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Further Characterization of Hemopressin Peptide Fragments in the Opioid and Cannabinoid Systems

Szlavicz, Eszter MD*; Perera, Pannilage Shiromi; Tomboly, Csaba PhD*; Helyes, Zsuzsanna PhD; Zador, Ferenc PhD*; Benyhe, Sandor PhD*; Borsodi, Anna PhD*; Bojnik, Engin PhD*

doi: 10.1213/ANE.0000000000000964
Anesthetic Pharmacology: Research Report

BACKGROUND: Hemopressin, so-called because of its hypotensive effect, belongs to the derivatives of the hemoglobin α-chain. It was isolated from rat brain membrane homogenate by the use of catalytically inactive forms of endopeptidase 24.15 and neurolysin. Hemopressin has antihyperalgesic features that cannot be prevented by the opioid receptor antagonist, naloxone.

METHODS: In the present study, we investigated whether hemopressin (PVNFKFLSH) and its C-terminally truncated fragment hemopressin 1–7 (PVNFKFL) have any influence on opioid-dependent signaling. Peptides have been analyzed using G-protein–stimulating functional and receptor bindings in this experimental setup.

RESULTS: These 2 compounds efficiently activated the G-proteins, and naloxone slightly blocked this stimulation. At the same time, they were able to displace radiolabeled [3H]DAMGO, a selective ligand for μ-opioid system, at micromolar concentrations. Displacement caused by the heptapeptide was more modest compared with hemopressin. Experiments performed on cell lines overexpressing μ-opioid receptors verified the opioid activity of both hemopressins. Moreover, the CB1 cannabinoid receptor antagonist, AM251, significantly decreased their G-protein stimulatory effect.

CONCLUSIONS: Here, we further confirm that hemopressins can modulate CB1 receptors and can have a slight modulatory effect on the opioid system.

From the *Laboratory of Opioid Research, Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary; Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka; and Department of Pharmacology and Pharmacotherapy, Medical School Pecs, Pecs, Hungary.

Engin Bojnik, PhD, is currently affiliated with Department of Surgery, Oncology and Gastroenterology Oncology and Immunology Division, University of Padova, Padova, Italy.

Accepted for publication June 25, 2015.

Funding: Supported by grants from Hungarian National Scientific Research Fund OTKA 108518, K77783, Hungarian National Development Agency TAMOP-2012-024/TAMOP-2012-052 and János Bolyai Research Scholarship of the Hungarian Academy of Sciences (C.T.).

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Engin Bojnik, PhD, Department of Surgery, Oncology and Gastroenterology Oncology and Immunology Division, University of Padova, via Gattamelata, 6435128 Padova, Italy. Address e-mail to

Recently, a new member of hemoglobin fragments has been discovered in rat brain extract. It is called “hemopressin,” based on its primary effect on systemic blood pressure.1 Hemopressin is a short peptide composed of 9 amino acids and has the sequence of PVNFKFLSH.1,2 Besides hemopressin, N-terminally extended forms have been described, such as RVD-HPα (RVDPVNFKLLSH) and VD-HPα (VDPVNFKLLSH).2,3 It is questionable to consider the hemopressin nonapeptide an endogenous compound because recent studies describe it as a hot acid extraction artifact.3,4

Many important physiologic changes were attributed to the hemopressin nonapeptide. It was shown to have a moderate blood pressure–reducing effect,5 and its hypertensive effects were linked to nitric oxide release, predominantly in the microcirculation.6 Nevertheless, the signaling mechanisms responsible for the blood pressure reduction are not fully understood and are currently the subjects of further investigations.7 Moreover, in experimental animal models, hemopressin demonstrated antinociceptive activity.8 It has been observed that bradykinin-induced hyperalgesia was significantly reduced by the application of hemopressin and that the opioid antagonist, naloxone, could not prevent the antihyperalgesia induced by hemopressin.8,9 Thus, the mode of action of hemopressin in pain sensitivity has so far been considered distinct from that of opioids.

Alongside opioids, the elements of the endogenous cannabinoid system can cause antinociception as well.10,11 There are 2 known cannabinoid receptors: the CB1 and CB2 receptors. CB1 receptors are localized particularly in the central nervous system, whereas CB2 receptors can be detected mainly on immune cells. Endocannabinoids are implicated in numerous physiologic processes, including pain sensation, inflammatory responses, neurodegeneration, appetite, and food intake.10 Several reviews have reported that, in addition to analgesic effects, hemopressin, like cannabinoids, is important in the regulation of food intake.9,12 Moreover, in vitro analyses have found CB1-dependent signaling for hemopressin.12,13 On the basis of these findings, hemopressin is considered the first-known peptide activator of the CB1 receptor.

Besides N-terminally extended forms, we also investigated C-terminally truncated derivatives of hemopressin. It was determined that the first 6 amino acids of the nonapeptide (PVNFKF) are essential for binding to the CB1 receptor and that the deletion of the C-terminal 3 residues did not affect receptor recognition,13 although the shorter fragment was found to adopt a different conformation than that of the nonapeptide in a cellular nuclear magnetic resonance study.14 The physiologic relevance of the shorter fragments is not clear. However, the ability of these shorter fragments to recognize the receptor and maintain bioactivity may be related to the reported oral activity of hemopressin,13 because the peptide may be able to withstand some proteolysis before adsorption and still exhibit activity at the receptor.

Although previous data support a mode of action rather distinct from opioids, we were interested in the further analysis of the opioid system. In our study, we investigated the affinity of the rat nonapeptide hemopressin (HP, PVNFKFLSH) and its C-terminally truncated fragment hemopressin 1–7 (HP 1–7, PVNFKFL) toward opioid receptors, by using competitive binding and G-protein–stimulating in vitro functional assays. G-protein activation caused by hemopressins also was characterized in CB1 and in the less studied CB2 systems.

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To perform this study, the approval of the Animal Care and Use Committee was obtained from the IRB of the Biological Research Centre, Hungarian Academy of Sciences. The animals used for all experiments were treated in accordance with ethical Animal Care and Use Commitee tenets, and the study was conducted in a manner that did not inflict unnecessary pain or discomfort for the animals.

CB1−/−/CB2−/− double knockout and the control CB1+/+/CB2+/+ C57BL/6J mice were provided by Dr. Zimmer’s laboratory.15 Wistar rats and guinea pigs were derived from the local animal house (BRC, Szeged, Hungary). All animals were housed under a 12:12-hour light/dark cycle at 21 to 24°C and were provided with food and water ad libitum. Animal handling was conducted in accordance with the European Communities Council Directives (86/609/ECC) and the Hungarian Act for the Protection of Animals in Research ( 32.§).

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Compounds, Chemicals, and Radioligands

Hemopressins were prepared by in situ neutralization solid-phase peptide synthesis, as described previously.16 Cannabinoid compounds were obtained from Tocris Bioscience (Bristol, United Kingdom). Guanosine-5′-diphosphate, guanosine-5′-O-(3-thiotriphosphate), guanosine-5′-O-[γ-thio]triphosphate) (GTPγS), and all other chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO). [3H]DAMGO ([D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin, 41 Ci/mmol), [3H]U69,593, and [3H]DIDI (Tyr1,Ile5,6deltorphin-2; 48 Ci/mmol) were prepared in the Laboratory of Chemical Biology of BRC. GTPγS (1200 Ci/mmol) was purchased from the Institute of Isotopes Co, Ltd (Budapest, Hungary).

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Rat and Mouse Brain Membrane Preparations

Membrane preparations were produced according to the method described previously.17 To summarize, brains were removed quickly from the decapitated animals and washed several times with cold 50 mM Tris-HCl (pH 7.4) buffer. Next, the brains were homogenized with an electrically driven homogenizer and then filtered to remove any larger aggregates. Homogenates were centrifuged at 40,000g for 20 minutes, and the pellet was resuspended in fresh buffer by a vortex. To remove any endogenous opioids, the suspension was incubated for 30 minutes at 37°C. Centrifugation was repeated, and the pellet was resuspended in 5 volumes of 50 mM Tris-HCl (pH 7.4) containing 0.32 M sucrose to give a final concentration of 3 to 4 mg/mL protein. Before being used, the membranes were stored in 5-mL aliquots at −70°C. The binding activity of the preparation remained stable for at least 6 months.

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Cell Culture and Cell Membrane Preparations

Chinese hamster ovary (CHO) cells overexpressing human μ-opioid (MOP) receptors were provided by Dr. Zvi Vogel (Rehovot, Israel) and have been described previously.18 Cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fisher Scientific, Waltham, MA), supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, 25 mg/mL Fungizone, and 0.5 mg/mL Geneticin. CHO cell lines were kept in culture in a humidified atmosphere (5% CO2 and 95% air), at 37°C.

Cell membranes were obtained from subconfluent cultures. Cells were washed 3 times with 10 mL phosphate-buffered saline and homogenized in 50 mM Tris-HCl buffer (pH 7.4). Homogenates were centrifuged 2 times (18,000g for 20 minutes). The pellet was resuspended in 50 mM Tris buffer (pH 7.4). Aliquots were stored at −80°C until use.

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Receptor Binding Assays

To remove the sucrose, membranes were thawed and resuspended in 50 mM Tris-HCl (pH 7.4) buffer and centrifuged at 40,000g for 20 minutes. Pellets were taken up in fresh buffer and used immediately. All binding assays were performed at 35°C for 45 minutes in 50 mM Tris-HCl buffer (pH 7.4) in a final volume of 1 mL that contained 1 mg BSA and 0.2 to 0.4 mg/mL membrane protein.19 Rat brain membranes were incubated with the selective opioid receptor agonists [3H]DAMGO, [3H]DIDI, and [3H]U69,593 (0.9–1.2 nM). Nonspecific binding was measured in the presence of 10 μM naloxone. Reaction was terminated by the separation of bound and free radioligands with Brandel M24R Cell Harvester (Brandel Harvesters, Gaithersburg, MD) using rapid filtration under vacuum through Whatman GF/C glass fiber filters (GE Healthcare, Little Chalfont, UK). Filters were washed 3 times with 5 mL ice-cold 50 mM Tris-HCl (pH 7.4) buffer and were inserted into an environmentally friendly, nonvolatile, toluene-free scintillation cocktail Ultima Gold™ MV (Perkin Elmer, Waltham, MA). Bound radioactivity was measured by Packard TriCarb 2300TR liquid scintillation analyzer (Perkin Elmer, Waltham, MA). Receptor binding experiments were performed in duplicates and repeated at least 3 times.

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[35S]GTPγS Binding Assays

Membrane fractions (10 μg of protein/sample) were incubated at 30°C for 60 minutes in Tris-EGTA buffer (pH 7.4) composed of 50 mM Tris-HCl, 1 mM EGTA, 3 mM MgCl2, and 100 mM NaCl containing [35S]GTPγS (0.05 nM). Compounds were tested in increasing concentrations (10−9 to 10−5 M, final volume of 1 mL) and in the presence of 30 μM guanosine-5′-diphosphate. Total binding was determined in the absence of test compound. Nonspecific binding was measured in the presence of 10 μM unlabeled GTPγS. To calculate the specific binding, the values of nonspecific binding were subtracted from the total binding. The reaction was started by the addition of [35S]GTPγS and terminated by the filtration through Whatman GF/B glass fiber filters (GE Healthcare, Little Chalfont, UK). Filters were washed with ice-cold 50 mM Tris-HCl buffer (pH 7.4) 3 times using Brandel M24R Cell Harvester. They were then dried, and bound radioactivity was measured in Ultima Gold™ MV scintillation cocktail. Binding assays were performed in triplicates and repeated at least 3 times. The agonist-induced G-protein stimulation is given as percentage over the specific [35S]GTPγS binding obtained in the absence of receptor ligands (basal activity).

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Statistical Analysis

Efficacy (Emax) and the potency (logEC50) values were expressed as means ± SEM obtained from at least 3 independent experiments, each performed in triplicate. The significance levels were determined by unpaired t test with 2-tailed P value statistical analysis, using GraphPad Prism 5.0 software (GraphPad Prism Software, San Diego, CA). Significant findings are supported by the statistical analysis at P < 0.01 level. Data were presented as means ± SEM in the presence of the applied concentration points in logarithm form, and a corresponding curve was fitted by nonlinear regression. In the figures, curves present the mean of at least 3 independent experiments, and each independent experiment was done in triplicate. Because authors transform data to stimulation which is given as percentage, curves are summed and describe a common stimulatory curve. GraphPad Prism 5.0, the professional curve fitting program, was used to automatically fit these curves using nonlinear regression, based on the data of separated experiments. During evaluation of GTPγS binding, Emax and logEC50 values were calculated by a “Sigmoid dose-response” fitting equation. In the case of receptor bindings, a “One-site competition” fitting equation was used.

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Hemopressins Can Specifically Stimulate CB1 Receptor–Mediated G-Proteins

At first, we intended to further characterize cannabinoid receptor affinity using a GTPγS assay in which the nucleotide exchange process is monitored by a nonhydrolyzable radiolabeled GTP analog. We investigated cannabinoid affinity of hemopressins by using several in vitro conditions. To this aim, rat whole brain membrane homogenates were prepared and hemopressin–induced, G-protein activation was measured by a [35S]GTPγS in vitro functional assay. At first, we detected the G-protein–stimulating effect of hemopressins in areas that highly expressed the CB receptors, such as the hippocampus, spinal cord, and cortex, and because the differences were modest between areas (data not shown), we continued the characterization of hemopressins using crude brain membrane homogenates.

Figure 1

Figure 1

In the [35S]GTPγS experiments, hemopressins efficiently stimulated the G-proteins, obtaining the Emax value between 110% and 120%. CB1 receptor–dependent signaling could be blocked by using a potent CB1 receptors antagonist AM251. The efficacy of HP (115.7 ± 1.32) was reduced by the addition of AM251 (92.95 ± 4.10; P = 0.001). Similar to HP, the decrease also was prominent when AM251 was coapplied with HP 1–7 (from 119.5 ± 2.05 to 92.42 ± 3.55; P = 0.0027; Fig. 1).

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CB1 Knockout Experiments

Figure 2

Figure 2

To further confirm the CB affinity of the HP, we used the mice deficient in CB1 receptors to study its efficacy. When we used the CB1 knockout animals, HP was not able to stimulate G-proteins, as in the case of wild-type mice, confirming the results of previously conducted CB1 antagonist experiments. On CB1−/− mice brain homogenates, the efficacy was significantly diminished both for arachidonyl-2'-chloroethylamide (ACEA) (113.17 ± 2.16; P = 0.0006) and HP (99.46 ± 0.86; P = 0.0019) compared with the CB1+/+ samples (Fig. 2). Through the use of mice deficient in CB receptors, we showed that G-protein stimulatory activity of hemopressin is lost in CB knockout animals. Because of technical complexity, the use of a competitive receptor binding assay for cannabinoid system was beyond the scope of the present study. In addition, we observed that the G-protein stimulation caused by hemopressin is significantly lower (121.9 ± 2.16; P = 0.0022) than activation induced by the selective CB1 agonist ACEA (145.8 ± 2.66).

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Receptor Binding and G-Protein–Stimulating Assays Reveals Slight MOP Affinity of Hemopressins

To determine the affinity toward opioid receptors, hemopressins were first tested in competitive receptor binding assays. MOP receptor–dependent signaling was determined by radiolabeled [3H]DAMGO, a selective ligand for the MOP system. HP was able to displace [3H]DAMGO at micromolar concentrations. Displacement caused by HP 1–7 was more modest compared with that of the nonapeptide. Furthermore, δ- (DOP) and κ-opioid receptor (KOP) affinity was tested by radiolabeled Ile5,6 deltorphin ([3H]DIDI) and [3H]U69,593, but our findings indicated that there was no detectable interaction even with the DOP/KOP opioid system and hemopressins (Fig. 3).

Figure 3

Figure 3

For further clarification, we examined whether the nonspecific, universal opioid antagonist, naloxone, could inhibit the G-protein–stimulating effects of hemopressins. The interactions between these compounds were studied on whole rat brain membrane homogenate. Our results indicated that incubation of HP or HP 1–7, with or without naloxone, caused slight nonsignificant changes in the G-protein stimulatory effect (HP: from 119.0 ± 1.44 to 116.7 ± 2.07, P = 0.41; HP 1–7: from 127.0 ± 2.96 to 125.3 ± 2.45, P = 0.68; Fig. 4).

Figure 4

Figure 4

To confirm MOP affinity found on whole brain membrane homogenates, we further aimed at evaluating the interaction of MOP receptors and hemopressin accurately; therefore, we decided to repeat competitive binding assays using CHO cell lines, which overexpress the MOP receptors. Displacement experiments performed using CHO cells revealed that both hemopressin and its derivative HP 1–7 (Fig. 5) are also able to bind to MOP receptors expressed in CHO cells. Verification experiments were also performed for G-protein stimulation on CHO cells.

Figure 5

Figure 5

Figure 6

Figure 6

We found that both HPs, compared with brain homogenates, were more efficient in stimulating the G-proteins in hMOPCHO cells (HP: 159.1 ± 8.035; P = 0.0060; HP 1–7: 135.8 ± 2.83; P = 0.0171). The G-protein stimulation caused by HP may be partially, but not significantly, antagonized by naloxone (HP: from 159.1 ± 8.03 to 126.4 ± 5.98; P = 0.030; Fig. 6).

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Hemopressin (PVNFKFLSH), a nonapeptide derived from the α-chain of hemoglobin, was isolated originally from rat brain homogenates. In vivo studies have demonstrated that administration of hemopressin causes significant nonopioid antinociceptive effects in rats.8,13 The cellular target of hemopressin was later identified to be the CB1 receptor.3,13 A docking study was performed to reveal binding sites and suggested that hemopressin binds to the CB1 receptor in the same binding pocket as the previously known CB1 antagonist drug, rimonabant.14

To date, several authors have investigated hemopressin and their N-terminally extended forms in different pain models16,20–22; however, results of these studies are contradictory. Dale et al.8 found that hemopressin was able to antagonize hyperalgesia induced by intraplantar administered carrageenan. Intrathecally and orally applied hemopressin was also effective in paw-pressure tests to prevent development of hyperalgesia.13 Petrovszki et al.16 used hemopressin in a wide dose range (0.3–30 μg), but they could not show that intrathecally administered hemopressin decreases carrageen-induced hyperalgesia. These controversial results might be because of the differences in the timing of the treatment and the applied pain models.16 Regarding acute heat pain test, observations of Hama and Sagen20 were in accordance with Petrovszki et al.16; however, their data were contradictory in the case of HP antagonism on CB1 receptors. Hama and Sagan20 showed that HP pretreatment was unable to block activation mediated by the CB agonist, WIN 55,212-2, in contrast to the pretreatment with rimonabant. They suggested that, despite the common in vitro characteristics of hemopressin and rimonabant, hemopressin might influence non-CB1 receptors as well.20

Although earlier articles claimed that the antinociceptive effect caused by hemopressin was naloxone-resistant, we were interested to see whether we could detect opioid-like signaling for hemopressin and its shorter derivative. Despite the naloxone insensitivity, we did not rule out the possibility of influencing the opioid system at least indirectly because opioids have extended relationships with other biological systems. It is well-known that cannabinoid compounds share several overlapping physiologic functions with opioids.23 Hemopressin was described as the first peptide ligand for cannabinoid receptors. By conformation with state-sensitive antibodies, an inverse agonistic/antagonistic feature was assumed.13 Later, using binding and functional assays, the N-terminally extended forms, RVD- and VD-hemopressin, were characterized as partial agonists.

We have observed that hemopressins efficiently activates G-proteins; however, the extent of stimulation is lower than that of the highly selective CB receptor agonist, ACEA. By applying the CB1 antagonist AM251, G-protein stimulation caused by both HP and HP 1–7 was markedly diminished on whole brain homogenates (Fig. 1). These results showed the ability of hemopressins to stimulate CB1 cannabinoid receptors. The sigmoid dose/response curves of CB1+/+ mice brain experiments also indicate that hemopressins act in a cannabinoid-like manner: G-protein stimulation was significantly diminished in the case of CB1 knockout animals (Fig. 2), which unequivocally confirmed CB1-mediated action.

To elucidate opioid binding affinity of hemopressin compounds, we performed competitive receptor binding assays using radiolabeled ligands specific for the main types MOP, KOP, and DOP receptors. A slight opioid affinity was shown only in the case of MOP receptors, with a minor difference between displacement caused by hemopressin and hemopressin 1–7: displacement by the shorter fragment was lower than that of the nonapeptide. DOP/KOP affinity was negligible (Fig. 3). In addition, at the level of the linkage between the G-protein and the receptors, the nonselective opioid antagonist, naloxone, slightly blocked the stimulatory effects of hemopressins in GTPγS in vitro functional assay (Fig. 4). We further aimed at evaluating the interaction of MOP receptors and hemopressin accurately; therefore, we decided to repeat competitive binding assays using CHO cell lines, which overexpresses only the MOP receptors and found that both hemopressins were able to activate the MOP receptors, which supported the results of whole brain membrane experiments (Fig. 5). We also measured G-protein stimulation and determined that stimulation of hemopressin was partially antagonized by naloxone (Fig. 6). These data indicate that hemopressin can stimulate MOP receptors, either directly or indirectly.

Partial antagonism by naloxone suggests the existence of direct binding; however, affinity for CB1 receptors is possibly greater than affinity for MOP receptors. However, other factors of activation may be attributed to different mechanisms. In the recent years, an increasing number of publications have reported that well-known CB1-specific compounds can exert their physiologic functions independent from CB receptors.24–26 Zádor et al.25 have shown that rimonabant inhibits MOP receptors and δ-specific ligand binding. In addition, prominent μ-inhibition was observable by the endocannabinoid noladin ether that was presented by Páldyová et al.24 Moreover, it has been shown that AM251 and rimonabant can act as direct antagonists at MOP receptors, as described in the study by Seely et al.26

Opioid receptors might possess secondary binding sites where ligands bind with a lower affinity—sites that cannot be antagonized by naloxone. Activation of the MOP system by hemopressins might be a novel example of cooperation between opioids and compounds acting on CB receptors. In addition, slight differences shown in receptor binding and the functional assays point out that the C-terminal of the nonapeptide moderately enhanced the affinity for μ-receptors. Besides the interaction at the level of ligand-receptor binding, other authors have suggested that opioids and cannabinoids might influence their signaling processes reciprocally: antinociception provoked by cannabinoid ligands depends on previous opioid release,27 and cannabinoids could promote the release of opioids as well.28 Thus, indirect activation via cellular signaling pathways could be another way to elucidate the MOP affinity of hemopressins.

Recent data indicate that hemopressins have a slight modulatory effect on the opioid system, which might be related to direct binding and naloxone-insensitive, secondary binding sites, respectively, or the consequence of signaling beginning at the CB receptors. However, the ability of hemopressins to modulate CB1 receptors has been repeatedly confirmed. We should emphasize that modulation of the CB system has an emerging importance in neurochemistry because the application of CB1 receptor agonists is unfortunately very limited because of their effect on the central nervous system. Taken together, our recent findings shed light on the complex mechanism of how hemopressins exert their effects and of how cannabinoid-like compounds interact with the opioid system.

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Name: Eszter Szlavicz, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Eszter Szlavicz has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Pannilage Shiromi Perera.

Contribution: This author helped design the study and analyze the data.

Attestation: Pannilage Shiromi Perera has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Csaba Tomboly, PhD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Csaba Tomboly has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Zsuzsanna Helyes, PhD.

Contribution: This author helped design the study and analyze the data.

Attestation: Zsuzsanna Helyes has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Ferenc Zador, PhD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Ferenc Zador has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Sandor Benyhe, PhD.

Contribution: This author helped design the study and analyze the data.

Attestation: Sandor Benyhe has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Anna Borsodi, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Anna Borsodi has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Engin Bojnik, PhD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Engin Bojnik has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.

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The authors thank Professor Maria Wollemann (BRC Biochemistry, Szeged, Hungary) and Erika Griechisch (Department of Medical Physics and Informatics, University of Szeged) for their helpful suggestions.

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