Determination of HOL Phosphatidyl Choline by an Enzymatic Method

Abstract

Summary: A routine method is described for the enzymatic determination of phosphatidyl choline in the apolipoprotein B-free supernatant after precipitation of blood sera with phosphotungstic acid/MgC! 2 . The principle of this method is based on the specific cleavage of phosphatidyl choline by purified phospholipase C from B. cereus, and the enzymatic determination of choline by choline kinase after hydrolysis of phosphorylcholine. The enzymatic method provides HDL phosphatidyl choline values which coincide with those of the conventional chemical method. Furthermore, the values obtained with the enzymatic method for the HDL fraction isolated by ultracentrifugation (1.063-1.21 kg/l) also closely coincide with those of the apolipoprotein B-free supernatant fraction. The precision, linearity and sample stability were also checked. The findings obtained show that the enzymatic assay introduced here is suitable for the routine determination of phosphatidyl choline in the apolipoprotein B-free supernatant.HDL phosphatidyl choline values of 1.18 ±0.37 mmol/1 (162 men) and 1.37 ± 0.38 mmol/1 (59 women) were obtained using this method in normal individuals. Zur Analytik von HDL Phosphatidylcholin mit einer enzymatischen BestimmungsmethodeZusammenfassung: Es wird eine Routinemethode für die enzymatische Bestimmung von Phosphatidylcholin im Apoliprotein B-freien Überstand nach Fällung von Blutseren mit Phosphorwolframsäure/MgCb beschrieben. Das Prinzip dieser Methode besteht in der spezifischen Spaltung von Phosphatidylcholin mit gereinigter Phospholipase C aus B. cereus und der anschließenden enzymatischen Bestimmung von Cholin mittels Cholinkinase nach Hydrolyse von Phosphorylcholin. Im Vergleich zur konventionellen chemischen Methode ergab die enzymatische Methode übereinstimmende HDL Phosphatidylcholinwerte. Weiterhin wurden über-einstimmende Ergebnisse mit der enzymatischen Methode zwischen der HDL-Fraktion, die durch Ultrazentrifugation (1,063-1,21 kg/l) isoliert wurde, und der Apolipoprotein Befreien Uberstandsfräktion erzielt. Die Präzision, Linearität und JProbenstabilität wurden ebenfalls untersucht. Die Ergebnisse zeigen, daß die eingeführte ehzymatische Methode für die routinemäßige Bestimmung von Phosphatidylcholin im Apolipoprotein B-freien Überstand 'geeignet ist. Mit dieser Methode fanden wir in der Normalbevölkerung für Män-ner (n = 162) einen HDL Phosphatidylcholinwert von 1,18 + 0,37 mmol/1 und bei Frauen (n = 59) einen HDL Phosphatidylcholinwert von 1,37 ± 0,38 mmöl/1. Introduktionconstituent of HDL. However, the HDL do not repIn recent years the quantitation of high density lipo-resent a single substance, but are a heterogeneous proteins (Ht>L) äs an indicator of the risk of coro-mixture of macromolecules, whose composition denary heart disease has gained particular importance pends to a considerable degree on the metabolism of (l, 2). The typical routine methods for determining HDL. For instance, in hypertriglyceridaemic sera the HDL involve quantitative analysis of the cholesterol cholesterol tonstit...

HDL Phosphatidyl Choline and Risk-Factors bf Coronary Heart Disease

“…A similar change in HDL composition in hypertriglyceridaemic individuals /has also been observed during analysis of HDL sphingomyelin levels in patients with a type IV hyperlipoproteinaemia (21). In these patients the relative sphingomyelin content of HDL was clearly higher than in normotrl· glyceridaemic individuals (21).…”

“…Since the introduction of commercially available phospholipases and the development of sensitive methods for choline assay, it is now possible to analyse phospholipid fractions äs routine Parameters. We have recently developed corresponding procedures for the analysis of HDL sphingomyeline (16) and HDL phosphatidyl choline (9,10). For the HDL phosphatidyl choline analysis in this study we employed an enzymatic colour test, using the analyser Cobas Bio (Hoffmann La Röche), äs previously described (10).…”

“…Recently, enzymatic methods have been developed in order to measure HDL phosphatidyl choline (9)(10)(11). In this report we present the first results of our epidemiological study on the relationship of HDL phosphatidyl choline to the various risk factors for coronary heart disease.…”

An Economical Assay for HDL Phosphatidyl Choline

“…We used samples of the serum collected for the 'Prospective epidemiological study of Company employees in Westfalia* reported elsewhere (8).…”

“…Only after the additional determination of other HDL components, such äs HDL apolipoproteins or HDL phospholipids, is it possible to gain any Information regarding HDL composition or HDL mass. This group recently reported pn an enzymatic method for determining HDL phosphatidyl choline (8). The principle of the method involves the specific cleavage Pf phosphatidyl choline by phospholipase C from B.cereus, followed by enzymatic determination of choline hydrolysed from phosphorylcholine by choline kinase, äs detected by an optical method.…”