Heme metabolism and erythropoiesis in abnormal iron states: role of delta-aminolevulinic acid synthase and heme oxygenase

Heme metabolism was examined in bone marrow and hepatic cells from iron-deficient and chronically iron-overloaded rats. Results indicate that the rate limiting enzymes delta-aminolevulinic acid synthase (ALAS) and heme oxygenase were significantly elevated in the iron-overloaded hepatic and bone marrow cells and near normal levels in cells from iron-deficient rats. Conversely, delta-aminolevulinic acid dehydrase (ALAD) was depressed in iron-overloaded cells and elevated in iron-deficient cells. Erythroid colony (CFU-E) cultures demonstrated that iron-overloaded bone marrow cells were poor hemin and CFU-E responders in vitro, whereas iron-deficient marrows grew exuberant numbers of CFU-E and responded to hemin. Succinylacetone (1 mM) inhibited ALAD activity and normal CFU-E growth, and CFU-E inhibition by succinylacetone was completely overcome by the addition of hemin. Results are discussed with respect to the regulation of hepatic and bone marrow heme metabolism in abnormal iron states and the possible role of iron in the induction of heme oxygenase in chronic iron overload.     

Mechanism of iron potentiation of hepatic uroporphyria: studies in cultured chick embryo liver cells

Effects of iron were studied in cultured chick embryo liver cells to help elucidate the effect of hepatic iron in the human disease porphyria cutanea tarda and in toxic porphyria caused by chemicals. These cultures have proven useful because (a) phenobarbital and phenobarbital-like drugs induce a common form(s) of cytochrome P-450 (P-450-phenobarbital) in these cultures; (b) 20-methylcholanthrene and certain other polycyclic hydrocarbons induce a different form(s) (P-450-methylchol-anthrene), and (c) uroporphyria can be produced rapidly by exposure to suitable chemicals. In these cultures, treatment with iron alone did not produce porphyrin accumulation, and treatment with iron + 5-aminolevulinate caused accumulation of protoporphyrin, as did treatment with 5-aminolevulinate alone. However, treatment with phenobarbital-like drugs and iron, the latter at a concentration as low as 0.2 microM, led to accumulation of uro- and heptacarboxylporphyrins. Potentiation of uroporphyrin accumulation by iron began before there was a detectable synergistic increase in activity of 5-aminolevulinate synthase, the rate-controlling enzyme of heme synthesis. In contrast, treatment of cultures with 20-methylcholanthrene, in the presence or absence of iron, did not result in uroporphyrin accumulation or an increase in the activity of 5-aminolevulinate synthase. Uroporphyrinogen decarboxylase activity was unchanged by drug and iron treatments. Inhibitors of P-450-phenobarbital, SKF525A and piperonyl butoxide, as well as cadmium and cycloheximide prevented the porphyrin accumulation produced by glutethimide + iron, even though, except with cycloheximide, these substances further increased 5-aminolevulinate synthase activity. In vitro, uroporphyrin was oxidized autocatalytically by iron. In intact hepatocytes, even low concentrations of iron (0.2 to 20 microM), in the presence of a form of cytochrome P-450 induced by phenobarbital-like chemicals, produces uroporphyria primarily by enhancing uroporphyrinogen oxidation, not by inhibition of the decarboxylase. Induction of 5-aminolevulinate synthase amplifies the porphyrin overproduction.     

Intravenous Heme-Albumin in Acute Intermittent Porphyria: Evidence for Repletion of Hepatic Hemoproteins and Regulatory Heme Pools

The purpose of this study was to assess effects of heme administered intravenously, complexed to human serum albumin, on activities of the hepatic hemoproteins, cytochrome(s) P-450, and tryptophan pyrrolase, and on the size of the heme pool that regulates activity of 5-aminolevulinate synthase. Effects were compared in six normal women and four women with acute intermittent porphyria. All porphyric subjects over-excreted heme precursors and had histories of acute neurovisceral porphyric attacks. All subjects were placed on a constant daily diet that included at least 3 g carbohydrate/kg body weight and sufficient total intake to provide 1.4 times the estimated resting energy expenditure. Urinary excretions of 5-aminolevulinate, porphobilinogen, porphyrins, and metabolites of tryptophan were measured daily before, during, and after infusions of heme-albumin. In the porphyric subjects, intravenous heme [4 mg (6.1 mumol)/kg body weight (BWt) with equimolar albumin], given daily for 4 days, markedly reduced overexcretion of 5-aminolevulinate, porphobilinogen, and porphyrins, indicating repletion of the regulatory heme pool. The heme infusions also decreased mean urinary excretion of 5-hydroxyindoleacetic acid from 4.9 to 2.9 mg/g creatinine per day, suggesting increased activity of hepatic tryptophan pyrrolase, the rate-controlling enzyme for metabolism of tryptophan to products not in the serotonin-5-hydroxyindoleacetic acid pathway. Heme-albumin infusions were without detectable effects on excretions of heme precursors or tryptophan metabolites in normal subjects. In contrast, in both normals and porphyrics, heme-albumin infusions significantly increased rates of antipyrine metabolism (by 159% and 330%, respectively), suggesting increased activities of cytochrome(s) P-450 were produced by the infusions. The infusions were well tolerated; no subject developed thrombophlebitis or bleeding. We conclude that such infusions are safe and effective in repleting deficient heme pools and hemoproteins in patients with acute porphyria, and that activities of cytochrome(s) P-450 in normal subjects may also be increased by heme administration. The therapeutic effect of heme in acute porphyria probably relates to its ability to decrease overproduction of precursors of heme or serotonin, as the result of its increasing critical cellular heme pools.     

Erythroid colony development as a function of age: the role of marrow cellular heme

The activities of the heme biosynthetic enzymes ALA synthase (ALAS) and ALA dehydrase (ALAD) and the heme degradative enzyme heme oxygenase were analyzed from bone marrow cells obtained from young, middle-aged, and senescent rats. There was age-related reduced activity of bone marrow ALAS but no age-related difference in the activity of ALAD. In contrast, heme oxygenase activity was 50% greater in the senescent marrow cells. Incorporation of 14C-glycine into heme was 45% less in senescent rat marrow cells, whereas incorporation of 14C-delta-aminolevulinic acid was not related to age. Senescent bone marrow cells demonstrated a marked reduction in 14C-leucine and 3H-uridine incorporated into protein and nucleic acid synthesis, respectively. In vitro erythroid colony (CFUE) growth by senescent bone marrow cells was as much as 40% less compared with young bone marrow cells. The decreased ability to form CFUE by the senescent bone marrow cells may be related to reduced ALAS activity and increased heme oxygenase activity. Thus, part of the aging process appears to involve fluctuations in the enzyme activities and protein synthesis involved with metabolism of heme.     

ALAS2 acts as a modifier gene in patients with congenital erythropoietic porphyria

Mutations in the uroporphyrinogen III synthase (UROS) gene cause congenital erythropoietic porphyria (CEP), an autosomal-recessive inborn error of erythroid heme biosynthesis. Clinical features of CEP include dermatologic and hematologic abnormalities of variable severity. The discovery of a new type of erythroid porphyria, X-linked dominant protoporphyria (XLDPP), which results from increased activity of 5-aminolevulinate synthase 2 (ALAS2), the rate-controlling enzyme of erythroid heme synthesis, led us to hypothesize that the CEP phenotype may be modulated by sequence variations in the ALAS2 gene. We genotyped ALAS2 in 4 unrelated CEP patients exhibiting the same C73R/P248Q UROS genotype. The most severe of the CEP patients, a young girl, proved to be heterozygous for a novel ALAS2 mutation: c.1757 A > T in exon 11. This mutation is predicted to affect the highly conserved and penultimate C-terminal amino acid of ALAS2 (Y586). The rate of 5-aminolevulinate release from Y586F was significantly increased over that of wild-type ALAS2. The contribution of the ALAS2 gain-of-function mutation to the CEP phenotype underscores the importance of modifier genes underlying CEP. We propose that ALAS2 gene mutations should be considered not only as causative of X-linked sideroblastic anemia (XLSA) and XLDPP but may also modulate gene function in other erythropoietic disorders.     

Isolation of a rat liver delta-aminolevulinate dehydrase (ALAD) cDNA clone: evidence for unequal ALAD gene dosage among inbred mouse strains.

We have isolated several cDNA clones encoding delta-aminolevulinate dehydrase [ALAD; porphobilinogen synthase; 5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24], the second enzyme in the heme biosynthetic pathway. We used a rabbit polyclonal antibody developed against the purified 35-kDa subunit of rat liver ALAD to screen a lambda gt11 cDNA expression library constructed from rat liver mRNA. A prototype clone (ALAD-1) contained a 680-base-pair insert and expressed a 140-kDa beta-galactosidase gene cDNA insert fusion protein immunoreactive with both polyclonal and monoclonal anti-ALAD. Identity of ALAD-1 was verified by hydridization to ALAD mRNA that had been enriched via immunopurification of rat liver polysomes with anti-ALAD. Intensity of such hybridization to a 1500-nucleotide-long mRNA was approximately equal to 200-fold greater than that realized with whole liver mRNA, a result consistent with the degree of immunoenrichment of ALAD mRNA found independently by analysis of cell-free translation products. A second ALAD cDNA clone (ALAD-3) was isolated when the rat liver cDNA expression library was rescreened with ALAD-1. The identity of both ALAD cDNA clones was established by correspondence between their nucleotide sequence and the reported amino-terminal protein sequence of bovine ALAD. Hybridization of ALAD cDNA to mouse genomic DNA indicates that the previously unexplained incremental differences in ALAD enzymatic activity among inbred mouse strains has arisen through alterations in ALAD gene dose.     

Elucidation of the mechanism of mitochondrial iron loading in Friedreich's ataxia by analysis of a mouse mutant

We used the muscle creatine kinase (MCK) conditional frataxin knockout mouse to elucidate how frataxin deficiency alters iron metabolism. This is of significance because frataxin deficiency leads to Friedreich''s ataxia, a disease marked by neurologic and cardiologic degeneration. Using cardiac tissues, we demonstrate that frataxin deficiency leads to down-regulation of key molecules involved in 3 mitochondrial utilization pathways: iron-sulfur cluster (ISC) synthesis (iron-sulfur cluster scaffold protein1/2 and the cysteine desulferase Nfs1), mitochondrial iron storage (mitochondrial ferritin), and heme synthesis (5-aminolevulinate dehydratase, coproporphyrinogen oxidase, hydroxymethylbilane synthase, uroporphyrinogen III synthase, and ferrochelatase). This marked decrease in mitochondrial iron utilization and resultant reduced release of heme and ISC from the mitochondrion could contribute to the excessive mitochondrial iron observed. This effect is compounded by increased iron availability for mitochondrial uptake through (i) transferrin receptor1 up-regulation, increasing iron uptake from transferrin; (ii) decreased ferroportin1 expression, limiting iron export; (iii) increased expression of the heme catabolism enzyme heme oxygenase1 and down-regulation of ferritin-H and -L, both likely leading to increased “free iron” for mitochondrial uptake; and (iv) increased expression of the mammalian exocyst protein Sec15l1 and the mitochondrial iron importer mitoferrin-2 (Mfrn2), which facilitate cellular iron uptake and mitochondrial iron influx, respectively. Our results enable the construction of a model explaining the cytosolic iron deficiency and mitochondrial iron loading in the absence of frataxin, which is important for understanding the pathogenesis of Friedreich''s ataxia.     

Selenium regulation of hepatic heme metabolism: induction of delta-aminolevulinate synthase and heme oxygenase.

Selenium was found to be a novel regulator of cellular heme methabolism in that the element induced both the mitochondrial enzyme delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating); EC 2-3-1-37] and the microsomal enzyme heme oxygenase [heme, hydrogen-donor:oxygen oxidoreductase(alpha-methene-oxidizing, hydroxylating); EC 1-14-99-3] in liver. The effect of selenium on these enzyme activities was prompt, reaching a maximum within 2 hr after a single injection. Other changes in parameters of hepatic heme metabolism occurred after administration of the element. Thirty minutes after injection the cellular content of heme was significantly increased; however, this value slightly decreased below control values within 2 hr, coinciding with the period of rapid induction of heme oxygenase. At later peroids heme content returned to normal values. Selenium treatment caused only a slight decrease in microsomal cytochrome P-450 content. However, drug-metabolizing activity was severely inhibited by higher doses of the element. Unlike other inducers of delta-aminolevulinate synthase, which as a rule are also porphyrinogenic agents, selenium induction of this enzyme was not accompanied by an increase in the cellular content of prophyrins. When rats were pretreated with selenium 90 min before administration of heme, a potent inhibitor of delta-aminolevulinate synthase production, the inhibitory effect of heme of formation of this mitochondrial enzyme was completely blocked. Selenium, at high concentrations in vitro, was inhibitory to delta-aminolevulinate synthase activity. It is postulated that selenium may not be a direct inducer of heme oxygenase as is the case with trace metals such as cobalt, but may mediate an increase in heme oxygenase through increased production and cellular availability of "free" heme, which results from the increased heme synthetic activity of hematocytes. Subsequently, the increased heme oxygenase activity is in turn responsible for the lack of increase in the microsomal heme content, thus maintaining heme levels at normal values despite the highly increased activities of both heme oxygenase and delta-aminolevulinate synthase. It is further suggested that the increase in delta-aminolevulinate synthase activity is not due to a decreased rate of enzyme degradation or an activation of preformed enzyme, but to increased rate of synthesis of enzyme protein. Although selenium in trace amounts has been postulated to be involved in microsomal electron transfer process, the data from this study indicate that excess selenium can substantially inhibit microsomal drug metabolism.     

Differential effects of partial hepatectomy on hepatic and renal heme and cytochrome P450 metabolism

Partial hepatectomy has been suggested to affect hepatic and renal cytochrome P450 content and the related drug metabolizing enzyme system. In addition, cytochrome P450 and its dependent activities have been shown to be regulated by the availability of cellular heme. We, therefore, studied cytochrome P450 in addition to the level of heme oxygenase, the rate-limiting enzyme of heme catabolism, and delta-aminolevulinic acid (ALA) synthase, the rate-limiting enzyme of heme synthesis, in the remnant liver and intact kidneys of rats after two-thirds hepatectomy. The level of hepatic heme oxygenase was elevated threefold in partially hepatectomized rats as compared to sham-operated rats, while ALA synthase was decreased by 40%. This was reflected in decreased hepatic cytochrome P450 content, ie, from 0.689 +/- 0.175 nmole/mg to 0.505 +/- 0.089 nmole/mg protein and associated decreased drug metabolizing enzymes: aryl hydrocarbon hydroxylase, 7-ethoxycoumarin-O-deethylase, and benzphetamine N-demethylase, by 40%, 40%, and 47%, respectively. In contrast, renal heme oxygenase was not changed after hepatectomy, whereas renal ALA synthase was increased by fourfold. Renal cytochrome P450, aryl hydrocarbon hydroxylase, 7-ethoxycoumarin-O-deethylase, and benzphetamine N-demethylase were increased after partial hepatectomy by 84%, 360%, 165% and 406%, respectively. These data indicate that partial hepatectomy decreases liver cytochrome P450 levels by inducing heme oxygenase and inhibiting ALA synthase activities. In this situation the kidney plays a substitutive role in metabolizing endogenous substrates oxygenated by cytochrome P450 isozymes.     

Hepatic microsomal function in rats with chronic dietary iron overload

We determined whether alterations in hepatic microsomal function occur in association with iron-induced lipid peroxidation in vivo in rats with chronic dietary iron overload. In rats fed a 2.0% carbonyl iron diet for a period of 20 wk, there was no significant microsomal conjugated diene formation (evidence of microsomal lipid peroxidation) or difference in cytochrome P450 concentration found at mean (+/- SEM) hepatic iron concentrations of 1210 +/- 92 micrograms/g liver (wet wt) or 2730 +/- 100 micrograms/g. At a hepatic iron concentration of 4090 +/- 245 micrograms/g, however, there was significant conjugated diene formation (p less than 0.001) and a 56% decrease in the cytochrome P450 concentration (p less than 0.001). In rats fed a 2.5% carbonyl iron diet for 10 wk, achieving a liver iron concentration of 4820 +/- 420 micrograms/g, there was significant microsomal conjugated diene formation (p less than 0.001), a 35% reduction in cytochrome P450 (p less than 0.005), and a 16% reduction in aminopyrine demethylase activity (p less than 0.025), but only an 8% reduction in glucose-6-phosphatase activity (p = not significant). Finally, in rats fed a 3.0% iron-supplemented diet for 7 wk, achieving a liver iron concentration of 2730 +/- 205 micrograms/g, there was a 23% reduction in cytochrome P450 (p less than 0.025), a 28% reduction in cytochrome b5 (p less than 0.001), and a 47% increase in heme oxygenase activity (p less than 0.025) (heme oxygenase activity measured in this group only). We conclude that oral iron loading can produce microsomal lipid peroxidation in vivo that is associated with selective decreases in microsomal hemoprotein concentrations and cytochrome P450-dependent enzymes.     

Effect of age on rat liver heme and drug metabolism

Old (24-months) rats have lower activities of hepatic delta-aminolevulinic synthase and the microsomal cytochrome P-450 monooxygenase activities--aminopyrine N-demethylase and aniline hydroxylase--as compared to young (2-months) animals. In contrast, the activity of the heme degradative enzyme, heme oxygenase, is higher in the old rats. Cytochrome P-450 and microsomal heme contents were maintained in the old. When inducibility and inhibition of these enzymes were studied, the old rats responded to the same degree as the young. These results indicate that the ability of the heme synthetic and degradative enzymes to respond to decreasing cellular heme levels is not significantly altered with age. The observations that there is a lower baseline activity of ALA-synthase and good maintenance of microsomal heme and cytochrome P-450 content, in spite of elevated heme oxygenase activity in the old, suggest that, at least in the senescent rat, hepatic heme utilization and degradation are only loosely coupled to heme production. It appears, therefore, that alternate sources of heme for cytochrome P-450 are available in the old animals. Furthermore, it is suggested that the old rat has a baseline change in ALA-synthase, heme oxygenase, and cytochrome P-450 that may be overcome under the appropriate conditions.     

Uroporphyria caused by ethanol in Hfe(-/-) mice as a model for porphyria cutanea tarda

Two major risk factors for the development of porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). To develop an animal model, Hfe knockout mice were treated continuously with 10% ethanol in drinking water. By 4 months, uroporphyrin (URO) was detected in the urine. At 6 to 7 months, hepatic URO was increased and hepatic uroporphyrinogen decarboxylase (UROD) activity was decreased. Untreated Hfe(-/-) mice or wild-type mice treated with or without ethanol did not show any of these biochemical changes. Treatment with ethanol increased hepatic nonheme iron and hepatic 5-aminolevulinate synthase activity in Hfe(-/-) but not wild-type mice. The increases in nonheme iron in Hfe(-/-) mice were associated with diffuse increases in iron staining of parenchymal cells but without evidence of significant liver injury. In conclusion, the results of this study suggest that the uroporphyrinogenic effect of ethanol is mediated by its effects on hepatic iron metabolism. Ethanol-treated Hfe(-/-) mice seem to be an excellent model for studies of alcohol-mediated PCT.     

Porphyrin Metabolism in Experimental Hepatic Siderosis in the Rat

S ummary . The combination of iron overload and hexachlorobenzene (HCB) feeding in rats produces an 'experimental symptomatic porphyria'. Studies of hepatic haem biosynthesis in this model showed a decrease in total liver haem and cytochromes P450 and b₅, together with decreased incorporation of ¹⁴C-succinate into liver haem and an absence of uroporphyrinogen decarboxylase activity. A marked alteration was found in the redox state of the liver cell cytoplasm both in siderotic and non-siderotic animals after HCB feeding; the ratio NAD: NADH was increased more than two-fold. These findings suggest that the primary effect of HCB with regard to liver haem biosynthesis may be this change in the redox state, possibly leading to decreased ability to maintain porphyrinogens in the reduced form and that the additional effect of iron overload is mediated by impairment of uroporphyrinogen decarboxylase activity and an increased rate of degradation of microsomal drug detoxifying cytochromes. It is probable that in human symptomatic porphyria a number of factors are similarly responsible for the excessive production and excretion of porphyrins.     

Hepatic mitochondrial oxidative metabolism in rats with chronic dietary iron overload

We examined the effects of chronic dietary iron overload on hepatic mitochondrial oxidative metabolism. Experimental iron overload was produced by feeding rats a chow diet supplemented with carbonyl iron over a 7-week period. Biochemical and histologic evaluations of liver tissue confirmed moderate degrees of hepatic parenchymal iron overload. Electron microscopy showed no abnormalities in hepatic mitochondrial ultrastructure in blocks of tissue or in mitochondrial fractions from iron-loaded liver. Studies of mitochondrial oxidative metabolism revealed a consistent and progressive decrease in state 3 (ADP-stimulated) respiration and in respiratory control ratios at hepatic iron concentrations above 1,000 micrograms per gm for all three substrates studied, glutamate, beta-hydroxybutyrate and succinate. Changes in state 4 (ADP-limited) respiration and ADP/O ratios were not progressive with increasing hepatic iron concentrations. At hepatic iron concentrations at which there were decreases in state 3 respiration and respiratory control ratios, there was also evidence of lipid-conjugated diene formation, indicative of mitochondrial lipid peroxidation. There were no changes in mitochondrial function when iron as either ferritin or hemosiderin or as a combination of ferritin, hemosiderin and ferric nitrilotriacetate was added in vitro to normal liver homogenates. Use of density gradient centrifugation to reduce iron and lysosomal contamination of mitochondrial fractions failed to prevent the reduction in mitochondrial function. We conclude that moderate degrees of chronic hepatic iron overload in vivo result in an inhibitory defect in the mitochondrial electron transport chain as evidenced by a decrease in state 3 respiration and respiratory control ratios.     

African iron overload

Iron overload is common in rural sub-Saharan African populations that have the custom of drinking a traditional fermented beverage with high iron content. As with both excessive alcohol exposure and HFE hemochromatosis, hepatic portal fibrosis and micronodular cirrhosis are prominent sequelae of African iron overload. Two observations are therefore important in characterizing iron overload in Africa. First, the hepatic iron concentrations associated with African iron overload often far exceed those seen in alcoholic liver disease and histologic changes of alcohol effect are almost always absent. Second, the pattern of iron accumulation in African dietary iron is prominent in both macrophages and hepatic parenchymal cells; this pattern is in contrast to HFE homochromatosis, which is marked by predominantly parenchymal iron-loading. For a long time, it was thought that African iron overload was purely dietary in nature, that increased iron and alcohol in the diet could fully explain markedly elevated tissue iron levels sometimes seen with this condition. Recent studies of pedigrees suggest that, in addition to high dietary iron content, a genetic defect may also be implicated in iron overload in Africans. These studies indicate that the possible defect is different from mutations in the HFE gene frequently found in Caucasians with iron overload, but the putative gene has not been identified. Recent studies also indicate that non-HFE iron overload occurs in African-Americans, but the prevalence and possible genetic basis is yet to be determined.     

Heme biosynthesis in Friend erythroleukemia cells: control by ferrochelatase.

The activities of the enzymes of heme biosynthesis (except protoporphyrin oxidase) have been followed during the induction of Friend cells in culture. All the enzyme activities increased after induction with dimethyl sulfoxide. The activities of the intermediate enzymes were much higher than those of delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37], the initial enzyme, or ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), the final enzyme of the pathway. Ferrochelatase activity was not detectable in the uninduced cell. delta-Aminolevulinate synthase activity increased during the first 24 hr of induction; porphobilinogen deaminase activity began to increase after 48 hr and ferrochelatase activity, after 72 hr. However, the induction of heme synthesis followed the same time course as that of ferrochelatase activity, not that of delta-aminolevulinate synthase activity. The cellular growth medium was found to contain traces of protoporphyrins. Thus, ferrochelatase is shown to be rate limiting for heme synthesis during early stages of Friend cell induction. A Friend cell variant (Fw), which is not inducible except in the presence of exogenous hemin, was also studied. All the enzymes of heme synthesis except ferrochelatase were inducible by butyric acid. Ferrochelatase was not inducible by butyric acid or hemin plus butyric acid. These cells also excrete protoporphyrin, The failure to induce ferrochelatase activity is believed to be the cause of, not a consequence of, the noninducibility of this cell line.     

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 α-thalassaemia had moderately raised reticulocyte counts, but were not anaemic and showed little, if any, iron loading. In contrast, mice with homozygous β-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 hypotransferrinaemic 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.