OBJECTIVE:The link between a reduced capacity for skeletal muscle mitochondrial fatty acid oxidation (FAO) and lipotoxicity in human insulin resistance has been the subject of intense debate. The objective of this study was to investigate whether reduced FAO is associated with elevated acyl CoA, ceramide, and diacylglycerol (DAG) in severely obese insulin resistant subjects. METHODS:Muscle biopsies were conducted in lean (L, 22.6 ± 0.5 kg/m(2) , n = 8), Class I (CI, 32.1 ± 0.4 kg/m(2) , n = 7) and Class II&III obese (CII&III, 45.6 ± 1.1 kg/m(2) , n = 15) women for acyl CoA, sphingolipid and DAG profiling. Intramyocellular triglyceride (IMTG) content was determined by histology. FAO was assessed by incubating muscle homogenates with [1-C]palmitate and measuring CO2 production. Cardiolipin content was quantified as an index of mitochondrial content. Lipid metabolism proteins, DGAT1, PLIN5, and PNPLA2 were quantified in biopsy samples by western blot. RESULTS:CII&III were more insulin resistant (HOMA-IR: 4.5 ± 0.5 vs. 1.1 ± 0.1, P < 0.001), and had lower FAO (∼58%, P = 0.007) and cardiolipin content (∼31%, P = 0.013) compared to L. IMTG was elevated in CI (P = 0.04) and CII&III (P = 0.04) compared to L. Sphingolipid content was higher in CII&III compared to L (13.6 ± 1.1 vs. 10.3 ± 0.5 pmol/mg, P = 0.031) whereas DAG content was not different among groups. DGAT1 was elevated in CII&III, and PLIN5 was elevated in CI compared to L. CONCLUSIONS:Severe obesity is associated with reduced muscle oxidative capacity and occurs concomitantly with elevated IMTG, ceramide and insulin resistance.

Cardiolipin is a phospholipid that is specific to the inner mitochondrial membrane and essential for numerous mitochondrial functions. Accordingly, a quantitative assay for cardiolipin can be a valuable aspect of assessing mitochondrial content and functional capacity. The current study was undertaken to develop a simple and reliable method for direct analysis of the major molecular species of cardiolipin and with particular application for analysis of human skeletal muscle. The method that is presented is based on derivatization of cardiolipin in a total lipid extract with 1-pyrenyldiazomethane (PDAM), to form stable, fluorescent 1-pyrenylmethyl esters. The derivatization reaction takes 30min on ice in a two-phase system (chloroform:methanol:H2O:H2SO4) containing 0.5–1.0mM PDAM and detergent. The contents of the major cardiolipin species in the derivatization mixture can be estimated by HPLC separation with fluorescent detection during a 20min run on a reverse phase column and with HPLC grade ethanol/0.5mM H3PO4 as the mobile phase. The recovery is about 80%. The method is specific and sensitive with quantitation limits of 0.5–1pmol cardiolipin. The response of the fluorescence detector (peak area) is linear across a range 5–40pmol. The assay is linear over the range between 0.3 and 3.0mg of tissue (R2=0.998). The assay provides good reproducibility and accuracy (within 5–10%).

The current study addresses a novel hypothesis of subcellular distribution of mitochondrial dysfunction in skeletal muscle in type 2 diabetes. Vastus lateralis muscle was obtained by percutaneous biopsy from 11 volunteers with type 2 diabetes; 12 age-, sex-, and weight-matched obese sedentary nondiabetic volunteers; and 8 lean volunteers. Subsarcolemmal and intermyofibrillar mitochondrial fractions were isolated by differential centrifugation and digestion techniques. Overall electron transport chain activity was similar in type 2 diabetic and obese subjects, but subsarcolemmal mitochondria electron transport chain activity was reduced in type 2 diabetic subjects (0.017 +/- 0.003 vs. 0.034 +/- 0.007 units/mU creatine kinase [CK], P = 0.01) and sevenfold reduced compared with lean subjects (P < 0.01). Electron transport chain activity in intermyofibrillar mitochondria was similar in type 2 diabetic and obese subjects, though reduced compared with lean subjects. A reduction in subsarcolemmal mitochondria was confirmed by transmission electron microscopy. Although mtDNA was lower in type 2 diabetic and obese subjects, the decrement in electron transport chain activity was proportionately greater, indicating functional impairment. Because of the potential importance of subsarcolemmal mitochondria for signal transduction and substrate transport, this deficit may contribute to the pathogenesis of muscle insulin resistance in type 2 diabetes.

This study addresses an application of pyridine nucleotide enzymatic analyses to evaluate the activity of the mitochondrial electron transport chain (reduced nicotinamide adenine dinucleotide (NADH) oxidase) and Complexes I and II in samples of human muscle as small as ∼10mg wet weight. Key aspects in this adaptation are the use of high-performance liquid chromatography with fluorescence detection of NADH and use of alamethicin, a channel-forming antibiotic that enables an unrestricted access of substrates into the mitochondrial matrix. The procedure includes disintegration of tissue by Polytron homogenizer, extraction of myosin from myofibrillar fragments by KCl/pyrophosphate to facilitate release of mitochondria, and preparation of fractions of subsarcolemmal and intermyofibrillar mitochondria. Oxidation of NADH or succinate is assayed in the presence of 40μg/ml alamethicin and the reaction is terminated by H2SO4, which also destroys the remaining NADH. Nicotinamide adenine dinucleotide (NAD) or fumarate concentrations are measured using alcohol dehydrogenase or fumarase plus malic dehydrogenase reactions, respectively. Generation of NADH, assessed in auxiliary reactions in the presence of hydrazine, is strictly proportional to NAD or fumarate content across a concentration range of 1–20μM. NADH is quantitatively analyzed with a detection limit of 3–5pmol by HPLC using a reverse-phase Hypersil ODS column connected to a fluorescence detector.

The current study was undertaken to address responsiveness of skeletal muscle mitochondrial electron transport chain (ETC) activity to weight loss (WL) and exercise in overweight or obese, sedentary volunteers. Fourteen middle-aged participants (7 male/7 female) had assessments of mitochondrial ETC activity and mitochondrial (mt)DNA in vastus lateralis muscle, obtained by percutaneous biopsy, before and after a 16-wk intervention. Mean WL was 9.7 (1.5%) and the mean increase in Vo(2 max) was [means (SD)] 21.7 (3.7)%. Total ETC activity increased significantly, from 0.13 (0.02) to 0.19 (0.03) U/mU creatine kinase (CK; P < 0.001). ETC activity was also assessed in mitochondria isolated into subsarcolemmal (SSM) and intermyofibrillar (IMF-M) fractions. In response to intervention, there was a robust increase of ETC activity in SSM (0.028 (0.007) to 0.046 (0.011) U/mU CK, P < 0.001), and in IMF-M [0.101 (0.015) to 0.148 (0.018) U/mU CK, P < 0.005]. At baseline, the percentage of ETC activity contained in the SSM fraction was low and remained unchanged following intervention [19 (3) vs. 22 (2)%], despite the increase in ETC activity. Also, muscle mtDNA content did not change significantly [1665 (213) vs. 1874 (214) mtDNA/nuclear DNA], denoting functional improvement rather than proliferation of mitochondria as the principal mechanism of enhanced ETC activity. Increases in ETC activity were correlated with energy expenditure during exercise sessions, and ETC activity in SSM correlated with insulin sensitivity after adjustment for Vo(2 max). In summary, skeletal muscle ETC activity is increased by WL and exercise in previously sedentary obese men and women. We conclude that improved skeletal muscle ETC activity following moderate WL and improved aerobic capacity contributes to associated alleviation of insulin resistance.

Skeletal muscle is strongly dependent on oxidative phosphorylation for energy production. Because the insulin resistance of skeletal muscle in type 2 diabetes and obesity entails dysregulation of the oxidation of both carbohydrate and lipid fuels, the current study was undertaken to examine the potential contribution of perturbation of mitochondrial function. Vastus lateralis muscle was obtained by percutaneous biopsy during fasting conditions from lean (n = 10) and obese (n = 10) nondiabetic volunteers and from volunteers with type 2 diabetes (n = 10). The activity of rotenone-sensitive NADH:O(2) oxidoreductase, reflecting the overall activity of the respiratory chain, was measured in a mitochondrial fraction by a novel method based on providing access for NADH to intact mitochondria via alamethicin, a channel-forming antibiotic. Creatine kinase and citrate synthase activities were measured as markers of myocyte and mitochondria content, respectively. Activity of rotenone-sensitive NADH:O(2) oxidoreductase was normalized to creatine kinase activity, as was citrate synthase activity. NADH:O(2) oxidoreductase activity was lowest in type 2 diabetic subjects and highest in the lean volunteers (lean 0.95 +/- 0.17, obese 0.76 +/- 0.30, type 2 diabetes 0.56 +/- 0.14 units/mU creatine kinase; P < 0.005). Also, citrate synthase activity was reduced in type 2 diabetic patients (lean 3.10 +/- 0.74, obese 3.24 +/- 0.82, type 2 diabetes 2.48 +/- 0.47 units/mU creatine kinase; P < 0.005). As measured by electron microscopy, skeletal muscle mitochondria were smaller in type 2 diabetic and obese subjects than in muscle from lean volunteers (P < 0.01). We conclude that there is an impaired bioenergetic capacity of skeletal muscle mitochondria in type 2 diabetes, with some impairment also present in obesity.

F(2)-isoprostanes are produced during free radical oxidation of cell phospholipids and are considered reliable biomarkers of oxidative stress. Currently, mass spectroscopy is the method of choice to detect F(2)-isoprostanes. However, due to numerous isomeric forms, analysis of F(2)-isoprostanes using MS detectors requires preliminary chromatographic separation. The current study was undertaken to develop a method of HPLC separation and quantification of different isomers of prostaglandin F(2alpha), including 8-iso-PGF(2alpha), following derivatization with 1-pyrenyldiazomethane (PDAM) to a highly fluorescent 1-pyrenylmethyl ester. HPLC separation and quantification of 1-pyrenylmethyl esters of PGF(2alpha) isomers at picogram level are complicated by numerous interfering products of the degradation of 1-pyrenyldiazomethane. A procedure of derivatization and purification was developed to reduce these interfering contaminants. The procedure of derivatization includes sorption of PGF(2alpha) isomers from solution in a hexane:ethyl acetate mixture (10:1) on a cellulose support prepared in the form of small (4 x 4mm) filter paper squares. Bound PGF(2alpha) isomers are derivatized by 1-pyrenyldiazomethane dissolved in the same hexane:ethyl acetate mixture. During subsequent washing of the cellulose squares by the hexane:ethyl acetate mixture (10:1), fluorescent derivatives of PGF(2alpha) remain bound to cellulose while a significant portion of the contaminants are washed out. The 1-pyrenylmethyl esters of PGF(2alpha) can be quantitatively extracted from cellulose by an ethyl acetate:methanol (1:1) mixture. The next step in eliminating interference is a solid-phase extraction on silica cartridges using ethyl acetate for application of the sample and an ethyl acetate:methanol (1:1) mixture for elution. Final purification is achieved by normal-phase HPLC with wet ethyl acetate as the mobile phase. This chromatographic method displays remarkable resolution in the separation of different PGF(2alpha) isomers and can be used not only for sample purification but also for pre-MS separation. The purified 1-pyrenylmethyl esters of PGF(2alpha) were quantitatively analyzed by reverse-phase HPLC with fluorescent detection, with a detection limit of 5-10 pg.

Various types of cancer occur in peroxidase-rich target tissues of animals exposed to aryl alcohols and amines. Unlike biotransformation by cytochrome P450 enzymes, peroxidases activate most substrates by one-electron oxidation via radical intermediates. This work analyzed the peroxidase-dependent formation of phenoxyl radicals in HL-60 cells and its contribution to cytotoxicity and genotoxicity. The results showed that myeloperoxidase-catalyzed redox cycling of phenol in HL-60 cells led to intracellular formation of glutathionyl radicals detected as GS-DMPO nitrone. Formation of thiyl radicals was accompanied by rapid oxidation of glutathione and protein-thiols. Analysis of protein sulfhydryls by SDS-PAGE revealed a significant oxidation of protein SH-groups in HL-60 cells incubated in the presence of phenol/H2O2 that was inhibited by cyanide and azide. Additionally, cyanide- and azide-sensitive generation of EPR-detectable ascorbate radicals was observed during incubation of HL-60 cell homogenates in the presence of ascorbate and H2O2. Oxidation of thiols required addition of H2O2 and was inhibited by pretreatment of cells with the inhibitor of heme synthesis, succinylacetone. Radical-driven oxidation of thiols was accompanied by a trend toward increased content of 8-oxo-7,8-dihydro-2′-deoxyguanosine in the DNA of HL-60 cells. Membrane phospholipids were also sensitive to radical-driven oxidation as evidenced by a sensitive fluorescence HPLC-assay based on metabolic labeling of phospholipids with oxidation-sensitive cis-parinaric acid. Phenol enhanced H2O2-dependent oxidation of all classes of phospholipids including cardiolipin, but did not oxidize parinaric acid–labeled lipids without addition of H2O2. Induction of a significant hypodiploid cell population, an indication of apoptosis, was detected after exposure to H2O2 and was slightly but consistently and significantly higher after exposure to H2O2/phenol. The clonogenicity of HL-60 cells decreased to the same extent after exposure to H2O2 or H2O2/phenol. Treatment of HL-60 cells with either H2O2 or H2O2/phenol at concentrations adequate for lipid peroxidation did not cause a detectable increase in chromosomal breaks. Detection of thiyl radicals as well as rapid oxidation of thiols and phospholipids in viable HL-60 cells provide strong evidence for redox cycling of phenol in this bone marrow-derived cell line.

Abstract: Interaction of hydrogen peroxide or organic hydroperoxides with hemoproteins is known to produce oxoferryl hemoprotein species that act as very potent oxidants. Since skeletal and cardiac muscle cells contain high concentrations of myoglobin this reaction may be an important mechanism of initiation or enhancement of oxidative stress, which may impair their Ca2+ transport systems. Using skeletal and cardiac sarcoplasmic reticulum (SR) vesicles, we demonstrated by EPR the formation of alkoxyl radicals and protein‐centered peroxyl radicals in the presence of myoglobin (Mb) and tert‐butyl hydroperoxide (t‐BuOOH). The low temperature EPR signal of the radicals was characterized by a major feature at g= 2.016 and a shoulder at g= 2.036 . In the presence of SR vesicles, the magnitude of the protein‐centered peroxyl radical signal decreased, suggesting that the radicals were involved in oxidative modification of SR membranes. This was accompanied by SR membrane oxidative damage, as evidenced by accumulation of 2‐thiobarbituric acid‐reactive substances (TBARS) and the inhibition of Ca2+ transport. We have shown that nitric oxide (NO), reacting with redox‐active heme iron, can prevent peroxyl radical formation activated by Mb/t‐BuOOH. Incubation of SR membranes with an NO donor, PAPA/NO (a non‐thiol compound that releases NO) at 200–500 μM completely prevented the t‐BuOOH‐dependent production of peroxyl radicals and formation of TBARS, and thus protected against oxidative inhibition of Ca2+ transport.

Reactive-oxygen species and organic free radicals are essential metabolic intermediates and have important regulatory functions. Overproduction of these reactive metabolites is implicated in the etiology of a host of degenerative diseases (1), such as cardiovascular disease and neurodegenerative disease, in acute conditions such as trauma and infection, and in aging. Of the various types of oxidative damage that cells undergo, lipid peroxidation is considered to be one of the major contributors to oxidative injury (2); thus, strict regulation of lipid peroxidation is extremely important for normal cell physiology. Quantitative assays of lipid peroxidation in intact cells are essential for evaluating oxidative damage from various sources, and in testing the efficacy of antioxidant interventions.

OverviewOxidative stress as a potential mechanism of Ca2+ disregulation in skeletal and cardiac musclesCalcium is central to numerous cellular functions and impairment of membrane Ca2+ transport results in tissue injury (Rasmussen and Barrett, 1984; Thomas et al., 1996). In striated muscle, the signal that triggers force generation is a rise in cytoplasmic free Ca2+-concentration (Cai), whereas the removal of Cai imparts a state of relaxation. Based on sophisticated biochemical and biophysical studies, a generally accepted picture of force generation has emerged. In brief, force generation is produced from a cyclic interaction between actin filaments and cross-bridges projecting from the myosin filaments. This interaction is controlled by a regulatory protein complex (tropomyosin—troponin) bound to the actin filament. When Ca2+ is low (<10-7 M), the cross-bridge interactions are inhibited by the regulatory complex. As Cai rises, Ca2+ ions bind to the regulatory binding site of the troponin C subunit, thereby activating cross-bridge cyclic and tension development. In striated muscle, the regulation of Cai is controlled by a combination of influx and efflux processes. The firing of an action potential produces an influx of Cai which is predominantly the result of a release of Ca2+ from the sarcoplasmic reticulum via the opening of Ca2+ release channels of ryanodine receptors, with minor contributions from voltage-gated Ca2+ channels in the surface membrane and from the Na+/Ca2+ exchanger, in heart muscle. The removal of Cai is primarily driven by the active uptake of Ca2+/Mg2+ ATPases on the sarcoplasmic reticulum, with minor contributions from Ca2+ ATPases on the sarcolemma and in the heart, the Na+/Ca2+ exchanger on the sarcolemma (Bers, 1991; Sneyd et al., 1995; Sutko and Airey, 1996). In skeletal muscle, myocardium and aortic smooth muscle, these Ca2+-regulating mechanisms are known to be susceptible to oxidative stress produced by organic free radicals and reactive oxygen intermediates (Kagan, 1988; Comporti, 1989; Keith, 1993). Not surprisingly, oxidative injury of various cells has been shown to be associated with increased intracellular concentrations of Ca2+ (Forman et al., 1987; Livingston et al., 1992; Kumar et al., 1996).

The antifungal activity of amphotericin B (AmB) and its side-effects (e.g. nephrotoxicity and hemolytic action) are suggested to be associated with its prooxidant effects in target cells. To test this hypothesis, we have undertaken studies to examine the role of AmB in oxidative stress in cultured rat aortic smooth muscle cells (SMC) incubated in the absence or in the presence of a lipid-soluble ate-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). No changes in the pattern of membrane phospholipids could be detected by two-dimensional high performance thin-layer chromatography (HPTLC) after oxidative stress induced by AMVN in which the cells remained viable, as judged by trypan blue exclusion. To improve the sensitivity of detection of oxidative stress in the cells, cis-parinaric acid (PnA) was incorporated biosynthetically into the membrane phospholipids [using PnA-human serum albumin (hSA) complex]. Incubation of the cells under aerobic conditions in the presence of up to 10 mu M AmB showed no significant change in the pattern of PnA-labeled phospholipids, suggesting that AmB was not affecting the oxidative state of the cells. In contrast, treatment with AMVN (0.5 mM, incubation in the dark for 2 hr at 37 degrees-conditions in which the viability of the cells was maintained) caused a significant reduction of all fluorescently labeled phospholipid fractions separated by HPLC. When PnA-labeled cells were subjected to oxidative stress by incubation with 0.5 mM AMVN in the presence of AmB, the loss of fluorescent phospholipids was reduced in a concentration-dependent manner over a concentration range of 0.25 to 10 mu M. Thus, AmB does not produce any prooxidant effect but rather acts as an intracellular antioxidant. (C) 1997 Elsevier Science Inc.

The antifungal effects of amphotericin B are believed to be due to two possibly interrelated mechanisms: an increase in permeation by binding to sterols in cellular membranes and a prooxidant effect causing oxidative damage in target cells. However, the seven conjugated double bonds in amphotericin B raise the possibility that it could be highly susceptible to autoxidation, causing an antioxidant effect. In the present study, we investigated the prooxidant and antioxidant properties of amphotericin B in a model system in which oxidation of a reporter molecule, cis-parinaric acid, was induced by azo initiators of peroxyl radicals. Since interactions of amphotericin B with sterols are essential for its pharmacological and toxic actions, we also studied the effects of cholesterol on the prooxidant and antioxidant properties of amphotericin B. Amphotericin B caused a noncollisional quenching of a characteristic fluorescence of cholesteryl cis-parinarate integrated in liposomes, suggesting the formation of amphotericin B-cholesteryl cis-parinarate complex. This effect of amphotericin B was ablated by increasing concentrations of cholesterol. We found that amphotericin B inhibited oxidation of cis-parinaric acid complexed with human serum albumin [using a water-soluble azo initiator, 2,2'-azobis(2aminopropane)dihydrochloride] and in liposomes [using a lipid-soluble azo initiator, 2,2'-azobis(2,4-dimethylvaleronitrile)]. The inhibitory effect of amphotericin B on 2,2'-azobis(2,4-dimethylvaleronitrile)-induced peroxidation of cis-parinaric acid in liposomes was also diminished by cholesterol. The antioxidant effect of amphotericin B in this model system suggests that amphotericin B does not exert its pharmacological and toxicological responses through a prooxidant effect to cause damage in target cells.