Tri-Herbal Medicine Divya Sarva-Kalp-Kwath (Livogrit) Regulates Fatty Acid-Induced Steatosis in Human HepG2 Cells through Inhibition of Intracellular Triglycerides and Extracellular Glycerol Levels.

Steatosis is characterized by excessive triglycerides accumulation in liver cells. Recently, application of herbal formulations has gained importance in treating complex diseases. Therefore, this study explores the efficacy of tri-herbal medicine Divya Sarva-Kalp-Kwath (SKK; brand name, Livogrit) in treating free fatty acid (FFA)-induced steatosis in human liver (HepG2) cells and rat primary hepatocytes. Previously, we demonstrated that cytosafe SKK ameliorated CCl4-induced hepatotoxicity. In this study, we evaluated the role of SKK in reducing FFA-induced cell-death, and steatosis in HepG2 through analysis of cell viability, intracellular lipid and triglyceride accumulation, extracellular free glycerol levels, and mRNA expression changes. Plant metabolic components fingerprinting in SKK was performed via High Performance Thin Layer Chromatography (HPTLC). Treatment with SKK significantly reduced the loss of cell viability induced by 2 mM-FFA in a dose-dependent manner. SKK also reduced intracellular lipid, triglyceride accumulation, secreted AST levels, and increased extracellular free glycerol presence in the FFA-exposed cells. SKK normalized the FFA-stimulated overexpression of SREBP1c, FAS, C/EBPα, and CPT1A genes associated with the induction of steatosis. In addition, treatment of rat primary hepatocytes with FFA and SKK concurrently, reduced intracellular lipid accumulation. Thus, SKK showed efficacy in reducing intracellular triglyceride accumulation and increasing extracellular glycerol release, along with downregulation of related key genetic factors for FFA-associated steatosis.

Validation of Putative Apicoplast-Targeting Drugs Using a Chemical Supplementation Assay in Cultured Human Malaria Parasites.

Malaria parasites contain a relict plastid, the apicoplast, which is considered an excellent drug target due to its bacterial-like ancestry. Numerous parasiticidals have been proposed to target the apicoplast, but few have had their actual targets substantiated. Isopentenyl pyrophosphate (IPP) production is the sole required function of the apicoplast in the blood stage of the parasite life cycle, and IPP supplementation rescues parasites from apicoplast-perturbing drugs. Hence, any drug that kills parasites when IPP is supplied in culture must have a nonapicoplast target. Here, we use IPP supplementation to discriminate whether 23 purported apicoplast-targeting drugs are on- or off-target. We demonstrate that a prokaryotic DNA replication inhibitor (ciprofloxacin), several prokaryotic translation inhibitors (chloramphenicol, doxycycline, tetracycline, clindamycin, azithromycin, erythromycin, and clarithromycin), a tRNA synthase inhibitor (mupirocin), and two IPP synthesis pathway inhibitors (fosmidomycin and FR900098) have apicoplast targets. Intriguingly, fosmidomycin and FR900098 leave the apicoplast intact, whereas the others eventually result in apicoplast loss. Actinonin, an inhibitor of bacterial posttranslational modification, does not produce a typical delayed-death response but is rescued with IPP, thereby confirming its apicoplast target. Parasites treated with putative apicoplast fatty acid pathway inhibitors could not be rescued, demonstrating that these drugs have their primary targets outside the apicoplast, which agrees with the dispensability of the apicoplast fatty acid synthesis pathways in the blood stage of malaria parasites. IPP supplementation provides a simple test of whether a compound has a target in the apicoplast and can be used to screen novel compounds for mode of action.

Curcumin improves alcoholic fatty liver by inhibiting fatty acid biosynthesis.

Alcoholic fatty liver is a threat to human health. It has been long known that abstinence from alcohol is the most effective therapy, other effective therapies are not available for the treatment in humans. Curcumin has a great potential for anti-oxidation and anti-inflammation, but the effect on metabolic reconstruction remains little known. Here we performed metabolomic analysis by gas chromatography/mass spectrometry and explored ethanol pathogenic insight as well as curcumin action pattern. We identified seventy-one metabolites in mouse liver. Carbohydrates and lipids were characteristic categories. Pathway analysis results revealed that ethanol-induced pathways including biosynthesis of unsaturated fatty acids, fatty acid biosynthesis and pentose and glucuronate interconversions were suppressed by curcumin. Additionally, ethanol enhanced galactose metabolism and pentose phosphate pathway. Glyoxylate and dicarboxylate metabolism and pyruvate metabolism were inhibited in mice fed ethanol diet plus curcumin. Stearic acid, oleic acid and linoleic acid were disease biomarkers and therapical biomarkers. These results reflect the landscape of hepatic metabolism regulation. Our findings illustrate ethanol pathological pathway and metabolic mechanism of curcumin therapy.

Antilipidemic effects and gene expression profiling of the glycosaminoglycans from cricket in rats on a high fat diet.

Glycosaminoglycan (GAG) from cricket (Gryllus bimaculatus) was studied as a potential health supplement. Antiatherosclerotic and antilipidemic effects of the GAG of G. bimaculatus (GbG, 5 or 10 mg/kg) were investigated in 15-week old Wistar rats treated with GbG for over a month. GbG produced a meaningful anti-edema effect with inhibition of C-reactive protein (CRP). Also, the weights of abdominal and epididymidal fat were also reduced in conjunction with a mild increase in body weight. Furthermore, the sero-biochemical parameters showed an antihyperlipidemic effect with decreased levels of phospholipid, AST, ALT, total cholesterol and glucose in a dose-dependent manner. In addition anticoagulant and antithrombotic effects were seen: platelet, thrombin time, prothrombin time and Factor I were increased with GbG treatment. Furthermore, the GbG treated rat group (at 10 mg/kg) compared to control, showed that 588 genes (test/control ratio >2.0) including lipocalin 2 (Lcn2) and alpha 2-macroglobulin (A2 m) were up-regulated, and 569 genes (test/control ratio >0.5) including stearoyl-coenzyme A desaturase 1 (Scd1) were down-regulated. Based on these results, GbG could potentially prevent or treat fatty liver or hyperlipidemia in rats on a high-fat diet.

Liquid fructose downregulates Sirt1 expression and activity and impairs the oxidation of fatty acids in rat and human liver cells.

Fructose ingestion is associated with the production of hepatic steatosis and hypertriglyceridemia. For fructose to attain these effects in rats, simultaneous induction of fatty acid synthesis and inhibition of fatty acid oxidation is required. We aimed to determine the mechanism involved in the inhibition of fatty acid oxidation by fructose and whether this effect occurs also in human liver cells. Female rats were supplemented or not with liquid fructose (10% w/v) for 7 or 14 days; rat (FaO) and human (HepG2) hepatoma cells, and human hepatocytes were incubated with fructose 25mM for 24h. The expression and activity of the enzymes and transcription factors relating to fatty acid ß-oxidation were evaluated. Fructose inhibited the activity of fatty acid ß-oxidation only in livers of 14-day fructose-supplemented rats, as well as the expression and activity of peroxisome proliferator activated receptor α (PPARα). Similar results were observed in FaO and HepG2 cells and human hepatocytes. PPARα downregulation was not due to an osmotic effect or to an increase in protein-phosphatase 2A activity caused by fructose. Rather, it was related to increased content in liver of inactive and acetylated peroxisome proliferator activated receptor gamma coactivator 1α, due to a reduction in sirtuin 1 expression and activity. In conclusion, fructose inhibits liver fatty acid oxidation by reducing PPARα expression and activity, both in rat and human liver cells, by a mechanism involving sirtuin 1 down-regulation.

Staphylococcus aureus fatty acid auxotrophs do not proliferate in mice.

Inactivation of acetyl-coenzyme A (acetyl-CoA) carboxylase confers resistance to fatty acid synthesis inhibitors in Staphylococcus aureus on media supplemented with fatty acids. The addition of anteiso-fatty acids (1 mM) plus lipoic acid supports normal growth of ΔaccD strains, but supplementation with mammalian fatty acids was less efficient. Mice infected with strain RN6930 developed bacteremia, but bacteria were not detected in mice infected with its ΔaccD derivative. S. aureus bacteria lacking acetyl-CoA carboxylase can be propagated in vitro but were unable to proliferate in mice, suggesting that the acquisition of inactivating mutations in this enzyme is not a mechanism for the evasion of fatty acid synthesis inhibitors.

Is bacterial fatty acid synthesis a valid target for antibacterial drug discovery?

The emergence of resistance against most current drugs emphasizes the need to develop new approaches to control bacterial pathogens, particularly Staphylococcus aureus. Bacterial fatty acid synthesis is one such target that is being actively pursued by several research groups to develop anti-Staphylococcal agents. Recently, the wisdom of this approach has been challenged based on the ability of a Gram-positive bacterium to incorporate extracellular fatty acids and thus circumvent the inhibition of de novo fatty acid synthesis. The generality of this conclusion has been challenged, and there is enough diversity in the enzymes and regulation of fatty acid synthesis in bacteria to conclude that there is not a single organism that can be considered typical and representative of bacteria as a whole. We are left without a clear resolution to this ongoing debate and await new basic research to define the pathways for fatty acid uptake and that determine the biochemical and genetic mechanisms for the regulation of fatty acid synthesis in Gram-positive bacteria. These crucial experiments will determine whether diversity in the control of this important pathway accounts for the apparently different responses of Gram-positive bacteria to the inhibition of de novo fatty acid synthesis in presence of extracellular fatty acid supplements.

Clerodendron glandulosum.Coleb extract ameliorates high fat diet/fatty acid induced lipotoxicity in experimental models of non-alcoholic steatohepatitis.

This study evaluates the protective role of Clerodendron glandulosum.Coleb (CG) aqueous extract against high fat diet/fatty acid induced lipotoxicity in experimental models of non-alcoholic steatohepatitis (NASH). Supplementation of NASH mice with CG extract (1% and 3% in high fat diet for 16 weeks) prevented high fat diet induced elevation in liver enzymes, plasma and hepatic lipids, mitochondrial oxidative stress and compromised enzymatic and non-enzymatic antioxidant status and histopathological damage to hepatocytes. Furthermore, results from in vitro study indicates, addition of CG extract (20-200 µg/ml for 24h) to HepG2 cells minimizes oleic acid induced lipid accumulation, higher lipid peroxidation, cytotoxicity and reduced cell viability. These in vivo and in vitro studies suggest that CG extract has the potential of preventing high fat/fatty acid induced NASH.

Novel antifungal agents, targets or therapeutic strategies for the treatment of invasive fungal diseases: a review of the literature (2005-2009).

BACKGROUND: The incidence and prevalence of serious mycoses continues to be a public health problem. Despite aggressive treatment with new or more established licensed antifungal agents, these infections are an important cause of morbidity and mortality, especially in immunocompromised patients. AIMS: To critically review the literature regarding important new developments in the field of antifungal therapy both in the English and Spanish versions. METHODS: The search of the literature focused on different antifungal targets or mechanisms of action as well as new agents or strategies that could improve antifungal therapy. RESULTS: The review produced a huge amount of information on the use of virulent factors such as growth, filamentation, pathogen tissue clearance, among others, as putative targets of antifungal activity. More recently, the chemical-genetic relationships for licensed agents as well as for other compounds have been provided by the identification of the genes related to the mechanism of action. CONCLUSIONS: Although the antifungal activity of numerous compounds has been examined, most of them are at the in vitro or animal models of efficacy stages. Therefore, further investigation should be carried out to realize the true clinical utility of these compounds.

Novel antifungal agents, targets or therapeutic strategies for the treatment of invasive fungal diseases: a review of the literature (2005-2009) / Nuevos antifúngicos, nuevas dianas y estrategias terapéuticas para el tratamiento de las micosis invasoras: revisión de la bibliografía (2005-2009)

Background: The incidence and prevalence of serious mycoses continues to be a public health problem. Despite aggressive treatment with new or more established licensed antifungal agents, these infections are an important cause of morbidity and mortality, especially in immunocompromised patients. Aims: To critically review the literature regarding important new developments in the field of antifungal therapy both in the English and Spanish versions. Methods: The search of the literature focused on different antifungal targets or mechanisms of action as well as new agents or strategies that could improve antifungal therapy. Results: The review produced a huge amount of information on the use of virulent factors such as growth, filamentation, pathogen tissue clearance, among others, as putative targets of antifungal activity. More recently, the chemical-genetic relationships for licensed agents as well as for other compounds have been provided by the identification of the genes related to the mechanism of action. Conclusions: Although the antifungal activity of numerous compounds has been examined, most of them are at the in vitro or animal models of efficacy stages. Therefore, further investigation should be carried out to realize the true clinical utility of these compounds (AU)

Antecedentes: La incidencia y la prevalencia de micosis invasoras continúa siendo un problema de salud pública. A pesar de los tratamientos más agresivos con los nuevos fármacos o los antifúngicos más establecidos, las infecciones fúngicas causan bastante mortalidad y morbilidad, especialmente en los pacientes inmunodeficientes. Objetivos: Revisar críticamente la bibliografía acerca de los nuevos desarrollos más importantes en el campo del tratamiento antifúngico en las versiones en español y en inglés. Métodos: Se enfocó la revisión en los estudios relacionados a dianas o mecanismos de acción diferentes a los actuales; también se revisaron los informes de fármacos nuevos, estrategias terapéuticas prometedoras o alternativas para los pacientes que presentan infecciones fúngicas invasoras. Resultados: En numerosos estudios se ha evaluado una variedad de factores de virulencia como posibles dianas de actividad antifúngica. Más recientemente, la relación química-genética de los antifúngicos aprobados y de otras moléculas se ha definido debido a la identificación de los genes relacionados con el mecanismo de acción correspondiente. Conclusiones: A pesar de los resultados favorables aportados en esos estudios, el desarrollo de la mayoría de estas moléculas está al nivel de su espectro in vitro o in vivo, pero en estudios de eficacia en modelos animales. Por lo tanto, deben realizarse más evaluaciones para que su desarrollo llegue al nivel de ensayos clínicos (AU)

Combined leptin actions on adipose tissue and hypothalamus are required to deplete adipocyte fat in lean rats: implications for obesity treatment.

Intense hyperleptinemia completely depletes adipocyte fat of normal rats within 14 days. To determine the mechanism, epididymal fat pads from normal wild-type (+/+) and obese (fa/fa) Zucker Diabetic Fatty (ZDF) donor rats were transplanted into normal +/+ and fa/fa ZDF recipients. Hyperleptinemia induced by adenovirus-leptin administration depleted all fat from native fat pads and from fat transplants from +/+ donors but not from transplants from ZDF(fa/fa) donors with defective leptin receptors. In both native and transplanted +/+ fat pads, large numbers of mitochondria were apparent, and genes involved in fatty acid oxidation were up-regulated. However, +/+ fat pads transplanted into fa/fa recipients did not respond to hyperleptinemia, suggesting lack of an essential leptin-stimulated cohormone(s). In +/+ but not in fa/fa rats, plasma catecholamine levels rose, and both P-STAT3 and P-CREB increased in adipose tissue, suggesting that both direct and indirect (hypothalamic) leptin receptor-mediated actions of hyperleptinemia are involved in depletion of adipocyte fat.

Inhibitory effects of green tea polyphenols on the production of a virulence factor of the periodontal-disease-causing anaerobic bacterium Porphyromonas gingivalis.

The effect of polyphenolic compounds isolated from green tea (Camellia sinensis) on the production of toxic end metabolites of Porphyromonas gingivalis was investigated. Green tea polyphenols completely inhibited the production of n-butyric acid and propionic acid at a concentration of 1.0-2.0 mg/mL in general anaerobic medium (GAM). (-)-Epigallocatechin gallate (EGCg), which is a major component of tea polyphenols also inhibited the production of phenylacetic acid at 0.5 mg/mL in GAM broth. In the experiment using resting cells of P. gingivalis, phenylacetic acid was produced from l-phenylalanine and phenylpyruvic acid, but this reaction was also inhibited by EGCg, (-)-epicatechin gallate, and (-)-gallocatechin gallate. However, (+)-catechin, (+)-gallocatechin, (-)-epicatechin, and (-)-epigallocatechin did not inhibit those reactions. These results indicate that the inhibitory effect on the production of toxic end metabolites of P. gingivalis can be attributed to the presence of the galloyl moiety, which is ester-linked with the 3-OH of the catechin moiety in the polyphenolic compounds. This study shows that continuous application of tea polyphenols on a daily basis can be considered as a useful and practical method for the prevention of periodontal diseases.

Inhibition of fatty acid synthesis in bovine mammary homogenate by palmitic acid is not a detergent effect.

Supplemental fat fed to dairy cows affects the fat composition of milk by reducing the yield of mammary synthesized fatty acids. The effect has been attributed to a potential allosteric inhibition of acetyl coenzyme-A, a key enzyme in fatty acid synthesis. In vitro experiments have demonstrated an inhibition of fatty acid synthesis when long-chain fatty acids are added to incubations. However, in vitro inhibition can result from a nonspecific detergent effect arising from an inherent physical property of fatty acids. An allosteric role for palmitic acid has not been tested in bovine mammary tissue. The objective of this experiment was to test the hypothesis that palmitic acid is an allosteric inhibitor of fatty acid synthesis in mammary tissue. We tested for a detergent effect by including a synthetic detergent, sodium dodecyl sulfate, under identical incubation conditions. A subcellular supernatant fraction of mammary tissue was used for incubations in the present experiment. The incubation system produced free fatty acids in a linear fashion for time and protein content. Results indicated that fatty acid synthesis was affected by the addition of palmitic acid to the incubations but not by caprylic acid, a short-chain fatty acid. Sodium dodecyl sulfate did not affect fatty acid synthesis at the concentrations used. The results of the present experiment indicate that palmitic acid inhibited fatty acid synthesis, and the effect was not the result of a detergent effect.

An unusual seed-specific 3-ketoacyl-ACP synthase associated with the biosynthesis of petroselinic acid in coriander.

Petroselinic acid (18:1 delta6) is the major component of the seed oil of Umbelliferae species such as coriander (Coriandrum sativum) as well as Araliaceae and Garryaceae species. This unusual fatty acid is synthesized in plastids by the delta4 desaturation of palmitoyl-acyl carrier protein (16:0-ACP) and subsequent elongation of delta4-hexadecenoyl (16:1 delta4)-ACP. To characterize the enzymatic nature of the elongation reaction, an in vitro assay was developed with 16:1 delta4-ACP and 16:0-ACP as substrates. Extracts from developing coriander seeds elongated 16:1 delta4-ACP in a competitive assay at rates ten-fold greater than that with 16:0-ACP. In contrast, extracts from castor seeds, which do not synthesize petroselinic acid, displayed a strong preference for the elongation of 16:0-ACP rather than 16:1 A4-ACP. In addition, the elongation of 16:1 A4-ACP and 16:0-ACP by coriander seed extracts was strongly inhibited by cerulenin at concentrations as low as 10 microM. This finding suggested that the elongation of 16:1 A4-ACP and 16:0-ACP in coriander seed is catalyzed by a 3-ketoacyl-ACP synthase (KAS) 1-type enzyme(s), rather than a KAS II-type activity that is typically associated with 16:0-ACP elongation. Consistent with this, a cDNA for a diverged form of KAS I was isolated from a cDNA library prepared from developing coriander seed. Using a variety of heterologous probing techniques, no KAS II-type cDNAs could be identified in this library. Multiple alignment of KAS amino acid sequences indicated that, although the polypeptide corresponding to the coriander cDNA is more closely related to KAS I. its active site motif deviates from those found in both KAS I and KAS II enzymes. Also suggestive of a possible role in petroselinic acid synthesis, antibodies raised to the recombinant protein recognize an abundant 45 kDa polypeptide in coriander endosperm that is not detected in coriander leaves. These antibodies also recognize a major band of similar size in developing seeds of English ivy (Hedera helix), an Araliaceae species that also accumulates petroselinic acid in a seed-specific manner.

Oral hypoglycemic agents: insulin secretagogues, alpha-glucosidase inhibitors and insulin sensitizers.

In this review we present the agents that are in use in the treatment of type 2 diabetes. Sulfonylureas of the 1st and 2nd generation increase insulin secretion but can induce hyperinsulinemia and sometimes prolonged hypoglycemia. Glimepiride is a new 3rd generation sulfonylurea with some advantages over the other members of this group, such as a lower risk of hypoglycemia, no interaction with cardiovascular KATP-channels and a possibility that it may increase insulin sensitivity. There are also newer insulin secretagogues (such as neteglinide and repaglinide) with a rapid onset of action on the beta-cell, therefore inducing a more physiological profile of insulin secretion during meals. The category of insulin sensitizers includes metformin and thiazolidinediones. Metformin effectively reduces hyperglycemia, hyperlipidemia and macroangiopathy in patients with type 2 diabetes. This agent increases the sensitivity of the liver and peripheral tissues to insulin and, therefore, it could be considered as a drug of choice for the prevention of type 2 diabetes. Thiazolidinediones (rosiglitazone and pioglitazone) increase the sensitivity of the tissues to insulin. This mechanism of action makes them powerful therapeutic tools for the treatment of type 2 diabetes (and possibly other insulin resistant states) either alone or in combination with other oral agents. The category of agents that interfere with the absorption of glucose and lipids includes alpha-glucosidase inhibitors (acarbose and miglitol) and lipase inhibitors (or-listat). alpha-Glucocidase inhibitors improve the time relationship between plasma insulin and glucose increases after a meal. Therefore, these agents may be used in the treatment of patients with type 2 diabetes, either alone at a very early stage of this disease (when insulin secretion is still adequate), or in combination with insulin secretagogues. alpha-Glucosidase inhibition may also prove useful as a supplement to insulin therapy in patients with type 1 diabetes mellitus. The inhibitor of gastrointestinal lipase orlistat may prove a useful adjunct to hypocaloric diets in patients with type 2 diabetes and obesity.

Identification of the conjugated linoleic acid isomer that inhibits milk fat synthesis.

Conjugated linoleic acids (CLA) are octadecadienoic fatty acids that have profound effects on lipid metabolism. Our previous work showed that CLA (mixture of isomers) markedly reduced milk fat synthesis. In this study, our objective was to evaluate the effects of specific CLA isomers. Multiparous Holstein cows were used in a 3x3 Latin square design, and treatments were 4-day abomasal infusions of 1) skim milk (control), 2) 9,11 CLA supplement, and 3) 10,12 CLA supplement. CLA supplements provided 10 g/day of the specific CLA isomer (cis-9,trans-11 or trans-10,cis-12). Treatments had no effect on intake, milk yield, or milk protein yield. Only the 10,12 CLA supplement affected milk fat, causing a 42 and 44% reduction in milk fat percentage and yield, respectively. Milk fat composition revealed that de novo synthesized fatty acids were extensively reduced. Increases in ratios of C(14:0) to C(14:1) and C(18:0) to C(18:1) indicated the 10,12 CLA supplement also altered Delta(9)-desaturase. Treatments had minimal effects on plasma concentrations of glucose, nonesterified fatty acids, insulin, or insulin-like growth factor-I. Overall, results demonstrate that trans-10,cis-12 CLA is the isomer responsible for inhibition of milk fat synthesis.

Mechanism for the inhibition of fat digestion by chitosan and for the synergistic effect of ascorbate.

We investigated the mechanism for the inhibition of fat digestion by chitosan, and the synergistic effect of ascorbate. The important inhibition characteristics of fat digestion by chitosan from observations of the ileal contents were that it dissolved in the stomach and then changed to a gelled form, entrapping fat in the intestine. The synergistic effect of ascorbate (AsA) on the inhibition of fat digestion by chitosan is thought not to be acid-dependent but due to the specificity of AsA itself, according to the data resulting from using preparations supplemented with sodium ascorbate (AsN). The mechanism for the synergistic effect is considered to be 1) viscosity reduction in the stomach, which implies that chitosan mixed with a lipid is better than chitosan alone, 2) an increase in the oil-holding capacity of the chitosan gel, and 3) the chitosan-fat gel being more flexible and less likely to leak entrapped fat in the intestinal tract.

Hyperbaric hyperoxia reversibly inhibits erythrocyte phospholipid fatty acid turnover.

The present study is one component of a comprehensive investigation of oxygen tolerance of tissues and organs in normal human subjects. The focus of this study was the acylation of membrane phospholipid in situ by erythrocytes. Activation of exogenous [9,10-3H]oleic acid to acyl thioester and transesterification of the acyl thioester into phospholipid by intact human erythrocytes incubated in vitro decreased 30% after exposure of 10 human subjects to hyperbaric hyperoxia (100% O2, 3 ATA, 3.5 h). Partial recovery of activity could be detected when additional cells were obtained from these subjects and assayed in vitro 24 h after cessation of exposure. No significant change in membrane phospholipid fatty acid composition was detected under these conditions. The reduced glutathione content of intact erythrocytes increased by 15% after hyperbaric hyperoxia and remained elevated 24 h after exposure. In isolated membranes prepared from the same cells activation of [9,10-3H]oleic acid to acyl thioester and its transesterification into phospholipid did not change after hyperoxia. Since the ability of intact cells to replace oxidized fatty acids in membrane phospholipids via deacylation and reacylation in situ may be necessary for the maintenance of membrane integrity during exposure to oxidative stress, the decrease in [9,10-3H]oleic acid incorporation by human erythrocytes detected in vitro after hyperbaric hyperoxia in vivo may reflect an early event in the pathogenesis of oxygen-induced cellular injury and may be a useful index for assessment of the tolerance of tissues to hyperoxia.