Biomedical subjects

M D Sitrin

Publications and source records attributed to M D Sitrin.

At least 37 records · Page 2Linked to original sources

PubMed1993

Characterization of phosphoinositide-specific phospholipase C in rat colonocyte membranes.

The phosphoinositide signal transduction pathway mediates important processes in intestinal physiology, yet the key enzyme, phosphoinositide-specific phospholipase C (PI-PLC), is not well-characterized in the colon. PI-PLC activity was examined in rat colonic membranes using exogenous [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate, and beta-glycerophosphate to suppress degradation of substrate or product. The activity of membrane PI-PLC increased 6-fold with the addition of alamethicin, and a further 2-3-fold enhancement was observed with 10 microM guanosine 5'-[gamma-thio]triphosphate (GTP[S]), suggesting the involvement of G-protein(s). The effect of GTP[S] appeared to be specific, as up to 100 microM adenosine 5'-[gamma-thio]-triphosphate failed to stimulate PI-PLC activity, and guanosine 5'-[beta-thio]diphosphate inhibited activity. The response of membrane PI-PLC to Ca2+ was biphasic, while > 0.5 mM Mg2+ was inhibitory with or without GTP[S]. Comparable total PI-PLC activities and responses to GTP[S] and Ca2+ were observed in purified brush-border and basolateral membranes. Western immunoblots probed with monoclonal antibodies to PLC isoenzymes PLC-beta 1, -gamma 1 and -delta 1 demonstrated that these antipodal plasma membranes contain predominantly the PLC-delta 1 isoform, with small amounts of PLC-gamma 1 present but no detectable PLC-beta 1. PLC-gamma 1 was the major isoform detected in cytosol.

Adenine Nucleotides↗

PubMed1993

Characterization of the 1,25-dihydroxycholecalciferol-stimulated calcium influx pathway in CaCo-2 cells.

The present studies were conducted to investigate the mechanisms underlying the 1,25-dihydroxycholecalciferol (1,25(OH)2D3)-induced increase in intracellular Ca2+ ([Ca2+]i) in individual CaCo-2 cells. In the presence of 2 mM Ca2+, 1,25(OH)2D3-induced a rapid transient rise in [Ca2+]i in Fura-2-loaded cells in a concentration-dependent manner, which decreased, but did not return to baseline levels. In Ca(2+)-free buffer, this hormone still induced a transient rise in [Ca2+]i, although of lower magnitude, but [Ca2+]i then subsequently fell to baseline. In addition, 1,25(OH)2D3 also rapidly induced 45Ca uptake by these cells, indicating that the sustained rise in [Ca2+]i was due to Ca2+ entry. In Mn(2+)-containing solutions, 1,25(OH)2D3 increased the rate of Mn2+ influx which was temporally preceded by an increase in [Ca2+]i. The sustained rise in [Ca2+]i was inhibited in the presence of external La3+ (0.5 mM). 1,25(OH)2D3 did not increase Ba2+ entry into the cells. Moreover, neither high external K+ (75 mM), nor the addition of Bay K 8644 (1 microM), an L-type, voltage-dependent Ca2+ channel agonist, alone or in combination, were found to increase [Ca2+]i. 1,25(OH)2D3 did, however, increase intracellular Na+ in the absence, but not in the presence of 2 mM Ca2+, as assessed by the sodium-sensitive dye, sodium-binding benzofuran isophthalate. These data, therefore, indicate that CaCo-2 cells do not express L-type, voltage-dependent Ca2+ channels. 1,25(OH)2D3 does appear to activate a La(3+)-inhibitable, cation influx pathway in CaCo-2 cells.

Calcitriol↗

PubMed1993

Biennial survey of physician clinical-nutrition training programs.

This is the fourth survey of physician clinical-nutrition training programs. As in previous reports, current fellowship training programs were identified, descriptive information obtained, and program content surveyed. In addition, a questionnaire developed by the American Board of Nutrition Committee on Fellowship Training Programs was used to determine the degree of emphasis given to content in the areas of basic nutrition science, clinical applications, and research training. Among the 38 programs identified, uniform ratings of importance were found in all major topic areas. There was also uniformity in most subtopics, with minor exceptions. As expected, in the area of nutrition in the life cycle, pediatric training programs emphasized infancy and childhood whereas medical-surgical programs emphasized adulthood and aging. Alcoholism was emphasized in medical-surgical training programs whereas cystic fibrosis and inborn errors of metabolism were emphasized in pediatric programs. Nutrition in burn patients received minor emphasis in all programs. The overall uniformity of curricular content in training programs confirms the contention that clinical nutrition has a defined clinical scope and should be considered for establishment as a recognized subspecialty in American medicine.

Education↗

PubMed1993

Supplemental dietary calcium fails to alter the acute effects of 1,2-dimethylhydrazine on O6-methylguanine, O6-alkylguanine-DNA alkyltransferase and cellular proliferation in the rat colon.

Prior studies from our laboratory have demonstrated that K-ras G to A mutations were detectable in a high percentage of carcinomas which developed in the colons of animals treated with the known colonic procarcinogen, 1,2-dimethyl-hydrazine (DMH). Moreover, in this model, the incidence of these mutations was decreased by a supplemental dietary calcium regimen which concomitantly decreased the frequency of rats with multiple tumors as well as tumor size. In an attempt to clarify the possible mechanism(s) involved in this antimutagenic effect of supplemental calcium, two groups of Sprague-Dawley rats were fed semisynthetic diets containing either 0.87 or 1.80% calcium by weight for 3 weeks, s.c. injected with 100 mg/kg of DMH and killed prior to and at various time periods (16-144 h) after injection. The colons of animals were analyzed and compared with respect to O6-methylguanine content in DNA, O6-alkylguanine-DNA alkyltransferase levels as well as cellular proliferation, as assessed by immunohistochemical staining of colonic crypts by bromodeoxyuridine. In certain experiments, these parameters were also analyzed in the proximal and distal colon before and at various times after administration of DMH. The results of these experiments demonstrated that supplemental dietary calcium was not found to influence significantly O6-methylguanine levels, alkyltransferase levels or cellular proliferation in the entire colon or in either colonic segment before or after the acute administration of DMH. DMH did, however, differentially alter all three of these biochemical parameters in the colonic segments (distal > proximal), possibly due to a greater degree of metabolic activation in the distal colon.

Animals↗

PubMed1993

Effect of 1,25(OH)2D3 and its analogues on membrane phosphoinositide turnover and [Ca2+]i in Caco-2 cells.

Our laboratory has recently reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates membrane polyphosphoinositide turnover and increases intracellular calcium concentration ([Ca2+]i) in Caco-2 cells. The role of binding to the vitamin D receptor (VDR) in the regulation of these rapid biochemical events, however, remains unclear. The present studies were, therefore, conducted using analogues of 1,25(OH)2D3, which differ markedly in their affinities for the VDR, to assess and compare their effects on [Ca2+]i and on inositol 1,4,5-trisphosphate (IP3) formation. Competitive binding studies performed with both intact cells and high-salt cytosolic extracts from Caco-2 cells demonstrated that 1,25(OH)2D3 and 1,24-(OH)2-22-ene-24-cyclopropyl-D3 (BT) have high affinities for the VDR; 25(OH)-16-ene,23-yne-D3 (AT), however, has a much lower affinity (approximately 1,000-fold less) for the VDR. Despite these large differences in binding affinities for the VDR, AT and BT produced similar concentration-dependent increases in [Ca2+]i and in IP3 formation while 1,25(OH)2D3 was approximately 10-fold less active. These results indicate that the structural requirements for the rapid action of these secosteroids on signal transduction in Caco-2 cells are different from those for receptor binding and transcriptional regulation.

Binding, Competitive↗

PubMed1993

1,25-Dihydroxycholecalciferol rapidly activates rat colonic particulate guanylate cyclase via a protein kinase C-dependent mechanism.

The present studies were performed to determine whether the major biologically active metabolite of vitamin D3, 1,25-dihydroxycholecalciferol [1,25(OH)2D3], could influence the activities of rat colonic particulate guanylate cyclase and adenylate cyclase. To address these issues, colonocytes were harvested from Sprague-Dawley rats and suspended in Krebs-Ringer bicarbonate buffer. The cells were then treated with 1,25(OH)2D3 or other agents (see below) and crude membranes were prepared and analyzed for particulate guanylate cyclase and adenylate cyclase activities. The results of these studies demonstrated that: 1) 1,25(OH)2D3, in a concentration-dependent manner, rapidly (within minutes) stimulated guanylate, but not adenylate cyclase activity; 2) preincubation of the cells with staurosporine, a protein kinase inhibitor, or U73122, an inhibitor of phosphoinositide-phospholipase C-dependent processes, blocked the increase in guanylate cyclase activity induced by 1,25(OH)2D3; and 3) 12-O-tetradecanoyl phorbol 13-acetate and 1,2-dioctanoyl-sn-glycerol, known activators of protein kinase C, also rapidly stimulated rat colonic particulate guanylate cyclase activity. Taken together, these results demonstrate that 1,25(OH)2D3 rapidly stimulates together, these results demonstrate that 1,25(OH)2D3 rapidly stimulates rat colonic particulate guanylate cyclase, at least in part, via a protein kinase C-dependent mechanism.

Adenylyl Cyclases↗

PubMed1993

Tissue and cell-specific patterns of expression of rat liver and intestinal fatty acid binding protein during development and in experimental colonic and small intestinal adenocarcinomas.

BACKGROUND: Mammalian small intestinal and colonic epithelium express members of a multigene family of hydrophobic ligand binding proteins of which liver and intestinal fatty acid binding protein represent among the most abundant intestinal gene products. These proteins are expressed in a cell- and region-specific manner and emerge in a temporally distinctive pattern. EXPERIMENTAL DESIGN: We have studied expression of these genes in the small intestine and colon of rats treated with 1,2-dimethylhydrazine since these animals develop a predictable pattern of both small and large bowel cancers. Studies were also undertaken in fetal and neonatal small intestine and colon. RESULTS: There was a 10- and 50-fold decrease, respectively, in mRNA abundance for intestinal and liver fatty acid binding protein in RNA from colon cancers compared with either uninvolved or control RNA. Immunocytochemical analysis revealed decreased staining for both proteins within their normal distribution together with ectopic clusters of cells reactive for liver fatty acid binding protein within colonic tumors. A more striking mosaic of immunocytochemical staining for both liver and intestinal fatty acid binding protein was found in small intestinal adenocarcinomas. Similar mosaic patterns of immunocytochemical staining were transiently detectable in rat fetal small intestine and neonatal colon. CONCLUSIONS: The region- and cell-specific expression of these genes, which may be linked temporally to events in intestinal differentiation, are subject to disruption in a cell-specific manner in the transformed, or dedifferentiated, phenotype.

1,2-Dimethylhydrazine↗

PubMed1992

Differential effect of 1,25-dihydroxycholecalciferol on phosphoinositide turnover in the antipodal plasma membranes of colonic epithelial cells.

1,25-dihydroxycholecalciferol stimulates membrane phosphoinositide turnover in colonic epithelial and other cells, but the effects of this hormone on phosphoinositide metabolism in specific antipodal plasma membranes has not been examined. In the present studies, addition of 10(-8)M 1,25-dihydroxycholecalciferol to rat colonic crypts for 90 seconds decreased the phosphatidylinositol-4,5-bisphosphate content and increased the diacylglycerol content of the baso-lateral, but not the brush border plasma membrane. Using Caco-2 cells grown as tight polarized monolayers, 1,25-dihydroxycholecalciferol reduced cellular phosphatidylinositol-4,5-bisphosphate and increased cellular inositol-1,4,5-triphosphate and diacylglycerol when added to the buffer bathing the baso-lateral, but not the brush border membrane surface. These data indicate, therefore, that 1,25-dihydroxycholecalciferol activates the phosphoinositol signal transduction cascade specifically in the baso-lateral cell membrane of colonic cells.

Animals↗

PubMed1992

Metabolism of 25-hydroxyvitamin D3 in rats: low-calcium diet vs. calcitriol infusion.

It has been proposed that the decreased serum level of 25-hydroxyvitamin D [25(OH)D] observed with dietary Ca restriction is mediated by an increase in circulating 1,25-dihydroxyvitamin D [1,25(OH)2D]. We compared the effects of endogenous and exogenous elevations in serum 1,25(OH)2D on the production rate (PR), metabolic clearance rate (MCR), and excretory pathways of [3H]25(OH)D3 in rats, with the use of steady-state techniques. Low-Ca diet and 1,25(OH)2D3 infusion caused comparable reductions in serum 25(OH)D and elevations in 1,25(OH)2D. Low-Ca diet lowered serum 25(OH)D by increasing MCR from 21.8 +/- 3.2 to 29.1 +/- 5.4 (SD) microliters.min-1.kg-1 (P less than or equal to 0.005) and decreasing PR from 944 +/- 161 to 663 +/- 163 pg.Ain-1.kg-1 (P less than or equal to 0.001). In contrast, 1,25(OH)2D3 infusion produced a dramatic rise in the MCR of 25(OH)D from 23.4 +/- 4.5 to 62.8 +/- 13.7 microliters.min-1.kg-1 (P less than or equal to 0.001) and also increased the PR from 943 +/- 165 to 1,500 +/- 337 pg.min-1.kg-1 (P less than or equal to 0.001). With 1,25(OH)2D3 infusion, urinary excretion of metabolites of [3H]25(OH)D3 rose rapidly, and kidney homogenates from these rats demonstrated vigorous side-chain oxidation of [3H]25(OH)D3. With low-Ca diet, urinary tritium excretion increased more gradually, and no direct side-chain oxidation of [3H]25(OH)D3 occurred in vitro. The increased MCR of 25(OH)D3 with low-Ca diet could be accounted for by enhanced synthesis of 1,25(OH)2D3 and subsequent degradation in target tissues.

Animals↗

PubMed1992

Effect of vitamin D status on the rapid actions of 1,25-dihydroxycholecalciferol in rat colonic membranes.

Recent studies from our laboratory have demonstrated that the in vitro addition of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] rapidly (seconds to minutes) stimulated membrane phosphoinositide turnover, translocated protein kinase C from the cytosolic to particulate fraction, increased cytosolic calcium ([Ca2+]i), and decreased cytoplasmic pH (pHi) via inhibition of Na(+)-H+ exchange in rat colonic epithelium of dietary vitamin D-sufficient rats and in Caco-2 cells. In contrast to these prior findings, in the present experiments, 1,25(OH)2D3 failed to elicit any of these colonic biochemical responses in vitamin D-deficient animals. Bethanechol chloride also failed to alter this signal transduction pathway, [Ca2+]i, or pHi. In vivo administration of this hormone for 5-7 days, moreover, to vitamin D-deficient animals restored the rapid biochemical effects of in vitro 1,25(OH)2D3 and bethanechol chloride. These studies, therefore, indicate that alterations in the vitamin D status of rats modulate the action of 1,25(OH)2D3 and other agents on the colonic phosphoinositide signal transduction system and on [Ca2+]i, which, in turn, may influence important cellular processes in this organ such as Na(+)-H+ exchange.

Animals↗

PubMed1992

Nutrition support in inflammatory bowel disease.

Many patients with the inflammatory bowel diseases, Crohn's disease, or ulcerative colitis have significant protein-calorie malnutrition and micronutrient deficiencies. Factors that contribute to these nutritional deficits include inadequate nutrient intake, malabsorption, excessive nutrient secretion across the diseased gastrointestinal tract, drug-nutrient interactions, and increased nutrient requirements. In this review, the use of enteral and parenteral nutrition support as primary therapy for active Crohn's disease and ulcerative colitis is discussed. Other roles for nutrition support in patients with inflammatory bowel disease, including preoperative nutrition support, nutritional treatment of intestinal fistulas and growth retardation, and home parenteral nutrition for gut failure, are also reviewed.

Deficiency Diseases↗

PubMed1992

1,25-dihydroxyvitamin D3 inhibits Na(+)-H+ exchange by stimulating membrane phosphoinositide turnover and increasing cytosolic calcium in CaCo-2 cells.

We have examined the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the phosphoinositol signal transduction pathway in the human colon cancer-derived cell line CaCo-2 and have studied the regulation of intracellular calcium ([Ca2+]i) and pH (pHi) by this secosteroid. CaCo-2 cells were prelabeled with [3H]myoinositol and treated with 10(-8) M 1,25-(OH)2D3 or vehicle for 90 sec. 1,25-(OH)2D3 caused a decrease in labeled phosphatidylinositol-4-5-bis-phosphate and an increase in labeled inositol 1,4,5-trisphosphate. Treatment with 10(-8) M 1,25-(OH)2D3 for 90 sec also raised the cellular content of diacylglycerol. In a dose-dependent manner, 1,25-(OH)2D3 caused the translocation of protein kinase-C activity from the cytosolic to the membrane fraction, which occurred after as little as 15 sec of exposure to the secosteroid, peaked at about 1-5 min, and then returned toward baseline values. In these CaCo-2 cells, baseline [Ca2+]i was 258 +/- 2 nM (mean +/- SE), as assessed using the fluorescent dye fura-2. After exposure to 10(-8) M 1,25-(OH)2D3, [Ca2+]i rapidly increased to 392 +/- 14 nM after 100 sec, fell, and then subsequently rose to a plateau of 350 +/- 3 nM after 400 sec. In Ca(2+)-free buffer, 1,25-(OH)2D3 caused only a transient rise in [Ca2+]i, indicating that 1,25-(OH)2D3 stimulated both the release of intracellular calcium stores and calcium influx. 1,25-(OH)2D3 caused a dose-dependent decrease in pHi in CaCo-2 cells, as assessed by the fluorescent dye BCECF, which was not observed in cells suspended in Na(+)-free buffer or pretreated with amiloride, indicating that the secosteroid inhibited Na(+)-H+ exchange. No effect of 1,25-(OH)2D3 on pHi was observed in cells in a Ca(2+)-free buffer or pretreated with the phospholipase-C inhibitor U-73,122, which also blocked the rise in [Ca2+]i, or in cells pretreated with the Ca2+/calmodulin inhibitor calmidazolium. Taken together, these studies indicate that 1,25-(OH)2D3 rapidly stimulates membrane phosphoinositide breakdown in CaCo-2 cells, generating the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, causing translocation of protein kinase-C to the membrane, and increasing [Ca2+]i by both releasing calcium stores and promoting calcium influx. Secondary to the rise in [Ca2+]i, Na(+)-H+ exchange is inhibited by a calcium/calmodulin-dependent pathway.

Amiloride↗

PubMed1991

Dietary calcium and vitamin D modulate 1,2-dimethylhydrazine-induced colonic carcinogenesis in the rat.

To determine whether supplemental dietary calcium and/or vitamin D deficiency are involved in modulating colon cancer induced by 1,2-dimethylhydrazine (DMH), Sprague-Dawley rats were fed diets containing either: (a) a normal content of calcium (0.87%) and phosphorus (0.60%) with 2.2 IU of vitamin D3 per g of feed (group A); (b) the same diet as group A, but with calcium and phosphorus increased to 1.80 and 0.80%, respectively (group B); or (c) a vitamin D-deficient diet with supplemental calcium (1.80%) and phosphorus (0.80%) (group C). After 6 weeks on their respective diets, one-half the animals in each group were given s.c. injections of either vehicle or DMH (20 mg/kg body weight/week) for 26 weeks. Animals were then sacrificed and the incidence of tumors as well as the number of tumors per tumor-bearing rat were determined. Colonic mucosal polyamine levels were measured after 15 weeks of exposure to vehicle or DMH, before development of histologically recognizable neoplasms. The results of these experiments demonstrated that neither calcium supplementation alone nor supplemental calcium in conjunction with vitamin D deficiency altered the incidence of colonic cancer induced by this carcinogen. Supplemental calcium, however, significantly decreased the number of rats with multiple tumors and reduced tumor size. Moreover, vitamin D deficiency abolished these protective effects of calcium on colon cancer in this experimental model. DMH treatment increased polyamine levels in the premalignant colonic mucosa in group A rats. This carcinogen-induced effect was blunted by high dietary calcium. Vitamin D-deficient, calcium-supplemented rats (group C) showed an increase in N1-acetylspermidine, but not the other polyamines, with DMH treatment.

1,2-Dimethylhydrazine↗

PubMed1991

K-ras mutations in 1,2-dimethylhydrazine-induced colonic tumors: effects of supplemental dietary calcium and vitamin D deficiency.

Recent studies from our laboratory have demonstrated that dietary supplemental calcium had no significant effect on the incidence of 1,2-dimethylhydrazine-induced colonic tumors, but did decrease the number of rats with multiple tumors and reduced tumor size. Moreover, concomitant vitamin D deficiency appeared to abolish these protective effects of calcium on colonic tumors in this experimental model. To date, however, the mechanism(s) involved in these phenomena remain unclear. In order to address these important issues, 1,2-dimethylhydrazine-induced colonic tumors from animals on control, Ca(2+)-supplemented, vitamin D-sufficient, and Ca(2+)-supplemented, vitamin D-deficient diets were examined for the presence of ras oncogene mutations. DNA was extracted from each of these tumors. Targeted areas of K-ras and H-ras genes were amplified by the polymerase chain reaction and analyzed for point mutations using allele-specific oligonucleotide hybridization and subsequent DNA sequencing. The results of these studies demonstrated that: (a) approximately one-third of 1,2-dimethylhydrazine-induced colonic carcinomas in the control group had K-ras G to A mutations; (b) no mutations, however, were detected in the cancers of the calcium-supplemented group; (c) concomitant vitamin D deficiency abolished the antimutagenic effect of dietary calcium supplementation (e.g., approximately one-third of cancers in this group again had detectable K-ras mutations); and (d) no H-ras point mutations were detected in colonic tumors from any group. These findings suggest that alterations in K-ras mutations may be one possible mechanism by which calcium and vitamin D status influence colonic carcinogenesis in this experimental model.

1,2-Dimethylhydrazine↗

PubMed1991

Down-regulation of protein kinase C activity in 1,2-dimethylhydrazine-induced rat colonic tumors.

Recent studies by our laboratory have indicated that alterations in protein kinase C activity may be involved in the early stage(s) of malignant transformation in the 1,2-dimethylhydrazine model of colonic adenocarcinoma. In order to further evaluate the possible role of protein kinase C in this multistage process, rats were given subcutaneous weekly injections of this procarcinogen (20 mg/kg body weight) or diluent for 26 weeks. One week after receiving the last injection, animals were killed and control colonic tissue, tumor tissue and tissue at least 1 cm away from these tumors ('uninvolved mucosa') were harvested. The activity and distribution of protein kinase C in the cytosolic and membrane fractions of these preparations as well as their 1,2-diacylglycerol mass were then examined and compared. The results of these studies demonstrated that: (1) total protein kinase C activity was reduced by approximately 35% and 60%, respectively, in the 'uninvolved' colonic mucosa and tumors of carcinogen-treated rats compared to their control counterpart values; (2) in the 'uninvolved' mucosa, this decrease in total activity was secondary to a decrease solely in cytosolic protein kinase C, whereas, in tumors both membrane and cytosolic activities were reduced; and (3) 1,2-diacylglycerol mass was significantly increased in colonic tumors versus control values. Based on these findings, it would appear that alterations in the cellular distribution and total activity of protein kinase C, possibly secondary to increases in 1,2-diacylglycerol mass, may also play a role in the latter stage(s) of malignant transformation in this experimental model.

1,2-Dimethylhydrazine↗

PubMed1991

Determination of the affinity of vitamin D metabolites to serum vitamin D binding protein using assay employing lipid-coated polystyrene beads.

We have developed an assay to measure the affinity of serum vitamin D binding protein for 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and vitamin D3, using uniform diameter (6.4 microns) polystyrene beads coated with phosphatidylcholine and vitamin D metabolites as the vitamin D donor. The lipid metabolite coated beads have a solid core, and thus all of the vitamin D metabolites are on the bead surface from which transfer to protein occurs. After incubating these beads in neutral buffer for 3 h, essentially no 3H-labeled vitamin D metabolites desorb from this surface. Phosphatidylcholine/vitamin D metabolite-coated beads (1 microM vitamin D metabolite) were incubated with varying concentrations of serum vitamin D binding protein under conditions in which the bead surfaces were saturated with protein, but most of the protein was free in solution. After incubation, beads were rapidly centrifuged without disturbing the equilibrium of binding and vitamin D metabolite bound to sDBP in solution was assayed in the supernatant. All three vitamin D metabolites became bound to serum vitamin D binding protein, and after 10 min of incubation the transfer of the metabolites to serum vitamin D binding protein was time independent. The transfer followed a Langmuir isotherm, and the Kd for each metabolite binding to serum vitamin D binding protein was derived by nonlinear least-squares fit analysis. From this analysis the following values for the Kd were obtained: 5.59 x 10(-6) M, 25-hydroxyvitamin D; 9.45 x 10(-6) M, 1,25-dihydroxyvitamin D; and 9.17 x 10(-5) M, vitamin D. In conclusion, we have developed a method which avoids problems encountered in previous assays and allows the precise and convenient determination of binding affinities of vitamin D metabolites and serum vitamin D binding protein.

Animals↗

PubMed1991

Intestinal absorption of cholecalciferol and 25-hydroxycholecalciferol in patients with both Crohn's disease and intestinal resection.

We compared the intestinal absorption of cholecalciferol and 25-hydroxycholecalciferol in patients with Crohn's disease and resections of the small bowel. Patients were subgrouped into those with small (less than 100 cm), intermediate (100-300 cm), and large (greater than 300 cm) resections. [3H]cholecalciferol or [3H]25-hydroxycholecalciferol were given orally and serial blood samples were taken for measurement of plasma radiolabeled vitamin. Absorption of both forms of the vitamin decreased with extent of resection but 25-hydroxycholecalciferol absorption was always greater than that of cholecalciferol. When compared with normal control subjects, 25-hydroxycholecalciferol absorption in these patients was better maintained than that of cholecalciferol. These data indicate that vitamin D malabsorption reflects the extent of distal small-bowel resection in Crohn's disease. Treatment with oral cholecalciferol is sufficient in those with small or moderate resections but oral 25-hydroxycholecalciferol supplementation may be preferred in those with a severe short-bowel syndrome.

Absorption↗

PubMed1991

Correction of enhanced Na(+)-H+ exchange of rat small intestinal brush-border membranes in streptozotocin-induced diabetes by insulin or 1,25-dihydroxycholecalciferol.

Diabetes was induced in rats by administration of a single i.p. injection of streptozotocin (50 mg/kg body wt). After 7 d, diabetic rats were further treated with insulin or 1,25-dihydroxycholecalciferol [1,25(OH)2D3] for an additional 5-7 d. Control, diabetic, diabetic + insulin, and diabetic + 1,25(OH)2D3 rats were then killed, their proximal small intestines were removed, and villus-tip epithelial cells were isolated and used to prepare brush-border membrane vesicles. Preparations from each of these groups were then analyzed and compared with respect to their amiloride-sensitive, electroneutral Na(+)-H+ exchange activity, using 22Na uptake as well as acridine orange techniques. The results of these experiments demonstrated that (a) H+ gradient-dependent 22Na uptake as well as Na+ gradient-dependent transmembrane H+ fluxes were significantly increased in diabetic vesicles compared to their control counterparts, (b) kinetic studies demonstrated that this enhanced 22Na uptake in diabetes was a result of increased maximal velocity (Vmax) of this exchanger with no change in apparent affinity (Km) for Na+, (c) serum levels of 1,25(OH)2D3 were significantly lower in diabetic animals compared with their control counterparts; and (d) insulin or 1,25(OH)2D3 treatment restored the Vmax alterations to control values, without any significant changes in Km, concomitant with significantly increasing the serum levels of 1,25(OH)2D3 in diabetic animals. These results indicate that Na(+)-H+ activity is significantly increased in proximal small intestinal luminal membranes of streptozotocin-induced diabetic rats. Moreover, alterations in the serum levels of 1,25(OH)2D3 may, at least in part, explain this enhanced antiporter activity and its correction by insulin.

Animals↗