Freddie L. Hill

Detection of sterol hydroperoxides on thin-layer chromatoplates by means of the wurster dyes

Abstract A specific and sensitive means of detection of sterol hydroperoxides on irrigated thin-layer chromatoplates using N,N-dimethyl-p-phenylenediamine dihydrochloride or N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride has been developed. Using the color test the presence of specific sterol hydroperoxides in aged samples of cholesterol, stigmasterol, β-sitosterol, lanosterol, 24,25-dihydrolanosterol, 3α, 5-cyclo-5α-cholestan-6β-ol, 5α-cholest-7-en-3β-ol, and 5α-cholest-8(14)-en-3β-ol and the absence of sterol hydroperoxides in similarly stored samples of 5α-cholestan-3β-ol and 5β-cholestan-3β-ol are demonstrated. Autoxidation of cholesterol in the solid state, in the irradiated solid state, in colloidal dispersion, and in aerated solution is shown to proceed via initial formation of sterol hydroperoxides.

Distribution of peptide YY in the gastrointestinal tract of the rat, dog, and monkey.

The objective of this study was to characterize the distribution and concentration of peptide YY (PYY) in the gastrointestinal tract of the rat, dog, and monkey. In the rat, the greatest concentration of PYY was detected in the ileum and colon. The concentrations of PYY in the ileum and colon were 72 +/- 49 and 768 +/- 180 ng/g tissue, respectively. In the dog, PYY was found primarily in extracts of the mucosal layer of the ileum and colon, with smaller amounts in the distal jejunum. The concentration of PYY in the mucosal layers of the canine distal jejunum was 113 +/- 25 ng/g tissue, proximal jejunum 302 +/- 56, mid jejunum 507 +/- 151, distal ileum 691 +/- 184, and colon 1706 +/- 774 ng/g tissue. In the monkey gastrointestinal tract, PYY was detected predominantly in mucosal extracts of the jejunum, ileum, and colon. The concentration of PYY in the mucosal layer extract of the jejunum was 92 +/- 23, ileum 615 +/- 127, and colon 1013 +/- 243 ng/g tissue.

Peptide YY, a new gut hormone (a mini-review)

This report describes our current knowledge of a new gut peptide hormone, peptide YY. The localization, action, and possible mechanisms that control release of peptide YY are described.

Distribution of bombesin-like peptides in the alimentary canal of several vertebrate species ☆

The objective of this study was to quantitate and characterize the variants of bombesin-like immunoreactivity in the alimentary canal of the rat, rabbit, hawk, owl, dog, monkey and human. Bombesin-like immunoreactivity was found throughout the entire gastrointestinal tract of all species studied. In the rat, the highest concentration of bombesin-like immunoreactivity was found in the colon. Gel chromatography showed that bombesin-like immunoreactivity corresponded to gastrin-releasing peptide (GRP-27) and GRP-10. In the dog, the greatest concentration of bombesin-like immunoreactivity was observed in the mucosal layer of the fundus, whereas the concentration of bombesin-like immunoreactivity in the muscle layer of the dog did not vary significantly from region to region. Gel chromatography showed that bombesin-like immunoreactivity in the dog corresponded to GRP-27, bombesin, GRP-10, and a smaller fragment. In the human, the concentration of bombesin-like immunoreactivity did not vary significantly from region to region in the mucosal and muscular layers. Gel chromatography of human fundal mucosa showed that bombesin-like immunoreactivity peaks occur in the regions of GRP-27, bombesin and GRP-10. These findings substantiate the observation that bombesin-like peptides play a variety of roles in the regulation of gut function.

Metabolic control mechanisms in human erythrocytes. The role of glyceraldehyde phosphate dehydrogenase.

Hemolyzates of human red cells have been utilized to evaluate the role of glyceraldehyde 3-phosphate dehydrogenase as a control enzyme in the glycolytic pathway. The kinetic data obtained at pH 7.3 indicate Km values of 21 μ m , 143 μ m , and 6.6m m , respectively, for glyceraldehyde 3-phosphate, NAD, and inorganic phosphate. NADH is a competitive inhibitor of NAD with a Ki of 9.1 μ m . At physiologic concentrations of substrates and NADH, glyceraldehyde phosphate dehydrogenase is operating at less than 1% of maximal velocity. Consequently, despite the relatively high activity reported for this enzyme in erythrocytes, it appears likely that it is a site of metabolic control. It is clearly the rate-limiting reaction when the pH of the cells is raised or when the phosphate concentration of the external medium is increased markedly.

Radioimmunoassay of pancreatic polypeptide in mammalian and submammalian vertebrates using a carboxyl-terminal hexapeptide antiserum

Pancreatic polypeptide (PP) immunoreactivity in acid-ethanol extracts of the pancreas of representative species of mammals, birds, reptiles, amphibians, and fish was studied by a radioimmunoassay (RIA) that utilizes an antiserum which cross-reacts exclusively with the COOH-terminal hexapeptide of PP (CTPP). PP immunoreactivity in acid-ethanol extracts of rat nonpancreas tissues (stomach, duodenum, skeletal muscle, brain) was also examined. Significant concentrations of PP immunoreactivity were detected in the pancreatic extracts of all species, except fish. Appreciable quantities of PP immunoreactivity were also found in the stomach and duodenum of rats. In all cases, tissue extracts showed parallelism with reference PP (bovine) in the RIA. Gel chromatography (Sephadex G-50sf) of tissue extracts (rat, turtle) demonstrated a major peak of PP immunoreactivity, which eluted in the region of the reference PP. Salamander PP immunoreactivity eluted after bovine PP. In addition, the CTPP RIA can be applied to measure plasma levels of PP in rats, dogs, and humans. By using this PP RIA, we observed that plasma PP levels increase significantly in dogs (P less than 0.05) after intravenous administration of neurotensin. In rats, administration of intravenous bombesin resulted in a significant elevation of plasma PP.

Phosphorylation of Tris(hydroxymethyl)aminomethane by human erythrocytes

Abstract Whole human blood was incubated for periods of up to 96 hr at 37 °. Control of pH within the limits of 7.1–7.5 was achieved by the addition of pH 8.5 isotonic Tris buffer. Glucose, inorganic phosphate, adenine, and pyruvate were added as necessary to maintain the intracellular ATP level near that of fresh erythrocytes. During the prolonged incubation, an unusual phosphate ester accumulated within the erythrocytes. In samples incubated for 2–4 days, the final intracellular level of this compound ranged from 3.1 to 5.8 μmoles/ml cells. These values represent concentrations two to five times that of ATP. Isolation and purification of the phosphate ester was accomplished using anion and cation-exchange chromatography. The isolated phosphate ester was shown to be identical to synthetic phosphoryl-Tris using: (1) anion-exchange chromatography; (2) paper chromatography; and (3) infrared spectrophotometry. The synthesis and properties of phosphoryl-Tris are described.

Comparison of the Actions of Bombesin, Gastrin-Releasing Peptide-27, Neuromedin B, and Gastrin-Releasing Peptide-10 in Causing Release of Gastrin and Gastric Inhibitory Peptide in Rats

Abstract The objective of this study was to compare the gastrin- and gastric inhibitory peptide (GIP)-releasing actions of bombesin, gastrin-releasing peptide (GRP)-27, neuromedin B, and GRP-10 in rats. Both bombesin and GRP-27 are potent stimulants of gastrin and GIP release, whereas neuromedin B and GRP-10 are less effective, on a molar basis.

Unstable hemoglobin hemolytic anemia: In vitro incubation studies on erythrocytes with hemoglobin Sabine☆

Abstract Additional biochemical studies have been carried out to investigate the effects of the presence of an unstable hemoglobin (Hb Sabine) on metabolism of erythrocytes. In vitro incubation under physiological conditions of these erythrocytes for periods of 5–15 hours has been utilized to evaluate effects of various additives on metabolism of the cells. The addition of adenine to the blood stimulates adenine nucleotide biosynthesis and is effective in maintaining levels of adenine nucleotides. An excessive rate of breakdown of adenine nucleotides to hypoxanthine has been shown previously to be a major factor in the inability of these cells to maintain ATP. The addition of azide during in vitro incubation proved detrimental to the metabolism of these abnormal erythrocytes. This finding indicates that the immature erythrocytes of the blood are relying on oxidative phosphorylation to meet a portion of their energy needs. Neither pyruvate nor sucrose provided significant beneficial effects on the metabolism of the cells. Methylene blue stimulated the formation of 2,3-diphosphoglycerate, but proved harmful to the maintenance of adenine nucleotides. Effects of precipitation of denatured globin from hemoglobin Sabine on reduced glutathione and on the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase, were also studied following in vitro incubation. The precipitating beta polypeptide chains apparently do form disulfide bridges with reduced glutathione. However, no disulfide bridge formation with glyceraldehyde-3-phosphate dehydrogenase was noted during a 15-hour in vitro incubation.

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<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<0.05) elevation of plasma levels of secretin.