newsletter2

The International Coenzyme Q10 Association not only has its headquarters in Ancona, Italy, but also carries out several research programs there, centered on the role of CoQ10 in biology and medicine. In this second newsletter I would like to trace the outlines of these studies.

Our laboratories are located within the Institute of Biochemistry of the Polytechnic University of the Marche which has most of its faculties in the city of Ancona.

An important abnormality in chronic heart failure (CHF) is endothelial dysfunction which contributes to functional impairment. In CHF, endothelial dysfunction may depend either on reduced nitric oxide synthesis, or increased nitric oxide inactivation, or both. In cooperation with Dr. Belardinelli, of the Lancisi Heart Institute here in Ancona, we have been studying the effect of CoQ10 administration on endothelial function for several years. At the dosage of 300 mg/day oral CoQ10 was found to improve functional capacity, endothelial function and left ventricle contractility in CHF. The combination of CoQ10 exercise training resulted in more pronounced effects on all of the above mentioned parameters (3) .

Our laboratory is actively collaborating with the Ancona Pediatric Hospital Salesi in a research aimed at studying the antioxidant effect of Coenzyme Q10 in Down Syndrome (DS). Oxidative stress is known to play a major role in this pathology both due to genetic and

epigenetic factors, suggesting that oxidative imbalance contributes to the clinical manifestation of DS. In particular, the implications of oxidative DNA damage in DS is a very debated argument and it has been

linked with an Alzheimer-type neurodegeneration associated with the syndrome. We previously demonstrated, using an optimized version of the single cell gel electrophoresis (Comet assay), an increased level of DNA damage in peripheral blood lymphocytes of pediatric DS patients compared to aged matched healthy controls (1). Recently in a double-blind randomized controlled trial on 40 patients supplemented on 4 mg/Kg/day Coenzyme Q10 we verified that ubiquinone has a protective effect on slightly damaged cells whilst it was not effective, in our experimental conditions, in decreasing overall levels of DNA damage (2).

Extracellular superoxide dismutase (ecSOD) is located in the extracellular matrix and on the surface of endothelial cells, where it exerts its protective effect on endothelial function. This effect is probably mediated through a decreased inactivation of nitric oxide by the superoxide anion. ecSOD can be tested in patients after an intravenous injection of heparin, which binds and mobilizes the enzyme making it measurable in plasma. Vascular ecSOD activity is substantially reduced in patients with coronary heart disease. Moreover a strong correlation was found between ecSOD and the flow dependent endothelial—mediated dilation (FMD) a functional parameter commonly used as a biomarker of vascular function. In a randomized control study we showed that oral CoQ10 was able to improve ecSOD and endothelial function in patients with coronary heart disease (4). The increase in the ecSOD activity as well as the improvement in the brachial artery ED relaxation were more pronounced in the subgroup of patients with low ecSOD levels on entry. These data suggest that CoQ10 is more effective in patients with decreased levels of antioxidant defences, presumably more prone to oxidative stress.

An important single nucleotide polymorphism, nucleotide 760 G>C in codon 213 in exon 3 of the ecSOD gene occurs with a relatively high frequency in the population: (4% of Swedes, 3% of Australians and 6% of Japanese). This variant does not affect ecSOD enzymatic activity, but reduces its affinity for the endothelium and consequently lowers tissue antioxidant levels with important clinical implications. Heterozygous carriers of this missense mutation has shown 2.3 fold increased risk of ischemic heart disease. Moreover, it has been suggested that the R213G gene variant accelerates the progression of renal failure and atherosclerosis in uremic patients. In particular, in (diabetic nephropathy) hemodyalisis patients with diabetes mellitus, a significantly lower 5-year survival rate was reported in mutated patients. It is therefore important to identify patients carrying this mutation in order to highlight lowered antioxidant defences at vascular levels which could lead to increased susceptibility toward coronary artery disease and atherogenesis. This could also address pharmacogenomical approaches to the therapy. In our laboratory we developed a method to detect R213 single nucleotide polymorphism based on a real time PCR strategy using Locked Nucleic Acid (LNA) probes. This homogeneous technique amplifies and detects the mutation in a single reaction tube combining rapidity and high specificity. A summary of this research was selected as oral presentation at the FEBS international conference in Athens in July 2008 (5).

Coenzyme Q10 biosynthesis is markedly active in testes and, as in other organs, this molecule plays a crucial role both in energy metabolism and as a lipophilic antioxidant. Our group has been working on the role of CoQ10 in sperm cell motility and antioxidant protection in seminal fluid for many years. After investigating the distribution of CoQ10 in sperm cells and seminal plasma, also in varicocele, and the effect of CoQ10 administration on seminal fluid antioxidant capacity, we recently published the results of a double blind, placebo-controlled study conducted in infertile men with idiopathic asthenozoospermia. It was found that exogenous administration of CoQ10 increases the level of the same in semen and is effective in improving sperm kinetic features in these patients (6).

Reduced Coenzyme Q10 (Ubiquinol-10) is the species endowed with antioxidant properties, at a mitochondrial and extramitochondrial level, and in plasma lipoprotein; furthermore it is able to regenerate the active form of Vitamin E from the tocopheryl-quinone. Among the CoQ10 reducing enzymes, NQO1 is the most studied, owing also to its broad substrate specificity. On the basis of these antioxidant and detoxification functions the presence of this enzyme might mean protection against oxidative stress and neoplasia. Conversely, it could be expected that individuals with low or absent NQO1 activity will have a higher susceptibility to developing tumors. Our lab has been studying CoQ10 reductase activity of different cell lines in vitro. Genotype analysis has shown the presence of some polymorphisms and in some lymphoma cell lines the NQO1 gene is not efficiently translated (7). A deficient CoQ10 reducing capacity might bear some practical implications in different pathological conditions.

CoQ10, as well as other antioxidants are often added to different foods, with the aim of decreasing susceptibility to oxidative stress. Our lab also addresses this line of research, by exploring the increase of plasma CoQ10 after taking foods enriched with the same molecule and measuring its biochemical effect. One of the main findings (8) was that percentage variation of plasma CoQ10 following the intake of the relatively low amounts of CoQ10 contained in these functional foods is remarkable. Moreover, there was also an increase in total antioxidant capacity of plasma. This aspect is now being further investigated by measuring the susceptibility to induced peroxidation of low density lipoproteins (LDL) isolated from plasma of subjects who have taken these CoQ10 enriched functional foods for several weeks.

Gian Paolo Littarru

References

1) Tiano L, Littarru GP, Principi F, Orlandi M, Santoro L, Carnevali P, Gabrielli O. Assessment of DNA damage in Down Syndrome patients by means of a new, optimized single cell electrophoresis technique. BioFactors, 2005; 25: 187-195

2) Tiano L, Carnevali P, Santoro L, Padella L, Principi F,

Brugè F, Carle F, Gesuita R, G abrielli O, Littarru GP. Effect of CoQ10 in mitigating oxidative DNA damage in Down Syndrome Patients. Submitted to Pediatric Research

3) Belardinelli R, Muçaj A, Lacalaprice F, Solenghi M, Seddaiu G, Principi F, Tiano L, Littarru GP. Coenzyme Q10 and exercise training in chronic heart failure. Eur Heart J. 2006 Nov;27(22):2675-81.

4) Tiano L, Belardinelli R, Carnevali P, Principi F, Seddaiu G, Littarru GP. Effect of coenzyme Q10 administration on endothelial function and extracellular superoxide dismutase in patients with ischaemic heart disease: a double-blind, randomized controlled study. Eur Heart J. 2007;28:2249-55.

5) Brugè F., Littarru G.P., Silvestrini L., Mancuso T., Tiano L. A novel real time-PCR strategy to detect ecSOD SNP using LNA probes. Mutation Res. May 2009 in press.

6) Balercia G, Buldreghini E, Vignini A, Tiano L, Paggi F, Amoroso S, Ricciardo-Lamonica G, Boscaro M, Lenzi A, Littarru G.P. Coenzyme Q10 treatment in infertile men with idiopathic asthenozoospermia: a placebo-controlled, double-blind randomized trial. Fertil Steril. 2008 Apr 5. [Epub ahead of print]

7) Bruge’ F, Virgili S, Cacciamani T, Principi F, Tiano L, Littarru GP. NAD(P)H:quinone oxidoreductase (NQO1) loss of function in Burkitt’s lymphoma cell lines. Biofactors 2008: in press

8) Arsenio L, Caronna S, Dall’Aglio E, Frega N, Pacetti D, Boselli E, Tiano L, Principi F, Littarru GP. Effect of antioxidant enriched foods on plasma Coenzyme Q10 and total antioxidant activity. Eur J Lipid Sci Technol. 2008: 110.