Iron metabolism and placental iron transfer in the guinea pig

The interrelationship between fetal iron uptake and maternal iron metabolism has been studied in the guinea pig in the course of pregnancy. The rapid increase of the maternal need for iron during the period of fast increasing rates of placental iron transfer is largely compensated for by increased intestinal absorption. No enhanced mobilisation of iron from the liver and spleen iron stores could be demonstrated. The plasma iron turnover, corrected for the transplacental iron transfer rate, remained constant during pregnancy. This means that not only the mobilisation of iron from the stores remains principally unchanged, but also the supply of iron to the maternal organs and tissues. The haemoglobin concentration decreased by about 15% during the period of rapid fetal growth and iron uptake. The maternal blood volume increased during this very period and explained most of the observed reduction. Intestinal iron absorption increases. At day 55 of pregnancy placental iron transfer is maximal. It could be shown that a day 55 the rate of intestinal iron uptake equals the rate of iron transfer across the placentas. It is evident that pregnancy effects a direct influence on intestinal iron absorption, independent of the magnitude of the maternal iron stores. How this influence is realized without changing the iron kinetics of the maternal stores, cannot be explained with the prevailing theory.     

Iron dextran treatment does not induce serum protein carbonyls in the newborn pig

Oxidation of serum proteins can lead to carbonyl formation that alters their function and is often associated with stress-related diseases. As it is recommended that all pigs reared in modern production facilities be given supplemental iron at birth to prevent anemia, and metals can catalyze the carbonylation of proteins, the primary objective of this study was to determine whether standard iron dextran treatment was associated with enhanced serum protein oxidation in newborn piglets. Piglets were treated with 100 mg of iron dextran intramuscularly either on the day of birth, or on the third day after birth. Blood samples were collected from piglets 48 or 96 h after treatment and serum was harvested. For quantification, serum protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and analyzed spectrophotometrically. To identify and determine relative distribution of carbonylated proteins, serum protein carbonyls were derivatized with biotin hydrazide, separated by two-dimensional polyacrylamide gel electrophoresis, stained with avidin-fluorescein and identified by mass spectrometry. The standard iron dextran treatment was associated with no increase in total oxidized proteins if given either on the first or third day of life. In addition, with a few noted exceptions, the overall distribution and identification of oxidized proteins were similar between control and iron dextran-treated pigs. These results indicate that while iron dextran treatment is associated with a marked increase in circulating iron, it does not appear to specifically induce the oxidation of serum proteins.     

Purified moniliformin does not affect the force or rate of contraction of isolated guinea pig atria

Aspergillus flavus Link ex Fries and A. parasiticus Speare can invade peanut kernels and under certain environmental conditions produce unacceptable levels of the mycotoxin aflatoxin. A concerted effort is underway to reduce aflatoxin contamination in peanut and peanut products. A potentially effective method of control in peanut is the discovery and use of genes for resistance to either fungal invasion or aflatoxin formation. The objective of the present experimental study was to develop an effective and efficient procedure for screening individual plants or pods of single plants for resistance to invasion by the aflatoxigenic fungi and subsequent aflatoxin production. Methods of obtaining adequate drought-stress and fungal infection were developed through this series of experiments. By completely isolating the pods from the root zone and imposing drought-stress only on pegs and pods, high levels of fungal infection were observed. High amounts of preharvest aflatoxin accumulation were also produced by completely isolating the pods from the root zone. Mid-bloom inoculation with A. parasiticus-contaminated cracked corn and drought-stress periods of 40 to 60 days were the most effective procedures. This technique was used to assess peanut genotypes previously identified as being partially resistant to A. parasiticus infection or aflatoxin contamination, and segregating populations from four crosses. Variability in aflatoxin contamination was found among the 11 genotypes evaluated, however, none were significantly lower than the standard cultivars. Broad-sense heritability of four crosses was estimated through evaluation of seed from individual plants in the F₂ generation. The heritability estimates of crosses GFA-2 × NC-V11 and Tifton-8 × NC-V11 were 0.46 and 0.29, respectively, but mean aflatoxin contamination levels were high (73,295 and 27,305 ppb). This greenhouse screening method could be an effective tool when genes for superior aflatoxin resistance are identified.Cooperative investigation of the USDA-ARS and the University of Georgia, College of Agriculture.     

Iron accumulation in tissues of magnesium-deficient rats with dietary iron overload

The mineral imbalances in magnesium-deficient rats with dietary iron overload were studied. Forty-four male Wister rats were divided into six groups and fed six diets, two by three, fully crossed: magnesium adequate or deficient, and iron deficient, adequate, or excess. The concentrations of iron, magnesium, calcium, and phosphorus in tissues of the rats were measured. The results were as follows: (1) The excess iron intake reinforced the iron accumulation in liver and spleen of magnesium deficient rats; (2) The saturation of iron binding capacity was enormously elevated in the magnesium deficient rats fed excess iron; and (3) Dietary iron deprivation diminished the degree of calcium deposition in kidney of magnesium deficient rats. These results suggest that magnesium-deprived-rats have abnormal iron metabolism losing homeostatic regulation of plasma iron, and magnesium deficient rats with dietary iron overload may be used as an experimental hemochromatosis model.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) does not destroy nigrostriatal neurons in the scorbutic guinea pig

Guinea pigs were injected subcutaneously with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in maximal tolerated doses (8 mg/kg, once daily) for 10 or 15 days. No neurological effects were noted, other than sedation and hypotonia lasting a few hours after each injection, either in animals maintained on normal diet or in animals fed an ascorbate-deficient diet and rendered severely scorbutic. Subsequent chemical analyses of the striatum showed no evidence of lasting damage to nigrostriatal dopaminergic neurons in MPTP treated guinea pigs on normal diet, and minimal evidence of permanent damage to these neurons in scorbutic animals. MPTP was undetectable in the urine of MPTP-treated animals, although a metabolite, presumably 1-methyl-4-phenylpyridinium ion (MPP+) was regularly present in urine. The relative lack of neurotoxicity of MPTP in the guinea pig remains unexplained. This species clearly is not a suitable small animal for MPTP-induced parkinsonism.     

Gnpat does not play an essential role in systemic iron homeostasis in murine model

The GNPAT variant rs11558492 (p.D519G) was identified as a novel genetic factor that modifies the iron-overload phenotype in homozygous carriers of the HFE p.C282Y variant. However, the reported effects of the GNPAT p.D519G variant vary among study populations. Here, we investigated the role of GNPAT in iron metabolism using Gnpat-knockout (Gnpat^−/−), Gnpat/Hfe double-knockout (Gnpat^−/−Hfe^−/− or DKO) mice and hepatocyte-specific Gnpat-knockout mice (Gnpat^fl/fl;Alb-Cre). Our analysis revealed no significant difference between wild-type (Gnpat^+/+) and Gnpat^−/− mice, between Hfe^−/− and DKO mice, or between Gnpat^fl/fl and Gnpat^fl/fl;Alb-Cre with respect to serum iron and tissue iron. In addition, the expression of hepcidin was not affected by deleting Gnpat expression in the presence or absence of Hfe. Feeding Gnpat^−/− and DKO mice a high-iron diet had no effect on tissue iron levels compared with wild-type and Hfe^−/− mice, respectively. Gnpat knockdown in primary hepatocytes from wild-type or Hfe^−/− mice did not alter hepcidin expression, but it repressed BMP6-induced hepcidin expression. Taken together, these results support the hypothesis that deleting Gnpat expression has no effect on either systemic iron metabolism or the iron-overload phenotype that develops in Hfe^−/− mice, suggesting that GNPAT does not directly mediate iron homeostasis under normal or high-iron dietary conditions.     

Dietary iron loading does not influence biliary iron excretion in rats

The effect of dietary iron loading on biliary iron excretion was investigated with male Wistar rats aged 6 wk. The rats were fed purified diets with either 174 or 1740 mg FeSO₄. 7H₂O/kg diet and demineralized water for 6 wk. Blood haemoglobin, hematocrit, and iron concentrations in kidney and heart were not affected and iron concentrations in liver, spleen, and tibia were significantly raised after feeding the high-iron diet. The high-iron diet did not raise biliary iron excretion, suggesting that biliary iron excretion does not play an important role in regulating iron metabolism in rat after dietary iron loading.     

Selected physical and biochemical parameters in the streptozotocin-treated guinea pig: insights into the diabetic guinea pig model

Since evidence suggests that ascorbic acid deficits may provoke certain diabetic complications, it becomes necessary to develop a diabetic animal model which, like man, is unable to synthesize this vitamin. To this end, the present study monitored the diabetogenic effects of streptozotocin (STZ, 150 mg/kg) in the male guinea pig, a species rarely used in diabetes research. Over a 3-week period, body weight and relative food intake were lower in the STZ group compared to controls. The mean daily water intake and urine volume of the STZ group after 1 week were 175 and 270% of their initial pretreatment values, respectively, while control values were unchanged. The STZ group also exhibited a persistent glycosuria throughout the study. At the end of 3 weeks, aldehyde fuchsin staining of pancreatic beta cell granules (an index of stored insulin) was 58% lower in the STZ group compared to controls. Plasma C-peptide (indicator of insulin secretion) was expressed in human equivalents (mean +/- SEM). C-peptide was reduced in the STZ group (103 +/- 65 pg/ml) compared to controls (549 +/- 96 pg/ml); however, no change in plasma glucose was observed. Plasma ascorbic acid levels also were lower for STZ animals (150 +/- 26 micrograms%) versus controls (410 +/- 28 micrograms%). This study 1) demonstrates a diabetic syndrome in the STZ-treated guinea pig based on a reduced growth rate, beta cell dysfunction, polydipsia, polyuria and glycosuria, and 2) suggests the usefulness of this diabetic model in studies of pathologic mechanisms influenced by ascorbic acid.     

An experimental model of ameboma in guinea pig.

Among the wide variety of clinicopathological manifestations of intestinal amebiasis, amebomas occur rarely and their pathogenesis is not well understood. When cholesterol-fed, 2- to 4-week-old guinea pigs were infected intracecally with a virulent, monoaxenic strain of Entamoeba histolytica, gross and histologically characteristic amebomas developed in 85% of the animals by the 3rd day, in 94% by the 9th day, and in 96% by the 12th day postinfection, by which time most of them had died. Amebomas were confirmed by histopathology. Thus, a model of consistent production of amebomas was documented.     

Pathogenesis of cytomegalovirus-associated labyrinthitis in a guinea pig model

Cytomegalovirus infects fetuses through the placenta, resulting in various congenital disorders in newborns, including hearing loss. We developed a monoclonal antibody to guinea pig cytomegalovirus (GPCMV) that was available for immunohistochemistry, and investigated the expression of the GPCMV antigen in animal models of direct and congenital infections. Injection of GPCMV, directly to the inner ear, increased the sound pressure level and resulted in labyrinthitis with severe inflammation. Immunohistochemistry detected GPCMV-infected cells mainly in the scala tympani, scala vestibule and spinal ganglion, but rarely in the cochlear duct. Injection of GPCMV to 5-week pregnant guinea pigs resulted in severe labyrinthitis in fetuses. Immunohistochemistry detected GPCMV-infected cells in the perilymph area and spinal ganglion, but not in the endolymph area, including hair cells. These data suggest that the virus spreads via the perilymph and neural routes in the inner ear of both models of direct and congenital infections.     

Iron supplementation moderates but does not cure the Belgrade anemia

Belgrade rats inherit microcytic, hypochromic anemia as an autosomalrecessive trait (gene symbol b). Erythrocytes and tissue are iron deficientin the face of elevated TIBC (total iron binding capacity) and percent ironsaturation; iron injections increased the number of erythrocytes but theirappearance remained abnormal. We have investigated iron supplements toimprove husbandry of b/b rats and to learn more about the underlying defectand its tissue distribution. Weekly IM (intramuscular) injections ofiron–dextran (Imferon at 30 mg kg) improved the anemia but did not alter thered cell morphology. Certain diets also improved the health of b/b rats whencompared to standard rat chows by the criteria of weight, survival toadulthood, hematology and reproduction. The critical nutritional factorturned out to be iron bioavailability, with ferrous iron added to the dietimproving the health of Belgrade rats without affecting the underlyingerythroid defect. Tissue iron measurements after dietary or parenteralsupplementation confirmed the iron deficient status of untreated b/b rats andestablished that dietary ferrous iron partially relieved this deficiency,with injections leading to greater amounts of tissue iron. Serum iron andTIBC were also found to be elevated in untreated b/b rats, with dietarysupplementation decreasing but not eliminating the elevation in TIBC. Thesestudies indicate that iron supplements can improve the health of b/b ratswithout altering the underlying defect and also suggest that the mutationcould alter iron uptake in the GI (gastrointestinal) tract.     

By-passing the sphincter of Oddi does not affect gallbladder emptying in the pig

A study of the relationship between bile secretion and nutrition in the pig requires a complete and continuous collection of the bile and its reinfusion to the animal. In most of the studies performed in different species, bile has been directly reinfused into the duodenum, leading to the exclusion of the sphincter of Oddi from the biliary pathway. It has been postulated that such an exclusion could inhibit gallbladder emptying. The aim of the present work was to study postprandial gallbladder emptying in the pig, depending on the site of bile reinfusion, i.e. the duodenum or the lower bile duct. The gallbladder bile was coloured with indocyanine green (ICG) and marker secretion was recorded after a test-meal. The results showed that after meal intake, the gallbladder emptied over a similar period of time and according to similar kinetics, whatever the site of bile reinfusion.     

Post-transfusional iron overload in the haemoglobinopathies

In this report, we review the recent advances in evaluation and treatment of transfusional iron overload (IO). Results of the French thalassaemia registry are described. According to the disease, thalassaemia major or sickle cell anaemia, mechanisms and toxicity of iron overload, knowledge about IO long-term outcome and chelation treatment results, respective value of IO markers, differ. The recent tools evaluating organ specific IO and the diversification of iron chelator agents make possible to individualize chelation therapy in clinical practice. The severity of IO and the level of transfusional iron intake, the preferential localization of IO (heart/liver) as well as the tolerance and adherence profiles of the patient can now be taken into account. Introduction of cardiac magnetic resonance imaging for the quantification of myocardial iron and use of oral chelators have already been reported as decreasing the cardiac mortality rate related to IO in thalassaemia major patients. Long-term observation of patients under oral chelators will show if morbidity is also improving via a more continuous control of toxic iron and/or a better accessibility to cellular iron pools.     

Hepcidin induction limits mobilisation of splenic iron in a mouse model of secondary iron overload

Venesection has been proposed as a treatment for hepatic iron overload in a number of chronic liver disorders that are not primarily linked to mutations in iron metabolism genes. Our aim was to analyse the impact of venesection on iron mobilisation in a mouse model of secondary iron overload. C57Bl/6 mice were given oral iron supplementation with or without phlebotomy between day 0 (D0) and D22, and the results were compared to controls without iron overload. We studied serum and tissue iron parameters, mRNA levels of hepcidin1, ferroportin, and transferrin receptor 1, and protein levels of ferroportin in the liver and spleen. On D0, animals with iron overload displayed elevations in iron parameters and hepatic hepcidin1 mRNA. By D22, in the absence of phlebotomies, splenic iron had increased, but transferrin saturation had decreased. This was associated with high hepatic hepcidin1 mRNA, suggesting that iron bioavailability decreased due to splenic iron sequestration through ferroportin protein downregulation. After 22 days with phlebotomy treatments, control mice displayed splenic iron mobilisation that compensated for the iron lost due to phlebotomy. In contrast, phlebotomy treatments in mice with iron overload caused anaemia due to inadequate iron mobilisation. In conclusion, our model of secondary iron overload led to decreased plasma iron associated with an increase in hepcidin expression and subsequent restriction of iron export from the spleen. Our data support the importance of managing hepcidin levels before starting venesection therapy in patients with secondary iron overload that are eligible for phlebotomy.     

Endogenous tachykinins cause bradycardia by stimulating cholinergic neurons in the isolated guinea pig heart

The purpose of this study was to determine if endogenous tachykinins can cause bradycardia in the isolated perfused guinea pig heart through stimulation of cholinergic neurons. Capsaicin was used to stimulate release of tachykinins and calcitonin gene-related peptide (CGRP) from cardiac afferents. A bolus injection of 100 nmol capsaicin increased heart rate by 26 +/- 7% from a baseline of 257 +/- 14 beats/min (n = 6, P < 0.01). This positive chronotropic response was converted to a minor bradycardic effect in hearts with 1 microM CGRP-(8-37) present to block CGRP receptors. The negative chronotropic response to capsaicin was markedly potentiated in another group of hearts with the further addition of 0.5 microM neostigmine to inhibit cholinesterases. In this group, capsaicin decreased heart rate by 30 +/- 10% from a baseline of 214 +/- 6 beats/min (n = 8, P < 0.05). This large bradycardic response to capsaicin was inhibited by 1) infusion of neurokinin A to desensitize tachykinin receptors or 2) treatment with 1 microM atropine to block muscarinic receptors. The latter observations implicate tachykinins and acetylcholine, respectively, as mediators of the bradycardia. These findings support the hypothesis that endogenous tachykinins could mediate axon reflexes to stimulate cholinergic neurons of the intrinsic cardiac ganglia.