Prognostic value of serum phospholipase A in acute pancreatitis

Summary Serum from 48 patients with acute pancreatitis (21 with interstitial-edematous and 27 with necrotizing pancreatitis) was monitored for immunoreactive (IR) phospholipase A₂ (PLA₂) protein concentration and PLA catalytic activity. In both groups within 48 h after start of acute pancreatitis an up to tenfold increase of IR-PLA₂ was demonstrable. Determination of IR-PLA₂ revealed no differences between the groups. In contrast, determination of PLA catalytic activity allowed us to differentiate between patients with interstitial-edematous and necrotizing pancreatitis.     

The role of phospholipase A2 in human acute pancreatitis

Summary Several studies suggest that the activation of pancreatic phospholipase A₂ (PLA₂) and its release from injured acinar cells play an important role in the pathogenesis of acute pancreatitis. Elevated catalytic activity of PLA₂ in serum is associated especially with severe forms of the disease. PLA₂ has been purified from human cadaver pancreas and an antiserum raised against the enzyme in rabbits. Immuno-histochemical localization of PLA₂ in pancreatic tissue was abnormal in acute pancreatitis. A time-resolved fluoroimmunoassay for human pancreatic PLA₂ has been developed. Increased serum concentrations of immunoreactive PLA₂ were found in acute pancreatitis during the first week after hospital admission. The values returned to normal somewhat more slowly than corresponding serum amylase values. The immunochemical determination of PLA₂ in serum provides a fast and specific detection of injury to pancreatic acinar cells. The pancreas is not the only source of PLA₂ in acute pancreatitis. The nonpancreatic PLA₂ may originate from various inflammatory cells, but this hypothesis remains to be proven.     

Diagnostic value of immunoreactive phospholipase A2 in acute pancreatitis

Summary In a prospective clinical trial 85 patients with acute pancreatitis were analysed for serum total amylase, pancreatic amylase, pancreatic lipase, trypsin, elastase 1, and immunoreactive phospholipase A₂ (IR-PLA₂). The diagnostic sensitivity of serum IR-PLA₂ was comparable to that of serum total amylase, pancreatic amylase, and trypsin. The specificity of IR-PLA₂ is superior to that of serum total amylase determination due to the fact that the IR-PLA₂ determination is based on an antibody against human pancreatic PLA₂.     

Inhibition of porcine pancreas phospholipase A2 activation by gabexate mesilate

Summary We investigated the effect of gabexate mesilate on the catalytic activity of phospholipase A₂ in homogenized porcine pancreatic tissue. Gabexate mesilate is a potent inhibitor of serine proteases. There is no direct inhibition of phospholipase A₂ catalytic activity in concentrations up to 6 mmol/l. Preincubation of homogenized pancreatic tissue with gabexate mesilate leads to a reduction of phospholipase A₂ activity even in concentrations as low as 6 µmol/l. The activation of purified porcine prophospholipase A₂ added to pancreatic tissue can be completely inhibited. Thus, gabexate mesilate might influence the activation of phospholipase A₂ administered in therapeutic concentrations in inflamed pancreatic tissue.Abbreviations PLA₂ Phospholipase A₂ - ProPLA₂ Prophospholipase A₂     

Lysolecithin concentration in pancreatic tissue during therapy with phospholipase A2-inhibitors in acute necrotizing pancreatitis

Summary Four hours after hemorrhagic necrotizing pancreatitis was induced in experimental animals the concentration of lysolecithin in pancreatic tissue rose (p=0.0025). Parenteral administration of unspecific inhibitors of phospholipase A₂, such as chlorpromazine, gabexat mesilat and CaNa₂ EDTA, can neither inhibit elevation of the enzyme nor influence the course of the disease.     

A radiochemical test for phospholipase A2 catalytic activity

Summary A position-specifically labelled phosphatidylcholine is the substrate for the selective determination of Phospholipase A₂ in serum, ascites and tissue samples. Optimal reaction conditions and simplifications of handling are discussed.A control group of human serum samples ranged up to 2.1 U/l. The maximum serum activity in samples of patients with acute pancreatitis was 126 U/l. In human ascites activities up to 380 U/l were measured. The method described here turned out to be a practicable instrument for the determination of phospholipase A₂ activity using only commercially available reagents.Abbreviation PlA₂ Phospholipase A₂     

A rapid phospholipase A2 bioassay using14C-oleate-labelledE. coli bacterias

Summary Two methods of phospholipase A₂ determination using¹⁴C-labelledE. coli bacterias as substrate were compared. One method works with a filter membrane for separation of cleaved¹⁴C-oleate from remaining phospholipids, the other uses the well-known thin-layer chromatography for lipid analysis. Some features of human serum phospholipase A₂ regarding pH and Ca²⁺ dependency were investigated. Possible sources of errors were discussed. It was shown that either method can differentiate between normal and pathologically elevated phospholipase A₂ levels, but that the filter method is superior in terms of sensitivity and workload.Abbreviations PLA₂ phospholipase A₂ - BSA bovine serum albumin - (F)FA (free) fatty acid - TLC thin-layer chromatography     

Bioactive Lysophosphatidylcholine 16:0 and 18:0 Are Elevated in Lungs of Asthmatic Subjects

Purpose

Asthma is a chronic inflammatory disease of the airways, and is associated with upregulation of phospholipase A₂ (PLA₂), the enzyme that hydrolyzes phosphatidylcholine, producing lysophosphatidylcholine (LPC) and free fatty acids. LPC is a lipid mediator with known pro-inflammatory and pro-atherogenic properties, and is believed to be a critical factor in cardiovascular diseases. We postulate that asthmatic subjects have an elevated content of LPC in the lung lining fluids.

Methods

Eight non-asthmatic controls and seven asthmatic subjects were recruited for broncho-alveolar lavage fluids (BALF) collection for analysis of LPC by high performance liquid chromatography-tandem mass spectrometry.

Results

LPC16:0 and LPC18:0 were significantly elevated in the BALF of asthmatics with impaired lung function characteristic of moderate asthma, but not mild asthma. The increased LPC content in BALF was accompanied by increased PLA₂ activity. Furthermore, qRT-PCR analysis of the BALF cell fraction indicated increased secretory PLA₂-X (sPLA₂-X).

Conclusions

The increased LPC content in the lung lining fluids is a potential critical lipid mediator in the initiation and/or progression of airway epithelial injury in asthma.     

Effect of serum from cardiovascular patients on catalytic activity of secretory phospholipase A2 (IIA)

Incubation of patients’ serum catalytically active by type IIA secretory phospholipase A₂ (SP-IIA) with serum containing the enzyme in a high concentration but exhibiting no catalytic activity in 1:1 volume ratio led to a significant inhibition of SP-IIA catalytic activity. Donor and patient sera with low levels of SP-IIA had no effect on the serum with SP-IIA activity under these conditions. However, the increase in the content of patients’ serum with a low level of SP-IIA in the incubation mixture to 1:2 (v/v) and of donor serum to 1:3 (v/v) also led to blockade of SP-IIA catalytic activity. These results indicate that human serum contains an SP-IIA inhibitor and its concentration decreases significantly in sera with SP-IIA activity. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 11, pp. 525–527, November, 2006     

Alterations in arachidonic acid metabolism in mouse mast cells induced to undergo maturation in vitro in response to stem cell factor

We studied arachidonic acid (AA) metabolism during the maturation of bone marrow–derived cultured mast cells (BMCMCs) into mast cells with phenotypic characteristics, which were more similar to those of connective tissue–type mast cells. BMCMCs were maintained in medium containing 100 ng/ml recombinant rat stem cell factor (SCF) for 1 to 6 weeks. After 3 to 4 weeks in SCF, BMCMCs acquired many phenotypic characteristics of maturation, including enlarged size, numerous electron-dense cytoplasmic granules, and a 50-fold elevation in histamine content. Maintenance in SCF for 6 weeks did not significantly alter the amounts or species of eicosanoids that were produced by BMCMCs stimulated with calcium ionophore A23187. However, SCF-treated mast cells released 2.6 ± 0.13 times more free AA and accumulated 6.4 ± 1.0 times higher levels of intracellular free AA than did immature BMCMCs not exposed to SCF. There was no increase in the mobilization of other fatty acids (e.g., linoleic or oleic acid), indicating specificity for AA. Moreover, there were no differences between the 5-lipoxygenase activities of SCF-treated or untreated cells, as assayed in cell homogenates prepared by nitrogen cavitation. Although the total AA content in SCF-treated cells was significantly elevated, the distribution of AA in phospholipid and neutral lipid classes was not altered by SCF treatment. Total phospholipase (PL)A₂ activity increased 85% ± 11.5% in SCF-treated cells. In homogenates of immature BMCMCs, 51.0% ± 13.7% of the PLA₂ activity was inhibited by 0.5 mmol/L dithiothreitol, whereas the same concentration of dithiothreitol caused only a 2.2% ± 10.7% reduction in the PLA₂ activity in homogenates of SCF-treated BMCMCs (p ≤0.05, n = 4). These findings suggest that SCF treatment induces a dithiothreitol-resistant PLA₂ and that this PLA₂ may contribute to the mobilization of AA that is not further metabolized to eicosanoids. (J ALLERGY CLIN IMMUNOL 1996;97:1329-41.)     

Involvement of oxygen radicals and phospholipase A2 in acute pancreatitis of the rat

Summary The purpose of this study was to assess the involvement of oxygen radicals and phospholipase A₂ in acute pancreatitis. Acute pancreatitis was induced in rats by the CCK-analogue cerulein (5 µg/kg · h) for 30 min, 3.5 h, and 12 h. At the end of the infusion, serum enzymes and the lipid peroxidation products conjugated dienes and malondialdehyde in the tissue were measured. Moreover, tissue samples underwent lightmicroscopical examination. After 3.5 h cerulein, an interstitial edema and a beginning accumulation of granulocytes in the pancreatic gland is observed. These changes are aggravating within 12 h, leading to tissue necrosis and migration of the granulocytes into the tissue. Concomitantly amylase and lipase increase by 15 and 35 times, respectively. Conjugated dienes and malondialdehyde increase already after 30 min cerulein and reach their highest levels after 3.5 h cerulein. At the same time the tissue activity of phospholipase A₂ is elevated three fold. Rats were treated with superoxide dismutase and catalase before cerulein infusion. Treatment significantly prevents tissue necrosis, granulocyte accumulation, and edema formation. The enhanced activity of phospholipase A₂, however, is unaffected by the treatment. Oxygen radicals seem to be instrumental in the development of the disease.     

Involvement of PKCα–MAPK/ERK-phospholipase A2 pathway in the Escherichia coli invasion of brain microvascular endothelial cells

Escherichia coli K1 is the most common Gram-negative organism that causes neonatal meningitis following penetration of the blood–brain barrier. In the present study we demonstrated the involvement of cytosolic (cPLA₂) and calcium-independent phospholipase A₂ (iPLA₂) and the contribution of cyclooxygenase-2 products in E. coli invasion of microvascular endothelial cells. The traversal of bacteria did not determine trans-endothelial electrical resistance (TEER) and ZO-1 expression changes and was reduced by PLA₂s siRNA. cPLA₂ and iPLA₂ enzyme activities and cPLA₂ phosphorylation were stimulated after E. coli incubation and were attenuated by PLA₂, PI3-K, ERK 1/2 inhibitors. Our results demonstrate the role of PKCα/ERK/MAPK signaling pathways in governing the E. coli penetration into the brain.     

Groups I,II and III extracellular phospholipases A2: Selective inhibition of group II enzymes by indomethacin but not other NSAIDs

The three types (groups I, II and III) of stable extracellular 14 kDa phospholipase A₂ enzymes differ in their primary amino acid sequences and their properties. It may thus be possible to design low-molecular weight inhibitors targeted to the secretory form of mammalian PLA₂. this enzyme has been implicated in inflammatory disorders. We have studied the inhibition of four distinct PLA₂ enzymes by a range of NSAIDs, using³H-oleate release from prelabelled membranes ofE. coli for assay. The enzymes used were cobra venom PLA₂ (Naja naja, a group I enzyme), bee venom PLA₂ (Apis mellifera, group III), recombinant human synovial PLA₂ (group II) and rat peritoneal PLA₂ (group II). Under the conditions of the³H-oleateE. coli assay, 1 mM concentrations of aspirin, sodium salicylate, paracetamol (acetaminophen), oxphenbutazone, ibuprofen, flurbiprofen and nabumetone failed to inhibit significantly any of the four enzymes. However, indomethacin inhibited all four enzymes, although effects were greatest on the two group II enzymes (rat peritoneal and human synovial PLA₂). Approximate IC₅₀ values were 28 and 35 M, respectively. Inhibition by indomethacin was not time dependent and was greater at micromolar rather than millimolar levels of calcium. We conclude that indomethacin but not the other tested classes of NSAID inhibits the group II PLA₂ enzyme in a selective manner and suggest that this may be relevant both to its clinical spectrum and to the design of novel pharmaceutical leads.     

Role of Phospholipase A2 in Activation of Isolated Cardiomyocyte Respiration in Postinfarction Cardiosclerosis

The rate of oxygen consumption by isolated cardiomyocytes was studied in rats with experimental postinfarction cardiosclerosis. The increase in oxygen consumption under these condition was comparable to that in melittin- and arachidonic acid-induced activation of phospholipase A₂ in cardiomyocytes of intact animals. Bromophenacyl bromide inhibition of phospholipase A₂ in cardiomyocytes of rats with postinfarction cardiosclerosis led to reduction of oxygen consumption rate to values characteristic of intact animal cardiomyocytes. The results confirm the hypothesis according to which high oxygen consumption in postinfarction cardiosclerosis is related to increased activity of phospholipase A₂. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 12, pp. 631–633, December, 2008     

Role of phospholipase A2 in prepulse inhibition of the auditory startle reflex in rats

High levels of calcium-independent phospholipase A₂ (iPLA₂) are present in the striatum and cerebral cortex [W.Y. Ong, J.F. Yeo, S.F. Ling, A.A. Farooqui, Distribution of calcium-independent phospholipase A₂ (iPLA₂) in monkey brain, J. Neurocytol. 34 (2005) 447–458], and several clinical investigations have suggested a possible role of altered iPLA₂ activity in neurodegenerative and psychiatric disorders. The present study was carried out to elucidate a possible effect of PLA₂ on prepulse inhibition (PPI) of the acoustic startle reflex. Rats that received intraperitoneal injection of the non-specific PLA₂ inhibitor, quinacrine, showed significantly decreased PPI at 76, 80, and 84 dB, compared to saline injected controls. In addition, rats that received intrastriatal injection of antisense oligonucleotide to iPLA₂ showed significant reduction in PPI at prepulse intensities of 76 and 84 dB compared to scrambled sense injected controls. Together, these findings point to a role of PLA₂ in PPI of the auditory startle reflex and sensorimotor gating.     

Phospholipase A2 activity and lipid peroxidation during endotoxemia under conditions of experimental peritonitis

The severity of endotoxemia in peritonitis depends on morphofunctional state of the intestine determined by the intensity of free-radical lipid peroxidation and phospholipase A₂ activity, which are the highest on day 1 postoperation. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 1, pp. 31–33, January, 2000     

Phospholipase A2 — Regulation and inhibition

Summary Phospholipase A₂ (PLA₂) has been implicated in the pathogenesis of different diseases. Thus, the pharmacological intervention of PLA₂ activity by specific inhibitors is of great therapeutical value in ameliorating pathological conditions. Despite a great number of published data regarding PLA₂ inhibitors none has reached clinical application. Since enzyme activity can be greatly influenced by the experimental conditions of the test system used, a potentin vitro enzyme inhibitor does not indicate therapeutic effectiveness per se. In order to enhance the predictable value of anin vitro screening system for PLA₂ inhibitors, a battery of test systems each measuring certain parameters should be applied. Considering the complex mechanism(s) of PLA₂ it is extremely important to elucidate the exact inhibition mechanism of those compounds, which have passed these first filters. True inhibitors of PLA₂ should then be evaluated in suitableex vivo, in vivo models.     

In vitro inhibition of phospholipase A2 by gabexate mesilate,camostate, and aprotinine

Summary Gabexate mesilate (FOY, ethyl-p-(6-guanidino-hexanoyl-oxy)-benzoate-methansulfonate), camostate (N,N-dimethyl-carb-amoylmethyl-4-(guanidinobenzoyloxy)-phenyl-acetate) methansulfonate and aprotinine (Trasylol) were tested for possible inhibition of phospholipase A₂. Gabexate mesilate at a concentration of 5×10^–4 mol/l and camostate at a concentration of 10^–3 mol/l caused a 50% reduction in enzyme activity. There was almost no inhibition by aprotinine at clinical doses; 40 million KIU/l were necessary to reduce phospholipase A₂ activity by 20%. From the therapeutic dose (4,000 mg/day per i.v. infusion) and the half-life of gabexate mesilate in blood circulation (1 min) it can be calculated that the in vitro concentration of gabexate mesilate is only 10^–6 to 10^–7 mol/l. Under these conditions gabexate mesilate cannot diminish the in vivo enzyme activity of phospholipase A₂.     

Action of lipases on low-density serum lipoproteins

During the action of phospholipase A₂ on low-density serum lipoproteins (LDL) in the presence of serum albumin as adsorbent for low-molecular-weight products of lipolysis a change takes place in the physicochemical properties of the LDL, expressed as a decrease in their flotation coefficients. Lipase hydrolyzes the triglycerides of LDL after preliminary treatment of the latter with phospholipase A₂. Under the influence of lipases the LDL residue becomes insoluble and a cholesterol-rich precipitate is formed. Loss of solubility of lipoproteins following treatment with lipases and proteases may take place in certain states in vivo and may provide the basis for the formation of atheromas, i.e., it may be one of the factors in the pathogenesis of atherosclerosis.Laboratory of Physical Chemistry of Biopolymers, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Orekhovich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 8, pp. 173–175, August, 1977.