Intravenous rutin in rat exacerbates isoprenaline-induced cardiotoxicity likely due to intracellular oxidative stress.

OBJECTIVES: Rutin, quercetin-3-O-rutinoside, a natural flavonol glycoside, has shown various in vitro benefits with potential use treating human diseases, especially cardiovascular system disorders. Antioxidant properties are assumed to underlie the majority of these benefits. Yet rutin pro-oxidant properties have been reported as well. Our research group has recently shown aggravating effects on isoprenaline (ISO)-induced cardiotoxicity in Wistar:Han rats after 24 hours. METHODS: This study was designed to examine in more detail the reasons for the negative effects of rutin (11.5 and 46 mg/kg, i.v.) after administration of ISO (100 mg/kg, s.c.) in rats within 2 hours of continuous experiment and in the H9c2 cardiomyoblast-derived cell line. RESULTS: Like our previous findings, rutin did not (11.5 or 46 mg/kg, i.v.) reduce the ISO-induced mortality within 2 hours although the lower dose significantly reduced cardiac troponin T (cTnT) and partly improved the histological findings. In contrast, the higher dose increased the mortality in comparison with solvent (1.26% w/v sodium bicarbonate). This was not caused by any specific haemodynamic disturbances. It appears to be associated with oxidative stress as rutin enhanced intracellular reactive oxygen species formation in vitro and had the tendency to increase it in vivo. CONCLUSIONS: Rutin, likely due to its pro-oxidative effects, can exacerbate catecholamine cardiotoxicity depending on the dose used.

Antiplatelet Effects of Flavonoids Mediated by Inhibition of Arachidonic Acid Based Pathway.

Flavonoids, important components of human diet, have been claimed to possess a significant antiplatelet potential, in particular due to their effects on the arachidonic acid cascade. Due to variable and incomplete results, this study was aimed at delivering a detailed analysis of the effects of 29 structurally relevant, mainly natural flavonoids on three consecutive steps of the arachidonic acid cascade.Only the isoflavonoids genistein and daidzein were shown to possess a marked cyclooxygenase-1 inhibitory activity, which was higher than that of acetylsalicylic acid using the isolated ovine enzyme, and physiologically relevant, although lower than acetylsalicylic acid in human platelets. None of the tested flavonoids possesses an effect on thromboxane synthase in a clinically achievable concentration. Contrarily, many flavonoids, particularly those possessing an isolated 7-hydroxyl group and/or a 4'-hydroxyl group, acted as antagonists on thromboxane receptors. Interestingly, the substitution of the free 7-hydroxyl group by glucose might not abolish the activity.In conclusion, the consumption of few flavonoids in a diet, particularly of the isoflavonoids genistein and daidzein, may positively influence platelet aggregation.

Isoflavones Reduce Copper with Minimal Impact on Iron In Vitro.

Isoflavones are commonly consumed in many Asian countries and have potentially positive effects on human being. Only a few and rather controversial data on their interactions with copper and iron are available to date. 13 structurally related isoflavones were tested in the competitive manner for their Cu/Fe-chelating/reducing properties. Notwithstanding the 5-hydroxy-4-keto chelation site was associated with ferric, ferrous, and cupric chelation, the chelation potential of isoflavones was low and no cuprous chelation was observed. None of isoflavones was able to substantially reduce ferric ions, but the vast majority reduced cupric ions. The most important feature for cupric reduction was the presence of an unsubstituted 4'-hydroxyl; contrarily the presence of a free 5-hydroxyl decreased or abolished the reduction due to chelation of cupric ions. The results from this study may enable additional experiments which might clarify the effects of isoflavones on human being and/or mechanisms of copper absorption.

Cardiovascular effects of coumarins besides their antioxidant activity.

Coumarins are a large group of substances, primarily of plant origin. Like their more intensively examined congeners flavonoids, many of them are antioxidants. Although such properties may be advantageous in cardiovascular diseases, it has been shown that coumarins exhibit direct effects on the cardiovascular system which are not based on antioxidant activity. The most common example is the well-known drug warfarin, a synthetic compound derived from natural dicoumarol. Moreover, other coumarins have been shown to possess antiplatelet and vasodilatory potential. Interestingly, the former effect may be mediated by the inhibition of various pathways leading to platelet aggregation, their differing effects on those pathways being due to structural differences between the various coumarins. Conversely, their vasodilatory potential is linked in the majority of cases to the inhibition of increases in intracellular calcium concentration in vascular smooth muscle cells, and in several coumarins also to NO-mediated vasodilatation. Available data on both activities are summarized in this review. At the end of this review, relevant data are provided from a few studies testing the in vivo effects of coumarins on major cardiovascular diseases; the clinical use of warfarin and other coumarin anticoagulants, as well as the limited data on the clinical use of coumarins in chronic venous insufficiency and the possible toxicological effects of coumarins.

Is a highly linear relationship between the dose of quercetin and the pharmacological effect possible? - a comment on Liu, et al. Evaluation of antioxidant and immunity activities of quercetin in isoproterenol-treated rats. Molecules 2012, 17, 4281-4291.

We wish to offer some comments on the article by H. Liu et al. entitled "Evaluation of antioxidant and immunity activities of quercetin in isoproterenol-treated rats", published in Molecules in 2012 [1]. [...].

Oral administration of quercetin is unable to protect against isoproterenol cardiotoxicity.

Catecholamines are endogenous amines that participate in the maintenance of cardiovascular system homeostasis. However, excessive release or exogenous administration of catecholamines is cardiotoxic. The synthetic catecholamine, isoprenaline (isoproterenol, ISO), with non-selective ß-agonistic activity has been used as a viable model of acute myocardial toxicity for many years. Since the pathophysiology of ISO-cardiotoxicity is complex, the aim of this study was to elucidate the effect of oral quercetin pretreatment on myocardial ISO toxicity. Wistar-Han rats were randomly divided into four groups: solvent or quercetin administered orally by gavage in a dose of 10 mg kg(-1) daily for 7 days were followed by s.c. water for injection or ISO in a dose of 100 mg kg(-1). Haemodynamic, ECG and biochemical parameters were measured; effects on blood vessels and myocardial histology were assessed, and accompanying pharmacokinetic analysis was performed. Quercetin was unable to protect the cardiovascular system against acute ISO cardiotoxicity (stroke volume decrease, cardiac troponin T release, QRS-T junction elevation and histological impairment). The sole positive effect of quercetin on catecholamine-induced cardiotoxicity was the normalization of increased left ventricular end-diastolic pressure caused by ISO. Quercetin did not reverse the increased responsiveness of rat aorta to vasoconstriction in ISO-treated animals, but it decreased the same parameter in the control animals. Accompanying pharmacokinetic analysis showed absorption of quercetin and its metabolite 3-hydroxyphenylacetic acid formed by bacterial microflora. In conclusion, a daily oral dose of 10 mg kg(-1) of quercetin for 7 days did not ameliorate acute ISO-cardiovascular toxicity in rats despite minor positive cardiovascular effects.

Effect of novel 1-phenyl-3-methyl-4-acylpyrazolones on iron chelation and Fenton reaction.

Iron is an essential element in many physiological processes due to its ability to easily convert between two oxidation states Fe(III)/Fe(II). However, at a pathological state, unbound iron may promote the production of highly toxic hydroxyl radicals via Fenton reaction, particularly when it is present in the excess.Iron chelators forming tight complexes with iron may prevent this reaction. In this study, novel synthetic 1-phenyl-3-methyl-4-acyl-pyrazol-5-ones were analyzed for their iron-chelating properties at four pathophysiologically relevant pH conditions (4.5-7.5) as well as for their effects on iron-based Fenton reaction. For the former competitive ferrozine spectrophotometric assay and for the latter HPLC method using salicylic acid as the indicator of hydroxyl radical production were used. All of the tested acylpyrazolones were efficient ferric chelators, however, their ferrous-chelating properties were clearly dependent on an acyl substitution. Interestingly, several acylpyrazolones had ferrous-chelating properties superior to those of the standard iron chelator - deferoxamine. Of particular interest is H2QpyQ, i.e. 2,6-bis[4(1-phenyl-3-methylpyrazol-5-one)carbonyl]pyridine, whose ferrous-chelating properties were increasing while pH was decreasing. In spite of large differences in ferrous chelation, a majority of the tested acylpyrazolones were powerful inhibitors of Fenton reaction as deferoxamine. In conclusion, the novel 1-phenyl-3-methyl-4-acyl-pyrazol-5-ones are efficient iron chelators and H2QpyQ may represent a prototype of specific iron chelators designed for chelation at acidic conditions in particular.

The relationship of oxidative stress markers and parameters of myocardial function in a rat model of cardiotoxicity.

Although a majority of studies related oxidative stress to cardiovascular diseases, the pathophysiological relevance has been remaining unknown. The aim of this study was to establish the relationship among different commonly used biomarkers of oxidative stress and cardiovascular dys/function in rats. A pathological state in many aspects similar to that of acute myocardial infarction was induced by administration of isoprenaline (100mg.kg(-1), s.c.) in Wistar:Han rats. Haemodynamic, biochemical and ECG parameters were measured in two sets of experiments: after 24hours and continuously during the first 2hours following the administration of isoprenaline. Serum cardiac troponin T (cTnT) correlated strongly with cardiac function, myocardial calcium levels, wet ventricles weight and relevant ECG parameters (T wave, R wave and J - junction - point amplitudes). However, only weak negative correlations were found for cTnT and total blood glutathione or serum vitamin C concentrations, while no significant associations were found with serum vitamin E and plasma TBARS. Although the oxidized form of glutathione correlated positively with heart rate, no correlation with the above-mentioned ECG parameters was found. However, correlations of in 8-isoprostane with both R wave and J-junction-point amplitudes were observed. Conclusively, more selective markers of oxidative stress may predict the functional status of the heart.

In vitro copper-chelating properties of flavonoids.

Copper is an indispensable trace element for human body and the association between a disruption of copper homeostasis and a series of pathological states has been well documented. Flavonoids influence the human health in a complex way and the chelation of transient metal ions indisputably contributes to their mechanism of the action, however, the information about their copper-chelating properties have been sparse. This in vitro study was thus aimed at the detailed examination of flavonoids-copper interactions by two spectrophotometric assays at four (patho)physiologically relevant pH conditions (4.5-7.5), with the emphasis on the structure-activity relationship. The tested flavonoids were compared with the clinically used copper chelator, trientine. Most of the 26 flavonoids chelated copper ions, however, in a variable extent. Only flavones and flavonols were able to form stable complexes with both cupric and cuprous ions. The 3-hydroxy-4-keto group and 5,6,7-trihydroxyl group represented the most efficient chelation sites in flavonols and flavones, respectively, and the 2,3-double bond was essential for the stable copper chelation. Interestingly, the 3´,4´-dihydroxyl (catechol) group was associated only with a weak activity. Although none of the tested flavonoids were more potent than trientine at physiological or slightly acidic conditions, 3-hydroxyflavone, kaempferol and partly baicalein surpassed trientine at acidic conditions. Conclusively, flavonoids containing appropriate structural features were efficient copper chelators and some of them were even more potent than trientine under acidic conditions.

Mathematical calculations of iron complex stoichiometry by direct UV-Vis spectrophotometry.

The effects of iron-chelating agents on miscellaneous pathologies are currently largely tested. Due to various indications, different properties for chelators are required. A stoichiometry of the complex in relation to pH is one of the crucial factors. Moreover, the published data on the stoichiometry, especially concerning flavonoids, are equivocal. In this study, a new complementary approach was employed for the determination of stoichiometry in 10 iron-chelating agents, including clinically used drugs, by UV-Vis spectrophotometry at relevant pH conditions and compared with the standard Job's method. This study showed that the simple approach based on absorbance at the wavelength of complex absorption maximum was sufficient when the difference between absorption maximum of substance and complex was high. However, in majority of substances this difference was much lower (9-73 nm). The novel complementary approach was able to determine the stoichiometry in all tested cases. The major benefit of this method compared to the standard Job's approach seems to be its capability to reveal a reaction stoichiometry in chelators with moderate affinity to iron. In conclusion, using this complementary method may explain several previous contradictory data and lead to a better understanding of the underlying mechanisms of chelator's action.

Common biomarkers of oxidative stress do not reflect cardiovascular dys/function in rats.

BACKGROUND: Predicting cardiovascular events remains challenging despite the range of known biomarkers. AIM: To establish relationships between various biochemical and functional parameters of the cardiovascular system. METHOD: The relationship between cardiovascular dys/function and various biomarkers was examined in 145 experimental rats half of which received isoprenaline 100 mg/kg s.c. to induce cardiac impairment. RESULTS: Serum concentration of cardiac troponin T (cTnT), a known marker of cardiac derangement, correlated strongly with degree of myocardial injury (e.g. calcium overload, stroke volume) but correlations between cTnT and oxidative stress parameters were weak (for glutathione and vitamin C) or not found (for serum vitamin E and plasma thiobarbituric acid reactive substances levels). Relationships between cTnT and other parameters were exponential with the exception of myocardial calcium, where a power function was found. CONCLUSIONS: Commonly used biomarkers of oxidative stress cannot reliably predict cardiovascular dys/function in experimental rats.

Novel method for rapid copper chelation assessment confirmed low affinity of D-penicillamine for copper in comparison with trientine and 8-hydroxyquinolines.

Copper is an essential trace element involved in many physiological processes. Since disorder of copper homeostasis is observed in various pathologies, copper chelators may represent a promising therapeutic tool. This study was aimed at: 1) formation of an in vitro methodology for screening of copper chelators, and 2) detailed analysis of the interaction of copper with clinically used D-penicillamine (D-PEN), triethylenetetramine (trientine), experimentally tested 8-hydroxyquinolines, and the disodium salt of EDTA as a standard chelator. Methodology based on bathocuproinedisulfonic acid disodium salt (BCS), usable at (patho)physiologically relevant pHs (4.5-7.5), enabled assessment of both cuprous and cupric ions chelation and comparison of the relative affinities of the tested compounds for copper. In the case of potent chelators, the stoichiometry could be estimated too. Clioquinol, chloroxine and EDTA formed very stable complexes with Cu(+)/Cu(2+) at all tested pHs, while copper complexes with trientine were stable only under neutral or slightly acidic conditions. Non-substituted 8-hydroxyquinoline was a less efficient copper chelator, but still unequivocally more potent than D-PEN. Both 8-hydroxyquinoline and D-PEN chelation potencies, similarly to that of trientine, were pH-dependent and decreased with pH. Moreover, only D-PEN was able to reduce cupric ions. Conclusively, BCS assay represents a rapid, simple and precise method for copper chelation measurement. In addition, lower binding affinity of D-PEN compared with 8-hydroxyquinolines and trientine was demonstrated.

In vitro platelet antiaggregatory properties of 4-methylcoumarins.

Platelets play a crucial role in physiological haemostasis. However, in coronary arteries damaged by atherosclerosis, enhanced platelet aggregation, with subsequent thrombus formation, is a precipitating factor in acute myocardial infarction. Current therapeutic approaches are able to reduce approximately one quarter of cardiovascular events, but they are associated with an increased risk of bleeding and in some resistant patients are not efficient. Some coumarins possess antiplatelet activity and, due to their additional antioxidant effects, may be promising drugs for use in combination with the present therapeutic agents. The aim of this study was to analyse a series of simple 4-methylcoumarins for their antiplatelet activity. Human plasma platelet suspensions were treated with different aggregation inducers [arachidonic acid (AA), collagen and ADP] in the presence of the 4-methylcoumarins. Complementary experiments were performed to explain the mechanism of action. 5,7-Dihydroxy-4-methylcoumarins, in particular those containing a lipophilic side chain at C-3, reached the activity of acetylsalicylic acid on AA-induced aggregation. Other tested coumarins were less active. Some of the tested compounds mildly inhibited either collagen- or ADP-induced aggregation. 5,7-Dihydroxy-4-methylcoumarins did not interfere with the function of thromboxane synthase, but were competitive antagonists of thromboxane A(2) receptors and inhibited cyclooxygenase-1 as well. 5,7-Dihydroxy-4-methylcoumarins appear to be promising candidates for the extension of the current spectrum of antiplatelet drugs.

Iron reduction potentiates hydroxyl radical formation only in flavonols.

Flavonoids, substantial components of the human diet, are generally considered to be beneficial. However, they may possess possible pro-oxidative effects, which could be based on their reducing potential. The aims of this study were to evaluate the ability of 26 flavonoids to reduce ferric ions at relevant pH conditions and to find a possible relationship with potentiation of hydroxyl radical production. A substantial ferric ions reduction was achieved under acidic conditions, particularly by flavonols and flavanols with the catecholic ring B. Apparently corresponding bell-shaped curves displaying the pro-oxidant effect of flavonols quercetin and kaempferol on iron-based Fenton reaction were documented. Several flavonoids were efficient antioxidants at very low concentrations but rather inefficient or pro-oxidative at higher concentrations. Flavonols, morin and rutin were progressively pro-oxidant, while 7-hydroxyflavone and hesperetin were the only flavonoids with dose-dependent inhibition of hydroxyl radical production. Conclusively, administration of flavonoids may lead to unpredictable consequences with few exceptions.

Dexrazoxane provided moderate protection in a catecholamine model of severe cardiotoxicity.

Positive effects of dexrazoxane (DEX) in anthracycline cardiotoxicity have been mostly assumed to be associated with its iron-chelating properties. However, this explanation has been recently questioned. Iron plays also an important role in the catecholamine cardiotoxicity. Hence in this study, the influence of DEX on a catecholamine model of acute myocardial infarction (100 mg/kg of isoprenaline by subcutaneous injection) was assessed: (i) the effects of an intravenous dose of 20.4 mg/kg were analyzed after 24 h, (ii) the effects were monitored continuously during the first two hours after drug(s) administration to examine the mechanism(s) of cardioprotection. Additional in vitro experiments on iron chelation/reduction and influence on the Fenton chemistry were performed both with isoprenaline/DEX separately and in their combination. DEX partly decreased the mortality, reduced myocardial calcium overload, histological impairment, and peripheral haemodynamic disturbances 24 h after isoprenaline administration. Continuous 2 h experiments showed that DEX did not influence isoprenaline induced atrioventricular blocks and had little effect on the measured haemodynamic parameters. Its protective effects are probably mediated by inhibition of late myocardial impairment and ventricular fibrillation likely due to inhibition of myocardial calcium overload. Complementary in vitro experiments suggested that iron chelation properties of DEX apparently did not play the major role.

In vitro characteristics of 1-phenyl-3-methyl-4-acylpyrazol-5-ones iron chelators.

Iron chelators represent a group of structurally different compounds sharing the ability of iron binding. The group has been evolving in recent years mainly due to novel experimental indications associated with variable requirements for iron chelators. A group of synthetic 1-phenyl-3-methyl-4-acyl-pyrazol-5-ones has been known for many years but data on their potential biological activity are rather limited. In this study, we analysed a series of these compounds for their iron-chelating properties as well as for their effects on iron based Fenton chemistry. For the former ferrozine spectrophotometric method and for the latter HPLC method with salicylic acid were used. All of the tested compounds were very efficient ferric chelators but their ferrous-chelating effects differed according to the acyl substitution. Notwithstanding various ferrous chelation activities, the individual Fe(2+)-affinities were not significantly different through pathophysiologically relevant pH conditions and some of the tested substances were more potent ferrous chelators at pH 4.5 than clinically used standard deferoxamine. Of particular interest is H(2)QpyQ /2,6-bis[4(1-phenyl-3-methylpyrazol-5-one)carbonyl]pyridine/ which iron-chelating affinity increased when pH was decreasing. In spite of ferrous chelation differences, most of the tested acylpyrazolones were similarly active powerful inhibitors of Fenton chemistry as deferoxamine. Conclusively, acylpyrazolones are efficient iron chelators and H(2)QpyQ may represent a prototype of novel specific chelators designated particularly for chelation at acidic conditions.

In vitro analysis of iron chelating activity of flavonoids.

Flavonoids have been demonstrated to possess miscellaneous health benefits which are, at least partly, associated with iron chelation. In this in vitro study, 26 flavonoids from different subclasses were analyzed for their iron chelating activity and stability of the formed complexes in four patho/physiologically relevant pH conditions (4.5, 5.5, 6.8, and 7.5) and compared with clinically used iron chelator deferoxamine. The study demonstrated that the most effective iron binding site of flavonoids represents 6,7-dihydroxy structure. This site is incorporated in baicalein structure which formed, similarly to deferoxamine, the complexes with iron in the stoichiometry 1:1 and was not inferior in all tested pH to deferoxamine. The 3-hydroxy-4-keto conformation together with 2,3-double bond and the catecholic B ring were associated with a substantial iron chelation although the latter did not play an essential role at more acidic conditions. In agreement, quercetin and myricetin possessing all three structural requirements were similarly active to baicalein or deferoxamine at the neutral conditions, but were clearly less active in lower pH. The 5-hydroxy-4-keto site was less efficient and the complexes of iron in this site were not stable at the acidic conditions. Isolated keto, hydroxyl, methoxyl groups or an ortho methoxy-hydroxy groups were not associated with iron chelation at all.

Cardiovascular effects of flavonoids are not caused only by direct antioxidant activity.

Epidemiological, as well as most in vivo, studies suggest that flavonoids have a positive influence on various cardiovascular diseases. Traditionally, these effects were only attributed to their antioxidant activity, which has been extensively studied. Apart from the direct antioxidant properties, which include direct reactive oxygen species scavenging activity and transient metal chelation, this review reports on many other effects that in pharmacologically achievable concentrations may also be responsible for their positive cardiovascular influence. These include direct inhibition of some radical-forming enzymes (xanthine oxidase, NADPH oxidase, and lipoxygenases), decreased platelet aggregation and leukocyte adhesion, and vasodilatory properties. For each of the aforementioned effects different structural features are necessary. Briefly, a catecholic B-ring is necessary for scavenging activity; hydroxyl groups in an ortho position, the 3-hydroxy-4-keto group, or the 5-hydroxy-4-keto group enable iron chelation; planar conformation with the 4-keto group and 2,3-double bond is essential for inhibition of leukocyte adhesion and platelet aggregation; specific hydroxy-methoxy ortho conformation in ring B is necessary for the inhibition of NADPH oxidase; and the 4-keto group is a requisite for vasodilatory action. This review shows that positive cardiovascular effects of flavonoids are achieved by various flavonoids via the interaction with different targets.

In vitro interactions of coumarins with iron.

Coumarins are a large group of natural substances with diverse pharmacological properties that may predetermine some of them for the prevention and/or treatment of cardiovascular diseases and also other pathologies. Free iron participates in the production of reactive oxygen species (ROS) and plays an important role in the pathogenesis of cardiovascular diseases. Therefore, chelation of iron may attenuate some ROS consequences, but on the other hand, reduction of ferric ions to ferrous ones is unfavourable and leads to intensification of ROS production. In this study, we have examined the interaction of iron with coumarins which has been rarely analyzed. A series of naturally occurring and chemically synthesized 4-methylcoumarins were analyzed for their ferrous and total iron-chelating properties and compared with standard iron chelator deferoxamine. The iron chelation activity was assessed by a simple spectrophotometric approach based on the specific indicator for ferrous ions--ferrozine. The methodology was also extended for the measurement of total iron. Among the tested coumarins, ortho-dihydroxyderivatives were the most potent iron chelators and 7,8-dihydroxy-4-methylcoumarin even reached the efficiency of deferoxamine in neutral pH. However, these ortho-dihydroxycoumarins did not bind iron firmly in acidic conditions (e.g., in acute myocardial infarction) and, moreover, they reduced ferric ions that could lead to intensification of the Fenton chemistry. Other tested coumarins did not substantially chelate iron with the exception of ortho-diacetoxycoumarins. Conclusively, the use of iron-chelating coumarins in acidic conditions may be disadvantageous in contrast to neutral conditions.

Cardiac biomarkers in a model of acute catecholamine cardiotoxicity.

Coronary heart disease and in particular its most serious form - acute myocardial infarction (AMI) - represents the most common cause of mortality in developed countries. Better prognosis may be achieved by understanding the etiopathogenetic mechanisms of AMI. Therefore, a catecholamine model of myocardial injury, which has appeared to be very similar to AMI in human in some aspect, was used. Male Wistar:Han rats were randomly divided into two groups: control group (saline) and isoprenaline group (ISO; synthetic catecholamine, 100 mg.kg(- 1) subcutaneously [s.c.]). After 24 hours, functional parameters were measured, biochemical markers in the blood and metals content in the heart tissue were analysed and histological examination was performed. ISO caused marked myocardial injury that was associated with myocardial calcium overload. Close correlation between myocardial impairment (i.e. serum TnT, stroke volume index and wet ventricles weight) and the levels of myocardial calcium was observed. Direct reactive oxygen species (ROS) involvement was documented only by non-significant increase in malonyldialdehyde 24 hours after ISO injury. Moreover, myocardial element analysis revealed no significant changes as for the content of zinc and iron while selenium and copper increased in the ISO group although it reached statistical significance only for the latter.