Bo E. Hedlund

Abnormal iron distribution in infants of diabetic mothers: Spectrum and maternal antecedents

Because chronic hypoxemia causes a redistribution of iron from serum and storage pools into an expanding erythrocyte mass, and because infants of diabetic mothers are often hypoxemic in utero and have a high prevalence of polycythemia at birth, we studied iron distribution in 43 term infants of diabetic mothers. Twenty-four infants were at an appropriate size for gestational age; 19 were large for gestational age. At birth, 28 infants (65%) had abnormal serum iron profiles; eight had decreased ferritin concentrations only (stage 1), nine had decreased ferritin and increased total iron-binding capacity values (stage 2), and 11 had these serum findings plus elevated free erythrocyte protoporphyrin concentrations (stage 3). The hypoglycemic infants who were large for gestational age (n = 14) had a higher prevalence of abnormal iron profiles than euglycemic infants who were appropriate in size for gestational age (n = 20; 93% vs 50%; p = 0.009). Progressively abnormal iron profiles were associated with higher glycosylated fetal hemoglobin values, greater degrees of macrosomia, increased hemoglobin and erythropoietin concentrations, and increased erythrocyte/storage iron ratios. Erythropoietin concentrations were inversely linearly correlated with serum iron values (n = 32, r = -0.54; p = 0.003). The combined erythrocyte and storage iron pools were significantly lower in infants with abnormal iron values whose mothers were diabetic, particularly in infants of women with confirmed diabetic vasculopathy. We speculate that these findings are likely due to (1) increased fetal iron utilization during compensatory hemoglobin synthesis in response to chronic hypoxemia and (2) reduced iron transfer during late gestation complicated by diabetes.

Iron deprivation increases erythropoietin production in vitro, in normal subjects and patients with malignancy

Although tissue hypoxia is the major stimulus for erythropoietin (EPO) production, serum EPO (sEPO) levels at any given Hb in iron‐deficiency anaemia are relatively higher than in other anaemias. Iron chelators stimulate erythropoiesis in anaemia of chronic disease via unknown mechanisms. A recent study suggested that deferoxamine (DFO) regulates steady‐state EPO RNA. Here we report that altered intracellular iron balance regulates EPO production both in vitro and in two unique clinical trials. In vitro, both iron chelation with DFO and blockade of Tf‐mediated iron uptake with anti‐Tf receptor antibody 42/6, stimulated EPO production in serum‐deprived hepatoma cells. Conversely, iron repletion by haemin, inhibited EPO production in these cells. In clinical studies, sEPO levels rose in adult volunteers treated with DFO coupled to hydroxyethyl starch (HES‐DFO) and in patients with advanced malignancy treated with anti‐Tf receptor antibody 42/6, in a time‐ and dose‐dependent manner. These studies indicate intracellular iron balance regulates EPO production in humans.

EXTRACELLULAR IRON CHELATORS PROTECT KIDNEY CELLS FROM HYPOXIA/REOXYGENATION

Iron is an important contributor to reoxygenation injury because of its ability to promote hydroxyl radical formation. In previous in vivo studies, we demonstrated that iron chelators that underwent glomerular filtration provided significant protection against postischemic renal injury. An in vitro system was employed to further characterize the protection provided by extracellular iron chelators. Primary cultures of rat proximal tubular epithelial cells were subjected to 60 min hypoxia and 30 min reoxygenation (H/R). During H/R, there was a 67% increase in ferrozine-detectable iron in cell homogenates and increased release of iron into the extracellular space. Cells pretreated with either deferoxamine (DFO) or hydroxyethyl starch-conjugated deferoxamine (HES-DFO), an iron chelator predicted to be confined to the extracellular space, were greatly protected against lethal cell injury. To further localize the site of action of DFO and HES-DFO, tracer quantities of 59Fe were added to DFO or HES-DFO, and their distribution after 2 h was quantitated. Less than 0.1% of DFO entered the cells, whereas essentially none of the HES-DFO was cell-associated. These findings suggest that iron was released during hypoxia/reoxygenation and caused lethal cell injury. Iron chelators confined to the extracellular space provided substantial protection against injury.

Hemoglobin: A Lifesaver and an Oxidant how to tip the Balance

Hemoglobin solutions were assessed in terms of their ability to promote lipid peroxidation, which was quantitated by measuring the formation of thiobarbituric acid reactive substances (TBARS) under specified conditions in murine brain homogenates. Solutions designed for use in acute treatment of hypovolemic shock and trauma should incorporate ingredients specifically aimed at decreasing oxygen and lipid radical mediated injury occurring secondary to ischemia and reperfusion. A number of strategies aimed at decreasing the oxidant effect of hemoglobin solutions and other blood and plasma substitutes have been evaluated. These include use of the naturally occurring anti-oxidants in human plasma, specifically transferrin and ceruloplasmin. Similarly, certain iron chelators, such as deferoxamine (Desferal, Ciba-Geigy), effectively prevent molecular and cellular damage caused by iron catalyzed formation of oxygen derived radicals.

Studies on the effect of reagent and protein charges on reactivity of the β93 sulfhydryl group of human hemoglobin using selected mutations

Abstract The reactivity of the β93 sulfhydryl (SH) group of human oxyhemoglobins with the negatively charged 5,5′-dithiobis(2,2′-nitrobenzoate) and the uncharged 2,2′-dithiodipyridine was determined as a function of pH. Selected mutant hemoglobins having increased oxygen affinity and having residue substitutions altering charge near the SH group (Wood, Malmo, Yakima, Kempsey, Andrew-Minneapolis, Osler, and Chesapeake) were compared to hemoglobin (Hb) A. Although both reagents reacted with GSH at the same rate and with the same enthalpies of activation, the rates with Hb were different and the difference showed a pronounced pH dependence. The charged reagent was sensitive to charges near the SH group; a positive charge increased the rate and a negative one decreased the rate. The uncharged reagent which reacted with Hb A with activation enthalpies similar to those for GSH was insensitive to neighboring charges, but was sensitive to tertiary and quaternary structural changes. The rates obtained with the latter reagent did not correlate with oxygen affinity. The evolutionary aspects of the β93 cysteine in relation to structure and function are reviewed.

Effect of carbohydrate on protein solubility

The effect of covalently attached carbohydrate on the solubility of a number of proteins has been examined by the PEG precipitation technique. Both increases and decreases in solubility are observed depending on the state of glycosylation, the type of protein, and temperature. It is concluded from this data and associated apparent thermodynamic parameters that a general role for carbohydrate in the solubilization of proteins is not currently experimentally supportable.

Thermodynamics of 2,3-diphosphoglycerate association with human oxy- and deoxyhemoglobin

Abstract The association of 2,3-diphosphoglycerate with oxy- and deoxyhemoglobin was studied by means of ultrafiltration and microcalorimetry. It was found that in addition to parameters that are known to influence the binding of 2,3-diphosphoglycerate to both species of hemoglobin (such as pH, temperature and concentration of competing anion), the association is also strongly dependent on the hemoglobin concentration. The difference between the apparent association constants for the formation of the complex of the organic phosphate with oxy- and deoxyhemoglobin is relatively small. At pH 7.3, 25° C and 0.154 M chloride this difference is only 0.6 kcal/mole of free energy favoring the Hb·DPG complex. This free energy difference increases with decreasing pH but is not strongly affected by hemoglobin concentration. The enthalpy change for the formation of the 2,3-diphosphoglycerate complex with deoxyhemoglobin is 8–10 kcal/mole more exothermic than the complex with oxyhemoglobin.

Partial reduction of aquomethemoglobin on a sephadex G-25 column as detected by magnetic circular dichroism spectroscopy and revised extinction coefficients for aquomethemoglobin

In a typical preparation of aquomethemoglobin, oxyhemoglobin is oxidized with potassium ferricyanide, and the resultant mixture of methemoglobin and potassium ferro- and ferricyanides is separated on a Sephadex G-25 column. We find that about 1% of the heme is reduced on the column and is eluted with the methemoglobin. Magnetic circular dichroism spectra show that the reduced species is oxyhemoglobin. Magnetic circular dichroism is more sensitive than is absorption spectroscopy to small amounts of oxyhemoglobin in such solutions; we can detect its presence at the 0.1% level. A redetermination of the extinction coefficients for methemoglobin gives a value of 0.80 for the absorbance ratio A570A630 at pH 6. This value lies clearly outside the currently accepted range of 0.83 to 0.87.

Hemoglobin Minneapolis-Laos [β-118 (GH1) Phe→Tyr] A New Hemoglobin Variant with Normal Functional Properties

Routine screening of hemoglobin samples from Southeast Asian immigrants by isoelectric focusing (1) revealed an individual with an abnormal hemoglobin component which migrated with approximately the same mobility as fetal hemoglobin. The proband was a 27 year old male of Laotian origin. His newborn son was found to be heterozygous for this variant and HbE. The probands wife was homozygous for HbE. The probands hematological findings were as follows: RBC -5.13 million, PCV -48.8%, MCV 95 fl, and Hb -16.7 g/dl. His red cells were normochromic and normocytic.

Application of a coulometric oxygen sensor to the determination of hemoglobin--oxygen binding isotherms.

Abstract In this report we describe the employment of a coulometric oxygen sensor for the measurement of hemoglobin-oxygen binding isotherms. The method is uniquely suited to the study of oxygen binding in highly concentrated solutions of hemoglobin. The sensor is absolute in that the oxygen in a given solution of hemoglobin is removed from the protein and measured in the gaseous form by reduction at a yield close to the maximum allowed by Faradays law.