Omoefe O. Abugo

Production of superoxide from hemoglobin-bound oxygen under hypoxic conditions

By low temperature electron paramagnetic resonance we have detected the formation of a free radical signal during incubation of partially oxygenated hemoglobin at 235 K. The observed signal has g parallel = 2.0565 and g perpendicular = 2.0043, consistent with the previously reported values for superoxide. The presence of additional EPR signals for oxygen-17 bound hemoglobin, with (017-017)A perpendicular = 63 G and (017-016)A perpendicular = 94 G under identical conditions, confirms the presence of a radical containing two nonequivalent oxygens as required for a superoxide in magnetically inequivalent environments. The superoxide radical has not previously been directly detected during hemoglobin autoxidation because of its rapid dismutation. Our ability to follow the formation of superoxide for more than 15 min is attributed to its production in the hydrophobic heme pocket where dismutation is slow. The enhanced production of this free radical at intermediate oxygen pressures is shown to coincide with enhanced rates of hemoglobin autoxidation for partially oxygenated intermediates. The formation of superoxide in the heme pocket under these conditions is attributed to enhanced heme pocket flexibility. Greater flexibility facilitates distal histidine interactions which destabilize the iron-oxygen bond resulting in the release of superoxide radical into the heme pocket.

Detection, formation, and relevance of hemichromes and hemochromes.

Publisher Summary Native hemoglobins contain one histidine coordinated to the iron on the proximal side of the heme and another histidine in the ligand pocket on the distal side of the heme. Oxygen located in this pocket in oxyhemoglobin can be replaced by other neutral ligands, such as carbon monoxide, nitric oxide, and alkylisocyanide in the reduced Fe(II) hemoglobins. The iron in deoxyhemoglobin is in a high-spin state, whereas all of the Fe(II) complexes are in a low-spin state. Oxidized Fe(III) hemoglobin ligands bound in this pocket include water, hydroxide, fluoride, azide, thiocyanate, imidazole, and cyanide. The spin state for these complexes depends on the strength of the axial ligand and can be high spin (example, fluoride), low spin (example, cyanide), or a mixture (example, hydroxide). Low-spin hemoglobin complexes also exist when the exogenous ligand is replaced by an endogenous amino acid side chain. These low-spin complexes define the Fe(II) hemochromes and Fe(III) hemichromes. For many other heme proteins, low-spin complexes involving axial coordination of two amino acid side chains are functional. This chapter discusses the methods for determining the presence of the low-spin complexes. The characterization of different types of hemichromes, primarily on the basis of electron paramagnetic resonance (EPR) is reviewed. The chapter discusses the different ways of forming hemi(hemo)chromes. The processes clearly dissociated with denaturation to those clearly indicative of native substates and the significance of hemi(hemo)chrome formation are also discussed. The information regarding hemoglobin that can be obtained from studies of hemi(hemo)chrome formation and the possible functional role of bis-histidine complex formation have also been described in the chapter.

The Reduction of Nitroblue Tetrazolium by Red Blood Cells: a Measure of Red Cell Membrane Antioxidant Capacity and Hemoglobin-membrane Binding Sites

The reduction of nitroblue tetrazolium (NBT) with intact Red Blood Cells (RBCs) is biphasic with an initial rapid reduction followed by a slower second phase. This biphasic kinetics has been explained with the initial rapid phase attributed to antioxidants in the red cell which reduce membrane bound NBT and the slower phase associated with the reaction of NBT with membrane bound hemoglobin. This model has been confirmed by a utilization of a number of red cell modifications which either increase the red cell antioxidants (vitamin C and vitamin E) or damage the red cell membrane (cumene hydroperoxide and N-ethylmaleimide). The utilization of this assay for human blood samples was investigated by studying a series of 20 human subjects ranging between 34 and 87 years of age. It was possible to fit all of these samples with two adjustable parameters which reflect the red cell membrane antioxidant capacity (x) and the hemoglobin membrane interactions (m). The antioxidant capacity shows a significant (p < 002; R = -.67) decrease with age. This finding is consistent with a decrease in the level of antioxidants in aged subjects. In addition, the number of hemoglobin membrane sites are negatively correlated with the antioxidant capacity (p < .02; R = -.52) suggesting that the oxidative stress associated with reduced antioxidants results in increased hemoglobin-membrane interactions.

Modulation Sensing of Fluorophores in Tissue: A New Approach to Drug Compliance Monitoring

We describe a method to detect the presence of fluorophores in scattering media, including intralipid suspensions and chicken muscle covered with skin. The fluorophores were rhodamine 800 (Rh800) and indocyanine green (IcG), both of which can be excited at long wavelengths where there is minimal absorption by tissues. These fluorophores were dissolved in intralipid or in chicken muscle under skin. A method to approximate the fluorophore concentration in such samples was developed using a long lifetime reference fluorophore in a polymer film placed immediately on the illuminated surface of the sample. Because of the long lifetime of the reference film, the modulation of its emission at low frequencies near 2 MHz is near zero. Since the lifetime of Rh800 and IcG are below 2 ns the modulation of the combined emission is a measure of the intensity of the fluorophore (Rh800 or IcG) relative to the long lifetime reference. Using this method we were able to measure the concentration-dependent intensities of Rh800 and IcG in an intralipid suspension. Additionally, micromolar concentrations of these probes could be detected in chicken muscles, even when the muscle was covered with a layer of chicken skin. The presence of an India ink absorber in the intralipid had only a moderate effect on the modulation values. We suggest the use of this transdermal detection of long-wavelength fluorophores as a noninvasive method to monitor patient compliance when taking medicines used for treatment of chronic diseases such as AIDS or tuberculosis.

Alterations in Erythrocyte Deformability Under Hypoxia: Implications for Impaired Oxygen Transport

Oxygen delivery to the tissues requires that the erythrocyte be highly deformable in order to pass through the narrow pores of the capillary bed. It has been shown that oxidative damage produces crosslinking of hemoglobin with membrane proteins (Shaklai et al., 1987; Snyder et al., 1985; Reinhart et al., 1986) resulting in a more rigid, less deformable membrane. Hypoxia as a source for oxidative damage and thereby a possible loss in erythrocyte deformability is based on recent studies which indicated enhanced autoxidation and superoxide formation (Rifkind et al., 1989, 1991) within erythrocytes at intermediate oxygen pressures.

Fluorescence properties of albumin blue 633 and 670 in plasma and whole blood

We have determined the fluorescence characteristics of two long wavelength dyes, albumin blue 633 (AB633) and 670 (AB670), in plasma and blood to evaluate the possibility of making direct fluorescence sensing measurements in blood. Using binding and lifetime measurements we were also able to show that these dyes bind selectively to human serum albumin (HSA) in plasma and blood. By measuring changes in the mean lifetime of AB670 with changes in the HSA concentration, we showed that lifetime-based sensing can be used to monitor HSA concentrations using these albumin blue dyes. Anisotropy measurements for AB633 and AB670 in plasma and blood revealed high anisotropy values for these dyes in these media. Exploiting these high anisotropies, we were also able to determine HSA concentrations in plasma and blood mimics using changes in AB670 anisotropy with HSA concentration. These results show that, apart from being able to make fluorescence measurements directly in plasma and blood, it is possible to sense directly for specific plasma/blood components using fluorescent probes that bind preferentially to them.

DIRECT MEASUREMENTS OF HEMOGLOBIN INTERACTIONS WITH LIPOSOMES USING EPR SPECTROSCOPY

Electron paramagnetic resonance (EPR) spectroscopy was used to compare the rates of autoxidation at 37°C of acellular and liposome-encapsulated hemoglobin (LEH) crosslinked between alpha chains with bis (3,5-dibromosalicyl) fumarate (ααHb). This method avoids the difficulties inherent in using conventional ultraviolet-visible (UV-vis) spectroscopy caused by the high turbidity of liposome suspensions. Rate constants of 0.039/h and 0.065/h were obtained for the ααHb and LEH samples, respectively. Similar oxidation measurements with ααHb using UV-vis spectroscopy gave a rate constant comparable to that obtained with EPR spectroscopy. Indirect measurement of the oxidation kinetics of LEH utilizing extraction of ααHb with chloroform from partially oxidized LEH samples was unreliable because the amount of extractable hemoglobin was inversely proportional to the degree of oxidation. EPR measurements showed a shift in the g value and substantial enhancement in the intensity of the bis-histidine low-spin B complex for the encapsulated hemoglobin, indicating a perturbation of this low-spin complex. We suggest that lipid-associated perturbations are responsible for the enhancement of the oxidation observed with the LEH samples compared to the unencapsulated material.

Fluorescent measurements in whole blood and plasma using red-emitting dyes

We have determined the fluorescence characteristics of albumin blue 670 and Rhodamine 800 in plasma and blood in order to test the feasibility of making direct fluorescence sensing measurements in blood. These dyes were used because of their absorption in the red/NIR where absorption by hemoglobin is minimized. Front face illumination and detection was used to minimize absorption and scattering during measurement. Fluorescence emission was observed for these dyes in plasma and blood. Attenuation of the fluorescence emission was observed in blood because of hemoglobin absorption. Using frequency domain fluorometry, we recovered the expected lifetime parameters for both dyes in blood and plasma. We were able to quantify HSA concentrations using changes in the mean lifetime of AB670, a dye previously shown to bind preferentially to HSA. Rh800 concentrations in plasma and blood were also determined using modulation sensing. Anisotropy measurements revealed high Anisotropy for these dyes in plasma and blood. It also showed an increase in the anisotropy of AB670 with increase in HSA concentration in the presence of red blood cells. These results indicate that qualitative and quantitative fluorescence measurements can be made directly in blood without the need to process the blood.

Preliminary studies on the use of modulation sensing for noninvasive monitoring of drug compliance

Presently two methods in concert, a marker method and an electronic monitoring method, have been emphasized for an objective monitoring of drug compliance in ambulatory care. While the marker method proves dose ingestion, the electronic monitoring method provides continuous record of timing of presumptive drug doses. The marker method is however time intensive with associated safety problems, and the electronic monitoring method is easily defeated. We here present preliminary results on modulation sensing, a new method that could be used to non-invasively monitor patient compliance. Measurement is based on observing the amplitude modulation of the emission from both a short lifetime marker fluorophore of interest and a long lifetime reference fluorophore contained in the monitoring device. At some intermediate frequencies, the observed modulation of the combined emission is nearly equivalent to the fractional intensity of the marker fluorophore. This method precludes problems associated with measuring fluorescence intensities in highly scattering media. Using this method we measured the presence of rhodamine 800 (Rh800) in intralipid suspensions and chicken tissue. Rh800 is excited at long wavelengths not absorbed by tissues. Micromolar concentrations of this dye were detected in intralipid and chicken muscle covered with a layer of chicken skin.