Gastric mucosal damage induced by combination of ethanol and lysophosphatidylcholine.

The purpose of this study was to determine the effect of lysophosphatidylcholine on the guinea pig stomach after dosing the animal with 20% ethanol by orogastric intubation. We studied four groups of animals; one control group received saline orogastrically followed by buffer and one test group received saline followed by buffer plus 1 mM lysophosphatidylcholine. Two other groups were challenged with 20% ethanol (5 ml) orogastrically followed by buffer or buffer plus 1 mM lysophosphatidylcholine. Compared to other groups, the stomachs of animals given ethanol followed by lysophosphatidylcholine displayed statistically significant increases in the number of gross hemorrhagic lesions, in back-diffusion of hydrogen ion, in net secretion of sodium ion, and in morphologic damage. Transmucosal potential differences in this group were also decreased. We conclude that 90 min after dosing with ethanol, the guinea pig stomach is more susceptible to damage by lysophosphatidylcholine. Our data further suggest that these agents cause mucosal damage by different mechanisms and that the combination acts synergistically.

Lysophosphatidylcholine-induced gastric injury and ulceration in the guinea pig.

The effect of lysophosphatidylcholine (lysoPC) on the guinea pig stomach was studied. At concentrations observed in gastric secretions of gastric ulcer patients (1 to 2 mM), lysoPC induced both functional and morphologic changes in the gastric mucosa. Two millimolar lysoPC caused back diffusion of H+ and forward diffusion of Na+, indicating impairment of the gastric mucosal barrier. In the lysoPC-treated stomachs, an increase was observed in 1) the number of mucosal erosions, 2) the intensity of inflammation, and 3) the degree of vascular congestion. Microvascular plugging by platelets, vascular stasis, and polymorphonuclear leukocyte margination were dose-responsive to lysoPC. Pretreatment of animals with aspirin (20 mg/kg) did not alter injury. These results indicate a role of lysoPC reflux in gastric mucosal injury, including mucosal erosions and ulcerations.

Duodenogastric reflux in patients with gastric ulcer disease.

We studied reflux of duodenal contents into the stomach in patients with gastric ulcers, patients with duodenal ulcers, and normal subjects. Duodenogastric reflux was assessed in the fasting state and after cholecystokinin octapeptide administration (0.02 micrograms/kg intravenously). Slight reflux was observed in the fasting state in all three groups. However, after cholecystokinin octapeptide administration, reflux was significantly greater in gastric ulcer patients than in control patients for pancreatic phospholipase A2 (p less than 0.01) and lysophosphatidylcholine (p less than 0.001). Also in gastric ulcer patients, the gastric contents were significantly more alkaline (pH 5.26 +/- 0.58, p less than 0.001) during duodenogastric reflux than in normal subjects (pH 3.65 +/- 0.50) or duodenal ulcer patients (pH 2.67 +/- 0.63). Our results suggest that reflux of both pancreatic and biliary secretions might contribute to the gastric mucosal injury in gastric ulcer patients and we postulate that pancreatic phospholipase A2 might have a greater role in this process than has been previously acknowledged.

Pancreatitis with arthropathy and subcutaneous fat necrosis. Evidence for the pathogenicity of lipolytic enzymes.

The occurrence of peripheral fat necrosis in exceptional cases of pancreatic disease is not well understood. We report studies on such a patient with arthropathy and subcutaneous nodules. Examination of serial serum samples demonstrated striking elevations of the pancreatic enzymes phospholipase A, 3-3.4 units/ml (normal 0.17-0.41); lipase, 7-39 Sigma-Tietz units/ml (normal less than 1); immunoreactive trypsin, 912-3,207 ng/ml (normal 12-41). The distinguishing characteristic of the patient's synovial fluid was a marked elevation of hydrolized fatty acids (680 mg/dl versus 19 +/- 19 in control inflammatory joint fluids). Synovial fluid fatty acid distribution was identical to values for tissue fat. In contrast, serum fatty acid levels and distribution were normal. No associated proteinase inhibitor or significant immunologic abnormality was detected. Certain properties of adipose cells and lipolytic enzymes may help explain the characteristically selective necrosis of fat cells observed in this syndrome.

The metabolic fate of double-labeled disulfiram.

The metabolic fate of disulfiram labeled with both 14C and 35S was studied in the rat. After administration of 50 mg of 14C, 35S-disulfiram dissolved in corn oil to rats by stomach tube, 95% of the radioactivity was recovered in urine, feces, and expired air at 144 hr. The major portion of the excreted radioactivity was in urine (75 +/- 6%). Feces and expired air contained 13 +/- 2% and 6 +/- 5% of the body organs or carcass. Pretreatment of rats with unlabeled disulfiram for 20 days prior to administration of 14C, 35S-disulfiram led to more rapid catabolism of the drug and more rapid excretion of radioactivity in the urine during the first 12 hr after dosing.

Enzymatic formation of cholesteryl ester from cholesterol by gallbladder mucosa.

The formation of cholesteryl ester from cholesterol and acyl CoA catalyzed by the enzyme acyl CoA:cholesterol acyltransferase (EC 2.3.1.26) was studied in guinea pig gallbladder mucosa homogenate and the subcellular fractions. The enzymatic activity was enriched in the microsomal fraction. Highest activity was observed with palmitoyl, stearoyl or oleoyl CoA as substrate. Lowest activity was observed with linoleoyl CoA. These data elucidate one mechanism for the formation of cholesteryl ester from cholesterol by the gallbladder wall.

The metabolism of 14C-disulfiram by the rat.

After administration of 14C-disulfiram to rats by stomach tube, we found that 87% of the radioactivity was excreted in urine and 7% in feces. Greater than 80% of the radioactivity was excreted by 48 hr. Small but measurable radioactivity was excreted in urine up to 144 hr after administration. Total recovery of radioactivity at 144 hr was 95% of the ingested dose with less than 1% in organs, blood, and carcass; the remainder was in urine and feces. Studies on specific radioactivity showed that diethylamine, a major urinary metabolite of disulfiram, is excreted in the urine undiluted with endogenous diethylamine. Pretreatment of rats with unlabeled disulfiram leads to a more rapid catabolism of the radioactive drug and more rapid excretion of radioactivity in the urine. Further, pretreatment appears to induce formation of a glucuronide conjugate of a disulfiram metabolite.

Lipid metabolism by The gall-bladder. II. The in vitro conversion of lysophosphatidylcholine to phosphatidylcholine.

Lysophosphatidylcholine acyltransferase, which catalyzes the acylation of lysophosphatidylcholine with fatty acid coenzyme A to form phosphatidylcholine, was assayed in gall-bladder mucosa. In guinea pig gall-bladder the activity parallels that of the microsomal enzyme, glucose-6-phosphatase with 3--4-fold enrichment of the activity in the microsomes. Studies with saturated and unsaturated substrates demonstrated highest activity when oleoyl coenzyme A and palmitoyl lysophosphatidylcholine were used and the lowest activity when palmitoyl coenzyme A and palmitoyl lysophosphatidylcholine were used. This activity was demonstrated in the dog, rabbit, cat, calf and human gall-bladder mucosa; however, a wide variation in the amount was observed. Lysophospholipase, which catalyzes the hydrolysis of lysophosphatidylcholine to glycerophosphorylcholine and fatty acid, was also demonstrated in gall-bladder mucosa.

Comparison of linoleic acid and stearic acid absorption by the gallbladder.

The absorption of [14C]linoleic acid and [14C]stearic acid in guinea pig bile by the in situ guinea pig gallbladder was compared. Linoleic acid was adsorbed at a faster rate than was stearic acid. Differences were also observed in the incorporation of these two fatty acids into complex lipids of the gallbladder mucosa. A greater portion of adsorbed linoleic acid was incorporated into triacylglycerol whereas a greater portion of stearic acid was incorporated into sphingomyelin. The significance of these findings in relation to the fatty acid composition of bile is discussed.

Absorption of cholesterol by the gallbladder.

To study whether cholesterol is absorbed by the gallbladder, we instilled from 1 to 300 nmoles of [4-14C]cholesterol dissolved in 1 ml of guinea pig bile into the in situ guinea pig gallbladder. The bile used in these experiments contained 7 mumole/ml bile acid, 27 nmole/ml lecithin, and 8 nmol/ml cholesterol. To this bile, we added the radioactive cholesterol, from 0 to 1 mumole/ml egg lecithin, and 100 mug/ml of bromosulfophthalein, a nonabsorbable marker. After 1 nmole [4-14C]cholesterol was instilled in bile without added lecithin, 77 +/- 8% of the radioactivity was demonstrated to be in the gallbladder wall 6 hours later. The proportion of cholesterol absorbed by the gallbladder decreased as the concentration of added lecithin or bile salts was increased. Radioautography showed radioactivity in the mucosa, and subcellular fractionation of homogenized cells by centrifugation showed cholesterol in the mitochondrial (23 +/- 4%) and microsomal (18 +/- 4%) fractions. Studies on specific activity suggested that there was net absorption of cholesterol and not merely an exchange of cholesterol in the contents for cholesterol in the wall. This study presents evidence that significant quantities of cholesterol but not cholesterol ester can be absorbed by the guinea pig gallbladder. We also found that the absorbed cholesterol can be converted to cholesterol ester and the relevance of these findings to cholesterosis in man are discussed.

Method for the detection of diethylamine, a metabolite of disulfiram, in urine.

Disulfiram is a drug used in the treatment of chronic alcoholism in man. Accurate assessment of patient compliance is important in this treatment. This paper describes a method for the detection and quantitative analysis of diethylamine, a metabolite of disulfiram, in urine. The method involves conversion of the water-soluble diethylamine in the urine to a derivative, N,N-diethyl-3,5-dinitrobenzamide, that is soluble in an organic solvent. This derivative is extracted from urine with diethyl ether and then subjected to thin-layer chromatography. A spectrophotometric procedure is used for quantification. This method provides a means of determining whether or not a patient is taking his prescribed disulfiram.