Comparison of serum phospholipase A2, polymorphonuclear granulocyte elastase, C-reactive protein and serum amyloid A with the APACHE II score in the prognosis of multiple injured patients.

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.

Determination of the catalytic activity of phospholipase A2: E. coli-based assay compared to a photometric micelle assay.

Phospholipase A2 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 [1-14C]oleic acid-labelled E. coli biomembranes. In the photometric assay the phospholipase A2 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 resistant to other lipases in human sera, i.e. lipoprotein lipases, hepatic triacylglycerolipase or pancreatic lipase and thus a very specific substrate for the phospholipase A2 of human serum. The photometric assay, though, is susceptible to other lipases in human serum. The ratio of [1-14C]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 A2 activity, but not in the normal range. The sensitivity is higher by at least two powers of 10. The human serum phospholipase A2 strongly preferred E. coli membranes as substrate to the mixed micelles containing phosphatidylcholine/phosphatidylethanolamine. In conclusion, the modified phospholipase A2 assay based on E. coli membranes is a sensitive, specific, reliable, and convenient method for the measurement of phospholipase A2 activity in human sera. The photometric assay suffers from low sensitivity but has the advantage of practicability in a normal routine laboratory, including the amenability to automation.

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.

Fast HPLC determination of serum free fatty acids in the picomole range.

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.

Dependence of fatty acid composition of Listeria spp. on growth temperature.

In Listeria spp., various fatty acids are produced; by far the most common members are C15 and C17 chain length fatty acids. This pattern is rather similar in all species. At low temperatures, most of the Listeria are able to change the relative composition whereby more of the C15 fatty acids are produced, which could increase the fluidity of the bacterial cell membrane under these conditions.

The influence of chloro-substituent sites of hexachlorobiphenyl on the respiration of rat liver mitochondria.

The actions of three hexachlorobiphenyls (HCBs) 2,3,4,2',3',4'-, 2,3,4,3',4',5'- and 3,4,5,3',4',5'-HCBs, on the respiration of rat liver mitochondria with succinate as the substrate were compared, and the effect of chloro-substitution sites in HCB on the respiration was examined. 2,3,4,2',3',4'-HCB strongly inhibited both state 3 and 2,4-dinitrophenol (DNP)-stimulated respiration with 50% inhibition dose of 52 and 54 microM for state 3 and DNP-stimulated respiration, respectively. The inhibitory action of 2,3,4,3',4',5'-HCB on both respiration was approximately half as potent as that of 2,3,4,2',3',4'-HCB. On the other hand, 3,4,5,3',4',5'-HCB did not inhibit any respiration at all. These results indicate that both inside (ortho) and outside (meta or para) positions in each phenyl ring of the biphenyl molecule should be replaced with chlorines for HCB to be an effective inhibitor. Either the actual position of chloro-substituent or steric conformation caused by its substitution or both can be considered as factors affecting the inhibition. On the basis of the conformational energy, calculated by AM1 (Austin model 1) method, with increases in chlorine number in ortho position, HCB molecule became angulated. Furthermore, calculated probability of the conformation distribution for HCB indicated that the probability of nonplanarity was higher for effective HCB than for less effective HCB. These structural features suggest the significance of steric conformation as well as chloro-substituent sites in determining the inhibitory ability of HCB.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential potency of atropisomers of polychlorinated biphenyls on cytochrome P450 induction and uroporphyrin accumulation in the chick embryo hepatocyte culture.

The atropisomers of 2,2',3,4,6-pentachlorobiphenyl (PeCB), 2,2',3,4,4',6-hexachlorobiphenyl (HeCB), and 2,2',3,3',4,4',6,6'-octachlorobiphenyl (OCB) were studied in the chick embryo hepatocyte culture to determine if chirality plays a role in the recognition events associated with the induction of cytochromes P450 and the accumulation of uroporphyrin (URO). Concentration-related induction of cytochrome P450 content, ethoxyresorufin-O-deethylase (EROD) and benzphetamine N-demethylase (BPDM) activities were measured. The rank order of potency for total cytochrome P450 induction was HeCB greater than OCB greater than or equal to PeCB. The (+)- and (-)-enantiomers of PeCB and OCB were of equal potencies as inducers of cytochromes P450, whereas the (+)-HeCB was greater than the (-)-HeCB. HeCB was a much more potent inducer of EROD activity than was either PeCB or OCB. EROD activity was induced to a much greater extent by the (+)-enantiomers of all compounds, with the (-)-enantiomers of PeCB and OCB being inactive. BPDM activity was induced by all three compounds in the order of OCB greater than or equal to HeCB greater than PeCB. The (-)-enantiomers were more potent inducers of BPDM activities than were the (+)-enantiomers, except for HeCB, in which the (+)- was more potent than the (-)-enantiomer. Analysis of porphyrin accumulation in cultures treated with delta-aminolevulinic acid revealed that (+)-HeCB caused the greatest percent URO accumulation, which also correlated with the greatest increase in EROD activity. All other enantiomers caused up to 47% URO accumulation, which did not correlate with an increase in EROD activity.

Chiral effects in the induction of drug-metabolizing enzymes using synthetic atropisomers of polychlorinated biphenyls (PCBs).

Atropisomers of the polychlorinated biphenyls 2,2',3,4,4',6-hexachlorobiphenyl (II) and 2,2',3,3',4,4',6,6'-octachlorobiphenyl (III), stable to racemization under physiological conditions, were administered to immature male Sprague-Dawley rats. The racemic hexachlorobiphenyl (II) was found to be a potent (phenobarbital-type) inducer, whereas (+)-II and (-)-II, administered at 100 mumol/kg, showed clearly differing potencies as inducers with (+)-II enhancing aminopyrine N-demethylase, aldrin epoxidase and cytochrome P-450 content more potently than (-)-II. In contrast, the racemic octachlorobiphenyl (III) and its individual enantiomers were only weak phenobarbital-type inducers of cytochrome P-450, and the enantiomers of III were equally (weakly) potent. Separate studies conducted to investigate pharmacokinetic influences on the differential potency of the enantiomers of II showed that after 5 days the concentration of (+)-II in the liver was twice as high as that of its antipode. Therefore, enantioselectivity in disposition as well as in recognition may be responsible for the differential potency seen.

Differential induction of cytochrome P-450 by the enantiomers of trans-stilbene oxide.

Optically pure (+)- and (-)-trans-stilbene oxide (TSO) enantiomers were administered to immature male Sprague-Dawley rats. (+)-TSO was the more potent inducer of liver microsomal cytochrome P-450-dependent monooxygenases. The greater potency of (+)-TSO may be explained on the basis of stereoselective metabolism since a far greater concentration of TSO was found in liver microsomes of (+)-TSO-treated rats. Furthermore, of the enzymes known to metabolize TSO, cytosolic epoxide hydrolase turned over the (-)-TSO enantiomer at a faster rate, consistent with the greater persistence of the (+)-enantiomer. Although this report is of chiral effects in potency of enzyme induction, stereoselective metabolism (i.e. disposition) rather than inherent structural characteristics (recognition) may be responsible for these effects.