Phytic acid in wheat bran affects colon morphology, cell differentiation and apoptosis.
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Wheat bran (WB) and its component phytic acid (PA) have both been shown to decrease early biomarkers of colon carcinogenesis, i.e. the PCNA labeling index of cell proliferation and certain aberrant crypt foci parameters. However, it is not known how WB and PA alter other biomarkers of colon cancer risk, such as rate of apoptosis and degree of differentiation, or how they affect colon morphology. Thus, the objectives of this study were to determine the effects of WB on these parameters, to see if PA contributes to these effects and whether there is a difference between endogenous and exogenously added PA. Five groups of azoxymethane-treated male Fischer 344 rats were fed a basal control diet (BD) or BD supplemented with either 25% wheat bran, 25% dephytinized wheat bran (DWB), 25% DWB plus 1.0% PA or 1.0% PA for 100 days. The WB, DWB and PA diets significantly increased the rate of apoptosis and cell differentiation in the whole crypt and the top 40% of the crypt. The WB, DWB and PA diets also significantly increased cell apoptosis in the bottom 60% of the crypt, while all the treatment groups significantly increased cell differentiation versus the BD group in the bottom 60% of the crypt. In addition, the WB, DWB and PA diets decreased the number of crypts per millimeter of colon, while the DWB and PA diets also decreased crypt height measured as number of cells. It is concluded that WB, partly due to its dietary fiber and endogenous PA, and exogenous PA when added to a low fiber diet can increase cell apoptosis and differentiation and favorably affect colon morphology. (+info)
Dietary phytic acid and wheat bran enhance mucosal phytase activity in rat small intestine.
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The aim of this work was to investigate the influence of dietary phytic acid (PA) on intestinal phytase activity in growing rats by in vitro determination of phytase activity in the three segments of the small intestine (duodenum, jejunum and ileum), and by in vivo intestinal perfusion of a solution rich in PA (diluted soymilk). Using the in vitro method, duodenal and jejunal activities were enhanced significantly by adaptation to purified PA (+44 and +145% respectively, compared with control rats). For the rats adapted to the wheat bran (WB) diet, the induction of intestinal phytase by the substrate compared with the control values (P < 0.001) was observed only in ileum. Using soymilk in perfusions, rats consuming PA or WB diets hydrolyzed more phytate (P < 0.001 and P < 0.05, respectively) than controls. Further, Mg absorption from diluted soymilk was not affected by food adaptation, whereas Ca absorption was greater in the PA and WB groups (P < 0.001 and P < 0.05, respectively) than in the control group. Thus, intake of pure PA by rats enhances phytase in the upper parts of the small intestine (duodenum and jejunum), whereas the WB diet activates ileal phytase. Furthermore, the induction of phytase activity is greater in magnitude in rats fed synthetic PA than that observed in rats fed the WB diet. The enhancement of phytase improves intestinal Ca absorption, thus showing the capacity of the small intestine to adapt to diets rich in PA and poor in Ca. (+info)
Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three-dimensional structure.
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Previously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49-68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the present investigation, to increase the specific activity of Aspergillus fumigatus phytase. The crystal structure of Aspergillus niger NRRL 3135 phytase known at 2.5 A resolution served to specify all active site residues. A multiple amino acid sequence alignment was then used to identify nonconserved active site residues that might correlate with a given favorable property of interest. Using this approach, Gln27 of A. fumigatus phytase (amino acid numbering according to A. niger phytase) was identified as likely to be involved in substrate binding and/or release and, possibly, to be responsible for the considerably lower specific activity (26.5 vs. 196 U x [mg protein](-1) at pH 5.0) of A. fumigatus phytase when compared to Aspergillus terreus phytase, which has a Leu at the equivalent position. Site-directed mutagenesis of Gln27 of A. fumigatus phytase to Leu in fact increased the specific activity to 92.1 U x (mg protein)(-1), and this and other mutations at position 27 yielded an interesting array of pH activity profiles and substrate specificities. Analysis of computer models of enzyme-substrate complexes suggested that Gln27 of wild-type A. fumigatus phytase forms a hydrogen bond with the 6-phosphate group of myo-inositol hexakisphosphate, which is weakened or lost with the amino acid substitutions tested. If this hydrogen bond were indeed responsible for the differences in specific activity, this would suggest product release as the rate-limiting step of the A. fumigatus wild-type phytase reaction. (+info)
Targeted deletion of Minpp1 provides new insight into the activity of multiple inositol polyphosphate phosphatase in vivo.
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Multiple inositol polyphosphate phosphatase (Minpp1) metabolizes inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) and inositol hexakisphosphate (InsP(6)) with high affinity in vitro. However, Minpp1 is compartmentalized in the endoplasmic reticulum (ER) lumen, where access of enzyme to these predominantly cytosolic substrates in vivo has not previously been demonstrated. To gain insight into the physiological activity of Minpp1, Minpp1-deficient mice were generated by homologous recombination. Tissue extracts from Minpp1-deficient mice lacked detectable Minpp1 mRNA expression and Minpp1 enzyme activity. Unexpectedly, Minpp1-deficient mice were viable, fertile, and without obvious defects. Although Minpp1 expression is upregulated during chondrocyte hypertrophy, normal chondrocyte differentiation and bone development were observed in Minpp1-deficient mice. Biochemical analyses demonstrate that InsP(5) and InsP(6) are in vivo substrates for ER-based Minpp1, as levels of these polyphosphates in Minpp1-deficient embryonic fibroblasts were 30 to 45% higher than in wild-type cells. This increase was reversed by reintroducing exogenous Minpp1 into the ER. Thus, ER-based Minpp1 plays a significant role in the maintenance of steady-state levels of InsP(5) and InsP(6). These polyphosphates could be reduced below their natural levels by aberrant expression in the cytosol of a truncated Minpp1 lacking its ER-targeting N terminus. This was accompanied by slowed cellular proliferation, indicating that maintenance of cellular InsP(5) and InsP(6) is essential to normal cell growth. Yet, depletion of cellular inositol polyphosphates during erythropoiesis emerges as an additional physiological activity of Minpp1; loss of this enzyme activity in erythrocytes from Minpp1-deficient mice was accompanied by upregulation of a novel, substitutive inositol polyphosphate phosphatase. (+info)
Iron bioavailability in humans from breakfasts enriched with iron bis-glycine chelate, phytates and polyphenols.
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This study was conducted to determine the bioavailability of iron amino acid chelate (ferrochel) added to fortify breads prepared from either precooked corn flour or white wheat flour + cheese and margarine compared with the same basal breakfast enriched with either ferrous sulfate or iron-EDTA. The inhibitory effect of phytate and polyphenols on iron absorption from ferrochel was also tested. A total of 74 subjects were studied in five experiments. Iron absorption from ferrochel was about twice the absorption from ferrous sulfate (P: < 0.05). When ferrous sulfate and ferrochel were administered together or in different meals, absorption from ferrochel was about twice the absorption from ferrous sulfate (P: < 0.05). Polyphenols present in coffee and tea inhibited iron absorption in a dose-dependent manner. American-type coffee did not modify iron absorption significantly, whereas both espresso-type coffee and tea reduced iron absorption from ferrochel by 50% (P: < 0. 05). Ferrochel partially prevented the inhibitory effect of phytates. Because of its high solubility in aqueous solutions even at pH 6, its low interactions with food and high absorption, ferrochel is a suitable compound for food fortification. (+info)
Biochemical and functional characterization of inositol 1,3,4,5, 6-pentakisphosphate 2-kinases.
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Synthesis of inositol 1,2,3,4,5,6-hexakisphosphate (IP(6)), also known as phytate, is integral to cellular function in all eukaryotes. Production of IP(6) predominately occurs through phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (IP(5)) by a 2-kinase. Recent cloning of the gene encoding this kinase from Saccharomyces cerevisiae, designated scIpk1, has identified a cellular role for IP(6) production in the regulation of mRNA export from the nucleus. In this report, we characterize the biochemical and functional parameters of recombinant scIpk1. Purified recombinant scIpk1 kinase activity is highly selective for IP(5) substrate and exhibits apparent K(m) values of 644 nm and 62.8 microm for IP(5) and ATP, respectively. The observed apparent catalytic efficiency (k(cat)/K(m)) of scIpk1 is 31,610 s(-)(1) m(-)(1). A sequence similarity search was used to identify an IP(5) 2-kinase from the fission yeast Schizosaccharomyces pombe. Recombinant spIpk1 has similar substrate selectivity and catalytic efficiency to its budding yeast counterpart, despite sharing only 24% sequence identity. Cells lacking sc-IPK1 are deficient in IP(6) production and exhibit lethality in combination with a gle1 mutant allele. Both of these phenotypes are complemented by expression of the spIPK1 gene in the sc-ipk1 cells. Analysis of several inactive mutants and multiple sequence alignment of scIpk1, spIpk1, and a putative Candida albicans Ipk1 have identified residues involved in catalysis. This includes two conserved motifs: E(i/l/m)KPKWL(t/y) and LXMTLRDV(t/g)(l/c)(f/y)I. Our data suggest that the mechanism for IP(6) production is conserved across species. (+info)
Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1.
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Phytic acid (myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate or Ins P(6)) typically represents approximately 75% to 80% of maize (Zea mays) seed total P. Here we describe the origin, inheritance, and seed phenotype of two non-lethal maize low phytic acid mutants, lpa1-1 and lpa2-1. The loci map to two sites on chromosome 1S. Seed phytic acid P is reduced in these mutants by 50% to 66% but seed total P is unaltered. The decrease in phytic acid P in mature lpa1-1 seeds is accompanied by a corresponding increase in inorganic phosphate (P(i)). In mature lpa2-1 seed it is accompanied by increases in P(i) and at least three other myo-inositol (Ins) phosphates (and/or their respective enantiomers): D-Ins(1,2,4,5,6) P(5); D-Ins (1,4,5,6) P(4); and D-Ins(1,2,6) P(3). In both cases the sum of seed P(i) and Ins phosphates (including phytic acid) is constant and similar to that observed in normal seeds. In both mutants P chemistry appears to be perturbed throughout seed development. Homozygosity for either mutant results in a seed dry weight loss, ranging from 4% to 23%. These results indicate that phytic acid metabolism during seed development is not solely responsible for P homeostasis and indicate that the phytic acid concentration typical of a normal maize seed is not essential to seed function. (+info)
Preventive potential of wheat bran fractions against experimental colon carcinogenesis: implications for human colon cancer prevention.
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Epidemiological studies suggest an inverse relationship between the intake of dietary fiber, particularly fiber from cereal grains, and colon cancer risk. Animal model assays have demonstrated that the protective effects of dietary fiber on colon cancer development depend on the nature and source of the fiber. Wheat bran (WB) appears to inhibit colon tumorigenesis more consistently than do oat bran or corn bran. This study was designed to determine whether specific WB fractions such as WB fiber, WB lipids, or phytic acid differentially affect colon carcinogenesis in a well-established colon cancer model. In addition, the modulating effect of specific fractions of WB on the activities of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-1 and COX-2 enzymes were assessed in colon tumors as those have been shown to play a role in tumor progression. At 5 weeks of age, groups of male F344 rats were assigned to one of six diets: a high-fat diet containing 10% WB (control diet) and experimental high-fat diets containing 10% dephytinized WB (WB-P), 10% defatted WB (WB-F), 10% dephytinized and defatted WB (WB-PF), 10% WB-PF fortified with 2% bran oil and/or with 0.4% phytate. At 7 weeks of age, all eats except those in the vehicle-treated groups were given two weekly s.c. injections of azoxymethane (AOM) at a dose rate of 15 mg/kg body weight/week. They continued to receive their respective diets until 50 weeks after carcinogen treatment and were then killed. Colon tumors were analyzed for iNOS, COX-1, and COX-2 expression and enzymatic activities. Colon tumors were evaluated histopathologically and classified as adenomas and adenocarcinomas. We found that removal of phytic acid (WB-P) or lipids (WB-F) from WB had no significant effect on colon tumor incidence (% animals with tumors) or multiplicity (tumors/ animal), whereas removal of both phytate and lipids from WB (WB-PF) significantly increased colon tumor multiplicity and volume. Interestingly, WB-PF fortified with excess bran oil or with bran oil plus phytate significantly inhibited colon tumor incidence, multiplicity, and volume; but supplementation of WB-PF with phytate alone had no significant effect on colon tumorigenesis in rats suggesting that lipid fraction of WB possesses tumor-inhibitory properties. Moreover, feeding WB-PF diet significantly increased iNOS, total COX and COX-2 enzyme activities, and iNOS protein expression in colon tumors as compared with wheat bran control diet. Feeding the WB-PF that was fortified with excess bran oil alone or with bran oil plus phytate significantly suppressed the activities of iNOS and COX-2 as well as the expression of iNOS and COX-2 in colon tumors compared with that in rats fed the WB diet or WB-PF diet. The study demonstrates for the first time that the lipid fraction of wheat bran has strong colon tumor inhibitor properties. The exact mechanism(s) by which the lipid fraction of WB inhibits colon carcinogenesis in addition to alteration of iNOS and COX activities remains to be elucidated. Additional studies are warranted to identify biologically active constituents of lipid fraction of WB and their relative role in colon tumor inhibition. (+info)