Hemolysis Of Human Erythrocytes Research Articles

Chemical Kinetic Behavior of Chlorogenic Acid in Protecting Erythrocyte and DNA against Radical-Induced Oxidation

As an abundant ingredient in coffee, chlorogenic acid (CGA) is a well-known antioxidant. Although some works have dealt with its radical-scavenging property, the present work investigated the protective effects of CGA on the oxidation of DNA and on the hemolysis of human erythrocytes induced by 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) by means of chemical kinetics. The inhibition period (t(inh)) derived from the protective effect of CGA on erythrocyte and DNA was proportional to its concentration, t(inh) = (n/R(i))[CGA], where R(i) refers to the radical-initiation rate, and n indicates the number of radical-propagation chains terminated by CGA. It was found that the n of CGA to protect erythrocytes was 0.77, lower than that of vitamin E (2.0), but higher than that of vitamin C (0.19). Furthermore, CGA facilitated a mutual protective effect with VE and VC on AAPH-induced hemolysis by increasing n of VE and VC. CGA was also found to be a membrane-stabilizer to protect erythrocytes against hemin-induced hemolysis. Moreover, the n of CGA was only 0.41 in the process of protecting DNA. This fact revealed that CGA served as an efficient antioxidant to protect erythrocytes more than to protect DNA. Finally, the reaction between CGA and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS(+*)) or 2,2'-diphenyl-1-picrylhydrazyl (DPPH) revealed that CGA was able to trap radicals by reducing radicals more than by donating its hydrogen atoms to radicals.

Human erythrocyte hemolysis induced by selenium and tellurium compounds increased by GSH or glucose: A possible involvement of reactive oxygen species

Oxidative stress can induce complex alterations of membrane proteins in red blood cells (RBCs) eventually leading to hemolysis. RBCs represent a good model to investigate the damage induced by oxidizing agents. Literature data have reported that chalcogen compounds can present pro-oxidant properties with potent inhibitory effects on cell growth, causing tissue damage and inhibit a variety of enzymes. In this study, human erythrocytes were incubated in vitro with various chalcogen compounds at 37 °C: diphenyl ditelluride ( 1), dinaphthalen diteluride ( 2), diphenyl diselenide ( 3), ( S)- tert-butyl 1-diselenide-3-methylbutan-2-ylcarbamate ( 4), ( S)- tert-butyl 1-diselenide-3-phenylpropan-2-ylcarbamate ( 5), selenium dioxide ( 6) and sodium selenite ( 7) in order to investigate their potential in vitro toxicity. After 6 h of incubation, all the tested compounds increased the hemolysis rate, when compared to control and compound ( 2) had the most potent hemolytic effect. The addition of reduced glutathione (GSH) or glucose to the incubation medium enhanced hemolysis caused by chalcogen compounds. The thiol oxidase activity of these compounds was evaluated by measuring the rate of cysteine (CYS) and dithiotreitol (DTT) oxidation. DTT and cysteine oxidation was increased by all the compounds tested. The results suggest a relationship between the oxidation of intracellular GSH and subsequent generation of free radicals with the hemolysis by chalcogen compounds.

Three anti-inflammatory stigmastane steroids from Alchornea floribunda leaves

The anti-inflammatory activity of Alchornea cordifolia has been reported in previous studies [1,2,3]. In the present study, bioactivity – guided fractionation led to the isolation of three stigmastane steroids from Alchornea floribunda leaves. The anti-inflammatory activities of these compounds were evaluated using in vitro and in vivo animal models. The compounds, 1, 2 and 3 at 5mg/ear inhibited xylene – induced ear edema in mice, with edema inhibitions of 59.9, 51.5 and 51.54% respectively. At 20mg/kg (ip), all the compounds significantly (p<0.05) inhibited acute inflammation induced by subplanta injection of undiluted egg albumen in rats' paw. Compound 1 exhibited a higher anti-inflammatory effect (% edema inhibition=50. 0) than prednisolone (% edema inhibition=48.0) at 3h. 1 and 3 at concentrations of 50 and 100µg/ml significantly (p<0.05) stabilized heat-induced haemolysis of human erythrocytes in vitro, but have no effect on hypotonicity-induced haemolysis. Spectral analysis elucidated the compounds as stigmastane –3, 6– dione (1), stigmastane –22– ene –3, 6– dione (2) and 3– hydroxystigmastane –22– ene (3). These compounds may, in part, account for the anti-inflammatory effect of Alchornea floribunda leaves. This is the first report on the isolation and structure elucidation of anti-inflammatory steroids from Alchornea floribunda leaves.

Chromobacterium haemolyticum sp. nov., a strongly haemolytic species

A Gram-negative bacterium, strain MDA0585(T), isolated from a sputum culture, was characterized by a polyphasic approach. The 16S rRNA gene and a conserved portion of the DNA gyrase A gene were sequenced and analysed phylogenetically. Strain MDA0585(T) showed the closest relationships with Chromobacterium violaceum ATCC 12472(T) and Chromobacterium subtsugae PRAA4-1(T) (96.1 % and 96.3 % 16S rRNA gene sequence similarity, respectively). The cellular fatty acids of strain MDA0585(T) consisted mainly of C(16 : 0), C(16 : 1)omega7c and C(16 : 1)omega6c (summed feature 3) and C(18 : 1)omega7c and C(18 : 1)omega6c (summed feature 8), a profile that was similar to, but distinguishable from, those of C. violaceum ATCC 12472(T) and C. subtsugae PRAA4-1(T). In culture, strain MDA0585(T) differed from C. violaceum and C. subtsugae in several ways: lack of violet pigmentation, the ability to haemolyse sheep blood, differences in several biochemical reactions and higher resistance to antibiotics. The culture supernatant of strain MDA0585(T) also caused remarkable haemolysis of human erythrocytes. These results suggest that strain MDA0585(T) represents a novel species within the genus Chromobacterium, for which the name Chromobacterium haemolyticum sp. nov. is proposed. The type strain is MDA0585(T) (=CCUG 53230(T)=JCM 14163(T)=DSM 19808(T)).

Antioxidant and prooxidant properties of a polyphenol‐rich extract from Geranium sanguineum L. in vitro and in vivo

A polyphenol-rich extract from Geranium sanguineum L. (PC) protected biological membranes due to its antioxidant capacity. PC caused a dose-dependent decrease of the osmotic hemolysis of human erythrocytes and increased their resistance against the toxic effect of H(2)O(2); no effect on catalase activity was observed. While PC reduced the accumulation of TBA-reactive products in rat liver microsomes in inducible lipid peroxidation (LPO), the non-induced LPO was not affected. Further the effect of PC on the products of LPO was investigated in the lungs, livers and sera of intact and influenza virus-infected mice (VIM). The infection enhanced LPO in the lungs and livers. In the group of PC-treated VIM, malondialdehyde (MDA) in the lungs and livers was brought to control levels. PC-treatment caused a significant increase of MDA in the lungs of intact mice, a slight one in the livers and did not affect MDA in the sera. Thus the extract exhibited prooxidant characteristics in intact animals as well as antioxidant properties in VIM. The reducing ability of PC on LPO could be an alternative mechanism of its protective effect in experimental influenza infection.

The protective effects of ginsenosides on human erythrocytes against hemin-induced hemolysis

Panax ginseng has been used in traditional Chinese medicine to enhance stamina and capacity to deal with fatigue and physical stress. Many reports have been devoted to the effects of ginsenosides on many in vitro or in vivo experimental systems. The major aim of this work is to investigate the protective effects of 12 individual ginsenosides including Rb1, Rb3, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1, Rh2, R1 and pseudoginsenoside F11, together with the central structures of aforementioned ginsenosides, 20(S)-protopanaxadiol (PD) and 20(S)-protopanaxatriol (PT), on hemin-induced hemolysis of human erythrocytes. This is because hemin can induce hemolysis by accelerating the potassium leakage, dissociating skeletal proteins and prohibiting some enzymes in the membrane of erythrocyte. Thus, the structure-activity-relationship (SAR) between ginsenosides and protective effects has been screened in this in vitro experimental system. It is found that Rh2 and Rg3 intensify hemolysis in the presence of hemin, and initiate hemolysis even in the absence of hemin. All the other ginsenosides protect human erythrocytes against hemin-induced hemolysis more or less. The overall sequence is Rc > Rd > Re ∼ Rb1 > Rg1 ∼ Rh1 > Rb3 ∼ Rg2 ∼ R1 ∼ F11 ∼ PT. In addition, the protective effects of PD and PT have been detected, and found that PD promotes hemolysis appreciably, whereas PT protects erythrocytes efficiently. Moreover, the protective effects of PT ginsenosides are similar to PT itself, and the protective effects of PD ginsenosides vary remarkably, demonstrating that the positions of the sugar moieties make the protective activities of ginsenosides complicated. Especially, sugar moiety at 20-position is critical for PD ginsenosides to inhibit hemolysis, whereas hydroxyl group at 3-position is important for PT ginsenosides. The present result may be useful for understanding the SAR of ginsenosides.

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 “double-faced” effect of VC-12 on free-radical-induced haemolysis of human erythrocytes: antioxidant and prooxidant

Vitamin C is a popular antioxidant; however, its water solubility limits its function in the lipid environment. As a result, the antioxidative properties of its lipophilic derivatives have aroused research attention, especially L-ascorbyl-6-laurate (VC-12). We have investigated the effect of a high concentration of VC-12 on 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced haemolysis of human erythrocytes. The findings indicated that VC-12 was capable of protecting erythrocytes against AAPH-induced haemolysis when its concentration was below 60 microM. With an increase in the concentration of VC-12 and a decrease in the concentration of AAPH, VC-12 promoted haemolysis remarkably, the mechanism of which has been proposed as VC-12-mediated peroxidation. When the concentration of VC-12 was increased to above 150 microM, VC-12 promoted haemolysis by its function as a surfactant, to unbalance the osmotic pressure within and outside erythrocytes. High concentrations of VC-12 may have generated radicals via autooxidation, resulting in eventual haemolysis. Therefore, the double-faced effect of VC-12 on haemolysis of human erythrocytes was due to its concentration. This information may be useful for the safe use of lipophilic vitamin C.

Inhibition of Monosodium Urate Monohydrate-mediated Hemolysis by Vitamin E

Microcrystals of monosodium urate monohydrate (MSUM) induce cytolysis and hemolysis in erythrocytes. In this report, we studied the effect of vitamin E on MSUM-mediated hemolysis in human erythrocytes. Vitamin E significantly inhibited hemolysis induced by MSUM. The hydroxyl group in the chromanol ring of vitamin E is dispensable for protecting erythrocytes against hemolysis induced by MSUM, indicating that the inhibitory effect of vitamin E is not due to its antioxidant properties. However, both the chromanol ring and the isoprenoid side chain are important for vitamin E to suppress MSUM-induced hemolysis. Our current study suggests that vitamin E inhibits hemolysis induced by MSUM as a membrane stabilizer.

Free-radical-scavenging effect of carbazole derivatives on AAPH-induced hemolysis of human erythrocytes

Since the research on antioxidants provides theoretical information for the medicinal development, and supplies some in vitro methods for quick-optimizing drugs, it attracts more scientific attention from bioorganic and medicinal chemists. In addition to the traditional O–H bond-type antioxidant, carbazole and its related tricyclic amines (Ar 2NHs), in which N–H bond functioned as the antioxidant, have attracted much research attention because Ar 2NHs have always been the central structure in many currently used drugs. Thus, the investigation on the structure–activity relationship (SAR) between Ar 2NHs and their free-radical-scavenging capacities in detail will benefit the development of novel radical-scavenging drugs containing Ar 2NHs as the central structure. Therefore, carbazole (CazNH) and its structural analogues including phenoxazine (PozNH), phenothiazine (PtzNH), iminostilbene (IsbNH) together with diphenylamine (DpaNH) were applied to protect human erythrocytes against 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced hemolysis in vitro. By introducing the chemical kinetic formula related to free radical reaction, namely, the quantitative relationship between inhibition period ( t inh) and the concentration of antioxidant (AH), t inh = ( n/ R i)[AH], into AAPH-induced hemolysis, the values of stoichiometric factor ( n) of Ar 2NHs indicated that the free-radical-scavenging sequence of Ar 2NHs is PozNH > DpaNH > CazNH > IsbNH > PtzNH > α-tocopherol (TocH). Another aim of this work was to investigate the antioxidative effect of Ar 2NHs used together with other antioxidants including Trolox (TroH), VC, l-ascorbyl-6-laurate (VC-12), and TocH. The obtained data revealed that n value of PozNH when used together with all the other antioxidants decreases, whereas, n values of CazNH, DpaNH, IsbNH, and PtzNH when used in combination with TroH increase, demonstrating that two different interaction styles existed in the case of Ar 2NHs used with other antioxidants. These findings may be useful for the development of agents for various ROS-mediated diseases in vivo.

S-Phase Accumulation of Candida albicans by Anticandidal Effect of Amentoflavone Isolated from Selaginella tamariscina

Amentoflavone, which is a biflavone derived from various plants, has been known to possess various biological activities including anti-tumor and anti-inflammatory activity. In the previous study, we reported antifungal effect of amentoflavone isolated from an ethyl acetate extract of Selaginella tamariscina on human pathogenic fungi. Amentoflavone significantly inhibited the growth of fungal cells without hemolysis of human erythrocytes. In the present study, we investigated antifungal acting mode of amentoflavone in human pathogenic yeast Candida albicans. Anticandidal activity was exerted in an energy-independent manner presented by an antifungal assay in the presence of NaN(3), which is ATP-depleting agent as a metabolic inhibitor. To investigate the effects of amentoflavone on cellular physiology in C. albicans, we performed cell cycle analysis, and the analysis showed that amentoflavone significantly arrested cell cycles during the S-phase. These results demonstrated that amentoflavone has potent anticandidal activity with significant physiological changes inducing S-phase arrest in intracellular environment. Therefore, amentoflavone may be applied to a lead compound for the development of therapeutic agents, which can treat candidiasis resulted from candidal infections.

The crude venom from the sea anemone Stichodactyla helianthus induces haemolysis and slight peroxidative damage in rat and human erythrocytes

The haemolytic and peroxidative effects of crude venom of the sea anemone Stichodactyla helianthus were evaluated in rat and human erythrocytes. Venom extract caused a significant concentration-dependent effect on haemolysis (release of haemoglobin). Human erythrocytes were more sensitive (0.094 mg protein/ml) than those of the rats (0.3787 mg protein/ml). In contrast, a light effect on lipid peroxidation (LP, an index of oxidative damage to membrane lipids) was recorded. The concentrations needed to produce a significant effect on LP in rat and human erythrocytes were, respectively, 2-fold and 7-fold higher than those required to produce significant haemolysis. The differential effect of S. helianthus venom on haemolysis and oxidation of membrane lipids is not common for venoms of other sea anemones, which usually show a tightly related effect on LP and haemolytic damage.

Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cell-penetrating peptide Pep-1

Here, we report the successful design of a novel bacteria-selective antimicrobial peptide, Pep-1-K (KKTWWKTWWTKWSQPKKKRKV). Pep-1-K was designed by replacing Glu-2, Glu-6, and Glu-11 in the cell-penetrating peptide Pep-1 with Lys. Pep-1-K showed strong antibacterial activity against reference strains (MIC=1–2μM) of Gram-positive and Gram-negative bacteria as well as against clinical isolates (MIC=1–8μM) of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. In contrast, Pep-1-K did not cause hemolysis of human erythrocytes even at 200μM. These results indicate that Pep-1-K may be a good candidate for antimicrobial drug development, especially as a topical agent against antibiotic-resistant microorganisms. Tryptophan fluorescence studies indicated that the lack of hemolytic activity of Pep-1-K correlated with its weak ability to penetrate zwitterionic phosphatidylcholine/cholesterol (10:1, w/w) vesicles, which mimic eukaryotic membranes. Furthermore, Pep-1-K caused little or no dye leakage from negatively charged phosphatidylethanolamine/phosphatidylglycerol (7:3, w/w) vesicles, which mimic bacterial membranes but had a potent ability to cause depolarization of the cytoplasmic membrane potential of intact S. aureus cells. These results suggested that Pep-1-K kills microorganisms by not the membrane-disrupting mode but the formation of small channels that permit transit of ions or protons but not molecules as large as calcein.

Molecular cloning and sequence analyses of preprotemporin mRNAs containing premature stop codons from extradermal tissues of Rana tagoi

The temporins are a family of hydrophobic, C-terminally α-amidated antimicrobial peptides that are synthesized in the skins of a wide range of species of frogs belonging to the genus Rana. In the present study, we investigated using RT-PCR the expression of preprotemporin mRNAs in extradermal tissues of Tago's brown frog Rana tagoi. cDNAs encoding temporin-1TGa (FLPILGKLLS 10GIL.NH 2), previously isolated from an extract of the skin of R. tagoi skin, were amplified and cloned from the stomach, liver, kidney, skeletal muscle. However, a net insertion of 10 nucleotides resulted in the presence of a premature stop codon in the open reading frame that was not present in the corresponding region of preprotemporin-1TGa from skin. The preprotemporin cDNA obtained from small intestine contained an additional 12 nucleotide insertion in the region that encodes the temporin sequence so that a novel peptide (FLPVILPVIG 10KLLSGIL.NH 2), termed temporin-1TGc, is specified. This cDNA also contained a premature stop codon in the open reading frame. Although it is unclear whether temporin-1TGc is produced in R. tagoi tissues, a synthetic replicate of the peptide of was biologically active, inhibiting the growth of Staphylococcus aureus (minimal inhibitory concentration = 37.5 μM) and producing hemolysis of human erythrocytes (LD 50 = 50 μM).

Evaluation of the free-radical-scavenging activity of diclofenac acid on the free-radical-induced haemolysis of human erythrocytes

Free-radical-induced peroxidation in-vivo is regarded as the aetiology of some diseases and free-radical-scavenging drugs, also called antioxidants (AH), have been widely used to overcome oxidative stress. An in-vitro experimental method, 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced haemolysis of human erythrocytes can be applied to assess the free-radical-scavenging activity of a drug. The major objectives of this work were focused on three aspects. Firstly, introduction of the chemical kinetic deduction of free-radical-initiating reaction to AAPH-induced haemolysis of human erythrocytes, by which the number of free radicals trapped by an antioxidant, n, can be obtained after finding the quantitative relationship between the inhibition period (t(inh)) and the concentration of the antioxidant, t(inh) = (n/Ri) [AH]. Ri, the free-radical-initiating rate, was initially confirmed by using alpha-tocopherol (VE) whose n was taken as 2. Secondly, the free-radical-scavenging activity of diclofenac acid (DaH) and its sodium salt (DaNaH) was assessed. It has been found that DaH and DaNaH protect human erythrocytes against AAPH-induced haemolysis dose-dependently. In particular, the n values of DaH and DaNaH (4.96 and 3.60) were much higher than some traditional antioxidants, such as 6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble structural analogue of VE, n = 0.30) and L-ascorbic acid (VC, n = 0.25), and L-ascorbyl-6-laurate (VC-12, a lipophilic structural analogue of VC, n = 1.11). Moreover, the free-radical-scavenging activity of lipophilic antioxidants is higher than the corresponding water-soluble species. Thirdly, the free-radical-scavenging activity of mixed antioxidants, VE + DaH, VC-12 + DaH, Trolox + DaNaH and VC + DaNaH, was revealed. The n value of VC, VC-12, VE and Trolox increase in the case of mixed usage with DaH and DaNaH, implying that diclofenac acid can repair the radical of these antioxidants. Thus, a mutual antioxidant effect between diclofenac acid and these antioxidants prolongs the lifespan of VC, VC-12, VE and Trolox, respectively.

Model function to calculate the refractive index of native hemoglobin in the wavelength range of 250-1100 nm dependent on concentration

The real part of the complex refractive index of oxygenated native hemoglobin solutions dependent on concentration was determined in the wavelength range 250 to 1100 nm by Fresnel reflectance measurements. The hemoglobin solution was produced by physical hemolysis of human erythrocytes followed by ultracentrifugation and filtration. A model function is presented for calculating the refractive index of hemoglobin solutions depending on concentration in the wavelength range 250 to 1100 nm.

Nutritive Value and Haemolytic Properties (in vitro) of the Leaves of Vernonia amygdalina on Human Erythrocyte

Vernonia amygdalina (Bitter leaf) is popularly consumed in many homes in Nigeria as a vegetable in soup preparation, while the infusion is used in folk medicine in the treatment of malaria fever. In this study, fresh leaves of Vernonia amygdalina were subjected to abrasion (to remove the bitter taste) as is normally done in soup preparation, subsequently the nutrient, anti-nutrient and zinc bioavailability of the processed and unprocessed leaf were determined. The heamolytic effect of the leaf infusion on human erythrocytes (Genotype-SS, AS, AA) was also determined. The study revealed that Vernonia amygdalina leaf has high protein (33.3%), fat (10.1%), crude fibre (29.2%), ash (11.7%), mineral (Na, K, Ca, Mg, Zn & Fe), phytate (1015.4mg/100g) and tannin (0.6%) content, while it contain low cyanide (1.1mg/kg). Abrasion of the leaf caused a significant decrease (P<0.05) in both the nutrient and antinutrient content of the leaf except in carbohydrate where there was a significant increase (P<0.05) and Mg where there was no significant change (P>0.05). The result of the calculated [Ca][phytate]/ [Zn] molar ratio (the best index for the prediction of Zn bioavailability) indicated that the phytate content of both the processed and unprocessed Vernonia amygdalina leaf will not reduce the Zn content to a critical level (0.5mol./kg). Further studies on the leaf revealed that the infusion induced the haemolysis of human erythrocyte (In vitro). Human genotype-SS (1024) were highly susceptible to haemolysis induced by Vernonia amygdalina infusion, genotype-AS moderately susceptible (512) while genotype-AA (256) is highly resistant to haemolysis induced by the same infusion. Bitter leaf has high nutrient and antinutrient content, however, abrasion will bring about a significant decrease (P<0.05) in both. The infusion of the Vernonia amygdalina leaf induces the haemolysis of mammalian erythrocyte in vitro, with Human-SS having the highest susceptibility.

The "Unexpected Role" of Vitamin E in Free Radical-Induced Hemolysis of Human Erythrocytes: α-Tocopherol-Mediated Peroxidation

Vitamin E involves a group of tocopherols and tocotrienols, in which alpha-tocopherol with the highest biological activity plays a more efficient role in advanced lesions with aged oxidized tissues. However, the results of the present study reveal that a large amount of endogenous alpha-tocopherol in human low-density lipoprotein (LDL) in the absence of any other antioxidants may initiate additional free radical propagation under low concentration of free radical initiator (i.e., 2,2'-azobis(2-amidinopropane hydrochloride) [AAPH], a water-soluble free radical source) to peroxide polyunsaturated fatty acids in LDL in the manner of alpha-tocopherol-mediated peroxidation (TMP). Whether the addition of high concentration of exogenous alpha-tocopherol to human erythrocytes under low concentration of AAPH can also drive TMP is the concern in this research work. Moreover, the hemolysis extent of human erythrocytes peroxidized by AAPH is followed easily by the determination of the hemoglobin outside the erythrocytes. A series of observations on various concentrations of AAPH-induced hemolysis in the presence of various concentrations of exogenous alpha-tocopherol demonstrates that the high concentration of exogenous alpha-tocopherol, coupled with low concentration of AAPH, can initiate TMP in the free-radical-induced peroxidation of human erythrocytes system as well. This result provides direct evidence to support TMP theory and expands its application into in vitro erythrocyte system.

Effect of triorganotin compounds on membrane permeability

Organotin compounds are widely distributed toxicants. They are membrane-active molecules with broad biological toxicity. In this contribution, we study the effect of triorganotin compounds on membrane permeability using phospholipid model membranes and human erythrocytes. Tribultyltin and triphenyltin are able to induce the release of entrapped carboxyfluorescein from large unilamellar vesicles. The rate of release is similar for phosphatidylcholine and phosphatidylserine systems and the presence of equimolar cholesterol decreases the rate of the process. Release of carboxyfluorescein is almost abolished when a non-diffusible anion like gluconate is present in the external medium, and it is restored by addition of chloride. Tributyltin is able to cause hemolysis of human erythrocytes in a dose-dependent manner. Relative kinetics determination shows that potassium leakage occurs simultaneously with hemoglobin release. Hemolysis is reduced when erythrocytes are suspended in a gluconate medium. These results indicate that triorganotin compounds are able to transport organic anions like carboxyfluorescein across phospholipids bilayers by exchange diffusion with chloride and suggest that anion exchange through erythrocyte membrane could be related to the process of hemolysis.

The antioxidant effect of hydroxyl‐substituent Schiff bases on the free‐radical‐induced hemolysis of human erythrocytes

The major objectives of the present work were focused on assessing the antioxidant capacities of two hydroxyl-substituent Schiff bases, 2-((o-hydroxylphenylimino)methyl)phenol (OSAP) and 2-((p-hydroxylphenylimino)methyl)phenol (PSAP) either used alone or in combination with some familiar water-soluble antioxidants i.e. 6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and L-ascorbic acid (VC), and lipophilic ones i.e. alpha-tocopherol (TOH) and L-ascorbyl-6-laurate (VC-12). 2,2'-Azobis(2-amidinopropane hydrochloride) (AAPH). Induced hemolysis of human erythrocytes functioned as the evaluation experimental system in this research. The present findings showed that either OSAP or PSAP not only was an antioxidant with high activity in protecting erythrocytes against AAPH-induced hemolysis concentration-dependently, but can also protect erythrocytes by acting with Trolox, TOH, VC and VC-12 synergistically. Based on chemical kinetic deduction, the number of trapping peroxyl radicals, n, of the above-mentioned antioxidants can be calculated in relation to Trolox that traps two peroxyl radicals; thus, TOH can trap 3.83 peroxyl radicals, VC-12 traps 2.87 and VC can only trap 1.08. As for OSAP and PSAP, 8.71 and 13.7 peroxyl radicals can be trapped, respectively, indicating that they were the most efficient inhibitors against AAPH-induced hemolysis. Moreover, the total number of peroxyl radicals trapped by OSAP+Trolox, OSAP+TOH, OSAP+VC and PSAP+VC were higher than the sum of the above individual antioxidant used alone, demonstrating that a mutual promotive effect existed in the above mixed antioxidants. In contrast, owing to the fact that the total number of peroxyl radicals trapped by OSAP+VC-12, PSAP+Trolox, PSAP+TOH and PSAP+VC-12 were less than the sum of the above individual antioxidant used alone, a mutual antagonistic effect was suggested in these combinative usages. This information may be helpful in the pharmaceutical application of two Schiff bases.