From the antioxidant and anticoagulant properties of myo–inositol to ex vivo vascular studies and GC–FID analysis

From the antioxidant and anticoagulant properties of myo–inositol to ex vivo vascular studies and GC–FID analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article From the antioxidant and anticoagulant properties of myo–inositol to ex vivo vascular studies and GC–FID analysis Agata Rolnik, Beata Olas, Joanna Szablińska–Piernik, Lesław Bernard Lahuta, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4403797/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract A potential protective effect of myo –inositol against pro–oxidant damage to human plasma and rat thoracic arteries has been demonstrated, suggesting that this nutraceutical agent plays a key role in the vasculature and may be beneficial for preventing harmful environmental effects. Aortic rings were isolated from aged (12–month–old) male Wistar rats and preincubated with myo –inositol (0.01–100 mg/L, 2 h). A stable thromboxane A 2 analog was added (0.1 nM, 2 h) to analyze vascular dysfunction. The concentration of myo –inositol in the organ baths was determined via GC–FID. In another experiment, human blood plasma was subjected to H 2 O 2 /Fe 2+ administration, and myo –inositol was added to analyze lipid and protein peroxidation processes. The hemostatic parameters were also studied. Myo –inositol (1–100 mg/L) administration protects proteins against oxidative stress, which modifies the vascular response. A dose above 100 mg/L additionally protected lipids and increased thrombin time. acetylcholine carbon monoxide myo–inositol reactive oxygen species thromboxane A2 U–46619 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Inositol (cyclohexane–1,2,3,4,5,6–hexol) is a natural cyclitol present in both animal and plant cells. The basic structure contains polyols in nine stereoisomeric forms depending on the spatial orientation of the six hydroxyl groups [ 1 ] . Among the nine possible geometrical isomers, seven are inactive. The remaining two form a chiral pair. Its predominant isomeric form present in nature and human food is myo –inositol, also known as cis –1,2,3,5– trans –4,6–cyklohexanehexol. It can have either a free form or can be bound to phospholipids or inositol phosphate derivatives. The greatest amount of myo –inositol in human food is found in products including seeds, fresh vegetables, and fruits. Citrus and cantaloupe have extraordinarily high levels of myo –inositol, whereas leafy vegetables are a poor source [ 1 , 2 ] . Cells produce inositol from three main sources. The first is based on de novo biosynthesis from glucose–6–phosphate by 1–ᴅ– myo –inositol–phosphate synthase (MIPS) and inositol monophosphatase (IMPase). The second is based on the dephosphorylation of inositol phosphates derived from the breakdown of inositol–containing membrane phospholipids, and the third is uptake from the extracellular fluid via specialized myo –inositol transporters [ 1 ] . All living cells contain inositol phospholipids in their membranes. Phytic acid, another form of inositol, is the principal storage form of phosphorus in many plant tissues, such as bran and seeds. Myo –inositol plays a crucial role in various cellular processes, such as comprising the structural basis for secondary messengers including inositol triphosphates, inositol glycans, and phosphatidylinositol phosphate lipids. Myo –inositol also plays an essential role in other cell functions, including cell growth, peripheral nerve development, reproduction, and osteogenesis [ 1 ] . Cellular myo –inositol concentration has an essential influence on various diseases [ 2 , 3 ] . Recently, Rodríguez-Nieto et al. [ 4 ] observed age–related differences in myo –inositol expression across brain regions, suggesting the occurrence of neurodegeneration and altered gliosis. In addition, our previous study demonstrated that myo –inositol exposure has a positive effect on the vascular system and blood plasma protein carbonylation under shared stress in vitro . Myo –inositol exposure improves vasodilator and vasoconstrictor response in arteries from young Wistar rats subjected to thromboxane A 2 mimetic administration. It also increases NO release/bioavailability and decreases H 2 O 2 production, which correlates to oxidative damage of blood plasma proteins [ 5 ] . Given the scarcity of information on the in vitro effect of myo –inositol on the arteries, we aimed to determine its effect on the regulation of vascular resistance; oxidative stress, including protein and lipid oxidation levels; and the coagulation process during aging, together with increased inflammation were also studied. Results Myo –inositol concentration The presence of the aortic rings in the preincubation medium did not significantly modify the concentration of myo –inositol (measured at three concentrations of 1.0, 10, and 100 mg/L of myo –inositol). Hence, myo –inositol was neither degraded in the solution nor taken up by the aortic rings in the metabolic processes during 6 h of incubation (Table 1 ). Table 1 Concentration of myo –inositol (mg/L) in the KHS with (+) or without (–) added aortic rings. Ten aortic rings per 5 mL chamber were incubated for 6 h (n = 4). myo –inositol (mg/L) aortic rings 0 1.0 10 100 – nd 0.98 8.07 106.1 + nd 0.87 7.13 113.8 P nd NS NS NS NS – not significant nd – below detection level Lipid and protein peroxidation Increased TBARS (5.26–fold) concentration was observed in the plasma preincubated with H 2 O 2 /Fe 2+ (Fig. 1A). Increasing concentrations of myo –inositol added to the preincubated plasma decreased TBARS concentrations by 0.84–fold (0.01 mg/L), 0.76–fold (0.1 mg/L), 0.76–fold (1.0 mg/L), 0.76–fold (10 mg/L), and 0.62–fold (100 mg/L), with a significant effect at the highest concentration of 100 mg/L (Fig. 1A). An increased carbonylation level (8.39–fold) was observed in the plasma preincubated with H 2 O 2 /Fe 2+ (Fig. 1B). Increasing concentrations of myo –inositol added to the preincubated plasma decreased the carbonyl groups in a dose–dependent manner by 0.79–fold (0.01 mg/L), 0.68–fold (0.1 mg/L), 0.62–fold (1.0 mg/L), 0.53–fold (10 mg/L), and 0.48–fold (100 mg/L), with significant effects at all studied concentrations ranging from 0.01–100 mg/L (Fig. 1B). A decreased level of thiol groups (0.27–fold) was observed in the plasma preincubated with H 2 O 2 /Fe 2+ (Fig. 1C). Increasing concentrations of myo –inositol added to the preincubated plasma increased the concentrations of the thiol groups in a dose–dependent manner by 1.90–fold (0.01 mg/L), 2.28–fold (0.1 mg/L), 3.36–fold (1.0 mg/L), 3.65–fold (10 mg/L), and 3.80–fold (100 mg/L), with a significant effect at 1–100 mg/L (Fig. 1C). Figure 1. Concentrations of ( A ) TBARS, ( B ) carbonyl groups, and ( C ) thiol groups in human blood plasma incubated with the pro–oxidant agent H 2 O 2 /Fe 2+ . P < 0.05 vs. H 2 O 2 /Fe 2+ positive control. Myo –inositol favorably decreased indices of lipid peroxidation (TBARS, 100 mg/L) and protein peroxidation (decreasing carbonyl groups at 0.01–100 mg/L and increasing thiol groups at 1.0–100 mg/L). For the four highest concentrations (0.1, 1, 10 and 100 mg/L of myo –inositol), the level of thiol groups recovered and was comparable to that of the negative control (plasma not treated with H 2 O 2 /Fe 2+ ), and this was not observed for TBARS and the carbonyl groups (Fig. 1S). Because we determined that the highest concentration of myo –inositol decreased the concentrations of TBARS exclusively (100 mg/L but not 10 mg/L), we have further analyzed the exact concentration of myo –inositol between 10 and 120 mg/L that induced the effect. A concentration equal to 120 mg/L (0.65–fold) decreased TBARS which is contrary to the other studied concentrations, 90 mg/L (0.77–fold), 60 and 30 mg/L (0.86–fold), and 10 mg/L (0.93–fold), which did not significantly modify TBARS (Fig. 2 ). Overall, our study confirmed that concentrations above 100 mg/L are able to decrease lipid peroxidation significantly. Coagulation processes Blood plasma activated partial thromboplastin time (APTT) did not significantly change when increasing concentrations of myo –inositol were added (0.01–100 mg/L, 0.97–fold for all studied groups) (Fig. 3A). Blood plasma prothrombin time (PT) was not significantly modified (0.01–100 mg/L, 1.05–fold for all studied groups) (Fig. 3B). Blood plasma thrombin time (TT) increased 1.10–fold only for the highest concentration of myo –inositol (100 mg/L) and was not modified in the other groups (0.01–10 mg/L, 1.05–fold increase only) (Fig. 3C). Vascular response Vascular contraction Aortic rings were obtained from aged (12–month–old) male Wistar rats and preincubated with myo –inositol (1–100 mg/L). further, a stable analog of thromboxane A 2 (TxB 2 , 0.1 nM) was added and icubated for 2 h. KCl–induced contraction did not change with increasing concentrations of myo –inositol (0.01–100 mg/L) added to the incubated aortic rings (Fig. 4 A). NA–induced contraction decreased with increasing concentrations of myo –inositol (as AUC) to 0.77–fold (1.0 mg/L), 0.76–fold (10 mg/L), and 0.76–fold (100 mg/L, Fig. 4 A and Table 2 ), but not with 0.01 or 0.1 mg/L (data not presented). Myo –inositol administration decreased the sensitivity to NA and thus shifted the cumulative concentration response curve to the right (Fig. 4 B). Table 2 Vasoconstrictor response to NA (determined as AUC) of isolated aortic rings from aged (12–month–old) male Wistar rats preincubated with myo –inositol (2 h) and exposed to TxB 2 (0.1 nM, 2 h) – ex vivo studies control (vehicle) myo –inositol (mg/L) Concentration 0 1.0 10.0 100.0 Mean 8513 6529* 6477* 6460* Std. Deviation 3213 2258 1783 2487 P 0.05 0.042 0.402 *vs. Control (Vehicle) Vasodilation myo –inositol added to the preincubated aortic rings, enhanced the vasodilator response of the isolated aortic rings to ACh at 1.0, 10 and 100 mg/L concetrations, but not at 0.01 or 0.1 mg/L (Fig. 5 A). This effect was most significant for 1.0 mg/L of myo –inositol, as both the AUC and LogEC 50 were changed (Table 3 and Table 4 ); for 10.0 mg/L of myo –inositol, only the AUC was modified (Table 3 and Table 4 ); and for 100 mg/L of myo –inositol, only one point from the CCRC was modified (0.1 µM, see Fig. 5 A). In all cases, Emax did not significantly change (Table 5 ). Next, the vasodilator response to CORMs was studied, and an increase in vasodilation was observed at 10 –5.5 M and 10 –5 M for 1.0, 10 and 100 mg/L of myo –inositol (Fig. 5 B). The vasodilatory effect was the most significant for 1.0 mg/L of myo –inositol, as both the AUC and LogEC 50 were changed (Table 3 and Table 4 ); for 10.0 mg/L of myo –inositol, neither the AUC nor the LogEC 50 was modified (Table 3 and Table 4 ); and for 100 mg/L of myo –inositol, both the AUC and LogEC 50 exhibited an increased response. In all cases, Emax did not significantly change (Table 5 ). The vasodilator response to SNP was studied, and an increase in vasodilation was observed at 10 nM SNP for 1.0, 10 and 100 mg/L myo –inositol (Fig. 5 C). However, neither the AUC nor the logEC 50 or the Emax were significantly modified (see Tables 3 – 5 ). Compared with that of the control group (vehicle), the vasodilator response to A231987 was not different (Fig. 5 D and Tables 3 – 5 ). However, significant differences were observed between the myo –inositol groups: 1.0 mg/L vs. 100 mg/L and 10 mg/L vs. 100 mg/L. The vasodilator responses to 8–bromo–cGMP and pinacidil were not modified (Fig. 5 E, F and Tables 3 – 5 ). Table 3 Vascular response (AUC) to the vasodilators ACh, CORM, SNP, A23187, 8–bromo–cGMP and pinacidil of isolated aortic rings preincubated with thromboxane B 2 (0.1 nM, 2 h) from aged (12–month–old) Wistar rats: ex vivo studies Control (Vehicle) Myo –inositol (mg/L) P # 0 1.0 10.0 100.0 1–100 10–100 1–10 ACh Mean 202.3 253.2* 239.1* 229.7 0.2448 0.8509 Std. Deviation 38.00 32.68 53.40 39.32 P ANOVA 0.0020 0.0337 0.1478 CORM Mean 124.1 162.5* 144.1 150.8* 0.3056 Std. Deviation 38.68 28.53 38.84 35.03 P ANOVA 0.0078 0.2569 0.0479 SNP Mean 329.8 348.5 356.4 350.9 Std. Deviation 41.13 31.80 32.59 33.96 P ANOVA 0.6849 0.4321 0.6048 A23187 Mean 114.0 129.5 132.1 80.25 0.2458 0.2132 Std. Deviation 32.46 29.12 63.53 22.97 P 0.9045 0.8598 0.5185 Pinacidil Mean 149.8 152.2 156.6 147.7 Std. Deviation 15.02 19.23 15.54 21.39 P ANOVA 0.9793 0.7031 0.9872 8–bromo–cGMP Mean 40.67 29.83 52.33 34.17 Std. Deviation 36.41 27.76 14.22 23.00 *vs. Control (Vehicle), # based on the results from Fig. 5 Table 4 Vascular response (logEC 50 ) to the vasodilators ACh, CORM, SNP, A23187, 8–bromo–cGMP and pinacidil of isolated aortic rings preincubated with thromboxane B 2 (0.1 nM, 2 h) from aged (12–month–old) Wistar rats – ex vivo studies Control (Vehicle) Myo –inositol (mg/L) P # 0 1.0 10.0 100.0 1–100 10–100 1–10 ACh 25% Percentile –7.380 –7.733 –7.666 –7.547 0.1145 > 0.9999 Median –7.230 –7.594* –7.490 –7.371 75% Percentile –7.109 –7.396 –6.998 –7.185 KW 0.0018 0.2544 0.7421 0.2441 CORM 25% Percentile –5.627 –6.041 –5.781 –5.709 Median –5.417 –5.661* –5.622 –5.633* 75% Percentile –4.854 –5.531 –5.234 –5.490 Shapiro–Wilk test KW 0.0120 0.7351 0.0498 SNP 25% Percentile –8.855 –8.782 –8.877 –8.726 Median –8.176 –8.478 –8.556 –8.467 75% Percentile –8.038 –8.352 –8.379 –8.314 Shapiro–Wilk test KW 0.7093 0.4249 > 0.9999 Pinacidil 25% Percentile –6.701 –6.813 –6.815 –6.724 Median –6.587 –6.621 –6.678 –6.579 75% Percentile –6.532 –6.543 –6.609 –6.501 Shapiro–Wilk test KW > 0.9999 0.7257 > 0.9999 # based on the results from Fig. 5 Table 5 Vascular response Emax (%) to the vasodilators ACh, CORM, SNP, A23187, 8–bromo–cGMP and pinacidil of isolated aortic rings preincubated with thromboxane B 2 (0.1 nM, 2 h) from aged (12–month–old) Wistar rats – ex vivo studies Control (Vehicle) Myo –inositol (mg/L) 0 1.0 10.0 100.0 ACh 25% Percentile –95.13 –97.49 –97.87 –98.91 Median –90.37 –94.62 –93.77 –95.05 75% Percentile –86.84 –91.79 –89.55 –90.07 Shapiro–Wilk test KW 0.1440 0.3145 0.2017 CORM 25% Percentile –103.9 –103.8 –103.9 –103.7 Median –99.29 –101.5 –102.1 –101.9 75% Percentile –95.79 –97.29 –99.95 –98.69 Shapiro–Wilk test KW > 0.9999 > 0.9999 > 0.9999 SNP Mean –98.94 –100.2 –101.0 –102.2 Std. Deviation 2.684 5.172 2.952 2.203 ANOVA 0.8727 0.6097 0.2576 Pinacidil 25% Percentile –101.3 –104.1 –103.1 –100.2 Median –97.96 –98.72 –98.57 –96.26 75% Percentile –96.28 –98.04 –94.70 –93.38 Shapiro–Wilk test KW > 0.9999 > 0.9999 > 0.9999 *vs. control Discussion Our findings revealed that different concentrations of myo –inositol have varying effects on blood stimulated with the pro–oxidant H 2 O 2 /Fe 2+ in vitro . All concentrations above 0.01 mg/L improved the carbonylation level, whereas concentrations above 1.0 mg/L improved the thiol content, both of which are markers of protein protection from oxidation. Lipid peroxidation, reflected as content of thiobarbituric acid reactive substances (TBARS), which measures malondialdehyde (MDA) levels, improved with the administration of above 100 mg/L of myo –inositol. Interestingly, for the thiol group levels, only concentrations above 0.1 mg/L corresponded to the values obtained for the blood that were not stimulated with H 2 O 2 /Fe 2+ (negative control with no added H 2 O 2 /Fe 2+ ). Neither was this observed with TBARS content nor with the carbonylation level, which did not recover to the control level. Similar to TBARS content, thrombin time (TT) was also modified with the highest concentration of 100 mg/L myo –inositol. Neither activated partial thromboplastin time (APTT) nor prothrombin time (PT) (INR determinant) were changed. The abovementioned results point to the differences between the mentioned concentrations, which to varying extents protect against oxidative stress. Vascular contraction in response to the adrenergic receptor agonist noradrenaline, which depends on DAG and IP3, decreased with increasing concentrations of myo –inositol (1.0–100 mg/L), but this effect was not observed at concentrations less than this level (0.01–0.1 mg/L). This is contrary to Ca 2+ –dependent KCl–induced contraction, which bypasses G protein–coupled receptors (GPCRs) [ 6 ] , as this was not altered. The ACh–induced vasodilator response was potentiated above 1.0 mg/L, and this effect was more significant at lower doses (1.0 mg/L and 10 mg/L) than at the highest dose (100 mg/L) (based on the AUC and Emax). The exogenous NO donor SNP potentiated vasodilation only to a small extent, and neither the AUC nor the Emax changed; therefore, we concluded that endothelial cells were not corrupted in a significant way and that the bioavailability of endogenous NO was maintained. Based on the AUC and Emax, another gasotransmitter, carbon monoxide (CO) [ 7 ] also induced the greatest effect at 1.0 mg/L myo –inositol, with a lesser effect at 100 mg/L myo –inositol, similar to that of ACh, with almost no effect at 10 mg/L. The Ca 2+ ionophore A–23187, which induces either contraction or relaxation of blood vessels, did not modify the response compared to that in the control group. However, A–23187 administration weakened the vasodilator response to 100 mg/L myo –inositol opposite to that to 1.0 and 10 mg/L, which may in part explain the response to the vasodilator ACh and/or CO–releasing molecule CORMs described above. Neither the cGMP–dependent protein kinase pathway nor ATP–sensitive potassium channels were involved in the vasodilation induced by myo –inositol. Our results point to a non–cGMP–dependent mechanism in which vascular reactivity is modulated by myo –inositol preincubation in aged rats treated with 0.1 nM thromboxane ex vivo . Here, for the first time, we discussed the direct influence of myo –inositol on vascular reactivity, coagulation, and antioxidant properties. Although myo –inositol has been widely studied in vivo (described later in the second part of the discussion section) due to its effectiveness in treating polycystic ovary syndrome [ 8 ][ 9 ] and in menstrual recovery [ 10 ] , little is known about its influence on vascular tone regulation in elderly subjects exposed to inflammation following vascular dysfunction [ 11 ] . For these reasons, arteries were taken from aged rats and exposed to a thromboxane A 2 analog, which is a well–known factor contributing to vascular dysfunction [ 11 ][ 12 ] . Indeed, myo –inositol administration modified vascular contraction and relaxation in the range of 1–100 mg/L; however, this effect was not dependent on the concentration, and with increasing doses starting at 1 mg/L, different effects were observed. No significant differences in vascular contraction were detected between the studied groups in response to high concentrations of KCl. High KCl concentration affects vascular smooth muscle (VSM) membrane depolarization, with a subsequent influx of Ca 2+ through voltage–dependent calcium channels (VDCCs). Interestingly, compared with the control group (without myo –inositol), myo –inositol decreased vasoconstriction in response to noradrenaline; but no significant differences were observed between the studied concentrations. Stimulation of vascular α1–adrenoreceptor with the agonist noradrenaline results in activation of Gq receptors via the phospholipase C (PLC) cascade, a subsequent increase in the levels of secondary messengers (IP 3 and DAG), and an increase in the intracellular Ca 2+ concentration through intracellular storage (due to IP 3 ) or VDCC activation (the PKC/CaMKII pathway), which results in VSM contraction. As the KCl–induced response was not altered, in contrast to the NA–induced contraction, we concluded that the influence of myo –inositol on this mechanism was dependent on these secondary messengers rather than the mechanical response of the VSM. ACh–induced vasodilation is mediated by endothelial M3 receptors and endothelial nitric oxide (eNO) synthase (eNOS), which impact the NO/GC/cGMP pathway through subsequent activation of K + channels (BK Ca and K ATP ) and VSM membrane hyperpolarization. Since eicosanoids and NO can induce vasodilation through the activation of K ATP channels [ 13 ] , this phenomenon has also been studied using the K ATP channel opener, pinacidil. However, pinacidil did not modify the response, suggesting that myo –inositol had no impact on K ATP channel function. In addition to K ATP channels, cGMP–dependent protein kinases are also involved in vasodilation [ 14 ] . 8–bromo cGMP is an analog of cGMP that activates PKG and protein phosphorylation [ 15 ] , resulting in VSM relaxation. However, in our study, the 8–bromo cGMP–induced response was not altered, suggesting that this pathway was not involved in myo –inositol–induced relaxation of aged arteries exposed to thromboxan A 2 analog, which confirmed that K ATP channels are not involved in myo –inositol–induced vasodilation. Unfortunately, BK Ca channels were not analyzed; however, the data mentioned above indicate a low probability that these channels are involved in vascular tone regulation. However, the participation of K + channels other than ATP–dependent K + channels cannot be ruled out. SNP is the exogenous donor of NO and is used when the bioavailability of endothelial NO is impaired due to eNOS dysfunction. However, in our study, myo –inositol had little effect on SNP–induced vasodilation, in contrast to ACh–induced relaxation; thus, we concluded that the endothelial eNOS/NO pathway was not impaired, and that NO is not the primary molecule responsible for the observed differences in VSM relaxation under ACh influence. Another gasomediator, CO, induces vasodilation via several mechanisms: (i) the eNOS/sGC/cGMP–dependent pathway, through the activation of eNOS by calcium and PI3K/Akt; (ii) a mechanism involving the activation of calcium–dependent potassium channels; and (iii) the anti–inflammatory effects of CO through the activation of the p38 mitogen–activated protein kinase (MAPK) pathway. Moreover, CO may have an inhibitory effect on numerous proteins, including cytochrome P450 and cytochrome c oxidase (iv) [ 7 ][ 16 ] . The first mechanism can be ruled out since neither SNP nor cGMP was involved in the vasodilator response to myo –inositol in this ex vivo model. The activation of calcium–dependent potassium channels seems to not be considered, as opposed to third and fourth mechanisms, which should be further studied. In the presence of myo –inositol, the endothelium–dependent relaxation in response to A23187 was comparable to that in the control group without myo –inositol exposure. However, significant differences were observed between different myo –inositol concentrations, with the greatest attenuation occurring at 100 mg/L compared to 1 and 10 mg/L. Since the aortic rings were obtained from aged rats and treated with a thromboxane analog (0.1 nM), this is the reason for the impaired vasodilation induced by A23187. Previously, reduced relaxant responses to A23187 have been observed in individuals with diabetes [ 17 ] and hypertension [ 18 ][ 19 ] . A23187 is an endothelium–dependent vasodilator [ 17 ] at a relatively low concentration (0.1 nM–0.1 µM). At higher doses (0.3–1 µM), A23187 induces secondary contraction due to the participation of contracting factors produced by cyclooxygenase and thromboxane receptor (TP) activation [ 17 ] . In our study, this secondary contraction was not observed, in response to either ACh or to A23187 (A23187 is an even more powerful agent than ACh), which induces endothelium–dependent contractions [ 17 ] . This secondary contraction depends on the animal model and the contracting factors used. In SHR rats, ACh induces secondary contraction, as endoperoxides and prostacyclin are involved in the activation of TP receptors in the rat aortae. Thromboxane A 2 , along with prostacyclin and endoperoxides, are implicated in the response to A23187. In diabetic rabbits, thromboxane A 2 or possibly its precursor, endoperoxide, is involved in vascular contraction [ 17 ] . Interestingly, endoperoxides influence the P450 pathway, which can also be a target of myo –inositol. In thoracic arteries, both NO and prostanoids are the main factors responsible for vascular relaxation dependent on endothelial cells. Since NO modulates arachidonic acid metabolism, further studies are needed to determine the eventual participation of proteinoids in the myo –inositol–induced effect on vasodilation. Moreover, other molecules, such as peroxynitrate (ONOO), hydrogen peroxide (H 2 O 2 ) and eicosanoids, might modulate vasodilation, which should also be analyzed in subsequent studies. A high level of ROS/RNS is involved in impaired vascular functioning. Increased TBARS concentration corresponds to increased MDA concentration and allows us to determine the level of lipid peroxidation. In our study, myo –inositol administration at a concentration of 100 mg/L but not 1–10 mg/L succeeded in protecting lipids against peroxidation processes dependent on H 2 O 2 /Fe 2+ , which may in part explain the observed differences in vascular function between the lower doses (0.01–10 mg/L) and the highest (100 mg/L). Like for TBARS concentration, TT was also increased at the highest concentration, but this effect was not observed at the lowest dose. Several substances or factors, such as heparins when overdosed, fibrinogen deficiency (following thrombolytic therapy), elevated levels of fibrin–(ogen) degradation products, and paraproteins, prolong TT. This effect of high myo –inositol on TT, whether beneficial or harmful, should be further studied. In addition to lipid peroxidation, protein damage is also observed during vascular dysfunction. In many cases, protein oxidation is irreversible and leads to protein dysfunction via the inhibition of enzymatic activity. Thus, in the present study, the oxidative damage of proteins was analyzed based on reduced levels of thiol residues and increased levels of carbonyl groups. Proteins such as membrane–related proteins, electron transport chain–related proteins, and endoplasmic reticulum–related proteins are subjected to ROS–induced carbonylation to a greater extent [ 20 ] . In our study, increasing concentrations of myo –inositol protected proteins against oxidative stress. The most sensitive groups were protein carbonyl groups (all studied concentrations above 0.01 mg/L myo –inositol), and the least sensitive were thiol groups (above 1.0 mg/L). A high level of myo –inositol has been observed in various diseases, including gliomatosis cerebri, diabetes mellitus, multiple sclerosis, and even Alzheimer’s disease. Conversely, a decrease in myo –inositol levels in the brain can be observed in chronic hepatic and hypoxic encephalopathy, stroke, cryptococcosis, and lymphoma [ 2 ] . Myo –inositol can reduce the accumulation of hepatic triglycerides in the liver, and deprivation of myo –inositol in daily diets results in fatty liver conditions. Therapy combined with myo –inositol helps remove cholesterol from the myocardium, which results in a reduction in lipid buildup in the heart, improving heart function [ 2 ] . Both myo –inositol and d– chiro –inositol also demonstrate insulin–like effects. Insulin linked to cells has an impact on the molecular inositol pathway because, during this process, inositol–secondary messengers are produced. d– Chiro –inositol–based secondary messengers promote glycogen synthesis, and myo –inositol secondary messengers regulate glucose intake, leading to an increase in the activity of glucose transport proteins [ 3 ] . Early diabetes–related hyperlipidemia and hyperglycemia can cause endothelial dysfunction. In addition, inositol phosphoglycans are generated in response to insulin. The level of chiro –inositol decreases in the urine of diabetic subjects, which is correlated with insulin resistance. Administration of d– chiro –inositol can effectively decrease hyperglycemia and hypertriglyceridemia in diabetic individuals [ 2 ] . Pintaudi et al. (2016) [ 3 ] showed that the oral administration of inositol can have a positive effect on the treatment of type 2 diabetes mellitus. Twenty subjects were involved in this study. After treatment, a significant decrease in blood glucose levels was observed. The results demonstrated that inositol could represent a valid strategy for improving glycemic control in patients with type II diabetes mellitus [ 3 ] . In addition, myo –inositol can inhibit carcinogenesis in various organs. It protects small airway epithelial cells against benzo[a]pyrene, which causes inhibition of metabolite differentiation. Benzo[a]pyrene is a carcinogen derived from tobacco. Administration of myo –inositol can decrease the multiplicity and size of surface tumors and the size of adenocarcinomas [ 2 ] . It is known that oxidative stress plays an important role in the development of various diseases, including cardiovascular diseases and polycystic ovarian syndrome, which are endocrine–metabolic diseases affecting approximately 10% of women. As an example, these patients demonstrate an increased risk of developing cardiovascular diseases, hypertension, dyslipidemia and insulin resistance. Oxidative stress in these patients also results in alterations in erythrocyte membranes, inducing structural modifications. Donà et al. [ 21 ] evaluated the effects of 12 weeks of dietary myo –inositol supplements (1200 mg/day powder) on various metabolic and hormonal parameters in patients with polycystic ovarian syndrome. In this study, the levels of diamide–related band 3 Tyr–P, glutathione and membrane glutathionylated proteins in erythrocytes were analyzed. The results indicated that treatment with dietary myo –inositol positively affects erythrocytes by preventing increases in various parameters of oxidative stress in the membranes. The authors observed that even a low dose of myo –inositol administered regularly for three months had therapeutic effects [ 21 ] . Reactive oxygen species production increases during sperm cryopreservation and thawing, which leads to oxidative damage in cells. Antioxidant supplementation is used to prevent this process. Myo –inositol not only has antioxidant properties but also has a positive effect on sperm motility. These studies suggest that myo –inositol acts directly on the mitochondrial level, which is one of the biggest sources of ROS in cells, causing a decrease in ROS production during the sperm cryopreservation and thawing process [ 22 ] . Interestingly, Benvenga et al. [ 23 ] demonstrated the antioxidant activity of myo –inositol in patients with Hashimoto’s thyroiditis. Their studies aimed to stress peripheral blood mononuclear cells (PBMC) with H 2 O 2 to understand whether, in the presence of H 2 O 2 and the addition of an equimolar concentration of myo –inositol, selenomethlonletol and their mixture would protect PBMCs from the effects of H 2 O 2 . In both women with Hashimoto’s thyroiditis and healthy controls, a dose–dependent decrease in toxicity caused by H 2 O 2 was observed. The results indicated that myo –inositol alone and in combination with selenomethlonletol demonstrated activity similar to that of antioxidant compounds [ 23 ] . Rolnik et al. [ 5 ] analyzed the antioxidant activity of myo –inositol in in vitro studies, including one regarding human plasma exposed to strong pro–oxidants. The authors observed that myo –inositol reduces oxidative damage, decreases plasma lipid peroxidation and plasma protein carboxylation, and increases the level of thiol groups in plasma proteins. Moreover, it also improved the vascular response in compromised arteries from rats subjected to a thromboxane A 2 receptor agonist. All of these findings confirmed that myo –inositol positively influences selected elements of the hemostasis process [ 5 ] . Inositol may participate in the activation of NO production pathways and increase superoxide scavenging, which leads to the prevention of endothelial dysfunction. Myo –inositol supplementation in women with gestational diabetes mellitus has been shown to have anti–inflammatory and antioxidant effects due to its ability to reduce monocyte cell adhesion and intercellular ROS levels in the basal state. Through modulation of the inflammatory and oxidative agents, myo –inositol improved endothelial stress/damage in vascular cells [ 24 ] . Regular supplementation with myo –inositol also has a positive effect on women with polycystic ovarian syndrome. In addition, it improves metabolic and hormonal parameters, including markers of cardiovascular diseases. It decreases the levels of plasma triglycerides, total cholesterol concentration and low–density lipoproteins and increases the level of high–density lipoproteins [ 1 ] . Healthy aging is associated with changes in the morphology and physiology of the brain, including changes in metabolite concentrations. Tunc–Skarka et al. [ 25 ] demonstrated that the level of N–acetylaspartate is correlated with both the aging process and several cognitive deficits. A lower concentration of N–acetylaspartate results in a higher myo –inositol concentration. This study included 83 healthy volunteers. The results showed significant positive associations between myo –inositol levels and healthy aging [ 25 ] . Conclusion Increasing concentrations of myo –inositol protect proteins against oxidative stress. The most sensitive substances were protein carbonyl groups and the least sensitive were thiobarbituric acid reactive substances (TBARS/MDA), which are markers of lipid peroxidation. However, only the highest dose of myo –inositol (100 mg/L) protected lipids (TBARS/MDA) against oxidative stress and exclusively increased the time of clot formation (thrombin time). Myo –inositol at a concentration range effective for thiols (1–100 mg/L) decreased vascular contraction to noradrenaline and potentiated vasodilation to ACh and CORMs, and this was the most effective at 1 mg/L myo –inositol. The NO/GC/cGMP–dependent pathway was not involved in the described effect of myo –inositol on vascular relaxation. Materials and methods Drugs and chemicals For vascular reactivity studies, we used the following chemicals: noradrenaline hydrochloride (NA), acetylcholine chloride (ACh), sodium nitroprusside (SNP), carbon monoxide (CO) releasing molecule (CORM), cGMP analog (8–bromo–cGMP), calcium ionophore (A23187), pinacidil, and thromboxane A 2 stable analog (U–46619) (Sigma–Aldrich, St. Louise, MO, USA), and potassium chloride (KCl) (Chempur, Piekary Slaskie, Poland). The stock solutions (10 mM) of drugs were prepared in DMSO, except for noradrenaline (NA), which was dissolved in a NaCl (0.9%) + ascorbic acid (0.01% w/v) solution. These solutions were kept at − 20°C, and appropriate dilutions were made in Krebs–Henseleit solution daily (KHS composition in mM: NaCl 115; CaCl 2 2.5; KCl 4.6; KH 2 PO 4 1.2; MgSO 4 1.2; NaHCO 3 25; glucose 11.1). DMSO at a concentration of 0.01% did not alter the reactivity of the isolated aortic rings. Standard of myo –inositol, 1–(trimethylsilyl) imidazole, pyridine, dichloromethane used for GC – FID were shipped from Sigma–Aldrich (St. Louis, MO, USA) Animal protocol Aged male Wistar rats (12 months old) weighing 562.1 ± 42.10 g (means ± SDs) or 513.0–652.0 g (min–max) were intraperitoneally injected with ketamine (100 mg/kg BW) and xylazine (10 mg/kg BW) and decapitated. The thoracic aortae were carefully dissected, cut into 4–5–mm rings and placed in an ice–cold KHS. Blood for in vitro experimentation was obtained from nine healthy human donors (nonsmoking women and men) from the Medical Center in Lodz, Poland. Blood was collected in tubes with citrate/phosphate/dextrose/adenine solution as blood/CPDA (8.5:1, v/v). Donors had not taken any medication or addictive substances (including tobacco, alcohol, or antioxidant supplements) for one week before donation. Blood plasma was isolated by differential centrifugation. Institutional Review Board Statement All experimental protocols were approved by the Ethics Committee for Animal Experiments in Olsztyn, Poland (protocol code 90/2019). All methods were carried out in accordance with relevant guidelines, and regulations and are in accordance with ARRIVE guidelines and the 3Rs rule. Gas chromatography with flame–ionization detection (GC–FID) Myo –inositol (1, 10 or 100 mg/L) was added to KHS (5 mL) and incubated with or without 10 aortic rings for 6 h. In the next step, the incubation solution (2 mL) was collected and frozen (–80°C). After freeze–drying, the precipitate was derivatized to determine the myo –inositol content. For this purpose, a mixture of 1–(trimethylsilyl) imidazole: pyridine (1:1, v:v) was added to the precipitate and heated at 80°C for 45 min. The presence of myo –inositol in the aortic rings was monitored using GC with a flame ionization detector (GC–FID, GC2010 Plus, Shimadzu, Kyoto, Japan) equipped with a Zebron ZB–1 capillary column (15 m length, 0.25 mm diameter, 0.1 µm film thickness, Phenomenex, Torrance, CA, USA). The injector temperature was 325°C, and the samples were loaded onto the column using the split method (10:1) as previously described [ 5 ] . Vascular reactivity studies As previously described [ 5 ] , aortic rings from the rat thoracic aortae were mounted in stagnant 5 mL organ baths (Graz Tissue Bath System, Barcelona, Spain) filled with KHS with or without added myo –inositol (1, 10 or 100 mg/L). Aortic rings were continuously aerated with carbogen gas and subjected to a preload tension of 1 g (TAM–A Hugo Sachs Elektronik, March, Germany). The thromboxane A 2 stable analog (U–46619, 0.1 nM) was added to the incubation chambers after aortic ring stabilization (30 min) and incubated for 2 h. The functional integrity of the aortic rings was tested with high concentrations of KCl (75 mM) and ACh (10 µM) [ 25 ] . Aortic rings were washed with KHS + myo –inositol at the appropriate concentration to recover to basal tension. Cumulative concentrations of either acetylcholine chloride (0.1 nM–10 µM), sodium nitroprusside (0.1 nM–10 µM), carbon monoxide (CO)–releasing molecule (CORM, 0.1–100 µM), calcium ionophore (A23187, 0.01 nM–1 µM), pinacidil (10 nM–10 µM), or cGMP analog (8–bromo–cGMP, 0.1–10 µM) were added to aortic rings precontracted with noradrenaline (0.1 µM). The effect of myo –inositol on the vasoconstrictor response was also investigated by analyzing the response generated by a single KCl (75 mM) dose and by cumulative concentrations of NA (0.1 nM–10 µM). Lipid and protein peroxidation Human plasma was preincubated (5 min, 37°C) with myo –inositol and treated with 4.7 mM H 2 O 2 :3.8 mM FeSO 4 :2.5 mM EDTA (25 min, 37°C). The levels of thiol groups (nmol/mg of plasma protein), carbonyl groups (nmol/mg of plasma protein), and TBARS/MDA (nmol/mL of plasma) were determined spectrophotometrically using a SPECTROstar Nano Microplate Reader (BMG LABTECH) for thiols, with a method involving 2,4–dinitrophenylhydrazine (DNPH) for carbonyl group measurement, and calculated based on absorbances measured at λ = 575 nm using a SPECTROstar Nano Microplate Reader (BMG LABTECH, Ortenberg, Germany) for TBARS as previously described [ 5 ] . Hemostasis parameters Prothrombin time, thrombin time, and activated partial thromboplastin time were determined coagulometrically with an Optic Coagulation Analyzer, model K–3002 (Kselmed, Grudziadz, Poland), according to the manufacturer’s instructions. Data analysis and statistics For the CCRCs, the area under the curve (AUC), maximal response (Emax, %) and potency (logEC 50 ) were calculated based on a nonlinear regression model. The Shapiro–Wilk test was used to analyze normally distributed data. Non–Gaussian data are presented as medians (25–75% percentiles). The Kruskal–Wallis test was used for multiple comparisons. Otherwise, ordinary one–way ANOVA with Dunnett’s multiple comparison test was used to compare the means and standard deviations (or the means and SEMs for CCRCs). A P value less than 0.05 was considered significant. Declarations Acknowledgements: This research was supported by the UWM Statutory fundings, No. 61.610.007-110, granted to M.S.M. Author contributions: Conceptualization, M.S.M; Funding Acquisition, M.S.M; Investigation, A.R., J.S-P., M.S.M; Methodology, B.O., L.B.L, L.G., M.S.M; Project Administration, M.S.M; Resources, B.O., L.B.L, L.G., M.S.M.; Software, M.S.M.; Supervision, M.S.M.; Visualization, M.S.M.; Writing—Original Draft, B.O., M.S.M.; Writing—Review and Editing, M.S.M.; All authors read and approved the final manuscript. Data availability statement: All data generated or analyzed during this study are included in this published article (and its supplementary information files). Competing Interests Statement: The authors declare no competing interests. Correspondence and requests for materials should be addressed to M.S.M. References Croze, M. L., & Soulage, C. O. Potential role, and therapeutic interests of myo-inositol in metabolic diseases. Biochimie 95 , 1811–1827 (2013). Chhetri, D. R. Myo-Inositol and Its Derivatives: Their Emerging Role in the Treatment of Human Diseases. Front Pharmacol . 10 (2019). Pintaudi, B., G. Di Vieste & M. Bonomo. The Effectiveness of Myo-Inositol and D-Chiro Inositol Treatment in Type 2 Diabetes. Int J Endocrinol . 9132052 (2016). Rodríguez-Nieto, G., et al. Organization of neurochemical interactions in young and older brains as revealed with a network approach: Evidence from proton magnetic resonance spectroscopy ( 1 H-MRS). Neuroimage 266 , 119830 (2023). Rolnik, A., et al. Beneficial in vitro effects of a low myo-inositol dose in the regulation of vascular resistance and protein peroxidation under inflammatory conditions. Nutrients 14 , 1–13 (2022). Ratz, P. H., Berg, K. M., Urban, N. H., & Miner, A. S. Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus. Am J Physiol Cell Physiol. 288 , 769–783 (2005). Yang, P. M., Huang, Y. T., Zhang, Y. Q., Hsieh, C. W., Wung, B. S. Carbon monoxide releasing molecule induces endothelial nitric oxide synthase activation through a calcium and phosphatidylinositol 3-kinase/Akt mechanism. Vascul Pharmacol. 87 , 209–218 (2016). Pkhaladze, L., et al. Treatment of lean PCOS teenagers: a follow-up comparison between Myo-Inositol and oral contraceptives. Eur Rev Med Pharmacol Sci. 25 , 7476–7485 (2021). Nordio, M., Basciani, S., & Camajani, E. The 40:1 myo-inositol/D-chiro-inositol plasma ratio is able to restore ovulation in PCOS patients: comparison with other ratios. Eur Rev Med Pharmacol Sci. 23 , 5512–5521 (2019). Zhao, H., Xing, C., Zhang, J., He, B. Comparative efficacy of oral insulin sensitizers metformin, thiazolidinediones, inositol, and berberine in improving endocrine and metabolic profiles in women with PCOS: a network meta-analysis. Reprod Health. 18 , 171 (2021). Cupitra, N. I., Calderón, J. C., Narvaez-Sanchez, R. Influence of Ageing on Vascular Reactivity and Receptor Expression in Rabbit Aorta: A Complement to Elastocalcinosis and Smooth Muscle Mechanisms. Clin Interv Aging. 15 , 537–545 (2020). Zhang, M., et al. Thromboxane-induced contractile response of mesenteric arterioles is diminished in the older rats and the older hypertensive rats. Front Pharmacol. 13 , 1019511 (2022). Shi, W. W., Yang, Y., Shi, Y., & Jiang, C. K(ATP) channel action in vascular tone regulation: from genetics to diseases. Sheng Li Xue Bao. 64 , 1–13 (2012). Surks H. K. cGMP-dependent protein kinase I and smooth muscle relaxation: a tale of two isoforms. Circ Res. 101 , 1078–1080 (2007). Francis, S. H., Busch, J. L., Corbin, J. D., & Sibley, D. cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action. Pharmacol Rev. 62 , 525–563 (2010). Kaczara, P., Przyborowski, K., Mohaissen, T., & Chlopicki, S. Distinct Pharmacological Properties of Gaseous CO and CO-Releasing Molecule in Human Platelets. Int J Mol Sci. 22 , 3584 (2021). Shi, Y., Feletou, M., Ku, D. D., Man, R. Y., Vanhoutte, P. M. The calcium ionophore A23187 induces endothelium-dependent contractions in femoral arteries from rats with streptozotocin-induced diabetes. Br J Pharmacol. 150 , 624–632 (2007). Yang, D., Gluais, P., Zhang, J. N., Vanhoutte, P. M., & Félétou, M. Endothelium-dependent contractions to acetylcholine, ATP and the calcium ionophore A 23187 in aortas from spontaneously hypertensive and normotensive rats. Fundam Clin Pharmacol. 18 , 321–326 (2004). Yang, D., Gluais, P., Zhang, J. N., Vanhoutte, P. M., & Félétou, M. Nitric oxide and inactivation of the endothelium-dependent contracting factor released by acetylcholine in spontaneously hypertensive rat. J Cardiovasc Pharmacol. 43 , 815–820 (2004). Lee, J., Song, C. H. Effect of Reactive Oxygen Species on the Endoplasmic Reticulum and Mitochondria during Intracellular Pathogen Infection of Mammalian Cells. Antioxidants (Basel) 10 , 872 (2021). Donà, G., et al. Inositol administration reduces oxidative stress in erythrocytes of patients with polycystic ovary syndrome. Eur J Endocrinol. 166 , 703–710 (2012). Ponchia, R., et al. Oxidative Stress Measurement in Frozen/Thawed Human Sperm: The Protective Role of an In Vitro Treatment with Myo-Inositol. Antioxidants (Basel) 11 , 10 (2021). Benvenga, S., et al. Favorable effects of myo-inositol, selenomethionine or their combination on the hydrogen peroxide-induced oxidative stress of peripheral mononuclear cells from patients with Hashimoto's thyroiditis: preliminary in vitro studies. Eur Rev Med Pharmacol Sci. 21 , 89–101 (2017). Baldassarre, M. P. A., et al. Myoinositol Reduces Inflammation and Oxidative Stress in Human Endothelial Cells Exposed In Vivo to Chronic Hyperglycemia. Nutrients 13 , 2210 (2021). Tunc-Skarka, N., et al. Effects of normal aging and SCN1A risk-gene expression on brain metabolites: evidence for an association between SCN1A and myo-inositol. NMR Biomed. 27 , 228–234 (2014). Majewski, M., Klett-Mingo, M., Verdasco-Martín, C. M., Otero, C., Ferrer, M. Spirulina extract improves age-induced vascular dysfunction. Pharm Biol. 60 , 627–637 (2022). Additional Declarations No competing interests reported. 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16:54:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1948794,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4403797/v1/5ca753ca-9e14-4541-98e2-c16b742cd96a.pdf"},{"id":56958913,"identity":"90290471-5ded-4959-b7dd-53cde1d375bd","added_by":"auto","created_at":"2024-05-22 16:46:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":177290,"visible":true,"origin":"","legend":"","description":"","filename":"suplfig.docx","url":"https://assets-eu.researchsquare.com/files/rs-4403797/v1/1a9240828669f341ca3e802d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"From the antioxidant and anticoagulant properties of myo–inositol to ex vivo vascular studies and GC–FID analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInositol (cyclohexane\u0026ndash;1,2,3,4,5,6\u0026ndash;hexol) is a natural cyclitol present in both animal and plant cells. The basic structure contains polyols in nine stereoisomeric forms depending on the spatial orientation of the six hydroxyl groups \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Among the nine possible geometrical isomers, seven are inactive. The remaining two form a chiral pair. Its predominant isomeric form present in nature and human food is \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, also known as \u003cem\u003ecis\u003c/em\u003e\u0026ndash;1,2,3,5\u0026ndash;\u003cem\u003etrans\u003c/em\u003e\u0026ndash;4,6\u0026ndash;cyklohexanehexol. It can have either a free form or can be bound to phospholipids or inositol phosphate derivatives. The greatest amount of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol in human food is found in products including seeds, fresh vegetables, and fruits. Citrus and cantaloupe have extraordinarily high levels of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, whereas leafy vegetables are a poor source \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCells produce inositol from three main sources. The first is based on \u003cem\u003ede novo\u003c/em\u003e biosynthesis from glucose\u0026ndash;6\u0026ndash;phosphate by 1\u0026ndash;ᴅ\u0026ndash;\u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol\u0026ndash;phosphate synthase (MIPS) and inositol monophosphatase (IMPase). The second is based on the dephosphorylation of inositol phosphates derived from the breakdown of inositol\u0026ndash;containing membrane phospholipids, and the third is uptake from the extracellular fluid \u003cem\u003evia\u003c/em\u003e specialized \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol transporters \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. All living cells contain inositol phospholipids in their membranes. Phytic acid, another form of inositol, is the principal storage form of phosphorus in many plant tissues, such as bran and seeds. \u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol plays a crucial role in various cellular processes, such as comprising the structural basis for secondary messengers including inositol triphosphates, inositol glycans, and phosphatidylinositol phosphate lipids. \u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol also plays an essential role in other cell functions, including cell growth, peripheral nerve development, reproduction, and osteogenesis \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Cellular \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol concentration has an essential influence on various diseases \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Recently, Rodr\u0026iacute;guez-Nieto et al.\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e observed age\u0026ndash;related differences in \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol expression across brain regions, suggesting the occurrence of neurodegeneration and altered gliosis. In addition, our previous study demonstrated that \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol exposure has a positive effect on the vascular system and blood plasma protein carbonylation under shared stress \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol exposure improves vasodilator and vasoconstrictor response in arteries from young Wistar rats subjected to thromboxane A\u003csub\u003e2\u003c/sub\u003e mimetic administration. It also increases NO release/bioavailability and decreases H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production, which correlates to oxidative damage of blood plasma proteins \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven the scarcity of information on the \u003cem\u003ein vitro\u003c/em\u003e effect of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol on the arteries, we aimed to determine its effect on the regulation of vascular resistance; oxidative stress, including protein and lipid oxidation levels; and the coagulation process during aging, together with increased inflammation were also studied.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMyo\u003c/strong\u003e \u003cstrong\u003e\u0026ndash;inositol concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe presence of the aortic rings in the preincubation medium did not significantly modify the concentration of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (measured at three concentrations of 1.0, 10, and 100 mg/L of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol). Hence, \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol was neither degraded in the solution nor taken up by the aortic rings in the metabolic processes during 6 h of incubation (Table \u003cspan\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eConcentration of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (mg/L) in the KHS with (+) or without (\u0026ndash;) added aortic rings. Ten aortic rings per 5 mL chamber were incubated for 6 h (n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (mg/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eaortic rings\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e106.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e113.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eNS \u0026ndash; not significant\u003c/p\u003e\n \u003cp\u003end \u0026ndash; below detection level\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eLipid and protein peroxidation\u003c/h2\u003e\n \u003cp\u003eIncreased TBARS (5.26\u0026ndash;fold) concentration was observed in the plasma preincubated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e (Fig. 1A). Increasing concentrations of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol added to the preincubated plasma decreased TBARS concentrations by 0.84\u0026ndash;fold (0.01 mg/L), 0.76\u0026ndash;fold (0.1 mg/L), 0.76\u0026ndash;fold (1.0 mg/L), 0.76\u0026ndash;fold (10 mg/L), and 0.62\u0026ndash;fold (100 mg/L), with a significant effect at the highest concentration of 100 mg/L (Fig.\u0026nbsp;1A).\u003c/p\u003e\n \u003cp\u003eAn increased carbonylation level (8.39\u0026ndash;fold) was observed in the plasma preincubated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e (Fig. 1B). Increasing concentrations of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol added to the preincubated plasma decreased the carbonyl groups in a dose\u0026ndash;dependent manner by 0.79\u0026ndash;fold (0.01 mg/L), 0.68\u0026ndash;fold (0.1 mg/L), 0.62\u0026ndash;fold (1.0 mg/L), 0.53\u0026ndash;fold (10 mg/L), and 0.48\u0026ndash;fold (100 mg/L), with significant effects at all studied concentrations ranging from 0.01\u0026ndash;100 mg/L (Fig.\u0026nbsp;1B).\u003c/p\u003e\n \u003cp\u003eA decreased level of thiol groups (0.27\u0026ndash;fold) was observed in the plasma preincubated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e (Fig. 1C). Increasing concentrations of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol added to the preincubated plasma increased the concentrations of the thiol groups in a dose\u0026ndash;dependent manner by 1.90\u0026ndash;fold (0.01 mg/L), 2.28\u0026ndash;fold (0.1 mg/L), 3.36\u0026ndash;fold (1.0 mg/L), 3.65\u0026ndash;fold (10 mg/L), and 3.80\u0026ndash;fold (100 mg/L), with a significant effect at 1\u0026ndash;100 mg/L (Fig. 1C).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 1.\u003c/strong\u003e Concentrations of (\u003cstrong\u003eA\u003c/strong\u003e) TBARS, (\u003cstrong\u003eB\u003c/strong\u003e) carbonyl groups, and (\u003cstrong\u003eC\u003c/strong\u003e) thiol groups in human blood plasma incubated with the pro\u0026ndash;oxidant agent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e positive control. \u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol favorably decreased indices of lipid peroxidation (TBARS, 100 mg/L) and protein peroxidation (decreasing carbonyl groups at 0.01\u0026ndash;100 mg/L and increasing thiol groups at 1.0\u0026ndash;100 mg/L).\u003c/p\u003e\n \u003cp\u003eFor the four highest concentrations (0.1, 1, 10 and 100 mg/L of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol), the level of thiol groups recovered and was comparable to that of the negative control (plasma not treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e), and this was not observed for TBARS and the carbonyl groups (Fig.\u0026nbsp;1S).\u003c/p\u003e\n \u003cp\u003eBecause we determined that the highest concentration of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol decreased the concentrations of TBARS exclusively (100 mg/L but not 10 mg/L), we have further analyzed the exact concentration of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol between 10 and 120 mg/L that induced the effect. A concentration equal to 120 mg/L (0.65\u0026ndash;fold) decreased TBARS which is contrary to the other studied concentrations, 90 mg/L (0.77\u0026ndash;fold), 60 and 30 mg/L (0.86\u0026ndash;fold), and 10 mg/L (0.93\u0026ndash;fold), which did not significantly modify TBARS (Fig. \u003cspan\u003e2\u003c/span\u003e). Overall, our study confirmed that concentrations above 100 mg/L are able to decrease lipid peroxidation significantly.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003eCoagulation processes\u003c/h2\u003e\n \u003cp\u003eBlood plasma activated partial thromboplastin time (APTT) did not significantly change when increasing concentrations of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol were added (0.01\u0026ndash;100 mg/L, 0.97\u0026ndash;fold for all studied groups) (Fig.\u0026nbsp;3A).\u003c/p\u003e\n \u003cp\u003eBlood plasma prothrombin time (PT) was not significantly modified (0.01\u0026ndash;100 mg/L, 1.05\u0026ndash;fold for all studied groups) (Fig.\u0026nbsp;3B).\u003c/p\u003e\n \u003cp\u003eBlood plasma thrombin time (TT) increased 1.10\u0026ndash;fold only for the highest concentration of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (100 mg/L) and was not modified in the other groups (0.01\u0026ndash;10 mg/L, 1.05\u0026ndash;fold increase only) (Fig.\u0026nbsp;3C).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eVascular response\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eVascular contraction\u003c/h2\u003e \u003cp\u003eAortic rings were obtained from aged (12\u0026ndash;month\u0026ndash;old) male Wistar rats and preincubated with \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (1\u0026ndash;100 mg/L). further, a stable analog of thromboxane A\u003csub\u003e2\u003c/sub\u003e (TxB\u003csub\u003e2\u003c/sub\u003e, 0.1 nM) was added and icubated for 2 h.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKCl\u0026ndash;induced contraction did not change with increasing concentrations of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (0.01\u0026ndash;100 mg/L) added to the incubated aortic rings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eNA\u0026ndash;induced contraction decreased with increasing concentrations of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (as AUC) to 0.77\u0026ndash;fold (1.0 mg/L), 0.76\u0026ndash;fold (10 mg/L), and 0.76\u0026ndash;fold (100 mg/L, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), but not with 0.01 or 0.1 mg/L (data not presented). \u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol administration decreased the sensitivity to NA and thus shifted the cumulative concentration response curve to the right (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVasoconstrictor response to NA (determined as AUC) of isolated aortic rings from aged (12\u0026ndash;month\u0026ndash;old) male Wistar rats preincubated with \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (2 h) and exposed to TxB\u003csub\u003e2\u003c/sub\u003e (0.1 nM, 2 h) \u0026ndash; \u003cem\u003eex vivo\u003c/em\u003e studies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003econtrol (vehicle)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6529*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6477*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6460*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStd. Deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1783\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2487\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.042\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.402\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e*vs. Control (Vehicle)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eVasodilation\u003c/h2\u003e \u003cp\u003e \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol added to the preincubated aortic rings, enhanced the vasodilator response of the isolated aortic rings to ACh at 1.0, 10 and 100 mg/L concetrations, but not at 0.01 or 0.1 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This effect was most significant for 1.0 mg/L of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, as both the AUC and LogEC\u003csub\u003e50\u003c/sub\u003e were changed (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e); for 10.0 mg/L of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, only the AUC was modified (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e); and for 100 mg/L of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, only one point from the CCRC was modified (0.1 \u0026micro;M, see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In all cases, Emax did not significantly change (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNext, the vasodilator response to CORMs was studied, and an increase in vasodilation was observed at 10\u003csup\u003e\u0026ndash;5.5\u003c/sup\u003e M and 10\u003csup\u003e\u0026ndash;5\u003c/sup\u003e M for 1.0, 10 and 100 mg/L of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The vasodilatory effect was the most significant for 1.0 mg/L of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, as both the AUC and LogEC\u003csub\u003e50\u003c/sub\u003e were changed (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e); for 10.0 mg/L of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, neither the AUC nor the LogEC\u003csub\u003e50\u003c/sub\u003e was modified (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e); and for 100 mg/L of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, both the AUC and LogEC\u003csub\u003e50\u003c/sub\u003e exhibited an increased response. In all cases, Emax did not significantly change (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe vasodilator response to SNP was studied, and an increase in vasodilation was observed at 10 nM SNP for 1.0, 10 and 100 mg/L \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). However, neither the AUC nor the logEC\u003csub\u003e50\u003c/sub\u003e or the Emax were significantly modified (see Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCompared with that of the control group (vehicle), the vasodilator response to A231987 was not different (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, significant differences were observed between the \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol groups: 1.0 mg/L vs. 100 mg/L and 10 mg/L vs. 100 mg/L.\u003c/p\u003e \u003cp\u003eThe vasodilator responses to 8\u0026ndash;bromo\u0026ndash;cGMP and pinacidil were not modified (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F and Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVascular response (AUC) to the vasodilators ACh, CORM, SNP, A23187, 8\u0026ndash;bromo\u0026ndash;cGMP and pinacidil of isolated aortic rings preincubated with thromboxane B\u003csub\u003e2\u003c/sub\u003e (0.1 nM, 2 h) from aged (12\u0026ndash;month\u0026ndash;old) Wistar rats: \u003cem\u003eex vivo\u003c/em\u003e studies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl (Vehicle)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100.0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u0026ndash;100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u0026ndash;100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u0026ndash;10\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e202.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e253.2*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e239.1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e229.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.2448\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.8509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStd. Deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eANOVA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCORM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e124.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e162.5*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e144.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e150.8*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.3056\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStd. Deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eANOVA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2569\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e329.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e348.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e356.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e350.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStd. Deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eANOVA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6849\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.4321\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.6048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA23187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e114.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e129.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e132.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e80.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.2458\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.2132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStd. Deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.8598\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePinacidil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e149.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e152.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e156.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e147.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStd. Deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eANOVA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9793\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.7031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9872\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u0026ndash;bromo\u0026ndash;cGMP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e52.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e34.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStd. Deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e*vs. Control (Vehicle), \u003csup\u003e\u003cb\u003e#\u003c/b\u003e\u003c/sup\u003e \u003cem\u003ebased on the results from\u003c/em\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVascular response (logEC\u003csub\u003e50\u003c/sub\u003e) to the vasodilators ACh, CORM, SNP, A23187, 8\u0026ndash;bromo\u0026ndash;cGMP and pinacidil of isolated aortic rings preincubated with thromboxane B\u003csub\u003e2\u003c/sub\u003e (0.1 nM, 2 h) from aged (12\u0026ndash;month\u0026ndash;old) Wistar rats \u0026ndash; \u003cem\u003eex vivo\u003c/em\u003e studies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl (Vehicle)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100.0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u0026ndash;100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u0026ndash;100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u0026ndash;10\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;7.380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;7.733\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;7.666\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;7.547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.1145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;7.230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;7.594*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;7.490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;7.371\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;7.109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;7.396\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;6.998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;7.185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2544\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.7421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.2441\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCORM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;5.627\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;6.041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;5.781\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;5.709\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;5.417\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;5.661*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;5.622\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;5.633*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;4.854\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;5.531\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;5.234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;5.490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShapiro\u0026ndash;Wilk test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.7351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0498\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;8.855\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;8.782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;8.877\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;8.726\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;8.176\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;8.478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;8.556\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;8.467\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;8.038\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;8.352\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;8.379\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;8.314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShapiro\u0026ndash;Wilk test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.4249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePinacidil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;6.701\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;6.813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;6.815\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;6.724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;6.587\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;6.621\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;6.678\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;6.579\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;6.532\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;6.543\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;6.609\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;6.501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShapiro\u0026ndash;Wilk test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.7257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ebased on the results from\u003c/em\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVascular response Emax (%) to the vasodilators ACh, CORM, SNP, A23187, 8\u0026ndash;bromo\u0026ndash;cGMP and pinacidil of isolated aortic rings preincubated with thromboxane B\u003csub\u003e2\u003c/sub\u003e (0.1 nM, 2 h) from aged (12\u0026ndash;month\u0026ndash;old) Wistar rats \u0026ndash; \u003cem\u003eex vivo\u003c/em\u003e studies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl (Vehicle)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol (mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100.0\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;95.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;97.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;97.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;98.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;90.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;94.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;93.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;95.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;86.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;91.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;89.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;90.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShapiro\u0026ndash;Wilk test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1440\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCORM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;103.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;103.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;103.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;103.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;99.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;101.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;102.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;101.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;95.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;97.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;99.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;98.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShapiro\u0026ndash;Wilk test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;98.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;100.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;101.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;102.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStd. Deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.684\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.952\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.203\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eANOVA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.8727\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.6097\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2576\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePinacidil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;101.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;104.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;103.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;100.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;97.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;98.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;98.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;96.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75% Percentile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;96.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;98.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;94.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;93.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShapiro\u0026ndash;Wilk test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e*vs. control\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings revealed that different concentrations of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol have varying effects on blood stimulated with the pro\u0026ndash;oxidant H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e \u003cem\u003ein vitro\u003c/em\u003e. All concentrations above 0.01 mg/L improved the carbonylation level, whereas concentrations above 1.0 mg/L improved the thiol content, both of which are markers of protein protection from oxidation. Lipid peroxidation, reflected as content of thiobarbituric acid reactive substances (TBARS), which measures malondialdehyde (MDA) levels, improved with the administration of above 100 mg/L of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol. Interestingly, for the thiol group levels, only concentrations above 0.1 mg/L corresponded to the values obtained for the blood that were not stimulated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e (negative control with no added H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e). Neither was this observed with TBARS content nor with the carbonylation level, which did not recover to the control level. Similar to TBARS content, thrombin time (TT) was also modified with the highest concentration of 100 mg/L \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol. Neither activated partial thromboplastin time (APTT) nor prothrombin time (PT) (INR determinant) were changed. The abovementioned results point to the differences between the mentioned concentrations, which to varying extents protect against oxidative stress.\u003c/p\u003e \u003cp\u003eVascular contraction in response to the adrenergic receptor agonist noradrenaline, which depends on DAG and IP3, decreased with increasing concentrations of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (1.0\u0026ndash;100 mg/L), but this effect was not observed at concentrations less than this level (0.01\u0026ndash;0.1 mg/L). This is contrary to Ca\u003csup\u003e2+\u003c/sup\u003e\u0026ndash;dependent KCl\u0026ndash;induced contraction, which bypasses G protein\u0026ndash;coupled receptors (GPCRs) \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, as this was not altered.\u003c/p\u003e \u003cp\u003eThe ACh\u0026ndash;induced vasodilator response was potentiated above 1.0 mg/L, and this effect was more significant at lower doses (1.0 mg/L and 10 mg/L) than at the highest dose (100 mg/L) (based on the AUC and Emax). The exogenous NO donor SNP potentiated vasodilation only to a small extent, and neither the AUC nor the Emax changed; therefore, we concluded that endothelial cells were not corrupted in a significant way and that the bioavailability of endogenous NO was maintained. Based on the AUC and Emax, another gasotransmitter, carbon monoxide (CO) \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e also induced the greatest effect at 1.0 mg/L \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, with a lesser effect at 100 mg/L \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, similar to that of ACh, with almost no effect at 10 mg/L. The Ca\u003csup\u003e2+\u003c/sup\u003e ionophore A\u0026ndash;23187, which induces either contraction or relaxation of blood vessels, did not modify the response compared to that in the control group. However, A\u0026ndash;23187 administration weakened the vasodilator response to 100 mg/L \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol opposite to that to 1.0 and 10 mg/L, which may in part explain the response to the vasodilator ACh and/or CO\u0026ndash;releasing molecule CORMs described above. Neither the cGMP\u0026ndash;dependent protein kinase pathway nor ATP\u0026ndash;sensitive potassium channels were involved in the vasodilation induced by \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol. Our results point to a non\u0026ndash;cGMP\u0026ndash;dependent mechanism in which vascular reactivity is modulated by \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol preincubation in aged rats treated with 0.1 nM thromboxane \u003cem\u003eex vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eHere, for the first time, we discussed the direct influence of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol on vascular reactivity, coagulation, and antioxidant properties. Although \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol has been widely studied \u003cem\u003ein vivo\u003c/em\u003e (described later in the second part of the \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003ediscussion\u003c/span\u003e section) due to its effectiveness in treating polycystic ovary syndrome \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e][\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e and in menstrual recovery \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, little is known about its influence on vascular tone regulation in elderly subjects exposed to inflammation following vascular dysfunction \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. For these reasons, arteries were taken from aged rats and exposed to a thromboxane A\u003csub\u003e2\u003c/sub\u003e analog, which is a well\u0026ndash;known factor contributing to vascular dysfunction \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e][\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Indeed, \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol administration modified vascular contraction and relaxation in the range of 1\u0026ndash;100 mg/L; however, this effect was not dependent on the concentration, and with increasing doses starting at 1 mg/L, different effects were observed. No significant differences in vascular contraction were detected between the studied groups in response to high concentrations of KCl. High KCl concentration affects vascular smooth muscle (VSM) membrane depolarization, with a subsequent influx of Ca\u003csup\u003e2+\u003c/sup\u003e through voltage\u0026ndash;dependent calcium channels (VDCCs). Interestingly, compared with the control group (without \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol), \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol decreased vasoconstriction in response to noradrenaline; but no significant differences were observed between the studied concentrations. Stimulation of vascular α1\u0026ndash;adrenoreceptor with the agonist noradrenaline results in activation of Gq receptors \u003cem\u003evia\u003c/em\u003e the phospholipase C (PLC) cascade, a subsequent increase in the levels of secondary messengers (IP\u003csub\u003e3\u003c/sub\u003e and DAG), and an increase in the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration through intracellular storage (due to IP\u003csub\u003e3\u003c/sub\u003e) or VDCC activation (the PKC/CaMKII pathway), which results in VSM contraction. As the KCl\u0026ndash;induced response was not altered, in contrast to the NA\u0026ndash;induced contraction, we concluded that the influence of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol on this mechanism was dependent on these secondary messengers rather than the mechanical response of the VSM.\u003c/p\u003e \u003cp\u003eACh\u0026ndash;induced vasodilation is mediated by endothelial M3 receptors and endothelial nitric oxide (eNO) synthase (eNOS), which impact the NO/GC/cGMP pathway through subsequent activation of K\u003csup\u003e+\u003c/sup\u003e channels (BK\u003csub\u003eCa\u003c/sub\u003e and K\u003csub\u003eATP\u003c/sub\u003e) and VSM membrane hyperpolarization.\u003c/p\u003e \u003cp\u003eSince eicosanoids and NO can induce vasodilation through the activation of K\u003csub\u003eATP\u003c/sub\u003e channels \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, this phenomenon has also been studied using the K\u003csub\u003eATP\u003c/sub\u003e channel opener, pinacidil. However, pinacidil did not modify the response, suggesting that \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol had no impact on K\u003csub\u003eATP\u003c/sub\u003e channel function. In addition to K\u003csub\u003eATP\u003c/sub\u003e channels, cGMP\u0026ndash;dependent protein kinases are also involved in vasodilation \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. 8\u0026ndash;bromo cGMP is an analog of cGMP that activates PKG and protein phosphorylation \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, resulting in VSM relaxation. However, in our study, the 8\u0026ndash;bromo cGMP\u0026ndash;induced response was not altered, suggesting that this pathway was not involved in \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol\u0026ndash;induced relaxation of aged arteries exposed to thromboxan A\u003csub\u003e2\u003c/sub\u003e analog, which confirmed that K\u003csub\u003eATP\u003c/sub\u003e channels are not involved in \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol\u0026ndash;induced vasodilation. Unfortunately, BK\u003csub\u003eCa\u003c/sub\u003e channels were not analyzed; however, the data mentioned above indicate a low probability that these channels are involved in vascular tone regulation. However, the participation of K\u003csup\u003e+\u003c/sup\u003e channels other than ATP\u0026ndash;dependent K\u003csup\u003e+\u003c/sup\u003e channels cannot be ruled out.\u003c/p\u003e \u003cp\u003eSNP is the exogenous donor of NO and is used when the bioavailability of endothelial NO is impaired due to eNOS dysfunction. However, in our study, \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol had little effect on SNP\u0026ndash;induced vasodilation, in contrast to ACh\u0026ndash;induced relaxation; thus, we concluded that the endothelial eNOS/NO pathway was not impaired, and that NO is not the primary molecule responsible for the observed differences in VSM relaxation under ACh influence.\u003c/p\u003e \u003cp\u003eAnother gasomediator, CO, induces vasodilation \u003cem\u003evia\u003c/em\u003e several mechanisms: (i) the eNOS/sGC/cGMP\u0026ndash;dependent pathway, through the activation of eNOS by calcium and PI3K/Akt; (ii) a mechanism involving the activation of calcium\u0026ndash;dependent potassium channels; and (iii) the anti\u0026ndash;inflammatory effects of CO through the activation of the p38 mitogen\u0026ndash;activated protein kinase (MAPK) pathway. Moreover, CO may have an inhibitory effect on numerous proteins, including cytochrome P450 and cytochrome c oxidase (iv) \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e][\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The first mechanism can be ruled out since neither SNP nor cGMP was involved in the vasodilator response to \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol in this \u003cem\u003eex vivo\u003c/em\u003e model. The activation of calcium\u0026ndash;dependent potassium channels seems to not be considered, as opposed to third and fourth mechanisms, which should be further studied.\u003c/p\u003e \u003cp\u003eIn the presence of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, the endothelium\u0026ndash;dependent relaxation in response to A23187 was comparable to that in the control group without \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol exposure. However, significant differences were observed between different \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol concentrations, with the greatest attenuation occurring at 100 mg/L compared to 1 and 10 mg/L. Since the aortic rings were obtained from aged rats and treated with a thromboxane analog (0.1 nM), this is the reason for the impaired vasodilation induced by A23187. Previously, reduced relaxant responses to A23187 have been observed in individuals with diabetes \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e and hypertension \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e][\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. A23187 is an endothelium\u0026ndash;dependent vasodilator \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e at a relatively low concentration (0.1 nM\u0026ndash;0.1 \u0026micro;M). At higher doses (0.3\u0026ndash;1 \u0026micro;M), A23187 induces secondary contraction due to the participation of contracting factors produced by cyclooxygenase and thromboxane receptor (TP) activation \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. In our study, this secondary contraction was not observed, in response to either ACh or to A23187 (A23187 is an even more powerful agent than ACh), which induces endothelium\u0026ndash;dependent contractions \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. This secondary contraction depends on the animal model and the contracting factors used. In SHR rats, ACh induces secondary contraction, as endoperoxides and prostacyclin are involved in the activation of TP receptors in the rat aortae. Thromboxane A\u003csub\u003e2\u003c/sub\u003e, along with prostacyclin and endoperoxides, are implicated in the response to A23187. In diabetic rabbits, thromboxane A\u003csub\u003e2\u003c/sub\u003e or possibly its precursor, endoperoxide, is involved in vascular contraction \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Interestingly, endoperoxides influence the P450 pathway, which can also be a target of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol. In thoracic arteries, both NO and prostanoids are the main factors responsible for vascular relaxation dependent on endothelial cells. Since NO modulates arachidonic acid metabolism, further studies are needed to determine the eventual participation of proteinoids in the \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol\u0026ndash;induced effect on vasodilation. Moreover, other molecules, such as peroxynitrate (ONOO), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and eicosanoids, might modulate vasodilation, which should also be analyzed in subsequent studies.\u003c/p\u003e \u003cp\u003eA high level of ROS/RNS is involved in impaired vascular functioning. Increased TBARS concentration corresponds to increased MDA concentration and allows us to determine the level of lipid peroxidation. In our study, \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol administration at a concentration of 100 mg/L but not 1\u0026ndash;10 mg/L succeeded in protecting lipids against peroxidation processes dependent on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e, which may in part explain the observed differences in vascular function between the lower doses (0.01\u0026ndash;10 mg/L) and the highest (100 mg/L). Like for TBARS concentration, TT was also increased at the highest concentration, but this effect was not observed at the lowest dose. Several substances or factors, such as heparins when overdosed, fibrinogen deficiency (following thrombolytic therapy), elevated levels of fibrin\u0026ndash;(ogen) degradation products, and paraproteins, prolong TT. This effect of high \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol on TT, whether beneficial or harmful, should be further studied.\u003c/p\u003e \u003cp\u003eIn addition to lipid peroxidation, protein damage is also observed during vascular dysfunction. In many cases, protein oxidation is irreversible and leads to protein dysfunction \u003cem\u003evia\u003c/em\u003e the inhibition of enzymatic activity. Thus, in the present study, the oxidative damage of proteins was analyzed based on reduced levels of thiol residues and increased levels of carbonyl groups. Proteins such as membrane\u0026ndash;related proteins, electron transport chain\u0026ndash;related proteins, and endoplasmic reticulum\u0026ndash;related proteins are subjected to ROS\u0026ndash;induced carbonylation to a greater extent \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. In our study, increasing concentrations of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol protected proteins against oxidative stress. The most sensitive groups were protein carbonyl groups (all studied concentrations above 0.01 mg/L \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol), and the least sensitive were thiol groups (above 1.0 mg/L).\u003c/p\u003e \u003cp\u003eA high level of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol has been observed in various diseases, including gliomatosis cerebri, diabetes mellitus, multiple sclerosis, and even Alzheimer\u0026rsquo;s disease. Conversely, a decrease in \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol levels in the brain can be observed in chronic hepatic and hypoxic encephalopathy, stroke, cryptococcosis, and lymphoma \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol can reduce the accumulation of hepatic triglycerides in the liver, and deprivation of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol in daily diets results in fatty liver conditions. Therapy combined with \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol helps remove cholesterol from the myocardium, which results in a reduction in lipid buildup in the heart, improving heart function \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBoth \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol and d\u0026ndash;\u003cem\u003echiro\u003c/em\u003e\u0026ndash;inositol also demonstrate insulin\u0026ndash;like effects. Insulin linked to cells has an impact on the molecular inositol pathway because, during this process, inositol\u0026ndash;secondary messengers are produced. d\u0026ndash;\u003cem\u003eChiro\u003c/em\u003e\u0026ndash;inositol\u0026ndash;based secondary messengers promote glycogen synthesis, and \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol secondary messengers regulate glucose intake, leading to an increase in the activity of glucose transport proteins \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Early diabetes\u0026ndash;related hyperlipidemia and hyperglycemia can cause endothelial dysfunction. In addition, inositol phosphoglycans are generated in response to insulin. The level of \u003cem\u003echiro\u003c/em\u003e\u0026ndash;inositol decreases in the urine of diabetic subjects, which is correlated with insulin resistance. Administration of d\u0026ndash;\u003cem\u003echiro\u003c/em\u003e\u0026ndash;inositol can effectively decrease hyperglycemia and hypertriglyceridemia in diabetic individuals \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Pintaudi et al. (2016) \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e showed that the oral administration of inositol can have a positive effect on the treatment of type 2 diabetes mellitus. Twenty subjects were involved in this study. After treatment, a significant decrease in blood glucose levels was observed. The results demonstrated that inositol could represent a valid strategy for improving glycemic control in patients with type II diabetes mellitus \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition, \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol can inhibit carcinogenesis in various organs. It protects small airway epithelial cells against benzo[a]pyrene, which causes inhibition of metabolite differentiation. Benzo[a]pyrene is a carcinogen derived from tobacco. Administration of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol can decrease the multiplicity and size of surface tumors and the size of adenocarcinomas \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is known that oxidative stress plays an important role in the development of various diseases, including cardiovascular diseases and polycystic ovarian syndrome, which are endocrine\u0026ndash;metabolic diseases affecting approximately 10% of women. As an example, these patients demonstrate an increased risk of developing cardiovascular diseases, hypertension, dyslipidemia and insulin resistance. Oxidative stress in these patients also results in alterations in erythrocyte membranes, inducing structural modifications. Don\u0026agrave; et al. \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e evaluated the effects of 12 weeks of dietary \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol supplements (1200 mg/day powder) on various metabolic and hormonal parameters in patients with polycystic ovarian syndrome. In this study, the levels of diamide\u0026ndash;related band 3 Tyr\u0026ndash;P, glutathione and membrane glutathionylated proteins in erythrocytes were analyzed. The results indicated that treatment with dietary \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol positively affects erythrocytes by preventing increases in various parameters of oxidative stress in the membranes. The authors observed that even a low dose of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol administered regularly for three months had therapeutic effects \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eReactive oxygen species production increases during sperm cryopreservation and thawing, which leads to oxidative damage in cells. Antioxidant supplementation is used to prevent this process. \u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol not only has antioxidant properties but also has a positive effect on sperm motility. These studies suggest that \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol acts directly on the mitochondrial level, which is one of the biggest sources of ROS in cells, causing a decrease in ROS production during the sperm cryopreservation and thawing process \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInterestingly, Benvenga et al. \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e demonstrated the antioxidant activity of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol in patients with Hashimoto\u0026rsquo;s thyroiditis. Their studies aimed to stress peripheral blood mononuclear cells (PBMC) with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to understand whether, in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and the addition of an equimolar concentration of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, selenomethlonletol and their mixture would protect PBMCs from the effects of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. In both women with Hashimoto\u0026rsquo;s thyroiditis and healthy controls, a dose\u0026ndash;dependent decrease in toxicity caused by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was observed. The results indicated that \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol alone and in combination with selenomethlonletol demonstrated activity similar to that of antioxidant compounds \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRolnik et al. \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e analyzed the antioxidant activity of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol in \u003cem\u003ein vitro\u003c/em\u003e studies, including one regarding human plasma exposed to strong pro\u0026ndash;oxidants. The authors observed that \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol reduces oxidative damage, decreases plasma lipid peroxidation and plasma protein carboxylation, and increases the level of thiol groups in plasma proteins. Moreover, it also improved the vascular response in compromised arteries from rats subjected to a thromboxane A\u003csub\u003e2\u003c/sub\u003e receptor agonist. All of these findings confirmed that \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol positively influences selected elements of the hemostasis process \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInositol may participate in the activation of NO production pathways and increase superoxide scavenging, which leads to the prevention of endothelial dysfunction. \u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol supplementation in women with gestational diabetes mellitus has been shown to have anti\u0026ndash;inflammatory and antioxidant effects due to its ability to reduce monocyte cell adhesion and intercellular ROS levels in the basal state. Through modulation of the inflammatory and oxidative agents, \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol improved endothelial stress/damage in vascular cells \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Regular supplementation with \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol also has a positive effect on women with polycystic ovarian syndrome. In addition, it improves metabolic and hormonal parameters, including markers of cardiovascular diseases. It decreases the levels of plasma triglycerides, total cholesterol concentration and low\u0026ndash;density lipoproteins and increases the level of high\u0026ndash;density lipoproteins \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHealthy aging is associated with changes in the morphology and physiology of the brain, including changes in metabolite concentrations. Tunc\u0026ndash;Skarka et al. \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e demonstrated that the level of N\u0026ndash;acetylaspartate is correlated with both the aging process and several cognitive deficits. A lower concentration of N\u0026ndash;acetylaspartate results in a higher \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol concentration. This study included 83 healthy volunteers. The results showed significant positive associations between \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol levels and healthy aging \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIncreasing concentrations of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol protect proteins against oxidative stress. The most sensitive substances were protein carbonyl groups and the least sensitive were thiobarbituric acid reactive substances (TBARS/MDA), which are markers of lipid peroxidation. However, only the highest dose of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (100 mg/L) protected lipids (TBARS/MDA) against oxidative stress and exclusively increased the time of clot formation (thrombin time).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol at a concentration range effective for thiols (1\u0026ndash;100 mg/L) decreased vascular contraction to noradrenaline and potentiated vasodilation to ACh and CORMs, and this was the most effective at 1 mg/L \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol. The NO/GC/cGMP\u0026ndash;dependent pathway was not involved in the described effect of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol on vascular relaxation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDrugs and chemicals\u003c/h2\u003e \u003cp\u003eFor vascular reactivity studies, we used the following chemicals: noradrenaline hydrochloride (NA), acetylcholine chloride (ACh), sodium nitroprusside (SNP), carbon monoxide (CO) releasing molecule (CORM), cGMP analog (8\u0026ndash;bromo\u0026ndash;cGMP), calcium ionophore (A23187), pinacidil, and thromboxane A\u003csub\u003e2\u003c/sub\u003e stable analog (U\u0026ndash;46619) (Sigma\u0026ndash;Aldrich, St. Louise, MO, USA), and potassium chloride (KCl) (Chempur, Piekary Slaskie, Poland). The stock solutions (10 mM) of drugs were prepared in DMSO, except for noradrenaline (NA), which was dissolved in a NaCl (0.9%)\u0026thinsp;+\u0026thinsp;ascorbic acid (0.01% w/v) solution. These solutions were kept at \u0026minus;\u0026thinsp;20\u0026deg;C, and appropriate dilutions were made in Krebs\u0026ndash;Henseleit solution daily (KHS composition in mM: NaCl 115; CaCl\u003csub\u003e2\u003c/sub\u003e 2.5; KCl 4.6; KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1.2; MgSO\u003csub\u003e4\u003c/sub\u003e 1.2; NaHCO\u003csub\u003e3\u003c/sub\u003e 25; glucose 11.1). DMSO at a concentration of 0.01% did not alter the reactivity of the isolated aortic rings.\u003c/p\u003e \u003cp\u003eStandard of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol, 1\u0026ndash;(trimethylsilyl) imidazole, pyridine, dichloromethane used for GC \u0026ndash; FID were shipped from Sigma\u0026ndash;Aldrich (St. Louis, MO, USA)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnimal protocol\u003c/h2\u003e \u003cp\u003eAged male Wistar rats (12 months old) weighing 562.1\u0026thinsp;\u0026plusmn;\u0026thinsp;42.10 g (means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs) or 513.0\u0026ndash;652.0 g (min\u0026ndash;max) were intraperitoneally injected with ketamine (100 mg/kg BW) and xylazine (10 mg/kg BW) and decapitated. The thoracic aortae were carefully dissected, cut into 4\u0026ndash;5\u0026ndash;mm rings and placed in an ice\u0026ndash;cold KHS.\u003c/p\u003e \u003cp\u003eBlood for \u003cem\u003ein vitro\u003c/em\u003e experimentation was obtained from nine healthy human donors (nonsmoking women and men) from the Medical Center in Lodz, Poland. Blood was collected in tubes with citrate/phosphate/dextrose/adenine solution as blood/CPDA (8.5:1, v/v). Donors had not taken any medication or addictive substances (including tobacco, alcohol, or antioxidant supplements) for one week before donation. Blood plasma was isolated by differential centrifugation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInstitutional Review Board Statement\u003c/h2\u003e \u003cp\u003e All experimental protocols were approved by the Ethics Committee for Animal Experiments in Olsztyn, Poland (protocol code 90/2019). All methods were carried out in accordance with relevant guidelines, and regulations and are in accordance with ARRIVE guidelines and the 3Rs rule.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGas chromatography with flame\u0026ndash;ionization detection (GC\u0026ndash;FID)\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol (1, 10 or 100 mg/L) was added to KHS (5 mL) and incubated with or without 10 aortic rings for 6 h. In the next step, the incubation solution (2 mL) was collected and frozen (\u0026ndash;80\u0026deg;C). After freeze\u0026ndash;drying, the precipitate was derivatized to determine the \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol content. For this purpose, a mixture of 1\u0026ndash;(trimethylsilyl) imidazole: pyridine (1:1, v:v) was added to the precipitate and heated at 80\u0026deg;C for 45 min. The presence of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol in the aortic rings was monitored using GC with a flame ionization detector (GC\u0026ndash;FID, GC2010 Plus, Shimadzu, Kyoto, Japan) equipped with a Zebron ZB\u0026ndash;1 capillary column (15 m length, 0.25 mm diameter, 0.1 \u0026micro;m film thickness, Phenomenex, Torrance, CA, USA). The injector temperature was 325\u0026deg;C, and the samples were loaded onto the column using the split method (10:1) as previously described \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eVascular reactivity studies\u003c/h2\u003e \u003cp\u003eAs previously described \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, aortic rings from the rat thoracic aortae were mounted in stagnant 5 mL organ baths (Graz Tissue Bath System, Barcelona, Spain) filled with KHS with or without added \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (1, 10 or 100 mg/L). Aortic rings were continuously aerated with carbogen gas and subjected to a preload tension of 1 g (TAM\u0026ndash;A Hugo Sachs Elektronik, March, Germany). The thromboxane A\u003csub\u003e2\u003c/sub\u003e stable analog (U\u0026ndash;46619, 0.1 nM) was added to the incubation chambers after aortic ring stabilization (30 min) and incubated for 2 h. The functional integrity of the aortic rings was tested with high concentrations of KCl (75 mM) and ACh (10 \u0026micro;M) \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Aortic rings were washed with KHS\u0026thinsp;+\u0026thinsp;\u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol at the appropriate concentration to recover to basal tension.\u003c/p\u003e \u003cp\u003eCumulative concentrations of either acetylcholine chloride (0.1 nM\u0026ndash;10 \u0026micro;M), sodium nitroprusside (0.1 nM\u0026ndash;10 \u0026micro;M), carbon monoxide (CO)\u0026ndash;releasing molecule (CORM, 0.1\u0026ndash;100 \u0026micro;M), calcium ionophore (A23187, 0.01 nM\u0026ndash;1 \u0026micro;M), pinacidil (10 nM\u0026ndash;10 \u0026micro;M), or cGMP analog (8\u0026ndash;bromo\u0026ndash;cGMP, 0.1\u0026ndash;10 \u0026micro;M) were added to aortic rings precontracted with noradrenaline (0.1 \u0026micro;M). The effect of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol on the vasoconstrictor response was also investigated by analyzing the response generated by a single KCl (75 mM) dose and by cumulative concentrations of NA (0.1 nM\u0026ndash;10 \u0026micro;M).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eLipid and protein peroxidation\u003c/h2\u003e \u003cp\u003eHuman plasma was preincubated (5 min, 37\u0026deg;C) with \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol and treated with 4.7 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e:3.8 mM FeSO\u003csub\u003e4\u003c/sub\u003e:2.5 mM EDTA (25 min, 37\u0026deg;C). The levels of thiol groups (nmol/mg of plasma protein), carbonyl groups (nmol/mg of plasma protein), and TBARS/MDA (nmol/mL of plasma) were determined spectrophotometrically using a SPECTROstar Nano Microplate Reader (BMG LABTECH) for thiols, with a method involving 2,4\u0026ndash;dinitrophenylhydrazine (DNPH) for carbonyl group measurement, and calculated based on absorbances measured at λ\u0026thinsp;=\u0026thinsp;575 nm using a SPECTROstar Nano Microplate Reader (BMG LABTECH, Ortenberg, Germany) for TBARS as previously described \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHemostasis parameters\u003c/h2\u003e \u003cp\u003eProthrombin time, thrombin time, and activated partial thromboplastin time were determined coagulometrically with an Optic Coagulation Analyzer, model K\u0026ndash;3002 (Kselmed, Grudziadz, Poland), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eData analysis and statistics\u003c/h2\u003e \u003cp\u003eFor the CCRCs, the area under the curve (AUC), maximal response (Emax, %) and potency (logEC\u003csub\u003e50\u003c/sub\u003e) were calculated based on a nonlinear regression model. The Shapiro\u0026ndash;Wilk test was used to analyze normally distributed data. Non\u0026ndash;Gaussian data are presented as medians (25\u0026ndash;75% percentiles). The Kruskal\u0026ndash;Wallis test was used for multiple comparisons. Otherwise, ordinary one\u0026ndash;way ANOVA with Dunnett\u0026rsquo;s multiple comparison test was used to compare the means and standard deviations (or the means and SEMs for CCRCs). A \u003cem\u003eP\u003c/em\u003e value less than 0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThis research was supported by the UWM Statutory fundings, No. 61.610.007-110, granted to M.S.M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e Conceptualization, M.S.M; Funding Acquisition, M.S.M; Investigation, A.R., J.S-P., M.S.M; Methodology, B.O., L.B.L, L.G., M.S.M; Project Administration, M.S.M; Resources, B.O., L.B.L, L.G., M.S.M.; Software, M.S.M.; Supervision, M.S.M.; Visualization, M.S.M.; Writing\u0026mdash;Original Draft, B.O., M.S.M.; Writing\u0026mdash;Review and Editing, M.S.M.; All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability statement:\u003c/strong\u003e All data generated or analyzed during this study are included in this published article (and its supplementary information files).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting Interests Statement:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Correspondence and requests for materials should be addressed to M.S.M.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCroze, M. 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Favorable effects of myo-inositol, selenomethionine or their combination on the hydrogen peroxide-induced oxidative stress of peripheral mononuclear cells from patients with Hashimoto\u0026apos;s thyroiditis: preliminary in vitro studies. \u003cem\u003eEur Rev Med Pharmacol Sci.\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 89\u0026ndash;101 (2017).\u003c/li\u003e\n\u003cli\u003eBaldassarre, M. P. A., et al. Myoinositol Reduces Inflammation and Oxidative Stress in Human Endothelial Cells Exposed In Vivo to Chronic Hyperglycemia. \u003cem\u003eNutrients\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 2210 (2021).\u003c/li\u003e\n\u003cli\u003eTunc-Skarka, N., et al. Effects of normal aging and SCN1A risk-gene expression on brain metabolites: evidence for an association between SCN1A and myo-inositol. \u003cem\u003eNMR Biomed.\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 228\u0026ndash;234 (2014).\u003c/li\u003e\n\u003cli\u003eMajewski, M., Klett-Mingo, M., Verdasco-Mart\u0026iacute;n, C. M., Otero, C., Ferrer, M. Spirulina extract improves age-induced vascular dysfunction. \u003cem\u003ePharm Biol.\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 627\u0026ndash;637 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"acetylcholine, carbon monoxide, myo–inositol, reactive oxygen species, thromboxane A2, U–46619","lastPublishedDoi":"10.21203/rs.3.rs-4403797/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4403797/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA potential protective effect of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol against pro\u0026ndash;oxidant damage to human plasma and rat thoracic arteries has been demonstrated, suggesting that this nutraceutical agent plays a key role in the vasculature and may be beneficial for preventing harmful environmental effects.\u003c/p\u003e \u003cp\u003eAortic rings were isolated from aged (12\u0026ndash;month\u0026ndash;old) male Wistar rats and preincubated with \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol (0.01\u0026ndash;100 mg/L, 2 h). A stable thromboxane A\u003csub\u003e2\u003c/sub\u003e analog was added (0.1 nM, 2 h) to analyze vascular dysfunction. The concentration of \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol in the organ baths was determined \u003cem\u003evia\u003c/em\u003e GC\u0026ndash;FID. In another experiment, human blood plasma was subjected to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e administration, and \u003cem\u003emyo\u003c/em\u003e\u0026ndash;inositol was added to analyze lipid and protein peroxidation processes. The hemostatic parameters were also studied. \u003cem\u003eMyo\u003c/em\u003e\u0026ndash;inositol (1\u0026ndash;100 mg/L) administration protects proteins against oxidative stress, which modifies the vascular response. A dose above 100 mg/L additionally protected lipids and increased thrombin time.\u003c/p\u003e","manuscriptTitle":"From the antioxidant and anticoagulant properties of myo–inositol to ex vivo vascular studies and GC–FID analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-22 16:46:39","doi":"10.21203/rs.3.rs-4403797/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-12T13:08:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-11T12:58:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-08T20:01:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-06T00:50:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234727098987690391951841086116712565224","date":"2024-06-04T16:31:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309069385259363296679092823995059058877","date":"2024-06-02T15:38:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43869770141204244697134068370979051616","date":"2024-06-01T21:47:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101258189904954127130220122826419138581","date":"2024-06-01T11:37:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"242884691834153887511445750798412906615","date":"2024-05-30T13:18:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95774881159091506614447250570940249511","date":"2024-05-30T12:18:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-30T05:53:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-28T13:47:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-14T06:56:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-14T04:36:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-05-11T06:15:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1fd5c0e-1bf5-46f0-898f-0f2eba78887d","owner":[],"postedDate":"May 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-10-15T03:38:42+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-22 16:46:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4403797","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4403797","identity":"rs-4403797","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}