Oxidative Stress Is a Concept, Not an Indication for Selective Antioxidant Treatment.

The steady-state redox status is physiologically important and therefore homeostatically maintained. Changes in the status result in signaling (eustress) or oxidative damage (distress). Oxidative stress (OS) is a hard-to-quantitate term that can be estimated only based on different biomarkers. Clinical application of OS, particularly for selective antioxidant treatment of people under oxidative stress, requires quantitative evaluation and is limited by the lack of universal biomarkers to describe it. Furthermore, different antioxidants have different effects on the redox state. Hence, as long as we do not have the possibility to determine and quantify OS, therapeutic interventions by the "identify-and-treat" approach cannot be assessed and are, therefore, not likely to be the basis for selective preventive measures against oxidative damage.

Do low molecular weight antioxidants contribute to the Protection against oxidative damage? The interrelation between oxidative stress and low molecular weight antioxidants based on data from the MARK-AGE study.

A redox steady state is important in maintaining vital cellular functions and is therefore homeostatically controlled by a number of antioxidative agents, the most important of which are enzymes. Oxidative Stress (OS) is associated with (or/and caused by) excessive production of damaging reactive oxygen and/or nitrogen species (ROS, RNS), which play a role in many pathologies. Because OS is a risk factor for many diseases, much effort (and money) is devoted to early diagnosis and treatment of OS. The desired benefit of the "identify (OS) and treat (by low molecular weight antioxidants, LMWA)" approach is to enable selective treatment of patients under OS. The present work aims at gaining understanding of the benefit of the antioxidants based on interrelationship between the concentration of different OS biomarkers and LMWA. Both the concentrations of a variety of biomarkers and of LMWA were previously determined and some analyses have been published by the MARK-AGE team. For the sake of simplicity, we assume that the concentration of an OS biomarker is a linear function of the concentration of a LMWA (if the association is due to causal relationship). A negative slope of this dependence (and sign of the correlation coefficient) can be intuitively expected for an antioxidant, a positive slope indicates that the LMWA is pro-oxidative, whereas extrapolation of the OS biomarker to [LMWA] = 0 is an approximation of the concentration of the OS biomarker in the absence of the LMWA. Using this strategy, we studied the effects of 12 LMWA (including tocopherols, carotenoids and ascorbic acid) on the OS status, as observed with 8 biomarkers of oxidative damage (including malondialdehyde, protein carbonyls, 3-nitrotyrosine). The results of this communication show that in a cross-sectional study the LMWA contribute little to the redox state and that different "antioxidants" are very different, so that single LMWA treatment of OS is not scientifically justified assuming our simple model. In view of the difficulty of quantitating the OS and the very different effects of various LMWA, the use of the "identify and treat" approach is questionable.

Gender- and age-dependencies of oxidative stress, as detected based on the steady state concentrations of different biomarkers in the MARK-AGE study.

Recently, Weber et al. published a thorough investigation of the age-dependency of oxidative stress (OS) determined by the steady state concentrations of different compounds - oxidation products and antioxidants - that are in common use as biomarkers of OS in 2207 healthy individuals of the cross-sectional MARK-AGE Project. The correlations among biomarkers were significant but weak. These findings may indicate different manifestations of OS and must further be evaluated. Here, we report a refined analysis of OS based on the above-mentioned original data. We show that malondialdehyde (MDA) appears to be sensitive to both gender and age. It is significantly lower and shows a greater age-dependence in women than in men. The age-dependency of MDA in women arises in a stepwise fashion. The age-dependent slope of the steady state concentration is maximal at the age between 50 and 55 years, indicating that it may be attributed to the change of metabolism in the post-menopause. Interestingly, total glutathione (GSH) decreased with age simultaneously with the increase in MDA. Different biomarkers yield different gender- and age-dependencies. Unlike the concentration of MDA, the concentrations of the other two oxidation products, i.e. protein carbonyls and 3-nitrotyrosine were similar in men and women and appeared to be independent of age in the healthy study population. The analyzed antioxidants exhibited different gender- and age-dependencies. In conclusion, it appears that all the biomarkers assessed here reflect different types of OS and that MDA and GSH reflect the same type of OS.

Oxidative stress, as assayed by a single test, cannot be used as a diagnostic tool.

The commonly used term "oxidative stress" (OS) is intuitively defined as an excess of pro-oxidative compounds, over antioxidants. The redox status is homeostatically controlled because on one hand, pro-oxidants are essential for normal body function, whereas, on the other hand, pro-oxidants (and OS) are associated with many diseases due to the risk of oxidative damage. One reason "to monitor the OS" is to identify people under OS and treat people under high OS by antioxidants, because it is believed that people under OS benefit from antioxidant supplementation more than others. This approach led to the production of many assay kits, based on the concentrations of different biomarkers in body fluids. Unfortunately, this expensive approach (evaluated at about a half a billion dollars per year) yields very limited results because: (i) the effect of antioxidants on the OS is not that simple and (ii) OS cannot be quantitated in terms of a universal criterion and the method-dependent OS yields different groups of people under OS. This conclusion gains strong support from analysis of the results of a previous study of the OS in more than 2000 participants, using many OS assays. The small overlapping between the "people under OS" as assayed by different biomarkers clearly shows that OS cannot be used as a diagnostic tool. © 2018 BioFactors, 44(3):222-223, 2018.

Deuterium kinetic isotope effect (DKIE) in copper-induced LDL peroxidation: Interrelated effects of on inhibition and propagation.

LDL peroxidation plays a major role in many physiological and pathophysiological processes. The mechanisms of LDL peroxidation induced by transition metal ions have therefore been studied intensively. It has been proposed that the mechanism involves free radical production that occurs via decomposition of hydroperoxides. This, in turn, requires the cleavage of O-H bonds. Cleavage of O-D bond is slower and comparison of the kinetics in D2O to the kinetics in H2O is therefore a straightforward way to test this aspect of the alleged mechanism. The kinetics of peroxidation exhibit marked deuterium kinetic isotope effects at all the stages of oxidation under all the studied conditions. We found that the rate of propagation of copper-induced peroxidation is a monotonically decreasing function of D2O fraction in D2O/H2O mixtures. The only elementary reaction that involves "exchangeable" hydrogen at this stage is copper-induced decomposition of conjugated hydroperoxides. Therefore, we conclude that the latter step is rate-limiting reaction including cleavage of oxygen-hydrogen bond of hydroperoxide. The lag preceding rapid peroxidation exhibits a biphasic dependence on the fraction of D2O. This may be understood on the basis of the effect of substituting hydrogen atoms by deuterium. Specifically, such substitution is expected to decrease both the rate of initiation of peroxidation and the potency of the antioxidant. We interpret our results in terms of the effects of isotopic substitution on the rates of the reactions that involve the abstraction of "exchangeable" hydrogen atoms of OH groups in tocopherol and hydroperoxides.

In Healthy Young Men, a Short Exhaustive Exercise Alters the Oxidative Stress Only Slightly, Independent of the Actual Fitness.

The aim of the present study was to evaluate the apparent disagreement regarding the effect of a typical cycling progressive exercise, commonly used to assess VO2max, on the kinetics of ex vivo copper induced peroxidation of serum lipids. Thirty-two (32) healthy young men, aged 24-30 years, who do not smoke and do not take any food supplements, participated in the study. Blood was withdrawn from each participant at three time points (before the exercise and 5 minutes and one hour after exercise). Copper induced peroxidation of sera made of the blood samples was monitored by spectrophotometry. For comparison, we also assayed TBARS concentration and the activity of oxidation-related enzymes. The physical exercise resulted in a slight and reversible increase of TBARS and slight changes in the activities of the studied antioxidant enzymes and the lag preceding peroxidation did not change substantially. Most altered parameters returned to baseline level one hour after exercise. Notably, the exercise-induced changes in OS did not correlate with the physical fitness of the subjects, as evaluated in this study (VO2max = 30-60 mL/min/kg). We conclude that in healthy young fit men a short exhaustive exercise alters only slightly the OS, independent of the actual physical fitness.

The effect of compartmentalization on the kinetics of transition metal ions-induced lipoprotein peroxidation.

In a previous study, we proposed characterizing the typically observed kinetic profiles of transition metal ion-induced lipid peroxidation in terms of a limited number of characteristic time-points. These time-points can be derived from experimental time-dependencies and be presented in terms of rate constants and concentrations as calculated based on mechanistic considerations. The critical part of that analysis was that we had to assume that the experimental system behaves as if it is homogeneous, i.e., as if the reaction occurs in a solution. In spite of the uncertainties due to the latter assumption, we obtained a reasonable agreement between the experimental data and the theoretically predicted dependencies, which supports our theoretical treatment. Yet, several previous findings could not have been explained in terms of our ('quasi-homogeneous') model, indicating that the model is valid not under all conditions. One example is that under certain conditions, rapid peroxidation occurs prior to complete consumption of LDL-associated tocopherol. This can be attributed to compartmentalization of residual tocopherol, namely, after the onset of propagation, part of the LDL particles contain tocopherol, whereas in the other, tocopherol-depleted particles, the PUFA may undergo rapid LOOH-accelerated peroxidation only if they contain at least two hydroperoxides molecules per particle. In the present investigation, we show that the results of all our kinetic studies can be understood if we consider compartmentalization. Specifically, for any given composition of the particles (LDL and/or HDL), the kinetic results may be governed by the distribution and rate of exchange of antioxidants and hydroperoxides between particles. Our analysis is of special importance for systems containing more than one population of lipoprotein particles.

Oxidative stress, the term and the concept.

The 30th birthday of a central concept in biomedicine, such as oxidative stress (OS) is a good time for re-evaluation of its contribution to science and particularly to the field of redox biology. In his recent communication, Sies described the history of the concept as well as the benefits and pitfalls of the term OS. In this mini-review, we discuss the problems associated with the still common perception of "bad OS, good antioxidants". Specifically, the term OS is an intuitively understood term originally used to describe an imbalance between pro-oxidative factors and anti-oxidative factors. It has no units, its level is dependent on the way it is measured and there is no correlation between various criteria of OS, which indicates that there are sub-classes (types) of OS (other than the classifications presented by Sies). In spite of these limitations, it is commonly regarded a measure of a person's probability to suffer from oxidative damages and is being held responsible for many diseases and antioxidants are predicted to be good to us. In fact, a "Basal OS" is vital and antioxidants may interfere with the mechanisms responsible for maintaining the oxidative status. We also discuss the linkage of OS to the outcome of antioxidant supplementation and comment on the importance of kinetic studies in evaluation of OS and on the ranking of antioxidants.

Prolongation of the lag time preceding peroxidation of serum lipids: a measure of antioxidant capacity.

Antioxidants inhibit oxidation processes and by this affect many biological processes. This, in turn, promotes continuing efforts to synthesize new efficient antioxidants and discover compounds of natural origin capable of preventing peroxidation. Although many assays have been developed to evaluate antioxidants, the search for improved protocols is still actual. The presented protocol is based on the effect of antioxidant on the kinetics of peroxidation of lipids in human blood serum. Specifically, we evaluate the capacity of antioxidant by the relative prolongation of lag phase (delay) of copper-induced peroxidation of lipids in unfractionated serum. The main advantage of the assay is that it implements inhibition of peroxidation in physiologically relevant system. We propose expressing the results of the assay either in terms of the relative prolongation of the lag per 1 µM of antioxidant or as the concentration of antioxidant required to double the lag. To allow for comparing the results with those of other assays, these results may be normalized and expressed in terms of the unitless "TROLOX equivalents."

The relationship between oxidative stress and exercise.

Physical exercise has many benefits, but it might also have a negative impact on the body, depending on the training level, length of workout, gender, age and fitness. The negative effects of physical exercise are commonly attributed to an imbalance between the levels of antioxidants (both low molecular weight antioxidants and antioxidant enzymes) and reactive oxygen and nitrogen species due to excessive production of free radicals during physical exercise. In this critical review, we look for answers for three specific questions regarding the interrelationship between physical exercise and oxidative stress (OS), namely, (i) the dependence of the steady-state level of OS on fitness, (ii) the effect of intensive exercise on the OS and (iii) the dependence of the effect of the intense exercise on the individual fitness. All these questions have been raised, investigated and answered, but the answers given on the basis of different studies are different. In the present review, we try to explain the reason(s) for the inconsistencies between the conclusions of different investigations, commonly based on the concentrations of specific biomarkers in body fluids. We think that most of the inconsistencies can be attributed to the difference between the criteria of the ill-defined term denoted OS, the methods used to test them and in some cases, between the qualities of the applied assays. On the basis of our interpretation of the differences between different criteria of OS, we consider possible answers to three well-defined questions. Possible partial answers are given, all of which lend strong support to the conclusion that the network responsible for homeostasis of the redox status is very effective. However, much more data are required to address the association between exercise and OS and its dependence on various relevant factors.

Analysis of the kinetics of lipid peroxidation in terms of characteristic time-points.

Measuring peroxidation of aggregated lipids in model systems (liposomes, micelles, emulsions or microemulsions) as well as in samples of biological origin ex vivo (isolated lipoproteins, blood sera or plasma) is widely used in medical and biological investigations, to evaluate the oxidative stress, antioxidants' efficiency and lipid oxidizability in different pathophysiological states. To avoid possible artifacts, such investigations must be based on the time course of peroxidation (i.e. on kinetic studies). To be able to compare complex kinetic profiles, it is important to characterize them in terms of mechanistically meaningful and experimentally unequivocal parameters. In this review, we characterize the typically observed continuous kinetic profiles in terms of a limited number of characteristic time-points (both commonly used and additional time-points and their combinations) that can be derived from experimental time-dependencies. The meaning of each of the experimentally observed characteristic parameters is presented in terms of rate constants and concentrations, derived on the basis of mechanistic considerations. Theoretical expressions for these characteristic parameters are based on a model that includes both the inhibited peroxidation and the uninhibited peroxidation occurring after consumption of the antioxidant(s). Comparison between theoretically predicted dependencies and experimental data support our treatment considered with special emphasis on transition metals-induced peroxidation of lipoproteins.

The mechanism of detergent solubilization of lipid bilayers.

Multiple data are available on the self-assembly of mixtures of bilayer-forming amphiphiles, particularly phospholipids and micelle-forming amphiphiles, commonly denoted detergents. The structure of such mixed assemblies has been thoroughly investigated, described in phase diagrams, and theoretically rationalized in terms of the balance between the large spontaneous curvature of the curvophilic detergent and the curvophobic phospholipids. In this critical review, we discuss the mechanism of this process and try to explain the actual mechanism involved in solubilization. Interestingly, membrane solubilization by some detergents is relatively slow and the common attribute of these detergents is that their trans-bilayer movement, commonly denoted flip-flop, is very slow. Only detergents that can flip into the inner monolayer cause relatively rapid solubilization of detergent-saturated bilayers. This occurs via the following sequence of events: 1), relatively rapid penetration of detergent monomers into the outer monolayer; 2), trans-membrane equilibration of detergent monomers between the two monolayers; 3), saturation of the bilayer by detergents and consequent permeabilization of the membrane; and 4), transition of the whole bilayer to thread-like mixed micelles. When the detergent cannot flip to the inner monolayer, the outer monolayer becomes unstable due to mass imbalance between the monolayers and inclusion of the curvophilic detergent molecules in a flat surface. Consequently, the outer monolayer forms mixed micellar structures within the outer monolayer. Shedding of these micelles into the aqueous solution results in partial solubilization. The consequent leakage of detergent into the liposome results in trans-membrane equilibration of detergent and subsequent micellization through the rapid bilayer-saturation mechanism.

Detergent solubilization of lipid bilayers: a balance of driving forces.

Although detergents are routine tools in biomembrane research, their use remains empirical. We propose that solubilization is the result of a balance between two parameters: (i) the energy associated with bending of phospholipid monolayers into a curved micellar surface, and (ii) the energy associated with filling the void in the center of the resultant mixed micelle. In this review, we show that reliable data on the phase boundaries, and their dependence on various conditions, are consistent with this hypothesis, even if the data might have been interpreted differently. Although most of the experimental data discussed here were obtained with the non-ionic detergent Triton X-100, the conclusions should be applicable to a wide variety of detergents.

[Who is likely to gain from high dose supplementation of vitamin E?].

Given the assumption that oxidative stress plays a pivotal role in atherogenesis, it could have been predicted that antioxidants will slow or even stop the development of atherotic plaques. The results of the latter prediction were disappointing. Moreover, previous meta-analyses concluded that indiscriminate supplementation of vitamin E at high dose (400 IU or more) results in increased mortality, both cardiovascular and all-cause. This conclusion raised serious criticisms, particularly on the choice of studies for meta-analyses, the end point (mortaLity) and the heterogeneity of the clinicaL studies with respect to both the population and the treatment. In Dr. Dotan's PhD thesis, conducted under my supervision, with the assistance of Drs. Leshno and Pinchuk, we analysed the results of the major cLinicaL studies within a Markov model. Using this approach enabled us to adjust the data for heterogeneities and assess the sensitivity of the outcome to the different affectors. The adjusted data were then used to assess the risk of transitions between predefined health state (healthy, dead or CVD patients, following a cardiac event). We also used a different 'end point', that is the quantity-adjusted life years (QALY), which reflects both morbidity and mortality. The major result of this study is that indiscriminate high dose supplementation of vitamin E is associated with an average loss of about 0.3 QALY. Yet, we think that some specific groups (presumably those that suffer from high oxidative stress) may gain from vitamin E supplementation. The existing data is insufficient to predict who is likely to benefit from vitamin E treatment. Until such data becomes available, we cannot recommend high dose vitamin E supplementation.

Ranking antioxidants based on their effect on human serum lipids peroxidation.

Evaluation of the activity of antioxidants is commonly based on measurements of the effect of a specific antioxidant on redox reactions conducted in a solution. Given the difference between reactions that occur in homogeneous solutions and those that occur at lipid-water interfaces, as in biological membranes and lipoproteins, the relevance of the commonly-used assays (such as TEAC and ORAC) to the antioxidative activity in biological systems is questionable. The aim of the present investigation is to develop a more relevant assay. Based on our results, we propose an assay based on prolongation of the lag preceding fast peroxidation of serum lipids. The assay employs our previously developed procedure for determination of susceptibility of serum lipids to peroxidation. The effect of antioxidants is expressed in terms of the relative prolongation of the lag preceding peroxidation. It can be considered reliable because it is only marginally dependent on the specific sera used for the assay. The resultant ranking of antioxidants may be expressed either as the relative prolongation of the lag per 1µM of antioxidant or as the concentration of antioxidant required to double the lag. As expected, the observed ranking order is very different from that reported for TEAC or ORAC assays, undermining the relevance of these assays for oxidation that occurs at interfaces.

[Medical training in Israel--can we realize the comments of Pazi's Committee and at what cost?].

In spite of the growth of Israel's population, the number of graduates from the four Israeli Schools of Medicine has changed very little in the last 30 years. Nevertheless, the annual number of new practitioners grew from about 300 to 900, due to Israeli graduates of European Schools and the large number of physicians among the new immigrants from the former USSR states in the early nineties. A committee, nominated by the National Board of Higher Education (MALAG), headed by the late Prof. Pazi, concluded that under steady state conditions we must train 800 medical students per year. MALAG adopted this recommendation. As a first step, MALAG approved two new programs: a special program for Academic Reserve candidates (Atudaim) and a new (5th) medical school in the Galilee. These two new programs, together with the new 4-year MD program in Tel Aviv, will add about 200 graduates to the list of medical students. Yet, the question of whether we can realize the recommendation of Pazi's Committee (to train 800 MDs annually) and at what cost, remains open. This preliminary review is devoted to the relevant factors that must be considered before the latter questions can be answered. First, we note that the limiting factor of the number of medical students is the ratio between the number of teaching departments in each of the medical disciplines and the number of weeks of Clerkships required in the given disciplines. Our main conclusion is that realization of the recommendation of Pazi's committee requires an increase in the number of teaching departments, preferably by upgrading the academic level of those departments that do not teach students, and increasing the teaching load of other departments. We may also have to reduce the number of weeks of 'bedside teaching' but should beware of reducing it to a minimum lower than about 70 weeks, as in North America. Regardless of the need to train more physicians, we must increase teaching in the community. Only a combination of all these measures will reduce our dependence on the education of Israeli physicians abroad.

Does skeletal muscle oxidative stress initiate insulin resistance in genetically predisposed individuals?

Reactive oxygen species (ROS) are postulated to be a common trigger of insulin resistance. For example, treatment of adipocytes with either tumor-necrosis factor-alpha or dexamethasone increases ROS before impairing glucose uptake. Similarly, treatment with mitochondria-specific antioxidants preserves insulin sensitivity in animal models of insulin resistance. However, it remains unclear whether ROS contribute to insulin resistance in humans. First-degree relatives (FDRs) of type 2 diabetes subjects are at increased risk of developing insulin resistance and type 2 diabetes. Here we review the documented metabolic impairments in FDRs that could contribute to insulin resistance via increased oxidative stress. We propose that lipotoxic intermediates and lipid peroxides in skeletal muscle interfere with insulin signaling and might cause insulin resistance in these 'at risk' individuals.

No evidence supports vitamin E indiscriminate supplementation.

For many years, the prevailing concept was that LDL oxidation plays the central role in atherogenesis. As a consequence, supplementation of antioxidants, particularly vitamin E, became very popular. Unfortunately, major randomized clinical trials yielded disappointing results and recent meta-analyses concluded that indiscriminate, high dose vitamin E supplementation results in increased mortality. This conclusion raised (quite reasonable) criticism, much of which referred to the characteristics of meta-analysis. In our recent study, we used a Markov-model approach, which is free of most of the limitations of meta-analyses. Our major finding was that the average quality-adjusted life years (QALY) of vitamin E- supplemented individuals was 0.30 QALY (95%CI 0.21 to 0.39) less than that of untreated people. In our view, this supports the view that indiscriminate supplementation of high dose vitamin E can not be recommended to the general public.In the present communication we address several recent studies that demonstrated negative effects of vitamin E and raise possible mechanisms that may be responsible for the harmful effects of vitamin E supplementation. We also review recent studies conducted with specific groups of patients that gained from vitamin E supplementation, indicating that although, on the average, indiscriminate supplementation of high dose vitamin E is not beneficial, specific populations may gain from vitamin E. The challenge is to establish selection criteria that will predict who is likely to benefit from vitamin E supplementation. Such criteria may be based either on the assumption that antioxidants are likely to be beneficial for people under oxidative stress or on knowledge regarding the benefit of sick people with certain diseases. In short, we adopt the view that vitamin E is a "double-edge sword" that should not be consumed until criteria are defined to predict who is likely to benefit from high dose supplementation of vitamin E.

Cholesterol reverts Triton X-100 preferential solubilization of sphingomyelin over phosphatidylcholine: a 31P-NMR study.

The distribution of phosphatidylcholine (PC) and sphingomyelin (SM) between the solubilized (micellar) and non-solubilized (lamellar) fractions arising from bilayers composed of PC and SM, with or without cholesterol (Chol) has been measured under conditions of partial, incomplete solubilization by Triton X-100. Quantitation is achieved by (31)P-NMR determination of the composition of mixed micelles in the range of bilayer-micelle coexistence. We find that the solubilized fraction of bilayers consisting of binary mixtures of PC and SM is rich in SM, as expected from previous data on solubilization of pure PC and pure SM liposomes. In contrast, after partial solubilization of ternary mixtures of PC, SM and Chol, the solubilized fraction becomes SM-poor, as observed in the partial solubilization of biomembranes.