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In a protein-protein electron transfer (ET) photocycle, the "forward" ET reaction is initiated with the excited complex, [3DA], in an equilibrium ensemble of configurations, the majority of which exhibit less than the maximal ET matrix element. In contrast, the charge-separated intermediate complex is formed in a nonequilibrium set of configurations with maximal ET matrix elements and would be expected to return to the ground state with the largest rate constant possible unless conformational interconversion first "breaks the connection" and the complex converts to less-reactive substates. According to this analysis, the forward and back ET reactions should show a differential response to viscosity, and the latter could even show an increased rate constant under conditions which suppress departure from the reactive configuration(s). We now report that the viscosity dependences of forward and back ET rate constants for the photocycle within the [alpha2(Zn),beta2(Fe3+N3-)] mixed-metal hemoglobin hybrid at pH 7 show the anticipated behavior: kf decreases as viscosity increases, but, in sharp contrast, kb increases strongly.
High resolution separation of metalloproteins and other iron compounds based on native gel electrophoresis followed by 59Fe autoradiography is described. Lysates of mouse spleen erythroid cells metabolically labeled with 59Fe-transferrin were separated on 3-20% polyacrylamide gradient gels in the presence of Triton X100 and detected by autoradiography. In addition to ferritin and hemoglobin, several compounds characterized by their binding of iron under different conditions were described. Iron chelatable by desferrioxamine migrated in the region where several high-molecular weight compounds were detected by silver staining. The technique is nondissociative, allowing identification of iron compounds with the use of specific antibodies. Cellular iron transport and the action of iron chelators on specific cellular targets can be investigated in many small biological samples in parallel.
Although intravenous iron has proved to optimize the efficacy of EPOrHu in hemodialysis patients, hitherto no consensus exists with respect to the best regimen of intravenous iron administration. We started a prospective randomized study in 26 patients undergoing chronic hemodialysis who had adequate iron metabolism indices (serum ferritin >100 microg/l; %TSAT >20%; %HypoE <10% and CHr >26 pg) and were in the maintenance phase of EPOrHu administration (target hemoglobin obtained >10 g/dl). All patients were receiving sodium ferric gluconate (Ferrlecit) intermittently prior to the study and after a 1-month wash-out period where iron was not administered patients were randomized to receive the same previous dose of intravenous iron either in a continuous (6.25-21.3 mg in every hemodialysis session) or an intermittent regimen (62.5 mg every 1-4 weeks, not modifying the previous schedule of administration). At 16 weeks, the continuous group showed a significant increment in serum Hb (11.83 +/- 1.12 g/dl) with respect to baseline (10.96 +/- 1.31 g/dl) (p < 0.05), whereas no differences were obtained in intermittent group (baseline: 11.16 +/- 1.03 g/dl; 16 weeks: 11.14 +/- 0.90 g/dl, NS). In contrast with the intermittent group, serum ferritin increased significantly in the continuous group (16 weeks: 508 +/- 157 microg/l; baseline: 368 +/- 56 microg/l; p < 0.05), whereas %TSAT and CHr did not modified during the study in both groups. %HypoE increased significantly with respect to baseline values in the continuous group (p < 0.05) and close to significantly different in the intermittent group (p = 0.06). Our study suggests that hemodialysis patients in the maintenance phase of EPOrHu administration would obtain further benefit in terms of serum hemoglobin level with a continuous intravenous serum ferric gluconate regimen, at least in the short term.
The effect of pH on the X-band electron paramagnetic resonance (EPR) spectrum of ferrous nitrosylated human adult tetrameric hemoglobin (HbNO) as well as of ferrous nitrosylated monomeric alpha- and beta-chains has been investigated, at -163 degrees C. At pH 7.3, the X-band EPR spectrum of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains displays a rhombic shape. Lowering the pH from 7.3 to 3.0, tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains undergo a transition towards a species characterized by a X-band EPR spectrum with a three-line splitting centered at 334mT. These pH-dependent spectroscopic changes may be taken as indicative of the cleavage, or the severe weakening, of the proximal HisF8-Fe bond. In tetrameric HbNO, the pH-dependent spectroscopic changes depend on the acid-base equilibrium of two apparent ionizing groups with pK(a) values of 5.8 and 3.8. By contrast, the pH-dependent spectroscopic changes occurring in ferrous nitrosylated monomeric alpha- and beta-chains depend on the acid-base equilibrium of one apparent ionizing group with pK(a) values of 4.8 and 4.7, respectively. The different pK(a) values for the proton-linked spectroscopic transition(s) of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains suggest that the quaternary assembly drastically affects the strength of the proximal HisF8-Fe bond in both subunits. This probably reflects a 'quaternary effect', i.e., structural changes in both subunits upon tetrameric assembly, which is associated to a relevant variation of functional properties (i.e., proton affinity).
Arterial baroreceptors are mechanosensitive nerve endings in the aortic arch and carotid sinus that play a critical role in acute regulation of arterial blood pressure. A previous study has shown that nitric oxide (NO) or NO-related species suppress action potential discharge of baroreceptors. In the present study, we investigated the effects of NO on Na+ currents of isolated baroreceptor neurons in culture. Exogenous NO donors inhibited both tetrodotoxin (TTX) -sensitive and -insensitive Na+ currents. The inhibition was not mediated by cGMP but by NO interaction with channel thiols. Acute inhibition of NO synthase increased the Na+ currents. NO scavengers (hemoglobin and ferrous diethyldithiocarbamate) increased Na+ currents before but not after inhibition of NO synthase. Furthermore, NO production in the neuronal cultures was detected by chemiluminescence and immunoreactivity to the neuronal isoform of NO synthase was identified in fluorescently identified baroreceptor neurons. These results indicate that NO/NO-related species function as autocrine regulators of Na+ currents in baroreceptor neurons. Modulation of Na+ channels may represent a novel response to NO.
Stable isotope tagging methods have enabled relative quantitation of proteins between samples in LC-MS/MS analyses. However, most such methods are not applicable to the differential quantitation of modified proteins because the isotope tagging reagents only react with certain peptides or because the reagents incorporate a mass increment that is too small to allow reliable quantitation on low resolution ion trap MS instruments. Here, we describe the use of d0- and d5-phenyl isocyanate (PIC) as N-terminal reactive tags for essentially all peptides in proteolytic digests. PIC reacts quantitatively with peptide N-terminal amines within minutes at neutral pH and the PIC-labeled peptides undergo informative MS/MS fragmentation. Ratios of d0- and d5-PIC-labeled derivatives of several model peptides were linear across a 10000-fold range of peptide concentration ratios, thus indicating a wide dynamic range for quantitation. Application of PIC labeling enabled relative quantitation of several styrene oxide adducts of human hemoglobin in LC-MS/MS analyses. PIC labeling offers a versatile means of quantifying changes in modified or variant protein forms in paired samples.
The use of hemoglobin as a predictor of response to iron therapy, for screening, and for prevalence estimates was studied. An Fe supplementation trial was performed in Quito, Ecuador, in which 412 pregnant women were randomly assigned to treatment and control groups. Women in the treatment group received 390 mg ferrous sulfate/d for 2 mo. The prevalence of Fe deficiency as defined by response to therapy was found to be 60.8%. Sensitivity and specificity were calculated at various cutoff points of hemoglobin. The estimates of sensitivity and specificity allow for the use of hemoglobin in screening for Fe deficiency anemia and in the estimation of the prevalence in populations with characteristics similar to those found in the sample of pregnant women in Quito. Hemoglobin was shown to be a good predictor of response to Fe treatment and a good estimate of prevalence of Fe deficiency when prevalence is high.
Native hemoglobin in the form of stroma-free hemoglobin cannot be used as blood substitute. Hemoglobin has to be modified either molecularly or encapsulated. First generation molecularly modified ultrapure hemoglobins are now in clinical trial--some in Phase III. There are a number of these. Polyhemoglobin is formed by crosslinking hemoglobin molecules intermolecularly and intramolecularly. A crosslinked single hemoglobin molecule is formed by crosslinking hemoglobin intramolecularly. Recombinant hemoglobin from E.coli is formed by fusion of the subunits of each hemoglobin molecule. Conjugated hemoglobin is formed by crosslinking each hemoglobin molecule to soluble polymers. A second generation system formed by crosslinking hemoglobin-superoxide dismutase-catalase is being developed. A third generation hemoglobin-based blood substitute is based on microencapsulated hemoglobin, artificial red blood cells, that more closely resemble a complete red blood cell.
OBJECTIVE: To assess the efficacy of oral iron therapy in the recovery of patients' hemoglobin levels after major surgery. DESIGN: Randomized controlled trial. SETTING: Private orthopedic practice confined to one large community hospital. PATIENTS: One hundred seventy consecutive elderly patients undergoing hip surgery; 75 failed to meet entry hematologic or medical criteria; 95 were randomized, with 16 withdrawn because of complications. INTERVENTION: Thirty-seven patients received ferrous sulfate orally four times a day for the duration of their hospitalization. Forty-two patients who received no iron supplement served as the control group. MAIN OUTCOME MEASURES: Changes in hemoglobin levels and reticulocyte counts over the 2- to 3-week follow-up period. RESULTS: There was no significant difference in mean hemoglobin levels between the treatment and control groups (95% confidence interval [CI] for difference of -6.6 to 5.4 g/L). Corrected reticulocyte fractions increased equally in both groups (95% CI for difference of -9 x 10(3) to 2 x 10(-3). The study was designed to detect a difference in mean hemoglobin levels of 8.5 g/L or greater or a difference in mean reticulocyte fraction of 10 x 10(-3) between the two groups with a power of 0.80 at the .05 (two-sided) level of significance. CONCLUSION: The administration of oral iron supplements to elderly, healthy orthopedic patients postoperatively did not hasten the recovery of hemoglobin levels, provided adequate tissue iron stores were present.
Levels of 4-aminobiphenyl-hemoglobin adducts in smokers of blonde (flue-cured) and black (air-cured) tobacco have been found to be proportional to bladder cancer risk. In addition, risk of bladder cancer due to exposure to occupational carcinogens is elevated in genetically determined slow acetylators. In this study of normal male volunteers, 4-aminobiphenyl-hemoglobin adducts were found to be related to both the quantity and the type of tobacco smoked, as well as to the acetylator phenotype (independently of smoking habits). The demonstration that both the genetically determined slow acetylator phenotype and tobacco smoking are independently associated with levels of the carcinogen 4-aminobiphenyl in adducted hemoglobin suggests a single mechanism to explain the contribution of genetic susceptibility and environmental exposure in bladder carcinogenesis.
To assess the effects of iron therapy on platelet monoamine oxidase (MAO) activity and urinary excretion of total metanephrines (MN) in infants and young children with iron deficiency anemia, 24 subjects were tested before and after one month of oral iron treatment. Thirteen healthy children comprised the control group. In the control group, platelet MAO level was 0.21 +/- 0.02 U/mg protein (mean +/- SE), urinary total metanephrine was 2.51 +/- 0.47 micrograms/mg creatinine. In cases with iron deficiency, mean platelet MAO level was 47.6% lower (p less than 0.005) whereas mean urinary metanephrine plus normetanephrine (MN-NMN) was only 20.7% lower (p greater than 0.05) than the control values. After one month, the anemic patients receiving oral iron therapy showed a significant increase in hemoglobin concentration, per cent transferrin saturation and platelet MAO activity (p less than 0.05). However, urinary metanephrine excretion was found to be lower in this group when compared to the metanephrine levels in iron deficiency before the medication (p less than 0.05). Although hemoglobin and transferrin saturation did not return to normal levels, these findings suggested that platelet MAO activity increased and urinary excretion of metanephrines decreased after iron medication.
Styrene is metabolized to styrene oxide, a direct-acting mutagen and carcinogen. Styrene oxide reacts with DNA mainly at the N-7 position in guanine, but also at other sites and with other bases. Substitution occurs at both the alpha- and beta-positions of the styrene molecule. Experiments with radiolabeled styrene and styrene oxide demonstrate that both have a low level of DNA binding activity in experimental animals. 32P-Postlabeling studies have demonstrated the potential of the technique to detect styrene-DNA adducts. Styrene oxide alkylates several nucleophilic sites in proteins, particularly cysteine sulfydryl, histidine imidazole, lysine amino, aspartic, and glutamic carboxylic groups, and the N-terminal position. In experimental animals, styrene oxide treatment results in cysteine adducts in hemoglobin and albumin, valine adducts in hemoglobin, and carboxylic acid adducts in hemoglobin. The extent of alkylation is low compared with that produced by ethylene oxide. The available evidence indicates, therefore, that styrene and styrene oxide have low DNA and protein binding activities in vivo. There is preliminary evidence for the presence of DNA adducts and for adducts in hemoglobin and albumin in blood cells of styrene-exposed workers. Nevertheless, the applicability and sensitivity of DNA and protein adduct detection methods for monitoring human exposure to styrene remain to be determined.
In order to explore the effect of sodium iron ethylenediaminetetraacetate (NaFeEDTA) on preventing rats from lead poisoning and reducing the lead toxicity, and at the same time to explore its impact on zinc and copper, sixty Wistar rats were randomly divided into six groups including blank control group, model control group, positive control group and three different level NaFeEDTA (high, middle and low) groups. With the exception of the blank control group, which only received double distilled water treatment throughout the experiment, all the other rats were continuously treated with lead solution. At the same time, with the exception of the blank and model control groups, the other four groups were treated with acidi dimercaptosuccinici (DMSA), high, middle and low level NaFeEDTA solutions respectively. At the end of the study, several indicators were determined. The results showed that the hemoglobin levels in the three different level NaFeEDTA groups were significantly higher than those of the other four groups and at the same time the lead levels in blood and organs were lower than those of model group significantly. There were significant reverse correlations between iron supplement and the lead levels in liver, kidney and tibia among the three different level NaFeEDTA groups, and the correlation coefficients were -0.4432, -0.6134 and -0.3878 respectively. There were no significant differences in zinc levels and copper levels between the three level NaFeEDTA groups and the negative control group except the brain zinc levels in the middle and high level NaFeEDTA groups were lower and the kidney copper level in the low level NaFeEDTA group was higher than those of the model control group. It is concluded that the NaFeEDTA can effectively improve hemoglobin level, prevent rats from lead poisoning and reduce the lead toxicity. At the same time, NaFeEDTA does not have significant influence on zinc and copper levels in rats.
In the context of limited effectiveness of iron supplementation programs, intermittent iron supplementation is currently under debate as a possible alternative strategy that may enhance the effectiveness of operational programs. This field-based trial assessed the outcome of twice weekly iron supplementation compared to daily in Pakistan. A double-blind, randomized, clinical trial was conducted in Northern Pakistan. Anemic pregnant women (n = 191) were assigned to receive daily (200 mg ferrous sulfate) or twice weekly (2 x 200 mg ferrous sulfate) iron supplementation. Hemoglobin was measured at baseline and at 4-wk intervals for up to 12 wk. Serum ferritin was measured at baseline and 8 or 12 wk. Analysis was by intention to treat. The two groups did not differ in age, parity, sociodemographic characteristics, hemoglobin or serum ferritin concentrations at baseline. Women who received iron daily had a greater rise in hemoglobin compared with women who received iron twice weekly (17.8 +/- 1.8 vs. 3.8 +/- 1.2 g/L, P < 0.001). The serum ferritin concentrations increased by 17.7 +/- 3.9 microgram/L (P < 0.001) in the daily supplemented group and did not change in the twice weekly group. Daily iron supplementation remained superior to twice weekly supplementation after controlling initial hemoglobin Z-scores and duration of treatment. The body mass index (BMI) modified the effect of daily versus twice weekly iron supplementation. For every unit increase in BMI, the difference between the two treatment groups was reduced by 0.0014 (final hemoglobin Z-score; P = 0.027). We recommend continuation of daily iron supplementation as opposed to intermittent iron supplementation in pregnant women in developing countries.
We automated the Jendrassik-Grof method for measurement of bilirubin with the Greiner Selective Analyzer GSA II D. Comparison with the manual Jendrassik-Grof method showed a highly significant correlation coefficient of 0.994 for total and 0.980 for "direct" bilirubin measurement. The automated method showed good precision and was linear to 667 mu mol/L, the highest concentration tested. Addition of hemolysate caused a greater effect on the measurement of direct bilirubin than on total bilirubin. The inhibitory effect on measurement of total bilirubin, though small, was statistically significant at hemoglobin concentrations greater than 2 g/L. Comparison with the method involving diazotized 2,4-dichloroaniline showed the Jendrassik-Grof method to be relatively less affected by hemolysis and turbidity in the measurement of total bilirubin.
Cytoplasmic monomeric hemoglobin I from the bacteria-harboring gill of the bivalve mollusc Lucina pectinata has been crystallized in a form suitable for atomic resolution X-ray structural investigations. The crystals have been grown at pH 4.8, in 0.05 M-acetate buffer, using 2.6 M-ammonium sulfate as precipitating agent. The crystals belong to the monoclinic space group P2(1), with unit cell constants a = 50.0 A, b = 38.6 A, c = 42.1 A, beta = 107.1 degrees, and contain one molecule (14,000 Mr) in the asymmetric unit. By means of single crystal microspectrophotometry it has been shown that the crystals contain the ferric form of L. pectinata "sulfide reactive" hemoglobin I. On the other hand, by careful control of the buffering medium composition, it has been possible to obtain stable crystals of the deoxy, oxy and sulfide forms of the protein.
We report a novel reagent containing ferric perchlorate, perchloric acid, and polyoxyethylene (23) lauryl ether (Brij 35) with which the concentration of chloride in serum can be measured. We applied this reagent to use with a centrifugal analyzer (CentrifiChem 400) in a dynamic bichromatic procedure, resulting in broad linearity of the standard curve (0-180 mmol/L), short analysis time (1 min), and little interference from bilirubin, hemoglobin, turbidity, or bromide ions. The reagent is simple, contains no mercury, and the combination of low acid concentration and surfactant prevents serum protein precipitation. Precision is good (for x- = 93 mmol/L, CV = 1.55%), and results correlate well with those obtained by coulometry (r = 0.974).