The effect of long-term treatment with coenzyme Q10 on nucleic acid modifications by

oxidation in children with Down syndrome

Emil List Larsena,b, Lucia Padellac, Helle Kirstine Mørup Bergholdta,b, Trine Henriksena,b, Lucia

Santoroc, Orazio Gabriellic, Henrik Enghusen Poulsena,b,d*, Gian Paolo Littarrue, Patrick

Orlandof, Luca Tianof

aLaboratory of Clinical Pharmacology, Rigshospitalet, University of Copenhagen,

Copenhagen, Denmark

bDepartment of Clinical Pharmacology, Bispebjerg Frederiksberg Hospital, University of

Copenhagen, Copenhagen, Denmark

cPediatric Clinic Laboratory, Department of Clinical Sciences, Children Hospital Salesi,

Polytechnic University of Marche, Ancona, Italy

dDepartment of Clinical Medicine, Faculty of Health and Medical Sciences, University of

Copenhagen, Copenhagen, Denmark

eDepartment of Clinical Sciences, Polytechnic University of Marche, Ancona, Italy

fDepartment of Life and Environmental Sciences, Polytechnic University of Marche, Ancona,

Italy

*Corresponding author:

Professor, Henrik Enghusen Poulsen, MD, DMSc

Laboratory of Clinical Pharmacology, Q7642

Rigshospitalet, University of Copenhagen

Ole Maaloeesvej 26, Entrance 76

DK-2200 Copenhagen N

Denmark

1

E-mail: hepo@rh.dk

Phone: + 45 3545 7671

Fax: +45 3545 2745

Keywords: Down syndrome, oxidative stress, oxidative nucleic acid modifications, 8-oxo-

7,8-dihydro-2’-deoxyguanosine, 8-oxo-7,8-dihydroguanosine.

Abbreviations:

8-oxo-7,8-dihydro-2’-deoxyguanosine, 8-oxodG; 8-oxo-7,8-dihydroguanosine, 8-oxoGuo;

Coenzyme Q10, CoQ10; Down syndrome, DS; Formamidopyrimidine Glycosylase, FPG;

Human serum albumin, HAS; Peripheral blood mononuclear cell, PBMC; Specific gravity, SG

Abstract word count: 170/170

Article word count: 3075

Number of references: 41

2

ABSTRACT

Elevated levels of nucleic acid modifications by oxidation in children with Down syndrome

(DS) have been proposed to be associated with some of the clinical characteristics of the

disease. Even though coenzyme Q10 (CoQ10) supplementation improves oxidative status in

children with DS by increasing the ubiquinol-10:total CoQ10-ratio, treatment with CoQ10 for

6 months does not affect DNA oxidation. Here we investigated the effect of long-term

treatment with CoQ10 on DNA- and RNA oxidation in children with DS. Thirty-two children

with DS were treated with 4 mg/kg/day CoQ10 for 4 years. Fourteen age-matched children

with DS were included as controls, receiving no intervention. DNA oxidation was determined

by urinary excretion of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and tail intensity of

Formamidopyrimidine Glycosylase comet assay in peripheral blood mononuclear cells,

whereas RNA oxidation was determined by urinary excretion of 8-oxo-7,8-dihydroguanosine

(8-oxoGuo). Using generalised linear mixed model by maximum likelihood we found no effect

of 4 years treatment with CoQ10 at the dosage of 4 mg/kg/day on DNA- and RNA oxidation

in children with DS.

3

Highlights

Nucleic acid modifications by oxidation are associated with Down syndrome (76/85)

CoQ10 (4 mg/kg/day) for up to 4 years did not affect DNA- and RNA oxidation status

(85/85)

DNA oxidation in PBMC does not correlate with urinary oxidised nucleic acids (79/85)

Studies using higher dosage of the active form of coenzyme Q10 are required (85/85)

4

1. INTRODUCTION

Down syndrome is the result of complete or partial trisomy of chromosome 21(Mikkelsen,

1977) and the most common chromosomal abnormality in live births(Parker et al., 2010)

resulting in e.g. mental retardation, early onset of Alzheimer’s disease, and high prevalence

of congenital heart disease(Freeman et al., 1998; Mann et al., 1990).

Individuals with Down syndrome presents elevated levels of oxidative

modifications compared to healthy individuals(Jovanovic et al., 1998; Maluf and Erdtmann,

2001; Morawiec et al., 2008; Pallardó et al., 2006; De Sousa et al., 2015). Studies have

shown that both mitochondrial dysfunction(Schuchmann and Heinemann, 2000) and

overexpression of genes on chromosome 21(De Haan et al., 1996) might explain the

elevated levels of oxidative modifications. The pro-oxidant state is hypothesised to

accelerate neuronal degeneration(Capone, 2001), and hereby induce some of the clinical

characteristics in individuals with Down syndrome(De Haan et al., 1996; Kedziora and

Bartosz, 1988; Zana et al., 2007).

Despite the pro-oxidant state, the level of DNA modifications by oxidation was

only higher in individuals ≤20 years of age with Down syndrome compared to disease free

controls(Pallardó et al., 2006). A high level of DNA modifications by oxidation has also been

reported in studies examining the autopsied brains of deceased older patients with

Alzheimer’s disease(Gabbita et al., 1998; Mecocci et al., 1994). Due to the high levels of

DNA oxidation in both children with Down syndrome and adults with Alzheimer’s disease,

and the early onset of Alzheimer’s disease in patients with Down syndrome, DNA oxidation is

hypothesised to influence the development of Alzheimer’s disease in patients with Down

syndrome.

In addition to the elevated levels of DNA modifications by oxidation,

accumulation of RNA modifications by oxidation are reported in individuals with Down

syndrome compared to disease free individuals, especially among those aged 10-30

5

years(Nunomura et al., 2000). Traditionally, focus has been on DNA oxidation rather than

RNA oxidation, despite the fact that RNA oxidation is also associated with neurodegenerative

diseases e.g. Alzheimer’s disease(Nunomura et al., 2006).

Coenzyme Q10 (CoQ10) is essential in the mitochondrial respiratory chain, has

an anti-oxidative effect on plasma lipids, and affects gene expression(Littarru and Tiano,

2010).The endogenous production of CoQ10 is sufficient to meet the physiologic demands;

however, in certain conditions of lowered synthesis or increased demand it might be useful to

increase the intake through dietary supplements, since the content of CoQ10 in food is low

(i.e. 3-5 mg/day)(Weber et al., 1997).

The endogenous synthesis of CoQ10 is carried out by the mevalonate pathway,

which is also responsible for production of cholesterol(Littarru and Tiano, 2010). We have

previously demonstrated that despite a normal level of cholesterol, the cellular content of

CoQ10 in peripheral blood mononuclear cells(PBMC) and platelets is lower in patients with

Down syndrome compared to healthy controls(Tiano et al., 2008).

The human organism produces the reduced and active form of CoQ10,

ubiquinol, as an antioxidant. In plasma samples approximately 95% of CoQ10 is present in

the form of ubiquinol, while the rest exists in the oxidised form (ubiquinone)(Bhagavan and

Chopra, 2007; Niklowitz et al., 2004). At cellular levels, there is an equilibrium of ubiquinone

and ubiquinol due to metabolic redox reactions(Niklowitz et al., 2004).

Oral intake of ubiquinone increases plasma content and reduces the absorption

of CoQ10 and thereby increases the active reduced state(Bhagavan and Chopra, 2007). The

daily dose of CoQ10 as a nutritional supplement is below 200 mg/day. The therapeutic dose

shown to produce an effect on extracellular superoxide dismutase in patients with

cardiovascular disease is around 300 mg/day(Littarru et al., 2011), but a safe dosage of 2400

mg/day was found in neurodegenerative disease trials(McGarry et al., 2017).

Oral supplementation with ubiquinol at 10 mg/kg/day has been shown to

improve plasma oxidative status by increasing the ubiquinol-10:total CoQ10-ratio in children

6

with Down syndrome(Miles et al., 2007). This finding was published while the present study

was already in progress. Previously, we showed that supplementation with CoQ10 in its

oxidised form (ubiquinone) at a lower dose (4mg/kg/day) for up to 6 months was not effective

in lowering DNA modifications by oxidation(Tiano et al., 2011). Here, our primary objective

was to investigate the effect of long-term treatment (up to 4 years) with ubiquinone at the

dosage of 4 mg/kg/day on nucleic acid modifications by oxidation, both DNA and RNA, in

children with Down syndrome.

7

2. MATERIAL AND METHODS

2.1 Trial design

The clinical trial was conducted at the Pediatric Clinic Laboratory at the Children Hospital

Salesi in Italy. We assigned 32 children with Down syndrome to the intervention group and

14 age-matched children with Down syndrome to the control group. The participants were

monitored every sixth month, concomitant with their outpatient consultation at the hospital.

Primary and secondary outcomes were measured before the initiation of treatment

(baseline), and again after 2 and 4 years. The primary outcome of this study was urinary

excretion of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and 8-oxo-7,8-

dihydroguanosine (8-oxoGuo). The secondary outcome was PBMC tail intensity in

Formamidopyrimidine Glycosylase (FPG) comet assays determined in peripheral blood

leucocytes. The FPG comet assay was performed to evaluate the correlation with urinary

excretion of 8-oxodG and 8-oxoGuo.

The trial was approved by the ethical committee on the Marche Polytechnic University and

Regional Hospital ”A.O. Ospedali Riuniti”(Protocol number 204244).The present study

originates from a short-term randomised, controlled trial(Tiano et al., 2011).

2.2 Participants

Children with Down syndrome, aged 2-17 years were included in the trial. The children were

included in relation to their outpatient consultation at the Salesi Children Hospital. We

excluded individuals with autoimmune diseases (i.e. diabetes, hypothyroidism, or celiac

disease), congenital cardiovascular disease, and individuals receiving pharmacological

therapy or the vitamin supplementation from the trial. Written informed consent was obtained

from the parents/legal guardian of all participants.

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2.3 Intervention

The participants in the treatment group received orally administrated coenzyme Q10

(Myoquinon, Pharma Nord, Vejle, Denmark) 4 mg/kg/day. The parents received the tablets in

relation to the outpatient consultation and administered the medicine. The age-matched

control group received no treatment and was instructed not to use CoQ10 supplementation

during the investigation period. In order to evaluate adherence to treatment, we counted the

remaining CoQ10 soft-gels and confirmed intake by measuring plasma levels of CoQ10 of

both participants in the intervention- and control group. In the present study, we used a dose

of 4 mg/kg/day in accordance with other therapeutic interventions(Miles et al., 2006) in the

form of ubiquinone.

2.4 Laboratory analyses

2.4.1 Urinary excretion of 8-oxodG and 8-oxoGuo

We measured urinary excretion of 8-oxodG and 8-oxoGuo to assess whole body DNA and

RNA oxidation, respectively(Poulsen et al., 2014). A freshly voided urine sample was

immediately well mixed, and an aliquot was stored at -20 ˚C until analysis. The urine samples

(stored on dry ice) were shipped to Denmark by air courier for analysis. The analyses of 8-

oxodG and 8-oxoGuo were performed using ultra-performance liquid chromatography

tandem mass-spectrometry (UPLC-MS/MS) and carried out at the Laboratory of Clinical

Pharmacology at Rigshospitalet in Copenhagen, Denmark. The analyses of samples and

quality control were performed as described by (Rasmussen et al., 2016), with the exception

that 8-oxodG was measured by electrospray in the positive mode, using the MS/MS

transitions m/z 284 →168 (quantification ion) and m/z 168 → 140. The UPLC-MS/MS

method is preferable to determine urinary excretion of 8-oxodG and 8-oxoGuo due to the

high specificity compared to the ELISA method(Weimann et al., 2012).

A study has demonstrated that urine density, measured by specific gravity

(SG), provides a higher stability compared to creatinine in children(Wang et al., 2015).

9

Therefore, to adjust the urinary excretion of 8-oxodG and 8-oxoGuo to urinary flow, we

standardised the results using urine density. Urine density was determined by a Mettler

Toledo, DA-110M Density Meter and reported as SG.

2.4.2 Formamidopyrimidine Glycosylase comet assay

Alkaline version of the comet assay with incubation of nucleoids with FPG was performed as

previously reported(Tiano et al., 2012). Lymphocytes were isolated by stratification over

Lymphoprep (FraseniusKabi, Oslo, Norway) and centrifugation at 693 x g for 20 min at 4°C.

After two washes cell suspensions in Phosphate-buffered saline were cryopreserved with an

equal volume of freezing mix containing Human Serum Albumin (HSA) 5%; HSA 20%,

Dimethyl Sulfoxide (3:1:1). Cryovials were chilled slowly (in boxes of expanded polystyrene)

to –80°C. After completing the freezing process, vials were transferred to liquid nitrogen for

long term storage. At the time of analysis, cells were rapidly thawed by immersion in a water

bath at 37°C and immediately supplemented with an equal volume of a solution containing

Eudextran (FraseniusKabi, Oslo, Norway) and HSA 5% (1:1).

Dehibernated cells were resuspended in low melting agarose at 0.7% and

stratified on a HT Trevigen slide and subsequently immersed in a alkaline lysis solution (pH

10) containing NaCl (2.5 M), Na2EDTA (0.1 M), TrisHCl (10 mM) Dimethyl Sulfoxide 10%,

Triton X-100 1%, and kept at 4°C for at least 1 hr. After lysis the slides were washed twice for

15 minutes with enzyme buffer (40 mMHepes, 0.1 M KCl, 0.5 mM EDTA and 1% HSA at pH

8) at 4°C. 50 μl of Enzyme buffer (used as control) was dropped onto each spot and

incubated at 37°C for 45 minutes. After 20 min unwinding in electrophoresis buffer (1 mM

EDTA and 300 mM sodium hydroxide, pH > 13) gels were electrophoretically processed at 1

V/cm, neutralized in trisHCl pH 7.5 for 5 min, dehydrated in methanol for 2 min and finally

dried at 60°C for 2 min and stored for microscopic examination.

10

2.5 Statistic

We investigated the difference in baseline characteristics of age, weight, cholesterol level, 8-

oxodG, and 8-oxoGuobetween the two groups using Student’s t-test and reported mean and

standard deviation. Difference in gender distribution (percentage) was investigated using

Chi-square test.

We analysed the effect of 4 years treatment with CoQ10 on our primary and

secondary outcomes using an intention-to-treat approach. Changes in the primary (urinary

excretion of 8-oxodG and 8-oxoGuo) and secondary outcomes (tail intensity in FPG comet

assay) were analysed using a generalised linear mixed model by maximum likelihood with

random effects, utilizing data from all three examinations.

Finally, we examined the correlation between median tail intensity and urinary

excretion of 8-oxodG and 8-oxoGuo, respectively, at baseline using the Pearson method.

In all the above mentioned analyses, we used an alpha level of 0.05. All data

management and analyses were performed using R version 3.2.3(R Core Team, 2014).

11

3. RESULTS

3.1 Study population

We screened 51 children for eligibility and subsequently enrolled the 48 children who met the

inclusion criteria (Figure 1). Two participants were lost to follow-up during the 4-year period.

Baseline characteristics of age, weight, cholesterol, 8-oxodG, and 8-oxoGuo were similar in

the two groups (Table 1).

3.2 Urinary excretion of 8-oxodG and 8-oxoGuo

The data of urinary excretion of 8-oxodG and 8-oxoGuo are presented in Figure 2. The effect

of treatment with CoQ10 on urinary excretion of 8-oxodG and 8-oxoGuo was analysed using

generalised linear mixed model by maximum likelihood with random effects. The model

showed that the level of DNA oxidation measured by 8-oxodG/SG changed by -0.16 nM

(95% confidence interval (CI): -2.64;2.31 nM, p= 0.90) per 2 years, whereas treatment with

CoQ10 increased the slope with 1.65 nM (95% CI: -1.31;4.60 nM, p=0.28).

The level of RNA oxidation measured by 8-oxoGuo/SG changed by -0.95 nM (95% CI:

-5.92;4.01 nM, p=0.71) per 2 years, whereas treatment with CoQ10 increased the slope with

2.41 nM (95% CI: -3.53;8.35 nM, p=0.43).

3.3 FPG Comet assay

The data of the tail intensity in FPG comet assay are presented in Figure 3. Considering the

non-normal distribution of tail intensity index, data are presented as box plot relative to the

distribution of tail intensity parameters i.e. 25th percentile, median tail intensity, and 75th

percentile tail intensity. Moreover, the effect of treatment with CoQ10 was analysed using

generalised linear mixed model by maximum likelihood with random effects. The model

showed that median tail intensity changed by -0.49 %-point (95% CI: -1.81;0.82 %-point,

p=0.47) per 2 years, whereas treatment with CoQ10 changed the slope with 0.43 %-point

12

(95% CI: -1.13;2.00, p=0.58). The results were verified by logarithm transforming data before

analysis.

3.4 Correlation between the FPG Comet assay and urinary excretion of 8-oxodG/8-

oxoGuo

The correlation between median tail intensity in the FPG comet assay and urinary excretion

of 8-oxodG or 8-oxoGuo is presented in Figure 4.

We found no significant correlation between urinary excretion of 8-oxodG and median tail

intensity in the FPG comet assay (r = 0.02, p = 0.89). Further, there was no significant

correlation between urinary excretion of 8-oxoGuo and median tail intensity in the FPG

comet assay (r = -0.11, p = 0.49).

13

4. DISCUSSION

This study demonstrates that long-term treatment with 4 mg/kg/day CoQ10 in the form of

ubiquinone does not reduce DNA modifications by oxidation, measured by urinary excretion

of 8-oxodG nor measured by PBMC tail intensity in the FPG comet assay in children with

Down syndrome. Further, long-term treatment with CoQ10 does not reduce RNA

modifications by oxidation, measured as urinary excretion of 8-oxoGuo.

The present study is the final act of a triple set of studies that originate from the original

experimental design, investigating the DNA protective effect of CoQ10 in children with Down

syndrome. The first two papers refer to shorter span of investigations i.e. 6 months and 20

months(Tiano et al., 2012, 2011). The analytical endpoints have been progressively

implemented in order to reach a more detailed investigation; starting from plain alkaline

comet assay, introducing oxidised base analysis with FPG, and in this version focussing on

whole organism production of oxidised nucleic acids adducts, both DNA and RNA, through

urine UPLC-MS/MS analysis. Although a promising trend toward improvements in certain

age groups has been observed in the 20 months study(Tiano et al., 2012), this evidence is

not confirmed in the present study with a longer time frame.

In general, oxidative modifications have been appointed as a characteristic in individuals with

Down syndrome that might account for accelerated aging and associated pathologies e.g.

Alzheimer’s disease(Kedziora and Bartosz, 1988; Zana et al., 2007). Therefore, antioxidant

supplements have been proposed as a relevant intervention, in some cases with promising

outcomes at behavioural level(de la Torre et al., 2016). However, our data seem to suggest

that CoQ10 in our experimental setting is not effective in lowering nucleic acid modifications

by oxidation. Nucleic acids represent a sensible and critical target of reactive oxygen species

with potential repercussions in the development of Down syndrome associated pathologies.

14

In contrast with our finding, another study showed that CoQ10 improved oxidative status in

plasma of children with Down syndrome(Miles et al., 2007). However, the study used higher

dose in accordance with that used in neurodegenerative disorders. Along with this, CoQ10

formulation was in the reduced form as ubiquinol, which only became available as an

ingredient in supplements while this trial was ongoing. Ubiquinol is characterised by higher

bioavailability and does not require being reduced, thus providing direct antioxidant

activity(Hosoe et al., 2007). Furthermore, previous studies have shown that CoQ10 redox

status plays a role in preventing DNA modifications by oxidation(Schmelzer and Döring,

2012).

We found no correlation between the FPG comet assay tail intensity and urinary excretion of

8-oxodG or 8-oxoGuo. Urinary excretion of 8-oxodG and 8-oxoGuo are markers of whole

body DNA and RNA oxidation, respectively(Poulsen et al., 2014). The FPG comet assay

evaluates the oxidative DNA modifications only in leukocytes in peripheral blood. The lack of

correlation between these markers might be explained by different distribution or

compartmentalisation of oxidative modifications within the organism. This explanation would

support the common perception that multiple markers of oxidative modifications are required

in order to fully describe the oxidative status of patients and in order to evaluate potential

effects of intervention strategies(Frijhoff et al., 2015). Another possible explanation for the

lack of correlation in the present study could be that not all adducts from the oxidised DNA

within the cells are repaired and excreted in the urine. Furthermore, urinary excretion of 8-

oxodG and 8-oxoGuo are the result of progressive accumulation over time, whereas the

comet assay quantifies the cellular steady-state of oxidative DNA damage that results from

DNA formation and repair at the time of the analysis.

The strengths of this study are the long treatment period and the reliability and precision of

methodologies used for the analysis. The present data originate from a follow-up of the

15

original short term randomised, controlled trial(Tiano et al., 2011). For ethical reasons and in

the light of the long duration of the study, we decided that the control group should be

independent, untreated controls, instead of a placebo treated control group. The study is

limited by the experimental design without randomisation and blinding of the treatment.

Studies without blinding and randomisation are generally considered to bias towards false

positive effect(Kjaergard et al., 2001). However, this seems not to be the case in the present

study.

In conclusion, our data demonstrated no effect of long-term treatment with 4 mg/kg/day

CoQ10 on nucleic acid modifications by oxidation in children with Down syndrome. However,

it is possible that the dosage and form of the active component used was not optimal in order

to modify nucleic acid modifications by oxidation. Thus, additional long-term studies are

required to evaluate the efficacy of ubiquinol supplementation at higher dose with respect to

these endpoints – and a combination of mitochondrial nutrients is more likely to have better

chance of producing an protective effect(Pagano et al., 2014a, 2014b).

16

Data access and responsibility: The principal investigator, Luca Tiano, had full access to

all of the data and takes responsibility for the integrity and accuracy of the data and the

decision to publish.

Conflict of interest

All authors declare no conflicts of interest.

All authors have completed the Unified Competing Interest form at

www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author).

Acknowledgement

We thank Pharma Nord, Denmark for kindly donating Myoquinon used for the study. We

thank Dr. Anna Tangorra for assisting with the comet assay analysis. Further, we thank

senior laboratory technician Lis K. Hansen and laboratory technicians Katja L. Christensen

for performing the urine analyses. Furthermore, we thank Dr. A. Collins for kindly donating

purified FPG.

We would also like to thank the participants and their families for making this study possible.

Funding

This study was supported by Marche Polytechnic University Atheneum funding to Gian Paolo

Littaru and Luca Tiano.

None of the funding sources were involved in the design, conduct of study, collection,

management, analysis, or interpretation of data, preparation or review of manuscript, and

had no right to approve or disapprove of the submitted manuscript.

17

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Table and figure legends

Table 1. Baseline characteristics of the participants. The values are presented as n(%) or

mean(sd); aChi square test; bStudent’s t-test. 8-oxodG = 8-oxo-7,8-dihydro-2’-

deoxyguanosine; 8-oxoGuo = 8-oxo-7,8-dihydroguanosine; SG = Specific gravity.

Figure 1. Participant flow.

Figure 2. Urinary excretion of 8-oxo-7,8-dihydro-2’-deoxyguanosine(8-oxodG)/specific

gravity (SG) and 8-oxo-7,8-dihydroguanosine(8-oxoGuo)/SG presented as mean (point) and

standard error (line).

Figure 3. Tail intensity (%) in the Formamidopyrimidine Glycosylase (FPG) comet assay

separated on 25th percentile, median, and 75th percentile.

Figure 4. A) There was no significant correlation between urinary excretion of 8-oxo-7,8-

dihydro-2’-deoxyguanosine (8-oxodG)/specific gravity (SG) and median tail intensity in the

Formamidopyrimidine Glycosylase (FPG) comet assay at baseline (r = 0.02, p=0.89). B)

There was no significant correlation between urinary excretion of 8-oxo-7,8-

dihydroguanosine (8-oxoGuo)/SG and median tail intensity in the FPG comet assay at

baseline (r=-0.11, p=0.49).

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Tables

Table 1

Control Coenzyme Q10 pTotal number 14 32Sex, male 11 (78.6 %) 18 (56.3 %) 0.27a

Age (years) 9.5 (5.1) 10.2 (3.0) 0.65b

Weight (kg) 35.2 (17.8) 35.4 (13.7) 0.97b

Cholesterol (mg/dL) 184.9 (27.9) 168.0 (28.6) 0.07b

8-oxodG/SG (nM) 17.1 (9.2) 16.4 (8.1) 0.80b

8-oxoGuo/SG (nM) 31.1 (14.7) 31.6 (15.7) 0.92b

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Figures

Figure 1

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Figure 2

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Figure 3

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Figure 4

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