Rapid Assessment of Hemoglobin-Oxygen Dissociation

Introduction:

Oxygen (O₂) delivery to tissues depends on the hemoglobin (Hb)-O₂ affinity and dissociation of O₂ from Hb in red blood cells (RBCs). Mutations that impact α and β-globin subunits of Hb often alter the Hb-O2 affinity of Hb and Hb-O₂ dissociation. A point mutation in the β-globin gene yields sickle hemoglobin (HbSS) which results in sickle cell disease (SCD). Recently, there has been an upsurge in improving SCD pathophysiology through modifying HbSS O₂ affinity. The ability to alter cellular phosphate such as 2,3-DPG that influences Hb-O₂ affinity is also under investigation. In this context, measuring Hb-O₂ affinity has gained additional significance as a clinical and laboratory parameter. Hb-O₂ affinity is determined by measuring Hb-O₂ saturation (SO₂, %) vs. partial pressure of oxygen (pO₂, mmHg). The Hb-O₂ affinity is commonly represented by the p50 value, which is the pO₂ value at 50% SO₂. p50 measurement is underutilized in clinical practice mainly due to cost, complexity, and limited throughput of existing instruments. Herein, we present a rapid assay that can detect Hb-O₂ affinity disorders, determine cellular oxygen availability, and examine the effectiveness of existing and novel therapies for SCD patients.

Methods:

In this assay, we optically measure the Hb-O₂ dissociation by induction of physiologic acidic pH (6.9). We exploited the acidic pH environment to expedite chemically induced partial deoxygenation of Hb (deoxy-Hb) during which the conformational changes in the tertiary structure of Hb are manifested as significant peak shifts in the optical absorption spectra. Hb absorption spectra in the 350-700 nm wavelength range primarily reflect the conformation of heme-heme interactions, which are sensitive indicators of changes in the Hb-O₂ affinity. We validated the assay by comparing it with the reference standard Hemox analyzer using blood from healthy individuals (HbAA) and from patients with SCD (HbSS) i.e., low Hb-O₂ affinity. Venous blood samples were collected in EDTA-coated tubes from subjects with HbSS (n=44), HbAS (n=14), and HbAA (n=17) following an IRB-approved study protocol. 75 µl of whole blood diluted 100-fold with pH 6.9 phosphate buffer was incubated for 2 minutes at room air to attain full oxygenation. The mixture of blood and the buffer was then deoxygenated with 0.053M of sodium metabisulphite (Na₂S₂O₅) for 3 minutes to achieve partial deoxy-Hb. We then obtained 350-450 nm spectra of deoxy-Hb using a spectroscopy microplate reader with a 2 nm resolution. We recorded each sample's Soret band shift in 4 minutes using the 414 nm Hb fully oxygenated absorption peak as the reference. Data were reported as SEM.

Results: Hb exhibits three characteristic light absorption peaks. The Soret peak is located at 414 nm under full oxygenation at pO₂ of 162 mmHg, and upon full deoxy-Hb at pO₂ of 5 mmHg, the peak shifts to 430 nm (Figure 1A). We initially tested different concentrations of Na₂S₂O₅ and the time of deoxygenation to achieve partial deoxy-Hb. At 75 mmHg we analyzed the Soret peak which was used as an optical marker of oxygen affinity, an equivalent to the p50 value. The peak shift significantly correlated to the p50 value measured by the Hemox device (PCC=0.81, p=0.004, n=28) (Figure 1A). We also observed significant differences between HbAA (n=17), HbAS(n=14), SCD samples on various treatments (transfusion (Tx) (n=8), and Hydroxyurea (n=15) and SCD treatment-naïve patients’ samples (n=20). HbAA vs. HbAS; p=0.01, HbAS vs. HbSS+Tx; p=0.02, HbSS+Tx vs. HbSS+Hu; p=0.04, HbAA vs. HbSS treatment naïve; p=0.000, HbSS treatment Naive vs. HbAS; p=0.001 (Figure 1B). Additionally, the peak shift for HbSS correlated with the percentage of HbSS (PCC = 0.87, p = 0.000, n=20, data not shown).

Discussion:

We demonstrate fast, effective differentiation of abnormal Hb-O₂ affinity and characterize the changes in the HbSS-O₂ affinity in response to treatments by using chemically induced partial deoxygenation of Hb at a physiologic acidic pH. The ubiquity of optical absorption spectrophotometers positions this assay as an accessible, equitable, and accurate Hb-O₂ dissociation measurement resource for research and clinical use. The assay will facilitate the diagnosis and prognosis of red blood cells and Hb disorders requiring minimal technical expertise and laboratory infrastructure and assist with testing the effect of new targeted and genome-editing-based therapies for SCD.