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Development and validation of an oxygen dissociation assay, a screening platform for discovering, and characterizing hemoglobin-oxygen affinity modifiers.

ABSTRACT: Introduction:Hemoglobin (Hb) is a critical molecule necessary for all vertebrates to maintain aerobic metabolism. Hb-oxygen (O2) affinity modifiers have been studied to address various diseases including sickle cell disease, hypoxemia, tumor hypoxia, and wound healing. However, drug development of exogenous Hb modifiers has been hindered by the lack of a technique to rapidly screen compounds for their ability to alter Hb-O2 affinity. We have developed a novel screening assay based upon the spectral changes observed during Hb deoxygenation and termed it the oxygen dissociation assay (ODA). Methodology:ODA allows for the quantitation of oxygenated Hb at given time points during Hb deoxygenation on a 96-well plate. This assay was validated by comparing the ability of 500 Hb modifiers to alter the Hb-O2 affinity in the ODA vs the oxygen equilibrium curves obtained using the industry standard Hemox Analyzer instrument. Results:A correlation (R2) of 0.7 indicated that the ODA has the potential to screen and identify potent exogenous Hb modifiers. In addition, it allows for concurrent comparison of compounds, concentrations, buffers, or pHs on the level of Hb oxygenation. Conclusion:With a cost-effective, simple, rapid, and highly adaptable assay, the ODA will allow researchers to rapidly characterize Hb-O2 affinity modifiers.

SUBMITTER: Patel MP

PROVIDER: S-EPMC5989706 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Development and validation of an oxygen dissociation assay, a screening platform for discovering, and characterizing hemoglobin-oxygen affinity modifiers.

Patel Mira P MP Siu Vincent V Silva-Garcia Abel A Xu Qing Q Li Zhe Z Oksenberg Donna D

Drug design, development and therapy 20180601

<h4>Introduction</h4>Hemoglobin (Hb) is a critical molecule necessary for all vertebrates to maintain aerobic metabolism. Hb-oxygen (O<sub>2</sub>) affinity modifiers have been studied to address various diseases including sickle cell disease, hypoxemia, tumor hypoxia, and wound healing. However, drug development of exogenous Hb modifiers has been hindered by the lack of a technique to rapidly screen compounds for their ability to alter Hb-O<sub>2</sub> affinity. We have developed a novel screen ...[more]

PMID: 29910606

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Project description:Purpose To generate magnetic resonance (MR) imaging-derived, oxygen-hemoglobin dissociation curves and to map fetal-placental oxygen-hemoglobin affinity in pregnant mice noninvasively by combining blood oxygen level-dependent (BOLD) T2* and oxygen-weighted T1 contrast mechanisms under different respiration challenges. Materials and Methods All procedures were approved by the Weizmann Institutional Animal Care and Use Committee. Pregnant mice were analyzed with MR imaging at 9.4 T on embryonic days 14.5 (eight dams and 58 fetuses; imprinting control region ICR strain) and 17.5 (21 dams and 158 fetuses) under respiration challenges ranging from hyperoxia to hypoxia (10 levels of oxygenation, 100%-10%; total imaging time, 100 minutes). A shorter protocol with normoxia to hyperoxia was also performed (five levels of oxygenation, 20%-100%; total imaging time, 60 minutes). Fast spin-echo anatomic images were obtained, followed by sequential acquisition of three-dimensional gradient-echo T2*- and T1-weighted images. Automated registration was applied to align regions of interest of the entire placenta, fetal liver, and maternal liver. Results were compared by using a two-tailed unpaired Student t test. R1 and R2* values were derived for each tissue. MR imaging-based oxygen-hemoglobin dissociation curves were constructed by nonlinear least square fitting of 1 minus the change in R2*divided by R2*at baseline as a function of R1 to a sigmoid-shaped curve. The apparent P50 (oxygen tension at which hemoglobin is 50% saturated) value was derived from the curves, calculated as the R1 scaled value (x) at which the change in R2* divided by R2*at baseline scaled (y) equals 0.5. Results The apparent P50 values were significantly lower in fetal liver than in maternal liver for both gestation stages (day 14.5: 21% ± 5 [P = .04] and day 17.5: 41% ± 7 [P < .0001]). The placenta showed a reduction of 18% ± 4 in mean apparent P50 values from day 14.5 to day 17.5 (P = .003). Reproduction of the MR imaging-based oxygen-hemoglobin dissociation curves with a shorter protocol that excluded the hypoxic periods was demonstrated. Conclusion MR imaging-based oxygen-hemoglobin dissociation curves and oxygen-hemoglobin affinity information were derived for pregnant mice by using 9.4-T MR imaging, which suggests a potential to overcome the need for direct sampling of fetal or maternal blood. Online supplemental material is available for this article.

Dataset Information

Development and validation of an oxygen dissociation assay, a screening platform for discovering, and characterizing hemoglobin-oxygen affinity modifiers.

Publications

Development and validation of an oxygen dissociation assay, a screening platform for discovering, and characterizing hemoglobin-oxygen affinity modifiers.

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