Thiols, gold-thiols, zinc-thiols and the redox state of hemoglobin.

The beta subunit of human hemoglobin can be oxidized site-specifically through beta-Cys-93 by Cu(II)(His)2. A series of thiol ligands, gold thiols and zinc(II) inhibit this oxidation. The thiol inhibitors formed a transient ternary intermediate involving Cu(I) with consequent inhibition of electron transfer from the Fe(II)-heme. The intermediate led to the formation of a disulfide at the beta-Cys-93 site. The most effective thiols achieved maximum inhibition at one equivalent per beta heme. Gold thiols and zinc complexes inhibited heme oxidation by competing with the Cu(II)(His)2 for the beta-Cys-93 site.

Time-dependent modifications of ferric-adriamycin.

The biological and chemical properties of the ferric-Adriamycin complex changed with time after its preparation. Our experiments demonstrated that the toxicity of the iron-chelate in mice decreased as a function of its age. The reduced toxicity can be correlated with changes in the difference spectrum of ferric-Adriamycin vs Adriamycin (ADR), where a peak around 610 nm shifted to the 570 nm region. When ferric-Adriamycin "aged", the affinity of the drug for iron increased. Concurrently, the redox properties of the chelate changed, such that the bound iron was no longer reduced by glutathione or cysteine. The time-dependent changes observed did not involve the formation of polynuclear iron, as shown by electron spin resonance. Thin-layer chromatography showed that ADR undergoes accelerated degradation in the presence of iron. The iron-catalyzed degradation was oxygen independent. The changes evolving in the spectral and chemical properties of the chelate were shown to stem from transfer of the iron from ADR to one of the degradation products.

Oxidative damage to human red cells induced by copper and iron complexes in the presence of ascorbate.

The role of trace metals in the generation of free radical mediated oxidative stress in normal human red cells was studied. Ascorbate and either soluble complexes of Cu(II) or Fe(III) provoked changes in red cell morphology, alteration in the polypeptide pattern of membrane proteins, and significant increases in methemoglobin. Neither ascorbate nor the metal complexes alone caused significant changes to the cells. The rate of methemoglobin formation was a function of ascorbate and metal concentrations, and the chemical nature of the chelate. Cu(II) was about 10-times more effective than Fe(III) in the formation of methemoglobin. Several metals were tested for their ability to compete with Cu(II) and Fe(III). Only zinc caused a significant inhibition of methemoglobin formation by Fe(III)-fructose. These observations suggest that site-specific as well as general free radical damage is induced by redox metals when the metals are either bound to membrane proteins or to macromolecules in the cytoplasm. The Cu(II) and Fe(III) function in two catalytic capacities: (1) oxidation of ascorbate by O2 to yield H2O2, and (2) generation of hydroxyl radicals from H2O2 in a Fenton reaction. These mechanisms are different from the known damage to red cells caused by the binding of Fe(III) or Cu(II) to the thiol groups of glucose-6-phosphate dehydrogenase. Our system may be a useful model for understanding the mechanisms for oxidative damage associated with thalassemia and other congenital hemolytic anemias.

Electron transfer from cytochrome b5 to iron and copper complexes.

The rates of electron transfer from the tryptic fragment of bovine liver cytochrome b5 to FeIIINTA, FeIIIATP, CuIINTA, CuIIATP, and CuIIHis have been measured by anaerobic stopped-flow techniques. The rates of reduction of the Fe(III) complexes are independent of ionic strength, enhanced at low pH, and slightly inhibited by ZnIINTA. Saturation kinetics are observed with CuIINTA (kappa et = 0.05 s-1, K = 8.6 M-1), CuIIHis (kappa et = 0.2 s-1, K = 2.6 X 10(3) M-1), and CuIIATP (kappa et = 0.6 s-1, K = 4.5 X 10(3) M-1), thereby indicating that binding of Cu(II) to the protein occurs prior to electron transfer. 1H NMR resonances of the three surface histidines and some neighboring residues have been assigned by two-dimensional NMR techniques. NMR titration experiments show unequivocally that CuIINTA binds preferentially at a site near His-26 and Tyr-27.

Retention and distribution of iron added to cow's milk and human milk as various salts and chelates.

Iron supplementation of infant formulas is recommended by most national and international organizations, but the optimal form of supplementation has not been determined. We have compared the bioavailability and tissue distribution of iron from four iron chelates and two commonly used iron salts. Weanling C57BL/6J mice were fed for 1 week an evaporated cow's milk diet supplemented with vitamins and minerals (except for iron). Following the adjustment period, mice were divided into 12 groups of 20 each. Six groups continued to receive the cow's milk diet for 18 hours, while the other six groups were fed a similar diet based on human milk. Individual groups received a single dose of milk radioactively labeled with Fe(II)Cl2, Fe(II)SO4, Fe(III)NTA, Fe(III)EDTA, Fe(III)citrate or Fe(III)lactobionate. Wholebody retention was measured after 4 days; animals were then killed and individual tissues were counted for radioactivity. Iron from FeCl2, FeSO4 and FeNTA were the best retained from both milk diets. Fe citrate had a significantly lower iron retention than all other groups in either diet and is probably not an effective chelate for delivering iron to milk diets. Iron bioavailability was higher from the human milk diets than from the cow's milk diets from all vehicles used except citrate and lactobionate. Absorption of Fe citrate was similar from the two milk diets, while percent retention from Fe lactobionate was higher from cow's milk than from human milk. Tissue distribution of retained iron was similar for the milk diets and among the groups, indicating that, once absorbed, iron from the different vehicles is metabolized in a similar manner.

Effects of iron deficiency and exercise on myoglobin in rats.

The effects of iron deficiency and endurance training on muscle myoglobin (Mb), body weights, and blood lactic acid concentration were studied in rats. Fifty animals were divided into four groups: anemic trained (AT), normal trained (NT), anemic sedentary (AS), and normal sedentary (NS). Following 5 weeks of dietary control, the mean hemoglobin values for the AT and AS rats were 0.013 +/- 0.002 mmol X l-1 (8.7 +/- 1.4 g X dl-1) and 0.014 +/- 0.003 mmol X l-1 (9.2 +/- 1.7 g X dl-1) respectively, and did not significantly change throughout the study. AT and NT rats were run on a motor driven treadmill 4 days/week for 6 weeks up to a pre-established time of 90 min. Following the training, body weights of the AT (157 +/- 13 g) and NT (153 +/- 13 g) rats were lower than their respective sedentary groups AS (172 +/- 9 g) and NS (176 +/- 15 g). Resting blood lactic acid concentration following training was lower in both trained groups, AT (3.3 +/- 2.0 mM) and NT (2.3 +/- 1.9 mM) compared to AS (8.2 +/- 2.6 mM) and NS (3.8 +/- 1.6 mM). Training increased Mb concentration in hearts of both the anemic and normal trained groups (AT, 0.66 +/- 0.13 mg X g-1; NT, 0.95 +/- 0.08 mg X g-1) compared to the sedentary groups (AS, 0.44 +/- 0.08 mg X g-1; NS, 0.70 +/- 0.13 mg X g-1). Only the AT rats showed an increase in skeletal muscle Mb. This study provides evidence that myoglobin may limit aerobic metabolism.

Iron is maintained as Fe(II) under aerobic conditions in erythroid cells.

It was found that in rabbit erythrocytes loaded with Fe(II) (using the ionophore, A23187) over 90% of this metal is maintained in its ferrous form. In rabbit reticulocytes more than 50% of the transferrin-donated nonheme iron proved to be divalent. These results strongly suggest that under aerobic conditions the intracellular environment of intact erythroid cells is reducing for iron.

Adaptations of lactate metabolism in iron-deficient rats.

Effects of dietary iron deficiency on lactate metabolism were studied in weanling female rats. Following an iron-deficient diet for 5 weeks, mean hemoglobin concentration was lowered to 6.4 g/dl relative to 12.2 in the control group. Mean plasma iron levels were 58 and 162 micrograms/dl, respectively. Significantly elevated resting lactate levels were observed in whole blood and plasma from iron-deficient anemic (relative to control) rats. Total activity of lactate dehydrogenase (LDH) was elevated in soleus and gastrocnemius muscles in response to iron deficiency from 269 +/- 51 to 364 +/- 60 (Mean +/- SD) and from 265 +/- 65 to 372 +/- 61 IU . 10(-3) . g-1, respectively. The LDH activity in heart was lowered from 700 +/- 61 to 593 +/- 45 IU . 10(-3) . g-1. The M3H and M2H2 isozymes in soleus were increased from 12.7 +/- 2.8 to 20.4 +/- 5.8% and from 19.4 +/- 6.1 to 28.2 +/- 3.6%, respectively. Similar increase was observed in M2H2 and MH3 in gastrocnemius from 9.8 +/- 0.9 to 14.8 +/- 2.0% and from 17.4 +/- 2.0 to 20.5 +/- 2.3%, respectively. The H4 isozyme was significantly reduced in soleus, gastrocnemius, and plantaris muscles from 27.7 +/- 4.7 to 12.4 +/- 4.4, from 15.8 +/- 1.9 to 7.2 +/- 2.9, and from 10.5 +/- 2.9 to 3.9 +/- 2.1%, respectively. It was suggested that iron-deficiency anemia induces an elevation of lactate production following an increase in total LDH activity and change in LDH isozyme patterns.

Transitory hematologic effects of moderate exercise are not influenced by iron supplementation.

A young women's exercise/fitness class tested the idea that administration of supplemental iron would prevent "sports anemia" that may develop during exercise and training and improve iron status of exercising females of menstrual age. Fifteen women (aged 18-37) were selected for each of three treatment groups: (1) no supplemental iron; (2) 9 mg X d-1 of Fe; and (3) 18 mg X d-1 of Fe (1 US Recommended Daily Allowance). Women exercised at approximately 85% of maximal heartrate for progressively increasing lengths of time in a jogging program and worked up to 45 min of exercise 4 d X week-1 for 8 weeks. Hematologic analysis was performed in weeks 1, 5, and 8. A significant decline in hemoglobin (Hb) concentration and hematocrit (Hct) was observed at week 5 when all data were examined without regard for iron intake; these red cell indices returned to pre-exercise levels by week 8. Reduction of mean cell hemoglobin concentration (MCHC) indicated that the midpoint decline was not caused by simple hemodilution during exercise. Serum ferritin (SF) concentration changed in parallel with Hb and Hct. Although the midpoint decline in SF was not statistically significant, it ruled out the possibility that turnover of red cell iron was directed to storage. Lowered MCHC and SF suggested lower availability of iron during the synthesis of a new generation of red cells. Few iron treatment effects of magnitude were observed. Iron did not prevent the midpoint decline in Hb concentration. Iron intake did not affect SF, serum iron, transferrin saturation, or final Hb, and Hct.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioavailability of iron- and copper-supplemented milk for Mexican school children.

Fortification of dairy products with trace metals requires use of assimilable compounds that do not catalyze off-flavors due to lipid peroxidation but show good biological availability. The Fe(III) and Cu(II) chelates of the promising chelator, lactobionic acid, have been compared to Fe(II) and Cu(II) salts for their ability to improve hematological status in a mildly anemic population. Fe- and Cu-fortified cow milk was administered to children (aged 6 to 15) in the Durango, Mexico, "school lunch" program. Children drank milk providing 20 mg Fe and 3 mg Cu as ferric/cupric lactobionate ("chelate") or ferrous/cupric chloride ("salt") for 5 of 7 days/wk for 3 months. Supplementation with "salt" and "chelate" raised Hb significantly by 1 and 0.3 g/dl, respectively, above the control (unsupplemented) group. No significant change was observed in incremental serum ferritin, serum Fe, or transferrin saturation, or in final serum Cu. Ferric lactobionate shows poorer bioavailability than ferrous ion in the presence of Cu, but milk can be an excellent vehicle for Fe or Cu supplementation.

Mitochondrial NADH dehydrogenase in iron-deficient and iron-repleted rat muscle: an EPR and work performance study.

Iron may affect both respiratory O2 transport and mitochondrial electron transport in the performance of muscle work. This study was designed to elucidate the molecular defect of iron-deficient work performance by identifying heretofore unmeasurable mitochondrial enzymes that are diminished by iron deficiency and may be restored by iron repletion. Female rats were made iron-deficient by dietary control and were repleted by oral iron. Iron deficiency reduced physical work capacity (treadmill running time), haemoglobin (Hb), and mitochondrial iron-sulphur (Fe-S) centres in heart and skeletal muscles; mitochondrial number was unaffected. Oral iron supplementation restored work capacity and Hb within 4 d to normal or near-normal levels, but in general Fe-S centres of mitochondria due to NADH dehydrogenase remained at iron-deficient levels. Subnormal concentrations of mitochondrial iron-dependent NADH dehydrogenase in muscle are not by themselves rate-limiting in work performance.

Distribution and metabolism of iron in muscles of iron-deficient rats.

Iron-deficiency anemia leads directly to both reduced hemoglobin levels and work performance in humans and experimental animals. In an attempt to observe a direct link between work performance and insufficient iron at the cellular level, we produced severe iron deficiency in female weanling Sprague-Dawley rats following five weeks on a low-iron diet. Deficient rats were compared with normal animals to observe major changes in hematological parameters, body weight, and growth of certain organs and tissues. The overall growth of iron-deficient animals was approximately 50% of normal. The ratio of organ weight: body weight increased in heart, liver, spleen, kidney, brain, and soleus muscle in response to iron deficiency. Further, mitochondria from heart and red muscle retained their iron more effectively under the stress of iron deficiency than mitochondria from liver and spleen.Metabolism of iron in normal and depleted tissue was measured using tracer amounts of(59)Fe administered orally. As expected, there was greater uptake of tracer iron by iron-deficient animals. The major organ of iron accumulation was the spleen, but significant amounts of isotope were also localized in heart and brain. In all muscle tissue examined the(59)Fe preferentially entered the mitochondria. Enhanced mitochondrial uptake of iron prior to any detectable change in the hemoglobin level in experimental animals may be indicative of nonhemoglobin related biochemical changes and/or decrements in work capacity.

Iron(III)--phosphoprotein chelates: stoichiometric equilibrium constant for interation of iron(III) and phosphorylserine residues of phosvitin and casein.

Estimates of the strength of iron binding to model phosphoproteins were obtained from equilibrium dialysis experiments. Iron-free phosvitin (chicken and frog) or alpha sl-casein (cow) was dialyzed against the iron(III) chelates of nitrilotriacetate (NTA), )ethylenedinitrilo)tetraacetate (EDTA), or citrate. Protein-bound metal was measured at equilibrium; competition of chelator and phosphoprotein for iron(III) was determined by reference to comprehensive equilibrium equations presented in the Appendix. Analysis of the iron-binding data for phosvitin suggested that clusters of di-O-phosphorylserine residues (SerP.SerP) were the most probable iron-binding sites. A stoichiometric equilibrium constant of 10(18.0) was calculated for the formation of the Fe3+(SerP.SerP) chelate. When comared on the basis of phosphate content, casein bound iron more weakly than phosvitin. However, if the stoichiometric equilibrium constant for the formation of the casein Fe3+(SerP.SerP) chelate (10(17.5) was adjusted to account for the fact that a smaller percentage of casein phosphoserines occurs in di-O-phosphorylserine clusters, the affinity of casein and phosvitin for iron was very similar. A theoretical comparison showed that the "strengths" of the ferric chelates can be ranked: EDTA greater than phosphoprotein di-O-phosphorylserine greater than citrate greater than NTA.

Degradation of ascorbic acid (vitamin C) in iron-supplemented cows' milk.

The fate of [6-carbon-14] ascorbic acid in iron-supplemented and unsupplemented raw milk was studied by anion-exchange chromatography, which permitted quantitative analysis of the conversion of ascorbate to dehydroascorbate and diketogulonate as a function of time. Iron catalyzed an increase in the rate of autoxidation of ascorbate to dehydroascorbate but did not alter the equilibrium concentrations of ascorbate, dehydroascorbate, and diketogulonate. The conversion of ascorbate to dehydroascorbate and of dehydroascorbate to diketogulonate occurred rapidly even in unsupplemented milk. Thus, trace metal supplementation may not affect materially the vitamin C content of stored milk.

Removal of cadmium(II) from crystallized ferritin.

The cadmium content of crystallized horse spleen ferritin, usually about 2% by weight without special treatment, can be substantially decreased by prolonged dialysis against certain chelating agents, chaotropic ions, or weakly reducing anions. For example, neutral bisulphite buffer (2M) removed 95% of the bound cadmium of crystallization without affecting the iron content, and may thus be valuable for preparing "metal-free" holoferritin for physical-chemical studies.

Correlation of serum ferritin and liver ferritin iron in the anemic, normal, iron-loaded rat.

We developed a two-site immunoradiometric assay for rat serum ferritin that uses antibody immobilized on agarose. Individual serum ferritin values were significantly correlated with iron stores as determined chemically by liver ferritin iron content. This group correlation was not sufficiently great, however, to allow confident prediction of iron stores in a given animal on the basis of serum ferritin alone. Significant differences in mean liver ferritin iron concentration between groups of rats raised on diets of differing iron content were not always reflected by differences in mean serum ferritin values. Data correlating the serum levels of ferritin and a liver-specific transaminase suggested that hepatocellular death may sometimes contribute ferritin to the serum. Strong postitive correlations between serum ferritin and iron stores in the rat were not observed when serum transaminase levels were in the normal range.