Albin Hermetter

Effect of hemodialysis on the antioxidative properties of serum.

In patients with chronic renal failure undergoing regular hemodialysis (HD), oxidative stress is involved in the development of dialysis-related pathologies. The aim of the study was to measure the effect of HD treatment on the general antioxidative status of serum with special consideration of the specific oxidizability of lipids and proteins. Indicators for the oxidative/antioxidative status of plasma were monitored at the beginning and at the end of a dialysis session on the arterial and venous side of the dialyzer. A decrease in the antioxidant status was accompanied by an increased oxidizability of proteins as well as lipids during HD treatment. During the first passage of the dialyzer, the lag time of lipid oxidation decreased from 114.0+/-19.8 to 81.5+/-18.9 min, the lag time of protein oxidation decreased from 105.0+/-24.6 to 72.9+/-21.3 min and the total antioxidative status decreased from 518+/-24 to 252+/-124 microM trolox equivalents. The carbonyl content of serum proteins was high in patients with end stage renal disease (ESRD) (3.9+/-1.1 vs. 0.9+/-0.1 nmol/mg in controls) but did not change significantly during dialysis procedure. Our data demonstrate that the susceptibility of serum lipids and proteins to oxidative modification is severely increased by HD treatment.

The alpha-amino group of l-arginine mediates its antioxidant effect

Antioxidant effects may constitute part of the possible antiatherogenic effects of the amino acid l‐arginine. These antioxidant properties were further characterized in a model of lipoprotein oxidation.

Powerful probes for glycosidases: novel, fluorescently tagged glycosidase inhibitors1

Amino-1,2,5-trideoxy-2,5-imino-D-mannitol was fluorescently tagged by reaction with dansyl chloride at N-1 or by attachment of a dansyl amide bearing spacer to this centre. Compounds obtained are highly potent inhibitors of beta-glucosidase exhibiting Ki values in the single figure nanomolar range. The 1-N-dansyl substituted inhibitor was successfully exploited for binding studies with beta-glucosidase from Agrobacterium sp. employing fluorescence spectrometric methods.

Single and multiple desipramine exposures of cultured cells: Changes in cellular anisotropy and in lipid composition of whole cells and of plasma membranes

Effects of the antidepressant drug desipramine (DMI) on fluorescence anisotropy were studied in living cultured human fibroblasts, rat brain astrocytes and rat ROC-1 hybridoma cells (oligodendrocytes x C6). Fluorescence anisotropy, a measure for fluidity, was measured by means of a fluorescence polarization technique using a set of n-(9-anthroyloxy) fatty acids as markers. Apparent fluorescence anisotropies were determined in cells following single or multiple dose exposures to 5 microM DMI at 37 degrees and compared to control cells. In all three cell types single doses of DMI led to significant decreases in anisotropies of the deeper layers (12-AS) of the membranes only, suggesting increases in fluidity. Repeated exposures to 5 microM DMI led to cell specific, significant changes in anisotropies of the superficial membrane layers, as determined by 2-AP, 6-, 7- and 9-AS. The resulting anisotropy values of the three different cell types became more alike than prior to DMI exposure. Alterations in anisotropies were accompanied with changes in the phospholipid patterns of whole cells and isolated plasma membrane vesicles. The changes of PC/PE ratios were consistent with changes observed in fluorescence anisotropies. Such alterations may be individual regulatory responses of the cells to the chronic presence of the drug within the membranes.

A NEW FLUORESCENCE METHOD FOR THE CONTINUOUS DETERMINATION OF SURFACE LIPID OXIDATION IN LIPOPROTEINS AND PLASMA

We report on a new method for the determination of lipid oxidation in lipoproteins and plasma. The biological lipid system is preloaded with a fluorescent analog of phosphatidylcholine containing diphenylhexatriene (DPH) propionic acid covalently linked to the sn-2 position. When externally added, the respective phospholipid label (DPHPC) localizes to the surface monolayer of a lipoprotein. Under oxidative conditions (e.g. in the presence of Cu2+ ions) the fluorophore undergoes decomposition, resulting in a continuous decrease of fluorescence intensity which reflects the oxidation of a chemically defined phospholipid molecule with well defined localization. When incorporated into LDL particles, the kinetics of the decrease in DPHPC fluorescence intensity upon exposure to Cu2+ us very similar to that of conjugated diene accumulation. Furthermore, our assay can be applied to follow the oxidation of lipids in diluted serum and may also be developed into a suitable test system for clinical studies of susceptibility of plasma lipids to oxidation.

Effects of culture and incubation conditions on membrane fluidity in monolayers of cultured cells measured as fluorescence anisotropy using trimethylammoniumdiphenylhexatriene (TMA-DPH)

Membrane fluidity of coverslip attached living cells was measured as fluorescence anisotropy using 5 microM trimethylammoniumdiphenylhexatriene (TMA-DPH) as fluorescent probe. Fluorescence anisotropy is inversely related to membrane fluidity. Cells were grown on glass coverslips that were inserted and directly incubated in quarz cuvettes. The coverslips were fixed with special holders at an angle of 30 degrees in respect to the incident light. Effects of incubation temperature, of cell growth and densities and of the ionic and nonionic composition of the incubation medium on membrane fluorescence anisotropy were measured. Membranes of growing cells were more fluid than those of stationary cells, while cell densities had no effect except at very low cell numbers. Calcium concentrations increasing from 0 to 8 mmol/l in the incubation medium proportionally decreased membrane fluidity. Hypotonicity of the incubation media increased membrane fluidity while hypertonicity compared to normotonicity had no effect. Differentiated human fibroblasts from different origins exhibited similar membrane fluidities. They were, however, different from those of rat cells. Membrane fluidity of rat brain tumor cells increased with age in culture while membrane fluidity of primary differentiating rat brain cells decreased in with age in culture. Measurement of fluorescence anisotropy in living cells attached to glass coverslips is a convenient tool to study effects of culture--as well as of environmental--conditions on membrane fluidity.

Reversible reduction of phospholipid bound arachidonic acid after low density lipoprotein apheresis. Evidence for rapid incorporation of plasmalogen phosphatidylethanolamine into the red blood cell membrane

In order to evaluate whether acute changes in fatty acids bound to phospholipids in plasma are transmitted into red blood cell membrane (RBCM) phospholipids, molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were analyzed after reduction of apo B containing lipoproteins through low density lipoprotein (LDL) apheresis in patients with severe hypercholesterolemia. As compared to the control, increases and decreases in molecular species with arachidonic acid (20:4) and with linoleic acid (18:2), respectively, at sn-2 of plasma diacyl-PC were seen in the patients before the apheresis. Directly after the procedure, the sum of species of plasma and RBCM PC plus PE with 20:4 were reduced. Two days after apheresis major species of plasma diacyl-PC reapproached preapheresis values while, in contrast, the composition of plasma alkenylacyl(plasmalogen)-PE was distinctly altered. In plasmalogen-PE of RBCM similar modifications were induced by the apheresis as in the same subgroup in plasma. In vitro experiments using vesicles with plasmalogen-PE labeled at sn-2 with either [14C]20:4 or a fluorescent pyrenedecanoyl residue indicated fast incorporation of the subgroup into the RBCM. In contrast, diacyl-PE was not taken up by the RBCM. In conclusion, apo B containing lipoproteins are partially responsible for the supply of phospholipids with arachidonic acid to RBCM, in particular by means of the fast incorporation of plasmalogen-PE. The transmission of changes induced by apheresis in plasma into those of the RBCM suggest that erythrocytes play an important role in the homeostasis of fatty acids bound to plasma phospholipids in vivo.

Dicyanmethylen-acenaphthenon-hydrazone — Polymethine mit dem Amino-dicyanallylidenimin-Chromophor

Acenaphthenequinone reacts with malononitrile and asymm. substituted hydrazines to deeply coloured 2-dicyanomethylene-1-acenaphthenone-hydrazones. They are classified as aza-cyanine type polymethine dyes with the amino-dicyanoallylidenimide chromophore.

Synthesis and intermembrane transfer of pyrene-labelled liponucleotides: ceramide phosphothymidines.

Phospholipid conjugates of 3-azido-3-deoxythymidine (AZT) show activity against human immunodeficiency virus (HIV) in vitro. Here we report on the synthesis and characterization of two pyrene containing conjugates: 2-N-(4-(pyren-1-yl)butanoyl)ceramide 5-phosphothymidine (Pbs-Cer-P-T) (XII) and 2-N-(10-(pyren-1-yl)decanoyl)ceramide 5-phosphothymidine (Pds-Cer-P-T) (XIII). These fluorescent labelled conjugates served as model compounds to study incorporation of sphingoliponucleotides into membranes. The complex compounds were prepared by condensation of 3-acetylthymidine and labelled ceramides using the phosphite triester coupling procedure. UV absorption, fluorimetry as well as 1H-, 31P-, 13C-NMR analyses were used for structure confirmation of the synthesized substances. When incorporated into small unilamellar 1-palmitoyl-2-oleoyl-glycerophosphatidyl-choline (POPC) vesicles and incubated with unlabelled acceptor POPC vesicles, the compounds (XII) and (XIII) exhibited spontaneous transfer. Kinetic data suggest that transfer from donor to acceptor vesicles occurred via the intervening aqueous phase. The non-specific lipid transfer protein from bovine liver stimulated the transfer of Pds-Cer-P-T between phospholipid vesicles in a concentration dependent manner.

Interaction of N-methyl-anthraniloyl-labelled porcine apolipoprotein A-I with porcine lecithin: Cholesterol acyltransferase: An energy transfer study

1. To investigate whether a direct protein-protein interaction between apoA-I and lecithin:cholesterol acyltransferase (LCAT) is necessary for the activation of the enzyme, apoA-I was labelled with N-methylisatoic anhydride at lysine residues. The intermolecular resonance energy transfer from tryptophan residues of LCAT (donor) to N-methyl-anthraniloyl (NMA)-labelled apoA-I (NMA-apoA-I) (acceptor) was used as a sensitive fluorescence method for studying molecular interactions. 2. In the absence of lipids no fluorescence energy transfer was measurable. 3. Fluorescence energy transfer occurred from LCAT to NMA-apoA-I in the presence of liposomes with phospholipid/cholesterol ratios ranging from 5:1 to 18:1 and regardless whether only 1 or up to 5 NMA-apoA-I molecules resided at the liposome surface. 4. This indicates a preferred binding of the enzyme directly to or in spatial proximity to the activator protein NMA-apoA-I even if enough space at the liposome surface is available to allow LCAT binding at a distance, where no energy transfer is measurable.