Oxygen, a paradoxical element?

Oxygen is an essential element for life on earth. No life may exist without oxygen. But in the last forty years, conclusive evidence demonstrated the double-edge sword of this element. In certain conditions, oxygen may produce reactive species, even free radicals. More, the production of reactive oxygen species (ROS) takes place everywhere: in air, nature or inside human bodies. The paradox of oxygen atom is entirely due to its peculiar electronic structure. But life began on earth, only when nature found efficient weapons against ROS, these antioxidants, which all creatures are extensibly endowed with. The consequences of oxygen activation in human bodies are only partly known, in spite of extensive scientific research on theoretical, experimental and clinical domains.

Is IGF-1 involved in the regulatory modifications of cholesterolemia following the administration of embryonary peptides?

Significant modifications of IGF-1 and cholesterol (total and LDL) were observed following the administration of an extract of embryonary peptides (EP) to old subjects for 60 days. For most of the subjects, due to the aging process, the initial values of the biochemical parameters were shifted towards pathological range. Following the administration of EP, the serum levels of IGF-1 and cholesterol (total and LDL) were shifted towards the physiological limits for their age. The most significant modifications towards physiological range were observed for subjects with high, initial levels of IGF-1, when the decrease was striking (1-2 orders of magnitude). For these subjects, significant modifications were observed simultaneously for cholesterol. The modifications induced following the administration of EP exhibit a regulatory feature, as they are dependent on the initial levels of these parameters. The action of EP on the levels of IGF-1 and cholesterol was significantly equal for both sexes, but the influence of EP was more clear-cut in men. In conclusion, our results support an implication of IGF-1 in the regulatory mechanisms of cholesterolemia in old subjects following the long-term administration of EP.

Significant modification of lipid metabolism in aged persons following the treatment with a nutritive supplement containing embryonary peptides--preliminary results.

HUMANOFORT is a nutritive supplement extracted and purified from embryonated chicken eggs according to an original procedure under licence. Humanofort received the suitable consent from the Romanian Ministry of Health. The main components of Humanofort are two oligopeptides of 5,000 and 10,000 D molecular weight. 40 subjects aged 50-75 years (18 men and 22 women) consumed, daily, 4 caps of Humanofort for 60 days. The samples of blood from each subject were obtained before and after treatment. Therefore, each subject was his own control. In all subjects, after treatment, total cholesterol and LDL-cholesterol decreased approximately 30% as compared with the initial values. In 80% of patients, an increase of HDL cholesterol and a decrease of the insulin level in blood were also observed. After treatment, the cardiac risk factors (Aethna 2000 Program), such as total cholesterol/HDL and Apolipoproteins B/A were shifted towards lower range. These long-lasting modifications have an adaptative-regulatory character and seem to be produced by the growth factors contained in Humanofort.

The formation of oxidative stress condition in the experimental chemically induced hepatotoxicity.

In spite of extensive studies concerning the chemically induced hepaototoxicity, no clear-cut practical conclusions were obtained. Wide chemical structure of compounds as well as the different way of administration are the most important factors. We used an acute administration of carbon tetrachloride and paracetamol and a chronic intoxication with acetylphenylhydrazine, cadmium chloride and ethanol. For the acute administration of chemicals in high doses, in the rat liver microsomes a decrease of cytochrome P450, aniline hydroxylase and glutathione (GSH) was noticed with an increase of lipid peroxides even at 2 hours after administration. 24 hours after the chemicals administration, in the blood of rats characteristic changes of hepatotoxicity such as increases glutamate-pyruvate transminase, triglycerides and lipid peroxides, while GSH was decreased. In chronic administration, the cummulated concentration of chemical was true main factor. For both ways of chemicals administration, as a critical concentration was reached, an oxidative stress was produced, demonstrated by the increase of lipid peroxides and subsequently decrease of GSH. In both ways of administration, the oxidative stress was the precocious biochemical event occurring even at 2 hours and it triggers other metabolic changes which favor fatty infiltration and liver damage.

Fibrinogen interaction with activated polymorphonuclear leukocytes studied by chemiluminescence.

Fibrinogen (FB) is an acute phase protein. It is recognized as an independent risk factor in the plasma of patients with coronary heart disease (CHD). The plasma FB level is also significantly increased in inflammation and neoplasms. We performed an in vitro study showing that the chemiluminescence (CL) emission produced by zymosan-activated polymorphonuclear leukocytes (PMNL) was directly related to the concentration of FB, fibrinopeptide A or FB-degradation products (FDP). Fibrin inhibited CL emission. Our results emphasized the role of FB as an independent risk factor in CHD but related to the level of activated polymorphonuclear leukocytes in the plasma. Activated PMNL may contribute to an elevated plasma level of FDP as a consequence of enhanced enzyme-dependent degradation of FB. Our results indicate that the role of FB as independent risk factor in CHD depends on the level of activated polymorphonuclear leukocytes in the plasma.

The levels of bilirubin may be related to an inflammatory condition in patients with coronary heart disease.

In agreement with previous reports, we found that the bilirubin level is significantly lower in the blood of patients with coronary heart disease (CHD) than in age and sex matched controls. However, we found that the level of bilirubin in the blood seemed to be an age-dependent phenomenon and closely related to the activation of leukocytes. In 1,000 cardiac catheterised patients from Urbana, USA suffering from CHD, the level of blood bilirubin was found to be lower than in age and sex-matched controls. The same results were obtained on 300 patients with acute ischemia from three hospitals from Bucharest, Romania. The activation of polymorphonuclear leukocytes increased in the catheterised patients, as well in Romanian patients. An activation of leukocytes triggered by a chronic inflammatory process may increase the lysis of erythrocytes. The erythrocytes of patients with 100% stenosis exhibited a higher rate of in vitro lysis in the presence of activated leukocytes and homocysteine. The increased hemolysis may trigger the activation and removal of the resulting bilirubin from blood. Such a mechanism may depend on the liver clearing function. This function was decreased in catheterized patients over 60 years of age, but had accelerated in younger patients. An individual variation in liver function may explain the widespread bilirubin levels in the blood of patients suffering from CHD.

Fibrinogen is an efficient antioxidant.

Fibrinogen has been included among the risk factors for vascular disease. Fibrinogen belongs with albumin, ceruloplasmin and transferrin to an acute phase protein group in the plasma. Albumin, ceruloplasmin and transferrin are already recognized as natural antioxidants. In the present study we used three different oxygen generating systems in order to test whether fibrinogen is able to act as an antioxidant in an in vitro system. We used 1) pyrogallol auto-oxidation, 2) the reaction catalysed by xanthine oxidase coupled with the reduction of ferricytochrome c and 3) chemiluminescence. We found that in a dose-dependent manner fibrinogen inhibited superoxide generation (pyrogallol and xanthine-xanthine oxidase reactions), ferrous ion oxidation and hydroxyl radical dependent degradation (of deoxyribose). Fibrinogen also inhibited LDL oxidation (copper and azo compound-induced), hydrogen peroxide oxidation and chemiluminescence produced by polymorphonuclear leukocytes. Fibrinogen, albumin, ceruloplasmin and transferrin act as a supplementary antioxidant defense mechanism against oxidative stress arising from inflammatory conditions.

The relationship of oxidized lipids to coronary artery stenosis.

A total of 1200 patients with angina were cardiac catheterized establishing that 63% had 70-100% stenosis, 12% had 10-69% stenosis of one or more of their coronary arteries and 25% had microvascular angina listed as 0% stenosis. Prior to catheterization 10 ml of blood was drawn and the plasma subjected to analysis for the concentration of cholesterol, lipid peroxides (LPX), total antioxidant capacity (TAOC), fibrinogen (FB), ceruloplasmin (CP) and activation of polymorphonuclear leukocytes (PMNLs). Comparisons were made to non-smoking controls without angina. Significant differences in LPX were found between the patients with 0 and 10-69% stenosis (P<0.001), with 10-69 and 70-100% stenosis (P<0.001), and with 0 and 70-100% stenosis (P<0.001). Under 70 years of age there was a significant difference in LPX between patients with all levels of stenosis and age and sex matched controls (P<0.001). Differences in the mean plasma cholesterol concentration for different levels in the degree of stenosis were not significant, indicating that LPX provided consistent data on the severity of stenosis while the plasma cholesterol concentration did not. Compared with controls an increase in activation of PMNLs (P<0.01), an increase in concentration of both FB and CP (P<0.01) and a decrease in total antioxidant capacity were noted in the plasma of catheterized patients. In summary the concentration of oxidation products rather than the concentration of cholesterol in the plasma identified stenosis in cardiac catheterized patients.

Stimulatory effect of some plant extracts used in homeopathy on the phagocytosis induced chemiluminescence of polymorphonuclear leukocytes.

Some plant extracts on a large range of dilutions as used in Homeopathy were tested on the chemiluminescence emission produced by polymorphonuclear leukocytes. The high stimulatory action was noticed when extracts from Uvae Ursi and Saponaria were tested, as the classical effect exerted by zymosan was exceeded. A moderate stimulatory action comparable with that of zymosan was found when extracts from Echmaceea, Aleo and Prumis were used, as well as in the case of Propolis. The relationship between stimulatory effect and the concentration range is modulated as function of the extract source, several peaks being observed for some dilutions (Saponana), but generally no quantitative relations were obtained. By studying the time when a chemiluminescence peak was observed, it is possible to estimate wether the weight of the NADPH oxidase or myeloperoxidase pathways are involved in the stimulatory effect on polymorphonuclear leukocytes.

Influence of low magnesium concentrations in the medium on the antioxidant system in cultured human arterial endothelial cells.

Using cultured human endothelial cells, we investigated the contribution of concentrations of magnesium to the antioxidant system and oxidative stress. Cells were cultured at decreasing magnesium levels (569, 380, 190 and 95 microM) for 72 h. We then measured the amount of released hydrogen peroxide (H2O2) from the cells, the consumption of exogenous H2O2, the intracellular reduced glutathione (GSH) and the oxidized glutathione (GSSG) contents and the activities of glutathione reductase and catalase. Magnesium at a level of 949 microM was used as a control. The effect of magnesium deficiency on cellular membrane permeability was determined by measurement of the amount of [14C] amino acid mixture released from the cells. The results showed that during 72 h of magnesium-deficient treatment, the H2O2 release from the cells gradually increased and consumption of exogenous H2O2 was enhanced during the first 48 h of treatment. GSH content gradually decreased but GSSG was not affected. The activity of glutathione reductase was first stimulated and then inhibited. Catalase activity was gradually reduced. [14C]Amino acid mixture release from the cells continuously increased. We suggest that magnesium deficiency affected the intracellular antioxidant system in cultured endothelial cells.

The levels of bilirubin may be related to an inflammatory condition in patients with coronary heart disease.

In agreement with previous reports, we found that the bilirubin level is significantly lower in the blood of patients with coronary heart disease (CHD) than in age and sex matched controls. However, we found that the level of bilirubin in the blood seemed to be an age-dependent phenomenon and closely related to the activation of leukocytes. In 1,000 cardiac catheterized patients from Urbana. USA suffering from CHD, the level of blood bilirubin was found to be lower than in age and sex-matched controls. The same results were obtained on 300 patients with acute ischemia from three hospitals from Bucharest, Romania. The activation of polymorphonuclear leukocytes increased in the catheterized patients, as well in Romanian patients. An activation of leukocytes triggered by a chronic inflammatory process may increase the lysis of erythrocytes. The erythrocytes of patients with 100% stenosis exhibited a higher rate of in vitro lysis in the presence of activated leukocytes and homocysteine. The increased hemolysis may trigger the activation and removal of the resulting bilirubin from blood. Such a mechanism may depend on the liver clearing function. This function had decreased in catheterized patients over 60 years of age, but had accelerated in younger patients. An individual variation in liver function may explain the widespread bilirubin levels in the blood of patients suffering from CHD.

The hemolytic activity of homocysteine is increased by the activated polymorphonuclear leukocytes.

Homocysteine is an accepted risk factor when its plasma level exceeds the physiological upper limit of 12 mumol/L. We found in vitro that homocysteine is able to lyse the erythrocytes (RBCs) at higher concentrations than 15 mumol/L only when activated polymorphonuclear leukocytes (PMNL) are present. The hemolytic effect of homocysteine was higher in RBCs obtained from cardiac catheterized patients with 100% stenosis of the coronary arteries. Homocysteine was also able to increase the activation in vitro of PMNLs triggered by opsonized zymosan. The hemolytic action of homocysteine was found to be dependent on the ratio of PMNLs to RBCs. This relationship may help to explain the great individual variations in the hemolytic activity noticed in blood obtained from cardiac catheterized patients, and also may explain the mild anemia in some patients suffering from cardiovascular disease.

Direct detection of the antioxidant activity of a new flavonic derivative using the chemiluminescence method.

The antioxidant potential of a new water soluble flavonic derivative, namely theophylline rutoside (TR-1722) has been tested using the chemiluminescence method. The method is based on the oxidative degradation of luminol by the hydrogen peroxide in Tris-HCl-buffer, when reactive species of oxygen are being obtained: O2-., HO., 1O2, and allows for the capability of substances to inhibit the free radical processes in this test system to be quantified and hence for their antioxidant properties in respect to a standard substance (in our case quercetin) to be compared. The results obtained reveal that TR-1722 has antioxidant action comparable to that of quercetin, the highest efficacity being registered at the concentration of 2 mumol/l, the conditions being: H2O2 16,2 mmol/l; luminol 2 mumol/l, in Tris-HCl buffer 20 mmol/l, pH 8.3. The antioxidant potential of TR-1722 is also maintained when the conditions of the system are modified, that is, the concentration of the hydrogen peroxide, the intensity of the action being dependent on the hydrogen peroxide concentration, but no direct proportionality is registered.

The influence of "selenium organicum" upon the hepatic function of carbon tetrachloride poisoned rats.

The hepatoprotective action of the Romanian preparation Orgasel containing selenium (Se) 5.01 mg/100 g autolysated yeast powder, was tested on adult Wistar rats poisoned with carbon tetrachloride (CCl4). The hepatotoxic agent (a 20% CCl4 solution in oil) was administered i.p. in a single dose of 0.3 ml CCl4 solution/100 g body weight, and the preparation tested (autolysate of seleniated yeast) was administered by gavage in 4 doses (of 100 mg Se powder/100 g animal each) along 2 days. After 48 hrs the animals were sacrificed, then their blood and liver were collected. The treatment with Orgasel significantly reduced the organs, morphological changes (fat liver degeneration, splenomegaly, testicle degeneration) induced by CCl4 poisoning in the rat, an effect found also at the biological parameters levels studied in plasma and liver. In the plasma, the high lipid peroxide concentrations, the increased activity of alkaline phosphatases, and the reduced antioxidative activity generated by CCl4 have been statistically significant brought to the normal range after Orgasel administration. At the liver this treatment significantly decreased the lipid peroxides production, the total lipids and cholesterol concentrations, and statistically significant increased the enzymes activity (alkaline phosphatases, GPT). The results obtained after Orgasel administration proved that this preparation has a global beneficial action upon the organism in the poisoned rat, as well as a strong antioxidative effect, confirming once again the essential role of Se in maintaining cells' integrity.

Comparative study of the presence of oxidative stress in sportsmen in competition and aged people, as well as the preventive effect of selenium administration.

The study demonstrates the presence of oxidative stress (OS) in physiological conditions in sportsmen in competition and in elderly subjects over 65 years. The OS was estimated by measuring some biochemical parameters, such as peroxides, total antioxidants, uric acid and free SH groups in plasma and peroxides in urine. OS produces a significant increase of peroxides in plasma and urine and a decrease of plasma antioxidants and SH groups. The presence of OS suggests an increased formation of free radicals in young subjects in sporting competition, This increase is subsequently compensated by an increase of antioxidants. In old agers, this unbalance remains unchanged. The level of OS is higher in sportsmen during competition, but the subsequent compensation depends on the degree of training, individual adaptation, diet, etc. The administration of organic selenium partially compensates and decreases the intensity of OS. This study confirmed the hypothesis that in old agers the formation of free radicals increases in the ageing process and recommends the prevention of OS in young sportsmen by an adequate protection with antioxidants, such as organic selenium.

Cigarette smoking causes biochemical changes in blood that are suggestive of oxidative stress: a case-control study.

Cigarette smoking has been shown to be a major risk factor for cardiovascular disease, lung cancer, and respiratory diseases. Due to its high content of oxidants, the cigarette smoke is bound to cause a prooxidant/antioxidant imbalance in the blood plasma and tissues of smokers. The study groups were selected from an apparently healthy population living in urban areas, comprising 200 subjects aged 18 to 80 years, half of whom were smokers. In smokers aged 18 to 45 years, the changes of the plasma prooxidant parameters (i.e., lipid peroxides, leukocyte activation, and the antioxidant ones [thiol concentration, total antioxidant capacity]) were not significantly different from those of the age-matched controls, whereas in the 46 to 80 age group they were. In smokers, both antioxidant erythrocyte enzymes, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), exhibited increased activity in the 18 to 45 age group and decreased activity in the 46 to 80 age group. The differences in enzyme activity between the smoking and nonsmoking groups were highly significant for SOD in all ages, whereas for GSH-Px the difference in activity was significant only in the case of older smokers. These findings would suggest that a process of adaptation takes place in younger smokers, in whom the antioxidant systems are able to counteract the oxidant factors, while in older smokers this process is no longer occurring and the plasma and tissues are under permanent oxidative stress. Our results clearly demonstrated that a prooxidant/antioxidant imbalance exists in the blood of smokers, and the determination of leukocytes stimulation index may be a useful and simple way of assessing the oxidative stress status of these individuals. A hypothesis regarding a possible mechanism linking cigarette smoking to the development of coronary heart disease is presented.

The antioxidative action of fibrinogen and the implications of this effect on platelet aggregation.

Purified fibrinogen strongly acts as an antioxidant by inhibiting the chemiluminescent emission developed in vitro, in a cell-free system composed of luminol and hydrogen peroxide. The antioxidative action of fibrinogen depends directly on its concentration, even in the presence of human serum. On the other hand, the platelet aggregation is an important source of oxygen free radicals. These radicals can also be measured by chemiluminescence, when the platelet aggregation is triggered by arachidonic acid. In a platelet suspension, fibrinogen inhibits both aggregation and the associated chemiluminescent emission. A possible correlation between the plasma level of lipid peroxides and fibrinogen was studied in different groups of patients, mostly with cardiovascular diseases and neoplasms. A weak correlation was found only in cardiovascular diseases, in which the tendency of fibrinogen increase could also be interpreted as an antioxidative action of peroxidation restriction, especially in ischemic conditions. In neoplasms, this correlation could not be found, in spite of the high level of fibrinogen associated with a decrease of peroxide formation, a characteristic feature of tumoral growth due to the change of fatty acids nature in the cellular membranes.

Tissue lipid peroxidation may be triggered by increased formation of bilirubin in vivo.

Following the administration of phenylhydrazine, cadmium chloride and ethanol to rats there was a marked increase in the concentration of liver lipid peroxides and a sharp decline in GSH levels. The oxidative stress generated by the action of these toxic compounds led to the induction of liver heme oxygenase, which exhibited a 3-fold increase in activity over the control value. Patients with various forms of liver disorders showed increased levels of plasma lipid peroxides as well as hyperbilirubinemia. In view of the known ability of bilirubin to cause lipid peroxidation in illuminated erythrocyte membranes, the results of the present paper suggest that in severely impaired liver, as in some liver diseases, lipid peroxides may be also produced by a mechanism involving heme oxygenase.

Estimation of oxidative stress in cardiovascular diseases.

Patients with mild and severe cardiovascular diseases showed a significant disturbance of the redox equilibrium, manifested by increases of lipid peroxides and decreases of plasma total antioxidants. When the resulting oxidative stress is associated with ECG changes characteristic of "coronary disease" it indicates an acceleration of atherosclerosis, in the initial phase (cellular involvement, migration, proliferation) and an additional thrombogenic risk in the "ischemic phase". When oxidative stress is associated with chronic heart failure, it indicates a progressive form with replacement fibrosis. These data suggest the necessity of adding to the usual therapy antioxidants, that reduce the oxidative stress, may slow down the pathologic changes and improve the patient's homeostatic systems.

Steroid hormones may modulate the chemiluminescence emission produced by polymorphonuclear leukocytes.

Steroid hormones, testosterone and estrone may modify the in vitro chemiluminescence (CL) produced by polymorphonuclear leukocytes (PMNL) during phagocytosis. These changes (increases or decreases) depended on the age and health condition of the donor, but not on sex. Generally, these steroid hormones increase the PMNL CL emission at concentrations lower than 10(-5) M, but decrease it at higher levels. In an acellular system, using the same CL technique, these hormones exhibit an opposite effect, by increasing the emission at higher concentrations. As the PMNL induced CL emission is a membrane-dependent process, strongly related to phagocytosis, the action of steroid hormones seems to be due to the receptors on the surface of leukocytes. In both systems, cellular and acellular, the effect of steroid hormones on the CL emission also depends on the concentration of hydrogen peroxide added or released during phagocytosis. The modulatory action of steroid hormones on phagocytosis may occur in certain conditions, such as infections diseases and hormone disturbances in the course of which there occurs a significant release of oxygen free radicals, concomitantly with variations of the levels of steroid hormones.