High-resolution 1H NMR study of membrane phospholipids in rat pancreas stimulated by caerulein.

In this study, the peak observed at 3.2 +/- 0.2 ppm in the 1H nuclear magnetic resonance (NMR) spectra recorded at 60 MHz of rat pancreas stimulated by caerulein was characterized at higher resolution (100 and 300 MHz). Whole pancreas from rats stimulated by caerulein was analyzed ex vivo by 1H NMR spectroscopy at 100 MHz. After suppression of the tissue water peak (OSIRIS method), a peak at 3.2 +/- 0.2 ppm appeared distinctly, along with two other weaker signals (2.8 and 3.5 ppm). No signals were observed in these regions in the spectra recorded from pancreas of 48-h fasted rats. The signal at 3.2 +/- 0.2 ppm was characterized by analysis of the high-resolution 1H NMR (300 MHz) spectra of lipid extracts of rat pancreas. Addition of various pure membrane phospholipids in extracts showed that the peak was due to the N(CH3)3 groups of choline-containing lipids. The weaker signals (2.8 and 3.5 ppm) were attributed to the methylene protons of fatty acids and the glycerol of glycerophosphorylcholine (GPC). A small decrease in phosphatidylcholine (PC) concentration was observed on analysis of these lipid extracts by high-performance liquid chromatography, indicating that the increase in intensity of the 3.2 +/- 0.2 ppm peak was not due to any increase in PC concentration, but rather to a change in conformation of PC, allowing higher mobility of the trimethylamino protons. Lorglumide, a specific inhibitor of caerulein, markedly reduced the intensity of the NMR peak, and pentagastrin, which also stimulates exocytosis of zymogen granules in the pancreas, had a similar but somewhat smaller effect than caerulein.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined magnetic resonance imaging and spectrometry for characterization of human pancreatic adenocarcinoma (Capan-1) heterotransplanted in nude mice.

Human pancreatic cancer cells (Capan-1 cell line) were heterotransplanted subcutaneously into nude mice. The resulting macroscopically visible tumours were analyzed by proton nuclear magnetic resonance spectrometry in a search for characteristic parameters. The Capan-1 xenografts were visualized by nuclear magnetic resonance imaging and were characterized by a significantly increased transverse relaxation time T2 (125 +/- 32 ms) compared with that recorded from ex vivo healthy human pancreatic tissue (60 +/- 11 ms). Three characteristic signals at 2.73, 3.23 and 3.5 ppm were observed by nuclear magnetic resonance spectrometry. They corresponded to unsaturated fatty acids, phosphatidylcholine, glycerophosphorylcholine and/or taurine respectively. These peaks were not observed in the spectra recorded from healthy human or mouse pancreas. On the basis of these parameters, small intraperitoneal Capan-1 xenografts (less than 0.5 cm diam), which were only visible at the macroscopic level after opening the abdominal cavity, were accurately localized by magnetic resonance imaging and spectrometry.

Characterization of a specific signal from human pancreatic tumors heterotransplanted into nude mice. Study by high resolution 1H NMR and HPLC.

In a previous study, we demonstrated the existence of a 3.2 +/- 0.2 ppm peak in the 1H NMR spectrum at 60 MHz from human pancreatic adenocarcinomas (Capan-1 cell) heterotransplanted into nude mice. This peak, which is not present in normal human pancreas, was attributed to enhanced membrane fluidity and/or or an increase in phospholipid turnover. The present study was designed to identify this signal by comparing the 1H NMR spectra recorded in vivo at 100 MHz from Capan-1 tumors, after suppression of the tissular water proton peak, to those recorded from normal pancreatic tissue, and to those recorded at 300 MHz from lipid extracts. The 1H NMR spectra at 100 MHz of the Capan-1 tumors in vivo exhibited three main peaks in the 3.2 +/- 0.2 ppm region: 1. A peak at 2.8 +/- 0.1 ppm from CH2 protons of the acyl chains of unsaturated phospholipids; 2. A peak at 3.2 +/- 0.1 ppm from the protons of the N(CH3)3 group of choline; and 3 A peak at 3.5 +/- 0.1 ppm attributed to GPC. The NMR 1H 300 MHz spectrum of phospholipid extracts of Capan-1 tumors displayed 12 principal resonances, of which only the N(CH3)3 peak of PC had a similar chemical shift to that observed at low resolution (3.2 +/- 0.2 ppm). This peak had a higher intensity in the xenografts than in normal human pancreatic tissue. HPLC analysis of the same lipid extracts from Capan-1 cells in culture, of tumors derived from these cells and from normal pancreas showed: 1. Identical concentrations of the different phospholipids from cancerous human pancreatic cells in vivo and in culture; and 2. A significantly higher level of PC in the extracts of normal human pancreatic tissue. The increase in intensity of the N(CH3)3 peak of PC in the Capan-1 tumors was not thought to be caused by an increase in PC concentration, but to a difference in conformation or mobility of the PC protons in the xenografts. The increase in relaxation time in cancerous tissue (from 60 to 125 ms) was also taken to be evidence in favor of a high mobility of protons.(ABSTRACT TRUNCATED AT 400 WORDS)

Creatine phosphate as energy source in the cerulein-stimulated rat pancreas study by 31P nuclear magnetic resonance.

Stimulation of the rat exocrine pancreas by cerulein induces a variety of cellular processes, some of which require the expenditure of energy. In this study, changes in the amounts of various energy metabolites, including creatine phosphate (PCr), ATP, and ADP were determined by high-resolution 31P NMR spectroscopy. The spectrum of a perchloric acid extract of pancreas from the 48 h fasted rat was taken as a reference for comparison of 31P NMR spectra recorded after stimulation by cerulein. The NMR results obtained from rat pancreas stimulated in vivo by cerulein (3, 5, 10, 20, 40 min) were compared to those determined by HPLC. We show that during hormonal stimulation, the relative concentrations of PCr in the pancreas of the fasted rat rise significantly (p less than 0.02), reach a maximum at 10 min, fall between the 10th and 20th min, and then return to the relatively low levels observed in controls. On the other hand, the relative concentrations of ATP fall during the first 10 min after stimulation by cerulein, then rise significantly between the 10th and 20th min, whereas the levels of ADP rise during the first 10 min and fall between the 10th and 20th min. The energy required for exocytosis was assumed to be supplied by ATP synthesized in acinar cells. The 31P NMR results indicated that this ATP was derived from phosphorylation of ADP by PCr, and that large amounts of PCr are synthesized during the first minutes after cerulein stimulation. In addition, a significant rise in glycerophosphocholine was observed after cerulein stimulation, which was attributed to an enhanced catabolism of membranes and an increase in phospholipid turnover. Injection of cerulein antagonists, such as asperlicin or lorglumide, inhibited the effects of cerulein stimulation on energy metabolites. Furthermore, no changes were observed after injection of secretin, a hormone that stimulates secretion of bicarbonate. However, the analog of cerulein, pentagastrin, produced the same effects as cerulein, although to a lesser extent.

Demonstration of a new resonance peak by proton NMR in rat pancreas stimulated with caerulein.

Low-resolution proton nuclear magnetic resonance (1H NMR) analysis of the pancreas of rats stimulated with caerulein, a cholecystokinin analog, was found to differ from that of control rats either fed ad libitum or fasted for two days. The hormonal stimulation induced (a) an increase in the longitudinal relaxation time T1; (b) a 1H NMR peak situated at 1.8 +/- 0.2 ppm from the resonance peak of tissular water. This resonance peak was not observed in the pancreas of fasted rats, although it could just be detected in the pancreas of rats fed ad libitum. These features were not observed after injection of lorglumide, an antagonist of cholecystokinin, followed by caerulein stimulation. On the other hand, stimulation with secretin induced a slight increase in T1 but did not lead to the appearance of the 1.8 +/- 0.2 ppm peak. The 1.8 +/- 0.2 ppm resonance peak thus appears to be related to the hormone-stimulated state of the exocrine pancreas and might be a useful indicator of the physiological state of exocrine pancreatic tissue. From ultrastructural examination of pancreatic cells and 1H NMR studies of solutions of the major membrane phospholipids in rat plasma, we concluded that the 1.8 +/- 0.2 ppm resonance peak stemmed from an alteration in the metabolism of membrane phospholipids and/or an increase in membrane fluidity after stimulation of acinar cells by caerulein.

Enhanced membrane phospholipid metabolism in human pancreatic adenocarcinoma cell lines detected by low-resolution 1H nuclear magnetic resonance spectroscopy.

The low-resolution proton nuclear magnetic resonance (NMR) spectra of human pancreatic adenocarcinomas maintained in nude mice and in culture exhibit characteristic features. First, the high values of longitudinal (T1) and transverse (T2) relaxation times were attributed to disturbances in the exchange of ions and water molecules in cancerous cells. Second, a new peak situated at about 1.8 +/- 0.2 ppm from the peak of tissular water was observed. It was higher in spectra recorded from the proliferative peripheral zone of the tumor than from the central necrotic zone and was not observed in healthy control pancreatic tissue. Histological examination of the xenografts by transmission electron microscopy indicated intense phospholipid metabolism with marked development of the plasma membrane and the presence of numerous secretory granules, lysosomes, and multivesicular bodies in the cytoplasm. The new 1H NMR low-resolution peak was thought to reflect an increase in membrane viscosity stemming from alterations in the structure and metabolism of membrane phospholipids. Whatever its origin, the 1.8-ppm peak is a particular feature of cancerous pancreatic cells, which should be readily detectable by NMR in vivo.