Kinetic investigation of myeloperoxidase upon interaction with copper, cadmium, and lead ions

Myeloperoxidase [MPO], which is abundantly expressed in neutrophils, catalyzes the formation of a number of reactive oxidant species. However, evidence has emerged that MPO-derived oxidants contribute to tissue damage and initiation and propagation of inflammatory diseases, particularly, cardiovascular diseases. Therefore, studying the regulatory mechanisms of the enzyme activity is of great importance. For clarifying some possible mechanism of the enzyme activity, kinetic investigations of MPO in the presence of Copper [Cu], Cadmium [Cd], and Lead [Pb] ions were carried out in vitro. MPO was partially purified from human white blood cells using ion-exchange and gel-filtration chromatography techniques. Its activity was measured spectrophotometrically by using tetramethyl benzidine [TMB] as substrate. Purified enzyme had a specific activity of 21.7 U/mg protein with a purity index of about 0.71. Cu inhibited MPO activity progressively up to a concentration of 60 mM at which about 80% of inhibition achieved. The inhibition was non-competitive with respect to TMB. An inhibitory constant [Ki] of about 19 mM was calculated from the slope of repot. Cd and Pb did not show any significant inhibitory effect on the enzyme activity. The results of the present study may indicate that there are some places on the enzyme and enzyme-substrate complex for Cu ions. Binding of Cu ions to these places result in conformational changes of the enzyme and thus, enzyme inhibition. This inhibitory effect of Cu on the enzyme activity might be considered as a regulatory mechanism on MPO activity

Comparative effects of copper, iron, vanadium and titanium on low density lipoprotein oxidation in vitro

Oxidation of low density lipoprotein [LDL] has been strongly implicated in the phathogenesis of atherosclerosis. The use of oxidants in dietary food stuff may lead to the production of oxidized LDL and may increase both the development and the progression of atherosclerosis. The present work investigated the effects of some elements including: copper [Cu], iron [Fe], vanadium [V] and titanium [Ti] on in vitro LDL oxidation quantitatively. The first LDL fraction was isolated from fresh plasma by single vertical discontinuous density gradient ultracentrifugation. The formation of conjugated dienes and thiobarbituric acid reactive substances and increase in electrophoretic mobility of LDL were monitored as markers of the oxidation of LDL. It was demonstrated that Cu, Fe, V and Ti exhibited strong oxidant activity in this respect [P<0.001]. Oxidation of LDL in the presence of Cu was more and appeared to be in this order Cu>Fe> V>Ti. Discussion: Cu, Fe, V and Ti are redox-active transition metals that may cause oxidative damage to lipids, proteins and DNA molecules. We suggest that these elements may also influence the oxidation of LDL in vivo, which could increase both the development and progression of atherosclerosis

Low doses of vanadyl sulfate protect rats from lipid peroxidation and hypertriglyceridemic effects of fructose-enriched diet

Insulin resistance, hyperinsulinemia and disturbances in lipid metabolism can be produced in healthy rats by feeding them a fructose-enriched diet. Vanadyl sulfate, an antidiabetic trace element, enhances insulin sensitivity in type 2 diabetic patients. The aim of this study was to determine the effect of vanadyl sulfate treatment [0.2 mg/ml in drinking water for 7 days] on plasma insulin, triglyceride concentration and plasma lipid peroxidation in rats that were fed a fructoseenriched diet that leads to insulin resistance. Male Wistar rats were divided into four groups: fructose-fed rats [FF]; vanadyl sulfate treated-fructose fed treated rats [FV]; control rats [C]; and vanadyl sulfate-treated control rats [CV]. Control and vanadyl sulfate-treated control rats were fed with standard laboratory chow. High fructose feeding resulted in hyperinsulinemia and hypertriglyceridemia, and the plasma lipid peroxidation marker TBARS [thiobarbituric acid reactive substances] was significantly elevated. Administration of vanadyl sulfate was associated with significant normalization of plasma insulin and triglyceride levels. These rats also showed significantly lower TBARS than untreated, fructose-fed rats. We conclude that enhanced lipid peroxidation occurs in addition to hypertriglyceridemia in fructose-fed rats. It is suggested that lipid peroxidation associated with hypertriglyceridemia may be responsible for the pathologies induced by high fructose consumption. The plasma insulin level probably contributes to this increased peroxidation. Improved insulin action in fructose-fed vanadyl sulfate treated rats could be responsible for the amelioration of those abnormalities induced by fructose feeding

Spectroscopic studies on titanium ion binding to the apolactoferrin

Titanium [Ti] is a relatively abundant element that has found growing applications in medical science and recently some of Ti compounds are introduced as anticancer drugs. In spite of very limited data which exist on the Ti metabolism, some proteins might be involved in the mechanism of action of Ti. Up to our knowledge, there is not any report in the literature concerning binding of Ti to apolactoferrin. Binding of apolactoferrin with Ti[IV]-citrate was studied by spectroflourimeterey and spectrophotometery techniques under physiological conditions. The spectroflourimeteric studies revealed a significant fluorescence quenching, that indicated binding of apolactoferrin with Ti[IV]. The same reaction was monitored through spectrophotometry technique; this represents a characteristic UV difference band at 267 nm, which is different from lac-Fe [III]. Titration studies show that lactoferrin specifically binds two moles Ti[IV] as complex with citrate per mol protein. Spectroflourimeterey and spectrophotometery techniques indicated that Ti[IV] ions cause a reduction [13%-14%] in binding of Fe[III] to lactoferrin. In overall, we may come to this conclusion that this element might be involved in the iron metabolism

Changes in biochemical parameters related to lipid metabolism following lithium treatment in rat

Lithium is widely used in medicine as an anti-depressive drug. In spite of abundant literature, questions on the side effects of lithium ions are far from being answered. In this study, the effects of lithium on biochemical parameters related to lipid metabolism were investigated. Male Wistar rats were treated with different doses of lithium for a period of up to 60 days. Blood samples were collected and livers were removed for analysis. Lipid related parameters in plasma and livers were measured by st and ard methods. Epididymal fat pads were used to investigate the mechanism of lithium action on lipolysis. It is shown that the major effect of lithium is reduction of HDL-C concentration and the increase of LDL-C only in high doses. Lithium treatment led to a decrease in liver content of triglycerides but phosphohpids increased significantly. Lithium also showed to inhibit lipoprotein lipase activity. This inhibitory effect is potentiated in the presence of citrate. Fat cell lipolysis is also inhibited by lithium, which is not reversed by alpha, and beta-receptor blockers indicating that lithium may exert its effect beyond these receptors. Lithium changes the metabolism of lipoproteins. The finding that lithium decreases HDL and increases LDL concentrations should be considered seriously, especially in patients using this drug for a long period

Effect of ATP-dependent K+ channel openers and blockers on serum concentration of aldosterone in rats

There are many reports for involvement of ATP-sensitive potassium channels in pancreatic, cardiac and vascular smooth muscle cells. This study examined the effect of single doses of K+ channel openers; diazoxide, minoxidil and K+ channel blockers; chlorpropamide, glibenclamide on serum concentration of aldosterone in male rats. Blood samples were obtained 60 minutes after drug treatment and serum aldosterone level was determined by RIA. The basal serum aldosterone was 659.32 +/- 71.48 pg/ml and after diazoxide or minoxidil administration increased to 1188.53 +/- 99.45 pg/ml and 1392.69 +/- 177.83 pg/ml, respectively. Chlorpropamide or glibenclamide treatment did not produce any change in basal serum aldosterone concentration, but in early streptozotocin-induced diabetic rats decreased serum aldosterone level significantly [P<0.001]. Pretreatment with glibenclamide blocked aldosterone response to diazoxide but did not affect aldosterone response to exogenous ACTH to the same extent. Effect of diazoxide in insulin-treated rats was approximately similar to that of normal rats. Comparison of blood glucose concentration determined in normal, insulin treated and diabetic rats after different drug administration showed that there is no correlation between blood glucose level and the responses observed in serum hormone concentration. The results indicate that regulatory processes involved in the secretion of aldosterone are responsive to drugs affecting glibenclamide-sensitive K+ channels