Ferrous Ions Detected in Iron-Overloaded Cord Blood Plasma From Preterm and Term Babies: Implications for Oxidative Stress

Redox active iron chelatable to bleomycin is often present in the plasma of cord blood samples taken from preterm and term babies. The low caeruloplasmin and high ascorbate levels in plasma at birth may allow this iron to exist in the reduced ferrous state. In support of this postulate thirteen cord blood samples showing the presence of low molecular mass iron were able to degrade DNA in the presence of bleomycin and plasma.     

Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis. Characterization by high performance liquid chromatography and nuclear magnetic resonance spectroscopy

The nature of non-transferrin-bound iron in the plasma or serum of iron-overloaded hemochromatosis patients was studied by high performance liquid chromatography (HPLC) and high resolution nuclear magnetic resonance (NMR). 500-MHz proton Hahn spin-echo NMR spectra of plasma or serum, combined with the use of the iron chelator desferrioxamine, suggests complexation of iron ions with citrate and a possible involvement of acetate. Addition of FeCl3 to hemochromatosis samples broadened the NMR signals from citrate. HPLC analysis rigorously confirmed the presence of an iron-citrate complex in ultrafiltrates of plasma or serum studies with added FeCl3 or desferrioxamine supported this conclusion. It is proposed that non-transferrin-bound iron in the plasma of iron-overloaded patients exists largely as complexes with citrate and possibly also as ternary iron-citrate-acetate complexes. The presence of such complexes would account for the ability of non-transferrin-bound iron to be measurable by the bleomycin assay and for its rapid clearance from the circulation by the liver.     

Bleomycin-Detectable Iron in Brain Tissue

The normal brain contains regions with high concentrations of iron, part of which appears to be in a low molecular mass chelatable form. Iron complexes with a molecular mass of below 10,000, were measured in ultrafiltrates of homogenized gerbil brains using the bleomycin assay, and were found to average 20.5 ± 3.5 μM (n = 8). As expected, no bleomycin detectable iron was found in the plasma of these animals.

No obvious difference in the tissue levels of bleomycin-detectable iron was recorded following ischaemia and reperfusion. This is probably due to the already abundant presence of iron in the brain and the likely release of iron from protected sites due to structural damage inherent in the preparative procedures used.     

The human lymphocyte micronucleus assay. Response of cord blood lymphocytes to gamma-irradiation and bleomycin

The induction of micronuclei in human cord blood lymphocytes by treatment with gamma-irradiation and bleomycin has been measured. Culture durations which gave peak MN frequencies were determined. The lowest tested doses, 0.1 Gy irradiation and 1.25 micrograms/ml bleomycin, produced significant increases in the frequency of micronuclei. The spontaneous frequency of micronucleated lymphocytes in 28 cord blood samples ranged between 0.5 and 9.5 per thousand lymphocytes, with a modal value of 2.5. The method is evaluated for its potential usefulness in monitoring populations for chromosome breakage.     

Bleomycin-detectable iron in the plasma of premature and full-term neonates.

The bleomycin assay measures non-transferrin-bound iron, able to catalyze free radical reactions, in human plasma. No bleomycin-detectable iron is present in plasma from healthy adults. However, plasma from 3/15 premature babies was positive in this assay. Plasma from 52 apparently-healthy term babies was analyzed and 11 were positive in the bleomycin assay. Hence not only some premature but also some full-term apparently-healthy babies may be at risk of severe oxidative damage.     

Inhibition of catalase-dependent ethanol metabolism in alcohol dehydrogenase-deficient deermice by fructose.

Some 40% of knee-joint synovial fluids from arthritic patients show the presence of bleomycin-detectable iron. This is released from a protein component of the fluid to bleomycin at acidic pH values. Patients whose fluids release iron have lower contents of transferrin, lactoferrin and caeruloplasmin than do patients whose fluids do not release iron to bleomycin. These proteins are important extracellular antioxidants, and measured antioxidant activities are extremely low in the iron-releasing fluids. The propensity of some fluids to release iron at low pH values, characteristic of the microenvironment beneath adherent macrophages, coupled with their decreased antioxidant protection against iron-stimulated oxygen-radical damage, might explain previously reported correlations between clinical disease severity, lipid peroxide content and the presence of bleomycin-detectable iron [Rowley, Gutteridge, Blake, Farr & Halliwell (1984) Clin. Sci. 66, 691-695].     

Bleomycin-metal interaction: ferrous iron-initiated chemiluminescence

Chemiluminescence often accompanies the spontaneous degradation of intermediates in an electronically excited state. The interaction of iron with bleomycin results in the activation of bleomycin to a reactive intermediate which can alter DNA or undergo self-inactivation. This report demonstrates that the interaction of ferrous iron with bleomycin results in chemiluminescence, that this response is iron-specific and that the presence of DNA prevents the generation of chemiluminescence. These observations suggest that the activated bleomycin intermediate may be in an electronically excited state.     

Reciprocal adaptive response of human peripheral lymphocytes induced by bleomycine or gamma rays.

The adaptive response and reciprocal adaptive response induced in vitro by exposure to low doses of gamma rays (0.05 Gy) or bleomycin (0.05 microg/ml) in human peripheral blood lymphocytes were assessed by the frequency of chromosome aberrations. Gamma rays (1.5 Gy) or bleomycin (1.5 microg/ml) were used as the challenge doses. In the experiments, blood samples from 5 healthy donors were investigated. It has been found that low doses of bleomycin and gamma rays induced a reciprocal adaptive response to high doses of gamma rays or bleomycin. Moreover, the results confirmed that the adaptive response did not correlate with the radiosensitivity of the peripheral blood lymphocytes.     

Characteristics of the changes in the natural killer activity of peripheral blood lymphocytes during pregnancy

The influence of blood plasma of women of the 1st and 3rd pregnancy trimester and of cord blood on natural killer (NK) activity of peripheral blood lymphocytes of non-pregnant women was investigated. The maximum suppressive activity on NK was observed with plasma of pregnant women of the 3rd trimester with low NK-activity. Data suggesting the involvement of estradiol in this suppressive activity was obtained. No suppressive activity of cord blood plasma was found despite the low NK-activity of cord blood.     

Reaction of FeBlm with DNA: Fe(II)Blm-NO.

The structure of the iron bleomycin nitric oxide complex is altered in the presence of calf thymus DNA as determined from epr studies. This altered structure predominates for one iron bleomycin nitric oxide molecule per coil of the DNA helix. In the absence of nitric oxide, as the pH is lowered, iron bleomycin dissociates in two steps, supporting the hypothesis that in-plane nitrogens may be easily perturbed.     

Maternal-Fetal Distribution of Calcium,Iron, Copper,and Zinc in Pregnant Teenagers and Adults

Adolescence is marked by intensive growth and development. When pregnancy occurs during this period of the mother’s growth, there is an increase in her nutritional needs. The aim of this study was to determine the levels of calcium, iron, copper, and zinc in maternal plasma, the placenta, and in the cord plasma of pregnant teenagers and adults. A total of 80 sets of maternal plasma, placentas, and cord plasma (40 from teenagers and 40 from adults) were analyzed using synchrotron radiation total reflection X-ray fluorescence. The levels of calcium, copper, and zinc in the maternal and cord plasma from teenagers were not significantly different than those from adults. Iron levels in the teenagers maternal and cord plasma were higher than in the adults. All of the minerals analyzed were present at higher levels in the placentas from adults than those from teenagers. However, the low quantities of placental calcium, iron, copper, and zinc in the teenagers do not compromise the levels of these minerals in the cord plasma. Future research regarding the placental transport of these minerals is recommended to investigate the efficiency of mechanisms of transfer of these minerals in pregnant teenagers.     

Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by peroxides

Hydrogen peroxide and organic hydroperoxides react with haemoglobin to release iron which can be complexed to apotransferrin, bleomycin and desferrioxamine. This released iron promotes deoxyribose degradation by a Fenton reaction, DNA degradation in the presence of bleomycin and stimulates lipid peroxidation. It is likely that iron released from haemoglobin is the true generator of hydroxyl radicals in the Fenton reaction.     

Effect of deoxyribonucleic acid on the production of reduced oxygen by bleomycin and iron

The binding of bleomycin to DNA in the presence and absence of ferric iron was measured by fluorescence spectroscopy. In millimolar concentrations of tris(hydroxymethyl)aminomethane, pH 7.5, approximately 80% of the bleomycin binds to DNA. Ferric iron seems to have no significant effect on the binding of DNA to bleomycin. The induction of oxygen uptake by ferrous iron and bleomycin was monitored in the presence and absence of DNA. DNA has no effect on the rate of oxygen uptake. Therefore, the iron binding site and the DNA binding site appear to be independent of each other. Under conditions where 80% of the bleomycin is bound to DNA, the ferrous iron-bleomycin-induced reduction of oxygen follows Michaelis-Menten kinetics. Ferrous iron autoxidation produces ethylene from methional. The addition of bleomycin greatly increases ethylene production. DNA, under conditions where 80% of the bleomycin is bound to DNA, inhibits ethylene production. Since ethylene is a measure of hydroxyl radical production, we conclude that DNA is able to compete with methional for the hydroxyl radical. We postulate a mechanism for DNA double-strand breaks in which the bleomycin selectively binds to DNA and recurrently produces the hydroxyl radical at that site. The localized generation of many hydroxyl radicals as provided by the proposed oxidation-reduction cycle mechanism may cause multiple strand breaks taking place on both strands of the DNA duplex leading to double-strand breaks. Since catalase, but not superoxide dismutase, is able to inhibit ferrous iron-bleomycin-induced products of the hydroxyl radical, hydrogen peroxide, but not the superoxide radical, is the immediate precursor of the hydroxyl radical.     

Light-induced nicking of deoxyribonucleic acid by cobalt(III) bleomycins

The anticancer drug bleomycin is a glycopeptide that causes strand scission of DNA both in vivo and in vitro. Cleavage of DNA by bleomycin has been studied extensively in vitro, with the findings that ferrous ion and molecular oxygen must be present and that addition of reducing agents greatly enhances the reaction. To date, only iron has been shown to be an effective metal cofactor for the cleavage of DNA by bleomycin. Here it is reported that two stable cobalt(III) complexes of bleomycin are strikingly effective in causing single-strand breaks (nicks) in supercoiled DNA in the presence of ultraviolet or visible radiation. For example, 366-nm light from an 18-W long-wavelength mercury lamp for 1 h causes 10(-6) M cobalt(III) bleomycin to completely convert supercoiled phi X174 DNA (10(-8) M DNA, 10(-4) M phosphate) into the nicked circular form. Furthermore, numerous alkali-labile sites are produced on the DNA during this treatment. The observed reactions are not caused by adventitious iron, and they occur only in the presence of cobalt(III) bleomycin and light.     

Non-protein-bound iron within bacterial cells and the action of bleomycin

Damage to DNA by bleomycin in vitro is entirely dependent on the presence of an iron salt and molecular oxygen. This same reaction has been used to measure non-protein-bound iron in different bacterial species. Although several of the species examined were insensitive to the toxic effects of bleomycin, they all contained a concentration of non-protein-bound iron which should have been sufficient to allow iron-dependent damage to DNA. Other factors such as permeability or inactivation of the drug must explain their apparent insensitivity.     

A high resolution PIXE measurement for blood plasma ultrafiltrate

A new technique for investigation of elemental concentrations in subfractions of blood plasma is presented. The method is composed of the ultrafiltration of plasma in the presence of ethylenediaminetetraacetic acid (EDTA) and the measurement of the elemental composition by proton-induced X-ray emission (PIXE). The blood samples were collected from both healthy persons and patients suffering from breast cancer. The main emphasis in this study was on the determination of loosely bound copper (Cu) in plasma subfractions containing substances with molecular mass under 10,000, but zinc (Zn) and iron (Fe) contents of these fractions were also determined. The detection limits obtained with this method for Cu, Zn and Fe were ≈10 ppb (wet wt).     

Iron Complexes and Their Reactivity in the Bleomycin Assay for Radical-Promoting Loosely-Bound Iron

The sensitivity of the bleomycin assay for loosely-bound iron depends on the concentration of bleomycin and ascorbic acid and the pH of the reaction. The non-haem-iron proteins transferrin, conalbumin and ferritin release iron at an acid pH value, whereas the haem-iron proteins release iron more readily at an alkaline pH. In addition, haem proteins are liable to release iron when peroxides are present. Organic peroxides and hydrogen peroxide can be produced during the bleomycin reaction leading to iron release from haem proteins. However, this can be prevented from reacting with bleomycin by adding zinc ions to the reaction following addition of the sample. Iron already bound to bleomycin is not displaced by zinc whereas zinc bound to bleomycin is not displaced by iron allowing 'free' and 'released' iron to be discriminated.     

Cord blood plasma lipoproteins inhibit mitogen-stimulated lymphocyte proliferation

Lipoproteins were isolated from adult plasma and the umbilical cord blood plasma of newborn infants and were compared for their capacity to inhibit mitogen-stimulated [3H]thymidine uptake of adult peripheral blood mononuclear cells in vitro. Relative to the comparable adult lipoproteins, cord blood low density lipoproteins and high density lipoproteins inhibited mitogen stimulation at twofold to fourfold lower total protein concentrations. Apoproteins AI, B, and E were quantitated by radioimmunoassay of each of the adult and cord blood lipoprotein fractions. A strong correlation was observed between inhibitory activity and the amount of apoprotein E in the cord blood low and high density lipoproteins. Further evidence that lipoproteins containing apoprotein E accounted for the difference in suppressive activity of cord blood low and high density lipoproteins relative to the adult lipoproteins was obtained by selective removal of the apoprotein E-containing lipoproteins by using immunoaffinity chromatography or heparin-agarose adsorption. The results indicated that cord blood lipoproteins containing apoprotein E in association with apoproteins AI or B are capable of suppressing lymphocyte proliferation in vitro.     

Risk of iron overload is decreased in beating heart coronary artery surgery compared to conventional bypass.

Conventional cardiopulmonary bypass surgery (CCPB) increases the iron loading of plasma transferrin often to a state of plasma iron overload, with the presence of low molecular mass iron. Such iron is a potential risk factor for oxidative stress and microbial virulence. Here we assess 'off-pump' coronary artery surgery on the beating heart for changes in plasma iron chemistry. Seventeen patients undergoing cardiac surgery using the 'Octopus' myocardial wall stabilisation device were monitored at five time points for changes in plasma iron chemistry. This group was further divided into those (n=9) who had one- or two- (n=8) vessel grafts, and compared with eight patients undergoing conventional coronary artery surgery. Patients undergoing beating heart surgery had significantly lower levels of total plasma non-haem iron, and a decreased percentage saturation of their transferrin at all time points compared to conventional bypass patients. Plasma iron overload occurred in only one patient undergoing CCPB. Beating heart surgery appears to decrease red blood cell haemolysis, and tissue damage during the operative procedures and thereby significantly decreases the risk of plasma iron overload associated with conventional bypass.     

Direct analysis of human blood (mothers and newborns) by energy dispersive X-ray fluorescence

This work is an application of energy dispersive X-ray fluorescence (EDXRF) as analytical technique for trace element determination in human tissues. Potassium (K), calcium (Ca), iron (Fe), copper (Cu), zinc (Zn), bromine (Br), rubidium (Rb) and lead (Pb) were determined directly in blood samples from 66 mothers at delivery after full-term pregnancies. The corresponding 66 cord-blood samples of the newborns were also analysed, in order to find element correlations between maternal and newborn blood at birth. The studied samples were obtained from mothers aged between 15 and 39 years old, the gestational age being between 35 and 41 weeks and the newborns' weight between 2.310 and 4.310 kg. Samples were lyophilised and analysed without any chemical treatment. Very low levels of Pb were found both in maternal and fetal cord blood samples. Cu values ranged from 3 to 13 microg g-1, both for mothers and children. A correlation between Cu and Fe concentrations in maternal and fetal cord blood was found. Zn is considered as one of the key elements in newborn health. Concentrations between 10 and 40 microg g-1 were measured. A positive correlation between Br levels in mothers and children was observed. Positive correlations for mothers were observed between Zn and Rb as well as K and Fe. The corresponding correlations in fetal cord blood samples were not observed, however positive correlations were found between Ca and K; Cu and Fe. The mean concentrations for each element were similar in maternal and in fetal cord blood, except for Cu and Zn, being higher in maternal samples. No correlations between element concentrations and pathologies of the mothers were observed.