Hepatic mitochondrial dysfunction in Friedreich ataxia.
ABSTRACT Mitochondrial dysfunction due to respiratory chain impairment is a key feature in pathogenesis of Friedreich ataxia. Friedreich ataxia affects the nervous system, heart and pancreas.
We assessed hepatic mitochondrial function by (13)C-methionine-breath-test in 16 Friedreich ataxia patients and matched healthy controls.
Patients exhaled significantly smaller amounts of (13)CO(2) over 90 minutes. Maximal exhaled percentage dose of (13)CO(2) recovery was reduced compared to controls.
(13)C-methionine-breath-test indicates subclinical hepatic mitochondrial dysfunction in Friedreich ataxia but did not correlate with GAA repeat lengths, disease duration or disease severity.
RESEARCH ARTICLEOpen Access
Hepatic mitochondrial dysfunction in Friedreich
Sven H Stüwe1, Oliver Goetze2,3, Larissa Arning4, Matthias Banasch2, Wolfgang E Schmidt2, Ludger Schöls5,6and
Background: Mitochondrial dysfunction due to respiratory chain impairment is a key feature in pathogenesis of
Friedreich ataxia. Friedreich ataxia affects the nervous system, heart and pancreas.
Methods: We assessed hepatic mitochondrial function by13C-methionine-breath-test in 16 Friedreich ataxia
patients and matched healthy controls.
Results: Patients exhaled significantly smaller amounts of13CO2over 90 minutes. Maximal exhaled percentage
dose of13CO2recovery was reduced compared to controls.
Conclusions:13C-methionine-breath-test indicates subclinical hepatic mitochondrial dysfunction in Friedreich ataxia
but did not correlate with GAA repeat lengths, disease duration or disease severity.
Keywords:13C-methionine, breath test, Friedreich, Ataxia, neurodegeneration
Friedreich ataxia (FRDA) is an autosomal recessive neu-
rodegenerative disorder caused by expanded GAA triplet
repeats located in the first intron of the FXN gene cod-
ing for frataxin on chromosome 9 . Frataxin is a
mitochondrial protein involved in biogenesis of iron-sul-
fur clusters (ISCs). ISCs serve as prosthetic group in
several enzymes of the mitochondrial energy metabolism
including aconitase and complexes I, II and III of the
respiratory chain that are impaired in FRDA . Fra-
taxin mRNA was found to show a broad expression pat-
tern, including tissues with a high metabolic rate, like
liver, kidney, brown fat and heart . In addition to
mitochondrial dysfunction, increase of reactive oxygen
species (ROS)s is regarded as key feature in FRDA
pathogenesis . The length of the shorter of the two
GAA repeats is thought to determine the residual
amount of frataxin and thereby influencing age at onset
of symptoms and disease severity [5,6]. Additionally,
repeat length influences the development of diabetes
and cardiomyopathy .
Several clinical rating scales have been proposed to
assess disease severity in FRDA. The “scale for the
assessment and rating of ataxia” (SARA) was evalutated
in FRDA with high interrater reliability and practicabil-
ity . For future trials, especially for a possible neuro-
protective treatment, there is a need for biomarkers
linked to pathogenesis of FRDA. Beside others, quantifi-
cation of frataxin, proton magnetic resonance spectro-
scopy (MRS) of the brain and phosphorus MRS in calf
muscle  as well as atrophy of spinal cord and the
medulla  were described as potential biomarkers in
Methionine, an essential amino acid, is mainly meta-
bolized in the liver which is remarkable rich in mito-
chondria . Excess of methionine methyl groups is
metabolised via sarcosine (N-methylglycine) and mito-
chondrial oxidation to CO2. Since the highest speci-
fic activity of methionine adenosyltransferase in
mammals occurs in hepatic tissue, methyl-13C-methio-
nine breath test (MeBT) has been proposed for the
assessment of hepatic mitochondrial function in vivo
. Meanwhile MeBT is established for non-invasive,
easy to perform and cheap quantification of oxidative
capacity of liver mitochondria and was used for drug
monitoring in HIV and Hepatitis C [12,13]. Further
* Correspondence: email@example.com
1Department of Neurology, Ruhr-University, St. Josef-Hospital, Bochum,
Full list of author information is available at the end of the article
Stüwe et al. BMC Neurology 2011, 11:145
© 2011 Stüwe et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
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any medium, provided the original work is properly cited.
applications include detecting of drug and alcohol
related liver toxicity, liver steatosis and cirrhosis [10,14].
The method has not been used in neurological disorders
The aim of this study was to investigate mitochondrial
liver function of FRDA patients and to explore if the
MeBT is feasible to reflect disease severity in FRDA.
16 patients all with genetically confirmed Friedreich
ataxia and homozygous for the GAA expansion were
recruited from the Department of Neurology, St. Josef-
Hospital, Ruhr-University Bochum and the Ataxia
Clinic, Department of Neurodegeneration, University of
Tübingen, Germany. Healthy volunteers were matched
to patients by age, gender and body surface area (BSA;
see Fischer Analysen Instrumente GmbH, Leipzig, Ger-
many). All participants gave written informed consent
to the study. The study was approved by the ethic com-
mittee of the Ruhr-University Bochum, (registration-
number 2718). Exact GAA repeat lengths in the FXN
gene were available in 14 of 16 FRDA patients. Two
patients had a late onset of the disease (28 and 30
years), only one was still able to walk independently.
Disease severity was assessed using SARA (table 1) .
Six FRDA participants were free of medication, four
were on proton pump inhibitors (PPIs) and beta-block-
ers, two respectively on baclofen 5 or 10 mg, ACE-inhi-
bitors, digitoxin and one respectively on buprenorphine
0.2 mg, ropinirole 4 mg, olmesartan 20 mg, trospium
chloride 60 mg, etoricoxib 60 mg, clonazepam 1 mg,
citalopram 10 mg, amitriptyline 50 mg, gabapentin 800
mg, prednisone 7.5 mg, hydrochlorothiazide 25 mg,
acetylsalicylic acid 300 mg daily. Three FRDA patients
got antioxidants; one took L-carnitine 1500 mg, coen-
zyme Q(10) 400 mg and vitamine E 1000 mg, one idebe-
none 2250 mg and one L-carnitine 1500 mg. All
participants paused with their medication for at least 12
hours prior to the MeBT. Exclusion criteria were con-
current liver diseases, excessive alcohol consumption (50
g/d of ethanol), severe other diseases, underage and
The13C-methionine breath test was prepared as estab-
lished by Banasch et al 2008 .
breath test was run after an overnight fasting. All sub-
(L-methionine-13C, 99% atom isotopic enrichment;
Cambridge Isotope, Andover, MA) in a dose of 2 mg/kg
of body weight dissolved in 100 ml of water. Breath
samples were collected before substrate administration
at baseline and then every 10 minutes for 90 minutes in
50 ml closed aluminized plastic breath bags. During the
test patients were requested to sit relaxed. The collected
breath samples were analysed by measuring the13C/12C
isotope ratio via nondispersive isotope selective infrared
spectroscopy (IRIS;13C Wagner Analysen Technik, Bre-
men, Germany). Results were expressed as delta (δ)13C/
12C and as delta over baseline (DOB). PDB-standard
from South Carolina (RatioPDB= 0.0112372) was used
for the analysis of the data. The results were expressed
as percentage dose of13C recovery (PDR) over time for
each time interval, maximum PDR (PDRmax) and cumu-
lative PDR (cPDR) after different minutes of testing
time up to 90 min (cPDR90).
Statistical analysis was performed using the software
program SPSS statistics 18. All measured parameters
and clinical data are presented as mean ± SD. Normality
of distribution of the data was tested by one-sample
Kolmogorov-Smirnov test. For testing the significance of
differences between the two groups, independent t test
procedure was used. Homogeneity of variance was
shown by Levene’s test. For specification of relationship
between13C-methionine breath test results and FRDA
rating scales as well as clinical data of patients a
Table 1 Baseline group statistics of FRDA patients and healthy controls
ParameterFRDA (n = 16) controls (n = 16)
GAA shorter allel *
GAA larger allel *
both alleles *
Onset of the disease [yr]
Disease duration [yr]
43.8 ± 9.3 (28-59)
1.72 ± 0.1 (1.59-1.83)
73.9 ± 9.9 (58-95)
1.89 ± 0,2 (1.61-2.20)
402 ± 127 (220-560)
798.5 ± 250.6 (250-1110)
394492.9 ± 245596.8
17.2 ± 5.9 (7-30)
26.7 ± 8.7 (8-42)
26.5 ± 7 (9.5-36)
43.1 ± 8.7 (28-60)
1.73 ± 0,1 (1.57-1.89)
74.8 ± 9 (56-94)
1.9 ± 0.2 (1.61-2.16)
Values are given as mean ± SD; range (min-max) in brackets; Abbreviations: BSA - body surface area, yr - years, SARA - scale for the assessment and rating of
ataxia. *Exact GAA allel length was available for n = 14
Stüwe et al. BMC Neurology 2011, 11:145
Page 2 of 4
stepwise linear regression model was performed. The
significance level of the F value for accepting an inde-
pendent variable to the linear regression model was cho-
sen as p = 0.05 and for exclusion as p = 0.1.
Demographic, clinical and genetic characteristics of
FRDA participants and matched healthy controls are
provided in table 1. All variables showed normal distri-
bution. Independent t-test for scale parameter and
Fisher exact homogeneity Chi-square test for categorical
parameter revealed no significant differences (t-test: p >
0.1, Chi-square test: p = 1) between the FRDA group
and the control group.
3.1.13C-Methionine Breath Test
Significant decay of mitochondrial function was found
in FRDA patients in comparison to controls for the
cumulative percentage dose rate over 90 minutes of
exhaled13CO2(cPDR90 [%] ± SEM: 5.61% ± 0.61% vs.
7.45% ± 0.4%; p = 0.018; Figure 1B). Additionally, the
maximal exhaled percentage dose of13CO2recovered
was reduced in the FRDA patient group (PDRmax
± SEM: 6.82%/h ± 0.81%/h vs. 9.71%/h ± 0.6%/h; p =
0.007; at 33 ± 10.5 min, see Figure 1A). In an addi-
tional explorative analysis, independent t-test was cal-
culated for the cPDR of each time point. Here
differences of cPDR between FRDA and matched con-
trols were found already after test duration of 20 min-
utes (Figure 1B). No differences were observed
between drug-free FRDA patients (n = 6; cPDR90 [%]
± SEM: 6.99% ± 0.67%, PDRmax± SEM: 8.65%/h ±
0.93%/h) and patients on medication (n = 10; cPDR90
[%] ± SEM: 4.79% ± 0.8%, PDRmax± SEM: 5.72%/h
± 1.05%/h). No differences were observed between
patients with antioxidants (n = 3; cPDR90 [%] ± SEM:
5.86% ± 1.24%, PDRmax± SEM: 6.86%/h ± 1.42%/h)
and patients without antioxidants (n = 13; cPDR90 [%]
± SEM: 5.56% ± 2.69%, PDRmax ± SEM: 6.81%/h
± 3.56%/h; p = 0.855, 0.979 respectively).
Liver metabolism as assessed by cPDR and PDRmax
did not correlate with genetic markers (expanded GAA
repeats and their interaction) nor with age, age at onset
of symptoms or duration of disease and did not reflect
disease severity as assessed by SARA using multiple lin-
ear regression analysis.
The present cross-sectional study is the first to show
subclinical liver affection in FRDA. Since participants
with concurrent liver diseases or excessive alcohol con-
sumption were excluded, none of the participants was
suffering from a clinical manifest liver affection. Using
clearly demonstrate mitochondrial dysfunction in the
liver in vivo. This is in accordance with histophalogical
findings, describing an increased iron deposition not
only in FRDA heart, but also in liver and spleen in a
pattern consistent with a mitochondrial location .
Down regulation or defect of citric acid cycle enzymes
like aconitase, defects in complex I/II/III of the endoxi-
dation and thus a failure of redox-equivalent oxidation
necessary for the citric acid cycle are potential mechan-
isms. The reduced mitochondrial function in MeBT
confirms the importance of frataxin for mitochondrial
energy metabolism in FRDA .
Subclinical, impaired mitochondrial liver function
should be considered when applying drugs with poten-
tial mitochondrial toxicity like valproate or propofol that
are known to exert critical effects in other mitochon-
drial disorders like polymerase gamma (POLG) muta-
tions . One case with fatal liver failure associated
with valproate therapy in a patient with FRDA has been
described so far .
Contrary to our expectation, no correlations between
MeBT and disease severity, age of onset or disease dura-
tion could be established. Although inverse correlation
of clinical severity and repeat lengths has repeatedly
been reported in FRDA,[5,6,19] we found no correlation
of MeBT results with the number of GAA repeats or
their interaction. Thus, measuring of hepatic mitochon-
drial activity by MeBT does not seem to be a good bio-
marker on the follow-up of natural history of the
disease and the response to drugs in clinical trials.
One limitation of our study is that an influence of co-
commitant medication on the MeBT outcome cannot
be completely excluded. However, no differences were
observed between drug-free FRDA patients (n = 6) and
patients on medication (n = 10) and an impairment of
mitochondrial metabolism is not described for the listed
13C-methionine breath test, however, we could
Figure 1 Mean recovered
[%/h]) (A) and mean cumulative exhaled13CO2(cPDR [%]) (B)
of 16 FRDA patients (circle) and 16 healthy controls (square);
Error bares = ± SEM; * - significant difference between the
groups (p < 0.05); ‡ - explorative difference between the
groups (p < 0.05).
13CO2as function of time (PDR
Stüwe et al. BMC Neurology 2011, 11:145
Page 3 of 4
Taken together, the data of our pilot study using the
MeBT in FRDA indicate that FRDA patients exhale sig-
nificantly smaller amounts of13CO2compared with
healthy controls indicating a subclinical liver affection in
We are grateful to all patients for participation.
Carsten Saft was supported by a FoRUM grand, University of Bochum (AZ:
K040-2009). Ludger Schöls was supported by a BMBF grant to mitoNET
(01GM0864). Oliver Götze was supported by DFG (Gö 13582/1).
The study was approved by the ethics committee of the University of
1Department of Neurology, Ruhr-University, St. Josef-Hospital, Bochum,
Germany.2Department of Internal Medicine I, Ruhr-University, St. Josef-
Hospital, Bochum, Germany.3Division of Gastroenterology and Hepatology,
University Hospital Zurich, Switzerland.4Department of Human Genetics,
Ruhr-University Bochum, Germany.5Department of Neurology and Hertie
Institute for Clinical Brain Research, Tübingen, Germany.6German Center for
Neurodegenerative Diseases (DZNE), Tübingen, Germany.
Stüwe 1C, 2B, 3A; Goetze 2A, 2B, 2C, 3B; Banasch 1C, 3B; Arning 1C, 2C, 3B;
Schmidt 1A, 2C, 3B; Schöls 1B, 2A, 2C, 3B; Saft 1A, 1B, 1C, 2A, 2C, 3A, 3B
1. Research project: A. Conception, B. Organization, C. Execution;
2. Statistical Analysis: A. Design, B. Execution, C. Review and Critique;
3. Manuscript: A. Writing of the first draft, B. Review and Critique;
All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Received: 25 August 2011 Accepted: 15 November 2011
Published: 15 November 2011
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