Flavonoids from italian multifloral honeys reduce the extracellular ferricyanide in human red blood cells.

In this study we investigated some biological properties of flavonoids recovered in the aqueous (AqE) and ether (EtE) extracts from four Italian multifloral honeys. In particular, a cell-free assay was employed to detect direct reduction of ferricyanide, whereas an assay using intact human erythrocytes was used to measure the ability to donate electrons to a trans-plasma membrane oxidoreductase. It was found that the AqE displays greater "in vitro" ferricyanide-reducing activity than the EtE but, unlike the latter, is virtually ineffective in the cell-based assay. Uptake studies employing high-performance liquid chromatography/mass spectrometry (HPLC/MS) showed that the different results were explained by the inability of AqE components to cross the erythrocyte plasma membrane and by the excellent uptake of EtE flavonoids, which, once within the cell, donate electrons to the membrane oxidoreductase to efficiently reduce extracellular oxidants. The latter property appears to depend on the content of ether-soluble flavonoids in the starting honeys.

Reactions of phenyldiazene and ring-substituted phenyldiazenes with ferrihemoglobin.

Two simultaneous reactions take place between ferrihemoglobin and phenyldiazene in the absence of excess ferricyanide or of oxygen, namely the reduction of ferrihemoglobin to ferrohemoglobin and the binding of an exogenous ligand by ferrihemoglobin to form a compound with the optical spectrum of a ferrihemochrome. In the presence of excess ferricyanide, only the formation of a ferrihemochrome is observed. This compound differs from the ferrihemochrome induced by salicylate or by benzoate with respect to optical spectrum, concentration of inducer, and stability. One phenyl group is bound per heme, probably as phenyldiazene. Phenyl groups are also bound to globin, but phenyldiazene does not react anaerobically with thiols. Each ring-substituted isomer of methylphenyldiazene or bromophenyldiazene yields a different ferrihemochrome spectrum with ferrihemoglobin in the presence of ferricyanide. Only reduction of ferrihemoglobin occurs with 2- or 4-diazenylbenzoic acid in the absence of excess ferricyanide, but partial formation of ferrihemochrome occurs with 4-diazenylbenzoic acid in excess ferricyanide. The ability of an aryldiazene to bind quantitatively to ferrihemoglobin parallels the ability of the corresponding arylhydrazine to induce in vivo hemolysis.

Redox properties of components I and IV of trout hemoglobins: kinetic and potentiometric studies.

Redox properties of component I and IV from trout hemoglobin (Salmo irideus) have been studied kinetically and at equilibrium. In the case of component I of trout hemoglobin, the mid-point potential (Eh) is pH independent below the acid-alkaline transition (pKa approximately equal to 8.6) and decreases at higher pH, following the deprotonation of the water molecule. Similarly to human hemoglobin, the mid-point potential of component IV of trout hemoglobin is pH-dependent, but the redox Bohr effect is extended to more acid pH. Moreover, the cooperativity of the redox equilibrium process is higher than in human hemoglobin. These features parallel the oxygen-binding properties of the same hemoglobin components from trout hemolysate. Differently from human hemoglobin, the oxidation kinetics of the two hemoglobins from trout by potassium ferricyanide show markedly biphasic progress curves with pH-independent second-order rate constants. This behavior suggests a different energy barrier for the interaction with ferricyanide in the two types of subunit of both Hb components from trout.

Transmembrane electron transfer in diabetic nephropathy.

OBJECTIVE: Erythrocytes (red blood cells [RBCs]) reduce extracellular ferricyanide by transmembrane transfer of reducing equivalents involving ascorbate recycling. RESEARCH DESIGN AND METHODS: Because ascorbate regeneration is glutathione (GSH) dependent and cells may be depleted of GSH in diabetes, we measured RBC GSH, plasma sulfhydryl (SH) groups, and RBC-mediated ferricyanide reduction in 30 type 1 diabetic patients (age 34 +/- 10 years, disease duration 20 +/- 8 years; no complications, n = 10; retinopathy, n = 10; nephropathy, n = 10), their 36 siblings (age 39 +/- 13 years), and matched healthy volunteers. RESULTS: Fasting plasma glucose was 15 +/- 7 mmol/l (vs. 5 +/- 1 in control subjects, P < 0.001), HbA1c 8.4 +/- 1.5% (vs. 5.4 +/- 0.3, P < 0.001), GSH 0.76 +/- 0.12 mg/ml packed RBCs (vs. 0.88 +/- 0.18, P < 0.01), SH groups 401 +/- 72 micromol/l (vs. 444 +/- 56, P < 0.05), and ferrocyanide generation 15 +/- 5 micromol/ml RBC per h (vs. 13 +/- 5, NS). In comparison with 10 normoalbuminuric diabetic subjects with retinopathy, 10 patients with diabetic nephropathy had similar fasting plasma glucose, HbA1c, and SH groups; lower RBC GSH (0.73 +/- 0.08 vs. 0.85 +/- 0.11, P < 0.05); and higher ferrocyanide generation (18 +/- 4 vs. 14 +/- 5, P < 0.05). The 10 patients without complications differed from the 10 healthy volunteers in glycemic control and RBC GSH. RBC electron transfer correlated with plasma lactate (r = 0.8, P = 0.01) only in the uncomplicated group. No difference was detected between siblings and healthy control subjects or between siblings of subjects in the nephropathy and retinopathy groups. Among diabetic patients, the rate of ferrocyanide generation was associated with urinary albumin excretion, plasma creatinine, and SH groups (multiple r = 0.6, P < 0.01). CONCLUSIONS: Transmembrane electron transfer is selectively increased in diabetic nephropathy, where RBC GSH is also depleted. The abnormality is peculiar to the nephropathy group and not contributed by familial or hereditary components because the electron flow was normal in siblings. The close relationship between cytosolic NADH and RBC electron transfer observed in diabetic patients without complications seems to be lost in the microangiopathic patients. Whereas patients with retinopathy alone still had normal activity of the RBC-reducing system, patients with nephropathy showed significantly increased activity, unrelated to metabolic parameters or plasma lactate concentration and correlated with renal function parameters and plasma thiols.

Automated determination of red cell methaemoglobin reductase activity by a continuous-flow system for screening hereditary methaemoglobinaemia.

A flow diagram for the automated determination of ferricyanide reductase activity in red blood cells was prepared in the modules from AutoAnalyzer AA I (Technicon Instruments Inc). Ferricyanide reductase assay can be substituted for assay of cytochrome b5 reductase (EC 1.6.2.2), which plays a major role in reducing methaemoglobin in erythrocytes, and is defective specifically in the erythrocytes of patients with hereditary methaemoglobinaemia. The effective sampling rate of the analysis is 30/h, and less than 0.05 ml of whole blood is required. Interference of haemoglobin with absorption by potassium ferricyanide at 420 nm is effectively exculded by dialysis. This automated method was compared with the accepted diaphorase method, and it distinguished clearly the ferricyanide reductase activity of cord bloods from that of adult bloods. The activity of the blood from a patient with hereditary methaemoglobinaemia was only residual. It is suggested that the method is useful as a mass screening test for hereditary methaemoglobinaemia.

[Kinetics of human haemoglobin oxidation in normal and pathological states]. / Kinetika okisleniia gemoglobina cheloveka v norme i pri patologii

Kinetics of human haemoglobin oxidation by ferricyanide was studied in the process of development and in hematological diseases. The rate of haemoglobin oxidation was found to be altered in different ages and pathological states. Possible correlation between a peculiarity of oxidation reaction and haemoglobin composition is discussed.

How do erythrocytes contribute to the ABTS* scavenging capacity of blood?

It has been suggested lately that erythrocytes contribute significantly to the oxidant scavenging capacity (OSC) of blood and that surface adsorption of polyphenols enhances the antioxidant capacity of erythrocytes. The aim of this study was to examine the contribution of erythrocytes to the OSC of whole blood measured with a substrate not penetrating into the cells. Comparison of reduction of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical (ABTS*) by whole blood and blood plasma indicates that erythrocytes do contribute to ABTS* reduction but their contribution is lower with respect to plasma. ABTS* reduction by erythrocytes and its enhancement by polyphenols were inhibited by thiol reagents (N-ethylmaleimide and iodacetate). These reagents inhibited also the reduction of extracellular ferricyanide by erythrocytes and its enhancement by polyphenols. On this basis we postulate that the contribution of erythrocytes to the blood OSC estimated by ABTS* decolorization is at least partly due to the transmembrane-reducing system, which activity is routinely assayed by ferricyanide reduction.

Detection of ferricyanide as a probe for the effect of hematocrit in whole blood biosensors.

Measurement of the concentration of an analyte in whole blood can be influenced by a range of factors; the red cell content or hematocrit (Hct) of the sample, the distribution and rate of movement of analyte between red cells and plasma, the amount of protein in solution, the viscosity of the sample and fouling of the sensor. The effect of the red cells is the major factor that must be taken into account. Using the analyte molality rather than the analyte molarity, the theoretical response for a range of analytes which are found in plasma and in the red cells can be calculated. For an analyte which is found in plasma alone, the effect of hematocrit is significant, with a bias of -1% per %Hct; if the analyte can freely and rapidly diffuse between the red cells and plasma, this bias is reduced to zero. Using ferrocyanide as a model analyte, the effects of fouling and reduced sample viscosity were measured to be -0.2% per %Hct, giving an overall bias of -1.2% per %Hct, a level of bias which is not clinically acceptable. This bias can be negated by measuring the hematocrit separately and incorporating it into the measurement algorithm. Such a correction is essential for the correct measurement of the concentration of an analyte in whole blood.

Sodium nitroprusside and cyanide release: reasons for re-appraisal.

The standard method for estimating cyanide liberated from sodium nitroprusside (SNP) has been colorimetric. Using a cyanide ion-selective electrode technique, it was found that SNP remained unchanged in the presence of whole blood, plasma and washed erythrocytes, no cyanide being detected. Previously reported releases of cyanide from SNP in vivo and in vitro are probably inaccurate. Values obtained could have arisen not from reaction with blood, but from photo-decomposition during the lengthy analysis required by the colorimetric method or during infusion, if the solution was left uncovered. Because of the supposed cyanide hazard, low doses of SNP have been recommended; it may now be possible to revise these.

Metabolism of sodium nitroprusside in dogs awake and anesthetized with halothane.

Sodium nitroprusside (SNP) is rapidly metabolized to cyanide (CN) and thiocyanate (SCN). The authors determined the rates of CN and SCN production during SNP infusion sufficient to maintain blood pressure at 80 per cent of baseline in the dog awake and during halothane anesthesia. Each dog served as its own control. The endogenous whole-blood CN concentration was significantly lower in anesthetized dogs (0.6 nmol/ml) than awake dogs (1.8 nmol/ml). CN concentration increased similarly during SNP infusion in awake (4.5 nmol/ml) and anesthetized dogs (2.3 nmol/ml). In another group of dogs, whole-blood CN concentration decreased significantly due to halothane anesthesia. The regression coefficient was -0.21 nmol CN/ml/hr. There was no significant difference in plasma SCN concentration following infusion of SNP in both awake (33 nmol/ml) and anesthetized dogs (26 nmol/ml). The cause of this decreased blood CN concentration with or without SNP infusion during halothane anesthesia is not known.

[Determination of serum nitroprusside level when used in the treatment of accelerated hypertension (author's transl)]. / Technique de dosage sérique du nitroprussiate de sodium au cours de son utilisation dans le traitement des hypertensions malignes.

Using the effect of soluble sulfides in an alkaline medium on sodium nitroprusside, the authors were able to describe a method for titrating this substance. So, they could determine serum nitroprusside levels in patients treated for accelerated hypertension.

Reduction of extracellular potassium ferricyanide by transmembrane NADH: (acceptor) oxidoreductase of human erythrocytes.

Reduction of extracellular ferricyanide by intact erythrocytes proceeds by a membrane bound, NADH-dependent reaction. It is depressed by a glycolysis inhibitor and a non penetrable sulfhydryl reagent, and activated by dehydroascorbate. Dehydroascorbate activation cannot be accounted for by release of reducing equivalents from the cells. It is concluded that the observed reaction is brought about by transmembrane NADH-acceptor oxidoreductase with donor binding at the inner and acceptor binding at the outer cell surface.

Determination of cyanide and nitroprusside in blood and plasma.

A procedure was refined for quantitative isolation of cyanide by gas transfer from acidified blood or plasma samples. The cyanide was trapped in dilute alkali and quantified as the pyridine/pyrazolone complex. The within-day coefficient of variation was 2%, which increased to about 2.5% for the day-to-day variation. Nitroprusside used as a hypotensive agent in clinical medicine provides a risk of cyanide toxicity when the rate of administration or the total amount of drug given is excessive. A procedure was developed for measuring nitroprusside in the plasma of man and animals. Nitroprusside in the sample is quantitatively converted to cyanide by incubation with cystein solution at slightly alkaline pH. Methemoglobin is added to combine with the cyanide formed and prevent its destruction. On acidification, the total amount of cyanide originally present as free cyanide or as nitroprusside is liberated as HCN, isolated by gas transfer into a sodium hydroxide trap, and quantified by spectrophotometry. Nitroprusside present in the sample is calculated from the increase in cyanide observed in the cysteine-treated sample compared to that obtained without cysteine treatment. The method has been used to estimate in vitro stability of nitroprusside in aqueous solution, blood, and plasma. Blood cyanide and plasma nitroprusside concentrations were measured when sodium nitroprusside was infused into a baboon. Over 90% of the nitroprusside in blood is present in the plasma, suggesting that the drug crosses the erythrocyte membrane slowly.

A comparison of the behaviour of human adult and foetal haemoglobins on oxidation.

The measurements of oxidation-reduction potentials at pH 7.0 and ionic strength of 0.1 revealed only a slight difference between human adult (HbA) and foetal haemoglobin (HbF) (161 and 145 mV), which disappeared on increasing the ionic strength to 2.1. Kinetic data for the reactions with potassium ferricyanide and sodium nitrite of both haemoglobins in oxy- and carbonmonoxi-forms are presented at different excessive molar concentrations of oxidizing agents, in different environments at pH 7.0 and temperature ranging from 10--30 degrees C. The rate of HbF oxidation was considerably higher with both agents, despite vast differences in the reaction mechanisms.

Activation of a NADH dehydrogenase in the human erythrocyte by beta-adrenergic agonists: possible involvement of a G protein in enzyme activation.

NADH dehydrogenase in the plasma membrane transfers electrons from NADH to external oxidants like ferricyanide, through pathways which are linked to metabolic processes in the cell. Hormone binding to specific sites (receptors) can modify the enzyme activity, suggesting a direct or indirect coupling between the redox system and the hormone receptors. Reduction of external ferricyanide to ferrocyanide by human erythrocytes was stimulated by beta-adrenergic agonists (adrenaline, ritodrine and isoxsuprine), this effect being dependent upon concentration and pH. The agonist-stimulatory effect was attenuated in the presence of metoprolol (10(-4) M), a beta-adrenergic antagonist, and was not modified in the presence of prazosin, an alpha-adrenergic antagonist, suggesting that modification of the redox activity is mediated by binding of the agonists to beta-adrenergic receptors present in the human erythrocytes. Basal and agonist-dependent activities were inhibited in the presence of sulfhydryl reagents p-chloromercuribenzoate (PCMB, 10(-5) M) and N-ethylmaleimide (NEM, 10(-3) M), indicating the involvement of -SH groups. Inactivation by NEM was reversed by washing the cells with GTP (10(-3) M) and GTP gamma S (10(-4) M), suggesting that the specific alkylated -SH group(s) is located on a G protein in the hormone-receptor-G-protein complex. The human erythrocytes contain G proteins, displaying both guanine-nucleotide-binding properties and GTPase activity. Fluoride (10(-2) M) and fluoroaluminate (AlF4- (F-, 10(-2) M + Al3+, 10(-5) M), G protein activators, enhanced the basal and agonist-dependent activities, suggesting the involvement of G proteins in this system. The overall results indicated that one of the coupling components between the hormonal receptors and the redox system is probably a G protein, and the mechanism of enzyme activation after hormone binding to the receptor is based on the redox state of cysteine residues probably within the receptor-G-protein complex.