Tsukasa Kodaira

Elevation of plasma CCK concentration after intestinal administration of a pancreatic enzyme secretion-stimulating peptide purified from rat bile-pancreatic juice: Analysis with N-terminal region specific radioimmunoassay

The rat plasma cholecystokinin (CCK) concentration was measured after intestinal administration of a peptide purified from rat bile-pancreatic juice, which has a stimulatory effect on pancreatic enzyme secretion. The plasma CCK concentration was measured by means of a radioimmunoassay using CCK-8 N-terminal specific antibody, OAL-656. In experimental rats with protease-free intestines, intraduodenal infusion of 10 micrograms of the purified peptide, which stimulates pancreatic enzyme secretion 2.0-2.5 fold, induced a significant increase in the plasma CCK level. Furthermore, after removal of CCK from the plasma by immunoabsorption with an OAL-656-bound Sepharose 4B column, the stimulatory effect of the plasma on pancreatic enzyme secretion was abolished when it was injected intravenously into recipient rats. It was concluded that this peptide stimulates the release of CCK in the intestine and that this is responsible at least in part for the pancreatic enzyme secretion-stimulating activity of the peptide.

Radioimmunoreactive plasma bradykinin levels and histological changes during the course of cerulein-induced pancreatitis in rats

The plasma bradykinin (BK) and serum amylase levels and histological changes in rats during the course of acute pancreatitis induced by a large dose of cerulein were examined. Animals were given four intraperitoneal injections of 20 μg/kg body wt of cerulein at hourly intervals. The plasma concentration of BK-like immunoreactivity (BK-LI), measured by a highly sensitive and specific radioimmunoassay established in this study, was found to reach a peak 6 h after the first injection of cerulein and then to remain elevated. On the other hand, the serum amylase and the histological alterations (i.e., interstitial edema, vacuolization, and inflammatory infiltration) were maximal 9 h after the first injection and returned to nearly normal after 24 h. These observations suggest that the BK generation is indicative of the participation of the kallikrein-kinin system in the pathophysiological change and that the plasma BK-LI level is a good marker of cellular damage and inflammation within the pancreas during the course of acute pancreatitis.

Development of an enzyme-linked immunosorbent assay for metallothionein-I and -II in plasma of humans and experimental animals

BACKGROUND An easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothionein-1 (MT-1) and 2 (MT-2) simultaneously in serum and other biological specimens in humans and experimental animals has not been developed yet. METHODS We developed a competitive ELISA, a specific polyclonal antibody against rat MT-2. The epitope mapping of the antibody was conducted using MTs in mouse, rat, rabbit, human and the fragment peptides of human MT-2. MT1/2 and MT-3 knock-out mice and cadmium treated mice were used for the evaluation of the ELISA. Pretreatment method of serum was examined to deplete blocking factors for this assay. RESULTS The antibody used for this ELISA had the same cross-reactivity with MT in humans and experimental animals. NH2 terminal peptide of MT with acetylated methionine was proved to be the epitope of this antibody. The reactivity of this ELISA system with liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6 ng/ml and the added MT-1 was fully recovered from serum. The mean MT concentration in our preliminary study was 23+/-4.6 ng/ml in human serum. Cadmium treatment to mice induced significantly higher amount of MT in serum, liver, kidney and spleen as reported previously by different established methods. CONCLUSION The proposed competitive ELISA is an easy and specific method for practical use, determining total MT-1 and -2 simultaneously in serum and other biological specimens of human and experimental animals.

Determination of the serum metallothionein (MT)1/2 concentration in patients with Wilson's disease and Menkes disease.

We have developed an easy and specific enzyme-linked immunoassay (ELISA) for the simultaneous determination of serum metallothinein-1 (MT-1) and 2 (MT-2) in both humans and experimental animals. A competitive ELISA was established using a specific polyclonal antibody against rat MT-2. The antibody used for this ELISA had exhibited the same cross-reactivity with MT in humans and experimental animals. The NH2 terminal peptide of MT containing acetylated methionine was shown to be the epitope of this antibody. The reactivity of this ELISA system with the liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in an MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6ng/ml and the spiked MT-1was fully recovered from the plasma. We investigated the normal range of MT1/2 (25-75%tile) in 200 healthy human serum and found it to be 27-48ng/ml, and this was compared with the serum levels in various liver diseases. The serum MT1/2 levels in chronic hepatitis C (HCV) patients were significantly lower than healthy controls and also other liver diseases. In the chronic hepatitis cases, the MT1/I2 levels increased gradually, followed by the progression of the disease to liver cirrhosis and hepatocellular carcinoma. In particular, we found significantly elevated MT1/2 plasma levels in Wilsons disease patients, levels which were very similar to those in the Long-Evans Cinnamon (LEC) rat (model animal of Wilsons disease). Furthermore, a significantly elevated MT1/2 level was found in patients with Menkes disease, an inborn error of copper metabolism such as Wilsons disease.

Meal-stimulated cholecystokinin release and exocrine pancreatic secretion in dogs.

SummaryTo confirm the role of cholecystokinin (CCK) and secretin in digestion, exocrine pancreatic secretion, plasma CCK, and secretin were measured simultaneously in six dogs prepared with gastric and pancreatic fistulas after feeding a solid meal. Plasma CCK concentration determined by radioimmunoassay increased significantly from the basal level, reached a peak 35 min after meal ingestion, and after a dip it further increased toward the end of the 3-h observation. Pancreatic protein output increased significantly, peaked at the fifth 10-min period, and then declined progressively. Plasma CCK concentration and pancreatic protein output correlated significantly during the first postprandial hour. Plasma secretion demonstrated significant elevation at 15 min and a peak at 25 min after a meal. Plasma secretin correlated significantly with both bicarbonate output and flow rate during the 3h. Simultaneous measurements of plasma CCK and secretin and of pancreatic secretion suggested that postprandial pancreatic secretion is primarily mediated by releases of CCK and secretin, but these hormones do not seem to be the only factors responsible for the secretion.

Stimulatory Effects of Bombesin on Plasma Trypsin Release and Exocrine Pancreatic Secretion in Dogs

We examined the effect of bombesin on plasma trypsin release and exocrine pancreatic secretion in dogs. Bombesin significantly increased plasma immunoreactive trypsin (IRT). Atropine significantly inhibited the response of plasma IRT to bombesin. Pancreatic trypsin secretion was also increased by bombesin, as well as bicarbonate and protein outputs. Atropine failed to inhibit pancreatic trypsin secretion. In conclusion, bombesin has a stimulatory effect on plasma trypsin release mediated by a cholinergic mechanism and different from pancreatic secretion.

The normal range of the plasma immunoreactive glucagon level during a 75 g oral glucose tolerance test

In order to establish a normal value of plasma glucagon immunoreactivity (GI) and glucagon-like immunoreactivity (GLI) during a newly adopted 75 g OGTT, 50 normals (N), 102 individuals with IGT and 20 diabetics (D) were subjected to the OGTT, and their plasma GI and GLI levels were determined at various intervals by radioimmunoassay using 2 kinds of the C-terminal region specific antibody, OAL123 and 30K, and of the antibody specific for the N-terminal and/or central region of glucagon, OAL196, respectively. The basal levels of OAL123-GI and 30K-GI and OAL196-GLI in the 3 groups were as follows; N, 114.3, 80.8, and 335.5; IGT, 107.6, 76.1, and 338.5; and D, 135.7, 76.9, and 342.2 pg/ml. After glucose administration, a significant decrease in plasma GI and increase in plasma GLI were observed in the 3 groups, although their changes from the basal levels were variable. The plasma samples of inexplicably high GI concentration were chromatographed to clarify the nature of the hyperglucagonemia. The apparent GI was mostly eluted in the Vo component, but negligibly at the 3500 mol.wt. glucagon fraction. There was a marked difference in the Vo peak depending upon the antiserum used. These facts suggest that plasma GI values are dependent on the amount of BPG present in particular samples, and the antibody used.