hem6: an ENU-induced recessive hypochromic microcytic anemia mutation in the mouse.

Mouse models have proven invaluable for understanding erythropoiesis. Here, we describe an autosomal recessive, inherited anemia in the mouse mutant hem6. Hematologic and transplantation analyses reveal a mild, congenital, hypochromic, microcytic anemia intrinsic to the hematopoietic system that is associated with a decreased red blood cell zinc protoporphyrin to heme ratio, indicative of porphyrin insufficiency. Intercross matings show that hem6 can suppress the porphyric phenotype of mice with erythropoietic protoporphyria (EPP). Furthermore, iron uptake studies in hem6 reticulocytes demonstrate defective incorporation of iron into heme that can be partially corrected by the addition of porphyrin precursors. Gene expression and enzymatic assays indicate that erythroid 5-aminolevulinic acid synthase (Alas2) is decreased in hem6 animals, suggesting a mechanism that could account for the anemia. Overall, these data lead to the hypothesis that hem6 encodes a protein that directly or indirectly regulates the expression of Alas2.

Heme oxygenase induction. A possible factor in aluminum-associated anemia.

The effect of repeated parenteral administration of aluminum (Al) was investigated to determine if a relationship exists between the severity of anemia and increase in hepatic heme oxygenase activity. Female Swiss Webster mice were dosed for 11 d with 50 mg Al/kg, as Al lactate, and sodium lactate was given to control mice. On d 12, hematocrit, hemoglobin, blood smears, hepatic heme oxygenase activity, and cytochrome P450 levels were assessed. Significant decreases in hematocrit (39.1 +/- 0.7 vs 43.1 +/- 0.3% in controls) and hemoglobin (13.1 +/- 0.4 vs 14.2 +/- 0.2 g/dL in controls) were produced by Al administration. Blood smears from Al-treated mice consistently showed smaller, more irregular red cells. Cytochrome P450 content was significantly decreased (0.443 +/- 0.043 vs 0.665 +/- 0.055 nmol/mg) whereas hepatic heme oxygenase activity was significantly increased (2.75 +/- 0.34 vs 1.66 +/- 0.20 nmol/mg/h) in Al-treated animals. The production of mild anemia by parenteral aluminum correlated significantly with the increase in heme oxygenase activity, which, although only 66% greater than in control, preceded a significant loss of cytochrome P450. The increased heme oxygenase activity, with subsequent increased destruction of heme and/or heme proteins is discussed as a possible mechanism for the microcytic, hypochromic anemia associated with Al overload.

Red cell lipid peroxidation and antioxidant enzymes in iron deficiency.

Whether iron deficient RBC in humans have a reduced, or an increased, susceptibility to lipid peroxidation was studied in the iron deficiency states of primary proliferative polycythaemia and iron deficiency anaemia and related to changes in the activities of iron-dependent and non-iron dependent antioxidant enzymes. Susceptibility of RBCs to lipid peroxidation was increased when expressed per g Hb. However, this was a result of the low RBC Hb giving an increased membrane lipid: Hb ratio in the incubations. Results were normal when expressed either per cell, or per ml, RBC. Glutathione reductase was normal. Increased RBC superoxide dismutase activity in iron deficiency may be explained by the younger RBC population and reductions in glutathione peroxidase and catalase activities by the microcytic hypochromic changes and the lack of availability of iron, respectively. There is no evidence of an increased susceptibility of RBC to lipid peroxidation in iron deficiency.

Changes in the cochlear iron enzymes and adenosine triphosphatase in experimental iron deficiency.

The influences of iron deficiency on the cochlear iron enzymes and adenosine triphosphatase were studied in 68 iron-deficient rats and 68 control rats (normal and with chronic anemia). A disorderly or topographic distribution and reduction or disappearance of the cochlear succinic dehydrogenase and peroxidase reaction products were found in 37.8% of the rats fed on a basic iron-deficient diet for 14 to 100 days. The activity of cochlear sodium-potassium-dependent adenosine triphosphatase in iron-deficient rats was slightly increased, compared to that in normal controls. These results suggest that iron deficiency would produce significant abnormalities of succinic dehydrogenase and peroxidase activity, which in turn would disturb cell respiration and initiate peroxidative damage to the inner ear cells, result in sensorineural hearing loss, or provide a pathologic basis for cochlear deafness.

Plasma ferritin concentrations in anemic children: relative importance of malaria, riboflavin deficiency, and other infections.

Anemia (hemoglobin less than 110 g/L) was documented in 36 children of both sexes aged 1-12 y who were divided into two groups: malaria and other infections. The control subjects were 10 children of similar age with no anemia and without any apparent infections. Plasma ferritin concentrations (median, range) were higher in the anemic patients (203 micrograms/L, 21-5000 micrograms/L) than in control children (52 micrograms/L, 25-239 micrograms/L) although ferritin concentrations in those with malaria were still within the normal range (99 micrograms/L, 21-205 micrograms/L). In the rest of the anemic group, five patients had plasma ferritin concentrations greater than 1000 micrograms/L. There was no difference in riboflavin status between control subjects and patients or between the two anemic groups. Severity of anemia was no different between the two anemic groups either. The data indicate that riboflavin deficiency makes no contribution to the infection-induced elevation in plasma ferritin and that the contribution of malaria is smaller than that of other unidentified factors.

[Influence of iron deficiency anemia on development of thymus and spleen and adenosine deaminase activity in rats].

Models of rats with iron deficiency anemia (IDA) were established, and changes in development of the thymus and spleen and adenosine deaminase (ADA) activity in them studied. The size and weight of the thymus in IDA rats were decreased, which suggested that the development of the thymus was lowered; but the size and weight of the spleen in IDA group were increased. ADA activities of the thymus and the spleen in IDA rats were significantly decreased by 32.98% and 25.89%, respectively. One week after iron supplement, the weight of the thymus and spleen in IDS group returned to normal level (P greater than 0.05), and their ADA activity also significantly increased, but ADA activity of the thymus tissue did not return to the normal. The results suggested that the changes in ADA activity of the thymus and spleen tissues were related to iron deficiency. Stepwise regression analysis suggested that there was a negative correlation between ADA activity of the thymus tissue and FEP/Hb ratio.

Phagocyte metabolic functions in iron deficiency anaemia of Indian children.

Nitroblue tetrazolium test (NBT) and bactericidal activity of polymorphonuclear leucocytes was studied in 40 patients with iron deficiency anaemia (aged 0-12 years). NBT test had a significant correlation with serum iron (P less than 0.001) in all cases of iron deficiency anemia. Haemoglobin levels less than 4 g/dl also correlated significantly with NBT score. Bactericidal activity with opsonin-coated Staphylococcus aureus was considerably decreased in severe anaemia, but no significant correlation was found with serum iron concentration (P less than 0.1). The phagocytic and bactericidal activity of polymorphonuclear leucocytes is dependent upon serum iron levels and is considerably reduced in iron deficient state in anaemic children.

Effects of different degrees of iron deficiency on cytochrome P450 complex and pentose phosphate pathway dehydrogenases in the rat.

Four groups of weanling male rats were fed one of three iron-deficient diets (6, 18 and 23 mg iron/kg diet) or a normal iron-containing diet (41 mg iron/kg diet) for 30 d. The effects of the diets on various iron status parameters were determined and four enzymes were assayed: cytochrome P450 (P450) and NADPH cytochrome P450 reductase (P450-RED) in liver and intestine microsomes, and glucose-6-phosphate dehydrogenase (G6P-DH) and 6-phosphogluconate dehydrogenase (6PG-DH) in liver, intestine and erythrocyte cytosol. Rats fed 6 mg iron/kg diet were severely anemic, whereas rats fed 18 or 23 mg iron/kg diet were moderately or mildly iron-deficient, as shown by their hemoglobin levels, hematocrit, red blood cell parameters, erythrocyte protoporphyrin and liver iron stores. P450 concentration and P450-RED activity in liver were unaffected by iron deficiency, but P450 concentration was markedly lower in the intestine of the three iron-deficient groups than in the controls. Activities of G6P-DH and 6PG-DH were not impaired in liver or intestine, except that liver 6PG-DH activity of severely anemic rats was less than that of control rats. However, severe and moderate iron deprivation resulted in a stimulation of G6P-DH and 6PG-DH activities per million erythrocytes. These results demonstrate that even moderate iron deficiency may alter fundamental enzymatic systems intervening in drug metabolism and in the pentose phosphate pathway.

Heme metabolism and in vitro erythropoiesis in anemia associated with hypochromic microcytosis.

Heme metabolism and in vitro erythropoietic growth (CFU-E, BFU-E) were examined in bone marrow cells taken from two siblings with apparent familial hypochromic microcytic anemia. Bone marrow cells from both patients grew adequate numbers of CFU-E and BFU-E colonies in culture in the presence of erythropoietin. In addition, small numbers of endogenous CFU-E were seen in 7-day cultures. Assays on bone marrow cells taken from both patients revealed that baseline delta-aminolevulinic synthase activity was considerably reduced, but increased six to seven fold (to normal levels) when patients' cells were exposed to pyridoxal phosphate (PLP). In both cases, ferrochelatase and delta-aminolevulinic acid dehydratase activities were normal. Bone marrow heme oxygenase showed no significant differences in activities between normals and patients values in the absence or presence of PLP. In contrast, heme synthesis by patients' bone marrow was less than that of normals. This study demonstrates that bone marrow cells from patients with this rare disorder have some disturbances in heme metabolism, whereas erythropoiesis appeared to be normal when cultured with adequate nutrients in vitro.

Induction of an increase in mitochondrial matrix enzymes in muscle of iron-deficient rats.

Young rats maintained on an iron-deficient diet developed severe anemia and had large decreases in the levels of the iron-containing flavoproteins and cytochromes of the mitochondrial respiratory chain in skeletal muscle. In contrast, the levels of a number of mitochondrial matrix marker enzymes, including citrate synthase, isocitrate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacid-CoA transferase, and aspartate aminotransferase, increased in red skeletal muscle but not in white muscle. Phosphocreatine concentration was decreased and inorganic phosphate concentration was increased in soleus muscle frozen in situ. We hypothesize that the increase in mitochondrial matrix enzymes reflects a stimulus to mitochondrial biogenesis in posture-maintaining and weight-bearing red muscle fibers in severely iron-deficient rats. It is our working hypothesis that this stimulus to mitochondrial biogenesis arises from mild activity of the red fibers and is due to the same perturbation in cellular homeostasis that is normally caused by vigorous exercise or hypoxia. In iron deficiency, the stimulus to mitochondrial biogenesis can induce an increase in only those enzymes not prevented from increasing by iron deficiency, resulting in formation of mitochondria of grossly abnormal composition.

Red-cell metabolism in patients with iron deficiency.

18 components of metabolism were determined in the red cells of iron-deficient patients and data were expressed per 10(12) red cells to avoid the complicating effects of hypochromia and microcytosis. Glucose consumption, AMP and ATP, glycolytic intermediates except 2,3-bisphosphoglycerate (2,3-DPG) and phosphoenolpyruvate (PEP), red-cell Na+ and the net passive leakage of Na+ and K+ at 4 degrees C were all normal. Creatine, 6-phospho-D-gluconate: NADP oxidoreductase (6PGD) activity and fresh red-cell K+ were raised, suggestive of a young cell population. However, ATP: D-fructose-6-phosphate 1-phosphotransferase (PFK) activity and ADP were low. An elevated 2,3-DPG level was attributable to the anaemia present but the somewhat raised PEP level is unexplained. It is concluded that red cells in iron deficiency show some characteristics of a young cell population; in other respects they appear normal, but in containing a low PFK activity they are abnormal.

Erythrocyte enzymes in polycythemia vera: a comparison to erythrocyte enzyme activities of patients with iron deficiency anemia.

Fifteen cytoplasmic erythrocyte enzyme activities were determined in patients with polycythemia vera (PV), iron deficiency anemia (IDA), and a group of healthy volunteers. Among the PV patients, the erythrocyte enzyme activities were compared between 2 groups: patients treated solely with phlebotomy and patients treated with phlebotomy, Myleran (busulfan) and/or radioactive phosphorus 32P. Significant reduction in glutathione reductase activity was found in the PV group of patients. This activity was normalized by the addition of flavin adenine dinucleotide. In contrast to previous reports, no other enzyme activity was found to be significantly reduced. The activities of the enzymes known to be age-dependent were significantly elevated in all the groups tested except for phosphofructokinase and 3-phosphoglycerate mutase. The former was not elevated in any of the groups studied, while the latter was elevated only in the group of patients treated with Myleran and/or 32P. It was concluded that glutathione reductase (GR) deficiency is the only acquired enzyme deficiency in our group of PV patients and that radiation and chemotherapy did not induce further reduction in the activities of any of the enzymes tested. The possible involvement of GR deficiency in the etiology of the red cell life span shortening was discussed.

Neutrophil glutathione peroxidase activity in iron deficiency anaemia.

Neutrophil glutathione peroxidase (GSH-Px) activity was studied in 30 children with nutritional iron deficiency anaemia and 25 healthy age-matched controls. Initially GSH-Px in iron deficient anaemic children was significantly (p less than 0.001) low compared to the control group. All patients were subjected to iron therapy but only 8 returned for follow-up. The GSH-Px activity in all 8 cases rose to normal levels after therapy. These results suggest a relationship between iron and GSH-Px activity.

Effects of oral contraceptive steroids (norethisterone/mestranol) on the activities of hepatic drug-metabolizing enzymes in iron-deficient anemic rats.

Either oral contraceptive steroid (norethisterone/mestranol; N/M) treatment or iron-deficiency (Fe(-] anemia alone caused an increase in NADPH cytochrome c reductase and in three hepatic microsomal mixed-function oxidase activities in female rats. When N/M treatment and the Fe(-) diet are combined, no further change in hepatic enzyme activity is seen compared with that with either treatment alone.