A Glycomic Approach to Hepatic Tumors in N -acetylglucosaminyltransferase III (GnT-III) Transgenic Mice Induced by Diethylnitrosamine (DEN): Identification of Haptoglobin as a Target Molecule of GnT-III

A glycomic approach to the identification of target molecules in glycosyltransferase gene targeting mice is a promising strategy to understand the biological significance of glycosyltransferase genes in vivo. In order to understand the biological effects of N -acetylglucosaminyltransferase III (GnT-III) on tumor formation in the liver, diethylnitrosamine (DEN) induced tumor formation in the GnT-III transgenic mice was examined. Our findings show that the incidence of hepatic tumor could be dramatically suppressed. A glycomic approach using two-dimensional gel electrophoresis followed by lectin blot analysis and sequence analysis revealed that haptoglobin, a radical scavenger molecule in serum was heavily glycosylated in hepatic tumor-bearing GnT-III transgenic mice that had been treated with DEN. Immunoprecipitation followed by E 4 -PHA lectin blot analysis also confirmed that the bisecting GlcNAc, a product of GnT-III was added to haptoglobin molecules. Since the use of DEN is known to lead to the production of lipid peroxidation products which facilitate this reaction and haptoglobin is an acute phase reactant, acting as a radical scavenger against hemoglobin or iron stimulated lipid peroxidation, a relationship between the glycosylation of haptoglobin and the suppression of hepatoma development can not be ruled out. This paper is the first report that shows a relationship between the sugar chains of glycoproteins with radical scavenger activity and hepatocarcinogenesis.     

A glycomic approach to hepatic tumors in N-acetylglucosaminyltransferase III (GnT-III) transgenic mice induced by diethylnitrosamine (DEN): identification of haptoglobin as a target molecule of GnT-III

A glycomic approach to the identification of target molecules in glycosyltransferase gene targeting mice is a promising strategy to understand the biological significance of glycosyltransferase genes in vivo . In order to understand the biological effects of N -acetylglucosaminyltransferase III (GnT-III) on tumor formation in the liver, diethylnitrosamine (DEN) induced tumor formation in the GnT-III transgenic mice was examined. Our findings show that the incidence of hepatic tumor could be dramatically suppressed. A glycomic approach using two-dimensional gel electrophoresis followed by lectin blot analysis and sequence analysis revealed that haptoglobin, a radical scavenger molecule in serum was heavily glycosylated in hepatic tumor-bearing GnT-III transgenic mice that had been treated with DEN. Immunoprecipitation followed by E 4 -PHA lectin blot analysis also confirmed that the bisecting GlcNAc, a product of GnT-III was added to haptoglobin molecules. Since the use of DEN is known to lead to the production of lipid peroxidation products which facilitate this reaction and haptoglobin is an acute phase reactant, acting as a radical scavenger against hemoglobin or iron stimulated lipid peroxidation, a relationship between the glycosylation of haptoglobin and the suppression of hepatoma development can not be ruled out. This paper is the first report that shows a relationship between the sugar chains of glycoproteins with radical scavenger activity and hepatocarcinogenesis.     

Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by peroxides

Hydrogen peroxide and organic hydroperoxides react with haemoglobin to release iron which can be complexed to apotransferrin, bleomycin and desferrioxamine. This released iron promotes deoxyribose degradation by a Fenton reaction, DNA degradation in the presence of bleomycin and stimulates lipid peroxidation. It is likely that iron released from haemoglobin is the true generator of hydroxyl radicals in the Fenton reaction.     

Cytochrome c-catalyzed membrane lipid peroxidation by hydrogen peroxide.

Cytochrome c(3+)-catalyzed peroxidation of phosphatidylcholine liposomes by hydrogen peroxide (H2O2) was indicated by the production of thiobarbituric acid reactive substances, oxygen consumption, and emission of spontaneous chemiluminescence. The iron chelator diethylenetriaminepentaacetic acid (DTPA) only partially inhibited peroxidation when H2O2 concentrations were 200 microM or greater. In contrast, iron compounds such as ferric chloride, potassium ferricyanide, and hemin induced H2O2-dependent lipid peroxidation which was totally inhibitable by DTPA. Cyanide and urate, which react at or near the cytochrome-heme, completely prevented lipid peroxidation, while hydroxyl radical scavengers and superoxide dismutase had very little or no inhibitory effect. Changes in liposome surface charge did not influence cytochrome c3+ plus H2O2-dependent peroxidation, but a net negative charge was critical in favoring cytochrome c(3+)-dependent, H2O2-independent lipid auto-oxidative processes. These results show that reaction of cytochrome c with H2O2 promotes membrane oxidation by more than one chemical mechanism, including formation of high oxidation states of iron at the cytochrome-heme and also by heme iron release at higher H2O2 concentrations. Cytochrome c3+ could react with mitochondrial H2O2 to yield "site-specific" mitochondrial membrane lipid peroxidation during tissue oxidant stress.     

Iron binding to microsomes and liposomes in relation to lipid peroxidation

The effects of ADP, ATP, citrate and EDTA on iron-dependent microsomal and liposomal lipid peroxidation, and on 59FeCl3 binding to the lipid membranes were measured. The aim was to test if initiation of lipid peroxidation is a site-specific mechanism requiring bound iron. In the absence of chelator, iron was bound to both membranes. EDTA and citrate removed the iron and inhibited peroxidation. ATP and ADP stimulated peroxidation, but whereas ADP allowed only half of the iron to remain bound, all was removed by ATP. Chelators, therefore, cannot be simply influencing a site-specific mechanism. Their effects must relate to the reactivities of the different iron chelates as initiators of lipid peroxidation.     

tert-butyl hydroperoxide-dependent microsomal release of iron and lipid peroxidation. II. Evidence for the involvement of nonheme, nonferritin iron in lipid peroxidation

In a previous study tert-butyl hydroperoxide (t-BOOH) was found to promote reductive release of nonheme, nonferritin iron from rat liver microsomes. The reaction was catalyzed by cytochrome P450 and was strictly contingent on the availability of ADP. In this study, t-BOOH was also found to promote microsomal lipid peroxidation, as evidenced by formation of malondialdehyde. t-BOOH-dependent lipid peroxidation was stimulated by ADP, and four lines of evidence suggested that such stimulation was mediated by reductive release and subsequent redox cycling of nonheme, nonferritin iron. First, lipid peroxidation was stimulated by the same concentration of ADP that promoted iron release. Second, depletion of nonheme, nonferritin iron by pretreatment of rats with phenobarbital decreased the stimulation of lipid peroxidation by ADP. Third, the effect of ADP was maximal when the concentration of t-BOOH was adjusted to values that yielded maximum iron release. Fourth, the effect of ADP was abolished by bathophenanthroline, which is known to chelate ferrous iron in a redox inactive form. These results suggest that the reductive release of nonheme, nonferritin iron exacerbates the deleterious effects of t-BOOH on microsomal lipids.     

Antioxidant protection by haemopexin of haem-stimulated lipid peroxidation.

Haem (ferrous protoporphyrin IX) is a reactive low-molecular-mass form of iron able to participate in oxygen-radical reactions that can lead to the degradation of proteins, lipids, carbohydrates and DNA. Oxygen-radical reactions are likely to occur upon tissue damage. Extracellular fluids rely on antioxidant mechanisms different from those found inside the cell, and circulating proteins limit radical reactions by converting pro-oxidant forms of iron into less-reactive forms. Of the compounds tested, only apohaemopexin and the chain-breaking antioxidant butylated hydroxytoluene inhibited (by more than 90%) haemin-stimulated peroxidation as measured by formation of conjugated dienes, thiobarbituric acid-reactive material from linolenic acid or peroxidation-induced phospholipid fluorescence. Haptoglobin, the haemoglobin-binding serum protein, was ineffective. Conversely, only haptoglobin significantly inhibited haemoglobin-stimulated lipid peroxidation. Iron-salt-induced lipid peroxidation was inhibited only by apotransferrin and the iron-chelator desferrioxamine. All lipid peroxidations were inhibited by the radical scavengers butylated hydroxytoluene and propyl gallate. These findings support the concept that transport and conservation of body iron stores are closely linked to antioxidant protection.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.     

Initiation of lipid peroxidation in submitochondrial particles: effect of respiratory inhibitors

Initiation of lipid peroxidation in the inner mitochondrial membrane was investigated using respiratory substrates and inhibitors and various iron chelates. An iron chelate was required for initiation of lipid peroxidation in the presence of either NADH or NADPH. The two nicotinamide nucleotides exhibited different activities in initiating lipid peroxidation with regard to concentration and to the effects of rotenone and rhein. Succinate and both nicotinamide nucleotides supported lipid peroxidation in the presence of thenoyl trifluoroacetone (TTFA), without a requirement for exogenously added iron. ADP stimulated lipid peroxidation in the case of NAD(P)H and TTFA, but inhibited it in the case of succinate and TTFA. Lipid peroxidation is thought to be enzymatically induced in both the NADH and the succinate dehydrogenase regions of the respiratory chain, and evidence is presented for a novel pathway of NADPH oxidation that may also be involved. Possible initiation mechanisms are discussed.     

Comparison of iron-catalyzed DNA and lipid oxidation

Lipid and DNA oxidation catalyzed by iron(II) were compared in HEPES and phosphate buffers. Lipid peroxidation was examined in a sensitive liposome system constructed with a fluorescent probe that allowed us to examine the effects of both low and high iron concentrations. With liposomes made from synthetic 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine or from rat liver microsomal lipid, lipid peroxidation increased with iron concentration up to the range of 10--20 microM iron(II), but then rates decreased with further increases in iron concentration. This may be due to the limited amount of lipid peroxides available in liposomes for oxidation of iron(II) to generate equimolar iron(III), which is thought to be important for the initation of lipid peroxidation. Addition of hydrogen peroxide to incubations with 1--10 microM iron(II) decreased rates of lipid peroxidation, whereas addition of hydrogen peroxide to incubations with higher iron concentrations increased rates of lipid peroxidation. Thus, in this liposome system, sufficient peroxide from either within the lipid or from exogenous sources must be present to generate equimolar iron(II) and iron(III). With iron-catalyzed DNA oxidation, hydrogen peroxide always stimulated product formation. Phosphate buffer, which chelates iron but still allows for generation of hydroxyl radicals, inhibited lipid peroxidation but not DNA oxidation. HEPES buffer, which scavenges hydroxyl radicals, inhibited DNA oxidation, whereas lipid peroxidation was unaffected since presumably iron(II) and iron(III) were still available for reaction with liposomes in HEPES buffer.     

Effects of Ceruloplasmin on Superoxide-Dependent Iron Release from Ferritin and Lipid Peroxidation

Ceruloplasmin (CP) effectively inhibited superoxide and ferritin-dependent peroxidation of phospholipid liposomes, using xanthine oxidase or gamma irradiation of water as sources of superoxide. In addition, CP inhibited superoxide-dependent mobilization of iron from ferritin. suggesting that CP inhibited lipid peroxidation by decreasing the availability of iron from ferritin. CP also exhibited some superoxide scavenging activity as evidenced by its inhibition of superoxide-dependent cytochrome c reduction. However, superoxide scavenging by CP did not quantitatively account for its inhibitory effects on iron release. The effects of CP on iron-catalyzed lipid peroxidation in systems containing exogenously added ferrous iron was also investigated. CP exhibited prooxidant and antioxidant effects; CP stimulated at lower concentrations, reached a maximum. and inhibited at higher concentrations. However. the addition of apoferritin inhibited CP and Fe(II)-catalyzed lipid peroxidation at all concentrations of CP. In addition, CP catalyzed the incorporation of Fe(II) into apoferritin. Collectively these data suggest that CP inhibits superoxide and ferritin-dependent lipid peroxidation via its ability to incorporate reductively-mobilized iron into ferritin.     

Acetaldehyde-mediated hepatic lipid peroxidation: role of superoxide and ferritin

Evidence in alcoholics as well as in experimental models support the role of hepatic lipid peroxidation in the pathogenesis of alcohol-induced liver injury, but the mechanism of this injury is not fully delineated. Previous studies of the metabolism of ethanol by alcohol dehydrogenase revealed iron mobilization from ferritin that was markedly stimulated by superoxide radical generation by xanthine oxidase. Peroxidation of hepatic lipid membranes (assessed as malondialdehyde production) was studied during in vitro alcohol metabolism by alcohol dehydrogenase. Peroxidation was initiated by acetaldehyde-xanthine oxidase, stimulated by ferritin, and inhibited by superoxide dismutase or chelation or iron with desferrioxamine. In conclusion, lipid peroxidation may be initiated during the metabolism of ethanol by alcohol dehydrogenase by an iron-dependent acetaldehyde-xanthine oxidase mechanism.     

Stimulation and inhibition of iron-dependent lipid peroxidation by desferrioxamine

Peroxidation of rat brain synaptosomes was assessed by the formation of thiobarbituric acid reactive products in either 50 mM potassium phosphate buffer (pH 7.4) or pH adjusted saline. In phosphate, addition of Fe2+ resulted in a dose-related increase in lipid peroxidation. In saline, stimulation of lipid peroxidation by Fe2+ was maximal at 30 uM, and was less at concentrations of 100 uM and above. Whereas desferrioxamine caused a dose-related inhibition of iron-dependent lipid peroxidation in phosphate, it stimulated lipid peroxidation with Fe2+ by as much as 7-fold in saline. The effects of desferrioxamine depended upon the oxidation state of iron, and the concentration of desferrioxamine and lipid. The results suggest that lipid and desferrioxamine compete for available iron. The data are consistent with the hypothesis that either phosphate or desferrioxamine may stimulate iron-dependent lipid peroxidation under certain circumstances by favoring formation of Fe2+/Fe3+ ratios.     

Effect of tocilizumab on haematological markers implicates interleukin-6 signalling in the anaemia of rheumatoid arthritis

Introduction

Our objective was to determine the interrelationships of interleukin (IL)-6 receptor inhibition with haemoglobin, acute-phase reactants and iron metabolism markers (including hepcidin) in patients with rheumatoid arthritis (RA).

Methods

Data of patients receiving tocilizumab or placebo in the MEASURE study were analysed. We investigated associations at baseline and during tocilizumab treatment among haemoglobin, parameters of haemoglobin and iron homeostasis [ferritin, total iron-binding capacity (TIBC), hepcidin, haptoglobin], IL-6 and acute-phase reactants [C-reactive protein (CRP), erythrocyte sedimentation rate (ESR)] to identify statistical correlates of rise in haemoglobin level.

Results

At baseline, CRP and haptoglobin were inversely correlated (modestly) with haemoglobin levels. After treatment with tocilizumab, CRP, hepcidin, ferritin and haptoglobin levels fell alongside increases in TIBC and haemoglobin. The falls in CRP, hepcidin and haptoglobin levels in the first 2 weeks correlated with a week 12 rise in TIBC and haemoglobin.

Conclusions

Inflammatory anaemia improves in patients with RA treated with tocilizumab. This improvement correlates with the degree of suppression of systemic inflammation, reduction in hepcidin and haptoglobin and increase in iron-binding capacity. These clinical data provide evidence of a role for IL-6 signalling in the inflammatory anaemia of RA.     

Role of hydroxyl radical in superoxide induced microsomal lipid peroxidation: Protective effect of anion channel blocker

Treatment of bovine pulmonary artery smooth muscle microsomes with the superoxide radical generating system hypoxanthine plus xanthine oxidase stimulated iron release, hydroxyl radical production and lipid peroxidation. Pretreatment of the microsomes with deferoxamine or dime thy lthiourea markedly inhibited lipid peroxidation, and prevented hydroxyl radical production without appreciably altering iron release. The superoxide radical generating system did not alter the ambient superoxide dismutase activity. However,addition of exogenous superoxide dismutase prevented superoxide radical induced iron release,hydroxyl radical production and lipid peroxidation. Simultaneous treatment of the microsomes with deferoxamine, dimethylthiourea or superoxide dismutase prevented hydroxyl radical production and liqid peroxidation. While deferoxamine or dimethylthiourea did not appreciably alter iron release, superoxide dismutase prevented iron release. However, addition of deferoxamine, dimethylthiourea or superoxide dismutase even 2 min after treatment did not significantly inhibit lipid peroxidation, hydroxyl radical production and iron release. Pretreatment of microsomes with the anion channel blocker 4,4’- dithiocyano 2,′- disulphonic acid stilbine did not cause any discernible change in chemiluminiscence induced by the superoxide radical generating system but markedly inhibited lipid peroxidation without appreciably altering iron release and hydroxial radical production.     

Effect of ferritin-containing fractions with different iron loading on lipid peroxidation.

Ferritin-containing fractions with different degrees of iron loading were prepared. All ferritin fractions stimulated the peroxidation of bovine brain phospholipid liposomes, as measured by the formation of thiobarbituric acid-reactive material. This stimulation was increased in the presence of ascorbate. Iron salts of equivalent concentration to those of the ferritin fractions were more stimulatory to lipid peroxidation at the higher iron concentrations. None of the fractions inhibited ascorbate-dependent peroxidation in the presence of added iron salts.     

Radiation-induced generation and properties of lipid hydroperoxide in liposomes

The generation of hydroxyl free radicals in 60Co gamma-irradiation of a dilute aqueous suspension of phosphatidyl choline liposomes, resulted in the rapid accumulation of lipid hydroperoxides (linearly with time), but only small concentrations of malondialdehyde. Incubation of the irradiated liposomes with ferric chloride was found to significantly increase the malondialdehyde, and evidence is presented that this resulted from iron catalysed decomposition of the lipid hydroperoxide. This suggests a role for free iron or iron chelates in the propagation of lipid peroxidation stimulated by other systems.     

An investigation into the role of hydroxyl radical in xanthine oxidase-dependent lipid peroxidation

A model lipid peroxidation system dependent upon the hydroxyl radical, generated by Fenton's reagent, was compared to another model system dependent upon the enzymatic generation of superoxide by xanthine oxidase. Peroxidation was studied in detergent-dispersed linoleic acid and in phospholipid liposomes. Hydroxyl radical generation by Fenton's reagent (FeCl₂ + H₂O₂) in the presence of phospholipid liposomes resulted in lipid peroxidation as evidenced by malondialdehyde and lipid hydroperoxide formation. Catalase, mannitol, and Tris-Cl were capable of inhibiting activity. The addition of EDTA resulted in complete inhibition of activity when the concentration of EDTA exceeded the concentration of Fe²⁺. The addition of ADP resulted in slight inhibition of activity, however, the activity was less sensitive to inhibition by mannitol. At an ADP to Fe²⁺ molar ratio of 10 to 1, 10 mm mannitol caused 25% inhibition of activity. Lipid peroxidation dependent on the enzymatic generation of superoxide by xanthine oxidase was studied in liposomes and in detergent-dispersed linoleate. No activity was observed in the absence of added iron. Activity and the apparent mechanism of initiation was dependent upon iron chelation. The addition of EDTA-chelated iron to the detergent-dispersed linoleate system resulted in lipid peroxidation as evidenced by diene conjugation. This activity was inhibited by catalase and hydroxyl radical trapping agents. In contrast, no activity was observed with phospholipid liposomes when iron was chelated with EDTA. The peroxidation of liposomes required ADP-chelated iron and activity was stimulated upon the addition of EDTA-chelated iron. The peroxidation of detergent-dispersed linoleate was also enhanced by ADP-chelated iron. Again, this peroxidation in the presence of ADP-chelated iron was not sensitive to catalase or hydroxyl radical trapping agents. It is proposed that initiation of superoxide-dependent lipid peroxidation in the presence of EDTA-chelated iron occurs via the hydroxyl radical. However, in the presence of ADP-chelated iron, the participation of the free hydroxyl radical is minimal.     

Biochemical evidence for chemical and/or topographic differences in the lipoperoxidative processes induced by CCl4 and iron

Isolated rat hepatocytes, treated with CCl4 or ADP-Fe3+ complex show an enhanced lipid peroxidation and a decreased glucose 6-phosphatase activity. Lipid peroxidation is much more stimulated by ADP-Fe3+ or Fe3+ than by CCl4, when the metal and the haloalkane are used at a similar concentration. Increasing rates of lipid peroxidation in the different experimental conditions do not correlate with the degree of glucose 6-phosphatase inactivation, which is produced by CCl4 and not by a similar amount of ferric iron. In the case of iron, its intracellular concentration must be higher to give the enzyme inactivation exerted by CCl4. Higher intracellular levels of iron are reached when the metal is added to the cell suspension together with ADP. Under these conditions there is inactivation of glucose 6-phosphatase. Possible mechanisms accounting for a different enzyme sensitivity to iron and CCl4 are discussed.     

Inhibition of the Fenton reaction by the protein caeruloplasmin and other copper complexes. Assessment of ferroxidase and radical scavenging activities

The copper-containing protein caeruloplasmin is an important biological extracellular protein. By catalysing the oxidation of ferrous ions to the ferric state (ferroxidase activity) it can inhibit lipid peroxidation and the Fenton reaction. This activity is readily destroyed by heat-denaturation. When a ferric-EDTA complex is added to hydrogen peroxide, OH X radicals are formed in a reaction inhibitable by superoxide dismutase (SOD). This reaction is also inhibited by caeruloplasmin both before and after heat-denaturation, suggesting a non-catalytic scavenging role for the protein. A combination of ferroxidase and radical scavenging activities in fluids containing iron complexes and hydrogen peroxide, but no SOD or catalase, would make caeruloplasmin an important extracellular antioxidant.