. Fernández, C. Bueno, J. López-Barneo, P. Navas, P. Menendez.

UBIQUINOL REDUCES PLASMA OXIDATIVE STRESS, HYPOXIA AND THE THICKNESS OF THE MICROVASCULATURE BASEMENT MEMBRANES IN THE HIPPOCAMPUS OF THE 3xTg-AD MOUSE MODEL OF ALZHEIMER DISEASE. F. J. Sancho-Bielsa, J. Frontiñan, R. Santiago-Mora, J. R. Peinado, L. Gimenez-Llort, M. Durán- Prado, F. J. Alcaín

UBIQUINOL-10 DECELERATES SENESCENCE AND AGE-RELATED DISORDERS VIA THE ACTIVATION OF THE AMPK-SIRT1 PATHWAY AND THEIR DOWNSTREAM MOLECULES J. Sawashita, G. Tian, Z. Xu, H. Kubo, M. Tsuruoka, K. Hosoe, K. Higuchi

PLASMA MEMBRANE NADH-CoQ REDUCTASE CONNECTS AEROBIC METABOLISM WITH AGEING E. Siendones, S. SantaCruz-Calvo, A. Martín-Montalvo, M. V. Cascajo, J. Ariza, G. López- Lluch, J. M. Villalba, C. Acquaviva-Bourdain, E. Roze, M. Bernier, R. de Cabo, P. Navas

INCREASE IN COENZYME Q BIOSYNTHESIS IN RAT LIVER EARLY AFTER BIRTH J. Spáčilová., M. Hůlková, D. Sedláčková, S. Knopová, J. Zeman, H. Hansíková

STUDY OF NUTRITIONAL GUIDANCE METHODS CONCERNING COENZYME Q10 INTAKE T. Suzuki, M. Otani, K. Kawai, M. Takahashi

LONG-TERM HOSPITALIZED OLDER PEOPLE HAVE LOWER COENZYME Q10 BLOOD LEVELS THAN NON-HOSPITALISED OLDER PEOPLE M. Takahashi, T. Suzuki, H. Matsumoto, H. Nukada, N. Hashizume

EFFECT OF UBIQUINOL ON REPRODUCTIVE HORMONES OF AMENORRHEIC PATIENTS: A HOSPITAL BASED STUDY A.S. Thakur, I. Funahashi, U.S. Painkara, N.S. Dange, P. Chauhan

COENZYME Q IN THE MITOCHONDRIAL RESPIRATORY CHAIN: INTER-ENZYME CHANNELING VS. POOL BEHAVIOUR. G. Tioli, Y. Birinci, M. Kopuz, A.I. Falasca, G. Lenaz, M.L. Genova

XIII 2-PHENOXY-1,4-NAPHTHOQUINONE AS A DUAL MOLECULE SIMULTANEOUSLY TARGETING BOTH GLYCOLYSIS AND MITOCHONDRIAL FUNCTION E. Uliassi, F. Prati, C. Bergamini, R. Amorati, A. Berteotti, A. Cavalli, M. Zaffagnini, R. Fato, G. Paredi, S. Bruno, M.L. Bolognesi

FUNCTIONAL ANALYSIS OF ADCK4 MUTATIONS CAUSING CoQ10 DEFICIENCY AND NEPHROTIC SYNDROME L. Vazquez-Fonseca, M. Doimo, M.A. Desbats, V. Morbidoni, L. Salviati

ASSESSMENT OF COENZYME Q10 TRANSPORT ACROSS THE BLOOD BRAIN BARRIER L. Wainwright, J. Preston, S. Heales, J. Abbott, I. Hargreaves

EFFECTS OF REDUCED COENZYME Q10 TREATMENT ON BONE METABOLISM IN RAT H. Wakabayashi, J. Kanda, N. Izumo, Y. Kobayashi, T. Shimakura, N. Yamamoto, K. Onodera

UBIQUINOL-10 MIGHT SUPPRESS THE AGING PROCESS BY INHIBITING TYPE 4 CAMP PHOSPHODIESTERASES Z. Xu, G. Tian, J. Huo, H. Kubo, K. Hosoe, K. Higuchi, J. Sawashita

A RANDOMIZED CLINICAL TRIAL ON THE THERAPEUTIC EFFECT OF COENZYME Q10 IN PATIENTS WITH HUMAN IMMUNODEFICIENCY VIRUS INFECTION F. Yousefi, F. Roozbeh, H. Rahmani

DETERMINATION OF URINARY CoQ10 BY HPLC WITH ELECTROCHEMICAL DETECTION: REFERENCE VALUES FOR A PEDIATRIC POPULATION. D. Yubero, R. Montero, V. Neergheen, I. P Hargreaves, R. Artuch

XIV

ABSTRACTS of ORAL PRESENTATIONS

IN MEMORY OF SVEND AAGE MORTENSEN

Gian Paolo Littarru

It is my great pleasure and honor to open this meeting in Bologna, where my research adventure with Coenzyme Q10 started. At that time, almost fifty years ago, Prof.Lenaz, the chairman of this meeting, introduced me to the research issue of specificity of different ubiquinone homologs for the inner membrane of yeast mitochondria. Soon after, as a young postdoc at the Institute for Biomedical Research, in Austin Texas, I shifted my attention to the human heart. For the first time it was possible to investigate the CoQ10 status in mitochondria isolated from the failing heart: a deficiency was found, which correlated with the extent of cardiac failure. Several years later Dr Folkers started his cooperation with a Danish cardiologist, Svend Aage Mortensen, who sadly left us earlier this year, and to whom this introductory lecture is dedicated. With the new HPLC technique it was possible to analyze the CoQ10 content of ventricular heart biopsies provided by Prof Mortensen. The CoQ10 content of the heart muscle of cardiac patients in NYHA class III and IV was lower than the corresponding ones obtained for patients in class I and II; this confirmed the values we had found about fifteen years earlier with the enzymatic differential assay. Encouraged by this data Prof.Mortensen extended his research to the effect of CoQ10 in animal models of cardiopathy, and in patients affected by heart failure. My cooperation and friendship with Prof. Mortensen began in 1985, and he was one of the founding members of the International Coenzyme Q10 association when, back in 1997, I started this endeavour. In those years the idea of a possible multicenter clinical study with CoQ10 in Heart failure began to develop. It was owing to the enthusiasm and great work of this esteemed friend that the Q SYMBIO study could initiate, several years later, together with the efforts of the International CoQ10 Association and of Pharma Nord. The laboratory of the International CoQ10 Association in Ancona was the reference lab where blood samples of this double blind multicenter study were sent. It is interesting to note that during those years when I analyzed the CoQ10 and NT-proBNP content in sera from the enrolled patients, we started a new research field in Ancona, aimed at elucidating the vascular implications of CoQ10. Thanks to the remarkable efforts of Dr Belardinelli and Dr Tiano the effects of Coenzyme Q10 on Flow Mediated Dilation and on extra cellular SOD were highlighted. The results of the Q Symbio study were made public after intensive, fruitful interchange with the referees of the paper. The findings of this study, positive and encouraging, were eventually published, just a few months before Prof. Mortensen started his final personal battle, after concluding the great work of his life.

1 METABOLIC REMODELING IN PHYSIOLOGY AND PATHOLOGY

Obre E., Bellance N., Esteves P., Brillac A., Lalou C., Imasawa T., Rossignol R.

Laboratory of Rare Diseases : Genetics and Metabolism, University of Bordeaux (Bordeaux ; France).

Human tissues differ by the type and the amount of energy substrate that they use and store, as dictated by their genetically-programmed « metabolic rigidity » which defines the expression of tissue-specific catabolic and anabolic enzymes. In contrast to normal tissues, most tumors develop a dramatic « metabolic flexibility », which could allow them to consume whatever energy substrate is available. Cancer cells also avoid the metabolic control by insulin, growth factors and energy substrate starvation or excess. Such flexibility is made possible by the molecular rewiring of several metabolic pathways, which undergo branching, truncation and reversal, as extensively documented for the Krebs cycle in the last decade. Nowadays, large-scale analyses of comprehensive proteomics, transcriptomics and metabolomics, potentially supported by computer modeling of metabolism and genetic approaches of sub-pathways validation, allow to decipher the fine changes in cell metabolism, both in physiology and pathology. Those patterns of metabolic deviations and the corresponding genetic signatures can be confirmed by flux analyses using 13C-labeled isotopes of relevant metabolites. Analysis of deviant metabolism can identify potential targets so that therapeutic strategies could be derived from this knowledge, and preclinical studies are ongoing to alter alternative metabolic pathways in cancer and other diseases. During this talk, I will present our work on metabolic remodeling in different models.

2 THE CENTRAL ROLE OF UBIQUINONE IN THE PROTON PUMPING MECHANISM OF RESPIRATORY COMPLEX I

Ulrich Brandt1,2

1Nijmegen Center for Mitochondrial Disorders, Radboudumc Nijmegen, Netherlands 2Cluster of Excellence Frankfurt "Macromolecular Complexes,” Goethe-University, Frankfurt am Main, Germany

Complex I (NADH:ubiquinone oxidoreductase), the largest component of the respiratory chain, contains one FMN and eight iron-sulfur clusters as prosthetic groups and is composed of some 40 subunits with a total mass of ~1,000 kDa (Brandt, 2006). Human complex I deficiencies are the most frequent cause of inherited mitochondrial disorders and have been implicated in different neurodegenerative disorders and biological ageing. Despite its central importance, our knowledge about complex I is still rather limited. Complex I uses the energy released by the transfer of two electrons from NADH to ubiquinone to pump four protons across the bacterial plasma membrane or the inner mitochondrial membrane. The ubiquinone chemistry that takes place in the peripheral domain plays a pivotal role in this mechanism of energy conversion, by providing the energy that drives vectorial proton transport at the four putative pump sites in the membrane domain complex I. By solving the X-ray structure of complete mitochondrial complex I from Yarrowia lipolytica to a resolution of 3.6-3.9 Å (Zickermann et al, 2015), we obtained deep insight into the molecular mechanism of redox-driven proton pumping and its regulation by the so-called active/deactive transition. The structure is in line with a two-state stabilization change mechanism involving a concerted structural rearrangement that is driven by the stepwise reduction and pronation of ubiquinone (Brandt, 2011). The mechanism has immediate implications for our understanding of different pathophysiological conditions involving mitochondrial function.

References Brandt U (2006) Energy converting NADH:quinone oxidoreductase (Complex I). Ann Rev Biochem 75:69-92 Brandt U (2011) A two-state stabilization-change mechanism for proton-pumping complex I Biochim Biophys Acta 1807:1364-1369 Zickermann V, Wirth C, Nasiri H, Siegmund K, Schwalbe H, Hunte C, Brandt U (2015) Mechanistic insight from the crystal structure of mitochondrial complex I Science 347:44-49

3 ROLE OF CARDIOLIPIN IN MITOCHONDRIAL BIOENERGETICS: MOLECULAR AND PHYSIOPATHOLOGICAL ASPECTS

G. Paradies, V. Paradies, F.M. Ruggiero and G. Petrosillo

Department of Biosciences, Biotechnologies and Biopharmaceutis, University of Bari and National Research Council , Institute of Biomembranes and Bioenergetics, Bari, Italy

Cardiolipin (Cl) is a unique phospholipid which is located at the level of the inner mitochondrial membrane (IMM), where it is biosynthesized. Considerable progress has recently been made in understanding the role of CL in mitochondrial function in wealth and disease. This phospholipid is known to be intimately involved in several mitochondrial bioenergetic reactions, including electron transport chain, metabolites translocation, binding of cytochrome c to IMM and functioning of the multiple other IMM proteins and enzymes. Cl is also emerging as an important player in the control of the mitochondrial phase of apoptosis, in mitochondrial membrane stability and dynamics and in protein import. Cl is particularly susceptible to ROS attack due to its high content of unsaturated fatty acids. Oxidative damage to CL would negatively impact the biochemical function of the IMM, altering ion permeability, structure and function of respiratory chain complexes and their organization into supercomplexes, cytochrome C binding to IMM, resulting in reduced mitochondrial oxidative phosphorylation efficiency. Alterations in Cl structure, content and acyl chains composition have been associated with mitochondrial dysfunction in multiple tissues in several physiopathological situations, including heart ischemia/reperfusion, different thyroid states, diabetes, aging, Barth syndrome as well as cell death. Preservation of mitochondrial CL content and integrity by chemical reagents may provide a potential treatment for these diseases.

4 COENZYME Q REDOX STATUS CONTROLS THE DYNAMIC CONFIGURATION OF THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN BY REVERSE ELECTRON TRANSPORT

José Antonio Enriquez

Fundación CNIC Carlos III - Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain.

Electrons feed into the mitochondrial electron transport chain (mETC) from NAD- or FAD- dependent enzymes. A shift from glucose to fatty acids increases electron flux through FAD, which can saturate the oxidation capacity of the dedicated coenzyme Q (CoQ) pool and result in the generation of harmful reactive oxygen species. To prevent this, the mETC superstructure can be reconfigured through the degradation of respiratory complex I, liberating associated complex III to increase electron flux via FAD at the expense of NAD. Here we demonstrate that this adaptation is driven by the ratio of reduced to oxidized CoQ. Saturation of CoQ oxidation capacity induces reverse electron transport from reduced CoQ to complex I, and the resulting local generation of superoxide oxidizes specific complex I proteins, triggering their degradation and the disintegration of the complex. CoQ redox status thus acts as a metabolic sensor that fine-tunes mETC configuration to match the prevailing substrate profile.

5 COENZYME Q IS AN IMPORTANT FACTOR IN AGING

Guillermo López-Lluch

Centro Andaluz de Biología del Desarrollo (CABD-CSIC), Departamento de Fisiología, Anatomía y Biología Celular, Universidad Pablo de Olavide, CIBERER, Instituto de Salud Carlos III, Seville, Spain.

Introduction. Aging is an irremediable process accompanied by the accumulation of damaged structures and oxidized molecules in cells, malfunction of metabolism and dysregulation of protective mechanisms such as antioxidants enzymatic activities, proteasome, autophagy, etc… In this process, malfunction of metabolic processes and the accumulation of oxidative damage due to dysregulation of antioxidant mechanisms seem to be key factors. Taken into consideration that coenzyme Q (Q) is an essential factor in both, metabolism and membrane-linked enzymatic activities, we consider Q as a key factor in aging. A brief revision of the last findings on the role of Q in aging permits us to reach this conclusion.

Age-dependent effect of caloric restriction on Q-dependent activities in plasma membrane. It is known that caloric restriction (CR) is one of the most effective methods to increase longevity or healthspan in different animal models from yeast to monkeys. Several yeast ago, we demonstrated that plasma membrane Q-dependent activities such as Cytochrome b5 Reductase and NQO1, increase in mice and rat liver but only in old animals (1,2). This same effect was found in brain (3). This effect has been associated not only with a higher protection of membrane against oxidation but also in metabolic control since the activity of this system in plasma membrane has been linked to the oxidative activity in mitochondria (4). However, to date, no information about how the age-related modulation of Q levels and activities in plasma membrane occurs and the mechanisms involved are known. Recently, we have studied the relationship among oxidative stress, ageing and coenzyme Q levels in mice liver. Oxidative stress was measured in mitochondrial fraction by determining levels of peroxidation and protein nytrotyrosine levels, in both cases increasing levels were found in mitochondria from aged animals (18 months) in comparison with young (1 month) or mature (6 months) animals. This higher oxidative profile was accompanied by a decrease in the glutathione peroxidase activity and an increase in Cytochrome b5 reductase activity. Levels of Q also decreased in mitochondria from livers of 18 months aged mice although mainly affecting Q9 levels whereas Q10 levels increased. This effect produced a clear decrease in the ratio Q9/Q10 in liver mitochondria with age. This tendency was accompanied by a lower level of CoQ7 protein in mitochondria. Furthermore, recent results indicate that the mRNA levels of many of the components of Q synthesis show a biphasic curve increasing in mature animals and decreasing in old animals that show similar levels of expression than young animals. This is important and obligates us to revise the evolution of the expression of key components during aging. Furthermore, recently we have shown that CR can modulate the expression of these genes in an organ-dependent effect indicating a more complex scenario where age of the individual and organ introduce new factors to be taken into consideration.

Q levels are affected by exercise in human blood plasma. Another important issue is the role of Q in the prevention of oxidative damage in blood plasma. Q10 in human plasma is mainly associated to lipoproteins and especially to LDL. We have determined the effect of physical activity on the levels of Q10 in human plasma and the peroxidation degree (5).

6 Interestingly, we have determined that physical activity decrease Q10 in plasma in young individuals, however, in old people, higher level of physical activity were accompanied by higher levels of Q10 and lower peroxidation of lipids and oxidation of LDL. Furthermore, our studies have demonstrated that sedentarism is associated with lower Q10 levels in plasma and higher oxidative markers (6).

Conclusions. All these studies demonstrate that Q must be taken into consideration as an important factor in aging because it seems to be related to the deterioration of mitochondrial activity during aging and in regulatory mechanisms involved in the protection of cell membranes and importantly plasma membrane during aging. Other important evidence of the role of Q in aging is that CR, exercise and even resveratrol are able to modulate its levels in cell membranes and lipoproteins and the expression of the components of the its synthesis. These evidences reinforce the need of a deeper research on the role of Q in aging.

Acknowledgements The research group is financed by the Andalusian Government as the BIO177 group financed by FEDER funds (European Commission). Research has been also financed by the Spanish Government grants DEP2012-39985.

References 1. De Cabo, R., Cabello, R., Ríos, M., López-Lluch, G., Ingram, D.K., Lane, M.A., and Navas, P. Calorie restriction attenuates age-related alterations in the plasma membrane antioxidant system in rat liver. 2004. Exp. Gerontol. 39(3):297-304. 2. López-Lluch G., Ríos, M., Lane, M:A., Navas, P., and De Cabo, R. Mouse liver plasma membrane redox system activity is altered by aging and modulated by calorie restriction.2005. Age. 27(2):153-160. 3. Hyun, D.H., Emerson, S.S., Jo, D.G., Mattson, M.P., and De Cabo, R. Calorie restriction up-regulates the plasma membrane redox system in brain cells and suppresses oxidative stress during aging. 2006. Proc. Natl. Acad. Sci. USA. 103(52):19908-19912. 4. Jiménez-Hidalgo, M., Santos-Ocaña, C., Padilla, S., Villalba, J.M., López-Lluch, G., Martín-Montalvo, A., Minor, R.K., Sinclair, D.A., de Cabo, R., and Navas, P. NQR1 controls lifespan by regulating the promotion of respiratory metabolism in yeast. 2009. Aging Cell. 8(2):140-151. 5. Del Pozo-Cruz, J., Rodríguez-Bies, E., Ballesteros-Simarro, M., Navas-Enamorado, I., Tung, B.T., Navas, P., and López-Lluch, G. Physical activity affects plasma coenzyme Q10 levels differently in young and old humans. 2014. Biogerontology. 15(2):199-211. 6. Del Pozo-Cruz, J., Rodríguez-Bies, E., Navas-Enamorado, I., Del Pozo-Cruz, B., Navas, P., and López-Lluch, G. Relationship between functional capacity and body mass index with plasma coenzima Q10 and oxidative damage in community-dwelling elderly-people. 2014. Exp. Gerontol. 52:46-54.

7 THE MITOCHONDRIA BEYOND OXPHOS

Claudio Franceschi

DIMES, University of Bologna and IRCCS, Institute of Neurological Sciences of Bologna, Bologna, Italy

Mitochondria are key organelles for cell bioenergetics, as they are involved in Oxidative Phosphorylation (OXPHOS) and beta-oxidation of fatty acids. Due to the OXPHOS activity, they are also the major source of Reactive Oxygen Species (ROS), that are a leading cause of damage to macromolecules. Finally, it is established that mitochondria play also a role in apoptosis (intrinsic pathway). However, beside these well-known activities, new emerging roles for mitochondria in cell biology have been discovered, in particular as far as the aging of the cell. Such a new role of mitochondria will be discussed in the presentation. In particular, the following topics will be covered: i. the role of mitophagy (a specialized form of autophagy aimed at disposing dysfunctional mitochondria) and of the complex interplay between mitochondria and telomeres in the process of cell senescence; ii. the mitochondria as core player in the activation of inflammasomes, and their possible role in inflammaging, the chronic, low-grade inflammatory process characterizing old persons; iii. the spreading of signals originating from mitochondria to other cells and tissues, such as humanin; allowing cells to communicate with each other; finally, the hypothesis that aging can spread from cell to cell and from tissue to tissue through this kind of signaling molecules will be discussed.

8 REDOX STATE OF MITOCHONDRIAL COENZYME Q REGULATES ROS PRODUCTION AND DETERMINES LIFESPAN IN ANIMALS.

Alberto Sanz

Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, Newcastle University, Newcastle upon Tyne, United Kingdom.

The mitochondrial electron transport chain (ETC) is the main generator of Reactive Oxygen Species (ROS) in most cellular types. ROS can cause oxidative damage if they are produced in excess, but are also essential cellular messengers implicated in maintaining homeostasis. The redox state of the ubiquinone pool (CoQ) determines the amount of electrons that leak ETC and univalently reduce oxygen to superoxide. We report that the ectopic expression of an alternative NADH dehydrogenase (NDI1) –in Drosophila melanogaster- increases the reduction of CoQ. This generates reverse electron transport (RET) at respiratory complex I, and greatly increases the levels of ROS. Interestingly, we show that these ROS extend lifespan since cancellation of this signal, expressing an alternative oxidase -that maintains the CoQ oxidized- suppresses the extension in lifespan. We demonstrate that RET is able to partially compensate the knockdown of superoxide dismutase 2, indicating that cells are able to distinguish ROS associated with RET. Finally, we show evidence that RET is able to prevent the onset of age-related diseases. Knockdown of PINK1 caused a Parkinson-like phenotype characterized by reduced complex I-linked respiration, reduced mobility and shorten lifespan. Expression of NDI1 in a Pink1 depleted background rescues all these three phenotypes, showing that RET may be a new therapeutic option.

9 REDUCED COENZYME Q10 (UBIQUINOL-10) ACTIVATES MITOCHONDRIAL FUNCTIONS AND DECELERATES SENESCENCE BY ACTIVATING SIRTUIN PATHWAYS AND INHIBITING cAMP PHOSPHODIESTERASES

Higuchi Keiichi1,2, Xu Zhe1, Tian Geng1, Huo Jia1, Kubo Hiroshi3, Hosoe Kazunori4 and Sawashita Jinko1,2

1Department of Aging Biology, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, Matsumoto, Japan. 2Department of Biological Sciences for Intractable Neurological Diseases, Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Matsumoto, Japan. 3Biotechnology Development Laboratories, Kaneka Corporation, Hyogo, Japan. 4Functional Food Ingredients Marketing Group, QOL Division, Kaneka Corporation, Osaka, Japan. [email protected]

Our studies have revealed significantly delayed senescence and age-related hearing loss (AHL) in Senescence-accelerated mouse prone-1 (SAMP1) mice1) given supplementation with reduced form 2,3) of coenzyme Q10 (ubiquinol-10) , but the mechanism of action of this effect has remained unclear. Here, we report that dietary ubiquinol-10 supplementation prevents age-related decreases in the expression of NAD+-dependent protein deacetylases (SIRT1 and SIRT3), which results in the activation of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), a major factor that controls mitochondrial biogenesis and respiration, as well as superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2), which are mitochondrial antioxidant enzymes4). Ubiquinol-10 supplementation prevented age-related decrease in mitochondria complex I activity and numbers of mitochondria and age-related increase in oxidative stress markers. Ubiquinol-10 suppressed the staining of senescence-associated β-galactosidase (SA-β-gal) in SAMP1 mouse embryonic fibroblast (MEF) cells. Furthermore, ubiquinol-10 increased levels of cAMP by activating adenylate cyclase (AC) and repressing phosphodiesterases (PDEs) in HepG2 cells. In particular, ubiquinol-10 decreased the expression levels of PDEs without affecting the specific activities of PDEs. The increased cAMP may activate cAMP response element-binding protein (CREB) and AMP-activated protein kinase (AMPK) and activate sirtuin pathway. These findings suggest that ubiquinol-10 supplementation to activate sirtuin pathway could prevent mitochondrial decay associated with aging and decelerate age-related diseases by increasing oxidative stress resistance in SAMP1 mice (Figure 1). Further studies should be performed to elucidate the mechanism by which ubiquinol-10 activates sirtuin pathway using SAMP1 and other strains of mice and various cell lines.

References 1. Takeda T: Senescence-accelerated mouse (SAM): Biogerontological resource in aging research. Neurobiol Aging 20: 105-110, 1999 2. Yan J, Fujii K, Yao J, Kishida H, et al. Reduced coenzyme Q10 supplementation decelerates senescence in SAMP1 mice. Exp Gerontol 41: 130–40, 2006 3. Schmelzer C, Kubo H, Mori M, et al. Supplementation with the reduced form of coenzyme Q10 decelerates phenotypic characteristics of senescence and induces a peroxisome proliferator- activated receptor-α gene expression signature in SAMP1 mice. Mol Nutr Food Res. 54: 805-15, 2010 4. Tian G, Sawashita J, Kubo H, el al. Ubiquinol-10 supplementation activates mitochondria functions to decelerate senescence in senescence-accelerated mice. Antioxid Redox Signal. 20: 2606-20, 2014

10

Figure 1. Possible mechanism of the anti-aging effects of ubiquinol-10 supplementation. Ubiquinol-10 may increase the level of cAMP by activating stimulatory G protein α-subunits (Gαs) and repressing inhibitory G protein α-subunits (Gαi) and PDEs. Increased cAMP levels activate SIRT1 and PGC-1α by increasing the levels of phosphorylated CREB, LKB1 (liver serine/threonine kinase B1) and AMPK in turn. SIRT1 deacetylates and activates PGC-1α that can then induce SIRT3 in mitochondria though ERRα (estrogen-related receptor α), whi ch activates SOD2, IDH2, and mitochondrial genes through NRF2 (nuclear factor erythroid 2-related factor 2) and TFAM (mitochondrial transcription factor A). Activated SOD2, IDH2, and GSH (reduced glutathione), decreases ROS (reactive oxygen species) levels and increases the activity of mitochondrial electron transport chain complexes I and IV. Together, these effects may protect tissues from oxidative stress and prevent the accelerated senescence and age-related diseases including AHL, seen in SAMP1 mice. PPARα peroxisome proliferator-activated receptor-α. Figure 1 was modified from Fig 8 in reference 4.

11 COENZYME Q10 CONTENT AND OXIDATIVE STRESS DURING AGEING IN HUMAN DERMAL FIBROBLASTS CULTURED UNDER DIFFERENT OXYGEN TENSION

Brugè F.a, Damiani E.b, Orlando P.a, Silvestri S.a, Marcheggiani F.a, Littarru GP.a, Tiano L.a

Dipartimento Scienze Cliniche Specialistiche ed Odontostomatologiche, Università Politecnica delle Marche, Ancona, Italy; b Dipartimento Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche, Ancona, Italy

The ageing process is due to different detrimental changes in cells and tissues, leading to an increase in proteins, nucleic acids and lipids oxidation. Indeed, during ageing, oxidative stress determined by reactive oxygen species (ROS) increases. However, recent reports highlight the potential beneficial effects of ROS that could be involved in cellular regulation by acting as signal mediators. From this point of view, ROS are considered agents that increase lifespan and represent adaptations to toxicity. In this context, mitochondria, which are the main source of ROS, play a pivotal role. Approximately 1% to 5% of the oxygen consumed by mitochondria is partially reduced and converted to these species. Coenzyme Q10 is involved in these processes both in relation to its bioenergetic and antioxidant role. The aim of our study was to evaluate the oxidative status and ageing markers in cultured human dermal fibroblasts (HDF) undergoing replicative senescence and grown both at 21% and 5% O2. Cell cultures are commonly maintained under standard atmospheric oxygen (20% O2, normoxia) which is far from the 5% physiological O2 tension (hypoxia) typically experienced by HDF in vivo. The results obtained highlight that the ageing process, assessed using the β-galactosidase assay and p16 gene expression, is faster in cells grown under normoxia. However, despite observing an higher production of superoxide anion at the mitochondrial level, particularly during early passages, cytoplasmic ROS levels were lower compared to those in HDF maintained under hypoxia. Our data demonstrate that antioxidant defences constitute a mechanism that is induced as an adaptive response which involves both enzymatic (SOD1) and non-enzymatic antioxidant systems (Coenzyme Q10). In conclusion, these processes represent a homeodynamic adaptation to mild oxidative stress and the underlying molecular mechanisms could be used to develop new anti- ageing strategies.

12 Notes

Notes

THE DUAL FUNCTION OF Coq4p IN YEAST: THE NUCLEATION OF COENZYME Q6 BIOSYNTHESIS COMPLEX AND THE ACTIVATION OF COMPLEX III

Elena García-Testón Páez, Plácido Navas Lloret and Carlos Santos-Ocaña

Centro Andaluz de Biología del Desarrollo-CSIC, CIBERER. Universidad Pablo de Olavide

Although the molecular function of the Coq4 protein in CoQ6 biosynthesis is not completely understood, much evidence has demonstrated a function of Coq4p on the nucleation and assembly of the CoQ6 biosynthetic complex [1,2]. In recent years growing evidence has indicated that protein phosphorylation is involved in the complex assembly and its regulation. In that way several phosphoproteins have been identified as components of the complex such as Coq3p, Coq5p and Coq7p, a phosphatase such as Ptc7p and also a kinase like Coq8p [3–7]. Although the real effect of activation or inactivation of phosphorylation is not clear, we have analyzed the function of protein phosphorylation on the complex nucleation through the role of Coq4p. Sequence analysis searching for phosphoprotein motifs allowed to find three putative residues located far from the zinc finger motifs required to the isoprenoid binding of Coq4p [8]. The modification of phosphoaminoacids to alanine produce a moderate decrease of in vitro phosphorylation but the expression of mutated versions of Coq4p abolished the growth in non- fermentable carbon sources. However the CoQ6 determination in mitochondrial samples indicated that these mutants are not deficient in CoQ6 biosynthesis. In parallel, respiratory O2 consumption in whole cells decreased compared to wild type cells while endogenous oxidative stress was unaffected. Mitochondrial respiratory chain activities remained intact with the exception of complex III and complex I+III. Since CoQ6 is produced at wild type levels and the ratio of Q6/Q6H2 remains constant in phosphomutants the defect is not produced due to the lack of CoQ6 or Q6H2. One option to explain the respiratory defect is the unavailability of CoQ6 for complex III. When cells are supplemented with exogenous CoQ6, phosphomutants are unable to restore the growth in YPG, contrary to other COQ4 mutants such as Coq4-1 defective in CoQ6 biosynthesis [9]. Surprisingly, the negative control (a COQ4 null mutant strain) did not recover the growth in YPG after CoQ6 supplementation while in other null mutant strains such as COQ5 growth recovering was successful. The recovery of YPG growth after CoQ6 supplementation is a general feature of null mutants of CoQ6 synthesis that it is not produced in COQ4 mutants. This supports the idea of the requirement of Coq4p to the nucleation and assembly of the biosynthetic complex. Our hypothesis is that Coq4p shows two roles on mitochondrial function: the nucleation of the complex and it is also required for the stabilization of complex III. The first function is probably carried out through the binding to the early CoQ6 precursor HHB. That function fails in previously described point mutants such as coq4-1 that can be recovered with exogenous CoQ6 while it was not produced in phosphomutants. The second function is interacting with respiratory complexes such as complex III to provide CoQ6, an endogenous component of this complex [10,11]. In this sense the steady state of mitochondrial respiratory complexes analyzed by BN-PAGE demonstrated that complex patterns are modified in phosphoprotein mutants of Coq4p. This opens a new window to the study of CoQ6 biosynthesis regulation, the possibility that Q synthesis is connected with mitochondrial respiratory chain activities directly, even physically producing large supercomplexes.

References 1. I. González-Mariscal, E. García-Testón, S. Padilla, A. Martín-Montalvo, T. Pomares-Viciana, L. Vazquez-Fonseca, et al., IUBMB Life. n/a–n/a, (2014).

13 2. C.M. Allan, A.M. Awad, J.S. Johnson, D.I. Shirasaki, C. Wang, C.E. Blaby-Haas, et al., J. Biol. Chem. jbc.M114.633131, (2015). 3. A. Martin-Montalvo, I. Gonzalez-Mariscal, S. Padilla, M. Ballesteros, D.L. Brautigan, P.P. Navas, et al., Biochem. J. 114, 107–114, (2011). 4. L.X. Xie, E.J. Hsieh, S. Watanabe, C.M. Allan, J.Y. Chen, U.C. Tran, et al., Biochim Biophys Acta. 1811, 348–360, (2011). 5. L.X. Xie, M. Ozeir, J.Y. Tang, J.Y. Chen, S.-K. Jaquinod, M. Fontecave, et al., J. Biol. Chem. 287, 23571–81, (2012). 6. A. Tauche, U. Krause-Buchholz, G. Rödel, G. Rodel, FEMS Yeast Res. 8, 1263–75, (2008). 7. A. Martin-Montalvo, I. Gonzalez-Mariscal, T. Pomares-Viciana, S. Padilla-Lopez, M. Ballesteros, L. Vazquez-Fonseca, et al., J. Biol. Chem. 288, 28126–37, (2013). 8. S.L. Rea, B.H. Graham, E. Nakamaru-Ogiso, A. Kar, M.J. Falk, Dev. Disabil. Res. Rev. 16, 200– 18, (2010). 9. B. Marbois, P. Gin, M. Gulmezian, C.F. Clarke, Biochim. Biophys. Acta - Mol. Cell Biol. Lipids. 1791, 69–75, (2009). 10. C. Santos-Ocaña, T.Q. Do, S. Padilla, P. Navas, C.F. Clarke, C. Santos-Ocana, J. Biol. Chem. 277, 10973–81, (2002). 11. S. Bartoschek, M. Johansson, B.H. Geierstanger, J.G. Okun, C.R. Lancaster, E. Humpfer, et al., J. Biol. Chem. 276, 35231–4, (2001).

14 BIOSYNTHESIS OF CoQ10 IN FISSION YEAST

Makoto Kawamukai

Faculty of Life and Environmental Science, Shimane University, Shimane 690-8504, Japan. E-mail: [email protected]

My group has been focused on the biosynthesis of CoQ10 and its roles in fission yeast Schizosaccharomyces pombe [1]. One of the reasons to study fission yeast is the status that this yeast has been extensively studied as a model organism for basic research with accumulating knowledge. Studies on such a model organisms contribute the understanding of roles of human genes involved in the CoQ biosynthesis, as we showed that human COQ gene counterparts mostly replaced the function of fission yeast coq gene knockout [1]. The other reason to study fission yeast is that this organism produces CoQ10, which means that it has potential for commercial production of CoQ10. The study of CoQ10 production was performed recently. In spite of the efforts to express various genes responsible for CoQ10 synthesis in fission yeast, it was difficult to overproduce CoQ10 because they generally end up to growth retardation. But the genes located in the upstream pathway of CoQ10 synthesis contribute significantly for production of CoQ10 [2]. We also found Pka1 (Protein kinase A) is an important regulator for the production of CoQ10. In S. pombe, CoQ is not essential for growth, but CoQ-deficient S. pombe displays many interesting phenotypes: It grows very slowly on minimum medium, is sensitive to oxidative stress, does not survive in the stationary phase (short life span) and produces a large amount of sulfide. Some of these phenotypes are partly suppressed by cyclodextrin encapsulated CoQ10 but not by ethanol solubilized CoQ10 [3]. I will also discuss on our recent analyses of intermediates accumulated in coq mutants. A CoQ-binding protein named Coq10 belongs to a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain superfamily. Deletion of the coq10 gene caused a respiratory defect in S. pombe, which indicated that Coq10 may support efficient electron transfer between the respiratory complexes; however, its physiological role remains elusive. To elucidate the role of Coq10, we attempted to identify the binding site of CoQ in recombinant S. pombe Coq10 expressed in Escherichia coli cell membrane through photoaffinity labeling with the photoreactive CoQ probe, UQ-1, in combination with biotinylation of the labeled peptide. Comprehensive proteomic analyses revealed that the quinone-head ring of UQ-1 specifically binds to the N-terminus region Phe39-Lys45 of Coq10, which corresponds to the ligand-binding pocket of many proteins containing the START domain. We provide the direct evidence for Coq10 accommodating the quinone-head ring of CoQ in its START domain [4].

References 1. Hayashi, K., Ogiyama, Y., Yokomi, K. Nakagawa, T., Kaino, T., Kawamukai, M. Functional conservation of coenzyme Q biosynthetic genes among yeasts, plants, and humans. PLoS ONE 9(6):e99038 (2014) 2. Moriyama, D., Hosono, K., Fujii, M., Washida, M., Nanba, H., Kaino, T., Kawamukai, M. Production of CoQ10 in fission yeast by expression of genes responsible for CoQ10 biosynthesis. Biosci. Biotechnol. Biochem. (in press) 3. Nishida, T., Kaino, T., Ikarashi, R., Nakata, D., Terao, K. Ando, M. Hamaguchi, H., Kawamukai, M. Yamamoto, T. The effect of coenzyme Q10 included by gamma-cyclodextrin on the growth of fission yeast studied by microscope Raman spectroscopy. J. Molecular Structure 375-381(2013) 4. Murai M, Matsunobu K, Kudo S, Ifuku K, Kawamukai M, Miyoshi H. Identification of the binding site of the quinone-head group in mitochondrial Coq10 by photoaffinity labeling. Biochemistry 53:3359-4003 (2014)

15 UBIAD1 IS AN ANTIOXIDANT ENZYME THAT REGULATES CoQ10 SYNTHESIS IN ENDOTHELIAL CELLS

Raj Sewduth1, Emiliano Panieri2, Carlo Millia1, Massimo M. Santoro1,2

1 Vesalius Research Center, VIB-KUL, Leuven, Belgium 2 Dept. of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Italy

Protection against an excess of reactive oxygen species (ROS) by an antioxidant network is essential for tissues, especially in the cardiovascular system. Here we identified a new gene with important anti-oxidant features by isolating a null allele of the zebrafish ubiad1 gene, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a non-mitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. The loss of UBIAD1 function reduces the cellular membrane pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation. Surprisingly, inhibition of eNOS rescues Ubiad1-dependent cardiovascular oxidative damage suggesting a crucial role for this enzyme and non-mitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a new CoQ10 prenyltransferase with specific cardiovascular protective function by controlling eNOS activity.

16 CHARACTERIZATION OF PROTEINS ASSOCIATED WITH THE MITOCHONDRIAL CoQ SYNTHOME IN SACCHAROMYCES CEREVISIAE

Catherine F. Clarke, Christopher M. Allan, Agape M. Awad, Cuiwen H. He, Alice Hsu, Hei Tong Lam, Anish Nag, and Hui S. Tsui

Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, CA, USA

Saccharomyces cerevisiae (baker’s yeast) serves as an excellent model for studies on CoQ biosynthesis and function because it is a simple model and is highly conserved with respect to human CoQ biosynthesis. In yeast, several of the eleven proteins (Coq1p-Coq9p, Yah1p, and Arh1p) necessary for the biosynthesis of CoQ6 have been shown to be associated in a high molecular mass complex that localizes in the inner mitochondrial membrane, and has been termed the ‘CoQ-synthome’. Here we identify other potential protein partners that co-purify with several of the Coq polypeptides and appear to be required for efficient Q biosynthesis. We use co- immunoprecipitation techniques, Western blotting, blue native PAGE, lipid analysis, yeast cell growth assays, phosphorylation enrichment, and liquid chromatography-tandem mass spectrometry to identify novel associated proteins and phosphorylation modifications sites of the Coq polypeptides. Elucidating their biosynthetic role in regulatory processes, such as phosphorylation, will aid our understanding and treatment of multiple diseases caused by CoQ-deficiencies and will provide novel insight regarding longevity and issues related to aging in humans.

17 ELUCIDATING THE ROLES OF COQ8 AND COQ9 IN COENZYME Q BIOSYNTHESIS

David J. Pagliarini

University of Wisconsin-Madison

Coenzyme Q (CoQ) is an integral piece of the mitochondrial machinery that produces over 90% of cellular ATP; yet, the biochemical functions of multiple proteins involved in the biosynthesis of CoQ remain unknown. Recently, we have gained novel biochemical and structural insights into two of these proteins, ADCK3 (aka COQ8) and COQ9. First, our crystal structures of the human ADCK3 reveal an atypical protein kinase-like fold with flexible features positioned to inhibit protein kinase activity. Mutating ADCK-specific features enables cis autophosphorylation in vitro, but inhibits ATPase activity in vitro and dampens CoQ biosynthesis in vivo. Our findings argue against ADCK3 acting as a typical protein kinase and demand a new model for how ADCK3 enables CoQ biosynthesis, which I discuss. Second, by solving the structure of human COQ9, we discovered that it has structural homology to the TetR family of bacterial transcription factors and, importantly, has a lipid-binding cavity. We also demonstrated that COQ9 interacts with another CoQ biosynthetic protein, COQ7, likely as part of a biosynthetic complex. Our recent work has revealed further insights into the nature of this interaction and into the endogenous COQ substrate, which I present. Collectively, our work provides new molecular insight into the roles of poorly characterized, essential members of the CoQ biosynthetic pathway.

18 ABOUT pABA AND 4-HYDROXYBENZOIC ACID IN YEAST COENZYME Q BIOSYNTHESIS

Laurie-Anne Payet1, Mélanie Leroux1, Mohammad Ozeir1, Ulrika Forsman2, Pavel Sindelar2, Ludovic Pelosi1, Alexandre Ismail3, Caroline Mellot-Drazniek3, Marc Fontecave3, Fabien Pierrel1, 2

1 Laboratoire d’adaptation et de pathogénie des microorganismes, UMR 5163 CNRS-UJF, Grenoble, France 2 Laboratoire Chimie et Biologie des Métaux, UMR5249 CNRS-CEA-UJF, F-38054 Grenoble, France 3 Laboratoire de Chimie des Processus Biologiques, UMR8229 CNRS, Collège de France, Paris Email : [email protected]

The yeast Saccharomyces cerevisiae can use 4-hydroxybenzoic acid (4-HB) and also para- aminobenzoic acid (pABA) as precursors of the aromatic ring of coenzyme Q [1, 2]. In order to synthesize Q from pABA, the amino group on carbon C4 must be replaced by an hydroxyl group via a C4-deamination reaction that takes place at an undefined step after the prenylation of pABA by Coq2. Coq6 is a flavin-dependent monooxygenase which catalyzes the hydroxylation reaction on C5 [3]. We show that C-terminal truncated forms of Coq6 or a Coq6 point mutant are able to support in vivo Q biosynthesis from 4-HB but not from pABA because the C4-deamination is impaired. Labelling experiments demonstrate that dioxygen is the source of the OH group on C4 which substitutes the amino group originally present on pABA, suggesting that Coq6 functions either as a monooxygenase or as a deaminating dioxygenase depending on whether 4-HB or pABA is used as a precursor of Q. In the light of these new data, I’ll discuss the capacity of different organisms to use or not pABA as a precursor of Q. The synthesis of 4-HB from tyrosine is not elucidated. Through a genetic screen, we have identified a yeast mutant that displays a very low level of Q unless 4-HB is added to the growth medium. Our results demonstrate that the gene product catalyzes the last reaction in the biochemical pathway which converts tyrosine into 4-HB. In addition, we also identify the first reaction of the pathway, namely, the deamination of tyrosine and we demonstrate that yeast is not able to generate 4-HB directly from chorismate unlike previously proposed.

References 1. Pierrel, F., Hamelin, O., Douki, T., Kieffer-Jaquinod, S., Muhlenhoff, U., Ozeir, M., Lill, R. & Fontecave, M. (2010) Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis, Chem Biol. 17, 449-459. 2. Marbois, B., Xie, L. X., Choi, S., Hirano, K., Hyman, K. & Clarke, C. F. (2010) para-aminobenzoic acid is a precursor in coenzyme Q(6) biosynthesis in Saccharomyces cerevisiae, J Biol Chem. 285, 27827-27838. 3. Ozeir, M., Muhlenhoff, U., Webert, H., Lill, R., Fontecave, M. & Pierrel, F. (2011) Coenzyme Q biosynthesis: Coq6 Is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency, Chem Biol. 18, 1134-1142.

19 THE ROLE OF COQ REDOX STATE IN MITOCHONDRIAL SUPEROXIDE PRODUCTION

Mike Murphy

MRC Mitochondrial Biology Unit, Cambridge, UK

Mitochondrial ROS have long been known to contribute to damage in conditions such as ischaemia- reperfusion (IR) injury in heart attack and stroke, and more recently to redox signalling. Over the past few years we have investigated the mechanism of the ROS production using metabolomic approaches. We found that the ROS production can be driven by reverse electron transport at complex I during reperfusion. This surprising mechanism indicates that the redox stste of the CoQ pool plays a key role in determining how mitochondrial ROS can be produced to act as redox signals.

20 AN IN VIVO-GENERATED METABOLITE OF IDEBENONE IS A PROMISING MOLECULE FOR TREATMENT OF LHON

Valentina Giorgio1,2, Marco Schiavone2,3, Marco Carini4, Tatiana Da Ros4, Maurizio Prato4, Francesco Argenton3, Valeria Petronilli1,2, Paolo Bernardi1,2

1CNR Neuroscience Institute and Departments of 2Biomedical Sciences and 3Biology, University of Padova, Padova, Italy; 4Department of Chemical and Pharmaceutical Sciences, University of Trieste, Trieste, Italy

Coenzyme Q (CoQ10) is a lipophilic molecule mostly, but not exclusively, localized in the inner mitochondrial membrane. Given that CoQ10 is very insoluble, several short chain hydrosoluble quinones have been tested in a variety of disease models, in particular 2,3-dimethoxy-5-methyl-6- (10-hydroxydecyl)-1,4-benzoquinone (idebenone). Idebenone has been used in several diseases associated to respiratory chain dysfunction like Leber's hereditary optic neuropathy (LHON), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes, Leigh syndrome, Friedreich's ataxia, as well as Huntington's and Alzheimer’s disease. More recently, a double blind, placebo-controlled, randomized trial with idebenone in LHON patients (RODHOS study) showed some efficacy, as also reported in the retrospective analysis of a large case series of LHON patients treated with idebenone during the acute phase of the disease. However, despite these encouraging results, the efficacy of idebenone in vivo remains debated. A potentially harmful effect of idebenone is opening of the permeability transition pore (PTP), an event that may actually play a causative role in the disease, and has been associated to retinal ganglion cell degeneration. Furthermore, the potential efficacy of idebenone in LHON relies on the ability of its reduced form (idebenol, which does not induce PTP opening) to mediate electron transfer to complex III of the respiratory chain, an effect that requires the presence of an idebenone reductase such as NAD(P)H:quinone oxidoreductase 1. Idebenone-generated metabolites have been shown to support mitochondrial respiration by donating electron at complex III in vitro, thus allowing maintenance of a membrane potential. We find that idebenone metabolites that are generated in vivo support respiration in LHON cybrids, yet at variance from idebenone do not induce PTP opening. Treatment of zebrafish (Danio rerio) with one such metabolite at 20 hours post fertilization significantly protects embryos from the aberrant phenotypes and death caused by rotenone, a selective inhibitor of complex I. These results suggest that idebenone-generated metabolites may represent a promising treatment for LHON.

21 EFFECT OF CoQ10 SUPPLEMENTATION ON CELLULAR BIOENERGETIC STATUS AND OXIDATIVE STRESS: AN IN VIVO AND IN VITRO STUDY

Christian Bergamini1, Anna Rita Fetoni2, Diana Troiani3, Giorgio Lenaz4 and Romana Fato1

1Dept.of Pharmacy and Biotechnology (FaBiT), university of Bologna, Italy. 2Institute of Otolaryngology, Catholic University School of Medicine, Rome, Italy. 3Human Physiology, Catholic University School of Medicine, Rome, Italy. 4Dipartimento di Scienze Biomediche e Neuromotorie, Alma Mater Studiorum, Università di Bologna , Italy .

Coenzyme Q is a crucial component of the oxidative phosphorylation process in mitochondria, key organelles of the eukaryotic cells where they play a double role being both the cellular powerhouse and the major source of reactive oxygen species. The reduced form of ubiquinone exerts its antioxidant effects either by direct reaction with radicals or by regeneration of tocopherol and ascorbate. Deficiency of coenzyme Q has been described in several pathological conditions and in ageing, strongly supporting a therapeutic role of coenzyme Q10 supplementation. Despite these potential beneficial effects, clinical studies showed controversial results that are mainly due to the high hydrophobicity of this compound, which reduces its bioavailability. For our experiments in 1 vitro and in vivo, we used a water-soluble CoQ10 formulation (Qter) . - In vitro studies The goal was to increase the mitochondrial content of CoQ10 in cultured cells (T67 and H9c2 cell lines) in order to improve their bioenergetics parameters. We found that the cellular and mitochondrial Coenzyme Q content in two cell lines was significantly increased after treatment with a water-soluble CoQ10 formulation. Moreover, we demonstrated that the increase of mitochondrial and cellular CoQ10 content correlates with an improved mitochondrial functionality (increased oxygen consumption rate, transmembrane potential, ATP synthesis) and resistance to oxidative stress 2. - In vivo studies Considering the promising results from our in vitro studies, we exploited the beneficial effects of Qter treatment in a rat model of repeated noise exposure. We addressed the relationship between cochlear oxidative damage and auditory cortical injury and we tested the protective effect of CoQ10 administration. The systemic administration of water-soluble CoQ10 increased the cochlear levels of CoQ9 and CoQ10. Furthermore, the ubiquinone administration reduced the oxidative-induced cochlear damage, hearing loss, and cortical dendritic injury. These findings indicate that CoQ10 treatment restores auditory cortical neuronal morphology and hearing function by reducing the noise-induced redox imbalance in the cochlea and the deafferentation effects upstream the acoustic pathway 3. The supplementation with coenzyme Q10 improves cellular energy metabolism both in in vitro and in vivo models, exerting a protective effect on redox imbalance related damages.

References 1. Carli F, Corvi Mora P, Canal T (2003) Co-grinding process for the preparation of a ternary composition. European patent office. Wipo website. Available: http://www.wipo.int/patentscope/search/en/WO2003097012. 2. A water soluble CoQ10 formulation improves intracellular distribution and promotes mitochondrial respiration in cultured cells. Bergamini C, Moruzzi N, Sblendido A, Lenaz G, Fato R. PLoS One. 2012;7(3):e33712 3. Noise-induced hearing loss (NIHL) as a target of oxidative stress-mediated damage: cochlear and cortical responses after an increase in antioxidant defense.Fetoni AR, De Bartolo P, Eramo SL, Rolesi R, Paciello F, Bergamini C, Fato R, Paludetti G, Petrosini L, Troiani D. J Neurosci. 2013 Feb 27;33(9):4011-23

22 COENZYME Q10 TARGETS MECHANISMS OF DIABETIC CARDIOMYOPATHY IN PRE-CLINICAL MODELS

Rebecca H Ritchie

Heart Failure Pharmacology, Baker IDI Heart & Diabetes Institute, Melbourne, Australia.

‘Diabetic cardiomyopathy’ is characterized by impaired left ventricular (LV) diastolic function, cardiomyocyte hypertrophy, LV fibrosis & cardiomyocyte apoptosis. Elevated oxidative stress and LV generation of the ROS superoxide are considered likely contributors to this pathophysiology1. Our laboratory has undertaken a number of studies aimed at assessing whether chronic supplementation of Coenzyme Q10 protects the diabetic heart against dysfunction and remodelling, using a range of preclinical in vivo rodent models of both type 1 (T1D) and type 2 diabetes (T2D). Firstly, in a mouse model of T1D using streptozotocin, Coenzyme Q10 supplementation (10 mg/kg/day for 8 weeks), significantly attenuated T1D-induced increases in LV NADPH oxidase gene expression and activity (lucigenin chemiluminescence), LV uncoupling protein UCP3 expression, and both LV and systemic oxidative stress (LV 3-nitrotyrosine and plasma malondialdehdye levels). This was accompanied by blunting of the T1D-induced impairments in LV diastolic function (E:A ratio and deceleration time by echocardiography, LV end-diastolic pressure, and LV −dP/dt by micromanometry), LV remodelling (cardiomyocyte hypertrophy, cardiac fibrosis, apoptosis), and LV expression of pro-inflammatory mediators (tumor necrosis factor-α, with a similar trend for interleukin IL-1β). Coenzyme Q10's actions were independent of 2 glycemic control, body mass, and blood pressure . Similarly, Coenzyme Q10 supplementation (10 mg/kg/day) for 10 weeks from 6 weeks of age significantly attenuated LV NADPH oxidase activity, diastolic dysfunction, cardiomyocyte hypertrophy and fibrosis in spontaneously type 2 diabetic (T2D) db/db mice. Phosphorylation of the cell survival kinase Akt, depressed in untreated db/db 3 mice, was also restored with Coenzyme Q10 . Notably in both models, Coenzyme Q10 was comparable in effectiveness as the ACE inhibitor ramipril. Lastly, we sought the effectiveness of chronic Coenzyme Q10 supplementation in a more severe model of T1D diabetic cardiomyopathy, a setting of reduced cardiac phosphoinositide 3-kinase p110α (PI3K) protective signalling. T1D was induced using streptozotocin; in both nontransgenic (Ntg) mice and transgenic mice expressive a cardiac-selective dominant negative PI3K mutant (dnPI3K). After 4 weeks of diabetes, Coenzyme Q10 supplementation (10 mg/kg/day for 8 weeks) commenced. At study end (12 weeks of diabetes), LV NADPH oxidase (Nox2 gene expression and activity, on lucigenin-enhanced chemiluminescence), as well as oxidative stress(3-nitrotyrosine, 4-hydroxynonenal), were increased by diabetes, exaggerated in diabetic dnPI3K mice, and attenuated by Coenzyme Q10. Diabetes- induced LV diastolic dysfunction (prolonged deceleration time, elevated end-diastolic pressure, impaired E/A ratio), cardiomyocyte hypertrophy and fibrosis, expression of atrial natriuretic peptide, connective tissue growth factor and β-myosin heavy chain, were all attenuated by 4 Coenzyme Q10 . Hence, chronic Coenzyme Q10 supplementation attenuates several aspects of diabetic cardiomyopathy. Further, inappropriate LV superoxide levels thus appear to play a key causal role in triggering diabetic cardiomyopathy. Given Coenzyme Q10 supplementation has been suggested to have positive outcomes in heart failure patients, chronic Coenzyme Q10 supplementation may represent an attractive adjunct therapy for diabetic heart failure.

REFERENCES 1. Huynh K, Bernardo BC, McMullen JR, Ritchie RH. Invited review: Diabetic cardiomyopathy: Mechanisms and new treatment strategies targeting antioxidant signaling pathways. Pharmacol Ther 2014; 142: 375-415.

23 2. Huynh K, Kiriazis H, Du XJ, Love JE, Gray SP, Jandeleit-Dahm KA, McMullen JR*, Ritchie RH*. Targeting the upregulation of reactive oxygen species subsequent to hyperglycemia prevents type 1 diabetic cardiomyopathy in mice. Free Rad Biol Med 2013; 60: 307-317. 3. Huynh K, Kiriazis H, Du XJ, Love JE, Jandeleit-Dahm KA, Forbes JM, McMullen JR*, Ritchie RH*. Coenzyme Q10 attenuates diastolic dysfunction, cardiomyocyte hypertrophy and cardiac fibrosis in the db/db mouse model of type 2 diabetes. Diabetologia 2012; 55: 1544-1553. 4. de Blasio MJ, Huynh K, Qin CX, Rosli S, Kiriazis H, Du XJ, McMullen JR*, Ritchie RH*. Coenzyme Q10 protects the heart against diabetes-induced diastolic dysfunction and structural remodelling in mice with diminished PI3K(p110α) signalling. Proc ASCEPT 2014 abstract 322

24 A ROLE FOR COENZYME Q IN CELLULAR METABOLISM

Anita Ayer1, Catherine F. Clarke2, Ian W. Dawes3 and Roland Stocker1

1Victor Chang Cardiac Research Institute, Sydney, NSW, Australia 2Department of Chemistry and Biochemistry, UC Los Angeles, CA, USA 3School of Biotechnology and Biomolecular Sciences, University of NSW, Sydney, NSW, Australia

Coenzyme Q (CoQ) is best known for its ability to participate in redox reactions. Indeed, this is the basis for CoQ’s role as an electron/proton transport agent, antioxidant and source of superoxide anion radical, a reactive oxygen species that rapidly dismutates to hydrogen peroxide (H2O2). H2O2 itself is increasingly recognized as a signalling molecule, regulating several enzymes and processes including the metabolic adaptation to hypoxia. Therefore, we investigated the role of CoQ on cellular metabolism using a transcriptional approach in yeast. Saccharomyces cerevisiae is one of the most studied organisms in terms of CoQ biosynthesis and regulation, and complete gene- deletion mutants are available for all non-essential genes. Microarray analyses were carried out on WT and selected CoQ biosynthesis deletion mutants grown in defined medium lacking p- aminobenzoic acid to examine the global transcriptional responses to changes in CoQ content. Petite cells were used as a control to delineate CoQ-mediated changes versus effects mediated by respiratory incompetence. Additionally, microarrays were carried out on WT and deletion mutants with exogenous CoQ added to the growth medium to better understand the role of CoQ itself on CoQ biosynthesis and the feedback regulation in place. The results of these microarray analyses will be presented.

25 Notes

Notes

SECONDARY COENZYME Q10 DEFICIENCIES

Rafael Artuch

Hospital Sant Joan de Déu, Barcelona

Coenzyme Q10 (CoQ) biosynthesis is an intricate biological process that it may be influenced by a large number of genetic or environmental conditions. Thus, after the identification of a CoQ- deficient status in patients, the elucidation of the biological process underlying this biochemical finding is difficult. There are several genes involved in the synthesis of CoQ, but few patients have been described harbouring mutations in such genes. However, a growing number of neurological and extra-neurological conditions may display a CoQ deficiency as a relevant biochemical feature. Among these diseases, mitochondrial DNA depletion syndromes usually present a CoQ deficiency in muscle and or fibroblasts, and it may be as frequent as a 70% of the patients. Furthermore, other mitochondrial diseases may also present CoQ deficiency. However, the pathophysiology of this biochemical feature is not well-known, although the correction of CoQ deficiency by means of CoQ supplementation seems advisable. Other non-mitochondrial disorders may also present CoQ deficiency in different biological fluids, such as phenylketonuria or lysosomal storage diseases. The causes and consequences of these deficiencies are not well understood, but it strongly support the key role that CoQ, as an essential molecule for different biological processes, may have in the pathophysiology of different diseases.

26 COQ4, A NOVEL DISEASE-GENE FOR MITOCHONDRIAL DISORDERS ASSOCIATED WITH CoQ10 DEFICIENCY

Daniele Ghezzi

Fondazione IRCCS Istituto Neurologico "C. Besta", Milano, Italy

Primary Coenzyme Q10 (CoQ10) deficiencies are rare, clinically heterogeneous disorders caused by mutations in several genes encoding proteins involved in CoQ10 biosynthesis. CoQ10 , a lipoidaquinone, is an essential component of the electron transport chain (ETC), where it shuttles electrons from complex I or II to complex III. By whole-exome sequencing we identified five individuals carrying biallelic mutations in COQ4. The precise function of human COQ4 is not known, but it seems to play a structural role in stabilizing a multiheteromeric complex that contains most of the CoQ10 biosynthetic enzymes. The clinical phenotypes of the five subjects varied widely, but four had a prenatal or perinatal onset with early fatal outcome. Two unrelated individuals presented with severe hypotonia, bradycardia, respiratory insufficiency, and heart failure; two sisters showed antenatal cerebellar hypoplasia, neonatal respiratory distress syndrome, and epileptic encephalopathy. The fifth subject had an early-onset but a slowly progressive clinical course dominated by neurological deterioration with hardly any involvement of other organs. The CoQ10 amount was reduced in all available specimens from affected subjects, which often displayed a decrease of CoQ10-dependent ETC complex. The pathogenic role of all identified mutations was experimentally validated in a recombinant yeast model. Recently, other patients with COQ4 mutations have been reported confirming that COQ4 is a disease-gene responsible for early-onset mitochondrial encephalo-cardio-myopathy and associated with CoQ10 deficiency.

27 PRIMARY COENZYME Q10 DEFICIENCY CAUSED BY COQ2 MUTATIONS: FUNCTIONAL CHARACTERIZATION OF THE HUMAN GENE AND GENOTYPE-PHENOTYPE CORRELATIONS

Eva Trevisson1,2, Maria Andrea Desbats1,2, Micol Silic-Benussi3, Placido Navas4 and Leonardo Salviati1, 2

1Clinical Genetics Unit, Department of Woman and Child Health, University of Padova, Italy, 2IRP Istituto di Ricerca Pediatrica, Città della Speranza, Padova, Italy, 3 Istituto Oncologico Veneto-IRCCS (IOV), Padova, Italy 4Centro Andaluz de Biologìa del Desarrollo, University Pablo de Olavide, Sevilla, Spain

COQ2 encodes the enzyme required for the second step of the final reaction sequence of Coenzyme Q10 biosynthesis [1]. Its mutations, which have been reported in 10 families so far [2, 3], cause primary CoQ10 deficiency and have been associated to a wide clinical spectrum both in the age of onset and in the severity of symptoms. In fact, COQ2-associated phenotypes include a rapidly fatal neonatal multisystemic disease [2, 4], a severe multisystemic disorder with steroid-resistant nephrotic syndrome (SRNS) [5], isolated SRNS [6], and finally a late-onset encephalopathy with retinopathy mimicking multiple system atrophy [7]. The reasons of this variability are still unknown and genotype-phenotype correlations remain to be elucidated. Human COQ2 was sequentially cloned joining by PCR the 5’ region amplified from genomic DNA and the 3’ region obtained from cDNA [1]. Since the reported human sequence shows four different in frame ATG codons and the N-terminus of the human COQ2 protein did not show any homology with other mammalian orthologues, we characterized the 5’ region of the human gene. Our data showed that the functional human COQ2 mRNA is shorter than what was previously thought. We also analyzed the topology and subcellular localization of COQ2. We then employed a yeast model to validate the pathogenicity of all the COQ2 missense mutations reported so far. Our system proved to be simple and reproducible and demonstrated that all the COQ2 alleles analyzed are hypomorphic. Furthermore, we showed that the level of residual activity correlates with the clinical phenotypes observed in patients, supporting the hypothesis of a different sensitivity to CoQ10 deficiency of the affected tissues.

References 1. Forsgren M, Attersand A, Lake S, Grünler J, Swiezewska E, Dallner G, Climent I. Biochem J. (2004) Isolation and functional expression of human COQ2, a gene encoding a polyprenyl transferase involved in the synthesis of CoQ. 382:519-26. 2. Desbats MA, Vetro A, Limongelli I, Lunardi G, Casarin A, Doimo M, Spinazzi M, Angelini C, Cenacchi G, Burlina A, Rodriguez Hernandez MA, Chiandetti L, Clementi M, Trevisson E, Navas P, Zuffardi O, Salviati L. (2015) Primary coenzyme Q10 deficiency presenting as fatal neonatal multiorgan failure. Eur J Hum Genet. In press. 3. Desbats MA, Lunardi G, Doimo M, Trevisson E, Salviati L. (2015) Genetic bases and clinical manifestations of coenzyme Q10 (CoQ 10) deficiency. J Inherit Metab Dis. 38:145-56. 4. Mollet J, Giurgea I, Schlemmer D, Dallner G, Chretien D, Delahodde A, Bacq D, de Lonlay P, Munnich A, Rötig A. (2007) Prenyldiphosphate synthase, subunit 1 (PDSS1) and OH-benzoate polyprenyltransferase (COQ2) mutations in ubiquinone deficiency and oxidative phosphorylation disorders. J Clin Invest. 117:765-72. 5. Quinzii C, Naini A, Salviati L, Trevisson E, Navas P, Dimauro S, Hirano M. (2006) A mutation in para- hydroxybenzoate-polyprenyl transferase (COQ2) causes primary coenzyme Q10 deficiency. Am J Hum Genet. 78:345-9. 6. Diomedi-Camassei F, Di Giandomenico S, Santorelli FM, Caridi G, Piemonte F, Montini G, Ghiggeri GM, Murer L, Barisoni L, Pastore A, Muda AO, Valente ML, Bertini E, Emma F. (2007) COQ2 nephropathy: a newly described inherited mitochondriopathy with primary renal involvement. J Am Soc Nephrol. 18:2773-80. 7. Multiple-System Atrophy Research Collaboration.(2013) Mutations in COQ2 in familial and sporadic multiple- system atrophy. N Engl J Med. 18;369:233-44.

28 THE ROLE OF PATIENT ADVOCACY GROUPS IN FACILITATING THE DEVELOPMENT OF THERAPEUTICS FOR MITOCHONDRIAL DISEASE

Philip E. Yeske, Ph.D.

Science & Alliance Officer, United Mitochondrial Disease Foundation

Patient advocacy groups have historically focused on patient support, education, awareness, advocacy and research funding as component s of their overall mission. While such programs remain important, there is increasing emphasis being placed by foundations on stewarding the therapeutic development process from the patient perspective. This talk will speak to the key pieces of infrastructure required to effectively facilitate such a process and the approach the United Mitochondrial Disease Foundation is taking, while also providing an overview of mitochondrial disease therapies currently in development in the US and Europe.

29 THE EFFECT OF COENZYME Q10 ON MORBIDITY AND MORTALITY IN CHRONIC HEART FAILURE

S.A. Mortensen, F. L. Rosenfeldt, K. Filipiak, G.P. Littarru., K. Overvad, F. Skjøth, C. D. Sindberg, P. Dolliner, G. Steurer, V. Nagy, J. Feher, G. Paragh, P. Fülop, A. Kumar, H. Kaur, C.S. Ping, A.A.A. Rahim, M. Bronisz, M. Stopinski, M. Marchel, A. Kaplon-Cieslicka, W. Sinkiewicz, B. Wozakowsk-Kaplon, M. Bzymek, H. Wysocki, M. Krzciuk, D. Pella, I. Lazurova, U. Alehagen

Objectives This randomized controlled multicenter trial evaluated coenzyme Q10 as adjunctive treatment in chronic heart failure.

Background Coenzyme Q10 (CoQ10 ) is an essential cofactor for energy production and also a powerful antioxidant. A low level of myocardial CoQ10 is related to the severity of heart failure. Previous randomized controlled trials of CoQ10 in heart failure have been underpowered to address major clinical endpoints.

Methods Patients with moderate to severe heart failure were randomly assigned in a two-year prospective trial to either CoQ10 100 mg three times daily or placebo in addition to standard therapy. The primary short term endpoints at 16 weeks were changes in NYHA classification, 6-minute walk test and levels of N-terminal pro-B type natriuretic peptide. The primary long term endpoint at 2 years was composite major adverse cardiovascular events using a time to first event analysis.

Results A total of 420 patients were enrolled. There were no significant changes in short term endpoints The primary long term endpoint was reached by 15% of the patients in the CoQ10 group vs. 26% in the placebo group; (hazard ratio 0.50; 95% confidence interval, 0.32 – 0.80); p=0.003) by intention to treat analysis. The following secondary endpoints were significantly lower in the CoQ10 group compared to the placebo group: cardiovascular mortality (9 % vs. 16%, p=0.026), all-cause mortality (10 % vs. 18%, p=0.018) and incidence of hospitalization for heart failure (p=0.033). Also, a significant improvement of NYHA Class was found in the CoQ10 group after 2 years (p=0.028).

Conclusions Long term CoQ10 treatment of patients with chronic heart failure is safe, improves symptoms and reduces major adverse cardiovascular events.

30 STATIN CARDIOMYOPATHY: THE INVISIBLE PANDEMIC Peter H. Langsjoen MD1, Jens O. Langsjoen MD*2, Alena M. Langsjoen MS3

11107 Doctors Drive, Tyler, Texas 75701, USA 2University of New Mexico, Department of Internal Medicine, Albuquerque, New Mexico 87106, USA 3 Coenzyme Q10 Laboratory, Inc., 1107 Doctors Drive, Tyler, Texas 75701, USA [email protected]

Overview Statin cardiomyopathy (SCM) can be defined as an impairment in heart muscle function of a severity to cause heart failure, consequent to statin drug therapy and not explainable by any other pathophysiology. HMG-CoA reductase inhibitors (statins) not only impair skeletal muscle function but there is growing evidence that they can also impair heart muscle function1, 2, 3.

Methods From April 2007 to April 2015 we prospectively identified 146 patients who presented on long term statin therapy with clinical heart failure not explainable by established pathophysiology. Patients with coronary artery disease, valvular heart disease, hypertensive heart disease, previous chemotherapy, any type of cardiomyopathy, myocarditis or chronic lung disease were all excluded from enrollment. Study interventions consisted of simultaneous discontinuation of statin therapy and supplementation with coenzyme Q10 (CoQ10) at the time of initial visit. Supplemental CoQ10 was initiated predominantly in the form of ubiquinol at an average dose of about 300 mg per day given in divided doses with meals. Baseline symptom scores, physical exams, echocardiograms, and laboratory testing of CoQ10 and total cholesterol blood levels were performed at or soon after the initial visit. A zero to three symptom severity scoring system was used to assess symptoms potentially attributable to statin adverse effects: 0 = no symptoms, 1 = mild symptoms, 2 = moderate symptoms, and 3 = severe symptoms. Scored symptoms included fatigue, myalgias, muscle weakness, peripheral neuropathy, and memory loss. Echocardiographic measurements included diastolic function and ejection fraction (EF). Diastolic function was scored as 0 = normal, 1 = mild or impaired relaxation, 2 = moderate or pseudo normal, and 3 = severe or restrictive. Patients were assessed in follow up clinic visits every 3 to 6 months with repeat symptom scoring, physical exams, echocardiograms, and CoQ10 blood levels.

Results Of the 146 initially enrolled patients follow up data is available for 133. Four patients died during the study period, two from unknown causes, one from sudden cardiac death, and one from lung cancer. One heart attack and one stroke occurred during the study period. Statin therapy at presentation was on average 40 mg per day normalized to atorvastatin potency taken for four years. Patients have now been followed for an average of 29 months and 70% of patients have now been followed for more than one year. All statin-attributable symptom scores improved significantly (p<0.0001) from time of initial visit to most recent visit with the following average improvements in symptom severity: fatigue from 2.3 down to 0.4, myalgias from 1.4 down to 0.1, muscle weakness from 1.2 down to 0.1, memory loss from 1.3 down to 0.3, peripheral neuropathy 0.9 down to 0.3. Eighty seven percent of patients presented with heart failure with preserved EF (HFpEF), with an average initial diastolic dysfunction grade of two. Thirteen percent of patients presented with a reduced EF, on average 43%. Follow up echocardiographic data was available for 125 patients.

31 Diastolic function improved significantly (p<0.0001) with average diastolic function grade improvement from 1.7 down to 0.8, giving an average diastolic grade improvement of about one. Fifty two patients had complete normalization of diastolic function, with an average grade change of two, 24 patients had improvement without normalization of diastolic function, 31 patients had no change in their diastolic function, and four patients had worsening of diastolic function. In the 17 patients that initially presented with a reduced EF, the EF improved by an average of 11% from 43% to 54% (p=0.0002). Initial and follow up laboratory data for CoQ10 blood levels was available for 49 patients. Total (oxidized and reduced) CoQ10 levels changed from an average of 1.2 µg/ml initially to 6.1 µg/ml at most recent measurement. Average CoQ10 to cholesterol ratio changed from 0.3 µmol/mmol to 1.2 µmol/mmol, average % oxidized CoQ10 from 2.9% to 1.5%, and average total cholesterol from 186 mg/dl to 235 mg/dl. All laboratory changes where statistically significant (p<0.002). No adverse effects where experienced over the study period by patients due to CoQ10 supplementation. Seven patients who initially recovered developed worsening heart function and/or worsening statin-associated symptoms after either stopping their CoQ10 supplementation or changing CoQ10 formulation from ubiquinol to ubiquinone.

Conclusions In outpatient clinical cardiology SCM may be a prevalent and unrecognized cause of HFpEF. The great majority of SCM patients show a gratifying amelioration of symptoms along with measurable improvement in heart function upon statin drug discontinuation along with the supplementation of CoQ10. However, there is a significant minority of patients in whom SCM appears to be permanent. We propose that statin cardiomyopathy is a significant factor in the current pandemic of heart failure and in particular the increasing prevalence of HFpEF4.

References 1. Suzuki T, Nozawa T, Sobajima M, et al. Atorvastatin-induced changes in plasma coenzyme q10 and brain natriuretic peptide in patients with coronary artery disease. Int Heart J. 2008;49(4):423-433. 2. Silver MA, Langsjoen PH, Szabo S, Patil H, Zelinger A. Effect of atorvastatin on left ventricular diastolic function and ability of coenzyme Q10 to reverse that dysfunction. Am J Cardiol. 2004;94(10):1306-1310. doi:10.1016/j.amjcard.2004.07.121. 3. Langsjoen PH, Langsjoen JO, Langsjoen AM, Lucas LA. Treatment of statin adverse effects with supplemental Coenzyme Q10 and statin drug discontinuation. BioFactors Oxf Engl. 2005;25(1- 4):147-152. 4. Kaila K, Haykowsky MJ, Thompson RB, Paterson DI. Heart failure with preserved ejection fraction in the elderly: scope of the problem. Heart Fail Rev. 2012;17(4-5):555-562. doi:10.1007/s10741-011-9273-z.

32 UBIQUINOL SUPPLEMENTATION IN THE ELDERLYPATIENTS UNDERGOING AORTIC VALVE REPLACEMENT: BIOCHEMICAL AND CLINICAL EFFECTS

Nicolini F1, Molardi A1, Gherli T1, Brugè F2, Orlando P2, Silvestri S2, Littarru G.P2 and Tiano L2

1 Cardiac Surgery Unit, University of Parma, Italy 2 Department of Dentistry and Clinical Sciences, Biochemistry Section, Marche Polytechnic University, Ancona, Italy

There is a steady rise in the mean age of patients affected by heart disease undergoing cardiac surgery. On the other hand senescent myocardium has reduced tolerance to ischemic stress and there are clear indications about age-associated deficit in myocardial performance after operatory stress. Coenzyme Q10 has been shown to ameliorate several conditions related to bioenergetic deficit or increased exposure to oxidative stress. The use of ubiquinol-10, the reduced form of CoQ10, is particularly promising in these clinical settings, on the basis of its superior bioavailability and of the alleged impaired CoQ10-reducing capacity in the elderly.

Here we show the results relative to 46 patients where we investigated clinical and biochemical effects of ubiquinol-10, administered in the perioperatory stage to elderly patients undergoing aortic valve replacement. Patients, affected by severe aortic stenosis, were randomized into 2 groups: one received placebo, the active group was given 400 mg/die of ubiquinol-10 (QH absorb, Jarrow Formulas, LA, USA) divided into 2 doses, starting 7 days before surgery and continuing for one month after cardiac intervention. Blood samples were withdrawn at time 0 before ubiquinol administration, one day before surgery, 1, 5 and 30 days after surgery. Primary endpoints were the following:

1. Plasma levels of CoQ10 2. Redox status of plasma CoQ10 throughout the study phases 3. Plasma concentration of IL-6, TNF-alpha, S100 protein

Secondary endpoints were: 1. Major cardiac adverse effects in the postoperatory phase 2. Quality of life 3. Evaluation of contractility and myocardial hypertrophy

Results obtained in treated patients and placebo controls indicate an increase in the percentage of oxidized CoQ10 in plasma following surgical intervention. Treatment with ubiquinol-10 was able to improve basal (before surgery) oxidative status of CoQ10 and mitigate increased oxidation related to the surgical intervention.

33 STATIN INTOLERANCE – DEFINITION, PATHOPHYSIOLOGY, RISK FACTORS AND MANAGEMENT - WHAT IS THE ROLE OF COENZYME Q10?

Daniel Pella

1st Internal Clinic Medical Faculty PJ Šafárik University and Louis Pasteur University Hospital Košice, Slovakia

Statins are one of the most commonly prescribed drugs in clinical practice. They are usually well tolerated and effectively prevent cardiovascular events. Most adverse effects associated with statin therapy are muscle related. Although muscle syndromes are the most common adverse effects observed after statin therapy, excluding other side effects might underestimate the number of patients with statin intolerance, which might be observed in 10–15% of patients. In clinical practice, statin intolerance limits effective treatment of patients at risk of, or with, cardiovascular disease. Knowledge of the most common adverse effects of statin therapy that might cause statin intolerance and the clear definition of this phenomenon is crucial to effectively treat patients with lipid disorders. Statin intolerance is the inability to tolerate a dose of statin required to reduce a person’s cardiovascular risk sufficiently from their baseline risk and could result from different statin related side effects including: muscle symptoms, headache, sleep disorders, dyspepsia, nausea, rash, alopecia, erectile dysfunction, gynecomastia, and/or arthritis. Statin intolerance has been also described as a clinical syndrome with the following characteristics: 1. incapacity to use statin therapy because of appearance of symptoms and/or abnormal values of biomarkers at the initiation or at dose escalation of statin therapy, 2. no presence of individual predispositions, such as drug-drug interactions, untreated hypothyroidism, febrile illness, that may contribute to statin intolerance.

In addition, two different clinical patterns have been identified: the complete intolerance, when a subject is intolerant to any statin at any dose, and the partial intolerance, when a subject is intolerant to some statins at some doses. CoQ10 (ubiquinone) is one of the key substances in myocardial energetic metabolism, and is also important for cell membrane stability, and with CoQ10 deficiency, myocytes could be prone to damage in the form of myopathy or myositis, or even rhabdomyolysis. Although CoQ10 depletion does not appear to play an etiopathogenic role in statin-induced myopathy, it is highly probable that it is a critical predisposing factor, especially in subjects for whom other CoQ10-depleting conditions coexist. Thus, CoQ10 supplementation may decrease the need to withdraw these drugs if adverse effects appear. This may be of great importance, owing to fact that statins represent the basic treatment for secondary prevention of atherosclerosis, and their prescription rate during recent decades has increased dramatically.

34 Notes

Notes

METABOLIC REGULATION BY THE ANTIOXIDANT RESPONSE AXIS: MITOCHONDRIAL AND COENZYME Q ALTERATIONS PRODUCED BY GENETIC DELETION OF Nrf2 OR NAD(P)H:QUINONE OXIDOREDUCTASE 1

J.M. Villalba1, J. Ariza1, L. Fernández del Río1, S.R. Levan2, E. Mercken2, H. Khraiwesh1, J.A. González-Reyes1, L. Jódar1, M.I. Burón1, M. Bernier2, R. de Cabo2

1 Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Universidad de Córdoba, E- 14014, and Campus de Excelencia Internacional Agroalimentario ceiA3, Córdoba, Spain; 2 Translational Gerontology Branch, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA

Nuclear factor E2-related factor-2 (Nrf2) is a cap’n’collar/basic leucine zipper (b-ZIP) transcription factor which acts as cellular sensor of oxidative and electrophilic stress generated from intracellular reactions and substances like chemicals, xenobiotics or drugs. Upon stress conditions, Nrf2 is activated by protein stabilization and then translocated to the nucleus where it activates its target genes. Nrf2 signaling thus abates the consequences of oxidative stress, including several aging- associated diseases like neurodegeneration, chronic inflammation and cancer in rodents and humans. The flavoenzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) is one of the best characterized detoxifying enzymes whose expression is regulated by Nrf2. NQO1 catalyzes the reduction of a variety of quinone substrates using both NADH and NADPH. A link between the Nrf2/NQO1 and energy metabolism balance also exists beyond its well-characterized role in detoxification and antioxidant protection of cells under stress conditions. It has been shown that Nrf2 is required for proper maintenance of mitochondria though a mechanism that relies on NQO1. Moreover, modifications of the Nrf2/NQO1 status have been related with the metabolic alterations induced by calorie restriction, a dietary intervention which extend longevity. Finally, our recent investigations has pointed out that overexpression of extra-mitochondrial oxidoreductases, including NQO1, produces several metabolic benefits in mouse tissues. We wanted to investigate the impact of Nrf2 or NQO1 genetic deletion on metabolism and several mitochondrial-linked processes in mouse embryonic fibroblasts derived from corresponding knockout mice. Since coenzyme Q (Q) links basic aspects of cell physiology such as energy metabolism, antioxidant protection, and the regulation of cell growth, we also wanted to investigate if Nrf2 or NQO1 genetic deletion affects Q homeostasis in these cells. Our results demonstrate that genetic deletion of Nrf2 or NQO1 produce profound alterations in mitochondria, with distinct modifications in metabolism, coenzyme Q system and cell behavior. The Nrf2-NQO1 axis is now emerging as part of the regulatory network that dictates the metabolic health of cells.

Funding This work was supported by grants to JMV from Spanish Junta de Andalucía Proyectos de Excelencia (P09-CVI-4887), Ministerio de Economía y Competitividad (BFU2011-23578) and BIO-276 (Junta de Andalucía and the University of Córdoba) and the Intramural Research Program of the NIA/NIH (to RdC). JA was supported by a FPU contract funded by the Spanish Ministerio de Educación, Cultura y Deporte and a research contract funded by P09-CVI-4887. LFdR was supported by a FPU contract funded by the Spanish Ministerio de Educación, Cultura y Deporte.

35 CYTOCHROME b5 REDUCTASE OVEREXPRESSION EXTENDS LIFESPAN

Michel Bernier

Translational Gerontology Branch, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224, USA [email protected]

Understanding the mechanisms by which metabolism is controlled is critical in order to achieve therapeutic strategies for the treatment of metabolic diseases and aging. Cytochrome b5 reductases (CYB5R) are required for the elongation and desaturation of fatty acids, cholesterol synthesis and mono-oxygenation of cytochrome P450 enzymes, all of which are associated with protection against metabolic disorders. To gain new insights into the physiological role of CYB5R in the context of aging and metabolism, we created a transgenic mouse line overexpressing the rat CYB5R3 gene (CYB5R3-Tg) and generated Drosophila melanogaster overexpressing Drosophila CYB5R (CYB5R-OE). Both experimental models were found to live longer than their wild-type counterparts, and partial protection from N-diethylnitrosamine (DEN)-induced hepatocarcinoma was noted in CYB5R3-Tg mice. Additional experiments showed that the lifespan extension stemmed from positive effects on mitochondrial function and several metabolic parameters, and improved antioxidant defenses with concomitant reduction in oxidative damage. Accumulation of high levels of long-chain polyunsaturated fatty acids and inhibition of chronic pro-inflammatory pathways occurred both in CYB5R3-Tg and CYB5R-OE. Taken together, these findings have implications for further development of interventions targeting CYB5R for the treatment and prevention of age-related diseases and extension of lifespan.

36 NQO1; STRUCTURE, FUNCTION, POLYMORPHISMS AND REDOX CHANGES David Ross and David Siegel

Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Center, Denver, Colorado, USA

NQO1 is an NAD(P)H and FAD-dependent homodimer that has been shown to catalyze the reduction of quinones directly to hydroquinones but has multiple additional functions in cells. One important role is protection against cellular redox stress and NQO1 is induced rapidly under stress conditions often to high levels. NQO1 can generate lipophilic antioxidants including reduced vitamin E derivatives and ubiquinol, is a component of the plasma membrane redox system, can function as a superoxide reductase under stress conditions and can bind to mitotic spindles potentially serving an antioxidant role during cell division. Emerging functions of NQO1 include its ability to bind to and protect a diverse range of proteins, including p53, from 20S proteasomal degradation and its ability to serve as a mRNA binding protein. NQO1 is polymorphic and the homozygous NQO1*2 null polymorphism is present in between 4-34 % of individuals depending on ethnic group. This polymorphism can modulate cellular functions of NQO1 including protein binding, metabolism of anticancer quinones and may influence CoQ10 status in humans. We have examined pyridine nucleotide dependent behavior of NQO1 which has implications for redox stress. Following binding of NAD(P)H, NQO1 underwent a conformational change leading to loss of immunoreactivity against antibodies that target the C-terminus. In cell free experiments using purified human recombinant NQO1 (rhNQO1), antibodies targeting the C-terminus could efficiently pull-down rhNQO1 in the absence of NAD(P)H but not in its presence. Oxidation of FADH2 to FAD in rhNQO1 by the addition of the redox cycling quinone β-lapachone re-established C-terminal immunoreactivity, demonstrating that the loss of immunoreactivity was reversible. These data demonstrate that NQO1 undergoes structural changes in an oxidative environment resulting in modulation of its ability to interact with antibodies and other proteins. This suggests that NQO1 may function as a redox sensor in cellular compartments dependent on pyridine nucleotide ratios and oxidative environment with a resultant change in its structure and functionality.

37 NOVEL METABOLIC ROLES OF NQO1 IN METABOLIC REGULATION

Rafael de Cabo

Translational Gerontology Branch, Biomedical Research Center, Baltimore, USA

Caloric restriction (CR) without malnutrition has consistently extended mean and maximal lifespan in most organisms tested. The plasma membrane redox system (PMRS), by virtue of its ability to lower oxidative stress and adjust the levels of intracellular NAD+, constitutes a key factor in the control of the molecular and physiological mechanisms that govern aging and age-related metabolic disorders. Herein, we evaluated whether the health benefits of CR could be dependent to some extent on the induction of NQO1, a known member of the PMRS involved in detoxification of pro- oxidant molecules associated with the development of cancer and diabetes. Transgenic mice overexpressing rat NQO1 gene (NQO1-Tg) that were maintained on a standard diet (SD) exhibited survival curves and body weight trajectories, body composition, food intake, and energy balance similar to their SD-fed wild-type counterparts, indicating that the pro-longevity benefits of CR are not related to NQO1. However, under metabolic stress conditions, overexpression of NQO1 lowered chronic inflammatory profile and enhanced insulin sensitivity while conferring protection from high-fat diet-induced morbidities, in part, by improving liver integrity. Taken together, these results highlight the contribution of NQO1 in the maintenance of glucose homeostasis through fine- tuning regulation of insulin signaling. The newly described role of NQO1 in energy control places it as a potential therapeutic target for the treatment and prevention of chronic metabolic diseases.

38 EFFECT OF MODIFIED TOCOTRIENOLS IN DIABETES Gustav Dallner and Kerstin Brismar

Department of Biochemistry and Biophysics, Stockholm University and Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden

Tocotrienols belong to the vitamin E family and have an all-trans polyisoprenoid side chain. Epoxidation one of the isoprenes increases coenzyme Q (CoQ) biosynthesis in tissue culture cell but not in vivo. Additional modification of the chromanol ring is required for increasing CoQ synthesis in mice which treatment does not affect cholesterol metabolism. When this compound is given ip to db/db diabetic mice, they lost weight and the MRI measurements demonstrated that the decrease of weight is elicited by a decrease of fat/lean ratio in spite of the unchanged food consumption. Studies with metabolic chambers show a decrease of CO2/O2 ratio indicating the elevated utilization of fatty acids. Triglycerides in the blood and liver are reduced. Fat accumulated in spheroids of primary human hepatocytes are substantially decreased after treatment with the compound. Arginase-1 activity is elevated in db/db mice, peroxynirate formed and heart perfusion with red blood cells from treated animals decreases reperfusion injury. We have synthesized substances similar to tocotrienols with various modifications. The biological effects of the modified substances were studied in tissue culture cells and an upregulation of a number of genes important for wound healing such as PDGFB and VEGF, was observed. An in vitro wound healing assay demonstrated an increased cell migration of fibroblasts. A greatly enhanced blood vessel formation was also observed in an in vitro model system of endothelial cells. For in vivo studies of wound healing in db/db mice we have used an established surgical procedure to remove a circular part of the skin (punch biopsy). The substances were applied on the wound of the mice skin and the progress of wound healing was followed continuously and registered. The healing process exhibited a considerably higher rate, above 30%, indicating the possible use of these compounds to treat a common complication of diabetes. Db/db mice were also investigated for renal function after prolonged treatment. The diabetic mice have a deficient renal function shown by an increased appearance of albumin in the urine. Significant changes were absent after four weeks of treatment but after four months albumin excretion was greatly decreased. ROS production is decreased and the genes important in renal protection, HPR1 and SLC12A3 were upregulated. Adaptation to hypoxia is reflected by the increase of HIF1α. Mitochondria were prepared after both time point and mitochondrial swelling, an indication of opening of the mitochondrial transition pore, was analyzed. After four weeks a small decrease of the swelling was observed and after four months of treatment the decrease of swelling was substantial. Mitochondria from diabetic kidney exhibited loosely coupled respiration when the electron transport chain was investigated, but after prolonged treatment the tightly coupled respiratory chain was reestablished.

39 EFFECTS OF COENZYME Q10 ON RETINAL-EVOKED AND CORTICAL- EVOKED RESPONSES IN PATIENTS WITH OPEN-ANGLE GLAUCOMA

Vincenzo Parisi

Fondazione G.B. Bietti-IRCCS, Rome, Italy

Purpose To evaluate pattern-evoked retinal and cortical responses [pattern electroretinogram (PERG) and visual-evoked potential (VEP), respectively] after treatment with coenzyme Q10 in open-angle glaucoma (OAG) patients.

Methods Forty-three OAG patients (mean age, 52.5±5.29y; intraocular pressure <18mm Hg with b-blocker monoterapy only) were enrolled. At baseline and after 6 and 12 months, simultaneous recordings of PERG and VEPs were obtained from 22 OAG patients who underwent treatment consisting of coenzyme Q10 and vitamin E (Coqun, 2drops/d) in addition to b -blocker monoterapy (GC group), and from 21 OAG patients who were only treated with b-blockers (GP group).

Results At baseline, intraocular pressure, PERG, and VEP parameters were similar in both GC and GP groups (analysis of variance, P >0.05). After 6 and 12 months, PERG and VEP response parameters of GP patients were unchanged when compared to baseline. In GC patients, PERG P50 and VEP P100 implicit times were decreased, whereas PERG P50-N95 and VEP N75-P100 amplitudes were increased (P <0.01) when compared to baseline. In the GC group, the differences in implicit times and amplitudes with respect to baseline were significantly larger (P<0.01) than those recorded in the GP group. The improvement (12 mo minus baseline) of VEP implicit time was significantly correlated with the changes of PERG P50-N95 amplitude (r =0.66171, P =0.0008) and P50 implicit time (r =0.68364, P =0.00045) over a period of 12 months.

Conclusions Coenzyme Q10 administration in OAG shows a beneficial effect on the inner retinal function (PERG improvement) with consequent enhancement of the visual cortical responses (VEP improvement)

40 INCREASED OXIDATIVE STRESS IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS: THE EFFECT OF EDARAVONE ADMINISTRATION AND A POSSIBLE NEED FOR COENZYME Q10 SUPPLEMENTATION

Yorihiro Yamamoto1, Midori Nagase1, Yusuke Miyazaki1, Hiide Yoshino2

1School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura-cho, Hachioji, Tokyo, Japan 2Yoshino Neurology Clinic, 3-3-16 Khonodai, Ichikawa, Chiba, Japan

Compared to age-matched healthy controls (n = 55), patients with amyotrophic lateral sclerosis (ALS) (n = 26) showed increased oxidative stress, as indicated by a significantly increased percentage of oxidized coenzyme Q10 in total plasma coenzyme Q10 (%CoQ10), significantly decreased level of plasma uric acid, and significantly decreased percentage of polyunsaturated fatty acids in total plasma free fatty acids (FFA). Therefore, the efficacy of edaravone, a radical scavenger, in these ALS patients was examined. Among the 26 ALS patients, 17 received edaravone (30 mg/day, 1-4 times a week) for at least 3 months and 13 continued for 6 months. Changes in revised ALS functional rating scale (ALSFRS-R) were significantly smaller in these patients than in edaravone-untreated ALS patients (n = 19). Edaravone administration significantly reduced excursions of more than one standard deviation from the mean for plasma FFA and contents of palmitoleic and oleic acids, plasma markers of tissue oxidative damage, in the satisfactory progress group (ΔALSFS-R ≥ 0) as compared to the ingravescent group (ΔALSFS-R < - 5). Edaravone increased plasma uric acid, suggesting that it is an effective scavenger of peroxynitrite. However, edaravone did not decrease % CoQ10. Therefore, combined treatment with agents such as CoQ10 may further reduce oxidative stress in ALS patients.

41 BENEFITS OF TOPICAL Q10 TREATMENT ON HUMAN SKIN

Anja Knott

Research & Development, Beiersdorf AG, Hamburg, Germany

Coenzyme Q10 (CoQ10) is present in every membrane of all cells in the body with a preferential accumulation in mitochondria, plasma membranes, Golgi vesicles and lysosomes (Gille & Nohl, 2000). It was originally shown to be an essential component of the mitochondrial respiratory chain (Bentinger, 2010; Mitchell, 1975). Since then it has been well established, that CoQ10 has many other important functions for example as a native constituent of the lysosomal electron transport chain, promoting proton translocation across the lysosomal membrane (Gille & Nohl, 2000) or as the only endogenously synthesized lipid soluble antioxidant exceeding both in amount and efficiency that of other antioxidants (Bentinger, 2007). In skin CoQ10 is not only found in living cells, but also in the skin surface lipids (SSL), that are a mixture of sebum and lipids originating from keratinizing epidermal cells, mainly corneocytes. On this outermost level of the body - which is in permanent contact with external stress factors causing reactive oxygen species (ROS) - CoQ10 is apart from vitamin E, the only lipophilic antioxidant serving important functions in the defense against oxidative stress. Both were found to synergically inhibit the UV induced depletion of squalene (Passi, 2002). Thus, CoQ10 is widespread in skin, both within living cells and skin lipids. However, endogenous CoQ10 levels decline with increasing age (Hoppe, 1999) and due to external insults like UV-damage (Podda, 1998). This lack in CoQ10 may have impact on the mitochondrial activity of skin cells, but also on the effectiveness of antioxidant protection against ROS within the tissue and on the skin surface. Along these lines two important points of action arise in order to strengthen skin in its natural functions. First, maintenance of sufficient cellular energy levels to stop the decline of mitochondrial activity and, second, antioxidant protection against ROS originating from any source. Our studies focus on the topical application of CoQ10 in cosmetic formulations and the resulting benefits on human skin. New data will be presented which point to the relevance of topical CoQ10 treatment to support skin’s natural functions at any age.

References 1. M. Bentinger, K. Brismar, G. Dallner, The antioxidant role of coenzyme Q, Mitochondrion 7S (2007) 41-50. 2. M. Bentinger, M. Tekle, G. Dallner, Coenzyme Q - Biosynthesis and functions, Biochem. Biophys. Research Comm. 396 (2010) 74-79. 3. L. Gille, H. Nohl, The Existance of a Lysosomal Redox Chain and the Role of Ubiquinone, Arch. Biochem. Biophys. 375 (2000) 347-354. 4. U. Hoppe, J. Bergemann, W. Diembeck, J. Ennen, S. Gohla, I. Harris, J. Jacob, J. Kielholz, W. Mei, D. Pollet, D. Schachtschabel, G. Sauermann, V. Schreiner, F. Stäb, F. Steckel, Coenzyme Q10, a cutaneous antioxidant and energizer. Biofactors 9 (1999) 371–378. 5. P. Mitchell, Protonmotive redox mechanisms of cytochrome b-c1 complex in the respiratory chain: protonmotive ubiquinone cycle, FEBS Lett. 56 (1975) 1-6. 6. S. Passi, O. de Pità, P. Puddu, G.P. Littarru, Lipophilic Antioxidants in Human Sebum and Aging, Free Radical Research 36 (2002) 471-477. 7. M. Podda, M.G. Traber, C. Weber, L.J. Yan, L. Packer, UV-irradiation depletes antioxidants and causes oxidative damage in a model of human skin. Free Radic. Biol. Med. 24 (1998) 55–65.

42 PROTECTIVE EFFECTS OF COENZYME Q10 AND ASPARTIC ACID ON OXIDATIVE STRESS AND DNA DAMAGE IN SUBJECTS AFFECTED BY IDIOPATHIC ASTHENOZOOSPERMIA.

Giacomo Tirabassi1, Arianna Vignini2, Luca Tiano2, Eddi Buldreghini1, Francesca Brugè2, Sonia Silvestri2, Patrick Orlando2, Antimo D’Aniello3, Laura Mazzanti2, Andrea Lenzi4 and Giancarlo Balercia1

1Division of Endocrinology, Department of Clinical and Molecular Sciences, School of Medicine, Umberto I Hospital, Polytechnic University of Marche, Via Tronto 10/A, 60126 Ancona, Italy, 2Department of Odontostomatologic and Specialized Clinical Sciences, Polytechnic University of Marche, Ancona, Italy, 3Laboratory of Biochemistry, ADIPHARM, Sant’Antimo, Naples, Italy, 4Andrology, Pathophysiology of Reproduction and Endocrine Diagnosis Unit, Policlinic Umberto I, University of Rome ‘‘La Sapienza’’, Rome, Italy

Objective To assess the effects of Coenzyme Q10 (CoQ10) and Aspartic acid (D-Asp) on some previously untested parameters of sperm oxidative stress and DNA damage. Design Observational longitudinal study. Setting Division of Endocrinology, Ancona, Italy. Patients Twenty patients affected by idiopathic asthenozoospermia. Intervention Administration of oral dietary supplement including CoQ10 and D-Asp. Main Outcomes CoQ10 and D-Asp levels, superoxide dismutases (SOD) activity, nitric oxide (NO) and peroxynitrite levels and DNA damage (comet assay) in sperm.

Results NO and peroxynitrite levels decreased, whereas SOD activity increased significantly after therapy. Furthermore, tail intensity, a marker of DNA damage, diminished significantly after treatment. Correlation analysis revealed a negative relationship between the increase of CoQ10 and the decrease of NO and tail intensity and a positive one between the increase of CoQ10 and the rise of SOD activity; no significant correlation was found between the increment of D-Asp and the changes of markers of oxidative stress and DNA damage. Increase of SOD activity and decrease of NO levels were negatively and positively correlated with the diminishment of tail intensity, respectively.

Conclusions Only CoQ10, and not D-Asp, seems to play a protective role against oxidative stress and DNA damage in sperm.

43 THERAPEUTIC EFFECTS OF UBIQUINOL ON FATIGUE AND CHRONIC FATIGUE SYNDROME

Yasuyoshi Watanabe1,2, Sanae Fukuda1-5, Kouzi Yamaguti2,5, Osami Kajimoto3, Kenji Fujii6, and Hirohiko Kuratsune4,5

1RIKEN Center for Life Science Technologies, Hyogo, 2Department of Physiology, Osaka City University Graduate School of Medicine, Osaka, 3Department of Medical Science of Fatigue, Osaka City University Graduate School of Medicine, Osaka, 4University of Kansai Welfare Sciences, Osaka, 5Fatigue Clinical Center, Osaka City University Hospital, Osaka, and 6Kaneka Corporation, Osaka, Japan.

We have been investigating the molecular and neural mechanisms of fatigue and chronic fatigue and also the pathophysiology and therapies of Chronic Fatigue Syndrome. In our previous study, we showed that oral administration of ubiquinone improved subjective fatigue sensation and physical performance during fatigue-inducing workload trials (1). Then, we have done the open trial of ubiquinol supplementation in the patients with Chronic Fatigue Syndrome (CFS). In an open trial with 20 CFS patients (5 males and 15 females), 8-week supplementation of ubiquinol (150 mg/day) showed the increase in plasma level of total ubiquinone in all patients, and resulted in positive effects on the depression score by CES-D, sleep time, and correct score of arithmetic task. Then, we proceeded to a randomized, double-blind, placebo-controlled trial with 31 patients (finally 17 ubiquinol group and 14 placebo group completed the study). In this second trial, 12-week supplementation of 150 mg/day ubiquinol has been performed in the ubiquinol group and the same type of capsules of placebo was taken by placebo group patients. In the ubiquinol group, the plasma level of total ubiquinone after 12-week supplementation increased around four times than the level before intake and also the level of placebo group before and after intake. The results from the second trial confirmed the supplementation effects in the open trial; namely, ubiquinol supplementation is effective in the parasympathetic nerve function, less awakening time at night sleep, and the correction rate of arithmetic task. Subjective symptoms of fatigue were improved well correlated with increment of plasma level of total ubiquinone in the patients.

References 1. Mizuno, K., et al.: Antifatigue effects of coenzyme Q10 during physical fatigue. Nutrition, 24: 293-299, 2008.

44 Notes

Notes

SURVIVAL TRANSCRIPTOME IN THE COENZYME Q DEFICIENCY SYNDROME IS ACQUIRED BY EPIGENETIC MODIFICATIONS.

Daniel José Moreno Fernández-Ayala and Plácido Navas Lloret

CABD-CSIC/UPO and CIBERER-ISCIII, Universidad Pablo Olavide, Seville, SPAIN

Objectives Coenzyme Q10 (CoQ10) deficiency syndrome is a rare condition that causes mitochondrial dysfunction and includes a variety of clinical presentations such as encephalomyopathy, ataxia and renal failure. We sought to set up what all have in common, and investigate why CoQ10 supplementation reverses the bioenergetics alterations in cultured cells but not all the cellular phenotypes. To do that, (1) we analyzed the common gene expression profile in primary cell cultures of dermal fibroblasts from patients suffering any of the clinical presentation, (2) we determined why CoQ10 treatment restored respiration but not all the clinical phenotypes, and (3) we investigated the stable genetic cause responsible for the survival adaptation to mitochondrial dysfunction owing to CoQ10 deficiency.

Methods  The expression profile of fibroblasts from CoQ10-deficient patients (3 had primary deficiency and 4 had a secondary form) and aged-matched controls, before and after supplementation with CoQ10 was characterized by microarray analysis. Results were validated by Q-RT-PCR.  The profile of DNA (CpG) methylation was evaluated for a subset of gene with displayed altered expression in the same samples described above.  The profile of histone methylation was analyzed by ChIP-Seq (deep-sequencing of immune- precipitated chromatin with any of H3K4me3 and H3K27me3 antibodies) in control fibroblast and in fibroblast from one of the best-studied patient. Parallel analyses were done independently for the histone-repressive mark (H3K27me3) and the histone-activating mark (H3K4me3) to select the putative regulated genes for any of the marks.

Results  CoQ10-deficient fibroblasts (independently from the etiology) showed a common transcriptomic profile that promotes cell survival by activating cell cycle and growth, cell stress responses and inhibiting cell death and immune responses. Most of the biological processes and pathways related with apoptosis, differentiation and immune response were repressed. Energy production was supported mainly by glycolysis.  The regulated genes shift the histone marks supporting this gene regulation: repressed genes lose the histone-activating mark H3K4me3 and a slight gain of the repressive H3K27me3 mark in patients, whereas activated genes increased the activating H3K4me3 mark and lose the repressive H3K27me3 one.  CoQ10 supplementation restored oxidative phosphorylation and partially recovered the expression of genes involved in cell death pathways. As described before, the regulated genes shift the histone marks supporting this partial recovery of the gene expression.  Genes involved in differentiation, cell cycle and growth were not affected after CoQ10 supplementation.

45  Unaffected genes by CoQ10 treatment corresponded to highly activated genes by DNA- demethylation in patients, whereas those genes with responsive expression to CoQ10 supplementation presented an unchanged DNA-methylation pattern. These results approach to explain the incomplete recovery of clinical symptoms after coenzyme Q treatment, at least in some patients.

Conclusion  Our results demonstrated that, among the genetic heterogeneity ongoing CoQ10 deficiency syndrome, all primary fibroblast showed a common transcriptomic profile that justified their pathological phenotype, responded equally to CoQ10 treatment and presented similar DNA methylation pattern.  The survival transcriptome in the CoQ10 deficiency syndrome is acquired by epigenetic modifications of chromatin, including DNA-demethylation and histone modifications in specific residues.

46 PHYSIOLOGICAL FUNCTIONS OF UBIQUINONE AND UBIQUINOL DEPENDENT GENE EXPRESSION SIGNATURES

Frank Döring

Institute of Human Nutrition and Food Science, University of Kiel

Coenzyme Q (CoQ) and its derivatives are evolutionary highly conserved lipophilic molecules that have a number of essential functions in almost all organisms, including in the respiratory chain, as a cofactor of pyrimidine biosynthesis and as an antioxidant in extra mitochondrial membranes. More recently, CoQ has been identified as a modulator of apoptosis and inflammatory processes. Based on studies in-silico, in-vitro and in-vivo we provide evidence that CoQ10 is involved in gene expression. A deficiency in CoQ in humans is a clinically and genetically heterogeneous disorder, with manifestations that may range from neonatal multisystem failure to adult-onset encephalopathy. The generation of animal models deficient in CoQ is important to further clarify the cellular function of CoQ and to unravel the complexity in the pathophysiological consequences of CoQ deficiency. Using C. elegans as model system, we established an experimental set-up to identify gene expression signatures induced by endogenous CoQ deficiency or exogenous ubiquinol supply. We found that a deficiency of endogenous CoQ-synthesis down-regulates a cluster of genes that are important for growth (i.e., RNA polymerase II, eukaryotic initiation factor) and up- regulates oxidation reactions (i.e., cytochrome P450, superoxide dismutase) and protein interactions (i.e., F-Box proteins). Exogenous supply of ubiquinol partially restores the expression of these genes as well as the growth retardation of CoQ-deficient mutants. Moreover, we found that CoQ- deficiency induces a gene expression signature that mimics the cellular surveillance-activated detoxification and defences response. Thus, we propose that ubiquinone and ubiquinol promotes growth and defence mechanisms via gene expression, which will have implications to understand the role of CoQ deficiency in humans.

This study is supported by Kaneka Corporation, Japan

47 EFFECTS OF THE MEDITERRANEAN DIET SUPPLEMENTED WITH COENZYME Q10 ON OXIDATIVE STRESS AND INFLAMMATORY RESPONSE IN ELDERLY MEN AND WOMEN

José López-Miranda

Lipids and Atherosclerosis Unit, IMIBIC/Reina Sofia University Hospital, University of Cordoba and CIBER Fisiopatología Obesidad y Nutrición, Instituto de Salud Carlos III, IMIBIC/Fundación para la Investigación Biomédica de Córdoba, Córdoba, Spain.

Background. Aging is a biological process characterized by time-dependent, progressive physiological declines accompanied by the increased incidence of age-related diseases. A growing body of evidence points toward the oxidative damage caused by reactive oxygen species (ROS) as one of the primary determinant of aging. However, recent scientific studies have advanced the notion of chronic inflammation, as other risk factors, underlying aging and age-related diseases as neurodegenerative disorders, type 2 diabetes, atherosclerosis, and cardiovascular diseases. Addressing the central mechanisms underlying these pathologies will have implications for aging and should lead to new therapeutic approaches for treating these conditions. We have investigated whether the quality of dietary fat modifies plasma biomarkers related to oxidative stress, expression of genes and protein involved in oxidative stress, inflammatory response and endoplasmic reticulum stress and p53-dependent DNA repair. In addition, we determined whether supplementation with coenzyme Q10 (CoQ), described as a powerful antioxidant that reduces oxidative stress, improves this situation in an elderly population. Methods. In this randomized cross-over study, 20 participants were assigned to receive three isocaloric diets for periods of 4 week each. 1. Mediterranean diet supplemented with CoQ (Med+CoQ diet). 2. Mediterranean diet (Med diet). 3. Saturated fatty acid-rich diet (SFA diet). After a 12-hour fast, volunteers consumed a breakfast with a fat composition similar to that consumed in each of the diets. We determined CoQ, lipid peroxides (LPO), oxidized LDL (oxLDL), protein carbonyl (PC), total nitrite, nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) plasma levels, catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, ischemic reactive hyperaemia (IRH), gene expression related to oxidative stress (Nrf2, p22phox and p47phox, SOD1 and 2, GPx1, thiorredoxin reductase (TrxR)), gene expression related to inflammatory response and endoplasmic reticulum stress (calreticulin, IL- , JNK-1, p65, IkB- - -9, IL-1b, JNK-1, sXBP-1, and BiP/Grp78), gene expression and protein levels related to both DNA damage and p53- dependent DNA repair (p53, p21, p53R2, mdm2, Gadd45a, Gadd45b, OGG1, APE-1/Ref-1, DNApolβ, and XPC). Results. Med diet produced a lower postprandial GPx activity and a lower decrease in total nitrite level compared to the SFA diet. Med and Med+CoQ diets induced lower fasting SOD2, calreticulin, IL-1b, and JNK-1 gene expression, lower postprandial Nrf2, p22phox, p47phox, SOD1, TrxR, p65, IKK-b, MMP-9, IL-1b, JNK-1, sXBP-1, and BiP/Grp78, p53, mdm2, Gadd45a, Gadd45b, XPC, DNApolβ and OGG1 gene expression, and higher postprandial IkB-a gene expression compared to the SFA diet. Also, Med and Med+CoQ diets induced a higher postprandial increase in IRH and a lower postprandial LPO, oxLDL and nitrotyrosine and 8-OHdG plasma levels than the SFA diet.

48 Med+CoQ diet produced a lower postprandial decrease in total nitrite and a greater decrease in PC levels and lower postprandial Nrf2 gene expression compared to the other two diets and lower SOD, CAT and GPx activities and lower GPx1 gene expression than the SFA diet. Conclusions. Our results support the antioxidant and anti-inflammatory effect of a Med diet and that exogenous CoQ supplementation has an additional effect against free radical overgeneration and oxidative DNA damage through the lowering of oxidative stress, increasing the action of antioxidant system and modulating the inflammatory response in elderly subjects. These results suggest a starting point for the prevention of oxidative processes associated with aging.

49 UBIQUINOL VERSUS UBIQUINONE

Gian Paolo Littarru

While the use of oxidized CoQ10 as a nutraceutical and in clinical practice has now a history of several decades, only a few years ago ubiquinol-10, the reduced form of CoQ10, was introduced for oral use. The rationale on which ubiquinol was proposed lies on two facts: -superior bioavailability compared to ubiquinone -possible decrease of ubiquinone-reducing capacity with age.

It is well known that when ubiquinone is orally administered its reduction must occur, in order to be absorbed. There is experimental evidence that bioavailability of ubiquinol-10, when given orally, is superior to the corresponding bioavailability of oxidized CoQ10. This can be verified with healthy people; moreover, several cases have been reported where some cardiac patients treated with CoQ10 experienced a worsening of their clinical condition, which was reversed when they replaced ubiquinone with ubiquinol. This improvement was accompanied by an increase of their blood CoQ10 levels.

In some animal models an age related CoQ10-reducing capacity was reported. DT-diaphorase (NQO1), is a well-known enzyme, important for detoxification of several compounds with benzene rings. It is also one of the main extra-mitochondrial enzymatic systems capable of + reducing CoQ10 to CoQ10H2. The reducing equivalents for this reaction come from NADPH+H . NQO1 specific activity significantly decreases with age in mouse and in rat liver. Activity of thioredoxin reductase also shows a marked decrease in kidneys of aged rats. On the other hand, levels of NADPH, an essential electron donor for NQO1 enzyme, were found to significantly decline with age. The SAMP1 strain is a mouse model for accelerated senescence and severe senile amyloidosis. CoQ10H2 was shown to have antiaging activity in this mouse model. In fact lifelong administration of reduced CoQ10 slowed down the increase of grading score with age. New conditions could be identified where the use of ubiquinol would be preferred.

Prof Mortensen was the first person to show improved mortality outcome from the administration of Q10 in heart failure. He will be remembered for his lifelong work towards this great accomplishment.

50 EFFECT OF UBIQUINOL ON MITOCHONDRIAL POTENTIAL AND OXIDATIVE STRESS IN RELATION TO INTENSIVE PHYSICAL EXERCISE L.Tiano, P. Orlando, S. Silvestri, F. Brugè, T. Bacchetti, GP Littarru

DISCO- Department of Clinical and Dental Sciences Polytechnic University of Marche, Via Ranieri Ancona

Regular exercise is able to modulate antioxidant defence system and improve energetic metabolism in mitochondria. On the other hand, exhaustive exercise might be associated with increased ROS production causing oxidative damage. Mitochondria, in this respect, represent a sensible target of oxidative damage and their impairment might lead both to decreased energy production and enhanced ROS formation. However, free radicals not only cause damage but also have a physiological role in cell signalling underlying adaptation to stress and training. A tight control of redox status, taking into account training experience, is therefore critical in preserving pivotal cellular functions. The aim of the present study was to investigate whether supplementation of mitochondrial nutrient Ubiquinol affected cellular antioxidant activity and mitochondrial functionality in trained individuals before and after a single bout of intense physical exercise. Investigation has been conducted on 21 athletes from a local Rugby team enrolled in a double blind crossover study. Athletes were randomized to take either 200 mg ubquinol per day for 3 weeks or placebo and then treatment was switched in a crossover manner, following a washout period of 2 weeks. Samples were obtained before and after a single bout of intensive exercise produced by 40 minutes run at 75% of the maximal heart rate on a treadmill. During the exercise indexes of physical performance such as speed and heart rate have been recorded and blood samples were collected at rest and immediately after the run. Moreover whole blood was maintained at 37°C for up to 210 minutes in order to evaluate biochemical indexes in an ex-vivo recovery phase. Biochemical analyses were conducted on plasma and included lipid profile, antioxidant levels and markers of muscular stress such as creatin kinase and mioglobin. At cellular level investigations were conducted on peripheral blood leukocyte that represent a easily accessible, but also relevant cell type to measure intracellular ROS levels and mitochondrial functionality using fluorescent probes and flowcytometric analysis. Moreover oxidative DNA damage was measured in the same cells using the comet assay. Finally, using quantitative real time PCR modulation of expression of genes involved in antioxidant defences, oxidant signaling and mitochondrial biogenesis was also evaluated. Data have shown a significant decrease of plasma CoQ10, even after plasma cholesterol normalization, after intense physical exercise associated with an enhancement of muscular stress markers. This might indicate an enhanced requirement of CoQ10 in cellular compartments. Ubiquinol supplementation was able to prevent such decrease although it was not able to modify plasma CK and Mb release. At cellular level a distinctive improvement in cellular oxidative status and mitochondrial functionality was observed.

51 ON THE REACTIONS OF ENDOGENOUS COENZYME Q10 WITH NITRIC OXIDE AND ITS METABOLITE NITROGEN DIOXIDE

Lucedio Grecia and Gian Paolo Littarrub

a, Università Politecnica delle Marche, Dipartimento DiSVA, via brecce Bianche, 60131 Ancona, Italy. b Università Politecnica delle Marche, Dipartimento DISCO, via Ranieri, 60131 Ancona, Italy

CoQ10 is present everywhere in the human body and in particular in mitochondria where it plays its well-known role in the mitochondrial respiratory chain. Nitric oxide (NO) is also an endogenous product, responsible for vasodilation. Its half-life is very short, because it reacts rapidly with oxygen generating nitrogen dioxide (NO2) and nitrous -. anhydride (N2O3), and even more rapidly with superoxide anion (O2 ) forming peroxynitrite. Very few researchers have addressed the reactions of oxidised CoQ10 and reduced CoQ10H2 with NO and NO2. The present communication describes the reactivity of these two natural species with CoQ10 and CoQ10H2. The studied reactions were carried out with pure CoQ10 or with CoQ10 contained in the blood and in mitochondria. Attention was focused not only on the most studied quinoid moiety, but also on the isoprenic chain. Results evidenced that nitric oxide in the presence of oxygen reacts with the isoprenic chain generating products characterised by the addition of nitrogen dioxide to the double bonds and also a nitroxide, detectable by EPR directly in biological systems. Studies are in progress to measure the rate constants of the interactions of these endogenous compounds.

52 POTENTIAL IMPACT OF IN VIVO UBIQUINOL SUPPLEMENTATION IN THE PREVENTION OF ATHEROTHROMBOSIS IN ANTIPHOSPHOLIPID SYNDROME PATIENTS. PRELIMINARY RESULTS OF A CLINICAL TRIAL

Ch. López-Pedrera1, C. Pérez-Sánchez1, M.A. Aguirre1, F. Velasco1, P. Ruiz-Limón1, N. Barbarroja1, Y. Jiménez-Gómez1, P. Segui1, E. Collantes-Estévez1, L. Fernández del Río2, J.A. González-Reyes2, M.J. Cuadrado3 and J.M. Villalba2

1IMIBIC/ Reina Sofía Hospital, Córdoba, Spain; 2 Department of Cell Biology, Physiology and Immunology, University of Córdoba, and Agrifood Campus of International Excellence, ceiA3, Spain; 3Lupus Research Unit, St Thomas Hospital, London, United Kingdom.

Background: We have recently investigated the role of oxidative stress and mitochondrial dysfunction in the pro-atherothrombotic status of APS patients induced by antiphospholipid antibodies (IgG-aPL) and the beneficial effects of supplementing cells with Coenzyme Q10 (Q, Pérez-Sanchez et al., Blood 2012). In that study, a significant increase in relevant prothrombotic and inflammatory parameters in 43 APS patients was found compared to 38 healthy donors. Increased peroxide production, nuclear abundance of Nrf2, antioxidant enzymatic activity, decreased intracellular glutathione and altered mitochondrial membrane potential (ΔΨm) were found in monocytes and neutrophils from APS patients. Accelerated atherosclerosis was found further associated to their inflammatory/oxidative status. Q preincubation of healthy monocytes before IgG-aPL treatment decreased oxidative stress, the percentage of cells with altered ΔΨm, and the induced expression of tissue factor (TF), vascular endothelial growth factor (VEGF) and its receptor Flt1. Also, Q significantly improved the ultrastructural preservation of mitochondria and prevented IgG-APS-induced fission mediated by Drp-1 and Fis-1 proteins. Thus, this study demonstrated the presence of an oxidative perturbation in APS patient’s leukocytes, which was directly related to an inflammatory and pro-atherothrombotic status, and relied on alterations on mitochondrial dynamics and metabolism, which was prevented and/or reverted by treatment with Q. Those results prompted us to evaluate the in vivo beneficial effects of Q supplementation in the prevention of athero-thrombosis in APS patients, by developing a clinical trial.

Objective: To investigate the beneficial effects of in vivo ubiquinol (QH2, reduced form of Q) supplementation on athero-thrombosis prevention in APS patients, through the implementation of a prospective, randomized, double-masked, placebo-controlled study.

Methods: To date, the study has been conducted on 20 APS patients randomized to receive either QH2 (200 mg/day) or placebo for one month. Blood was drawn at time 0 and at the end of the treatment. Studies were performed in plasma and purified leukocytes subsets. Total plasma Q levels, various prothrombotic/proinflammatory parameters, and oxidative stress biomarkers were evaluated. Ultrastructural evaluation of mitochondria in isolated monocytes was performed by electron microscopy. Endothelial activity analysis was performed by Laser-Doppler measurement of post ischemic reactive hyperemia. Carotid intimae media thickness (CIMT) was measured as an early atherosclerosis marker.

Results: Seventeen out of 20 patients completed the intervention, which increased significantly total plasma Q levels. Endothelial function improved notably, as shown by the amelioration in the

53 highest perfusion value after occlusion was released, expressed as a percentage of change vs. rest flow value (RF-PF). CD14highCD16- classical monocyte count was not changed, but CD14highCD16+ intermediate monocytes and CD14dimCD16+ non-classical monocytes were decreased. QH2 treatment decreased Tissue Factor (TF) expression levels in total monocytes, and more notably in intermediate monocytes, which also displayed a more robust reduction of intracellular IKK levels. Only this monocyte subset exhibited IL-8 reduction after intervention. QH2 supplementation produced a reduction in both the levels of peroxides and the percentage of monocytes with altered mitochondrial membrane potential (ΔΨm). A number of proinflammatory mediators were found further reduced in CD16+ monocytes and neutrophils by effect of in vivo QH2 treatment, including VEGF, CCL3, IL-1ß, IL6, and TNFα.

Correlation studies showed that reduced monocyte TF expression after QH2 treatment were related to decreased peroxides levels, increased total plasma Q levels and improved endothelial function. Improved endothelial function further correlated with IL8 levels. The decrease of non-classical monocytes count was related to reduction in the percentage of cells with altered ΔΨm as well as with the increase in RF-PF value. QH2 effects were particularly relevant in APS patients showing pathologic CIMT and thrombotic recurrences (both of them directly associated to the inflammatory and oxidative status of the patients). In our cohort, 8 out of 11 patients showing atheromatous plaques had also suffered at least one thrombotic recurrence. These pathologic processes were further linked to a poorer endothelial function compared to the remaining APS patients. Of note, particularly these patients were the ones showing a better response to QH2 treatment, with a significant increase in endothelial parameters such as hyperemia area and perfusion value after ischemic occlusion.

Conclusion: QH2 supplementation at 200 mg/d significantly improved endothelial function, and reduced mitochondrial dysfunction, oxidative stress, and the expression of prothrombotic/ proinflammatory proteins. Our results support the potential impact of QH2 in the prevention of atherothrombosis in APS patients.

Supported by: CTS-7940, PI12/01511, Spanish Rheumatology Society, KANEKA.

54 WHY IS THE BIOAVAILABILITY OF UBIQUINOL GREATER THAN THAT OF UBIQUINONE?

Mark L. Failla and Chureeporn Chitchumroonchokchai

Human Nutrition Program, Department of Human Sciences, The Ohio State University, Columbus, OH 43210, USA

The bioavailability of orally administered ubiquinol (CoQH) in healthy adults has been reported to be markedly greater than that of ubiquinone (CoQ10) (Miles et al. J. Functional Foods I, 65, 2009). The basis for the influence of redox state on the absorption of CoQ was investigated using the coupled in vitro digestion/Caco-2 human intestinal cell model. CoQH and CoQ10 were solubilized in hydrogenated castor oil before addition to fat-free yogurt. Partitioning of CoQH in the aqueous (i.e., bioaccessible) fraction of chyme after simulated gastric and small intestinal digestion significantly exceeded that of CoQ10. When the bioaccessible fraction generated during digestion was added to cultures of differentiated Caco-2 cells, uptake and trans-epithelial transport of CoQH were 2.3 and 3.4 times, respectively, greater than that of CoQ10. Glutathione reductase, NQO1 and thioredoxin reductase have been proposed to have a central role in the maintaining a high intracellular ratio of CoQH to CoQ10. The impact of these enzymes on the uptake and trans- epithelial transport was examined. Inhibition of GSH reductase and decreased cellular GSH status resulted in a greater decline in the uptake and transport of CoQH than CoQ10, suggesting that a reduced intracellular state is essential for efficient absorption of CoQ across the small intestinal epithelium. Auranofin-mediated inhibition of thioredoxin reductase activity also decreased apical uptake and transport of CoQH, but not CoQ10. However, this differential effect was associated with auranofin-associated oxidation and degradation of micellar CoQH in apical medium. NQO1 activity was not detected in Caco-2 cells. These data suggest that the increased bioavailability of CoQH compared to CoQ10 results from the more efficient micellarization during small intestinal digestion and enhanced uptake and trans-epithelial transport of the former. Moreover, apical uptake and basolateral secretion are dependent on a high intracellular ratio of GSH to GSSG in order to maintain supplemental CoQ in the reduced state.

Bioaccessibility and basolateral secretion of CoQH > CoQ10

Supplements Digestion Micelles Enterocytes Lymph

CoQH CoQH CoQH CoQH CoQH CoQH CoQH

CoQ10 CoQ10 CoQ10 CoQ10

55 Notes

Notes

ABSTRACTS of POSTER PRESENTATIONS

P1. INTRAVENOUS ADMINISTRATION OF SOLUBILIZED COENZYME Q10 PROVIDES NEUROPROTECTION IN RAT MODEL OF PERMANENT CEREBRAL ISCHEMIA

Belousova M.A., Medvedev O.S., Gorodetskaya E.A., Kalenikova E.I.

Lomonosov Moscow State University, Moscow, Russia.

Objective: In our previous study in rats it was shown that intravenous injection of solubilized coenzyme Q10 (CoQ10) solution is followed by its passage through the blood-brain barrier and increase in CoQ10 brain level [1]. This finding opened the opportunity to investigate effects of CoQ10 in acute cerebral ischemia. The purpose of the study was to estimate the neuroprotective efficacy of single intravenous injection of solubilized CoQ10 in rat model of middle cerebral artery occlusion. Methods: The experiments were performed on 49 male Wistar rats weighed 310 to 390g. Cerebral focal ischemia was induced by permanent left middle cerebral artery occlusion (pMCAO). Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and subjected to pMCAO. The middle cerebral artery was blocked by threading silicone-coated 4-0 nylon monofilament through the external carotid to the internal carotid artery, blocking the origin of middle cerebral artery. All the animals were randomly divided into 4 groups: 1- intact rats (n=3); 2 – sham-operated (n=8); 3 – pMCAO+Vehicle (n=12); 4 – pMCAO+CoQ10 (n=11). 60 minutes after the onset of occlusion animals were treated with intravenous injection of CoQ10 (30 mg/kg) or Vehicle (2 ml/kg). 24 hours after the onset of occlusion the neurological deficit was evaluated using the mNSS grading system with a scale from 0 to 18, the lower the score, the greater the neurological deficit. After evaluating the neurological deficit rats were euthanized, brains were collected. Infarction volume was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The infarct percentage was calculated as: 100 x infarct volume/ipsilateral hemisphere volume. CoQ10 tissue level was evaluated by HPLC with electrochemical detection separately in ipsilateral (i/l) and contrlateral (c/l) hemispheres. Data are shown as mean ± SD. Statistical analysis was performed by nonparametric Mann-Whitney U-test. A difference with p < 0.05 was considered statistically significant. Results: There was no neurological deficit and no brain damage in the sham-operated group. All rats from pMCAO groups exhibited the severe reduction in neurologic score in comparison with sham operated. The neurological score in pMCAO+CoQ10 group was significantly higher compared with pMCAO+Vehicle group (10 vs 7 score respectively, p<0.01). The infarct volume in pMCAO+CoQ10 group was 28.4±9.8% and was significantly reduced by 49% compared to pMCAO+Vehicle group (57.2±9.6%), p<0.05. CoQ10 brain level in intact and sham-operated rats was 22,1±1,0 mkg/g and 22,5±1,8 mkg/g resp., and was not significantly different, p>0.05. In pMCAO+Vehicle group level of CoQ10 significantly decrease in both hemisphere, mostly in ipsilateral (i/l – 16,9±2,1 mkg/g; c/l- 18,4±2,3 mkg/g), p<0.05. Single intravenous injection of CoQ10 in pMCAO+CoQ10 group leads to the restoration of CoQ10 brain level in ipsilateral hemisphere (23,9±2,6 mkg/g) and to its accumulation in c/l- 24,4±1,2 mkg/g, p<0.05 compared with intact rats. Conclusion: single intravenous injection of solubilized coenzyme Q10 is followed by its accumulation in ischemic brain and the neuroprotective effect by improving neurological outcomes and reduction in stroke infarct volume.

References 1. Gorodetskaya E., Kalenikova E., Medvedev O., 2010 Intravenous bolus of solubilized coenzyme Q10 increased rapidly its myocardial and brain level. Journal of Hypertension 28, E196. Supported by grant from the Russian Scientific Foundation (project № 14-15-00126 56 P2. AGE-DEPENDENT CHANGES IN COENZYME Q-SYNTHESIS COMPLEX TRANSCRIPTOME

Carmen Campos-Silva, Iván Reyes-Torres, Maximiliano Rivera, Catherine Meza-Torres, Elisabet Rodriguez-Bies, Plácido Navas, Guillermo López-Lluch

Centro Andaluz de Biología del Desarrollo (CABD-CSIC), Departamento de Fisiología, Anatomía y Biología Celular, Universidad Pablo de Olavide, CIBERER, Instituto de Salud Carlos III, Seville, Spain.

Regulation of gene expression is an important process in the maintenance of homeostasis during aging. In many studies, mRNA and protein levels are compared between young and old animals; however, our results demonstrate that differences can be higher when mature animals are considered. In our study, mRNA levels of all the known components of the coenzyme Q-synthesis machinery were analysed in different organs such as brain, kidney and liver by qPCR from animals aged 8 (young), 18 (mature) and 24 (old) months. Interestingly, liver showed higher changes in mRNA levels of the different components. In most of the cases, mRNA levels of the different components were higher in mature animals in comparison with young and old animals. When mRNAs of young and old animals were compared, only minor reduction of mRNA levels was found. In brain, changes were minor although most of the changes showed a similar pattern than in liver. In order to determine the effect of two hormetic factors such as resveratrol and physical exercise in old animals, we compared mRNA levels in 24 month old control animals, animals fed with resveratrol during 6 months or animals trained during 1.5 months. Interestingly, only COQ10 and mDLP1 responded to both, physical exercise and resveratrol in brain whereas the other components were not affected significantly. Interestingly, in liver, the effects of exercise and resveratrol were more evident and affected more components of the synthesis machinery such as COQ2, COQ6 and COQ9 whereas COQ10 was affected similarly to brain but mDLP1 was not affected. Interestingly, COQ7 mRNA was reduced by exercise and resveratrol. Our results indicate that age is an important factor affecting COQ gene expression but its effect depends on the organ and that mature animals show higher levels of mRNA than young and old animals. The effect of hormetic factors such as calorie restriction (previously published Parrado- Fernández et al., 2011), physical exercise or resveratrol is organ-dependent and probably age- dependent since these factors have shown higher effect in old than in young animals (Tung et al., 2014). Probably, Q is an important factor in age progression and a deeper study of the response of COQ synthesis machinery to age and the regulatory factors involved is needed.

Acknowledgements. The research group is financed by the Andalusian Government as the BIO177 group financed by FEDER funds (European Commission). Research has been also financed by the Spanish Government grants DEP2012-39985.

References. 1. Parrado-Fernández, C., López-Lluch, G., Rodríguez-Bies, E., Santa-Cruz, S., Navas, P., Ramsey, J.J. and Villalba, J.M. Calorie rstriction modifies ubiquinone and COQ transcript levels in mouse tissues. Free Rad Biol Med. 50: 1728- 1736 (2011). 2. Tung, B.T., Rodríguez-Bies, E., Ballesteros-Simarro, M., Motilva, V., Navas, P., López-Lluch, G. Modulation of endogenous antioxidant activity by resveratrol and exercise in mouse liver is age dependent. J Gerontol Biol Sci Med Sci. 69:398-409.

57 P3. RNA-BINDING PROTEINS REGULATE CELL RESPIRATION AND COENZYME Q BIOSYNTHESIS BY POST-TRANSCRIPTIONAL REGULATION OF COQ7

María V. Cascajo1,3, Emilio Siendones1,3, José M. Cuezva2,3, Daniel M. Fernández-Ayala1,3, Plácido Navas1,3 and Myriam Gorospe4

1Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-CSIC-JA, E-41013 Sevilla, Spain 2 Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain 3Centre for Biomedical Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain 4Laboratory of Genetics, National Institute on Aging-Intramural Research Program, NIH, Baltimore, Maryland, USA

Coenzyme Q (CoQ) is synthesized by a multi-peptide complex (COQ1-COQ10) and COQ7 is a central regulatory element of the biosynthetic pathway. CoQ concentration is highly regulated in cells because either a deficit or an excess of about 30% has negative consequences to cell metabolism. It has been indicated that tissue concentration of CoQ is modified in aging and pathophysiological conditions. However, the knowledge of the biosynthesis and regulation of this lipid in human is quite limited. We have previously demonstrated that COQ7 is transcriptional and post-translationally regulated. Indeed NF-kB regulates COQ7 gene as a response to oxidative stress, and it is also regulated under a nutrient-dependent phosphorylation-dephosphorylation cycle. In this work, we have focus in the post-transcriptional regulation of COQ7 and we have observed that 3’UTR of COQ7 mRNA contains cis-acting elements involved in the post-transcriptional control of this gene. HuR and hnRNP C1/C2 are RNA-binding proteins that bind to 3’UTR of COQ7 mRNA. HuR regulates many essential genes involved in stress response, cell division cycle, carcinogenesis, immune cell activation and replicative senescence. The hnRNP C1/C2 function is still quite unknown. The absence of HuR destabilizes the COQ7 mRNA and reduces both its expression and CoQ biosynthesis rate. By contrast, the lack of hnRNP C1/C2 increases the half-life of COQ7 mRNA. Both COQ7 and HuR knockdown in human cells affected mitochondrial respiration, by decreasing both oxygen consumption rates and ATP production, and increasing lactate levels. We show that cells modulate respiration rates through the levels of CoQ, which depends on the regulated protein- mRNA COQ7 translation. Funded by FIS PI14-01962 grant.

58 P4. VITAMIN K2 CANNOT SUBSTITUTE COENZYME Q10 AS ELECTRON CARRIER IN THE MITOCHONDRIAL RESPIRATORY CHAIN OF MAMMALIAN CELLS

Cristina Cerqua1,2, Alberto Casarin1,2 Leonardo Salviati1,2 and Eva Trevisson1,2

1 Clinical Genetics Unit, Department of Woman and Child Health, University of Padova, Padova, Italy 2 IRP Città della Speranza, Padova, Italy

Defects of ubiquinone biosynthesis cause coenzyme Q10 (CoQ) deficiencies, a group of clinically and genetically heterogeneous conditions. Patients with primary CoQ deficiency respond well to ubiquinone oral administration if treated prematurely, but there are some pharmacokinetic constraints related to the difficulty of CoQ to reach the mitochondrial respiratory chain (MRC), due to its extreme hydrophobicity. Therefore, in the last years many studies have been performed to identify more water-soluble product formulations or other substances that could mimic its function, and different CoQ analogs are being used in clinical trials. Recently, vitamin K2 (menaquinone-4, MK-4) was reported to act as a mitochondrial electron carrier, similar to CoQ, in the MRC of D. melanogaster (Vos et al., 2012). We tested whether vitamin K2 could mimic CoQ function in a human cell model of CoQ deficiency. At this purpose we used human fibroblasts isolated from skin biopsies of patients with primary CoQ deficiency harbouring mutations in COQ2, one of the gene required for CoQ biosynthesis. Our data show that vitamin K2 cannot substitute CoQ in the MRC of these human cells since it is not able to restore the decreased Complex II+III activity. We also found that the growth of yeast Coq mutants on a non-fermentable carbon source cannot be rescued by the addition of MK-4 to the medium. Our results suggest that vitamin K2 cannot functionally replace CoQ as electron carrier in the MRC of human cells.

59 P5. MOLECULARLY IMPRINTED SOLID PHASE EXTRACTION BEFORE COQ10 ANALYSIS FROM TWO COMPLEX MATRICES

M. Contin1, C. Garcia Becerra2, M. Moretton1,3, S. Flor1,2,3, D. Chiappetta1,3, S. Lucangioli1,3, V. Tripodi1,3

1 Department of Pharmaceutical Technology, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina 2 Department of Analytical Chemistry and Physicochemistry, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina 3 Consejo Nacional de Investigaciones Científicas y Tecnológicas, CONICET, Buenos Aires, Argentina

Molecular imprinting is a strategy to introduce a 'molecular memory' in a polymeric system obtaining materials with specific recognition properties (1). Molecularly imprinted polymers (MIPs) are usually developed by mixing a template molecule with functional monomers, a cross-linker and an initiator. After polymerization, the template molecules are removed making the binding sites and the cavities, which are complementary to the template in size, shape and functionality, accessible. The MIP possesses a molecular “memory”, and thus, it is able to specifically recognize and bind the target molecule. MIPs are use as sensors, for chromatography, immunoassays, controlled drug delivery and catalysis. In a previous work we have reported for first time the use of CoQ0 as a template in the development of a molecularly imprinted polymer with the ability to recognize CoQ10 (2). In this work we present the use of this polymer in solid phase extraction (SPE) procedure before CoQ10 analysis by HPLC. In this opportunity the clean up after SPE of two matrices is presented. The first matrix is a bovine liver, were a high number of interferences were found which can led to electrodes passivate and a damage to the chromatographic column. The second matrix is CoQ10 nano-encapsulated in a polymeric nano-particle (NPs). It is known that those NPs are extremely harmful to the chromatographic column.

Experimental The polymer was synthesized by bulk polymerization using methacrylic acid (MAA) and ethylene glycol dimethaclylate (EGDMA) as monomers, CoQ0 as template and benzoyl peroxide as initiator. It was characterized by scanning electron microscopy (SEM), and superficial area (BET area). The SPE consisted in three steps, first the sample loading, then the washing step and finally the elution step. The proportion of 1-propanol: water in each step was optimized in order to favor a specific interaction. All experiments were performed with a polymer control (NIP) which is synthesized under the same conditions in the absence of the template molecule. An additional binding experiment was performed employing CoQ10 or ubichromenol (UC) in order to evaluate the specificity of the interaction expressed in terms of the “imprinting-induced promotion of binding” (IPB), a parameter that allows the study of the efficiency of the imprinting effect.

Results and discussion The SEM analysis revealed an internal structure composed of interconnected granules, and the BET area was around 24 m2 g-1 for the MIP and 8 m2 g-1 for the NIP. The IPB values obtained when CoQ10 were employed in the binding assay was six time higher than when UC were employed, which mean that the polymer is able to bind CoQ10 selectively.

60 The clean-up of the liver extract with the polymer as stationary phase was compared to the clean-up employing a C-18 commercial particle; the purification factor was around four times higher when the polymer was used. The MIP showed the capacity to bind CoQ10 over others compounds present in the liver. The chromatograph from the analysis of CoQ10 from the CoQ10 nano-encapsulated revealed a significant improvement when a clean-up is previously performed with the polymer. The recovery of CoQ10 was higher than 73% and a CV% lower than 8% in the biological matrix and nearly 100% of recovery and a CV% lower than 2% in the pharmaceutical matrix. Both assays where performed by triplicate in three different concentrations.

Conclusion The ability of the molecularly imprinted polymer to bind CoQ10 was successfully used in a solid phase extraction which led to improve the chromatographic analysis. The elimination of nano-particles from the pharmaceutical matrix will be important in order to expand the life time of the column and the elimination of compounds from the biological matrix as well as the pharmaceutical matrix will be important to determine CoQ10 correctly.

References 1. Gagliardi, M; Mazzolai, B Future Medical Chemistry 7 (2), 2015, 123-138. 2. Contin, M; Flor, S; Martinefski, M; Lucangioli, S; Tripodi, V Analytical Chimica Acta 807 (7), 2014, 67-74.

61 P6. UBIQUINOL SUPPLEMENTATION REDUCES OXIDATIVE DAMAGE ASSOCIATED TO STRENUOUS EXERCISE

Diaz-Castro J1,2, Sarmiento A1,2, Kajarabille N1,2, Pulido-Morán M1,3, Moreno-Fernández J1,2, Osa VM4, Hijano S1,2, Ochoa JJ1,2

1 Institute of Nutrition and Food Technology; 2 Department of Physiology. University of Granada; 3 Department of Biochemistry and Molecular Biology II. University of Granada; 4Servicio Andaluz de Salud. Hosp. Univ. Granada.

Introduction Regular, moderate and planned exercise has many health benefits (1). However, exhaustive exercise could induce fatigue resulting in alterations of several hormonal processes, transient immunosuppression, increased free radicals output and an increased susceptibility to infection (2). Antioxidants for sports people may reduce the muscle damage induced by free radicals, increase endurance and achieve performance improvements (3). In this sense, ubiquinol; anti-oxidant form of CoQ10, is considered a safe compound, without any side effects, legally authorized, non-doping (4).

Material and methods 100 fireman were divided into two groups: ubiquinol (Kaneka) group (n=50), and placebo (control) group (n=50). The Ubiquinol group received oral administration of 200 mg/day during 2 weeks and the control group took a capsule of placebo. After supplementation period subjects performed a strenuous exercise protocol consisted of conducting two identical strenuous exercise tests, with a rest period between tests within 24 hours, in order to cause muscle damage. The workload corresponded approximately to 60-70 % of the Dynamic Maximum Force. Five blood and urine samples were taken: before supplementation (T1), after supplementation (T2), after first physical exercise test (T3), after 24h of rest (T4), after second physical exercise test (T5). Urinary 8-OHdG was measured using a commercial kit (8-OHdG Check, Japan Institute for the Control of Aging, Shizuoka, Japan). 8-iso-Prostaglandin F2α in urine were measured using a commercial kit Enzyme Immunoassay for Urinary Isoprostane (Oxford Biomedical Research, Oxford, England). Oxygen Radical Antioxidant Capacity (ORAC) was measured using a commercial kit (OxiSelect™ Oxygen Radical Antioxidant Capacity (ORAC) Activity Assay, Cell Biolab, San Diego, USA).

Results The physical tests increased urinary 8-OHdG at T4 with respect to T3 and T5 in control group. A significant difference between groups was observed in the sample T4, after the 24 hours of rest and before the second physical test with a major content in 8-OHdG in the control group (Figure 1A). A higher level of urinary 15-F2t-Isoprostanes was observed in T3 and T5 (after strenuous exercise), though in the supplemented group it was only significant in the T5, whereas in the group control there were differences with regard to the T3 and T5. In the sample T4 also major values were observed, though it was only significant in the control group. Between the groups we found differences in T2, T3 and T4 (Figure 1B).In the control group, a maximum antioxidant capacity was observed in the sample T3, just after performing the first physical test, with significant differences with regard to T1. A decrease was also observed in the following samples, T4 and T5 with significant differences in comparison with sample T3. In the supplemented group a higher antioxidant capacity was observed in the samples T2, T3 and T4 in comparison with T1, without the previously commented decrease in the control group. Between experimental groups significant differences were shown in T2 and T4 with higher values in the supplemented group (Figure 1C).

62 Conclusions Ubiquinol supplementation diminished urinary 8-OHdG and isoprostanes indicating lower DNA and prostaglandins damage as a result of oxidative stress and increased antioxidant capacity after intense exercise sessions, therefore we can conclude that ubiquinol is a safe compound, efficient ameliorating the oxidative stress induced by the strenuous exercise.

A) 30,0 Control UG B) 4,5 Control ubiquinol 4,0 C C 25,0 A A A a 3,5 a a A a a* b 20,0 A 3,0 B

a* (ng/ml) B (ng/ml) 2,5 a* 15,0 a 2,0 a*

OHdG OHdG A - 10,0 1,5 1,0

Urinary 8 Urinary 5,0 Urinary Isoprostane Urinary 0,5 0,0 0,0 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 C) 8,0 Control ubiquinol b 7,0 A b* B A b* A ab

6,0 a A )

/L 5,0

4,0

3,0

2,0 ORAC (mmoles ORAC 1,0

0,0 T1 T2 T3 T4 T5 Fig. 1. Effect of exercise and ubiquinol supplementation on 8-OHdG urinary (A), urinary isoprostanes (B) and ORAC in plasma (C).

Figure Legends: Data shown are the mean ± SD (n= 50 subjects). *Mean values differ between groups in this sample taking, P<0.05. Capital letters “ABCDE” indicate the existence of significant differences between the diverse ubiquinol intakes in control group (the existence of not coincidental letters between different samples taking indicates the existence of significant differences between these samples, P<0.05). The lowercase letters “abcde” indicate differences between the diverse samples of the ubiquinol group, and the existence of not coincident letters means statistical differences, P<0.05.

References 1. Haskell, WL; Lee, IM; Pate, RR; Powell, KE; Blair, SN; Franklin, BA; Macera, CA; et al. Physical activity and public health: updated recommendation for adults from the American College of Sports Medicine and the American Heart Association. Circulation, 2007, 39(8), 1423-34. 2. Radak, Z; Chung, HY; Koltai, E; Taylor, AW; Goto, S. Exercise, oxidative stress and hormesis. Ageing Res. Rev, 2008, 7, 34-42. 3. Díaz-Castro, J; Guisado, R; Kajarabille, N; García, C; Guisado, IM; de Teresa, C; Ochoa, JJ. Coenzyme Q10 supplementation ameliorates inflammatory signaling and oxidative stress associated with strenuous exercise. European Journal of Nutrition, 2012, 51, 791-799. 4. Ochoa, JJ; Díaz-Castro, J; Lambrechts, P. CoQ10 and Ubiquinol: Novel, safe dietary supplementation for trained and untrained athletes. Agro FOOD Industry Hi Tech, 2013, 24(6), 31-34.

63 P7. PLASMATIC AND INTRACELLULAR MARKERS OF OXIDATIVE STRESS IN NORMAL WEIGHT AND OBESE PATIENTS WITH POLYCYSTIC OVARY SYNDROME

Chantal Di Segni1, Sebastiano Raimondo1, Antonio Mancini1, Francesco Guidi2, Daniela Romualdi3, Rosanna Apa3, Antonio Lanzone3, Christian Bergamini4, Francesco Volta5 and Romana Fato4

1Dept. of Medical Sciences, Division of Endocrinology, Catholic University of Sacred Heart, Rome, Italy 2Institute of Hematology, Catholic University of Sacred Heart, Rome, Italy 3Dept. Of Obstetrics and Gynecology, Catholic University of Sacred Heart, Rome, Italy 4Dept. Of Pharmacology and Biotechnology (FaBiT), University of Bologna, Bologna, Italy 5Helmholtz Zentrum München, German Research Center for Environmental Health

Insulin resistance (IR) is associated with polycystic ovary syndrome (PCOS). Oxidative stress (OS) is, in turn, related to IR. Studies in PCOS have remarked abnormal OS markers, mainly in obese patients. In order to investigate OS parameters in PCOS and relationship with hormonal and metabolic picture, we performed a case-control study on Outpatients afferent to the Department of Endocrinology and Gynecology at our hospital. We studied 2 PCOS groups: normal weight, (n=21, age 20-30ys, mean ± ES BMI 20.7±0.2 kg/m2) and obese (n=15, 19-23 ys, BMI 32.8 ± 1.1) compared with two control groups: matched for BMI (normal (n=10, 19-23 ys, BMI 21.6 ± 0.9) and obese (n=20, 21-31ys, BMI 36.8 ± 1.0). Malondialdehyde (MDA) in blood plasma and peripheral mononuclear cells, obtained by density- gradient centrifugation, was assayed spectrophotometrically at 535nm by TBARS assay. CoenzymeQ10 in plasma and cells was assayed by HPLC. PCOS patients of both groups exhibited higher Testosterone levels than controls and obese patients had a higher HOMA index, as expected. Despite plasma MDA levels were not significantly different (N-PCOS 3380 ± 346.94 vs normal women 7120 ± 541.66; OB-PCOS 5517.5 ± 853.9 vs OB. 3939.66 ± 311.2 pmol/ml), intracellular MDA levels were significantly higher in N-PCOS than controls (mean 3259 ± 821.5 vs 458 ± 43.2 pmol/106/cells) and higher than OB-PCOS, although not 6 significantly (1363.1 ± 412.8 pmol/10 /cells). Intracellular Coenzyme Q10 was similarly higher in N-PCOS, but also in OB controls. Results about evaluation of CoQ10, both intracellular and plasmatic, in each group, are shown in the table below.

Plasmatic CoQ10 (pmol/ml) Intracellular CoQ10 (pmol/106/cells) Normal weight controls 472.85 ± 40.23 17.03 ± 3.04

Normal weight PCOS 536.25 ± 17.13 30.99 ± 3.11

Obese controls 851.55 ± 54.32 33.46 ± 3.44

Obese PCOS 570.33 ± 63.7 37.32 ± 8.77

These data show that OS could be present in tissue even if not revealed in plasma, especially in N- PCOS. The relationship with metabolic status remains to be established, but it could be a physiopathological basis for antioxidant treatment in such patients.

64 P8. LOSS OF MITOCHONDRIAL UBIQUINONE AS A DRIVER OF INSULIN RESISTANCE

Daniel J. Fazakerley1*, Rima Chaudhuri1*, Ghassan J. Maghzal2, Cacang Suarna2, Pengyi Yang3, Christopher C. Meoli1, Sean J. Humphrey4, Kelsey H. Fisher-Wellman5, James R. Krycer1, Benjamin L. Parker1, Kristen C. Thomas1, Jagath R. Junutula6, Zora Modrusan6, Ganesh Kolumam6, Jean Y. Yang7, Kyle L. Hoehn8, Roland Stocker2 and David E. James1 [email protected]

1, Charles Perkins Centre, School of Molecular Bioscience, University of Sydney, Camperdown, NSW, Australia 2, Vascular Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia 3, Systems Biology Group, Biostatistics Branch, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, NC, USA 4, Max Planck Institute of Biochemistry, Department of Proteomics and Signal Transduction, Munich, Germany 5, Duke Molecular Physiology Institute, Duke University School of Medicine, Durham, NC, USA 6, Genentec Inc., South San Francisco, CA, USA 7, School of Mathematics and Statistics, University of Sydney, Camperdown, NSW, Australia 8, School of Biotechnology and Biomedical Sciences, University of New South Wales, Sydney, Australia.

Insulin resistance (IR) is one of the earliest detectable risk factors for a range of metabolic diseases, including type 2 diabetes, cardiovascular disease and some cancers. IR is a state where the peripheral insulin-responsive tissues (adipose, muscle and liver) fail to respond appropriately to insulin. As IR is associated with changes in diverse cellular pathways, it is challenging to identify which changes drive IR pathogenesis. To comprehensively capture this complexity and to identify the key common drivers of IR, we used multiple distinct in vitro models of IR and integrated observed transcriptomic and proteomic changes. ‘Driver pathways’ identified were then verified in vivo in insulin resistant adipose tissue from mice fed a high fat diet (HFD). Using this approach, we identified the mevalonate biosynthetic pathway (that produces ubiquinone, dolichol and cholesterol) as a putative ‘driver’. While the mevalonate pathway was altered differently between models, a decrease in mitochondrial ubiquinone content was observed consistently in every model. For example, in mice fed a HFD, mitochondrial ubiquinone in adipose tissue was decreased significantly despite an increase in total tissue ubiquinone. Functional studies revealed that replenishment of ubiquinone restored insulin sensitivity, whilst inhibition of ubiquinone biosynthesis induced IR, indicating that loss of ubiquinone was both necessary and sufficient for adipocyte IR. Mechanistically, loss of ubiquinone increased mitochondrial hydrogen peroxide, as assessed by an increase in the dimer-to-monomer ratio of peroxiredoxin 3. Our findings highlight the power of integrating different models to pinpoint drivers of IR, and they implicate a specific defect in the synthesis or retention of ubiquinone in mitochondria and resulting increase in mitochondrial oxidants in the aetiology of adipose tissue IR.

65

P9. UBIQUINONE AND UBIQU61INOL DEPENDENT GENE EXPRESSION SIGNATURES UNDER AD LIBITUM AND DIETARY RESTRICTION CONDITION IN THE MODEL ORGANISM C. ELEGANS

Alexandra Fischer1, Petra Niklowitz2, Thomas Menke2, Frank Döring1

1Institute of Human Nutrition and Food Science, University of Kiel 2Children’s Hospital of Datteln, University of Witten/Herdecke

Dietary restriction (DR) is a robust intervention that extends both health span and life span in many organisms. In humans DR decreases the incidence of age-dependent diseases, including atherosclerosis and cancer. Ubiquinone (coenzyme Q, CoQ) plays a central role in energy metabolism and functions in several cellular processes including gene expression. CoQ can be acquired through the diet and is also synthesised de novo in the body. Little is known whether DR influences level and redox-state of CoQ. Here we addressed this question in the nematode C. elegans, as both DR responsive pathways (i. e. AMPK pathway, insulin receptor pathway) and CoQ-synthesizing enzymes are conserved from yeast to man. We found that DR down-regulates the steady-state expression levels of several evolutionary conserved genes (i. e. coq-1) that encode key enzymes of the mevalonate and CoQ-synthesizing pathways. In line with this, DR decreases the levels of total CoQ and ubiquinol. This CoQ-reducing effect of DR is obvious in adult worms but not in L4 larvae and is also evident in the eat-2 mutant, a genetic model of DR. In conclusion, we propose that DR reduces the level of CoQ and ubiquinol via gene expression in the model organism C. elegans. Translating our findings into the situation in humans, one could speculate that dieting besides the overall health benefits may enhance the risk of an undesirable CoQ10 status including low levels of ubiquinol-10.

This study is supported by Kaneka Corporation, Japan

66

P10. CONTROL OF MITOCHONDRIAL SUPEROXIDE ION GLIOBLASTOMA MULTIFORME: IMPACT ON TUMOR METABOLISM, NEOVASCULARIZATION AND INFLAMMATION

Javier Frontiñán-Rubio1, Raquel Santiago-Mora1, María Victoria Gómez-Almagro2, Margarita Villar-Rayo3, Gustavo Ferrín-Sánchez4, Juan Ramón Peinado1, José Manuel Villalba5, Francisco Javier Alcaín1 and Mario Durán-Prado1#

1Dept. of Medical Sciences, Faculty of Medicine of Ciudad Real, University of Castilla la Mancha, Spain; 2IRICA, University of Castilla-La Mancha, Ciudad Real, Spain; 3IREC, CSIC-University of Castilla-La Mancha, Ciudad Real, Spain; 4IMIBIC, Córdoba, Spain; 5Dept. of Cell Biology, Physiology and Immunology, University of Córdoba, Spain. [email protected]

.- .- Elevated mitochondrial superoxide radical (O2 ) is linked to the aggressive features of GBM. O2 dismutates to H2O2, translocates to the cytosol and triggers pathways that favor tumor progression. .- Increased O2 and H2O2 levels stabilize HIF1-α, even in normoxia, which promotes the “glycolitic switch” and initiates neovascularization, which, added to the attraction of inflammatory cells, facilitates tumor growth and invasion. Herein, we show how coenzyme Q10 (CoQ),normalizes the .- level of O2 , H2O2, HIF1-α and several other key signaling molecules governing GBM progression. To that purpose, using molecular and cell biology approaches as well as “omics”, we have tested the effect of CoQ using human GBM cell lines in vitro, and in vivo, xenografted subcutaneously or .- ortotopically implantated into the striatum of nu/nu mice. Treatment with CoQ reduced O2 and H2O2 levels in 50%, accompanied by an increase in complex II-associated respiration. These results are mirrored by a decrease in cell viability and motility, as consequence of the modification of the full proteome and to a decrease in the aerobic glycolisys rate. These results were reproduced in vivo. In subcutaneous xenografts of GBM, CoQ reduced tumor volume by a direct action on tumor cells but also by regulating neovascularization and local inflammation. Indeed, similar results were obtained in ortotophic models, where CoQ not only reduced the volume of primary tumors, also, and dramatically, impeded tumor cells infiltration. As a whole, our results presented herein appoint towards CoQ as a pleiotropic regulator of GBM progression and pave the way for the implementation of further therapeutic approaches to manage this, nowadays, devasting pathology.

Aknowledgements. The authors’ work is supported by Grants 220020351 (James S. MacDonnell Foundation), GI20152960 (Universidad de Castilla-La Mancha) and PPII-2014-010-P (Junta de Comunidades de Castilla-La Mancha). We thank Kaneka Corporation, who generously provided the CoQ used in these experiments.

67

P11. NONLINEAR PHARMACOKINETIC OF SOLUBILIZED UBIDECARINONE AFTER INTRAVENOUS ADMINISTRATION

Gorodetskaya E.A., Kalenikova E.I.., Medvedev O.S.

Lomonosov Moscow State University, Moscow, Russia

Objective The cardio- and neuroprotective effectiveness of ubidecarenone (coenzyme Q10) was demonstrated in numerous experimental and clinical investigations. The need to develop ubidecarenone forms for parenteral and, primarily, i.v. administration for application in urgent situations (ischemic damage, myocardial infarct) has now become obvious. Pharmacokinetic studies are an important phase of preclinical trials of a new drug form. The goal of this pilot study was to estimate the linearity of ubidecarenone pharmacokinetics after i.v. injection. Design and Methods The studies used male Wistar rats. The dynamics of coenzyme Q10 tissue levels were studied after i.v. injection of a solution of solubilized ubidecarenone (Qudesan, Rusfik) at doses of 10 and 30 mg/kg. Samples of rat blood plasma and liver were collected during 48 h after the injection using 4- 9 animals for each time point. Coenzyme Q10 baseline level was measured by collecting tissue samples from control animals after injection of normal saline. Coenzyme Q10 contents in the samples were measured by HPLC with electrochemical detection. Areas under concentration-time curves were calculated by a trapezoid method. Results Coenzyme Q10 blood plasma levels were elevated through the whole observation period and exceeded the control levels by 200 and 850 times (for the 10 and 30 mg/kg doses, respectively) by 48 h. The data plotted in semi-logarithmic scale demonstrated the exponential nature of the concentration decrease in the time interval studied. The areas under the concentration–time curves for the 10 (2620 mg*h/mL) and 30 mg/kg (22658 mg*h/mL) doses differed by 8.6 times, not 3 times. Furthermore, normalization of the kinetic data to the dose did not superimpose the concentration–time curves. These results indicated that coenzyme Q10 pharmacokinetics were nonlinear. Nonlinear coenzyme Q10 pharmacokinetics after i.v. injection in our investigation may have resulted from the particulars of its in vivo distribution. The liver is the main organ for accumulation and excretion of ubidecarenone and also for its secretion into the systemic circulation. The fastest concentration increase was observed in the first hours after the injection and slowed down considerably after the first day. Thus, ubidecarenone after i.v. injection was distributed for at least 48 h. The drug accumulated gradually in liver with the areas under concentration–time curves differing by a factor of 4.4. Conclusion These data indicates that ubidecarenone pharmacokinetics in plasma and liver were nonlinear after i.v. injection.

Supported by grant from the Russian Scientific Foundation (Project No. 14-15-00126).

68

P12. THE COENZYME Q6 SYNTHESIS REGULATION BY THE COUPLE Coq7p/Ptc7p INTEGRATES THE CELL BIOENERGETICS STATE WITH THE MITOCHONDRIAL HOMEOSTASIS IN SACCHAROMYCES CEREVISIAE

Gutiérrez Ríos P., González-Mariscal I., Martín-Montalvo A., Navas P., and Santos-Ocaña C.

Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-CSIC, CIBERER Instituto de Salud Carlos III, Sevilla, 41013, Spain. The synthesis of coenzyme Q in yeast is produced by the establishment of an enzyme complex wherein the Coq7 protein has a double catalytic and regulatory function. Coq7p modulates the levels of coenzyme Q using a phosphorylation cycle where the dephosphorylation of three amino by the mitochondrial phosphatase Ptc7p increases the levels of coenzyme Q [1]. The conversion of the three non-phosphorylatable amino acids (Ser or Thr to Ala) leads to a permanently active state of Coq7p that significantly increases the levels of coenzyme Q up to 250% [2]. However, this modification produces a decrease in the activity of the mitochondrial respiratory chain, a lower resistance to oxidative stress, the increase of the endogenous production of ROS, a carbonylated protein accumulation and a significant decreased of chronological life span (CLS) compared to the wild strain. The analysis of Coq7p in a phosphorylated state was studied in a null mutant strain of the Ptc7p phosphatase [3]. In this case there was a decrease in the synthesis of coenzyme Q associated with a significant shortening of the CLS that was not reversed after adding exogenous coenzyme Q. This finding suggests an additional function of Ptc7p beyond the regulating of coenzyme Q synthesis. An effect on autophagy was discarded but the absence of Ptc7p prevents mitophagy activation in conditions of nitrogen deprivation. The overexpression of Ptc7p not only restored normal levels of CLS but also increased mitophagy in a wild type strain. We propose a model where the couple Coq7p/Ptc7p initially modulates the adaptation of yeast to a respiratory metabolism by supplying coenzyme Q involving mitochondrial biogenesis. Following the establishment of the stationary phase Ptc7p would be required to activate mitophagy and remove the excess of mitochondria produced that is required to establish an extended CLS in yeast.

References 1. González-Mariscal, E. García-Testón, S. Padilla, A. Martín-Montalvo, T. Pomares-Viciana, L. Vazquez-Fonseca, et al., Regulation of coenzyme Q biosynthesis in yeast: A new complex in the block, IUBMB Life. (2014). 2. A. Martin-Montalvo, I. Gonzalez-Mariscal, S. Padilla, M. Ballesteros, D.L. Brautigan, P.P. Navas, et al., Respiratory- induced coenzyme Q biosynthesis is regulated by a phosphorylation cycle of Cat5p/Coq7p., Biochem. J. 114 (2011) 107–114. 3. A. Martin-Montalvo, I. Gonzalez-Mariscal, T. Pomares-Viciana, S. Padilla-Lopez, M. Ballesteros, L. Vazquez- Fonseca, et al., The phosphatase Ptc7 induces coenzyme Q biosynthesis by activating the hydroxylase Coq7 in yeast., J. Biol. Chem. 288 (2013) 28126–37.

69

P13. COENZYME Q10 LEVELS ARE DECREASED IN THE CEREBELLUM OF MULTIPLE-SYSTEM ATROPHY PATIENTS

Iain Hargreaves1,3 Lucia V Schottlaender1, Conceicao Bettencourt1, Aoife P Kiely2, Annapurna Chalasani3, Viruna Neergheen3, Janice L Holton2; Henry Houlden1,4,5

1Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, UK. 2Reta Lila Weston Institute for Neurological Studies and The Queen Square Brain Bank, London, UK. 3Neurometabolic Unit, National Hospital, London, UK; Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK. 4Neurogenetics Laboratory, The National Hospital for Neurology and Neurosurgery, Queen Square, London, UK. 5The MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology, London, UK.

Multiple system atrophy (MSA) is a fatal late onset neurodegenerative condition characterized by cerebellar ataxia and autonomic dysfunction. Pathologically the disorder presents with widespread alpha-synuclein glial cytoplasmic inclusions together with neurodegeneration. MSA can be subdivided into MSA-OPCA (olivopontocerebellar), MSA-SND (striatonigral) and MSA-mixed according to the affected brain regions. Through linkage-analysis and whole-genome-sequencing, variants in COQ2 gene have been linked to clinically diagnosed Japanese MSA patients in a multicenter study.1 The authors of that study, by analysing a small patient group ( n = 3) suggested an association between MSA and decreased brain CoQ10 status. At present, we have been unable to replicate the genetic finding of the Japanese study. However, in view of the brain tissue result, we decided to investigate evidence of cerebral CoQ10 deficiency in MSA. We determined the level of CoQ10 in brain tissue from pathologically confirmed MSA cases and compared these to age matched controls as well as to patients with other neurodegenerative disorders. Flash-frozen brain tissue of 22 pathologically confirmed MSA patients [9 MSA-OPCA, 6 MSA- SND, and 6 MSA-mixed subtype] was used for this study. Elderly controls (n=37) as well as Idiopathic Parkinson's disease (IPD; n=7), dementia with Lewy bodies (DLB; n=20), corticobasal degeneration (CBD; n=15) and cerebellar ataxia (CER-ATX; n=18) patients were used as comparison groups. In this study we detected a statistically significant decrease in the levels of CoQ10 in the cerebellum of MSA cases (p=0.001), specifically in OPCA (p=0.001) and mixed cases (p=0.006), when compared to controls as well as to other neurodegenerative diseases [DLB (P<0.001), IPD (p=0.001), CBD (p<0.001), and CRB-ATX (p=0.001)]. Our results suggest that a perturbation in CoQ10 biosynthesis may be involved in the pathogenesis of MSA.

References 1. Multiple-System Atrophy Research Collaboration. Mutations in COQ2 in familial and sporadic multiple-system atrophy. N Engl J Med. 2013 Jul 18;369(3):233–44.

70

P14. TISSUE DISTRIBUTION AND PHARMACOKINETIC PROFILE OF COENZYME Q10 DURING 8 DAYS AFTER INTRAVENOUS ADMINISTRATION

Kalenikova E.I., Gorodetskaya E.A., Medvedev O.S.

Lomonosov Moscow State University, Moscow, Russia

Objective Our previous investigation have shown that the single intravenous (i.v.) injection of coenzyme Q10 (CoQ10) after myocardial infarct results in limitation of necrosis volume and improvement of cardiac function in rats. At the same time the increased CoQ10 plasma levels were detected up to 21 days after injection. So, the aim of the present study was to examine the concentration–time CoQ10 profile for multiple days and to calculate the main pharmacokinetic parameters after single i.v. administration. Design and Methods CoQ10 (solubilized ubidecarenone solution, Qudesan, Rusfik) was administered i.v. to Wistar rats at a dose of 30 mg/kg. Five rats were assigned to each group representing 0 min, 15 min, 30 min, 1 h, 2 h, 6 h, 8 h, 12 h, 24h, 48h, 4d, 8d and 16d (only for blood). The blood samples (300 μL) were drawn from the abdominal aorta of each rat and placed into heparinized microfuge tubes. All of the collected blood samples were immediately centrifuged to obtain plasma, which was frozen at −20 °C until analysis. The tissues, including heart, liver, brain and kidney, were excised immediately, thoroughly rinsed with ice-cold physiological saline solution to remove blood or content and then blotted dry with filter paper and stored at −20 °C until treatment. Individual plasma and tissue samples were analyzed using HPLC methods. Results: Pharmacokinetics of ubidecarenone in plasma is described by the two compartment model and by bi-exponential function curve. The first phase fits the distribution in internal organs and tissues and lasts 96h followed by the elimination phase that was traced up to 16 days. The following pharmacokinetic parameters were calculated: time of half-distribution ((Т 1/2α) = 14.2 h, time of half-elimination (Т ½ el) = 117.5h, distribution constant (k α) = 0.05 h-1, elimination constant (k el) = 0.006 h-1, distribution volume (Vd) = 20.4 l/kg, total clearance (Cl t) = 0.39 ml/h. In 16 days after i.v. administration the concentration of CoQ10 was twice as high compared with the baseline level. Tissue distribution has shown that increased concentrations of CoQ10 take place as early as in 15 min in heart, brain, kidney, liver. Elevated levels of CoQ10 in the myocardium were maintained at least for 4 days, in the liver – 8 days, in the kidneys – 2 days, in the brain – 12 hours. Tissue bioavailability was 0.075 for myocardium, 0.02 for brain, 0.082 for kidney, 4.36 for liver. Conclusion So, i.v. administration of ubidecarenone is followed by the fast increase and long-term maintenance of the high levels of CoQ10 in plasma, myocardium, liver, kidneys and the brain. These results on pharmacokinetics and tissue distribution may be useful for the clinical application of CoQ10. Results of our studies confirm the perspectiveness of the development of parenteral form of CoQ10 to be used in acute ischemic conditions.

Supported by grant from the Russian Scientific Foundation (Project No. 14-15-00126).

71 P15. THE EFFECT OF LONG-TERM INTAKE OF UBIQUINOL ON IMPROVING QUALITY OF LIFE FOR COMMUNITY RESIDENTS

Tetsu Kinoshita1)2), Koutatsu Maruyama3), Takeshi Tanigawa3)

1Department of Epidemiology and Preventive Medicine, Graduate School of Medicine, Ehime University, Toon, Ehime, Japan 2Institute of Community Life Sciences Co., Ltd., Matsuyama, Ehime, Japan 3Department of Public Health, Graduate School of Medicine, Juntendo University, Bunkyo-ku, Tokyo, Japan

Objective Ubiquinol (reduced form of Coenzyme Q10) is widely used as anti-ageing material for supplement and cosmetics. In this study, we evaluated the effect of long-term intake of ubiquinol on improving or maintaining of the subjective QOL (Quality of Life), bone quality, and fatigue degree in community residents.

Method This field trial was conducted in Kamijima-town which is consisted of 25 small islands, Ehime Japan. Total of 124 residents (36 male and 88 female aged 22-85 y) participated in this study. Participants consumed 100-120mg ubiquinol per day for 6 months (n=22) or 12 months (n=102). We measured serum ubiquinol, bone quality markers (homosysteine and pentosidine) levels, subjective QOL score (SF-36) and fatigue degree (autonomic nerve function test) at before and after of intervention. We analyzed mean differences of those measurements and correlations between the change of ubiquinol level and the changes of index.

Result Serum ubiquinol level was increased significantly. In female, the scores of Role Physical (+3.1, P=0.04), Vitality (+3.0, P<0.01), Social Functioning (+3.2, P<0.01), Mental Health (+2.3, P=0.02), and Mental Component Score (+1.7, P=0.03) were also increased significantly. Meanwhile, in male, the significant score changes were not observed. Bone quality and fatigue degree did not show significant changes in both sexes. The correlations between the change of ubiquinol level in serum and the changes of QOL score were not reached statistically significant.

Conclusion This trial indicated the probable effects of ubiquinol on the female’s mental condition. We would continue to follow the participants, and analyze the differences of effects by intake period.

72 P16. EVALUATION OF PLASMA COENZYME Q10 IN ADULTS PATIENTS WITH GROWTH HORMONE DEFICIENCY

A. Mancini1, C. Di Segni1, S. Raimondo1, P. Orlando2, S. Silvestri2, L. Tiano2 and G.P. Littarru2

1Dept. Of. Medical Sciences, Division of Endocrinology Catholic University of the Sacred Heart, Rome, Italy 2Institute of Biochemistry, Polytechnic University of the Marche, Ancona, Italy

It is known that growth hormone deficiency (GHD) is associated with oxidative stress, both in children and adults, but discordant data concern GH effects on antioxidant levels. Results in experimental animal models are also contradictory: GHR-KO mice have decreased antioxidant defence (but with differential pattern in relation to sex and tissues); in df/df (mutant mice with congenital PRL and GH deficiency) a discrepancy has been demonstrated between enzymatic antioxidants (which appear to be increased) and scavengers, which are on the contrary decreased. In humans, GH treatment seems to increase antioxidant capacity, but other studies show reduced lipid peroxidation in GHD adults, which is reverted by GH treatment. Moreover oxidative stress could play an important role in metabolic syndrome (MS)-related manifestations (atherosclerosis, hypertension, type 2 diabetes, contributing to insulin resistance. GHD patients exhibit some clinical features of MS, such as truncular obesity and insulin resistance. Oxidative stress could be involved in cardiovascular risk in these patients.

We have therefore evaluated the levels of total, reduced and oxidized Coenzyme Q10 in a group of 52 patients, with GHD (aged 75-17 ys) compared with a group of 36 patients with MS diagnosed by ATPIII criteria. GHD was documented by GH peak after GHRH+arginine lower than 9 ng/ml; the deficiency was due to empty sella or previous transsphenoidal surgery; it was not associated with other hormone deficiencies (gonadotropin or thyroid) known to influence CoQ10 levels. Hormone levels were assayed by chemiluminescence; CoQ10 was assayed by electrochemical method.

Results are reported in the table below (mean ± SEM).

CoQ10 (µg/mL) Qox/Qtot (%) HOMA Index

Patients with GHD (n=52) 0.57 ± 0.03 9.55 ± 0.65 2.20 ± 0.3

Patients with MS (n=36) 0.75 ± 0.03 4.70 ± 0.70 3.72 ± 0.62

These data confirm a lower CoQ10 plasma concentration with higher percentage of oxidized form.

Our data, in the context of metabolic alterations and cardiovascular risk of patients of both groups, seem to suggest the possible usefulness of CoQ10 therapy together with replacement therapy to obtain better metabolic control.

73 P17. COENZYME Q10 SUPPLEMENTATION IMPROVES ETHYNIL ESTRADIOL-INDUCED CHOLESTASIS IN RATS

Martinefski Manuela1, Rodriguez Myrian2,3, Lucangioli Silvia1,3, Bianciotti Liliana2,3 and Tripodi Valeria1,3

1 Department of Pharmaceutical Technology, Faculty of Pharmacy and Biochemistry, Universidad de Buenos Aires, Buenos Aires, Argentina 2 Pathophysiology-INIGEM-CONICET, Faculty of Pharmacy and Biochemistry, Universidad de Buenos Aires, Buenos Aires, Argentina 3 Consejo Nacional de Investigaciones Científicas y Tecnológicas, CONICET, Buenos Aires, Argentina email: [email protected]

Intrahepatic cholestasis of pregnancy (ICP) is a high risk liver disease given the eventual deleterious consequences that may occur in the fetous. ICP is characterized by the accumulation of bile acids, particularly hydrophobic bile acids such as litocholic acid (LCA), which induces oxidative stress and apoptosis. We previously reported that women with IPC show in plasma decreased coenzyme Q10 (CoQ10) and enhanced bile acids. These findings were also observed in ethynil estradiol (EE)- induced cholestasis in rats. Furthermore, cholestatic rats also showed decreased CoQ10 content in diverse organs including the liver. Here, we evaluated the effect of CoQ10 supplementation in EE- induced cholestasis in rats. Cholestasis was induced in Sprague-Dawley rats by a daily intraperitoneal injection of 5 mg/kg EE for 5 days (EE). A group of these rats also received daily oral 250 mg/kg CoQ10 supplementation for 5 days (EE+CoQ10). Another group of rats received only CoQ10 supplementation for the same period of time (CoQ10). Serum, bile acids (total and LCA), coenzyme Q9 (CoQ9) and CoQ10 were assessed. Bile flow was also measured. No differences were observed between CoQ10 and control groups except for an increase in plasma CoQ10 (p<0.001). However, CoQ10 supplementation in cholestatic rats (EE+CoQ10) increased both CoQ10 and CoQ9 plasma levels (p<0.05), and enhanced bile flow (p<0.05) as compared with EE. Furthermore it also decreased plasma bile acids, particularly LCA levels. These findings show that CoQ10 administration improved cholestasis and further suggest that CoQ10 supplementation may be a potential therapeutic strategy in estrogen-induced cholestasis in humans as ICP.

74 P18. EFFECT OF SHORT- AND LONG-TERM COMPLETE STARVATION AND REGENERATION DIET ON ANTIOXIDANTS, OXIDATIVE STRESS AND KIDNEY FUNCTION

1Mojto V, 2Gvozdjáková A., 2Kucharská J, 3Mišianik J, 4Rausová Z, 5Valuch J

1Comenius University in Bratislava, Medical Faculty, Department of Third Medical Clinic, Bratislava, Slovakia, 2Comenius University in Bratislava, Medical Faculty, Pharmacobiochemical Laboratory of Third Medical Clinic, Bratislava, 3Oncology Department of St. Elizabeth, Bratislava, 4Institute of Automation, Measurement and Applied Informatics, Faculty of Mechanical Engineering, Slovak University of Technology, 5Forensic Medicine, Bratislava

Long-term restriction of calories, with current adequate nutrition, is the result of metabolic adaptation of the body that reduces the risk of developing type 2 diabetes, hypertension, cardiovascular diseases and cancer(1). Nutrition experts have different opinions on the starvation in the healthy and sick organism. On the one hand, is expected to improve the health of patients, cleansing the body from unwanted metabolites, on the other hand, starvation represents a burden on the body. Complete starvation may impaire antioxidant defense status and kidney function in human subjects. The aim of this study was to compare effect of short- and long-time complete starvation and diet regeneration on the antioxidants, oxidative stress, lipids fraction and kidney function in human subjects. Methods 1. Short-term complete starvation and regeneration: Ten human subjects lived in spa, were included in complete starvation (with drinking only water) and diet regeneration during 11 days(2); five men, mean age: 46,4 years (range 31-58 years); five women, mean age: 53,6 years (range 44-63 years). All drugs (decreasing blood pressure, lipid fraction, improving coxarthrosis and hypothyreosis) were omitted. Subjects walked 6–10 km daily, swimmed, received massages. Three times (before, after starvation and regeneration) in plasma were monitored: antioxidants (coenzyme Q10-TOTAL, γ-tocopherol, α-tocopherol, β-carotene), peroxidation of lipids (TBARS), lipids fractions and kidney functions (creatinine, uric acid, urea and glomerular filtration - GFR). For statistical analysis multiple one-way analysis of variance (ANOVA) with Bonferoniho correction was used. 2. Long-term complete starvation and regeneration: Case report: Men aged 61 years after nephrectomy from oncology reason in year 2009, was on complete starvation and diet regeneration several times, during 25 – 50 days in years 2009 – 2013. In 2014 the men was on complete starvation during 43 days (with drinking only water) and regeneration diet during 43 days. Antioxidants, oxidative stress, lipid fractions and kidney functions were measured before starvation, every week and finished after 43 days of diet regeneration. Results 1. Short-term of complete starvation and regeneration diet: Starvation decreased lipid peroxidation (P<0.015) and stimulated concentration of γ-tocopherol from 3,74±0,27 µmol/l to 6,30±1,04 µmol/l. α-tocopherol and coenzyme Q10-TOTAL remained reduced after regeneration (from baseline values 0,655±0,059 to 0,420±0,037µmol/l CoQ10-TOTAL). Complete starvation increased Chol-total and LDL-Chol, which returned to the reference values after diet regeneration. HDL-Chol and TAG were in reference values. Complete starvation increased serum creatinine from baseline values 45,5±2,87 to 73,8±3,63 µmol/l (p<0.0002), after regeneration returned to baseline values. Uric acid was increased from baseline values 295,48±28,20 µmol/l to 821,88±39,70 µmol/l after starvation (p<0.0001) and returned to baseline values after regeneration. Concentrations of urea were not influenced. After starvation GFR was decreased and returned to baseline values after regeneration.

75 2. Long-term of complete starvation and regeneration diet: Starvation and diet regeneration decreased TBARS. CoQ10-TOTAL, decreased from baseline plasma level 0,623 µmol/l to 0,317 µmol/l and returned to the baseline values after regeneration. Concentrations of α-tocopherol and β- carotene were occasionally decreased. Starvation stimulated γ-tocopherol concentration from 3,31 to 11,24 µmol/l during the first 15 days and decreased during next weeks of starvation and regeneration. Starvation stimulated LDL-cholesterol concentration during first seven days (from 4,48 mmol/l to 6,11 mmol/l), after next days decreased to baseline values. Complete starvation worsened parameters of kidney function: creatinine concentration, which returned after diet to reference values and significantly increased uric acid concentration from 368±13,59 µmol/l to 943,3±23,63 and 823,3±35,95 µmol/l, which returned to the reference values after regeneration. Concentrations of urea were not influenced. Conclusions Short– (11 days) and long-time (43 days) complete starvation and regeneration diet modified antioxidants and oxidative stress in organism. Decreased CoQ10 concentrations contribute to the decreasing of energy production and antioxidant protective system in organism against oxidative stress. We suppose, that increased γ-tocopherol concentration in plasma could be the result of cyt P450 inhibition by ketone bodies in the liver during the first 15 days of starvation and reversion can be explained by adaptation of organism. Ketone bodies compete with uric acid for a common tubular secretion site and starvation impaired the ability of the kidney of excrete uric acid. It is essential in the prophylaxis of renal complication during starvation to ensure that a sufficient fluid intake is maintained and the urinary output is controlled. Further studies with higher number of patients are recommended.

Acknowledgements: Ministry of Education of Slovak Republic, Grant VEGA 1/0614/12 and 1/0058/15.

References: 1. Rizza W, Veronese N, Fontana L: What are the roles of calorie restriction and diet quality in promoting healthy longevity? Ageing Res Rewiews 2014; 13: 38-45. 2. Nikolajev JS, Nilov EI, Čerkasov VG: Golodanie radizdorovja Sovetskaja Rossija, Moskva, 1988.

76 P19. PURIFICATION AND CHARACTERIZATION OF HUMAN RECOMBINANT COQ4

1L. Morbiato, 3F. Tonello, 3P. Berto, 3G. Zanotti, 1MA. Desbats, 3C. Montecucco, 2L. Salviati

1 IRP (Pediatric research institute), Città della Speranza, Padova 2 Clinical Genetics Unit, Dept. of Pediatrics, University of Padova 3 Dept. of Biomedical Sciences, Padova

Defects in genes involved in coenzyme Q (CoQ) biosynthesis cause primary CoQ deficiency, a clinically heterogeneous disorder with clinical manifestation ranging from fatal neonatal multisisytem disorders to adult-onset isolated encephalopathy or nephropathy. COQ4 codes for an ubiquitously expressed 265 amino acid protein that is peripherally associated with the mitochondrial inner membrane on the matrix side (ref 1,3); the precise function of human COQ4 is not known, but the yeast ortholog Coq4p seems to play a structural role crucial in the stabilization of a multiheteromeric complex including several, if not all, of the CoQ biosynthetic enzymes (ref 1). It has also been proposed that yeast Coq4p could be a zinc protease, based on the presence of the zinc binding motif HDxxH (ref 2). In this view the protein of interest could also play a role in the maturation of other COQ polypeptides. At the beginning the protein (devoid of the N-terminal mitochondrial targeting sequence) was expressed in E.Coli attached to a 6 His-Tag and a consensus for enterokinase (DDDK). We purified the protein using a Ni affinity cromatografy on an AKTA-FPLC and a gel filtration chromatography, in order to increase the purity. The yield and the purity of recombinant human COQ4 were satisfactory. Biochemical characterization of the protein was carried out using different approaches like: circular dichroism (CD), analysis of zinc content, blue native gel, and electron microscopy. We found by CD that COQ4 binds zinc, increasing the stability of the recombinant protein, but the addition of precursors of the quinone ring, or of soluble CoQ analogues had no effect. We observed both by BN-PAGE and by gel filtration that our protein forms a high molecular weight complex, a multimeric complex comprised of 5-6 monomers. Altogether these data provide new insights on the role of COQ4 in CoQ biogenesis. Further work will be aimed at obtaining crystals in order to solve the three-dimensional structure of the protein.

References 1. Brea-Calvo G, Haack TB, Karall D, Ohtake A, Invernizzi F, Carrozzo R, Kremer L, Dusi S, Fauth C, Scholl-Bürgi S, Graf E, Ahting U, Resta N, Laforgia N, Verrigni D, Okazaki Y, Kohda M, Martinelli D, Freisinger P, Strom TM, Meitinger T, Lamperti C, Lacson A, Navas P, Mayr JA, Bertini E, Murayama K, Zeviani M, Prokisch H, Ghezzi D. 2015. The American Journal of Human genetics. COQ4 mutations cause a broad spectrum of mitochondrial disorders associated with CoQ10 deficiency. 2. Marbois B., Gin P., Gulmezian M., Catherine F. Clark CF. 2008 BBA, The yeast Coq4 polypeptide organizes a mitochondrial protein complex essential for coenzyme Q biosynthesis 3. Casarin A., Jimenez-Ortega JC., Trevisson E., Pertegato V., Doimo M., Ferrero-Gomez ML., Sara Abbadi S., Artuch R., Quinzii C., Hiran M., Basso G., Santos Ocaña C., Navas P., Salviati L. 2008 BBRC; Functional characterization of human COQ4, a gene required for Coenzyme Q10 biosynthesis.

77 P20. UBIQUINOL SUPPLEMENTATION POSITIVELY CORRELATES WITH REDUCED SKELETAL MUSCLE DAMAGE IN PROFESSIONAL FOOTBALL PLAYERS

I. Navas-Enamorado1,2, A. Sánchez-Cuesta1, P. Bjork2, J. del Rio2, T. Viar3, J. Lekue3, F. Angulo3, P. Navas1, G. López-Lluch1

1 Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-CSIC-JA, and CIBERER, Instituto de Salud Carlos III, Sevilla, Spain 2 100% Natural, Madrid, Spain 3 Athletic Club Bilbao, Bilbao, Spain

Ubiquinol is the reduced form of coenzyme Q10 (CoQ10), which is a lipid electron carrier that drives electrons from complex I, and complex II to complex III in the mitochondria respiratory chain, which is required for the synthesis of most of the ATP in cells. Deficiency of CoQ10 causes severe neuromuscular diseases particularly severe myopathies that courses with muscle damage that can reach rhabdomyolysis [1]. Professional football players have extreme physical activities during the season through the daily trainings but also by the regular games, which are really competitive and mainly when these players participate in different national and international leagues as it happens to Athletic Club Bilbao, Spanish First League Team. In order to study if a higher concentration of ubiquinol in blood would help the performance of players, we design a protocol to supply a blind selection of players with 300 mg ubiquinol/day (Kaneka Ubiquinol) and different extractions to study the evolution of blood content: 1) pre- season starting, final of the training pre-seasonal period, mid-season and final-season. Levels of ubiquinol in plasma after the pre-season was decreased relative to the basal content after vacations indicating that endurance exercise induced a decrease in blood as it was previously demonstrated in healthy young people [2]. The middle and final season extractions demonstrated that some of players were taken ubiquinol because these ones showed a significant increase independent of the exercise endurance preventing the decrease previously observed. The analysis of plasma demonstrated that cholesterol and triglycerides were unaffected during all the study and we observed a light increase of lipoperoxides as it was previously observed in non-professional young players short-term study [3], but changes do not correlate with ubiquinol levels of players activities. We also analyzed creatine kinase (CK) as a marker of skeletal muscle damage and found that there was a negative correlation between the levels of both ubiquinol and CK in the player’s plasma of the third extraction indicating that ubiquinol would protect skeletal muscle cells from the extreme exercise caused by both daily training and weekly game.

References 1. Desbats, M.A., et al., Genetic bases and clinical manifestations of coenzyme Q10 (CoQ 10) deficiency. J Inherit Metab Dis, 2015. 38(1): p. 145-56. 2. Del Pozo-Cruz, J., et al., Physical activity affects plasma coenzyme Q10 levels differently in young and old humans. Biogerontology, 2014. 15(2): p. 199-211. 3. Tauler, P., et al., Supplementation with an antioxidant cocktail containing coenzyme Q prevents plasma oxidative damage induced by soccer. Eur J Appl Physiol, 2008. 104(5): p. 777-85.

78 P21. IDENTIFICATION OF POPULATION SUBGROUPS WITH CRITICAL UBIQUINOL STATUS

Simone Onur1, Alexandra Fischer1, Petra Niklowitz2, Thomas Menke2, Frank Döring1

1Institute of Human Nutrition and Food Science, University of Kiel 2Children’s Hospital of Datteln, University of Witten/Herdecke

Ubiquinone and ubiquinol are lipid soluble antioxidants that are synthesized endogenously in almost all species and function as an obligatory cofactor of the respiratory chain. The reduced form of Coenzyme Q10 (CoQ10), ubiquinol, serves as a potent antioxidant in mitochondria and lipid membranes. To identify population subgroups with critical ubiquinol status we established a large cohort containing more than 1900 persons. First, we examined the activity of the serum gamma- glutamyltransferase (GGT), a potential early and sensitive marker of inflammation and oxidative stress. We found a strong positive association between CoQ10 status and serum GGT activity in our cohort. However, a gender-specific examination revealed differences between male and female volunteers regarding the association between CoQ10 status and serum GGT activity. Interestingly, ubiquinol supplementation caused a significant decrease in serum GGT activity. Thus, we provide evidence that higher ubiquinol levels improve oxidative stress via reduction of serum GGT activity in humans. We also investigated the relationship between serum levels of CoQ10 and NT-proBNP, a biomarker for chronic heart failure, in healthy volunteers of an elderly study population (mean age 52 years, n=871), a subpopulation from our cohort. We found a negative association between serum levels of ubiquinol and NT-proBNP (p<0.001). Accordingly, the CoQ10 redox state (% oxidized form of CoQ10) is positively associated with serum NT-proBNP level (p<0.001). Compared to patients who survived a myocardial infarction, healthy subjects have lower NT-proBNP level (500.39±631.28 pg/ml vs. 76.90±120.27 pg/ml, p<0.001), higher ubiquinol serum level (0.43±0.19 µmol/l vs. 0.71±0.32 µmol/l; p<0.001) and a lower CoQ10 redox state (27.6±13.8% vs. 17.6±10.1%; p<0.001). In summary, higher serum levels of ubiquinol are associated with lower serum NT-proBNP levels in healthy elderly subjects. However, to what extent a high serum level of ubiquinol is a protective factor for heart failure remains to be elucidated in prospective studies.

This study is supported by Kaneka Corporation, Japan

79 P22. THE EFFECT OF DIETARY INTAKE OF COENZYME Q10 ON SKIN PARAMETERS AND CONDITION: RESULTS OF A PLACEBO-CONTROLLED HUMAN STUDY

Tina Pogačnik, Janko Žmitek, Katja Žmitek

VIST - Higher School of Applied Sciences, Gerbičeva 51a, Ljubljana, Slovenia email: [email protected]

Background Coenzyme Q10 (CoQ10) is a natural antioxidant with an important role in cellular bioenergetics. It is known that CoQ10 levels in skin decline with age and therefore CoQ10 is being used as a functional ingredient of both cosmetics and foods in order to reduce signs of ageing. As shown by several in vitro experiments, CoQ10 is able to protect skin from reactive oxidative species (ROS), reduce DNA damage triggered by UVA irradiation, inhibit MMP-1 enzymes that degrade extracellular matrix components, accelerate the production of epidermal basement membrane components, and decrease levels of superoxide generation by ArNOX proteins.1-4 While the beneficial effect of topical application of CoQ10 on ageing skin has been shown in some controlled studies, the existing data about the effect of dietary intake of CoQ10 on skin parameters and condition is scarce. To gain an insight into this issue, we conducted a placebo-controlled experiment with 33 healthy volunteers.

Methods A randomised, placebo-controlled, double-blind study was conducted in 33 healthy female volunteers aged 52.6±4.2 (SD) years, with skin phototypes II and III (Fitzpatrick classification). Subjects were randomly assigned to a placebo group, a low-dose (LD) group receiving 50 mg CoQ10/day and a high-dose group (HD) receiving 150 mg CoQ10/day (11 subjects per group). The placebo or CoQ10 were administered in the form of a syrup; all subjects consumed 5 mL of syrup daily for 12 weeks. A water-soluble form of CoQ10 with improved bioavailability (Q10vital®, Valens Int. d.o.o., Slovenia) was used.5 Wrinkle, evenness and microrelief analysis, expert evaluations and self-evaluations of face skin conditions were conducted before supplementation (the baseline), after 6 and after 12 weeks of supplementation. In addition, the photoprotective potential of CoQ10 was evaluated with a determination of the minimal erythema dose (MED) before (the baseline) and after 12 weeks of the supplementation. The study was approved by a local ethics committee and all volunteers gave their informed consent.

Results Thirty-two subjects completed the study (11 in the LD group, 11 in the placebo group, 10 in the HD group). Preliminary results of the expert evaluations and measurements show there was a significant improvement in skin evenness in both groups receiving CoQ10 compared to the placebo group, as it was observed for 70% of subjects in the HD group and 82% of subjects in the LD group in contrast to 0% of subjects in the placebo group. In the HD and SD groups, the microrelief also became notably smoother (in 60% and 63% of volunteers, respectively) in comparison to the control group (9%). Periorbital wrinkles became less expressed in the HD group (60% of volunteers), while in the LD and placebo groups no significant changes in periorbital wrinkles were observed. Each subject completed a self-assessment questionnaire evaluating efficacy attributes using a 5-point continuous scale at the baseline and after 12 weeks of CoQ10 supplementation. The results are in accordance with the expert assessments.

80 After 12 weeks, subjects in the HD and LD groups noticed a significant improvement in skin evenness and firmness as the change from the baseline was 1.10±0.23 (Mean±SEM) for the LD group and 0.50±0.16 for the HD group for evenness, and 1.10±0.28 and 0.45±0.28 for firmness, while no significant changes in either attribute were noticed in the placebo group (0.1±0.18; 0.09±0.16). There were no significant changes in determined MED values between the control and LD/HD groups.

Conclusions The study shows that long-term oral CoQ10 supplementation improves some skin conditions, particularly evenness, firmness and subsequently the skin microrelief. Effects are dose-related as a higher dose (150 mg CoQ10/day) proved more effective than a lower (50 mg/day) dose. In the HD group, periorbital wrinkles were also significantly reduced, while those effects were not observed for the lower-dose and placebo groups. However, it can be speculated that a similar effect on wrinkles is likely to show over a longer supplementation period. On the other hand were unable to demonstrate the photoprotection potential of CoQ10 supplementation.

References 1. H. Masaki, Role of antioxidants in the skin: Anti-aging effects. J. Dermatol. Sci., 2010, 58, 85-90. 2. Y. Ashida, Inhibitory effects of Coenzyme Q10 on skin aging. In: Nutritional cosmetics: Beauty from within. A. Tabor, R. M. Blair (eds.) (William Andrew Inc, 2009), pp. 199-215. 3. D. H. Morre, D. J. Morre, W. Rehmus, D. Kern, Supplementation with CoQ10 lowers age-related (ar) NOX levels in healthy subjects. BioFactors, 2008, 32, 221-230. 4. M. Inui, M. Ooe, K. Fujii, H. Matsunaka, M. Yoshida, M. Ichihashi, Mechanisms of inhibitory effects of CoQ10 on UVB-induced wrinkle formation in vitro and in vivo. BioFactors, 2008, 32, 237-243. 5. J. Zmitek, A. Smidovnik, M. Fir, M. Prosek, K. Zmitek, J. Walczak, I. Pravst. Relative bioavailability of two forms of a novel water-soluble coenzyme Q10. Ann. Nutr. Metab. 2008, 52, 281-287.

81 P23. INVERSE METABOLIC ENGINEERING OF SPORIDIOBOLUS JOHNSONII ATCC-20490 FOR IMPROVED PRODUCTION OF COENZYME Q10 AND ITS PROCESS OPTIMIZATION

Prafull Ranadive, Alka Mehta and Saji George

School of Bio Sciences and Technology, VIT University, Vellore-632014, Tamil Nadu, INDIA e-mail: [email protected]

Coenzyme Q10 (CoQ10) is an industrially important molecule having nutraceutical, pharmaceutical and cosmeceutical applications. CoQ10 is mainly produced by microbial fermentation; hence the process demands the use of microbial strains with high productivity and yields of CoQ10 [1]. Sporidiobolus johnsonii ATCC 20490, a heterobasidiomycetes yeast strain was selected for generating mutant phenotypes with high CoQ10 content, as a part of doctoral studies. This strain becomes a promising source of natural CoQ10 derived from eukaryote kingdom, having high degree of conservation with mammalian system and its biosynthetic pathways [1]. With the first report, an atorvastatin resistant mutant strain-UF16 (phenotype) was generated from S. johnsonii ATCC 20490 by sequential induced mutagenesis, rational selection and screening process, resulting in 2.3-fold improvement in CoQ10 content [2, 3]. The glycerol and tryptone based -1 -1 medium was designed for optimum expression of CoQ10 (13.35 mg l , 0.834 mg g ) by UF16. UF16 showed improved flux of isoprene precursors on the mevalonate pathway leading to overproduction of CoQ10 (2.3-fold), ergosterol (2.6-fold), and total carotenoids (1.4-fold), whereas down-regulation of fatty acid biosynthetic pathway was observed. The HMG-CoA reductase gene (HMG1), which is a gene coding for the rate limiting enzyme in isoprenoid pathway, was partially sequenced and reported for the first time in Sporidiobolus strain. No mutations were detected on this gene but it was found to be up-regulated in UF16 during submerged fermentation, as seen from the gene expression studies using RT-qPCR. Overall, acquired atorvastatin resistance in UF16 due to induced mutagenesis facilitated the overexpression of HMG-CoA reductase gene, resulting in improved flux of isoprene units to key downstream metabolites like CoQ10 leading to overproduction [Unpublished]. The fermentation and downstream purification process was optimized for production of CoQ10 by UF16. Under optimized fermentation condition in laboratory fermentor (10L), UF16 produced -1 -1 20.90 mg l of CoQ10 having specific CoQ10 content of 0.86 mg g of DCW. Further process improvement by precursors feeding is on-going. The CoQ10 was extracted and isolated from UF16 using chromatographic separations with ~95% purity and characterized by UV, Mass and 1H NMR spectroscopy. Ergosterol and carotenoid pigments were isolated as a by-products of this fermentation process [Unpublished]. Investigating mutations and gene expressions from whole CoQ10 pathway genes of novel strain- UF16 is on-going, as a post-doctoral research work, in order to design the inverse metabolic engineering strategy for further improvement of CoQ10 and other terpenoids production in S. johnsonii. The outcome of this academic research may lead to an industrial process for commercial production of CoQ10 derived from eukaryote kingdom.

References 1. Makoto Kawamukai (2009) “Biosynthesis and International Journal of Chemical Engineering and bioproduction of Coenzyme Q10 by yeasts and other Applications. June;Vol. 2, No.3 :216-220. organisms”, Biotechnol Appl. Biochem, 53:217–226 3. Ranadive, P., A. Mehta, Y. Chavan, A. Marx and S. DOI:10.1042/BA20090035. George (2014). Morphological and molecular 2. Ranadive, P., A. Mehta and S. George (2011). “Strain differentiation of Sporidiobolus johnsonii ATCC 20490 improvement of Sporidiobolus johnsonii ATCC 20490 and its coenzyme Q10 overproducing mutant strain UF16. for biotechnological production of coenzyme Q10.” Indian J Microbiol. Sep;54(3):343-57 DOI 10.1007/s12088-014-0466-8, Springer Publication.

82 P24. MODELING COENZYME Q10 DEFICIENCY USING IPS CELLS

Damià Romero-Moya1, Carlos Santos-Ocaña2, Patricia González-Rodríguez3, Jose A. Rodríguez-Gómez3, Iván Velasco1, Julio Castaño1, Alessandra Giorgetti1, Mario F Fraga4, Agustín Fernández4, Clara Bueno1, Jose López-Barneo3, Plácido Navas2, Pablo Menendez1,5

1Josep Carreras Leukemia Research Institute. University of Barcelona. Barcelona. Spain.2Centro Andaluz de Biología del Desarrollo. Universidad Pablo de Olavide. Sevilla. Spain.3Instituto de Investigación Biomédica de Sevilla (IBIS). Sevilla. Spain.4Instituto Oncológico del principado de Asturias (IUOPA). Oviedo. Spain.5Institucio Catalana de Recerca i Estudis Avançats (ICREA). Barcelona. Spain. Contact: [email protected]

Primary Coenzyme Q10 (CoQ10) deficiency is a rare autosomal metabolic syndrome, which originates from mutations in genes responsible for CoQ biosynthesis. It is clinically and genetically heterogeneous and begins during childhood leading to a disorder associated to encephalopathies, seizures, myopathies and ataxias, all clinical features affecting tissues with high-energy demand such as brain and skeletal muscle. Mutations in COQ4 causes a very variable pathological phenotype associated to CoQ10 deficiency (Brea-Calvo et al. 2015). We recently diagnosed a 4-year old patient with CoQ10 deficiency carrying a novel heterozygous c.483 G>C (E161D) mutation in COQ4 gene. Metabolically, patient’s fibroblasts showed a 75% and 87% reduction in the concentration of CoQ10 and the rate of CoQ10 biosynthesis, respectively, and a mitochondrial respiratory chain (MRC) defect of 25% and 54% in complexes I+III and II+III, respectively. Clinically, the patient presented weakness, mild psychomotor retardation, clumsiness, drooling and myoglobinuria due to a massively elevated levels of creatine kinase in serum with eventual lethal rhabdomyolysis. Our understanding of the functional consequences of CoQ10 deficiency in the affected tissues is very limited. The generation of patient-specific induced pluripotent stem cells (iPSC) is a unique system to model human disease. Here, we generated iPSCs from the diagnostic COQ4 mutated primary fibroblasts. Multiple iPSC clones harboring the c.483 G>C mutation were generated using OKSM-expressing Sendai virus (SeV). iPSC efficiency was 0,17% and 0,8%, respectively, for CQ4-mutated and aged-matched control fibroblasts. Several clones were fully characterized and after >20 passages resulted to be transgene-independent bona fide iPSC as demonstrated by teratoma formation, expression of pluripotency surface markers and transcription factors, demethylation of pluripotency genes, and normal karyotype. Interestingly, metabolic analyses revealed that COQ4-iPSCs retained CoQ10 deficiency but reestablished the MRC function, likely reflecting the glycolytic nature of iPSC. To elucidate the pathogenesis and developmental impact of primary CoQ10 deficiency, iPSCs were differentiated into disease-affected tissues. iPSCs were re-differentiated into motor neurons (Amoroso et al. 2013) and dopaminergic neurons (Kriks et al. 2011). By confocal analyses and RT-PCR no differences were observed between COQ4- and Ctrl-iPSC in their specification into neural progenitors (NESTIN+SOX2+), total neurons (TUJ1+), motor neurons (TUJ1+HB9+ or ISL1+), and dopaminergic neurons (TUJ1+TH+). Although dopamine release and electrophysiology experiments are underway, this iPSC model does not seem to reproduce the mild neurologic phenotype observed in the patient. This is supported by the diagnosis 4 years later of a sister with the same neurological symptoms but lacking COQ4 mutation, which indicates that the COQ4 mutation is not uniquely responsible of this disease phenotype. We are currently differentiating the iPSCs into skeletal muscle (SKM) which is the most affected tissue in this patient who suffered a lethal rhabdomyolysis. Transgenic iPSCs expressing the inducible PAX7, an early pan-SKM factor, have been generated for SKM differentiation. The generation of PAX7+ SKM precursors and their proliferation and differentiation potential (MHC+MYOGENIN+) will be assessed as per Darabi et al. 2012. iPSC-derived SKM will also be metabolically characterized (CoQ10 levels and mitochondrial complexes I+III and II+III).

83 Release of creatine kinase will also be measured from Ctrl- and COQ4-iPSCs as an indirect measure of SKM damage/rhabdomyolysis. Finally, we will attempt to repair the c.483 G>C mutation using the CRISPR/Cas9 system to rule out the potential effect of inter-individual variation by using syngenic iPSCs.

84 P25. UBIQUINOL REDUCES PLASMA OXIDATIVE STRESS, HYPOXIA AND THE THICKNESS OF THE MICROVASCULATURE BASEMENT MEMBRANES IN THE HIPPOCAMPUS OF THE 3xTg-AD MOUSE MODEL OF ALZHEIMER DISEASE Francisco J Sancho-Bielsa1*, Javier Frontiñan1*, Raquel Santiago-Mora1, Juan Ramón Peinado1, Lydia Gimenez-Llort2, Mario Durán-Prado1 and Francisco J. Alcaín1#

1Dept. of Medical Sciences, Faculty of Medicine of Ciudad Real, University of Castilla la Mancha, Spain. 2 Dept. Psychiatry and Forensic Medicine. Institut of Neurosciencies Institut. University Autonoma of Barcelona, Spain. [email protected]

Numerous structural and functional abnormalities in the cerebral microvasculature have been described in animal models of Alzheimer disease (AD), some of them associated to a disturbance of the oxidative balance to pro-oxidant conditions. Soluble circulating amyloid β-peptide (Aβ) damages endothelial cells in asymptomatic Alzheimer’s stages preceding Aβ deposition and a thickness of the epithelial basal membrane and a greater collagen-IV deposition occurs around microvasculature, probably affecting soluble exchanges. The goal of our work is to analyze the role of ubiquinol in the homeostasis of the cerebrovascular system. In vitro, the oxidized form, ubiquinone, prevented Aβ-induced damage in human umbilical vein endothelial cells (HUVECs). The deleterious effect of Aβ to HUVECs depended on Ca2+ release from mitochondria followed by necrosis and apoptosis. Pretreatment with CoQ prevented these deleterious effect due to the inhibition of Aβ entry and accumulation into endothelial cell mitochondria. The 3xTg-AD mice mimic critical hallmarks of the human disease with a temporal- and regional specific profile. When mice were orally supplemented with ubiquinol from 3 month-old to sacrifice at 12 months-old, the concentration of coenzyme Q10 in plasma increased from 0.2-0.3 pmoles/μg protein to 2.5-3.3 pmoles/μg protein. Ubiquinol was stabilized with the Kaneka QH P30 powder. In this model, 12 months-old mice present high level of oxidative stress in plasma compared to age-matched no- transgenic (NTg) mice. No differences were found between genders. Ubiquinol treated 3xTg-AD mice showed levels of carbonyl groups and lipid peroxidation in plasma, similar to NTg animals. No change was detected in the level of oxLDL. In addition, oxidative stress in leucocytes, an accurate peripheral indicator of oxidative stress in brain, increased in 3xTg-AD mice compared to NTg animals. In ubiquinol-treated animals, oxidative stress decreased to values close to NTg animals. Conversely, Kaneka QH P30 powder also reduced these parameters but a lesser extent than ubiquinol. Noteworthy, 3xTg-AD females displayed higher levels of Aβ-plaques in the hippocampal area than males. Areas with higher Aβ burden also showed hypoxic regions that colocalize with Aβ plaques. In ubiquinol-treated females, the burden of Aβ were reduced significantly and almost non-hypoxic regions were detected. Moreover, a greater collagen-IV deposition occurred around vessels in both, 3xTg-AD males and females, independently of the Aβ burden. No differences were found among genders for this parameter, but in ubiquinol-treated animals collagen-IV deposition in basal membrane was similar to NTg animals. All these parameters were also reduced in 3xTg-AD mice treated with Kaneka QH P30 powder but at a lesser extent. Overall, the present results in 3xTg-AD mice reveal disruption of the homeostasis of hippocampal microvasculature and the preventive benefits of ubiquinol.

Acknowledgement: This study was supported by Kaneka Corporation, Japan.

85 P26. UBIQUINOL-10 DECELERATES SENESCENCE AND AGE-RELATED DISORDERS VIA THE ACTIVATION OF THE AMPK-SIRT1 PATHWAY AND THEIR DOWNSTREAM MOLECULES

Sawashita Jinko1,2, Tian Geng2, Xu Zhe2, Kubo Hiroshi3, Tsuruoka Mineko3, Hosoe Kazunori4, and Higuchi Keiichi1,2

1Department of Biological Sciences for Intractable Neurological Diseases, Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Matsumoto, Japan. 2Department of Aging Biology, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, Matsumoto, Japan. 3BioTechnology Development Laboratories, Kaneka Corporation, Hyogo, Japan. 4Functional Food Ingredients Marketing Group, QOL Division, Kaneka Corporation, Osaka, Japan. e-mail; [email protected]

1 Coenzyme Q10 (CoQ10) or Q9 (CoQ9) levels decrease with age in many human and rodent tissues . Ubiquinol-10, which is the reduced form of CoQ10, has a greater reducing power compared with its oxidized form, and it is the predominant form present in the body. Thus, ubiquinol-10 is expected to have more beneficial effects against oxidative stress and age-related diseases than its oxidized form. In fact, we previously showed that supplementation with ubiquinol-10 significantly delayed senescence in Senescence-accelerated mouse prone-1 (SAMP1) mice, and ubiquinol-10 functioned more effectively compared with its oxidized form2,3. In our recent study, we found that ubiquinol-10 can also delay progression of age-related hearing loss in SAMP1 mice, and that ubiquinol-10 may enhance mitochondrial activity in the liver and the inner ear by increasing levels of sirtuins and their downstream molecules such as PGC-1α, SIRT3, SOD2, and IDH24-6. Here, we evaluated the hypothesized mechanisms of ubiquinol-10 using the human hepatoma HepG2 cell line. Cells were treated with solubilized ubiquinol-10 (Kaneka Corp., Osaka, Japan) at 0.5 - 10 µM and incubated for 48 h. Ubiquinol-10 decreased production of reactive oxygen species (ROS) and reduced the cell-senescence marker SA-β Gal. Ubiquinol-10 also increased mitochondrial metabolic activity via activations of SIRT1 and its downstream molecule peroxisome proliferator-activated receptor coactivator 1α (PGC-1α). Ubiquinol-10 increased the level of the upstream molecule AMP-activated kinase (AMPK). These results suggest that ubiquinol-10 may increase the levels of AMPK and its downstream sirtuins, and then activate further downstream molecules. These molecules enhance mitochondrial activity and protect against the progression of aging and symptoms of age-related diseases4.

Note: Additional new data will be reported in another poster presented by our colleague, Z. Xu (First presenter).

References 1. Kalen A, Appelkvist EL, and Dallner G. (1989) Age-related changes in the lipid compositions of rat and human tissues. Lipids 24:579-84. 2. Yan J, Fujii K, Yao J, et al. (2006) Reduced coenzyme Q10 supplementation decelerates senescence in SAMP1 mice. Exp Gerontol 41:130-40. 3. Schmelzer C, Kubo H, Mori M, et al. (2010) Supplementation with the reduced form of coenzyme Q10 decelerates phenotypic characteristics of senescence and induces a peroxisome proliferators-activated receptor-α gene expression signature in SAMP1 mice. Mol Nutr Food Res 54:805-15. 4. Tian G, Sawashita J, Kubo H, et al. Mechanisms of reduction of age-related disease in senescence-accelerated strain (SAMP1) mice by supplementation with reduced form of coenzyme Q10. (2012) Programme and Abstracts of the 7th conference of the international coenzyme Q10 association, Seville, Spain 53-6. 5. Sawashita J, Tian G, Kubo H, et al. Diets supplemented with reduced coenzyme Q10 decreased age-related senescence and hearing loss in senescence-accelerated mice prone 1 (SAMP1) mice. (2012) Programme and Abstracts of the 7th conference of the international coenzyme Q10 association, Seville, Spain 127-8. 6. Tian G, Sawashita J, Kubo H, et al. (2014) Ubiquinol-10 supplementation activates mitochondria functions to decelerate senescence in senescence-accelerated mice. Antioxid Redox Signal. 20: 2606-20.

86 P27. PLASMA MEMBRANE NADH-CoQ REDUCTASE CONNECTS AEROBIC METABOLISM WITH AGEING

Emilio Siendones1,2, Sara SantaCruz-Calvo1,2, Alejandro Martín-Montalvo3, María V. Cascajo1,2, Julia Ariza4, Guillermo López-Lluch1,2, José M. Villalba4, Cécile Acquaviva-Bourdain5, Emmanuel Roze6, Michel Bernier3, Rafael de Cabo3 and Plácido Navas1,2

1Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-CSIC-JA, Sevilla, Spain. 2Centre for Biomedical Research on Rare Diseases (CIBERER), ISCIII, E-41013 Sevilla, Spain. 3Translational Gerontology Branch, National Institute on Aging, NIH, Baltimore, Maryland. 4Departamento de Biologıía Celular, Fisiología e Immunología, Universidad de Córdoba, Córdoba, Spain. 5Service des Maladies Héréditaires du Métabolisme et Depistage Neonatal, Centre de Biologie et de Pathologie, Lyon, France. 6Centre de Recherche de l’Institut du Cerveau et de la Moelle épinière (CRICM), INSERM U975, CNRS UMR 7225, UPMC Université Paris 6 UMR_S 975, Paris, France.

NADH-CoQ reductase (NADH-cytochrome b5 oxidoreductase 3, CYB5R3) is a key component of the trans-plasma membrane redox system (PMRS), which protects against exogenous oxidants. PMRS is also activated in mitochondrial DNA-depleted (rho) cells probably by the inability of these cells to oxidize accumulated NADH in mitochondria. PMRS-CYB5R3 consumes NADH to reduce CoQ producing NAD+, and intracellular NAD+/NADH ratio can be boosted. CYB5R3 deficiency in human causes recessive hereditary methemoglobinemia, an incurable disease characterized by several neurological disorders. We explored the role of CYB5R3 on mitochondria and ageing by analyzing the phenotype exhibited by both CYB5R3-mutant cells from methemoglobinemia patients and CYB5R3-silenced cells. We found that NAD+/NADH ratio, ATP levels and oxygen consumption rate were dramatically decreased and a senescence phenotype is induced in CYB5R3- deficient cells, suggesting a control on mitochondrial homeostasis and a role on ageing. We also found that serum withdrawal, AKT inhibition, hydrogen peroxide or diquat treatment induced CYB5R3 gene and protein expression in concomitant with FOXO3a and Nrf2 activation, respectively. ChIP analysis revealed the cooperation between Nrf2 and FOXO3a to regulate CYB5R3 gene transcription. We’ve established that CYB5R3 gene is regulated by the coordination of both Keap1/Nrf2/ARE and AKT/FOXO3 pathways giving a whole protecting response to both nutrient- and oxidative-dependent conditions. This way, CYB5R3 provides a key regulatory function in the maintenance of cellular health and contributes a new approach to understanding the pathophysiology of recessive hereditary methaemoglobinaemia and other disorders associated with energy depletion and oxidative stress.

87 P28. INCREASE IN COENZYME Q BIOSYNTHESIS IN RAT LIVER EARLY AFTER BIRTH

Spáčilová J., Hůlková M., Sedláčková D., Knopová S., Zeman J., Hansíková H.

Laboratory for Study of Mitochondrial Disorders, Department of Pediatrics and Adolescent Medicine, First Faculty Medicine, Charles University in Prague and General University Hospital in Prague

Mitochondrial biogenesis in mammals is a crucial step of metabolic switch, which proceeds rapidly after birth. Prenatally, the partial oxygen pressure in tissues is lower and metabolism is mainly glycolytic. In hours after birth, metabolism becomes more oxidative with full activation of oxidative phosphorylation (OXPHOS). A proper adaptation to cover ATP demands and antioxidant protection in extremely quickly changing conditions during parturition are necessary for newborn survival. The aim of the study was to analyse (1) coenzyme Q9 (CoQ9) concentration, (2) expression of coenzyme Q2 4-hydroxybenzoate polyprenyltransferase (Coq2) and (3) aarF domain containing kinase 3 (Adck3), an atypical kinase with predicted regulatory rate-limiting role in CoQ biosynthesis, in a rodent model (Norway rat) using late prenatal and early postnatal liver and skeletal muscle tissues. The sets of 53 rat liver and 33 skeletal muscle samples were collected between 16th fetal and 4th postnatal day. The mRNA was quantified by microarray analysis (GeneChip® RNA Rat Gene 1.0 ST; Affymetrix) and qPCR (TaqMan®; Applied Biosystems). The data analysis was accomplished by STEM 1.3.8, TIGR MeV 4.9 (betr), STATISTICA 12 and GenEx. CoQ9 content was analysed by HPLC, OXPHOS activities spectrophotometrically. We identified major biochemical pathways changing during rat development. Out of 1546 mitochondrial genes, 1119 genes (72 %) were significantly changed in liver and 827 genes (53 %) in skeletal muscle. The major expression shift occurred more than two days before birth. Among analysed genes, some genes involved in CoQ biosynthesis were significantly changed in both tissues (Adck3, Coq9), other showed tissue specificity (significant increase of Coq3 and Coq5 expression in liver, whereas Coq4 and Coq6 increase in skeletal muscle). Significant acceleration (p<0.05) in Adck3 expression was validated by qPCR in both tissues (Coq2 remained unchanged). In agreement with the expression data, CoQ9 concentration and coupled OXPHOS activities (I + III, II + III) were increased after birth in rat liver. In rat perinatal liver, we showed that expression of mitochondrial and CoQ-biosynthetic genes is significantly changing. Increasing CoQ9 concentration and OXPHOS activation are probably illustrating acceleration of mitochondrial metabolism and necessary protection from oxidative stress. This might be helpful in the adaptation mechanism needed for transition to extra-uterine conditions after birth.

Supported by: GACR 14-36804G, Centre of mitochondrial biology and pathology (MITOCENTRE), UNCE 204011/2015, SVV UK 260148/2015, RVO-VFN64165, First Faculty of Medicine, Charles University in Prague - GAUK667612.

88 P29. STUDY OF NUTRITIONAL GUIDANCE METHODS CONCERNING COENZYME Q10 INTAKE

Toshikazu Suzuki1,2, Miharu Otani1, Kotomi Kawai1, Michiyo Takahashi2

1Department of Health and Nutrition, Wayo Women’s University, Chiba, Japan 2Graduate School of Human Ecology, Wayo Women’s University, Chiba, Japan

Background As a vitamin like nutrient, reduced form of coenzyme Q10 (Ubiquinol) is a key element in mitochondrial energy production and antioxidant protection. Because Ubiquinol is biosynthesized in the body, daily intake of Ubiquinol is not considered in nutritional guidance or menu planning in Japan. We found that hospitalized older people have lower blood levels of Ubiquinol, but not lower levels of oxidized form of CoQ10 (Ubiquinone). It might be caused by a decreased intake of Ubiquinol from food/enteral nutrition compared to healthy older people, suggesting the possibility that adequate intake of Ubiquinol maintains wellness in older people. In this study, we designed a food intake guide for ingestion of increased amounts of Ubiquinol and we evaluated the usability in a diet intervention trial.

Materials and Methods The participants were 24 women, aged 20–30 years who studied nutrition/dietetics. The amounts of food consumption were measured by weight, and nutrient intakes were calculated using computer software (Eiyo Navi). The intake of Ubiquinol was calculated manually using the published data of Ubiquinol contents of 70 food items. Three-day averages of Ubiquinol intake before and after the food intervention were compared.

Results The average calorie intakes before and after the intervention were 1595 ± 251 and 1576 ± 258 kcal/day, and the values were 98.0% and 96.8% of the mean value of women in their 20s in the National Health and Nutrition Survey 2014 in Japan, respectively. Protein, fat, and carbohydrate (PFC) balance of the participants’ diets were within the Dietary Reference Intakes for Japanese 2015. Ubiquinol intake before and after intervention was 3.4 ± 1.3 and 3.8 ± 1.3 mg/day, respectively. Fifteen out of 24 participants succeeded in increasing Ubiquinol intake after the intervention, although the increase was not statistically different.

Discussion Food intervention using our food intake guide may be effective in increasing Ubiquinol intake from food while maintaining PFC balance. However, the guide worked differently for different participants. For example, intake of food items according to food intervention was sometimes a burden to the participants. Development of improved intervention methods using a Ubiquinol- fortified food may be necessary for consuming adequate amounts of Ubiquinol each day more easily, especially for older people.

89 P30. LONG-TERM HOSPITALIZED OLDER PEOPLE HAVE LOWER COENZYME Q10 BLOOD LEVELS THAN NON-HOSPITALISED OLDER PEOPLE

Michiyo Takahashi1, Toshikazu Suzuki1,2, Hikaru Matsumoto1,2, Hitoshi Nukada3, Naotaka Hashizume4

1 Graduate School of Human Ecology, Wayo Women’s University 2 Department of Health and Nutrition, Wayo Women’s University 3 The Nukada Institute for Medical and Biological Research 4 Department of Health and Nutrition, University of Human Arts and Sciences

Background The reduced form of Coenzyme Q10 (Ubiquinol) has attracted much attention as a supplement for anti-aging and improvement of metabolism. It is considered a vitamin-like nutrient, and can be synthesized by the body. However, up to 40% of CoQ10 in the body is thought to be derived from the diet. Therefore, intake of Ubiquinol from foods and/or dietary supplements is important, especially in older persons whose ability to synthesize Ubiquinol seems to decline. There is no report comparing the trophic state of CoQ10 between hospitalized and non hospitalized older people. In the present study, we determined the blood levels of CoQ10 of long-term hospitalized and non hospitalized older people with their nutritional status.

Materials and methods The sample of participants comprised 15 aged people (4 men and 11 women, mean age 76.5 ± 6.2 years) who were hospitalized long-term. Thirty healthy non hospitalized older people (9 men and 21 women, mean age 77.6 ± 6.5 years) were also analyzed as controls. The long-term hospitalized older people were divided by mode of obtaining nourishment into oral- and enteral- feeding inpatients. Body composition, estimated nutrient intakes, and blood levels of albumin and CoQ10 were measured.

Results The long-term hospitalized aged people were considered malnourished based on body composition assessment, estimated nutrient intakes, and serum albumin levels. Non hospitalized older people were assessed as well-nourished. The blood levels of total CoQ10 of oral- and enteral- feeding inpatients were 59.7% and 47.0% lower, respectively, as compared to those of non hospitalized people. Redox ratio of CoQ10 (% of Ubiquinol versus total CoQ10) in serum of non hospitalized older people, oral- and enteral-feeding inpatients were 96.4%, 91.4%, and 88.3%, respectively.

Discussion Both the blood levels and redox ratios of CoQ10 (% of Ubiquinol ratio versus total CoQ10) in long-term hospitalized older people were lower than those of non hospitalized older people, and the values of enteral-feeding inpatients were the lowest among the three groups. These results imply the possibility not only of a decline in the synthesis but also that reduced food intakes that affect blood levels of Ubiquinol, and that hospitalized older people were under more intense stress than non hospitalized older people. Intake of sufficient amounts of Ubiquinol from dietary supplements and/or enteral formulae might be effective for preventing deterioration in quality of life of long- term hospitalized older people.

90 P31. EFFECT OF UBIQUINOL ON REPRODUCTIVE HORMONES OF AMENORRHEIC PATIENTS: A HOSPITAL BASED STUDY

A.S. Thakur1, I. Funahashi3, U.S.Painkara4, N. S. Dange5, P. Chauhan6

1. Corresponding Author, Prof and Head, Dept. of Biochemistry, Lt. BRKMGMCJ, C.G, India.. 2 Director, Research, Kaneka Corporation., Japan, 3 Dean, Lt. BRKM Govt. Medical College, Jagdalpur (C.G), India. 4 Associate Prof. Dept. of Biochemistry, Lt. BRKMGMCJ, C.G, India. 6 Associate Prof. Dept. of Gynecology, Lt. BRKMGMCJ, C.G, Inida .

Organisms survive by maintaining a dynamic equilibrium with their environment. (1) Oxidative stress, a condition defined as unbalancing between production of free radicals and antioxidant defenses, is an important pathogenetic mechanism in different diseases. (2) Glucocorticoids released from the adrenal gland in response to stress–induced activation of the hypothalamic–pituitary adrenal (HPA) axis induce activity in the cellular reduction–oxidation (redox) system. Glucocorticoids induce neuronal oxidative stress directly through enhanced mitochondrial respiration and oxidative phosphorylation. The increase in oxidative status is produced by a glucocorticoid–dependent and transcriptional increase in pro-oxidative drive, with concurrent inhibition of the antioxidant defense system, ultimately leading to increased neuronal cell death. These observations highlight that maintenance of a balanced redox state is critical for normal cellular homeostatic function within the neuroendocrine system.(3) Functional hypothalamic disturbances and neuroendocrine aberrations have both short and long term consequences for reproductive health. Understandably, an impaired or diminished hypothalamic–pituitary–ovarian axis leads to anovulation and hypoestrogenism. Lack of cyclical changes of estradiol and progesterone concentrations leads to abolished endometrial cyclicity and typically the endometrium is in the persistent early proliferative phase. Anovulation is directly linked to the neurohormonal and hormonal background of FHA(Functional Hypothalamic Amenorrhea). Impairment of pulsatile GnRH secretion causes the impairment of pulsatile LH & FSH secretion (4). Infertility can be defined as a lack of pregnancy after one year of regular unprotected intercourse. Approximately 15 – 20% of couples of reproductive age are infertile, which can be attributed equally to both male and female factors. Recent research on the role of reactive oxygen species (ROS) in human infertility has received a great deal of attention from scientists and medical practitioners, leading to the development of newer therapeutic approaches to treat infertility. (5) Coenzyme Q10 (CoQ10), also known as ubiquinone for its presence in all body cells, is an essential part of the cell energy – producing system. However, it is also a powerful lipophilic antioxidant protecting lipoproteins and cell membranes. Due to these two actions, Ubiquinol (the reduced form of CoQ10) is an active form of CoQ10. It is commonly used in clinical practice in chronic heart failure, infertility, and neurodegenerative diseases. (6) The importance of oxidative stress in various pituitary disorders suggests a possible clinical usefulness of antioxidant molecules like the lipophilic antioxidant Ubiquinol.(2) In order to identify the role of Ubiquinol on reproductive hormones FSH and LH, we identified 50 infertile patients aged between 20 to 40, who were mostly amenorrheic. Out of the 50 only 30 patients were in continuous follow up after supplementation with 150 mg of Ubiquinol every day for 4 months. The hormonal levels were estimated by ELISA technique at follicular phase.

91 The data were observed as follows: Hormone Subjects (Normal Controls After 2 mnths After 4 mnths (0days) range in (Mean ± of Q10 suppl of Q 10 suppl P Value (Mean ± Follicular S.D) (Mean ± S.D) (Mean ± S.D) S.D) Phase) FSH (3.0 to 12.0 7.2 ± 1.61 3.10 ± 2.70 10.08 ± 6.93 10.09 ± 6.95 * mIU/ml) LH 14.83 ± (0.5 to 10.5 5.3 ± 1.45 27.85 ± 22.30 28.02 ± 21.18 * 10.48 mIU/ml) Prolactin 34.71 ± (1.2 to 19.5 9.6 ± 1.61 32.26 ± 19.88 31.03 ± 19.53 ns 27.88 ng/ml) Progesterone (0.15 to 0.70 0.45 ± 0.11 1.72 ± 1.30 2.40 ± 1.45 2.40 ± 1.43 ns ng/ml) *(Significant), ns(Not Significant).

Results suggest that FSH concentration is increased but remains within the normal limit. LH values were found doubled the normal range. Prolactin values were decreased while Progesterone values were high but not in the significant range. This increase in concentration of FSH and LH may be due to reduced oxidative stress by Ubiquinol which could have normalized the function of the hypothalamic–pituitary–ovarian axis. As a pilot study, we are getting promising results; hence the study requires a large number of subjects, which is in our future plan.

References 1. T.M. O’Connor, D.J. O’Halloran and F.Shananhan et. al., The stress response and the hypothalamic –pitutary- adrenal axis: from molecule to melancholia. Q J Med 200; 93:323-333. 2. Mancini A, Festa R, Di Donna V, Leone E, Littarru GP, Silvestrini A, Meucci E, Pontecorvi A. et al., Hormones and antioxidant systems: role of pituitary and pituitary-dependent axes. J Endocrinol Invest. 2010 Jun;33(6):422-33. 3. Jereme G. Spiers, Hsiao-Jou Cortina Chen, Conrad Sernia and Nickolas A. Lavidis. et, al., Activation of the hypothalamic-pituitary-adrenal stress axis induces cellular oxidative stress. Frontiers in Neuroscience.2015 Jan; 8: 456. 4. B. Meczekalski, K. Katulski, A. Czyzyk, A. Podifigurna-Stopa, M. Maciejewska-jeske, et, at., Functional hypothalamic amenorrhea and its influence on women’s health. J Endocrinol Invest 2014; 37: 1049 – 1056. 5. Ashok Agarwal, Shyam S.R. Allamaneni, et, al., Oxidants and antioxidants in human infertility. Middle East Ferility Society Journal 2004; 9:3.

92 P32. COENZYME Q IN THE MITOCHONDRIAL RESPIRATORY CHAIN: INTER-ENZYME CHANNELING VS. POOL BEHAVIOUR

Gaia Tioli1, Yelda Birinci2, Müge Kopuz3, Anna Ida Falasca4, Giorgio Lenaz1 and Maria Luisa Genova1

1 Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, 40126 Bologna, Italy 2 Faculty of Engineering and Natural Sciences, Sabancı University, 34956 Tuzla-Istanbul, Turkey 3 Medical Biochemistry Department, Medicine Faculty of Karadeniz Technical University, 61080 Trabzon, Turkey 4 Department of Pharmacy, University of Parma, 43124 Parma, Italy

A large proportion of the mitochondrial respiratory chain complexes in a variety of organisms is arranged in supramolecular assemblies called supercomplexes (SCs) or respirasomes [1]. However, the factors able to modulate SC association and the mechanisms through which they act are still unclear. In the reconstructed models of the SC I1III2IV1, only few interaction sites exist between the neighbouring protein complexes. Some of the gap volumes may be lipid filled and it is worth noting that cardiolipin, the signature phospholipid of mitochondria, can also integrate into the structure of the respiratory complexes and act as a critical determinant of the respiratory function. Actually, there is growing awareness that free complexes can co-exist with SCs. In this context, the plasticity model has been proposed for the organization of the mitochondrial respiratory chain [2], thus considering the previous opposed models, solid vs. fluid, as two possible extreme situations of a dynamic range of different molecular associations between respiratory complexes. The natural assembly of the respiratory complexes I, III, and IV into supramolecular stoichiometric entities is not just a mere structural feature, but has deep functional implications on the properties of the respiratory chain (reviewed in [3]). The most striking and obvious functional consequence of SC formation is enzymatic channeling, which provides kinetic advantage for the inter-enzyme electron transport mediated by Coenzyme Q (CoQ) molecules bound to the SC. Our studies in proteoliposomes enriched in SC I1III2 and CoQ10 demonstrate that electron transfer between Complex I and Complex III (NADH-cytochrome c oxidoreductase) is more efficient than predicted by a random diffusional behaviour of CoQ10 on the basis of the activities of the individual complexes. Conversely, dissociation of the SC I1III2 into its individual enzyme components (e.g. in vitro, by lipid dilution of the proteoliposomes and by treatment of the mitochondrial membrane with detergents, or in vivo as a consequence of genetic and metabolic alterations) leads to resumption of the less efficient diffusional behaviour of CoQ in the membrane, exerting much lower rates of NADH-cytochrome c oxidoreductase activity and the dependence of electron transfer on the collisional encounters of the free CoQ molecules (pool function) with the partner enzymes [4]. We provide new kinetic evidence that electrons from NADH and from succinate reach cytochrome c through separate pathways that are respectively mediated by SC I1III2 and by free molecules of Complex II/Complex III and that involve separate compartments of CoQ10, being the two streams of electrons independent and additive. Moreover, we suggest that bound CoQ is still in dissociation equilibrium with the CoQ pool, thus exhibiting Michaelis-Menten kinetics but with lower apparent Km compared to the condition of free and randomly distributed respiratory enzymes. This reasoning allows us to conclude that the amount of bound CoQ molecules, at steady state, would be dictated by the size of the quinone pool and that the latter is still necessary in order to guarantee both CoQ compartmentation within the SC I1III2 and efficient channeling of electrons. Accordingly, CoQ10 supplementation is bound to enhance the NADH-dependent respiratory activity of SCs when the endogenous quinone levels in the mitochondrial membrane are impaired.

93 REFERENCES 1. Schagger H. Respiratory chain supercomplexes of mitochondria and bacteria. Biochim Biophys Acta 1555:154-159, 2002 2. Acin-Perez R, Fernandez-Silva P, Peleato ML, Perez-Martos A, and Enriquez JA. Respiratory active mitochondrial supercomplexes. Mol Cell 32:529-539, 2008. 3. Lenaz G, and Genova ML. Structure and organization of mitochondrial respiratory complexes: a new understanding of an old subject. Antioxid Redox Signal 12: 961- 1008, 2010. 4. Maranzana E, Barbero G, Falasca AI, Lenaz G, and Genova ML. Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from Complex I. Antioxid Redox Signal 19: 1469-1480, 2013

94 P33. 2-PHENOXY-1,4-NAPHTHOQUINONE AS A DUAL MOLECULE SIMULTANEOUSLY TARGETING BOTH GLYCOLYSIS AND MITOCHONDRIAL FUNCTION

E. Uliassi(a), F. Prati(a), (b), C. Bergamini(c), R. Amorati(d), A. Berteotti(b), A. Cavalli(a), (b), M. Zaffagnini(e), R. Fato(c), G. Paredi(f), S. Bruno(f), M.L. Bolognesi(a)

(a)Department of Pharmacy and Biotechonology, University of Bologna, via Belmeloro 6, 40126 Bologna, Italy; (b)Department of Drug Discovery and Development, Italian Institute of Technology, via Morego 30, 16163 Genova, Italy; (c)Department of Biochemistry “G. Moruzzi”, University of Bologna, via Irnerio 48, 40126 Bologna, Italy; (d)Department of Organic Chemistry “A. Mangini”, University of Bologna, via S. Giacomo 11, 40126 Bologna, Italy; (e)Department of Experimental Evolutionary Biology, University of Bologna, via Irnerio 42, 40126 Bologna, Italy; (f)Department of Pharmacy, University of Parma, Parco area della Scienza, 27/A, Parma Italy [email protected]

The natural-inspired 2-phenoxy-1,4-naphthoquinone B6 is an attractive anti-trypanosomatid1 and anti-cancer2 lead compound. Performing a through investigation aimed to evaluate its molecular mechanism of action, we disclosed for B6 a multitarget profile directed at both glycolysis and mitochondrial function. In particular, by chemical proteomics and bioenergetics studies, we identified B6 as able to inhibit the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme and to produce reactive oxygen species (ROS) at mitochondrial level.3 To specifically dissect the mechanism(s) underlying ROS production, the capability of B6 to interfere with the mitochondria electron transport chain (ETC) have been evaluated. Interestingly, B6 displayed no effect on Complex I and Complex II, while it was able to generate ROS during respiration in a mitochondrial fraction and additionally to function as NAD(P)H:quinone oxidoreductase 1 (NQO1) substrate. Moreover, the GAPDH inhibition mechanism was further investigated by kinetic studies. These suggested that B6 may act as a covalent GAPDH inhibitor, probably through a cysteine trapping mechanism. This hypothesis is conceivable with the presence in B6 of a Michael acceptor motif, which can act as a warhead and react irreversibly to form a covalent bond. Accordingly, preliminary docking studies3 showed that B6 could undergo a nucleophilic attack from the catalytic Cys166 via three possible mechanisms: I) 1,4 Michael addition with formation of the corresponding thioether-substituted quinone; II) substitution reaction with phenate displacement; III) 1,4 Michael addition followed by elimination of the phenate group. To verify the possible formation of such a covalent adduct and clarify the mechanism by which this occurs, the reactivity of B6 against thiol groups was investigated in a model system and through in silico and biochemical studies. The overall results confirmed that B6 covalently modifies the active site cysteine and suggested the substitution mechanism as the most accredited one. This peculiar mechanism of action makes B6 a promising starting point for the development of more effective compounds targeted to two distinct nodes of cancer or parasite metabolism (glycolysis and mitochondria) and with therapeutic potential against two devastating diseases.

95

References 1. Bolognesi, M. L.; Lizzi, F.; Perozzo, R.; Brun, R.; Cavalli, A. Bioorg Med Chem Lett 2008, 18, 2272-6. 2. Prati, F.; G.; Bergamini, C.; Molina, M.T.; Kaiser, M.; Brun, R.; Fato, R.; Bolognesi, M. L. J Med Chem 2015, (manuscript under revision). 3. Pieretti, S.; Haanstra, J. R.; Mazet, M.; Perozzo, R.; Bergamini, C.; Prati, F.; Fato, R.; Lenaz, G.; Capranico, G.; Brun, R.; Bakker, B. M.; Michels, P. A.; Scapozza, L.; Bolognesi, M. L.; Cavalli, A. PLoS Negl Trop Dis 2013, 7, e2012.

96 P34. FUNCTIONAL ANALYSIS OF ADCK4 MUTATIONS CAUSING CoQ10 DEFICIENCY AND NEPHROTIC SYNDROME

Vazquez-Fonseca L1, Doimo M1, Desbats MA1, Morbidoni V1, Salviati L1

1 Clinical Genetics Unit, Department of Woman and Child Health, University of Padova, Italy

The coenzyme Q biosynthesis pathway has been well studied in yeast and comprises at least 12 COQ genes. COQ8 encodes an atypical protein kinase that appears to be essential for the phosphorylation of several Coq polypeptides. Yeast coq8 mutants lack CoQ6 and are not able to growth in non-fermentable carbon media sources as glycerol. Vertebrates harbor 2 paralogous genes ADCK3 and ADCK4 which display significant similarity with yCOQ8. Mutations in ADCK3 cause CoQ10 deficiency and cerebellar ataxia while mutations in ADCK4 cause a predominantly renal phenotype. yCOQ8 and human ADCK3 and ADCK4 differ in the N-terminus. COQ8 and ADCK4 have a typical mitochondrial targeting sequence, while ADCK3 has a longer sequence, which does not present the characteristics of standard mitochondrial targeting sequences (MTS) and a hydrophobic transmembrane domain. Carbonate extraction experiments show that ADCK4 is a peripheral membrane protein, with a similar behavior to other COQ proteins like COQ4 and COQ5. Moreover, we observed complementation of yeast deletion strain yCOQ8 when the ADCK4 MTS was replaced by the yCOQ3 (or yCOQ8) MTS but not when the MTS was simply added to the N-terminus as in the case of ADCK3. This tool allowed to confirm the pathogenicity of missense ADCK4 mutants identified in patients with CoQ deficiency. Interestingly, there is no clear genotype-phenotype correlation as with other COQ genes like COQ2. Finally, yeast complementation allowed us to examine the functional consequences of a common ADCK4 polymorphism, which was found to significantly affect ADCK4 function. This finding has important implications to explain the pathogenesis of secondary CoQ deficiencies

97 P35. ASSESSMENT OF COENZYME Q10 TRANSPORT ACROSS THE BLOOD BRAIN BARRIER

Luke Wainwright1,3, Jane Preston3, Simon Heales2, Joan Abbott3, Iain Hargreaves1,2

1 Department of Molecular Neuroscience, Institute of Neurology, University College London, London, UK; 2 Neurometabolic Unit, National Hospital of Neurology and Neurosurgery, London, UK; 3 Institute of Pharmaceutical Sciences, King’s College London, London, UK.

Since the first description of human (coenzyme Q10) CoQ10 deficiency in 1989, over 149 cases have been reported, 94 of these presenting with cerebellar ataxia which is the most common clinical presentation of this disorder. Once a deficiency is diagnosed CoQ10 supplementation is commenced. However, only 49% of patients with the cerebellar ataxic phenotype have been reported to show improvement/stabilisation in their ataxic symptoms following supplementation (Emmanuele et al., 2012). The refractory nature of neurological symptoms to treatment has also been reported for the encephalomyopathic phenotype of CoQ10 deficiency (Emmanuele et al. , 2012). The reasons for this remain to be elucidated, however a major contributory factor may be the poor transfer of CoQ10 across the blood brain barrier (BBB) resulting in insufficient CoQ10 availability for the deficient neurons. Currently, there is a paucity of information available on the ability of CoQ10 to cross the BBB and the optimum dose of CoQ10 required to have therapeutic potential in the treatment of the neurological dysfunction associated with this disorder. Using a well-established primary porcine endothelial cell model of the BBB this study evaluates the permeability of the BBB to CoQ10 by determining the degree of transport across this system. Furthermore, the effect of a pharmacologically induced BBB CoQ10 deficiency is assessed on the transport of CoQ10 across the barrier.

References Emmanuele V, López LC, Berardo A, et al. Heterogeneity of Coenzyme Q10 deficiency: Patient Study and Literature Review. Arch Neurol. 2012; 69: 978-83.

98 P36. EFFECTS OF REDUCED COENZYME Q10 TREATMENT ON BONE METABOLISM IN RAT

Hiroyuki Wakabayashi1, Junkichi Kanda1, Nobuo Izumo2, Yoshiko Kobayashi3, Taketoshi Shimakura4, Noriaki Yamamoto4, and Kenji Onodera5

1 Dept. Clin. Pharmacother., Niigata Univ. Pharm. Appli. Life. Sci., 265-1 Higashizima, Akiha-ku, Niigata, 956-8603, Japan. 2 Dept. Clin. Pharmacol., Yokohama Col. Pharm., 601 Matano-cho, Totuka-ku, Yokohama, Kanagawa, 245-0066, Japan. 3 Dept. Rad. Sci., Yokohama Col. Pharm., 601 Matano-cho, Totuka-ku, Yokohama, Kanagawa, 245-0066, Japan. 4 Niigata Bone Sci. Inst., 761 Kizaki, Kita-ku, Niigata, 950-3304, Japan. 5 Lab. Pharmacother. Yokohama Col. Pharm., 601 Matano-cho, Totuka-ku, Yokohama, Kanagawa, 245-0066, Japan.

In nature, there are two forms of coenzyme Q10 (CoQ10), oxidized form (Ubiquinone) and reduced form (Ubiquinol). It is well known that Ubiquinol has an antioxidant protection action, cell activator action, hypotensive action, etc. It is clear that the CoQ10 content in human organ is reduced by aging, thus it is recommended to take Ubiquinol as a supplement. In this study, to evaluate the effects of Ubiquinol on rat bone metabolism, male Wister rats were administered with Ubiquinol (150, 300, 450 mg/kg) orally every day for 12 weeks. As a result, the serum osteocalcin levels in Ubiquinol (450 mg/kg) treated group was significantly higher than in the control group, while significant changes for bone mineral density (BMD) in Ubiquinol treated groups were not found. In histomorphometric analyses of tibia metaphysis, MS/BS, BFR/BV, Oc.S/BS and Oc.N/BS in the both 300 and 450 mg/kg treated groups were significantly increased. In addition to these results, the maximum load and breaking energy in bone strength analysis was significantly increased compared to control group. These results indicate that bone quality was improved by Ubiquinol treatment with accelerated bone turnover.

99 P37. UBIQUINOL-10 MIGHT SUPPRESS THE AGING PROCESS BY INHIBITING TYPE 4 CAMP PHOSPHODIESTERASES

Xu Zhe1, Tian Geng1, Huo Jia1, Kubo Hiroshi2, Hosoe Kazunori3 Higuchi Keiichi1,4 and Sawashita Jinko1,4

1Department of Aging Biology, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, 3-1-1 Asahi Matsumoto, 390-8621 Japan. 2 BioTechnology Development Laboratories, Kaneka Corporation, Hyogo, Japan. 3Functional Food Ingredients Marketing Group, QOL Division, Kaneka Corporation, Osaka, Japan. 4Department of Biological Sciences for Intractable Neurological Diseases, Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Matsumoto, Japan. e-mail; [email protected]

Our recent studies revealed significantly delayed senescence in Senescence-accelerated mouse prone 1 (SAMP1) mice that were fed diets supplemented with the reduced form of coenzyme Q10 (ubiquinol-10). Ubiquinol-10 appeared to achieve this by inducing pathways that activated SIRT1 and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Induction is attributed to ubiquinol-10 increasing the level of cyclic adenosine monophosphate (cAMP) and activating cAMP response element-binding protein (CREB) and AMP-activated protein kinase (AMPK) 1-3). Here, we report that ubiquinol-10 can enhance the survival rate and slow the aging induced by paraquat (PQ) in the human hepatoma HepG2 cell line. Ubiquinol-10 at low concentrations (5μM) upregulated the expression of Sirt1, Sod2 and Ppargc1a to suppress the oxidative stress induced by PQ. Ubiquinol- 10 elevated intracellular cAMP levels and this effect was not completely prevented by MDL- 12,330A, an inhibitor of adenylate cyclase (AC), a key enzyme during cAMP synthesis. Ubiquinol- 10 also suppressed gene expression of the type 4 cAMP phosphodiesterases (Pde4a to Pde4d). Using Promoter Analysis Pipeline (PAP) 4), we found that the transcription factor c-Fos might be involved in transcriptional regulation of the Pde4 genes. Application of ubiquinol-10 to cultured HepG2 cells markedly inhibited the expression of the Fos gene. This change seems to be achieved by the inhibition of voltage dependent Ca2+ channels (VDCCs) on the cell membrane by ubiquinol- 10. In conclusion, this study showed that ubiquinol-10 can suppress the VDCCs to reduce intracellular calcium ion concentration and then impact the expression of the Fos gene. All of these processes led to a decline of Pde4 genes expression, an increase in the intracellular cAMP level and enhancement of Sirt1, Sod2 and Ppargc1a expression. With these changes, cellular anti-oxidation capacity may be improved and senescence may be delayed.

References 1) Yan J, Fujii K, Yao J, Kishida H, et al. Reduced coenzyme Q10 supplementation decelerates senescence in SAMP1 mice. Exp Gerontol 41: 130–40, 2006 2) Schmelzer C, Kubo H, Mori M, et al. Supplementation with the reduced form of coenzyme Q10 decelerates phenotypic characteristics of senescence and induces a peroxisome proliferators-activated receptor-α gene expression signature in SAMP1 mice. Mol Nutr Food Res. 54: 805-15, 2010 3) Tian G, Sawashita J, Kubo H, el al. Ubiquinol-10 supplementation activates mitochondria functions to decelerate senescence in senescence-accelerated mice. Antioxid Redox Signal. 20: 2606-20, 2014 4) Chang LW, Nagarajan R, Magee JA, el al. A systematic model to predict transcriptional regulatory mechanisms based on overrepresentation of transcription factor binding profiles. Genome Res. 16(3): 405-13, 2006

100 P38. A RANDOMIZED CLINICAL TRIAL ON THE THERAPEUTIC EFFECT OF COENZYME Q10 IN PATIENTS WITH HUMAN IMMUNODEFICIENCY VIRUS INFECTION

Yousefi F1,2, Roozbeh F2, Rahmani H3

1Health Research Institute, Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 2Department of Infectious and Tropical Diseases, Razi Hospital, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 3Ahvaz Behavioral Diseases Consultation Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

Objectives Despite many developments in the treatment of human immunodeficiency virus (HIV) infection, efforts for finding complementary therapies, including those that could enhance the immunologic status of HIV-infected patients, continue. So, we planned this study to evaluate the effects of coenzyme Q10 (CoQ10), a substance with known effects on the immune system, on the occurrence of opportunistic infections and CD4+ T-cell count of HIV-infected patients as markers of their immunologic status. Methods This was a parallel, double-blind, placebo-controlled, randomized clinical trial on adult (>18 years old) patients with HIV infection on antiretroviral therapy (ART) referring to Ahvaz Behavioral Diseases Consultation Center. Participants were allocated to two groups (cases and controls) by the use of a random number table. The case patients were given CoQ10, one 200 mg capsule per day, and the controls received placebo, each for 3 months. Patients with autoimmune diseases, tuberculosis, or with other concurrent immunodeficiency states like lymphoma or other malignancies, pregnant patients, and patients consuming drugs affecting the immune system like corticosteroids, were excluded. The participants were observed for the occurrence of any opportunistic infection and their CD4+ T-cell count, complete blood cell count (CBC), and serum levels of blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured at the beginning and the end of the study. All the participants, caregivers, and the person assessing the outcomes were blinded to group assignment. Results Seventy-four patients were participated in the study; of these, 73 patients (37 cases, 36 controls) remained to the end of the study and were analyzed. One patient was lost to follow-up. The mean age of the cases and the controls was 39 and 41 years, respectively (p=0.423). Thirty-one case patients (83.8%) and 25 control patients (69.4%) were male (p=0.175). No opportunistic infections were observed among the participants during the study period. There was no statistically significant difference in the mean CD4+ T-cell count at the beginning of the study (p=0.232) and also in it’s increase after 3 months (p=0.114) between the two groups; however, the mean CD4+ T-cell count increased significantly by the end of the study in each group (p=0.045 for cases, p=0.001 for controls). There was no statistically significant difference in CBC parameters and in the mean serum levels of BUN, Cr, ALT, AST, and ALP between the two groups. No adverse effects attributable to CoQ10 consumption were found. Conclusion This study suggested that CoQ10 had no remarkable effect on the CD4+ T-cell count and the incidence of opportunistic infections in adult HIV-infected patients on ART.

Trial Registration: IRCT201108027197N1, IRC

101 P39. DETERMINATION OF URINARY CoQ10 BY HPLC WITH ELECTROCHEMICAL DETECTION: REFERENCE VALUES FOR A PEDIATRIC POPULATION

Dèlia Yubero1, Raquel Montero1, Viruna Neergheen2, Iain P Hargreaves2, Rafael Artuch1

1Genetics and Molecular Medicine Department, Hospital Sant Joan de Déu and CIBERER-ISCIII, Barcelona, Spain; 2Neurometabolic unit, National Hospital for Neurology and Neurosurgery, London, UK.

Coenzyme Q10 (CoQ) deficiency diagnosis requires the biochemical determination of CoQ in biological fluids and different cell types. Plasma CoQ measurement is reliable for CoQ monitoring treatment, but its status may not truly reflect tissue levels [1], therefore CoQ determination in cells is the gold standard for the identification of most of the CoQ deficient syndromes. To our knowledge, urinary CoQ analysis has not been documented. The rationale basis of our study would be that as in mitochondrial disorders kidney involvement associated with CoQ deficiency is being recognized, kidney epithelial cells would be an adequate sample for CoQ determination ‒not only for diagnosis but also for treatment monitoring purposes. Our aim was to standardize a new procedure for urinary CoQ determination and to establish reference values for a paediatric population. A total of 135 fresh urine samples were collected, centrifuged and washed with 9 mg/mL saline. The reconstituted pellet was stored at -20ºC until the moment of analysis. Total protein content of the pellet was analyzed by Lowry procedure, and total CoQ measurement was quantified by reverse-phase HPLC with electrochemical detection (Coulochem II, ESA, Chelmsford, MA, USA) as previously reported [2], with slight modifications. Age dependent reference intervals were estrablished from 43 control subjects (age range=2-20 years; average=7.76; SD=4.63): from 2-10 years (n=30; average= 61; SD = 24.5; Q10 range values: 24-109 µmol/g protein) and 11-17 years (n=13; average=84; SD=32.9; Q10 range values: 43-139 µmol/g protein). Moreover, different preanalytical conditions were studied to assess the optimal conditions for CoQ epithelial kidney cells quantification. CoQ epithelial urinary tract cells analysis has the great advantage that it is a non-invasive procedure, ideal for the study in paediatric patients. The growing implication of kidney in mitochondrial diseases, specially in primary CoQ deficiencies [3,4], supports the usefulness of this procedure. In addition, urinary CoQ analysis (in renal diseases, but in other neurological conditions too) may represent a good option for CoQ treatment monitoring, especially considering that the clinical outcome after CoQ supplementation in patients with renal disease and CoQ deficiency may be excellent.

References 1. Duncan AJ, Heales SJ, et al. Determination of Coenzyme Q10 in blood mononuclear cells, skeletal muscle and plasma by HPLC using di-propoxy-Coenzyme Q10 as an internal standard. Clin Chem 2005 Dec;51(12):2380-82. 2. Montero R, Sánchez-Alcázar JA, et al. Analysis of coenzyme Q10 in muscle and fibroblasts for the diagnosis of CoQ10 deficiency syndromes. Clin Biochem. 2008 Jun;41(9):697-700. 3. McCarthy HJ, Bierzynska A, et al. Simultaneous sequencing of 24 genes associated with steroid-resistant nephrotic syndrome. Clin J Am Soc Nephrol. 2013 Apr;8(4):637-48. 4. Heeriga SF, Chernin G, et al. COQ6 mutations in human patients produce nephrotic syndrome with sensorineural deafness. J Clin Invest. 2011 May;121(5):2013-24.

102 Notes

INDEX

Abbott J 98 Chauhan P 91 Garcia Becerra C 60 Acquaviva-Bourdain C 87 Chiappetta D 60 García-Testón Páez E 13 Aguirre MA 53 Chitchumroonchokchai C 55 Genova ML 93 Alcaín FJ 67, 85 Clarke CF 17, 25 George S 82 Alehagen U 30 Collantes-Estévez E 53 Gherli T 33 Allan CM 17 Contin M 60 Ghezzi D 27 Amorati R 95 Cuadrado MJ 53 Gimenez-Llort L 85 Angulo F 78 Cuezva JM 58 Giorgetti A 83 Apa R 64 D’Aniello A 43 Giorgio V 21 Argenton F 21 Da Ros T 21 Gómez-Almagro MV 67 Ariza J 35, 87 Dallner G 39 González-Mariscal I 69 Artuch R 26, 102 Damiani E 12 González-Reyes JA 35, 53 Awad AM 17 Dange NS 91 González-Rodríguez P 83 Ayer A 25 Dawes IW 25 Gorodetskaya EA 56, 68, 71 Bacchetti T 51 de Cabo R 35, 38, 87 Gorospe M 58 Balercia G 43 del Rio J 78 Greci L 52 Barbarroja N 53 Desbats MA 28, 77, 97 Guidi F 64 Bellance N 2 Di Segni C 64, 73 Gutiérrez Ríos P 69 Belousova MA 56 Diaz-Castro J 62 Gvozdjáková A 75 Bergamini C 22, 64, 95 Doimo M 97 Hansíková H 88 Bernardi P 21 Dolliner P 30 Hargreaves I 7, 98, 102 Bernier M 35, 36, 87 Döring F 47, 66, 79 Hashizume N 90 Berteotti A 95 Durán-Prado M 67, 85 He CH 17 Berto P 77 Enriquez JA 5 Heales S 98 Bettencourt C 70 Esteves P 2 Higuchi K 10, 96, 100 Bianciotti L 74 Failla ML 55 Hoehn KL 65 Birinci Y 93 Falasca AI 93 Holton JL 70 Bjork P 78 Fato R 22, 64, 95 Hosoe K 10, 86, 100 Bolognesi ML 95 Fazakerley DJ 65 Houlden H 70 Brandt U 3 Feher J 30 Hsu A 17 Brillac A 2 Fernández A 83 Hůlková M 88 Brismar K 39 Fernández del Río L 35, 53 Humphrey SJ 65 Bronisz M 30 Fernández-Ayala DJM 45, 58 Huo J 10, 100 Brugè F 12, 33, 43, 51 Ferrín-Sánchez G 67 Imasawa T 2 Bruno S 95 Fetoni AR 22 Ismail A 19 Bueno C 83 Filipiak K 30 Izumo N 99 Buldreghini E 43 Fischer A 66, 79 James DE 65 Burón MI 35 Fisher-Wellman KH 65 Jiménez-Gómez Y 53 Bzymek M 30 Flor S 60 Jódar L 35 Campos-Silva C 57 Fontecave M 19 Junutula JR 65 Carini M 21 Forsman U 19 Kajarabille N 62 Casarin A 59 Fraga MF 83 Kajimoto O 44 Cascajo MV 58, 87 Franceschi C 8 Kalenikova EI 56, 68, 71 Castaño J 83 Frontiñán-Rubio J 67, 85 Kanda J 99 Cavalli A 95 Fujii K 44 Kaplon-Cieslicka A 30 Cerqua C 59 Fukuda S 44 Kaur H 30 Chalasani A 70 Fülop P 30 Kawai K 89 Chaudhuri R 65 Funahashi I 91 Kawamukai M 15 Khraiwesh H 35 Mišianik J 75 Pogačnik T 80 Kiely AP 70 Miyazaki Y 41 Prati F 95 Kinoshita T 72 Modrusan Z 65 Prato M 21 Knopová S 88 Mojto V 75 Preston J 98 Knott A 42 Molardi A 33 Pulido-Morán M 62 Kobayashi Y 99 Montecucco C 77 Rahim AAA 30 Kolumam G 65 Montero R 102 Rahmani H 101 Kopuz M 93 Morbiato L 77 Raimondo S 64, 73 Krycer JR 65 Morbidoni V 97 Ranadive P 82 Krzciuk M 30 Moreno-Fernández J 62 Rausová Z 75 Kubo H 10, 86, 100 Moretton M 60 Reyes-Torres I 57 Kucharská J 75 Mortensen SA 30 Ritchie RH 23 Kumar A 30 Murphy M 20 Rivera M 57 Kuratsune H 44 Nag A 17 Rodriguez M 74 Lalou C 2 Nagase M 41 Rodriguez-Bies E 57 Lam HT 17 Nagy V 30 Rodríguez-Gómez JA 83 Langsjoen AM 31 Navas Lloret P 13, 28, 45, 57, Romero-Moya D 83 Langsjoen JO 31 58, 69, 78, 83, Romualdi D 64 Langsjoen PH 31 87 Roozbeh F 101 Lanzone A 64 Navas-Enamorado I 78 Rosenfeldt FL 30 Lazurova I 30 Neergheen V 70, 102 Ross D 37 Lekue J 78 Nicolini F 33 Rossignol R 2 Lenaz G 22, 93 Niklowitz P 66, 79 Roze E 87 Lenzi A 43 Nukada H 90 Ruggiero FM 4 Leroux M 19 Obre E 2 Ruiz-Limón P 53 Levan SR 35 Ochoa JJ 62 Salviati L 28, 59, 77, 97 Littarru GP 1, 12, 30, 33, 50, Onodera K 99 Sánchez-Cuesta A 78 51, 52, 73 Onur S 79 Sancho-Bielsa FJ 85 López-Barneo J 83 Orlando P 12, 33, 43, 51, 73 SantaCruz-Calvo S 87 López-Lluch G 6, 57, 78, 87 Osa VM 62 Santiago-Mora R 67, 85 López-Miranda J 48 Otani M 89 Santoro MM 16 López-Pedrera Ch 53 Overvad K 30 Santos-Ocaña C 13, 69, 83 Lucangioli S 60, 74 Ozeir M 19 Sanz A 9 Maghzal GJ 65 Pagliarini DJ 18 Sarmiento A 62 Mancini A 64, 73 Painkara US 91 Sawashita J 10, 86, 100 Marcheggiani F 12 Panieri E 16 Schiavone M 21 Marchel M 30 Paradies G 4 Schottlaender LV 70 Martinefski M 74 Paradies V 4 Sedláčková D 88 Martín-Montalvo A 69, 87 Paragh G 30 Segui P 53 Maruyama K 72 Paredi G 95 Sewduth R 16 Matsumoto H 90 Parisi V 40 Shimakura T 99 Mazzanti L 43 Parker BL 65 Siegel D 37 Medvedev OS 56, 68, 71 Payet LA 19 Siendones E 58, 87 Mehta A 82 Peinado JR 67, 85 Silic-Benuss M 28 Mellot-Drazniek C 19 Pella D 30, 34 Silvestri S 12, 33, 43, 51, 73 Menendez P 83 Pelosi L 19 Sindberg CD 30 Menke T 66, 79 Pérez-Sánchez C 53 Sindelar P 19 Meoli CC 65 Petronilli V 21 Sinkiewicz W 30 Mercken E 35 Petrosillo G 4 Skjøth F 30 Meza-Torres C 57 Pierrel F 19 Spáčilová J 88 Millia C 16 Ping CS 30 Steurer G 30 Stocker R 25, 65 Tsui HS 17 Wysocki H 30 Stopinski M 30 Tsuruoka M 86 Xu Z 10, 86, 100 Suarna C 65 Uliassi E 95 Yamaguti K 44 Suzuki T 89, 90 Valuch J 75 Yamamoto N 99 Takahashi M 89, 90 Vazquez-Fonseca L 97 Yamamoto Y 41 Tanigawa T 72 Velasco F 53 Yang JY 65 Thakur AS 91 Velasco I 83 Yang P 65 Thomas KC 65 Viar T 78 Yeske PE 29 Tian G 10, 86, 100 Vignini A 43 Yoshino H 41 Tiano L 12, 33, 43, 51, 73 Villalba JM 35, 53, 67, 87 Yousefi F 101 Tioli G 93 Villar-Rayo M 67 Yubero D 102 Tirabassi G 43 Volta F 64 Zaffagnini M 95 Tonello F 77 Wainwright L 98 Zanotti G 77 Trevisson E 28, 59 Wakabayashi H 99 Zeman J 88 Tripodi V 60, 74 Watanabe Y 44 Žmitek J 80 Troiani D 22 Wozakowsk-Kaplon B 30 Žmitek K 80