In vivo effect of pancreatic phospholipase A2 on the arachidonic acid cascade.

BACKGROUND: In acute pancreatitis, pancreatic phospholipase A2 increases in systemic circulation. Yet the pathophysiological significance is controversial, because previous in vitro studies have shown that the enzyme has little cytotoxicity or ability to activate the arachidonic acid cascade by itself in contrast to other isozymes. AIM OF THE STUDY: The aims of this study are to examine the effect of pancreatic phospholipase A2 on the arachidonic acid cascade in vivo; to explain the discrepancy, if present, between in vitro and in vivo findings; and to reassess the pathophysiological significance of circulating pancreatic phospholipase A2. METHODS: Pancreatic phospholipase A2 was infused intravenously in guinea pigs, and changes in the arachidonic acid cascade, plasma lipoprotein, and cardiopulmonary function were investigated. RESULTS: Plasma concentrations of 6-keto-prostaglandin F1alpha, prostaglandin E2, and thromboxane B2 increased after intravenous (iv) infusion of pancreatic phospholipase A2. Some of the plasma phospholipids such as phosphatidylcholine and phosphatidylethanolamine decreased, and free dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid were detected in plasma. These changes were accompanied with decreases in blood pressure, heart rate, and base excess. CONCLUSION: Circulating pancreatic phospholipase A2 activates the arachidonic acid cascade, probably by supplying free eicosanoid precursors from plasma lipoprotein to eicosanoid-producing cells. It is supposed to be a cause of systemic complications in acute pancreatitis.     

Effect of a selective thromboxane A2 synthetase inhibitor on the systemic changes induced by circulating pancreatic phospholipase A2

Background In acute pancreatitis, pancreatic phospholipase A₂ (PLA2) in the circulating blood hydrolyzes phospholipids contained in plasma lipoproteins, liberating eicosanoid precursors that are subsequently converted to various eicosanoids. The pathophysiological significance of eicosanoid synthesis via this pathway is unknown. The aim of this study was to clarify the role of thromboxane A₂ (TXA₂) synthesis by circulating pancreatic PLA₂ in the pathogenesis of the systemic complications of acute pancreatitis. Methods Guinea pigs were divided into two groups: a control group and an ozagrel group, which received intravenous administration of ozagrel, a selective TXA₂ synthetase inhibitor. Pancreatic PLA₂ was infused intravenously in both groups for 30 min, and systemic changes during the infusion were examined. Results In the control group, there was an increase in plasma thromboxane B₂ (TXB₂) concentration, a decrease in mean arterial pressure and heart rate, a decrease in arterial base excess (BE), bicarbonate concentration (HCO₃ ⁻), and pH, a decrease in platelet count and plasma fibrinogen concentration, and a shortened prothrombin time during the infusion of pancreatic PLA₂. In the ozagrel-treated group, changes in plasma TXB₂ concentration, BE, HCO₃ ⁻, and platelet count were significantly inhibited. Conclusions TXA₂ synthesis by circulating pancreatic PLA₂ contributes to metabolic acidosis and thrombocytopenia during acute pancreatitis.     

Small-Bowel Mucosal Inflammation in Reticulin or Gliadin Antibody-Positive Patients without Villous Atrophy

According to recent studies, phospholipase A₂ (PLA₂) may be an important factor in the pathogenesis and pathophysiology of acute pancreatitis. Increased serum PLA₂ activities and concentrations have been measured in patients with acute pancreatitis. Serum PLA₂ activities have been shown to correlate with the severity and prognosis of the disease. To study the different methods of PLA₂ determination, we measured the PLA₂ activity by means of an isotopic assay method and the concentration by a radioimmunologic method in several body fluids of 52 consecutive patients with acute pancreatitis. PLA₂ activity and concentration were detected in all of the patient body fluids. The serum PLA, activities were 2.5-fold higher (mean ± SD, 7.6 ± 6.0) than normal activities, and the concentrations were 9.6-fold higher (mean ± SD, 41 ± 88). The enzyme activities and concentrations correlated well in ascites, fluids from the pleural cavity, and peritoneal lavation and poorly in serum, urine, and fluid from pancreatic pseudocyst.     

The diagnostic value of serum pancreatic phospholipase A2 (PLA2) in pancreatic diseases

The diagnostic significance of serum immunoreactive pancreatic phospholipase A₂ (PLA₂) was studied in 119 patients with pancreatic disease, 200 with various non-pancreatic disease, and 203 healthy controls using radioimmunoassay (RIA) specific to human pancreatic PLA₂. This newly developed RIA using monoclonal antibody was satisfactorily sensitive and reliable. Serum PLA₂ was elevated in all six patients with acute pancreatitis. Frequency of abnormal serum PLA₂ levels was 60% in chronic pancreatitis (n=52) and 67% in pancreatic cancer (n=61). Serum PLA₂ levels were low in chronic pancreatitis with severe exocrine insufficiency and advanced pancreatic cancer. In chronic pancreatitis, patients with low serum PLA₂ level showed lower enzyme output in secretin test than patients with normal or high serum PLA₂ level. Frequency of abnormal PLA₂ levels was 27% in non-pancreatic disease and, in particular, patients with renal failure showed high PLA₂ levels. Sensitivity (62%) and efficiency (69%) of serum PLA₂ assay in pancreatic disease were superior to those of amylase. In conclusion, serum PLA₂ determination using RIA was useful for the diagnosis of acute pancreatitis by high serum PLA₂ levels and the diagnosis of severe exocrine pancreatic insufficiency by low serum PLA₂ levels. This work was supported in part by a research grant for intractable pancreatic disease and a pancreatic cancer grant from the Ministry of Health and welfare of Japan.     

Increased activity of group II phospholipase A2 in plasma in rat sodium deoxycholate induced acute pancreatitis

Background—Two different types of secretoryphospholipase A₂ (PLA₂), pancreatic group I(PLA₂-I) and non-pancreatic group II (PLA₂-II),have been identified and postulated to be associated with thepathogenesis of various diseases, such as acute pancreatitis, septicshock, and multiple organ failure.
Aims—To investigate the type of secretoryPLA₂ responsible for its catalytic activity found in plasmaand ascites of experimental acute pancreatitis.
Methods—Acute pancreatitis of differing severitywas induced by the injection of different concentrations (1% or 10%)of sodium deoxycholate (DCA) into the common biliopancreatic duct inrats, and catalytic PLA₂ activity in plasma and asciteswere differentiated by anti-PLA₂-I antibody and specificinhibitor of PLA₂-II. Survival rate and plasma amylase,aspartate aminotransferase (AST), and alanine aminotransferase (ALT)were also measured.
Results—In 1% and 10% DCA induced acutepancreatitis, plasma amylase values as well as PLA₂activity in ascites were greatly increased. PLA₂ activityin plasma was also notably increased in 10% DCA induced acutepancreatitis, but not in 1% DCA induced acute pancreatitis.PLA₂-I specific polyclonal antibody significantly inhibitedPLA₂ activity in ascites but not that in plasma. In contrast, plasma PLA₂ activity was completely suppressed byPLA₂-II specific inhibitor. In addition, a high mortality(93% at five hours) and a significant increase in plasma AST and ALTwere noted in 10% DCA induced pancreatitis.
Conclusion—Ascites PLA₂ activity ismainly derived from PLA₂-I, whereas plasma PLA₂activity is mostly derived from PLA₂-II in severe acutepancreatitis, suggesting that increased plasma PLA₂-IIactivity might be implicated in hepatic failure arising after severeacute pancreatitis.

Keywords:acute pancreatitis; phospholipase A₂; sodium deoxycholate pancreatitis; hepatic failure

     

EDITORIAL

The changes in contents of pancreatic carboxyl ester lipase, phospholipase A₂, and lingual lipase in rats with streptozotocin (STZ)-induced diabetes have been studied. The contents of pancreatic carboxyl ester lipase and phospholipase A₂ decreased by 40% and 45%, respectively, 5 days after injection of STZ, whereas pancreatic lipase steadily increased to 100% over control. The content of lingual lipase decreased sharply by more than 90% 2 days after STZ injection, followed by a tendency to recover slightly. Insulin treatment at a dose abolishing the urine glucose in diabetic rats for 3 days restored the contents of pancreatic lipase, carboxyl ester lipase, and lingual lipase but not pancreatic phospholipase A₂. The results indicate that lack of insulin action induces an anticoordinate change in gastrointestinal lipolytic enzymes, with decreases in pancreatic carboxyl ester lipase, phospholipase A₂, and lingual lipase contents and an increase in pancreatic lipase content.     

Role of various phospholipases A2 and inhibitors in the pathogenesis and prevention of pancreatic acinar cell necrosis

Summary Background. Phospholipase A₂ (PLA2) may play a central role in the pathogenesis of pancreatic acinar cell necrosis. Several questions, however, are unsolved: Is acinar cell necrosis caused by PLA2 derived from infiltrating leukocytes or from pancreatic PLA2 itself? Does PLA2 cause cellular lysis by the release of lysolecithin from lecithin or by generation of free radicals? The aims of this study were to determine which form of PLA2 is responsible for cellular damage and how to inhibit its action. Methods. Isolated rat pancreatic acini were prepared by collagenase digestion. Newly synthesized proteins were labeled by ³⁵S-methionine. Acini were incubated in buffer to which various factors, such as porcine pancreatic PLA2 or bee venom PLA2, homogenates of either leukocytes or pancreatic homogenates, all with or without lecithin and with or without potential inhibitors (aprotinin, 4-bromophenacylbromide, BM 16.2115, quinacrine, various analogs of arachidonic acid), or free radicals (hydrogen peroxide, xanthine/xanthine oxidase) with or without allo-purinol or dismutase/catalase were added. Cellular destruction was measured by the release of radiolabeled proteins. Results. PLA2 alone, free radicals, and granulocytes were not harmful to acini within 30 min of incubation. Free radicals caused significant release of radiolabeled proteins only after 3 h of incubation; this release could be inhibited by scavengers. Incubation of pancreatic acini with PLA2 in combination with lecithin caused rapid release of radiolabeled proteins. Addition of high concentrations of enterokinase activated pancreatic homogenates both alone and with lecithin caused release of cellular proteins, suggesting that pancreatic PLA2 uses lecithin from pancreatic membranes as substrate. Almost all tested potential inhibitors of PLA2 were unable to prevent the destruction caused by either pancreatic or bee venom PLA2 and lecithin. However, HK 42, a polyunsaturated fatty acid analog, was able to reduce dose dependently the release of acinar proteins caused by pancreatic PLA2 and lecithin. Conclusion. Pancreatic PLA2 and not PLA2 from infiltrating leukocytes may play a role in pancreatic acinar cell necrosis. Cellular lysis is caused upon the action of lysolecithin and probably not via the action of free radicals.     

Serum Phospholipase A2, Immunoreactive Trypsin,and Trypsin Inhibitors During Human Acute Pancreatitis

Serum immunoreactive trypsin and phospholipase A₂ were analyzed at regular intervals in seven patients hospitalized as a result of acute hemorrhagic pancreatitis. α₁-Antitrypsin and α₂-macroglobulin levels and trypsin-inhibitor capacity of serum were determined simultaneously. Serum trypsin concentrations were markedly raised in all patients. The levels of immunoreactive trypsin remained elevated for longer periods than those of urinary amylase. α₁-Antitrypsin and trypsin-inhibitor capacity were also significantly increased as compared with the post-illness values, but α₂-macroglobulin decreased considerably, reaching the lowest levels on the 5th day after admission. Consequently, phospholipase and trypsin are released to the circulation during hemorrhagic pancreatitis, but the increase in trypsin is compensated for by an increase in trypsin-inhibitor capacity of serum due to elevated α₁-antitrypsin levels. The decrease of α₂-macroglobulin in hemorrhagic pancreatitis was one of the most interesting findings, and it is proposed that this inhibitor may be consumed in the elimination of proteases through the reticuloendothelial system. The two patients who died had higher phospholipase values than those who recovered, but the prognosis could not be predicted from the values of the other measured variables.     

Dual pathways for carbamylcholine-stimulated arachidonic acid release in rat pancreatic acini

Recent studies suggested the involvement of arachidonic acid in the mediation of pancreatic amylase release. However, an effect of carbamylcholine on arachidonic acid release has not yet been reported in the exocrine pancreas. This study was performed to evaluate the effect of carbamylcholine on arachidonic acid release and determine the underlying intracellular mechanisms. From enzymatic assays, phospholipase A₂ and diacylglycerol lipase were activated by carbamylcholine and these activations were inhibited by the phospholipase A₂ inhibitors, mepacrine and aristolochic acid, and by the diacylglycerol lipase inhibitor RHC 80267. Carbamylcholine also increased arachidonic acid release in a concentration-dependent manner. Both phospholipase A₂ and diacylglycerol inhibitors partially inhibited carbamylcholine-stimulated arachidonic acid release. The phospholipase C inhibitor U73122 and the protein kinase C inhibitor staurosporine also caused partial inhibition. Arachidonic acid release by carbamylcholine was suppressed by the simultaneous addition of RHC 80267 with either phospholipase A₂ inhibitors. Our data demonstrate that phospholipase A₂ and diacylglycerol lipase are activated and arachidonic acid is released in pancreatic acini by carbamylcholine. Dual pathways are responsible for carbamylcholine-induced arachidonic acid release. One such pathway involves the sequential action of phospholipase C, protein kinase C and diacylglycerol lipase, whereas the other involves phospholipase A₂ activation.     

Hydrocortisone Treatment of Early SIRS in Acute Experimental Pancreatitis

This work studied the effects of hydrocortisone treatment in experimental acute pancreatitis on cytokines, phospholipase A₂, and breakdown products of arachidonic acid and survival. Edematous and necrotizing pancreatitis were induced in Wistar rats by cerulein hyperstimulation and retrograde intraductal infusion of sodium taurocholate, respectively. Hydrocortisone (10 mg/kg) was administered intravenously 10 minutes after induction of acute pancreatitis. Serum was assayed for phospholipase A₂; interleukin (IL) 1, IL-6, IL-10, thromboxane B₂; Prostaglandin E₂; and leukotriene B₄ at five different time points. A significant release of inflammatory mediators was seen only in the severe model. Hydrocortisone powerfully suppressed arachidonic acid breakdown products and only mildly attenuated the systemic increase of phospholipase A₂ and pro- and antiinflammatory cytokines. The mortality rate after 72 hr in the severe model was 86%. Hydrocortisone treatment reduced mortality to 13% (P = 0.001; Fisher's exact test). Hydrocortisone seems to be effective in the treatment of the early systemic inflammatory response syndrome associated with severe acute pancreatitis.     

Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase: a potential new risk factor for coronary artery disease

A specific and robust immunoassay for the lipoprotein-associated phospholipase A₂ (Lp-PLA₂), platelet-activating factor acetylhydrolase, is described for the first time. The immunoassay was used to evaluate possible links between plasma Lp-PLA₂ levels and atherosclerosis risk amongst susceptible individuals. Such an investigation was important because Lp-PLA₂ participates in the oxidative modification of low density lipoprotein by cleaving oxidised phosphatidylcholines, generating lysophosphatidylcholine and oxidised free fatty acids. The majority of Lp-PLA₂ was found associated with LDL (approximately 80%) and, as expected, enzyme levels were significantly positively correlated to LDL cholesterol. Plasma Lp-PLA₂ levels were significantly elevated in patients with angiographically proven coronary artery disease (CAD) when compared with age-matched controls, even though LDL cholesterol levels did not differ significantly. Indeed, when included in a general linear model with LDL cholesterol and other risk factors, Lp-PLA₂ appeared to be an independent predictor of disease status. We propose, therefore, that plasma Lp-PLA₂ mass should be viewed as a potential novel risk factor for CAD that provides information related to but additional to traditional lipoprotein measurements.     

The role of phospholipase a2 in pancreatic acinar cell injury

The integrity of rat pancreatic acinar cells under the influence of human phospholipase A₂ (PLA2) was studied. Isolated pancreatic acini showed no increased discharge of aspartylaminotransferase (ASAT) when incubated either in solutions containing human pancreatic PLA₂ or the bile salt sodium deoxycholate (DEC), the latter in concentrations that augment PLA₂ activity but have no destructive detergent effect. When human pancreatic PLA₂ was injected into the rat pancreatic duct, uneven distribution was observed at 15 min and 3 h in immunohistochemical sections. Edema and a mild inflammatory reaction were the main changes in the pancreas. The necrotic areas seen by light and electron microscopy were quite small and located mostly at the periphery of lobules corresponding the spread of the injected material. Necrosis was of the coagulation type and showed equal extent after the injection of PLA₂ with or without DEC. Internalized human pancreatic PLA₂ was present already 15 min after the injection in the cytoplasm of some intact acinar cells, indicating a functioning protective mechanism. It was concluded that pancreatic acinar cells are quite resistant to PLA₂-catalyzed hydrolysis of membrane phospholipids in vitro, but additional trauma, e.g., pressure caused by intraductal injection, and tissue related factors, such as the mediators of the inflammatory reaction, make acinar cells susceptible to the effect of PLA₂.     

Duodenal secretion of phospholipase A2, amylase, and bicarbonate in chronic pancreatitis

Phospholipase A₂ has been suggested to be involved in the pathogenesis and pathophysiology of acute pancreatitis. We determined phospholipase A₂ and amylase activities in duodenal juice collected during a secretin test from 30 consecutive patients who were suspected to have chronic pancreatitis or biliary disease. The patients underwent endoscopic retrograde cholangiopancreatography (ERCP) the following day. In the 8 patients with ERCP findings of advanced chronic pancreatitis, the mean outputs of phospholipase A₂, amylase, and bicarbonate were reduced by 74%, 72%, and 60% compared to the respective values in the 13 (control) patients without a diagnosis of any pancreatic disorder or jaundice. In the 3 patients with recurrent pancreatitis but normal ERCP findings and in the 6 patients with jaundice the output values were not significantly reduced compared to those in the patients without any pancreatic disorder or jaundice. The outputs of amylase and phospholipase A₂ were not significantly interrelated, whereas the outputs of phospholipase A₂ and bicarbonate correlated well. Receiver characteristic (ROC) curves confirmed the high specificity and sensitivity of phospholipase A₂ or bicarbonate output in patients with ERCP findings of advanced chronic pancreatitis compared to those with no changes in pancreatic ducts, with similar probability values of 0.880 ± 0.111 (SEM), compared to the respective lower value of amylase, 0.676 ± 0.118. Phospholipase A₂ and bicarbonate output proved of equal value as markers of chronic pancreatitis and were superior to amylase output in the secretin test. (Received Mar. 12, 1997; accepted Aug. 22, 1997)     

Role of Phospholipase A2 in Cholesterol Gallstone Formation Is Associated with Biliary Phospholipid Species Selection at the Site of Hepatic Excretion

Phospholipase A₂ plays a role in cholesterol gallstone development by hydrolyzing bile phospholipids into lysolecithin and free fatty acids. Lysolecithin and polyunsaturated free fatty acids are known to stimulate the synthesis and/or secretion of gallbladder mucin via a prostanoid pathway, leading to enhancing cholesterol crystal nucleation and growth, and therefore, the action of phospholipase A₂ is associated, in part, with bile phospholipid fatty acid. To clarify this hypothesis, we evaluated the effect on bile lipid metastability in vitro of replacing phospholipids with lysolecithin and various free fatty acids. Supersaturated model biles were created with an identical composition (cholesterol saturation index, 1.8; egg yolk lecithin, 34 mM; taurocholate, 120 mM; cholesterol, 25 mM) except for 5%, 10%, or 20% replacement of egg yolk lecithin with a combination of palmitoyl–lysolecithin and a free fatty acid (palmitate, stearate, oleate, linoleate, or arachidonate), followed by time-sequentially monitoring of vesicles and cholesterol crystals using spectrophotometer and video-enhanced differential contrast microscopy. Replacement with hydrophilic fatty acids (linoleate and arachidonate) reduced vesicle formation and promoted cholesterol crystallization, whereas an enhanced cholesterol-holding capacity was evident after replacement with hydrophobic fatty acids (palmitate and stearate). These results indicate that the effect of phospholipase A₂ on bile lithogenecity is modulated by the fatty acid species in bile phospholipids, and therefore, that the role of phospholipase A₂ in cholesterol gallstone formation is dependent, in part, on biliary phospholipid species selection at the site of hepatic excretion.     

Sustained activity of luteal cytosolic phospholipase A2 during luteolysis in pseudopregnant rats

We investigated the expression and activity of cytosolic phospholipase A₂ (cPLA₂) in the corpus luteum during spontaneous and induced luteolysis in pseudopregnant rats. In both models, luteal PLA₂ activity rose in association with functional regression and persisted during the following structural regression. Tissue concentration of prostaglandin F_2α with a luteolytic potency showed a similar fluctuation. The enzyme activity was almost completely suppressed by a cPLA₂-specific inhibitor. Expression of cPLA₂, analyzed by immunohistochemistry, became enhanced during luteolysis with preferential localization to phagocytotic and fibrotic replacement sites. Taken together with our previous finding, the data indicate a persistent elevation in luteal cPLA₂ expression and activity that may affect tissue involution in vivo.     

Ischemia-reperfusion arrhythmias and lipids: Effect of human high- and low-density lipoproteins on reperfusion arrhythmias

The effect of high- and low-density lipoproteins separated from human serum on the postischemic reperfusion arrhythmias was investigated. The hearts were perfused by working heart mode with Krebs Henseleit bicarbonate buffer containing arachidonic acid (1 μg/ml) for 5 minutes. Whole heart ischemia was induced by the use of a one-way ball valve, and hearts were perfused for 15 minutes followed by 20 minutes of reperfusion. Physiologic concentrations of high- and low-density lipoproteins were constantly infused through the atrial route during ischemic perfusion. Coronary effluent was collected via pulmonary artery cannulation for subsequent radioimmunoassay of thromboxane B₂ and 6-keto-prostaglandin F_1α, the major stable metabolites of thromboxane A₂ and prostacyclin, respectively. The incidence of ventricular arrhythmias during reperfusion was 6/6 (100%), 1/6 (17%), and 6/6 (100%) in control, high-density lipoprotein and low-density lipoprotein infusion groups, respectively. There was no significant difference in coronary flow among the three groups throughout the perfusion. Both thromboxane B₂ and 6-keto-prostaglandin F_1α increased significantly during ischemia compared with preischemic values in all groups of hearts. However, the ratio of these two parameters varied in control and low-density lipoprotein infusion groups during ischemia, while there was no significant change in the high-density lipoprotein infusion group. These results provide the possibility that arachidonate metabolites may be involved in the regulation of ischemia-reperfusion arrhythmias and that high-density lipoprotein that was infused during ischemia markedly inhibits the incidence of ischemia-reperfusion-induced ventricular arrhythmias, due in part at least, to stabilizing the arachidonate metabolites during ischemic perfusion.     

Lipoprotein Associated Phospholipase A<Subscript>2</Subscript> Inhibition Reduces Generation of Oxidized Fatty Acids

Purpose Lipoprotein associated phospholipase A₂ (Lp-PLA₂) is an emerging cardiovascular risk marker. After low-density lipoprotein (LDL) oxidation, Lp-PLA₂ generates oxidized nonesterified fatty acids and lysophosphatidylcholine, both of which have demonstrated proinflammatory and proapoptotic activities. Through the use of a selective inhibitor of Lp-PLA₂ (SB-677116), we investigated whether Lp-PLA₂ participates in the ex vivo generation of oxidized fatty acids (ox-FA). Methods Due to the higher correlation between Lp-PLA₂ activity and small LDL particles, we investigated the effects of a selective Lp-PLA₂ inhibition on production of ox-FA in metabolic syndrome subjects with small LDL size <20.5 nm. Whole blood samples were incubated with vehicle (0 μM) or SB-677116 for 6 h at two different concentrations (0.3, 3.0 μM) to determine the effects of inhibitor on Lp-PLA₂ activity, the formation of oxidized esterified and nonesterified hydroxy-fatty acid (OH-FA) or ox-FA in 24 subjects. Results Whole blood incubation with Lp-PLA₂ inhibitor (0.3, 3.0 μM) reduced multiple C-18 OH-FA subclasses (p < 0.05 versus control). For the highly redox-sensitive 9-OH-FA, there was a concentration-dependent reduction in Lp-PLA₂ activity and 9-OH-FA (p _trend = 0.0016) Conclusions In conclusion, selective inhibition of Lp-PLA₂ reduced levels of OH-FA generated in whole blood of metabolic syndrome patients. These novel findings suggest that Lp-PLA₂ inhibition may attenuate some noxious downstream effects of lipid peroxidation that potentially include inflammatory responses. Dr. Rosenson has stock ownership in LipoScience and he serves as a Consultant to LipoScience.     

Phospholipase Activation and Arachidonic Acid Release in Intestinal Epithelial Cells from Patients with Crohn's Disease

A method for studying the mobilization of free arachidonic acid (AA) in viable isolated human intestinal epithelial cells has been developed and applied to the study of patients with Crohn's disease. Cells were isolated from morphologically unaffected parts of the distal ileum and incubated with ¹⁴C-AA; most of the incorporated ¹⁴C-AA was then found in phospholipids (mainly phosphatidylcholine) and in a pool of neutral lipids (mainly triacylglycerols). Cells from patients with Crohn's disease incorporated more ¹⁴C-AA into their neutral lipids than did cells from control patients. When the labeled cells were stimulated with phospholipase C from Clos-tridium perfringens or with the calcium ionophore A23187, they released significant amounts of AA, mainly from phosphatidylcholine. There was no difference between cells from Crohn patients and controls in the ¹⁴C-AA amounts released, but unstimu-lated and phospholipase C-stimulated cells from prednisolone-treated Crohn patients released less AA than cells from control patients. The A23187-stimuiated AA release was completely inhibited by the phospholipase A₂ inhibitor 4-bromophenacyl bromide, whereas the phospholipase C-stimulated release was not. These findings suggest that AA release in human small-intestinal epithelial cells may be caused by calcium-mediated phospholipase A₂ activation or by products of microbial phospholipase C activity and that prednisolone reduces the mobilization of free AA in intestinal epithelial cells. They also illustrate the potential use of isolated epithelial cells for revealing mechanisms underlying AA release in the intestinal mucosa in different disease states.     

Imbalance in A₂ and A₂B phenotype frequency of ABO group in South India

Background

The heterogeneity of A and B alleles results in weak variants of these antigens. Subgroups of A differ from each other quantitatively and qualitatively. The expected frequencies of A₁ and A₂ subtypes will be in Hardy-Weinberg equilibrium for the Mendelian inheritance of the allelic A₁ and A₂ genes. The frequency of A subgroups in the population from south India is not known. The aim of our study was to study the frequency of A subtypes and the prevalence of anti-A₁ antibody among this population.

Methods

Over a period of 3 years, patients’ blood group was typed using a standard tube technique. Anti-A₁ lectin studies were done for all patients with groups A and AB. Based on serological reactivity the samples were classified into A₁/A₁B, A₂/A₂B and weak A subgroups. The prevalence of A subgroups was determined. The significance of differences in proportions was analysed using the chi-square test.

Results

A total of 40,113 patients’ samples were typed for ABO, Rh group and A subgroups in our blood bank attached to a tertiary care hospital. Among 10,325 group A samples, 98.14% classified as A₁, 1.07% as A₂, and 0.01% as weak A; the remaining group A samples were from neonates and reacted poorly with anti A₁-lectin. The majority of AB samples (n=2,667) were of A₁B type (89.28%). However, the proportion of A₂B (8.99%) among AB samples was significantly higher than that of A₂ in group A samples (p < 0.0001). The prevalence of anti-A₁ antibodies among A₂ and A₂B samples was 1.8% and 3.75%, respectively, and none of them showed reactivity at 37°C.

Conclusion

The results of our study show a significantly higher proportion of A₂B subtypes than A₂ subgroups. A similar imbalance is seen in blacks and Japanese. The incidence of anti-A₁ antibodies is also higher among A₂B patients.