Intravascular hemolysis renders hemoglobin (Hb), usually protected by the red blood cell environment, susceptible to oxidation to its ferric (metHb) form. Haptoglobin (Hp) provides first aid to protect the vasculature from acellular metHb havoc. However, once Hp is consumed, ferrous Hb readily undergoes oxidation to metHb, in which the globin to heme affinity is reduced. MetHb releases the loosely bound heme allowing it to execute its oxidative activity. While nucleated cells, like endothelial cells, are equipped with the heme oxygenase-1 defence system, the nucleus lacking red blood cells (RBC) and low density lipoproteins (LDL) particles have no defence from heme-induced oxidation. Therefore the sole defence from heme oxidative damage remains the plasma protein hemopexin (Hx), which binds hemin with high affinity comparable to that of globin. Following hemin binding, Hx undergoes conformational changes rendering the heme inaccessible thereby inactive.
The current study is focused on the ability of plasma Hx to defend the vasculature from acellular Hb induced damage.
Hx defence from metHb damage was studied in the following projects: a) preventing in vitro LDL oxidation by metHb, b) decreasing in vitro lysis of red cells created by hypotonic and mechanical stress. (c) Exploring consumption of Hx in hemodialysis (HD) treated patients by measuring their Hx levels. (a) Methemoglobin iron is a central cause of LDL oxidation. This process initiates formation of blood vessel plaques and atherosclerosis development. This part of the study examined the effect of apoHx (lacking heme) on LDL oxidation by metHb. Incubation of isolated native LDL (nLDL) or vitamin E depleted (dLDL) with low concentration of metHb range (1-10μM) resulted in covalent apoB inter-particle crosslinking in both LDL types, dLDL being more sensitive than nLDL to the oxidative crosslinking. Addition of apoHx abolished metHb-induced oxidation even in the dLDL. This Hx activity (comparable to that of haptoglobin) was confirmed by both SDS-PAGE and fluorescent assay of bityrosines formation. The fact that Hx protects LDL, especially vitamin E depleted particles, shed light on the controversial antioxidant vitamin E properties by indicating its efficacy in defence against acellular Hb induced LDL oxidation. (b) Like LDL the red cell membrane is exposed to metHb-induced oxidative damage. Acellular Hb was formed by hypotonic shock or mechanical shear stress to fresh whole blood of healthy donors. The effect of Hx on acellular Hb levels was measured spectrophotometrically. Reduction of osmotic pressure in fresh blood to 40% of its normal value resulted in formation of ∼300 μM acellular Hb. Supplementation of exogenous apoHx (20μM) to blood before exposure to hypotonicity reduced acellular Hb formation by half. The specificity of Hx was proved by addition of up to 80μM albumin, which had no effect on acellular Hb concentration. Acellular Hb was formed also by applying shear stress on circulating fresh blood in narrow channels using a peristaltic pump. Within 4 hours acellular Hb reached ∼500μM. However, in presence of 20μM apoHx hemolysis was significantly reduced (up to 50%). This reduction may be attributed to Hx's ability to block the hemolysis amplifying effect of metHb on the RBC membrane. (c) In HD treated patients RBCs are exposed to the effects of recurrent mechanical shear stress. The last part of the research was dedicated to quantisation of apoHx levels (by titration with hemin) in sera of HD patients (n=60) prior to and immediately after a single dialysis procedure versus normal controls (n=20). Sera apoHx levels in HD patients prior to a dialysis session were lower compared to healthy controls (10.91±3.19 μM vs. 14.95±2.63 μM, p<0.05) and were even lower (7.49±3.8 μM) immediately after the treatment. These findings indicate that in the plasma of HD treated patients Hx consumption rate exceeds synthesis rate leading to a chronically low level of Hx. Moreover, reduction of Hx was even more prominent in patients on long term HD treatment of above five years.
Hx is specifically effective in attenuation of acellular Hb damage to the vasculature. These results point to the option that treating HD patients with exogenous Hx may slow down the rate of atherosclerotic cardiovascular disease development, the major cause of morbidity and mortality in these patients.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.