The physiological role of the respiratory hemoproteins (RH), hemoglobin and myoglobin, is to deliver O2 via its binding to their ferrous (FeII) heme-iron. Under variety of pathological conditions RH proteins leak to blood plasma and oxidized to ferric (FeIII, met) forms becoming the source of oxidative vascular damage. However, recent studies have indicated that both metRH and peroxides induce Heme Oxygenase (HO) enzyme producing carbon monoxide (CO). The gas has an extremely high affinity for the ferrous heme-iron and is known to reduce ferric hemoproteins in the presence of suitable electron donors. We hypothesized that under in vivo plasma conditions, peroxides at low concentration can assist the reduction of metRH in presence of CO. The effect of CO on interaction of metRH with hydrophilic or hydrophobic peroxides was analyzed by following Soret and visible light absorption changes in reaction mixtures. It was found that under anaerobic conditions and low concentrations of RH and peroxides mimicking plasma conditions, peroxides served as electron donors and RH were reduced to their ferrous carboxy forms. The reaction rates were dependent on CO as well as peroxide concentrations. These results demonstrate that oxidative activity of acellular ferric RH and peroxides may be amended by CO turning on the reducing potential of peroxides and facilitating the formation of redox-inactive carboxyRH. Our data suggest the possible role of HO/CO in protection of vascular system from oxidative damage.

The method is valid for accurate quantification of Hb at a wide concentration range (>0.1 μm/L) in erythroid precursors or plasma and is optional for other biological fluids.

Outside their cellular environments, hemoglobin (Hb) and myoglobin (Mb) are known to wreak oxidative damage. Using haptoglobin (Hp) and hemopexin (Hx) the body defends itself against cell-free Hb, yet mechanisms of protection against oxidative harm from Mb are unclear. Mb may be implicated in oxidative damage both within the myocyte and in circulation following rhabdomyolysis. Data from the literature correlate rhabdomyolysis with the induction of Heme Oxygenase-1 (HO-1), suggesting that either the enzyme or its reaction products are involved in oxidative protection. We hypothesized that carbon monoxide (CO), a product, might attenuate Mb damage, especially since CO is a specific ligand for heme iron. Low density lipoprotein (LDL) was chosen as a substrate in circulation and myosin (My) as a myocyte component. Using oxidation targets, LDL and My, the study compared the antioxidant potential of CO in Mb-mediated oxidation with the antioxidant potential of Hp in Hb-mediated oxidation. The main cause of LDL oxidation by Hb was found to be hemin which readily transfers from Hb to LDL. Hp prevented heme transfer by sequestering hemin within the Hp-Hb complex. Hemin barely transferred from Mb to LDL, and oxidation appeared to stem from heme iron redox in the intact Mb. My underwent oxidative crosslinking by Mb both in air and under N2. These reactions were fully arrested by CO. The data are interpreted to suit several circumstances, some physiological, such as high muscle activity, and some pathological, such as rhabdomyolysis, ischemia/reperfusion and skeletal muscle disuse atrophy. It appear that CO from HO-1 attenuates damage by temporarily binding to deoxy-Mb, until free oxygen exchanges with CO to restore the equilibrium.

Carbon monoxide releasing molecules (CORMs) have been recently developed for research and pharmacological purposes. A considerable amount of studies demonstrated a wide spectrum of biological activities for lipophilic CORM-2 (tricarbonyldichlororuthenium (II) dimer). It is generally accepted that the liberated gas provides the specific activities to CORMs, with a little attention paid to any possible effect of complementary core molecules. However, the versatile repertoire of actions attributed to CORM-2 is surprisingly wide for CO, a molecule with the sole chemical activity of binding to ferrous iron in protein prosthetic groups. The study was designed to analyze CORM-2 and its core molecule (“i”CORM) activities at a molecular level. With respect to the hydrophobic nature of the compounds, we followed their interactions with several amphipathic entities: the heme sites of hemoproteins, heme binding proteins and cell membranes. CORM-2/“i”CORM decreased the Soret optical density of hemoglobin and myoglobin, indicating that both compounds interact with the protein amphipathic site in the heme pocket. Pre-addition of CORM-2/“i”CORM to the apo-forms of the plasma heme binding proteins, hemopexin and albumin, partially abolished their heme binding capacity. In contrast, the compounds had no effect on the preformed heme-protein complexes. Addition of CORM-2/“i”- CORM to blood or isolated erythrocytes revealed aggregation of the cells or lysis, depending on the rea-gent-to-cells ratio. It was concluded that the ruthenium containing core molecule of CORM-2 may be physiologically active due to non-specific hydrophobic interactions. As each type of CORMs is expected to have a different mode of action beyond CO activity, their potential therapeutic uses will require clarification

3207 Background: 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. Aim: The current study is focused on the ability of plasma Hx to defend the vasculature from acellular Hb induced damage. Results: 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. Conclusion: 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. Disclosures: No relevant conflicts of interest to declare.