Free radical scavenging activity of silibinin in nitrite-induced hemoglobin oxidation and membrane fragility models

Free radical formation in heme proteins is recognized as a factor in mediating the toxicity of many drugs. Xenobiotics and drug therapy-related toxicity, due to oxidative modification of hemoglobin (Hb), has been attributed in part to the uncontrolled oxidative reactions. A variety of antioxidant strategies to ameliorate potential oxidative damage in vivo have been suggested. The present study was designed to evaluate the dose–response relationship of the free radical scavenging properties of silibinin dihemisuccinate (SDH) in nitrite-induced Hb oxidation in vitro and in vivo. Different concentrations of SDH were added, before and after different intervals of inducing Hb oxidation in erythrocytes lysate, and formation of methemoglobin (MetHb) was monitored spectrophotometrically; the same approach was utilized to evaluate the effect of the same doses of SDH on the integrity of erythrocytes after induction of hemolysis. Moreover, the most effective dose of SDH was administered in rats before challenge with toxic dose of sodium nitrite, and MetHb formation was monitored as mentioned before. The results showed that in both in vitro and in vivo models, SDH successfully attenuates Hb oxidation after challenge with sodium nitrite; this protective effect was not related to the stage of the catalytic stage of Hb oxidation, though the effect was more prominent when the compound was administered before nitrite. In conclusion, SDH can effectively, in concentration-dependent pattern, attenuate sodium nitrite-induced Hb oxidation and maintain integrity of red blood cells both in vitro and in vivo.     

Comparison of histamine release in cord blood and adult blood.

Histamine release (HR) after stimulation with anti-IgE, concanavalin A (ConA) and Formyl-Met-Leu-Phe (FMLP) from 97 cord blood samples was compared to results obtained in identically treated blood samples from adults. The maximal HR obtained with anti-IgE did not differ significantly from the values obtained in adult blood, although a ten times higher concentration of anti-IgE was required for maximum release. Passive sensitization with IgE-rich plasma caused a significant increase in maximal anti-IgE-induced HR in the majority of cord blood samples, and the dose-response curve was similar to that obtained in adult blood. Challenge with ConA and FMLP caused a HR similar to that seen in adult blood, but the close correlation between anti-IgE- and concanavalin A-induced HR seen in adult blood was absent in cord blood.     

Comparison of soluble and membrane bound HLA-class I and DR levels in umbilical cord blood and adult peripheral blood

HLAs are membrane bound (membrane HLA mHLA) or secreted as soluble forms(soluble HLA = sHLA) in plasma or serum. Umbilical cord blood (UCB) mHLA, from mononuclear cells (MNCs), and sHLA were quantified by flow cytometry and enzyme linked immunosorbant assay (ELISA), respectively, and compared to levels of MNC-mHLA and sHLA in adult peripheral blood (PB). The mean fluorescence intensity (MFI) of mHLA-I was 3-fold lower in UCB-MNCs than in adult PB-MNCs, however, due to higher cell numbers in UCB, total mHLA-I quantities per ml of blood were not different in UCB and adult PB. In addition, sHLA-I in UCB was significantly lower than in adult PB. The MFI of mHLA-DR from UCB-MNCs was significantly higher than the MFI of mHLA-DR in adult PB-MNCs, and the total mDR quantity (per ml of blood) in UCB was 3-fold higher than in adult PB. sHLA-DR concentrations, however, were equivalent in UCB and adult PB. These findings indicate that fetal tissue or cells might excrete smaller quantities of both sHLA-I and sDR antigens than adult tissues or cells. Alternatively, fetal sHLA antigens might be unstable and degradated compared to those of adult.     

Effects of amyl nitrite on circulation,respiration and blood homoeostasis in cyanide poisoning

The effects of intravenously (i.v.) administered or inhaled amyl nitrite (AN) were followed under chloralose anaesthesia in intact and cyanide-poisoned, spontaneously breathing beagles. The i.v. doses of AN were 0.03 and 0.15 mmol/kg and the i.v. dose of KCN was 0.06 mmol/kg. AN was inhaled in a closed system at 0.15 mmol/kg without previous poisoning and, in addition, at 0.074 mmol/kg (two ampoules at 0.3 ml AN) during artificial ventilation after poisoning with 0.045 mmol KCN/kg i.v., Mean arterial pressure decreased by 15 and 40 mmHg, respectively, after i.v. injection of AN, associated with bradycardia and lowered peripheral blood flow. Respiratory minute volume rose by 65% with the higher dose. Arterial pO₂ decreased by 20 mmHg while pCO₂ rose by 6 mmHg. Within 30 min of injection, these changes were only partially reversible. Similar results were obtained following inhalation of AN in a closed system. Lactic acidosis and lowering of pH were produced by the i.v. route, but not by inhalation. Total haemoglobin increased. The lethality of KCN was abolished with AN doses that produced 10–30% ferrihaemoglobin. Artificial ventilation and simultaneous inhalation of AN after poisoning with lethal doses of KCN turned out to be ineffective therapeutic measures. The findings are compared with those of other papers dealing with cyanide poisoning and AN. It is pointed out that, for the present, there is no experimental proof for another antidotal mechanism of action of AN than ferrihaemoglobin formation.     

白桦酯醇的氧化动力学性质及含量分析

目的:测定白桦酯醇清除羟自由基的能力以研究白桦酯醇的氧化动力学性质及其含量分析。方法:根据I3-与罗丹明B(λm ax=556 nm)形成离子缔合物,使体系的吸收峰降低。而白桦酯醇能与羟自由基反应,从而抑制了I3-的生成,使得556 nm处吸光度上升,吸光度的变化与白桦酯醇的浓度相关。结果:白桦酯醇浓度在0~30.94μg.mL-1范围内符合比耳定律,相关系数为0.9991,回收率为98.9%(n=6),RSD为2.1%。该法适合测定白桦酯醇含量。白桦酯醇的氧化反应为一级反应,其表观活化能为84.25 kJ.mol-1,反应速率常数为3.3×10-3m in-1。它可用作潜在的天然抗氧剂。     

Phosphine-mediated Heinz body formation and hemoglobin oxidation in human erythrocytes

In this work the in vitro reactions of phosphine with intact red blood cells and membrane-free hemoglobin extracts are reported. We demonstrate that phosphine or phosphine derivatives induce dense aggregates of denatured hemoglobin known as 'Heinz bodies' in intact red blood cells. The reaction products include irreversible hemichrome formation. We further demonstrate an oxygen requirement for these effects. PH3 appears to act as a novel type of O2 radical chain initiator or propagator with heme proteins.     

Analysis of hemoglobin adducts from acrylamide, glycidamide, and ethylene oxide in paired mother/cord blood samples from Denmark

The knowledge about fetal exposure to acrylamide/glycidamide from the maternal exposure through food is limited. Acrylamide, glycidamide, and ethylene oxide are electrophiles and form adducts with hemoglobin (Hb), which could be used for in vivo dose measurement. In this study, a method for analysis of Hb adducts by liquid chromatography-mass spectrometry, the adduct FIRE procedure, was applied to measurements of adducts from these compounds in maternal blood samples (n = 87) and umbilical cord blood samples (n = 219). The adduct levels from the three compounds, acrylamide, glycidamide, and ethylene oxide, were increased in tobacco smokers. Highly significant correlations were found between cord and maternal blood with regard to measured adduct levels of the three compounds. The mean cord/maternal hemoglobin adduct level ratios were 0.48 (range 0.27-0.86) for acrylamide, 0.38 (range 0.20-0.73) for glycidamide, and 0.43 (range 0.17-1.34) for ethylene oxide. In vitro studies with acrylamide and glycidamide showed a lower (0.38-0.48) rate of adduct formation with Hb in cord blood than with Hb in maternal blood, which is compatible with the structural differences in fetal and adult Hb. Together, these results indicate a similar life span of fetal and maternal erythrocytes. The results showed that the in vivo dose in fetal and maternal blood is about the same and that the placenta gives negligible protection of the fetus to exposure from the investigated compounds. A trend of higher levels of the measured adducts in cord blood with gestational age was observed, which may reflect the gestational age-related change of the cord blood Hb composition toward a higher content of adult Hb. The results suggest that the Hb adduct levels measured in cord blood reflect the exposure to the fetus during the third trimester. The evaluation of the new analytical method showed that it is suitable for monitoring of background exposures of the investigated electrophilic compounds in large population studies.     

不同孕周新生儿脐血血红蛋白电泳结果分析

【摘要】目的 了解贵阳市户籍人群α-地中海贫血（α-地贫）的发生率,探讨不同孕周新生儿各种血红蛋白（Hb）相对含量的变化情况。方法 收集贵阳市户籍人群不同孕周新生儿脐血样本1 222份,采用Sebia全自动电泳仪对新生儿脐血进行血红蛋白电泳分析,根据Hb Bart’s检出率及相对含量,了解本地α-地贫的发生率及其类型。将新生儿按孕周分为5个组,比较不同孕周新生儿各种血红蛋白相对含量的差异。结果 在1 222份脐血样本中,检出Hb Bart’s阳性,即α-地贫36例,检出率为2.95%,其中标准型23例,静止型12例,中间型1例,未检出重型患儿;检出异常血红蛋白2例,均为Hb J,检出率为0.16%。新生儿脐血中Hb成分主要为Hb F,各组Hb F相对含量的均值都在75%以上。随着孕周的增加,Hb F相对含量逐渐降低,而Hb A相对含量逐渐增加。结论 贵阳市是α-地贫的高发区,不同孕周新生儿血红蛋白各成分的相对含量呈动态变化。     

Pharmacokinetics of sodium nitrite-induced methemoglobinemia in the rat.

A biologically based mathematical model was created to characterize time and dose-dependent relationships between exposure to nitrite and induction of methemoglobinemia. The model includes mass action equations for processes known to occur: oral absorption of nitrite, elimination from the plasma, partitioning between plasma and erythrocytes, binding of nitrite to hemoglobin and methemoglobin, and the free radical chain reaction for hemoglobin oxidation. The model also includes Michaelis-Menten kinetics for methemoglobin reductase-catalyzed regeneration of hemoglobin. Body weight-scaled rate constants for absorption (k(a)) and elimination (k(e)), the effective erythrocyte/plasma partition coefficient (P), and the apparent K(m) for methemoglobin reductase were the only parameters estimated by formal optimization to reproduce the observed time course data. Time courses of plasma nitrite concentrations and blood levels of hemoglobin and methemoglobin in male and female rats that had received single intravenous or oral doses of sodium nitrite were measured. Peak plasma levels of nitrite were achieved in both sexes approximately 30 min after oral exposure, and peak methemoglobin levels were achieved after 100 min. The model predicts that 10% of the hemoglobin is oxidized to the ferric form after oral doses of 15.9 mg/kg in male rats and 11.0 mg/kg in female rats and after intravenous doses of 8.9 and 7.1 mg/kg in male and female rats, respectively. The t(1/2) for recovery from methemoglobinemia was 60 to 120 min depending on dose and route of administration. A sensitivity analysis of the model was performed to identify to which parameters the predictions of the model were most sensitive and guide attempts to simplify the model. Replacement of the V(max) of methemoglobin reductase with a value representative of humans predicted a 10% methemoglobinemia following an intravenous dose of 5.8 mg/kg, in close agreement with an observed value of 5.7 mg/kg for humans.     

Kinetics of salicylates in blood and joint fluid

Summary Samples of blood and joint fluid from 30 patients who had taken buffered acetylsalicylic acid were examined for concentrations of total salicylates (TSA), acetylsalicylate (ASA) and salicylate (SA). The data were arranged in groups according to diagnosis of the joint disease. Analysis of the data did not show significant difference in the kinetics of TSA into blood. In groups the time to first appearance of 0.3 mg/l averaged 6.3 min for TSA; these values averaged 7.7 min for ASA and 10.9 min for SA. Close to maximum concentrations in blood averaged 18.9 mg/l for TSA, 3.3 mg/l for ASA, and 23.3 mg/l for SA. The time for first appearance of 0.3 mg/l of total salicylates in joint fluid ranged from 10 to 34 min with an average of 18.1 min; the values of ASA averaged 19.4 min and those of SA 21.9 min. The maximum concentration in joint fluid averaged 15.7 mg/l for TSA, 2.5 mg/l for ASA, and 14.5 mg/l for SA. Transport of salicylates from blood to joint fluid showed a pattern consistent with the type of joint disease. Support was found for the hypothesis that diffusion was the major factor in the movement of salicylates from blood to joint fluid.This study was partly supported by grants of U.S. Public Health Service AM 01431 and Bristol-Myers Company.     

Kinetics of hypobromous acid-mediated oxidation of lipid components and antioxidants

Hypohalous acids are generated from the oxidation of halide ions by myeloperoxidase and eosinophil peroxidase in the presence of H2O2. These oxidants are potent antibacterial agents, but excessive production can result in host tissue damage, with this implicated in a number of human pathologies. Rate constants for HOCl with lipid components and antioxidants have been established. Here, the corresponding reactions of HOBr have been examined to determine whether this species shows similar reactivity. The second-order rate constants for the reaction of HOBr with 3-pentenoic acid and sorbate, models of unsaturated lipids, are 1.1x10(4) and 1.3x10(3) M(-1) s(-1), respectively, while those for reaction of HOBr with phosphoryl-serine and phosphoryl-ethanolamine are ca. 10(6) M(-1) s(-1). The second-order rate constants (M(-1) s(-1)) for reactions of HOBr with Trolox (6.4x10(4)), hydroquinone (2.4x10(5)), and ubiquinol-0 (2.5x10(6)) were determined, as models of the lipid-soluble antioxidants, alpha-tocopherol, and ubiquinol-10; all of these rate constants are ca. 50-2000-fold greater than for HOCl. In contrast, the second-order rate constants for the reaction of HOBr with the water-soluble antioxidants, ascorbate and urate, are ca. 10(6) M(-1) s(-1) and closer in magnitude to those for HOCl. Kinetic models have been developed to predict the sites of HOBr attack on low-density lipoproteins. The data obtained indicate that HOBr reacts to a much greater extent with fatty acid side chains and lipid-soluble antioxidants than HOCl; this has important implications for HOBr-mediated damage to cells and lipoproteins.     

Mononuclear cells from peripheral blood of adult donors and from cord blood are equally protected by alpha- and beta-interferons against infection with HTLV-I

Human mononuclear cells derived from peripheral blood of adult donors (PBMC) or from neonatal cord blood (CBMC) were found to be equally sensitive to the protective effect of alpha- and beta-interferons (IFNs) against the infection with HTLV-I during long-term culture. The effect of IFNs was evidenced by a remarkable reduction of the number of virus-positive cells during culture as evaluated by indirect immunofluorescence for the p19 virus core protein. Moreover, the appearance of p19-positive immortalized clones was inhibited by IFNs in PBMC co-cultures, whereas it was delayed in CBMC cultures. These kinetics are in relation with the higher permissivity of CBMC to the virus in comparison with PBMC, since in CBMC cultures infected cells can be clearly detected starting already 1 week post-infection (p.i.), whereas in PBMC cultures their appearance time is approximately at the 6th week p.i. IFNs acted by 'priming' PBMC and CBMC to an active antiviral competence, since one single treatment with 1000 IU/ml of alpha- or beta-IFN at the onset of the co-culture of mononuclear cells with irradiated virus-donor cells was able to maintain very low levels of infection for approximately 6 weeks in CBMC cultures and at least for 18 weeks in PBMC cultures. As a consequence, it seems likely that IFN action is mediated by the expression of a defined, although not completely identified, set of genes in the host cells.