UNLABELLED:Retinoic acid (RA) mediates most of the biological effects of vitamin A that are essential for vertebrate survival. It acts through binding to receptors that belong to the nuclear receptor transcription factor superfamily (Mangelsdorf et al. 1994). It is also a highly potent vertebrate teratogen. To determine the function and effects of endogenous and exogenous RA, it is important to have a highly specific, sensitive, accurate, and precise analytical procedure. Current analyses of RA and other retinoids are labor intensive, of poor sensitivity, have limited specificity, or require compatibility with RA reporter cell lines (Chen et al. 1995. BIOCHEM: Pharmacol. 50: 1257-1264; Creech Kraft et al. 1994. BIOCHEM: J. 301: 111-119; Lanvers et al. 1996. J. Chromatogr. B Biomed. Appl. 685: 233-240; Maden et al. 1998.DEVELOPMENT:125: 4133-4144; Wagner et al. 1992.DEVELOPMENT:116: 55-66). This paper describes an HPLC/mass spectrometry/mass spectrometry product ion scan (HPLC/MS(n)) procedure for the analysis of retinoids that employs atmospheric pressure chemical ionization MS. The retinoids are separated by normal-phase column chromatography with a linear hexane-isopropanol-dioxane gradient. Each retinoid is detected by a unique series of MS(n) functions set at optimal collision-induced dissociation energy (30% to 32%) for all MS(n) steps. The scan events are divided into three segments, based on HPLC elution order, to maximize the mass spectrometer duty cycle. The all-trans, 9-cis, and 13-cis RA isomers are separated, if desired, by an isocratic hexane-dioxane-isopropanol mobile phase. This paper describes an HPLC/MS(n) procedure possessing high sensitivity and specificity for retinoids.

Pancreatic polypeptide (PP) increases hepatic insulin receptor (IR) binding activity in fasted PP-deficient rats, but not fasted normal animals. PP-induced alteration of hepatic IR levels in normal animals may be detectable in the fed state when IR concentrations are lower than during fasting. In the current study, the effect of exogenous PP on IR concentrations in the fed and fasted states was determined in healthy 300- to 350-g male Sprague–Dawley rats. Ten animals were administered PP 100 μg/kg/day for 3 days by intraperitoneal injection and 10 weight-matched control animals received saline vehicle. Five PP- and five saline-administered rats were fasted for 12 hr prior to organ procurement, while 5 PP- and 5 saline-treated rats were given free access to food for this period. Livers were removed and snap-frozen. IRs were isolated from solubilized hepatocyte membranes by affinity chromatography with agarose-bound wheat germ agglutinin. Western blots were performed using a specific antibody to the β subunit of the IR, which was detected by a chemiluminescence technique after 45-min exposure to X-ray film. Exposed films were examined by scanning densitometry and IR concentration was expressed as absorbance units per milligram of hepatic protein (mean ± SE). Statistical comparisons were by Student'sttest with significance taken atP< 0.05. Feeding was associated with a significantly lower IR concentration in saline-administered animals compared with the fasted state (24.2 ± 4.0 vs 53.3 ± 11.1). PP administration in fed rats resulted in significantly increased IR concentration as compared with that seen in saline-administered fed animals (43.8 ± 8.9 vs 24.2 ± 4.0). This difference may be due to increased IR synthesis with long-term PP administration, and supports the role of PP as a regulatory factor in hepatic carbohydrate metabolism.

BACKGROUND: Hepatic insulin resistance has previously been demonstrated in chronic pancreatitis, and has been shown to be, ameliorated by pancreatic polypeptide administration. Insulin binding was investigated in chronic pancreatitis induced by infusion of oleic acid into the pancreatic duct of rats.METHODS: Acute pancreatitis was induced in 12 200 to 225 gr 8-week-old male Sprague-Dawley rats by intubation of the main bile duct at its junction with the duodenum through a small mid-line abdominal incision, and infusion of 99% oleic acid 0.015 mL/min for 4 minutes, with an additional 4 minutes dwell-time after infusion. Sham-operated animals served as controls, After 6 weeks, chronic pancreatitic and sham-operated animals received either intraperitoneal bovine pancreatic polypeptide or saline vehicle for 5 days, Intraduodenal glucose tolerance tests (GTT) were performed in fasted animals, after which tissues were procured. Insulin receptors were isolated from solubilized hepatocyte and rectus abdominus membranes and competitive-binding studies were performed by incubation with I-125-insulin. Dissociation coefficients (K-d) and maximum binding capacities (B-max) for high-affinity receptors were derived from Scatchard analyses.RESULTS: B-max and K-d in muscle were not altered in animals with chronic pancreatitis. In liver, B-max was significantly less in rats with chronic pancreatitis given saline than in sham-operated rats given saline (17.0 +/- 6.3 versus 47.6 +/- 13.1 fmol/mg protein; data are mean +/- SEM), Pancreatic polypeptide administration increased hepatic B-max in rats with chronic pancreatitis (to 47.2 +/- 9.8 fmol/mg protein), but had no significant effect in sham-operated rats. Receptor affinity was not significantly different in rats with chronic pancreatitis or rats who underwent sham operations and was unaltered by the administration of pancreatic polypeptide. The integrated plasma glucose response during the GTT was reduced by pancreatic polypeptide administration in rats with chronic pancreatitis (29.5 +/- 15.0 mg/dL per minute versus 69.0 +/- 21.8 in chronic pancreatitis without pancreatic polypeptide), but was not significantly altered in sham-operated animals.CONCLUSION: Diminished expression of high-affinity receptors on the hepatocyte membrane may contribute to hepatic insulin resistance in chronic pancreatitis. In this model, pancreatic polypeptide improved glucose tolerance and increased receptor capacity to the level observed in livers from nonpancreatitic animals.