The effects of heme-binding proteins on the peroxidative and catalatic activities of hemin.

The plasma proteins hemopexin (Hx) and albumin (Alb) are known to bind heme with high and medium affinity, respectively. To study how this binding modifies heme catalytic reactivity, the effects of Hx, human serum Alb (HSA), and bovine serum Alb (BSA) on the peroxidase- and catalaselike activities of hemin were investigated. These hemin activities were found to be inhibited by 50 to 60% with either HSA or BSA, and by 80 to 90% with Hx. The heme complexes with Hx or Alb (1:1 = protein:heme) therefore had a much lower reactivity toward H2O2 and Cum-OOH than the nonprotein heme. A kinetic analysis suggested that binding to Hx or Alb inhibited the primary activation of heme by H2O2, the step common for both peroxidase- and catalaselike activities of hemin. It is thought that by complexing heme, the Hx and Alb can prevent the toxic effects of extracellular heme in blood plasma.

Protective effects of tea polyphenols against oxidative damage to red blood cells.

Tea polyphenols (TPP) from black and green teas were evaluated for their antioxidant effects on normal red blood cells (RBC) and beta-thalassemic RBC membranes challenged with exogenous oxidants in vitro. The TPP of both types protected RBC against primaquine-induced lysis; they also protected the whole cells and the membranes against H2O2-induced lipid peroxidation so that about 80% protection was reached at [TPP] = 10 microg/mL. TPP from black tea at the same concentration protected normal RBC from morphological alterations caused by the peroxide treatment. The mechanism of the effects of TPP was investigated using a chemical system generating .OH (iron + ascorbic acid). TPP from both black and green teas inhibited the .OH fluxes in a concentration-dependent manner, indicating the possibility of iron chelation by TPP. Spectrophotometric titration revealed that TPP could stoichiometrically bind ferric iron to form a redox-inactive Fe-TPP complex. Quantitative analysis suggests that one or more major catechins from the TPP preparations are the likely iron-binding compounds accounting for the antioxidant effects of TPP on RBC.

Hydroxyl radical generation in beta-thalassemic red blood cells.

To provide more experimental evidence for the proposed role of oxygen free radicals in red blood cell (RBC) damage in beta-thalassemia, hydroxyl radical generation was studied in thalassemic (Th) vs. normal (N) RBC. .OH fluxes were quantified by the conversion of salicylic acid (SA) into its hydroxylated products, 2,3- and 2,5-dihydroxybenzoic acids (DHBA) and catechol, assayed with HPLC coupled to electrochemical detection. No significant difference in spontaneous .OH generation between N-RBC and Th-RBC was found. Ascorbic acid (0.5-3.0 mM) induced many-fold increases in SA hydroxylation in a dose-dependent manner in both types of cells. In the presence of ascorbate (1.0 mM), the SA hydroxylated products were determined in Th-RBC vs. N-RBC as follows (nmol/ml): 2,5-DHBA, 1.45 +/- 0.06 vs. 1.81 +/- 0.05 (p = 0.001); 2,3-DHBA, 1.89 +/- 0.21 vs. 1.15 +/- 0.08 (p = 0.008) and catechol, 0.87 +/- 0.13 vs. 0.38 +/- 0.05 (p = 0.006). The results showed significant increase in the total SA hydroxylation in Th-RBC as compared to N-RBC with a tendency to form 2,3-DHBA and catechol at the expanse of 2,5-DHBA. The excessive .OH generation in Th-RBC is attributed to the abnormally high content of redox active iron in the cytosolic and/or membrane compartments of these cells.

Nitroxide stable radical prevents primaquine-induced lysis of red blood cell.

Primaquine (PQ), an antimalarial drug, is known to produce multiple oxidative effects in red blood cells (RBC). Because H2O2, OH and intracellular superoxide are implicated in this oxidation, the effect of cell-permeable nitroxide 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) capable of scavenging O2.- has been studied. PQ caused RBC lysis and facilitated the oxidation of oxyhemoglobin (oxyHb) to methemoglobin (MetHb). The lysis was partially inhibited by catalase and by the metal chelating agent 2,2-dipyridyl. TEMPO blocked PQ-induced RBC lysis in dose-dependent manner (2 mM IC50) but enhanced the oxidation of oxyHb to MetHb. PQ facilitated the lysis also in the presence of CO but without effecting Hb oxidation. This hemolysis, however, was inhibited by TEMPO. The results indicated that: (a) no causative relationship exists between PQ-induced Hb oxidation and RBC lysis; (b) TEMPO can directly oxidize heme-iron without causing membrane injury; (c) the aerobic toxicity of PQ in this system is mediated by O2.- and H2O2 and possibly by redox-active labile metals (d) TEMPO can protect by detoxifying O2.- and oxidizing reduced labile metal ions and thus blocking their participation in Fenton reaction.

Protective effects of rutin against hemoglobin oxidation.

A prooxidant drug, primaquine (PQ) was used to produce oxidative stress in human red blood cells (RBC) in vitro. Rutin, a plant flavonoid, did not prevent PQ-induced cell lysis but protected against hemoglobin (Hb) oxidation inside RBC. After PQ removal, rutin failed to reduce preformed met-Hb indicating that the rutin protective effect manifests only in the presence of PQ. Since H2O2 was proved to mediate PQ-induced Hb oxidation, authentic Hb was studied for its reaction with H2O2 and rutin in solution. Rutin partially protected oxy-Hb against H2O2-induced oxidation and heme loss. Rutin was also shown to delay H2O2-induced met-Hb oxidation to ferryl-Hb. Rutin directly reduced ferryl-Hb to met-Hb in stoichiometric (1:1) reaction characterized by a rate constant of 100 to 130/M/sec. It is assumed that by reducing ferryl-Hb, rutin prevents oxy-Hb from reacting with ferryl-Hb (comproportionation reaction), thus preventing half of the oxy-Hb molecules from being converted to met-Hb. This mechanism is consistent with 50% inhibition by rutin (at the maximum of its activity) of PQ-induced oxy-Hb oxidation in RBC. The present results demonstrate new antioxidant properties of rutin that may be useful in diminishing oxidative damage to pathological red blood cells.

Primaquine-induced superoxide production by beta-thalassemic red blood cells.

Primaquine, a prooxidant antimalarial drug, incubated with human red blood cells (RBC) induced marked superoxide generation in the cells as detected by exogenous cytochrome c reduction. In the presence of primaquine, beta-thalassemic RBC produced significantly more superoxide than normal RBC, thus reflecting the vulnerability of beta-thalassemic cells to oxidative stress.

[Synthesis of acridine derivatives of amino acid hydrazines and their antimalarial activity]. / Sintez akridinovykh proizvodnykh gidrazidov aminokislot i ikh protivomaliariinaia aktivnost'.

2-Methoxy-6-chloroacridine-9-yl- and 2-ethoxy-6-nitroacridine-9-yl-hydrazides of glycine, alpha- and beta-alanines, gamma-aminobutiric acid, epsilon-aminocaproic acid have been synthesized and their antimalarial activity has been investigated. The compounds were found to inhibit the growth of malaria parasite P. falciparum in in vitro cultures. Fifty per cent inhibitory concentrations ranged from 2 x 10(-7) to 6 x 10(-7) M and corresponded to therapeutic concentrations of known quinoline and acridine antimalarial drugs. The beta-alanine and gamma-aminobutiric acid derivatives were the most active and showed high activity against a chloroquine resistant strain of P. falciparum.

[Biochemical aspects of the interaction of the malarial parasite, Plasmodium berghei, with erythrocytes of the host]. / Biokhimicheskie aspekty vzaimodeistviia maliariinogo parazita Plasmodium berghei s éritrotsitom khoziaina.

Mice erythrocytes, infected with Plasmodium berghei, were studied as a model of integrated cell system "parasite-host erythrocytes". The rate of blood oxygenation, content of methhemoglobin and methhemoglobin reductase activity maintained at the initial level up to the lethal outcome of the infectious process. A steady-state kinetics of glycolysis was studied in the impaired erythrocytes by means of automatic potentiometric titration of lactic acid during incubation of the cells. Distinct 15-20-fold activation of glycolysis occurred as the parasite grew and developed in erythrocytes. Stationary concentration of ATP was decreased at the moment of extensive P. berghei development, accompanied by accumulation in blood of the most mature forms of parasite. At the same time, this step of infection was characterized by a 2-fold increase in content of reduced glutathione and by appearance of of lipoxygenase in the impaired erythrocytes. Activities of superoxide dismutase as well of oxidative pentosephosphate shunt were kept at the initial level. The data obtained are discussed on the basis of current concepts on biochemistry of blood forms of malarial parasite.

Hemoglobinopathies and glucose-6-phosphate dehydrogenase deficiency in one of the regions of Azerbaijan: mass screening and laboratory investigations.

Mass screening in the Geok-chai region of Azerbaijan covered 264 persons. 2 families with abnormal hemoglbin were detected. Electrophoresis in PAG and on cellulose acetate films as well as sickling test showed that in 3 out of 9 members of one of the families HbS was detected. 6 out of 8 members of the others family appeared to be HbD-carriers. In 4 members of this family abnormal hemoglobin was found out in combination with G-6-PDH deficiency. No clinical manifestations were found. A number of beta-thalassemia cases were detected in screened children as well as in adults. Hemoglobin oxygen equilibrium curves were studied in patients with heterozygous beta-thalassemia. In case of G-6-PDH deficiency when it is not possible to obtain active enzyme zones on the columns with PAG these zones can be detected when CoCl2 (2 mM) is added into the incubation medium.