Field strength dependence of R1 and R2* relaxivities of human whole blood to ProHance, Vasovist, and deoxyhemoglobin.

This study has measured the longitudinal and transverse (T2* relaxivity curves for ProHance (Gadoteridol), Vasovist (Gadofosveset) and deoxyhemoglobin at 1.5, 3.0, and 7.0 Tesla. The plots of R(1) versus both contrast agent and deoxyhemoglobin concentration were linear. The plots of R2* versus deoxyhemoglobin concentration showed a quadratic dependence. R2* versus contrast agent concentration showed a parabolic dependence with a minimum occurring at contrast agent concentrations of approximately 1.5 mM, corresponding to an accessible concentration in vivo. Monte Carlo simulations were performed to support the hypothesis that the minimum results from the susceptibility of the red blood cells being matched to the susceptibility of the plasma. Relaxivity values (s(-1)mM(-1)) for R2* and R1 for all agents and all three field strengths are given.

Universal metastability of sickle hemoglobin polymerization.

Sickle hemoglobin (HbS) polymerization occurs when the concentration of deoxyHbS exceeds a well-defined solubility. In experiments using sickle hemoglobin droplets suspended in oil, it has been shown that when polymerization ceases the monomer concentration is above equilibrium solubility. We find that the final concentration in uniform bulk solutions (i.e., with negligible boundaries) agrees with the droplet measurements, and both exceed the expected solubility. To measure hemoglobin in uniform solutions, we used modulated excitation of trace amounts of CO in gels of HbS. In this method, a small amount of CO is introduced to a spatially uniform deoxyHb sample, so that less than 2% of the sample is liganded. The liganded fraction is photolyzed repeatedly and the rate of recombination allows the concentration of deoxyHbS in the solution phase to be determined, even if polymers have formed. Both uniform and droplet samples exhibit the same quantitative behavior, exceeding solubility by an amount that depends on the initial concentration of the sample, as well as conditions under which the gel was formed. We hypothesize that the early termination of polymerization is due to the obstruction in polymer growth, which is consistent with the observation that pressing on slides lowers the final monomer concentration, making it closer to solubility. The thermodynamic solubility in free solution is thus achieved only in conditions with low polymer density or under external forces (such as found in sedimentation) that disrupt polymers. Since we find that only about 67% of the expected polymer mass forms, this result will impact any analysis predicated on predicting the polymer fraction in a given experiment.

Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature.

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3+/-0.2-microm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (Tc = 36.4+/-0.3 degrees C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 degrees C were non-linear and showed accelerated denaturation between 35 and 39 degrees C with a midpoint at 37.2+/-0.6 degrees C. The transition was reversible below 39 degrees C and independent of solution pH (pH 6.8-7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N2 showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.

Carbon monoxide religation kinetics to hemoglobin S polymers following ligand photolysis.

The re-equilibration rate of carbon monoxide binding to hemoglobin S polymers is determined by time-resolved measurements of linear dichroism spectra. Linear dichroism is used to detect religation to hemoglobin in the polymer in the presence of rebinding to free hemoglobin S tetramers. Measurement of the linear dichroism resulting from photolysis of the small percentage of ligand bound to the polymer is accomplished through the use of an ultrasensitive, ellipsometric linear dichroism technique developed for this purpose. The major finding is that the return of the polymer phase to its equilibrium ligation state is much slower than that of the solution phase hemoglobin tetramers. Assuming all hemes in the polymer are equally likely to participate in rebinding, the re-equilibration rate for carbon monoxide religation to hemoglobin S polymers is found to be 0.14 +/- 0.07 (s-1 mM-1), about 1000 times slower than the rebinding rate of carbon monoxide to T-state monomer hemoglobin. Several interpretations of this result are discussed. An understanding of the ligand binding kinetics to hemoglobin S polymers could have pathophysiological significance in its relevance to polymer formation and melting during red blood cell circulation.

Polymer domains, gelation models and sickle cell crises.

The multistranded fibers formed by deoxygenated sickle hemoglobin form well ordered spherulitic arrays called domains. Employing laser photolysis of the carboxy derivative to initiate polymerization and domain formation, we have monitored the evolution of the spatial character of domains by light scattering and birefringence. Spatial growth is analyzed by observing a 62.5 microns square area of the sample and storing the image as 2500 discrete elements. Domain formation appears to consist of two phases. In the early phase even symmetric domains are not radially isotropic. Although the domain expands during this period, the internal polymer density as inferred by the light scattering intensity also increases. In the second phase, the domain becomes structurally symmetric and spatially homogeneous, and changes in the scattered intensity including pronounced overshoots occur uniformly across the monitored region. In this phase, the birefringence increases as the scattering decreases, with the same apparent rate.