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Biomedical subjects

J L Rosenblum

Publications and source records attributed to J L Rosenblum.

At least 19 recordsLinked to original sources
PubMed1994

Rapid measurement of serum pancreatic amylase.

A simple, rapid assay for the pancreatic isoenzyme of human serum amylase was developed. The assay utilized an immunoabsorbent prepared by coating latex beads with a monoclonal antibody specific for pancreatic amylase. Treatment of patient serum with immunoabsorbent removed pancreatic amylase, and measurement of residual amylase activity with standard total amylase methodology allowed estimation of the pancreatic amylase content. Extraction efficiency of pancreatic amylase was consistent at amylase concentrations up to 1,000 U/L (y = 0.97 x +16.7 U/L; r = 0.9995). The assay was standardized with purified pancreatic amylase added to neonatal serum (low endogenous activity). A comparison of patient specimen results with the results of a standard technique (cellulose acetate electrophoresis) yielded an excellent correlation (immunoabsorption result = 0.96 electrophoresis result + 1.2 U/L; r = 0.987). Salivary amylase did not interfere with the assay until levels exceeded 1,000 U/L. Daily analysis of a frozen serum pool yielded a coefficient of variation of 9.2% at mean pancreatic amylase value of 54 U/L (+/- 5 U/L). A normal range study found a strong influence of age, with pancreatic amylase levels increasing dramatically in the first 3 years of life, to stabilize at a range of 0-66 U/L thereafter.

Amylases↗
PubMed1992

Macroamylases: differences in activity against various-size substrates.

Hyperamylasemia caused by macroamylases can lead to the overdiagnosis of acute pancreatitis. We examined whether interference from macroamylase is less in assays that use high-molecular-weight rather than oligosaccharide substrates. We hypothesized that high-molecular-weight substrates would be sterically excluded from macroamylasemic complexes and thus would be hydrolyzed less efficiently. Eighteen macroamylasemic samples were assayed by using red-dyed amylopectin or blue-dyed starch as polysaccharide substrates or by using maltoheptaose or maltotetraose as oligosaccharide substrates. The oligosaccharide substrates gave comparable results (y = 0.81x + 83); we observed consistently lower activities for amylopectin than for maltotetraose (y = 0.32x + 38). We observed no bias among methods when nonmacroamylasemic specimens were analyzed. The mechanism of this difference was examined by adding anti-human pancreatic amylase antibodies to hyperamylasemic serum samples from patients without macroamylasemia and purified human pancreatic or salivary isoamylases. In each case, polyclonal and monoclonal antibodies lowered amylase activity more in assays with complex polysaccharides than in those with oligosaccharides. The use of high-molecular-weight substrates diminishes interference, and detection of suspected macroamylasemia may be possible through comparison of activities determined from automated methods that use different substrates.

Amylases↗
PubMed1992

Macroamylases: differences in activity against various-sized substrates.

Hyperamylasemia caused by macroamylases can lead to the overdiagnosis of acute pancreatitis. We examined whether interference from macroamylase is less in assays that use high-molecular-mass (high-M(r)) substrates rather than oligosaccharide substrates. We hypothesized that high-M(r) substrates would be sterically excluded from macroamylasemic complexes and thus would be hydrolyzed less efficiently. Eighteen macroamylasemic samples were assayed by using red-dyed amylopectin or blue-dyed starch as polysaccharide substrates or by using maltoheptaose or maltotetraose as oligosaccharide substrates. The oligosaccharide substrates gave comparable results (y = 0.81x + 83), but we observed consistently lower activities for amylopectin than for maltotetraose (y = 0.32x + 38). We observed no bias among methods when nonmacroamylasemic specimens were analyzed. The mechanism of this difference was examined by adding antihuman pancreatic amylase antibodies to hyperamylasemic serum samples from patients without macroamylasemia and to purified human pancreatic or salivary isoamylases. In each case, polyclonal and monoclonal antibodies lowered amylase activity more in assays with complex polysaccharides than in those with oligosaccharides. The use of high-M(r) substrates diminishes interference, and detection of suspected macroamylasemia may be possible through comparing activities determined from automated methods that use different substrates.

Amylases↗
PubMed1990

Cloning and characterization of the human colipase cDNA.

Pancreatic lipase hydrolyzes dietary triglycerides to monoglycerides and fatty acids. In the presence of bile salts, the activity of pancreatic lipase is markedly decreased. The activity can be restored by the addition of colipase, a low molecular weight protein secreted by the pancreas. The action of pancreatic lipase in the gut lumen is dependent upon its interaction with colipase. As a first step in elucidating the molecular events governing the interaction of lipase and colipase with each other and with fatty acids, a cDNA encoding human colipase was isolated from a lambda gt11 cDNA library with a rabbit polyclonal anti-human colipase antibody. The full-length 525 bp cDNA contained an open reading frame encoding 112 amino acids, including a 17 amino acid signal peptide. The predicted protein sequence contains 100% of the published protein sequence for human colipase determined by chemical methods, but predicts the presence of five additional NH2-terminal amino acids and four additional COOH-terminal amino acids. Comparison of the predicted protein sequence with the known sequences of colipase from other species reveals regions of extensive identity. In vitro translation of mRNA transcribed from the cDNA gave a protein of the expected molecular size that was processed by pancreatic microsomal membranes. Sequence analysis of the in vitro translation product after processing demonstrated signal peptide cleavage and the presence of a human procolipase, as exists in the pig and horse colipases. DNA blot analysis was consistent with the presence of a single gene for colipase. RNA blot analysis demonstrated tissue-specific expression of colipase mRNA in the pancreas. Thus, we report, for the first time, a cDNA for colipase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Cloning and characterization of human pancreatic lipase cDNA.

Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) hydrolyzes dietary long chain triacylglycerol to free fatty acids and monoacylglycerols in the intestinal lumen. In the presence of bile acids, the activity of lipase is stimulated by colipase. As a prelude to studying the relationship of the protein structures to the functional properties of lipase and colipase, a cDNA encoding human pancreatic lipase was isolated from a lambda gt11 cDNA library screened with a rabbit polyclonal anti-human pancreatic lipase antibody. The full length cDNA clone of 1477 base pairs contained an open reading frame encoding a 465-amino acid protein, including a 16-amino acid signal peptide. The nucleotide sequence was 69% identical to the dog pancreatic lipase cDNA. The predicted NH2-terminal protein sequence agreed with the published NH2-terminal sequence of human pancreatic lipase and the predicted protein sequence was 85 and 70% identical to the protein sequences of pig and dog pancreatic lipase, respectively. A region of homology around Ser-153 is conserved in a number of lipid-binding proteins. Human hepatic lipase and lipoprotein lipase share extensive homology with pancreatic lipase, suggesting that the three proteins are members of a small gene family. In vitro translation of mRNA transcribed from the cDNA resulted in a protein of the expected molecular size that could be processed by microsomal membranes to yield a glycolated protein with proper signal peptide cleavage. RNA blot analysis demonstrated tissue specificity for pancreatic lipase. Thus, for the first time, a full length human pancreatic lipase cDNA has been isolated and characterized. The demonstrated regions of homology with other lipases will aid definition of interactions with substrate and colipase through site-specific mutagenesis.

Amino Acid Sequence↗
PubMed1988

Granulomatous hepatitis associated with cat scratch disease.

In three patients with cat scratch disease the liver was affected. All three had high fever (39 degrees C) for more than 3 weeks. Two of them had no peripheral adenopathy. Computed tomography of the abdomen revealed focal hepatic defects in two patients and periportal and periaortic adenopathy in the third. At laparotomy, there were nodules on the liver surfaces of all patients and histological examination revealed necrotising granulomata. The Warthin-Starry silver stain showed organisms consistent in appearance with the cat scratch bacillus in the liver and a periaortic lymph node of one patient, in the liver of the second patient, and in the axillary lymph node of the third. In all three patients the clinical findings and radiological abnormalities improved without specific therapy. A review of the surgical pathology files of Washington University revealed only two other cases of granulomatous hepatitis in children over a 6-year period. These findings indicate that cat scratch disease should now be included in the differential diagnosis of granulomatous hepatitis, at least in children. The absence of peripheral adenopathy in two of the three patients with granulomatous hepatitis suggests that the clinical spectrum of cat scratch disease may be broader than previously appreciated.

Cat-Scratch Disease↗
PubMed1988

Starch and glucose oligosaccharides protect salivary-type amylase activity at acid pH.

Salivary-type amylase may significantly contribute to duodenal starch hydrolysis in exocrine pancreatic insufficiency, provided that gastric inactivation does not occur. We investigated the effect of starch and its hydrolytic products, therefore, on salivary amylase activity in vitro at low pH. When incubated at pH 3 in the presence of 1% starch, 56% of the initial activity of amylase purified from saliva remained after 60 min at 37 degrees C compared with only 6% without starch. Similar protection of amylolytic activity was observed using human milk, which also contains a salivary-type amylase. In addition, partially hydrolyzed starch protected salivary amylase activity at pH 3, and purified glucose oligomers ranging in length from two to seven glucose molecules protected amylase in a concentration-dependent manner. Lactose, sucrose, and glucose, however, were ineffective in sparing amylase. Starch protected amylase activity even in the presence of pepsin, and maltotriose conferred striking protection below pH 3. These studies indicate that salivary-type isoamylases are protected in a simulated gastric environment by substrates of amylase as well as its end products of hydrolysis.

Adult↗
PubMed1988

Role of terminal fucose residues in clearance of human glycosylated alpha-amylase.

Human glycosylated alpha-amylase contains a single biantennary N-linked oligosaccharide that terminates with the structure Fuc alpha 1,3(Gal beta 1,4)GlcNAc. To examine the role of terminal fucose in the clearance of alpha-amylase, we used lectin affinity chromatography to isolate an alpha-amylase fraction that contains two terminal fucoses (one in each branch of the oligosaccharide) and a fraction from which both terminal fucoses have been enzymatically removed. In the rat, the rate of clearance of the radioiodinated fraction with terminal fucoses is rapid (t1/2 = 12 min) and is slowed by mannosylated but not galactosylated bovine serum albumin. The rate of clearance of the radioiodinated alpha-amylase fraction with no terminal fucoses is also rapid (t1/2 = 9.5 min), but the clearance is now slowed by galactosylated bovine serum albumin. These findings indicate that the fucosylated and defucosylated alpha-amylase fractions are recognized by different carbohydrate-specific receptors. We conclude therefore that terminal fucose is the recognition marker that effects the physiological clearance of this glycoprotein.

Carbohydrate Conformation↗
PubMed1988

Direct, rapid assay of pancreatic isoamylase activity by use of monoclonal antibodies with low affinity for macroamylasemic complexes.

Two Monoclonal Antibodies produced against Pancreatic Amylase have been produced and used to develop immunoassays (MAPA assays) for serum isoamylases. One antibody binds pancreatic isoamylase specifically and neither antibody inactivates amylolytic activity. MAPA assays that measure pancreatic and total enzymatic activities directly and rapidly in hyperamylasemic serum are described in this report. The influence of isoamylase determination on diagnosis is also investigated in 35 hyperamylasemic patients. The percentage for pancreatic amylase in serum as determined by immunoassay correlates well with results obtained by conventional electrophoresis (r = 0.90). Chart review indicates that use of MAPA assays would have potentially changed the diagnosis in 27% of patients. Five of six additional patients with hyperamylasemia caused by macroamylasemia had normal immunoreactive amylase values, and in all six patients the values were strikingly lower than predicted. The ability to assay organ-specific pancreatic isoamylase directly and early in the clinical course should reduce the risks of misdiagnosis and inappropriate therapy, especially in salivary-type hyperamylasemia or macroamylasemia.

Amylases↗
PubMed1985

Ulcerative and granulomatous colitis in children. Comparison of double- and single-contrast studies.

The authors reviewed 49 double-contrast and 27 single-contrast barium-enema examinations (BE) in 71 children with suspected inflammatory bowel disease. The diagnosis was proved in 41 patients, of whom 36 underwent proctoscopy and 5 had advanced disease which was treated surgically within 3 to 30 days after BE; the other 30 children served as controls. BE and proctoscopy were compared with regard to sensitivity (detection of disease) and accuracy (categorization as ulcerative or granulomatous colitis). Sensitivity was 93% for double-contrast and 82% for single-contrast BE. Both detected all cases of advanced disease; in early disease, double-contrast BE detected 91% of cases and single-contrast BE 70%. Colitis was correctly categorized as ulcerative or granulomatous in 93% with double-contrast BE and 86% with single-contrast BE. Specificity within the control group was 100% with both techniques. The authors conclude that both types of BE exhibit similar sensitivity in advanced disease, but the double-contrast technique is more accurate in detecting early disease.

Adolescent↗
PubMed1984

Metabolism of glycosylated human salivary amylase: in vivo plasma clearance by rat hepatic endothelial cells and in vitro receptor mediated pinocytosis by rat macrophages.

Salivary-type amylase normally comprises about 60% of the amylase activity in human serum, but only a small fraction is a glycosylated isoenzyme (amylase A). In contrast, 1/3 of amylase in human saliva is glycosylated. Since glycosylation can affect circulatory clearance, we studied the clearance of amylase A in rats and its uptake by rat alveolar macrophages. Following intravenous injection, 125I-labeled amylase A disappeared rapidly from plasma (t 1/2 = 9 min) and accumulated in the liver. Simultaneous injection of mannose-albumin slowed its clearance to a rate comparable to that of 125I-labeled nonglycosylated salivary amylase (t 1/2 = 45 min). In contrast, galactose-albumin had no effect. Electron microscope autoradiography of the liver following injection of 125I-labeled amylase A revealed a localization of grains over the hepatic endothelial cells. In vitro studies indicated that amylase A is taken up by alveolar macrophages via receptor-mediated pinocytosis. Uptake was linear over time, saturable, and inhibited by mannan and mannose-albumin, but not by galactose-albumin. We conclude that amylase A, which is a naturally occurring human glycoprotein with at most three terminal L-fucose residues per molecule, is recognized in rats by a mannose receptor located on hepatic endothelial cells. We speculate that this receptor, by rapidly clearing circulating amylase A, may be responsible for the low level of amylase A in human serum.

Amylases↗
PubMed1984

Metabolism of human isoamylases in the rabbit.

Human isoamylases were purified from pancreas and saliva with separation of salivary isoenzymes into glycosylated and nonglycosylated forms. After iodination, bolus injection of each purified isoamylase into conscious rabbits revealed biexponential disappearance from plasma. Glycosylated salivary amylase had a metabolic clearance (t 1/2 = 35 min) that was three to four times faster than nonglycosylated salivary (t 1/2 = 139 min) or pancreatic (t 1/2 = 111 min) amylase. In contrast to our previous studies on homologous (rabbit) isoamylases, human pancreatic isoamylase was excreted into urine as protein-bound radioactivity more readily than salivary isoamylase. No organ contained more than 18% of the injected dose of radioactivity except for a striking accumulation of radioactivity in the liver after injection of 125I-labeled glycosylated salivary amylase. We concluded that 1) human nonglycosylated salivary amylase and pancreatic amylase are cleared from the rabbit circulation at rates comparable with those reported for homologous isoamylases in the rabbit and baboon, 2) the rabbit kidney excretes human pancreatic amylase into urine more readily than human salivary isoamylases, and 3) with the notable exception of glycosylated salivary isoamylase, human isoamylases do not appear to be metabolized primarily by any single organ.

Animals↗
PubMed1983

Fate of circulating isoamylases in the rabbit.

Amylase purified from the pancreas and parotid gland of rabbits was labeled with 125I. Plasma disappearance was biexponential after bolus injection. Half-time of metabolic clearance was 97 and 95 min, respectively, for pancreatic and salivary 125I-amylase. About 20 and 30% of the injected pancreatic and salivary 125I-amylase, respectively, appeared intact in the urine within 24 h. Organs were assayed for radioactivity at specified times. No organ at any time contained more than 20% of injected radioactivity. At 24 h, more than 90% of radioactivity in organs was acid soluble. Bilateral nephrectomy significantly slowed plasma disappearance, although 90% hepatectomy did not. These studies in the rabbit indicate that 1) homologous isoamylases are cleared from plasma at rates comparable with those reported in primates; 2) in contrast to primates, urinary excretion of salivary amylase is greater than pancreatic amylase; and 3) within 24 h, virtually all of injected 125I-labeled isoamylases were catabolized or excreted.

Amylases↗