Antioxidant and anticoagulant properties of myo-inositol determined in an ex vivo studies and gas chromatography analysis | Scientific Reports

Scientific Reports volume 14, Article number: 25633 (2024) Cite this article

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Myo-inositol plays a key role in the vasculature and may be beneficial for preventing harmful environmental effects. In this study aortic rings were isolated from middle-aged (12-month-old) male Wistar rats and preincubated with myo-inositol (0.01–100 mg/L) for 2 h. A stable thromboxane A2 analog was added (0.1 nM, 2 h) to analyze vascular dysfunction. The concentration of myo-inositol in the organ baths was determined via gas chromatography. In another experiment, human blood plasma was subjected to pro-oxidant - hydrogen peroxide administration, and myo-inositol was added to analyze lipid and protein oxidation processes. The thromboplastin time, prothrombin time, and thrombin time were also studied. Myo-inositol administration protected thiol groups against oxidative stress, meanwhile decreased vascular contraction and potentiated vasodilation (concentrations 1–100 mg/L, but not ≤ 0.1 mg/L), and changed the level of 8-isoprostane (concentrations: 0.1–100 mg/L, but not 0.01 mg/L) in plasma treated with H2O2/Fe2+. A dose above 100 mg/L additionally protected lipids (measured as thiobarbituric acid reactive substances) and increased thrombin time. Moreover, significant differences in vascular relaxation were observed between the studied myo-inositol concentrations (1 vs. 10 vs. 100 mg/L), which was not detected under the 0.1 mg/L. The concentration of myo-inositol in the organ baths determined via gas chromatography revealed that this nutraceutical agent was not used by the aortic rings during the incubation period in physiological processes. A protective effect of myo-inositol against prooxidant damage to human plasma and rat thoracic arteries has been demonstrated.

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 groups1. 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 source1,2.

Cells produce inositol from three main sources. The first is based on de novo biosynthesis from glucose-6-phosphate by 1-D-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 transporters1. 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 (IP3), 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 osteogenesis1. Cellular myo-inositol concentration has an essential influence on various diseases2,3. 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. 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 A2 mimetic administration. It also increases nitric oxide (NO) release/bioavailability and decreases hydrogen peroxide (H2O2) production, which correlates to oxidative damage of blood plasma proteins5.

Given the scarcity of information on the in vitro effect of myo-inositol on the arteries, and metabolic disorders 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 connected to the increased inflammation. Little is known about myo-inositol supplementation in patients with cardiovascular dysfunction, as presented latter on in the "Discussion" section.

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).

Thiobarbituric acid reactive substances (TBARS) increased by 5.26-fold in the plasma preincubated with H2O2/Fe2+ (donor of hydroxyl radical), see 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 H2O2/Fe2+ (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 to 100 mg/L (Fig. 1B).

A decreased level of thiol groups (0.27-fold) was observed in the plasma preincubated with H2O2/Fe2+ (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).

Concentrations of A TBARS, B carbonyl groups, and C thiol groups in human blood plasma incubated with the pro-oxidant agent H2O2/Fe2+. ANOVA, P < 0.05 vs. H2O2/Fe2+ 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 H2O2/Fe2+), 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.

TBARS concentrations in human blood plasma incubated with the pro-oxidant agent H2O2/Fe2+and myo-inositol (10-120 mg/L). ANOVA, P ≤ 0.05 vs. H2O2/Fe2+ positive control. Myo-inositol (120 mg/L) decreased the TBARS concentration (P=0.0103). A concentration range of 10-90 mg/L did not significantly modify TBARS.

We observed that the tested concentrations (0.1–100 mg/L) of myo-inositol changed the level of 8-isoprostane in plasma treated with H2O2/Fe2+ (Table 2).

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). Plasma prothrombin time (PT) was also not significantly modified (0.01–100 mg/L, 1.05-fold for all studied groups) (Fig. 3B).

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).

Hemostatic parameters: A activated partial thromboplastin time (APTT), B prothrombin time (PT), and C thrombin time (TT) in human blood plasma. ANOVA, P ≤ 0.05 Addition of myo-inositol (100 mg/L) increased TT.

Aortic rings were obtained from middle-aged (12-month-old) male Wistar rats and preincubated with myo-inositol (1-100 mg/L). further, a stable analog of thromboxane A2 (TxB2, 0.1 nM) was added and incubated for 2 h.

Vasoconstrictor response to A high 75 mM KCl and B cumulative concentrations of noradrenaline (NA) in aortic rings isolated from middle-aged (12-month-old) male Wistar rats preincubated with myo-inositol (2 h) and exposed to TxA2 analog (0.1 nM, 2 h) - ex vivo studies. ANOVA, P ≤ 0.05. All of the studied concentrations (1.0, 10 and 100 mg/L) of myo-inositol decreased vasoconstriction in response to NA but did not modify the KCl-induced response. Lower concentrations (0.1 and 0.01 mg/L) of myo-inositol did not modify that response (data not presented)

KCl-induced contraction did not change with increasing concentrations of myo-inositol (0.01-100 mg/L) added to the incubated aortic rings (Fig. 4A).

NA-induced contraction decreased in a dose-independent manner with myo-inositol (as Area Under the Curve, AUC) to 0.77-fold (1.0 mg/L), 0.76-fold (10 mg/L), and 0.76-fold (100 mg/L, Fig. 4A; Table 3), but not with 0.01 or 0.1 mg/L (data not presented). Myo-inositol administration decreased the sensitivity to noradrenaline (NA) and thus shifted the cumulative concentration response curve to the right (Fig. 4B).

Myo-inositol added to the preincubated aortic rings, enhanced the vasodilator response of the isolated aortic rings to acetylcholine (ACh) in a dose-independent manner at 1.0, 10 and 100 mg/L concentrations, but not at 0.01 or 0.1 mg/L (Fig. 5A). This effect was most significant for 1.0 mg/L of myo-inositol, as both the AUC and LogEC50 were changed (Tables 4 and 5); for 10.0 mg/L of myo-inositol, only the AUC was modified (Tables 4 and 5); and for 100 mg/L of myo-inositol, only one point from the CCRC was modified (0.1 µM, see Fig. 5A). In all cases, Emax did not significantly change (Table 6).

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. 5B). The vasodilatory effect was the most significant for 1.0 mg/L of myo-inositol, as both the AUC and LogEC50 were changed (Tables 4 and 5); for 10.0 mg/L of myo-inositol, neither the AUC nor the LogEC50 was modified (Tables 4 and 5); and for 100 mg/L of myo-inositol, both the AUC and LogEC50 exhibited an increased response. In all cases, Emax did not significantly change (Table 6).

Vasodilator response of isolated aortic rings from middle-aged (12-month-old) Wistar rats to A acetylcholine, B CORMs, C sodium nitroprusside (SNP), D the calcium ionophore A23187, E 8-bromo-cGMP, and F pinacidil ex vivo. ANOVA, P ≤ 0.05. The three highest studied concentrations, 1.0, 10 and 100 mg/L (but not 0.1 and 0.01 mg/L, data not presented), of myo-inositol potentiated vasodilation in response to ACh and CORMs, with little effect on the SNP- and calcium ionophore (A23187)-induced response. A23187 calcium ionophore, ACh acetylcholine, CORMs CO-releasing molecule, SNP sodium nitroprusside

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. 5C). However, neither the AUC nor the logEC50 or the Emax were significantly modified (see Tables 3, 4 and 5).

Compared with that of the control group (vehicle), the vasodilator response to A231987 was not different (Fig. 5D; Tables 3, 4 and 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. 5E, F; Tables 3, 4 and 5).

Our findings revealed that different concentrations of myo-inositol have varying effects on plasma stimulated with the pro-oxidant H2O2/Fe2+ (donor of hydroxyl radical) 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 H2O2/Fe2+ (negative control with no added H2O2/Fe2+). 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) (International Normalized Ratio - 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 diacylglycerol (DAG) and inositol trisphosphate (IP3), decreased in a dose-independent manner of myo-inositol (1.0-100 mg/L). No significant change was observed at concentrations less than 0.1 mg/L (0.01-0.1 mg/L). Interestingly, the Ca2+-dependent KCl-induced contraction was not altered (the mechanism of KCl bypasses G protein-coupled receptors)6.

The ACh-induced vasodilator response was potentiated above 1.0 mg/L in a dose-independent manner. The observed vasodilation 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 Ca2+ ionophore A-23,187, which induces either contraction or relaxation of blood vessels, did not modify the response compared to that in the control group. However, A-23,187 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 cyclic guanosine monophosphate (cGMP)-dependent protein kinase pathway nor adenosine triphosphate (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 middle-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 syndrome8,9 and in menstrual recovery10, little is known about its influence on vascular tone regulation in elderly subjects exposed to inflammation following vascular dysfunction11. For these reasons, arteries were taken from middle-aged rats and exposed to a thromboxane A2 analog, which is a well-known factor contributing to vascular dysfunction11,12. 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 Ca2+ 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 (IP3 and DAG), and an increase in the intracellular Ca2+ concentration through intracellular storage (due to IP3) 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 (BKCa and KATP) and VSM membrane hyperpolarization.

Since eicosanoids and NO can induce vasodilation through the activation of KATP channels13, this has also been studied using the KATP channel opener, pinacidil. However, pinacidil did not modify the response, suggesting that myo-inositol had no impact on KATP channel function. In addition to KATP channels, cGMP-dependent protein kinases are also involved in vasodilation14. 8-bromo cGMP is an analog of cGMP that activates protein kinase G (PKG) and protein phosphorylation15, 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 middle-aged arteries exposed to thromboxane A2 analog, which confirmed that KATP channels are not involved in myo-inositol-induced vasodilation. Unfortunately, BKCa 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 middle-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 diabetes17 and hypertension18,19. A23187 is an endothelium-dependent vasodilator17 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) activation17. 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 contractions17. 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 A2, along with prostacyclin and endoperoxides, are implicated in the response to A23187. In diabetic rabbits, thromboxane A2 or possibly its precursor, endoperoxide, is involved in vascular contraction17. Interestingly, endoperoxides influence the cytochrome 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 (H2O2) and eicosanoids, might modulate vasodilation, which should also be analyzed in subsequent studies.

A high level of reactive oxygen and nitrogen species (ROS/RNS) is involved in impaired vascular functioning. Increased TBARS concentration corresponds to increased MDA and allows 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 H2O2/Fe2+, which may in part explain the observed differences in vascular function between the lower (0.01-10 mg/L) and the highest (100 mg/L) doses. Moreover we observed significant difference in the level of 8-isoprostane (concentrations 0.1-100 mg/L, but not 0.01 mg/L) in plasma treated with H2O2/Fe2+. 8-isoprostane, a marker of oxidative stress, is the stable end-product of lipid peroxidation and is widely used to characterize lipid peroxidation in the blood. 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 extent20. 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 lymphoma2. 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 function2.

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 proteins3. 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 individuals2. Pintaudi et al.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 mellitus3.

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 adenocarcinomas2.

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 effects21.

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 process22.

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 H2O2 to understand whether, in the presence of H2O2 and the addition of an equimolar concentration of myo-inositol, selenomethlonletol and their mixture would protect PBMCs from the effects of H2O2. In both women with Hashimoto’s thyroiditis and healthy controls, a dose-dependent decrease in toxicity caused by H2O2 was observed. The results indicated that myo-inositol alone and in combination with selenomethlonletol demonstrated activity similar to that of antioxidant compounds23.

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, for example it 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 A2 receptor agonist. All of these findings confirmed that myo-inositol positively influences selected elements of the hemostasis process5.

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 cells24. 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 lipoproteins1.

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 aging25.

Hormesis as a response to increasing doses of myo-inositol cannot be ruled out, however more concentrations and additional studies such as analysis of lipid peroxidation products should be conducted to further consider hormesis as a term referred to a biphasic dose response to myo-inositol. Future research directions should focus on myo-inositol supplementation in metabolic disorders, where an increased production of ROS and vascular dysfunction exist. Also, a possible alternative to the treatment of cardiovascular dysfunction is to slow down aging processes and focus on erectile dysfunction or prostate enlargement treatment, which is also related to vascular complications.

A protective effect of myo-inositol against pro-oxidant damage to human plasma and rat thoracic arteries has been demonstrated. Increasing concentrations of myo-inositol protected 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. The concentration of myo-inositol in the organ baths determined via gas chromatography-flame ionization detection also revealed that this nutraceutical agent was not used by the aortic rings during the incubation period but modulated the processes in the arteries. Myo-inositol is an interesting agent which should be further investigated in form of supplementation under metabolic disorders or when vascular dysfunction exists, in case of erectile dysfunction, prostate enlargement or brain disorders related to vascular degeneration.

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 A2 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, 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; CaCl2 2.5; KCl 4.6; KH2PO4 1.2; MgSO4 1.2; NaHCO3 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 were shipped from Sigma-Aldrich (St. Louis, MO, USA).

Middle-aged male Wistar rats (12 months old) weighing 562.1 ± 42.10 g (means ± SDs) with min-max 513.0–652.0 g were fed a standard rat chow. At the end of the experimentation rats were injected with ketamine (100 mg/kg BW) and xylazine (10 mg/kg BW) intraperitoneally 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.

All experimental protocols were approved by the Ethics Committee for Animal Experiments in Olsztyn, Poland (protocol code 90/2019), and the bioethics committee of the University of Łódź (2/KBBN-UŁ/III/2014) for human blood collection. Informed consent was obtained from all human subjects. All methods were carried out in accordance with relevant guidelines, and regulations and are in accordance with ARRIVE guidelines, the 3Rs rule, and the Helsinki Declaration.

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 (at − 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 described5.

As previously described5, aortic rings obtained 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 A2 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)25,26. 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).

Human plasma was preincubated (5 min, 37 °C) with myo-inositol and treated with 4.7 mM H2O2:3.8 mM FeSO4:2.5 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 (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 described5.

The level of free 8-isoprostane in human plasma was determined with an 8-isoprostane ELISA Kit (Item No. 516360, Cayman Chemical) according to the standard protocol described by the manufacturer. Myo-inositol was added to human plasma at the final concentrations of 0.01 and 100 mg/L. The concentration of free 8-isoprostane in the samples was calculated with a pre-configured analysis tool according to the manufacturer’s instructions.

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.

For the CCRCs, the area under the curve (AUC), maximal response (Emax, %) and potency (logEC50) 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 ≤ 0.05 was considered significant.

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

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This research was supported by the UWM Statutory fundings, No. 61.610.007-110, granted to M.S.M. and was funded by The Minister of Science under “The Regional Initiative of Excellence Program”.

Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Łódź, 90-236, Łódź, Poland

Agata Rolnik & Beata Olas

Department of Structural Biology, Faculty of Biomedical Sciences and Postgraduate Education, Medical University of Łódź, 90-752, Łódź, Poland

Agata Rolnik

Department of Botany and Evolutionary Ecology, University of Warmia and Mazury in Olsztyn, 10-721, Olsztyn, Poland

Joanna Szablińska-Piernik

Department of Plant Physiology, Genetics and Biotechnology, University of Warmia and Mazury in Olsztyn, 10-719, Olsztyn, Poland

Lesław Bernard Lahuta

Department of Cardiology and Internal Medicine, Faculty of Medicine, University of Warmia and Mazury in Olsztyn, 10-082, Olsztyn, Poland

Leszek Gromadziński

Department of Pharmacology and Toxicology, Faculty of Medicine, University of Warmia and Mazury in Olsztyn, 10-082, Olsztyn, Poland

Michał S. Majewski

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Conceptualization, MSM; Funding Acquisition, MSM; Investigation, AR, JSP, MSM; Methodology, BO, LBL, LG, MSM; Project Administration, MSM; Resources, BO, LBL, LG, MSM; Software, MSM; Supervision, MSM; Visualization, MSM; Writing—Original Draft, BO, MSM; Writing—Review and Editing, MSM; All authors read and approved the final manuscript.

Correspondence to Michał S. Majewski.

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Rolnik, A., Olas, B., Szablińska-Piernik, J. et al. Antioxidant and anticoagulant properties of myo-inositol determined in an ex vivo studies and gas chromatography analysis. Sci Rep 14, 25633 (2024). https://doi.org/10.1038/s41598-024-76527-2

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Received: 11 May 2024

Accepted: 15 October 2024

Published: 27 October 2024

DOI: https://doi.org/10.1038/s41598-024-76527-2

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