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Biomedical subjects

K B Raja

Publications and source records attributed to K B Raja.

At least 37 records · Page 2Linked to original sources
PubMed1995

The hypotransferrinaemic mouse: ultrastructural and laser microprobe analysis observations.

Homozygote hypotransferrinaemic mice (hpx/hpx) have cytopathological features similar to those of human congenital atransferrinaemia, genetic haemochromatosis, and neonatal haemochromatosis. These conditions all have in common high levels of cytotoxic non-transferrin-bound serum iron. This study describes the ultrastructural features of iron overload in liver, pancreas, heart, and small intestine of 2- and 12-month-old hypotransferrinaemic mice. Electron microscopic studies of unstained sections showed early parenchymal cell siderosis, with accumulation of numerous ferritin particles and clusters in the cytosol, as well as ferritin and haemosiderin in lysosomes (siderosomes). In the 12-month-old animals, iron was also found in Kupffer cells and macrophages in other tissues. In addition, there were conspicuous iron-containing compounds in the bile canaliculi, and marked iron deposition in the pancreas and heart. Laser microprobe mass analysis (LAMMA) enabled localization and relative quantitation of iron deposition in subcellular compartments providing in situ documentation of iron accumulation in siderosomes and contributed in assessing total cytosolic iron in various cell types. Moreover, it demonstrated the importance and magnitude of the biliary route for iron excretion in these animals.

Animals↗
PubMed1995

In vitro duodenal iron uptake and serum and mucosal iron protein levels, with special reference to rheumatoid arthritis.

Duodenal biopsies from control subjects, patients with iron-deficiency anaemia and rheumatoid arthritic patients with anaemia of chronic disorders (ACD) were investigated for their ability to take up 59Fe from iron ascorbate. Additionally, duodenal tissues were analysed for iron and immunoreactive ferritin and transferrin. Biopsies from iron-deficient subjects showed a 2- to 3-fold increase in the apparent Vmax for 59Fe uptake, compared to control values. Uptake was inversely related to body iron stores. ACD patients showed similar rates of 59Fe uptake to controls; the rates were independent of the degree of anaemia or serum ferritin levels. Tissue analysis showed reductions in mucosal iron and ferritin levels in iron-deficient patients, whilst transferrin levels were within the normal range. ACD patients also exhibited lower mucosal iron levels, but had iron protein levels within the normal range. It is suggested that factors distant from the intestinal mucosa influence iron absorption in ACD.

Adult↗
PubMed1995

Duodenal expression of NF-E2 in mouse models of altered iron metabolism.

This study investigated the relationship between duodenal mucosal mRNA levels of the transcription factor, NF-E2, H-ferritin (a putative NF-E2 regulated gene) and iron absorption in mice. CD1-strain mice with normal and altered iron metabolism (hypoxic, iron-deficient, iron-loaded) and animals with genetic defects of iron metabolism (hypotransferrinaemia, beta-thalassaemia) were studied. Tissue RNA from these mouse models was subjected to reverse transcription and PCR amplification for NF-E2 and a stable ribosomal protein (S14) and the products analysed with an automated laser fluorescent sequencer. Duodenal NF-E2 mRNA levels were generally low and decreased in the hypoxic and iron-deficient groups, both of which exhibited elevated iron absorption as compared to controls. A modest increase in the NF-E2 mRNA level was seen in the iron-loaded mice, whose iron absorption was decreased. In contrast, both the genetic strains showed elevated NF-E2 mRNA levels in conjunction with raised iron absorption values. Only the iron-deficient group exhibited an alteration in the duodenal mucosal H/L ferritin ratio. Hence, no relationship was evident between the NF-E2 mRNA levels and the H/L ferritin ratio. These data indicate that NF-E2 is not the primary regulator of intestinal iron absorption.

Animals↗
PubMed1995

Assessment of intestinal blood-flux by laser Doppler fluxmetry in mice with altered intestinal iron absorption.

Laser Doppler fluxmetry (LDF) has been used to assess mucosal blood-flux in the intestine of normal CD1 mice and groups of animals with altered iron metabolism (i.e. iron-deficient, hypoxic and hypotransferrinaemic [hpx/hpx]). All experimental animals showed a 2-3-fold increase in duodenal iron absorption, mainly due to changes in the 'mucosal transfer' phase. However, only the hypoxic mice exhibited any increases in duodenal blood-flux. In the hpx/hpx group, blood flux was maintained at control levels despite the anaemia and without any significant alterations in red blood cell velocity. Moreover, these two groups demonstrated a further capacity to increase blood-flux above basal levels during onset of acute hypoxia (inhalation of 10% O2). Changes in duodenal blood-flux were apparent on both the mucosal and serosal surfaces, suggesting that the LDF signal reflects flux throughout the thickness of the mouse intestine. Iron absorption is likely to be more related to plasma flux changes than to blood-flux changes. Deduction of plasma flux changes from blood-flux changes is complicated by vasodilation effects on capillary haematocrits. It is clear from the data, however, that plasma flux changes do not parallel changes in iron absorption in the experimental models. The lack of correlation between the duodenal blood-flux and iron absorption values in the experimental models argues against the possible involvement of blood-flux in the control of duodenal iron absorption.

Anemia, Iron-Deficiency↗
PubMed1995

Plasma clearance of transferrin in control and hypotransferrinaemic mice: implications for regulation of transferrin turnover.

Kinetic studies were performed to determine the clearance of iodinated transferrin in hypotransferrinaemic mice, as compared to normal animals. Clearance of i.v. (and i.p.) administered radiolabelled protein in homozygous (hpx/hpx) mice was significantly faster than in heterozygous (hpx/+) and wild-type control (+/+) groups. A comparable t1/2 value for transferrin clearance in hpx/hpx mice was derived from a study in which immunoassay was performed on serum samples obtained at various times post-injection with normal mouse serum, indicating that the clearance of 125I reflected true clearance of transferrin protein. The clearance rate in the hpx/+ group was significantly slower than in +/+ mice. Calculation of transferrin synthesis rates in these two groups suggested that transferrin levels do not regulate transferrin synthesis rates, but may affect degradation; this observation is consistent with the fact that transferrin levels in hpx/+ mice are > 50% of the values in +/+ mice, and indicates a partial compensation for reduced synthesis. The rapid clearance in hpx/hpx mice is an additional factor in determining the low levels of circulating transferrin in these synthesis-impaired mutants.

Anemia, Hypochromic↗
PubMed1994

Intestinal iron absorption studies in mouse models of iron-overload.

Three mouse strains have been evaluated as suitable models for investigations into the pathogenesis of iron-overload syndromes. Mice with hereditary heterozygous alpha-thalassaemia had moderately raised reticulocyte counts, but were not anaemic and showed little, if any, iron loading. In contrast, mice with homozygous beta-thalassaemia showed microcytic anaemia, reticulocytosis and splenomegaly. Iron-loading was marked, progressive with age and mainly confined to the spleen. Liver iron-loading increased until the age of 7-8 weeks, with no further increase over successive weeks. Although intestinal iron absorption was modestly increased due to enhanced mucosal uptake, the majority of the 'excess' liver and spleen iron could be accounted for by re-distribution of iron from the erythrocytic compartment. Homozygous hypotransferrinaemic mice, with approximately 1-2% of normal plasma transferrin levels, were markedly anaemic with hypochromic microcytic erythrocytes. Intestinal iron absorption increased 3-4-fold (predominantly due to changes in mucosal transfer), as compared to wild-type controls and heterozygotes, and was ascertained to be a major factor causing the marked hepatic iron overload. Heterozygous hypotransferrinamic mice, with over half normal plasma transferrin levels and a mild degree of hepatic iron loading, showed very similar characteristics to wild-type controls. Thus, of the three models, hpx/hpx mice showed the greatest enhancement in intestinal iron absorption and net iron-loading and provides a suitable animal model of spontaneous iron-overload. Comparison of iron absorption values between the models suggests that reticulocytes cannot account for the enhanced absorption seen in the hpx/hpx mice.

Animals↗
PubMed1993

Tissue iron loading and histopathological changes in hypotransferrinaemic mice.

Tissue iron loading in hypotransferrinaemic (hpx/hpx) mice was investigated as a model for genetic (primary) haemochromatosis. Iron loading of liver preceded that in the pancreas and heart. One-year-old hpx/hpx mice showed iron staining in exocrine pancreas, liver parenchymal cells, and cardiac and intestinal smooth muscle cells. Iron-loaded macrophages were observed in all these tissues. Islets of Langerhans, biliary epithelial cells, and spleen were iron-free. The pancreas was fibrotic with massive macrophage infiltration and loss of secretory epithelium. Liver showed evidence of chronic inflammatory infiltration with increased collagen fibres in the parenchymal region but no cirrhosis. Serum aspartate aminotransferase activity and plasma glucose were increased in hpx/hpx compared with wild-type mice. Heavy iron loading with haemosiderin deposition in the liver could be demonstrated in hpx/hpx mice from 6 weeks of age. Heterozygous hypotransferrinaemic mice showed minor increases in liver iron stores at 6-12 weeks, but not at 1 year of age. Serum ferritin levels in heterozygous mice were also increased at 6-8 weeks of age. It was concluded that 1-year-old hpx/hpx mice showed evidence of liver and pancreatic damage secondary to tissue iron overload. The iron loading pattern and tissue damage showed some features which were distinct from those observed in haemochromatosis.

Animals↗
PubMed1993

Rate of 59Fe uptake into brain and cerebrospinal fluid and the influence thereon of antibodies against the transferrin receptor.

Uptake of 59Fe from blood into brains of anaesthetized rats and mice has been studied by intravenous infusion of [59Fe]ferrous ascorbate or of 59Fe-transferrin, the results not being significantly different. Uptakes in the rat were linear with time, but increased at longer times in the mouse. Transfer constants, K(in) (in ml/g/h x 10(3)), for cerebral hemispheres were 5.2 in the adult rat and 5.6 in the mouse. These K(in) values corresponded to 59Fe influxes of 145 and 322 pmol/g/h, respectively. 59Fe uptake into the mouse brain occurred in the following order: cerebellum > brainstem > frontal cerebral cortex > parietal cortex > occipital cortex > hippocampus > caudate nucleus. In genetically hypotransferrinaemic mice, 59Fe uptake into brain was 80-95 times greater than in To strain mice. Pretreatment of young rats and mice with monoclonal antibodies to transferrin receptors, i.e., the anti-rat immunoglobulin G OX 26 and the anti-mouse immunoglobulin M RI7 208, inhibited 59Fe uptake into spleen by 94% and 98%, respectively, indicating saturation of receptors. The antibodies reduced 59Fe uptake into rat brain by 35-60% and that into mouse brain by 65-85%. Although a major portion of iron transport across the blood-brain barrier is normally transferrin-mediated, non-transferrin-bound iron readily crosses it at low serum transferrin levels.

Animals↗
PubMed1992

Non-transferrin-bound iron species in the serum of hypotransferrinaemic mice.

Serum from homozygous hypotransferrinaemic mice (a mixed group of males and females, aged 6-8 wk) was found to contain low levels of iron (mean 0.9 +/- 0.5 microM (SEM, n = 4), as assayed by conventional serum iron assays. Similarly, low levels of non-transferrin-bound iron were determined with a nitrilotriacetate chelation assay (1.3 +/- 0.4 microM, n = 4) (Singh, S., Hider, R.C. and Porter, J.B. (1990) Analytical Biochemistry 186, 320-323). Mononuclear Fe (citrate) was undectable by electron paramagnetic resonance spectroscopy (EPR). Significantly larger quantities of iron (16 +/- 5 microM, n = 8) were detected by the bleomycin assay (Gutteridge, J.M.C., Rowley, D.A. and Halliwell, B. (1981) Biochemical Journal 199, 263-265), while non-haem iron assay or atomic absorption spectrophotometry revealed up to 96 microM iron. Haemoglobin iron was detectable at approximately 10 microM by spectrophotometry. Ferri-haem was undetectable by EPR spectroscopy. Serum ferritin levels of 641 +/- 128 micrograms/l (n = 14) in hypotransferrinaemic mice (wild-types 44 +/- 6 micrograms/l, n = 14) were observed and these cannot account for the non-transferrin-bound iron. Hypotransferrinaemic mouse serum therefore contains large quantities of non-transferrin-bound iron which is unreactive in some assays used to detect such iron in human iron overload. Fractionation by Sephadex G200 chromatography revealed three distinct species with apparent molecular weights of > or = 150 kDa, 40-80 kDa and 1-5 kDa. The iron may be distinguished from known extracellular iron proteins and haem-proteins by its availability to hot acid extractions.

Animals↗
PubMed1992

Investigation of a role for reduction in ferric iron uptake by mouse duodenum.

59Fe uptake rates by mouse duodenal fragments incubated in vitro were markedly reduced by non-permeable reagents, ferricyanide (oxidising agent) and ferrozine (Fe2+ chelator), in the medium; ferrocyanide had no effect. Reduction of Fe3+, as reflected by an increase in ferrozine-(Fe2+)-chelatable iron, was observed in the presence of the tissue fragments. The generation of Fe2+ occurred linearly with time, was independent of the medium ferrozine concentration, and was not due to release of reducing factors from the duodenal fragments. Fe(3+)-reducing activity was mainly present on the mucosal surface and was localised primarily to the proximal region of the small intestine. Changes in Fe3+ reduction rates closely parallelled the changes in duodenal 59Fe uptake, when metabolic inhibitors or modulators of membrane potential were included in the medium. The enhancement in duodenal mucosal 59Fe uptake in chronic hypoxic and iron-deficient mice parallelled the changes in the tissue reduction of medium Fe3+. Moreover, the rates of reduction were quantitatively similar to rates of uptake. These observations indicate that a sequential reduction and uptake process operates for Fe3+ uptake in mouse duodenum.

Animals↗
PubMed1992

Regulation of iron absorption in iron loaded subjects with end stage renal disease: effects of treatment with recombinant human erythropoietin and reduction of iron stores.

The effects on iron absorption of variation in erythroid activity, haemoglobin and iron stores were studied in six anaemic dialysis-dependent subjects in whom iron stores were increased from previous red cell transfusions. Gastrointestinal mucosal uptake and whole body retention of oral iron were measured at the beginning of the study, after starting treatment with recombinant erythropoietin (but before significant increase in haemoglobin), after partial correction of anaemia, after further reduction of iron stores by repeated phlebotomy, and when erythropoiesis decreased from the discontinuation of treatment with erythropoietin. Between successive measurements, valid comparisons were made in five subjects. Correction of anaemia decreased whole body retention of iron through decreased mucosal uptake (P = 0.032). Further reduction in iron stores through repeated phlebotomy whilst the increase in haemoglobin was maintained by treatment with erythropoietin, tended to increase whole body retention of iron through an increase in mucosal transfer (P = 0.010). With initial enhancement of erythropoiesis in anaemic iron-loaded subjects there was no change in any measured component of iron absorption. However, after correction of anaemia and reduction of iron stores, a decrease in erythropoiesis was associated with decreased whole body iron retention in all subjects through decreased mucosal transfer (P = 0.028). The data suggest that anaemia upregulates mucosal iron uptake, and that erythroid activity upregulates mucosal transfer but that this latter effect may be counter-balanced by iron overload which downregulates mucosal transfer.

Adult↗
PubMed1992

Intestinal iron absorption in chronic alcoholics.

Chronic alcohol misusers frequently accumulate significant amounts of excess iron, but the mechanism of this loading is unknown. In vivo whole-body retention studies demonstrated, on average, a two-fold increase in intestinal iron absorption in six male chronic alcoholics. Degrees of iron loading as assessed by serum ferritin or hepatic iron levels did not correlate with alcohol consumption or liver function tests. In vitro studies of iron uptake at varying medium iron concentrations by duodenal mucosa biopsies showed increased iron uptake by tissue from the chronic alcoholics, particularly at the highest medium iron concentration used. Analysis of the uptake data showed similar Michaelis-Menten kinetic constants for uptake by tissue from control subjects and alcoholics. The analysis showed, in addition, a linear component for 59Fe uptake. This component was five-fold greater for the tissue from the chronic alcoholics compared to the controls at the highest medium iron concentration. 57Co-cyanocobalamin was included in the incubation medium as a tissue extracellular fluid marker (ECF). It was found that the apparent distribution volume of the ECF marker, reflecting tissue permeability, was 75% higher for the biopsies from the alcoholics compared to control subjects. These results, together with the previous reports of enhanced in vitro and in vivo intestinal permeability to 51Cr-EDTA in chronic alcoholics, indicate that unregulated increased iron absorption via the non-carrier-mediated paracellular route contributes to the iron overload in chronic alcoholics.

Adult↗
PubMed1991

Iron absorption by hypotransferrinaemic mice.

Iron absorption rates by homozygous and wild-type mice from a hypotransferrinaemic mouse colony were examined with in vivo tied-off duodenal segments and in vitro incubated duodenal fragments. Enhanced initial rates of mucosal uptake and carcass transfer by homozygotes, compared to wild-types, were observed. The changes in vivo and in in vitro uptake kinetics resemble changes seen in iron deficient or hypoxic mice, suggesting that the liver iron loading shown by homozygotes is due to a failure of the normal mechanism for regulation of iron absorption. In vivo mucosal uptake and carcass transfer of radioiron showed an inverse correlation with liver non-haem iron content in homozygous hypotransferrinaemic mice, suggesting that some degree of control of absorption, albeit at markedly reduced sensitivity, can operate in these mice. No correlation between haemoglobin level and iron absorption was observed in homozygous hypotransferrinaemic mice, suggesting that this regulator of iron absorption does not function in these mice. The precise pathogenic mechanism of the enhanced iron absorption in hypotransferrinaemia remains to be determined. Mucosal transferrin levels were found to parallel serum transferrin levels in homozygotes, heterozygotes and wild-type mice. This supports previous suggestions that mucosal transferrin is derived from plasma transferrin and that the enhancement of iron absorption, by physiological mechanisms, does not require the presence of mucosal transferrin.

Absorption↗
PubMed1991

Absorption of iron from iron succinyl-protein complexes by mouse small intestine.

The absorption of iron from iron succinyl-protein complexes was investigated in mice. 59Fe-labelled succinyl-casein and -albumin complexes, [59Fe]ferritin and 59FeSO4, at doses of 20 or 200 micrograms of iron, were administered orally to normal mice or mice with absorption enhanced by chronic hypoxia. 59Fe from iron succinyl-protein was well absorbed in normal mice (greater than 10% of dose) and showed enhanced absorption in hypoxic mice (greater than 40% of dose). Intestinal uptake was predominantly by the duodenum for all compounds studied. In-vivo absorption of 59Fe from an iron succinyl-protein complex was studied using tied-off segments of mouse duodenum, jejunum or ileum of normal or hypoxic mice. Incubation for up to 15 min in duodenum or 60 min in ileum showed very little absorption of 59Fe. No enhancement of absorption was seen in hypoxic mice. It was concluded that absorption of the intact iron succinyl-protein complex cannot explain absorption seen after oral dosing.

Animals↗