Carbonyl Adducts Research Articles

Screening and Characterization of Drugs That Protect Corneal Endothelial Cells Against Unfolded Protein Response and Oxidative Stress.

PurposeTo screen for and characterize compounds that protect corneal endothelial cells against unfolded protein response (UPR) and oxidative stress.MethodsBovine corneal endothelial cells (BCECs) were treated for 48 hours with 640 compounds from a Food and Drug Administration (FDA)-approved drug library and then challenged with thapsigargin or H2O2 to induce UPR or oxidative stress, respectively. Cell viability was measured using the CellTiter-Glo survival assay. Selected “hits” were subjected to further dose-response testing, and their ability to modulate expression of UPR and oxidative stress markers was assessed by RT-PCR, Western blot, and measurement of protein carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) adducts in immortalized human corneal endothelial cells (iHCECs).ResultsForty-one drugs at 20 μM and 55 drugs at 100 μM increased survival of H2O2-challenged cells, and 8 drugs at 20 μM and 2 drugs at 100 μM increased survival of thapsigargin-challenged cells, compared with untreated control cells. Nicergoline, ergothioneine, nimesulide, oxotremorine, and mefenamic acid increased survival of both H2O2- and thapsigargin-challenged cells. Oxotremorine altered DNA damage inducible 3 (CHOP) gene expression, glucose-regulated protein 78 kDa (GRP78) and activating transcription factor 4 (ATF4) protein expression, and protein carbonyl and 8-OHdG levels. Mefenamic acid altered GRP78 protein expression and protein carbonyl and 8-OHdG levels.ConclusionsOxotremorine and mefenamic acid are potential survival factors for corneal endothelial cells under UPR and oxidative stress. The described assay can be further expanded to screen additional drugs for potential therapeutic effect in corneal endothelial diseases such as Fuchs' endothelial corneal dystrophy.

Substrate-Induced Carbon Monoxide Reactivity Suggests Multiple Enzyme Conformations at the Catalytic Copper M-Center of Peptidylglycine Monooxygenase.

The present study uses CO as a surrogate for oxygen to probe how substrate binding triggers oxygen activation in peptidylglycine monooygenase (PHM). Infrared stretching frequencies (ν(C ≡ O)) of the carbonyl (CO) adducts of copper proteins are sensitive markers of Cu(I) coordination and are useful in probing oxygen reactivity because the electronic properties of O2 and CO are similar. The carbonyl chemistry has been explored using PHM WT and a number of active site variants in the absence and presence of peptidyl substrates. We have determined that upon carbonylation (i) a major CO band at 2092 cm-1 and a second minor CO band at 2063 cm-1 are observed in the absence of peptide substrate Ac-YVG; (ii) the presence of peptide substrate amplifies the minor CO band and causes it to partially interconvert with the CO band at 2092 cm-1; (iii) the substrate-induced CO band is associated with a second conformer at CuM; and (iv) the CuH-site mutants, which are inactive, fail to generate any substrate-induced CO bands. The total intensity of both bands is constant, suggesting that the Cu(I)M-site partitions between the two carbonylated enzyme states. Together, these data provide evidence for two conformers at CuM, one of which is induced by binding of the peptide substrate with the implication that this represents the conformation that also allows binding and activation of O2.

Synthesis of 1,4-Dicarbonyl Compounds from Silyl Enol Ethers and Bromocarbonyls, Catalyzed by an Organic Dye under Visible-Light Irradiation with Perfect Selectivity for the Halide Moiety over the Carbonyl Group.

We report the visible-light-induced radical coupling reaction of silyl enol ethers with α-bromocarbonyl compounds to give 1,4-dicarbonyls. The reaction was effectively accelerated using an inexpensive organic dye (eosin Y) as a photoredox catalyst. 1,4-Dicarbonyl compounds alone were afforded, without the generation of carbonyl adducts of the α-halocarbonyls, which are usually generated in the presence of fluoride anions or Lewis acids. A variety of silyl enol ethers, α-bromoketones, α-bromoesters, and α-bromoamides were applied to this system to produce the coupling compounds.

Biowaste material reinforced cyanate ester based epoxy composites for flame retardant applications

This article reports the development of halogen-free flame-retardant amine-terminated cyclophosphazene (ATCP) and functionalized rice husk ash (FRHA)-reinforced cyanate ester (CE; 2,2-bis(4-cyanatophenyl)propane)-based epoxy (EP) composite material through the ring formation of cyanurate and imino carbonyl adduct formation. The ATCP/FRHA/CE-EP composites were characterized using Fourier transform infrared spectroscopy, scanning electron microscope, X-ray diffraction analysis and contact angle measurements. A plausible fire-retardant mechanism has been hypothesized based on the analysis of decomposition temperature, char yield, heat release rate, total smoke production, smoke production rate and carbon monoxide production. These results suggest that the ATCP/FRHA/CE-EP composites can be used as hydrophobic, electrical-resistant and improved fire-retardant materials. Hence, ATCP/FRHA/CE-EP flame-retardant system paves new possibility of high-performance non-halogen flame-retardant polymeric materials for electronic and microelectronic applications.

Magnetic Hydrogels from Alkyne/Cobalt Carbonyl-Functionalized ABA Triblock Copolymers.

A series of alkyne-functionalized poly(4-(phenylethynyl)styrene)-block-poly(ethylene oxide)-block-poly(4-(phenylethynyl)styrene) (PPES-b-PEO-b-PPES) ABA triblock copolymers was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. PESn[Co2(CO)6]x-EO800-PESn[Co2(CO)6]x ABA triblock copolymer/cobalt adducts (10-67 wt % PEO) were subsequently prepared by reaction of the alkyne-functionalized PPES block with Co2(CO)8 and their phase behavior was studied by TEM. Heating triblock copolymer/cobalt carbonyl adducts at 120 °C led to cross-linking of the PPES/Co domains and the formation of magnetic cobalt nanoparticles within the PPES/Co domains. Magnetic hydrogels could be prepared by swelling the PEO domains of the cross-linked materials with water. Swelling tests, rheological studies and actuation tests demonstrated that the water capacity and modulus of the hydrogels were dependent upon the composition of the block copolymer precursors.

Phase Behavior of Alkyne-Functionalized Styrenic Block Copolymer/Cobalt Carbonyl Adducts and in Situ Formation of Magnetic Nanoparticles by Thermolysis

A series of polystyrene-block-poly(4-(phenylethynyl)styrene) (PS-b-PPES) diblock copolymers with a range of compositions were prepared by reversible addition–fragmentation chain transfer (RAFT) polymerization. Block copolymer/cobalt carbonyl adducts (PSx-PPESy[Co2(CO)6]n) were subsequently prepared by reaction of Co2(CO)8 with the alkyne groups of the PPES block. Phase behavior of the block copolymer/cobalt carbonyl adducts (PSx-PPESy[Co2(CO)6]n, 8% ≤ wt % PS ≤ 68%) was studied by small-angle X-ray scattering and transmission electron microscopy (TEM). As the composition of PSx-PPESy[Co2(CO)6]n copolymers was shifted from PS as the majority block to PPESy[Co2(CO)6]n as the majority block, the morphology was observed to shift from lamellar with larger PS domains to cylindrical with PS as the minority component and then to spherical with PS as the minority component. These observations have been used to map out a partial phase diagram for PSx-PPESy[Co2(CO)6]n diblock copolymers. Heating of PSx-PPESy[Co2(CO)...

Role of membrane cholesterol and lipid peroxidation in regulating the Na+/K+-ATPase activity in schizophrenia.

Background:Na+/K+-ATPase (NKA) activity is compromised in several neuropsychiatric disorders. Oxidative stress and membrane lipid composition play important roles in regulating NKA activity.Aims:The present study was undertaken to evaluate the effects of oxidative stress-induced membrane lipid damage and membrane cholesterol composition on NKA pump activity in schizophrenia.Settings and Design:It was a hospital-based, cross-sectional, observational study in 49 cases and 51 controls for 1 year.Materials and Methods:NKA pump activity in red blood cell membrane, serum levels of thiobarbituric acid reactive substances (TBARS), protein carbonyl (PC) adducts, and cholesterol were measured by standard spectrophotometric techniques in newly diagnosed schizophrenia patients by Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, Text Revision criteria. Membrane cholesterol was analyzed by chloroform and isopropanol extraction followed by measuring the cholesterol concentration by spectrophotometric technique.Statistical Analysis and Results:Mean values for NKA pump activity, membrane cholesterol level, and serum cholesterol levels were significantly lower in the case group (P < 0.001). The activity of NKA pump was found to be directly correlated to membrane cholesterol level rather than with the serum cholesterol values. Although the NKA pump activity showed inverse relationship with the serum values of TBARS and PC products both, on multiple linear regression analysis, it was found to be significantly positively dependent on the membrane cholesterol (β = 0.268, P = 0.01) and negatively dependent on the serum TBARS (β = −0.63, P < 0.001) levels only.Conclusion:Reduced membrane cholesterol and oxidative stress-induced damage to membrane lipids play crucial roles in decreasing the NKA activity in schizophrenia. Hence, for a better prognosis and treatment, measures are required to maintain optimum levels of cholesterol in neuronal tissues along with a proper control on oxidative stress.

Abstract 13651: Angiotensin-Converting Enzyme Inhibitors Preserve Limb Function and Decrease Myofibrosis and Oxidative Damage in the Calf Muscle of Patients With Peripheral Artery Disease

Introduction: A six-month treatment with the Angiotensin-Converting Enzyme Inhibitor (ACEI) Ramipril improved the walking ability of Peripheral Artery Disease (PAD) patients, but the mechanism remains unknown. ACEI have been shown to reduce fibrosis in heart and kidney and improve arterial compliance. We have identified a myopathy in calf muscles of PAD patients, characterized by fibrosis and increased TGF-β1 produced exclusively in smooth muscle cells (SMC) of microvessels. These events were linked to myofiber oxidative damage and reduced myofiber cross-sectional area. Hypothesis: The myopathy of PAD patients taking ACEI is improved compared to patients who are not taking ACEI and this improvement is associated with improved walking ability. Methods: Calf muscle biopsies, from PAD patients (Fontaine Stage II) taking ACEI (ACEI+) and those who were not (ACEI-), were fixed, embedded in paraffin, sectioned at 4 microns and slide mounted. Oxidative damage (carbonyl groups) (N=13 for ACEI- and 22 for ACEI+) and TGF-β1 (N=13 for ACEI- and 20 for ACEI+) were analyzed by quantitative fluorescence microscopy and expressed as grey scale units (gsu). Myofiber cross-sectional area was determined from fluorescence-labeled sarcolemma. Fibrosis was determined as collagen density (gsu) by multi-spectral analysis of slide specimens (N=13 for ACEI- and 20 for ACEI+) stained with Mason Trichrome. Limb function was assessed by the six-minute walking distance (N=28 for ACEI- and 20 for ACEI+). Results: Compared to ACEI- patients, ACEI+ patients had reduced TGF-β1 (4.25 gsu +/- 0.69 vs. 1.51 gsu +/- 0.23; p = 0.001) and decreased collagen density (2067 gsu +/- 88 vs. 1671 gsu +/- 51; p &lt; 0.001). Carbonyl adducts were reduced in myofibers of ACEI+ compared to ACEI- patients (1584 gsu +/- 55 vs. 1779 gsu +/- 58; p = 0.019) and myofiber cross sectional area was increased (4629 μm2 +/- 285 vs. 3816 μm2 +/- 267; p = 0.023). Six-minute walking distance was greater in ACEI+ compared to ACEI- patients (333 m +/- 20 vs. 266 m +/- 16; p = 0.011). Conclusions: Improved walking distance of PAD patients taking ACEI is associated with improved calf muscle myopathy, determined as reduced TGF-β1 production, collagen density, and myofiber oxidative damage, and increased myofiber cross-sectional area.

Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition

Non-enzymatic collagen cross-linking and carbonyl adduct deposition are features of Bruch's membrane aging in the eye, and disturbances in extracellular matrix turnover are considered to contribute to Bruch's membrane thickening. Because bisretinoid constituents of the lipofuscin of retinal pigment epithelial (RPE) cells are known to photodegrade to mixtures of aldehyde-bearing fragments and small dicarbonyls (glyoxal (GO) and methylglyoxal (MG)), we investigated RPE lipofuscin as a source of the reactive species that covalently modify protein side chains. Abca4(-/-) and Rdh8(-/-)/Abca4(-/-) mice that are models of accelerated bisretinoid formation were studied and pre-exposure of mice to 430 nm light enriched for dicarbonyl release by bisretinoid photodegradation. MG protein adducts were elevated in posterior eyecups of mutant mice, whereas carbonylation of an RPE-specific protein was observed in Abca4(-/-) but not in wild-type mice under the same conditions. Immunolabeling of cryostat-sectioned eyes harvested from Abca4(-/-) mice revealed that carbonyl adduct deposition in Bruch's membrane was accentuated. Cell-based assays corroborated these findings in mice. Moreover, the receptor for advanced glycation end products that recognizes MG and GO adducts and glyoxylase 1 that metabolizes MG and GO were up-regulated in Abca4(-/-) mice. Additionally, in acellular assays, peptides were cross-linked in the presence of A2E (adduct of two vitamin A aldehyde and ethanolamine) photodegradation products, and in a zymography assay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the matrix metalloproteinases MMP2 and MMP9. In conclusion, these mechanistic studies demonstrate a link between the photodegradation of RPE bisretinoid fluorophores and aging changes in underlying Bruch's membrane that can confer risk of age-related macular degeneration.

Virgin coconut oil maintains redox status and improves glycemic conditions in high fructose fed rats.

Virgin Coconut Oil (VCO), extracted from fresh coconut kernel possess similar fatty acid composition to that of Copra Oil (CO), a product of dried kernel. Although CO forms the predominant dietary constituent in south India, VCO is being promoted for healthy life due to its constituent antioxidant molecules. High fructose containing CO is an established model for insulin resistance and steatohepatitis in rodents. In this study, replacement of CO with VCO in high fructose diet markedly improved the glucose metabolism and dyslipidemia. The animals fed VCO diet had only 17% increase in blood glucose level compared to CO fed animals (46%). Increased level of GSH and antioxidant enzyme activities in VCO fed rats indicate improved hepatic redox status. Reduced lipid peroxidation and carbonyl adducts in VCO fed rats well corroborate with the histopathological findings that hepatic damage and steatosis were comparatively reduced than the CO fed animals. These results suggest that VCO could be an efficient nutraceutical in preventing the development of diet induced insulin resistance and associated complications possibly through its antioxidant efficacy.

ChemInform Abstract: Iron‐Facilitated Oxidative Radical Decarboxylative Cross‐Coupling Between α‐Oxocarboxylic Acids and Acrylic Acids: An Approach to α,β‐Unsaturated Carbonyls.

The first Fe-facilitated decarboxylative cross-coupling reaction between α-oxocarboxylic acids and acrylic acids in aqueous solution has been developed. This transformation is characterized by its wide substrate scope and good functional group compatibility utilizing inexpensive and easily accessible reagents, thus providing an efficient and expeditious approach to an important class of α,β-unsaturated carbonyls frequently found in bioactive compounds. The synthetic potential of the coupled products is also demonstrated in subsequent functionalization reactions. Preliminary mechanism studies suggest that a free radical pathway is involved in this process: the generation of an acyl radical from α-oxocarboxylic acid via the excision of carbon dioxide followed by the addition of an acyl radical to the α-position of the double bond in acrylic acid then delivers the α,β-unsaturated carbonyl adduct through the extrusion of another carbon dioxide.

The Pyridyl Functional Groups Guide the Formation of Pd Nanoparticles Inside A Porous Poly(4‐Vinyl‐Pyridine)

AbstractThe reactivity of palladium acetate inside a poly(4‐vinylpyridine‐co‐divinylbenzene) polymer is strongly influenced by the establishment of interaction between the Pd precursor and the pyridyl functional group in the polymer. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and simultaneous X‐ray absorption near edge structure (XANES) and small angle X‐ray scattering (SAXS) techniques have been applied to monitor the reactivity of palladium acetate in the presence of H2 and CO as a function of temperature. H2 reduces palladium acetate to Pd nanoparticles and acetic acid. The pyridyl groups in the polymer play a vital role both in stabilizing the formed acetic acid, thus allowing its detection by means of DRIFTS, and the final Pd nanoparticles, which are extremely small and mono‐dispersed. On the contrary, CO does not reduce palladium acetate. Rather, it forms Pd2+ carbonyl adducts, which favor the detachment of the acetate ligands and their thermal degradation. These adducts are well observable by means of SAXS because they cause an important local change of the electronic density.

Iron-Facilitated Oxidative Radical Decarboxylative Cross-Coupling between α-Oxocarboxylic Acids and Acrylic Acids: An Approach to α,β-Unsaturated Carbonyls

The first Fe-facilitated decarboxylative cross-coupling reaction between α-oxocarboxylic acids and acrylic acids in aqueous solution has been developed. This transformation is characterized by its wide substrate scope and good functional group compatibility utilizing inexpensive and easily accessible reagents, thus providing an efficient and expeditious approach to an important class of α,β-unsaturated carbonyls frequently found in bioactive compounds. The synthetic potential of the coupled products is also demonstrated in subsequent functionalization reactions. Preliminary mechanism studies suggest that a free radical pathway is involved in this process: the generation of an acyl radical from α-oxocarboxylic acid via the excision of carbon dioxide followed by the addition of an acyl radical to the α-position of the double bond in acrylic acid then delivers the α,β-unsaturated carbonyl adduct through the extrusion of another carbon dioxide.

Heterogeneity of peptide adducts with carbonylated lipid peroxidation products.

Highly reactive lipid peroxidation-derived carbonyls (oxoLPP) modify protein nucleophiles via Michael addition or Schiff base formation. Once formed, Michael adducts can be further stabilized via cyclic hemiacetals with or without loss of water. Depending on the mechanism of their formation, peptide-oxoLPP can carry aldehyde or keto groups and thus be a part of the total protein carbonylation level. If a carbonyl function is lost during consecutive reactions, the oxoLPP-peptide adducts will not be detected using the common carbonyl labeling protocols. Because of the differences in adduct stabilities, it is possible to address the heterogeneity of peptide/protein-oxoLPP adducts by careful evaluation of tandem mass spectra of modified peptides. Here, we used hydrophilic interaction liquid chromatography-tandem mass spectrometry analysis of lysine, cysteine and histidine containing model peptides co-incubated with oxidized 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine to characterize the collision-induced dissociation behavior of peptide-carbonyl adducts. Numerous modifications were detected based on the analysis of tandem mass spectra, including Schiff bases on lysine (two), Michael adducts on lysine (six), cysteine (eleven) and histidine (two), as well as 4-hydroxy-2-aldehydes derived dehydrated cyclic hemiacetals on cysteine (five) and histidine (one). Additionally, cysteine and histidine side chains were modified by lipid-bound aldehydes as Michael adducts and dehydrated hemiacetals. The tandem mass spectra revealed collision-induced dissociation characteristics specific for each class of oxoLPP-peptide adducts.

The synthesis of 2-halopentacenes and their charge transport properties

Three 2-halo-subsituted pentacene derivatives were prepared from stable and soluble carbonyl adduct precursors of high purity in high yields. These precursors were synthesized from the corresponding anthraquinones in overall yields about 30%. The crystals of 2-substituted pentacenes were grown by using a method of physical vapor transport. Organic field effect transistors made with these crystals displayed p-type charge carrier characteristics with the highest hole mobility of 5.0 cm2 V−1 s−1.

Chiral Transfer in the Reaction of Aminoallylic Stannanes with Carbonyls in Two Different Modes using Tin(II) and Indium(III) Halides for the Synthesis of Each Enantiomer

The reaction of homochiral aminoallylic stannanes with aldehydes gave carbonyl adducts of amino alcohols in the presence of either SnCl2 or InBr3. Both additives afforded the products in opposite absolute stereochemical configurations. The controlled chirality was ascribed to the different transmetalation pathways of SnCl2 and InBr3.

Quantitative analysis of exhaled carbonyl compounds distinguishes benign from malignant pulmonary disease

The analysis of exhaled breath is a promising noninvasive tool for the diagnosis of lung cancer, but its clinical relevance has yet to be established. We report the analysis of exhaled volatile carbonyl compounds for the identification of specific carbonyl cancer markers to differentiate benign pulmonary disease from early-stage lung cancer and to compare its diagnostic accuracy with positron emission tomography (PET) scans. Aminooxy-coated silicon microchips were used for the selective capture of exhaled carbonyls by an oximation reaction. Breath samples were collected then directed through the silicon chips by applying a vacuum. Carbonyl adducts were analyzed by Fourier transform mass spectrometry. Eighty-eight control subjects, 107 patients with lung cancer (64 stage 0, I, or II), 40 patients with benign pulmonary disease, and 7 patients with a solitary pulmonary metastasis participated. Analysis of cancer markers was performed blinded to the pathologic results. Four carbonyls were defined as cancer markers with significantly higher concentrations in patients with lung cancer. The number of increased cancer markers distinguished benign disease from both early and stage III and IV lung cancer. For early-stage disease, defining greater than 2 increased markers as diagnostic of lung cancer resulted in 83% sensitivity and 74% specificity. PET scans for this same cohort resulted in 90% sensitivity but only 39% specificity. Markers normalized for 3 of the 4 markers after resection of the lung cancer. Analysis of specific exhaled carbonyls can differentiate early lung cancer from benign pulmonary disease. Breath analysis was more specific than PET for a lung cancer diagnosis. Judicious use of these data may expedite the care of patients with lung cancer.

Synthesis, Structural Characterization, Reactivity, and Catalytic Properties of Copper(I) Complexes with a Series of Tetradentate Tripodal Tris(pyrazolylmethyl)amine Ligands

Novel tris(pyrazolylmethyl)amine ligands Tpa(Me3), Tpa*(,Br), and Tpa(Br3) have been synthesized and structurally characterized. The coordination chemistries of these three new tetradentate tripodal ligands and the already known Tpa and Tpa* have been explored using different copper(I) salts as starting materials. Cationic copper(I) complexes [Tpa(x)Cu]PF6 (1-4) have been isolated from the reaction of [Cu(NCMe)4]PF6 and 1 equiv of the ligand. Complexes 2 (Tpa(x) = Tpa*) and 3 (Tpa(x) = Tpa(Me3)) have been characterized by X-ray studies. The former is a 1D helical coordination polymer, and the latter is a tetranuclear helicate. In both structures, the Tpa(x) ligand adopts a μ(2):κ(2):κ(1)-coordination mode. However, in solution, all of the four complexes form fluxional species. When CuI is used as the copper(I) source, neutral compounds 5-8 have been obtained. Complexes 6-8 exhibit a 1:1 metal-to-ligand ratio, whereas 5 presents 2:1 stoichiometry. Its solid-state structure has been determined by X-ray diffraction, revealing its 3D polymeric nature. The polymer is composed by the assembly of [Tpa2Cu4I4] units, in which Cu4I4 presents a step-stair structure. The Tpa ligands bridge the Cu4I4 clusters, adopting also a μ(2):κ(2):κ(1)-coordination mode. As observed for the cationic derivatives, the NMR spectra of 5-8 show the equivalence of the three pyrazolyl arms of the ligands in these complexes. The reactivities of cationic copper(I) derivatives 1-4 with PPh3 and CO have been explored. In all cases, 1:1 adducts [Tpa(x)CuL]PF6 [L = PPh3 (9-11), CO (12-15)] have been isolated. The crystal structure of [Tpa*Cu(PPh3)]PF6 (9) has been obtained, showing that the coordination geometry around copper(I) is trigonal-pyramidal with the apical position occupied by the tertiary amine N atom. The Tpa* ligand binds the Cu center to three of its four N atoms, with one pyrazolyl arm remaining uncoordinated. In solution, the carbonyl adducts 13-15 exist as a mixture of two isomers; the four- and five-coordinate species can be distinguished by means of their IR νCO stretching bands. Finally, the catalytic activities of complexes 1-4 have been demonstrated in carbene- and nitrene-transfer reactions.

Protein Carbonylation and Heat Shock Proteins in Human Skeletal Muscle: Relationships to Age and Sarcopenia

Aging is associated with a gradual loss of muscle mass termed sarcopenia, which has significant impact on quality-of-life. Because oxidative stress is proposed to negatively impact upon musculoskeletal aging, we investigated links between human aging and markers of oxidative stress, and relationships to muscle mass and strength in young and old nonsarcopenic and sarcopenic adults. Sixteen young and 16 old males (further subdivided into “old” and “old sarcopenic”) were studied. The abundance of protein carbonyl adducts within skeletal muscle sarcoplasmic, myofibrillar, and mitochondrial protein subfractions from musculus vastus lateralis biopsies were determined using Oxyblot immunoblotting techniques. In addition, concentrations of recognized cytoprotective proteins (eg, heat shock proteins [HSP], αβ-crystallin) were also assayed. Aging was associated with increased mitochondrial (but not myofibrillar or sarcoplasmic) protein carbonyl adducts, independently of (stage-I) sarcopenia. Correlation analyses of all subjects revealed that mitochondrial protein carbonyl abundance negatively correlated with muscle strength ([1-repetition maximum], p = .02, r 2 = −.16), but not muscle mass (p = .13, r 2 = −.08). Abundance of cytoprotective proteins, including various HSPs (HSP 27 and 70), were unaffected by aging/sarcopenia. To conclude, these data reveal that mitochondrial protein carbonylation increases moderately with age, and that this increase may impact upon skeletal muscle function, but is not a hallmark of (stage-I) sarcopenia, per se.

Myofiber Type Sensitivity of Gastrocnemius Muscle to Oxidative Stress in Peripheral Arterial Disease Patients

Peripheral arterial disease (PAD) affects approximately 8.5 million Americans and is characterized by the formation of atherosclerotic plaques in the arteries limiting the blood supply in the legs. Previous studies have demonstrated that oxidative damage (carbonyl and 4-hydroxyl-2-nonenal adducts) in PAD muscle fibers is higher than control gastrocnemius and correlates with disease progression. In this study, we investigated the effect of oxidative damage in myofiber type (slow and fast twitch) of gastrocnemius muscle while the disease is progressing. Muscle biopsies were collected with a Bergstrom needle from PAD-claudication patients (PAD-C; n = 28), PAD-critical limb ischemia patients (PAD-CLI; n = 25), and control subjects (n = 25). Quantitative fluorescence microscopy was used to label the myofiber sarcolemmas, carbonyl adducts, and the slow/fast/hybrid twitch fibers. Differences between and within the groups of carbonyl adducts and myofiber cross-sectional area of each myofiber type were measured. A discriminant model was used to categorize muscle specimens on the basis of the disease progression. Carbonyl adducts in gastrocnemius fibers were higher in PAD-C and PAD-CLI patients when compared with control fibers for all myofiber types. Cross-sectional area of fast twitch and hybrid fibers was significantly decreased when compared with control fibers (P < .05). Fast twitch and hybrid fibers demonstrated a significant increase of oxidative damage (P < .01) when compared with slow twitch fibers for both the PAD-C and PAD-CLI groups. Fast twitch fibers frequency was decreased while the disease was progressing, while slow and hybrid twitch fibers frequency was increased. The discriminant model demonstrated that ankle-branchial index, oxidative damage, and cross-sectional area of fast twitch fibers can accurately identify controls (100% accuracy), PAD-C (78.6% accuracy), and PAD-CLI (84% accuracy) with an overall classification accuracy of 87.2%. Oxidative damage is myofiber-type-sensitive towards fast twitch and hybrid fibers. The discriminant model establishes that fast twitch fibers are more susceptible to degeneration in association with increased oxidative damage.