Investigating the role of transferrin in the distribution of iron, manganese, copper, and zinc

Abstract

The essential role of transferrin in mammalian iron metabolism is firmly established. Integral to our understanding of transferrin, studies in hypotransferrinemic mice, a model of inherited transferrin deficiency, have demonstrated that transferrin is essential for iron delivery for erythropoiesis and in the regulation of expression of hepcidin, a hormone that inhibits macrophage and enterocyte iron efflux. Here we investigate a potential role for transferrin in the distribution of three other physiologic metals, manganese, copper, and zinc. We first assessed metal content in transferrin-rich fractions of wild-type mouse sera and demonstrate that although both iron and manganese cofractionated predominantly with transferrin, the absolute levels of manganese are several orders of magnitude lower than those of iron. We next measured metal content in multiple tissues in wild-type and hypotransferrinemic mice of various ages. Tissue metal imbalances were severe for iron and minimal to moderate for some metals in some tissues in hypotransferrinemic mice. Metal levels measured in a transferrin-replete yet hepcidin-deficient and iron-loaded mouse strain suggested that the observed imbalances in tissue copper, zinc, and manganese levels were not all specific to hypotransferrinemic mice or caused directly by transferrin deficiency. Overall, our results suggest that transferrin does not have a primary role in the distribution of manganese, copper, or zinc to tissues and that the abnormalities observed in tissue manganese levels are not attributable to a direct role for transferrin in manganese metabolism but rather are attributable to an indirect effect of transferrin deficiency on hepcidin expression and/or iron metabolism.

Figure 1. Metal content in transferrin-rich fractions of mouse sera

Wild-type (wt) and hypotransferrinemic (hpx) sera were pooled from five to ten two- to four-month-old mice. Sera were applied to albumin affinity (Blue), immunoglobulin affinity(A/G) and anion exchange (Q) columns in series then eluted from individual columns. Proteins were eluted from the Q column with 100 mM NaCl (Q100) then 1000 mM NaCl (Q1000). Column flow through (FT) and all eluates were washed and concentrated to original serum volume. (a) Fractions (left two panels) and larger volumes of select fractions (right panel) were electrophoresed under denaturing, reducing conditions on polyacrylamide gels then stained to visualize proteins. * indicates transferrin band. (b) Fractions were immunoblotted with a transferrin (Tf)-specific antibody. Upper and lower images correspond to short and long exposures respectively. Iron (c) and manganese (d) levels in wt and hpx sera and fractions were measured by GF-AAS in triplicate. Serum* indicates sera diluted then concentrated without fractionation. ‘n.d.’ indicates value was below the lower limit of detection.

Figure 2. Tissue iron levels in hpx mice

Iron levels were measured in nitric acid-digested liver (a), spleen (b), kidney (c), lung (d), heart (e) and pancreas (f) from wt (filled circles) and hpx (open circles) mice by ICP-AES and GF-AAS and expressed as μg metal per g wet tissue relative to mouse age in days.* indicates statistically significant difference between wt and hpx values at same age. ↑ and ↓ indicate increase or decrease respectively in level at indicated age vs. one time point earlier for mouse of same genotype. Statistical significance determined by P<0.05 (Student’s t-test, two-tailed, unequal variance). Error bars indicate standard deviation. Each point represents 5–12 mice. All values were measured in triplicate.

Figure 3. Tissue manganese levels in hpx mice

Manganese levels in liver (a), spleen (b), kidney (c), lung (d), heart (e) and pancreas (f) were measured and plotted as described in Figure 2.

Figure 4. Iron and manganese levels in hpx muscle and bone

Iron (a) and manganese (b) levels were measured by GF-AAS in quadriceps muscle and femurs from five four-month-old wt and hpx mice. * indicates statistically significant difference between wt and hpx mice. Statistical significance determined by P<0.05 (Student’s t-test, two-tailed, unequal variance). Error bars indicate standard deviation.

Figure 5. Liver metal levels in Hjv mice

Liver iron (a) and manganese (b) levels were measured by ICP-AES in five to six two-month-old wild-type (wt), transferrin-deficient (hpx), Hjv-deficient (Hjv) and transferrin-/Hjv-deficient (hpx Hjv) mice. Levels were expressed as μg metal per g wet tissue. * indicates statistically significant difference between bracketed groups. Statistical significance determined by P<0.05 (Student’s t-test, two-tailed, unequal variance). Error bars indicate standard deviation.