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09/04/16 - Can heavy isotopes increase lifespan? (deuterium oxide)
Can heavy isotopes increase lifespan? Studies of relative abundance in various organisms reveal chemical perspectives on aging - Stable heavy isotopes co-exist with their lighter counterparts in all elements commonly found in biology. These heavy isotopes represent a low natural abundance in isotopic composition but impose great retardation effects in chemical reactions because of kinetic isotopic effects (KIEs). Previous isotope analyses have recorded pervasive enrichment or depletion of heavy isotopes in various organisms, strongly supporting the capability of biological systems to distinguish different isotopes. This capability has recently been found to lead to general decline of heavy isotopes in metabolites during yeast aging. Conversely, supplementing heavy isotopes in growth medium promotes longevity. Whether this observation prevails in other organisms is not known, but it potentially bears promise in promoting human longevity.

Are there aging factors that are independent of organismal fitness rectification? Some hints can be garnered from a classical aging assay: the yeast replicative lifespan assay. In this assay, individual cells are cultured with infinite supply of nutritional resources on a solid surface. Whereas nutritional effects on fitness are essentially masked, the ability of cells to produce progeny slows and stops after 20–30 cell divisions [4]. Clearly, something has diminished irreversibly over time, even when cells are maintained in nearly “perfect” conditions. These underlying factors may be intrinsic features of the process we call “living,” and explain, at least in part, the inevitable reason for aging. And because yeast cells cease to divide, despite ideal conditions, it is likely that such factors bear characteristics that are out of reach of genetic regulation, a feature reminiscent of aging. To find out what they are, scientists have to look at more fundamental aspects of life.

The ability of biological systems to discriminate among isotopes has been widely documented by comparing the isotope abundance in a specific organism with its surrounding environment (Table 1). In plants, carbon isotopes are discriminated in photosynthesis, a natural chemical process of carbon fixation, and the entry point of carbon into the whole biosphere [7]. Notably, carbon-13, the heavy isotopes with 1% natural abundance, is discriminated against in typical C3 plants, which results in its depletion [7]. Conversely, plants enrich oxygen-18 in their leaves, an observation thought to be related to transpiration or water movement in the plant body [8].

Intriguingly, heavy isotope enrichment seems to occur in many animals on a temporal scale. In one study, both carbon-13 and nitrogen-15 spiked in the fingernails of human infants at early stages (solely breast feeding) but declined with onset of weaning [9]. Conversely, human infants excrete urine that is depleted in both deuterium and oxygen-18, suggesting that human bodies tend to retain heavy isotopes of oxygen and hydrogen [10]. In addition, breast milk feeding has been found to cause a 25–50% boost in both urinal oxygen-18 and deuterium in babies [10]. Similar enrichment was also observed in sole fish larvae, where carbon-13 content increased by 25% in the first 4 weeks of their lives after hatching [14] and in various other animals fed with different diets [15]. It should also be noted that carbon-13 does not appear to be further depleted in animals after its entry into the biosphere through carbon-13 depleting photosynthesis (Table 1), which is probably due to the fact that no extensive metabolic rearrangement of carbon skeletons occurs in metazoan consumers. This observation is also well in line with the general trend of heavy isotope enrichment in various organisms.

Heavy isotopes profoundly modify biological functions - Despite extensive experimental and theoretical studies on stable isotopes in the chemistry field, there exist only limited – but intriguing – functional studies addressing heavy isotopes in the biological contexts. Most of these studies used deuterium, probably because deuterium could produce the most obvious KIE (Table 1) and is abundantly available as heavy water (D2O) at a reasonable price.

Water is the most abundant and most used metabolite in living organisms on Earth (Fig. 3), which makes it an appealing vector to introduce heavy isotopes into biological subjects. Shortly after its discovery by Harold Urey, deuterium was used in the form of heavy water to treat various organisms including plants, yeast, and animals [17, 18] and lately fruit flies [19, 20]. Amazingly, feeding experimental organisms with heavy water, despite its pronounced KIE effects, does not seem to have outstanding negative effects on life, and it merely slows down biological processes, such as seed germination, seedling development, fermentation in yeast, motion ability in flatworms, and produces tolerable behavioral changes in mice that are thought to be non-toxic [17].

Heavy water treatment at high dosage (50% or above) does not alter cell size, growth, metabolome structure, or oxygen consumption in yeast [5, 18]. Different species exhibit different levels of tolerance to heavy water uptake, e.g. a level above 20% of heavy water by weight was thought to be toxic in mammals, and only produced negligible growth effects in yeast and algae [17, 18, 21]. In most cases, the adverse effects caused by heavy water treatment can be reversed upon treatment withdrawal. These studies suggest that heavy isotopes, in the case of high dose of deuterium, are well tolerated in biological systems, and thus, do not seem to do serious harm to basic biological activities. Also, as suggested by the fact that heavy water only elicits metabolic slowdown that is akin to low temperature preservation [18, 20, 22], the largely reversible effects caused by heavy water treatment may also find applications in hibernation-like lifespan extension, as would be needed for interstellar travel.

More evidence about the biological effects of deuterium comes from animal studies. In isolated rat liver mitochondria, depleting deuterium in natural water by distillation to one third of the original abundance has been shown to boost succinate-dependent H2O2 generation by 67% in maximum velocity and enhance the substrate affinity by 57% in Michaelis constant Km [28]. Since the monitored reaction is an indicator of overall respiration function in mitochondria, an intracellular hub for energy and material metabolism, one would expect that deuterium in natural water can greatly impact the overall metabolism in any eukaryotes. This notion is also supported by our recent observation in yeast, where heavy water treatment remarkably suppresses the endogenous generation of reactive oxygen species (ROS) by mitochondria [5, 28]. Another line of evidence comes from the protective effects of metabolite deuteration. A recent study has found that deuterated polyunsaturated fatty acids, even supplied in a minor fraction, protected mammalian cells from various damage associated with oxidative stress, such as lipid peroxidation and mitochondrial uncoupling [29]. Deuteration of lipids, a major group of metabolites that are mainly metabolized in mitochondria, is thought to stabilize the aerobic metabolism and prevent uncoupling of respiration from ADP phosphorylation, a major source of endogenous ROS generation and chemical damages [29-31]. The chemical basis for these benefits originates exclusively from KIE.

Perplexingly, contradictory reports have emerged from deuterium depletion studies that may suggest heavy isotopes are detrimental. Tumor regression was reported to result from applying deuterium-depleted water in dogs and humans, and cancer cell growth in vitro was inhibited by deuterium depletion as well [32-34]. The benefits of deuterium depletion in cancer treatment are linked to suppression of oncogenic genes and the promotion of apoptosis, and exhibited strong differences in efficacy between sexes [33, 34].

As the first study of its kind, we have found all three common heavy isotopes in amino acids declined in yeast undergoing chronological aging [5]. This decline can be effectively retarded by the supplementation of heavy isotopes, which, consistent with our hypothesis, also extends lifespan. Although only a small group of metabolites were covered, our study has provided molecular evidence that the temporal change in heavy isotope content is real and relevant in aging. Whether similar isotopic declines occur in other organisms, such as mammals, is still pending. Nevertheless, it would be of interest to compare the heavy isotope decline in long-lived model animals, such as naked mole rat, with that in other rodents of much shorter lifespan.

In lieu of the observed heavy isotope decline with age, the next question is: where do they go? Two possibilities exist: increased excretion or decreased retention. We favor the latter because so far there is no chemical evidence indicating that heavy isotopes can be actively enriched in a well-defined biochemical process. In contrast, decreased retention may originate from deficient assimilation through anabolism and internal partitioning into inactive stock molecules. Depending on how heavy isotopes are introduced, in general they should retard catabolic reactions but produce much milder effects on the anabolic reactions (see Fig. 3 for an example of deuterium and hydrogen). Therefore, it is more likely that the observed heavy isotope decline derives from internal partitioning, a notion that is supported by observation that the heavy isotope content declines in the same metabolites found in both the cytosol and media during yeast aging, as demonstrated for glutamine [5].

Promoting longevity and health by heavy isotope supplementation - Before our observation of heavy isotope decline during organismal aging, deuterium-bearing heavy water has been shown to promote longevity or improve certain health aspects in several organisms, including fruit flies, rodents, and humans [19, 20, 29, 37, 38]. In fruit flies, transient exposure to heavy water at juvenile stages extends lifespan, and the exposure does not affect the health and reproduction [19]. However, a dosage of 50% heavy water shortens the lifespan [20], and the relative lifespan shortening by heavy water was ameliorated by temperature elevation from 10 to 30°C, suggesting a protective effect of heavy water on fruit fly survival in hot conditions where accelerated metabolic rate normally reduces longevity. Improved thermoresistance was indeed observed at the protein, cell, and organism levels in fruit flies upon heavy water treatment [22]. Similarly, a driving factor in temperature-compensated effects by heavy water was observed to alter the phase relation in circadian oscillation [39]. The heavy water effect is increasingly more pronounced with rising temperature. However, the mechanism is still unknown. The similarity in the biological responses between heavy water and low temperature also correlate well with the general observation that fruit flies and worms have longer lifespan, and retarded brain degeneration when maintained at low temperature [40, 41].

Several functional studies have shown that deuterated polyunsaturated fatty acids, even supplied in a minor fraction (20–50%), can protect yeast and mammalian cells from ROS damage to mitochondria [29, 37]. In whole animals, 25% heavy water was able to normalize high blood pressure induced by high salt diet in rats, possibly through suppressing hypertension-related elevation in calcium uptake [38]. These effects would surely extend lifespan.

In yeast, we also showed that heavy water extends chronological lifespan in a dosage-dependent manner [5]. This pro-longevity effect could be essentially abrogated by mild dietary restriction or mitochondrion removal. Heavy water also suppresses the endogenous ROS generation, which could ameliorate the background chemical damages from ROS and lead to long-term improvement in fitness and survival rate. All these protective effects indicate that heavy water functions as a metabolism modifier to promote longevity, a feature that could be amenable to implementation in the context of other well-known anti-aging interventions [1].

One remarkable feature of heavy water in biology has emerged from several earlier observations [17], in which heavy water at high dosage (50–90%) was found to suppress seed germination, retard seedling germination, disrupt flatworm activities, and even stimulate hyperactivity in mice fed with one volume equivalent of their total body fluid daily. However, heavy water was found to produce only reversible or non-accumulative effects, in other words, no long-term toxicity. If these observations apply to long-term processes, then heavy water seems merely to elicit a hibernation-like dormancy, without producing long-lasting adverse effects. These reversible effects would make the application of heavy water even more appealing in retarding aging and preserving vitality in human activities involving survival over extraordinary time periods, such as interstellar travel.

Although heavy water dominates in our current understanding and appreciation of heavy isotopes in biology, because of the prevalence of water in biochemistry (Fig. 3), other deuterium-bearing metabolites and other elements may also prove useful. Deuterated lipids have shown great healthcare promise in protecting cells from ROS damage [29, 31, 37]. Deuterated drugs are also superior to their non-deuterated peers in safety, efficacy and tolerability, because deuteration alters their metabolic and pharmaceutical profiles in a favorable way [42]. The more abundant and prevalent an element is in biology, the more effects its heavy isotope likely produces (such as hydrogen in water, see Fig. 3). In fact, augmentation of overall heavy isotope uptake through diet has been speculated to provide health benefits over a wide spectrum [6]. It would be fascinating to see whether isotopes of other elements have similar effects: oxygen-18, carbon-13, and nitrogen-15 are especially relevant for their respective elemental prevalence in organic matter.

Despite their potential benefits over the long term, heavy isotopes are generally associated with growth and development retardation, as described earlier in this manuscript. How these adverse effects modify the long-term aging outcome remains to be evaluated, and more mechanistic insights are needed. But innovative approaches, such as deuteration of polyunsaturated fatty acids and transient exposure to heavy water, have already provided promising means to overcome the potential caveats and to maximize the beneficial outcome from heavy isotope supplementation.

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It also covers oil/petroleum and how it can be used to produce energy and products, weather control for cancelling earthquakes, tsunamis, fires, floods and to produce rain or clear weather on demand, oxygen/ozone therapy, nitrogen as a motor driver, water generation and manipulation via steam and vacuum, ecological restoration techniques, biophysics, rejuvenation and an unending list of other subjects, most of which are accepted by 'orthodox' science.

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