Hans-Joachim Freisleben

[44] Antioxidant radical-scavenging activity of carotenoids and retinoids compared to α-tocopherol

Publisher Summary Free radicals are potentially dangerous for the cell, although in vivo they may be generated by metabolism. These radicals can be classified into two groups: (1) oxygen-derived radicals and active oxygen species, including superoxide anion radical, hydroxyl radical, hydrogen peroxide, and singlet molecular oxygen and (2) organic radicals that usually originate in the course of lipid peroxidation. According to the location in which they are generated, oxygen-derived radicals react mainly with components in the aqueous phases, such as extra- and intracellular fluids, while organic radicals interact mainly with constituents of the hydrophobic membranous phase. This chapter investigates the antioxidant activity of carotenoids and retinoids compared to α -tocopherol and its homologs. For the assay in the aqueous phase, a modified method of a phycoerythrin fluorescence-based procedure is used, whereas for the assay in the hydrophobic phase, a new cis-parinaric acid fluorescence-based assay is developed.

Black lipid membranes of tetraether lipids from Thermoplasma acidophilum

Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. Measurements of the specific membrane capacitance (Cm = 0.744 microF/cm2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 nm no matter from which solvent. This finding corresponds to the results of Gliozzi et al. for the lipids of another archaebacterium, Sulfolobus solfataricus. Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraether-lipid itself at room temperature and at defined concentration. In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin. At 10(-7) M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200-1200 microS/cm2) than from bilayer-forming lipids (125-480 microS/cm2). Nonactin, at 10(-6) M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 x 10(-11) M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness. Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments. Poly(L-lysine) destroyed black lipid membranes. Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves. The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes. This influence was stronger than that of the polysaccharide dextran.

Lipoic acid reduces ischemia-reperfusion injury in animal models

Hypoxia and reoxygenation were studied in rat hearts and ischemia and reperfusion in rat hindlimbs. Free radicals are known to be generated through these events and to propagate complications. In order to reduce hypoxic/ischemic and especially reoxygenation/reperfusion injury the (re)perfusion conditions were ameliorated including the treatment with antioxidants (lipoate or dihydrolipoate). In isolated working rat hearts cardiac and mitochondrial parameters are impaired during hypoxia and partially recover in reoxygenation. Dihydrolipoate, if added into the perfusion buffer at 0.3 microM concentration, keeps the pH higher (7. 15) during hypoxia as compared to controls (6.98). The compound accelerates the recovery of the aortic flow and stabilizes it during reoxygenation. With dihydrolipoate, ATPase activity is reduced, ATP synthesis is increased and phosphocreatine contents are higher than in controls. Creatine kinase activity is maintained during reoxygenation in the dihydrolipoate series. Isolated rat hindlimbs were stored for 4 h in a moist chamber at 18 degrees C. Controls were perfused for 30 min with a modified Krebs-Henseleit buffer at 60 mmHg followed by 30 min Krebs-Henseleit perfusion at 100 mmHg. The dihydrolipoate group contained 8.3 microM in the modified reperfusate (controlled reperfusion). With dihydrolipoate, recovery of the contractile function was 49% (vs. 34% in controls) and muscle flexibility was maintained whereas it decreased by 15% in the controls. Release of creatine kinase was significantly lower with dihydrolipoate treatment. Dihydrolipoate effectively reduces reoxygenation injury in isolated working rat hearts. Controlled reperfusion, including lipoate, prevents reperfusion syndrome after extended ischemia in exarticulated rat hindlimbs and in an in vivo pig hindlimbs model.

Ursodeoxycholate stabilizes phospholipid-rich membranes and mimics the effect of cholesterol: investigations on large unilamellar vesicles

Ursodeoxycholate is used to treat primary biliary cirrhosis and is incorporated into hepatocyte plasma membranes. Its steroid nucleus binds to the apolar domain of the membrane, in a similar position to cholesterol. Therefore the question arises whether ursodeoxycholate has a similar effect on membrane structure and stability as cholesterol. Using differential scanning calorimetry the thermotropic behavior of egg phosphatidylcholine and dimyristoylphosphatidylcholine were studied after incubation with cholesterol or ursodeoxycholate. Large unilamellar vesicles were prepared with cholesterol contents of 0-50%. Following incubation of these vesicles with different amounts of ursodeoxycholate, vesicle stability in a gravitational field was investigated by measuring the phospholipid and cholesterol release. Vesicle size was studied by laser light scattering after incubation with cheno- and ursodeoxycholate, and the release of entrapped carboxyfluorescein was measured by means of fluorescence spectroscopy. Increasing cholesterol diminished the enthalpy of the phase transition in the membrane. Ursodeoxycholate decreased the enthalpy of the phase transition at even lower concentrations. Lipid release from vesicles in a high gravitational field diminished with increasing cholesterol content of the vesicles. Ursodeoxycholate had a comparable effect, which increased as the cholesterol content of the vesicles was decreased. Chenodeoxycholate damaged vesicles, whereas ursodeoxycholate did not. Cholesterol and ursodeoxycholate (below its critical micellar concentration) decreased the carboxyfluorescein release from vesicles induced by chenodeoxycholate. Thus like cholesterol, ursodeoxycholate is incorporated into phospholipid model membranes and reduces the change in enthalpy of the gel to liquid-crystalline phase transition. Like cholesterol ursodeoxycholate also maintains membrane stability and prevents membrane damage induced by mechanical and chemical stress.

Reconstitution of bacteriorhodopsin and ATP synthase from Micrococcus luteus into liposomes of the purified main tetraether lipid from Thermoplasma acidophilum : proton conductance and light-driven ATP synthesis

The archaebacterium Thermoplasma acidophilum is cultivated at 59 degrees C in a medium containing sulfuric acid of pH 2. The purified bipolar membrane spanning main phospholipid (MPL) of this organism can be used to produce stable liposomes of 100-500 nm in diameter either using a French pressure cell detergent dialysis or sonication. Despite a potassium diffusion potential of 186 mV very low ionic permeability of sonicated MPL liposomes was measured using the potassium binding fluorescent indicator benzofuran isophthalate PBF1, which measures net K+ uptake. The latter also remained very low, in the presence of the K(+) ionophore valinomycin and palmitic acid. Addition of valinomycin and the potent uncoupler carbonylcyanid-p-trifluormehoxyphenyl-hydrazone (FCCP), led to a stimulation in potassium uptake. The rate of proton flux can be calculated from the net K(+) uptake. Under these conditions MPL liposomes are 1-2 orders of magnitude less permeable than egg yolk lecithin vesicles. The difference in proton permeability becomes even more pronounced with increasing temperature, examined using the fluorescent pH indicator pyranine. Purified bacteriorhodopsin from Halobacterium halobium was reconstituted into MPL liposomes in order to study the light-driven proton uptake in 150 mM KCl following addition of valinomycin, gramicidin, FCCP and Triton X-100. The light-driven proton transport into the liposomes was increased 30-fold by addition of valinomycin decreased by gramicidin and FCCP, and abolished by Triton X-100. Co-reconstituted MPL proteoliposomes containing bacteriorhodopsin and ATP synthase from Micrococcus luteus were capable of light-driven ATP synthesis demonstrating the functional coupling of proton transport and nucleotide generation in liposomal MPL membranes.

ANTIOXIDANT ACTIVITY OF ALPHA -TOCOPHEROL, BETA -CAROTENE, AND UBIQUINOL IN MEMBRANES : CIS-PARINARIC ACID-INCORPORATED LIPOSOMES

Publisher Summary Owing to their high reactivities, oxygen free radicals generated by various biological and chemical processes in vivo , are potentially dangerous to living cells. These radicals can induce oxidative destruction of the polyunsaturated fatty acyl chains of membrane lipids by the processes known as “lipid peroxidation.” The resultant loss of membrane integrity and function is implicated in pathological conditions, such as inflammation, diabetes, reperfusion injury, radiation damage, cancer, aging, and neurological diseases. However, in healthy living cells, oxidative processes can be ingeniously intercepted by a network of interacting antioxidants. This chapter discusses the antioxidant activity of α-tocopherol, β-carotene, and ubiquinol in membranes. α-Tocopherol, a lipid-soluble membrane constituent is an essential factor in the cellular antioxidant defense system. By donating a hydrogen atom, it functions as an efficient chain-breaking antioxidant that blocks lipid peroxidation. Other biological compounds, including carotenoids and ubiquinones, also play a role in the protection of biological membranes against oxygen free radicals.

Lifespan of immunosuppressed NMRI-mice is increased by deprenyl

Immunosuppressed NMRI-mice (nu/nu) were raised and kept under germ-reduced conditions and fed with a germ-reduced diet (14 animals = controls). For another 14 mice 4 mg of selegiline were admixed to 10 kg of the diet. The 50% survival rate of the latter group was 160% from birth or 220% from the beginning of the study. The survival rate in weeks finally reached 350%, and the area under the curve 250%. The last mouse in the control group died at the age of 5 months, 2.5 months after the study was started; the last mouse in the selegiline group died at the age of 14.5 months, 1 year after the beginning of the study.

Toxicity and Biodistribution of Liposomes of the Main Phospholipid from the Archaebacterium Thermoplasma Acidophilum in Mice

AbstractToxicity and biodistribution of negatively charged liposomes of the main phospholipid (MPL) from the archaebacterium Thermoplasma acidophilum were tested in mice. MPL liposomes with a diameter of 160–220 nm were prepared by extrusion through polycarbonate filters, or by means of a French pressure cell and screened for central nervous system effects after intraperitoneal (i.p.) injection of 4–324 mg of liposomes per kg body weight in NMRI-mice. Besides increased behavioural activity no pharmacological or toxic effects were detected. No alterations were seen in the morphology of the tissues analyzed. Longterm toxicity after life-long oral application of 30 mg MPL per kg body weight per day starting at the age of 10 weeks was tested in immunosuppressed NMRI-mice. Again, there were no toxic effects on survival. Biodistribution of MPL liposomes labeled with 111In-diethylenetriaminepentaacetic acid stearylamide was examined 15 min and 2.5 h after intravenous injection into ICR-mice. The liposomes were r...

Fatty acid binding site of the mitochondrial uncoupling protein: Demonstration of its existence by EPR spectroscopy of 5-DOXYL-stearic acid

Fatty acid binding site on isolated mitochondrial uncoupling protein (UcP) is demonstrated using EPR spectroscopy of 5-DOXYL-stearic acid (5-SASL), which also activated H+ transport in proteoliposomes containing UcP. In the presence of UcP the EPR spectrum showed reproducible broadening of the low field peak as well as an increase in h+1I/h+1M ratio, rotational correlation time and in order parameter. The half-height width of the low field peak was even doubled in the presence of another UcP ligand, GDP. Palmitic acid reversed the effect of 5-SASL and non-ionizable 5-DOXYL-decane did not exhibit it.Fatty acid binding site on isolated mitochondrial uncoupling protein (UcP) is demonstrated using EPR spectroscopy of 5‐DOXYL‐stearic acid (5‐SASL), which also activated H+ transport in proteoliposomes containing UcP. In the presence of UcP the EPR spectrum showed reproducible broadening of the low field peak as well as an increase in h +1I/h +1M ratio, rotational correlation time and in order parameter. The half‐height width of the low field peak was even doubled in the presence of another UcP ligand, GDP. Palmitic acid reversed the effect of 5‐SASL and non‐ionizable 5‐DOXYL‐decane did not exhibit it.

The effects of glucose, insulin and metformin on the order parameters of isolated red cell membranes: An electron paramagnetic resonance spectroscopic study

Human red blood cell (RBC) membranes (RBC ghosts) were treated with glucose, insulin and metformin. The order parameters of RBC membranes were determined by 5- and 16-doxyl-stearic acid spin labels. Metabolic effects were excluded using an isolated system of RBC membranes. The membranes were incubated with glucose in physiological (5 mM), renal threshold (10 mM) and manifested diabetic (20 mM) concentrations for limited times. High concentrations of glucose (10, 20, 100 mM) increase the order parameters of RBC membranes significantly. Insulin by itself has a similar effect which is, however, not strictly concentration-dependent. By contrast, metformin at therapeutic concentrations (0.5 and 5.0 microM) decreases the order parameters. At 50 microM concentration the metformin effect is expressed less and recurs at 100 microM concentration. The effects are significant with 5-doxyl-stearic acid, but are not significant with the 16-doxyl derivative. When RBC membranes are co-incubated with 20 mM glucose and metformin at 0.5 and 5.0 microM concentrations the order parameters as determined by 5-doxyl-stearic acid remain normal (= control values). Higher concentrations of metformin (50 and 100 microM) cause an overshoot to very low order parameters. Insulin at 10, 100 and 200 mU/L does not influence significantly the effects of metformin. Addition of physiological amounts of bovine serum albumin does not abolish the effects of metformin. Metformin, at therapeutic concentrations (0.5 and 5.0 microM), maintains the normal fluidity at the polar interface of isolated RBC membranes by counterbalancing non-enzymatic glycosylation with 20 mM glucose in vitro.