Summary:A new enzymatic method for the determination of cholesterol in serum and plasma was evaluated in 8 separate laboratories in comparison with routine and reference methods.Investigation of the analytical reliability in the 2-26 mmol/1 measurement ränge showed the following results:1. At the set reading points (10min at 25 °C and 5min at 37 °C) the reaction shows complete Substrate conversion. The colour complex is stable over a period of 60 min.2. The response to cholesterol is linear up to 26 mmol/1.3. Precision within the series was 0.6-2.8% in 20 determinations (coefficient of Variation).
We developed a method for determining individual free fatty acids in serum by using a modified one-step Dole extraction, derivatization, and a new high-performance liquid chromatographic (HPLC) separation. Sample handling is minimized to a single transfer of the fatty acids (upper layer of the Dole extract), which are readily derivatized at 85 degrees C with p-bromophenacyl bromide without significant hydrolysis of esterified fatty acids. The derivatization mixture is directly injected into the HPLC apparatus. The new method, which uses C6 (3-microns particle) column material and an isocratic acetonitrile-water eluent, separates nearly to baseline 12 of the physiologically most abundant long-chain fatty acids (C12-C22) in < 20 min with a detection limit of approximately 2 pmol. It is therefore suitable for routine analysis even with basic HPLC equipment and can easily analyze a series of 10-20 samples in about 2 h including extraction until first results are available. The method is also applicable to other matrices than serum, e.g., for determination of precursor fatty acids such as arachidonic acid in platelets or of fatty acid patterns liberated by lipases or phospholipases A1/A2 in test systems.
A female patient with the following symptoms has been observed: complete absence of subcutaneous fat on the arms and legs, well developed adipose tissue on the trunk and face, severe hyperlipidemia, eruptive xanthomas, insulin resistant diabetes mellitus with lack of ketoacidosis, hepatomegaly and elevated basal metabolic rate. The patient thus exhibited all characteristics of lipatrophic diabetes (Lawrence type of diabetes). The mother and a sister of the patient were found to have the same peculiar appearance and a slight hyperlipidemia but no diabetes mellitus. The combination of this type of partial lipodystrophy with severe hyperlipidemia, insulin resistant diabetes mellitus without ketoacidosis and elevated basal metabolic rate was further observed in 2 unrelated patients without known familial occurrence. Thus partial lipodystrophy of the extremities is another, previously undescribed, syndrome associated with the Lawrence type of diabetes mellitus. In the 1 family the syndrome of lipodystrophy and hyperlipidemia is dominantly inherited. Besides the autosomal recessively inherited syndrome of congenital generalized lipodystrophy there is a heterogenous group of dominantly inherited syndromes with various types of lipodystrophy.
In experimental bile obstruction the serum activities of the membrane-bound liver enzymes, alkaline phosphatase, 5'-nucleotidase and γ-glutamyltransferase are greatly increased, whereas in the liver only the alkaline phosphatase activity is elevated. After partial hepatectomy or tetrachloride poisoning the alkaline phosphatase activity in the regenerating liver is increased to the same extent as in cholestasis without an accompanying elevation in serum activity. The following results support the hypothesis of a bile salt-mediated solubilization of membrane-bound enzymes in cholestatic liver: (1) 30 min after bile duct ligation the total bile acids in the liver were increased 5-fold, 2 h later as much as 10-fold. After 1 day, the bile acid concentration was still 4 times above normal. (2) Isolated plasma membranes from normal and obstructed livers were incubated in vitro with increasing amounts of tri- and dihydroxycholanic acids. At a final concentration of 1 mmol/1 taurochenodeoxycholate significant amounts of membrane-bound enzymes were released into the 12,000-g supernatant. (3) In the regenerating liver, where tissue phosphatase activity was high and serum phosphatase activity unchanged, the bile salt concentration was not increased.
An männlichen Wistar-Ratten wurde durch intraperitoneale Injektion von 0,50-0,75 g/kg D-Galaktosamin eine Hepatitis und durch einmalige, orale Applikation von 150 mg/kg -Naphthylisothiocyanat eine intrahepatische Cholestase erzeugt. Nach 12, 24, 48 und 72 Stdn. wurden die Konzentrationen des gesamten und freien Cholesterins in Leber und Plasma sowie die Aktivität der LecithinCholesterin-Acyl-Transferase im Plasma zusammen mit anderen Enzymaktivitäten gemessen: 1. Bei Galaktosamin-Hepatitis trat ein rascher Abfall der Aktivität auf 10% der Norm auf, während die Alanin-und AspartatTransaminase auf das 100-fache des Kontrollwertes anstiegen. 2. Parallel zum Abfall der Lecithin-Cholesterin-Acyl-Transferase sanken die Cholesterinester im Plasma auf 15% des Normwertes ab und blieben während der gesamten Beobachtungszeit erniedrigt, obwohl das freie Cholesterin nach 48-72 Stdn. deutlich anstieg. Dabei wurde auch ein Anstieg der Cholesterinester in der Leber bei gleichzeitiger Triglycerideinlagerung beobachtet. 3. Bei Cholestase nach -Naphthylisothiocyanat zeigte sich im Plasma nur ein relatives Absinken der Esterfraktion von 67 auf 43%, wegen der ungleich stärkeren Vermehrung des freien Cholesterins. In der Leber fand sich ein gegensinniges Verhalten, d. h. die Cholesterinester nahmen stärker zu als das freie Cholesterin. Die Lecithin-Cholesterin-Acyl-Transferase-Aktivität war dabei gegenüber der Norm nicht erniedrigt, sondern eher etwas erhöht. Diese Befunde sprechen dafür, daß die Bildung der Cholesterinester im Plasma von der Sekretion eines spezifischen Enzyms durch die Leber abhängt. Die Aktivität dieser Lecithin-Cholesterin-Acyl-Transferase im Plasma nimmt daher bei experimenteller Leberschädigung durch Galaktosamin rasch ab mit nachfolgender Erniedrigung der Cholesterinester. Cholesterol metabolism and plasma lecithin-cholesterol acyl transf erase in experimental hepatitis and cholestasis in the ratA hepatitis was produced in male Wistar rats by the intraperitoneal injection of 0.50-0.75 g D-galactosamine per kg, and an intrahepatic cholestasis was produced by a single peroral application of 150 mg -naphthyl-iso-thiocyanate per kg. After 12, 24, 48 and 72 hr, the concentrations of total and free cholesterol in the liver and plasma and the activity of the plasma lecithin-cholesterol acyl transferase were measured, together with other enzyme activities. 1. In galactosamine hepatitis there was a rapid fall in the activity of the lecithin-cholesterol acyl transf erase activity to about 10% of normal, while the alanine and aspartate transaminase increased 100-fold over the control values. 2. Parallel to the decrease of lecithin-cholesterol acyl transf erase, the plasma cholesterol esters fell to about 15% of the normal value and remained low for the duration of the observed period. The free cholesterol, however, showed a marked increase after 48-72 hr and there was also an increase in the cholesterol esters in the liver, accompanied by the deposition of triglycerides. 3. After -naphthyl-iso-thiocyanate there was only...
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