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.

No abstract

This prospective study of 35 multitraumatized intensive care unit patients requiring mechanical ventilation examined the relative utility of four biochemical parameters with a physiological scoring system for predicting lethal outcome. Levels of serum phospholipase A2 (PLA2), serum amyloid A (SAA), polymorphonuclear granulocyte elastase (PMN elastase), and C-reactive protein (CRP) were determined at short intervals during the patient's hospitalization. The first specimen was obtained at the time of admission, and subsequent specimens were drawn at 8 h intervals for the first 48 h and then twice daily until death or convalescence. Calculations of the APACHE II score used the most deranged variables during the first 24 h of admission to assess patient outcome. Additional calculations of the APACHE II score at the time of each blood draw served as an indicator of patient status. The results indicate that during the first 24 h after admission none of the four examined biochemical parameters gives reliable information about the outcome. The APACHE II score provided the earliest indicator of patient outcome (83% sensitivity, 65% specificity). PMN elastase provided useful information first at 32 h (83% sensitivity, 45% specificity) and better at 132 h (86% sensitivity, 86% specificity). CRP was of intermediate use in predicting outcome initially at 72 h (83% sensitivity, 50% specificity) and later at 132 h (86% sensitivity, 93% specificity). PLA2 and SAA were not useful as early indicators of lethal outcome.

Summary: Phospholipase A 2 activity in human sera was determined of the basis of the E. coli assay and compared to a photometric micelle assay. The E. coli assay is based on the hydrolysis of phospholipids from [l-14 C]oleic acid-labelled E. coli biomembranes. In the photometric assay the phospholipase A 2 acts on mixed phospholipid micelles. The amount of fatty acid produced is quantitated in a subsequent photometric assay by coupling in the reaction to the coenzyme A metabolism. The E. coli membranes are essentially resistent to other Upases in human sera, i. e. lipoprotein upases, hepatic triacylglycerolipase or pancreatic lipase and thus a very specific substrate for the phospholipase A 2 of human serum. The photometric assay, though, is susceptible to other upases in human serum. The ratio of (l-14 C]oleic acid to released total fatty acids served as the basis for the calculation of the true enzymatic activity. The assay closely correlated with the photometric assay based on mixed micelles in the higher ranges of phospholipase A 2 activity, but not in the normal range. The sensitivity is higher by at least two powers of 10. The human serum phospholipase A 2 strongly preferred E. coli membranes as substrate to the mixed micelles containing phosphatidylcholine/phosphatidylethanolamine. In conclusion, the modified phospholipase A 2 assay based on E. coli membranes is a sensitive, specific, reliable, and convenient method for the measurement of phospholipase A 2 activity in human sera. The photometric assay süffers from low sensitivity but has the advantage of practicability in a normal routine laboratory, including the amenability to automation. Introduction... . , , , ,

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.