Characterization of plasma labile heme in hemolytic conditions

Abstract

Extracellular hemoglobin, a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro-oxidant manner and regulates cellular metabolism while exerting pro-inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here, we developed and characterized a panel of heme-specific single domain antibodies (sdAbs) that together with a cellular-based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches, we demonstrate that when generated during hemolytic conditions labile heme is bound to plasma molecules with an affinity higher than 10−7 m and that 2–8% (∼ 2–5 μm) of the total amount of heme detected in plasma can be internalized by bystander cells, termed here bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme-binding capacity, that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10−7 m. The heme-specific sdAbs neutralize the pro-oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme-specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme-specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme.

Antigenic response to CT‐P13 and infliximab originator in inflammatory bowel disease patients shows similar epitope recognition

“…The Fab lambda (λ) naïve library was constructed as described with slight modifications . RNA was isolated from Peripheral Blood Mononuclear Cells (PBMCs) from fresh blood of 18 healthy volunteers, and the primers used for PCR amplification of human heavy (HC) and light‐chain (LC) V‐regions, were constructed according to the literature . All primary PCRs were carried out with separate reverse primers and combined forward primers, to maintain maximal diversity.…”

“…The upper limit of normal for each assay was calculated using the mean Ab index with three standard deviations (SD) of the population of 10 healthy individuals. Any sample with an Ab index above the upper limit of normal was designated as positive for antibody reactivity to the infliximab peptide …”

Formation and Detection of Highly Oxidized Hemoglobin Forms in Biological Fluids during Hemolytic Conditions

“…The presence and the pathophysiologic effects of "free" heme have been also established in severe sepsis caused by polymicrobial infections or sickle cell disease caused by mutations in the gene encoding the β chain of Hb [20][21][22][23]. Heme is a wellknown prooxidant, regulates cellular metabolism while exerting proinflammatory and cytotoxic effects [24,25], but according to a recent study, not all the "free" heme is bioavailable [26]. In fact, its bioavailable concentration under strong hemolytic conditions remains below 5 μmol/L [26].…”

“…Heme is a wellknown prooxidant, regulates cellular metabolism while exerting proinflammatory and cytotoxic effects [24,25], but according to a recent study, not all the "free" heme is bioavailable [26]. In fact, its bioavailable concentration under strong hemolytic conditions remains below 5 μmol/L [26].…”

Heme induces innate immune memory

“…Hemolysis or other forms of tissue damage can cause accumulation of cellfree labile heme in plasma 2 . As it becomes loosely bound to plasma acceptor proteins or macromolecules, these reduce but do not prevent labile heme redox activity that underlies its pathological effects 2,3 . As such, labile heme acts as a highly pro-oxidant agonists that contributes critically to the pathogenesis of severe infections, as demonstrated for malaria [4][5][6][7] and for bacterial sepsis [8][9][10] .…”