Erectile dysfunction (ED) remains one of the most frequent disturbances of male health with great negative impact on psychological, behavioral, and social conditions of the affected subject [1]. Moreover, a close correlation between ED and the risk of cardiovascular diseases has recently become evident [1,2]. The search for safe and efficient approaches to combat and control ED has been growing steadily.

Current pharmacological means target two major causes of nonandrogen ED revealed so far: the impaired signalling from central nervous system and/or peripherally from penile tissue, to improve local endogenous metabolic pathways that control the erection process [3,4]. The drugs/supplements inducing penile smooth muscle relaxation are numerous, the best known being prostaglandin E1, NO donors, phosphodiesterase inhibitors, bioactive peptides, excitatory amino acids, and antagonists/agonists of neuro-transmitting and other receptors [3].

It is worthwhile to mention that practically all preparations with pro-erectile action implicate NO-dependent mediation although by different mechanisms [5]. The nitric oxide synthases, heavily expressed in the penile tissue (endothelial e-NOS, inducible i-NOS, and neuronal n-NOS), nitric oxide produced by these enzymes, and the connected downstream molecular targets-effectors like cGMP are at the crossroad of a number of routes leading to a cascade of events: vascular smooth muscle relaxation, increased intra-tissue blood flow, and eventually penile erection [3,5].

In accord with a common view, exclusively eNOS and nNOS expressed locally in the nerves or vascular endothelium of the penile tissue participate in penile erection [6], and are involved in the mechanisms of action of different anti-ED drugs [3]. No other cells bearing the enzymes have been considered so far, and never iNOS was suspected to have a potential positive role in the control of erectile function.

In general, iNOS has been broadly condemned as a proinflammatory enzyme producing large amounts of NO that causes damage to bioactive molecules and structures and is implicated in a number of pathologies including endothelial dysfunction in resistance and conduit vasculature [7].

However, classical pioneering studies from Moncada’s group [8] have demonstrated that initial moderate up-regulation of iNOS in macrophages is an essential impetus for further concerted activation of constitutive eNOS and nNOS. NO produced by iNOS in macrophages regulates the activities of these two constitutive enzymes and, being produced in excess, is inhibitory for its parent enzyme iNOS.

Moreover, a leading current concept of hormesis [9] considers macrophage-located iNOS and NO produced by macrophages as a double-edged molecule with potential for beneficial as well as for deleterious effects [8].

Both conditions, with too much and too little NO may have negative effects on protective immunity, on vascular tone, on nervous tissue degeneration, and on diabetic complications [8-10]. Hence, the search for hormesis-inducing substances as remedies from some human diseases has been gaining more and more grounds.

Marine fish collagen and collagen peptides have been widely used as functional foods or dietary supplements due to their homology to human collagen structure [11], their safety profile [12], stability, biocompatibility, high bioavailability through gastrointestinal barrier [13], and potent bioactivities [14]. Marine collagen peptides (MCPs) obtained by enzymatic digestion of fish skin have been shown to exert several health effects mainly in two directions: metabolic disorders and skin/bone repair.

Thus, they positively affected glucose and lipid metabolism in patients with type II diabetes mellitus [15], improved lipid metabolism in obese people [16] and genetically modified mice [17], ameliorated early alcoholic liver injury [18], and possessed hypotensive and lipid normalising action in patients with primary hypertension [19].

In a recent publication [20], results of a randomized single-blind self-controlled clinical investigation have shown that dietary supplementation with MCPs in combination with selected antioxidants remarkably enhanced nitrite/nitrate plasma levels and ATP content in erythrocytes, along with skin rejuvenation, and general energizing (hormesis-like) effects.

Taking into consideration the leading role of NO in penile erection, and the recognized NO-independent involvement of ATP through adenosine or purine receptors [3], the present pilot study was designed to reveal possible clinical actions of this food supplement towards ED, and to investigate possible mechanisms.

Study design

The clinical study protocol was conceived and developed at the Urology Department of the Medical Faculty, Kabardino Balkar State University named by Berbekov, Nal’chik, Russia. The Protocol and Case Report Forms were discussed and approved by the local Ethical Committee (Protocol N 44-02-2016, Medical Faculty, Kabardino Balkar State University named by Berbekov, Nal’chik, Russia). The study was performed during March-May, 2016.

Recruited patients and healthy controls

The pilot clinical-laboratory study enrolled an experimental group (EXP) of 15 adult Caucasian male volunteers (age 55-61 years; mean age 58,7±2,1 years) recruited at the Urology Department, Medical Faculty, Kabardino Balkar State University named by Berbekov, Nal’chik, Russia, according to the inclusion criteria.

Inclusion criteria comprised subjects: (i) of male sex, with age range 55-65 years, having a constant sexual partner, (ii) suffering a functional impotence confirmed by the International Index of Erection Function (IIEF-5) lower or equal to 20 since at least 1 year, (iii) without acute or chronic disorders/diseases of male sexual organs/hypogonadism, iv) without acute bacterial or viral infections, (v) without cardio-vascular and other internal diseases in the stage of decompensation, (vi) agreeing to interrupt any intake of antioxidant nutraceuticals/drugs for at least 1 week before and during the entire duration of the trial, (vii) agreeing to donate their blood samples (volume 20 mL) for further procession and analyses at enrollment and at trial cessation, (viii) without any difficulty in understanding and following the instructions received by the clinical investigator.

Subjects with allergic/intolerance reactions to any component of the tested product, subjects with diabetes/pre-diabetes, subjects with metabolic syndrome, subjects on any other nutraceutical interventions or/and therapies, and subjects simultaneously engaged in other clinical trials were excluded from the study. Demographic distribution, baseline clinical conditions, and laboratory markers of the EXP group relevant to the study are shown in Table 1.

Table 1: Demographic, clinical, and laboratory data of the the experimental group with erectile dysfunction (ED).

Several laboratory parameters presumably connected to ED were determined. Pre-selected ED patients (n = 15) resulted to be free from diabetes/pre-diabetes (normal levels of fasting glucose, glycosylated haemoglobin, and insulin, data not shown) and hypogonadism (total testosterone levels were within the normality range).

Two patients were obese (BMI 39, class II, and BMI 31, class I) with higher-than-normal levels of cholesterol (metabolic syndrome), the former also with moderate hyperhomocysteinaemia. Four patients were diagnosed with arterial hypertension although well controlled by blood pressure lowering drugs.

The participants were informed that they could interrupt clinical trial at any moment, without any explanation of causative reason for their action, or if they noticed any adverse reaction to the tested product, or had any sensation that the product intake affected negatively their sexual performance.

All recruited patients gave their informed consent to personal and anamnestic data collection and biological material sampling. The guidelines of Helsinki Declaration for human experimentation were strictly followed during the conduct of clinical trial.

Eighteen age-matched (55-65 y) men without ED (CTR1), and 41 healthy subjects of both sexes with age range 20-65 years (CTR2) served as controls, having agreed to donate 20 mL of venous blood for laboratory analyses.

Food supplement under investigation

A food supplement containing marine collagen peptides derived from skin of deep sea fish (MCPs, 570 mg), grape skin extract (10 mg), coenzyme Q10 of plant origin (10 mg), luteolin (10 mg), and selenium (0.05 mg) of plant origin was formulated in soft gelatine capsules. As inactive vehicles, refined and partly hydrogenated soybean oil and small admixture of pure soybean lecithin were used. The product, under the commercial name of CELERGEN^® (manufacturer: Laboratories-Dom, Carouge, Switzerland), was kindly provided by Suisse Ueli Corporation. According to manufacturer’s information, the deep sea fish sources, i.e., Pollachius vireos , Hippoglossus hippoglossus , and Pleuronectes platessa , originated from the French coast of North Sea.

Fish skin was homogenised in distilled water, with addition of a protease complex. The enzymatic proteolytic process was carried out at 40°C and pH 8.0 for 3 h, after which the proteases were inactivated by short-term heating (56°C for 10 min). The liquid was sterilized by Millipore filtration (pore size 0.02 mm), and spray-dried to prepare MCP powder, as described in detail previously [21,22]. Chemical analysis by a Kjeldalh assay of the powder confirmed a > 90% content of collagen peptides, with moisture and ash content < 10%. According to previous publications [22,23], the molecular weight distribution of MPCs after the described enzymatic digestion process was within the range of 10-60 Da, and MCPs were enriched in glycine, glutamine, proline, hydroxyproline, asparagine, alanine, and arginine.

The aqueous extract of grape skin was obtained from Vitis vinifera Linn. fruit and contained at least 70% of polyphenols and 20% of procyanidins as per UV/Vis spectrophotometry data. The coenzyme Q10 component of plant origin was of highest purity (100±3%), confirmed by both IR-spectrophotometry and high performance liquid chromatography (HPLC) methods. Food-quality luteolin was extracted from Marigold plant petals, and the extract contained 20% of luteolin and 1% of zeaxanthin evaluated by HPLC analysis. Selenium in the form of selenite (according to gravimetric method) was extracted from plant bulbs and leaves. Acute and chronic toxicity data, and documents of Certificates of Analyses, Security, and Registration in Switzerland were duly provided by the manufacturer.

Supplementation with Celergen^®

All recruited patients eligible for the clinical study were instructed to take 1 capsule of Celergen^® twice a day, in the morning at breakfast and in the evening at dinner time, for 30 consecutive days. In the case of unwanted adverse effects or ineffectiveness of the supplementation, the participants were free to interrupt it and were advised to report their feelings to the principal clinical investigator.

Clinical assessment

Clinical assessment of the state of functional impotency was performed two times, in the beginning (EXP0) and at the cessation of the clinical study (EXP30), by answering the validated questionnaire for the determination of the International Index of Erection Function-5 (IIEF-5), based on 5 questions. The results were expressed as a final score. The final erection score equal to 5 corresponded to severe erection dysfunction while erection score equal to 25 corresponded to normal erection function.

Reagents and KIT assays

The majority of chemical reagents, mediums, solvents, and luciferinluciferase for ATP assay were from Sigma Chemical Co. (St. Louis, MO, USA); kits for enzyme activity assays, and Griess reagent for nitrites/nitrates determination were from Cayman Chem. Co. (Ann Arbor, MI, USA). Manufacturers of other reagents are mentioned within the respective methods.

Biological material and processing

Peripheral venous blood (20 mL) was drawn after overnight fasting into vacutainers with ethylene diamine tetra-acetic acid disodium salt (EDTA) as anti-coagulant. Both patient and donor samples were processed and analyzed in parallel. Circulating granulocytes and mononuclear leukocytes were obtained by double density gradient centrifugation of 15 mL of total blood (Histopaque, d = 1.077 and 1.199 g/mL). Peripheral blood mononuclear leukocytes were collected from the upper interface, while granulocytes from the lower interface. The lower layer containing red blood cells (RBC) was stored at -80°C after thorough washing in saline, for glutathione peroxidase (GPX) activity assay.

Mononuclear leukocytes were left to adhere in Petri dishes in RPMI medium complemented with 2 mM glutamine, 1 mM sodium pyruvate, 1% non-essential amino acids, 0.05 mM 2-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin (RPMI complete; all from Invitrogen, San Giuliano Milanese, Italy) and 5% human plasma (all reagents from Sigma Chemicals, Milan, Italy). After 2 h at 37°C, the non-adherent cells, essentially composed of T-cells, were discarded. Adherent monocytes were re-suspended in phosphate buffer saline, centrifuged at 1650 rpm for 10 min and then aliquoted at 5 x 10⁶ cells/ vial. Granulocytes were used for phagocytosis assays, and monocytes for the determination of iNOS mRNA expression and ATP content.

Samples of whole blood (5 mL) were allowed to sediment for 40 min at room temperature and plasma supernatant was collected, aliquoted and stored at -80°C until quantitative analyses of nitrites/nitrates and cytokines.

Phagocyte function assays

Intensity of phagocytosis and intracellular killing were assessed by routine methods commonly used in clinical bacteriology laboratories. Briefly, 1 mL of granulocyte suspension (10⁶ cells/mL) was mixed with 1 mL of Staphylococcus aureus bacterial suspension (10⁷ cells/mL). The mixture was incubated under continuous mixing at 37°C for 30 min. Then, smears were prepared on microscopic slides, fixed, and stained by Romanovsky - Gaemsa dye. The smears were examined under microscope. Per cent of phagocytosing granulocytes and number of bacterial particles inside phagocyte were determined. The remaining mixture was used to assess intracellular bacterial killing. It was centrifuged at 1500x g for 10 min. Bacterial sediments were collected and diluted to an OD₆₀₀ of 0.1 with fresh medium and spread onto Petri dishes with appropriate medium and agar. They were allowed to grow at 37°C for 24 hours. Bacteria survival rates were calculated as colony-forming-units (CFU) of cells co-incubated with granulocytes divided by that of untreated bacteria. The results were expressed in per cent.

Intensity of reactive oxygen and nitrogen species by granulocytes was measured by luminol-dependent chemiluminescence. The suspension of granulocytes (10 μL, 10⁵ cells) was added to 1 mL of preheated Hanks’ Balanced Salt Solution (pH 7.4) containing 0.2 mM luminol. Then, phorbol-12-myristate-13-acetate (PMA) (10 ng/mL) was added and the luminol-dependent chemiluminescence response was monitored for 5 min with a Victor₂ (Wallac Oy) 1420 multi-label counter, equipped with Wallac 1420 Software (Perkin Elmer). Each measurement was repeated 3-5 times. Results were expressed in counts per second (cps) per 10⁶ granulocytes [24].

Nitric oxide synthases (NOS) mRNA expression and plasma levels of nitrites/nitrates

For the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assays, total RNA was extracted from isolated blood monocytes, reverse transcribed into DNA, and then amplified by PCR techniques using iNOS-specific or eNOS-specific primers.

Total RNA was isolated using the GenElute Mammalian Total RNA Kit (Sigma-Aldrich, Milan, Italy) in accordance with the manufacturer's instructions. The amount of RNA was determined by the absorbance at 260 nm. Total RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) at 42°C for 30 min. Then, cDNA was amplified with IQ SYBR green Supermix (Bio- Rad, Hercules, CA) using the MiniOpticon Real-Time PCR Detection System (Bio-Rad, Hercules, CA) in accordance with the manufacturer's instructions. Briefly, PCR was performed in a 25 μL volume containing cDNA equal to 100 ng of total RNA using a temperature program as follows: 36 cycles of denaturation at 95°C for 15s, annealing and extension at 60°C for 120s. Melt curve analysis was performed to confirm the specificity of the amplified products. Signal of two housekeeping genes, ribosomal 18S and beta-actin, was chosen as reference and expression of the other genes studied was normalised to each of these reference genes. Fold changes were calculated with the comparative C_t method (ΔΔC_t) according to [25] and were expressed as the percentage of mRNA in monocytes after the trial to that in monocytes before the trial. Total RNA from each treatment well was reverse-transcribed in duplicate and all PCR amplifications repeated twice from each RT reaction. The primer sets were designed using Primer-BLAST (NCBI) and were synthesised by Eurofins MWG Operon (Ebersberg, Germany).

The primer sets used for the quantitative RT-PCR were as follows: β- actin fwd: 5′-AAATCTGGCACCACACCTTCTAC-3′; β-actin rev: 5′- ATAGCACAGCCTGGATAGCAAC-3′; 18S rRNA fwd: 5'- TCCCCCAACTTCTTAGAGG-3';18S rRNA rev: 5'- GCTTATGACCCGCACTTAC-3′; iNOS fwd: 5'- TACTCCACCAACAATGGCAA-3′; iNOS rev: 5'- ATAGCGGATGAGCTGAGCAT-3′; eNOS fwd: 5'- GAGCTCTGCATTCAGCACG-3′; eNOS rev: 5'- AGAGTTCTGGGGGCTCATCT-3′.

Plasma levels of nitrites/nitrates (NO2¯/NO3¯, expressed as μM) were measured spectrophotometrically by Griess reagent [26]. Protein content was measured according to Bradford [27], using a microplate assay kit (Bio-Rad, Hercules, CA, USA).

Cytokine assays

The plasma levels of IL-1β, IL-10, TNF-α, and INF-γ were measured by enzyme-linked immunosorbent assay (ELISA) purchased from R&D Systems (Minneapolis, MN), following manufacturer’s instructions. Each measurement was repeated three times. Cytokine concentrations were expressed in pg/mL of plasma, and each factor was quantified in the linear range of its calibration curve.

ATP assay

One hundred μl of (monocyte+granulocyte) pellet was stored on ice until analysis. Ice-cold water (990 μl) was added to 10 μl of monocytes, mixed and the lysed erythrocytes were kept on ice. The principle of ATP assay is based on the quantitative bioluminescent determination of adenosine 5'-12 triphosphate (ATP), assessed by the Bioluminescence Assay Kit. In the assay, ATP is consumed when firefly luciferase catalyses the oxidation of D-luciferin to adenyl-luciferin which, in the presence of oxygen, is converted to oxyluciferin with light emission. This second reaction is essentially irreversible. When ATP is the limiting reagent, the light emitted is proportional to the ATP present. The measurements of luciferin-luciferase chemiluminescence were performed on a Victor2 1420 multi-label counter, equipped with Wallac 1420 Software (Perkin Elmer, MA, USA). Results were expressed as mmoles/L.

Glutathione peroxidase assay

RBC glutathione peroxidase (GPX, U/g Hb) activity was determined using Cayman Chemical kit, according to the method [28].

Statistical evaluation

Statistical analysis of clinical data was carried out using WINSTAT program for personal computers (Statistics for Windows 2007, Microsoft, USA). All biochemical and molecular measurements were done in triplicate and data were statistically evaluated. Values were presented as mean and standard error of the mean. Differences between initial/final data for a single participant were analysed by paired t test and by Mann-Withney test for changes from baseline. All reported P values are from two-tailed tests, and P values of less than 0.05 were considered to indicate statistical significance.

Safety and clinical efficacy of Celergen^® supplementation

There were no complaints from the participants about any allergic reactions or gastric intolerance to the intake of Celergen^®. There were no drop-outs from the trial due to reported supplement inefficiency or toxicity. Routine haematological and biochemical analyses, which were carried out after blood donation in the beginning and after the cessation of the study, did not show statistically significant changes possibly reflecting adverse consequences of the test nutraceutical in the prescribed dosages (data not shown).

All 15 recruited men (100%) with moderate insufficiency of erection (initial IIEF-5 scores ranged from 16 to 20) reported a significant improvement of their ED reflected by remarkably increased individual IIEF-5 score. Statistical evaluation of the scores showed a statistically significant increase in their mean value (P < 0.05; Table 2), which largely overlapped the IIEF-5 range of values characteristic for the group of healthy age matched males (CTR1). Both doctors and patients expressed their deep satisfaction with the effects of supplementation towards men’s erectile function.

Table 2: Effects of Celergen^® supplementation (30 days, 2 capsules/day) on the erectile function as measured by the International Index of Erection Function-5 (IIEF-5).

Effects on circulating phagocyte functions and levels of phagocyte-polarising cytokines

To prove the assumption that biological activities of MCPs target the phagocytes, the functions of circulating granulocytes and monocytes were determined before (EXP0) and after (EXP30) the clinical trial. The same parameters were tested in the group of age-matched males without complaints to ED (CTR1) and in a larger age-unrelated group of healthy subjects of both sexes (CTR2). It was shown that the supplementation with Celergen^® statistically significantly enhanced (P < 0.05) the phagocytosing index of bacterial particles reflecting a number of phagocytes (Figures 1A and 1B). Also, we registered increased (P < 0.05) intracellular bacterial killing (Figure 1C), and reactive oxygen (ROS) and nitrogen species (RNS) production by phagocytes as revealed by the PMA-activated luminol-dependent chemiluminescence (Figure 1D). These data provided a clear-cut proof that a major component of the supplement MCPs targeted circulating phagocytes, pre-conditioning them for better defence against bacterial invasion through ROS+RNS-dependent intracellular killing mechanism.

Figure 1: Effects of Celergen^® supplementation (30 days, 2 capsules/ day) on circulating phagocyte functions. (A) Index of phagocytosis (%, mean ± S.E.M); (B) Number of bacteria within one phagocyte (n. of units, mean ± S.E.M); (C) Efficiency of intracellular killing of bacteria (%, mean ± S.E.M); (D) PMA-stimulated luminoldependent chemilulinescence of neutrophils (arbitrary units, mean ± S.E.M).
CTR1 = Healthy male, age-matched with EXP group (55-65 y), n = 18
CTR2 = Healthy subjects of both sexes (20-45 y), n = 41
EXP0 = Males with mild erectile dysfunction (55-61 y), before clinical trial beginning, n = 15 EXP30 = Males with mild erectile dysfunction (55-61 y) , at clinical trial cessation, n = 15 LDC: luminol-dependent chemiluminescence; a.u.: arbitrary units
#P < 0.05 vs. EXP0
*P < 0.05 vs. CTR1

It has been also suggested that phagocyte activation may be effectuated through Toll-like receptors (TLRs) by the innate immunity pathways. For example, MCPs of different origin have been shown to activate innate immune response of macrophages and neutrophils through TLR4, that leads to NADPH-oxidase (NOX4) activation and ROS overproduction [29,30]. Stimulation of TLR-related innate immunity response in phagocytes leads to monocyte/macrophage polarisation towards the M1 (pro-inflammatory) type, characterised by increased production of tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), but not of interleukin 10 (IL-10) cytokines [31]. These M1 phagocytes produce increased amounts of NO synthesised by the up-regulated inducible nitric oxide synthase (iNOS), with anti-bacterial and anti-viral capacity [32].

The results obtained here indicate that supplemented ED patients had higher than the baseline levels of both TNF-α and IFN-γ (P < 0.001) (Figures 2A and 2B), while the levels of IL-10 remained unchanged (Figure 2C). Although the plasma levels of two proinflammatory cytokines were increased after supplementation, their values remained within the normal range detected in the control groups of healthy subjects. The levels of IL-1β were also elevated vs. baseline values (P < 0.05, Figure 2D).

Figure 2: Effects of Celergen^® supplementation (30 days, 2 capsules/ day) on the plasma levels of phagocyte-released inflammatory interleukins (pg/mL). (A) Tumor necrosis factor alpha (TNF-α); (B) interferon gamma (IFN-); (C) Interleukin 10 (IL-10); (D) Interleukin 1 beta (IL-1β).
CTR1 = Healthy male, age-matched with EXP group (55-65 y), n =
18
CTR2 = Healthy subjects of both (20-65 y), n = 41
EXP0 = Males with mild erectile dysfunction (55-61 y), before clinical trial beginning, n = 15
EXP30 = Males with mild erectile dysfunction (55-61 y), at clinical trial cessation, n = 15
#P < 0.05 vs. EXP0
##P < 0.001 vs. EXP0
**P < 0.00 vs. CTR1

As expected, plasma content of nitrites and nitrates as a measure of NO production were statistically increased (P < 0.05) in the experimental group after supplementation (Table 3). Finally, they reached normal range of values while the baseline ones were significantly lower-than-normal age-unrelated controls (CTR2). In concomitance, iNOS in circulating phagocytes was up-regulated at transcriptional level since iNOS mRNA content was nearly two-fold increased versus baseline (P < 0.05), again reaching the upper normal values of the CTR2 (Table 3). No up-regulation of constitutive endothelial nitric oxide synthase (eNOS) was observed (data not shown).

Table 3: Effects of Celergen^® supplementation (30 days, 2 capsules/day) on the plasma levels of nitrates/nitrites (NO2¯/NO3¯) and the expression of inducible nitric oxide synthase gene (iNOS) in circulating phagocytes.

In this clinical-laboratory part of the study, the metabolic and immunological outcomes of the supplementation indicated that circulating phagocytes as major cellular targets. The phagocytes were moderately pre-conditioned for more pronounced anti-bacterial defence with the help of ROS+RNS and of the pro-inflammatory cytokine-based armament. The interaction of MCPs of the supplement with circulating phagocytes occurred through TLR4 pathway, as it has been shown previously in animal experiments [29,30,33]. In these M1 type phagocytes, iNOS was up-regulated and the total amount of NO metabolites in plasma was enhanced versus its baseline value although it did not over-run the upper limit of normality. This situation was in favour of hormesis-like action of the supplement towards innate immunity and NO production.

The study has the limitation of not evaluating in detail the protective response by endogenous antioxidant systems. The only hydroperoxide detoxifying enzyme studied here was glutathione peroxidase (Table 4). Its activity did not change, consistently with our previous clinical data and their explanation in terms of the absence of lipid peroxides as a substrate for the enzyme [20]. However, this could be also a result GPX inhibition by NO [34].

Table 4: Effects of Celergen^® supplementation (30 days, 2 capsules/day) on the activity of glutathione peroxidase (GPX) in erythrocytes.

Supplementation effects on ATP levels in circulating phagocytes

The analysis of ATP content in circulating phagocytes (granulocytes and monocytes) showed that the baseline values in the experimental group of males were much lower than in the age-unrelated healthy subjects and slightly lower than that in the age-matched group of men without complaints of ED (Table 5). A one month period of supplementation with Celergen^® resulted in the remarkable increase in the levels of ATP (P<0.01 vs. the beginning of the trial). In the previous clinical trial, we have found highly increased levels of ATP in circulating erythrocytes of subjects supplemented with Celergen^® for two months [20].

ATP, adenosine, and other purines decreased both basal and induced tension in isolated rabbit corpus cavernosum preparations presumably through purinergic transmission, that seems to be important for the initiation and maintenance of penile erection [3,35]. However, some experimental data has strongly suggested a local regulating role of ATP and adenosine on erection through stimulation of A2a adenosine receptors [36]. ATP was then found to be a potent NO-independent relaxant for human and rabbit corpus cavernosum [37]. Contrasting data were reported, showing that a relaxing action occurred through purinoreceptors P2Y via NO release [38]. Notwithstanding existing uncertainties regarding possible involvement of ATP or its metabolites in the physiological mechanisms of erection, increased levels of ATP in circulating leukocytes (Table 5) and erythrocytes [20] of subjects supplemented with MCP+antioxidants could be a promising biochemical marker of improved ED.

Another promising mechanistic clue of observed Celergen^® effects could connect the improvement in bioenergetics with the moderate induction of NO production and endogenous cytoprotective mechanisms of stress resistance (hormesis). ATP deficiency due to impaired mitochondrial metabolism has been implicated in human ageing and age-related pathologies, such as neuro-degeneration and diabetes [39,40]. The decrease of energy levels severely affects the cellular functions requiring ATP, such as biosynthesis, receptor, and channel activation, signal transduction, and cell division [41]. On the other hand, effective ATP synthesis through activation of electron transfer in mitochondria and oxidative phosphorylation is tightly connected with electron leakage and mitochondria-induced oxidative stress, which is compensated by endogenous protection systems, such as Nrf2-dependent cytoprotective defence [42]. As a positive feedback loop, Nrf2 activation is essential for increased ATP synthesis [40]. This mechanism of protection from mild stresses is initiated by NO therefore one could predict that the mild induction of NO synthesis and its moderately increased levels would lead to the activation of protective mechanisms (hormesis-like action) and to the increased energy stores in human organism [43]. Such a hormesis-stimulating and ATP restoring strategy has been recently suggested as a promising therapeutic approach to treat complication of diabetes [10], and obesity [44], as well as age-related pathologies in general [9]. ED is one of the most frequent disorders in ageing men [45], and in the complications of metabolic diseases [2,46].

For the first time, marine collagen peptides combined with selected low-dose antioxidants have shown pro-erectile effects through different (phagocyte produced NO)-mediated mechanisms than those described previously. This supplementation could be considered as a safe and effective alternative to anti-ED drugs in the case of mild-tomoderate ED. Necessarily, more mechanistic studies are needed, and larger scale multi-centre placebo-controlled clinical trials should follow.

The authors gratefully acknowledge Suisse Ueli Corporation for providing free of charge the product for the clinical study and for covering the costs of reagents and analyses.

ZFK and IAM equally contributed to the work. They were principal clinical and laboratory investigators, conceived the protocol, organised clinical trial, collected clinical material, and discussed the data and conclusions. DHS carried out immunological and biochemical analyses. MP corrected the protocol, evaluated clinical data, and draw the conclusions. CD adapted basic research methods for clinical conditions, evaluated laboratory data, and performed statistical analysis. LK produced a general idea of the trial, analysed the data obtained, and wrote the manuscript.

The authors declare the absence of any commercial conflict of interests.