Ed.- We are pleased to include for the first time in the Anti-Aging Bulletin, an article by the renowned Imre Zs.-Nagy, MD. Professor Nagy is head of the Department of Gerontology at the University of Debrecen, Hungary. He is also the Editor in Chief of the Journal of Gerontology and the principle behind the Membrane Hypothesis of Aging. Furthermore, Professor Nagy is the recipient of the Monte Carlo Antiaging Conference ™ Award for Excellence in Anti-Aging Medicine.
I became involved in the gerontological basic research during the mid-sixties, mainly under the influence of Fritz Verzár. He was born in Hungary (1886), became the first professor of physiology at my University of Debrecen (1918). He won the chair of physiology in 1932 at the University of Basel (Switzerland), and lived there permanently since 1938. At his age of 70 (1956), when he had to retire by law from the University of Basel, founded the first basic research institute for experimental gerontology of the world in Basel, headed it for 21 years, and died in 1979 in his 93rd year of age. I am proud of the fact that during the last 15 years of his life I could be in a close scientific and human contact with him in Hungary, Switzerland and Italy.
Verzár was the best tutor I have ever met in my life. It was an absolute evidence for him that the science has to ask first valid and meaningful questions of the most general significance. Only finding the answers to those questions may open the way to a wider understanding of the living systems, to an intervention into the non-desirable processes occurring in the human body, like diseases and aging. He has not only taught us for this, but in more than 600 publications has demonstrated that he was dealing all his life with the most significant problems of physiology, like the hormones of the adrenal cortex, the absorption from the intestine, the electro-physiology of the nervous system, and at last with aging as a biological phenomenon. At the same time, he was able to formulate the main questions also in a widely comprehensive way. For example, in the fifties he said, somewhat jokingly, that the main question of the basic aging research was, to find out why an old chicken requires a much longer time of cooking, than a young one! He has also given the answer on the basis of his heat denaturation experiments on the collagen, and also on chromatin proteins, in terms of the age-dependent transformation of weak intermolecular bonds (H-bridges) into stronger, covalent ones (1). It should be stressed, however, that all this have been experimented and evaluated by Verzár during the late fifties, when molecular biology had not even been invented. It was a great achievement that analytical biochemistry could later identify many of those intermolecular cross-links; the existence of which has been so clearly predicted by Verzár.
The main roots of the theoretical gerontology
When I made my first steps in the experimental gerontology (1967-68), there were already various aging theories in the literature. As a young man, who intended to dedicate a life-long activity to this research field, I collected all the available literature, and tried to learn and understand those theories. Nevertheless, when analyzing the theories and comparing them to each other and also to the available general cell biological knowledge, I could establish only a very few solid points, which could be of help in my future research. There is no room here to list all the relevant problems of this field, however, some main items should be mentioned.
It was evident to me that the very-very first problem we have to face is the definition of the aging process. Such a definition at phenomenological level existed already in the ancient Greek philosophy. Namely, according to Aristotle (cited in: 2) the human body is composed of the 4 main components of the world (ancient elements: humid, dry, warm and cold) so that the humid and warm prevail until we are young, while the dry and cold take the predominance when we get old. Of course, this was a very much empirical approach, but it should be recognized that it had described the main aspects of human aging quite realistically.
Modern biology produced a more sophisticated phenomenological definition of aging. Strehler (3) suggested that the aging process had 4 main characteristics, as follows:
It should be stressed that this definition of aging (3) has never been contested by anyone, because it summarizes really well the main issues. Consequently, to be in harmony with this definition is an implicit requirement toward any serious aging theory. However, when creating various aging theories, many authors simply disregarded this requirement. Below I will list the main disappointing examples, without being complete.
The theory of somatic mutations of aging (4-7) and the modified version of it, called the theory of genetic mutations, or even later, the error catastrophe theories (8-11), have been in the center of interest for many years. Nevertheless, they have been abandoned because of the lack of any unquestionable experimental evidence. They were also in contradiction with the intrinsic character of aging: they assumed that the reason for aging is the cosmic radiation. The main argument against these theories is that although both young and old individuals really may have various damages in the genetic code from external radiations, the repair capacity of the living systems is by far exceeding the usually existing rate of damage. Consequently, these hypotheses may not represent a valid mechanism of aging.
The wear and tear explanations of aging go back to Erasmus Darwin (12), and were maintained in different forms in various ages until today (see for details: 13). This approach to aging is more realistic, in so far as it does not blame any external factor for aging, but emphasizes a gradually increasing incapacity of the organisms for auto reparation. Nevertheless, the essential features of the loss of auto reparative ability remain unclear.
The cross-linking explanation of aging tried to attribute the wear and tear phenomena to the formation of intra- and/or intermolecular cross-links, which are able to alter the structure of the macromolecules to such an extent that even their functions become compromised. This explanation was suggested by Björksten (14) and King (15), first merely on a theoretical basis. However, unquestionable experimental demonstration of the validity of cross-link formation was offered only by the heat-denaturation experiments of Verzár (1, 16, 17) on the collagen and chromatin. It was an extremely important step forward in this field when Harman (18, 19) proposed the free radical theory of aging (FRTA). He suggested that the free radicals deriving from the oxygen metabolism might have been involved in the age-dependent deterioration of the molecular structure and functions of the living systems. It should be emphasized that the polymer chemistry had accumulated by that time a lot of knowledge on the in vitro polymerizing effects of certain free radicals (see for details: 20), but most of these data were unknown for the biomedical science. Denham Harman himself could reach the basic idea of FRTA only because he studied not only medicine, but also organic chemistry. Verzár (1) itself has quoted the basic papers of Harman, however he did not perform any detailed analysis of this possibility. By the end of sixties, it became evident for me that the cross-linking hypothesis and the FRTA should be connected somehow in the further basic gerontological research. Unfortunately, however, the further development encountered an immediate difficulty the content of which has been called paradoxon of the FRTA (21). This paradoxon derives from the fact that the young individuals of any species consume more oxygen per unit mass and time, than the old ones, i.e., there must be an even higher rate of free radical formation at younger ages. Yet, the young organisms are able to grow and proliferate, i.e., they remain apparently untouched by the free radical attacks, while the older organisms become progressively deteriorated in their structure and function by the very same type of oxygen free radicals, even at a lower rate of their formation. The existence of this paradoxon dictated that the general statement of FRTA, according to which aging is caused by the oxygen free radicals remains shaky, unless we can find an explanation for this apparent contradiction in the biological structure itself. It will be shown later in this paper that the membrane hypothesis of aging (MHA) (13) is able to resolve this contradiction in a logical and realistic way, if one is considering the main physicochemical changes of the biological systems during their maturation and aging.
The accumulation theories of aging assume that aging is caused by accumulation of certain substances, like lipofuscin (age pigment), aged collagen, damaged neurofibrils in the brain, damaged enzymes, etc. Among these, the most extensive literature is available on lipofuscin, which has been considered as the most significant biomarker of aging (see for details: 13). Chemically speaking, the lipofuscin is a strongly altered, extensively cross-linked, mainly insoluble, auto fluorescing lipoprotein mass, localized in the lysosomes. It is not a toxic substance in itself, and it was assumed that it may inhibit the cell functions simply by occupying a great part of the useful cell volume. In the formation of lipofuscin, a great significance has been attributed to the lipidperoxidation, initiated by oxygen free radicals, and resulting in malondialdehyde, and at last Schiff base formation (22-25). It has been described, however, that that other ways of lipofuscin formation may also exist, like a series of cross-linking processes, leading to dolichol formation, etc (see for details: 13). It has also been shown long ago that the formation of lipofuscin takes place also in the young organisms, its just not accumulated until the lysosomal enzyme supply is sufficient for its elimination. The inhibition of the lysosomal thiolproteinases by leupeptin has clearly shown that lipofuscin-like products do accumulate in the nerve cells, myocardium, liver, epithelial cells, etc. even of young animals; in 1-2 weeks of application of leupeptin (26-28). It has also been demonstrated that one of the main components of lipofuscin derive from the cell membranes (29, 30). These observations have thrown a new light on the lipofuscin formation mechanisms, indicating that they are parts of a more general cell biological procedure performing the elimination of the biological waste products (30).
The theories of aging listed so far were classified as "causal explanations of aging" by Esposito (31). The very same author created also two other classes of aging explanations, namely the "systemic" and the "evolutionary " explanations of aging.
The systemic theories are of great significance, because some of them are followed by a wide interest among the biomedical scientists. The main difference between the causal and systemic explanations would be that the former assume the existence of damaging reactions (mainly at a molecular level), while the latter speak about some interactions between certain organs. Although this definition also contains some elements of contradiction, since the organ interactions inevitably involve some molecular reactions, one can use this classification, if being aware of the facts.
The program theory of aging assumes that aging is governed by a few genes being under the control of some positive or negative auto regulative mechanisms. The main version of this concept is represented by the hypothesis of Hayflick (32, 33), according to which the possible number of cell divisions is limited, to about a total of 50 doublings in case of the human WI-38 fibroblasts, and this limitation leads to aging of the organism. The great number of established cell lines which are doubling without such a limit even in-vitro, were declared to be abnormally transformed, tumor cell lines (33, 34).
It should be mentioned, however, without going into details, that the "Hayflick dogma" has been criticized very strongly. Numerous authors have shown that:
All these arguments render the situation so unclear that one cannot even start to prolong the life span of animals or humans through any experimental approach based on the Hayflick dogma. Nowadays the overwhelming majority of the gerontologists is of the opinion that this approach cannot be considered as a serious basis of the theoretical gerontology.
Other systemic theories of aging can be listed here as the theory of decomposed program, the theory of autoimmunization, the organic explanations of aging (31). All of them contain some elements, which are realistic in certain aspects and for certain animal strains. The problem is that these elements are absolutized and extrapolated (e.g., the caloric restriction hypothesis, the neuroendocrine theories of aging, the effects of melatonin, etc.), neglecting all the aspects in which they lack their validity, and this way apparently autonomous theories of aging are created. One approach is called "dysdifferentiation hypothesis of aging and cancer" (DHAC) (37,38) assumes that aging is largely the result of an improper gene regulation caused by oxygen free radicals, acting directly on the genetic apparatus. It has been shown however, that the "improper gene regulation" is nothing else than a quantitative reduction of the synthetic velocities of RNA and proteins, due to the unfavorable changes of the intracellular physicochemical conditions with time, as described in the MHA (39).
Lastly, we have to mention the evolutionary explanations of aging. Esposito (31) listed the main features of them. The most important aspect is that these are largely speculative, and there is practically no way to perform any experiments in this regard. From a medical point of view, it is still more hopeless to apply these explanations for life prolonging or anti-aging purposes (13).
The above outline gives the impression that most of the suggestions of the theoretical gerontology have resulted in frustrations during the last decades. The situation was deteriorating so deeply that basic concepts like the "biomarkers of aging" could not be defined unanimously, and the question could be asked: "Do we know what to look for?" (40).
This frustration is present even today in the gerontology and geriatric medicine. As a proof for this statement, it is sufficient to look at the actually applied anti-aging medicine: It is based mainly on the replacement of the missing active substances in the old age (hormones, vitamins, melatonin, DHEA, etc.), as testified, e.g., by the program of the last Monte Carlo Antiaging Conference ™ (2002). Of course, the replacement therapies may be of use in improving some actually lost functions, but until we do not have a really solid and generally accepted aging theory, or at least a considerable progress in this field, no real chance of a smart, preventive anti-aging intervention may be designed (41). This author had been deeply disappointed already during the early seventies by the frustrating divergences and contradictions of theoretical gerontology. So I started to develop step by step a more comprehensive, synthetic approach to the problem of biological aging, considering all the neglected, but evidently important aspects of cell biology, and all the well established facts. The result of this research activity is the MHA (13), which will be summarized briefly below.
The conceptual basis of the Membrane Hypothesis of Aging (MHA)
As regards the neglected aspects of cell biology, one can list the following main points.
It is a basic knowledge that the whole ontogenesis of the biological systems can be described as a process of accumulating dry mass and losing intracellular water content. However, due to the fact that most of the analytical biochemical research is done in test tubes at extremely high dilutions, almost nobody has ever considered the eventual effect of the increasing physical density of the living cells. It is very surprising, because all the molecular enzyme kinetic considerations have shown that the speed of enzyme functions is inversely proportional to the density of the environment, where the enzyme molecules are located (see for ref.: 42).
The FRTA has always suggested the use of free radical scavengers to prolong life span. However, it has never considered that the most efficient radical scavenger is the KCN, which is one of the most toxic substances, killing living beings immediately, much before the energy reserves are exhausted (13, 43, 44). The paradoxon of the FRTA was already mentioned before, but it had never been explained by the FRTA.
There is no doubt that the oxygen free radicals, particularly the OH( free radicals being extremely quick electron acceptors (i.e., oxidants) play a role of vital importance in a great number of processes, among others in the polymerization of the biological components (45). It had also been well established many decades ago that their polymerizing efficiency is largely density-dependent, but this feature had never been considered by the FRTA.
The electric properties of living cells are well explored and studied in many details. Nevertheless, it has never been considered that the electric polarity of the cell membrane and particularly its frequent discharge at each action potential, may represent an extra damaging factor of the membrane components through the discharge-induced heating (13).
My basic idea was to analyze the interactions of the known and neglected features for the cell biology in a complex way, in order to understand whether we can have a clearer idea about the mechanisms of cell maturation and aging. The MHA was born during the late seventies (46, 47) on the basis of a series of cell biological experiments, and developed during the last 25 years to a considerable extent. Nowadays one can state that MHA is a reasonably complete and comprehensive hypothesis of cellular differentiation and aging. Details of MHA have been described in numerous publications (13, 46-52), therefore, this present paper will only outline the main aspects of MHA, which permitted a successful, preventive, anti-aging intervention in animal experiments.
The main points of the MHA
Figure 1 shows a schematic outline of MHA. A primary role in differentiation and the aging process is attributable to the plasma membrane, which is the densest structure of the cells. The alterations are due greatly to OH( free radical induced molecular damage (44), and also to the "residual heat" formed during each discharge of the resting potential (13). In agreement with this statement, protein fractions of the shortest half-life have always been found in the plasma membrane. As a consequence, continuous and inevitable structural and functional alterations of the plasma membrane structure occur throughout life. Although these membrane alterations are repaired through a continuous replacement of the damaged components by de novo synthesis, for a number of simple reasons, the replacement can never be a perfect 100 %. Therefore, a certain accumulation of residual damage can be observed in the plasma membrane mainly in the postmitotic cells (like neurons), or relatively post-mitotic cells (like hepatocytes, muscle fibers, etc.). Since, however, all types of cells have a basically identical structure, this phenomenon takes place in every cell, although the quantitative relationships may be different in various cell types. The life-long accumulation of these cell membrane alterations causes a lot of consequent functional changes in the cells (Figure 1). Among those the most important ones are: a gradual decrease of the passive potassium permeability, with a consequent increase of intracellular potassium content and a consequent colloid condensation, a loss of intracellular water content, and an increase of intracellular dry mass content.
It should be noted here that the accumulation of dry mass is an implicit requirement during development and maturation of practically each living being. Namely, it is an even empirically clear fact that the life of any living individual is a process during which a highly hydrated state of fertilized oocytes, embryos, newborns, etc. is transformed into a gradually more and more dehydrated one (see for details: 13). This dehydration process is a useful one until the individual reaches their optimum performance requiring a given amount of enzymes, muscle fibers, collagen, neuro-filaments, etc. However, because it is of an ever-ongoing character, the further accumulation of intracellular dry mass has very serious consequences. First it slows down, and then stops the growth of the organisms. Still later, above a certain physical density of cell colloids, it compromises even the basic functions of the cells, because it increases the damaging efficiency of the free radicals, and the in-situ enzyme catalytic rate constants are strongly dependent on the density of their micro-environment (42). All this causes the slowing down of the rates of RNA and protein synthesis, as well as the total protein turnover, inducing also the waste product accumulation. The functional losses of aging (destructive and progressive character) find their explanation in these phenomena, simply because all living functions are realized directly or indirectly by enzymes.
Apart from our experimental findings described in the cited literature, MHA has gained a strong support from the recent developments of molecular genetics. Namely, the great majority of the products of oncogenes or anti-oncogenes, involved in the processes of mitotic regulation, cell differentiation and senescence have a more or less close plasma membrane localization (see for details: 51, 52). These facts confirm the central role of the plasma membrane played in the realization of practically each main biological regulation. Thus, MHA offers a deeper, synthetic understanding of the function of the cell plasma membrane, its governing role in the development and aging process, and the possibilities of an eventual intervention to prolong the useful life span. It is interesting to note that the aging process of the erythrocytes in the peripheral blood displays exactly the process described by the MHA, with the only difference, that because of the lack of protein synthesis, there is no possibility for replacement of the damaged components. The young, adult and old erythrocytes can be separated from each other by density gradient centrifugation, because they have an increasing density, being the oldest ones of the highest density.
To date, no serious criticism has so far been raised against the validity of the MHA, although it is also true, that only few people consider it really seriously. The problem is most probably related to the synthetic character of this idea, which is by far more complex than the average horizon of the contemporary, usually overspecialized biologists. There was only one apparent contradiction on the validity of certain aspects of the MHA (53), but it was based on a fatal misunderstanding of some basic concepts of enzyme kinetics (54). On the other hand, MHA does not contradict any well-established data or concept regarding biological aging. Conclusions of MHA permitted also a successful prevention of aging in animal experiments, including even a new drug design.
Life prolonging animal experiments based on the MHA
The MHA offers a good chance for a useful anti-aging intervention. Namely, if the most vulnerable structure of the cells is the cell plasma membrane, one can try to improve the defense of this structure against the damaging factors, and this should be beneficial for the maintenance of the cell integrity for a longer time. This approach is favored also by the fact that most pharmacia can reach the cell membrane. In addition, it is also a very attractive hypothesis that the large differences between the survival times of various species can be due to various levels of efficiency of the membrane-protecting mechanisms.
The logical way of any anti-aging intervention should be, therefore, to increase the available number of loosely bound electrons inside the plasma membrane, which are easily accessible for OH• free radical scavenging. First we tested the effects and nature of some available membrane-related anti-aging pharmacia, like centrophenoxine (CPH), in both animal experiments and human clinical trials. It turned out from these experiments that centrophenoxine really efficiently protects the neuronal cell membranes against OH• free radical induced damage, and at the same time, prolongs the life span of experimental animals. It proved to be also beneficial on the brain performance in human clinical trials of 2 months, performed on a 75-year-old population (55, 56). Chronic, life-long human voluntary applications of CPH are in course since 1976, so far without any side-effects and with apparently good results (41).
On the basis of those results, we have modified the CPH molecule, increasing the number of loosely bound electrons from 2 to 4 per molecule, leaving all other parts of the molecule intact. This new molecule has been coded as BCE-001 (57, 58). These modifications have resulted in a 1.9-fold faster rate constant of the OH• free radical scavenging reaction, and an even better life-prolonging effect in-vivo of old rats of an up to 40% increase in their medium life span. (13, 41, 57, 58). This drug is now under development for human testing.
It seems to be evident from the presented data that the basic concept of MHA realistically represents the basic process of cell differentiation and aging. One should consider the normal cell functioning as a continuous formation, damage and elimination cycle of the cell components, which are exposed to a continuous influence of damaging affects during their life. The highest rate of damage is involving the cell plasma membrane components, and consequently, this structure governs the entire machinery of protein synthesis very seriously. This assumption explains all the known characteristics of the maturation and aging process, explains the age-dependent decline of turnover of proteins, the accumulation of any natural or experimental "age pigment," and what is more, it opened the way of the preventive, anti-aging pharmacological interventions. Serious further research is needed to convert this small, but exclusive hope into prolongation of the human life span during this century.
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EXPLANATION TO FIGURE 1
The conceptual outline of the MHA, in part explained in the text. Arrows indicate causal interrelationships between the events described. This cycle is assumed to be the primary physicochemical driving mechanism for cell maturation and aging: it turns around with the same frequency as the proteins are replaced in the cell plasma membrane. Therefore, even a minor residual imperfection in the replacement of damaged membrane components per cycle may accumulate a considerable damage during a life-long repetition of the cycle in the postmitotic cells. Specific gene products are needed to maintain the membrane integrity against this general trend. The more sophisticated is the protective molecular mechanism, the longer is the survival of the given cell type.
Reproduced from (13), with permission.