metHb Formation Research Articles

Comparison of the effect of Aminopielik D pesticide and its active components on human erythrocytes

In the present work, the effect of Aminopielik D [417.5 g/l of dimethylamino salts of 2,4-dichlorophenoxyacetic acid (2,4-D) and 32.5 g/l of 3,6-dichloro-2-metoxybenzoic acid (Dicamba)] and its active components (used separately and in mixture) on human erythrocytes was examined. The parameters studied were: lipid peroxidation, metHb formation and catalase activity. Aminopielik D used at doses of 100–1000 ppm was found to increase lipid peroxidation, decrease of catalase activity and oxidation of haemoglobin. 2,4-D and Dicamba are present in Aminopielik D in the dimethylamino form; their sodium salts in solution (separately and as a mixture) did not cause such strong effects. A synergistic action of 2,4-D and Dicamba was excluded as the individual compounds caused the same effects as their mixture. Aminopielik D provoked slightly higher changes in the lipid peroxidation and catalase activity than its active components alone and in mixture, which was probably a result of the properties of the additives and interaction of tested systems with the dimethylamino group.

The effects of nitroglycerine on the redox status of rat erythrocytes and reticulocytes

The effects of nitroglycerine (NTG) are mediated by liberated nitric oxide (NO) after NTG enzymatic bio-transformation in cells. The aim of this study was to evaluate some products of NTG bio-transformation and their consequences on the redox status of rat erythrocytes and reticulocytes, considering the absence and presence of functional mitochondria in these cells, respectively. Rat erythrocyte and reticulocyte-rich red blood cell (RBC) suspensions were aerobically incubated (2 h, 37 degrees C) without (control) or in the presence of different concentrations of NTG (0.1, 0.25, 0.5, 1.0 and 1.5 mM). In rat erythrocytes, NTG did not elevate the concentrations of any reactive nitrogen species (RNS). However, NTG robustly increased concentration of methemoglobin (MetHb), suggesting that NTG bio-transformation was primarily connected with hemoglobin (Hb). NTG-induced MetHb formation was followed by the induction of lipid peroxidation. In rat reticulocytes, NTG caused an increase in the levels of nitrite, peroxinitrite, hydrogen peroxide, MetHb and lipid peroxide levels, but it decreased the level of the superoxide anion radical. Millimolar concentrations of NTG caused oxidative damage of both erythrocytes and reticulocytes. These data indicate that two pathways of NTG bio-transformation exist in reticulocytes: one generating RNS and the other connected with Hb (as in erythrocytes). In conclusion, NTG bio-transformation is different in erythrocytes and reticulocytes due to the presence of mitochondria in the latter.

Electron Paramagnetic Resonance Study of Cell-Free Hemoglobin in Sickle Cell Disease: Potential Antioxidant Role of Haptoglobin.

It has previously been shown that red blood cell-free oxyhemoglobin (oxyHb) can affect endothelial function in Sickle Cell Disease (SCD) by virtue of its NO scavenging properties (Reiter et al, Nat Med, 2002). This reaction may have down-stream consequence via formation of metHb which has been demonstrated to have pro-oxidant properties and can oxidize low-density lipoprotein in vitro. In this study we have examined the fate of metHb in SCD vs normal plasma by examining the characteristic single line EPR signature of metHb in the g = 6 region. We show that the EPR signature of metHb differs in SCD compared with normals and exhibits a split EPR line indicative of a more rhombic heme geometry (See Figure). [Display omitted] Addition of purified metHb to SCD plasma results in a conversion of the single line EPR spectrum to the rhombic form. By addition of hemin and hemoglobin to plasma constituents we have determined that the rhombic EPR spectrum is consistent with heme bound to serum albumin. In addition, immunoprecipitation of serum albumin from SCD plasma with EPR analysis of the IP fraction revealed the presence of albumin associated metHb. Interestingly, the transfer of heme from metHb to albumin is accelereated by the presence of low-density lipoprotein and inhibited by the addition of haptoglobin suggesting that plasma components can modulate heme release from metHb and that the lack of haptoglobin in SCD plasma allowed the transfer of heme from metHb to albumin. To confirm this we added metHb to SCD plasma in the presence and absence of haptoglobin and observed that the addition of haptoglobin prevented conversion of the metHb spectrum to the albumin associated split (rhombic) species. From these studies we conclude that plasma haptoglobin not only binds hemoglobin but also limits heme release from metHb. To examine potential oxidative consequences of this effect, we incubated LDL with metHb in the presence and absence of haptoglobin. Haptoglobin inhibited LDL oxidation in a dose-dependent manner. Interestingly, the 1-1 isoform of haptoglobin was much more effective than the 2-2 isoform, in agreement with published studies on the efficacy of hemoglobin binding to the two haptoglobin varieties. Additionally, we have measured levels of 8-isoprostanes in the plasma of SCD patients as an index of lipid oxidation and show that 8-isoprostane levels are significantly elevated in SCD patients (295 209 pg/ml, mean S.D., n= 13, vs. 69 37 pg/ml, mean S.D. n= 5 for normal controls). In conclusion, these studies provide evidence that the saturation of the haptoglobin clearance mechanisms in hemolytic disorders such as SCD can not only affect the NO-axis of endothelial function due to increases in cell-free hemoglobin, but can also lead to altered heme metabolism leading to lipid oxidation and formation of pro-inflammatory lipids which have the potential to alter both endothelial and red cell function.

Various Drugs with or without an Antisickling Effect in the In Vitro Tests Showed a Strong Antisickling Effect in the In Vivo Studies in Transgenic Sickle Mice.

Since its inception in 1997, the NHLBI Sickle Cell Disease (SCD) Reference Laboratory (Ref Lab) has received over 150 candidate drugs from over 30 researchers, universities and companies for evaluation. All candidate drugs are evaluated for both their beneficial and adverse effects in vitro using more than 10 sophisticated methods. Specifically, each candidate drug is studied to see whether it inhibits cell sickling, prolongs the delay time prior to deoxy-hemoglobin (Hb) S polymerization, increases the solubility of deoxy-Hb S, hydrates red blood cells (RBCs), prevents dehydration of RBCs, shifts the oxygen equilibrium curve (OEC) toward the left, reduces the adhesion of RBCs to endothelial cells, promotes the synthesis of Hb F, causes hemolysis, promotes formation of met-Hb and increases the denaturation of intracellular Hb S. With this new approach, we have found more than 10 new antisickling agents that showed beneficial effects without significant adverse effects in the in vitro tests. Of these, five drugs were further studied in vivo using transgenic mice that produce human Hb S, and all five drugs inhibited the formation of sickled cells in the blood and significantly prolonged their survival time under acute hypoxic conditions. The five drugs are NIPRISAN (plant extracts) [Brit. J. Haematol. (BJH) 122:1001,2003], MX-1520 (prodrug of vanillin) (BJH 125: 788, 2004), 5-hydroxymethyl-2-furfural (5HMF) (BJH 128:552, 2005), NS3623 (Blood 97: 1461,2001, in vivo results not shown), and FLOCOR (surfactant) (Blood 94:420a, 1999). Of these 5 drugs, FLOCOR and NS3623 did not have an antisickling effect in vitro. It is quite interesting that FLOCOR and NS3623 which showed no direct antisickling effect in vitro, showed a strong antisickling effect in vivo. Some of our results suggest that combined use of some of these drugs may have a synergistic effect in vivo. Studies on the combined use of these drugs are planned. Researchers with candidate drugs that may have beneficial effects for SCD are welcome to contact the SCD Ref Lab for free in vitro evaluation of their agents (for more information, please see our website at www.tatcom.com/sickle-cell ).

Hypoxia-Induced Post-Translational Changes in Red Blood Cell Protein Map of Newborns

Tyrosine (Tyr) phosphorylation is implicated in the modification of several erythrocyte functions, such as metabolic pathways and membrane transport, as well as in signal transduction systems. Here we describe the map of Tyr-phosphorylated soluble proteins of newborn red blood cells (RBC) using an in vitro model simulating RBC reoxygenation at birth after an intrauterine hypoxic event. We tested the hypothesis that a hypoxic environment and subsequent reoxygenation promote post-translational changes in the RBC protein map of newborns, in addition to desferrioxamine (DFO)-chelatable iron (DCI) release and methemoglobin (MetHb) formation. Umbilical cord blood RBC were incubated under hypoxic conditions for 16 h at 37 degrees C, and subsequently for 8 h under aerobic conditions. Control erythrocytes were incubated under aerobic conditions at 37 degrees C for the period of the experiment, i.e. for 24 h. Tyr-phosphorylation proteins were assessed using advanced high-resolution two-dimensional electrophoresis, 2-D immunoblot analysis with anti-phosphotyrosine (anti-pTyr) antibodies, and computer-aided electrophoretogram analysis. Higher DCI release and MetHb formation were observed in newborn RBC incubated under hypoxic conditions than in those incubated aerobically. Different immunoreactivity patterns with anti-pTyr antibodies were also observed between newborn RBC incubated under hypoxic conditions and controls. A hypoxic environment is a factor promoting DCI release, a well-known condition of oxidative stress. This is the first map of Tyr-phosphorylated soluble proteins of newborn RBC obtained using an in vitro model simulating RBC reoxygenation at birth after an intrauterine hypoxic event. Our results suggest that hypoxia increases Tyr-phosphorylation of antioxidant proteins, protecting RBC against oxidative stress.

Concentration-dependence of aniline-induced methemoglobinemia in dogs: A derivation of an acute reference concentration

Four male Beagle dogs per group were head-only exposed to aniline vapor in concentrations ranging from 15.8 to 493.6 mg/m 3. Each exposure session was for 4 h. Additionally, one group of dogs was both head-only and whole-body exposed to approximately 241 mg/m 3 to better appreciate the contribution of dermal uptake on methemoglobin (MetHb) induction. The exposure paradigm chosen was comparable to a dose-escalation type of study, however, providing adequate recovery periods between each exposure session to exclude ‘carry-over’ effects from one exposure session to another. This was verified by the determination of standard hematology endpoints before and after each exposure session. The time- and concentration-dependence of MetHb was analyzed as the key endpoint. The results of this study show that MetHb was maximal around the end of the 4 h exposure period. Significantly increased MetHb was observed at concentrations equal and above 30.3 mg/m 3 at a duration of exposure of at least 60 min. The extent of MetHb formation observed in some hyperventilating dogs suggests that the dosing-rate is important for the outcome of test. Whole body exposed dogs displayed markedly higher MetHb values when compared to head-only exposed dogs. Taking into account the entire concentration–time-effect relationship 23.6 and 20.6 mg/m 3 were estimated to be the threshold concentrations to cause 0.8% MetHb following a 4 and 8 h exposure period, respectively. The C n × t analysis of MetHb formation is consistent with a concentration-dependent restoration kinetics of MetHb suggesting that results from high level exposures cannot readily be extrapolated to low-level exposures. In summary, this study demonstrates that for aniline, an agent known to be bioactivated by a hepatic first-pass metabolism, the rate of delivery and uptake appears to be decisive for the extent of MetHb formation. Carry-over effects related to erythrotoxicity or Heinz body formation were not observed at any exposure level.

Nitrite transport into pig erythrocytes and its potential biological role

To study nitrite transport and its oxygenation dependency in pig erythrocytes, as this is fundamental to the possible participation of nitrite in blood flow regulation via its reduction to nitric oxide by deoxygenated haemoglobin (Hb). Pig red blood cells (RBCs) were tonometer-equilibrated to physiological pCO2 in oxygenated and deoxygenated states. Nitrite was added and the kinetics of NO2- influx and methaemoglobin (metHb) formation were assessed at variable temperature and haematocrit. Nitrite quickly permeated and equilibrated across the membrane, and then continued to enter RBCs as a consequence of its intracellular removal (via reactions with Hb to form nitrate and metHb in oxygenated cells, and NO and metHb in deoxygenated cells). The membrane permeation as such showed little oxygenation dependency, but as metHb formation was significantly higher in oxygenated than deoxygenated RBCs, nitrite transport tended to be largest into oxygenated RBCs. This contrasts with a preferential permeation of deoxygenated RBCs in some fish species. Nitrite transport showed low temperature sensitivity but was speeded up at low haematocrit via more rapid intracellular nitrite removal (metHb formation). Nitrite influx was not affected by inhibitors of facilitated diffusion (DIDS, phloretin and PCMB) and may occur via conductive transport. Extracellular pH was stable during nitrite transport. Nitrite extensively permeates both oxygenated and deoxygenated pig RBCs, which may enable a dual function of nitrite entry: viz. conversion to NO at low pO2 to promote blood flow and detoxification to non-toxic nitrate at inappropriate high nitrite levels.

Iron nitrosyl hemoglobin formation from the reaction of hydroxylamine and hemoglobin under physiological conditions

Sickle cell disease patients receiving hydroxyurea (HU) therapy have shown increases in the production of nitric oxide (NO) metabolites, which include iron nitrosyl hemoglobin (HbNO), nitrite, and nitrate. However, the exact mechanism by which HU forms HbNO in vivo is not understood. Previous studies indicate that the reaction of oxyhemoglobin (oxyHb) or deoxyhemoglobin (deoxyHb) with HU are too slow to account for in vivo HbNO production. In this study, we show that the reaction of methemoglobin (metHb) with HU to form HbNO could potentially be fast enough to account for in vivo HbNO formation but competing reactions of either excess oxyHb or deoxyHb during the reaction reduces the likelihood that HbNO will be produced from the metHb-HU reaction. Using electron paramagnetic resonance (EPR) spectroscopy we have detected measurable amounts of HbNO and metHb during the reactions of oxyHb, deoxyHb, and metHb with excess hydroxylamine (HA). We also demonstrate HbNO and metHb formation from the reactions of excess oxyHb, deoxyHb, or metHb and HA, conditions that are more likely to mimic those in vivo. These results indicate that the reaction of hydroxylamine with hemoglobin produces HbNO and lend chemical support for a potential role for hydroxylamine in the in vivo metabolism of hydroxyurea.

Decreased deformability of donor red blood cells after intrauterine transfusion in the human fetus: possible reason for their reduced life span?

The life span of donor red blood cells (RBCs) is reduced in the fetus with Rh hemolytic disease. This may have resulted from donor or recipient factors. Studied in vitro was the effect of gamma irradiation on hemolysis, methemo-globin (metHb), and lipid peroxidation of donor RBCs and the ability of fetal and adult plasma to protect irradiated RBCs from induced lipid peroxidation. Also studied in vivo were the effects after the time that donor RBCs reside in the fetus by measuring its lipid peroxidation, cholesterol-to-phospholipid ratios, and deformability of RBCs. Irradiation barely increased hemolysis and metHb formation and did not increase lipid peroxidation. Plasma samples of D+ fetuses inhibited induced oxidative stress less than plasma samples of adults. Nevertheless, in vivo lipid peroxidation of the donor RBC membrane had not increased, whereas the molar cholesterol-to-phospholipid ratio increased from 1.08 +/- 0.11 to 1.38 +/- 0.12. It became identical to that of the fetal RBCs (1.44 +/- 0.12). Before transfusion, the deformability of the adult RBCs (elongation index, 0.578 +/- 0.013) was better than that of the fetal cells (elongation index, 0.494 +/- 0.027), but decreased to fetal levels after transfusion (elongation index, 0.518 +/- 0.039). Irradiation of the RBCs and a reduced fetal antioxidant capacity do not lead to in vivo lipid peroxidation. The shorter life span of donor cells in the fetus probably results from a decreased deformability of the RBCs after transfusion, most likely owing to an increased cholesterol-to-phospholipid ratio.

Potential of peroxynitrite to promote the conversion of oxyhemoglobin to methemoglobin.

Superoxide anion and NO can react to form the highly oxidizing species peroxynitrite (ONOO-) which can react directly with hemoglobin (Hb) even in the presence of physiological concentration CO2. This research was to determine the ONOO- -mediated oxidation damage to the heme of oxyhemoglobin (oxyHb) under conditions expected in blood. Results showed that 8-10 mol ONOO- was needed to quickly and completely convert 1 mol oxyHb to methemoglobin (metHb). ONOO- (20-140 microM) caused rapid and extensive formation of metHb from oxyHb (50 microM) mainly occurring within first 5-20 min of incubation. The conversion efficiency reached 16%, 48%, 60%, 79% and 88% output of metHb after 90 min of incubation at 0, 20, 40, 100, and 140 microM ONOO- respectively. 1 mM CO2 caused a small decrease in the ability of ONOO- to oxidize oxyHb, and ONOO- -promoted conversion of oxyHb to metHb increased when pH decreased from 8.0 to 6.0. Relatively lower temperature in blood condition will inhibit this reaction in some degree. We postulate that ONOO- can mediate oxidation damage to the heme, and cause heme loss from the hydrophobic cavity of Hb when its concentration exceeded 90 microM. These results indicated that ONOO- could convert oxyHb to metHb under the conditions expected in blood, and this reaction was regulated by CO2 concentration, reaction time, temperature and pH value.

Verapamil prevents impairment in filterability of human erythrocytes exposed to oxidative stress.

Effects of oxidative stress on intact human erythrocytes were investigated using tert-butyl hydroperoxide (tBHP). Exposure of erythrocytes to tBHP caused a marked decrease in filterability in a time-dependent manner. Erythrocytes exposed to tBHP also show an increase in mean corpuscular volume and a remarkable formation of methemoglobin (met-Hb) without any appearance of hemichromes that form Heinz bodies. High performance liquid chromatography demonstrated that the tBHP-treated erythrocytes exhibited an apparent decrease in the membrane phospholipid, phosphatidylethanolamine (PE). The decrease in PE was inhibited by pretreatment with ascorbate, but not with verapamil. SDS-polyacrylamide gel electrophoresis of the tBHP-treated erythrocyte membrane showed a degradation of spectrin, band 3, band 4.2, and band 4.5, accompanied by the appearance of low-molecular-weight products. The degradation of the membrane proteins was not prevented by pretreatment with verapamil or ascorbate. However, the pretreatment with verapamil but not with ascorbate revealed significant inhibition of the tBHP-induced impairment in filterability in the presence of extracellular Ca2+. Thus, the present study shows that verapamil, a potent drug in reperfusion therapy, plays an important role in protection against oxidative injury, based on a close linkage among decreased filterability, met-Hb formation, and impaired membrane integrity.

Oxidation and nitrosylation of oxyhemoglobin by S-nitrosoglutathione via nitroxyl anion

The reaction between low molecular weight S-nitrosothiols and hemoglobin is often used to synthesize S-nitrosohemoglobin, a form of hemoglobin suggested to be involved in the regulation of vascular oxygen delivery. However, this reaction has not been studied in detail, and several groups have reported a variable co-formation of oxidized methemoglobin (metHb) during synthesis. This study examines the mechanism of metHb formation and shows that nitrosylhemoglobin (HbNO) can also be formed. Generation of metHb and HbNO is largely dependent on the presence of protein thiol groups. We present evidence for a mechanism for the formation of metHb and HbNO involving the intermediacy of nitroxyl anion. Specifically, the reaction of nitroxyl with S-nitrosothiols to liberate nitric oxide and reduced thiol is proposed to be central to the reaction mechanism.

Oxidation and nitrosylation of oxyhemoglobin by S-nitrosoglutathione via nitroxyl anion

The reaction between low molecular weight S-nitrosothiols and hemoglobin is often used to synthesize S-nitrosohemoglobin, a form of hemoglobin suggested to be involved in the regulation of vascular oxygen delivery. However, this reaction has not been studied in detail, and several groups have reported a variable co-formation of oxidized methemoglobin (metHb) during synthesis. This study examines the mechanism of metHb formation and shows that nitrosylhemoglobin (HbNO) can also be formed. Generation of metHb and HbNO is largely dependent on the presence of protein thiol groups. We present evidence for a mechanism for the formation of metHb and HbNO involving the intermediacy of nitroxyl anion. Specifically, the reaction of nitroxyl with S-nitrosothiols to liberate nitric oxide and reduced thiol is proposed to be central to the reaction mechanism.

Superoxide dismutase targets NO from GSNO to Cysbeta93 of oxyhemoglobin in concentrated but not dilute solutions of the protein.

The role of hemoglobin (Hb) in transmitting the vasodilatory property of NO throughout the vascular system is of much current interest. NO exchange between Hb and low-molecular-weight nitrosothiols such as S-nitrosoglutathione (GSNO) has been speculated and reported in vitro. Previously, we reported that NO delivery from GSNO to Cysbeta93 of human oxyHb is prevented in the presence of the Cu chelators, neocuproine, and DTPA.(1) In the present work, 5 mM solutions of commercial human Hb were found by ICP-MS to contain approximately 20 microM Cu and Zn, suggesting the presence of Cu,Zn-superoxide dismutase (CuZnSOD), which was confirmed by Western blotting. SOD activity measurements were consistent with the presence of approximately 20 microM CuZnSOD monomer in 5 mM Hb solutions, which is the physiological concentrations of these proteins in the red blood cell. Incubation of 3.75 mM oxyHb (15 mM heme; 7.5 mM Cysbeta93) with 3.75 or 7.5 mM GSNO gave rise to 50% or 100% S-nitrosation, respectively, of Cysbeta93 as monitored by FTIR nu(SH) absorption, whereas excess GSNO over Cysbeta93 converted oxyHb to metHb due to the reaction, oxyHb + NO<==>metHb + NO(3)(-). Removal of CuZnSOD by anion-exchange chromatography yielded an oxyHb sample that was unreactive toward GSNO, and replacement with bovine CuZnSOD restored reactivity. Addition of 1 microM GSNO (Cysbeta93/GSNO = 1) to solutions diluted 10(4)-fold from physiological concentrations of oxyHb and CuZnSOD resulted largely in metHb formation. Thus, this work reports the following key findings: CuZnSOD is an efficient catalyst of NO transfer between GSNO and Cysbeta93 of oxyHb; metHb is not detected in oxyHb/GSNO incubates containing close to the physiological concentration (5 mM) of Hb and CuZnSOD when the Cysbeta93/GSNO molar ratio is 0.5 to 1.0, but metHb is detected when the total Hb concentration is low micromolar. These results suggest that erythrocyte CuZnSOD may play a critical role in preserving the biological activity of NO by targeting it from GSNO to Cysbeta93 of oxyHb rather than to its oxyheme.

Prolonged Oxygen-Carrying Ability of Hemoglobin Vesicles by Coencapsulation of Catalase in Vivo

Hemoglobin (Hb) vesicles (particle diameter, ca. 250 nm) have been developed as Hb-based oxygen carriers in which a purified Hb solution is encapsulated with a phospholipid bilayer membrane. The oxidation of Hb to nonfunctional ferric Hb (metHb) was caused by reactive oxygen species, especially hydrogen peroxide (H(2)O(2)), in vivo in addition to autoxidation. We focused on the enzymatic elimination of H(2)O(2) to suppress the metHb formation in the Hb vesicles. In this study, we coencapsulated catalase with Hb within vesicles and studied the rate of metHb formation in vivo. The Hb vesicles containing 5.6 x 10(4) unit mL(-1) catalase decreased the rate of metHb formation by half in comparison with Hb vesicles without catalase. We succeeded in prolonging the oxygen-carrying ability of the Hb vesicle in vivo by the coencapsulation of catalase.

Using a "non uniform pulse sequence" can improve selective coagulation with a Nd:YAG laser (1.06 microm) thanks to Met-hemoglobin absorption: a clinical study on blue leg veins.

Evaluation of the efficacy, on 1-2 mm blue leg telangiectasia, of a 1,064 nm Nd:YAG laser emitting in a non uniform pulse sequence calculated to consider Met-Hb formation during laser irradiation of a blood vessel. A 1,064 nm Nd:YAG laser (Quantel Medical, Athos, France) was used in a non uniform pulse sequence mode, fluences: 300-360 J/cm(2) spot: 2 mm, + 5 degrees C contact cooling. The clinical evaluation was performed on 11 female patients, average age: 43 (25-57) years, phototype I-VI. All subjects were previously examined with Doppler ultrasound. A treatment site (6 x 4 cm) was selected on each patient. The topography of the vessels network was reported on a tracing plastic frame before each session and 6 weeks after the last one. These frames were digitized and the number of vessels was determined using the Digitized Tracing Frames Technique. Side effects were noted before and after every treatment, and 6 weeks after the last one. This study lasted for 10 months. Patients tolerated the procedure without anesthesia. Moderate pain, transient erythema and edema, one hyperpigmentation and one matting were noted. There was no hypopigmentation. 55% (P < 0.002) vessels clearance after one session, 86% after two sessions (P < 0.001), and 98% (P < 0.001) after three sessions were obtained. On two patients, the treatment was completed after two sessions with a full clearance. Data reported in this study were obtained thanks to a computerized calculation of vessels clearance. They are similar or superior to those reported in the literature about 1,064 nm Nd:YAG lasers and leg telangiectasia. Since, it was developed to consider the modification of blood absorption and the methemoglobin formation which leads to an increase of the 1.06 microm wavelength absorption, the non uniform pulse mode emphasizes the efficacy of this 1,064 nm Nd:YAG laser concerning the treatment of blue leg veins telangiectasia between 1 and 2 mm. This mode gives the possibility to deliver high energy while preserving the surrounding tissue and leads to a rapid vessel clearance with reduced pain and few side effects when compared to previously published clinical studies using a 1.06 microm laser.

Heme Nitrosylation of Deoxyhemoglobin byS-Nitrosoglutathione Requires Copper

NO reactions with hemoglobin (Hb) likely play a role in blood pressure regulation. For example, NO exchange between Hb and S-nitrosoglutathione (GSNO) has been reported in vitro. Here we examine the reaction between GSNO and deoxyHb (HbFe(II)) in the presence of both Cu(I) (2,9-dimethyl-1, 10-phenanthroline (neocuproine)) and Cu(II) (diethylenetriamine-N,N,N',N",N"-pentaacetic acid) chelators using a copper-depleted Hb solution. Spectroscopic analysis of deoxyHb (HbFe(II))/GSNO incubates shows prompt formation (<5 min) of approximately 100% heme-nitrosylated Hb (HbFe(II)NO) in the absence of chelators, 46% in the presence of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, and 25% in the presence of neocuproine. Negligible (<2%) HbFe(II)NO was detected when neocuproine was added to copper-depleted HbFe(II)/GSNO incubates. Thus, HbFe(II)NO formation via a mechanism involving free NO generated by Cu(I) catalysis of GSNO breakdown is proposed. GSH is a source of reducing equivalents because extensive GSSG was detected in HbFe(II)/GSNO incubates in the absence of metal chelators. No S-nitrosation of HbFe(II) was detected under any conditions. In contrast, the NO released from GSNO is directed to Cysbeta(93) of oxyHb in the absence of chelators, but only metHb formation is observed in the presence of chelators. Our findings reveal that the reactions of GSNO and Hb are controlled by copper and that metal chelators do not fully inhibit NO release from GSNO in Hb-containing solutions.

Heme Redox Properties of S-Nitrosated Hemoglobin A0 and Hemoglobin S: IMPLICATIONS FOR INTERACTIONS OF NITRIC OXIDE WITH NORMAL AND SICKLE RED BLOOD CELLS

S-Nitrosated hemoglobin is remarkably stable and can be cycled between deoxy, oxygenated, or oxidized forms without significant loss of NO. Here we show that S-nitrosation of adult human hemoglobin (Hb A(0)) or sickle cell Hb (Hb S) results in an increased ease of anaerobic heme oxidation, while anions cause redox shifts in the opposite direction. The negatively charged groups of the cytoplasmic domain of Band 3 protein also produce an allosteric effect on S-nitrosated Hb. Formation and deoxygenation of a SNO-Hb/Band 3 protein assembly does not in itself cause NO release, even in the presence of glutathione; however, this assembly may play a role in the migration of NO from the red blood cells to other targets and may be linked to Heinz body formation. Studies of the anaerobic oxidation of Hb S revealed an altered redox potential relative to Hb A(0) that favors met-Hb formation and may therefore underlie the increased rate of autoxidation of Hb S under aerobic conditions, the increased formation of Heinz bodies in sickle cells, and the decreased lifetime of red cells containing Hb S. A model for the interrelationships between the deoxy, oxy, and met forms of Hb A(0) and Hb S, and their S-nitrosated counterparts, is presented.

Complete deoxygenation from a hemoglobin solution by an electrochemical method and heat treatment for virus inactivation.

Hemoglobin (Hb) has been widely studied as a raw material for various types of oxygen carriers. In the purification of Hb from red blood cells including virus inactivation and denaturation of other proteins and the long-term storage of Hb vesicles (HbV), a deoxygenation process is one of the important processes because of the high stability of deoxygenated Hb to heating and metHb formation. Though an oxygenated Hb solution can be deoxygenated with an artificial lung, it is difficult to reduce the oxygen partial pressure of the Hb solution to less than 10 Torr. We developed an electrochemical system for complete deoxygenation of the Hb solution at the cathode compartment using hydrogen containing nitrogen gas at the anode compartment. Oxygen in the Hb solution was reduced to OH(-) at the cathode compartment within several minutes at a potential value of -1.67 V and was finally converted to water by neutralization with H(+) from the anode in the whole system. The resulting completely deoxygenated Hb could tolerate heat treatment at 62 degrees C for 10 h with no denaturation of deoxygenated Hb. The metHb formation rate of reoxygenated Hb at 37 degrees C was not changed after heat treatment. Furthermore, vesicular stomatitis virus (VSV) could be inactivated at an inactivation degree of more than 5.96 log by heat treatment.

ANILINE-INDUCED METHEMOGLOBINEMIA IN DOGS: PITFALLS OF ROUTE-TO-ROUTE EXTRAPOLATIONS

Two groups of four beagle dogs each were exposed head-only to aniline vapor in concentrations of 155 or 174 mg/m 3 air for a duration of 4 h in order to attain a targeted total exposure dose of approximately 15 mg aniline/kg body weight. One group of dogs received this calculated dose also by gavage. Dose estimates were made either by the determination of aniline-serum albumin adducts or by repeated measurements of the respiratory minute volume during the inhalation exposure period. The magnitude of methemoglobin (MetHb) produced following each route served as the basis for comparison. MetHb levels were maximal at the end of the inhalation exposure period, attaining approximately 5%, whereas administration by gavage produced a maximum MetHb response of 26%. Measurements of respiratory minute volumes in individual dogs appear to suggest that variability in MetHb formation depends solely on the rate of uptake (ventilation). The concentration of aniline-serum albumin adducts and the extent of MetHb formation showed a nonproportional relationship. In summary, this study demonstrates that for aniline, an agent known to be bioactivated by a hepatic first-pass metabolism, the conversion of findings obtained from oral dosing to inhalation exposure concentrations is subject to overestimating dramatically the magnitude of MetHb formation likely to occur following inhalation exposure. As to whether the fivefold lower potency by inhalation is solely related to the hepatic first-pass bioactivation, to the rate of delivery, or to a less than 100% retention of the inhaled vapor within the respiratory tract remains to be elucidated. This study demonstrates some of the pitfalls that can occur in context with route-to-route extrapolations and reinforces many of the concerns related to such extrapolations.