Search PubMed⌕ Search

Biomedical subjects

J Aufenanger

Publications and source records attributed to J Aufenanger.

28 records · Page 2Linked to original sources
PubMed1993

Characteristics and clinical application of a radiometric Escherichia coli-based phospholipase A2 assay modified for serum analysis.

Determination of activities of phospholipase A2 (PLA2) in human sera was based on the hydrolysis of phospholipids from [1-14C]oleic acid-labeled Escherichia coli biomembranes. The E. coli membranes served as substrate specifically for the PLA2 of human serum and were essentially resistant to other lipases in human sera, i.e., lipoprotein lipases, hepatic triacylglycerolipase, or pancreatic lipase in acute pancreatitis. Exchange of phospholipids between the serum and the biomembrane compartment aggravates the determination of PLA2 activity in human serum, which is naturally rich in phospholipids. In our modified E. coli assay, which overcomes these difficulties, the main substrate components phosphatidylethanolamine (70%) and cardiolipin (25%) were > 90% labeled in the sn-2 position. Fatty acids released by PLA2 activity were eluted from an aminopropyl solid-phase column directly into scintillation vials, where the radioactivity was counted. The ratio of [1-14C]oleic acid to released total fatty acids was used to calculate true enzymatic activity. The linear assay range extended from 0 to 3.6 U/L (0-60 nkat/L), with a detection limit of < 0.03 U/L (< 0.5 nkat/L). Within-assay imprecision (CV) was < 6% and between-assay is < 10% over the whole activity range. The normal range for men was 0-0.44 U/L (0-7.33 nkat/L) and for women 0.044-1.11 U/L (0.73-18.4 nkat/L). Patients with septicemia, pancreatitis, acute respiratory distress syndrome, or other severe diseases had PLA2 values up to 540 U/L (9000 nkat/L).

Carbon Radioisotopes↗
PubMed1993

Increased phospholipase A activities in sera of intensive-care patients show sn-2 specificity but no acyl-chain selectivity.

Phospholipase A (PLA) activities were measured by high-performance liquid chromatography in two enzyme preparations purified from human duodenal juice and a serum pool as well as in 52 sera from 31 intensive-care patients with various diseases. On the basis of a position-specific fatty acid analysis of the natural substrate ("soybean lecithin") from a commercial PLA kit, serum activities of PLA1 could be clearly distinguished from those of PLA2, which is not possible in the usual measurements made with single-label radioactive substrates. Independent of the type of disease, all sera with highly increased PLA activities (40-200 U/L) showed nearly pure PLA2 characteristics without any preference among oleic, linoleic, and linolenic acid in the sn-2 position of the glycerophospholipid substrate. Nevertheless, very low PLA1 activities (< or = 5 U/L, most likely due to heparin perfusion therapy) could also be detected by palmitic and stearic acid release from the sn-1 position, leading to small changes in fatty acid release patterns of sera with low PLA activities. Measurements with sera from heparin-treated volunteers demonstrated that heparin therapy may initially contribute as much as 22 U/L to increased PLA1 activities but is not important under prolonged therapy. The absence of selectivity with respect to acyl-chain desaturation supports the concept of serum PLA2 as an acute-phase protein rather than a regulator of the arachidonic acid cascade.

Adult↗
PubMed1991

The effect of impaired lipid metabolism on the smooth muscle cells of rabbits.

Our clinical data enabled us to demonstrate a correlation between impaired lipid metabolism and vasculogenic impotent men. Our aim was to evaluate the effect of an impaired lipid metabolism on the smooth muscle of the corpus cavernosum. A total of 16 rabbits were given a cholesterol-enriched diet for 3 months, and 8 of these received additional thromboxane A2 receptor antagonist; 10 other rabbits (control) were fed a normal diet. Subsequently, cavernous tissue biopsies were taken, and tissue lipid extractions and electron microscopic evaluation were made from 3 rabbits in each group. In the untreated high-cholesterol diet group, cholesterol levels reached approx. 2.1 micrograms/mg body weight compared with 1.07 micrograms/mg b.wt. in the thromboxane A2 receptor antagonist-treated group and elevated levels compared with control group. Similar results were found for the triglyceride and free fatty acid levels. Lecithin tissue levels in treated rabbits were distinctly elevated against those of other 2 groups. Ultramorphological examination of the control group disclosed normal smooth muscle cell (SMC) architecture with numerous sites of intercellular contacts. These findings contrasted with those of the high-cholesterol diet groups which showed significant SMC degeneration with loss of intercellular contacts. Our data imply that impaired lipid metabolism causes cavernous SMC degeneration which plays a major role in the pathogenesis of erectile dysfunction. The thromboxane A2 receptor antagonist seems to produce a protective metabolic effect on the erectile tissue which may have some consequences future treatment strategies.

Animals↗
PubMed1989

Improved method for enzymic determination of cholesterol in lipoproteins separated by electrophoresis on thin layer agarose gels.

The cholesterol of lipoproteins, separated electrophoretically on thin layer agarose films, is visualised and quantitated by incubating the gels in an enzymic reagent containing cholesterol esterase and cholesterol dehydrogenase. The individual fractions are quantitated by scanning densitometry. No sample pretreatment is necessary. All major fractions are detected readily. The accuracy of the determination is similar to that of ultracentrifugation. On average, imprecision is 3.1% for beta-, 7.0% for pre beta-, and 4.8% for alpha-lipoprotein cholesterol. Concentration and colour development are linear up to 8 mmol/l cholesterol in a given lipoprotein fraction. The results from the direct enzymic procedure for beta-, pre beta- and alpha-lipoprotein cholesterol are compared with those from quantitative lipoprotein electrophoresis after precipitation with phosphotungstic acid and bivalent cations and with those from different precipitation methods using dextran sulphate and polyethylene glycol. The new method has several advantages: high specificity; lack of dependence on the actual composition of the lipoproteins; lack of interference from coprecipitated proteins in the gel, e.g. fibrinogen or paraproteins; and insensitivity to lipolysis and high free fatty acid concentrations caused by heparin application or ageing of the specimen (at least for alpha-lipoprotein cholesterol quantitation). In its convenience and simplicity of operation, and the simple calculation of results, the method is similar to standard protein electrophoresis. The proposed method is therefore suggested as a standard method for elucidating lipoprotein disorders.

Cholesterol↗
PubMed1989

Enzymatic determination of lipids in human bile without bilirubin interference: reliable assessment of the cholesterol saturation index (CSI).

We describe a simple and rapid, but nevertheless precise and accurate method for the enzymatic determination of the main lipid constituents in human bile. Interfering bile pigments, especially bilirubin are eliminated by the use of aminopropyl bonded phase columns ("Bond-Elut") prior to the enzymatic measurement of cholesterol and lecithin. Intra-assay imprecision was between 3.1 and 4.9% CV, while the inter-assay figures were rather higher at 4.6 to 7.5% CV. Recoveries of bile salts, lecithin and cholesterol were between 94 and 103%. In contrast, the direct enzymatic determination in native bile produces falsely low results: lecithin from 5 to 20%, cholesterol from 25 to 40% of the true value. The results of both enzymatic methods correlated well with commonly accepted procedures for phospholipid and cholesterol determination. When compared with methods of bile lipid analysis involving solvent extraction, the column separation followed by enzymatic determination has the advantage of being simpler and less time consuming, without need of high-cost equipment, e.g. gas chromatography.

Alcohol Oxidoreductases↗
PubMed1988

A specific method for the direct determination of lipoprotein cholesterol in electrophoretic patterns.

Lipoproteins are separated electrophoretically and cholesterol is visualised with an enzymic reagent specific for cholesterol in which the gels are incubated. Quantitation of the individual fractions is accomplished by scanning densitometry. No sample pretreatment is necessary. All major fractions are detected readily. Accuracy agrees favorably with results from the ultracentrifugation. On the average, imprecision is 3.1% for beta-, 6.9% for prebeta-, and 5.2% for alpha-lipoprotein cholesterol. Concentration and color development are linear up to 8 mmol/l cholesterol in a given lipoprotein fraction. The results of the direct enzymic procedure for beta-, prebeta- and alpha-lipoprotein cholesterol are compared to the quantitative lipoprotein electrophoresis after precipitation with phosphotungstic acid and bivalent cations.

Cholesterol↗
PubMed1986

The effects of BM 15.766, an inhibitor of 7-dehydrocholesterol delta 7-reductase, on cholesterol biosynthesis in primary rat hepatocytes.

The effect of the piperazine derivative BM 15.766 (4-(2-[1-(4-chlorocinnamyl)piperazin-4-yl]ethyl]-benzoic acid) on the biosynthesis of sterols was investigated in adult rat hepatocytes in primary monolayer culture. The substance led to a dose-dependent reduction of cholesterol in the serum of various species of animals such as rat, dog and marmoset. BM 15.766 showed a dose-dependent action on the incorporation of 14C-acetate in neutral, nonsaponifiable lipids. The inhibition of the overall incorporation was 10-12% (10(-5) M). No inhibition was observed in the hepatocytes over the entire dose range of 10(-8) M to 2 X 10(-5) M, while the release of the neutral lipids from the hepatocytes into the culture medium was reduced by up to 40%. The biosynthesis of cholesterol could be reduced by more than 90%. Simultaneously, 7-dehydrocholesterol levels rose in the cells and, to a less marked extent, in the medium. This can be interpreted as an indication that 7-dehydrocholesterol is incorporated into the cell membrane, which results in a lower release of 7-dehydrocholesterol into the medium in comparison with controls. The site of attack is the inhibition of the delta 5.7-sterol delta 7-reductase. The formation of desmosterol and cholestatrienol as well as other 7-dehydrocholesterol precursors could also be demonstrated. After longer incubation, there was an additional accumulation of squalene and lanosterol with simultaneous reduction of 7-dehydrocholesterol by BM 15.766, whereas the total 14C-acetate incorporation in neutral lipids was increased.

Acetates↗
PubMed1985

Inhibition of cholesterol biosynthesis by BM 15.766.

BM 15.766 (4-[2-[1-(4-chlorocinnamyl)piperazin-4-yl]ethyl]benzoic acid) showed a dose dependent action on 14C-acetate incorporation in cholesterol and intermediates including squalene by adult rat hepatocytes in primary monolayer culture. The biosynthesis of cholesterol could be reduced by more than 90%. Simultaneously, the 7-dehydrocholesterol level rose in the cells and, to a less marked extent, in the culture medium.

Animals↗