Development and age-related changes in pancreatic cholecystokinin receptors and duodenal cholecystokinin in guinea pigs.

We have investigated the changes associated with development and aging on the interrelationships between cholecystokinin (CCK) and the pancreas in the guinea pig. Three groups (1 month old, 1 year old, and 3 years old) of male guinea pigs were sacrificed while feeding in order to measure food-stimulated levels of CCK in blood and in duodenal mucosa by radioimmunoassay (RIA), as well as the pancreatic concentrations of CCK receptors. Systemic blood concentrations of CCK did not change with age. However, the concentration and content of CCK in duodenal mucosa increased more than 3-fold with age. A single class of high-affinity (KD less than or equal to 0.1 nM) CCK-receptor was found on the pancreatic membranes. The concentration (fmol/mg protein) of these receptors significantly diminished by one-half with increasing age. We also found an apparently similar fall in the receptor-binding affinity, but the difference was not significant. We conclude that in the guinea pig, duodenal content of CCK increases so as to compensate for the decreasing concentration of pancreatic CCK receptors, or, perhaps, vice versa. The diminished exocrine function of the pancreas, seen with increasing age, may well reflect both the diminished number of CCK-receptors and the reduction of pancreatic acinar cells.

Vapor pressure and freezing point osmolality measurements applied to a volatile screen.

This is a report of a rapid and precise screening procedure, developed for the determination of ethanol in serum using osmolality measurements. The osmolality of the patient is determined by freezing point method (freezing point osmometry) and dew point (water vapor pressure osmometry) method. The difference between freezing point osmolality and vapor pressure osmolality (delta osm) is due to the presence of volatiles in the serum, because the volatiles are not measured by vapor pressure osmometry. The amount of ethanol (mg/dL) in serum is estimated by multiplying delta osm by a factor of 4.2. As a comparison method, ethanol also is measured by a spectrophotometric alcohol dehydrogenase method. In addition, a significant difference between an osmometric alcohol assayed value and enzymatic spectrophotometric measurement indicates the presence of volatiles, other than ethanol. In addition to ethanol there is a linear relationship between osmolality and isopropanol or methanol when added in vitro to serum.

Bile enhances release of insulin: an incretin-mediated effect.

Ingestion of food stimulates secretion of bile and release of gut hormones that enhance nutrient-stimulated release of insulin. The extent of physiologic participation of bile in the enteroinsular axis was examined in seven conscious dogs (weight: 20 +/- 2 kg) that were prepared for study with chronic cannulas placed in the duodenum opposite the ampulla of Vater. On separate days, a meal consisting of 10 gm (MG-10), 25 gm (MG-25), or 62 gm (MG-62) of glucose (dextrose) was given orally in the presence of normal bile flow or during bile diversion. Bile diversion was achieved by catheterization of the common bile duct via the duodenal cannula. The insulin responses (given as ng [0-120] min/ml) to the different glucose meals, with bile present (BP) or absent (BA) in the lumen, were as follows: MG-10, 81 +/- 8 BP and 72 +/- 11 BA; MG-25, 172 +/- 25 BP and 100 +/- 6 BA (p less than 0.05); and MG-62, 390 +/- 79 BP and 153 +/- 32 BA (p less than 0.05). Only MG-25 and MG-62 produced a significant elevation of plasma glucose concentrations. Release of gastrin was not affected by either the presence of bile or the glucose content of the meal. We conclude that endogenous bile enhances nutrient-stimulated release of insulin, this effect is glucose-dependent with a threshold of approximately 1 gm/kg of glucose, and bile may facilitate the release of insulinotropic hormones other than gastrin.

Quality control for a 14- to 18-megohm-cm deionized water supply.

We studied the quality control for a 14- to 18-megohm-cm deionized water system with multiple outlets. The quality of water was determined by measuring specific resistance, performing a total colony count, trace metal analysis, and high-performance liquid chromatographic analysis. The specific resistivity of water was at the level of 17 to 18 megohms-cm at the recirculation loop and greater than 10 megohms-cm at the individual faucets. The microbial content was consistently less than 100 colony-forming units/mL. Periodic trace-metal analysis indicated less than 1 microgram/L of copper, bismuth, zinc, cadmium, mercury, thallium, and lead. High-performance liquid chromatographic analysis, as determined by the absorbance of the highest peak by ultraviolet and fluorescence spectroscopy, indicated less than or equal to 0.001 absorbance unit. Our experience suggests that maintaining laboratory water at 14 to 18 megohms using a plastic system with plastic faucets eliminates the likelihood of bacterial organic and trace-metal contamination of the water.

Effect of age and sensitivity to cholecystokinin on gallstone formation in the guinea pig.

The purpose of this study was to evaluate the effect of age and the role of cholecystokinin therapy on gallstone formation in guinea pigs. Guinea pigs (31 1-mo-old, 31 1-yr-old, and 23 3-yr-old) were placed on a cholelithogenic diet for 2 wk while another 10 guinea pigs of each age group remained on regular chow. Half of each group received a daily injection of cholecystokinin (0.5 nmol/kg). After 2 wk, guinea pigs were killed and the gallbladders were examined for gallstones. The concentrations of bile constituents were determined. The prevalence of gallstones was: 1-mo-old, control 0 out of 16, cholecystokinin 1 out of 15; 1-yr-old, control 3 out of 14, cholecystokinin 5 out of 16; 3-yr-old, control 10 out of 11, cholecystokinin 3 out of 8. Gallstone formation was significantly greater in 3-yr-old controls than in the two younger control groups, and cholecystokinin treatment significantly reduced the incidence of gallstones to near the level seen in younger guinea pigs. In the two younger age groups (but not in the 3-yr-old group), the cholelithogenic diet significantly reduced the concentration of bile salts in bile below that of guinea pigs on a normal diet. The cholelithogenic diet and treatment with cholecystokinin did not alter the relative compositions of bile lipids from that of guinea pigs on a normal diet in any of the three ages studied. In the second experiment we measured gallbladder emptying in response to exogenous infusion of cholecystokinin-8 (100 fmol/kg/h-100 nmol/kg/h) in the same three age groups of guinea pigs in vivo that had been maintained on regular chow. There was no difference in cholecystokinin sensitivity between the two younger age groups, but both were significantly more sensitive to cholecystokinin than the 3-yr-old guinea pigs in rate of gallbladder emptying in the dose range 1 pmol/kg/h-1 nmol/kg/h. We conclude that a major factor in the increased incidence of gallstone formation in the aged guinea pig gallstone model is decreased gallbladder emptying due to decreased gallbladder sensitivity to cholecystokinin.

Effect of oral fat on plasma levels of neurotensin and neurotensin fragments in humans. Characterization by high-pressure liquid chromatography.

The effect of ingestion of fat (Lipomul 1 g/kg) on the circulating levels of neurotensin (NT1-13) and amino-terminal fragments (NT1-8, NT1-11) and carboxy-terminal fragment (NT8-13) of NT were investigated in six healthy male volunteers. NT and NT fragments were extracted from plasma collected at 0, 15, 30, and 60 min after ingestion of fat, and the plasma levels of NT1-13 and NT fragments were characterized using high-pressure liquid chromatography and radioimmunoassay techniques. Significant elevations of plasma levels of NT1-8, NT1-11, and NT1-13 were observed at 15, 30, and 60 min after fat ingestion. The maximum elevations were 273% for NT1-8, 234% for NT1-11, and 54% for NT1-13. NT8-13 levels failed to rise significantly when compared to basal levels. These findings indicate that both the amino-terminal and carboxy-terminal fragments of NT are either released along with intact NT or are formed as metabolites from NT1-13 in response to ingestion of fat in man.

Characterization of secretin release in response to food and intraduodenal administration of fat and hydrochloric acid.

The development and validation of a radioimmunoassay that detects release of secretin in plasma in response to low doses of secretagogues [intraduodenal HCl (0.033 meq/min); intraduodenal sodium oleate (0.04 mmol/min)] or an oral mixed meal in conscious dogs is described. Plasma secretin levels increased significantly (P less than 0.05) in response to an oral mixed meal in conscious dogs from a basal level of 4.0 to a peak level of 12.3 pg/ml at 15 min. Infusion of graded doses of HCl (2, 4, 8, 16, meq/hr for 30 min) intraduodenally in six dogs resulted in significant elevation of plasma secretin levels in a dose-dependent manner. The pancreatic bicarbonate and volume outputs correlated with the dosage of HCl administered and with the elevations in plasma secretin concentrations. Intraduodenal infusion of increasing doses of sodium oleate (2.4, 4.8, 9.6, and 19.2 mmol in 15-min periods) resulted in a significant (P less than 0.05) elevation of plasma levels of secretin.

Expression of cholecystokinin forms by medullary thyroid cancer.

We have studied the production and release of cholecystokinin (CCK) forms by rat medullary thyroid cancer (MTC) in vivo. MTC cells were inoculated s.c. into 8 Wag-Rij rats. One month later, after i.v. injection of calcium plasma levels of CCK, calcitonin and tissue contents of gastrin and calcitonin were determined by radioimmunoassay (RIA), immunocytochemistry, and high-pressure liquid chromatography (HPLC). The tumors were passed 4 times to 8 rats in 1-month intervals fasting levels of CCK in control rats were unaffected by calcium stimulation, and in tumor-bearing rats, plasma CCK was elevated in 3 out of 4 passages, falling to normal levels at the end of passage 4. Hypercalcemia had no effect on plasma levels of CCK in tumor-bearing rats, but did stimulate the release of calcitonin in both control and some tumor-bearing rats in later passages. CCK-8 sulfate was found in all 4 tumor passages but not CCK-33/39. We conclude that rat MTC synthesizes and releases CCK-8, but unlike calcitonin, release of CCK appears unresponsive to calcium stimulation.