12/15-lipoxygenase translocation enhances site-specific actin polymerization in macrophages phagocytosing apoptotic cells.

The enzyme 12/15-lipoxygenase (12/15-LO) introduces peroxyl groups in a position-specific manner into unsaturated fatty acids in certain cells, but the role of such enzymatic lipid peroxidation remains poorly defined. Here we report a novel function for 12/15-LO in mouse peritoneal macrophages. When macrophages were coincubated with apoptotic cells, the enzyme translocated from cytosol to the plasma membrane and was more extensively concentrated at sites where macrophages bound apoptotic cells, colocalizing with polymerized actin of emerging filopodia. Disruption of F-actin did not prevent the 12/15-LO translocation. In contrast, inhibition of the 12/15-LO activity, or utilization of genetically engineered macrophages in which the 12/15-LO gene has been disrupted, greatly reduced actin polymerization in phagocytosing macrophages. Lysates of 12/15-LO-deficient macrophages had significantly lower ability to promote in vitro actin polymerization than the lysates of wild type macrophages. These studies suggest that the 12/15-LO enzyme plays a major role in local control of actin polymerization in macrophages in response to interaction with apoptotic cells.

Kinetics of hemin distribution in plasma reveals its role in lipoprotein oxidation.

Hemin is a powerful in vitro inducer of low-density lipoprotein (LDL) oxidation, implicated in development of atherosclerosis. To support the proposed role of hemin in atherogenesis, the question of whether hemin has any chance of getting together with LDL in vivo, must be addressed. A stopped-flow technique was employed in order to investigate the fast kinetics of hemin binding to LDL and to other plasma hemin-binding proteins: high-density lipoprotein (HDL), albumin and hemopexin. Based on the measured rate constants of hemin association with and dissociation from each of these proteins, time-dependent hemin distribution in plasma was analyzed. The analysis shows that as much as 80% of total hemin binds initially to LDL and HDL, the plasma components which are most susceptible to oxidation. Only then hemin partially transfers to the antioxidants albumin and hemopexin. The half time of the hemin-LDL complex in plasma, initially comprising 27% of total hemin, was more than 20 s. Not only transient, but also oxidatively active steady-state hemin-lipoprotein complexes in plasma were both predicted from the kinetic analysis and found in experiment. Our data suggest that the hemin-LDL complex may exist in vivo and that its oxidative potential should be considered pro-atherogenic.

A fluorescence method for estimation of toxemia: binding capacity of lipoproteins and albumin in plasma.

The hazard of toxemia, a condition resulting from the spread of toxins by the bloodstream, is regulated by plasma proteins capable of binding with free toxins. As toxin binding results in a reduction of available binding sites, measuring the proteins' binding capacity can be used to estimate toxemia severity. Suggested by this approach, a novel fluorescence method was developed to determine lipoprotein and albumin binding capacities in whole plasma. The method entails two steps: specific binding of N(n-carboxy)phenylimide-4-dimethyl-aminonaphthalic acid with albumin followed by addition of 12-(9-anthroyloxy)stearic acid which, under these conditions, binds mostly with lipoprotein. Reduced fluorescence intensity of the probes in plasma of patients compared to that of healthy donors reflected saturation of binding sites by toxins, thereby estimating toxemia severity. Poor correlation was found between the lipoprotein and albumin binding abilities, suggesting their independent diagnostic values. The simplicity and rapidity of this method are advantageous for its clinical application.

Oxidation of low-density lipoprotein by hemoglobin stems from a heme-initiated globin radical: antioxidant role of haptoglobin.

Hemoglobin, known as a poor peroxidase, has been recently found to be a highly reactive catalyzer of low-density lipoprotein (LDL) oxidation resulting in oxidation of LDL lipids and covalent cross-linking of the LDL protein, apo B. We evaluated three possible mechanisms that may account for hemoglobin reactivity: oxidative activation by globin-dissociated hemin following its transfer to LDL; peroxidase-like reactivity of the ferryl iron active state in intact hemoglobin; and oxidation by a globin radical formed in oxidized hemoglobin. The first mechanism was ruled out because only a minor fraction of hemin was actually transferred to LDL in the process of oxidation. The second mechanism was excluded because hemoglobin ferryl, unlike ferryl of horseradish peroxidase, was not consumed in the process of LDL oxidation. Haptoglobin completely inhibited cross-linking of globin in hemoglobin/H2O2 mixtures but not in myglobin/H2O2, as well as cross-linking of apo B and oxidation of LDL lipids. Haptoglobin could not however abolish the hemoglobin ferryl state, a finding that further supported exclusion of the second mechanism. We conclude that the active species in hemoglobin-induced LDL oxidation is the globin radical, as suggested in the third mechanism. The present findings also show that haptoglobin functions as a major antioxidant thus protecting the vascular system.

Role of hemopexin in protection of low-density lipoprotein against hemoglobin-induced oxidation.

Globin-free hemin and certain hemoproteins, predominantly hemoglobin, are active triggers of low-density lipoprotein (LDL) peroxidation, a contributing cause of atherosclerosis. The role of the plasma heme-binding protein, hemopexin, in protecting apolipoprotein B and LDL lipids from oxidation triggered by either hemin or hemoglobin in the presence of low amounts of H2O2, was investigated at physiological pH and temperature. Significantly, hemopexin prevented not only hemin-mediated modification of LDL but also LDL peroxidation induced by hemoglobin, both by met and oxy forms. Analysis of the data revealed that the rate of heme transfer from methemoglobin to hemopexin was highly dependent upon temperature: only minimal heme transfer occurred at 20 degrees C, whereas at the physiological temperature of 37 degrees C, heme transfer was rapid, within the lag phase of LDL oxidation, regardless of the presence or absence of H2O2. Heme did transfer to hemopexin from oxyhemoglobin as well, but only in the presence of H2O2. The proposed mechanism of the inhibition of oxyhemoglobin oxidative reactivity by hemopexin involves peroxidation of oxyhemoglobin (Fe(II)) to ferrylhemoglobin (FeIV), followed by a comproportionation reaction (FeIV+FeII-->2FeIII), yielding methemoglobin (FeIII) from which heme is readily transferred to hemopexin. Taken together, the data demonstrate that hemopexin can act as an extracellular antioxidant against hemoglobin-mediated damage in inflammatory states, which is especially important when haptoglobin is depleted or absent.

Hemoglobin induced apolipoprotein B crosslinking in low-density lipoprotein peroxidation.

Oxidative modification of human low-density lipoprotein (LDL) is thought to play a major role in the development of atherosclerosis. Free hemin, hemoglobin, myoglobin, and horseradish peroxidase (HRP) were reported in different studies as promoters of LDL lipid oxidation. Based on our previous finding that hemin induced oxidative crosslinking of the LDL protein, apolipoprotein B (apo B) (Y. I. Miller and N. Shaklai (1994) Biochem. Mol. Biol. Int. 34, 1121-1129), we compared the ability of free hemin and the above hemoproteins to induce peroxidation modification of apo B using SDS-PAGE. The levels of the final products of lipid peroxidation were determined as thiobarbituric acid-reactive substances. Hemoglobin and myoglobin were found to be as active as free hemin and all these were much more active than the classic peroxidase HRP. Moreover, the products of oxidized apo B differed: hemoglobin, myoglobin, and hemin induced mostly covalent aggregates, while HRP caused fragmentation of apo B. Hemoglobin reactivity was expressed at low H2O2 concentrations even in the absence of molecular oxygen. Desferal, along with other antioxidants, inhibited the hemoglobin-induced LDL oxidation independently of its iron-chelating property. The high peroxidative reactivity of hemoglobin is explained by its ability (unlike HRP) to transfer the oxidative equivalents from the heme active site, through the globin, to LDL. The apo B radicals thus formed are terminated, yielding intermolecular crosslinked protein. It is suggested that small amounts of the highly reactive hemoglobin in plasma, suffice to trigger LDL protein oxidation (along with its lipid oxidation), thereby inflict the atherosclerosis precondition.

The involvement of low-density lipoprotein in hemin transport potentiates peroxidative damage.

Hemin binds to isolated low-density lipoprotein (LDL) and thereby triggers LDL oxidation. In this study we investigated whether hemin can get together with LDL under physiological conditions. The relative affinity of three blood components to free hemin was as follows: RBCM < LDL < albumin. At physiological molar ratio of LDL/albumin all the hemin was bound to albumin. In molar excess of albumin over hemin, existing even under pathological conditions, albumin served as an efficient antioxidant for the plasma hemin-induced LDL oxidation. RBCM-embedded hemin, unlike plasma hemin, affected LDL: the mobile hemin was transferred from RBCM to LDL in the absence of albumin, whereas in the presence of albumin most of the mobile hemin finally reached the albumin but partially via LDL. Thus, a transient hemin is built up in LDL. This transient hemin triggered LDL oxidation which was not inhibited but rather promoted by albumin. The involvement of albumin in this oxidation was explained by its acting as a pump thereby increasing the transient hemin in LDL. It is suggested that increased membrane hemin level as in hemoglobinopathies and/or excess LDL in dyslipidemia provide conditions for hemin-induced LDL oxidation.

Oxidative crosslinking of LDL protein induced by hemin: involvement of tyrosines.

This study investigated free hemin induced modifications in low density lipoprotein (LDL). By use of fluorescent probes hemin was found to associate with LDL thereby inducing peroxidation of both lipids and protein. Upon LDL peroxidation, covalent crosslinking of apolipoprotein B (Apo B) occurred as judged by SDS-PAGE. Concomitantly, a multifluorophore emission developed, which included contribution of bityrosines. The simultaneous formation of protein aggregates and bityrosines was interpreted as the involvement of intermolecular bityrosines in the hemin induced crosslinking of Apo B. Since LDL protein aggregation relates to conversion of macrophages into foam cells, hemin should be considered as an endogenous trigger of atherosclerosis.