Quantitative structure-activity relationship of hydroxyl-substituent Schiff bases in radical-induced hemolysis of human erythrocytes

The major objective of this work was to explore the quantitative structure-activity relationship (QSAR) of hydroxyl-substituent Schiff bases in protecting human erythrocytes against 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)- induced hemolysis, in which 10 Schiff bases including 4-phenyliminomethylphenol (PIH); 4-((4-hydroxybenzylidene) amino)phenol (PAH); 2-methoxy-4-((4-hydroxyphenylimino)methyl)phenol (PMH); 4-((furan-2-ylmethylene)amino) phenol (FAH); 4-((4-N,N-dimethylaminobenzylidene)amino)phenol (PDH); 2-((4-N,N-dimethylaminobenzylidene)amino) phenol (ODH); 2-(naphthalene-1-yliminomethyl)phenol (NAH); 2-(benzyliminomethyl)phenol (BPH); 1,4-di((2-hydroxyphenylimino) methyl)benzene (DOH); 1,4-di((4-hydroxyphenylimino)methyl)benzene DPH, were available for this in vitro experimental system. The results revealed that the radical-scavenging activity of the --OH attached to the para position of methylene in Schiff base was much lower than that attached to the ortho position of the N atom. The large conjugate system and low steric hindrance in the framework of Schiff base benefit the Schiff base to trap radicals. Meanwhile, since a Schiff base, even without any substituent, can also play an antioxidative role in this experimental system, the QSAR results suggest that hydroxyl-substituent Schiff bases are potential drugs in the treatment of radical-related diseases, and provide more information for designing novel drugs.     

The malonyl gramicidin channel: NMR-derived rate constants and comparison of calculated and experimental single-channel currents

Summary Malonyl gramicidin is incorporated into lysolecithin micelles in a manner which satisfies a number of previously demonstrated criteria for the formation of the transmembrane channel structure. By means of sodium-23 nuclear magnetic resonance, two binding sites are observed: a tight site and a weak site with binding constants of approximately 100m ^–1 and 1m ^–1, respectively. In addition, off-rate constants from the two sites were estimated from NMR analyses to bek _off ^t 3×10⁵/sec andk _off ^w 2×10⁷/sec giving, with the binding constants, the on-rate constants,k _on ^t 3×10⁷/msec andk _on ^w 2×10⁷/m sec.Five different multiple occupancy models with NMR-restricted energy profiles were considered for the purpose of calculating single-channel currents as a function of voltage and concentration utilizing the four NMR-derived rate constants (and an NMR-limit placed on a fifth rate constant for intrachannel ion translocation) in combination with Eyring rate theory for the introduction of voltage dependence.Using the X-ray diffraction results of Koeppe et al. (1979) for limiting the positions of the tight sites, the two-site model and a three-site model in which the weak sites occur after the tight site is filled were found to satisfactorily calculate the experimental currents (also reported here) and to fit the experimental currents extraordinarily well when the experimentally derived values were allowed to vary to a least squares best fit. Surprisingly the best fit values differed by only about a factor of two from the NMR-derived values, a variation that is well within the estimated experimental error of the rate constants.These results demonstrate the utility of ion nuclear magnetic resonance to determine rate constants relevant to transport through the gramicidin channel and of the Eyring rate theory to introduce voltage dependence.     

Inhibition of free radical-induced erythrocyte hemolysis by 2-O-substituted ascorbic acid derivatives

Inhibitory effects of 2-O-substituted ascorbic acid derivatives, ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S), on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of ascorbic acid (AA) and other antioxidants. The order of the inhibition efficiency was AA-2S> or =Trolox=uric acid> or =AA-2P> or =AA-2G=AA>glutathione. Although the reactivity of the AA derivatives against AAPH-derived peroxyl radical (ROO(*)) was much lower than that of AA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis and membrane protein oxidation. In addition, the AA derivatives were found to react per se with ROO(*), not via AA as an intermediate. These findings suggest that secondary reactions between the AA derivative radical and ROO(*) play a part in hemolysis inhibition. Delayed addition of the AA derivatives after AAPH-induced oxidation of erythrocytes had already proceeded showed weaker inhibition of hemolysis compared to that of AA. These results suggest that the AA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AA in vivo.     

Inhibitory effects of cocoa flavanols and procyanidin oligomers on free radical-induced erythrocyte hemolysis

Excessive peroxidation of biomembranes is thought to contribute to the initiation and progression of numerous degenerative diseases. The present study examined the inhibitory effects of a cocoa extract, individual cocoa flavanols (-)-epicatechin and (+)-catechin, and procyanidin oligomers (dimer to decamer) isolated from cocoa on rat erythrocyte hemolysis. In vitro, the flavanols and the procyanidin oligomers exhibited dose-dependent protection against 2,2'-azo-bis (2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis between concentrations of 2.5 and 40 microM. Dimer, trimer, and tetramer showed the strongest inhibitory effects at 10 microM, 59.4%, 66.2%, 70.9%; 20 microM, 84.1%, 87.6%, 81.0%; and 40 microM, 90.2%, 88.9%, 78.6%, respectively. In a subsequent experiment, male Sprague-Dawley rats (approximately 200 g; n = 5-6) were given a 100-mg intragastric dose of a cocoa extract. Blood was collected over a 4-hr time period. Epicatechin and catechin, and the dimers (-)-epicatechin-(4beta>8)-epicatechin (Dimer B2) and (-)-epicatechin-(4beta>6)-epicatechin (Dimer B5) were detected in the plasma with concentrations of 6.4 microM, and 217.6, 248.2, and 55.4 nM, respectively. Plasma antioxidant capacity (as measured by the total antioxidant potential [TRAP] assay) was elevated (P < 0.05) between 30 and 240 min following the cocoa extract feeding. Erythrocytes obtained from the cocoa extract-fed animals showed an enhanced resistance to hemolysis (P < 0.05). This enhanced resistance was also observed when erythrocytes from animals fed the cocoa extract were mixed with plasma obtained from animals given water only. Conversely, plasma obtained from rats given the cocoa extract improved the resistance of erythrocytes obtained from rats given water only. These results show cocoa flavanols and procyanidins can provide membrane protective effects.     

Computer-determined rate constants of hemolysis induced by Prymnesium parvum toxin

Rates of hemolysis of rabbit erythrocyte suspensions induced by P. parvum (prymnesin) have been measured colorimetrically at 25.5°C and pH 5.5. The data have been treated previously as consecutive first-order rate processes associated with the prolytic and lytic periods from which two specific rate constants have been obtained, k′ and kψ, respectively. These constants have been related to those obtained by a computer-generated fit of the rate data (absorbance A_t, as a function of time t) with the rate equation $\text{Y =}\text{D}\text{[1 +}\text{exp}\text{((X ? B)}\text{C)}\text{]}\text{+ E}$. Here Y equals A_t, X = time, t; D is equal to a spread factor, A_i ? A_∞; C is the slope of the curve at the inflection point; B is the midpoint time value, i.e., the time at which $\text{A}{}_{\mathrm{t}}\text{=}\text{D}\text{2}$; E is termed the off-set constant and is equal to A_∞. Of these constants, B is directly related to the length of the prolytic period, and C^?1 is directly related to the specific first-order rate constant for hemolysis, k_ψ.     

Superoxide dismutase: a comparison of rate constants

O₂^?was introduced, at a constant rate, into buffered aqueous solutions, either by mechanical infusion of KO₂, dissolved in tetrahydrofuran, or by the in situ action of xanthine oxidase on xanthine plus oxygen. This O₂^? was allowed to react with ferricytochrome c or with tetranitromethane and the formation of the reaction products, ferrocytochrome c or nitroform, respectively, was monitored spectrophotometrically. That concentration of Superoxide dismutase, which competed equally with given levels of cytochrome c or tetranitromethane and which thus caused 50% inhibition of the rates of accumulation of ferrocytochrome c or of nitroform, was determined. The rate constant for the enzymatic dismutation of O₂^? by the copper and zinc containing enzyme from bovine erythrocytes was then calculated from the known rate constants for the reaction of O₂^? with ferricytochrome c and with tetranitromethane and was found to be 2 × 10⁹m^?1 sec^?1 at pH 7.8 and 8.5. This rate constant was obtained at steady-state concentrations of O₂^? in the 10^?8m → 10^?13m range and is in full agreement with the results of pulse radiolytic investigations which were performed at O₂^? concentrations in the 10^?5m range. The second order rate constant for the enzymatic dismutation of O₂^? is thus independent of the concentration of O₂^? in the range 10^?5 → 10^?13m.Several distinct types of Superoxide dismutase have been described. These include the mangano-enzymes from Escherichia coli and from chicken liver mitochondria and the iron-enzyme from E. coli. The rate constants for the dismutations catalyzed by these enzymes have also been investigated as a function of pH.     

Protective effects of flavonols and their glycosides against free radical-induced oxidative hemolysis of red blood cells

The in vitro oxidative hemolysis of human red blood cells (RBCs) was used as a model to study the free radical-induced damage of biological membranes and the protective effect of flavonols and their glycosides (FOHs), i.e., myricetin (MY), quercetin (Q), morin (MO), kaempferol (K), rutin (R), quercetin galactopyranoside (QG), quercetin rhamnopyranoside (QR), and kaempferol glucopyranoside (KG). The hemolysis of RBCs was induced by a water-soluble free radical initiator 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). It was found that addition of AAPH at 37 degrees C to the suspension of RBCs caused fast hemolysis after a short period of inhibition period, and addition of FOHs significantly suppressed the hemolysis. The FOHs (MY, Q, R, QG and QR) which bears an ortho-dihydroxyl functionality showed much more effective anti-hemolysis activity than that of the other FOHs (MO, K and KG) bearing no such functionality.     

Oxidative hemolysis of erythrocytes and its inhibition by free radical scavengers

The oxidative hemolysis of rabbit erythrocytes induced by free radicals and its inhibition by chain-breaking antioxidants have been studied. The free radicals were generated from either a water-soluble or a lipid-soluble azo compound which, upon its thermal decomposition, gave carbon radicals that reacted with oxygen immediately to give peroxyl radicals. The radicals generated in the aqueous phase from a water-soluble azo compound induced hemolysis in air, but little hemolysis was observed in the absence of oxygen. Water-soluble chain-breaking antioxidants, such as ascorbic acid, uric acid, and water-soluble chromanol, suppressed the hemolysis dose dependently. Vitamin E in the erythrocyte membranes was also effective in suppressing the hemolysis. 2,2,5,7,8-Pentamethyl-6-chromanol, a vitamin E analogue without phytyl side chain, incorporated into dimyristoylphosphatidylcholine liposomes, suppressed the above hemolysis, but alpha-tocopherol did not suppress the hemolysis. Soybean phosphatidylcholine liposomes also induced hemolysis, and a lipid-soluble azo initiator incorporated into the soybean phosphatidylcholine liposomes accelerated the hemolysis. The chain-breaking antioxidants incorporated into the liposomes were also effective in suppressing this hemolysis.     

Delayed hemolysis of human erythrocytes in solutions of glucose

There has been described a type of hemolysis which occurs under certain defined conditions when erythrocytes are suspended in glucose solution. It consists of a prolytic phase lasting about an hour, followed by a hemolytic phase lasting about 2 hours. The physical factors controlling this delayed hemolysis have been investigated. The system is especially sensitive to changes of pH and of temperature. This type of hemolysis is inhibited by increased osmotic pressure and by phlorhizin, but not, as far as can be ascertained, by fluoride or iodoacetate. It is possible, but not yet proved, that delayed hemolysis in glucose solution is dependent on enzymic activity. Phosphorylation may be the limiting factor. During the prolytic phase the cells are easily permeable to potassium. It is concluded that the development of cation permeability is not a direct cause of hemolysis.     

Studies on hemolysis of human erythrocytes by linoleic acid

The results presented in this paper show that lysis of human erythrocytes by linoleic acid is not caused by peroxidation of the fatty acid. Peroxidase, superoxide dismutase and scavengers of O ₂ ⁻ and OH had no effect on the lysis while catalase showed only marginal inhibition suggesting that O ₂ ⁻ , OH, O ₂ ⁻ and H₂O₂ do not play any direct role in hemolysis by linoleic acid. Generators of H₂O₂ inhibited the lysis completely and methemoglobin cells were more resistant to hemolysis by linoleic acid. The fatty acid did neither bind to nor fomed complex with red cell ghosts. Membrane oxidation of sulphydryl groups was also not involved in the lysis. Β-Carotene, retinol and bile salts enhanced the lysis, while, cholesterol but not cholesterol acetate, inhibited it. Taurocholate-pretreated cells were more susceptible to linoleic acid lysis. These observations suggested-that lysis by linoleic acid may be due to its detergent property.     

Effect of dose-rate and dose fractionation on radiation-induced hemolysis of human erythrocytes.

Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit 2%) were exposed under air to gamma radiation at a dose rates of 2.2 kGy.h-1 and 4.2 kGy.h-1. The dose-response curves for hemolysis of erythrocytes indicated that the process of hemolysis is inversely related to the dose-rate. At both dose-rates we observed a reduced level of hemolysis, when erythrocytes were irradiated with a split dose (0.4 kGy + 2.3 kGy with an interval time between the subsequent exposures from 1 to 4 h) in comparison with the same single dose (2.7 kGy). The maximal effect of fractionation was observed when the interfraction time was equal to 3.5 h. The influence of the interfraction temperature on this effect was observed. The results obtained indicate that enucleated human erythrocytes under suitable radiation conditions are capable of repairing radiation damage which leads to hemolysis.