Guenther Gercken

Lipid, sterol and fatty acid composition of antarctic krill (Euphausia superba Dana)

The lipid classes, fatty acids of total and individual lipids and sterols of Antarctic krill (Euphausia superba Dana) from two areas of the Antarctic Ocean were analyzed by thin layer chromatography (TLC), gas liquid chromatography (GLC) and gas liquid chromatography/mass spectrometry (GLC/MS). Basic differences in the lipid composition of krill from the Scotia Sea (caught in Dec. 1977) and krill from the Gerlache Strait (caught in Mar. 1981) were not observed. The main lipid classes found were: phosphatidylcholine (PC) (33–36%), phosphatidylethanolamine (PE) (5–6%), triacylglycerol (TG) (33–40%), free fatty acids (FFA) (8–16%) and sterols (1.4–1.7%). Wax esters and sterol esters were present only in traces. More than 50 fatty acids could be identified using GLC/MS, the major ones being 14∶0, 16∶0, 16∶1(n−7), 18∶1(n−9), 18∶1(n−7), 20∶5(n−3) and 22∶6(n−3). Phytanic acid was found in a concentration of 3% of total fatty acids. Short, medium-chain and hydroxy fatty acids (C≤10) were not detectable. The sterol fraction consisted of cholesterol, desmosterol and 22-dehydrocholesterol.

Toxicity of metallic ions and oxides to rabbit alveolar macrophages

The effects of soluble compounds and oxides of As, Cd, Cu, Hg, Ni, Pb, Sb, Sn, V, and Zn on oxidative metabolism and membrane integrity of rabbit alveolar macrophages were studied by 24-hr in vitro exposure. Oxidative metabolism induced by phagocytosis of opsonized zymosan was measured by H2O2 and O2- release and by chemiluminescence in the presence of luminol. Membrane integrity was estimated by extracellular LDH activity. Metallic ions and oxides inhibited the release of active oxygen species. Cd(II), As(III), and V(V) were the most toxic elements as measured by all investigated parameters. Cu(II) decreased O2- release and chemiluminescence effectively but H2O2 release and membrane integrity less. Chemiluminescence was decreased strongly by Hg(II) while O2- and H2O2 release were depressed moderately. Zn(II) and Sb(III) compounds caused medium toxicity and the tested Sn, Ni, and Pb compounds showed only faint toxic effects.

Depression of alveolar macrophage hydrogen peroxide and superoxide anion release by mineral dusts: Correlation with antimony, lead, and arsenic contents

Activated rabbit alveolar macrophages were incubated with airborne dusts from four West German sites (1 to 200 micrograms/10(6) cells) and waste incinerator fly ash fractions (50 to 500 micrograms/10(6) cells). Quartz dust DQ 12 (5 to 200 micrograms/10(6) cells) and Fe2O3 (0.05 to 50 micrograms/10(6) cells) were used as control dusts. The zymosan-stimulated hydrogen peroxide and superoxide anion release of the macrophages were not affected significantly by Fe2O3. All other investigated dusts decreased the two cell functions which were correlated negatively with surfaces, particle numbers, and antimony, lead, and arsenic contents of the dusts. The influence of heavy metal antagonisms and dust surfaces on dust toxicity against alveolar macrophages is discussed.

Cytotoxicity of dust constituents towards alveolar macrophages: interactions of heavy metal compounds.

The interactions between different heavy metal compounds which affect their cytotoxicity towards rabbit alveolar macrophages were investigated. The cells were exposed in vitro to combinations of As3+, Cd2+, Hg2+, Ni2+, or V5+ with different concentrations of another heavy metal compound. Toxicity was determined as the depression of zymosan-induced release of superoxide anion radicals. Significant antagonisms occurred in the combinations Cd2+/Zn2+, Hg2+/As3+, and Hg2+/Se4+, while significant synergisms were exhibited by the combinations Cd2+/Cu2+, Cd2+/Sn2+, Hg2+/Cu2+, Ni2+/Cd2+, Ni2+/Cu2+, Ni2+/Sn2+ and V5+/Cu2+. In the combinations As3+/Zn2+, Hg2+/Cd2+ and Hg2+/Zn2+, both kinds of interactions were observed depending on the concentrations of the heavy metal compounds. An interpretation of the measured heavy metal interactions with reference to the toxicity of heavy metal-containing dusts is attempted.

Cytotoxicity to alveolar macrophages of airborne particles and waste incinerator fly-ash fractions

A waste incinerator fly ash was separated into different grain-size fractions by sieving and sedimentation in butanol. The element content of each fraction was determined by atomic absorption and emission spectrometry. The fly-ash fractions, an eluted fine fly-ash fraction and an eluted airborne dust were analysed microscopically for particle size and numbers, together with standard quartz DQ 12 and three element-analysed airborne dusts. Rabbit alveolar macrophages, isolated by lung lavage, were incubated for 24 h with the particulates, the two eluates and a mixed element compound solution corresponding to the element concentrations of one airborne dust. At the end of incubation, the activities of lactate dehydrogenase, N-acetyl-beta-glucosaminidase, beta-galactosidase and acid phosphatase were determined in medium and cell lysates. Cytotoxicity was expressed as ratio of extracellular to total LDH (lactate dehydrogenase) activity. Release of N-acetyl-beta-glucosaminidase and beta-galactosidase was correlated positively with LDH release, whereas the total activity of acid phosphatase decreased with increasing LDH release. Cytotoxicity of the dusts was correlated with particle numbers, and As, Sb and Pb contents. The contribution of As to particle toxicity is discussed. Eluates of dusts did not affect rabbit alveolar macrophage viability.

Incorporation of [1-14C]octadecanol into the lipids ofLeishmania donovani

After incubation of stationary phaseLeishmania donovani with [1-14C] octadecanol, about 70% of the precursor was taken up within 3 hr. Wax esters and acyl moieties of glycerolipids contained most of the14C-activity from 3 to 6 hr, because octadecanol was partly oxidized to stearate. Ether moieties were only weakly labeled. After 40 hr, 1-0-aklyl and 1-0-alk-1′-enyl diacylglycerols as well as 1-0-alkyl and 1-0-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamines contained nearly all of the radioactivity. Most of the label in the neutral ether lipids was located in the alkyl ether side chain, whereas, in the phosphatidylethanolamine fraction, most of the label was found in the alkenyl ether side chain. Administration of 1-0-[1-14C] hexadecyl glycerol resulted in rapid labeling of the vinyl ether side chain of phosphatidylethanolamine plasmalogen (1 hr) increasing further at 2.5 hr. Most of the radioactivity in the alkoxy diacylglycerols was found in the 1-0-alkyl moiety.

Measurement of phospholipase A2 and 1-alkylglycerophosphocholine acetyltransferase activities in stimulated alveolar macrophages by HPLC analysis of NBD-labeled ether lipids

The importance of phospholipases in cellular signaling and 1-alkylglycerophosphocholine acetyltransferase in the formation of platelet-activating factor (PAF) has stimulated demand for methods to measure these enzyme activities in inflammatory cells. Most of the assays currently used rely on radiolabeled substrates. We have synthesized NBD-labeled ether lipids as substrates for measuring enzyme activities of the PAF cycle and of lysosomal phospholipase A2 (PLA2). The fluorescent lipids were incubated with homogenates of stimulated bovine alveolar macrophages. The generated products were separated from the substrates by HPLC on a normal phase and monitored with a fluorescence detector. NBD-lyso-PAF was well accepted by acetyl- and acyltransferases of the cell-free preparations, which metabolized the substrate into NBD-PAF and NBD-alkyl-acylglycerophosphocholines. Homogenates of stimulated cells showed an enhanced production of NBD-PAF. The increased formation of the biological mediator was dependent on the nature of the stimuli and the time of stimulation. Lysosomal PLA2 was measured with 1-O-(12-NBD-aminododecyl)-2-acyl-sn-glycero-3-phosphocholine as substrate. By varying the pH and the calcium concentration, it was possible to distinguish between the cytosolic PLA2 and the lysosomal PLA2 activity. Optimal conditions for the determination of the lysosomal PLA2 were obtained at pH 4.5 and in the presence of EDTA. Stimulation with particulate agonists induced an enhancement of the lysosomal PLA2 activity in macrophages.

Carbamoylcholine-induced accumulation of inositol mono-, bis-, tris- and tetrakisphosphates in isolated cardiac myocytes from adult rats

The effect of carbamoylcholine on the phosphoinositide cycle in isolated ventricular myocytes from adult rats was studied. Separation of the phosphoinositides by high-performance thin-layer chromatography showed a constant ratio of incorporation of myo-[2-3H]inositol into phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate of cultured cardiac myocytes after at least 2 h. Carbamoylcholine caused a dose-dependent and time-dependent accumulation of inositol mono-, bis- and trisphosphates, which was antagonized by atropine. Using anion-exchange HPLC the existence of inositol 1,4,5-trisphosphate, inositol 1,3,4-triphosphate and inositol 1,3,4,5-tetrakisphosphate was confirmed in rat ventricular myocytes. Inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate accumulated within 20 s, while inositol 1,3,4-trisphosphate, inositol 1,4-bisphosphate and inositol monophosphate increased within 5 min.

ZYMOSAN-INDUCED CHEMILUMINESCENCE OF ALVEOLAR MACROPHAGES : DEPRESSION BY INORGANIC DUST CONSTITUENTS

A method has been developed which allows the incubation of alveolar macrophages as weakly affixed monolayers in siliconized glass dishes. Without vigorous mechanical agitation and without using proteolytic enzymes, these cells were subsequently transferred to cuvettes where the zymosan-stimulated chemiluminescence of the suspended cells was measured.In vitro incubations of activated rabbit alveolar macrophage monolayers with airborne dusts from four West German sites (1 to 200 μg/106 cells), fly ash fractions of a special waste incinerator at Hamburg (50 to 1,000 μg/106 cells), and quartz dust DQ 12 (5 to 200 μg/106 cells) resulted in a dose-dependent depression of the zymosan-stimulated chemiluminescence. The depression of chemiluminescence was correlated with particle numbers, estimated dust surface, and antimony and lead masses of the dusts to which the cells were exposed. Cytotoxicity was better correlated with these parameters than with dust mass.

Effects of quartz, airborne particulates and fly ash fractions from a waste incinerator on elastase release by activated and nonactivated rabbit alveolar macrophages.

Elastase release from cultured, activated and nonactivated rabbit alveolar macrophages (AM) was investigated after stimulation by different environmentally related mineral dusts (50-1000 micrograms/10(6) cells). Eight different dusts were analyzed for element contents and grain size: one rural and three urban airborne dusts, a coarse and a fine fraction of a sieved waste incinerator fly ash, a sonicated coarse fly ash fraction, and the standard quartz dust DQ 12. The fine fly ash fraction, the sonicated coarse fly ash fraction, and the quartz dust DQ 12 enhanced elastase release by activated AM. Only one of the tested airborne dusts effected a comparable elastase release. The untreated coarse fraction of the fly ash did not cause a significant increase of extracellular elastase activities. Elastase release was dependent on particle numbers and chemical composition and correlated best with barium and tin contents. Nonactivated AM released higher elastase activities than activated AM at low-dose levels. The possible role of dust-induced elastase secretion in the pathogenesis of emphysema is discussed.