Part2: 3-Hydroxyphenylacetic Acid: A Blood Pressure-Reducing Flavonoid Metabolite

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4. Discussion

Screening of different phenolic metabolites of flavonoids showed that 4 of 22 were potent vasodilators. Following on from these results, the cardiovascular effects of 3-(3-hydroxyphenyl)propionic acid, 34-dihydroxyphenylacetic acid (DHPA), and 4-methyl catechol were confirmed in vivo, and possible mechanisms of their actions were studied ex vivo [14,15]. Another flavonoid metabolite, 3-HPAA, was temporarily excluded from further investigation since it was not able to produce full relaxation in the initial screening of the rat aorta. In this work, we decided to extend our previous studies and focus on 3-HPAA as well. We aimed to(1) confirm its vasodilatory effect both in vivo in spontaneously hypertensive rats and ex vivo in another experimental model(porcine coronary arterial rings), and (2) study the mechanism of this action.

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Effect of 3-HPAA on the Blood Pressure and Heart Rate In Vivo in SHR

While 3-HPAA was a less potent vasodilator in the previous ex vivo screening [14], within this work, it was clearly confirmed that is able to decrease blood pressure in vivo in spontaneously hypertensive rats after intravenous administration. When administered as a bolus, the mean, systolic and diastolic blood pressures decreased. Quite unexpectedly, a significant effect on diastolic blood pressure was already observed after a very low dose of 10 ugs.kg-1.The extent of this response was relatively high (approx.20%). With increasing doses of 3-HPAA, the effect increased only slightly to approx.25%. The same was true in the case of systolic blood pressure when a significant decrease (approx. 15%)was present after the dose of 100ug.kg-I, and the maximal decrease found was approx.25%. There was variability among animals resulting in variable values; however, the dose-dependency of the effect was obvious. In contrast, no changes in heart rate were observed after intravenous bolus application. This can mean that the heart was not involved in the blood pressure-decreasing effect and the effect was solely based on peripheral relaxation. The absence of heart rate changes is important also from a safety point of view since the feedback increase in heart rate caused by the sympathetic nervous system, which follows a pronounced decrease in arterial blood pressure, is clinically unsuitable, as was well reported with the calcium channel blocker nifedipine [24].

In real situations, the colonic metabolites of ingested flavonoids are continuously absorbed from the GIT. To mimic this, slow intravenous infusions of 3-HPAA at different rates were administered in another set of experiments. Analogously to previous bolus application, a decrease in mean, systolic, and diastolic blood pressures was observed. The effect was dose-dependent, and a significant effect was brought about by the doses of 1 and 5mg.kg-1·min-Ireaching maximally approx.50% drop in blood pressure. Again, no significant changes in heart rate were observed during the 5 min-lasting-infusion, nor during the next 10 min of animal monitoring.

This effect on blood pressure can have a real impact. Flavonoids from the diet are rather poorly absorbed in the small intestine. They reach the colon and undergo microbial metabolism. The bacterial degradation process consists of a reduction of the double bond in the 2,3-position followed by the C-ring fission. The next step depends on the presence or absence of the 3-hydroxyl group. Flavones that do not possess this hydroxyl give origin to hydroxyphenylpropionic acid derivatives, while flavonols with the 3-hydroxyl give origin to derivatives of hydroxyphenyl acetic acids [25]. However, 3-HPAA arises as a ring-fission product derived from microbial catabolism of many parent flavonoids, not only flavonols such as quercetin. Moreover, an important metabolite with vasodilatory activity, DHPA, gives origin to 3-HPAA after its dehydroxylation and to a smaller extent to another vasorelaxant compound,3,4-dihydroxybenzoic acid/protocatechuic acid.3-HPAA is further catabolized into vaso-inactive hippuric and benzoic acids(Figure 1)[15,26]. In general, small flavonoid metabolites show higher plasma levels than their parent flavonoids and can reach peak concentrations usually in the range of 1 to 615 nM or even 42.9 uM in some cases [17]. The same seems to be true for 3-HPAA. In animal studies, administration of Calafate berries extract (providing~2.6 mg of phenolics)through gavage to gerbils resulted in maximal plasma concentrations of 3-HPAA of approx. 300 nM after 4h[27]. Other authors reported that a single 3-HPAA intravenous bolus of 2 and 4 mg. kg-Ito rats resulted in maximal plasma concentrations of approx.6mg.L-1(~40 uM and 16 mg.L-1（~100 μM） 【28】.In our study， the maximal dose administered intravenously as a bolus was 10 mg.kg-1. Thus, we can estimate [28] that the maximal plasma level achieved could be between 100 and 200 μM， which is 11-22 times higher than was physiologically detected [17]. Importantly, significant effects on the diastolic blood pressure were observed already at the dose of 10 μg.kg-1， which could roughly correspond to an achievable concentration of 100-200 nM. Analogous is true for administration via infusion. A dose of 1 mg·kg-I.min-could result in a plasma concentration of roughly 10 μM 【28】， which is within the range of total levels produced by diet [17]. In contrast, the dose of 5 mg.kg-.min-I would be difficult to achieve through a diet rich in polyphenols, and in this case, 3-HPAA maybe, instead, be applicable as a drug or supplement. Extrapolation of the animal data to humans is not easy; however, these concentrations are achievable in humans after consumption of a diet rich in flavonoids and might be associated with the effect on the vascular system[17]. Unfortunately, the kinetic data on 3-HPAA are still limited. An 8-week placebo-controlled dietary trial with72 participants showed a significant increase in plasma levels of 3-HPAA(from~180 to~250 nM) after consumption of berries, which provided about 837 mg of polyphenols per day. Moreover, there was also an increase of 87% in the urinary excretion of 3-HPAA [29]. Another study detected 60 different phenolic metabolites in plasma and urine in 10 volunteers after consumption of cranberry juice containing 787 mg of polyphenols. 3-HPAA was among the metabolites determined in plasma and reached a maximum concentration of ~600 nM after 10 h approximately [17]. Another kinetic study with nine healthy young men showed that the bioavailability of(poly)phenols does not depend solely on the amount taken. The amount of 766 mg of polyphenols led to maximum 3-HPAA plasma concentrations of~260 nM, whereas ingestion of more than double the amount gave rise to even lower levels(~240 nM) [30]. Nevertheless, in both cases, the plasma concentrations reached roughly 250 nM. 

Mechanism of the Vascular Effects of 3-HPAA Studied Ex Vivo

We performed a data search in the PubMed database with the keyword"3-hydroxyphenyl acetic acid".Analysis of the 110 articles found allowed us to conclude that data on 3-HPAA pharmacokinetics are limited and pharmacodynamic studies, to our best knowledge, do not exist. Only one study showed that3-HPAA decreases COX-2 protein levels in colon cancer cells, but without an effect on PGE2 production [31].

As in our in vivo experiments, 3-HPAA decreased blood pressure dose-dependently and had no impact on heart rate, we hypothesized that the mechanism of the observed effects could lie in the direct action of 3-HPAA on the vasculature. Therefore, we performed various complementary ex vivo experiments to explore the mechanism of action. As there is an apparent appeal in the Czech Republic to decrease the use of laboratory animals in line with the 3Rs (Replacement, Reduction, and Refinement), we selected an alternative model consisting of the use of porcine coronary arteries from fresh hearts, which were obtained from a local slaughterhouse. This model was not optimal. First, vasodilation was observed at a much higher concentration than in rat aorta, and second, this concentration is roughly 100 times higher than the concentration achievable through diet [17]. Notwithstanding this limitation, this setting allowed us to see differences between experimental groups and, hence, it served sufficiently for the determination of the mechanism of action. On the other hand, the advantage was that pigs and humans apparently share numerous similarities concerning the cardiovascular system [32]. It was shown that 3-HPAA produces dose-dependent vasodilation of pig coronary arteries ex vivo. This effect was at least partially mediated by endothelium with the participation of the endothelium-derived NO. In contrast to NO, we did not confirm the participation of endothelial M receptors, COX, SKca, and IKca channels, nor direct effects on smooth muscle Cay1.2 channels (L-type).

NO was previously referred to as an endothelium-derived relaxing factor and its role in vascular physiology is well known. Briefly, after being synthesized by eNOS in endothelial cells, NO diffuses to vascular smooth muscle cells where it activates soluble guanylate cyclase (sGC)and the cGMP-PKG pathway (Figure 8). PKG, in turn, activates various Kt channels present on the smooth muscle, namely large-conductance calcium-activated (BKca), ATP-sensitive (KATP), inward rectifier(KIR), and voltage-gated (Kv), thus allowing the transfer of K+ ions. This leads to an increase in negative membrane potential with the consequent inhibition of voltage-gated calcium channels (mainly L-type)and blockade of extracellular Ca2+ influx. The levels of intracellular Ca2+ are also regulated through activation of SERCA either by PKG or directly by NO [33], and by inhibition of IP3R channels. The lack of NO-mediated effects is related to various pathologies. A decreased expression and activity of eNOS was found in aortas from SHR 34 and impaired NO production was demonstrated in endothelial and vascular smooth muscle cells from mesenteric arteries and aorta of genetically modified hypertensive rats [35]. In human studies, an abnormal endothelial function was reported in patients with essential hypertension [36]. Endothelial dysfunction has been associated with impaired vascular bioavailability of NO[37] without specifying whether this mechanism is based on a reduction in synthesis, release, or diffusion of NO. Recently, the mechanisms postulated also include an increase in oxidant status that promotes NO breakdown [38,39]. Thus, the mechanisms described above might contribute to the vasodilatory effect of 3-HPAA (Figure 8), and its potential protective vascular effects. Importantly, this can be the reason for the large differences between a very high sensitivity of SHR to 3-HPAA vasodilatory effects and the low sensitivity of healthy coronary vessels from pig hearts, although this theory must be confirmed in additional studies.

If this is true, the question of 3-HPAA-induced NOsynthesis arises. The pKa value of 3-HPAA is 4, which means that under physiological pH, the substance is mostly ionized. This hinders passive transmembrane transport; however, the presence of a transporting system was not verified and cannot be excluded. The activation of eNOS is often triggered by an increase in cytosolic Ca2t.In our experiments, 3-HPAA vasodilation was not dependent on the activity of the endothelial IKCa and SKCa channels. As both these channels are Ca2+-sensitive [40, the previous increase in cytoplasmatic Ca2+ levels is not probable. Accordingly, endothelial M-receptors, which are GPCRs coupled to the second messengers'DAG+IP,/PKC+ cytosolic Ca2+ increase, were not involved, as their blockade by atropine did not modify the vasodilation caused by 3-HPAA. This observation, together with the fact that no changes occurred in the heart rate of rats, suggests that a direct cholinomimetic activity for 3-HPAA is unlikely. Of note, there is a large homology in the muscarinic receptors described among mammals: M1 to Ms in rats, and M1 to M in pigs, have revealed a homology greater than 90% with the human amino acid sequences of these receptors [41]. Last but not least, in addition to NO, endothelial vasodilation can also be mediated by other endothelial products, among them a mediator from cyclooxygenase-pathway prostacyclin (PGI2). This is not probable in the case of 3-HPAA because the presence of indomethacin, a cyclooxygenase blocker, had no effect.

Previously, we studied another important colonic metabolite, DHPA, on the rat aorta ex vivo. Its vasodilatory effect was also partially dependent on endothelium, but with the involvement of endothelial IKc。channels and COX, thus Cabling dependent [15]. This would imply that vasodilatory colonic metabolites of flavonoids may act through different mechanisms of action. In real conditions, the presence of different mechanisms and the interplay of several colonic metabolites might facilitate vasodilation. This hypothesis is in accordance with our in vivo study on mixtures of colonic metabolites [42]. Interestingly, some studies evidenced that the parent flavonoid, quercetin, is also vasoactive. It activates eNOS, and this action is mediated by an increase in cytosolic Ca2, activating thereafter the Ca2+-activated K channels, mainly SKca, and causing hyperpolarization of endothelial cells[43,44]. Quercetin, hence, acts in a different way. However, the bioavailability of parent quercetin is low [45,46] and, thus, its direct impact on vasodilation is probably not crucial.

The research published in this paper has several limitations. For instance, the porcine coronary artery is not a resistance vessel, while the arterial blood pressure-decreasing effect is likely associated with the dilation of resistance vessels. An i.v.administration of a single bolus or even slow i.v. infusion does not mimic a real exposure scenario in which humans ingest multiple doses of flavonoids, mainly in the form of glycosides, throughout the day. Thus, the plasma profile of metabolites may differ. Importantly, the flavonoids ingested are metabolized not into one but into a mixture of metabolites. Many of them may be vasoactive and the interplay among them influences the final effect. Furthermore, i.v.application does not allow for assessment of the role of the intestinal microbiota and variability in the production of 3-HPAA and other metabolites from parent flavonoids. Future studies should try to address these issues to better understand the bioactivities and the mechanism of action of 3-HPAA in the cardiovascular system, including the possible role of NO.

5. Conclusions

The data provided strong evidence that the flavonoid metabolite, 3-HPAA, formed by the human gut microbiota, is vasoactive and decreases blood pressure. In addition, the results suggest that a decrease in blood pressure can be attained at achievable concentrations. Along with these findings, we demonstrated that the hypotensive effect was not the result of direct action on the heart but was more likely vascular-based. Finally, the 3-HPAA-induced vasodilation was, at least partially, mediated by endothelium, where NO-dependent effects might play a role.

Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nul4020328/s1. Figure S1.Changes in mean blood pressure after bolus i.v.administration of 3-HPAA. Figure S2. Changes in heart rate after bolus i.v. administration of 3-HPAA.Figure S3. Effect of 3-HPAA infusions(0.05,0.25,1 and 5 mg.kg-1min-1) on systolic, diastolic and mean blood pressures in spontaneously hypertensive rats. Figure S4. Effect of 3-HPAA infusions (0.05, 0.25,1 and 5 mg.kg-1.min-l) on heart rate in spontaneously hypertensive rats.

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