A second-derivate fitting algorithm for the quantification of free hemoglobin in human plasma.

BACKGROUND: Assessment of hemolysis in vivo is becoming increasingly relevant in critical care. Current methods (Harboe, 1959) for quantifying the free hemoglobin (fHb) content produce unsatisfactory results in case of hyperbilirubinemia, a frequent condition in patients at risk for intravascular hemolysis. METHODS: A novel evaluation method based on second-derivative fitting to quantify fHb content was developed. The method uses spectrophotometric data from 350 to 650 nm recorded with standard instruments as input. To evaluate the power of the new method, plasma of patients and non-icteric plasma of healthy volunteers were spiked with fHb concentrations up to 2000 mg/L and compared to methods described in the literature by Harboe, Noe and Fairbanks. All measurements were done in compliance with the bioanalytical method validation protocol from the European Medicines Agency. RESULTS: Both the second-derivative fitting algorithm as well as the methods of Harboe, Noe and Fairbanks quantified fHb accurately in non-icteric samples, with inaccuracy and imprecision below 10%. For icteric specimen, false high results were obtained with the established formulas for fHb concentrations below 700 mg/L. In contrast, no interference was found with the second-derivate fitting method for bilirubin concentrations up to 465 µmol/L. The lower limits of quantifications for the second-derivative fitting algorithm were specified in agreement with the EMA guideline with 25 mg/L fHb for both non-icteric and icteric specimens. CONCLUSIONS: A user-friendly, computer-based algorithm is reported that allows the accurate quantification of fHb concentrations in the presence of high bilirubin concentrations. The new method allows for uniform sample preparation with only a single dilution step and can be readily implemented in any laboratory on standard spectrophotometers using the provided supplementary Microsoft Excel macro.

Evaluation of Antitrypanosomal Dihydroquinolines for Hepatotoxicity, Mutagenicity, and Methemoglobin Formation In Vitro.

N1-Benzylated dihydroquinolin-6-ols and their corresponding esters display exceptional activity against African trypanosomes in vitro, and administration of members of this class of compounds to trypanosome-infected mice results in cures in a first-stage African trypanosomiasis model. Since a quinone imine intermediate has been implicated in the antiparasitic mechanism of action of these compounds, evaluation of the hepatotoxic, mutagenic, and methemoglobin-promoting effects of these agents was performed. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride and 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate showed outstanding in vitro selectivity for Trypanosoma brucei compared to the HepG2, Hep3B, Huh7, and PLC5 hepatocyte cell lines. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride and 1-(2-methoxybenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate were not mutagenic when screened in the Ames assay, with or without metabolic activation. The latter 2 compounds promoted time- and dose-dependent formation of methemoglobin when incubated in whole human blood, but such levels were below those typically required to produce symptoms of methemoglobinemia in humans. Although compounds capable of quinone imine formation require careful evaluation, these in vitro studies indicate that antitrypanosomal dihydroquinolines merit further study as drug candidates against the neglected tropical disease human African trypanosomiasis.

Pro-stimulatory role of methemoglobin in inflammation through hemin oxidation and polymerization.

Inflammation or vascular occlusion by parasitized red blood cell contributes to the pathogenesis of cerebral malaria. The current study aimed to characterize the role of major pro-oxidant factor methemoglobin present in the malaria culture supernatant contributing in inflammation during malaria. Heme and heme polymer stimulate macrophage to secrete large amount of reactive oxygen species into the external micro-environment. The addition of methemoglobin along with heme or heme polymer amplifies production of ROS from macrophages several folds. Methemoglobin mediated stimulatory effect is not due to release of iron, enhanced production of H2O2 or mutual interaction of reaction components. Spectroscopic studies show that methemoglobin accepts heme as a substrate and oxidizes it through a single electron transfer mechanism. Heme oxidation product is a heme polymer with similar chemical and structural properties to synthetic ß-hematin. Phenyl N-t-butylnitrone inhibits heme polymerization (IC50=30 nM) and indicates the absolute necessity of heme oxidation and heme free radical generation for heme polymerization. Methemoglobin produced heme polymer is a potent pro-inflammatory factor to release ROS into external microenvironment. Interestingly, methemoglobin not only produces pro-inflammatory heme polymer, but it also amplifies the potential of heme or preformed heme polymer (haemozoin or ß-hematin) to produce several folds high ROS production from macrophages. This study illustrates the pro-inflammatory effect of methemoglobin, the underlying novel mechanism by which this occurs and a possible clinical intervention. Based on the results, we recommend methemoglobin directed peroxidase inhibitors as an adjuvant therapy during malaria.

Composite films of lecithin and heme proteins with electrochemical and electrocatalytic activities.

Functional composite films made from lecithin micelles and the two heme proteins of met-myoglobin (Mb) and met-hemoglobin (Hb) are reported in this paper. Proteins in functional composite films have much higher rates of electron transfer than proteins in solutions on carbon paste (CP) electrodes. Cyclic voltammograms (CVs) all give a pair of well-defined and quasi-reversible peaks, corresponding to the heme FeIII/FeII redox couple of proteins. Differential pulse voltammograms (DPVs) also show the same formal potential (E0') values of proteins under identical conditions. Electronic and vibrational spectra indicate that proteins in these films are not denatured, but their conformational differences from native states may exist. The E0' value for Mb in the lecithin film is found to be pH dependent. The Mb lecithin film can catalytically reduce O2 and H2O2, and its analytical application to H2O2 determination is established.

Hemoglobin polymerized with a naturally occurring crosslinking agent as a blood substitute: in vitro and in vivo studies.

A naturally occurring crosslinking agent, genipin, extracted from the fruits of Gardenia jasminoides ELLIS was used by our group to chemically modified biomolecules. Genipin and its related iridoid glucosides have been widely used as an antiphlogistic and cholagogue in herbal medicine. Our previous study showed that the cytotoxicity of genipin is significantly lower than glutaraldehyde. The study was to investigate the feasibility of using genipin to polymerize hemoglobin as a blood substitute. The results indicated that the rate of hemoglobin polymerization by glutaraldehyde was significantly faster than that by genipin and it readily produced polymers with molecular masses greater than 500,000 Da. It was found that the maximum degree of hemoglobin polymerization by genipin was approximately 40% if over-polymerization is to be prevented. With increasing the reaction temperature, hemoglobin concentration, and genipin-to-hemoglobin molar ratio, the duration taken to achieve the maximum degree of hemoglobin polymerization by genipin became significantly shorter. The P50 value of the unmodified hemoglobin was 9 mmHg, while that of the genipin-polymerized PLP-hemoglobin increased to 21 mmHg. It was found in a rat model that the genipin-polymerized PLP-hemoglobin resulted in a longer circulation time than the unmodified hemoglobin. In conclusion, the results of the study indicated that the genipin-polymerized hemoglobin solution has a lower oxygen affinity and a longer vascular retention time than the unmodified hemoglobin solution.

Internal intensity standards for heme protein UV resonance Raman studies: excitation profiles of cacodylic acid and sodium selenate.

We examine the utility of SO4(2-), ClO4-, cacodylic acid, and SeO4(2-) as internal intensity standards for Raman spectral measurements of protein structure. We find that 0.1 M SO4(2-) and ClO4- perturb the protein tertiary structure of aquomethemoglobin (met-Hb) and its fluoride (met-HbF) and azide (met-HbN3) complexes. Changes occur for the tryptophan near-UV absorption bands, the iron spin state is altered, and the fluoride ligand affinity decreases. Concentrations of ClO4- and SO4(2-) as low as 0.1 M suppress the met-HbF quaternary R----T transition induced by the allosteric effector inositol hexaphosphate (IHP). In contrast, similar concentrations of cacodylic acid and SeO4(2-) show little effect on the hemoglobin tertiary or quaternary protein structures or upon the R----T transition induced by IHP. We measure the Raman cross sections of cacodylic acid and SeO4(2-) between 218 and 514.5 nm and find that for UV excitation they are ca. 5-fold larger than ClO4- or SO4(2-). Thus, cacodylic acid and selenate can be used at lower concentrations. Cacodylic acid and SeO4(2-) are superior Raman internal intensity standards for protein structural studies.