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J Selhub

Publications and source records attributed to J Selhub.

At least 163 records · Page 9Linked to original sources
PubMed1986

Competitive inhibition of folic acid absorption in rat jejunum by triamterene.

Triamterene, a diuretic agent, has been reported to cause megaloblastic anemia in some patients. Because this drug is a pteridine derivative, we investigated its effect on folic acid absorption in the rat jejunum. In an in vivo intestinal loop method, triamterene inhibited the intestinal absorption of folic acid in a dose-dependent fashion, with 50% inhibition of systemic absorption occurring at a luminal concentration of 0.01 mmol/L of triamterene. Kinetic analysis using the influx chamber method demonstrated that triamterene is a competitive inhibitor of intestinal folate transport, with a Ki of 0.125 mmol/L. Because therapeutic doses can result in luminal concentration of the drug approximating or exceeding the Ki, the interaction between triamterene and folate absorption is potentially of clinical interest.

Animals↗
PubMed1985

Folate transport.

Although a matter of some controversy in the face of differing experimental systems employing a wide range of substrate concentrations and conditions, there is a growing consensus that monoglutamyl folates are taken up by the intestinal epithelial cell by a structure-specific and energy-mediated transport process coded-for genetically. This "carrier-mediated" transport system can be demonstrated in several mammalian species in vitro and can be shown to be the property of the intestinal microvillus membrane in studies of isolated membrane vesicles. The transport system is highly pH-dependent with an optimum slightly below pH 6 which is very close to the pH of the proximal small intestine. At or near optimal pH the folate transport system exhibits saturation kinetics, competition among different forms of monoglutamyl folates and methotrexate, counter transport, and sensitivity to metabolic inhibitors. This system is functional in the physiological range of folate concentration under 10 microM. At high concentrations of folate in the intestine which can be achieved by folate therapy, folate uptake by diffusion is demonstrable. This latter process predominates at pharmacologic concentrations of folate above 10 microM.

Animals↗
PubMed1985

Folate binding protein and the estrogen receptor in breast cancer.

Folate binding protein (FBP) and estrogen receptor (ER) content were determined in primary breast cancers of 48 patients. The mean FBP level was significantly higher in ER-negative tumors than in ER-positive tumors and largely independent of the degree of tumor involvement or menopausal status. FBP correlated negatively with ER and this was most marked for tumors from postmenopausal women. Since FBP may decrease available intracellular folate the present data support clinical findings that chemotherapeutic agents may be more effective for ER negative tumors.

Breast Neoplasms↗
PubMed1984

The folate-binding protein of rat kidney. Purification, properties, and cellular distribution.

Folate-binding protein (FBP) from rat kidney was isolated, and its properties and location in the kidney were determined. The particulate fraction of rat kidney homogenate was freed of its bound folate, solubilized with Triton X-100, and the FBP was purified using a combination of DEAE-cellulose and affinity chromatography. The purified protein migrated as a single band on sodium dodecyl sulfate-disc gel electrophoresis, has an isoelectric point of 5.7, contains 21.7% carbohydrate, and has an Mr of 28,500-30,000. The purified protein retained its affinities for different folate derivatives and its sensitivity to inorganic anions. Inorganic anions enhanced the binding of 5-methyltetrahydrofolate; chloride ion was the most effective, followed by Br- greater than I- greater than SO2-4. Chloride ion was also found to lower the dissociation constant of the folic acid-FBP complex at 50 degrees C by about 10-fold. This effect is thought to derive from the formation of a ternary FBP-folic acid-Cl- complex which is more stable than the binary FBP-folic acid complex. An antiserum raised against the purified protein in rabbits was used to determine the location of FBP in the kidney by immunofluorescence. Intense fluorescence staining for FBP was localized at the apices (brush border) of proximal tubules. The choroid plexus, an organ previously shown to contain FBP, also exhibited intense fluorescent staining.

Amino Acids↗
PubMed1984

Intestinal transport of 5-methyltetrahydrofolate.

The mechanism of intestinal absorption of 5-methyltetrahydrofolate (5- CH3THF ) has been the topic of some controversy. In the present study, we have used enzymatically prepared 5- CH3THF to characterize transport by rat intestinal loops in vivo and everted jejunal sacs in vitro. Transport of 5- CH3THF is saturable (Km = 5.2 microM) and highly pH dependent, with the rate of maximal transport occurring at pH 5.8. Transport is competitively inhibited by folic acid (Ki = 4.2 microM) and methotrexate (Ki = 4.65 microM). Metabolic poisons and anaerobiosis greatly reduce 5- CH3THF transport. We conclude that 5- CH3THF transport in the rat intestine occurs by the same structure-specific mechanism responsible for the transport of unreduced folic acid and other monoglutamyl folates.

Animals↗
PubMed1982

The hydrolysis of nicotinamide adenine nucleotide by brush border membranes of rat intestine.

The hydrolysis of NAD by rat intestine was studied to determine the subcellular site of this hydrolysis and to identify the niacin-containing products that are formed. Using [nicotinamide-14C]NAD as substrate, and high pressure liquid chromatography for identification and quantification of products, the present study demonstrates two independent reactions for the hydrolysis of NAD; one that forms nicotinamide through hydrolysis of the ribosyl-pyridinium bond and one that forms nicotinamide mononucleotide through the hydrolysis of the pyrophosphate bond. The nicotinamide mononucleotide is subsequently dephosphorylated to nicotinamide riboside. Enzymes which release nicotinamide mononucleotide and nicotinamide riboside are associated with the brush border membrane as determined by analysis of fractionated intestinal homogenates. The enzyme activity which releases nicotinamide from NAD is associated with the brush border membrane fraction and also with a second cellular particulate fraction. Between pH5 and pH6 NAD is hydrolysed principally to nicotinamide. At pH 7.0 rates of nicotinamide and nicotinamide mononucleotide formation are the same. Above pH 7.0 the formation of nicotinamide mononucleotide is preferred.

Animals↗
PubMed1982

FMN phosphatase and FAD pyrophosphatase in rat intestinal brush borders: role in intestinal absorption of dietary riboflavin.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are two major coenzyme forms of dietary riboflavin. Yet little attention has been given to the release of the vitamin from its coenzyme forms during the absorptive process. Homogenates from rat intestine catalyze the hydrolases of these flavin coenzymes. To determine the location of FMN and FAD hydrolases, homogenates of intestinal mucosa were fractioned. FMN and FAD phosphatases were localized in brush border membranes. FAD pyrophosphatase activity was maximal at pH between 6.5 and 8.5 while FMN phosphatase has a pH optimum of 7.5 - 8.0. FAD pyrophosphatase is more stable to heat. The two enzymes separate on ion exchange chromatography of an isobutanol extract of intestinal brush border membrane fraction. Inhibition of 14C-riboflavin uptake by FMN and FAd in everted rings of rat intestine is directly related to the amount of conversion of these coenzymes to free riboflavin by intestinal enzymes. When FMN and FAD conversion to riboflavin is inhibited by EDTA, competition with 14C-riboflavin for transport was correspondingly decreased. These studies are best explained by a sequential process in which hydrolysis of FMN and FAD by enzymes of the intestinal brush border is followed by absorption of free riboflavin.

Animals↗
PubMed1981

Folate transport in isolated brush border membrane vesicles from rat intestine.

The transport of folates by the intact intestine consists of a saturable component with a luminal pH optimum of 6.0 and a nonsaturable component which predominates at high folate concentrations and/or high luminal pH. We sought to determine whether these properties reflect the behavior of the brush border membrane, free of intracellular enzyme activities, organelles, and intercellular junctions. We have studied, therefore the transport characteristics of folic acid (PteGlu) and methotrexate in isolated brush border membrane vesicles from rat intestine. Both PteGlu and methotrexate were found to be taken up by these vesicles by a pH-dependent process with a maximum uptake at a medium pH near 5.0. Studies at pH 5.5 demonstrated both saturable and nonsaturable components for the uptake of methotrexate and PteGlu. For methotrexate, Km, corrected for the nonsaturable component, was 1.5 microM and Vmax = 3.08 pmol/mg of protein/0.5 min. For PteGlu, the Km was 0.42 microM and Vmax = 0.67 pmol/mg of protein/0.5 min. Methotrexate uptake was competitively inhibited by PteGlu (Ki = 0.6 microM and by 5-methyltetrahydropteroylglutamic acid (5-methyl-H4PteGlu; Ki = 1.35 microM). Brush border membrane vesicles exhibit folate transstimulation: those preloaded with PteGlu, 5-methyl-H4PteGlu or unlabeled methotrexate took up tritium labeled methotrexate at more rapid rates than did control vesicles. The data presented are consistent with a pH-dependent, structure-specific carrier or channel for folate transport across the luminal membrane of the intestinal cell shared by the three folate derivatives tested.

Animals↗
PubMed1981

Antifolate actions of sulfasalazine on intact lymphocytes.

SASP, the drug most widely used for the treatment of Crohn's disease and ulcerative colitis, is a competitive inhibitor of intestinal folate metabolism and transport. Some of the therapeutic effects of the drug could be related to antifolate actions on lymphocytes, which predominate in the inflammatory reaction in inflammatory bowel diseases. Experiments were designed to examine the effect of SASP on folate-dependent systems in cultured lymphocytes. In rat spleen lymphocytes, THF-dependent conversion of glycine to serine was inhibited by SASP, with 50% inhibition occurring at 0.1 mM. Further evidence of folate antagonism was obtained with the dU suppression test, which depends on the function of a folate-dependent pathway in the de novo synthesis of DNA. Folate antagonists like methotrexate or folate depletion reduces the incorporation of dU into DNA and thus favors incorporation of [3H]thymidine into DNA by an alternate pathway. SASP inhibited the folate-dependent pathway in proliferating virally transformed human lymphocytes (Raji cells). To confirm that SASP acts as a folate antagonist in this system, THF was demonstrated to partly reverse the action of SASP. The significance of this antifolate action by SASP in intact lymphocytes deserves further study in relation to the actions of SASP in patients with inflammatory bowel disease.

Animals↗
PubMed1979

Effect of anions on folate binding by isolated brush border membranes from rat kidney.

The characteristics of folate binding by brush border membranes from rat kidney homogenates were investigated. At pH 7.4, binding of [3',5',9-3H]-pteroylglutamic acid to membranes containing endogenous folate is inhibited by anions, with chloride being most effective followed by bromide, thiocyanate, iodide, phosphate and sulfate. A maximum inhibition of 70-75% is attained at a concentration of 0.1 M chloride and an incubation time of 30 min. The inhibition diminishes with increased incubation time and at 24 h is negligible. The binding of [3',5',9-3H]pteroylglutamic acid to brush border membranes stripped of endogenous folate by acid treatment is not inhibited by anions. Anion sensitivity can be restored to these treated membranes by reconstitution with membrane-derived folate, particularly 5-methyltetrahydropteroyl-glutamic acid, or by preincubation with synthetic 5-methyltetrahydropteroyl-glutamic acid. Inhibition of [3',5',9-3H]pteroylglutamic acid binding by anions in membranes with endogenous folate is best explained by an anion-induced stabilization of endogenous folate-binding protein complex resulting in a decreased rate of exchange with exogenous [3',5',9-3H]pteroylglutamic acid.

Animals↗
PubMed1978

Demonstration of high-affinity folate binding activity associated with the brush border membranes of rat kidney.

Folate binding activity of high affinity was identified in the particulate fractions of rat kidney homogenates. This binding activity cofractionated with alkaline phosphatase and maltase, two brush border membranes markers. With an enriched preparation of brush border membranes, freed of endogenous folate by acid treatment, the binding of [3H]olate was found to be saturable (Kb = 4.2 X 10(-11)M) and rapid. Binding was optimal at pH 6.4-7.7. At neutral pH, competition for binding with [3H]folic acid required 1.45 equivalents of pteroylheptaglutamate, 6.25 equivalents of N5-methyltetrathydrofolate, 29 equivalents of methotrexate, and 125 equivalents of N5-formyltetrahydrofolate. At alkaline pH, N5-methyltetrahydrofolate was as effective a competitor as folic acid. In view of reports that renal tubular reabsorption of folate includes an initial tight binding step, the binding activity associated with the brush border membranes may participate in this process.

Animals↗
PubMed1978

Inhibition of folate enzymes by sulfasalazine.

Sulfasalazine (salicylazosulfapyridine), an agent widely used for the treatment of ileitis and colitis, is also a competitive inhibitor of intestinal folate transport (1, 2). The mechanism of action of sulfasalazine remains uncertain. To further explore the mechanism of sulfasalazine action, the interaction of the drug with the folate recognition site was tested with three enzymes: dihydrofolate reductase, methylenetetrahydrofolate reductase, and serine transhydroxymethylase, each catalyzing a reaction involving a different folate derivative. Each of these enzymes was inhibited by sulfasalazine in the same concentration range as that previously observed to inhibit intestinal folate transport; the kinetic data are consistent with a competitive mode of inhibition. Specificity of inhibition was demonstrated by the finding that the reduction of the pteridine ring of pteroylheptaglutamic acid by dihydrofolate reductase was subject to inhibition, whereas the hydrolysis of the gamma-glutamyl peptide side chain by chicken pancreas conjugase was not affected. These results are interpreted to indicate that sulfasalazine interferes with a folate recognition site which is common to these enzymes and to the intestinal transport system. Sulfasalazine, therefore, has certain properties of an antifolate drug.

Folic Acid↗
PubMed1976

Fractionation of serum transcobalamins on charged cellulose filters.

A simple and rapid fractionation procedure of the three transcobalamins, TCI, TCII, and TCII, of human serum was achieved by filtration through a stack of charged cellulose filters composed of one cellulose-nitrate and three DEAE-cellulose (DE-81) disks. A reaction mixture containing microliter amounts of serum was incubated with excess of 57Co B12 of high specific activity, diluted with 0.1 M sodium borate buffer (pH 8.5), and passed through the filter stack by applying vacuum. Under these conditions TCII is selectively and quantitatively adsorbed to the cellulose-nitrate filter while both TCI and TCIII adsorb to the DE-81 filters. In the second step TCIII is selectively desorbed from the latter filters by a 0.05 M monopotassium phosphate solution of pH 4.6. Using sera of different distribution of transcobalamins the data obtained were comparable to those determined by the more laborious methods employing DE-52 column chromatography combined with procedures to remove TCII.

Blood Proteins↗