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Nuclear magnetic resonance studies of hemoglobins. 3. Evidence for the nonequivalence of alpha- and beta-hains in azide derivativeof methemoglobins.

Nuclear magnetic resonance spectroscopy (100-MHz proton) was used to study the low-spin (S = 1/2) azide derivatives of human adult (alpha(2)beta(2)), human fetal (alpha(2)gamma(2)), Zürich (alpha(2)beta(2) (63 His --> Arg)), and horse (alpha(2)'beta(2)') methemoglobins, as well as whale metmyoglobin in 0.1 M deuterated phosphate at pD 7 and at 31 degrees C. The experimental results indicate that the azide-bound heme groups of the alpha- and beta-chains in human adult methemoglobin and of the alpha- and gamma-chains in fetal methemoglobin are not equivalent. The affinity of the beta- or gamma-chain for azide ion appears larger than that of the alpha-chain. The nuclar magnetic resonance spectrum of hemoglobin Zürich shows that the environment of the azide-heme complex in the abnormal beta-chain is altered by the substitution of arginine for histidine in the beta-63 position, while the alpha-heme environment remains unaffected.

Animals↗

Volume changes in binding of ligands to methemoglobin and metmyoglobin.

The volume changes for the binding of various ligands to metmyoglobin and methemoglobin have been determined from the effect of pressure on the binding constants (for metmyoglobin) and by direct dilatometry (for methemoglobin). The volume changes associated with the binding of cyanide and azide ions to methemoglobin are pH-dependent. The volume change for the binding reaction is evidently affected by the same subtle structural variations that have been judged to be present from the variation with pH of enthalpy and entropy for the binding reactions in these proteins. Hydration changes and spin state changes which have been postulated to be linked with structural variations in these proteins must be pH-dependent.

Atmospheric Pressure↗

Instability of the oxy form of sickle hemoglobin and of methemoglobin in isopropanol.

Oxygenated hemoglobin from water-lysed sickle erythrocytes preciptated more rapidly than hemoglobin from normal cells when exposed to 17% isopropanol in 0.1 M tris, pH 7.4, buffer (the isopropanol solubility test of Carrell and Kay). Precipitates became visible after approximately 20 minutes of incubation with SS, AS, or SC blood, while AC hemoglobin and normal adult A hemoglobin required 40-60 minutes of precipitate. Methemoglobin comprised less than 1.5% of the total hemoglobin present in all samples under study. Significant preciptation of methemoglobin did not occur unless methemoglobin levels were greater than 8%. Solubilization of the precipitate in 6M urea-barbital buffer and subsequent electrophoresis demonstrated that the insoluble material was sickle hemoglobin (Betas chain). The results of these experiments confirm previous findings indicating that even fully oxygenated sickle hemoglobin is unstable.

1-Propanol↗

Determination of methemoglobin and total hemoglobin in toxicological studies by derivative spectrophotometry.

A method for the determination of methemoglobin in the presence of other hemoglobin subforms (i.e., oxy-, deoxy-, and carboxyhemoglobin) by use of derivative spectrophotometry is proposed. The method, which uses the first-derivative of the spectrum at 645 nm, is straightforward and expeditious, so it is of special interest to forensic toxicology laboratories as it allows the simultaneous determination of the methemoglobin saturation percentage and the hemoglobin concentration. This facilitates interpretation of the results and provides a better understanding of the significance of methemoglobin saturation in specific cases. Based on an analysis of interferences, the presence of other hemoglobin subforms or of endogenous components of plasma does not detract in any way from the performance of the method.

Forensic Medicine↗

Spectral, conformational and chemical properties of opossum methemoglobin.

Opossum methemoglobin differs from methemoglobin A in spectral, spin state, conformational and chemical properties. The primary structural alterations in opossum hemoglobin, including the critical substitution at alpha 58 (E7) His leads to Gln result in the following properties. (a) Major contribution of the spectral transitions due to inositol hexakisphosphate binding arises from the alpha chains. (b) The aquomet to hydroxymet (high-spin to low-spin) transition as a function of pH is slightly retarded resulting in considerable high spin at alkaline pH. (c) The tertiary conformation (t) around the beta hemes, upon transition to a T quaternary state, differs from the known hemoglobin t tertiary structure. (d) Both alpha and beta hemes are susceptible to rapid reduction by ascorbic acid (the reduction rate being tenfold faster than that of methemoglobin A). These properties suggest that the heme environments in both the alpha and beta subunits of opossum hemoglobin are different from those of human hemoglobin A.

Animals↗

Effect of methemoglobin formation on the MR appearance of subarachnoid hemorrhage.

Subarachnoid hemorrhage has a much higher intensity in magnetic resonance (MR) images with the passage of time. Acute subarachnoid hemorrhage is difficult to see; within 1 week its appearance has become intensified on T1-weighted images. Different concentrations of blood and lysed red blood cells in cerebrospinal fluid (CSF) were examined spectroscopically but did not significantly alter T1 and T2 relaxation of CSF acutely. Ultraviolet visible spectroscopy of bloody CSF stored hypoxically for 3 days showed the presence of methemoglobin. The iron in methemoglobin is paramagnetic; in combination with water this facilitates T1 relaxation. It is concluded that methemoglobin formation with T1 shortening at least partially accounts for the increasing intensity of the MR appearance of subarachnoid hemorrhage over time in the central nervous system and may also explain the intense appearance of subacute hemorrhage in MR images elsewhere in the body.

Acute Disease↗

Altered heme environments in opossum and rabbit methemoglobins.

The effect of pH and inositol hexaphosphate (IHP) on the symmetry of the heme environments in opossum (Didelphius marsupialis) and new Zealand White rabbit hemoglobins has been studied using electron spin resonance (ESR). Each methemoglobin is found to contain two different heme environments as detected by the rhombicity observed in the ESR spectrum. In both cases the rhombic nature of the ESR spectrum is influenced by pH and IHP binding, although in the case of rabbit methemoglobin the high spin ESR signal disappears above pH 8.0. In both hemoglobins, amino acid alterations in the alpha-chains are known to affect the properties of the ferrous derivatives. It is concluded that these alterations also provide the basis for the ESR spectral properties observed with the methemoglobins.

Animals↗

Methemoglobin formation by paraquat.

Paraquat is a broad spectrum herbicide known to be highly lethal to man and animals. Its toxicity is characterized by acute lung injury. Paraquat produces such toxic effects through the generation of the superoxide anion according to one proposed mechanism. The present experiment, methemoglobin formation was demonstrated after incubation of oxyhemoglobin with paraquat. The generation of the superoxide anion through the interaction of oxyhemoglobin with paraquat was suggested by chemiluminescence of luminol. Superoxide dismutase (SOD) and catalare inhibited methemoglobin formation. The generation of the superoxide anion is discussed in regard to methemoglobin formation by paraquat.

Chromatography, Ion Exchange↗

Intravascular reduction of methemoglobin in plasma of the rat in vivo.

The formation and reduction of extracellular methemoglobin (metHb) in plasma was studied in vivo in conscious rats after isovolemic exchange transfusions with polymerized hemoglobin solutions. After exchange transfusions of 40 and 70% of the blood volume with hemoglobin solutions, containing less than 6% methemoglobin, the methemoglobin level remained below 15%, whereas exchange transfusions of greater than 90% resulted in an increase in the metHb level to about 30% after 24 hours. The reduction of metHb was studied after exchange transfusions with fully oxidized hemoglobin. A gradual decrease in the metHb level to 20% was observed after exchanges of 5 or 40%. A higher exchange transfusion (70%) resulted also in a decrease in the metHb level but only to approximately 40% in 24 hours. In another series of experiments the reduction of metHb was studied in vitro with isolated human erythrocytes. Incubation of the erythrocytes in the presence of oxidized polymerized hemoglobin (3 g%) resulted in a decrease in the percentage of metHb from 91% to 64%. In the presence of 0.2 mM ascorbic acid the metHb level declined to 22%, suggesting a synergistic effect. These results indicate (1) that there is a potent reducing mechanism present in blood that can reduce extracellular oxidized polymerized hemoglobin and (2) that isolated erythrocytes have a large capacity to reduce extracellular metHb, and may also play an important role in the reduction of extracellular metHb in vivo.

Animals↗

Effects of p-hydroxymercuribenzoate binding on the visible absorption spectrum of methemoglobin.

The binding of p-hydroxymercuribenzoate to human methemoglobin causes a perturbation of the visible heme abosrption spectrum which is expressed by an increase in absorbance in the high spin band regions, 480 to 510 nm and 590 to 640 nm, concomitant with a decrease in absorbance in the alpha- and beta-band absorption regions. The pH dependence of the p-hydroxymercuribenzoate-induced difference spectrum can be accounted for quantitatively by a 5% shift toward higher spin of the aquo form of methemoglobin, a 15% shift toward higher spin of the hydroxide form, and a shift in the apparent pKa for the water to hydroxide transition from 7.92 to 8.04 when mercurial is bound. The rate of these heme abosrbance changes is consistent with the rapid second order formation of the beta93 cysteine, mercury-mercaptide bond and does not represent a change due to the dissociation of methemoglobin tetramers into dimers, even though the latter, slow process does follow mercurial binding. The observation of an increase in spin produced by the binding of a reagent which also promotes dimer formation argues strongly against any direct correlation between an increase in spin and the appearance of deoxyhemoglobin-like conformations.

Adult↗

[Methemoglobin-induced lipid peroxidation in model membranes].

Lipid peroxidation induced by methemoglobin in liposomes prepared from lecithin, cardiolipin and their mixtures has been investigated. Using absorption spectroscopy technique it was shown that in the bilayers with low initial oxidation methemoglobin caused the formation of diene conjugates. In the bilayers with high degree of oxidation protein activated cleavage of the available fatty acid hydroperoxides. Hydroperoxides were found to induce the reduction of methemoglobin absorption in the Soret band.

Lipid Peroxidation↗

[Methemoglobin and osmoresistance of erythrocytes in patients with different types of goiter].

The purpose of this study was to determine the level of methemoglobin in the blood and osmoresistance of erythrocytes in patients with different types of goiter and control subjects. Investigated parameters indicate to the antioxidant status of human organism. We examined 12 healthy controls and 49 hyperthyroid patients: 17 patients with nodular goiter (mild thyrotoxicosis), 22 patients with diffuse toxic goiter (high level of thyrotoxicosis) and 10--with euthyroid nodule goiter. Osmoresistance of erythrocytes was measured by differential photoelectrocolorymetric method and methemoglobin--by spectroscopic method. We had received significant correlation between the level of methemoglobin, osmoresistance of erythrocytes and the degree of thyrotoxicosis. The results once again prove sensitivity of antioxidant status in response to toxicity, in particular against thyrotoxicosis.

Adult↗

[Dependence of human erythrocyte methemoglobin reductase on temperature].

The effect of temperature on the activities of cytoplasmic and membrane-bound fractions of NADH-cytochrome beta 5 reductase on the total activity of methemoglobin reductase in intact human erythrocytes was studied within the temperature range of 20-50 degrees C. The above three activities showed a break in the Arrhenius plots at 42 degrees C which was attributed to irreversible inactivation of the enzymes. Thermal inactivation of methemoglobin reductase in erythrocytes was found to increase the methemoglobin content concomitantly with a decrease in the osmotic stability and activation of spontaneous cell hemolysis.

Cytochrome Reductases↗

[The phosphate and methemoglobin content of the erythrocytes in patients with ischemic heart disease].

Cardiac insufficiency due to chronic ischemic heart disease is accompanied by a reduction of total phosphate at the expense of its organic part with a simultaneous increase of the role of inorganic phosphate during increase of methemoglobin in the erythrocytes. After traditional therapy the methemoglobin level continued to rise while inorganic phosphate and the level of 2,3-DPH decreased. Treatment including myldronate caused an increase of 2,3-DPH in erythrocytes and a decrease of methemoglobin indicating an improvement of the oxygen transport function of the blood.

2,3-Diphosphoglycerate↗

Inhibition by superoxide dismutase of methemoglobin formation from oxyhemoglobin.

The formation of methemoglobin from oxyhemoglobin in a solution containing photoreduced riboflavin and oxygen was inhibited by superoxide dismutase. The rate of the reaction was pH-dependent in the range of 6.8 to 7.8, increasing as the pH was reduced. Inhibition by superoxide dismutase was enhanced as the EDTA concentration increased and was dependent on enzymatic activity. Under conditions in which superoxide dismutase inhibition was incomplete, catalase inhibited the reaction but mannitol had no effect. The data support the mediation of methemoglobin formation by superoxide. The hypothesis is offered that superoxide anion reduced the heme-bound oxygen in oxygemoglobin by one electron, permitting the subsequent dissociation of ferrihemoglobin and peroxide. The ability of superoxide dismutase to inhibit the formation of methemoglobin may represent one of its functions in the mature erythrocyte.

Animals↗

[Does the development of methemoglobin in the newborn infant affect the suitability of prilocaine for pudendal anesthesia? A clinical study in the peripartum phase].

Pudendal block is a well established method of achieving analgesia during the second stage of labor. Whenever a large amount of a local anesthetic has to be injected in well vascularized tissue, local anesthetic drugs with low systemic toxicity should be used, to minimize side effects. This means that prilocaine is the drug of choice. It is well known that the metabolites of prilocaine induce methemoglobinemia, and thus the question arises as to whether the methemoglobinemia affects the fetus. PATIENTS AND METHODS. Pudendal block was achieved with 2 x 10 ml prilocaine 1% in each of 17 mothers. Plasma concentrations of the local anesthetic in the second stage of labor were determined by gas chromatography in blood samples drawn from the mother and the newborn at the moment of childbirth. In addition, the time course of methemoglobinemia was determined by capillary blood samples from the neonate up to 6 h. To evaluate methemoglobinemia in the newborn, 125 microliters heparinized capillary blood was diluted with 200 microliters 0.9% sodium chloride; methemoglobin was detected by absorbance spectrometry. RESULTS. Before the pudendal block maternal methemoglobin concentrations were about 0.2% of the total hemoglobin concentration and within the physiological range. At the moment of delivery it was increased only to a small extent, without statistical significance. In the neonates mean methemoglobin concentrations were about 1% of total hemoglobin immediately after delivery, increasing up to 1.8% in the next 2 h and then decreasing continuously in all. At the moment of childbirth maternal mean prilocaine concentrations were 0.57 micrograms/ml on an average and 0.29 micrograms/ml in the newborn. DISCUSSION. With respect to systemic toxicity, prilocaine is the drug of choice in local anesthetic procedures when a long duration of anesthesia is not required; it guarantees short latency and adequate relief of pain. Methemoglobinemia induced by its metabolites is not a contraindication for its use in humans. Formerly prilocaine was judged to be contraindicated in pregnant women during delivery because of the small redox capacity of fetal erythrocytes. Our study, however, demonstrates that 200 mg prilocaine for pudendal block does not induce methemoglobinemia in newborns to any significant extent. One explanation for this may be the increased renal elimination of local anesthetics in newborns and the low fetomaternal ratio.

Anesthesia, Obstetrical↗

[Effect of glucocorticoids on the formation of methemoglobin in rats].

Experiments on rats showed that treatment of rat with hydrocortisone, dexamethasone and diplacin for 14 days increases the content of methemoglobin in blood. In vitro experiments revealed that dexamethasone in a concentration of 10(-6) M increased the amount of methemoglobin in the whole blood and did not change its amount in hemolysate. The data obtained show the participation of erythrocyte membranes in methemoglobin-forming effect of the studied substances.

Animals↗

Relationship between cyclic guanosine 3':5'-monophosphate formation and relaxation of coronary arterial smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric oxide: effects of methylene blue and methemoglobin.

The purpose of the present study was to determine time course relationship between cyclic GMP accumulation and relaxation in bovine coronary artery and evaluate the effects of recently identified inhibitors, methylene blue and methemoglobin, on these relationships. Arterial strips were suspended in specially mounted tissue baths which permitted continuous recording of isometric tension until rapid freeze-clamping for subsequent determination of cyclic GMP levels by radioimmunoassay. Relaxation and cyclic GMP levels were measured in submaximally contracted strips at zero time (untreated) or 5-sec to 5-min intervals after exposure to 0.5 microliter of nitric oxide, 1 microM glyceryl trinitrate, 1 microM sodium nitroprusside of 1 mM sodium nitrite in the absence or presence of 10 mM methylene blue or 1 microM methemoglobin. Cyclic GMP accumulation preceded onset of relaxation elicited by nitric oxide and glyceryl trinitrate and temporally correlated with relaxation induced by sodium nitroprusside and sodium nitrite. Methylene blue simultaneously inhibited cyclic GMP accumulation and relaxation induced by all four relaxants. In contrast to methylene blue, methemoglobin abolished cyclic GMP accumulation and relaxation elicited by nitric oxide without altering responses to glyceryl trinitrate, sodium nitroprusside and sodium nitrite. These findings are consistent with and strongly support an involvement of cyclic GMP formation in vascular smooth muscle relaxation elicited by nitrogen oxide-containing vasodilators.

Animals↗