Decreased lysophospholipase and increased phospholipase A2 activity in ileal mucosa from patients with Crohn's disease

Lysophospholipase (EC 3.1.1.5) and phospholipase A2 (EC 3.1.1.4) were determined in ileal mucosa from patients with Crohn's disease (CD) and non-inflammatory bowel diseases ( NIBD ). In addition, the activities of alkaline phosphatase, sucrase, maltase, and lactase were determined. The lysophospholipase activity, like that of alkaline phosphatase, sucrase and maltase, was decreased in affected areas of CD, whereas the phospholipase A2 activity was rather increased. Lysophospholipase and phospholipase A2 activities in apparently unaffected mucosa from CD patients were in between those in healthy mucosa from NIBD patients and those in affected mucosa from CD patients. These findings point to the possibility that the mucosal activity of lysophospholipase, like that of other brush border enzymes, is decreased in CD. This may render the mucosa less capable to handle lysolecithin, a potentially harmful agent formed in the intestine and known to induce inflammation in a number of experimental systems.     

Increased phospholipase A2 activity of Ileal mucosa in Crohn's disease

The activity of phospholipase A2 (EC 3.1.1.4) in endoscopic biopsy specimens of intestinal mucosa was compared in patients with Crohn's disease and controls without inflammatory bowel disease. In postresection Crohn patients there was significantly enhanced phospholipase A2 activity proximal to the anastomosis in the neoterminal ileum, whether or not the mucosa was inflamed at the time of biopsy. Highly elevated ileal phospholipase A2 activity had a predictive value for symptomatic relapse within 1 year after resection. Patients with concomitant Crohn's colitis, in whom the risk of ileal recurrence is particularly high, had greater ileal phospholipase A2 activity than noncolitis Crohn patients. Association thus was demonstrated between activity of phospholipase A2 in ileal mucosa and proneness to ileal inflammation in Crohn's disease.     

Increased sedentary time and decreased physical activity increases lipoprotein associated phospholipase A2 in obese individuals

Background and aimsLipoprotein-associated Phospholipase A₂ (Lp-PLA₂) is a protein produced by inflammatory cells in circulation and is associated with cardiovascular disease (CVD) risk. Physical activity (PA) is known to reduce inflammation and risk for CVD. However, Lp-PLA₂ has yet to be examined in relation to PA and sedentary time. The purpose of this study was to determine if PA and sedentary time impacts Lp-PLA₂ mass. A total of 25 subjects with an average BMI of 30.6 ± 5.7 were included in the data analysis.Methods and resultsData collected included anthropometric data, Lp-PLA₂ mass, peak oxygen uptake (VO₂peak), resting heart rate and blood pressure, obstructive sleep apnea (OSA) risk, and assessment of PA using an accelerometer. Sedentary minutes per day was positively associated with Lp-PLA₂ (r = 0.41, P < 0.05). Light intensity PA was negatively associated (r = ?0.51. P = 0.01) with Lp-PLA₂. When subjects were divided into 2-quantiles by Lp-PLA₂, the group with the higher Lp-PLA₂ mass accumulated more sedentary time per day (P < 0.001) and less light intensity PA per day (P = 0.001). OSA risk and Lp-PLA₂ showed no relationship. Sedentary behavior was higher, and light intensity PA was lower in subjects with hiLp-PLA₂ mass. No difference was seen in moderate-to-vigorous intensity PA or steps per day.ConclusionsThis suggests that, total PA habits, including time spent sedentary and lower intensity PA, impacts the levels of Lp-PLA₂, an important inflammatory marker and marker of CVD risk.     

Calcium-binding activity of rat intestinal mucosa after massive small bowel resection.

Male Sprague-Dawley rats underwent resection of 50 cm of either proximal or distal small intestine or sham-operation. 6-7 weeks after operation mucosal calcium-binding activity was measured in segments of duodenum ileum and remaining 'midgut'. Similar measurements were obtained from weight and age-matched unoperated rats. There was no difference in calcium-binding activity between unoperated and sham-operated animals. After proximal resection the binding activity increased significantly in duodenum and midgut but did not change in ileum. After distal resection the binding activity decreased in duodenum but was unchanged in midgut and ileum. These studies show that mucosal calcium-binding activity undergoes changes but alteration of the binding activity in remaining gut varies with the location of the small bowel resection.     

Increased muscular phospholipase A2 activity in diabetic rats.

Anomalies in prostaglandin (PG) synthesis have been suggested in both experimental and human diabetes mellitus; increased levels of plasma and tissue eicosanoids has been recently reported by several investigators. One step in prostaglandin synthesis is the enzymatic hydrolysis of membrane phospholipids by Phospholipase A2 (PLA2). Nevertheless the alternative pathway involving Phospholipase C must be considered. An evaluation of PLA2 activity is therefore a useful method for studying prostaglandin synthesis in the peripheral target tissues of insulin activity. We studied PLA2 activity in normal and diabetic rat muscle. Streptozotocin-induced diabetic rats showed significantly higher muscular PLA2 activity when compared with controls (3.04 x 10(-2) +/- 0.50 x 10(-2) versus 1.34 x 10(-2) +/- 0.35 x 10(-2) arachidonic acid pMol.mg protein-1.min-1 (p less than 0.01). This effect was not observed in diabetic animals successfully treated with insulin (1.78 x 10(-2) +/- 0.5 x 10(-2) versus 1.34 x 10(-2) +/- 0.35 x 10(-2) arachidonic acid pMol.mg protein-1.min-1), and a significant correlation was found between blood glucose and muscular PLA2 activity (r = 0.42; p less than 0.05). Our results clearly show that in streptozotocin-induced diabetic rats muscular PLA2 activity is significantly higher. The relationship between blood glucose levels and muscular PLA2 activity and the decrease of PLA2 activity after insulin treatment suggest that these changes may be related to a defect in insulin effect.     

Increased activity of lipoprotein-associated phospholipase A2 in non-severe asthma

BackgroundGiven increased risk of cardiovascular events in asthma we hypothesized that lipoprotein-associated phospholipase A₂ (Lp-PLA₂), an enzyme involved in atherosclerosis, is associated with proinflammatory and prothrombotic blood alterations in this disease.MethodsIn 164 adult asthmatics (63 with severe asthma) we measured plasma Lp-PLA₂ activity using the PLAC test. We determined its relations to inflammation and prothrombotic blood alterations.ResultsIn asthma, Lp-PLA₂ was inversely related to the age (β = ?0.1 [?0.18 to ?0.02]) and was lower in women (n = 122 [74%], 205 [182–242] vs. 243 [203–262] nmol/min/ml, p = 0.001). Interestingly, Lp-PLA₂ correlated negatively with the asthma severity score (β = ?0.15 [?0.23 to ?0.07]), being 10.3% higher in those with non-severe (mild or moderate) asthma (n = 101, 62%) as compared to the severe disease subtype (224 [191–261] vs. 203 [181–229], p = 0.006 after adjustment for potential confounders). Lp-PLA₂ activity was positively related to the levels of low-density lipoprotein (β = 0.1 [0.02–0.18]), triglycerides (β = 0.11 [0.03–0.19]) and glucose (β = 0.1 [0.02–0.18]) and inversely to the tumor necrosis factor α (β = ?0.27 [?0.35 to ?0.2]), high sensitivity C-reactive protein (β = ?0.1 [?0.19 to ?0.02]) and fibrinogen (β = ?0.12 [?0.21 to ?0.03]), as well as prothrombin (β = ?0.16 [?0.24 to ?0.08]), and parameters describing thrombin generation potential, such as endogenous thrombin potential (β = ?0.14 [?0.21 to ?0.06]) and peak thrombin generated (β = ?0.2 [?0.28 to ?0.12]).ConclusionsElevated Lp-PLA₂ activity in non-severe asthmatics suggests increased atherosclerotic risk in this group. Lower Lp-PLA₂ activity accompanied by its inverse relationship to inflammatory or prothrombotic blood biomarkers observed in turn in severe asthmatics might be related to the pathogenesis of more severe asthma phenotype.     

Studies of the phospholipase A2 activity of rat ileal mucosa

A rapid and simple procedure has been used to determine phospholipase A2 activity (EC 3.1.1.4) in rat ileal mucosa. We used 14C-oleate-labeled Escherichia coli as substrate for the phospholipase activity and a 0.45-micron Millipore filter to separate the product of hydrolysis--the 14C-oleic acid--from the unhydrolyzed substrate. The phospholipase A2 activity was optimal at pH 9.8 and at 2 mM Ca2+, but another peak of activity appeared at pH 7.2. In addition, cell fractionation revealed yet another phospholipase A2 activity at pH 5.0 in the absence of Ca2+. These findings suggest the presence of more than one phospholipase A2 in the ileal mucosa and points to the possible use of a simple procedure for studying their distribution and properties.     

Phospholipase A2 inhibition prevents mucosal damage associated with small intestinal ischaemia in rats.

The influence of various inflammatory inhibitors on the damaging effects of ischaemia in the small intestinal mucosa has been investigated. A rat experimental model was used, in which a ligated loop of the distal ileum was subjected to ischaemia and revascularisation and the ensuing mucosal damage assessed by lysosomal enzyme release and intestinal permeability measurements. The mucosal content of malondialdehyde - a lipid peroxidation product - and its activity of myeloperoxidase - a neutrophil granulocyte marker was also determined. In the absence of inhibitor, ischaemia and revascularisation caused increased mucosal permeability to sodium fluorescein, increased N-acetyl-beta-glucosaminidase release from the mucosa into the lumen, increased malondialdehyde content in the mucosa and increased myeloperoxidase activity in the mucosa. All these effects were inhibited by the phospholipase A2 inhibitors, quinacrine and nordihydroguaiaretic acid (NDGA), while the lipoxygenase inhibitor, BW755C, had no influence and the cyclooxygenase inhibitor, indomethacin, potentiated the increases in mucosal permeability and N-acetyl-glucosaminidase release. BN 52021, a specific platelet activating factor antagonist, did not influence the myeloperoxidase activity, but it decreased the formation of malondialdehyde and the increases in mucosal permeability and N-acetyl-beta-glucosaminidase release, although not to the same extent as quinacrine and NDGA. These findings indicate that phospholipase A2 inhibition prevents mucosal damage associated with small intestinal ischaemia and suggest that at least part of the ischaemic damage is mediated by products of phospholipase A2 activity that are not arachidonic acid metabolites.     

Increased group II phospholipase A2 in colonic mucosa of patients with Crohn's disease and ulcerative colitis.

The immunochemical protein content of group II phospholipase A2 (PLA2) and PLA2 enzymatic activity were measured for colonic mucosal biopsy samples obtained from patients with either Crohn's disease of the colon or ulcerative colitis, and control patients without inflammatory bowel disease. Immunoreactive group II PLA2 (IR-PLA2 II) content and PLA2 activity in actively inflamed colonic mucosa of Crohn's disease patients were significantly higher than those in inactively inflamed mucosa of Crohn's disease patients and the colonic mucosa of controls. IR-PLA2 II content and PLA2 activity in severely inflamed mucosa of ulcerative colitis patients were significantly higher than those in the colonic mucosa of the controls. Mucosal PLA2 enzymatic activity was closely correlated with mucosal IR-PLA2 II content in patients with Crohn's disease and ulcerative colitis. These results suggest that an increase in PLA2 enzymatic activity in inflamed colonic mucosa of Crohn's disease and ulcerative colitis was mainly attributed to increased protein content of group II PLA2, and that an increase in mucosal group II PLA2 may be involved in the pathogenesis of intestinal inflammation of Crohn's disease and ulcerative colitis.     

Increase in permeability and phospholipase A2 activity of colonic mucosa in Crohn's colitis

Patients with Crohn's colitis were investigated regarding the relationship between intestinal inflammation, mucosal activity of phospholipase A2 and intestinal permeability to different-sized polyethylene glycols (PEG). Mucosal specimens for phospholipase A2 were obtained at colonoscopy and PEG (590-942 daltons) were deposited in the descending colon. Colonic absorption was measured as urinary output of deposited PEG. Colonic absorption of PEG was higher in these patients, even when the colitis was in remission at colonoscopy, than in ileal Crohn's disease or control patients. Mucosal phospholipase A2 activity was increased in active colitis, but in the patients with endoscopic remission it was at the same level as in the controls or in the patients with ileal Crohn's disease. The observations suggest that increased intestinal permeability may be a primary factor in activation of mucosal phospholipase A2 activity and in intestinal inflammation.     

Increased glomerular cytosolic phospholipase A2 activity of OLETF rats with early diabetes.

In view of the potential role of prostaglandins (PGs) in development of glomerular hyperfiltration leading to diabetic nephropathy, we studied the temporal relationship of the activity of cytosolic phospholipase A2 (cPLA2), a rate-limiting enzyme for eicosanoid biosynthesis, with hyperfiltration and the histological changes in glomeruli using OLETF rats, a model for non-insulin-dependent diabetes mellitus (NIDDM). Diabetes mellitus and associated histopathological changes, which developed spontaneously by 30-46 weeks after birth of OLETF rats, were accompanied by approximately 65% increase in glomerular cPLA2 activity that showed significant correlations with elevated plasma glucose levels and creatinine clearance. Moreover, mesangial cells cultured for 5 days with high glucose exhibited approximately 2-fold higher cPLA2 activity than those cultured with physiologic level of glucose. These data suggest that increased glomerular cPLA2 activity leads to production of PGs, which may promote the progression of early diabetic glomerular hyperfiltration and subsequent diabetic nephropathy.     

Increased small intestinal apoptosis in coeliac disease.

BACKGROUND: Coeliac disease (CD) mucosa is flattened despite epithelial hyperproliferation. AIMS: To establish mechanisms of cell loss in CD. PATIENTS: 14 controls, 17 active CD patients, and 16 maintained with gluten free diet. METHODS: Programmed cell death was examined in small intestinal biopsy specimens by staining fragmented DNA using terminal uridine deoxynucleotidyl nick end labelling (TUNEL), in comparison with haematoxylin and eosin stained adjacent sections. Double staining with anti-CD45 antibodies determined the origin of apoptotic cells. Apoptosis was graded from 1-3 (< 5, 5-20, > 20% respectively). Proliferating cells, immunostained by Ki-67 (MIB-1) antibody, were counted. RESULTS: Apoptotic cells were seen rarely by haematoxylin and eosin but more readily by TUNEL. In controls, 1.4 +/- 0.2% of epithelial cells were apoptotic (mean grade 1.1), mainly located in the upper villus. In active CD, frequent apoptotic cells were distributed throughout the crypt-villus unit (mean grade 2.4), decreasing after treatment to 1.1 (p < 0.001) even when still histologically abnormal. CD45 antibodies rarely stained apoptotic cells in active CD. The number of TUNEL positive cells correlated with proliferating cell number (p < 0.001). CONCLUSION: Enterocyte apoptosis is greatly increased in untreated CD, correlates with proliferation, and falls to normal with a gluten free diet, before histological improvement. Increased apoptosis may be responsible for villous atrophy in CD.     

Association of lipid accumulation in small intestinal mucosa with decreased serum triglyceride and cholesterol levels in AIDS

Lipid accumulation has been described in the duodenal lamina propria of human immunodeficiency virus (HIV)-infected patients with diarrhea and malabsorption. Using light and electron microscopy, we studied duodenal biopsies obtained from 54 consecutive HIV-infected patients by means of upper gastrointestinal endoscopy after an overnight fast. The presence of diarrhea and weight loss were recorded, and all the patients had standard stool study for ova, parasites, and bacteria. Serum levels of albumin, triglycerides, and cholesterol were obtained within one week of the endoscopy. Fecal fat and fecal ₁-antitrypsin clearance were measured in 11 patients. Lipid accumulation was observed in nine patients (16.6%). Fat droplets were seen in enterocytes, in their basolateral membrane spaces, and in the lamina propria. The mean serum levels of triglycerides (1.85±0.20 mmol/liter) and cholesterol (2.81±0.30 mmol/liter) were significantly lower in the patients with enteric steatosis than in patients without this anomaly (respectively, 3.38±0.39 and 3.97±0.18 mmol/liter,P<0.001P<0.01). The mean amount of fecal fat in the three patients with lipid accumulation (16±1.60 g/24 hr) was significantly larger than in the eight patients without lipid accumulation (4.50±0.62 g/24 hr,P<0.01). These findings suggest that fat malabsorption in HIV-infected individuals is due to a blockage of transport through the duodenal mucosa. The frequency of diarrhea, weight loss, or identified enteric pathogens did not differ significantly between patients with and without enteric steatosis. Both the etiology and the pathophysiology of these alterations remain to be documented.     

Cryptosporidium, chronic diarrhoea and the proximal small intestinal mucosa.

The association between Cryptosporidium, chronic diarrhoea and a proximal small intestinal mucosal enteropathy was reviewed over a six and a half year period. One hundred and twenty three children with cryptosporidiosis and no clinical evidence of immune deficiency were identified. 50% of children excreting only Cryptosporidium had chronic diarrhoea. Most cases (63%) of chronic diarrhoea occurred in the first two years of life. A mild to moderate enteropathy was present in all nine children undergoing a small intestinal biopsy and seven showed the presence of Cryptosporidium adhering to villous epithelium. All patients eventually recovered spontaneously. Cryptosporidium is a cause of chronic diarrhoea and a proximal small intestinal mucosal enteropathy in children without immune deficiency. Screening for the parasite should be part of the investigative procedures in children with chronic diarrhoea.     

Mechanisms of lipid loss from the small intestinal mucosa.

Many water-soluble compounds have been shown to pass from the small intestinal mucosa into the lumen. In this work, the loss of lipids from the mucosa was investigated by perfusion experiments in rats, using 0-15M NaCl or buffer solutions over range of pH, with or without the addition of 5-7 or 11-4mM taurocholic acid. Perfusates were extracted for the estimation of individual lipids and for DNA, which is a measure of cell loss. The results suggest that free fatty acids reach the lumen by diffusion and that their solubility in the luminal fluid is a factor determining their rate of loss. Triglycerides, cholesterol, phosphatidyl ethanolamine, and phosphatidly choline are present onlyas the result of desquamation of mucosal cells.     

Effect of dietary fat on the small intestinal mucosa.

The presence of food within the small intestinal lumen promotes mucosal cell proliferation. To define the trophic role of triglycerides, three groups of eight female Wistar rats were isocalorically fed for four weeks with either Vivonex, or Vivonex with 50% calorie substitution with an essential fatty acid mixture, or Vivonex with 50% calorie substitution with a saturated fatty acid mixture. Although Vivonex caused greater body weight gain, both essential fatty acids and saturated fatty acids increased small intestinal weight, mucosal weight, protein and DNA overall, and in each of three intestinal segments (proximal, middle and distal), compared with Vivonex. Mucosal indices were similar for essential fatty acids and saturated fatty acids. These results show that triglycerides, regardless of essential fatty acid content, are trophic to the rat small intestinal mucosa.