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     

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.     

Characterization of phospholipase A2 activity in aspirates of human pancreatic pseudocysts after isolation by reversed-phase high performance liquid chromatography

Summary Phospholipase A (PLA) is able to attack membrane phospholipids and thereby plays a putative role in the pathogenesis of pancreatic pseudocysts. We looked for PLA₂-like activity in aspirates from human pancreatic pseudocysts. In material originating from one cyst which occurred shortly after an acute pancreatitis attack, hydrolyzing enzymatic activity measured by a sensitive bioassay system for PLA₂ activity was found without prior trypsin activation (67×10³ U/min/100 µl). A biochemical characterization of this hydrolyzing enzymatic activity was provided after resolution of the respective proteins contained in the cyst fluid by HPLC. High hydrolyzing activities were found in correspondence to one specific, early eluting peak. The purified enzyme had pH optima at 3.5 and 6. Addition of EDTA (5 mM) to the test system abolished the enzymatic activity which mirrored the requirement for calcium ions. The activity was optimal at calcium concentrations ranging from 1–2 mM. Higher calcium concentrations reduced the enzymatic activity. The enzyme showed high heat stability. SDS-gel analysis of the peak showed one single band with a molecular weight of about 20,000 Daltons. Our findings demonstrate the possibility of activated, PLA-like activity in human pancreatic pseudocyst fluid. We speculate that an inappropriate activation of this enzyme in peri- or intrapancreatic fluid collections could account for pseudocyst formation after an acute pancreatitis attack.     

Differential inhibition of human secretory and cytosolic phospholipase A2

The roles and relative contributions of secretory and cytosolic phospholipases A₂ in physiology and pathology are not precisely known. In a search for differential inhibitors of these enzymes, which could serve as tools to clarify this issue, we evaluated the potencies of reference compounds and three series of new compounds, viz. substrate analogues, 1,2-amino alcohols and enolized -tricarbonyl derivatives, as inhibitors of secretory phospholipase A₂ from human polymorphonuclear leukocytes (sPLA₂) and of cytosolic phospholipase A₂ from human U937 cells (cPLA₂). With few exceptions, the compounds selected are potent inhibitors of sPLA₂ with IC₅₀ values (concentration inhibiting 50%) in the low micromolar range. Inhibition of cPLA₂ was only observed with some phosphate-free substrate analogues, with 1,2-amino alcohols and two of seven reference compounds. These results suggest that inhibition of secretory and of cytosolic phospholipases A₂ are independent effects. Several inhibitors could be identified with a marked selectivity for sPLA₂.This work has been presented in part at the Symposium Phospholipase A₂, Basic and Clinical Aspects in Inflammatory Diseases Reisensburg Castle (FRG), November 18–20, 1992.     

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.     

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     

Serum phospholipase A2 in intensive care patients with peritonitis,multiple injury,and necrotizing pancreatitis

Summary To study the source and role of circulating phospholipase A₂ (PLA₂) catalytic activity we monitored the serum from patients with necrotizing pancreatitis (n=8), diffuse peritonitis (n=6), and multiple injuries (n=11). Immunoreactive PLA₂ serum protein concentration was analysed using a fluoroimmunoassay based on an antibody against human pancreatic PLA₂. Serum PLA₂ catalytic activity was analysed using a radiochemical method based on a substrate with tritiated palmitic acid in beta position. In necrotizing pancreatitis immunoreactive PLA₂ and PLA₂ catalytic activity both increased. Obviously, in necrotizing pancreatitis the major part of serum catalytic activity stems from the pancreas. In patients with diffuse peritonitis and multiple injuries, as a rule, immunoreactive phospholipase A₂ serum concentration appears to be within the normal range. In contrast, in these patients we demonstrated high serum catalytic PLA₂ activity comparable to that in necrotizing pancreatitis. The source of catalytic PLA₂ activity in peritonitis and multiple injuries seems not to be the pancreas. There was a correlation between pulmonary insufficiency and serum PLA₂ catalytic activity in patients with necrotizing pancreatitis, peritonitis, and multiple injuries.     

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.     

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.     

Hyperphospholipasemia A2 in human volunteers challenged with intravenous endotoxin

Phospholipase A₂ (PLA₂) activity was measured in the serum of 23 individuals infused intravenously with endotoxin (EN) at a dose of 4 ng/kg body weight. A marked increase in PLA₂ was noted 3 h after EN challenge (mean 828 ± 513 units/ml), reached its maximum at 24 h after the challenge (mean 2667 ± 2442 units/ ml), and was still evident at 48 h (mean 763 ± 366 units/ml). In contrast, TNF levels were maximal (mean 712 ± 375 pg/ml) 90 min after the EN challenge and subsided to very low values (5 ± 5 pg/ml) 5 h after the challenge. There was a positive correlation between the maximum response of TNF and that of PLA₂ (r = 0.82,P < 0.01). Administration of ibuprofen or pentoxifylline did not alter the PLA₂ response. EN challenge did not affect serum pancreatic PLA₂ concentration or that of the lysosomal cationic enzyme, lysozyme. Neutralizing antibody against human recombinant (synovial type) PLA₂ completely abolished PLA₂ activity in the sera tested. We conclude that EN infusions cause marked intravascular release of nonpancreatic secretory PLA₂ and that the magnitude of this response seems to be related to the prior generation of TNF.     

Phospholipase A2 induced diffuse alveolar damage — Effect of indomethacin and dexamethasone upon morphology and plasma-histamine level

Summary Phospholipase A₂, injected as a bolus into the jugular vein of adult male Wistar rats was investigated with respect to its action upon lung morphology and blood- plasma- histamine. In comparison with the injection of saline, phospholipase A₂ causes hyperemia of the lungs, sequestration of granulocytes and intraalveolar pulmonary edema; the histamine level is increased to the sixfold. Pretreatment of the animals with indomethacin diminishes the toxic effect of phospholipase A₂ upon the lungs and the histamine level.Supported by J. u. F. Marohn Stiftung an der Friedrich Alexander Universität Erlangen-Nürnberg     

Acid phospholipase A1 in liver — A brief survey

Summary Acid phospholipase A₁ activity in liver (rat, human) is predominantly localized in lysosomes. A minor proportion (less than 3% of the total activity) is also present in the Golgi apparatus and the endoplasmic reticulum, presumably due to enzymatically active precursors of the corresponding lysosomal enzyme. Lysosomal phospholipase A₁ is the most important enzyme initiating the intralysosomal catabolism of diacylphosphoglycerides. It has been purified 50,600-fold, with a yield of about 26%. The enzyme prefers phosphatidylethanolamine as a substrate, which at 200 µM and pH 4.5, is hydrolysed at a rate of approximately 8.2 U/mg. Lysosomal phospholipase A₁ is a glycoprotein of about 29 kDa with an isoelectric point of pH 5.3. Unspecific extralysosomal endogenous inhibitors of this enzyme are pH range, inorganic cations, and various proteins. Divalent cations are more potent inhibitors than monovalent ones. Most endogenous intra- and extracellular proteins inhibit the enzyme, the cationic species exhibiting high inhibitory potencies, glycoproteins only little. Inhibitory proteins act through their binding to the substrate. Lysosomal phospholipase A₁ seems to be an important target in drug-induced lipidosis. This lipid storage disease is caused by various cationic amphiphilic drugs that are trapped intralysosomally by protonation. In lysosomes such compounds raise the pH, interact with the polar lipids to be degraded and the lysosomal lipolytic enzymes, such as phospholipase A₁. These mechanisms result in impaired intralysosomal phospholipid degradation and hence in intralysosomal phospholipid accumulation.Dedicated to Professor Walther Vogt on the occasion of his 70th birthday     

STUDY OF THE α-N-ACETYLGALACTOSAMINYLTRANSFERASE IN SERA AND RED CELL MEMBRANES OF HUMAN A SUBGROUPS

The product of the A blood group gene in the erythrocyte membrane and serum from ‘weak A’ variants was investigated using low and high molecular weight acceptors and compared with common A₁ and A₂ blood samples. A reduced enzyme activity was detected, but only in some of the A variants, namely the A₃ (eight out of eleven), A_m or A_y samples. Other sera from A₃ (three out of eleven), A_x, A_end or A_el individuals were apparently devoid of enzyme activity. A kinetic study by temperature inactivation of A blood group sera also showed that A enzymes are more labile than B enzymes from B_I or B_II sera. Moreover, in one case (A₃ sample Del.), a very fast inactivation of the A enzyme was observed, suggesting the occurrence of a variant enzyme qualitatively different from the others so far studied. The erythrocyte membrane preparations from all A variants contained no detectable A enzyme activity except for the A₁ and A₂ samples, the former being three to five times more active than the latter. The B enzyme activities from four samples of B RBC tested were comparatively stronger than the A enzyme activity of A₁ RBC. The results were discussed and it was suggested that the synthesis and/or the secretion of the A enzyme in the organism is not uniform from one tissue to another, but could depend on which of the ‘weak A’ alleles or modifier genes is involved.     

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.     

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.     

Immunocytochemical evidence of phospholipase A2 in pancreatic tumors — Diagnostic values

Summary Phospholipase A₂ is an enzyme which is produced in acinar cells, and persists even in regressive states of chronic pancreatitis, when the production of other enzymes diminishes. We therefore tested this enzyme as a marker of acinar descent in various pancreatic tumors. This enzyme could be seen in 50% of the acinar-cell carcinomas, in 60% of solid and papillary pancreatic tumors, and in 50% of microglandular carcinomas. Ductal cancers and isletcell cancers were negative. In contrast to other markers of acinar matrix (amylase, antitrypsin), phospholipase A₂ gave fewer false-positive or false-negative results.Supported by a grant of the J. and F. Marohn Stiftung at the University of Erlangen/Nürnberg     

Expression of Human Secretory Group IIA Phospholipase A2 is Associated with Reduced Concentrations of Plasma Cholesterol in Transgenic Mice

It is well known that acute and chronic inflammatory reactions are accompanied by markedly decreased concentrations of plasma total cholesterol. However, the mechanisms underlying this hypocholesterolemia are not yet completely understood. To explore the question of whether an increased serum activity of secretory group IIA phospholipase A₂ (sPLA₂) may contribute to the development of hypocholesterolemia during inflammation, the lipids and lipoprotein patterns in the plasma of transgenic mice overexpressing the human sPLA₂ gene were studied and compared with those of nontransgenic controls. The mean plasma enzyme activities determined by using [¹⁴C]-oleate labeled Escherichia coli-membranes were found to be 331 ± 262 U/l in transgenic mice while the catalytic activity in plasma of controls was below the analytical sensitivity of the assay (0.5 U/l). Compared to nontransgenic littermates, sPLA₂-transgenic mice exhibited significantly lower plasma concentrations of total cholesterol (2.53 ± 0.37 mmol/l vs. 3.49 ± 0.44 mmol/l, p < 0.0001). The reduction of total cholesterol was due to decreased HDL and LDL cholesterol levels (1.21 ± 0.10 mmol/l vs. 1.78 ± 0.37 mmol/l, and 0.28 ± 0.02 mmol/l vs. 0.69 ± 0.23 mmol/l, respectively, p < 0.05). The analysis of lipoprotein composition indicated that the LDL of transgenic mice were selectively depleted in free and esterified cholesterol, whereas HDL of the two animal groups contained comparable percentages of cholesterol. The triglycerides were significantly enriched in LDL and HDL, but tended to be less in VLDL of transgenic mice. In conclusion, the results of the study have demonstrated that the expression of sPLA₂ may influence the metabolism of lipoproteins, possibly contributing to the development of hypocholesterolemia during inflammation.     

Human Hemoglobin Shares Bioactivities Ascribed to Human Tumor Necrosis Factor‐α

Hemoglobin mediated cytotoxicity and apoptosis has been evaluated in Tumor necrosis factor‐α (TNF‐α) sensitive cell line, U937 and compared with TNF‐α. Both species of hemoglobin, Hemoglobin A₂ and Hemoglobin A₀ induced apoptosis and cytotoxicity in U937 cell as measured by flow cytometry and 3‐(4,5‐dihydro‐6‐(4‐(3,4‐dimethoxybenzooyl)‐1‐piperazinyl)‐2(1H)‐quinoline (MTT) assay respectively. Different concentration of Hemoglobin A₀ (4 ng/mL to 4000 ng/mL) induced apoptosis ranging from 9% to 16% in U937 cells. 4000 ng/mL hemoglobin A₀ showed maximal apoptotic cells. TNF‐α showed 87% apoptotic U937 cells at concentration of 1 pg/mL. HbA₀ displayed cytotoxicity in U937 cell line at higher concentration in comparison to TNF‐α. 4000 ng/mL of hemoglobin A₀ showed optimal cytotoxic response in U937 cells. A dose response curve was also observed with varying doses of hemoglobin A₀. U937 cells pretreated with serum activated LPS for 1 hr and incubated with different concentration of hemoglobin or human TNF‐α for 24 h reduced the cytotoxic effect on U937. Dexamethasone treatment of U937 cells helped in protecting the HbA₀ and HbA₂ mediated cytotoxicity and anti‐TNF‐α antibody neutralized the hemoglobin mediated apoptosis and cytotoxicity. It is therefore apparent that human hemoglobin shares some of the bioactivities previously ascribed to TNF‐α. Sharing of bioactivities of TNF‐α by hemoglobin is interesting and suggests that cell free hemoglobin can mimic TNF‐α functionally.     

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     

The Inhibitory Properties of Group A and B Non-Secretor Saliva

The existence of some form of specifically active A or B blood group substance in the saliva of group A₁, A₂ and B non-secretors was demonstrated. Significant inhibition of agglutination results were obtained when the salivas of these non-secretors were examined in a test procedure using anti-A+B(group O) serum and group A_x red cells. Native saliva specimens from group A₁, A₂ and B secretors and non-secretors were filtered on Sephadex G-200® columns. The eluates were tested for blood group activity by inhibition of agglutination and for relative carbohydrate and protein content. A main excluded glycoprotein, blood group-active fraction was detected in all samples of saliva examined.