The effect of beta blocking drugs on lipid peroxidation in rat heartin vitro

Beta-adrenergic receptor blocking drugs include a structurally related class of drugs that are employed clinically to treat a variety of cardiovascular disorders. Since these drugs exert additional nonspecific effects including membrane stabilization, representative samples including atenolol, dilevolol, labetolol, metoprolol and propranolol were studied to determine their influence on lipid peroxidation. Homogenates or liposomes of adult rat hearts were incubated in the presence of various concentrations of propranolol or equivalent concentrations of dilevolol, labetolol, metoprolol or atenolol. Lipid peroxidation was stimulated with 50 μM FeSO₄, 5 μMt-butyl hydroperoxide (homogenates) or 0.2 mM citrate FeSO₄ (liposomes) plus O₂. Lipid peroxidation, as assessed by both the thiobarbituric acid reaction and chemiluminescence, was reduced in a dose-dependent manner as the propranolol concentration was increased from 1 to 10 mM. The five beta-adrenergic receptor blocking drugs reduced lipid peroxidation both in crude homogenates and in liposomes; their effectiveness was related to their lipophilicity. Dilevolol, propranolol, labetolol and metoprolol at a concentration of 20 mM reduced lipid peroxidation by 45%, 37%, 35% and 28%, respectively. The hydrophilic blocker atenolol was ineffective in reducing lipid peroxidation event at elevated concentrations. Lipophilic beta-blocking drugs apparently are capable of exerting an antioxidant effect in protecting membrane lipids against peroxidation.     

Thiobarbituric acid-reactive malondialdehyde formation during superoxide-dependent,iron-catalyzed lipid peroxidation: Influence of peroxidation conditions

A systematic study of the influence of biological lipid peroxidation conditions on lipid hydroperoxide decomposition to thiobarbituric acid-reactive malondialdehyde is presented. A superoxide-dependent, iron-catalyzed peroxidation system was employed with xanthine oxidase plus hypoxanthine plus ferric iron-adenosine diphosphate complex as free radical generator. Purified cardiac membrane phospholipid (as liposomes) was the peroxidative target, and 15-hydroperoxy-eicosatetraenoic acid was used as a standard lipid hydroperoxide. Exposure of myocardial phospholipid to free radical generator at physiological pH (7.4) and temperature (37°C) was found to support not only phospholipid peroxidation, but also rapid lipid hydroperoxide breakdown and consequent malondialdehyde formation during peroxidation. Under lipid peroxidation conditions, oxidative injury to the phospholipid polyunsaturated fatty acids required superoxide radical and ferric iron-adenosine diphosphate complex, whereas 37°C temperature and trace iron were sufficient for lipid hydroperoxide decomposition to malondialdehyde. Harsh thiobarbituric acid-test conditions following peroxidation were not mandatory for either lipid hydroperoxide breakdown or thiobarbituric acid-reactive malondialdehyde formation. However, hydroperoxide decomposition that had begun in the peroxidation reaction could be completed during a subsequent thiobarbituric acid test in which no lipid autoxidation took place. Iron was more critical than heat in promoting the observed hydroperoxide decomposition to malondialdehyde during the lipid peroxidation reaction at 37°C and pH 7.4. These data demonstrate that the radical generator, at physiological pH and temperature, serves a dual role as both initiator of membrane phospholipid peroxidation and promotor of lipid peroxide breakdown and thiobarbituric acid-reactive malondialdehyde formation. Consequently, peroxidation reaction conditions can directly influence lipid hydroperoxide decomposition, malondialdehyde production and system thiobarbituric acid-reactivity. In vivo, decomposition of lipid peroxides to malondialdehyde during radical-mediated, metal-catalyzed membrane peroxidation may represent an integral component of oxidative tissue injury rather than a mere consequence of hydrolyzing the peroxidized biological sample in a thiobarbituric acid test.     

Influence of vitamin E and nitrogen dioxide on lipid peroxidation in rat lung and liver microsomes

Rat lung and liver microsomes were used to examine the effects of dietary vitamin E deficiency on membrane lipid peroxidation. Microsomes from vitamin-E-deficient rats displayed increased lipid peroxidation in comparison to microsomes from vitamin-E-supplemented controls. The extent of lipid peroxidation, as determined by measurement of thiobarbituric acid reacting materials, was enhanced by addition of reduced iron and ascorbate (or NADPH). Rats fed a vitamin-E-supplemented diet and exposed to 3 ppm NO₂ for 7 days did not exhibit increases in microsomal lipid peroxidation compared to air-breathing controls. However, increases were found in microsomes prepared from rats fed a vitamin-E-deficient diet and exposed to NO₂. Lung microsomes from vitamin-E-fed rats contained almost 10 times as much vitamin E as liver microsomes when expressed in terms of polyunsaturated fatty acid content. The extent of lipid peroxidation was, in turn, considerably less in lung than in liver microsomes. Lipid peroxidation in lung microsomes from vitamin-E-deficient rats was comparable to liver microsomes from vitamin-E-supplemented rats as was the content of vitamin E in these respective microsomal samples. A combination of vitamin E deficiency and NO₂ exposure resulted in the greatest increases in lung and liver microsomal lipid peroxidation with the largest relative increases occurring in lung microsomes. An inverse relationship was found between the extent of lipid peroxidation and vitamin E content. Most of the peroxidation in lung microsomes appeared to proceed nonenzymatically whereas peroxidation in liver was largely enzymatic. Vitamin E appears to be assimilated by the lung during oxidant inhalation, but with dietary vitamin E deprivation, the margin for protection in lung may be less than in liver.     

Urinary malondialdehyde as an indicator of lipid peroxidation in the diet and in the tissues

Although malondialdehyde (MDA) is extensively metabolized to CO₂, small amounts are nevertheless excreted in an acid-hydrolyzable form in rat urine. In this study, urinary MDA was evaluated as an indicator of lipid peroxidation in the diet and in the tissues. MDA was released from its bound form(s) in urine by acid treatment and determined as the TBA-MA derivative by HPLC. MDA excretion by the rat was found to be responsive to oral administration of the Na enol salt and to peroxidation of dietary lipids. Urinary MDA also increased in response to the increased lipid peroxidation in vivo produced by vitamin E deficiency and by administration of iron nitrilotriacetate. Chronic feeding of a diet containing cod liver oil led to increases in MDA excretion which were not completely eliminated by fasting or feeding a peroxide-free diet, indicating that there was increased lipid peroxidation in vivo. MDA excretion was not responsive to Se deficiency or CCl₄ administration. DPPD, a biologically active antioxidant, but not BHA, a non-biologically active antioxidant, prevented the increase in MDA excretion in vitamin E deficient animals. The results indicate that MDA excretion can serve as an indicator of the extent of lipid peroxidation in the diet and, under conditions which preclude a dietary effect, as an index of lipid peroxidation in vivo. Part of this research was performed in fulfillment of the requirements for the MSc degree in nutrition.     

Measurement of lipid peroxidation in vivo: A comparison of different procedures

A study was undertaken to investigate whether some of the methods commonly used to detect lipid peroxidation of cellular membranes in vivo correlate with each other. The study was performed with the livers of bromobenzene-intoxicated mice, in which lipid peroxidation develops when the depletion of glutathione (GSH) reaches a threshold value. The methods tested and compared were the following: i) measurement of the malondialdehyde (MDA) content of the liver; ii) detection of diene conjugation absorption in liver phospholipids; iii) measurement of the loss of polyunsaturated fatty acids in liver phospholipids; and iv) determination of carbonyl functions formed in acyl residues of membrane phospholipids as a result of the peroxidative breakdown of phospholipid fatty acids. Correlations among the values obtained with these methods showed high statistical significances, indicating that the procedures measure lipid peroxidation in vivo with comparable reliability. Analogously, the four methods appeared also to correlate when applied to in vitro microsomal lipid peroxidation.     

Quinones and quinols as inhibitors of lipid peroxidation

The influence of biological quinonoid compounds upon oxidative polymerization of lipids has been compared with that of simple quinones and antioxidants. A new procedure for the accelerated production and measurement of oxidative polymerization was used for this comparison. The biological quinones were found to be relatively ineffective as retarders of oxidative polymerization. Heme-catalyzed lipid peroxidation, as measured by oxygen uptake, was inhibited by ubiquinone and ubiquinol, both having about one fourth of the antioxidant capacity ofa-tocopherol. The peroxidation of mitochondrial lipid in vitro was inhibited by the presence of exogenous ubiquinone indicating that this compound may contribute towards the protection of the organelle in vivo.     

Role of lipid structure in the activation of phospholipase A2 by peroxidized phospholipids

The time course of hydrolysis of a mixed phospholipid substrate containing bovine liver 1,2-diacyl-sn-glycero-3-phosphocholine (PC) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE) catalyzed byCrotalus adamanteus phospholipase A₂ was measured before and after peroxidation of the lipid substrate. The rate of hydrolysis was increased after peroxidation by an iron/adenosine diphosphate (ADP) system; the presence of iron/ADP in the assay had a minimal inhibitory effect. The rate of lipid hydrolysis was also increased after the substrate was peroxidized by heat and O₂. Similarly, peroxidation increased the rate of hydrolysis of soy PC liposomes that did not contain PE. In order to minimize interfacial factors that may result in an increase in rate, the lipids were solubilized in Triton X-100. In mixtures of Triton with soy PC in the absence of PE, peroxidation dramatically increased the rate of lipid hydrolysis. In addition, the rate of hydrolysis of the unoxidizable lipid 1-palmitoyl-2-[1-¹⁴C]oleoyl PC incorporated into PC/PE liposomes was unaffected by peroxidation of the host lipid. These data are consistent with the notions that the increase in rate of hydrolysis of peroxidized PC substrates catalyzed by phospholipase A₂ is due largely to a preference for peroxidized phospholipid molecules as substrates and that peroxidation of host lipid does not significantly increase the rate of hydrolysis of nonoxidized lipids.     

The effect of glutathione on the vitamin E requirement for inhibition of liver microsomal lipid peroxidation

Vitamin E dependent inhibition of rat liver microsomal lipid peroxidation in an NADPH and ADP-Fe⁺³ containing system occurred at lower vitamin E concentrations in the presence of glutathione (GSH). Using microsomes from rats fed a vitamin E deficient diet, vitamin E was shown to be rquired for inhibition. Inhibition also required the presence of a storage labile microsomal component, since no inhibition was observed when using microsomes that had been stored for one moth. This observation provides evidence that direct reduction of reversibly oxidized vitamin E by GSH does not appear to contribute significantly to inhibition of peroxidation. During GSH and vitamin E dependent inhibition of lipid peroxidation, vitamin E (reduced form) concentrations remained constant, indicating that GSH maintained vitamin E concentrations. Without GSH, vitamin E concentrations dropped rapidly. By adding vitamin E to microsomes, it was found that inhibition of lipid peroxidation in the presence of GSH occurred at about five-fold less vitamin E than in the absence of GSH. Inhibition at these lower levels of vitamin E was 85–90% complete. Results indicate that GSH can be used to maintain vitamin E (reduced form) concentrations, thereby lowering the concentration of vitamin E necessary to inhibit microsomal lipid peroxidation.     

Peroxidation of microsomal membrane protein—Lipid complexes

Nonenzymatic lipid peroxidation was studied using the TBA test on rat liver microsomal fractions, lipid micelles and structural protein-lipid micelle complexes. The kinetics, response to divalent cations, and iron-ascorbate catalysis were alike in the microsomal fraction and in the complex, but different in lipid micelles. The structural protein represented 41% of the total membrane protein, had a S_20,obs of 3.5 and was hydrophobic. The binding of lipid micelles by structural protein proceeded in two steps, with an initial fast rate followed by a slower rate. The binding appeared to involve a hyrophobic association between lipid and protein as evidenced by insensitivity to pH, ionic strength and lack of preference for the individual classes of phospholipid micelles. Deoxycholate caused an increase in the initial peroxidation rate in microsomal fractions. Iron and ascorbate catalyzed lipid peroxidation in both the microsomal fraction and in the complex. Iron catalyzed lipid peroxidation but calcium, cobalt and copper inhibited the reaction in the SP-lipid micelle complex. Lipid peroxidation in microsomal suspensions, therefore, appears to be determined, in part, by the hydrophobic nature of the protein-lipid association found in membranes.     

Novel interference in thiobarbituric acid assay for lipid peroxidation

The thiobarbituric acid test for lipid peroxidation, when applied to a mixture of acetaldehyde and sucrose, produces a 532 nm aborbing chromogen which is indistinguishable from that formed by malonaldehyde and thiobarbituric acid. Unless special procedures are adopted to correct for this effect, the combined action of acetaldehyde and sucrose interferes seriously with the assay of lipid peroxidation reactions, notably those implicated in alcohol-induced liver injuries. However, this unusual thiobarbituric acid effect also can be used as a sensitive method for the detection of acetaldehyde.     

Cholesterol autoxidation in phospholipid membrane bilayers

Lipid peroxidation in unilamellar liposomes of known cholesterol-phospholipid composition was monitored under conditions of autoxidation or as induced by a superoxide radical generating system, γ-irradiation or cumene hydroperoxide. Formation of cholesterol oxidation products was indexed to the level of lipid peroxidation. The major cholesterol oxidation products identified were 7-keto-cholesterol, isomeric cholesterol 5,6-epoxides, isomeric 7-hydroperoxides and isomeric 3,7-cholestane diols. Other commonly encountered products included 3,5-cholestadiene-7-one and cholestane-3β,5α,6β-triol. Superoxide-dependent peroxidation required iron and produced a gradual increase in 7-keto-cholesterol and cholesterol epoxides. Cholesterol oxidation was greatest in liposomes containing high proportions of unsaturated phospholipid to cholesterol (4∶1 molar ratio), intermediate with low phospholipid to cholesterol ratios (2∶1) and least in liposomes prepared with dipalmitoylphosphatidylcholine and cholesterol. This relationship held regardless of the oxidizing conditions used. Cumene hydroperoxide-dependent lipid peroxidation and/or more prolonge oxidations with other oxidizing systems yielded a variety of products where cholesterol-5β,6β-epoxide, 7-ketocholesterol and the 7-hydroperoxides were most consistently elevated. Oxyradical initiation of lipid peroxidation produced a pattern of cholesterol oxidation products distinguishable from the pattern derived by cumene hydroperoxide-dependent peroxidation. Our findings indicate that cholesterol autoxidation in biological membranes is modeled by the peroxide-induced oxidation of liposomes bearing unsaturated fatty acids and suggest that a number of cholesterol oxidation products are derived from peroxide-dependent propagation reactions occurring in biomembranes.     

Antioxidant status and immune activity of glycyrrhizin in allergic rhinitis mice

Oxidative stress is considered as a major risk factor that contributes to increased lipid peroxidation and declined antioxidants in some degenerative diseases. Glycyrrhizin is widely used to cure allergic diseases due to its medicinal properties. In the present study, we evaluated the role of glycyrrhizin on lipid peroxidation and antioxidant status in the blood and nasal mucosa of allergic rhinitis (AR) mice. Mice were divided into six groups: normal control mice, model control (MC) mice, three glycyrrhizin-treated mice groups and lycopene-treated mice. Sensitization-associated increase in lipid peroxidation was observed in the blood and nasal mucosa of MC mice. Activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (TAOC) and levels of glutathione (GSH) were found to be significantly decreased in the blood and nasal mucosa in MC mice when compared to normal control mice. However, normalized lipid peroxidation and antioxidant defenses were reported in the glycyrrhizin-treated and lycopene-treated mice. Moreover, glycyrrhizin treatment still enhanced IFN-γ and reduced IL-4 levels in glycyrrhizin-treated mice. These findings demonstrated that glycyrrhizin treatment enhanced the antioxidant status and decreased the incidence of free radical-induced lipid peroxidation and improved immunity activities in the blood and nasal mucosa of AR mice.     

Promotion of iron-induced rat liver microsomal lipid peroxidation by copper

Although copper has been demonstrated to promote lipid peroxidation in a number of systems, the mechanisms involved have not been fully defined. In this study, the role of copper in modifying lipid peroxidation has been explored in rat hepatic microsomes. In an in vitro system containing reduced glutathione (GSH, 200 μM) and Tris buffer, pH 7,4, cupric sulfate (1–50 μM) potentiated lipid peroxidation induced by ferrous sulfate (10 μM) but was unable to elicit peroxidation in the absence of iron. Higher levels of cupric sulfate (100 μM or greater) were inhibitory. The nature as well as the extent of the peroxidative response of microsomes to cupric sulfate were dependent on glutathione levels in addition to those of iron. Cupric sulfate (100 μM) strongly potentiated ferrous ion-induced lipid peroxidation in the presence of 400–800 μM GSH, while it inhibited peroxidation at lower levels of GSH (0–200 μM) and did not affect ferrous ion-induced peroxidation with glutathione levels of 3–10 mM. The potentiating effect of copper on ferrous ion-induced lipid peroxidation was further explored by investigating: (1) potential GSH-mediated reduction of cupric ions; (2) potential copper/GSH-mediated reduction of ferric ions (formed by oxidation during incubation); and (3) possible promotion of propagation reactions by copper/GSH. Our results indicate that cupric ions are reduced by GSH and thus are converted from an inhibitor to an enhancer of iron-induced lipid peroxidation. Cuprous ions appear to potentiate lipid peroxidation by reduction of ferric ions, rather than by promoting propagation reactions. Iron (in a specific Fe⁺²/Fe⁺³ ratio) is then an effective promoter of initiation reactions.     

Interplay between Vitamin E,Glutathione and Dihydrolipoic Acid in Protection against Lipid Peroxidation

Free radicals can disturb the intracellular homeostasis by either modification of essential free sulfhydryl groups or by inducing lipid peroxidation. The damage provoked by oxidation of sulfhydryl groups might be reversible but the damage induced by the process of lipid peroxidation is probably not reversible. The main protective constituents of the cell are thiols and vitamin E. Thiols, especially glutathione, protect the cytosol while vitamin E protects the lipid membranes against free radicals. In the scavenging of free radicals in the lipid membrane, vitamin E becomes oxidized. However continuous recycling of vitamin E by a reductase, with the cytosolic thiol glutathione as cofactor, will keep the vitamin E levels high enough to protect against lipid peroxidation. In the recycling glutathione is oxidized. Dihydrolipoic acid cannot provide directly reducing equivalents for the recycling of vitamin E by the free radical reductase. However indirectly, via the reduction of oxidized glutathione, dihydrolipoic acid can mediate the regeneration of vitamin E. One of the secondary mechanisms that mediates free radical induced damage is the rise in intracellular free Ca²⁺-concentration caused by inactivation of the endoplasmic reticulum ca²⁺-ATPase. The Ca²⁺-ATPase can be inactivated either by sulfhydryl alkylation or by lipid peroxidation. The authors used the thiol-alkylating agent N-ethylmaleimide, cystamine and ebselen. Dithiothreitol reversed the inhibition caused by all the three agents, while dihydrolipoic acid reversed the inhibition caused by ebselen. Glutathione was not able to reverse the effects of the sulfhydryl reactive agents. The reactivation of the microsomal Ca²⁺-ATPase by dihydrolipoic acid, may – besides the reduction of oxidized glutathione – contribute to the protective effect of dihydrolipoic acid on lipid peroxidation.     

Fractionation and analysis of fluorescent products of lipid peroxidation

The fluorescence excitation spectrum of model conjugated Schiff base compounds that arise from the reaction of malonaldehyde with amino acids was shown to contain a maximum at 260–280 nm in addition to the previously observed maximum at 350–390 nm. Excitation at either maximum results in emission at a single maximum at 440–480 nm. The excitation and emission maxima of the model fluorescent compounds, together with the characteristic reductions in fluorescence intensity caused by alkaline pH or heavy metal coordination, provide criteria with which to examine lipid peroxidation products for the presence of the conjugated Schiff base fluorophore. Silicic acid column chromatography and silica gel thin layer chromatography were employed to fractionate the fluorescent products of model lipid peroxidation systems and of rat testicular lipid soluble extracts. These products contained large families of compounds whose fluorescence characteristics were the same as those of the Schiff base floorophores. The fractionation methods used enabled more thorough fluorescence characterization of many of the products of lipid peroxidation, but the fluores-cence criteria available do not provide definitive proof of structure.     

Evaluation of a model system for the selective study of the lipid peroxidation process

The effect of molecular environment on the peroxidation of linoleic acid (LA), a polyunsaturated fatty acid (PUFA), initiated by ferrous ions was investigated in acidic and neutral pH conditions. Mixed nonionic surfactants TWEEN®‐20/LA micelles were established as a model system to obtain a surfactant‐in‐lipid aqueous system at high acidity level. The peroxidation of LA was induced by ferrous ions and the kinetics of the produced conjugated dienes was followed by UV measurements and the ferric thiocynate method. Ferrous ions were oxidized only by the preformed LA hydroperoxides, which under established conditions produced lipid alkoxyl and peroxyl radicals as the sole initiators of propagation. The results revealed the LA peroxidation process remained mainly unaffected within the 2.5<pH<5.5 range, while highly pH sensitive around pH 7. The propagation process prevailed at optimal concentrations of 500 µM of LA and 280 µM TWEEN®‐20, and at the ferrous ion concentration up to 75 µM, irrespective of the buffer used. Practical applications: A simple model system in water, suitable for the selective study of the lipid peroxidation propagation phase induced by ferrous ion is presented here. Fatty acids serve as model compounds susceptible to processes associated with oxidative radical initiated‐modifications of lipids. The obtained results contribute to a better understanding of the oxidative behavior of lipids, particularly those soluble in nonionic surfactant micelles in acidic medium. The oxidative stability of the PUFA in model systems containing TWEEN®‐20 and ferrous ion at low pH could be predicted and controlled by measuring the lipid hydroperoxide formation. The experimental conditions presented may also provide a suitable system for the study of the termination phase of lipid peroxidation.     

Inhibition of ferrous-induced lipid peroxidation by pyrimido-pyrimidine derivatives in human liver membranes

The effects of pyrimido-pyrimidine derivatives (dipyridamole, RA-642, and RA-233) on lipid peroxidation, using d-α-tocopherol as standard, were studied in enriched membrane fractions from human and rat hepatocytes. Equimolar concentrations of ferrous sulfate and ascorbic acid were used to induce lipid peroxidation. The amount of peroxidized lipids observed in membrane fractions from human liver was smaller than in those from rat liver. In both species, however, pyrimido-pyrimidine derivatives, except for RA-233 in rat liver, inhibited lipid peroxidation dose-dependently in the following sequence: RA-642 > dipyridamole > d-α-tocopherol RA-233.     

An esterification protocol for cis-parinaric acid-determined lipid peroxidation in immune cells1,2

Loss of fluorescence from cis-parinaric acid (cPnA) is a sensitive indicator of lipid peroxidation. The purpose of this study was to utilize cPnA to determine, at the level of the intact immune cell, whether enrichment of membranes with polyunsaturated fatty acids (PUFA) increased lipid peroxidation. P388D1 macrophages were labeled by addition of cPnA as an ethanolic solution. Within two minutes of addition, in the absence of serum, cPnA rapidly intercalated into the plasma membrane. Lipid peroxidation was initiated by addition of Fe²⁺-EDTA resulting in a dose-dependent decrease in fluorescence with increased oxidant concentration. Cells previously enriched with PUFA and labeled by intercalation showed no differences in spontaneous or Fe²⁺-induced lipid peroxidation. In separate experiments, 20 μM cPnA in ethanolic solution was injected into cell culture media containing 0.1% essentially fatty acid free bovine serum albumin (BSA). Cells were resuspended and incubated for 90 min at 37°C. After washing with BSA to remove cPnA which had not incorporated, 0.5% (0.1 μM) of the added cPnA was found esterified within cellular lipids. This level of cPnA provided a 100-fold increase over basal autofluorescence levels. Cells labeled in this manner also lost fluorescence in a dose-dependent manner as levels of oxidant stress increased. Cells enriched with PUFA and labeled by esterification had significantly increased rates and total amounts of lipid peroxidation. Co-incubation with α-tocopherol and PUFA resulted in a decrease in lipid peroxidation which was not significantly different from control cells. In conclusion, esterification of cPnA into membrane phospholipids can sensitively detect changes in lipid peroxidation induced by alteration of membrane PUFA and/or vitamin E content. Presented in part at the Experimental Biology Meetings, Anaheim, California, April 1994. Contribution from the Missouri Agriculture Extention Station, Journal #12,495.     

Fluorospectroscopic analysis of the fluorescent substances in peroxidized microsomes of rat liver

The fluorescent substances formed in rat liver microsomes in the course of lipid peroxidation were investigated by fluorescence techniques. The fluorescence emitted from peroxidizing microsomes continuously increased as lipid peroxidation progressed, while the steady-state fluorescence anisotropy increased and then reached a plateau. A similar increase was observed in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in peroxidizing microsomes. The fluorescence from peroxidized microsomes consisted of at least three species having short, middle or long fluorescence lifetimes. The lifetimes and relative amplitudes of fluorescence were unaffected by the extent of lipid peroxidation. Both fluorescence of the chromolipids extracted and the proteins isolated from peroxidized microsomes had the same characteristics in fluorescence lifetimes as the fluorescence from whole peroxidized microsomes. Thus, these lipids and proteins appear to be the major biological substances responsible for the fluorescence emanating from whole peroxidized microsomes. Furthermore, fluorescent substances formed in microsomes seem to increase in quantity rather than change in quality as lipid peroxidation progresses.