The Effect of Dietary Iron on Tissue Iron Levels in Intact and Splenectomized Mice Affected by β-Thalassemia.
Authors: Marongiu Maria Franca , Muirhead Kristen , Gardenghi Sara , Guy Ella , Rivella Stefano , Grady Robert W 
Published: April 2020

Abstract
Patients with β-thalassemia hyper absorb dietary iron, most of which is stored in the liver. They also suffer from ineffective erythropoiesis (IE) which leads to hepatosplenomegaly, often requiring a splenectomy. We have been conducting a series of studies utilizing the th3/+ mouse model of thalassemia intermedia to investigate the absorption, distribution and erythroid utilization of iron. Here we focus on changes in the iron content of liver and spleen resulting from diets containing low (2.5 ppm), sufficient (35 ppm) and high (200 ppm) levels of iron, and assess the impact of splenectomy on its distribution. The high iron diet was standard rodent chow while the others were defined diets. Th3/+ mice were either bred or generated by transplantation of th3/+ hematopoietic stem cells from E14.5 fetal livers into lethally irradiated wild type (+/+) recipients. Wild type controls were similarly obtained. Splenectomy of bred and recipient mice was performed at 5 weeks of age and bone marrow transplantation (BMT) at 8 weeks. Non-transplanted mice were placed on the test diets at 8 weeks of age, and transplanted mice at 11 weeks. All animals were sacrificed after 4 weeks on the test diets, and livers and spleens harvested for determination of their iron content by atomic absorption. Group sizes ranged from 3 to 10 mice (median 7). In general, the mean organ iron content of mice fed the high iron diet was not significantly different from that of the animals fed the iron sufficient diet, while those fed the low iron diet had reduced levels of tissue iron. Over the course of the 4-week feeding study, the iron content of the livers and spleens of +/+ mice fed the 35-ppm diet increased 39% and 202%, respectively, while the corresponding values of those fed the 2.5-ppm diet were −21% and 30%. The changes in the liver and spleen of th3/+ mice were 79% and 32% (35-ppm diet) and 14% and 12% (2.5-ppm diet) compared to the values at baseline. The latter values, those at 8 weeks of age, were 1.8- and 30-fold higher in the th3/+ mice, the massive accumulation of iron in the spleen undoubtedly resulting from IE. Where iron intake (liver plus spleen) was low, it went preferentially to the spleen, undoubtedly to sustain erythropoiesis. Groups of splenectomized +/+ mice were also fed the three diets for 4 weeks. The mean iron content of their livers was similar to that of non-splenectomized animals. Similar studies of th3/+ mice are now in progress. A second set of studies is being conducted in transplanted +/+ and th3/+ mice, the goal being to determine whether or not the absorption and distribution of iron is the same as in bred animals. Again, the organ iron content of those mice fed the high iron diet was similar to that of the animals fed the iron sufficient diet. In the case of the transplanted +/+ animals fed iron sufficient diets, the mean iron contents of the livers and spleens were 64% and 186% increased after 4 weeks of feeding, values not markedly different from those of bred animals. The corresponding values on the 2.5-ppm diet were 27% and 72%, again the pattern being similar. The transplanted th3/+ animals accumulated significantly less iron in these organs than those that were bred. However, the rate at which they accumulated this iron was 10 to 20 times higher than that of the other groups studied, including the transplanted +/+ mice, perhaps reflecting a synergistic effect of BMT and IE on iron absorption. Mice fed the 35-ppm diet had only 75% and 46% as much iron in their livers and spleens, the animals fed the 2.5-ppm diet having even less (35% and 23%) while again showing preferential diversion of iron to the spleen. Splenectomizing the animals resulted in further increasing the liver iron, more that 2.5-fold in those fed the low iron diet. The hemoglobin levels of all the mice evaluated were unchanged as a result of the dietary studies, except for a 20% decrease seen in bred +/+ mice fed the low iron diet. We are currently studying splenectomized transplanted th3/+ mice as well as doing feeding studies of 5-months duration. In summary, a low iron diet has a marked effect on the iron levels of liver and spleen, which are accentuated under conditions of IE. Secondly, more iron is absorbed under conditions of IE than is needed for erythropoiesis, the excess being shuttled to the liver for storage.