Bile acids: effects on absorption of 1,2-dimethylhydrazine and 7,12-dimethylbenz[a]anthracene in the colon of the rat and guinea pig.

The specific effects of bile acids as cocarcinogens were investigated. Absorptions of [14C]7,12-dimethylbenz[a]anthracene, [14C]dimethylhydrazine ([14C]DMH), and [3H]inulin from loops of colons from outbred Sprague-Dawley rats and Hartley guinea pigs were determined. In each animal absorption of one carcinogen and inulin was studied in one control loop and in an experimental loop containing either deoxycholic acid (DOC) or chenodeoxycholic acid (CDOC). DOC had a more pronounced effect on increasing loss of carcinogen from the intestinal lumen than did CDOC. This role of bile acids was consistent with their known effect of increasing intestinal permeability. Less carcinogen remained in the colon mucosa when DOC was present in the intestinal lumen. Although [14C]DMH was absorbed more rapidly from the intestinal lumina of guinea pigs than from those of rats, the rat accumulated more of the carcinogen in the intestinal mucosa and liver.

Solubility properties of reduced and oxidized ascorbate as determinants of membrane permeation.

The oil/water distribution coefficients of ascorbic acid and dehydro-L-ascorbic acid have been determined and compared with values for mannitol and lauric acid. In general, the relative degrees of hydrophobicity of the compounds evaluated are lauric acid much greater than mannitol approximately equal to dehydro-L-ascorbic acid greater than ascorbic acid. These findings and recent reports from transport studies do not support the concept that dehydro-L-ascorbic acid is very hydrophobic and crosses cell membranes rapidly by simple diffusion.

Transport of L-ascorbic acid and dehydro-L-ascorbic acid across renal cortical basolateral membrane vesicles.

The uptake of L-ascorbic acid and dehydro-L-ascorbic acid into renal cortical basolateral membrane vesicles has been characterized. The uptake systems for both solutes demonstrate saturation kinetics. The presence of structural analogs of L-ascorbic acid and dehydro-L-ascorbic acid results in cis-inhibition and trans-stimulation. Uptake of each substrate is Na+-independent, proceeding to an endpoint of substrate equilibrium across the vesicular membrane. The transport mechanism(s) for L-ascorbic acid and dehydro-L-ascorbic acid appears to be facilitated diffusion.

Na+-independent dehydro-L-ascorbic acid uptake in renal brush-border membrane vesicles.

A membrane preparation enriched in the brush-border component of the plasma membrane was isolated from rat renal superficial cortex by a divalent cation precipitation procedure. Uptake of dehydro-L-ascorbic acid, the oxidized form of L-ascorbic acid, by the brush-border membrane vesicles was studied. The uptake mechanism was found to be sodium-independent and insensitive to the trans-membrane electrical potential difference. Uptake was saturable and subject to cis-inhibition. Concentrative uptake was demonstrated only under conditions of trans-stimulation by structural analogs. The results suggest a mechanism of facilitated diffusion for the uptake of dehydro-L-ascorbic acid in renal brush-border membranes.

Effective use of renal cortical slices in transport and metabolic studies.

The uptake and metabolism of two water-soluble vitamins were measured in rat renal cortical slices, isolated tubules, and vesicles of the brush-border and basolateral cell membranes to determine (a) whether it is possible to produce slices that have open tubules and, (b) whether slices and tubules metabolize vitamins similarly. Transport of ascorbic acid is sodium-dependent in slices and in brush-border vesicles but is sodium-independent in basolateral vesicles, suggesting that the brush-border membrane of slices is accessible to components of the bathing solution. Nicotinic acid was metabolized similarly (97-98%) in both slices and isolated tubules. Oxygen consumption by slices maintained in a closed chamber was constant as pO2 decreased from 88% to 58%. Slices are concluded to be a suitable model for transport and metabolic studies providing that care is taken in their preparation and use.

Absorption of 2-acetylaminofluorene in the guinea-pig colon.

Absorption of the known chemical carcinogen, 2-acetylaminofluorene has been measured in the colon of guinea pigs. Unidirectional influx across the luminal cell membrane was determined in vitro, and transmural absorption across colonic mucosa was evaluated in vivo. The kinetics of unidirectional influx into colon in vitro do not indicate that absorption proceeds by simple diffusion. The observed saturable uptake is indicative of binding of 2-acetylaminofluorene to a cellular component. With 2-acetylaminofluorene present in the lumen in vivo at an initial concentration of 3.5 microM, the rate of absorption decreases over a 20 min period, which also indicates some form of specific interaction between 2-acetylaminofluorene and the intestinal mucosa. We have evaluated the hypothesis that surfactants and a bile salt act as cocarcinogens by increasing the rate of intestinal absorption of 2-acetylaminofluorene. The results lend no support to this possibility.

Choline influx across the brush border of guinea pig jejunum.

Choline uptake across the mucosal border of guinea pig jejunum was measured to determine the characteristics of this step in intestinal absorption. Unidirectional influx of [14C]choline appears to proceed primarily by a saturable, carrier-mediated process at low mucosal choline concentrations; at high concentrations (greater than 4 mM) the influx rate is approximately linearly related to the mucosal choline concentration, suggesting that absorption by passive diffusion predominates. Influx was only minimally reduced by elimination of Na+ from the mucosal test solution or by reduction of the intracellular Na+ concentration. Preincubation of tissue samples with metabolic inhibitors or with ouabain did not markedly reduce influx. These results are consistent with a model of choline transport across the brush border membrane by a carrier-mediated mechanism which is similar to that involved in fructose absorption but different from the Na+-dependent mechanism which participates in active transport of sugars and amino acids. At low lumenal choline concentrations, influx into colonic mucosa is slower than in jejunum and appears to be attributed solely to simple diffusion.

Studies on the electrical potential profile across rabbit ileum. Effects of sugars and amino acids on transmural and transmucosal electrical potential differences.

When isolated strips of mucosal rabbit ileum are bathed by physiological electrolyte solution the electrical potential difference (PD) across the brush border (psi(mc)) averages 36 mv, cell interior negative. Rapid replacement of Na in the mucosal solution with less permeant cations, Tris or choline, results in an immediate hyperpolarization of psi(mc). Conversely, replacement of choline in the mucosal solution with Na results in an abrupt depolarization of psi(mc). These findings indicate that Na contributes to the conductance across the brush border. The presence of actively transported sugars or amino acids in the mucosal solution brings about a marked depolarization of psi(mc) and a smaller increase in the transmural PD (Deltapsi(ms)). It appears that the Na influx that is coupled to the influxes of amino acids and sugars is electrogenic and responsible for the depolarization of psi(mc). Under control conditions Deltapsi(ms) can be attributed to the depolarization of psi(mc) together with the presence of a low resistance transepithelial shunt, possibly the lateral intercellular spaces. However, quantitatively similar effects of amino acids on psi(mc) are also seen in tissues poisoned with metabolic inhibitors or ouabain. Under these conditions Deltapsi(mc) is much smaller than under control conditions. Thus, the depolarization of psi(mc) might not account for the entire Deltapsi(ms), observed in nonpoisoned tissue. An additional electromotive force which is directly coupled to metabolic processes might contribute to the normal Deltapsi(ms).

Glutathione-dependent ascorbate recycling activity of rat serum albumin.

An efficient regeneration of vitamin C (ascorbate) from its oxidized byproduct, dehydroascorbate (DHAA), is necessary to maintain sufficient tissue levels of the reduced form of the vitamin. Additionally, the recycling may be more significant in mammals, such as guinea pigs and humans, who have lost the ability to synthesize ascorbate de novo, than it is in most other mammals who have retained the ability to synthesize the vitamin from glucose. Both a chemical and an enzymatic reduction of DHAA to ascorbate have been proposed. Several reports have appeared in which proteins, including thioltransferase, protein disulfide isomerase, and 3-alpha-hydroxysteroid dehydrogenase, characterized for other activities have been identified as having DHAA reductase activity in vitro. Whether these previously characterized proteins catalyze the reduction of DHAA in vivo is unclear. In the present study, a 66 kD protein was purified strictly on the basis of its DHAA-reductase activity and was identified as rat serum albumin. The protein was further characterized and results support the suggestion that serum albumin acts as an antioxidant and exerts a significant glutathione-dependent DHAA-reductase activity that may be important in the physiologic recycling of ascorbic acid.

Fever in hospitalized patients. With special reference to the medical service.

Fever (oral temperature of 38 degrees C or more on two or more consecutive days) during the hospital stay of 4,065 patients admitted to Grady Memorial Hospital during an 11-week period was studied. At least one episode of fever occurred in 1,194 patients (29 percent). Rates of fever were highest on medical and surgical services. Review of 341 episodes of fever in 302 patients on the medical service identified a single potential cause in 56 percent. Multiple factors were present in 26 percent, and no potential causes were found in 18 percent. Of 390 factors identified, 44 percent were community-acquired infections, 9 percent were nosocomial infections, 20 percent possibly involved infection, and 26 percent were noninfectious processes. Fever is a frequent finding in hospitalized patients. Both infectious and noninfectious processes play important roles. Determining the cause of fever is complicated by the multiplicity of possible causes.

Ascorbic acid uptake and metabolism by corneal endothelium.

Ascorbic acid is concentrated in various ocular compartments where it is thought to protect diurnal animal species against damaging effects of ultraviolet radiation. The authors evaluated the possibility that corneal endothelial cells have specific transport and/or metabolic properties that deliver ascorbic acid to the stroma. Bovine corneal endothelial cells were grown to confluence in multiple-well plates. Individual groups of cells (approximately 10(4)) were then incubated at various times at 34 degrees C in a physiologic buffer that contained a 10 microM level of 14C-labeled ascorbic acid or the oxidized product, dehydro-L-ascorbic acid. Endothelial cells take up dehydro-L-ascorbic acid at least seven times as rapidly as they take up ascorbic acid. After 30 sec of incubation with 14C-dehydro-L-ascorbic acid, most of the label accumulated in the cell is in the reduced form. Uptake is inhibited by cyanide and iodoacetamide but is unaffected by ouabain. Exposure of cultured cells to various intermediates in the energy metabolism pathways reduced uptake of ascorbic acid but had a minor effect on uptake of the oxidized molecule. These results suggest that the cornea has transport and metabolic capacity to extract dehydro-L-ascorbic acid from aqueous humor and reduce it, thus providing a source of ascorbic acid for corneal protection. This also would maintain "total" ascorbic acid of aqueous humor in the reduced state.

Changes in hepatic bile secretion following cholecystectomy.

The volume and compositon of hepatic bile was studied in anesthetized dogs before and approximately 18 weeks after cholecystectomy. Dogs had common duct cannulation, cystic duct ligation, and temporary occlusion of the pylorus. An intravenous infusion of sodium taurocholate, 9 muEq per minute, was given throughout the experiment. Bile volume, electrolyte composition, bile salt output, and 14C-mannitol clearance were determined during infusion of sodium taurocholate alone and after addition of secretin, 4 U. per kilogram per hour, to the infusion. Cholecystectomy then was done and the same experiment was repeated 18 weeks later. Cholecystectomy significantly increased bile volume, sodium and chloride output, and 14C-mannitol clearance both in response to taurocholate alone and to taurocholate plus secretin. Bile salt output and bicarbonate output were unchanged. The study shows that cholecystectomy significantly alters the volume and composition of hepatic bile. The data indicate that cholecystectomy does not affect ductular function or hepatic secretion of bile salts but that canalicular function is altered significantly.

Characteristics of inhibition of pancreatic secretion by isoproterenol.

The effect of isoproterenol on pancreatic secretion stimulated by the octapeptide of cholecystokinin (OP-CCK) was studied in dogs with gastric and pancreatic fistulas. Special precautions were taken to prevent entry of gastric acid into the duodenum and reflux of duodenal content into the stomach. OP-CCK increased volume and the outputs of bicarbonate and protein from the pancreas. Isoproterenol inhibited these pancreatic responses to OP-CCK, and the degree of inhibition was dose related. Propranolol blocked the inhibitory action of isoproterenol completely. Analysis of data by Michaelis-Menten kinetics revealed that there was competitive inhibition of protein output with noncompetitive inhibition of bicarbonate output. The experiments show that isoproterenol inhibits OP-CCK--stimulated pancreatic secretion by a beta-adrenergic mechanism. The kinetic analysis suggests that the intimate mechanisms involved in inhibition of protein and bicarbonate output by isoproterenol are different.

Effect of histamine on technetium-99m excretion by gastric mucosa.

The ability of histamine to increase excretion of 99mtechnetium by gastric mucosa was investigated in dogs with Heidenhain pouches and denervated antral pouches. Histamine increased Heidenhain pouch 99mtechnetium output in a dose-related manner, and 99mtechnetium output was related linearly to acid output. Antral pouch 99mtechnetium output was unchanged by increasing doses of histamine. The study suggests that concomitant use of histamine may improve the accuracy of 99mtechnetium scanning in the clinical diagnosis of conditions caused by ectopic gastric mucosa.

Metabolism and transport of dehydroascorbic acid in erythrocytes of "spontaneous diabetic BB/W" Wistar rats.

Rates of uptake and reduction of dehydroascorbic acid in erythrocytes of "Spontaneous diabetic BB/W" and control Wistar rats were determined. Lysed cells reduced 14C-dehydroascorbic acid more rapidly than intact cells did, suggesting that membrane transport is a rate-limiting step. Diabetic rats had lower plasma levels of ascorbic acid but more rapid reduction of dehydroascorbic acid than control animals. The results indicate more rapid transport of dehydroascorbic acid into erythrocytes of prediabetic "BB/W" rats than Wistar rats.

Cerebral metabolism of oxidized ascorbate.

The brain has a high level of ascorbic acid which is thought to act as a reducing agent, e.g. in protecting tissues against oxidative stress. The mechanism by which ascorbate is maintained in the useful, reduced state in the CNS is evaluated herein. Cerebrum from rat or calf was minced and homogenized in buffer. The endogenous levels of ascorbic acid, dehydro-L-ascorbic acid (DHAA) and reduced glutathione (GSH) were determined by HPLC with coulometric electrochemical detection. We also quantitated tissue capacity to regenerate ascorbic acid from DHAA, which is a product of electron transfer reactions of ascorbic acid. The homogenate was fractionated by centrifugation in steps up to 110,000 x g and dialyzed free of low molecular weight components. The activity for reducing DHAA was approximately equal in the various supernatants; resuspended pellets had little activity. The active component has several properties of a protein, including being precipitated by solid ammonium sulfate addition to the tissue extract; most activity appeared in the 40-80% saturated fraction. The activity was stable up to a temperature of 80 degrees C, but was lost at 95 degrees C. The protein was digested by trypsin. The results suggest that a cytosolic component of cerebrum regenerates ascorbic acid in a step that preferentially uses GSH and NADPH as reducing cofactors. At least one form of DHAA reductase exists in brain.

Water soluble antioxidants in mammalian aqueous humor: interaction with UV B and hydrogen peroxide.

HPLC/electrochemical detection was used to identify five major low MW water soluble electrochemically active molecules from the aqueous humor of three species of mammals: New Zealand White rabbits and humans (diurnal) and Sprague-Dawley rats (nocturnal). These molecules are L-cysteine (CYS), L-ascorbic acid (AA), glutathione (GSH), uric acid (UA) and L-tyrosine (TYR); all of these molecules have known antioxidant properties. Nocturnal rat aqueous humor is concentrated in two thiols: GSH (125 microM; n = 24 pooled eyes) and CYS (63 microM), in contradistinction to diurnal species which have high concentrations of AA. No deterioration of any of these antioxidants occurs in a synthetic aqueous humor mixture irradiated with a physiologically relevant spectral UV B dose of 30 mJ/cm2/h (5.5 UV equivalent sunlight hours). The same result occurred with addition of the endogenous aqueous humor UV B photosensitizer L-tryptophan. In a second set of experiments, human synthetic aqueous humor was subjected to hydrogen peroxide induced oxidant stress. The decay of antioxidants was CYS > GSH > AA > UA > TYR. The second highest concentrated antioxidant in human aqueous humor is TYR. Yet TYR failed to protect AA against H2O2-induced free radical damage in a synthetic aqueous humor model system (P = 0.10; ANOVA). The existence of multiple electrochemically active constituents and their thermodynamic interactions must be recognized when choosing animal models to evaluate human aqueous humor antioxidant defense.

Dehydroascorbic acid and cell membranes: possible disruptive effects.

Exposure of cellular membranes to dehydroascorbic acid can result in a loss of membrane integrity. Renal brush border or basolateral membrane vesicles pre-incubated with dehydroascorbic acid demonstrate a decrease in initial transport rates of D-glucose and a loss of intravesicular volume. The activity of brush border membrane specific leucine aminopeptidase is increased in vesiculated membrane preparations following exposure of the vesicles to either dehydroascorbic acid or Triton X-100. Erythrocytes in isotonic buffer with dehydroascorbic acid lose membrane integrity as demonstrated by a release of hemoglobin.

Ascorbic acid uptake in guinea pig intestinal mucosa.

Cellular accumulation of ascorbic acid was investigated in vitro in distal intestinal mucosa of guinea pig. With 14C-ascorbic acid present at 8 microM/L in the bathing media, tissue/media (T/M) concentration ratios of at least 5 were routinely achieved. Recently absorbed ascorbic acid appeared to be free in solution in the cellular fluid in that it diffused from tissue exposed to poisons with a disappearance half-time of approximately 10 minutes. Ascorbic acid uptake was highly dependent on the presence of sodium in the bathing media; total Tris substitution resulted in a 97% decrease in uptake. Also, metabolically depleted tissue did not accumulate ascorbic acid against a concentration gradient. Uptake of 14C-ascorbic acid from a bathing solution concentration of 8 microM/L was reduced 67% in the presence of 0.8 mM/L nonlabeled ascorbic acid. Recently absorbed 14C-ascorbic acid moved more rapidly back into the lumen when the luminal solution contained nonlabeled ascorbic acid (5 mM) than when it contained mannitol (5 mM). This demonstration of counter transport substantiates a carrier mechanism in the brush border.

Short term effects of oxidized ascorbic acid on bovine corneal endothelium and human placenta.

Studies on the toxic effects of dehydro-L-ascorbic acid (DHAA) have been extended to include evaluations over time periods up to 3 hr. and to test for specific effects on a membrane transport protein, a membrane-bound enzyme and a soluble intracellular enzyme. In studies on cultured corneal endothelial cells, DHAA concentrations of 1, 2, and 5 mM over 3 hr. had an inhibitory effect on subsequent uptake of DHAA present at a tracer level. Surviving fragments of human placenta and alkaline phosphatase activity of the placental brush-border membrane were susceptible to the effect of DHAA at a high concentration (10 mM). Because intracellular metabolism of DHAA was not affected, and an increase in membrane permeability was not detected, it is concluded that a specific membrane transport protein might be the site of DHAA-induced damage. These studies support the concept that the oxidized form of ascorbic acid (vitamin C) has potential toxic effects on biological systems and suggests that proteins that mediate transport and metabolism may be sites where DHAA causes damage.