Bile salt-induced calcium fluxes in artificial phospholipid vesicles.

The ionic permeability of selected biological membranes is increased by bile salts. To examine changes in calcium permeability during the exposure of artificial membranes to bile salts, we investigated calcium uptake by unilamellar and multilamellar phospholipid vesicles. In the presence of 750 microM taurodeoxycholate, uptake of radiolabelled calcium by unilamellar vesicles increased 2.5-fold over control values. Calcium uptake by multilamellar vesicles as measured with a free calcium indicator, arsenazo III, increased 2.2- or 21-fold in the presence of 60 microM lithocholate or 3 beta-hydroxy-5-cholenoate, respectively. Results were directly influenced by experimental variables such as bile salt hydrophobicity, external calcium concentration, and the bile salt/lipid molar ratio. Observed membrane solubilization was minimal despite increased calcium permeability. Comparison of radiolabelled calcium uptake with radiolabelled sodium or radiolabelled rubidium uptake indicated that bile salt-dependent calcium uptake was 60-140-times greater than bile salt-dependent uptake of either monovalent cation. In an effort to delineate forces affecting calcium translocation, vesicles were exposed either to valinomycin, which induced an electrochemical gradient across the membrane, or to nigericin, which induced a proton gradient. Exposure to valinomycin minimally influenced bile salt-induced calcium uptake while exposure to nigericin significantly promoted uptake by 40-70%. The results suggest that bile salts promote calcium uptake by a mechanism which may be similar to those of other carboxylic ionophores.

Overnight plasma profiles of melatonin and certain adenohypophyseal hormones in men.

Melatonin levels exhibited a day-night rhythm with highest levels at night. Nocturnal plasma melatonin concentrations were unrelated to sleep stages, whereas secretion of GH was temporally related to slow wave sleep. Levels of corticotropin rose in the later sleep cycles. We found no relationship between endogenous nocturnal melatonin and adenohypophyseal hormone levels. The results indicate that in young men nocturnal levels of melatonin are controlled separately from those of LH, PRL, corticotropin, and GH.

Circadian rhythm of pineal uptake of 32P in domestic fowl.

The uptake of radioactive phosphorus by the pineal gland in White Leghorn cockerels (Gallus domesticus) showed a diurnal variation with maxima in the light phase and minima in the dark phase of the light: dark cycle. Constant light caused the rhythm to disappear while constant dark had no effect other than lowering the amplitude of the variations. These data indicate that the rhythm in pineal uptake of 32P is circadian.

Effect of abdominal transplanted mammary tumor on peritoneal fluid cytology in mice.

In this study C3H/HeJ female mice received an intraperitoneal injection of mammary tumor suspended in saline; After 14 days, the presence of mammary neoplasm was detected by a significant change in the cytodifferential counts of aspirated peritoneal fluid. Previous studies suggest that ovarian cancer can be detected in mice and in women by the characteristic changes produced in abdominal cellular fluid distribution.

The effect of luteineizing hormone on chicken testis and pineal gland uptake of 32P.

The glandular uptake of radioactive phosphorus (32P), administered carrier-free, was used as an endpoint for the study of the effects of luteinizing hormone (LH) on the testis and pineal gland of 3-, 10- and 13-day-old White Leghorn cockerels. Pineal uptake of 32P of the 13-day-old birds decreased and testis uptake of 32P increased following LH treatment. Maximum effects were observed when 20 mug LH was administered 4.0 h before autopsy. Although testis uptake of 32P increased following LH treatment in 3- and 10-day-old cockerels, pineal uptake of 32P remained unchanged.

Mechanism of increase in steatorrhea with calcium and magnesium in exocrine pancreatic insufficiency: an animal model.

We used a rat model to investigate the phenomenon of increased steatorrhea associated with administration of calcium or magnesium containing antacids in humans with pancreatic insufficiency. Adult male rats with bile and pancreatic duct ligation were fed test meals containing 56 mumol [14C]triolein (0.5 ml), synthetic human bile (1.0 ml, 100 mumol bile salts, 75% glycine and 25% taurine conjugates, and 14.5 mumol lecithin), pancreatic enzymes (0.5 ml), and antacids (1.0 ml). The percent lipid malabsorbed when antacids were fed in addition to the test meal was: control 19.3 +/- 1%, NaHCO3 15.3 +/- 1% (P less than 0.05 vs. control), Al(OH)3 18.3 +/- 2%, Mg(OH)2 38.2 +/- 2% (p less than 0.001 vs. control), and CaCO3 42.4 +/- 1% (p less than 0.001 vs control). With NaCl, Al(OH)3, and NaHCO3 the malabsorbed fat was primarily triolein, whereas with Ca++ or Mg++ the majority of the lipid recovered was oleic acid. Calcium or magnesium administration was associated with precipitation of glycine-, but not taurine-, conjugated bile salts in the small intestine. When calcium was administered to animals in which the bile consisted entirely of glycine-conjugated bile salts, the lipid recovered (64.0 +/- 3% malabsorption) was almost entirely triolein suggesting reduced lipolysis. These studies suggest that these divalent cations exert their deleterious effect on replacement enzyme therapy by formation of poorly soluble calcium or magnesium soaps and precipitation of glycine conjugated bile salts.

Comparison of phenolphthalein and phenolphthalein glucuronide on net water transport in rat ileum and colon.

Phenolphthalein is an odorless, tasteless compound, poorly soluble in water, but readily soluble in alcohol or ether. Although its laxative activity was discovered in 1902, the mechanism of action remains unclear. Phenolphthalein has been found to be an ineffective laxative in both jaundiced patients and in experimental animals with ligated common bile ducts. In contrast to the poor solubility of phenolphthalein, phenolphthalein glucuronide is highly water-soluble. In addition, phenolphthalein glucuronide is found in bile following oral phenolphthalein administration, suggesting an enterohepatic circulation. We compared the effect of phenolphthalein and phenolphthalein glucuronide on net water absorption of the ileum and colon of Sprague-Dawley rats using a single-pass perfusion technique. Perfusion of phenolphthalein (10 mg/dl in 154 mM NaCl with 0.5% ethanol and 0.1 g/l gum arabic) resulted in significantly reduced water absorption in both the ileum and colon (66% and 64%, respectively, P less than 0.05). In contrast, phenolphthalein glucuronide did not have a significant effect on net water flux compared to control perfusion in either the ileum or colon. In the ileum only 77 +/- 3% and colon 71 +/- 1.2% of the phenolphthalein infused was recovered, suggesting that much of the infused phenolphthalein was absorbed. In contrast there was no evidence of absorption of phenolphthalein glucuronide, as 98.9 +/- 1.2% was recovered from ileal and colon perfusions. Phenolphthalein glucuronide is not the active form of phenolphthalein, although it may be an effective laxative after deconjugation by colonic bacteria.

Pathogenesis of rotavirus-induced diarrhea. Preliminary studies in miniature swine piglet.

The pathogenesis of diarrhea caused by rotavirus infection was studied in miniature swine piglets. The animals were inoculated orally with 2 X 10(7) plaque-forming units of porcine rotavirus (OSU strain). During the height of diarrhea, intestinal function was investigated by in vivo perfusion of a 30-cm segment of proximal jejunum and a 30-cm segment of distal ileum. Absorption of Na+ and water decreased and 3-O-methylglucose transport was markedly reduced, P less than 0.01 compared to control animals. Mucosal lactase and sucrase levels were depressed in both the jejunum and ileum, P less than 0.001. Na+,K+-ATPase activity was significantly depressed only in the ileum, P less than 0.001. These changes were associated with a marked reduction in villous height, suggesting that the diarrhea could be an osmotic diarrhea due to nutrient (carbohydrate) malabsorption. Fresh stool samples were obtained and analyzed immediately for NA+,K+, osmolarity, glucose, and lactose; the osmotic gap was also determined. Stool osmolarity continually increased from 248 +/- 20 mosm/liter prior to inoculation to 348 +/- 20 mosm/liter at 75 +/- 1 hr postinoculation (P less than 0.005); the majority of the fecal osmotic gap could be accounted for by the amount of lactose present in the stools. Stool sodium increased from 34 +/- 6 mM prior to inoculation to a maximum of 65 +/- 4 mM at 53 +/- 1 hr postinoculation, P less than 0.001. There was no significant change in potassium concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Does mixing pancreatic enzyme microspheres (Pancrease) with food damage the enteric coating?

Supplemental pancreatic enzymes are required for the treatment of malabsorption associated with cystic fibrosis. It is a general practice for enzymes to be sprinkled on, or administered in, food given to infants or children who require pancreatic enzyme replacement. We tested the effect of in vitro incubation of enteric-coated pancreatic enzyme microspheres with infant foods on total recoverable enzyme activity and on the enteric coating. Incubation of enzyme beads with foods with pH greater than 5 (i.e., chicken pH 6.5, veal pH 6.3, and green beans pH 5.6) was without significant effect on either parameter. There was a statistically significant, albeit minor, reduction in maximum enzyme activity recovered following incubation with applesauce (pH 3.5). In addition, the thickness of the enteric coating was significantly reduced. Whether changes in the enzyme beads results in improved or diminished efficiency of therapy remains to be determined. Lipase, unprotected by enteric coating, was rapidly inactivated in applesauce, emphasizing the need to instruct parents about which foods to avoid when administering pancreatic enzymes without enteric coating.

Effects of blinding, pinealectomy and superior cervical ganglionectomy on free thyroxine index of male golden hamsters.

Blinding young male golden hamsters led to a depression of the plasma free thyroxine index after an 8 week period. Removal of either the pineal gland or the superior cervical ganglia of blind hamsters reversed the effects of blinding on free thyroxine index. No significant effects were observed 2 weeks after treatment. Previously reported effects of blinking, and removal of the pineal gland or the superior cervical ganglia on testicular weight were confirmed. A similar mechanism of pineal inhibition of thyroid function and gonadal function is suggested.

A simple staining method for vaginal smears using red ink.

In adult rhesus monkeys each phase of 37 menstrual cycles was accurately classified by cellular identification of vaginal smears stained with red ink. This technic was simple, quick, and inexpensive, and produced well-stained slides in a few seconds.

The Hemo-matic Analyzer: a new occult blood testing device.

It is now recognized that screening for fecal occult blood is useful to alert the clinician to possible colon cancer in an early stage. The guaiac impregnated card is not widely used because patients must maintain strict diets omitting red meat, peroxidase-containing foods, iron supplements, and ascorbic acid. The Hemo-matic Analyzer uses a special filter to adsorb fecal Hb which is then automatically washed free of interfering substances and quantitatively identified. The filter electrostatically binds Hb and not pepsinized blood or substances that interfere with occult blood testing. We evaluated the sensitivity of this device and compared it to the Hemoccult II test in vivo and in vitro. It was approximately six times more sensitive than Hemoccult II and was not influenced by the presence of ascorbic acid, plant peroxidase, or ingestion of a large quantity of rare red meat. In addition, the samples remained positive more than 10 wk after preparation. The Hemo-matic Analyzer is a useful new device that offers many advantages over Hemoccult card testing for screening for colon cancer.

In vitro adsorption of bile salts and aspirin to sucralfate.

Sucralfate, an aluminum salt of sulfated sucrose, is a new drug designed for the treatment of peptic ulcer. Sucralfate has been reported to be useful in a variety of situations including prevention of aspirin-induced gastric mucosal damage. We investigated the in vitro adsorption of bile salts or aspirin to sucralfate in environments simulating the stomach (pH 1.5), small intestine (pH 7), and colon (pH 7.8). Bile salts were incubated with sucralfate, and the quantity of bile salt adsorbed was calculated by subtraction from the amount remaining in solution after centrifugation at 12,500g for 30 min. Adsorption experiments were performed in bile salt solutions at pH 1.5 and 7.0 with 0-10 g/dl sucralfate using glycocholate, glycochenodeoxycholate, taurocholate, taurodeoxycholate, or taurochenodeoxycholate. The dihydroxy-unconjugated bile salts, deoxycholic, and chenodeoxycholic salts were tested at pH 7.8. Binding capacity (micromoles per gram sucralfate) was calculated from the linear regression of micromoles bound vs grams sucralfate incubated. Sucralfate adsorbed all bile salts tested (except taurocholic acid at pH 1.5) but was less effective than cholestyramine. Sucralfate does not adsorb sufficient bile salts at neutral pH to cause bile salt depletion. Aspirin was minimally adsorbed by sucralfate [7.5 mumol (1.4 mg)/g sucralfate, pH 1.5], and thus adsorption of aspirin does not explain the protective effect of sucralfate against aspirin injury.

Bile salts induce calcium uptake in vitro by human erythrocytes.

At high concentrations, bile salts induce hemolysis by comicellization of lipid components of the cell membrane. However, bile salts are also associated with hemolysis at lower concentrations by mechanisms which have not been characterized. To investigate the possibility that bile salts promote calcium uptake by red blood cells and that bile salt-associated hemolysis is, in part, calcium-mediated, calcium uptake by red blood cells was measured in the presence of individual bile salts, and hemolysis dependence upon calcium availability was examined. Washed human red blood cells with or without ATP depletion were incubated with 1 mM CaCl2 and tracer amounts of 45CaCl2 in the presence of selected bile salts at concentrations (0.01 to 0.3 mM) reported to be below critical micellar concentrations. Calcium uptake (defined for the purposes of this study as 45Ca retained in red blood cells) was monitored over 5 hr, after which hemolysis and membrane phospholipid content were determined. The presence of bile salts stimulated calcium uptake 4- to 25-fold--the magnitude of which was partly related to the lipid solubility of the bile salts. ATP depletion or exposure to trifluoperazine, procedures which inhibit calcium pump activity in red blood cells, enhanced bile salt-induced calcium uptake relative to controls. The percentage of associated hemolysis (2 to 14%) at the end of 5 hr correlated directly with the observed calcium uptake. Removal of calcium from the extracellular space reduced hemolysis in the presence of bile salts to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of pineal indoles given late in the light period on reproductive organs and pituitary prolactin levels in male golden hamsters.

Intact and pinealectomized adult male hamsters kept in light:dark cycles of 14:10 (in hours) were given daily injections of either 25 microgram melatonin, 5-methoxytryptophol, N-acetylserotonin, or 6-hydroxymelatonin at 19.00 hours (13h after lights on). The injections were continued for 50 consecutive days. The daily melatonin injections significantly elevated body weights and depressed the growth of the testes and accessory sex organs and pituitary prolactin levels in intact hamsters. By comparison, in pinealectomized animals daily afternoon melatonin injections failed to alter body or testicular weights or pituitary prolactin levels. These findings indicate that for single injections of melatonin to be antigonadotrophic in the male hamster, the pineal must be intact. In intact animals, 5-methoxytryptophol and N-acetylserotonin slightly depressed the growth of the reproductive organs and pituitary prolactin levels, respectively. Again, these effects were negated by removal of the pineal gland. 6-Hydroxymelatonin was without influence on any of the parameters of reproduction that were measured.

Subcutaneous melatonin implants inhibit reproductive atrophy in male hamsters induced by daily melatonin injections.

Daily afternoon (at 7 p.m.) injections of melatonin (25 microng in oil) into adult male hamsters for 50 days led to atrophy of the testes and accessory sex organs (seminal vesicles and coagulating glands) and in a significant depression in pituitary LH and prolactin content and concentration. These actions of melatonin were prevented if the animals had been pinealectomized before the daily melatonin injections were begun. Likewise, if hamsters received a weekly subcutaneous implant of melatonin in beeswax (1 mg melatonin in 24 mg beeswax) the daily melatonin injections failed to inhibit the growth of the reproductive organs and to depress pituitary LH and prolactin levels. Beeswax by itself had no such effect.

Influence of pinealectomy on serum estrogen and progesterone levels in blind-anosmic female rats.

Previous studies show that the suppression of gonadal function in blind-anosmic rats is dependent on the pineal gland. The present results demonstrate that in young female rats both the pineal gland and dual sensory deprivation have additional independent antigonadal effects.