Biomedical subjects
Publications and source records attributed to R Wiesner.
The oxygenated lipids of human aortas with atherosclerotic lesions were analysed by RP-, SP- and Chiral Phase-HPLC and compared with those obtained after treatment of human LDL with pure 15-lipoxygenase (LOX) of rabbit reticulocytes. The data suggest an indirect role of 15-LOX in the atherogenesis in aortas.
Patients with hepatic cirrhosis develop widespread abnormalities in kidney function and vasoactive hormones. These change rapidly after liver transplantation during immunosuppression with cyclosporine. The role of changing eicosanoid excretion and endothelin levels in regulating renal function after transplantation in humans remains uncertain. We studied 32 patients with regard to renal hemodynamics, glomerular filtration, urinary prostacyclin (6-keto-PG-F1-alpha), thromboxane (TBX2), and endothelin before and during the first four weeks after orthotopic liver transplantation. Arterial pressure rose from 106 +/- 2/61 +/- 2 to 146 +/- 4/81 +/- 2 mmHg, (P less than .001), while renal blood flow fell (686 +/- 38 to 453 +/- 24 ml/min/1.73 m2, P less than .05), as did GFR. Pretransplant excretion of 6-keto and TBX2 was above that of normal subjects and fell progressively after transplant, as did plasma renin activity and aldosterone. The 6-keto levels fell below normal after two weeks. The ratio of TBX2/6-keto remained elevated compared with normal subjects throughout the month after transplant (1.54 +/- 0.38 vs. 0.54 +/- 0.07, P less than .01). Endothelin levels rose during the first week (7.4 +/- 1.4 vs. 12.4 +/- 2.7 pg/ml, P less than .05), but fell back to baseline thereafter. These results indicate that high levels of urinary eicosanoids in patients with liver disease fall rapidly after liver transplantation during CsA immunosuppression. Unlike results in many experimental models, these data suggest that renal vasoconstriction in humans may be associated primarily with suppression in renal prostacyclin excretion rather than stimulation of thromboxane.
Oxidative modification of low density lipoproteins and tissue lipids has been proposed to be involved in the pathogenesis of atherosclerosis. We examined human atherosclerotic lesions of various stages from fifteen victims of acute heart failure and detected substantial amounts of oxygenated fatty acids in the tissue ester lipids. The degree of lipid oxygenation correlated with the stage of advancement of the lesion. More than 85% of the oxygenated fatty acids were localized in the cholesterol esters, whereas phospholipids contained only small amounts. Structure elucidation of the oxygenation products indicated a nonspecific product pattern of various isomers of keto- and hydroxy-octadecadienoic acid. The data presented suggest an involvement of lipid peroxidation in the pathogenesis of atherosclerosis and indicate that the majority of the oxygenation products are formed via nonspecific, non-enzymatic reactions possibly initiated by the action of a 15-lipoxygenase.
The arachidonate 15-lipoxygenase from rabbit reticulocytes oxygenates cholesterol esters containing polyenoic fatty acids. Cholesterol esterified with saturated fatty acids is not oxygenated. The structures of the oxygenation products formed from various cholesterol esters have been identified by high pressure liquid chromatography, UV-spectroscopy and gas chromatography/mass spectroscopy. Oxygenated cholesterol esters have been detected in atherosclerotic plaques of human aortas.
We report the first two cases of apparent azathioprine hepatotoxicity occurring after liver transplantation. The two patients exhibited jaundice, elevated serum transaminase activities and histopathological features of sinusoidal congestion and centrilobular hepatocellular degeneration 17 and 61 days after transplantation. After withdrawal of azathioprine, liver test results improved immediately in both patients. Recurrence of liver injury after another challenge with azathioprine was seen in the first case. Previously, fatal venoocclusive disease occurring after kidney transplantation had been attributed to azathioprine. Irreversible venoocclusive disease and the reversible hepatotoxicity described in this report (i.e., sinusoidal congestion with centrizonal necrosis) most likely represent different stages of hepatic endotheliitis caused by azathioprine. Thus early diagnosis of azathioprine hepatotoxicity is of great importance.
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A pure lipoxygenase from dried green pea seeds (isoenzyme 1) oxygenates linoleic acid to 9(S/R)-hydroperoxy-10E,12Z-octadecadienoic acid (9-HPODE) and 13(S/R)-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPODE). Furthermore (10E,12Z)-9-keto-10,12-octadecadienoic acid (9-KODE) and (9Z,11E)-13-keto-9,11-octadecadienoic acid (13-KODE) in a ratio of 1:1 were formed. Uv-spectroscopic measurements and HPLC data indicated a hydroperoxy fatty acid: keto fatty acid ratio of about 2:1. The product mixture formed from arachidonic acid was even more complex. 15-, 11-, 9- and 5-H(P)ETE1 and their corresponding keto derivatives have been detected. The chemical structures of the compounds have been identified by HPLC analysis, by uv- and ir-spectroscopy and gas chromatography/mass spectrometry of the native compounds and their hydrogenated derivatives. The data presented indicate that a pure lipoxygenase catalyzes the formation of both hydroperoxypolyenoic fatty acids and ketopolyenoic fatty acids from linoleic acid and arachidonic acid. The possible mechanism of the formation of the keto compounds is discussed.
Lipoxygenase was purified from wheat kernels by means of ammonium sulfate precipitation, gel chromatography on Sephadex G-200 and anion exchange chromatography on DEAE-Sephadex A-50. Arachidonic acid was mainly converted by the wheat lipoxygenase to 5D-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5D8-HPETE) with other HPETE isomers including 8-HPETE being minor products. At higher concentrations of lipoxygenase, multiple oxygenation products such as 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) and, to a lower extent, 8,15-diHETE and lipoxin isomers were detected after reduction of the hydroperoxy derivatives primarily formed. Similar results were obtained with 5D8- or 15L8-hydroxyeicosatetraenoic acid as substrate. Moreover, evidence was obtained for leukotriene A4 synthase activity of the wheat lipoxygenase.
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We find that the reticulocyte lipoxygenase can oxygenate rat liver mitochondrial membranes, beef heart submitochondrial particles, rat liver endoplasmic membranes, and erythrocyte plasma membranes (inside-out and right side-out ghosts) without prior action of a phospholipase. After alkaline hydrolysis of the ester lipids, the main products were identified as 15S-hydro(pero)xy-5Z,8Z,11Z,13E-eicosatetr aenoic acid, 17S-hydro(pero)xy-4Z,7Z,10Z,13Z,15E, 19Z,-docosahexaenoic acid, 13S-hydro(pero)xy-9Z,11E-octadecadienoic acid, 9(S/R)-hydro(pero)xy-10E,12Z-octadecadienoic acid as well as the two all-E hydro(pero)xy octadecadienoic acid isomers. At low membrane concentrations (1 mg of protein/ml), the enzyme maintains a high stereospecificity for the S-configuration, but at higher concentrations (20 mg/ml), the products were virtually racemic. Addition of the antioxidant 2,6-ditert-butyl-p-cresol counteracted this tendency to lose stereospecificity. During these enzyme-catalyzed reactions, substantially more oxygen is consumed than can be accounted for as the hydro(pero)xy products. This discrepancy is due to secondary reactions which lead to the decomposition of the primary oxygenation products, the hydroperoxy lipids, and to oxidative modifications of membrane proteins. These data indicate that the reticulocyte lipoxygenase can oxygenate polyenoic fatty acids in various types of biological membrane and that the oxidative modifications are not restricted to the membrane lipids. The results are discussed in terms of the proposed role of the enzyme in the breakdown of mitochondria and other intracellular organelles during the maturation of red blood cells.
Mitochondrial membranes and plasma membranes of rabbit reticulocytes contain oxygenated polyenoic fatty acids such as (9Z,11E)-(13S)-13-hydroxy-9,11-octadecadienoic acid, 9S and 9R isomers of (10E,12Z)-9-hydroxy-10,12-octadecadienoic acid and their all-E isomers. Furthermore (5Z,8Z,11Z,13E)-(15S)-15-hydroxy-5,8,11,13-icosa tetraenoic acid, 9- and 13-oxooctadecadienoic acid were detected as minor products. The chemical structure of these products has been identified by co-chromatography with authentic standards, by ultraviolet and infrared spectroscopy, and by gas chromatography/mass spectrometry of the native compounds and their hydrogenated derivatives. The oxygenated fatty acids originate most probably from the intracellular action of the erythroid arachidonate 15-lipoxygenase. In membranes of the mature erythrocyte only small amounts of hydroxy fatty acids were detected. Young peripheral reticulocytes contain more oxygenated polyenoic fatty acids in their membranes than older cells. In mixed cell populations, about 85% of the lipoxygenase products were found esterified to the membrane ester lipids, whereas 15% were associated as free hydroxy fatty acids with the membranes. The hydroxy fatty acid content of the mitochondrial membranes is more than threefold higher than that of the plasma membranes. The pattern of the products isolated from plasma membranes shows a high specificity with (9Z,11E)-(13S)-13-hydroxy-9,11-octadecadienoic acid as the main product. In contrast, the pattern found in the mitochondrial membranes was much more unspecific: a complex mixture of all positional and optical isomers was detected. The data presented indicate that the reticulocyte lipoxygenase in vivo acts on both plasma membranes and mitochondrial membranes. The results are discussed in the light of the involvement of the lipoxygenase in the breakdown of mitochondria and other organelles in reticulocytes during maturation.
Membranes of intact rabbit reticulocytes and rat liver mitochondrial membranes oxygenated by the pure reticulocyte lipoxygenase contain 13-keto-9Z,11E-octadecadienoic acid and 9-keto-10E,12Z-octadecadienoic acid. In mitochondrial membranes not treated with lipoxygenase and in rabbit erythrocyte membranes these products were not detected. The chemical structure of the compounds has been identified by cochromatography with authentic standards on various types of HPLC columns, by uv and ir spectroscopy and GC/MS. In the membranes of rabbit reticulocytes up to 2% of the linoleate residues are present as its 9- and 13-keto derivatives. Most of the keto compounds (up to 90%) are esterified in the membrane ester lipids, only about 10% were found in the free fatty acid fraction. It is proposed that the keto dienoic fatty acids are formed via decomposition of hydroperoxy polyenoic fatty acids originating from the oxygenation of the membrane lipids by the reticulocyte lipoxygenase.
Rabbit reticulocytes metabolize exogenous polyenoic fatty acids via three different pathways. (i) incorporation into the cellular ester lipids, predominantly into phospholipids, (ii) beta-oxidation forming CO2 and (iii) lipoxygenase reaction. The lipoxygenase pathway contributes to about 30% to the metabolism of exogenously added linoleic acid. The endogenous substrates of the lipoxygenase pathway are not only the free polyenoic fatty acids bound to the cellular membranes but also the membrane phospholipids. The lipoxygenase products detected in the membrane lipids have been isolated and their complete chemical structure has been identified as 13-hydroxy-9Z,11E-octadecadienoic acid. 9-hydroxy-10E,12Z-octadecadienoic acid, their all E isomers and 15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid. Subcellular fractionation of different cellular membranes indicated that these products occur in both the mitochondrial membranes and the plasma membrane. By quantitative HPLC analysis it has been shown that the mitochondrial membranes contain about 3 times more oxygenated fatty acids than the plasma membranes; one out of ten linoleic acid residues in the mitochondrial membranes is present as hydroxylated derivative. The pattern of lipoxygenase products detected in the mitochondrial membranes was much more unspecific than that of plasma membranes. These data are discussed in the light of the involvement of the lipoxygenase pathway in the degradation of mitochondria during the maturation of red blood cells.
The oxygenation of different types of biological membranes (rat liver mitochondria, rat liver endoplasmic membranes, inside-out erythrocyte ghosts, right side-out erythrocyte ghosts) was studied with respect to products formed during the reaction. In all cases a very similar product pattern was observed with 15S-hydroperoxy-5Z.8Z.11Z.13E-eicosatetraenoic acid (15-HETE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid, (13-HODE) being the major products. Comparison of the amount of lipoxygenase products formed with the oxygen uptake measured during the reaction indicated an excessive oxygen uptake. With mitochondrial membranes the oxygen consumption was almost one order of magnitude higher than the amount of the products detected. The origin of the excessive oxygen uptake remains unclear. These data, however, indicate that the oxygen consumption with complex substrates is not a reliable measure for the lipoxygenase activity.
The reticulocyte lipoxygenase is able to oxygenate mitochondrial membranes. The main products formed during this reaction are 15S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HETE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HODE). The oxygenation of mitochondrial membranes is accompanied by an inactivation of enzymes localized in the outer and inner mitochondrial membranes, by a drastic change of the passive electric properties and by a destruction of the iron-sulfur clusters of the outer mitochondrial membrane. The possible mechanism of the processes leading to these functional changes is discussed.
The pure reticulocyte lipoxygenase oxygenates free arachidonic acid to 15S- and 12S-HPETE in a ratio of about 10:1 15S-H(P)ETE or its methyl ester can be further converted by the lipoxygenase via three different types of reactions: (i) oxygenase reaction, (ii) hydroperoxidase reaction and (iii) leukotriene synthase reaction. Here we summarize the products formed from arachidonic acid by the reticulocyte lipoxygenase via these three types of reactions.
Leaves of Glechoma hederacea L. and other Labiatae contain (9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid, (10E,12Z,15Z)-9-oxo-10,12,15-octadecatrienoic acid, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid and (10E,12Z)-9-oxo-10,12-octadecadienoic acid in a ratio of 71/14/12/3 (by mass), predominantly esterified in the membrane ester lipids. The leaves contain the highest level of these products, whereas only small amounts were found in the stalk and the roots. The chemical structures of these compounds were established by ultraviolet and infrared spectroscopy, by co-chromatography with authentic standards on various types of HPLC columns including chiral-phase HPLC and gas chromatography/mass spectrometry. The stereochemical specificity indicates the enzymatic origin of the products, most probably via a lipoxygenase reaction. Freshly harvested specimens of G. hederacea L. contain only small amounts of hydroxy-polyenoic fatty acids. Air-drying causes a strong increase in the content of free and esterified (9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid. Up to 80% of the hydroxy fatty acids of the total lipid extracts were esterified in the cellular lipids. The data presented indicate that lipoxygenase products occur in the cellular ester lipids of G. hederacea L. and other Labiatae. The results are discussed in the light of a possible involvement of the lipoxygenase pathway in the natural senescence of leaves.