04125 Detection and quantification of labile intravascular heme in the presence of hemoglobin
Year: 2024
Type: conference-paper
Abstract: Heme, iron protoporphyrin IX, is one of the most abundant molecules of aerobic life. As a prosthetic group of proteins it is highly important for numerous biological reactions and processes, such as oxygen transport, electron transfer, and catalysis. Heme also has modulating properties that go beyond the regulation of its own biosynthesis. In addition, serious conditions are provoked in case of malfunctioning endogenous heme production. Hemolytic events caused by various disorders (e.g., sickle cell disease), infections (e.g., malaria) or injuries and trauma lead to an accumulation of intravascular labile heme, whereas the different types of porphyrias are characterized by a lack of heme. Porphyria is the umbrella term for a group of hereditary metabolic disorders that involve one of the eight enzymatic steps of the heme biosynthesis pathway. Specific for each impaired enzyme is the accumulation of certain heme precursors in the blood, urine or stool, accompanied by various symptoms. As a possible treatment strategy, the substitution of heme is utilized in acute porphyria incidences or prophylactically. The accumulation of heme precursors is stopped e.g. by end product inhibition of 5-aminolevulinic acid synthase and at the same time restores the essential heme pool. Monitoring the administered heme and therefore, an individualized treatment can be targeted for advanced therapy. Until recently there was no quantification method available, which distinguished between labile heme and protein-bound heme and hence could help to monitor labile heme after administration. To close this gap of knowledge, we conducted a study to evaluate the validity and applicability of methods for quantification of heme levels in the presence of hemoglobin as well as to assess the differentiation of hemoglobin-bound heme and labile heme. In total, 10 direct and indirect primarily spectroscopic methods were examined regarding their linearity, accuracy, and precision, as well as their ability to distinguish between hemoglobin-bound heme and labile heme. Thereupon, the different approaches have been used to quantify heme and Hb solutions as well as mixtures thereof. The determination of a broad concentration range, heme ~0.02–45 µM and hemoglobin ~0.002–17 µM, was possible using the investigated indirect and direct methods. Nevertheless, a clear distinction between hemoglobin-bound heme and labile heme applying only one method was, however, not feasible, suggesting the use of a combined approach. In consequence, a combination of two spectroscopic methods, one direct and one indirect, including a newly established equation was used to quantify the labile heme content in porcine plasma samples as well as human plasma samples in healthy and varying hemolytic states. In conclusion, our study can accelerate porphyria treatment by aiding to monitor the administration of the proper heme dosage, improving the compatibility to and compliance of the patients.
Authors: Marie-T Hopp, Sonali M Vaidya, Karina M Grimmig, Xiaojing Yuan, Iqbal Hamza, Diana Imhof
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4402696170