50 replies to this topic

[Jeoshua]

There has been a lot of talk about C60, it's potential benefits, it's side effects, and various studies done on rats that show it may be good for extending their lives. This is all very good stuff. It's interesting, thought provoking, and radical science. I am impressed. But one thing bothers me:

How does it work?

I have seen no real evidence on how C60-EVOO is supposed to work. No information on it's metabolism. No information on what avenue that it enters the body by. The only bit of information I have seen that made any sense was an "in virtuo" study about the lipophillicity of C60 and how it would be preferentially attracted to cell walls, by virtue of them being extremely lipid. But that same information could mean that the prefered route of C60 into the human body would be through the lymphatic system.

I am looking for information on the "life cycle" of C60 or C60-EVOO administered to a mammalian patient orally. Where does it go? What does it do? How does it act in the body, and where? Theories about Electron Transport chains or Superoxide Dismutase are okay, but what evidence is there of this? Has anyone found any real information that proves any of those mechanisms are really in play?

[AdamI]

The rat study.... and also the on going very big studies on rats.

[niner]

Has anyone found any real information that proves any of those mechanisms are really in play?

Proves it? No. We have plausible hypotheses. That's all. "Proof" is hard to come by in the biological sciences. Mechanisms will be ironed out in the fullness of time, but it's going to take years.

[Jeoshua]

Perhaps I came off a bit too hostile. I genuinely want some kind of explanation for what happens after one takes C60. Where does it go, most of all?

[Hebbeh]

http://www.longecity...ic-discussions/

http://www.longecity...periments-home/

http://www.longecity...eory-of-action/

http://www.longecity...lifespan-study/

Start reading. Everything we know and then some will be in these threads.

[Turnbuckle]

There has been a lot of debate here on how it works and I've thrown out my own ideas, which are summarized on my profile page. However, this is a relatively new discovery, not even replicated yet, so you may have to wait a while for someone to nail down the mechanism.

[geo12the]

Perhaps I came off a bit too hostile. I genuinely want some kind of explanation for what happens after one takes C60. Where does it go, most of all?

You are not going to find this kind of information because it doesn't exist. There is only one published study on C60-OO. It showed a strong effect on life extension in a rat study. Lots of people here are putting a lot of faith into that study but its possible the effects they saw were entirely by chance because of the size of the study. These kinds of studies are notoriously difficult and sometimes scientists get over excited about results without trying to reproduce them. But other studies have shown health benefits (neuroprotective effects) of other forms of C60 but these were in-vitro mostly I think. Lots of anecdotal reports of positive health effects on this forum. But some of that may be placebo. The bottom line is that not a lot is known. Personally I have tried the stuff but I am on the fence if this stuff is beneficial. I have no idea if any scientists are working on trying to reproduce the original life extension study. That would be useful.

[niner]

Lots of people here are putting a lot of faith into that study but its possible the effects they saw were entirely by chance because of the size of the study.

This is a common misconception. Because the effects were both huge and uniform, the odds of it being due to chance are extremely small. In their 2012 paper, Baati et al. reported the probability that c60-oo extended lifespan as 0.999 with respect to the water placebo, and 0.995 with respect to the olive oil placebo.

I'm aware of one current placebo controlled study of lifespan enhancement by c60-oo. This is being performed in Ukraine, but it is not a replication study, because the rats were started in middle age.

Edited by niner, 24 February 2014 - 09:06 PM.

[geo12the]

Because the effects were both huge and uniform,

I am more skeptical because the effects were so huge. In science if something seems to good to be true it often is.

The past year or two there have been a few papers and opinion articles in the scientific literature that highlight how many, perhaps most, scientific studies these days are not reproducible.

In their 2012 paper, Baati et al. reported the probability that c60-oo extended lifespan as 0.999 with respect to the water placebo, and 0.995 with respect to the olive oil placebo.

Statistics are frequently misused and misrepresented by researchers. With the small sample size of the study its meaningless.

I'm aware of one current placebo controlled study of lifespan enhancement by c60-oo. This is being performed in Ukraine, but it is not a replication study, because the rats were started in middle age.

Are there other labs looking at this at all?

I'm not saying the stuff does not work, There is good evidence in addition to the Baati study that there are health benefits of this stuff. But I would definitely like to see more studies.

[Hebbeh]

Who's going to do studies and why would they want to do studies? It's not patentable so there is no money or profit to interest big pharma in investing in studies. It would be a waste of time and investment for them. And even if somebody had decided to pursue studies, they're not going to broadcast that until they're in a position to take advantage of the results.

Edited by Hebbeh, 25 February 2014 - 08:46 PM.

[Jeoshua]

While C60 itself might not be patentable, various derivatives and combinations of it would be. And "Big Pharma" is not the only group who could benefit from general studies on it. Actual manufacturers of C60 might be able to show that C60, due to it's metabolic pathways, is not toxic as some would assume, and thereby put themselves in a better position as far as continued sales go. And if it proves to have beneficial actions, then that means more sales for the manufacturers, as well.

Long story short, there is more than enough reason for research, beyond just curiosity.

[Hebbeh]

The toxicity studies have already been done. That was how the potential was discovered. There is nothing more to prove there warranting further investment for studies already completed. And they can't patent C60 for a use they didn't discover since the horse is already out of the barn and it's being freely sold as a supplement by a number of vendors. And it would be a trivial market for manufacturers of C60. Bottom line is there is no financial incentive for anybody to do studies.

[niner]

In their 2012 paper, Baati et al. reported the probability that c60-oo extended lifespan as 0.999 with respect to the water placebo, and 0.995 with respect to the olive oil placebo.

Statistics are frequently misused and misrepresented by researchers. With the small sample size of the study its meaningless.

It's not meaningless unless you just refuse to believe in statistics. If the sample size were larger, then the probability would be 0.9999 or 0.99999 instead of 0.999. In most of science, statistical significance is considered to be 0.95, the point at which people start to consider things to be "meaningful". In this case, the sample size isn't a problem.

I'm aware of one current placebo controlled study of lifespan enhancement by c60-oo. This is being performed in Ukraine, but it is not a replication study, because the rats were started in middle age.

Are there other labs looking at this at all?

You mean besides the lab in Ukraine? That's the only one I'm aware of. Actually, they're using mice, not rats; I misspoke above. The trouble with lifespan experiments is that they take years to complete, so it's going to be a long time until we get more data.

[geo12the]

It's not meaningless unless you just refuse to believe in statistics. If the sample size were larger, then the probability would be 0.9999 or 0.99999 instead of 0.999. In most of science, statistical significance is considered to be 0.95, the point at which people start to consider things to be "meaningful". In this case, the sample size isn't a problem.

I've seen too many studies where statistics are used to support data based on flawed experiments and weak controls.

These particular kinds of experiments can be influenced by weird environmental and other effects that the statistics don't effect. I was listening to a podcast on reproducibility in science where one mouse researcher was discussing how he used to select mice for experiments by picking them out of a cage. He would pick 10 for one group. then another 10 for a different group etc. But the act of picking the mice was skewing his data because the first ones would be the easiest to pick (less afraid, slower) compared to the last group (harder to catch, more aggressive) he picked.

Context is important. In the context of their experiment where they looked at only 6 rats in each group I just don't feel like you can really say anything about significance. It's very intriguing but I will believe it when the life extension results are reproduced, in rats or another animal system. Let's keep our fingers crossed.

[niner]

Significance is about whether or not the result is due to normal random fluctuation. It says nothing about whether the experiment was flawed or not. Whatever problems this study may have, sample size isn't one of them. You need a large sample size in order to tease out small effects, and everyone seems to have latched onto that meme very tightly. What many people seem to miss is that these are not small effects. They are large effects. Everyone is free to make their own decisions regarding the probability that this experiment was run correctly or not, but anyone who bases that decision on the sample size is making a mistake. If there is something wrong with this experiment, it's a systematic error. If they had used 12 rats, or 1200 rats, the systematic error would still be there.

[shifter]

While it may not be proper 'science', lets look at all rats everywhere who have not been given c60 and call them 'controls'. Has any rat (especially among the strain used) ever been known to live 5+years?

But we have 6 rats all next to each other that survived up to this long and remained in good health while others (both next to and around the world) die long before them and riddled with tumours or disease


So long as evryone is saying 'cant be bothered doing life extention studies, it'll take too long', it will never be done. If it were started years ago when c60 potenitial benefits came to light, we'd almost be done by now.

[geo12the]

If they had used 12 rats, or 1200 rats, the systematic error would still be there.

You can't run statistics with a small sample size (as in this study) and really say things are significant. Statistics are so misused in Science these days. People say things are statistically significant and its seen as magically validating their work.

For me the olive oil alone effects (I have only seen hand waving about) are a red flag and suggest that there is lifespan variation in the rat population unlinked to treatments. The control population may have gotten a skewed amount of the more short lived rats. With only 6 rats per treatment this can easily happen. Had they used 60 rats it would be more believable.

[ambivalent]

You can't run statistics with a small sample size (as in this study) and really say things are significant.

You can. Suppose, I give c60 to one c-elegan worm and it lived to 150 days. What odds would you like to receive (or be indiffferent to backing or laying) that the next c-elegan given c60 would live less than 60 days? If there were 6 from 6 c-elegans living to over 100 days, what odds that 5 from the next 10 live less than 60 days? As niner says, when the effects are large the sample doesn't need to be.

[geo12the]

You can't run statistics with a small sample size (as in this study) and really say things are significant.

You can. Suppose, I give c60 to one c-elegan worm and it lived to 150 days. What odds would you like to receive (or be indiffferent to backing or laying) that the next c-elegan given c60 would live less than 60 days? If there were 6 from 6 c-elegans living to over 100 days, what odds that 5 from the next 10 live less than 60 days? As niner says, when the effects are large the sample doesn't need to be.

I don't agree. You can't be sure what is causing that C.elegans to live longer is the c60 or that it has a mutation that causes it to live longer or it was in a part of the incubator with the optimal temperature for it to live longer. When the effects are large is exactly when you should be questioning the science. Not saying it's not true, just saying it needs to be taken with a grain of salt.

[niner]

For me the olive oil alone effects (I have only seen hand waving about) are a red flag and suggest that there is lifespan variation in the rat population unlinked to treatments. The control population may have gotten a skewed amount of the more short lived rats. With only 6 rats per treatment this can easily happen. Had they used 60 rats it would be more believable.

There was a significant lifespan extension from olive oil alone. While we know that high quality olive oil (such as the Tunisian Chemlali that they used) leads to modestly longer lifespans in humans, I don't know that anyone has ever run a rat experiment with good olive oil and an intermittent dosing such as was used here. At any rate, there are three different groups of rats with three different treatments, and three very different outcomes. What part of that makes you think that the treatments aren't related to the outcomes? The water controls lived normal lifespans for Wistar rats. That tells us there was not a selection of uniquely short-lived rats in the control group.

[Turnbuckle]

Who's going to do studies and why would they want to do studies? It's not patentable so there is no money or profit to interest big pharma in investing in studies. It would be a waste of time and investment for them. And even if somebody had decided to pursue studies, they're not going to broadcast that until they're in a position to take advantage of the results.

Patent applications are generally published these days, so you can find who is working on what. One company with a strong interest in C60 is Luna. Here's a list of their applications--

PUB. APP. NO. Title
1 20110251158 FULLERENE THERAPIES FOR INFLAMMATION AND INHIBITION OF BUILD-UP OF ARTERIAL PLAQUE
2 20110178316 NOVEL FULLERENE CYCLOPROPANATION REACTION
3 20110065944 NEW REACTIONS OF FULLERENES
4 20110062390 DERIVATIVES OF NANOMATERIALS AND RELATED DEVICES AND METHODS
5 20110028522 FULLERENE THERAPIES FOR INFLAMMATION
6 20110003773 USING FULLERENES TO ENHANCE AND STIMULATE HAIR GROWTH
7 20080214514 Steroid derivatives of fullerenes
8 20080213352 Liposome carriers for in vivo delivery of fullerenes
9 20080213324 Amphiphilic or lipophilic polar functionalized fullerenes and their uses
10 20080166285 Pegylation and Hydroxylation of Trimetallic Nitride Endohedral Metallofullerenes
11 20080085234 Chemical separation method for fullerenes
12 20050085051 Tunable dielectric device and process relating thereto

[ambivalent]

You can't run statistics with a small sample size (as in this study) and really say things are significant.

You can. Suppose, I give c60 to one c-elegan worm and it lived to 150 days. What odds would you like to receive (or be indiffferent to backing or laying) that the next c-elegan given c60 would live less than 60 days? If there were 6 from 6 c-elegans living to over 100 days, what odds that 5 from the next 10 live less than 60 days? As niner says, when the effects are large the sample doesn't need to be.

I don't agree. You can't be sure what is causing that C.elegans to live longer is the c60 or that it has a mutation that causes it to live longer or it was in a part of the incubator with the optimal temperature for it to live longer. When the effects are large is exactly when you should be questioning the science. Not saying it's not true, just saying it needs to be taken with a grain of salt.

All this is independent of sample of size, its not down to variance: you would bet that someting exceptional is occurring. It is much more likely, I would suggest, that something is very wrong experimentally, than this is flukey data. If, given the same sample size, this was replicated independently it would be highly conclusive.

[Adaptogen]

Yesterday Anti-Aging Firewalls posted a detailed update in regards to their theorized MOA.
http://www.anti-agin...s-in-olive-oil/

[xEva]

Yesterday Anti-Aging Firewalls posted a detailed update in regards to their theorized MOA.
http://www.anti-agin...s-in-olive-oil/

Thank you for linking it. It's a great summary of the latest research. Makes quite a rollercoaster ride though. Obviously the dust has not settled yet. It would be very interesting to see what mechanism of action it will turn out.

I had some problems with his first part where he discusses the Russian paper proposing that C60 acts as a charge transporter. He says:

As I understand it, the process goes like this:

1. The outer mitochondrial membrane is charged positively and the inner membrane is charged negatively. This is because free electrons are spun off in the complexes in the electron transfer chain. As the chain becomes less efficient and there is decline in expression of mitochondrial antioxidants, the more there is a charge differential.

That's not how I understand it. Maybe I'm nitpicking, but I can't help it.

First, the outer mitochondrial membrane has negative charge, just like bacteria. As for the inner mitochondrial membrane (the one that surrounds the matrix) its outer side is positively charged while its inner side has negative charge, which creates a difference in electric potentials -- and then he even quotes the Russian paper that says just this -?

Second, the difference in charge is due not to "electrons in the complexes" but to the number of protons on both sides of the membrane.

Third, a higher "charge differential" corresponds to higher efficiency in ATP production which is accompanied by higher ROS generation. In healthy mitos this should lead to upregulation of mitochondrial antioxidants rather than to their decline as he writes. The greater difference in electric potentials corresponds to a 'tighter' membrane. Uncoupling proteins (and Skulachev ions) make 'holes' in it, allowing some protons to escape to the other side without participating in ATP production. This is called "mild uncoupling of respiration" and it diminishes both ATP production and ROS generation. This is how I understand it and I sure hope I got it right -?


He seems very excited about this proposed role for C60 as a charge transporter, which makes it act essentially as a special kind of mitochondrial membrane uncoupler. But then what about the other, well known uncouplers? First, there are endogenously produced UCPs and then there is a number of various substances, like metformin and BHT just from the top of my head, not to mention Skulachev ion -- and none of them ever demonstrated the phenomenal life extension of c60oo. And it does not matter even if "fullerenes accumulate in the middle layer of the mitochondrial membrane where they can remain for a very long time", as he proposes, because those other uncouplers are taken daily, or even twice a day like with metformin. Something does not compute in this proposed role for fullerene as a charge transporter / uncoupler -?


All this contradictory research is perplexing. I thought it was cute how toward the end, in part 11, first he quotes a 2010 publication: "It summarized the biological activities of fullerenes derivatives ... as reactive oxygen species scavenger and lipid peroxidation inhibitor..." -- and then starts the very next paragraph with "The proposed mechanism of action is the generation of ROS." -?! Can c60 really be both ROS scavenger and lipid peroxidation inhibitor and ROS generator?

lol it's like a rollercoaster ride. In the end of part 11 he quotes the paper that proposes c60 as a ROS generator that binds antioxidant enzymes "which might lead to accumulation of reactive oxygen intermediates.” And part 12 is dedicated to a paper proposing the opposite: "The results obtained from this study clearly suggest that fullerol treatment suppresses the inflammatory responses ... by decreasing the level of ROS and potentially enhancing anti-oxidative enzyme gene expression".

He tries to explain this:

These seemingly contradictory conclusions require more detailed explanation since both of these observations could well be correct. [no way!] For example, production of ROS could be a cellular response to C60, and above we related how C60 might suppress the generation of ROS by inhibiting the formation of superoxide as well as by antioxidant effects.

But I thought the difference was in 'pristine' c60 vs 'nanocrystals', the latter being pro-oxidant -? He does not seem to make this distinction.

His conclusion: "Fullerenes may make a key difference in animal health, but figuring out how may require thinking “out of the box", for which he offers "Jim's" hypothesis: they quench "bad ROS damage" but produce "good ROS signaling.

Edited by xEva, 28 February 2014 - 06:36 AM.

[niner]

But I thought the difference was in 'pristine' c60 vs 'nanocrystals', the latter being pro-oxidant -? He does not seem to make this distinction.

His conclusion: "Fullerenes may make a key difference in animal health, but figuring out how may require thinking “out of the box", for which he offers "Jim's" hypothesis: they quench "bad ROS damage" but produce "good ROS signaling.

Yeah. Vince is a powerhouse, god bless him, but he's not a chemist. He's fairly new to molecular biology, too. The primary source of confusion here is that he's not appreciating the difference between the [aggregated / nanoparticle / nanocrystal / "nano-c60" / nc60] forms and the molecular forms. They are polar opposites as far as free radicals are concerned.

He's also putting way too much stock in that goofy paper that used density functional theory to arrive at an improbable structure with protons inside the c60 sphere, and proposed uncoupling as a mechanism. I posted on that a few months ago. That's one of those papers where I ask myself "how did this thing get published?"....

[xEva]

I have a couple of questions re these 'good' and 'bad' ROS. First, can mitochondria really produce 'good ROS'? In my understanding, all ROS they produce is bad, and high ROS production is the hallmark of their dysfunction. Healthy mitochondria should make very little or no ROS -?

Second, how exactly white blood cells (natural killers and such) produce their 'good ROS'? I am very vague on the mechanisms of their production of NO, O2-, H2O2- and... some other things? Does it involve their mitochondria?

As for 'signaling ROS', isn't it just an indicator of cellular damage, sort of like smoke for fire, which does not make it, in this particular role, either 'good' or 'bad' -?

Edited by xEva, 02 March 2014 - 04:14 PM.

[niner]

ROS are ROS, by and large. In some cases they are doing something useful as well as harmful, but the potential for harm is always there. Some ROS, like the hydroxyl radical, are really reactive and nasty, while superoxide is less bad. The ROS that constitute the "respiratory burst" phenomenon used as an anti-pathogenic mechanism are apparently mitochondrial in origin. Bear in mind that the "good ROS bad ROS" hypothesis was based on a misunderstanding of the behavior of aggregated versus molecular forms of C60.

[xEva]

Thank you niner!

Bear in mind that the "good ROS bad ROS" hypothesis was based on a misunderstanding of the behavior of aggregated versus molecular forms of C60.

Yes, of course. I was just wondering. First, all ROS were bad, lately some of them are becoming increasingly good (hence "Jim's hypothesis" in Vince's blog) but there is no clarity what exactly makes ROS good or bad. Following your link, I found this in Nature:

http://www.nature.co...nri2975_F3.html

Phagocytized bacteria activate Toll-like receptors and this promotes the translocation of TNF receptor-associated factor 6 (TRAF6) to mitochondria, where it engages evolutionary conserved signalling intermediate in Toll pathways to potentiate mROS generation from oxidative phosphorylation complexes. This leads to increased ROS-dependent bactericidal responses and/or activation of NF-κB and mitogen-activated protein kinase (MAPK) signalling to augment pro-inflammatory cytokine production. IFNγ signalling can also promote mROS generation...

I always thought that increased inflammation in the older age was actually the proper immune response to slowly accumulating intracellular pathogens (even though we still only recognize acute infections and tend to ignore smoldering ones). In this particular role, as innate immune response to bacteria, ROS is both good and bad. Or rather as you said, "ROS are ROS, by and large."

But then, if C60 is a mitochondrial antioxidant (electron donnor/acceptor) --I believe that's the best hypothesis so far-- then would not it impede this antibacterial innate immune response?

[niner]

But then, if C60 is a mitochondrial antioxidant (electron donnor/acceptor) --I believe that's the best hypothesis so far-- then would not it impede this antibacterial innate immune response?

That's something I've wondered about as well. So far, no one seems to be dropping dead from sepsis, so there must be more to it. Maybe the respiratory burst (RB) is sufficient to overwhelm the c60? Maybe the c60 and the ROS from RB are in different compartments? Maybe RB is a minor component of the overall immune response? Maybe the improved barrier function that c60 appears to provide is giving us a compensating protection? Some combination of any or all of these? Something else entirely?...

[xEva]

That's something I've wondered about as well. So far, no one seems to be dropping dead from sepsis, so there must be more to it. Maybe the respiratory burst (RB) is sufficient to overwhelm the c60? Maybe the c60 and the ROS from RB are in different compartments? Maybe RB is a minor component of the overall immune response? Maybe the improved barrier function that c60 appears to provide is giving us a compensating protection? Some combination of any or all of these? Something else entirely?...

Indeed, there are so many still unknown variables. And it is also true that the immune sys has many redundancies, so many different ways of dealing with the same thing. I certainly have not noticed increased susceptibility to bacterial infections. What I have noticed is a reduced susceptibility to viruses.

Also, reading up on these 'mitochondria-generated ROS-dependent bactericidal responses' just now, I came across info that some pathogens, like chlamydia, actually exploit this ROS-generated inflammatory response for their own growth. It appears the same is true for a number of viruses. Thriving on inflammation, instead of being killed by it, seems a hallmark of really nasty, difficult to rid of bugs. But the vast majority of viruses and bacteria present no problem for the fully functional immune sys, exactly because it has so many different tricks up its sleeve.