Luteinizing hormone activity in plasma during the menstrual cycle.

Daily determinations of luteinizing hormone activity in plasma throughout a menstrual cycle in ten young women showed a sharp peak of activity lasting less than 48 hours around midcycle and higher mean values during the follicular phase than during the luteal phase in nine instances.

Radioimmunoassay for luteinizing hormone in human plasma or serum: physiological studies.

It is not practical to quantitate gonadotropin in the blood of normal men and women by utilizing bioassays. We have developed a method for sensitive, precise, and specific radioimmunoassay of luteinizing hormone (LH) in human serum or plasma. Antisera were developed against human chorionic gonadotropin, and one of these was selected for extensive cross-reaction with human LH. Highly purified LH was radioiodinated by the method of Greenwood, Hunter, and Glover. Separation of antibody-bound from free LH-(131)I was accomplished by a double antibody technique. Dose-response curves for the purifed LH, an impure urinary LH preparation, pituitary powder, and LH in plasma were all identical. Immunoassay and bioassay of impure urinary and pituitary gonadotropin preparations in terms of a common standard resulted in an index of discrimination of close to unity. LH levels in plasma from 32 adult men and 30 women outside the midcycle ranged from 0.6 to 3.2 mmug per ml (1 mmug of our laboratory LH standard is equivalent to 8 mU of the Second International Reference Preparation of Human Menopausal Gonadotropin). Levels were remarkably constant in men from day to day and in women except at midcycle, when a sharp peak occurred lasting less than 24 hours. In all women studied who had a midcycle LH peak, mean plasma LH levels during the follicular phase of the menstrual cycle were higher than mean values obtained during the luteal phase. Prepubertal children had detectable plasma LH, and mean values were only slightly less than in adults. Plasma from castrate men or women or postmenopausal women contained 4.5 to 10.5 mmug per ml. Clomiphene treatment of four men resulted in a doubling of plasma LH in 5 days.

Effects of gastrin on circulating levels of somatostatin, pancreatic polypeptide, and vasoactive intestinal peptide in dogs.

The effect of synthetic human gastrin I, infused at two doses, on the concentrations of somatostatin, pancreatic polypeptide, and vasoactive intestinal peptide in portal and systemic blood was studied in six anesthetized dogs. Intragastric pH was maintained at 5.5, and acid output was measured by intragastric titration. Significant increases in somatostatin and pancreatic polypeptide concentrations in portal blood were found with the lower dose of gastrin (0.5 microgram/kg . h) infused for 40 min. When gastrin was infused at 1.5 microgram/kg . h for 100 min, both portal and systemic blood concentrations of somatostatin and pancreatic polypeptide rose significantly. Cimetidine (300 mg, as an iv bolus), given 40 min after the beginning of the second infusion, did not affect gastrin-stimulated release of somatostatin and pancreatic polypeptide, whereas acid output was completely abolished. Vasoactive intestinal peptide concentrations did not change with the infusion of either dose of gastrin. This daily shows that gastrin releases somatostatin and pancreatic polypeptide in a dose-dependent fashion, and since this release was not acid mediated, it appears likely to be a direct action of gastrin.

Action of pancreatic polypeptide on exocrine pancreas and on release of cholecystokinin and secretin.

We have studied the effect of exogenous porcine pancreatic polypeptide (PP; 0.8 and 2.1 microgram/kg . h, iv) on endogenously stimulated pancreatic exocrine secretion in five pancreatic-fistula dogs. Plasma levels of cholecystokinin (CCK), secretin, and PP were measured in addition to pancreatic secretion of water, bicarbonate, and protein. Intraduodenal infusions of acid and a mixture of phenylalanine and tryptophan were used to stimulate hormone release. PP caused a dose-dependent inhibition of endogenously stimulated pancreatic secretion, whereas the release of CCK and secretin was not affected. Duodenal acidification and intraduodenal infusion of phenylalanine and tryptophan caused a significant release of PP. This study shows that: 1) PP suppresses pancreatic secretion by means of a mechanism that is probably direct; this effect is not mediated through inhibition of release of CCK or secretin, and 2) phenylalanine and tryptophan, both strong stimulants of CCK release, cause a substantial rise in PP in peripheral blood. The mechanism of PP release may involve CCK (in previous studies, we have shown a rise in circulating PP levels after iv CCK infusion).

Specificity of cholecystokinin antibody may influence choice of tracer for radioimmunoassay.

When CCK33 was iodinated at His 20 with lactoperoxidase, the labelled hormone was less immunoreactive under radioimmunoassay conditions than CCK39 which is readily iodinated at Tyr 1. Since the difference in immunoreactivities could not be due to different degrees of oxidative damage, the regional specificity of the assay antibody (UT122, from the laboratory of J.C. Thompson, University of Texas) was re-examined. A synthetic parital sequence, CCK33 6-16, which had similar conformation to the same sequence in the intact peptide, as shown by CD analysis, was devoid of immunoreactivity. Results with sulphated and non-sulphated C-terminal octapeptides of the hormone, CCK8 (CCK33 27-33), indicate limited dependence of the C-terminal region for binding to antibody. Thus, the lower binding with 125I-CCK33 than with 125I-CCK39 indicates that His 20 is an important feature of the immunogenic site and that the iodine atom is large enough to cause steric hindrance. This explains why the method of iodination might have to be varied depending on the regional specificity of antibodies.

Increased plasma levels of pancreatic polypeptide and decreased plasma levels of motilin in encopretic children.

OBJECTIVE: Abnormalities of hormones affecting gastrointestinal motility have been found in "functional" disorders of the gastrointestinal system in adults. One such disorder of childhood, encopresis, is frequently associated with constipation, the treatment of which often eliminates the soiling. We hypothesized that hormones affecting gastrointestinal motility were different between encopretic patients and matched controls. METHODS: Ten encopretic patients were matched by age, race, and sex with controls who had no history of encopresis or constipation. After an overnight fast, each child consumed a meal of Ensure, the amount of which was based on body weight. Plasma levels of gastrin, pancreatic polypeptide, cholecystokinin, motilin, thyroxine, estrogen, and insulin were measured 20 and 5 minutes before the meal, and 5, 10, 15, 30, 45, 60, 90, 120, 150, and 180 minutes after the meal. RESULTS: Postprandial levels of pancreatic polypeptide remained consistently higher and peaked earlier (P < .05) for encopretic patients. The motilin response was lower (P < .03) for encopretic children than for controls. CONCLUSIONS: We conclude that pancreatic polypeptide and motilin responses to a meal are different in encopretic children than in children in the control group. These gastrointestinal hormone findings may in part explain and/or be the result of the severe constipation that frequently underlies the fecal soiling found in these patients. These findings also suggest the motility of the stomach and small intestine may be abnormal in encopresis.

Hormonal (gastrin, secretin, cholecystokinin) and secretory effects of bombesin and duodenal acidification in dogs.

We have studied the influences of duodenal acidification on the effects of bombesin on gastric and pancreatic secretion and on blood levels of gastrin, secretin, and cholecystokinin (CCK) in six conscious dogs with chronic gastric and pancreatic fistulas. Duodenal acidification suppressed bombesin-stimulated gastric acid output (from 20.1 +/- 4.2 to 8.2 +/- 1.9 mEq/120 min) and gastrin release (from an integrated 2-hour output of 16.0 +/- 1.2 to 11.1 +/- 1.1 ng-min/ml). On the other hand, duodenal acidification augmented bombesin-stimulated secretion of pancreatic bicarbonate (from 0.75 +/- 0.12 to 7.81 +/- 2.0 mEq/120 min) and protein (from 0.57 +/- 0.10 to 1.00 +/- 0.18 gm/120 min). Blood levels of CCK (but not of secretin) were increased with bombesin alone, whereas blood levels of secretin (but not CCK) were increased by duodenal acidification alone. Bombesin plus duodenal acidification resulted in increase of both CCK and secretin. This increase of secretin may be responsible, in part, for suppression of both gastric acid output and gastrin release, as well as for increases in pancreatic secretion of bicarbonate and protein.

Effect of parenteral L-amino acids on gastric secretion and serum gastrin in normal dogs and dogs with portacaval transposition.

The effect of intravenous infusion of L-amino acids (FreAmine II) on gastric secretion and on circulating levels of gastrin was studied in five gastric fistula dogs and three dogs with portacaval transposition. Significant increases in gastric acid secretion were found after infusions which delivered 2.0, 4.0, and 8.0 gm/hr of L-amino acids in five gastric fistula dogs. After portacaval transposition, administration of amino acid solutions via the hindleg (through the liver) resulted in a great fall in the acid secretory response. Gastrin levels were significantly elevated after 8.0 gm/hr of amino acids. After 2.0 and 4.0 gm/hr, there was a slight but insignificant increase in serum gastrin levels. Gastrin levels were unchanged after infusion of 8.0 gm/hr of L-amino acids through the liver. We conclude that L-amino acids given intravenously stimulate gastric acid secretion in a dose-dependent manner by a mechanism which does not involve gastrin. At the highest dosage of amino acids, some gastrin was released which might have stimulated acid output further.

Relationship of omeprazole-induced hypergastrinemia to gastric pH.

The increase in gastrin caused by the gastric proton pump inhibitor, omeprazole, is presumably secondary to inhibition of gastric acid secretion but could also be due to a direct effect on the gastrin cells. This experiment was designed to determine whether gastrin elevations caused by omeprazole are related to intragastric pH. We studied gastrin release and acid output in response to 10% peptone broth (400 ml) in five dogs with gastric fistulas. The broth, at pH 5.5 or 2.5, was instilled into the stomach through the cannula, and the desired pH was maintained by intragastric titration with 0.1N NaHCO3 for 2 hours. Studies at each pH level were performed on separate days before, during, and after omeprazole (10 mumol/kg daily for 20 days). Omeprazole increased intragastric pH to greater than or equal to 3.5 for 24 hours. At pH 5.5 omeprazole inhibited acid secretion and increased gastrin levels; however, setting the intragastric pH at 2.5 completely blocked omeprazole's effect on gastrin release. Therefore these data support the hypothesis that the hypergastrinemia caused by omeprazole is dependent on gastric pH and gastric acid suppression.

Plasma levels of secretin in man and dogs: validation of a secretin radioimmunoassay.

We have developed and validated a secretin radioimmunoassay that is sufficiently sensitvie to measure circulating levels of secretin in the plasma of man and dogs. At a final dilution of 1:50,000, the antibody bound 30 percent to 40 percent of radioiodinated (125 I) 6-tyrosyl synthetic secretin. Pure natural porcine secretin was used as a reference standard and a linear dose-response curve was generated with 10 to 1,000 pg. of the polypeptide. Little or no cross-reactivity was found when graded doses of other gastrointestinal polypeptides were assayed in the radioimmunoassay and immunoreactive secretin (IRS) in volumes of serum up to 300 mul could be measured accurately.

Effect of vagal stimulation on pancreatic secretion and on blood levels of gastrin, cholecystokinin, secretin, vasoactive intestinal peptide, and somatostatin.

We investigated in dogs the effect of graded frequencies of electrical vagal stimulation (1.5, 3, 6, and 12 cps) on pancreatic exocrine secretion and on portal blood levels of gastrin, secretin, cholecystokinin (CCK), vasoactive intestinal peptide (VIP), and somatostatin (STS). Stimuli of all four frequencies, each with a duration of 5 minutes, were applied with a secretin background of 0.25 CU/kg-hr, and one stimulatory period of 12 cps was applied without a secretin background. With secretin, a significant, frequency-dependent increase of volume and of pancreatic protein secretion occurred from 3 to 12 cps. Gastrin values increased significantly at all frequencies. VIP and STS increased significantly with 3, 6, and 12 cps. Maximal responses for gastrin, VIP, and STS were observed with 6 cps. Peak values for gastrin and VIP were found during stimulation, whereas STS peaked after the end of the stimulatory period. The integrated responses of gastrin and STS showed significant correlation (P less than 0.01). The results suggest that vagally induced pancreatic response is only partially mediated by gastrin and perhaps VIP, and that endogenous gastrin may be one of the releasing factors for somatostatin. Plasma levels of CCK and secretin did not change after electrical stimulation, which provides direct evidence that their release is unlikely to be under vagal control, and that CCK does not mediate the protein secretion obtained after electrical stimulation of the vagus.

Concentrations of gastrin and secretin in the alimentary tract of the cat.

The distribution of gastrin and secretin in the alimentary tract of the cat was determined from the esophagogastric junction to the ileocecum. The total content and concentration of each of these hormones in mucosal extracts taken from the gastric fundus, gastric antrum, proximal and distal duodenum, proximal and distal jejunum, and ileum were measured with specific radioimmunoassays. The gastric antrum contained the highest concentration of gastrin, but appreciable amounts also were found in the duodenum. The highest concentrations of secretin were found in the duodenum, but impressive quantities were measured in the jejunum. The role of extra-antral gastrin and of those stores of secretin beyond the proximal duodenum (where the pH probably never falls to levels associated with release of secretin) currently are unknown.

Influence of vagus on mechanisms for stimulation and inhibition of gastrin release.

In studies in dogs the gastrin response to food, to bombesin (1 micrgoram/kg-hr), and to somatostatin (2.5 and 5.0 microgram/kh-hr) plus food before and after truncal vagotomy was determined. Vagotomy caused an increase in basal levels of gastrin and in the release of gastrin after bombesin and food. Vagotomy augmented somatostatin suppression of food-stimulated gastrin release in a dose-dependent manner. We suggest that vagotomy causes a loss of both stimulatory and inhibitory vagal effects on gastrin release. Loss of vagal inhibition results in increased gastrin release to bombesin and food. Loss of vagal stimulation results in intensification of somatostatin-induced inhibition of postprandial gastrin release.

Effect of alcohol on the release of secretin and pancreatic secretion.

The effect of alcohol on the release of secretin was studied in man by radioimmunoassay, and in dogs and cats by radioimmunoassay and bioassay (secretion of water and bicarbonate by the pancreas). In man plasma secretin levels increased significantly from a basal of 121 +/- 14 to 164 +/- 24 pg/ml at 60 minutes after oral ingestion of alcohol. Intraduodenal administration of alcohol in man, dogs, and cats did not release secretin or affect pancreatic water and bicarbonate secretion in dogs and cats, but a moderate increase in pancreatic protein output was observed in these two species. Plasma gastrin levels rose significantly from a basal of 52 +/- 4 to 64 +/- 3 pg/ml after ingestion of alcohol in man and from 33 +/- 5 to 51 +/- 8 pg/ml after administration of alcohol into the stomach of dogs. Administration of alcohol into the duodenum in man, dogs, and cats did not release gastrin. We conclude that alcohol probably does not release secretin directly. The mechanism of release of secretin in man is unknown; it clearly involves the stomach and may be mediated via the release of gastric acid.

Rate of disappearance of circulating endogenous gastrin in dogs.

In five awake mongrel dogs, endogenous gastrin was released by continuous irrigation of the antrum with acetylcholine. After 60 minutes of antral perfusion, the entire vascular supply of the antrum was suddenly and totally occluded, and serial samples of peripheral blood were taken for measurement of gastrin. The rate of disappearance of endogenous gastrin was caluclated by standard linear regression analysis; the calculated half-life of endogenous gastrin was 8.62 minutes. Analysis of the data suggests that the disappearance rate of endogenous gastrin could be explained by two distinct half-lives: one of 2.8 minutes (which is similar to the half-time in dogs of both 14- and 17 -amino acid gastrin), and another of 15.4 minutes (which is similar to the half-time of 34 -amino acid gastrin). Physiologically released gastrin is a mixture of three or more molecular forms of gastrin and the half-life of 8.62 minutes probably represents the disappearance half-time of this mixture.

Release of antral gastrin in response to an intestinal meal in dogs.

This study was designed to determine whether gastrin is released by the antrum in response to an intestinal meal in dogs. Two groups of anesthetized dogs were prepared with innervated antral pouches. The antrum and duodenum were separated by complete division at the pylorus to prevent duodenoantral reflux. The duodenum and proximal jejunum were perfused with 10% liver extract at 200 ml/hr. In one group of six dogs a significant elevation of antral vein gastrin levels was observed after 45 minutes. Gastrin levels in portal and peripheral blood were not significantly elevated. In another group of eight dogs, in which antral veins were not cannulated, a significant rise in peripheral gastrin concentration was noted after 60 minutes. We conclude that gastrin is released by the antrum during the intestinal phase of gastric acid secretion; significantly increased levels of gastrin are detected in both antral and peripheral venous blood. Duodenoantral reflux, as a possible cause of this release, is ruled out by complete surgical separation between duodenum and antrum.

Catabolism of secretin by the liver and kidney.

We have investigated the roles of the liver and the kidney in the catabolism of secretin, using a specific and sensitive radioimmunoassay. Dogs were prepared with sampling catheters in the aorta, hepatic vein, portal vein, and renal vein and with electromagnetic flow probes on the portal vein, hepatic artery, and renal artery. Secretin levels in the vessels entering and leaving the liver and kidney were determined by radioimmunoassay and the total mass of secretin [concentration (picograms per milliliter) X plasma flow rate (milliliter per minute)] was calculated during an intravenous infusion of exogenous secretin and during release of endogenous secretin by acidification of the proximal intestine. The total masses of secretin entering and leaving the liver were the same during secretin infusion and during the release of endogenous secretin. Under conditions of elevation of plasma secretin, however, the kidney extracted 30 percent of arterial secretin during secretin infusion and 45 percent during release of endogenous secretin. Clearly the kidney is a major site of secretin catabolism.

Pancreatic polypeptide. A review.

Pancreatic polypeptide (PP), 36-amino acid peptide, may function as an important feedback inhibitor of pancreatic secretion after a meal. It arises from both islet and acinar cells of the pancreas. Release of PP by a meal, primarily protein, occurs in a biphasic manner. The first rapid release occurs as a result of vagal stimulation; the second, more prolonged rise (the so-called intestinal phase) occurs in response to hormonal stimulation, predominantly cholecystokinin. Plasma PP levels increase with age; PP levels are elevated above those of age-controlled normal subjects in diabetic patients and in some patients with pancreatic amine precursor uptake decarboxylase tumors. The value of plasma PP as a possible marker for pancreatic tumors is as yet unsettled but may be a valuable tool.

Effect of nicotine on basal and bombesin stimulated canine plasma levels of gastrin, cholecystokinin and pancreatic polypeptide.

The influence of nicotine on the basal and bombesin (BBS) stimulated plasma levels of gastrin, cholecystokinin (CCK) and pancreatic polypeptide (PP) was investigated in conscious dogs. Plasma levels of nicotine and gastrointestinal (GI) hormones were measured by employing gas liquid chromatography and specific radioimmunoassay (RIA). The basal levels of gastrin, CCK and PP were found to be in pg/ml (pmol/l) (mean +/- S.E.), 28 +/- 5 (13 +/- 3), 252 +/- 32 (66 +/- 8) and 347 +/- 136 (83 +/- 32), respectively and these values remained unchanged with nicotine. Significant increases in levels of gastrin, CCK and PP were, however, found with infusions of BBS alone or with BBS in combination with nicotine. Gastrin levels were higher whereas CCK and PP levels were lower with BBS alone than with BBS plus nicotine. The peak values for CCK and PP, but not gastrin, were less during second BBS infusion. These results indicate that nicotine, in presence of bombesin, has an inhibitory effect on the release of gastrin and a stimulatory effect on the release of PP and CCK.

L364718, a new CCK antagonist, inhibits biological actions of CCK in conscious dogs.

The effects of L364718, a new CCK receptor antagonist, on CCK-8 stimulated pancreatic secretion and PP release were examined in three conscious dogs with pancreatic fistulas. L364718 (20 nmol/kg) caused a potent inhibition of CCK-8 stimulated pancreatic protein, amylase and trypsin secretion but not of volume and bicarbonate secretion. Release of PP by CCK was also significantly suppressed by L364718. The degree of inhibition by L364718 was dependent upon the amount of CCK-8 infused. This study demonstrates that L364718 acts as a potent antagonist of CCK's action on pancreatic enzyme secretion and PP release in dogs and suggests that this agent might be a useful tool for studying the physiological role of CCK in conscious animals.