Function of Hemoglobin-Based Oxygen Carriers: Determination of Methemoglobin Content by Spectral Extinction Measurements

Abstract

Suspensions of hemoglobin microparticles (HbMPs) are promising tools as oxygen therapeutics. For the approval of clinical studies extensive characterization of these HbMPs with a size of about 750 nm is required regarding physical properties, function, pharmaco-kinetics and toxicology. The standard absorbance measurements in blood gas analyzers require dissolution of red blood cells which does not work for HbMP. Therefore, we have developed a robust and rapid optical method for the quality and functionality control of HbMPs. It allows simultaneous determination of the portion of the two states of hemoglobin oxygenated hemoglobin (oxyHb) and deoxygenated hemoglobin (deoxyHb) as well as the content of methemoglobin (metHb). Based on the measurement of collimated transmission spectra between 300 nm and 800 nm, the average extinction cross section of HbMPs is derived. A numerical method is applied to determine the composition of the HbMPs based on their wavelength-dependent refractive index (RI), which is a superposition of the three different states of Hb. Thus, light-scattering properties, including extinction cross sections can be simulated for different compositions and sizes. By comparison to measured spectra, the relative concentrations of oxyHb, deoxyHb, metHb are accessible. For validation of the optically determined composition of the HbMPs, we used X-ray fluorescence spectrometry for the ratio of Fe(II) (oxyHb/deoxyHb) and Fe(III) (metHb). High accuracy density measurements served to access heme-free proteins, size was determined by dynamic light scattering and analytical centrifugation and the shape of the HbMPs was visualized by electron and atomic force microscopy.

Keywords: HbMP; artificial blood substitute; hemoglobin-based oxygen carrier; light scattering; methemoglobin determination; spectral extinction; spectral refractive index; sub-micrometer particle characterization.

Figure 1

(a) Production scheme of HbMPs by co-precipitation and cross linking. (b,c) Images of HbMPs taken by scanning electron microscopy. (d,e) Atomic force microscopy of HbMPs attached with two different orientations to the surface. The white arrows correspond to a length of 200 nm in each image.

Figure 2

Results of spectral extinction measurements and simulations. (a) Volume-specific spectral extinction cross sections measured for oxyHbMP (dot-dashed red line) and calculated (red line) using 35% fraction of metHb and 65% oxyHb. (b) Volume-specific spectral extinction cross sections measured for metHbMP (dashed brown line) and calculated for 100% metHbMP (brown line). (c) Comparison of the measured spectral cross section in the Soret band and cross sections calculated for various metHb fractions. (d) Reduced wavelength range to show the Soret band of the measured and calculated extinction spectra.

Figure 3

Volume-specific spectral extinction cross sections measured (dash-dotted and dashed lines) in air and using argon for deoxygenation. Calculated, volume-specific spectral extinction cross sections (solid lines) are included to illustrate the change of oxygenation of the HbMPs.

Figure 4

(a) NEXAFS fluorescence spectra of an oxygenated HbMP suspension (dot-dashed red trace) and metHbMP (dashed brown graph). For comparison, the absorbance of reference spectra for Fe(II) and Fe(III) [18] are included as blue and green lines, respectively. (b) Analysis of the oxygenated HbMPs (dot-dashed red curve) spectra as superposition of HbMPs containing 35% metHb and 65% oxyHb.

Figure 5

Complex refractive index increments of the Hb components and HSA used in our calculations of the extinction cross sections of HbMPs.