Amino Acids

, Volume 50, Issue 10, pp 1391–1406 | Cite as

Effects of single and combined metformin and l-citrulline supplementation on l-arginine-related pathways in Becker muscular dystrophy patients: possible biochemical and clinical implications

  • Erik Hanff
  • Patricia Hafner
  • Alexander Bollenbach
  • Ulrike Bonati
  • Arslan Arinc Kayacelebi
  • Dirk Fischer
  • Dimitrios TsikasEmail author
Original Article


The l-arginine/nitric oxide synthase (NOS) pathway is considered to be altered in muscular dystrophy such as Becker muscular dystrophy (BMD). We investigated two pharmacological options aimed to increase nitric oxide (NO) synthesis in 20 male BMD patients (age range 21–44 years): (1) supplementation with l-citrulline (3 × 5 g/d), the precursor of l-arginine which is the substrate of neuronal NO synthase (nNOS); and (2) treatment with the antidiabetic drug metformin (3 × 500 mg/d) which activates nNOS in human skeletal muscle. We also investigated the combined use of l-citrulline (3 × 5 g/d) and metformin (3 × 500 mg/d). Before and after treatment, we measured in serum and urine samples the concentration of amino acids and metabolites of l-arginine-related pathways and the oxidative stress biomarker malondialdehyde (MDA). Compared to healthy subjects, BMD patients have altered NOS, arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) pathways. Metformin treatment resulted in concentration decrease of arginine and MDA in serum, and of homoarginine (hArg) and guanidinoacetate (GAA) in serum and urine. l-Citrulline supplementation resulted in considerable increase of the concentrations of amino acids and creatinine in the serum, and in their urinary excretion rates. Combined use of metformin and l-citrulline attenuated the effects obtained from their single administrations. Metformin, l-citrulline or their combination did not alter serum nitrite and nitrate concentrations and their urinary excretion rates. In conclusion, metformin or l-citrulline supplementation to BMD patients results in remarkable antidromic changes of the AGAT and GAMT pathways. In combination, metformin and l-citrulline at the doses used in the present study seem to abolish the biochemical effects of the single drugs in slight favor of l-citrulline.


ADMA AGAT Guanidinoacetate Homoarginine Metformin Nitric oxide 



Asymmetric dimethylarginine


Arginine:glycine amidinotransferase


Adenosine monophosphate-activated protein kinase


Alkaline phosphatase


Becker muscular dystrophy


Coronary artery disease


Coactivator-associated arginine methyltransferase


Cationic amino acid transporters



CITR group

Citrulline group


Creatine kinase


Creatinine kinase muscle b


Dimethylarginine dimethylaminohydrolase




Duchenne muscular dystrophy




Global l-arginine bioavailability ratio


Guanidinoacetate methyltransferase


Gas chromatography–mass spectrometry




Interquartile range


Multidrug and toxin extruders





MET group

Metformin group


6-min walking distance


Nitric oxide


Nitric oxide synthase


Neuronal nitric oxide synthase


Organic cation transporters




Peripheral artery occlusive disease


Protein arginine methyltransferases


Quality control


Symmetric dimethylarginine


Type 2 diabetes mellitus



We are grateful to Bibiana Beckmann for excellent laboratory assistance and to Dr. Anika Großhennig from the Institute for Biometry of the Hannover Medical School (Hannover, Germany) for her excellent advice in statistical analyses. Dirk Fischer was supported by the Tomi-Hopf-Stiftung, Switzerland (; the Lorenzo-Piaggio Foundation, Switzerland; the Neuromuscular Research Association Basel, Switzerland (; the University of Basel Children’s Hospital; and the Department of Neurology, University Hospital Basel.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

Ethics and health authority approvals were obtained from the local Ethics Committee (Pilotstudie zur Untersuchung der Wirksamkeit von l-Citrullin und Metformin bei Erwachsenen mit Muskeldystrophie Becker; reference No. EKBB EK17/13) and the National Swiss Drug Agency (Swissmedic: Pilotstudie bei Muskeldystrophie Becker, reference No. 2013DR2067, release date 30 May 2013). This pilot study entitled was conducted in accordance with the ethical principles of Good Clinical Practice (GCP) that have their origins in the Declaration of Helsinki.

Supplementary material

726_2018_2614_MOESM1_ESM.docx (43 kb)
Supplementary material 1 (DOCX 42 kb)


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Erik Hanff
    • 1
  • Patricia Hafner
    • 2
  • Alexander Bollenbach
    • 1
  • Ulrike Bonati
    • 2
  • Arslan Arinc Kayacelebi
    • 1
  • Dirk Fischer
    • 2
  • Dimitrios Tsikas
    • 1
    Email author
  1. 1.Core Unit ProteomicsInstitute of Toxicology, Hannover Medical SchoolHannoverGermany
  2. 2.Division of Paediatric NeurologyUniversity of Basel Children’s HospitalBaselSwitzerland

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