Lifespan of Harold Katcher’s Rats

Preliminary results from lifespan studies with E5

Harold Katcher has developed a protocol for lab rats using intravenous injection with a blood plasma fraction he calls “E5”. Three years ago, he announced that treated rats evinced many features of rejuvenation, including improvements in grip strength, endurance, and learning capacity. Two years ago, he announced that treated rats also were epigenetically younger, according to a rodent methylation clock developed by Steve Horvath.

This year, with a grant from Heales Foundation, Harold and his partner Akshay Sanghavi have supervised a trial in which older rats were treated with E5 and then allowed to live out their full lifespans so we might know whether epigenetic and phenotypic rejuvenation translate into increased life expectancy. Just this week, I obtained from them birth and death data for the experimental rats. There were 8 control rats, untreated, all dead, and 8 treated rats, 5 dead and 3 still living.


Executive summary of my findings: At any given age, treated rats are 4x less likely to die; but translated into life expectancy, this is less impressive. The rats are living a little longer, but not nearly so much as their methylation age would have predicted. There is good evidence for compressed morbidity — treated rats are healthier later in life, and their deaths are less spread out in time than control rats. Caveats: All rats in the epigenetic experiment were male, while all rats in the lifespan study were female. Also, the protocol was initiated at a later age in the lifespan study compared to the epigenetic study.


Raw data: Time is the rats’ age in days. “Death” =0 indicates that 3 of the rats are still living. Group 1 is control, Group 2 is rats treated with E5.

Time Death Group
1034 1 1
1064 1 1
1069 1 1
1155 1 1
1158 1 1
1159 1 1
1161 1 2
1173 1 2
1179 0 2
1183 1 2
1193 1 2
1197 1 1
1200 1 1
1209 0 2
1209 0 2
1218 1 2

How this data is analyzed

It is conventional and, IMO, also reasonable that the data on age at death are interpreted as a “probability of mortality”. Of course, deaths spread over a greater time period indicates a lower rate of mortality. Less intuitive, the rate of mortality is based on the number of rats that remain alive at any given time, and not on the total number of rats. Thus, when the first rat dies, its probability of mortality is just ⅛, but when the last rat dies, its probability of mortality is 1.0.

If a rat is still living it may contribute to the denominator only for rats who died earlier, and not for rats who died later.

Using these conventions, I produced the following plot for probability of mortality for the two groups. I have plotted probability of mortality on a log scale because it is an empirical fact that probability of death increases exponentially with age. This is called the “Gompertz rule”. If the Gompertz rule holds, then we expect the plot on a log scale to be a straight line. I have drawn the best straight line through the two sets of points.

The Gompertz distribution is characterized by two numbers. One is the base mortality rate, which is related to how early the animals start dying. The other is the mortality rate doubling time. The probability of death doubles again and again over the life of the animals. A short doubling time indicates that the deaths are all bunched together, and a long doubling time indicates that the deaths are spread out over a broader range of ages.

You can see that, compared to controls, the treated rats started dying later and that their mortality doubling time is shorter, with deaths bunched more closely in age.

There is substantial uncertainty in these conclusions because of the small number of rats, but there is enough data here to give us confidence in the basic conclusions:

  • Treated rats are less likely to die young
  • Once they begin dying, treated rats die faster than controls
  • It is unclear from data so far whether maximum lifespan has been increased. We will have a better handle on this question when we see how long the remaining rats live.

One more concern about the experiment: Rats are social. Treated rats were housed separately from control rats, 2 or 3 to a cage. Just like people, rats are more likely to die after their cage mates die. I don’t have information about which rats were housed with which, but the death dates show some signs of being bunched together. This social effect could amplify the difference in mortality patterns between treated and control rats.

Cox proportional hazard

The most conventional way to analyze contingent survival data is called the “Cox proportional hazard model”, a relatively new statistical innovation introduced by David R. Cox in 1972. Many drug treatments and environmental hazards are reported on the basis of Cox models.

Result of the Cox model is reported as a “hazard ratio”, interpreted to mean that “if you do X you will be Y% more (or less) likely to die at any given time.”

The Cox model has the advantage that it is independent of the Gompertz rule or any other assumption about how mortality risk changes over time. It has the disadvantage that it can be misleading if the two different groups have qualitatively different mortality patterns.

The Cox model assumes that the difference between the two groups can be expressed as a simple ratio. If the Gompertz rule holds, a simple ratio translates (using the mortality rate doubling time) into an age change. For example, for humans in modern Western cultures, mortality doubles every 7 years. A Cox ratio of ½ is thus equivalent to rejuvenation by 7 years.

I’ve done the Cox analysis for Katcher’s rats because it is conventional, but my opinion is that its assumptions are not satisfied in this case. The mortality rate doubling time seems to change in the treated rats, indicated by the fact that the slopes of the two lines are different. So interpret the Cox results with this in mind.

Cox analysis indicates that the hazard ratio for treated rats is 0.24, meaning that treated rats are 4x less likely to die. The p value = 0.02, indicating confidence in the conclusion that treated rats are living longer than untreated. Increase in life expectancy is about 7%, which is 85 days for the treated rats. Again, these numbers can change when we see how the remaining 3 rats fare.

Conclusions

I have been committed to the idea that methylation clocks provide a real indication of biological age, and that reduction in methylation age will translate to a longer lifespan. My DataBETA study is premised on this hypothesis. There is good theoretical and indirect experimental support for the idea that epigenetics is a driving force behind aging (last week’s blog).

On their face, these new results suggest the possibility that methylation age might be decoupled from life expectancy. This is worrisome, but there are other possible interpretations of the situation.

We don’t have methylation results for the actual animals in the lifespan study. I’ve heard there was some mixup sending tissue samples to Horvath’s lab for analysis. There are various reasons these animals may not have responded to E5 treatment as well as the previous group.

Katcher’s rats are our best opportunity to answer this urgent question about a causal link between methylation status and lifespan. Fortunately, he is beginning another lifespan study with both male and female rats, which will follow more closely the protocol of the original study, but will extend in time to offer lifespan data. Unfortunately, the composition of E5 is still proprietary, so the minds of other scientists and the resources of other laboratories are not available to study the remarkable effects reported from E5. Wider collaboration is urgently needed to study lifespan and also to optimize dosage, timing, and delivery procedures. A collaboration with Johns Hopkins University has been announced by Katcher’s company (called Yuvan), but we have as yet no details.

Possible theoretical interpretation

I have written in the past about the Achilles heel of methylation clocks. Aging is like a civil war within the body. In youth, all metabolic systems are protective, but with age there are systems that attack and destroy the body. Examples are autoimmunity and inflammation.

Typically, methylation sites (CpG’s) chosen for inclusion in a clock algorithm are correlated with age. There are two possible reasons that an epigenetic change might be correlated with age, depending on which side of the civil war the system is fighting for. A given CpG might be associated with a self-destruction gene, or it might be a protective response to the body sensing higher levels of damage. The training algorithm, based on correlation with chronological age or even with mortality, is generally unable to distinguish between these two possibilities.

I have proposed on theoretical grounds that drivers of aging ought to be more common than responses to damage. Methylation clocks are only useful for evaluating anti-aging interventions to the extent that they are based on genes that drive aging. It’s only through experiments like Katcher’s that we can learn if our methylation clocks have been contaminated with genes that protect from damage.

These preliminary results are a signal of caution and a call for more research, but the evidence is indirect and the results are too thin to change theoretical perspectives now.

61 thoughts on “Lifespan of Harold Katcher’s Rats

  1. Interesting results. Seems the common theme after all these years is that Significant maximal lifespan increase is no easy feat to obtain.

  2. Rat maximum lifespan is 45 months, and this data is from 40 months. 11% to go.
    How many of us have felt the need to pull it all up when challenged? Or will the remaining three subjects just give up?

  3. Josh’s analysis was 7% increased in life span.
    That seemed consistent with Fisetin and Dasatinib and Quercetin studies when started in older mice; but far short of 26% for female mice on Rapamycin.

    Very nice that Josh was able to cut through all the mumbo jumbo and come up with a real number 7%.

  4. Brillians analysis, Josh. Thank ypu. There are still few such experiments but in the late times there have been other experiments backing/ showing that the theoretical basis you and Harold pursue is the right one. One of them is the rejuvenation of brain or at least its learning improvement after young CSF is infused in old subjects. The other one is the rejuvenation of an old subject graft of skin when implanted on a young receptor (who is inmunosuppresed, of coures). So somehow we are getting closer to understand ageing or at least, to find astrategies against it. Despite they don’t seem as close as we would like to be. All the best.

  5. Although they aren’t living that much longer, from what I understand, Harold’s “elderly” rats are cavorting around like teenagers with thick fur coats and plenty of energy – basically right up until they suddenly croak (right, Harold?).

    Many of us here would be delighted with the increased “healthspan” these rats are enjoying, even if it came with only modest effects on lifespan. But even leaving that aside: can an argument be made that these rats have “lived” more within the same calendar span of time?

    I don’t mean philosophically, I mean metabolically.

    Given the enhanced vim & vigor they enjoy, if you were to consider the total energy throughput for their bodies over their lifetimes, wouldn’t that be much higher?

    Or if that number isn’t higher (because they are eating about the same), perhaps the total energy that *goes to useful activities* (muscles, etc) is higher? Call that maybe “available energy”?

    Or, flipping it around: are they wasting less calories on useless inflammation and other waste?

    Could we measure this? Can we measure total metabolic waste from “inflammaging”, maybe proxy it via area under the curve for C-reactive protein or other inflammation markers? Or could we measure it via mitochondria counts, mitochondrial turnover, approximate measures of calories expended by overactive T-cells and other unnecessary auto-immune activity?

    I do see there is at least some fledgling research on caloric cost of immune responses..

    There are two reasons I’m focusing on this:

    1. I am worried that Katcher & team’s results will be under-appreciated if the lifespan numbers turn out to be ~10%, which is less than CR. In reality, the same results in humans would be revolutionary, but may not be understood that way because of that headline ~10% number

    2. I took resveratrol & pterostilbene for a bit, and then stopped because I felt as though it was just slowing down my metabolism, which is also what one might expect from CR (calorie restriction) generally. I didn’t want to live like that, sluggish.

    I think we need some kind of objective way to measure healthspan improvements, and ideally, an approximate way to compare it to lifespan improvements. So, for example, you live 20% longer, but you do that at reduced “available energy”, well, we consider that equivalent to a 5% lifespan extension where “available energy” is never reduced.

    If we could measure caloric loss due to inflammaging, we might have something close to an objective measure here, and we will definitely need that in comparing therapeutic approaches in the future.

    • Completely agree with your assessment. I’ve made this point here on several occasions: Dying at 100 years of age isn’t something I’m overly worried about (I’m 56 now). My main motivation is to enjoy my remaining years to the fullest of my abilities. Free of pain and with maximum mobility would be a major bonus.

    • Thank you, Josh, for the thoughtful observations in your article.

      Charles:

      I also like the observation that the treated rats had a longer health span and then died more rapidly than the controls.

      I would much rather be relatively healthy, and then experience a sudden (or good death) rather than one where I would linger in a debilitated state for too long.

      People can sometimes linger for years in a physically and mentally debilitated state. That is not something that appeals to me, personally.

      An increased health and life span would be wonderful. Still, if I could only choose one, it would be health span.

    • Hi Charles, funny thing is that I’ve taught the “Biology of Aging” for several years and was surprised that most students did want hugely extended lives, but would rather live to 100 in good health and enjoy the limited lives we are given. Well, it seems E5 will do that. However, that is very far from the end of our quest, but the intermediate product (a topical application) will hopefully soon be available. Josh, who I consider a friend believes that pro-aging factors are more prevalent than repair systems, but you know (I’m not suggesting this as the reality), but a very simple model where the continued depletion of “E5” results in aging works very nicely. That is, we have proven that there are anti-aging substances in young blood, and also that they are dominant to or ‘upstream’ to the pro-aging substances, as injections of E5 restore youth with a minimal (less than 10%) dilution effect. Not that it was a well-controlled experiment – but the fact that simply rubbing a bit of E5 on my hand caused an obvious reversal of aging shows that it is the depletion of anti-aging factors rather than the increase of pro-aging factors that are dominant; there was certainly no dilution of pro-aging factors involved. The best example of this is that E5 decreases the levels of the inflammatory cytokines that increase with aging – that alone shows that the increase in pro-aging factors (inflammatory cytokines) depends on or is ‘downstream’ of the decrease of anti-aging factors (E5). Also, remember in this study we use female rats which showed a much lower response to E5 than male rats in terms of lowering epigenetic age – so we are also working on aging in females (which seems to use different mechanisms – seemingly related to the state of the ovary) a more direct approach to life-extension and I feel fairly certain (Insh’Allah) that we will succeed.

      • But didn’t the Conboys show similar rejuvenation from just plasma replacement with saline+albumen? That could only have removed pro-aging factors, which implies to me that pro-aging factors are also very strong, and that E5 shifts a balance, rather than either pro- or anti-aging factor being dominant or upstream.

        I wonder what would happen if you replaced plasma with saline+albumen+E5? You might eliminate that 10% dilution, and might get an additional lifespan boost, or something completely different might happen. It certainly doesn’t make sense for your team to do that experiment (doesn’t really lead to a more appealing product) but I hope someone does it. Maybe give the Conboys some E5 and encourage them to check it out 🙂

        Also, as far as dominance of pro- or anti-aging factors, I think cellular senescence and SASP must be considered: as I understand it, E5 rejuvenates the immune system, causing mass clearing of senescent cells, which presumably removes a lot of pro-aging components from plasma as well. Perhaps E5’s anti-aging factors are dominant, because, as an indirect senolytic, it is actually removing pro-aging factors as well.

        If so, this might be visible as a “tipping point” in the dose-response relationship of E5: you might observe that various small amounts have almost no effect, then suddenly, there is enough E5 to cause senescent cells to be cleared out, causing a sudden spike in efficacy, and then additional amounts of E5 have little additional effect. Have you observed anything like that? Do you have enough data to plot the dose-response curve for E5?

  6. “Once they begin dying, treated rats die faster than controls”

    Not sure if I agree on that end. The death rate among the treated rats is more clustered together, indicating that max lifespan is being reached. So we are talking a more left skewed distribution in comparison to the control rat group.

    Do you mean that there may be a cause as to why the treated rats die in relative short succession?

    • Ill give my take

      Could be cell loss in respiratory reflex nucleus or cancer. If it is cell loss in respiratory reflex attempts to wake the rats up during sleep, if they cease breathing, might lengthen lifespan, as voluntary breathing can occur even after involuntary breathing becomes probabilistic.

      As for cancer Im curious how this would fare with cancer resistant mice which seem to have immunity to multiple types of cancer.

      Also Ill say people have to keep in mind that the epigenetic change was more modest in females,, iirc which were the ones tested for lifespan, if im not mistaken. Males had larger epigenetic rejuvenation it seems.

      • reduce breathing make them live longer?

        Rather that the older they get the more reduction in respiration, less oxygen less life

        And, WHY on earth would harold get blood from pigs … when he can get blood from beef. Just look at ancestral supplements, organ caps are from beef, bennefit would be alot less with pig glandular, thats why nobody sells it or uses it

        Why on earth do they take pig blood ….

  7. Gustavo Barja, Professor Of Physiology, UCM Univ. of Madrid, Spain. Coordinator of non-profit LMRG research group on said:

    Josh,
    thanks for the effort to summarize, analyze, and interpret Harold’s data. The result you got is what I expected (only around 7%). Because to me it is the nuclear aging program (AP) what drives aging. It is a very complex hierarchical net of genes (Barja, Biogerontology, 2008) interrelated through many molecules including TFs, small RNAi, epigenetic modifications of genes, and now it seems epigenetic modifications of many kinds of mRNAs too. Its final target genes (1 000?) synthesize the proteins that determine the activity of the aging effectors (mitochondrial ROS production,  fatty acid double bond index of cellular membranes, autophagy, apoptosis, proteostasis, telomere shortening rate? (in mitotic tissues), and others still to be uncovered. Inflammaging is likely a secondary effect of some of these aging effectors although we cannot discard it as a
    further aging effector on its own right. The global effect of all these aging effectors (classically called separated “theories of aging) is to determine the final integrated value of the aging rate of the individual or the species (Barja, Exper. Gerontol. 2019).

    Methylation clocks are very nice to know your biological age. But to me, concerning solving the aging problem, epigenetics is much more important than that, Steve! It is essential to modify the final global activity of the AP (1.000? AP target genes and their synthesized proteins, and post-transcriptional and post-traductional modifications). Such activity (which determines the integrated aging rate) depends on the Continuous Interrelationships between the hierarchically organized AP genes as well as, most important, Steve!: the constant interrelationships between epigenetic and genetic factors inside the cell nucleus throughout life.

    Only if well granted and good Gero-Scientists finally  decide to “Enter the Nucleus” (forgetting considering it a TABU: the result of non-PA mainstreamer censorship at anglo-journals I do not why they obey non sense no-AP theories which huge amount of data discard…), to clarify, at least generally, broadly, how the AP is hierarchically organized, what are (most important!) its MASTER GENES (e.g. Hox genes, since aging follows development, could be involved; Barja, Biogerontology, 2008) and how the  Epigenetics-Genetics Interrelationship continuously regulate each other mainly inside the nucleus every minute of our lives.  Only after knowing that we could surpass the maximum number achieved by CR (1,4 fold life extension = 40%) and go towards the only 10 fold life extension (1.000% increase in maximum longevity) we sorely need to defeat aging. Then we will get negligible senescence and the new human could live 1.000 years minimum!, if he takes good care of avoiding external damage he could live 2.000, 3.000, theoretically forever….as Bob said on his so beautiful song, “Forever Young”:

    But to get that we must not run too much (trying to selfishly save ourselves from death) looking for “magical potions” made of a few chemicals. Such a hurry and selfish-oriented approach will not work. In Science the first thing is to know the CAUSE of the process, AGING in this case. So we need before trying any “therapy”(which like Harold´s or senolytic fisetin give even less result than CR!) to know where and how is the AP general structure and organization, uncover its Master genes and its control through Genetic-Epigenetic crosstalk, and do that across species Steve Horvath!. Your terrific data on 128 species will mean even much more “GOLD” if you take them again and instead of representing separate curves individualized per species, try to COMPARE your epigenetics BETWEEN SPECIES with different longevities. That is what I would do, no doubt, even with 10 times less species. Please compare between the 128 species, you, the consortium that has the data! Do it before publishing the final paper instead of being limited to the “BioRxiv preprint” which lacks this essential issue and that is  full of curves of species but separated all from each other, species by species each alone! Steve needs to understand the huge importance of the COMPARATIVE APPROACH IN GERONTOLOGY not to WASTE such huge 128 species material! It is a shame that after the huge effort of obtaining samples from 128 different animal species!, they are not compared to each other! A lot of relevant information will be obtained if you do that. You can know if you compare rats and humans, if the epigenetic state of a rat at 6 months of age is similar or not to the epigenetic state of a human at 20 years of age!

    In summary I published my Barja Experimental Gerontology (2019) paper mainly to shout aloud!!! to the big well-granted laboratories who could approach the problem in the lab. (costly) interested in really untangling the causes of aging: stop looking only at the pre- or post-nuclear and organelles cytoplasm, PLEASE LOOK AT THE NUCLEUS! (looking for the Aging Program and its control) to finally solve aging. I know it is complicated. But complicated was also Complex I and I did not hear Siren sing to me “Dr. Barja look at CxIII because it is simpler!”. I did not pay any attention to these songs because I knew from many published papers demonstrating us that the ROS than matter for aging are those coming from Complex I. CxI is one of the biggest proteins in the cell 900.000 Daltons in weight and composed at least by 46 polypeptides (and corresponding genes) and experts had reached after decades its structure at 3 Amstrong resolution only in Thermus thermophilus bacteria after decades! So no hope? Will I be buried for a century before they reach mammalian CxI structure?  But nevertheless, I went on reading the (complicated to me who am not a molecular structuralist) papers of the only two scientists in the world attempting to solve CxI mammalian structure, John Walker (Cambridge, UK) and Leonidas Sazanov (Austrian Institute of Technology) and without any rational hope, but stubbornly as good student of Santiago Ramón y Cajal -the  Spanish Nobel Prize discoverer of the neuron together with the Italian Golgi. Cajal advised to insist for years in your ideas if you think they are correct, no matter what the critics or the abounding pessimists say, etc. So, I read all the papers from these two authors which dealt with Cx I structure until, suddenly, in only a couple of years, around 2015 everything changed, a new technique (cryomicroscopy instead of classic X-ray crystallography) finally solved the structure of Complex I in mammals!, both in sheep and cow at 3 Amstrong resolution!

    And, that, together with many other things, allowed us finally to localize the CxI mitROS generator linked to aging at FeS cluster N1a (inside the NDUFV2 polypeptide codified by the nuclear single gene ndufv2; and we suspect and study now too the FeS cluster N3 plus the flavin FMN which are both inside NDUFV1 polypeptide too. See: Mota-Martorell et al., 2020; and,: Pamplona and Barja, FEBS J. 2021). Both are at the tip of the hydrophilic domain of CxI which makes sense because there the midpoint-redox potential and tendency to reduce oxygen to superoxide radical is greater (among other most important details concerning interrelationship between the mitROS generator and the mitDNA).  I mention this as an example to this blog participants than in Science to be in a hurry and trying to get the solution without first knowing how the system is organized and works almost always necessarily leads to failure in biological systems which are very complex. And in the case of aging rate, which is controlled by many hundreds, perhaps 1,000 nuclear genes, no doubt any potion or “elixir” will get, even if were by chance correctly designed, negative Feedback Corrections from the AP, because the species Must Control its aging rate to avoid extinction in its ecosystem, so that the final longevity will not move. That is the Cause of the 1,4 fold limit (CR the best) of all anti-aging interventions tried so far.

    Geroscientists must look and understand the nuclear AP and how it works before trying to extend animal or human aging (“we should not “start building the house by the roof”: Spanish saying). With the potion-elixir approach we will never get negligible senescence nor the new non aging man living young at least for 1,000 years (A huge jump for humanity not only to free it from 90% of all the degenerative diseases, but because it is one of the huge evolutionary leaps needed, together with approaching or surpassing the speed of light, for humanity jump to the stars, its real destiny? (with the permission of… especially, NATO (90% USA, 10% EU-Biden and Russia- Putin who are the only ones that can kill all Eukaryotic life in 5 minutes…).
    And to solve aging we have not still started! Please “LOOK at the Nucleus”! no matter what the Neo-Darwinian- based mainstream evolutionists, in this case incredibly rejecting natural selection!!!! of aging!. They behave like Evolutionary dogmatic “priests” denying the evidence, saying that aging genes cannot exist! Heh heh! we already know around 1 hundred! Saying that old animals do not exist in the wild He heh! Field working (instead of seated in chairs) Zoologists found lots of them depending on the species He, He! And postulating DS theories that do not fit with almost any real data in Nature or the laboratory.

    These evo-mainstream priests who neither study nature in the wild or in the laboratory, know nothing about the internal biology of organisms or their ecosystems, and only sit in their offices and think up theories, which will be  most likely wrong using only such a procedure. These are just prejudices purposedly invented to avoid the otherwise obvious result that: “Aging has been naturally selected…at GROUP LEVEL”…that is the key point that their selfish gene reductionistic (philosophically ridiculous) theory and stubborn individualism tries hard to negate for already one whole century maintaining the standard Theory of Evolution frozen in that of 1930´s “Modern Synthesis” (I agree goo, d for those times in which the true priests of that time wanted to eliminate Darwin´theory (to save literally the Biblical I find nice metaphor of Adam and Eve), but ridiculous not accepting the huge number of advances of molecular biology concerning evolution: duplications, polyploidy, TEs, HGT, Evo-Devo mechanisms, Species Hybridization, Symbiogenesis, or even the small detail of Punctuated Equilibria of Gould and Eldredge, etc. For this, those interested please see Chapter 5 of my book “Longevity and Evolution” Barja, 2010 New York Nova Sci. Pubs.). If some of you wants it I will send it to you for free, just write to: gbarja@bio.ucm.es, because the editorial broke our contract selling it at 180 US$!! when I had agreed with them to sell it at only 20 US$, plus other dirty things they did to me and prefer not to comment now…! 
    But why do experimental gerontologists listen to those mainstream “Evolutionists” about non-existence of the AOP if they know nothing about biology, how the physiology and biochemistry of animals is, or how their bodies work? Why are we in the same problem (analogy) as in the times of Galileo when the high priests at Rome did not want to look through Galileo’s telescope pointing to the moons of Jupiter and threatening to burn him in the bonfire as they did to Giordano Bruno? There is no bonfire now, Gerontologists, all these guys can do to you is to censor your ms. (I have plenty of experience on this but I am courageous enough not to retract!) and there is widespread evidence and notion now that a lot of aging genes, and old animals in the wild do exist! And that trajectories of aging curves differ a lot among species which cannot be explained by the wear and tear of inanimate objects. The fact that each species has his longevity and that it can vary up to 1 million fold! (200 fold in mammals) is the best proof that the AP necessarily exists! There is no need to do any experiment to know that. Being a good biologist is enough. Denying the genetic character of longevity is like denying the genetic character of bull horns or elephant trump! This confusion (non-PA theory) can only be passed to non-biologists!

    So, well granted gerontologists, please Start to Look at the Nucleus! It is long work, perhaps for a whole century, because regulation of gene expression complicates each time further (e.g. now epigenetic modification of RNAs is also emerging….). But the sooner you start, the sooner Gerontology will get his final objective: to defeat aging.

    And perhaps there is not a century left to our jump to the stars if we continue like now with  social systems almost all based on Competition (inevitably leading to the3 war of all against all..) instead of also. Balancing it, its Heraclitian Opposite, “COLABORATION” which should be the motto of the XXIcentury.

    There is not much time left before nuclear megatonic war (a risk now with the Ukraine war more evident than ever..), Climate Change, Overpopulation (no one regulates that?, crazy humanity! Can Earth support even more than 7.000 million people living decent lives?, I do not think so…  Wars and millions migrating; Wars by Countries Pirate-like looking for resources first and then by simply water! will finish humanity as a civilized society.

    In my humble opinion we need a new UTOPY composed of 3 key traits (Gustavo´s own Earth-Wide Utopy, with the permission of Thomas Moro; and of Biden-NATO and Putin-Russia…):

    1) Only one single and truly social and democratic country will be allowed. Its name: EARTH. So all borders between preset countries will be erased (then obviously there will be no more need of any army because there will be no other enemy Country possible. Consequence: War will be impossible. Only regular Earth police will be needed for individuals breaking Earth law).

    2) There will be only one single “race” because “races do not exist at all” (Cavalli-Sforza the Italian  Anthropologist demonstrated it with simple blood electrophoresis in the 1970´s and told all the planet giving talks all around it).

    3) Never again will any man exploit any other man (to live without working).

    Obviously this needs an absolute Change of Social System that does not and has never existed on Earth (the present widespread Capitalism now in regression from the welfare state of the second part of the XXth century starting with  Ronald Reagan and Margaret Thatcher Neoliberal turn to its more savage form from the XIX century -now we have in EU a lot of Poor people with a Work!- will obviously lead us to extinction. A better social system is urgently needed to confront the too many global problems we face now. See how the Ukrainian war has paralyzed the efforts to solve climate change for instance.. If we continue with our wrong political systems (I include ALL present countries as full wrong, socially) there is, absolutely sure, no time for defeating aging (we have not still started to know the AP, and it is very complex in structure and function indeed!, that will take much time and money) and there is a sure outcome: EXTINCTION.

    Extinction sounds terrible and non-realistic but not to a biologist. Homo sapiens extinction would not be anything strange, because biologists know well that 99,9999…% of all the species that have once lived on Earth since the beginning of life 4.000 my ago have been extinct. So extinction is the rule rather than the exception. Human extinction will be the response of the ecosystem to a species that has over-polluted the environment. Ecosystems always behave that way. If a species does not fit in the new environment and ecosystem, it is simply eliminated and life goes on…This has happened billions of times in evolutionary history and is good because it preserved the existence of the only Gustavo´s 4 dimensions species on Earth: LIFE (Longevity and Evolution, NY, 2010)   

    Coming back to Harold´s results I repeat that they are nice but small results, E5 composition unknown (problem with private companies research), and no serious Gerontologist (nor NIH  ITP program) will ever accept any claimed life extension without full survival curves (from born to dead animals) and at least a minimum of 40 animals per group!

     In Science things must be DEMONSTRATED and in gerontology you never demonstrate increased longevity using surrogates, only full life span survival curves with appropriate controls give the answer Recently big people like the “Famous” here Spanish Researcher Juan Carlos Izpisua Belmonte e.g. pretended in a recent heavily commented paper to have rejuvenated mice when the survival curves in their paper stop at middle age ¿?¿? (is it the richest-granted ones who are more miserly expending on their own experiments?, or are they too much in a hurry to claim they delayed aging when it is false!  And Nature accepts that gerontological bull shit only for the name of the author!? I know by my own experience that to do the full curves well done is long and expensive (Spanish grants are now 50.000 Euros per group for 4 years if you associate with others…In the 90´s they normally were 100.000 to 150.000 depending on group size) but if you want something you must take the effort! I only could do just two aging experiments in my whole scientific gerontological life of already 42 years in Spain, a very poor science supporting country (a shame for EU standards), one at the beginning in Rana perezi frogs and we discovered hormesis! (but did not give it a name: three final papers published for six years old frogs experiment with 220 frogs, 110 control 110 treated (duplicated in young and old frogs starting with): López-Torres et al.Free Rad. Biol. Med.15: 133-142, 1993: López-Torres et al., J. Neurosci. Res. 34:233-242, 1993) and one at the end (Gomez et al. Aging Cell 2014, 40 mice control 40 C57BL6. mice treated). I preferred to do only 2 but well done..

    Well done aging experiments as Richard Miller one (Harrison et al, Nature 2009) in the case of rapamycin and not in all the others still unproven life span extensors: melatonin, metformin, resveratrol, etc. with results obtained in CH3 short lived control mice (e.g. By Vladimir Anisimov at Saint Petersburg) but which were not reproduced when repeated at NIH under the ITP program with appropriate longer lived control mice. Using short-lived controls is a well known trick that old gerontologists all know and modern molecular biologists seem to ignore when entering the field (you must study a lot when you change field, and you must first learn the basics! As if you were a bachelor student, no matter if you have published in Science, Nature etc.) 
     
    Finally Josh, are these provisional again, Harold’s results published in some place?

    Sorry again for my too long comment

    Gustavo Barja

    • Gustavo, if you saw what happened to hand after a single application of E5 – you would have no doubt that there is no comparison to rapamycin (which I took for a while – until I found it was contraindicated for Irritable Bowel disease because it prevents the regrowth of the intestinal lining. E5 is what keeps animals young naturally so the only side effect is youthfulness.

      • Harold,
        Excuse me if I contact you this why but I invited you on July 12th to write an article on “Heterochronic Parabiosis” for an Special Issue of the Journal Experimental Gerontology on “Physioloģical and Evolutionary Mechanisms of Aging” which I was invited to Edit and is composed of 15 articles (Josh will wrte one of them) but I have not received any answer from you (I used the two different emails I have from you to invite you). Could you please give me an answer as soon as you can to my email? : gbarja@bio.ucm.es

      • Harold, why dont you use blood from a beef instead?

        Why use pig when it will be alot less quality vs beef.

        Look at all the companies selling organ glandular, NOBODY uses pig .. everyone uses a better animal aka beef. please look for a grass fed beef vendor

        and not GMO fed pig

  8. I always recollect Didier Coeurnelle emphasizing testing max lifespan when commenting, since its inception, on the E5 research, so I was not surprised when HEALES supported this year test. And btw this last post is witnessing again Josh being a wonderful human being and humble scientist, honestly hypothesizing and analyzing data. Thank you and all the best! I envy his and other brain here. Even if eventually lifespan translating to humans does not pan out as one would hope I belong to that group still appreciating the potential healthspan increase (am now 67!). I am still trying to live a decent lifestyle and keeping testing myself, e.g. using Phenotypic Age and Aging.Ai (despite losing a bit my trust in the latter due to the lack of their debugging the version 1.0), but still not sure if and which DNA methylation clock I could make good use of.

  9. Information for everyone.
    Real rejuvenation of rats is possible when giving rats water or a solution of malic acid, in which I can increase the tension of hydrogen atoms to a certain value by information activation using information field technology. These substances will rejuvenate rats until their perfection time after birth.
    To get such water, I need to send a photo of the water and from the photo I can charge the water right away. I already use this approach to treat and rejuvenate people. You can try it on rats, it is absolutely safe and this is the only way to rejuvenate rats.
    My address ErmakovP@i.ua
    Ermakov Petr, professor, Ukraine, Mirgorod

  10. I don’t think we have enough data yet to be certain of any theoretical concepts yet. After all, there has been clear evidence that some tissues “age” faster than others. (The thymus, for example.) So even if there is a single master aging clock, that does not mean that – 1. all tissues age at the same rate based on the status of that clock. and 2. Turning back that clock does not necessarily mean that all tissues will respond to the same extent.

    This is testable with the current technology. Take a cohort of young rats and start the E5 treatment for the majority of their lifespan. See how long they live. Will it block aging of those tissues that age faster? Will this have any effect on maximal lifespan? If so, what tissues are being affected? That would provide a roadmap for more targeted research. If the maximal lifespan does not increase, then there is some other general aging process that has yet to be discover.

  11. Thank you Charles, Mehrle, Ines, Albedo. Although healthspan improvements are quite attractive- I am one of those who have seen my parents suffer from diseases, we are hoping that E5 will increase maximum lifespan in a youthful, healthy body. As a biotechnology company our goal is to produce therapeutics that reverse diseases onset by aging and protect against them by reversing biological age. So Lifespan wasn’t of primary interest at this stage when we are planning to move from preclinical to clinical. But Didier by sponsoring this study has piqued our interest in lifespan as well. At some point Harold and I do want to see a lifespan study where we see the response we saw in our earlier male rat study. If the rats of both genders get young by 50% do they live longer?

  12. And of course thank you Josh for applying your brilliant talent as a statistician to this research 🙏 I know even better results in future studies will delight and vindicate you more.

    • Received the Yuvan update, thanks. With a rat maximum lifespan of 45 months, and the three subjects now at 41 months, will they get one or two more treatments before age 45?

  13. Actually, the company is “Yuvan Research” (“Yuvan” from the Sanskrit meaning “youth” (as in “juvenal” – Sanskrit is an Indo-European language)), and thank you for your statistical analysis. This was not a well-designed experiment, and it will be repeated, but performed correctly. While the male rats had their epigenetic age reduced by more than 50%, the females had their epigenetic ages reduced by a tenth of that. However in my last trip to Mumbai, a couple of months ago, I was actually able to see the rats, and it was clear to everyone which rats were treated or not, glossy, thick fur, more interest in their surroundings, more activity. in the treated rats. But what really made that agonizing trip worthwhile was the casual experiment I tried wherein I applied an old sample of E5 to the back of my hand and in three or four days, say my hand was apparently decades younger. So, I’m not submitting this as a valid experiment – it was not, but it does tell me (who knows it’s true and it will be repeated so everyone will) that E5 works on humans, at least as a topical application. We also know it acts where applied, rather than systemically.
    The question of whether there is a causal relationship between the rate of aging and life-span is what was investigated in two Nature papers this week – one by Caleb Finch and Austin two greats, that concluded that at least among exotherms like salamanders and turtles the answer was no – showing minimal aging, but relatively short lifespans, so that aging might have a function other than simply ending life. It also seems to be an evolved trait – in primitive chordates, like sharks, the physical limits of its size (the ratio of gill area to body mass) limits lifespan – as organisms, particularly terrestrial vertebrates evolved on the more resource-limited land, mechanisms evolved for limiting lifespan.
    There is however evidence that maximum life-span can be increased, in the experiment of Ludwig using heterochronic parabiosis the maximum life-span was increased by ~30% and the young parabiotic partner was old by the time the experiment ended, we (our group) can give the old rat the equivalent of serial young parabiotic ‘partners’. Oh, and one other very important learning (if you can believe it); there are beyond a doubt youth-producing substances in young blood, we have proven it.
    By the way, I don’t liken aging to war, but to an inherited progression of post-adult life stages, ie. young adulthood to old, old age. In a war, there is a chance either side could win, but there WAS no chance of life winning. Life seems to be more like a play. (I believe Shakespeare noted that).

    • “…By the way, I don’t liken aging to war, but to an inherited progression of post-adult life stages, ie. young adulthood to old, old age. In a war, there is a chance either side could win, but there WAS no chance of life winning. Life seems to be more like a play. (I believe Shakespeare noted that)…”
      So lucky and grateful to have met Josh’s blog. It’s a dream to read online replies directly from people like him and Katcher (and many more here). I just wonder if I am too astray from linking this quote to Katcher’s own theory (leveraging David Neill’s) as explained in his wonderful small book: I mean is the quote related to the “life’s timer” at p. 121 and Fig. 12 representing life phase as oscillations? I understand Fontana countered the vision of a clock or timer or oscillations though. Thank you!

    • =While the male rats had their epigenetic age reduced by more than 50%, the females had their epigenetic ages reduced by a tenth of that.=

      Why do you think it happened, Dr. Katcher?

      Also, what is your opinion on the recent finding that naked mole rats age epigenetically but not phenotypically? Another finding is that NMR have their t-cells mature outside of the thymus. Could it be that thymic rejuvenation is crucial?

    • If, as you say, the female rats in this experiment has their epigenetics reversed by ~5% to 10%*, then the maximum lifespan increased almost linearly.
      Consider: only 5 out of 8 treated rats died. When the other 3 treated rats will die, the maximum lifespan increase of treated rats will increase from 7% to probably 10-15%.

      This is strong evidence that epigenetics correlates not only with healthspan, but with maximum lifespan.

      *The percentage of the reversal of epigenetics is essential information, that should be in our host’s blog post, not only in the comments.

  14. Very impressive analysis, Josh! Thanks for sharing them.
    These results (specifically the mortality compression) vaguely suggest to me that Maximum Lifespan is an independent process controlled through a different clock mechanism, other than the known epigenetic clocks that present relevant correlation or even causation to the aging process. There’s apparently an “exponential wall” which defines Max Lifespan that seems not to be fully connected to the aging process itself as measured by the epigenetic clocks or even other general health indicators.
    How would you otherwise explain that physiologically “younger” rats are dying at about the same age as the physiologically older (untreated) ones?

  15. Based on the tiny and fragmented data that we have so far, it is looking like long term life extension will require a group of different solutions, all necessary but individually insufficient, to seriously extend life span.

    On the other hand, this is not cause for negativity, inasmuch as there are multiple, completely different, pathways being researched. I suspect that within 10 years, the results of the various pathway will be known and the combinations will be being tested.

    And along the way, some will provide greater healthspan, while the research goes on.

  16. I was very surprised to learn that the rats were living in separate groups. This seems an obvious stressor – easily avoided.

    Would it not make sense to do things that make all the rats more likely to live longer? Why not have them live together?

    • Gustavo Barja, Professor Of Physiology, UCM Univ. of Madrid, Spain. Coordinator of non-profit LMRG research group on said:

      In experimenatl science the two groups are almost always living in separated cages to avoid any confucsion, especially in long-life experiments, although I understand your point. Also living in cages of a limited surface is not like free living of course. That´s lab. science. If results are good one can go to field “experiments” in the wild under not well controlled conditions of course….same problem with human studies.

      Anyway, with that system experimental physiology and science has advanced hugely in a century since Claude Bernard´s times “Introduction au Methode Experimental en Physiologie et Medicine” (the true father of Homeostasis concept, “La constance du milieu Interieur” as Claude called it and explained at large on its seminal foundation books on what Experimental Science is in general..and the main basis of Physiology too, 50 years before Walter cannon coined the name “Homeostasis”. This is always “forgotten” in all textbooks books of Physiology Worldwide much to my chagrin.

    • It might be interesting to create a third grouping in which the treated and control rats live together, to see if it changes anything in either the treated or control rats.

  17. I assume that everyone tracking this study believed that the treatment arm of the study would outlive the untreated controls by a much longer interval of time than actually occurred.
    I believe that one explanation for the unexpected result of the most recent E5 rat study may be a self-sustaining epigenetic / transcriptional feedback loop. If Harold’s plasma fractions are epigenetically reversing a cell fate transcription determination that falls into the early life category, then the mechanism for inactivation of the longevity effect of E5 is known.
    [2022] Transcriptional and epigenetic control of early life cell fate decisions
    From a very cursory search for epigenetic control by feedback loops, multiple mechanistic systems have been identified that reverse epigenetic marks by erasing or writing methylation marks as a direct counter-intervention from the environment, genetic, drug, biological, or disease processes.

    On stark element of the results that Josh has shared looks like a pharmacological result of introducing a toxin. I am referring to the compressed window of time that the treated rats all died within. This small window increases the possibility that a feedback loop, take your pick as to which one gets invoked, becomes activated and this leads to a reversal of the longevity effect from the administration of E5.

    Epigenetic results from this study at multiple time points are the only way to begin to nail down this mechanism. Hopefully, that data can be analyzed.

  18. I don’t really think there’s a reason for discouragement.

    The first trial suggested that E5 might (possibly) make males live longer (based on the reduction in their epigenetic age.)

    That test should have either been repeated with male animals, letting the animals live their full lifespans this time, or repeated with equal numbers of males and females. Instead, all the rats in this trial were female, a totally different test.

    What this trial illustrates, if anything, is that males and females are different in key biological ways. It may turn out that the path to life extension is different for males than it is for females.

    E5 may turn out to be a path to male life extension. If so, we should perfect it, while simultaneously working to figure out a path to female life extension, which could turn out to be a completely different compound.

    • Gustavo Barja, Professor of Physiology, UCM Univ. of Madrid, Spain. Coordinator of non-profit LMRG research group on said:

      This experiment further illustrates that in spite of almost perfect correlation of methylation clocks, the whole survival curve with at least 40 animals per group and good longevity of the controls continues to be needed to validate any life extension. Neither methylation or any other future surrogate will ever substitute appropriately performed aging experiments with good and whole survival curves.

  19. Much focus on cells and mitochondria, but very litlle on the extra cellular matrix (ECM). I’m sure most of you above the age of 50 knows what I’m talking about. On a personal level, I do not lack either energy or muscle mass, but the persistent pain from tendons and ligaments (especially from running and sprinting) is the first step towards becomming less active and more sedentary. Healthspan is my primary focus, and I wonder, if there is any preliminary data on protein crosslinking in the ECM from E5 treated rats vs control rats?

  20. Thank you for your analysis, Josh. I came across some lifespan extension percentage comparisons after publishing those results, commenting things like” A 7% extension in mice would be a ho-hum result, considering much less invasive, and way lower cost treatments can currently produce a 23% life extension. Rapaymycin comes in at around 23%” and so on, but, as I have said many times before on our previous age reduction breakthrough blog, we are not simply a mechanism assembled in parts and we need to address ovarian aging, brain aging, muscle aging separately. We need to do it as a class, systemically to aging and until we have opportunity to discovering the region of non coding DNA that gives birth to elements that trigger the various changes that lead to the aging phenotype, so called conductor, I consider hacking the factors and proteins that cause progressive age related changes in the activation and repression of genes and replace them with factors and proteins either from a young environment or removing sole negative ones stopping us repairing, the only solution. This will change the gene expression signature back to what it was in youth and hopefully, lead us to rejuvenation. I am sure Harold and Akshay will be able to have a gender specific version based on the same principle, if at all needed in this newest mixed gender study. As Akshay said, E5 is still a crude biology, needing to be perfected, which will happen by this outstandingly fortitudinous team, I am 100% sure. Why we still can’t see a radical, dramatic life extension with the particular pathways fully solved and treated/activated? Just like the developmental program, the aging program too is global, unfolding in every cell in our body. Change is constant in our lifecycle. If there were no changes we would remain an egg. We would remain a baby if changes stopped after we developed into one. These changes make us an adult and then begin aging till we die. With E5, you are hacking biology itself, not a particular symptom, pathway or metabolism. You can lower your epigenetic age simply by taking absorbable form of calcium AKG, raise your glutathione level with acetylated glutathione, lower triglycerides, raise SOD, NAD levels and etc, but you will still die because of the transcriptional onslaught regulated by aging. Therefore, I do not think it’s a good idea to compare E5 with anything, even though, this experiment was not as impressive as the previous one.
    According to our DNA, the maximum lifespan of humans should be 38 years.
It is very chilling to read that based upon DNA epigenetic clock, humans would be expected to have a maximum lifespan of 38 years.
As some programmed theory believer scientists suggest, which I do agree and completely share that humans have modified the program and turned dying into senescence. I think the DNA study explains why humans have a very long period of senescence.
    After age 38, We are living on borrowed time.
In view of these mass extinctions, it is peculiar that aging theory is all about how animals should focus on longer lifespan as opposed to how animals should try to avoid extinction. The best way to meet the challenges is to evolve new traits to adapt. Understanding the best way to evolve new traits involves mathematical reasoning.
    Finally, I am more and more impressed by Harold and Akshay’s orientation and inclination, the conception of comparing and translating things from the anthropological standpoint, looking at super diversity and complexity of the living organisms and studying their conserved and unique mechanisms, not to mention their undaunted nature and the passion of being into dimension as its best, all the fears coming from the extreme complexity of the aging process fade away and make us to remain, at least not pessimistic, if not optimistic.

      • Thank you, Josh, I greatly appreciate. As for the “conductor”, I wish I could, but, as Sinclair said: “someone discovering the conductor, most definitely deserves the Nobel Prize. Most of the beliefs I do have, come from Akshay, my favorite and a true genius. To me, there should be no single one, like the hypothalamus. Apparently, each of our 30 trillion cells has its own manager that releases time regulated instructions or temporal regulation. So it seems there is no single chief but 30 trillion managers each managing their own cell and coordinating with other managers, maybe by our circulation to out those regulatory changes. But as you said, a faster ticking, oldest clock is going to win the battle and kill us.

    • Who said our lifespan is 38 ? …. dumbest thing ive heard

      Harvard study last year showed our lifespan is in reality 120 years.
      xx months later a new study came out showing that if we just have better dna repair, our dna can hold 150 years

      about the 120 tho, we are so toxic that we die 40 years before we are suppose to. guiess what toxins …

  21. Above else what this shows is that there is a massive need for more basic research into aging. One can hope with the recent launch of Hevolutiion that has a billion dollar per year pledge and the mounting efforts in the US to lobby Congress for more funds for basic research in aging, that will can get a clear picture of what causes aging, at least in a single cell organism, within ten years or so. Even that is far from certain though. We must all continue to help the effort in whatever form we best can.

    • Gregory, thanks for the link.

      The patent is 100 pages not including multiple addendums.

      The patent provides a roadmap for dramatically accelerating and improving the ability of all of us to access the dramatic and important opportunities that have been demonstrated from studies on parabiosis and young blood transfusion. Harold has stated many times that his insight into developing E5 came from a 2005 Conboy study. Reading the entire patent is very informative and opens the door to addressing multiple disease processes utilizing a xenographic interventional approach.
      I have produced a synopsis of the most important points as disclosed in the patent on this page of the ARC website.
      https://salmon-bluebird-ese8.squarespace.com/config/pages
      Scroll down this page to the pictures of Harold and Akshay.

  22. Here’s a thought I recently had. Since E5 is a plasma fraction that contains pro-youth factors, would its effect be even more potent if combined with plasma dilution, which reduces pro-aging factors?

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