Tanshinone II-A inhibits low density lipoprotein oxidation in vitro

Tanshinone II-A (TSII-A) is a major component of Salvia miltorrhiza Bunge which has long been used for preventing and ameliorating anginal pain in China. However the effect of TSII-A on low density lipoprotein (LDL) oxidation has not been studied. The present study was performed to investigate the effects of TSII-A on LDL oxidation using four oxidizing systems, including copper-, peroxyl radical- and peroxynitriteinitiated and macrophage-mediated LDL oxidation. LDL oxidation was measured in terms of formation of thiobarbituric acid-reactive substances (TBARS), relative electrophoretic mobility (REM) on agarose gel and lag time. In all four systems, TSII-A has apparent antioxidative effects against LDL oxidation, as evidenced by its dose-dependent inhibition of TBARS formation, prolongation of lag time and suppression of increased REM.

Regarding the mechanism underlying its antioxidative effect, TSII-A neither scavenged superoxide nor peroxynitrite. It also did not chelate copper. But it has mild peroxyl radical scavenging activity. The direct binding to LDL particles and conformational change of LDL structure by TSII-A were suggested, because it increased negative charge of LDL which was shown by increased REM on agarose gel. In conclusion, TSII-A is an effective antioxidant against LDL oxidation in vitro. The underlying mechanism appears to be related to its peroxyl radical scavenging and LDL binding activity.     

Resveratrol inhibits copper ion-induced and azo compound-initiated oxidative modification of human low density lipoprotein.

To investigate whether resveratrol, a polyphenolic compound in red wine, affects the oxidation of human low density lipoprotein (LDL), LDL purified from normolipidemic subjects was subjected to Cu(2+)-induce and azo compound-initiated oxidative modification, with and without the addition of varying concentrations of resveratrol. Modification of LDL was assessed by the formation of thiobarbituric acid reactive substances (TBARS) and changes in the relative electrophoretic mobility (REM) of LDL on agarose gels. Resveratrol (50 microM) reduced TBARS and REM of LDL during Cu(2+)-induced oxidation by 70.5% and 42.3%, respectively (p < 0.01), and prolonged the lag phase associated with the oxidative modification of LDL by copper ion or azo compound. These in vitro results suggest that resveratrol may afford protection of LDL against oxidative damage resulting from exposure to various environmental challenges, possibly by acting as a free radical scavenger.     

Antioxidative activity on human low density lipoprotein (LDL) oxidation by pentagalloic acid

The aim of this study was to investigate the efficiency of the pentagalloic acid compound in inhibiting the metal ions and cell lines that mediate in low density lipoprotein (LDL) oxidation. Pentagalloic acid prolonged the lag time preceeding the onset of conjugated diene formation. In chemically induced LDL oxidation by Cu²⁺ plus hydrogen peroxide or peroxyl radical generated by 2, 2′-azo-bis (2-amidino propane) hydrochloride (AAPH), pentagalloic acid inhibited LDL oxidation as monitored by measuring the thiobarbituric acid reactive substances (TBARS), malondialdehyde (MDA), and gel electrophoretic mobility. The physiological relevance of the antioxidative activity was validated at the cellular level where pentagalloic acid inhibited mouse macrophage J774 and endothelial cell-mediated LDL oxidation. When compared with several other antioxidants, pentagalloic acid showed a much higher ability than naturally occuring antioxidants, α-tocopherol and ascorbic acid, and the synthetic antioxidant, probucol.     

Antioxidant activities of a new lignan and a neolignan from Saururus chinensis

A new diarylbutane lignan, 2'-hydroxy dihydroguaiaretic acid (4), and a known 8-O-4'-type neolignan, machilin D (5), were isolated from the ethyl acetate extracts of the underground parts of Saururus chinensis. Compounds 4 and 5 exhibited low-density lipoprotein (LDL)-antioxidant activity in the thiobarbituric acid-reactive substances (TBARS) assay (4: IC(50)=3.3 microM and 5: IC(50)=3.8 microM), the lag time of conjugated diene production, the relative electrophoretic mobility (REM) of ox-LDL, the apoB-100 fragmentation on copper-mediated LDL oxidation and the macrophage-mediated LDL oxidation, and radical DPPH scavenging activity.     

Inhibitory effects of multi-substituted benzylidenethiazolidine-2,4-diones on LDL oxidation

Multi-substituted benzylidenethiazolidine-2,4-diones 3a-h were synthesized by Knoevenagel condensation of di- or tri-substituted 4-hydroxybenzaldehydes [or 1-(3,5-di-tert-butyl-4-hydroxyphenyl)ethanone] 1 with thiazolidine-2,4-dione (2) and evaluated for antioxidant activities of Cu(2+)-induced oxidation of human low-density lipoproteins (LDL). Among compounds 3a-h, 3a was superior to probucol in LDL-antioxidant activities and found to be ninefold more active than probucol. Due to its potency, compound 3a was tested for complementary in vitro investigations, such as TBARS assay (IC(50) = 0.1 microM), lag time (240 min at 1.5 microM), relative electrophoretic mobility (REM) of ox-LDL (inhibition of 83% at 10 microM), fragmentation of apoB-100 (inhibition of 61% at 5 microM), and radical DPPH scavenging activity on copper-mediated LDL oxidation. In macrophage-mediated LDL oxidation, the TBARS formation was also inhibited by compound 3a.     

Glucose enhancement of LDL oxidation is strictly metal ion dependent

Recent evidence suggests that lipoprotein oxidation is increased in diabetes, however, the mechanism(s) for such observations are not clear. We examined the effect of glucose on low-density lipoprotein (LDL) oxidation using metal ion-dependent and -independent oxidation systems. Pathophysiological concentrations of glucose (25 mM) enhanced copper-induced LDL oxidation as determined by conjugated diene formation or relative electrophoretic mobility (REM) on agarose gels. Similarly, iron-induced LDL oxidation was stimulated by glucose resulting in 4- to 6-fold greater REM than control incubations without glucose. In contrast, glucose had no effect on metal ion-independent LDL oxidation by aqueous peroxyl radicals. The effect of glucose on metal ion-dependent LDL oxidation was associated with enhanced reduction of metal ions, and in the case of iron-induced LDL oxidation, was completely inhibited by superoxide dismutase. The effect of glucose was mimicked by other reducing sugars, such as fructose and mannose, and the extent to which each sugar enhanced LDL oxidation was closely linked to its metal ion-reducing activity. Thus, promotion of LDL oxidation by glucose is specific for metal ion-dependent oxidation and involves increased metal ion reduction. These results provide one potential mechanism for enhanced LDL oxidation in diabetes.     

Evaluation of antioxidant activity of epigallocatechin gallate in biphasic model systems in vitro

The antioxidant activity of epigallocatechin gallate (EGCG) was studied in different in vitro model systems, which enabled evaluation of both chemical and physical factors involved in assessing the role of EGCG in oxidative reactions. EGCG suppressed the initiation rate and prolonged the lag phase duration of peroxyl radical-induced oxidation in a phospholipid liposome model to a greater extent (p < 0.01) compared to both Trolox and -tocopherol. Effectiveness of these antioxidants to prolong the peroxyl radical-induced lag phase was inversely related to lipophilic character. EGCG also protected against both peroxyl radical and hydroxyl radical-induced supercoiled DNA nicking. The rate constant describing EGCG reaction against hydroxyl radical was 4.22 ± 0.07 × 10¹⁰ M^–1·sec^–1, which was comparable to those of Trolox and -tocopherol, respectively. EGCG exhibited a synergistic effect with -tocopherol in scavenging 1,1-diphenyl-2-picylhydrazyl (DPPH) radical, thus displaying a direct free radical scavenging capacity. In vitro Cu²⁺-induced-human LDL oxidation was accelerated in the presence of EGCG and attributed to the conversion of Cu²⁺ to Cu⁺. We conclude that the particularly effective antioxidant properties of EGCG noted in both chemical and biological biphasic systems were related to a unique hydrophilic and lipophilic balance which enabled effective free radical scavenging. The same chemical-physical properties of EGCG also enabled prooxidant activity, only when in contact with unbound transition metal ions in a multiphasic system.     

Inhibition of peroxyl radical-mediated lipid oxidation by plasmalogen phospholipids and alpha-tocopherol

The recently discovered peroxyl radical scavenging properties of plasmalogen phospholipids led us to evaluate their potential interactions with alpha-tocopherol. The oxidative decay of plasmalogen phospholipids and of polyunsaturated fatty acids as induced by peroxyl radicals (generated from 2,2'-azobis-2-amidinopropane hydrochloride; AAPH) was studied in micelles using 1H-NMR and chemical analyses. In comparison with alpha-tocopherol, a 20- to 25-fold higher concentration of plasmalogen phospholipids was needed to induce a similar inhibition of peroxyl radical-mediated oxidation of polyunsaturated fatty acids. Plasmalogen phospholipids and alpha-tocopherol protected each other from oxidative degradation. In low-density lipoproteins (LDL) and micelles supplemented with plasmalogen phospholipids plus alpha-tocopherol, the peroxyl radical-promoted oxidation was additively diminished. The differences in the capacities to inhibit oxidation processes induced by peroxyl radicals between the plasmalogen phospholipids and alpha-tocopherol were less pronounced in the LDL particles than in the micelles. In conclusion, plasmalogen phospholipids and alpha-tocopherol apparently compete for the interaction with the peroxyl radicals. Oxidation processes induced by peroxyl radicals are inhibited in an additive manner in the presence of the two radical scavengers. The contribution of the plasmalogen phospholipids to the protection against peroxyl radical promoted oxidation in vivo is expected to be at least as important as that of alpha-tocopherol.     

Protection of endogenous beta-carotene in LDL oxidized by oxygen free radicals in the presence of supraphysiological concentrations of melatonin

This study was designed to evaluate the effect of high concentrations of melatonin on the peroxidation of human low density lipoproteins (LDLs) initiated by O(2)(*-) and ethanol-derived peroxyl radicals (RO(2)(*)) from water gamma radiolysis in the presence of ethanol. LDL (3 g/l; total LDL concentration) was oxidized in the absence of melatonin or in its presence at three concentrations (50 x 10(-6), 100 x 10(-6) or 250 x 10(-6) mol/l) in ethanol. Radiolytic yields (i.e. number of mole consumed or produced per Joule) of the markers of lipid peroxidation were determined (i.e. decrease in the endogenous antioxidants alpha-tocopherol and beta-carotene, formation of conjugated dienes and of thiobarbituric acid-reactive substances [TBARS]). Melatonin decreased the yields of lipid peroxidation products and delayed the onset of the propagation phase for conjugated dienes and TBARS in a concentration-dependent manner. Nevertheless, melatonin did not protect endogenous alpha-tocopherol against peroxyl-induced oxidation (probably due to a lower scavenging capacity than that of alpha-tocopherol towards peroxyl radicals), but delayed the consumption of LDL endogenous beta-carotene and decreased its rate of disappearance. The effect of melatonin seemed to be the highest for a melatonin concentration of 250 x 10(-6) mol/l.     

Inhibition of human low density lipoprotein oxidation by active principles from spices

Spice components and their active principles are potential antioxidants. In this study we examined the effect of phenolic and non-phenolic active principles of common spices on copper ion-induced lipid peroxidation of human low density lipoprotein (LDL) by measuring the formation of thiobarbituric acid reactive substance (TBARS) and relative electrophoretic mobility (REM) of LDL on agarose gel. Curcurriin, capsaicin, quercetin, piperine, eugenol and allyl sulfide inhibited the formation of TBARS effectively through out the incubation period of 12 h and decreased the REM of LDL. Spice phenolic active principles viz. curcumin, quercetin and capsaicin at 10 M produced 40–85% inhibition of LDL oxidation at different time intervals while non-phenolic antioxidant allyl sulfide was less potent in inhibiting oxidation of LDL. However, allyl sulfide, eugenol and ascorbic acid showed pro-oxidant activity at lower concentrations (10 M) and antioxidant activity at higher concentrations (50 M) only. Among the spice principles tested quercetin and curcumin showed the highest inhibitory activity while piperine showed least antioxidant activity at equimolar concentration during initiation phase of oxidation of LDL. The inhibitory effect of curcumin, quercetin and capsaicin was comparable to that of BHA, but relatively more potent than ascorbic acid. Further, the effect of curcurnin, quercetin, capsaicin and BHA on initiation and propagation phases of LDL oxidation showed that curcurnin significantly inhibited both initiation and propagation phases of LDL oxidation, while quercetin was found to be ineffective at propagation phase. These data suggest that the above spice active principles, which constitute about 1–4% of above spices, are effective antioxidants and offer protection against oxidation of human LDL.     

Water-soluble Hexasulfobutyl[60]fullerene inhibit low-density lipoprotein oxidation in aqueous and lipophilic phases

Oxidative modification of low-density lipoprotein (LDL) plays a pivotal role in the pathogenesis of atherosclerosis. Increasing the resistance of LDL to oxidation may therefore mitigate, or even prevent, atherosclerosis. A new water-soluble C60 derivative, hexasulfobutyl[60]fullerene [C60 - (CH2CH2CH2CH2-SO3Na)6; FC4S], consisting of 6 sulfobutyl moieties covalently bound onto the C60 cage is a potent free radical scavenger. This study explored the antioxidative effect of sulfobutylated fullerene derivatives (FC4S) on LDL oxidation. FC4S was found to be effective in protecting LDL against oxidation induced by either Cu2+ or azo peroxyl radicals generated initially in the aqueous or lipophilic phase, respectively. Levels of the oxidative products, conjugated diene and thiobarbituric acid-reactive substances, and the relative electrophoresis mobility of the LDL were decreased. The addition of 20 microM FC4S at the early stage of oxidation increased the kinetic lag time from 69 +/- 11 to 14 +/- 10 min (P < 0.05) and decreased the propagation rate from 17.1 +/- 2.6 to 6.3 +/- 1.0 mOD/min (P < 0. 005). Persistent suppression of peroxidation reaction was observed upon further addition of FC4S after full consumption of all endogenous antioxidants during the propagation period. Intravenous injection of hypercholesterolemic rabbits with FC4S (1 mg/kg/day) efficiently decreased atheroma formation. Data substantiate the use of FC4S as an excellent hydrophilic antioxidant in protecting atheroma formation, via removing free radicals, in either aqueous or lipophilic phase.     

Low-density lipoprotein (LDL)-antioxidant lignans from Myristica fragrans seeds

Six diarylbutane lignans 1-5 and one aryltetralin lignan 6 were isolated from the methanol (95%) extracts of Myristica fragrans seeds and then 7-methyl ether diarylbutane lignan 4 has proven to be new a compound. Their compounds 1-7 were evaluated for LDL-antioxidant activity to identify the most potent LDL-antioxidant 3 with an IC50 value of 2.6 microM in TBARS assay. Due to its potency, compound 3 was tested for complementary in vitro investigations, such as lag time (140 min at 1.0 microM), relative electrophoretic mobility (REM) of ox-LDL (inhibition of 80% at 20 microM and 72% at 10 microM), and fragmentation of apoB-100 (inhibition of 93% at 20 microM) on copper-mediated LDL oxidation. In macrophage-mediated LDL oxidation, the TBARS formation was also inhibited by compound 3.     

The influence of medium components on Cu(2+)-dependent oxidation of low-density lipoproteins and its sensitivity to superoxide dismutase.

The extent of in vitro Cu(2+)-dependent oxidation of low-density lipoproteins (LDL) has been reported to vary widely depending upon reaction conditions. In this study, the effect of proteins and amino acids on Cu(2+)-induced LDL oxidation was examined. Treatment of LDL with 5 microM CuSO4 for 18 h in either phosphate-buffered saline (PBS) or Ham's F-10 medium resulted in extensive oxidation as determined by the content of thiobarbituric acid reactive substances (TBARS) and by increased lipoprotein electronegativity. In PBS, oxidation was entirely blocked by histidine and the tripeptide, gly-his-lys (GHK). Oxidation was also prevented by bovine serum albumin, but superoxide dismutase (SOD) provided only 20% protection. Both proteins bound similar amounts of Cu2+, but albumin appeared to be a more effective peroxyl radical trap as evidenced by its ability to prevent LDL oxidation induced by 2,2'-azo-bis(2-amidinopropane hydrochloride). In F-10 medium, SOD had marked inhibitory effects, in contrast to PBS. The addition of disulfides to PBS markedly enhanced the ability of SOD to inhibit oxidation. These results indicate that medium components which affect Cu2+ availability influence LDL oxidation and suggest that albumin is ideally suited as a plasma antioxidant to prevent oxidative modification of LDL. Furthermore, in certain instances, the inhibitory effects of SOD may be attributable to effects such as Cu2+ binding rather than dismutation of superoxide.     

Products of the reaction of HOCl with tryptophan protect LDL from atherogenic modification

Hypochlorite (HOCl) attacks amino acid residues in LDL making the particle atherogenic. Tryptophan is prone to free radical reactions and modification by HOCl. We hypothesized, that free tryptophan may quench the HOCl attack therefore protecting LDL. Free tryptophan inhibits LDL apoprotein modification and lipid oxidation. Tryptophan-HOCl metabolites associate with LDL reducing its oxidizability initiated by endothelial cells, Cu(2+) and peroxyl radicals. One tryptophan-HOCl metabolite was identified as 4-methyl-carbostyril which showed antioxidative activity when present during Cu(2+) mediated lipid oxidation, but did not associate with LDL. Indole-3-acetaldehyde, a decomposition product of tryptophan chloramine (the product of the tryptophan-HOCl reaction) was found to associate with LDL increasing its resistance to oxidation. Myeloperoxidase treatment of LDL in the presence of chloride, H(2)O(2) and tryptophan protected the lipoprotein from subsequent cell-mediated oxidation. We conclude that, in vivo, the activated myeloperoxidase system can generate antioxidative metabolites from tryptophan by the reaction of hypochlorite with this essential amino acid.     

Paradoxical protective effect of aminoguanidine toward low-density lipoprotein oxidation: inhibition of apolipoprotein B fragmentation without preventing its carbonylation. Mechanism of action of aminoguanidine

Oxidative modification of low-density lipoproteins (LDLs) is an important feature in the initiation and progression of atherosclerosis. Aminoguanidine (AMG), classically described as an inhibitor of advanced glycation end products, turned out to be also efficient in animal models as an antioxidant against lipid peroxidation. The originality of the present study was based on the simultaneous assessment of the oxidation of LDL lipid and protein moieties in order to characterize the molecular sites of AMG protection. Oxidation of the LDL lipid moiety was monitored by measuring conjugated dienes (CD) and hydroperoxide molecular species from cholesteryl esters (CEOOH) and phosphatidylcholines (PCOOH). LDL protein oxidative modifications were assessed by evaluating apoB carbonylation and fragmentation. The LDL oxidation was mediated by water gamma radiolysis, which has the advantage of being quantitative and highly selective with regard to the free radicals produced. Here, we reported that AMG resulted in a protection of LDLs against lipid peroxidation (both in the lag phase and in the propagation phase) and against apoB fragmentation in a concentration-dependent manner, due to the scavenging effect of AMG toward lipid peroxyl radicals. Paradoxically, AMG was poorly efficient against apoB carbonylation that began during the lag phase. We hypothesize that, even in the presence of AMG, a nonnegligible proportion of (*)OH radicals remained able to initiate oxidation of the LDL protein moiety, leading to apoB carbonylation.     

Novel 3,5-diaryl pyrazolines and pyrazole as low-density lipoprotein (LDL) oxidation inhibitor

Compounds 4a-j and 5 were synthesized by cyclocondensation of 3a-j and hydrazine and showed significant LDL-antioxidant activities in the TBARS assay, the lag time of conjugated diene production, the relative electrophoretic mobility (REM) of ox-LDL, the apoB-100 fragmentation, and the macrophage-mediated LDL oxidation. Among compounds 4a-j and 5, 4a was found to be the most active compound as an inhibitor of LDL oxidation and 4a (IC50 = 0.1 microM) was 6-fold more potent than probucol (IC50 = 0.6 microM) in the TBARS assay.     

Ferulic acid antioxidant protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture systems in vitro: structure-activity studies

In this study, free radical scavenging abilities of ferulic acid in relation to its structural characteristics were evaluated in solution, cultured neurons, and synaptosomal systems exposed to hydroxyl and peroxyl radicals. Cultured neuronal cells exposed to the peroxyl radical initiator AAPH die in a dose-response manner and show elevated levels of protein carbonyls. The presence of ferulic acid or similar phenolic compounds, however, greatly reduces free radical damage in neuronal cell systems without causing cell death by themselves. In addition, synaptosomal membrane systems exposed to oxidative stress by hydroxyl and peroxyl radical generators show elevated levels of oxidation as indexed by protein oxidation, lipid peroxidation, and ROS measurement. Ferulic acid greatly attenuates these changes, and its effects are far more potent than those obtained for vanillic, coumaric, and cinnamic acid treatments. Moreover, ferulic acid protects against free radical mediated changes in conformation of synaptosomal membrane proteins as monitored by EPR spin labeling techniques. The results presented in this study suggest the importance of naturally occurring antioxidants such as ferulic acid in therapeutic intervention methodology against neurodegenerative disorders such as Alzheimer's disease in which oxidative stress is implicated.     

Inhibition of protein hydroperoxide formation by protein thiols

Abstract

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2′ azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by β-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

Protective effects of endomorphins, endogenous opioid peptides in the brain, on human low density lipoprotein oxidation

Neurodegenerative disorders are associated with oxidative stress. Low density lipoprotein (LDL) exists in the brain and is especially sensitive to oxidative damage. Oxidative modification of LDL has been implicated in the pathogenesis of neurodegenerative diseases. Therefore, protecting LDL from oxidation may be essential in the brain. The antioxidative effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid peptides in the brain, on LDL oxidation has been investigated in vitro. The peroxidation was initiated by either copper ions or a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). Oxidation of the LDL lipid moiety was monitored by measuring conjugated dienes, thiobarbituric acid reactive substances, and the relative electrophoretic mobility. Low density lipoprotein oxidative modifications were assessed by evaluating apoB carbonylation and fragmentation. Endomorphins markedly and in a concentration-dependent manner inhibited Cu2+ and AAPH induced the oxidation of LDL, due to the free radical scavenging effects of endomorphins. In all assay systems, EM1 was more potent than EM2 and l-glutathione, a major intracellular water-soluble antioxidant. We propose that endomorphins provide protection against free radical-induced neurodegenerative disorders.     

Inhibition of protein hydroperoxide formation by protein thiols

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2' azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by beta-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.