Intestinal barrier function in patients undergoing colectomy.

AIM: The aim of this pilot study was to determine whether the type of approach (open or laparoscopic) and the order of devascularization during laparoscopic colectomy affect intestinal barrier function, local inflammatory response and clinical outcome. METHOD: Twenty-two patients undergoing elective colectomy from April 2006 to July 2008 were randomized to two sequences of vascular ligation, starting with either the inferior mesenteric artery or the ileocolic artery. Eighteen patients scheduled for open surgery served as a prospective control group. To assess the intestinal barrier function, release of intestinal fatty-acid binding protein (I-FABP; a marker of mucosal injury and ischaemia) was measured pre- and postoperatively. Mesenteric lymph nodes were harvested to assess the expression of inflammatory mediator-related genes using multiplex ligation probe amplification. The study was registered under NTR1025. RESULTS: Laparoscopic devascularization starting at the ileocolic artery resulted in a significantly increased excretion of I-FABP over time (P = 0.002). In this group, the I-FABP levels were significantly increased on postoperative days 1 and 3 compared with preoperative values (P = 0.011 and P = 0.001, respectively). There were no differences in expression of inflammatory mediator-related genes or postoperative morbidity among the groups. CONCLUSIONS: In this pilot study, devascularization commencing at the ileocolic artery during laparoscopic colectomy was associated with prolonged intestinal mucosal ischaemia.

Transmembrane orientation of palmitoyl-CoA: lysophosphatidylcholine acyltransferase in microsomes isolated from an alveolar type II cell adenoma and rat liver.

1. The transverse localization of palmitoyl-CoA : lysophosphatidylcholine acyltransferase in the membrane of microsomal vesicles isolated from mouse lung adenomas and rat liver was studied by treating intact and deoxycholate-disrupted microsomes with trypsin and pronase. 2. The latency of mannose-6-phosphatase was preserved during protease treatment, suggesting that membrane integrity was not affected. 3. In adenoma microsomes 35-50% and in liver microsomes 35% of lysophosphatidylcholine acyltransferase activity is accessible to the action of the proteases. Our results suggest that at least a sizable portion of the active center of the enzyme that is responsible for remodeling phospholipids is embedded in the membrane interior. 4. Since enzymes involved in de novo lipid synthesis are reported to be located at the cytoplasmic surface of the microsomal membrane, our results support the notion that in lipid metabolism distinct metabolic pools might exist at opposite sides of the microsomal membrane.

Biosynthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor and a hypotensive lipid) by cholinephosphotransferase in various rat tissues.

The unique alkyl phospholipid, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, has been reported to exhibit powerful antihypertensive activity (Blank, M.L., Snyder, F., Byers, L.W., Brooks, B. and Muirhead, E.E. (1979) Biochem. Biophys. Res. Commun. 90, 1194-1200) and appears to be an extremely potent platelet-activating factor (Demopoulos, C.A., Pinckard, R.N. and Hanahan, D.J. (1979) J. Biol. Chem. 254, 9355-9358). In the present study, microsomal preparations from several rat tissues were found to catalyze the synthesis of 1-alkyl-1-acetyl-sn-glycero-3-phosphocholine by 1-alkyl-2-acetyl-sn-glycerol:CDPcholine cholinephosphotransferase reaction. Optimal conditions to measure enzyme activity were established. A subcellular survey of this cholinephosphotransferase activity showed that the enzyme was of microsomal origin. Enzyme activity was found in microsomes from several tissues; however, spleen has the highest activity of the tissues examined. Three different species of 1-alkyl-2-acetyl-sn-glycerol were all found to be substrates. The 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine synthesized in the microsomes could be hydrolyzed by adding the 100,000 x g supernatant fraction to the incubation medium. The optimum pH for formation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine was 8.0, which was different from the pH optimum of 8.5 observed for the long-chain diacylglycerol cholinephosphotransferases. Activity of cholinephosphotransferase towards 1-alkyl-2-acetyl-sn-glycerol was slightly enhanced and stabilized by dithiothreitol, whereas the activity towards a diacylglycerol was inhibited by dithiothreitol. The possible involvement of two different enzymes in the conversion of 1-alkyl-2-acetyl-sn-glycerol and diacylglycerol to their respective phospholipid products is discussed.

The transposition of molecular classes of phosphatidylcholine across the rat erythrocyte membrane and their exchange between the red cell membrane and plasma lipoproteins.

1. The molecular composition of phosphatidylcholine is similar in the inner and the outer layer of the rat erythrocyte membrane. 2. The rate of exchange of the various molecular classes of phosphatidylcholine between rat plasma and the red cell membrane does not depend on the degree of unsaturation of the different classes. 3. The transposition of the molecular classes of phosphatidylcholine between the inner and the outer layer of the rat erythrocyte membrane is more pronounced for the more unsaturated classes.

Asymmetry in the renewal of molecular classes of phosphatidylcholine in the rat-erythrocyte membrane.

1. Rat-blood phospholipids were labeled in vivo with [32P]phosphate. The erythrocytes were treated with phospholipase A2 plus sphingomyelinase to discriminate between the labeling patterns of the phospholipids from the inner and outer layer of the membrane. 2. The specific activities of the more unsaturated classes of phosphatidylcholine were higher in the outer layer of the erythrocyte membrane than in the inner layer. The disaturated class, however, had the highest specific activity in the inner layer. 3. After incubating 32P-labeled erythrocytes in unlabeled plasma, the labeling pattern recovered in the molecular classes of plasma phosphatidylcholine was very similar to that of the phosphatidylcholines in the outer layer of the erythrocyte membrane. 4. It is proposed that the exchange of phosphatidylcholines between plasma and the outer layer of the erythrocyte is mainly responsible for the renewal of the unsaturated phosphatidylcholines of the erythrocyte, and that the acylation activity of the erythrocyte is directed towards the formation of disaturated phosphatidylcholines at the inside of the membrane.

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.

The effects of bile salt hydrophobicity on model bile vesicle morphology.

The addition of bile salts to vesicles supersaturated with cholesterol induces cholesterol precipitation, an important step in the formation of cholesterol gallstones. To investigate the effects of bile salt hydrophobicity on vesicle morphology, vesicles obtained from supersaturated model bile by density gradient ultracentrifugation, were incubated with mixtures of deoxycholate (DC) and ursodeoxycholate (UDC) with a constant total bile salt concentration of 30 mM but with a varying hydrophobicity index ranging from -0.31 (UDC alone) up to +0.72 (DC alone) depending on the composition of the mixture. Five days after addition of bile salts to vesicles, cholesterol precipitation was determined microscopically and incubation samples were again subjected to ultracentrifugation to assess the lipid distribution among residual vesicles, mixed micelles, and cholesterol crystals. Structure and size of the isolated residual vesicles were studied by freeze fracture electron microscopy. The control, and samples exposed to hydrophilic bile salt mixtures, consisted of unilamellar vesicles of which more than 75% had a diameter of 50-80 nm. After addition of increasingly hydrophobic bile salt mixtures, multilamellar vesicles with progressively greater diameters (up to 1300 nm) were found, suggesting that vesicle fusion and aggregation took place and might hence be important in the cholesterol precipitation process. Accordingly, crystallization was positively correlated with bile salt hydrophobicity. We conclude that cholesterol crystallization from vesicles depends on the hydrophobicity of the bile salts added, and apparently occurs from fused or aggregated vesicles of extended magnitude and with a multilamellar constitution.

Bone healing during lower limb lengthening by distraction epiphysiolysis.

A study of limb lengthening by distraction epiphysiolysis in the rabbit tibia is presented. A special external distraction device was developed that allowed 10 mm lengthening of the leg. Bone formation in the elongated zone was studied by computed tomography and [99mTc] methylene diphosphonate (MDP) scintigraphy. Computed tomography showed bone formation proceeding for several weeks after the end of the distraction period, followed by a decrease in the amount of bone during a remodeling phase leading to the formation of a solid cortical structure. The uptake of [99mTc]MDP increased parallel to, but preceeding the actual accretion of bone, followed by a decrease during the bone remodeling phase. Uptake of the tracer will partly reflect bone metabolism, but other factors, like trauma, determine much of the uptake.

A randomized trial in primary biliary cirrhosis comparing ursodeoxycholic acid in daily doses of either 10 mg/kg or 20 mg/kg. Dutch Multicentre PBC Study Group.

BACKGROUND: Ursodeoxycholic acid (UDCA) prolongs transplantation-free survival in primary biliary cirrhosis (PBC). However, the optimal therapeutic dose has not been established. AIM: To compare the effects of UDCA administered in daily doses of 10 vs. 20 mg/kg on symptoms, liver biochemistry and biliary UDCA enrichment. METHODS: A 6-month multicentre randomized open controlled trial was conducted to assess the effects of an increase in the dose of UDCA to 20 mg/kg/day vs. continuation of 10 mg/kg/day for patients who had not achieved biochemical normalization during treatment for at least 6 months with the 10 mg/kg dose. Clinical and laboratory evaluations were performed at entry and at 3-month intervals. The percentage UDCA in duodenal bile was assessed at entry and at 6 months. RESULTS: Sixty-one patients were enrolled. No side-effects of UDCA were observed. Within the 20 mg/kg/day group significant decreases were found for alkaline phosphatase (- 8%; P = 0.003), aspartate aminotransferase (- 11%; P = 0.01), alanine aminotransferase (- 17%; P < 0.001), gamma-glutamyl transferase (- 34%; P < 0.001), immunoglobulin M (- 11%; P = 0.002) and cholesterol (- 8.1%; P < 0.001). In the 10 mg/kg group none of these parameters differed significantly from baseline. No significant differences between dose groups for symptom scores or serum bilirubin were found. Biliary enrichment with UDCA increased from 37% to 46% in the 20 mg/kg group (P = 0.02) while remaining stable in the 10 mg/kg group. CONCLUSIONS: Liver biochemistry improved in PBC patients receiving UDCA 20 mg/kg/day compared to a dose of 10 mg/kg/day. Both doses were equally well tolerated. These results indicate that UDCA 10 mg/kg/ day is a suboptimal dose for treating PBC.

Effect of direct current stimulation on bone growth after distraction epiphysiolysis of the rabbit tibia.

The effect of direct current stimulation on bone formation during limb lengthening was tested in a lower leg lengthening model in the rabbit. Limb lengthening was performed by distraction epiphysiolysis. A specially designed external distraction device was placed at the tibia. The distractor allowed 10 mm of lengthening in 4 weeks. Two weeks after starting the distraction, a platinum electrode was passed through the anterior cortex below the tibial tuberosity and advanced via the medullary cavity so that the tip rested in the elongated zone. Stimulation started at the time of placement of the electrodes and was continued for 3 weeks. The electrode in the elongated zone served as the cathode; the anode was placed subcutaneously. A 20 microA stimulus was selected. A control group received the same treatment without stimulation. Bone formation in the elongated zone was evaluated by histology and scintigraphy. The data from this experiment show that direct current stimulation in the early phase of a limb lengthening procedure had no effect on the extent of bone formation in the elongated zone.

Continuous monitoring of forces during tibial lengthening by distraction epiphysiolysis.

A leg length inequality of 5 cm was compensated by lengthening of the proximal tibia by distraction epiphysiolysis. During the first 7 weeks of lengthening the distraction force was monitored continuously by strain gauges attached to the extension bars of the external distraction device. The increase in length was 1.25 mm a day. The force registration showed a visco-elastic response of the tissue to distraction. In the first week the distraction force increased up to 791 N. Then lysis occurred, followed by a sudden decrease in force to an average level of 150 N. During subsequent distraction, pain and clinical symptoms could be associated with increasing tension and inadequate stress relaxation of soft tissues. Adjusting the distraction rate decreased the build-up of force in the tissue and ameliorated the clinical symptoms. These results show that force measurements can be clinically useful for appropriately adjusting the distraction rate.

Uptake of [99mTc]technetium methylene diphosphonate in the growth plates of the rabbit tibia during the final part of epiphyseal growth activity.

Uptake of 99mTc-MDP was measured in the proximal and distal growth plate of the rabbit tibia during the final phase of growth. Scintigraphic data were compared with roentgenographically measured tibial length and signs of bony fusion of the growth plates. Uptake by the proximal tibial growth plate was higher than in the distal tibial plate. Uptake decreased on cessation of growth and during subsequent bony fusion. In particular in the proximal tibial region, uptake was still high after cessation of longitudinal growth, which illustrates that during the final period of growth uptake of 99mTc-MDP by a roentgenographically still open growth plate does not necessarily indicate that such a growth plate still contributes to longitudinal growth. Decrease in uptake during the final phase of growth should be considered, when bone scanning is part of a research protocol.

Indomethacin enhances bile salt detergent activity: relevance for NSAIDs-induced gastrointestinal mucosal injury.

Gastroduodenal toxicity of nonsteroidal anti-inflammatory drugs (NSAIDs) is partly independent from cyclooxygenase inhibition, possibly related to increased intermixed micellar-vesicular (nonphospholipid-associated) bile salt concentrations thought to be responsible for bile salt cytotoxicity. We evaluated the effects of indomethacin on bile salt cytotoxicity with complementary in vitro and ex vivo systems. In the erythrocyte model, indomethacin alone did not induce hemolysis. In contrast, indomethacin enhanced and phospholipids decreased hemolysis induced by hydrophobic taurodeoxycholate (TDC). Hydrophilic tauroursodeoxycholate (TUDC) enhanced rather than decreased TDC-induced hemolysis in the presence of indomethacin. Indomethacin did not affect intermixed micellar-vesicular bile salt concentrations or compositions. Indomethacin also increased TDC-induced lactate dehydrogenase release in CaCo-2 cells and bile salt-induced rat colonic mucosal injury, and prevented potential protective effects of TUDC in these systems. Our data show that indomethacin enhances bile salt-induced cytotoxicity without affecting intermixed micellar-vesicular bile salt concentrations or compositions. These findings may be relevant for gastroduodenal injury during NSAID therapy.

Phospholipids from rat, human, and canine gastric mucosa. Composition and metabolism of molecular classes of phosphatidylcholine.

To validate a recent proposal that a phospholipid lining with a high content of dipalmitoylphosphatidylcholine may protect gastric mucosa against luminal acid, it was decided to study composition and metabolism of phospholipids in the gastric mucosa. Phospholipids were analyzed in rat, human, and dog gastric mucosal surface tissue and in a chloroform/methanol-lavage of rat and canine stomach. Phosphatidylcholine and phosphatidylethanolamine were the main components. Saturated fatty acids were almost exclusively esterified at the sn-1 position of the glycerol moiety of phosphatidylcholine, and unsaturated fatty acids mainly at the sn-2 position. The disaturated class of phosphatidylcholine comprised 2%-6% of total phosphatidylcholine. Precursors of phosphatidylcholine, i.e., [32P]orthophosphate and [methyl-14C]choline, were preferentially incorporated into the disaturated molecular class 0.5-6 hours after IV administration. It can be speculated that disaturated phosphatidylcholine, although quantitatively a minor component, is specifically triggered in mucosal renewal processes.

Normal cations and abnormal membrane lipids in the red blood cells of dogs with familial stomatocytosis-hypertrophic gastritis.

Examination of the red blood cells (RBCs) of eight dogs with familial stomatocytosis-hypertrophic gastritis (FS-HG), a multiorgan disease associated with hemolytic anemia, hereditary stomatocytosis (HSt), and hypertrophic gastritis resembling Ménétrier's disease in man, showed abnormal osmotic fragility, normal mean corpuscular volume, slightly increased cell water, and normal cation content and cation fluxes. Cholesterol was decreased in RBC and increased in plasma. In both RBCs and plasma, total phospholipid (PL) was normal, phosphatidylcholine (PC) decreased, and sphingomyelin increased. The palmitic acid content of PC was increased, and the stearic acid content of PC was decreased. Sodium dodecyl sulfate electrophoresis of RBC membrane proteins was normal. These findings have not been described previously in HSt. They suggest that in FS-HG, abnormal composition of the PL in RBCs secondary to abnormal PL in plasma causes defective membrane function and stomatocytic shape-change. This conclusion was supported by a shortened half-life of 51Cr-labeled RBCs from normal dogs after transfusion in dogs with FS-HG. It was concluded (1) that not all hereditary forms of stomatocytosis are necessarily associated with an intrinsic structural defect of the RBC membrane, but that the change in shape of RBC may also be induced by abnormal composition of the plasma; (2) that stomatocytosis may be caused by loss of membrane surface area rather than by the increased cation uptake such as has been shown in some human kindreds with HSt, (3) that FS-HG is a disorder of lipid metabolism, and by consequence, (4) that abnormal lipid metabolism might be involved in the pathogenesis of Ménétrier's disease.

Gastric mucosal phospholipids in dogs with familial stomatocytosis-hypertrophic gastritis.

In dogs, hypertrophic gastritis, which resembles Ménétrier's disease in man, has been demonstrated to be part of a hereditary syndrome called familial stomatocytosis-hypertrophic gastritis. In addition to hypertrophic gastritis, affected dogs exhibit abnormal blood phospholipid composition. Phospholipids may play a role in maintaining gastric mucosal integrity, and this may be compromised in gastritis. The question arises whether the differences in blood phospholipids may result from a disorder that might also be revealed in the composition of gastric mucosal phospholipids. We analysed the phospholipid composition of gastric mucosa from four dogs with familial stomatocytosis-hypertrophic gastritis. The general phospholipid composition and the molecular composition of phosphatidylcholine from mucosal tissue in the corpus of the stomach where hypertrophic gastritis was evident were not different from that of the antrum, where the tissue was normal. These results do not corroborate a relation between the gastric mucosal phospholipid composition and hypertrophic gastritis.

Topological asymmetry of phospholipid metabolism in rat erythrocyte membranes. Evidence for flip-flop of lecithin.

1. The distribution of phospholipids between inside and outside of rat erythrocyte membranes was studied by incubating the cells with phospholipase A2 from Naja naja venom and sphingomyelinase from Staphylococcus aureus. 2. Choline-containing phospholipids were found to comprise the majority of the outer layer of the membrane. 3. The incorporation of radioactive fatty acids into phospholipids occurred predominantly at the inside of the membrane. 4. Exchange of phospholipids between red cell membranes and plasma lipoproteins occurred at the outside of the membrane. 5. Indications were found for a rather slow flip-flop of lecithin across the membrane.

Long-term study of large ceramic implants (porous hydroxyapatite) in dog femora.

Blocks of porous ceramic hydroxyapatite (dimensions, 2.5 X 1.25 X 0.5 cm; sintering temperature, 1300 degrees; macroporosity, 56%; average pore size 0.18 mm2) were implanted into surgically created defects in dog femora. The implants were retrieved up to 3.5 years after implantation. The implants were 3.5 years after implantation. The implants were firmly attached to the bone. Histologic evaluation suggests that optimal contact between bone and implant should be provided to accelerate bone ingrowth. Bone growth in the pores reached a maximum level after 35 weeks, at which time about one-third of the pore space was filled with bone. When measuring the relative surface areas of bone and ceramic on histologic slides, no change in ceramic mass could be detected, indicating that hydroxyapatite ceramics are not affected by biodegradation processes. The implants effectively provided a scaffold for bone growth bridging a larger bone defect.