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

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.

. Oxygenation -deoxygenation and effect of particle loss. "air 1" denotes the measurement before bubbling with argon, "argon" denotes the measurement directly after and "air 2" the measurement after the sample was re-exposed to air. The latter two curves were rescaled to have the lowest deviation from curve "air 1".
RI determination of HSA. Literature values are available for the real RI increment of HSA in aqueous solutions. However, these were either determined using white light or a single wavelength (typically the sodium D-line at 589 nm). Hence, we determined the wavelengthdependence of the real part of the RI increment by own measurements. We used a HSA infusion solution (Human Albumin 200 g L -1 Baxalta Infusionslösung) as the sample, which contains proteins from human plasma, of which at least 95% are albumin (HSA).
Firstly, absorption spectra were recorded with a Cary6000i UV-Vis-NIR spectrophotometer (Agilent) using multiple dilutions with ultrapure water in pair-matched 10mm cuvettes. This yields the imaginary part HSA ( ) of the RI increment. At wavelengths higher than 700 nm, the absorbance is too low to be measured for the undiluted 200 g / L solution in a 10 mm cuvette.
Here, we set HSA ( ) = 0 in the simulations (compare Figure 5). Furthermore, HSA ( ) is significantly lower than Hb ( ) everywhere in the spectral range of 300 nm to 800 nm. The ( )  curves come close to each other in the UV below 300 nm, where the spectrum of the HSA infusion solution also exhibits a significant absorbance contribution due to its content of 16 mmol/L sodium acetyltryptophanate, making up an estimated 50% of the total absorbance at 279 nm (the absorbance peak of the amino acid tryptophan). However, this is outside the spectral range under consideration. Hence, the error due to the presence of sodium acetyltryptophanate has no significant effect on the simulation results.
The real part of the spectral RI increment HSA ( ) was determined using the same measurement setup as for the extinction measurements for the HbMPs. To this end, quasimonodisperse polystyrene beads with 2.539 µm nominal diameter (Lot. PS-ST-L2552, microparticles GmbH, Germany) were suspended in the 200 g L HSA solution at different particle concentrations and the collimated transmittance was measured. Given the spectral RI of the particles, obtained from an extinction measurement in pure water S1 , the real part of the spectral RI of the suspending fluid (i.e., the HSA solution) can be obtained from these measurements. For this purpose, the method described in Ref. S1 was modified S2 . The fact that the HSA solution absorbs light in UV limits the wavelength range available for transmittance measurements to λ ≥ 300 nm, but otherwise does not hamper the data analysis which is based on the assumption of a non-absorbing fluid S2 . Hence, the RI of the aqueous HSA solution is For the model fit of the measured ̅ ( ) the spectral dependence of the RI increment HSA ( ) was expressed by a 2-term Cauchy equation, i.e.
The result for the coefficients is = 0.180 mL g and = 3.2 × 10 mL g µm for 300 nm ≤ ≤ 1100 nm. The estimated uncertainty of HSA ( ) is HSA ( ) = 0.008 mL g -1 . For verification purposes, the RI was also determined with an Abbe refractometer at 590 nm, which resulted in 0.190(4) mL g compared to 0.188 (8)