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Biomedical subjects

D H Alpers

Publications and source records attributed to D H Alpers.

At least 19 recordsLinked to original sources
PubMed1992

The intrinsic factor (IF)-cobalamin receptor binding site is located in the amino-terminal portion of IF.

Intrinsic factor has two binding sites, one each for cobalamin and for the ileal receptor recognizing the intrinsic factor-cobalamin complex. To obtain initial functional mapping of these domains, cDNAs encoding intact rat and human intrinsic factor or fragments thereof were expressed transiently in COS-1 cells or in an in vitro transcription/translation system. Deletion of as little as 12% of the amino acids from the carboxyl terminus resulted in loss of cobalamin binding activity. On the other hand, the receptor binding region of intrinsic factor appears localized to a restricted region in the amino-terminal portion of the protein. Only those transcription/translation fragments of rat or human intrinsic factor tested that contained amino acid residues 25 to 62 (out of 399) showed calcium-dependent binding to isolated kidney brush borders, the shortest sequence corresponding with 20 consecutive amino acids. In contrast, a 232-amino acid carboxyl-terminal fragment of rat intrinsic factor and 243- and 338-amino acid carboxyl-terminal fragments of human intrinsic factor showed no receptor binding activity.

Animals↗
PubMed1979

Intestinal calcium binding protein in uremia.

In order to assess the usefulness of intestinal biopsies as indicators of end-organ responsiveness to vitamin D in uremic patients, calcium binding activity and calcium binding protein (CaBP) content were measured in intestinal biopsies from 12 uremic patients (glomerular filtration rate less than 5.0 ml/min) and 12 adult controls. Values for both were found to vary with the site of biopsy, highest values being obtained in the duodenal bulb, with lower values distally. Values for activity correlated with values for CaBP content in both normals and uremics and no difference was observed between groups. Levels of calcium binding activity and content of CaBP did not correlate with serum immunoreactive parathormone levels, but were directly related to circulating 25-hydroxycholecalciferol (25-OHD) levels. The data show that intestinal CaBP is normal in activity, quantity, and affinity for calcium in malabsorbing uremic patients, and are consistent with the hypothesis that calcium malabsorption in uremia is unrelated to deficiency of intestinal calcium binding protein.

Biopsy↗
PubMed1979

Successful maintenance of suckling rat ileum in organ culture.

Ileum from rats 4, 9, 11, 12, and 15 days old can best be maintained for 24 hours in a system using Hanks' Balanced Salt Solution without fetal calf serum, at 25 degrees C and 21% O2. Suckling rat duodenum and jejunum were difficult to maintain well for 24 hours in this system or a variety of other systems that were tried. A temperature of 37 degrees C hastened deterioration of duodenum, jejunum or ileum. With ileum, 3H-thymidine and 14C-leucine were increasingly incorporated into DNA and protein over the 24-hour period. Light microscopy, as well as scanning and transmission electron microscopy, showed very good preservation of the ileum after 24 hours. The addition to the medium of hydrocortisone, 1 micron, and thyroxine, 0.01 micron, alone or in combination, did not change DNA or protein synthesis, or morphology, possibly because of the relatively short (24 hour) time period. Our organ culture system emphasizes the differences between suckling rat ileum and the rest of the intestine, and provides a new tool for evaluating, over a 24-hour period, the developing rat small intestine.

Animals↗
PubMed1979

Sulfapyridine metabolites in children with inflammatory bowel disease receiving sulfasalazine.

Fifteen children followed as outpatients with chronic inflammatory disease of the colon were given sulfasalazine in doses from 1 to 4 gm/day (22 to 68 mg/kg, or 0.69 to 2.33 gm/m2). No correlation was found between the dose/m2 administered and the total serum sulfapyridine levels. However, 11 of 15 patients achieved SP levels greater than or equal to 17 micrograms/ml, a level approximating that reputedly associated with therapeutic efficacy. Patients who were either slow acetylators or slow hydroxylators of sulfapyridine had total SP levels significantly higher than patients who were both rapid acetylators and hydroxylators (20.0 +/- 1.2 vs 14.6 +/- 1.6). Total SP serum levels were not correlated with the activity of the disease. No toxic levels (greater than 50 micrograms/ml of SP) were encountered. We conclude that a dose of SASP in the range of 1.5 to 2.0 gm/m2 can be safely administered to children and is usually associated with serum SP levels considered in the therapeutic range. Although one-third of children are both rapid acetylators and hydroxylators and will have somewhat lower SP levels, the routine monitoring of SASP therapy with SP levels is not necessary for management of disease.

Acetylation↗
PubMed1978

Effect ob broad-spectrum antibiotics on in vitro synthesis of DNA in the rabbit colon.

Rabbit colonic mucosa was cultured in vitro in the presence of broad-spectrum antibiotics. DNA synthesis, as determined by [3H]thymidine incorporation into DNA, was inhibited by clindamycin (32%) and tetracycline (28%). Penicillin G, ampicillin, and metronidazole had no effect. When bile acids were added to the incubation at a concentration of 10 mM, the more lipophilic drug, lincomycin, inhibited DNA synthesis by 38% (cholic acid) and by 23% (chenic acid). Bile acids alone did not alter thymidine incorporation. Inhibition by clindamycin demonstrated a gradual dose-response curve, with inhibition first noted at about 20 microgram of antibiotic/ml and maximal inhibition at 1,000 microgram/ml. The more biologically active metabolite of clindamycin, N-demethylclindamycin, inhibited DNA synthesis at concentrations of as low as 10 microgram/ml. The inhibition was reversible, and the drugs did not affect thymidine uptake, thymidine pool concentrations, or protein or mucopolysaccharide synthesis at concentrations of up to 300 microgram/ml. These data demonstrate a direct but reversible toxic action on mammalian colonic mucosa by certain antibiotics implicated in the production of pseudomembranous colitis and are consistent with the hypothesis that such a toxic action may be one factor in the pathogenesis of antibiotic-associated colitis.

Animals↗
PubMed1978

Effect of proteolytic enzymes on the binding of cobalamin to R protein and intrinsic factor. In vitro evidence that a failure to partially degrade R protein is responsible for cobalamin malabsorption in pancreatic insufficiency.

Cobalamin (Cbl; vitamin B(12)) malabsorption in pancreatic insufficiency can be partially corrected by bicarbonate and completely corrected by pancreatic proteases but the mechanisms involved are unknown. Because saliva contains enough R-type Cbl-binding protein (R protein) to bind all of the dietary and biliary Cbl, it is possible that R protein acts as an inhibitor of Cbl absorption and that pancreatic proteases are required to alter R protein and prevent such inhibition. To test this hypothesis we studied the ability of R protein and intrinsic factor (IF) to compete for Cbl binding and ability of pancreatic proteases to alter this competition. Human salivary R protein bound Cbl with affinities that were 50- and 3-fold higher than those of human IF at pH 2 and 8, respectively. Cbl bound to IF was transferred to an equal amount of R protein with t((1/2))'s of 2 and 90 min at pH 2 and 8, respectively, and within several hours respective ratios of R protein-Cbl/IF-Cbl of 50 and 2 were observed. Cbl bound to R protein was not transferred to IF at either pH 2 or 8. Incubation of R protein with pancreatic proteases at pH 8 led to a 150-fold decrease in its affinity for Cbl. Incubation of R protein-Cbl with pancreatic proteases led to complete transfer of Cbl to IF within 10 min. Gel filtration studies with R protein-[(57)Co]Cbl and (125)I-R protein showed that pancreatic proteases partially degraded R protein. Pancreatic proteases differed in their ability to effect these changes with trypsin > chymotrypsin > elastase. Pancreatic proteases did not alter IF in any of the parameters mentioned above. Pepsin failed to alter either R protein or IF. THESE STUDIES SUGGEST THE FOLLOWING: (a) that Cbl is bound almost exclusively to R protein in the acid milieu of the stomach, rather than to IF as has been assumed previously; (b) that Cbl remains bound to R protein in the slightly alkaline environment of the intestine until pancreatic proteases partially degrade R protein and enable Cbl to become bound exclusively to IF; and (c) that the primary defect in Cbl absorption in pancreatic insufficiency is a lack of pancreatic proteases and a failure to alter R protein and effect the transfer of Cbl to IF. These studies also suggest that the partial correction of Cbl malabsorption observed with bicarbonate is due to neutralization of gastric HCl, since at slightly alkaline, pH IF can partially compete with R protein for the initial binding and retention of Cbl.

Carrier Proteins↗
PubMed1978

Intestinal apoproteins during fat absorption.

To compare the roles of apolipoprotein (Apo) A-I, B, and E (or arginine-rich apoprotein, ARP) in the intracellular production of intestinal chylomicrons (and/or VLDL), these apoproteins were localized in rat intestinal mucosa by the light microscope method of indirect immunofluorescence. In addition, tissue levels of ApoA-I and ApoB were measured during fat absorption by radioimmunoassay. Antisera were produced using ApoA-I isolated from rat plasma high density lipoprotein, and ApoB and ARP from plasma VLDL by column chromatography. The apoproteins yielded single bands on polyacrylamide disc gel electrophoresis in urea and in sodium dodecyl sulfate. Anti-apoprotein antisera were produced in rabbits. These antisera appeared to be monospecific on double-antibody immunoprecipitation of 125I-labeled apoproteins. In fasted animals granular staining of ApoA-I was noted in the supranuclear (Golgi) regions of epithelial cells in the top third of the villus. At 30 min, when fat droplets were seen in the supranuclear cytoplasm of the cells along the top two-thirds of the villus, intense ApoA-I staining surrounded droplets in the cytoplasm. At later times when epithelial cells and lamina propria both contained fat droplets, bright ApoA-I stain surrounded many droplets in the supranuclear cytoplasm of cells and in the lamina propria. Over the same period of time, tissue levels of ApoA-I rose 10-fold. The distribution and time-course of ApoB staining was nearly identical with that of ApoA-I. Concomitantly, tissue ApoB levels doubled. By contrast, in fasting rat intestine, staining of ARP was sparse, punctate, and confined to the lower quarter of the villus. After fat feeding, stained droplets were seen only in the lamina propria near the base of the villus even though abundant ARP was found in cells along most of this length of the villus. Stain was never seen to surround any droplets inside cells. Thus, ApoA-I and ApoB appeared to participate in the intracellular assemply of lipoproteins in gut, whereas ARP did not, although ARP was found within mucosal cells. Liver and intestine differed in their stainable contents of ApoA-I and ARP. Whereas intestine stained heavily for ApoA-I and lightly for ARP, liver stained heavily for ARP and lightly for ApoA-I. Both organs stained for ApoB. These findings suggest that there may be some quantitative "specialization" of the two organs which secrete lipoproteins.

Animals↗
PubMed1978

Correction of cobalamin malabsorption in pancreatic insufficiency with a cobalamin analogue that binds with high affinity to R protein but not to intrinsic factor. In vivo evidence that a failure to partially degrade R protein is responsible for cobalamin malabsorption in pancreatic insufficiency.

In vitro studies indicate that [(57)Co]cobalamin (Cbl) is preferentially bound to salivary R protein as opposed to intrinsic factor (IF) and that [(57)Co]Cbl bound to R protein is not transferred to IF at either pH 2 or pH 8. Incubation of R protein-[(57)Co]Cbl with pancreatic proteases causes a partial degradation of the R protein moiety and a rapid transfer of [(57)Co]Cbl to IF. We have postulated that the etiology of Cbl malabsorption in pancreatic insufficiency is an inability to partially degrade R protein because of a lack of pancreatic proteases. We have tested this hypothesis by determining the ability of a nonradioactive Cbl analogue, bound with high affinity by R protein but not by IF, to correct the malabsorption of [(57)Co]Cbl in patients with pancreatic insufficiency.R protein bound the Cbl analogue known as cobinamide with affinities that were the same and only 14-fold lower than those for Cbl at pH 8 and pH 2, respectively. Cobinamide was bound by IF with affinities that were 600,000- and 10,000-fold lower than those for Cbl at pH 8 and 2, respectively. The addition of 125 pmol of nonradioactive cobinamide to 0.5 pmol of [(57)Co]Cbl before being added to 1 pmol of R protein and 1 pmol of IF, markedly inhibited the ability of R protein to compete with IF for binding the [(57)Co]Cbl. Similar results were obtained with freshly aspirated gastric juice. This change was essentially indistinguishable from that observed previously when R protein or R protein-[(57)Co]Cbl was incubated in vitro with trypsin. The oral administration of 100 nmol of nonradioactive cobinamide in Schilling tests was equivalent to trypsin in its ability to completely correct the malabsorption of 0.4 nmol of [(57)Co]Cbl in three patients with pancreatic insufficiency. The fact that both trypsin and nonradioactive cobinamide inhibit the ability of R protein to compete with IF for [(57)Co]Cbl binding in vitro, and correct the mal-absorption of [(57)Co]Cbl in patients with pancreatic insufficiency in vivo, supports our hypothesis that the primary defect in Cbl absorption in this disease is an inability to partially degrade R protein because of a lack of pancreatic proteases.

Adolescent↗
PubMed1978

Effect of exchange exocrine pancreatic insufficiency on small intestine in the mouse.

A genetically conditioned mouse model of exocrine pancreatic insufficiency (epi) has been used to study the effect of the absence of lumenal proteases on small intestinal mucosal proteins. The small bowel was divided into eight equal segments. Enzyme activity was increased only in the first three segments in the case of maltase, sucrase, and lactase (all mol wt above 200,000). Alkaline phosphatase (mol wt 145,000), trehalase (mol wt 95,000), and peptidase (mol wt 175,000) activities were unaffected in proximal segments from epi mice. Proximal brush border proteins were identified and measured quantitatively by sodium dodecyl sulfate acrylamide gel electrophoresis. Those enzymes with increased activity were associated with increased amounts of protein in epi mice. Double labeled studies of protein turnover revealed a longer half-life for large brush border proteins (mol wt above 175,000) in epi mice than in normal mice. Enterokinase activity (a marker for duodenal mucosa) was nearly absent from the duodenum of epi mice. Receptors for the intrinsic factor-vitamin B12 complex (markers for ileal mucosal) were present in the ileum equally in normal and in epi mice. Enterokinase activity can be induced in epi mice by feeding its substrate trypsinogen, but not by trypsin or chymotrypsinogen. Epi mice thus retain the ability to synthesize enterokinase. Pancreatic proteases play an important role in the turnover of certain large mucosal proteins and in the induction of enterokinase.

Animals↗
PubMed1978

Role of caloric intake in the weight loss after jejunoileal bypass for obesity.

Sixty-five patients were studied prospectively after jejunoileal bypass for obesity. Dietary intake pre- and postoperatively was measured either directly by weighing food or by a research dietary history. Of 65 measurements, 59 were made at least 6 months after operation, when over 75% of weight loss had been achieved. Fat absorption was measured in 42 of the patients. The entire group ate fewer calories (mean +/- SE = 2595 +/- 135) postoperatively than preoperatively (mean +/- SE = 3261 +/- 138). This difference was highly significant (P less than 0.001). Forty-eight patients ate less after their operation. The caloric deficit calculated from the observed weight loss could be accounted for entirely by the estimated decrease in intake in 22 of the 48 patients who ate less postoperatively. Moreover, measured fat malabsorption accounted for only 31% of the observed weight loss in those who ate more postoperatively and 21% in those who ate less. We conclude that a decrease in caloric intake, along with malabsorption, is an important factor in the long term postoperative weight loss (1-9 months) after jejunoileal bypass for obesity.

Body Weight↗
PubMed1977

Development of intestinal brush border membrane proteins in the rat.

1. The proteins of the intestinal microvillus membrane have been studied during post-natal development in the rat (days 12--37). 2. In suckling animals (up to age 20 days), the majority of alkaline phosphatase, glucoamylase and lactase activities in the distal half of the intestine were located in the supernatant fraction (100000 X g, 60 min). These enzymes were attached to the membrane from the proximal intestine at all ages. 3. Alkaline phosphatase, maltase and lactase activities in the supernatant fractions chromatographed in Sephadex G-200 in positions similar to the corresponding membrane enzyme. Corresponding activities for lysosomal counter-parts of maltase and lactase present in the supernatant fraction chromatographed differently. Moreover, pH optimum of the soluble enzymes was 9.2 for phosphatase and 5.5--6.0 for glycoamylase and lactase. The soluble lactase and alkaline phosphatase were inhibited minimally by p-chloromercuribenzoate, and sodium fluoride respectively. L-Phenylalanine (20 mM) did inhibit the soluble phosphatase by 90%. Thus, the soluble enzymes are not mainly of the lysosomal origin, but have characteristics of membrane-bound enzymes. 4. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed 18 protein bands which were present in adult membranes. Two other proteins were unique for membranes of distal intestine in suckling rats. The proteins corresponding to known enzyme activity changed as expected with age (e.g. sucrase, maltase increased, lactase decreased). Most of the other proteins were also altered in amount during development. Thus, the changes in the microvillus membrane during development in the rat are not limited to specific enzymes.

Aging↗
PubMed1977

Distribution of intrinsic factor-vitamin B12 receptors in human intestine.

A new rapid and accurate method for detecting vitamin B12-intrinsic factor (IF-B12) receptors was used to study the distribution of mucosal IF-B12 receptors in three human small intestines. IF-B12 receptors in human intestine are present in significant amounts in the entire distal three-fifths of intestine. Triokinase activity is absent in the distal two-fifths of intestine, and tryptophan oxygenase is present only in the distal one-sixth, or most terminal part of the ileum. These data suggest (1) biochemical functions in the ileum have different anatomical distributions, (2) IF-B12 receptors correspond most closely with the accepted anatomical definitions of the ileum, and (3) vitamin B12 malabsorption seen in resection or disease of terminal ileum may not be attributable solely to a deficiency of ileal receptors.

Adult↗
PubMed1976

Hartnup disease. Clinical, pathological, and biochemical observations.

Hartnup disease is a rare genetic disorder of amino acid transport associated with variable and intermittent clinical abnormalities. A family is described in which three siblings had an intermittently progressive neurological disease and two of the affected siblings had the Hartnup-pattern aminoaciduria. Neuropathological examination of one case showed severe diffuse atrophy, generalized neuronal loss in the cortex, and Purkinje cell loss in the cerebellum. In vivo and in vitro studies of intestinal amino acid transport in the surviving sibling indicated a partial defect in the transport of several neutral amino acids (tryptophan, alanine, serine, and methionine) with normal transport of other neutral amino acids (threonine, phenylalanine, histidine, tyrosine, and isoleucine). Transport of glycine, proline, hydroxyproline, and the basic amino acids appeared normal.

Adolescent↗
PubMed1976

Psychiatric illness and the irritable bowel syndrome. Practical implications for the primary physician.

Twenty-nine consecutive outpatients with irritable bowel syndrome were given structured psychiatric interviews, as were 33 consecutive medical controls who did not have irritable bowel syndrome. All were from an internal medicine group practice. Seventy-two percent of irritable bowel syndrome subjects had psychiatric illness, with hysteria and depression the most prevalent syndromes. Only 18% of controls had psychiatric illness. The primary physician made an accurate psychiatric assessment in only 28% of the subjects. An awareness of his patient's psychiatric illness is necessary for the physician to provide effective treatment, as for depression, and to spare the patient needless medications, hospitalizations, and surgery, as with hysteria.

Colonic Diseases, Functional↗