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Biomedical subjects

K B Raja

Publications and source records attributed to K B Raja.

At least 19 recordsLinked to original sources
PubMed2004

Effect of altered iron metabolism on markers of haem biosynthesis and intestinal iron absorption in mice.

In this study, well-characterised animal models of altered iron metabolism were used to investigate link(s) between haem biosynthesis and intestinal iron absorption. Mice rendered iron deficient by feeding a low-iron diet for 3-4 weeks showed low levels of hepatic non-haem iron and hepcidin mRNA, with reduced urinary 5-aminolaevulinic acid (ALA) excretion and enhanced intestinal iron absorption. Hepatic ALA synthase activity was reduced while ALA dehydratase activity was increased. Iron-loaded mice had markedly increased liver non-haem iron and hepcidin mRNA, with increased urinary ALA excretion. Intestinal iron absorption was decreased mainly due to a reduction in transfer of absorbed iron from mucosa to the carcass. Hepatic ALA synthase activity was increased and ALA dehydratase activity moderately reduced. Mice exposed to hypoxia (0.5 atm) for 1-3 days had reduced hepatic hepcidin mRNA and urinary ALA excretion, while intestinal iron absorption was increased. Hepatic ALA synthase activity was reduced. The ALA dehydratase activity in liver and spleen was markedly enhanced. Injection of ALA to iron-deficient mice or hypoxic mice reduced their intestinal iron absorption to normal levels. This study further supports the hypothesis that alterations in haem biosynthesis influence duodenal iron absorption. ALA in particular appears to function as a modulator in controlling intestinal iron absorption.

5-Aminolevulinate Synthetase↗
PubMed2001

An iron-regulated ferric reductase associated with the absorption of dietary iron.

The ability of intestinal mucosa to absorb dietary ferric iron is attributed to the presence of a brush-border membrane reductase activity that displays adaptive responses to iron status. We have isolated a complementary DNA, Dcytb (for duodenal cytochrome b), which encoded a putative plasma membrane di-heme protein in mouse duodenal mucosa. Dcytb shared between 45 and 50% similarity to the cytochrome b561 family of plasma membrane reductases, was highly expressed in the brush-border membrane of duodenal enterocytes, and induced ferric reductase activity when expressed in Xenopus oocytes and cultured cells. Duodenal expression levels of Dcytb messenger RNA and protein were regulated by changes in physiological modulators of iron absorption. Thus, Dcytb provides an important element in the iron absorption pathway.

Amino Acid Sequence↗
PubMed2000

Involvement of iron (ferric) reduction in the iron absorption mechanism of a trivalent iron-protein complex (iron protein succinylate).

Iron protein succinylate is a non-toxic therapeutic iron compound. We set out to characterise the structure of this compound and investigate the importance of digestion and intestinal reduction in determining absorption of the compound. The structure of the compound was investigated by variable temperature Mössbauer spectroscopy, molecular size determinations and kinetics of iron release by chelators. Intestinal uptake was determined with radioactive compound force fed to mice. Reduction of the compound was determined by in vitro incubation with intestinal fragments. The compound was found to contain only ferric iron, present as small particles including sizes below 10 nm. The iron was released rapidly to chelators. Digestion with trypsin reduced the molecular size of the compound. Intestinal absorption of the compound was inhibited by a ferrous chelator (ferrozine), indicating that reduction to ferrous iron may be important for absorption. The native compound was a poor substrate for duodenal reduction activity, but digestion with pepsin, followed by pancreatin, released soluble iron complexes with an increased reduction rate. We conclude that iron protein succinylate is absorbed by a mechanism involving digestion to release soluble, available ferric species which may be reduced at the mucosal surface to provide ferrous iron for membrane transport into enterocytes.

Animals↗
PubMed1999

Importance of anemia and transferrin levels in the regulation of intestinal iron absorption in hypotransferrinemic mice.

The hypotransferrinemic mouse (trf (hpx)) is a mutant strain exhibiting transferrin deficiency, marked anemia, hyperabsorption of iron, and elevated hepatic iron stores. We set out to investigate the relative roles of anemia and of transferrin in the malregulation of intestinal iron absorption in these animals. Transfusion of erythrocytes obtained from littermate controls increased hemoglobin levels and reduced reticulocyte counts in recipient animals. Although mucosal to carcass (59)Fe transfer was reduced, total duodenal iron uptake was not significantly affected. Iron absorption in homozygotes, in contrast to littermate controls, was not reduced by hyperoxia. Mouse transferrin injections, in the short term, increased delivery of iron to the marrow and raised hemoglobin levels. Although mucosal transfer and total iron uptake were reduced at the higher transferrin doses, total uptake was still higher than in controls. Daily injections of mouse/human transferrin for 3 weeks from weaning, normalized hemoglobin values, and markedly reduced liver iron and intestinal iron absorption values in trf (hpx) animals. When such daily-injected mice were left for a week to allow transferrin clearance, iron absorption values were significantly enhanced; hemoglobin or hepatic iron levels were, however, not significantly altered. These data indicate that hyperabsorption of iron in trf (hpx) mice is not solely because of the anemia; transferrin levels per se do affect iron absorption, possibly via a direct effect on the intestinal mucosa.

Anemia↗
PubMed1999

The ferric-reducing activity of duodenal brush-border membrane vesicles is associated with a b-type haem.

Rabbit brush-border membrane vesicles possess ferricyanide reducing activity. This activity is preferentially dependent on NADH as reductant, and can be stimulated by the addition of FMN. The latency of activity observed following vesicle solubilisation suggests that the responsible component is transmembranous, and partially sequestered on the inner-face of the vesicles prior to full solubilisation. Subsequent increases in detergent concentration (> 0.3% w/v lauryl maltoside) were found to be inhibitory. Ferricyanide reducing activity was effectively inhibited by the sulphydryl modifying reagents N-ethyl malemide and p-chloromercuribenzoate, but not by the flavin analogue diphenylene iodonium. The ferric-reducing activity co-purified with a b-type haem when applied to Sephacryl S-200 columns. The putative cytochrome was found to be immunologically distinct from neutrophil cytochrome b558.

Animals↗
PubMed1999

Iron proteins of duodenal enterocytes isolated from mice with genetically and experimentally altered iron metabolism.

The molecular basis for the control of iron absorption by the duodenum remains unknown: however, ferritin (Ft) and the iron status of enterocytes have been suggested as regulatory factors. We determined the iron and Ft status of duodenal enterocytes from mice with hypotransferrinaemia, a genetic defect leading to greatly enhanced iron absorption, and for comparison we also investigated mice with experimentally-altered iron absorption. Duodenal enterocytes were isolated and analysed for Ft and non-haem iron content and for transferrin binding (as a measure of transferrin receptor activity). RNA was extracted from the duodenal mucosa and examined for transferrin receptor and H- and L-Ft mRNA levels by Northern hybridization analysis. Ft levels were elevated in enterocytes of hypotransferrinaemic mice, similar to that seen in iron dextran-injected mice of the CD1-strain. Enterocyte Ft levels were reduced in mice fed a diet diminished in iron, but unchanged in hypoxic mice enterocytes. Enterocytes of hypotransferrinaemic mice had normal non-haem iron levels and transferrin binding; however, enterocytes from CD-1 mice fed a low iron diet had increased transferrin binding and a decreased non-haem iron content. Duodenal mRNA levels for transferrin receptor and H-Ft were unchanged in hypotransferrinaemic mice, whereas L-Ft was increased. We conclude from the Ft and non-haem iron contents and transferrin binding that duodenal enterocytes from hypotransferrinaemic mice are not simply iron deficient, leading to increased expression of iron carriers proteins. Duodenal iron absorption can be enhanced in mice even when enterocyte Ft levels are raised or unchanged, suggesting that iron absorption is regulated by developmentally programmed expression of iron transporters by enterocytes.

Animals↗
PubMed1998

Monocyte-macrophage ferric reductase activity is inhibited by iron and stimulated by cellular differentiation.

The enzyme ferric reductase catalyses the reduction of Fe(III) as a prerequisite to its transportation across the cell membrane. Duodenal mucosal biopsies from iron overloaded patients with genetic haemochromatosis (GH) have increased ferric reductase activity and iron absorption compared with controls, yet the GH mucosa is iron deficient. A similar GH-related iron deficiency is also seen in macrophages. The aim of this study was to investigate whether macrophage ferric reductase activity is altered in GH, and to determine ferric reductase activity in monocytes and differentiated macrophages. The erythroleukaemic K562 cell line was studied as a clonal reference cell line. The basal K562 ferric reductase activity is characteristic of a membrane bound enzyme, being both temperature and protease sensitive. Ferric reductase activity was also demonstrated in human leucocyte, monocyte and macrophage preparations. Assays of K562 and macrophage cell supernatants confirmed that the ferric reductase activity was not due to a secreted factor. Assay of ferric reductase in normalized-iron and iron-enriched (100 microM ferric citrate) conditions showed no significant difference between Cys282Tyr (Cys282-->Tyr) homozygous GH macrophages and Cys282-Tyr negative control activities (P>0.05). However, a 900% increase in ferric reductase activity was observed during monocyte to macrophage differentiation (P<0.05), possibly reflecting the co-ordinate up-regulation of iron metabolism in these cells. The demonstration of approx. 25% activity after macrophage differentiation at high free-iron concentrations compared with 'normalized' iron is consistent with repression of human ferric reductase activity by iron. The identification of the human ferric reductase gene and its protein will ultimately provide insight into its regulation and role in mammalian iron metabolism.

Cell Differentiation↗
PubMed1997

Uptake of 26-Al and 67-Ga into brain and other tissues of normal and hypotransferrinaemic mice.

Aluminium uptake from blood into tissues of control and homozygous hypotransferrinaemic (hpx/hpx) mice, following continuous intravenous infusion of 26Al and 67Ga, has been compared with that of gallium, a proposed tracer for aluminium. 26Al uptake into tissues of control (hpx/+ and +/+) mice occurred in the order (expressed as a space): bone 464.7 ml 100 g-1; renal cortex 102.9 ml 100 g-1; liver 13.0 ml 100 g-1; spleen 8.4 ml 100 g-1 and brain 0.8 ml 100 g-1. 67Ga uptakes were similar in liver, spleen and brain, but smaller in the renal cortex and bone, at one-third and one-fifth of the values for 26Al, respectively. In the hypotransferrinaemic mice, uptake of 67Ga into all tissues was increased, especially in renal cortex (ninefold) and bone (twentyfold) as compared with the controls. Increases in 67Ga uptakes into cerebral hemisphere, cerebellum and brain stem of the hypotransferrinaemic mice were 3.8, 4.2 and 2.8 fold, respectively. 26Al uptake into tissues of the hypotransferrinaemic mice was similar to control values except in bone where it was three times greater. Pre-treatment of control animals with the anti-transferrin receptor antibody, RI7 208, enhanced 67Ga uptake in all tissues, the effect being greatest in renal cortex (tenfold) and bone (ninefold). 67Ga uptakes into cerebral hemisphere, cerebellum and brain stem in the mice pre-treated with RI7 208 were 6.4, 6 and 10 times greater than in untreated mice, respectively. No influence of antibody on 26AI uptake into mouse tissues was observed except in spleen where it was three times greater than in untreated mice. Hence, transport of aluminium and gallium into mouse tissues is not similar under all conditions. Non-transferrin mediated transport of each metal can occur into all tissues, especially in renal cortex and bone, where gallium may be a suitable marker for aluminium.

Aluminum↗
PubMed1997

Time-course of iron overload and biochemical, histopathological and ultrastructural evidence of pancreatic damage in hypotransferrinaemic mice.

1. The time course of iron overload of the pancreas in hypotransferrinaemic mice maintained on a standard rodent diet was compared with biochemical and histological markers of tissue damage. 2. Pancreatic iron levels increased linearly from weaning till 9 months of age [73.3 nmol/mg of tissue (SEM 9.9; n = 5) compared with 0.9 nmol/mg of tissue (SEM 0.1; n = 4) in age-matched controls] then decreased linearly till at least 18 months of age. 3. Investigation of tissue distribution of newly absorbed radioiron suggested that significant redistribution of iron from liver to pancreas (rather than direct dietary iron sources) must be invoked to explain the rate of pancreatic iron loading in hypotransferrinaemic mice. 4. Pancreatic epithelial cells first showed altered morphology at 9 months of age. At 12 months of age, the pancreatic epithelium had developed a micronodular appearance, with large numbers of acini replaced by atrophic, degenerated acinar cells. Increased collagen fibre deposition was evident by trichrome staining and by electron microscopy. Biochemical markers of pancreatitis (serum lipase, tissue pancreatitis-associated protein mRNA) were elevated before 9 months of age, whereas the levels of pancreatic amylase mRNA declined from 9 months of age. 5. The data suggest that iron loading of hypotransferrinaemic mouse pancreas proceeds up to a threshold level at 9 months of age followed by a progressive atrophy of secretory epithelium. The hypotransferrinaemic mouse pancreas is a useful model system for investigation of parenchymal cell damage by iron.

Acute-Phase Proteins↗
PubMed1997

Iron metabolism in transgenic mice with hypoplastic anaemia due to incomplete deficiency of erythropoietin.

Iron overload is a serious complication of many forms of anaemia, arising in part from mechanisms associated with compensatory increases in erythropoiesis. To investigate other mechanisms by which anaemia itself may perturb iron metabolism, without the confounding effects of compensatory erythropoiesis, we studied transgenic mice with a partially disabling insertion in the erythropoietin gene, which manifested as incomplete erythropoietin deficiency. Mice were studied aged 7-8 weeks. Haemoglobin concentrations were 6.6 +/- 0.8 g/dl in mice homozygous for the modified erythropoietin gene, 12.9 +/- 2.2 g/dl in heterozygous mice and 14.1 +/- 1.0 g/dl in controls. Homozygous mice showed significant hepatic iron loading (2-fold increase in liver non-haem iron, compared with heterozygous mice and normal controls, with iron staining principally in the periportal hepatocytes). Absorption studies using 59Fe showed increased uptake from the lumen of an in vivo isolated duodenal segment in homozygous mice, although at this point in time overall transfer of radioiron to the circulation and other tissues (mucosal transfer) was not different from controls. These observations demonstrate that anaemia can lead to hepatic iron loading even in the absence of increased erythropoiesis, and are consistent with the possibility that anaemic hypoxia can enhance mucosal iron uptake by the duodenal enterocyte.

Absorption↗
PubMed1996

Expression of H and L ferritin mRNAs in mouse small intestine.

Regulation of iron absorption occurs mainly at the level of duodenal enterocytes. Several proteins including ferritin, the iron-storing molecule, have been implicated in the uptake, cellular processing, and transfer of iron by the mucosal cells. H and L ferritin subunits assemble in various proportions to form a 24-subunit protein shell which can store up to 4500 iron atoms. Although tissue-specific distribution of H and L ferritin mRNAs has been widely described, little is known of ferritin gene expression in duodenal cells. In this study, we performed quantitative measurements of H and L ferritin mRNAs levels in mouse duodenum, ileum, and liver by ribonuclease protection assay. In addition, we assessed the relative subcellular distribution of these two mRNAs in mouse duodenal and ileal sections by in situ hybridization. The results show that in duodenal cells, the level of H ferritin mRNA is higher than the L ferritin level (H/L ratio of about 5). Moreover, expression of the H mRNA is regulated along both axes of the small intestine: the level increases sharply from the crypt to the apex of the villus, thus following the general differentiation pathway of these cells, and decreases from the proximal to the distal small intestine. In contrast, the L ferritin mRNA level does not change along the cryptovillus axis and increases in value in the ileum. These results suggest that expression of the H ferritin gene is dependent on the differentiation of the enterocytes but, as yet, the regulatory elements remain to be identified.

Animals↗
PubMed1996

Mucosal surface ferricyanide reductase activity in mouse duodenum.

Mouse duodenum possesses mucosal surface ferricyanide reductase activity. The reducing activity, determined in vitro by measuring ferrocyanide production from ferricyanide, was found to be greater in duodenal fragments when compared with ileal fragments. Experiments with right-side out tied-off duodenal sacs show that reduction occurs mainly on the mucosal side and indicates that the reducing activity is associated with the brush border membrane. Experiments using mice with increased levels of iron absorption (hypoxic, iron-deficient) showed corresponding increases in reducing activity. The increase was present in duodenal but not ileal fragments. Inhibitor studies showed no effect of several compounds which inhibit other, more characterized, transplasma membrane reductases. In particular, doxorubicin (10 microM) and quinacrine (1mM) were without effect on duodenal mucosal transplasma membrane reducing activity. Depolarization of the membrane potential with high medium K+ inhibited reducing activity. N-ethyl malemide (1 mM) was a potent inhibitor, but iodoacetate was found to be less inhibitory. Comparison with inhibitory effects on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) demonstrated that the effect of N-ethyl malemide on reducing activity was not secondary to GAPDH. Collectively these results indicate that mouse duodenum possesses mucosal surface transplasma membrane ferricyanide reductase activity and that the activity is correlated with the process of intestinal iron absorption. Furthermore, the reducing activity appears to be distinct from other reported transplasma membrane reductases.

Animals↗
PubMed1996

A duodenal mucosal abnormality in the reduction of Fe(III) in patients with genetic haemochromatosis.

BACKGROUND: Previous in vitro studies have shown that the uptake of Fe(III) by freshly isolated duodenal mucosal biopsy specimens is increased in patients with genetic haemochromatosis. Moreover, in the mouse it has recently been found that reduction of Fe(III) to Fe(II) is a prerequisite for iron uptake by the proximal intestine. AIMS/METHODS: This study used the in vitro technique to investigate the rates of reduction and uptake of 59Fe(III) by duodenal mucosal biopsy specimens obtained at endoscopy from treated and untreated patients with genetic haemochromatosis. RESULTS: The rate of reduction of iron in the medium was proportional to the incubation time and was not caused by the release of reducing factors from the tissue fragments. Ferrozine, a specific Fe(II) chelator and ferricyanide, a non-permeable oxidising agent, inhibited uptake of 59Fe showing that reduction of Fe(III) precedes uptake. The rates (all values given as pmol/mg/min) of reduction (152 (49) v 92 (23)) and uptake (8.3 (4.0) v 3.6 (1.3), mean (SD)), were significantly increased in biopsy specimens from the untreated group (n = 6) compared with those from 10 control subjects (p < 0.04). Furthermore, the reduction and uptake rates were still increased in five patients in whom iron stores were normal after venesection treatment. CONCLUSIONS: These results show that there is a persistent abnormality in the reduction and uptake of iron by the intestine in genetic haemochromatosis.

Analysis of Variance↗
PubMed1996

Expression of genes involved in iron metabolism in mouse intestine.

Intestinal nonheme iron levels and mRNA levels of genes implicated in iron metabolism were measured in mice with altered iron metabolism [chronic (4 wk) and acute (4 days) dietary iron deficiency; iron overload and hypoxia] to investigate their role in the process and regulation of intestinal iron absorption. Mucosal nonheme iron levels were decreased by both chronic and acute iron deficiency and increased by iron overload but were not affected by hypoxia. There was evidence of a gradient of mucosal nonheme iron along the small intestine (duodenum, jejunum > ileum). There were also regional differences in H-ferritin (duodenum > ileum) and transferrin receptor (ileum > duodenum) mRNA levels. Iron overload produced a decrease in transferrin receptor (TfR) mRNA in the duodenum, with ferritin mRNA levels unaffected in both the duodenum and ileum. Chronic iron deficiency induced a twofold increase in TfR mRNA levels in both the duodenum and ileum, whereas H- and L-ferritin mRNA levels did not change significantly. The ratio of H- to L-ferritin mRNA decreased significantly during exposure to hypoxia; however, individual ferritin and TfR mRNA levels were not significantly altered. Calreticulin (mobilferrin), cysteine-rich intestinal protein, and H(+)-adenosinetriphosphatase mRNA levels were virtually unchanged in all models. A comparison with previously published data on changes in iron absorption leads us to conclude that 1) iron absorption can be altered independently of effects on transcripts of genes for iron-related proteins, and 2) it is not essential for iron absorption to be coordinated with regulation of mucosal iron metabolism.

Animals↗
PubMed1995

Cytokine-mediated regulation of transferrin synthesis in mouse macrophages and human T lymphocytes.

Transferrin (Tf) plays an important role during immunologic activation by donating iron to activated lymphocytes. Therefore, synthesis by lymphomyeloid cells has been investigated. Mouse macrophages and macrophage cell lines synthesized Tf, with levels being markedly increased by gamma-interferon (gamma-IFN) and, to a lesser extent, by interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF alpha). Tf was also produced by phytohemagglutinin-stimulated human T cells and two T-cell lines and was increased by IL-2. Even after appropriate activation, none was synthesized by human macrophages or monocytic cell lines or by mouse T cells, T-cell lines, or thymus cells. In both species, B-lineage cell lines were negative. Tf was also synthesised by macrophages from congenitally hypotransferrinemic mice and was responsive to gamma-IFN, but levels were lower than those from normal controls. Synthesis by human and murine hepatoma cells was increased by IL-6 but unaffected by IL-1, TNF alpha, or gamma-IFN. Iron decreased synthesis by hepatoma cells but had no effect on the lymphomyeloid cells. Tf mRNA levels paralleled protein synthesis, suggesting that regulation was pre-translational. Thus, Tf synthesis by lymphomyeloid cells is regulated differently from hepatic synthesis, which is consistent with the suggestion that Tf may act in a paracrine (mouse) or autocrine (human) manner on activated lymphocytes.

Animals↗