Correlations among different platelet aggregation pathways in a group of healthy volunteers.

Platelet aggregation is a complicated process mediated by different signaling pathways. As the process is highly complex and apparently redundant, the relationships between these pathways are not yet fully known. The aim of this project was to study the interconnections among seven different aggregation pathways in a group of 53 generally healthy volunteers aged 20 to 66 years. Platelet aggregation was induced with thrombin receptor activating peptide 6 (TRAP), arachidonic acid (AA), platelet activating factor 16 (PAF), ADP, collagen, thromboxane A2 analogue U46619 or ristocetin (platelet agglutination) ex vivo in fasting blood samples according to standardized timetable protocol. Additionally, some samples were pre-treated with known clinically used antiplatelet drugs (vorapaxar, ticagrelor or acetylsalicylic acid (ASA)). Significant correlations among all used inducers were detected (Pearson correlation coefficients (rP): 0.3 to 0.85). Of all the triggers, AA showed to be the best predictor of the response to other inducers with rP ranging from 0.66 to 0.85. Interestingly, the antiplatelet response to ticagrelor strongly predicted the response to unrelated drug vorapaxar (rP = 0.71). Our results indicate that a response to one inducer can predict the response for other triggers or even to an antiplatelet drug. These data are useful for future testing but should be also confirmed in patients.

What is the context?• Platelet activation is a complicated process with multiple signaling cascades involved.• A total of seven common platelet triggers (ADP, collagen, TRAP-6, PAF, arachidonic acid/AA/, ristocetin and U46619) were tested.• The process is dependent on many factors including sex, age, concomitant disease(s), pharmacotherapy.What is new?• There were significant correlations between all tested aggregatory cascades.• AA has the highest rate of response predictability in our heterogeneous generally healthy volunteer group.• There was no correlation between impedance aggregometry in whole blood and turbidimetric measurement with platelet-rich plasma.What is the impact?• The effect of antiplatelet drugs can be assessed from the reaction to different trigger(s) at least in this group of healthy patients.• Future studies must test these relationships in patients with different diseases.

Analysis of vitamin K1 and major K2 variants in rat/human serum and lipoprotein fractions by a rapid, simple, and sensitive UHPLC-MS/MS method.

Determination of the various forms of vitamin K, which are involved in coagulation and other physiological processes in humans, is challenging and no standardized method is yet available. Therefore, a reliable and practical method was developed to quantify vitamin K levels in serum and additionally in lipoprotein fractions to clarify its distribution. The LC-MS/MS method for the determination of vitamin K1 and the three main isoforms of vitamin K2 (MK-4, MK-7, MK-9) was combined with a gradient ultracentrifugation technique to allow the separation of lipoprotein fractions. The chromatographic separation was carried out on a Kinetex™ C18 column using a mobile phase consisting mainly of methanol. The target analytes were detected by electrospray ionization mass spectrometry. The separation of all four substances was achieved after a simple sample preparation technique based on miniaturized liquid-liquid extraction. Our method of only 8.5 min revealed the levels of the major forms of vitamin K in 59 human and 12 rat sera and confirmed our hypothesis that vitamin K is primarily (about 50 %) found in the high-density lipoprotein fraction. The median concentrations of vitamin K1, MK-4, MK-7, and MK-9 were found to be 1.19, 2.98, 0.43, and < 0.71 nmol/L in human serum and 1.74, 6.75, less than 0.2, and less than 0.5 nmol/L in rat serum, respectively.

Head-to-head ex vivo comparison of clinically used direct anticoagulant drugs.

An imbalance in coagulation is associated with cardiovascular events. For prevention and treatment, anticoagulants, currently mainly xabans and gatrans, are used. The purpose of the present study was to provide a head-to-head comparison since there are no studies directly evaluating these novel anticoagulants. An additional aim was to find whether selected anthropological and biochemical factors can affect their anticoagulant properties as they are used in fixed doses. In this cross-sectional study, blood from 50 generally healthy donors was collected, and coagulation responses to dabigatran, argatroban, rivaroxaban, and apixaban, at a concentration of 1 µM, were analyzed. Heparin was used as a positive control. Prothrombin time (PT) expressed as international normalized ratio (INR) and activated partial thromboplastin time (aPTT) were measured and compared. Rivaroxaban was the most active according to PT/INR while argatroban according to aPTT. The ex vivo anticoagulant effect measured by INR correlated inversely with body mass index (BMI) in all four anticoagulants tested. Shortening of aPTT was associated with higher cholesterol and triglyceride levels. No sex-related differences were observed in response to the anticoagulant treatments. As this was an ex vivo study and pharmacokinetic factors were not included, the influence of BMI is of high therapeutic importance.

Pharmacokinetic and pharmacodynamic herb-drug interactions-part I. Herbal medicines of the central nervous system.

Unlike conventional drug substances, herbal medicines are composed of a complex of biologically active compounds. Therefore, the potential occurrence of herb-drug interactions is even more probable than for drug-drug interactions. Interactions can occur on both the pharmacokinetic and pharmacodynamic level. Herbal medicines may affect the resulting efficacy of the concomitantly used (synthetic) drugs, mainly on the pharmacokinetic level, by changing their absorption, distribution, metabolism, and excretion. Studies on the pharmacodynamic interactions of herbal medicines and conventional drugs are still very limited. This interaction level is related to the mechanism of action of different plant constituents. Herb-drug interactions can cause changes in drug levels and activities and lead to therapeutic failure and/or side effects (sometimes toxicities, even fatal). This review aims to provide a summary of recent information on the potential drug interactions involving commonly used herbal medicines that affect the central nervous system (Camellia, Valeriana, Ginkgo, Hypericum, Humulus, Cannabis) and conventional drugs. The survey databases were used to identify primary scientific publications, case reports, and secondary databases on interactions were used later on as well. Search keywords were based on plant names (botanical genera), officinal herbal drugs, herbal drug preparations, herbal drug extracts.

Proposed mechanisms of action of herbal drugs and their biologically active constituents in the treatment of coughs: an overview.

Various medicinal plants find their use in cough treatment, based on traditions and long-term experience. Pharmacological principles of their action, however, are much less known. Herbal drugs usually contain a mixture of potentially active compounds, which can manifest diverse effects. Expectorant or antitussive effects, which can be accompanied by others, such as anti-inflammatory or antibacterial, are probably the most important in the treatment of coughs. The aim of this review is to summarize the current state of knowledge of the effects of medicinal plants or their constituents on cough, based on reliable pharmacological studies. First, a comprehensive description of each effect is provided in order to explain the possible mechanism of action in detail. Next, the results related to individual plants and substances are summarized and critically discussed based on pharmacological in vivo and in vitro investigation.

3-Hydroxyflavone is a mildly active and safe cobalt chelator while cobalt markedly enhances baicalein toxicity toward erythrocytes.

Cobalt intoxication can occur after its release from metal-based prostheses, which is generally clinically severe. Therefore, there is a need for the development of a cobalt chelator since there are currently no approved drugs for cobalt intoxication. As flavonoids are known for their metal chelating properties and safety, the screening of cobalt chelating properties was performed in a total of 23 flavonoids by our recently developed new spectrophotometric assay. Further assessment of positive or negative consequences of cobalt chelation was performed both in vitro and ex vivo. Six and thirteen flavonoids significantly chelated cobalt ions at pH 7.5 and 6.8, respectively. Baicalein demonstrated a significant activity even at pH 5.5; however, none of the flavonoids showed chelation at pH 4.5. In general, baicalein and 3-hydroxyflavone were the most active. They also mildly decreased the cobalt-triggered Fenton reaction, but baicalein toxicity toward red blood cells was strongly increased by the addition of cobalt. Quercetin, tested as an example of flavonoid unable to chelate cobalt ions significantly, stimulated both the cobalt-based Fenton reaction and the lysis of erythrocytes in the presence of cobalt. Therefore, 3-hydroxyflavone can serve as a potential template for the development of novel cobalt chelators.

The Impact of Convertase Subtilisin/Kexin Type 9 Monoclonal Antibodies with and without Apheresis on Platelet Aggregation in Familial Hypercholesterolemia.

BACKGROUND AND AIMS: It is well known that elevated cholesterol is associated with enhanced platelet aggregation and patients suffering from familial hypercholesterolemia (FH) have a high risk of thrombotic cardiovascular events. Although decreasing cholesterol level is associated with attenuation of platelet hyperactivity, there are currently no data on the effect of convertase subtilisin/kexin type 9 monoclonal antibodies (PCSK9ab) on platelet reactivity in FH. The aim of the study was to analyse the impact of different therapies including PCSK9ab on platelet aggregation in FH. METHODS: This study enrolled all 15 patients treated in the University Hospital Hradec Králové for FH. PCSK9ab have been administered in 12 of 15 patients while 8 patients were also undergoing lipid apheresis. Blood samples from all patients including pre- and post-apheresis period were tested for platelet aggregation triggered by 7 inducers, and the effect of 3 clinically used drugs (acetylsalicylic acid, ticagrelor and vorapaxar) was compared as well. RESULTS: Although apheresis decreased the reactivity of platelets in general, platelet responses were not different between non-apheresis patients treated with PCSK9ab and apheresis patients (post-apheresis values) with the exception of ristocetin. However, when compared to age-matched healthy population, FH patients had significantly lower platelet aggregation responses to 4 out of 7 used inducers and higher profit from 2 out of 3 used antiplatelet drugs even after exclusion of FH patients regularly receiving conventional antiplatelet treatment. CONCLUSION: This study showed for the first time the suitability of PCSK9ab treatment for reduction of platelet reactivity in FH patients.

The Effect of 4-Methylcatechol on Platelets in Familial Hypercholesterolemic Patients Treated with Lipid Apheresis and/or Proprotein Convertase Subtilisin Kexin 9 Monoclonal Antibodies.

Elevated low-density lipoprotein (LDL) cholesterol levels lead to atherosclerosis and platelet hyperaggregability, both of which are known culprits of arterial thrombosis. Normalization of LDL cholesterol in familial hypercholesterolemia (FH) is not an easy task and frequently requires specific treatment, such as regularly performed lipid apheresis and/or novel drugs such as proprotein convertase subtilisin kexin 9 monoclonal antibodies (PCSK9Ab). Moreover, a high resistance rate to the first-line antiplatelet drug acetylsalicylic acid (ASA) stimulated research of novel antiplatelet drugs. 4-methylcatechol (4-MC), a known metabolite of several dietary flavonoids, may be a suitable candidate. The aim of this study was to analyse the antiplatelet effect of 4-MC in FH patients and to compare its impact on two FH treatment modalities via whole-blood impedance aggregometry. When compared to age-matched, generally healthy controls, the antiplatelet effect of 4-MC against collagen-induced aggregation was higher in FH patients. Apheresis itself improved the effect of 4-MC on platelet aggregation and blood from patients treated with this procedure and pretreated with 4-MC had lower platelet aggregability when compared to those solely treated with PCKS9Ab. Although this study had some inherent limitations, e.g., a low number of patients and possible impact of administered drugs, it confirmed the suitability of 4-MC as a promising antiplatelet agent and also demonstrated the effect of 4-MC in patients with a genetic metabolic disease for the first time.

Toxicity overview of endocrine disrupting chemicals interacting in vitro with the oestrogen receptor.

The oestrogen receptor (ER) from the nuclear receptor family is involved in different physiological processes, which can be affected by multiple xenobiotics. Some of these compounds, such as bisphenols, pesticides, and phthalates, are widespread as consequence of human activities and are commonly present also in human organism. Xenobiotics able to interact with ER and trigger a hormone-like response, are known as endocrine disruptors. In this review, we aim to summarize the available knowledge on products derived from human industrial activity and other xenobiotics reported to interact with ER. ER-disrupting chemicals behave differently towards oestrogen-dependent cell lines than endogenous oestradiol. In low concentrations, they stimulate proliferation, whereas at higher concentrations, are toxic to cells. In addition, most of the knowledge on the topic is based on individual compound testing, and only a few studies assess xenobiotic combinations, which better resemble real circumstances. Confirmation from in vivo models is lacking also.

SC-560 and mofezolac isosteres as new potent COX-1 selective inhibitors with antiplatelet effect.

Selective cyclooxygenase (COX)-1 inhibitors can be employed as potential cardioprotective drugs. Moreover, COX-1 plays a key role in inflammatory processes and its activity is associated with some types of cancer. In this work, we designed and synthesized a set of compounds that structurally mimic the selective COX-1 inhibitors, SC-560 and mofezolac, the central cores of which were replaced either with triazole or benzene rings. The advantage of this approach is a relatively simple synthesis in comparison with the syntheses of parent compounds. The newly synthesized compounds exhibited remarkable activity and selectivity toward COX-1 in the enzymatic in vitro assay. The most potent compound, 10a (IC50 = 3 nM for COX-1 and 850 nM for COX-2), was as active as SC-560 (IC50  = 2.4 nM for COX-1 and 470 nM for COX-2) toward COX-1 and it was even more selective. The in vitro COX-1 enzymatic activity was further confirmed in the cell-based whole-blood antiplatelet assay, where three out of four selected compounds (10a,c,d, and 3b) exerted outstanding IC50 values in the nanomolar range (9-252 nM). Moreover, docking simulations were performed to reveal key interactions within the COX-1 binding pocket. Furthermore, the toxicity of the selected compounds was tested using the normal human kidney HK-2 cell line.

Sex-Related Differences in Platelet Aggregation: A Literature Review Supplemented with Local Data from a Group of Generally Healthy Individuals.

The process of platelet aggregation is often influenced by several factors including sex and age. A literature review confirmed the existence of sex-related differences in platelet aggregation. Although 68 out of 78 papers found such differences, there are still some controversies regarding these differences, which can be due to multiple factors (age, trigger, concomitant disease, sample handling, etc.). These outcomes are discussed in line with novel results obtained from a local study, in which blood samples from a total of 53 overall healthy women and men with ages ranging from 20 to 66 years were collected. Aggregation was induced with seven different triggers (ristocetin, thrombin receptor activating peptide 6 [TRAP-6], arachidonic acid [AA], platelet-activating factor 16 [PAF-16], ADP, collagen, or thromboxane A2 analog U-46619) ex vivo. In addition, three FDA-approved antiplatelet drugs (vorapaxar, ticagrelor, or acetylsalicylic acid [ASA]) were also tested. In general, women had higher aggregation responses to some agonists (ADP, TRAP), as well as lower benefit from inhibitors (ASA, vorapaxar). The aggregatory responses to AA and TRAP decreased with age in both sexes, while responses to ADP, U-46619, and PAF were affected by age only in women. In conclusion, more studies are needed to decipher the biological importance of sex-related differences in platelet aggregation in part to enable personalized antiplatelet treatment.

A Complex Methodological Approach for the Screening of Efficient and Safe Cobalt Chelators.

BACKGROUND: Cobalt is an essential trace element, but it can also rarely cause cobalt toxicity due to its release from cobalt-containing medical devices. Currently, there are no approved selective cobalt chelators, which would represent an optimal treatment modality. OBJECTIVE: This study aimed to develop a simple and complex methodological approach for screening potential cobalt chelators and evaluating their potential toxicity. METHODS: Firstly, a simple spectrophotometric assay employing 1-nitroso-2-naphthol-3,6- disulfonic acid disodium salt (NNDSA) for screening cobalt chelation was standardized at a pathophysiologically relevant range of pH 4.5-7.5. Then, the suitability of the method was verified using four known metal chelators (EDTA, 8-hydroxyquinoline, chloroxine and nitroxoline). As cobalt can catalyse the Fenton reaction, the potential toxicity of cobalt-chelator complexes was also determined by employing a novel HPLC method with coulometric detection. The effect on erythrocyte haemolysis was tested as well. RESULTS: The NNDSA method had high sensitivity enabling the detection of 25-200 nM of cobalt ions depending on pH conditions. Measurements could be carried out in a wide range of wavelengths from 470 to 540 nm. All tested complexes of the selected chelators decreased the rate of the Fenton reaction. Interestingly, chloroxine mixed with cobalt ions caused marked lysis of erythrocytes in contrast to the other compounds. CONCLUSION: The described complex methodological approach could serve as a simple yet precise tool for evaluating novel, effective and safe cobalt chelators.

The effects of bisphenols on the cardiovascular system ex vivo and in vivo.

The human population is regularly exposed to bisphenols. The first compound of this class, bisphenol A, is burdened by numerous reports of its potential toxicity and has been hence replaced by its analogues, so-called next generation bisphenols. Their widespread use has made them pervasive throughout the environment. These endocrine disrupting chemicals can affect the cardiovascular system, and hence the aim of this study was to test 14 bisphenols (A, AF, AP, B, BP, C, E, F, G, M, P, PH, S and Z), and compare their effects in vitro (human and rat cell lines), ex vivo (isolated rat aorta) and in vivo (Wistar Han rats, acutely or chronically exposed to low environmental and high toxic doses). The majority of the tested bisphenols relaxed rat aorta, but their potency varied markedly. The most potent compound, bisphenol AF, had an EC50 of 57 µM. The mechanism of action was likely based on the inhibition of calcium influx via L-type calcium channels. The cytotoxicity of bisphenols towards 4 human and rat cell lines (H9c2, A-10, MCF7/S0.5 and MCF7/182R-6) showed variable potencies ranging from units of micromolar to millimolar concentrations. Based on these data, an effect on arterial blood pressure and possible cardiotoxicity was expected. Contrarily, the in vivo acute effects of three doses (0.005, 0.05 and 2.5 mg/kg) of bisphenol AF and 3 other analogues (A, S and F) on the cardiovascular system were rather biologically negligible. The most potent bisphenol, AF, was also administered chronically at a dose of 2.5 mg/kg for 4 weeks to rats, but had no impact on arterial blood pressure. Our results showed that bisphenols can relax vascular smooth muscles, but the effective concentrations are too high to produce clear cardiovascular effects in relation to common biological exposure as was confirmed with the most potent bisphenol AF.

Natural Sympathomimetic Drugs: From Pharmacology to Toxicology.

Sympathomimetic agents are a group of chemical compounds that are able to activate the sympathetic nervous system either directly via adrenergic receptors or indirectly by increasing endogenous catecholamine levels or mimicking their intracellular signaling pathways. Compounds from this group, both used therapeutically or abused, comprise endogenous catecholamines (such as adrenaline and noradrenaline), synthetic amines (e.g., isoproterenol and dobutamine), trace amines (e.g., tyramine, tryptamine, histamine and octopamine), illicit drugs (e.g., ephedrine, cathinone, and cocaine), or even caffeine and synephrine. In addition to the effects triggered by stimulation of the sympathetic system, the discovery of trace amine associated receptors (TAARs) in humans brought new insights about their sympathomimetic pharmacology and toxicology. Although synthetic sympathomimetic agents are mostly seen as toxic, natural sympathomimetic agents are considered more complacently in the terms of safety in the vision of the lay public. Here, we aim to discuss the pharmacological and mainly toxicological aspects related to sympathomimetic natural agents, in particular of trace amines, compounds derived from plants like ephedra and khat, and finally cocaine. The main purpose of this review is to give a scientific and updated view of those agents and serve as a reminder on the safety issues of natural sympathomimetic agents most used in the community.

The Antiplatelet Effect of 4-Methylcatechol in a Real Population Sample and Determination of the Mechanism of Action.

A polyphenol-rich diet has beneficial effects on cardiovascular health. However, dietary polyphenols generally have low bioavailability and reach low plasma concentrations. Small phenolic metabolites of these compounds formed by human microbiota are much more easily absorbable and could be responsible for this effect. One of these metabolites, 4-methylcatechol (4-MC), was suggested to be a potent anti-platelet compound. The effect of 4-MC was tested ex vivo in a group of 53 generally healthy donors using impedance blood aggregometry. The mechanism of action of this compound was also investigated by employing various aggregation inducers/inhibitors and a combination of aggregometry and enzyme linked immunosorbent assay (ELISA) methods. 4-MC was confirmed to be more potent than acetylsalicylic acid on both arachidonic acid and collagen-triggered platelet aggregation. Its clinically relevant effect was found even at a concentration of 10 µM. Mechanistic studies showed that 4-MC is able to block platelet aggregation caused by the stimulation of different pathways (receptors for the von Willebrand factor and platelet-activating factor, glycoprotein IIb/IIIa, protein kinase C, intracellular calcium elevation). The major mechanism was defined as interference with cyclooxygenase-thromboxane synthase coupling. This study confirmed the strong antiplatelet potential of 4-MC in a group of healthy donors and defined its mechanism of action.

Interactions of Isoquinoline Alkaloids with Transition Metals Iron and Copper.

Data on alkaloid interactions with the physiologically important transition metals, iron and copper, are mostly lacking in the literature. However, these interactions can have important consequences in the treatment of both Alzheimer's disease and cancer. As isoquinoline alkaloids include galanthamine, an approved drug for Alzheimer's disease, as well as some potentially useful compounds with cytostatic potential, 28 members from this category of alkaloids were selected for a complex screening of interactions with iron and copper at four pathophysiologically relevant pH and in non-buffered conditions (dimethyl sulfoxide) by spectrophotometric methods in vitro. With the exception of the salts, all the alkaloids were able to chelate ferrous and ferric ions in non-buffered conditions, but only five of them (galanthine, glaucine, corydine, corydaline and tetrahydropalmatine) evoked some significant chelation at pH 7.5 and only the first two were also active at pH 6.8. By contrast, none of the tested alkaloids chelated cuprous or cupric ions. All the alkaloids, with the exception of the protopines, significantly reduced the ferric and cupric ions, with stronger effects on the latter. These effects were mostly dependent on the number of free aromatic hydroxyls, but not other hydroxyl groups. The most potent reductant was boldine. As most of the alkaloids chelated and reduced the ferric ions, additional experimental studies are needed to elucidate the biological relevance of these results, as chelation is expected to block reactive oxygen species formation, while reduction could have the opposite effect.

Can Isoquinoline Alkaloids Affect Platelet Aggregation in Whole Human Blood?

Isoquinoline alkaloids have multiple biological activities, which might be associated with positive pharmacological effects as well as negative adverse reactions. As bleeding was suggested to be a side effect of the isoquinoline alkaloid berberine, we decided to ascertain if different isoquinoline alkaloids could influence hemocoagulation through the inhibition of either platelet aggregation or blood coagulation. Initially, a total of 14 compounds were screened for antiplatelet activity in whole human blood by impedance aggregometry. Eight of them demonstrated an antiplatelet effect against arachidonic acid-induced aggregation. Papaverine and bulbocapnine were the most potent compounds with biologically relevant IC50 values of 26.9 ± 12.2 µM and 30.7 ± 5.4 µM, respectively. Further testing with the same approach confirmed their antiplatelet effects by employing the most physiologically relevant inducer of platelet aggregation, collagen, and demonstrated that bulbocapnine acted at the level of thromboxane receptors. None of the alkaloids tested had an effect on blood coagulation measured by a mechanical coagulometer. In conclusion, the observed antiplatelet effects of isoquinoline alkaloids were found mostly at quite high concentrations, which means that their clinical impact is most likely low. Bulbocapnine was an exception. It proved to be a promising antiplatelet molecule, which may have biologically relevant effects.

The effect of flavonoids on the reduction of cupric ions, the copper-driven Fenton reaction and copper-triggered haemolysis.

Flavonoids are considered beneficial, but they may exhibit pro-oxidative effects likely due to metal reducing properties. For the first time, 24 structurally related flavonoids were compared for copper reduction, and modulation of the copper-triggered Fenton reaction and lysis of erythrocytes. The vast majority of flavonoids reduced cupric ions; their behaviour ranged from progressive gradual reduction through bell-shaped, neutral, to a blockade of spontaneous reduction. Similarly, different behaviours were observed with the Fenton reaction. Flavone was the only flavonoid that potentiated copper-triggered haemolysis (155 ± 81 % at twice the amount of Cu2+), while 18 flavonoids were at least partly protective in some concentrations. Only 5-hydroxyflavone did not reduce Cu2+ and behaved as an antioxidant in both assays (reduction of 60 ± 10 % and 88 ± 1%, respectively, at an equimolar ratio with Cu2+). In conclusion, relatively subtle structural differences resulted in very different anti/prooxidant behaviour depending on the model.

Interaction of Flavonoids with Zinc and Zinc-Containing Enzymes.

The current chelation therapy has several drawbacks, including lack of selectivity, which could lead to trace metal depletion. Consequently, the proper function of metalloenzymes can be disrupted. Flavonoids possess chelating properties and hence interfere with the homeostasis of essential metals. We focused on zinc, an important trace metal required for the function of many enzymes and transcription factors. After making an initial evaluation of the Zn2+-chelating properties of a series of flavonoids, the effect of these compounds on various zinc-containing enzymes was also investigated. We performed enzyme inhibition assays spectrophotometrically using yeast and equine alcohol dehydrogenases and bovine glutamate dehydrogenase. Nine of the 21 flavonoids tested were capable of chelating Zn2+. Baicalein and 3-hydroxyflavone were the most potent Zn2+ chelators under slightly acidic and neutral pH conditions. This chelation was also confirmed by the ability to reverse Zn2+-induced enzymatic inhibition of bovine glutamate dehydrogenase. Although some flavonoids were also able to inhibit zinc-containing alcohol dehydrogenases, this inhibition was likely not caused by Zn2+ chelation. Luteolin was a relatively potent inhibitor of these enzymes regardless of the presence of Zn2+. Docking studies confirmed the binding of active flavonoids to equine alcohol dehydrogenase without any significant interaction with the catalytic zinc.

Vitamin D: sources, physiological role, biokinetics, deficiency, therapeutic use, toxicity, and overview of analytical methods for detection of vitamin D and its metabolites.

Vitamin D has a well-known role in the calcium homeostasis associated with the maintenance of healthy bones. It increases the efficiency of the intestinal absorption of dietary calcium, reduces calcium losses in urine, and mobilizes calcium stored in the skeleton. However, vitamin D receptors are present ubiquitously in the human body and indeed, vitamin D has a plethora of non-calcemic functions. In contrast to most vitamins, sufficient vitamin D can be synthesized in human skin. However, its production can be markedly decreased due to factors such as clothing, sunscreens, intentional avoidance of the direct sunlight, or the high latitude of the residence. Indeed, more than one billion people worldwide are vitamin D deficient, and the deficiency is frequently undiagnosed. The chronic deficiency is not only associated with rickets/osteomalacia/osteoporosis but it is also linked to a higher risk of hypertension, type 1 diabetes, multiple sclerosis, or cancer. Supplementation of vitamin D may be hence beneficial, but the intake of vitamin D should be under the supervision of health professionals because overdosing leads to intoxication with severe health consequences. For monitoring vitamin D, several analytical methods are employed, and their advantages and disadvantages are discussed in detail in this review.