Reticulocyte Enrichment of Zinc Protoporphyrin/Heme Discriminates Impaired Iron Supply During Early Development

Abstract

ABSTRACT:In infants and children, elevated whole blood zinc protoporphyrin/heme (ZnPP/H) measures iron-deficient (ID) erythropoiesis. Because immature erythrocytes are less dense than mature erythrocytes, we hypothesized that the sensitivity of ZnPP/H is improved if measured in the least dense cells. Blood was collected from control suckling, mildly and severely ID suckling rats. Cord blood was collected after uncomplicated pregnancies (control), diabetic pregnancies (severe ID) and after pregnancies at-risk for iron deficiency (mild ID). ZnPP/H was measured before and after a two-step density centrifugation to obtain the lightest 6.25% of erythrocyte (top fraction). The difference between whole blood and top fraction was defined as ⌬ZnPP/H. In rats, although the whole or top ZnPP/H differed by postnatal age, ⌬ZnPP/H was greatest after the interval with least body iron accrual. In either rats or humans with mild ID, whole blood ZnPP/H was similar to, but ⌬ZnPP/H was greater than controls. In rats and newborn humans, ⌬ZnPP/H is more sensitive than whole blood ZnPP/H in identifying conditions associated with impaired erythrocyte iron delivery and may become a useful tool in measuring erythrocyte iron incorporation in early development. T here is need for a sensitive clinical index of iron-deficient (ID) erythropoiesis in growing premature infants. In premature infants, clinical practices, such as conservative transfusion criteria and erythropoietin therapy may deplete tenuous iron stores (1-4). When iron supply is limited, zinc replaces iron in the protoporphyrin IX ring to form zinc protoporphyrin (ZnPP) (5). An increased ratio of whole blood ZnPP-to-heme (ZnPP/H) reflects impaired erythrocyte iron incorporation (5,6). In older infants, whole blood zinc protoporphyrin/heme (ZnPP/H) is a more sensitive indicator of early, preanemic iron deficiency than Hb or ferritin (7). Whole blood ZnPP/H from cord blood or ill newborns also reflects iron status (4,8). ZnPP/H is higher in newborns born small for dates and offspring of mothers with insulin-treated diabetes (IDM), two conditions commonly exhibiting severely impaired tissue iron content, i.e., severe iron deficiency (severe ID) at birth (4,8). In premature infants, whole blood ZnPP/H falls in response to iron therapy (9) or with indirect delivery of iron via transfusion (3). In premature infants, whole blood ratios are inversely related to gestation (8), a clinical disadvantage because of gestation-based normal values are necessary to guide interpretation (4,8 -10).In mild ID, when Hb levels have not yet fallen, employing a more sensitive test of iron incorporation would be useful to facilitate earlier treatment. When iron demands are great and iron is limited, testing the iron status of reticulocytes, the most recently produced erythrocytes, would decrease the number of false negative assessments and improve the sensitivity of ZnPP/H to detect impaired iron incorporation. Previous work supports that measuring iron incorporation into reticulocytes (reticulocyte Hb,...

Iron Deficiency and Renal Development in the Newborn Rat

“…In normal dam-reared rats at P4, we previously observed a physiologic nadir in relative body iron content (15,19), due to no appreciable net iron absorption before P4. Because of the tenuous iron status at P4, we simultaneously rescued iron status by daily gavaged ferrous sulfate and restored body iron, Hb, RBC, and erythrocyte indices, glomerular generations and nephron density, supporting the independent contribution of iron in late nephrogenesis.…”

“…Another limitation is our sampling at only one point in time (P12), and more points in time can be studied. Only one iron dosage, 6 mg ⅐ kg Ϫ1 ⅐ d Ϫ1 of ferrous sulfate, was used because previously that dose restored Hb (15,19). Another dosage or form of iron may exhibit better bioavailability and organ trafficking.…”

“…Body iron content was determined after removing gastrointestinal tracts (esophagus to colon) to eliminate any unabsorbed, intraluminal iron from any remaining oral iron supplements. Carcasses or kidneys were weighed and iron content determined by acid digestion and atomic absorption (19). Tissue samples were placed in Teflon tubes engineered at UW Department of Engineering for acid digestion.…”

“…Tissue samples were placed in Teflon tubes engineered at UW Department of Engineering for acid digestion. Nitric then perchloric acids were added and tubes brought up to the same volume with 3M HCl (19). Iron standards and samples were read using an atomic absorption flame spectrophotometer (Perkin Elmer 2280, Waltham, MA).…”

Endogenous Siderophore 2,5-Dihydroxybenzoic Acid Deficiency Promotes Anemia and Splenic Iron Overload in Mice

“…Plasma was removed, and the cell pellet was washed three times with phosphate-buffered saline (PBS) to remove interfering pigments. Washed RBCs were then resuspended in PBS, and the whole-blood Zn protoporphyrin/heme (ZnPP/H) ratio was determined using a hematofluorometer as described previously (23).…”