I think that precautionary removal of dissolved O2 in the Olive Oil before the C60 is added is a good idea in principle. But it brings up a few questions that I don't have the answers to. 1. Is dissolved O2 actually significant in off the shelf olive oil? Would dissolved O2 be more common in EVOO or less processed OO? 3. Does putting it under vacuum, or vacuum filtering, actually remove O2 in a timely manner, or is degassification of oil a very slow process that takes days? If one is getting a different color from the degassed EVOO/C60 mix then you are from the natural EVOO/C60 mix then I tend to think the process is important and effective. My question then becomes, do I just want to do a simple vacuum filter of the raw OO, or do I want to do something more involved such as a Freeze-Pump-Thaw cycle and/or using Nitrogen as a replacement for the O2. At this point I think it will just be easier to do the precautionary vacuum filtering of the raw OO for an extended period of time rather than do a great deal of testing, and that is what I will do. That seems much simplier than answering all those complicated questions. They sell the bottle top vacuum rigs by the dozen, so I will have excess vacuum rigs anyway, of course this will mean starting over and just feeding my current batch of OO/C60 to the dog.A note on freezing the C60 solution:
I prepared a new sample of Pompeian Organic EVOO by first vacuum filtering to remove as much O2 as possible, then magnetically stirring with .5g/L C60 in a covered jar without air or light. In 24 hours there was no visible sediment so I filtered it and placed it in the freezer. Initially it had the classic magenta color. In 2 hours it had taken on a strawberry look when backlit (picture attached), and a dark brown, almost black appearance without backlighting. The viscosity was that of a thick honey. Returning it to room temperature restored the original clarity and color. There are two problems I saw with freezing. First, the C60 becomes non-homogenous because some components of the OO freeze out before others, and second, I expect it to eventually get too hard. I'd imagined you could eat it like ice cream but that now seem unlikely. I've had the base oil in the freezer for two days and it's hard as a brick. So I'm going to keep it in the refrigerator section instead.
A note on oxygen and browning:
If epoxides are forming with time due to the presence of dissolved O2, this will make the C60 more hydrophillic and thus reduce its oil solubility. Which might explain the change of a purple solution to a brown one that I observed previously, if fullerene epoxides began clumping. Processing without O2 and keeping the solution refrigerated ought to help.
C60 experiments @ home
#631
Posted 08 June 2012 - 03:57 PM
#632
Posted 08 June 2012 - 04:10 PM
#633
Posted 08 June 2012 - 04:50 PM
http://www.springerl...lk227584231.pdf
Remember, as time goes on, reaction products form, and this cannot be prevented at room temperature and pressure.
We don't know if these reaction products are the active molecule. In all probability, they are.
The situation is made more complex by the presence of chlorophyll in the olive oil, and many other organic impurities, all of which may be involved in the reaction chain. So, we have the Diels-Alder reaction taking place, and radical addition (page 332)
Freezing the oil might prevent these reaction products from forming, and you might not get the result that was observed in the rat study.
This isn't simple solubility. The dark red-purple-brown colour may in fact be highly desirable. If I read the study correctly, the more fullerene adducts form, the browner the liquid gets ( end paragraph just before 13.3.7)
I see no reference in the paper to clumping, just indications that the longer the fullerene is in contact with the oil, the more various posited reactions take place, and that these reactions even take place under an inert atmosphere, although they do take longer.
To reiterate: I would advise doing anything additional to the oil that wasn't done in the original study.
Edited by Metrodorus, 08 June 2012 - 05:02 PM.
#634
Posted 08 June 2012 - 05:20 PM
#635
Posted 08 June 2012 - 06:41 PM
C60 may be attracted to GC-rich regions, (depending on how much credence you put in molecular dynamic free energy perturbation theory), but how does C60 abstract a methyl group? Is there any evidence for such chemistry? I've not run across it.
I don't believe C60 has been studied as a demethylating agent, but similar longevity claims have been made for demethylating agents. In particular, procaine--
Anna Aslan also produced further evidence for her drug after 1962. In JOURNAL OF GERONTOLOGY 20(1965)1-8, she reports her results in a long study of the effect of procaine on the lifespan of Wistar white rats. She used 1840 rats, 920 treated animals and 920 controls, and studied not merely lifespans, but electrocardiograms, ability to learn mazes, and cell pathology of her animals. In all these parameters, treated animals did significantly better than untreated ones. The male rats (not the females) lived 20 percent longer than untreated rats. Both male and female treated rats did better on maze learning and had less heart disease than controls: almost 80 percent of control animals at 24 months showed signs of heart trouble, but only 30 percent of treated animals did so.
Two other studies of procaine in animals exist. One by Verzar (GERONTOLOGIA 3(1959) 350-358) using six times the dosage that Aslan used, found no effect on longevity or on one measure of aging (crosslinkage of collagen). A second study by Berger (PATH BIOL (PARIS) 8(1960) 1162-1166) found an average increase in survival of five months, agreeing with Aslan's work.
Life Extension
There is some debate about co-factors, especially those enhancing fat solubility, which has parallels to C60 in OO. Benzoic acid was part Aslan's formulation, for instance, and is also present in olive oil, but wasn't present in other procaine formulations.
Procaine with benzoic acid is sold today in formulations to "slow down the aging process."
#636
Posted 08 June 2012 - 09:13 PM
I don't believe C60 has been studied as a demethylating agent, but similar longevity claims have been made for demethylating agents. In particular, procaine--
There is some debate about co-factors, especially those enhancing fat solubility, which has parallels to C60 in OO. Benzoic acid was part Aslan's formulation, for instance, and is also present in olive oil, but wasn't present in other procaine formulations.
Procaine with benzoic acid is sold today in formulations to "slow down the aging process."
Well, maybe procaine is a demethylator, but the fact that it was once reported to mildly increase the lifespan of rats isn't any reason to expect C60 to be a demethylator. Obama and I are both male, but I'm not The President of the United States... It's the same logic.
#637
Posted 08 June 2012 - 10:10 PM
Well, maybe procaine is a demethylator, but the fact that it was once reported to mildly increase the lifespan of rats isn't any reason to expect C60 to be a demethylator. Obama and I are both male, but I'm not The President of the United States... It's the same logic.
There is simply no information to be found on fullerenes in this regard. However, we know that they are attracted to specific parts of DNA, especially those parts that aren't supposed to be methylated, and we know that good anti-oxidants can promote demethylation. Even vitamin C can do this, thus it isn't a big jump to hypothesize that a good antioxidant delivered into the mitochondria and which likes DNA might also grab hold of methyl groups--
Vitamin C (ascorbate) is a widely used medium supplement in embryonic stem cell culture. Here, we show that ascorbate causes widespread, consistent, and remarkably specific DNA demethylation of 1,847 genes in human embryonic stem cells . . .
Source
#638
Posted 08 June 2012 - 11:01 PM
Even vitamin C can do this, thus it isn't a big jump to hypothesize that a good antioxidant delivered into the mitochondria and which likes DNA might also grab hold of methyl groups--
Add the concept of an antitumor "memory" response to the potential Methylation-related Explanation Lexicon...
"...We show that transient exposure of cultured and primary leukemic and epithelial tumor cells to clinically relevant nanomolar doses, without causing immediate cytotoxicity, produce an antitumor “memory” response, including inhibition of subpopulations of cancer stem-like cells. These effects are accompanied by sustained decreases in genomewide promoter DNA methylation, gene reexpression, and antitumor changes in key cellular regulatory pathways. Low-dose decitabine and azacitidine may have broad applicability for cancer management.
Transient Low Doses of DNA-Demethylating Agents Exert Durable Antitumor Effects on Hematological and Epithelial Tumor Cells
Edited by wccaguy, 08 June 2012 - 11:02 PM.
#639
Posted 08 June 2012 - 11:20 PM
"... Pathway analysis of the genes with altered methylation patterns identifies the involvement of a cancer related network of genes whose activity may be heavily regulated by TNF-α in prostate tumorigenesis. Our results suggest that epigenetic dysregulation of cellular processes relevant to TNF-α-dependent apoptosis and electrophile detoxification may be intimately involved in prostate carcinogenesis..."
Genome-wide methylation analysis identifies involvement of TNF-α mediated cancer pathways in prostate cancer
Age-related loss of CpG methylation in the tumour necrosis factor promoter
Effect of polymorphisms of IL-1β and TNF-α genes on CpG island hyper methylation (CIHM) in the nonneoplastic gastric mucosa
... and a lot more with the simple search phrase "tnf methylation"
Edited by wccaguy, 08 June 2012 - 11:20 PM.
#640
Posted 08 June 2012 - 11:20 PM
There is simply no information to be found on fullerenes in this regard. However, we know that they are attracted to specific parts of DNA, especially those parts that aren't supposed to be methylated, and we know that good anti-oxidants can promote demethylation. Even vitamin C can do this, thus it isn't a big jump to hypothesize that a good antioxidant delivered into the mitochondria and which likes DNA might also grab hold of methyl groups--
We don't 'know' that fullerenes are attracted to specific parts of DNA, we have a result from one set of calculations that suggests this to be the case. Those kind of calculations just aren't that reliable. In order to get that ascorbate result, cells were continually bathed in it. That's nothing like what happens in an organism, but more importantly, just because one antioxidant is a demethylator certainly doesn't mean they all are. Is there any evidence that demethylation is a general property of antioxidants? That it extends over all chemical and structural classes of antioxidant?
It's not just a jump, it's an astonishing LEAP to think that on this basis, fullerenes will engage in chemistry that may well be physically impossible.
#641
Posted 08 June 2012 - 11:56 PM
We don't 'know' that fullerenes are attracted to specific parts of DNA, we have a result from one set of calculations that suggests this to be the case. Those kind of calculations just aren't that reliable. In order to get that ascorbate result, cells were continually bathed in it. That's nothing like what happens in an organism, but more importantly, just because one antioxidant is a demethylator certainly doesn't mean they all are. Is there any evidence that demethylation is a general property of antioxidants? That it extends over all chemical and structural classes of antioxidant?
It's not just a jump, it's an astonishing LEAP to think that on this basis, fullerenes will engage in chemistry that may well be physically impossible.
It's my hypothesis that it is possible and that it happens. Do you have any evidence to back up your assertion that it may be impossible?
#642
Posted 09 June 2012 - 02:27 AM
Not only that, the calculations may well be irrelevant for the fullerene adjuncts that form when fullerene reacts with olive oil.We don't 'know' that fullerenes are attracted to specific parts of DNA, we have a result from one set of calculations that suggests this to be the case.
We need to stop talking about 'fullerene' as though it were one molecule. We are dealing with fullerenes as a broad class, and the fullerene sub-group that forms when fullerene reacts with olive oil is quite a specific sub-group.
That being said, the DNA de-methylisation mechanism proposed could be tested for.
It isn't such a far-fetched extrapolation, and there may even be a study on methyl groups and fullerene lurking somewhere in the literature. It is more plausible than some of the proposed mechanisms of action that have been posited here to date. It is certainly a testable hypothesis.
Also, has anyone found any papers that discuss fullerene clumping when organic oils are the solvent? I have looked and found nothing in the literature.
Clumping occurs in water based solutions, because the hydrogen bonds between the fullerenes are quite strong, and water is a poor solvent for fullerene or fullerne derivative molecules.
Olive oil appears to be a strong solvent - does it prevent the hydrogen bonds from stabilising allowing the formation of nano-fullerene clumps? Any info anyone?
#643
Posted 09 June 2012 - 04:38 AM
That being said, the DNA de-methylisation mechanism proposed could be tested for.
Great. I'm unfamiliar with how that testing is done. Could you describe how that is done a bit? That would be a relatively short experiment right? I mean, this wouldn't require a wait until the life span of the animals was over right?
Or, perhaps, a life span study of some animals is begun and the de-methylisation study piggy-backs on that life span while yielding some interim useful data? I guess I'm asking: Do the animals have to be sacrificed to do a de-methylisation study of them?
Edited by wccaguy, 09 June 2012 - 04:47 AM.
#644
Posted 09 June 2012 - 05:46 AM
It's my hypothesis that it is possible and that it happens. Do you have any evidence to back up your assertion that it may be impossible?
Well, all I can do is point out the logical and evidentiary gaps. It's impossible to prove a negative, but there are a couple things I could point out. First, as far as I know, all biological methylation/demethylation reactions are enzymatic; none are chemical. Second, methyl is the epitome of a bad leaving group. Chemists categorize different functional groups as to their ability to leave a reacting center; methyl sucks. Of course it could be removed with the right reagents and sufficiently harsh conditions, but 37C isn't exactly harsh.
#645
Posted 09 June 2012 - 06:40 AM
Also, has anyone found any papers that discuss fullerene clumping when organic oils are the solvent? I have looked and found nothing in the literature.
Clumping occurs in water based solutions, because the hydrogen bonds between the fullerenes are quite strong, and water is a poor solvent for fullerene or fullerne derivative molecules.
Olive oil appears to be a strong solvent - does it prevent the hydrogen bonds from stabilising allowing the formation of nano-fullerene clumps? Any info anyone?
The use of triglyceride oils as a fullerene solvent is relatively new, so the odds aren't great. The behavior of hydrophobes in water is kind of complicated, but essentially, they clump. The attraction between two hydrophobes in water is called 'the hydrophobic effect', cleverly enough. It's actually entropically driven, which is a little counter-intuitive. A single hydrophobe orders the water around it, thus lowering the entropy of the water. If there are two hydrophobes, they will order the water less if they are in contact, because they present less surface to the water. This is the source of the free energy minimum in the clumped state. If the fullerene were decorated with hydroxyl groups, then there would be a large enthalpic term that would rescue the situation, as water molecules would interact strongly with the -OH groups on the fullerene.
Triglycerides are a really interesting case. They are about the right size for the three fatty acid chains to drape over the fullerene, forming a little cage. That's just a thought; I haven't seen any structural explanation for the remarkable solubility of fullerenes in OO. Once the fullerene is dissolved in the OO, I can't think of any driving force for it to crystallize or aggregate unless there was too much in solution. I could be missing something, because I think some people were seeing aggregation over time. Maybe it's just a function of the concentration. Maybe the oil is taking on water or oxygen, and you're getting reaction products. Once the fullerene reacts with the fatty acid chain, then I think it's likely to stay in solution.
#646
Posted 09 June 2012 - 12:06 PM
Hi Niner - my thinking has been along the same lines as yours - that clumping isn't really something to worry about.Once the fullerene reacts with the fatty acid chain, then I think it's likely to stay in solution.
There might be some tangentially related studies, as much work has been done on fullerene as a lubricant. I was wondering whether anyone had bumped into something that mentions this aspect....but like you, I am not hopeful.....still, never hurts to ask enquiring minds. A nice searchable database of all articules ever published that mention the word 'fullerene' would come in handy.....alas open access science is not yet a reality.
I'm not sure if anyone has reported precipitation as such, have they?
Some of the larger flakes of fullerene that are present in some of the reagent supplied ( even my SES fullerene had some quite large crystals) are dissolving / reacting much more slowly, as would be expected. I expect given time they will eventually dissolve/ react.
#647
Posted 09 June 2012 - 02:56 PM
...First, as far as I know, all biological methylation/demethylation reactions are enzymatic; none are chemical....
As I pointed out before, there appear to be some that act non-enzymaticly, in part by binding directly to the DNA--
Following this need to find new demethylating agents, we have tested the potential use of procaine, an anesthetic drug related to procainamide. Using the MCF-7 breast cancer cell line, we have found that procaine is a DNA-demethylating agent that produces a 40% reduction in 5-methylcytosine DNA content as determined by high-performance capillary electrophoresis or total DNA enzyme digestion. Procaine can also demethylate densely hypermethylated CpG islands, such as those located in the promoter region of the RARβ2 gene, restoring gene expression of epigenetically silenced genes. This property may be explained by our finding that procaine binds to CpG-enriched DNA.
Source: http://cancerres.aac...63/16/4984.long
This is the same region of the DNA that the theoretical study suggested would be energetically favorable for C60 binding. And both materials have been shown to extend the lifespan of Wistar rats.
#648
Posted 09 June 2012 - 03:24 PM
As I pointed out before, there appear to be some that act non-enzymaticly, in part by binding directly to the DNA--
[procaine]
This is the same region of the DNA that the theoretical study suggested would be energetically favorable for C60 binding. And both materials have been shown to extend the lifespan of Wistar rats.
Procaine doesn't act chemically. It's a DNA Methyltransferase inhibitor.
#649
Posted 09 June 2012 - 03:35 PM
Procaine doesn't act chemically. It's a DNA Methyltransferase inhibitor.
You have a source for that?
#650
Posted 09 June 2012 - 03:40 PM
I can not be sure that oxygenated fullerene is not important. I just find it highly improbable given that oxygenation is almost universally associated with spoilage, cross linking and aging that it would have a beneficial effect in this situation. A problem with reproducing the original study exactly is that we do not live in France where the study was conducted. All olive oil sold in my area is imported from Europe, shipped here on giant boats, maybe sitting for months in the hot sun, thus not as fresh as one could get in Europe. In researching my questions on OO purity and freshness I came across this interesting study on OO purity and freshness, http://www.olivecent...nal reduced.pdf The study included a couple of CA OOs that scored highly for purity and freshness, that is the direction I have decided to go, mail order EVOO from a CA grower.. The discussion in the study gave me enough information that I think I can skip the precautionary vacuum step i had considered in my prior post.My question would be, how do we know that the oxygenated fullerene is not important? As the original study didn't do this, I would not do it either.
#651
Posted 09 June 2012 - 04:11 PM
But they say, "Procaine...had no significant effect on the genomic methylation level, even at concentrations that were previously reported to be effective..."
Source: http://cancerres.aac...4.full.pdf html
In any case, could C60 be a methyltransferase inhibitor? A bulky C60 molecule stuck to the DNA next to a methyl group would get in the way of the methyltransferase, wouldn't it? And this would achieve the same effect as a demethylator as mitochondria replicate in a matter of days. The original DNA would still be methylated, but the copy would not.
C60's function would have two parts:
1. A tendency to attach to DNA as expected theoretically, and
2. A physical size large enough to get in the way of methyltransferase.
Of course, the attachment to DNA has to be temporary, otherwise the stuff would be deadly. But the lack of toxicity argues that it doesn't stick around for very long, which is in line with the rat study. And if size has something to do with it, then the higher fullerenes ought to be even more effective.
Edited by Turnbuckle, 09 June 2012 - 04:43 PM.
#652
Posted 09 June 2012 - 06:23 PM
A large-scale association study for nanoparticle C60 uncovers mechanisms of nanotoxicity disrupting the native conformations of DNA/RNA
http://nar.oxfordjou...nar.gks517.full
What is your take on this one?
The problem with a study like this is that we are most likely not dealing with naked C60 even in the body. Instead, we probably have some molecule consisting of olive oil lipids bound electrostatically (van der waalls most likely) to the C60.
The question, then, is figuring out the energetics of the system. For example, what is what the binding affinity of the olive oil lipids to the C60 compared to the binding affinity of C60 for other molecules, such as DNA, RNA, membrane phospholipids, etc.
If the C60 is more weakly bound to the olive oil lipids than it would bind to other molecules, the energetics would favor C60 binding to the other molecules. If C60 is more strongly bound to the olive oil lipids, it should tend to stay bound to them.
---
With regard to DNA binding and methylation, the situation is much more complex than some people here appear to think it is.
DNA in chromosomes is typically looped onto nucleosomes, which are then packaged into chromatin. Most DNA sits bound up like this unless it's actively being expressed (ie: transcribed to make proteins). It's protected while it's bound up like this, and would not be likely to be altered in any way unless the choromatin and then nucleosomes were unlooped.(See: http://upload.wikime..._Structures.png)
Methylation is not the uniformly bad thing it's being described as by some people here, and in fact if DNA was entirely de-methylated you'd expect tons of problems. Methylation is one of the major ways that DNA transcription is controlled, and hence one of the major ways that cell differentiation occurs. If you actually were able to strip off all the methyl groups, you'd end up with some weird kind of cell, not the tissue you started with at all.
So it's very unlikely that C60 is doing anything like demethylating the animal's DNA, because if it was, you would not be seeing increases in lifespan: you'd be seeing death.
Edited by smithx, 09 June 2012 - 06:36 PM.
#653
Posted 09 June 2012 - 06:37 PM
Aging and Chronic Sun Exposure Cause Distinct Epigenetic Changes in Human Skin
Evidence of increased DNA methylation of the androgen receptor gene in occipital hair follicles from men with androgenetic alopecia
No time now to look for the 3rd, i.e., profound muscle fatigue during exercise.
Edited by wccaguy, 09 June 2012 - 06:37 PM.
#654
Posted 09 June 2012 - 06:43 PM
DNA Methylation implicated in 2 of Turnbuckle's n=1 Observations... This isn't "proof," just data points that don't Falsify the DNA Methylation Hypothesis.
Aging and Chronic Sun Exposure Cause Distinct Epigenetic Changes in Human Skin
Actually, if you read the article it states that sun exposure caused HYPOmethylation. In other words, the sun damaged areas had LESS methylation.
The people in general had more methylation as they got older, but less in sun exposed areas. This indicates that if the wrong areas of DNA are demethylated, problems can result. So a non-specific DNA de-methylator would be expected to cause damage, not fix things.
However, I don't think we can draw any conclusions related to C60 from this study.
Edited by smithx, 09 June 2012 - 06:50 PM.
#655
Posted 09 June 2012 - 06:52 PM
Actually, if you read the article it states that sun exposure caused HYPOmethylation. In other words, the sun damaged areas had LESS methylation.
The people in general had more methylation as they got older, but less in sun exposed areas.
I don't think that you can draw many conclusions from this, in relation to what we're discussing.
I understand that point and saw it before I made the post. I'm still getting up to speed so I'm not using the precise language in the right way quite yet. I should have said something like "Methylation-related processes" are implicated... My purpose is to post puzzle pieces that are increasingly useful...
#656
Posted 09 June 2012 - 06:53 PM
You keep persisting in positing these modes of action, when we don't even know what molecule(s) we are dealing with!!!!!
It is all, quite frankly, rather far fetched.
First, we need to characterise the molecules that form in the olive oil.
I assume there are several reaction products.
Then, the metabolic effect of these need to be teased out, and that will take years, as if there are several reaction products, there will be several variables.
If the molecules work synergetically, then it will be even more difficult to untangle, as designing experiments that deal with multiple variables is fraught with difficulty.
The mechanisms of action posited so far in the extant literature are sufficient to explain what Mr Turnbuckle has felt - simply increasing the efficiency of the electron transport chain in the mitochondria will account for that. And we already know that fullerene in some of its forms has this effect. We do not need to posit any epigenetic changes. We do not even have to posit a direct effect on electron transfer, simply physical blockage of reactive species from access.
At this point we do not actually need any explanations. We simply need the original experiment to be reproduced, preferably more than once. Once that has been done, we will see whether the result was an artefact or not.
Edited by Metrodorus, 09 June 2012 - 06:54 PM.
#657
Posted 09 June 2012 - 07:29 PM
So it's very unlikely that C60 is doing anything like demethylating the animal's DNA, because if it was, you would not be seeing increases in lifespan: you'd be seeing death.
That's true if it were the nuclear DNA but the DNA we're discussing here is the mitochondrial DNA. The mitochondria is where the C60 is seen to end up in the rat paper. Also, if C60 acts anything like procaine, then it doesn't demethylate everything.
Edited by Turnbuckle, 09 June 2012 - 07:40 PM.
#658
Posted 09 June 2012 - 07:51 PM
Also, if C60 acts anything like procaine, then it doesn't demethylate everything.
I'd put that idea in the realm of "wild speculation" since it is based on exactly zero data.
By the way, non-specific demethylation of mitochondrial DNA would also be likely to cause big problems. See:
http://www.pnas.org/...108/9/3630.full
and others. Methylation in mitochondria also is used for controlling gene expression, likely in essential ways.
#659
Posted 09 June 2012 - 08:04 PM
I'd put that idea in the realm of "wild speculation" since it is based on exactly zero data.
By the way, non-specific demethylation of mitochondrial DNA would also be likely to cause big problems. See:
http://www.pnas.org/...108/9/3630.full
and others. Methylation in mitochondria also is used for controlling gene expression, likely in essential ways.
I don't see in that paper where hypomethylation of the mtDNA causes any specific problem.
#660
Posted 09 June 2012 - 08:11 PM
http://www.idspackag...g_supplier.html
Just thought I would post this here also in case it is of interest to anyone.
Scratch that, Miron lets UVA in, looks like black glass would be best...
Edited by zorba990, 09 June 2012 - 08:12 PM.
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