Dataset Information

Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry.

ABSTRACT: Methemoglobin (MetHb) is a hemoglobin (Hb) derivative with the heme iron in ferric state (Fe³⁺), unable to deliver oxygen. Quantification of methemoglobin is a very important diagnostic parameter in hypoxia. Recently, novel hemoglobin microparticles (Hb-MP) with a narrow size distribution around 700 nm, consisting of cross-linked Hb were proposed as artificial oxygen carriers. The cross-linking of Hb by glutaraldehyde (GA) generates a certain amount of MetHb. Due to the strong light scattering, quantitative determination of MetHb in Hb-MP suspensions by common spectrophotometry is not possible. Here, we demonstrate that ¹H₂O NMR relaxometry is a perfect tool for direct measurement of total Hb and MetHb concentrations in Hb-MP samples. The longitudinal relaxation rate 1/T₁ shows a linear increase with increasing MetHb concentration, whereas the transverse relaxation rate 1/T₂ linearly increases with the total Hb concentration. In both linear regressions the determination coefficient (R²) is higher than 0.99. The method does not require time-consuming pretreatment or digestion of the particles and is not impaired by light scattering. Therefore, it can be established as the method of choice for the quality control of Hb-MP and similar hemoglobin-based oxygen carriers in the future.

SUBMITTER: Kaewprayoon W

PROVIDER: S-EPMC7730817 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry.

Kaewprayoon Waraporn W Suwannasom Nittiya N Kloypan Chiraphat C Steffen Axel A Xiong Yu Y Schellenberger Eyk E Pruß Axel A Georgieva Radostina R Bäumler Hans H

International journal of molecular sciences 20201126 23

Methemoglobin (MetHb) is a hemoglobin (Hb) derivative with the heme iron in ferric state (Fe<sup>3+</sup>), unable to deliver oxygen. Quantification of methemoglobin is a very important diagnostic parameter in hypoxia. Recently, novel hemoglobin microparticles (Hb-MP) with a narrow size distribution around 700 nm, consisting of cross-linked Hb were proposed as artificial oxygen carriers. The cross-linking of Hb by glutaraldehyde (GA) generates a certain amount of MetHb. Due to the strong light s ...[more]

PMID: 33256027

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Project description:Nitrite has recently been recognized as a storage form of NO in blood and as playing a key role in hypoxic vasodilation. The nitrite ion is readily reduced to NO by hemoglobin in red blood cells, which, as it happens, also presents a conundrum. Given NO's enormous affinity for ferrous heme, a key question concerns how it escapes capture by hemoglobin as it diffuses out of the red cells and to the endothelium, where vasodilation takes place. Dinitrogen trioxide (N(2)O(3)) has been proposed as a vehicle that transports NO to the endothelium, where it dissociates to NO and NO(2). Although N(2)O(3) formation might be readily explained by the reaction Hb-Fe(3+)+NO(2)(-)+NO?Hb-Fe(2+)+N(2)O(3), the exact manner in which methemoglobin (Hb-Fe(3+)), nitrite and NO interact with one another is unclear. Both an "Hb-Fe(3+)-NO(2)(-)+NO" pathway and an "Hb-Fe(3+)-NO+NO(2)(-) " pathway have been proposed. Neither pathway has been established experimentally. Nor has there been any attempt until now to theoretically model N(2)O(3) formation, the so-called nitrite anhydrase reaction. Both pathways have been examined here in a detailed density functional theory (DFT, B3LYP/TZP) study and both have been found to be feasible based on energetics criteria. Modeling the "Hb-Fe(3+)-NO(2)(-)+NO" pathway proved complex. Not only are multiple linkage-isomeric (N- and O-coordinated) structures conceivable for methemoglobin-nitrite, multiple isomeric forms are also possible for N(2)O(3) (the lowest-energy state has an N-N-bonded nitronitrosyl structure, O(2)N-NO). We considered multiple spin states of methemoglobin-nitrite as well as ferromagnetic and antiferromagnetic coupling of the Fe(3+) and NO spins. Together, the isomerism and spin variables result in a diabolically complex combinatorial space of reaction pathways. Fortunately, transition states could be successfully calculated for the vast majority of these reaction channels, both M(S)=0 and M(S)=1. For a six-coordinate Fe(3+)-O-nitrito starting geometry, which is plausible for methemoglobin-nitrite, we found that N(2)O(3) formation entails barriers of about 17-20 kcal mol(-1) , which is reasonable for a physiologically relevant reaction. For the "Hb-Fe(3+) -NO+NO(2) (-) " pathway, which was also found to be energetically reasonable, our calculations indicate a two-step mechanism. The first step involves transfer of an electron from NO(2)(-) to the Fe(3+)-heme-NO center ({FeNO}(6)) , resulting in formation of nitrogen dioxide and an Fe(2+)-heme-NO center ({FeNO}(7)). Subsequent formation of N(2)O(3) entails a barrier of only 8.1 kcal mol(-1) . From an energetics point of view, the nitrite anhydrase reaction thus is a reasonable proposition. Although it is tempting to interpret our results as favoring the "{FeNO}(6)+NO(2)(-) " pathway over the "Fe(3+)-nitrite+NO" pathway, both pathways should be considered energetically reasonable for a biological reaction and it seems inadvisable to favor a unique reaction channel based solely on quantum chemical modeling.

Project description:BackgroundCerebral intraventricular hemorrhage (IVH) is a major cause of severe neurodevelopmental impairment in preterm infants. To date, no therapy is available that prevents infants from developing serious neurological disability following IVH. Thus, to develop treatment strategies for IVH, it is essential to characterize the initial sequence of molecular events that leads to brain damage. In this study, we investigated extracellular hemoglobin (Hb) as a causal initiator of inflammation in preterm IVH.MethodsUsing a preterm rabbit pup model, we investigated the molecular mechanisms and events following IVH. We also characterized the concentrations of cell-free Hb metabolites and pro-inflammatory mediators in the cerebrospinal fluid (CSF) of preterm human infants and rabbit pups. Finally, Hb metabolites were evaluated as causal initiators of inflammation in primary rabbit astrocyte cell cultures.ResultsFollowing IVH in preterm rabbit pups, the intraventricular CSF concentration of cell-free methemoglobin (metHb) increased from 24 to 72 hours and was strongly correlated with the concentration of TNFα at 72 hours (r2 = 0.896, P <0.001). Also, the mRNA expression of TNFα, IL-1β, and Toll-like receptor-4 and TNFα protein levels were significantly increased in periventricular tissue at 72 hours, which was accompanied by extensive astrocyte activation (that is, glial fibrillary acidic protein (GFAP)staining). Furthermore, exposure of primary rabbit astrocyte cell cultures to metHb caused a dose-dependent increase in TNFα mRNA and protein levels, which was not observed following exposure to oxyhemoglobin (oxyHb) or hemin. Finally, a positive correlation (r2 = 0.237, P <0.03) between metHb and TNFα concentrations was observed in the CSF of preterm human infants following IVH.ConclusionsFollowing preterm IVH, increased metHb formation in the intraventricular space induces expression of pro-inflammatory cytokines. Thus, the formation of metHb might be a crucial initial event in the development of brain damage following preterm IVH. Accordingly, removal, scavenging, or neutralization of Hb could present a therapeutic opportunity and plausible approach to decreasing the damage in the immature brain following preterm IVH.

Dataset Information

Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry.

Publications

Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry.

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