A realistic evaluation of the action of ozone on whole human blood

We have clarified the role of the ozone concentration in relation to the resistance of human erythrocytes in whole human blood or in blood diluted either in saline or in distilled water.

Spectrophotometric data related to haemoglobin were evaluated by exposing samples of fresh human blood directly to ozone doses (ratio 1:1 volume), within the therapeutic range (0.21–1.68 mM) and to one toxic dose (3.36 mM). Furthermore, the same determinations have been carried out after previous dilution of the same blood with either pure water or physiological saline (1 ml blood + 29 ml diluent) followed by ozonation with the above reported ozone doses. Addition of either saline or water implies a dilution of plasma antioxidants and also total haemolysis after water dilution. Particularly the latter case represents a most unphysiological situation because the osmotic shock causes the solubilization of the erythrocytic content. While it is possible to demonstrate that after haemolysis there is an ozone-concentration dependent transformation of some oxyhaemoglobin to methaemoglobin, no such process occurs after ozonation of whole blood.

The results of this study fully confirm our previous data that judicious ozone doses neither damage erythrocytes, nor induce oxidation of intracellular haemoglobin. We hope that our conclusions will definitively clarify the absence of toxicity of ozonetherapy.     

In vitro metabolism of selenite in sheep blood: factors controlling the distribution of selenium and the labelling of plasma protein.

Some factors controlling the distribution of Na275SeO3 in sheep blood were studied in vitro. After centrifuging Na275SeO3-incubated blood most of the radioactivity was found in the plasma. The labelling of plasma protein by 75Se was dependent on the presence of erythrocytes. The degree of labelling of plasma protein increased with erythrocyte concentration. When phosphate-buffered saline-washed erythrocytes were suspended in phosphate-buffered saline and incubated with Na275SeO3 the majority of the 75Se was detected in the erythrocytes. On incubating these labelled erythrocytes with unlabelled plasma there was a transfer of radioactivity to the plasma. The calculated activation energy for the labelling of plasma was 107.52 kJ/mol. Albumin was shown not to be a principal acceptor of 75Se from the erythrocytes by ammonium sulphate precipitation of radioactive plasma. Addition of Na2SeO3 to the labelled blood resulted in the transfer of 75Se from plasma to the erythrocytes. Radioactive plasma incubated at 37 degrees C was thermolabile with respect to its 75Se content whereas in whole blood the degree of 75Se binding to plasma protein did not vary suggesting that a recycling of selenium was occurring in blood. From the results presented an in vitro model of selenium metabolism in blood is postulated.     

The nutritional importance of dietary blood components for reproduction in the tsetse fly, Glossina morsitans

Females of Glossina morsitans morsitans were fed diets of different composition and their performance in terms of survival, fecundity and offspring size, used as a basis for assessing the nutritional importance of the dietary constituents for reproduction. Diets comprised defibrinated bovine blood from which individual components had been removed, or various fractions of bovine blood mixed in different ratios and combinations.Flies fed serum-free diets (comprising saline-washed bovine erythrocytes) failed to reproduce and their ovaries were atrophied. Attempts were made to correct these symptoms by adding serum components to washed erythrocytes. Results showed that serum albumin and lipoproteins were vital for ovarian growth. The erythrocytes in defibrinated bovine blood could be replaced by preparations of haemoglobin. Dialysis against isotonic sodium chloride, removal of gamma globulins, removal of lymphocytes or heating to 55°C for 2 h did not alter the nutritional quality of defibrinated bovine blood. The qualitative importance of dietary albumin associated lipids and lipoproteins for oöcyte growth suggests a specialisation which may be peculiar to viviparous Diptera. Results are discussed in terms of the development of synthetic diets for tsetse.     

Correction of Metabolic Indicators of Erythrocytes and Myocardium Structure with Ozonized Red Blood-Cell Mass

The early posttransfusion period after acute blood loss using erythromass without ozone was characterized by decreased electrophoretic mobility of erythrocytes (EPME), ATP, 2,3-diphosphoglycerate (2,3- DPG) concentration, Na⁺-K⁺-ATPase activity, and increased concentration of malonic dialdehyde (MDA) and catalase activity in erythrocytes. Most parameters gradually were restored over 5 days, but a reduced concentration of 2,3-DPG was observed throughout the entire experiment with transfusion of erythromass without ozone. Transfusion of the ozonized erythrocyte mass 1 h after its administration caused an increase in 2,3- DPG concentration in erythrocytes circulating in the vascular bed. Twenty-four hours later, increased catalase activity, EPPE, and ATP concentration were registered, and, after 5 days, enhanced Na⁺-K⁺-ATPase activity was registered. Their values were maintained until the end of the experiment at a higher level than in animals that had undergone transfusion of the nonozonized erythromass. Ozonized erythrocyte mass improves the functional metabolic state of erythrocytes, promotes an earlier recovery of the oxygen transport in blood, and limits the damage to the microcirculatory bed of the myocardium and cardiomyocytes during blood loss.     

Oxidative stress and potential free radical damage associated with photocopying. A role for ozone?

OBJECTIVE: To study the relationship between oxidative stress and potential free radical damage associated with photocopying and to explore a role for ozone emitted during the photocopying process. METHODS: 80 photocopying operators (PO) and 80 healthy volunteers (HV) were enrolled in a random control study design, in which the level of lipoperoxide (LPO, thiobarbituric acid reactive substances, TBARS) in erythrocytes and the levels of vitamin C (VC), vitamin E (VE) and beta-carotene (beta-CAR) in plasma as well as the activities of superoxide dismutase (SOD) and catalase (CAT) in erythrocytes were determined by spectrophotometric methods. RESULTS: Compared with the average values of the above biochemical parameters in the HV group, the average value of LPO (TBARS) in erythrocytes in the PO group was significantly increased (P < 0.0001), while the average values of VC, VE and beta-CAR in plasma as well as those of SOD and CAT in erythrocytes in the PO group were significantly decreased (P < 0.0001). Pearson product-moment correlation analysis showed that with the increase of the ozone level in photocopying sites and the PO duration of exposure to ozone, the level of LPO in erythrocytes in the operators was increased (P < 0.001), while the levels of VC, VE and beta-CAR in plasma as well as the activities of SOD and CAT in erythrocytes in the operators were decreased (P < 0.01-0.0001). CONCLUSION: The findings in this study suggest that ozone causes oxidative damage in copier operatives.     

Increased Release of Free Fe Ions in Human Erythrocytes During Aging in the Circulation

We investigated whether free Fe ions were released in erythrocytes during aging process in the circulation. Young and senescent erythrocytes were separated from freshly drawn human blood by Percoll density gradient centrifugation. Two different methods were employed for determination of free Fe ions in erythrocytes, desferrioxamine (DFO) method and bleomycin method. DFO-chelatable Fe ions were detected in whole erythrocytes from 2 donors, and the DFO-chelatable free Fe ion levels in senescent erythrocytes were higher than those in young erythrocytes. Bleomycin-sensitive Fe ions, which was rather lower than DFO-chelatable Fe ions, were also detected in whole erythrocytes from 5 donors, and the free Fe ion levels in senescent erythrocytes were also higher than those in young erythrocytes. Free Fe ions may be derived from oxidative damage of hemoglobin, because treatment of whole erythrocytes or purified oxyhemoglobin with hydrogen peroxide gave increased free Fe ions. The results indicated that free Fe ions were released from erythrocytes during aging process in the circulation. Released free Fe ions would promote oxidative damages of the cells during aging process.     

Increased release of free Fe ions in human erythrocytes during aging in the circulation

We investigated whether free Fe ions were released in erythrocytes during aging process in the circulation. Young and senescent erythrocytes were separated from freshly drawn human blood by Percoll density gradient centrifugation. Two different methods were employed for determination of free Fe ions in erythrocytes, desferrioxamine (DFO) method and bleomycin method. DFO-chelatable Fe ions were detected in whole erythrocytes from 2 donors, and the DFO-chelatable free Fe ion levels in senescent erythrocytes were higher than those in young erythrocytes. Bleomycin-sensitive Fe ions, which was rather lower than DFO-chelatable Fe ions, were also detected in whole erythrocytes from 5 donors, and the free Fe ion levels in senescent erythrocytes were also higher than those in young erythrocytes. Free Fe ions may be derived from oxidative damage of hemoglobin, because treatment of whole erythrocytes or purified oxyhemoglobin with hydrogen peroxide gave increased free Fe ions. The results indicated that free Fe ions were released from erythrocytes during aging process in the circulation. Released free Fe ions would promote oxidative damages of the cells during aging process.     

Chemical kinetics measurements on the reaction between blood and ozone

The pseudofirst-order ozonization rate constant of whole bovine blood has been measured in comparison to that of free haemin. The free prosthetic group haemin (which has also the central iron atom in the oxidized form) shows k values in the range of 0.20-0.03 s(-1) while the haeme groups inside haemoglobin protein and contained in the whole blood sample show slightly lower k values, just in the range of 0.10-0.02 s(-1). It has been found that ozone even with whole blood reacts specifically with haemoglobin of the red cells because it is adsorbed selectively on the iron atoms of the haeme prosthetic groups of haemoglobin. The absorption implies the oxidation of the central iron atom of the haeme groups with formation of methaemoglobin followed by an oxidative fission of the haeme rings. The other blood components do not exert any significant protection to the reaction between ozone and haemoglobin, which appear extremely specific and selective like the reaction between CO or HCN and haemoglobin. By analogy with the behaviour of these other gases ozone may be classified as a blood poison. The results of this work are discussed in the frame of the risks connected to the ozonotherapy and autohaemotherapy involving the blood ozonization of human or animal subjects and the re-injection of ozonized blood into the bodies.     

Reaction of ozone with glycophorin in solution and in lipid vesicles.

Glycophorin from human red blood cells was exposed to ozone in aqueous solution. Amino acid analysis of glycophorin exposed to a 10-fold molar excess of ozone showed that the only residue affected was methionine. Both methionine residues of the protein were oxidized to methionine sulfoxide. Exposure of the oxidized protein to cyanogen bromide caused no cleavage of the polypeptide chain. Glycophorin was incorporated into unilamellar lipid vesicles made from phosphatidylcholine. The protein containing vesicles were exposed to ozone in a 10-fold molar excess to the glycophorin. Gas chromatography of the methyl esters showed negligible change in the fatty acid composition. Amino acid analysis of the ozone-treated protein showed the oxidation of only one methionine residue per polypeptide chain to methionine sulfoxide. Ghosts of human erythrocytes were exposed to ozone. Cyanogen bromide treatment of the oxidized glycophorin yielded fragments showing that the only methionine residue oxidized by ozone was residue 8. These results indicate that in this membrane model (a) amino acid is more susceptible to ozone than is the lipid, and (b) amino acids external to the membrane are more susceptible than those in the polypeptide chain spanning the membrane.     

Protective effect of (−)-epigallocatechin-3-gallate on capsaicin-induced DNA damage and oxidative stress in human erythrocyes and leucocytes in vitro

The aim of this study is to show that protective effects of the main catechin (−)-epigallocatechin-3-gallate (EGCG) against capsaicin (CAP) induced oxidative stress and DNA damage in human blood in vitro. Superoxide dismutase, catalase, glutathione peroxidase and malondialdehyde (MDA) level were studied in erythrocytes and leucocytes with increased concentrations of CAP. DNA damage in leucocytes was measured by the comet assay. Human blood cells have been administered with doses between 0 and 200 μM of CAP and/or EGCG (20 μM) for an hour at 37 °C. Treatment with CAP alone has increased the levels of MDA and decreased antioxidant enzymes in human blood cells. A significant increase in tail DNA%, mean tail length and tail moment indicating DNA damage has been observed at the highest dose of CAP treatment when compared to controls. Treatment of cells with CAP plus EGCG prevented CAP-induced changes in antioxidant enzyme activities and MDA level and mean tail lenght indicating DNA damage. A significant increase in mean tail lenght was observed at high doses of CAP. These data suggest that EGCG can prevent toxicity to human erythrocytes and leucocytes caused by CAP, only at low doses.     

Estimation of some molecular effects of gaseous nitrogen oxide on human blood in vitro

The aim of this work is complex estimation of the nitric oxide action on whole blood of healthy people. We tested the reaction of whole human blood (n = 14) to the processing of it with cold NO-containing plasma. We performed direct sparging of blood samples by gaseous flow with NO in a special plant. Cold NO-containing plasma was generated by apparatus “Plazon” (Russia). We tested lactate dehydrogenase activity in direct and reverse reactions, aldehyde dehydrogenase and superoxide dismutase activity, total protein and lactate level, acid-base balance and the partial pressure of gases in blood. For integral assessment of energy metabolism changes a number of derivative parameters (coefficients of energy reaction balance and substrate provision) were used. Our experiments showed that the processing of whole human blood with NO-containing gas flow (NO concentration — 800 ppm) results in significant changes of its physical and chemical parameters. This exposure leads to inhibition of erythrocytes energy metabolism, decreasing plasma antioxidant reserves, developing moderate ionic disorders and acid-base disbalance in blood samples in vitro.     

Studies on the methodology of the carboxyfluorescein assay and on the mechanism of liposome stabilization by red blood cells in vitro

The stability of small unilamellar liposomes was investigated in human blood, in vitro. Using the carboxyfluorescein technique, interaction between the dye, the detergent Triton X-100, and an as yet unidentified component of human serum grossly interferes with the experiment and necessitates the use of other detergents, preferably sodium deoxycholate. Separation of liposomes and blood cells by centrifugation induces a small leakage from the liposomes and can lead to an underestimation of the real liposome stability. Upon incubation with whole blood, intact liposomes are absorbed nonspecifically to erythrocytes and internalized by leukocytes, the extent and kinetics of the former process being insenstive to the presence of metabolic inhibitors. The stability of liposomes is significantly enhanced in whole blood or in serum containing washed erythrocytes. Similarly, liposome stability in serum could be augmented be presaturating the serum lipoproteins with excess phospholipid. Our work adds support to previous notions that stable liposomes with high affinities for certain blood-cell components might be developed as suitable carrier systems for drug targetting in pathological disorders within the blood stream.     

Whole blood leukocytes isolation with microfabricated filter for cell analysis

The flow cytometric analysis of leukocytes in whole blood usually requires isolation of leukocytes from other components of whole blood. Density gradient centrifugation and red blood cell lysis are the most commonly used methods to separate leukocytes but come with significant limitations. We report the results of the evaluation of a microfabricated filtration device for blood preparation that separates erythrocytes from leukocytes based on their size and mechanical properties. The microfabricated filter evaluated here requires a rapid and simple procedure and results in high leukocytes recovery without introducing bias among the leukocyte subpopulations. The filter removes erythrocytes, platelets, plasma proteins, and unbound staining reagent. This gentle filtration process produces very clean stained leukocytes for cytometric analysis without any apparent damage to leukocytes.     

Ultrastructural changes of erythrocytes in whole blood after exposure to prospective in silico-designed anticancer agents: a qualitative case study

Background

Novel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample.The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female’s erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C.

Results

No significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress.

Conclusions

From this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.     

Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment

The development of comet assay for aquatic organisms is of particular relevance in light of the importance of coastal fisheries to several countries around the world. Two of the most common fish species native to southern Brazil are the gray mullet (Mugil sp.) and sea catfish (Netuma sp.) for which we have produced a standardized comet assay using whole erythrocytes taken from samples of these fish. We investigated the potential of the comet assay for monitoring genotoxicity in mullet and sea catfish and made a preliminary investigation of the baseline levels of DNA damage in the erythrocytes of samples of these fish from non-polluted areas as well as assessing the in vitro sensitivity of erythrocyte exposed to 2, 4 and 8 x 10(-5) M of methyl methanesulfonate (MMS) for 1, 2, 6 and 24h at 25 and 37 degrees C. Our results show that there was an increase in baseline DNA damage at higher temperatures and that the amount of MMS-induced DNA damage also increased at higher temperatures and that there was a clear dose/time response to treatment with MMS. To assess the possibility of using fish for environmental biomonitoring we also used the comet assay to investigate the in vitro genotoxic effect of MMS on whole blood cells from human donors and found a clear concentration-related effect at all exposure times, findings which agree with those of other workers. This study demonstrates the potential application of the comet assay to erythrocytes of mullets and sea catfish. However, these findings also suggest that temperature could alter both baseline DNA damage in untreated animals and in vitro cell sensitivity towards genotoxic pollutants.     

Changes in allosteric properties of phosphofructokinase bound to erythrocyte membranes.

Human and rabbit erythrocyte membranes prepared by hypotonic hemolysis contained 5 to 15% of the phosphofructokinase in the erythrocytes. The membrane-bound phosphofructokinase can be eluted by a saline wash. Human erythrocyte and rabbit muscle phosphofructokinase bind to the saline-washed membranes. This binding is specific for the inner surface of the membrane. The amount of phosphofructokinase bound is dependent on pH; at pH 7, 6 times more enzyme is bound than at pH 7.5. Unlike free phosphofructokinase, the membrane-bound phosphofructokinase is not inhibited by ATP or 2,3-diphosphoglycerate, and its fructose-6-P saturation curve is nonsigmoidal.     

Thixotropic properties of whole blood from healthy human subjects

The steady state non-Newtonian viscosity of whole human blood has been widely studied as a function of the shear rate; and used to characterize the blood in various pathological disorders. In our previous studies, we demonstrated that blood is a thixotropic fluid. Its time-dependency and shear rate dependency of rheological behavior can be represented by an equation developed by Huang. Parameters of the equation can be used for the characterization of an individual's blood. They provide information, such as the kinetic rate constant of breakdown of RBC rouleaux to individual erythrocytes and the relative amount of rouleau formation in the dynamic equilibrium between rouleaux and individual erythrocytes. In this communication, the thixotropic parameters from blood samples of fifteen apparently healthy human subjects were investigated. When compared to the use of apparent viscosity values for the correlation with a pathological disorder, thixotropic parameters are preferable. The mean values of thixotropic parameters obtained from apparently healthy human subjects provide a base for comparison with the same parameters as obtained from blood samples of patients with certain pathological disorders involving the circulation.     

Hydrolysis of guanosine and adenosine 3',5'-monophosphates by rat blood.

Cyclic nucleotide phosphodiesterase activity was measured in whole blood, plasma, and suspensions of platelets and erythrocytes from rats. In fresh whole blood, apparent phosphodiesterase activity was low, but it rose strikingly during the hour after blood withdrawal. The apparent phosphodiesterase activity in platelet-free plasma showed no such increase, but that in platelet-enriched plasma increased in parallel with that in whole blood. The apparent phosphodiesterase activity of blood or of platelet-enriched plasma also was increased markedly by sonication. The increase in rat blood phosphodiesterase activity with aging thus appeared to be due to damage of platelets. Most of the phosphodiesterase activity in rat erythrocytes and platelets was located in the soluble fraction of sonicated preparations, but the total enzyme activities from the two sources exhibited marked differences in substrate specificity. With erythrocyte preparations, the rate of hydrolysis of muM concentrations of cyclic AMP was approx. 50 times that of cyclic GMP, while with platelet preparations, cyclic GMP was hydrolyzed about 20 times faster than cyclic AMP at muM levels. The activity of phosphodiesterase in platelets was much greater than that in erythrocytes at all concentrations of both substrates.     

Effect of 2,3-diphosphoglycerate concentration on the alkaline fragility of phosphofructokinase-deficient canine erythrocytes

1. Erythrocytes in whole blood samples from dogs with phosphofructokinase (PFK) deficiency had lower 2,3-diphosphoglycerate (2,3-DPG) concentrations, higher ATP concentrations, and were more alkaline fragile than normal canine erythrocytes. 2. Reticulocytes from a PFK-deficient dog contained nearly three times the ATP concentration of normal canine erythrocytes, and had 2,3-DPG concentrations similar to normal canine erythrocytes. 3. PFK-deficient reticulocytes are not alkaline fragile. 4. The erythrocyte 2,3-DPG concentration in whole blood samples from PFK-deficient dogs was increased to normal by in vitro incubation with dihydroxyacetone, pyruvate and phosphate. This incubation resulted in only a slight increase in ATP concentration. 5. The alkaline fragility of these 2,3-DPG replenished PFK-deficient erythrocytes was normal. 6. Findings in this study indicate that the increased alkaline fragility of canine PFK-deficient erythrocytes is the result of decreased intracellular 2,3-DPG concentration.     

Acetaminophen protects human erythrocytes against oxidative stress

Acetaminophen protects human erythrocytes against various modes of oxidative stress. Protection against ozone-induced damage can be explained by a direct scavenging reaction between the drug and ozone. With t-butylhydroperoxide acetaminophen appeared to be an effective scavenger of radicals, generated in secondary reactions. The protection by acetaminophen against t-butylhydroperoxide- and hydrogen peroxide-induced lipid peroxidation and K+-leakage can be explained along these lines. In all cases the protective effect of acetaminophen was attended with covalent binding of acetaminophen to membrane proteins.