33 replies to this topic

[maxwatt]

A forward to this topic: the paper cited to start this thread has been reported to contain fabricated and falsified data by the University of Connecticut's investigation report.


This paper found negative effects from resveratrol in high doses in rats:

Resveratrol, a unique phytoalexin present in red wine, delivers either
survival signal or death signal to the ischemic myocardium
depending on dose
Jocelyn Dudley, Samarjit Das, Subhendu Mukherjee, Dipak K. Das⁎
PMID: 18789672

Das found that 5 mg/kg administered by gavage exerts a survival signal by up-regulating anti-apoptotic and redox proteins Akt and Bcl-2, while at higher doses (>25 mg/kg), it potentiates a death signal by down-regulating redox proteins and up-regulating pro-apoptotic proteins. (After 14 days, isolated working hearts were prepared from both experimental and control animals, and the hearts were subjected to 30-min global ischemia followed by 2 h of reperfusion.)

I found this curious, as Lagouge administered 400mg/kg to mice with only positive effect. (Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Lagouge M, Argmann C, et al. Cell. 2006; 127(6): 1109-22). 400 mg is the dose that made athletes out of couch potato rats.

The metabolic difference between mice and rats is such that 400 mg/day resulted in a plasma level of 120 ng/ml (Lagouge) albeit in chow, vs by gavage in the rats. I had to search for serum levels in rats, but found that administering 2mg/kg resulted in a peak plasma level of 550 ng.ml (Planas, Food Research International,Volume 35, Issues 2-3, 2002, Pages 195-199, Plasmatic levels of trans-resveratrol in rats.). This is a huge difference in levels for a given dose. It may reflect metabolic differences between rats and mice, or may be due to the difference between an acute dose, and a dowse mixed in food.

We have data for humans from Boocock's paper, where he found peak blood serum levels in humans of 117 ng/ml at a dose of 1 g (14 mg/kg for a 70 kg human) effectively the same as a mouse gets from a dose 400mg/kg. ( Phase I dose escalation pharmacokinetic study in healthy volunteers of resveratrol, a potential cancer chemopreventive agent. Boocock DJ, Faust GE, Patel KR, Schinas AM, Brown VA, Ducharme MP, Booth TD, Crowell JA, Perloff M, Gescher AJ, Steward WP, Brenner DE., Cancer Epidemiol Biomarkers Prev. 2007 Jun;16(6):1246-52.)

I concluded that Das' rats were receiving the equivalent of at least 10 times Lagouge's mouse dose, even though the mice were receiving the equivalent of 16 times as much resveratrol as Das' rats. Boocock's paper(*) found peak blood serum levels in humans of 117 ng/ml at a dose of 1 g (14 mg/kg for a 70 kg human) effectively the same as a mouse gets from a dose 400mg/kg when mixed in food. Rats appear to have much higher serum levels than mice from a given dose, though the difference may be at least partially due to the method of administration. J

Judging by peak plasma levels, it does seem that humans can achieve sufficiently high blood serum levels that resulted in mitochondrial and other metabolic improvements in mice. It also appears the human serum levels even with gram doses are below that which produced adverse effects in rats.

Edited by maxwatt, 17 January 2012 - 05:19 AM.

[2tender]

Thanks for posting the good news.

[niner]

This paper found negative effects from resveratrol in high doses in rats:

Resveratrol, a unique phytoalexin present in red wine, delivers either
survival signal or death signal to the ischemic myocardium
depending on dose
Jocelyn Dudley, Samarjit Das, Subhendu Mukherjee, Dipak K. Das⁎
PMID: 18789672

Das found that 5 mg/kg administered by gavage exerts a survival signal by up-regulating anti-apoptotic and redox proteins Akt and Bcl-2, while at higher doses (>25 mg/kg), it potentiates a death signal by down-regulating redox proteins and up-regulating pro-apoptotic proteins. (After 14 days, isolated working hearts were prepared from both experimental and control animals, and the hearts were subjected to 30-min global ischemia followed by 2 h of reperfusion.)

I found this curious, as Lagouge administered 400mg/kg to mice with only positive effect. (Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Lagouge M, Argmann C, et al. Cell. 2006; 127(6): 1109-22). 400 mg is the dose that made athletes out of couch potato rats.

The metabolic difference between mice and rats is such that 400 mg/day resulted in a plasma level of 120 ng/ml (Lagouge) albeit in chow, vs by gavage in the rats. I had to search for serum levels in rats, but found that administering 2mg/kg resulted in a peak plasma level of 550 ng.ml (Planas, Food Research International,Volume 35, Issues 2-3, 2002, Pages 195-199, Plasmatic levels of trans-resveratrol in rats.). This is a huge difference in levels for a given dose. It may reflect metabolic differences between rats and mice, or may be due to the difference between an acute dose, and a dowse mixed in food.

That Planas data seems wrong. From a Sirtris patent that Hedgehog once posted, rats getting unprocessed (non-micronized) resveratrol with Methylcellulose, orally dosed at 50mg/kg gave a Cmax of 2.2 uM. Planas' Cmax at 550ng/ml = 2.41 uM is about 25 times as potent, based on the claimed dose of 2mg/kg. I don't have access to Planas' M&M, so don't know if any formulation was involved. Cyclodextrin, perhaps? Planas' animals were fasted, so there will be a significant difference in Cmax between them and animals dosed in food. Even considering fasting and the unknown formulation, it's still kind of an outlier.

Das' results are interesting, at any rate. Sounds like you can take a lot of resveratrol if you have cancer, but you'd better not have a heart attack. Lagouge (a paper many know as Auwerx) didn't see anything bad happening at the high dose, but they didn't induce ischemia/reperfusion injury. To me, this is another nail in the coffin of very high (multi-gram) resveratrol doses, unless one has cancer and is interested in ramping up apoptosis. It does look, at least at first blush, that humans should be able to hit the plasma levels of the Auwerx mice with a sub-gram dose, assuming a good formulation, but this is putting a lot of importance on the plasma Cmax. Cmax is so transitory that I suspect it's not really what we want to look at. What's going to matter is likely to be tissue concentration, which I think will correlate better with AUC than with Cmax. There have been some interesting studies using radiolabeled resveratrol in rodents that looked at tissue concentration, but it's late...

[maxwatt]

Didn't find AUC studies in rats. I suspect the inter-species AUC in mammals should prove closely correlated to peak levels.
I did find another Planas rat study, and this one is available on-line.

The Daily Oral Administration of High Doses of trans-Resveratrol to Rats for 28 Days Is Not Harmful

M. Emília Juan, M. Pilar Vinardell and Joana M. Planas2
Departament de Fisiologia–Divisió IV, Facultat de Farmàcia, Universitat de Barcelona, E-08028 Barcelona, Spain

Abstract

trans-3,5,4'-Trihydroxystilbene (trans-resveratrol) is a phytochemical present in peanuts, grapes and wine with beneficial effects such as protection against cardiovascular disease and cancer prevention. The purpose of this study was to evaluate whether high doses of trans-resveratrol have harmful effects on Sprague-Dawley rats. trans-Resveratrol was administered orally to male rats for 28 d at a dose of 20 mg/(kg · d), 1000 times the amount consumed by a 70-kg person taking 1.4 g of trans-resveratrol/d. Body weight, and food and water consumption did not differ between rats treated with trans-resveratrol and the control group. Hematologic and biochemical variables were not affected by the treatment. Histopathologic examination of the organs obtained at autopsy did not reveal any alterations. These results support the view that repeated consumption of trans-resveratrol at 20 mg/(kg · d) does not adversely affect the variables tested in rats.

The serum levels were measured 24 h after oral administration, the measurements indicated that trans-resveratrol was the main compound with concentrations of 96.5 ± 12.3 nmol/L (n = 20) determined in 4 rat. Rats received 20 mg/kg of trans-resveratrol suspended in 10 g/L carboxymethylcellulose every d for 28 d

They found no significant difference between with the controls and the resveratrol-fed rats. Nada. Nothing bad, but also nothing good. But they did not isolate and reperfuse the hearts, not did they put the rats on a treadmill and measure mitochondrial density. 28 days in growing rats is more like 20 years in humans, though the generalization is risky.

Edited by maxwatt, 22 July 2009 - 03:46 AM.
spelling

[niner]

at a dose of 20 mg/(kg · d), 1000 times the amount consumed by a 70-kg person taking 1.4 g of trans-resveratrol/d.

I had to check the paper just to make sure this wasn't a typo or something. Since 1.4 g/d in a 70kg person is 20mg/(kg*d), where do they get the factor of 1000? Did they mean a 70kg person getting 1.4 milligrams per day, as in from wine?

Aside from that, the Cmax is very dependent on feeding/fasting status, and on formulation. The AUC is a lot less dependent on those things.

[maxwatt]

at a dose of 20 mg/(kg · d), 1000 times the amount consumed by a 70-kg person taking 1.4 g of trans-resveratrol/d.

I had to check the paper just to make sure this wasn't a typo or something. Since 1.4 g/d in a 70kg person is 20mg/(kg*d), where do they get the factor of 1000? Did they mean a 70kg person getting 1.4 milligrams per day, as in from wine?

Aside from that, the Cmax is very dependent on feeding/fasting status, and on formulation. The AUC is a lot less dependent on those things.

Planas lab seem to be producing some odd data.

For a given mode of administration (eg, oral with chow, or by gavage) I'd expect AUC to correlate with Cmax. Sirtris seems to aim fr a high Cmax with their resveratrol formulations; do they think a 'threshold' level is necessary for their formuations to show effectiveness, I wonder?

Das used gavage on his rats. I have not been successful so far at finding reliable rat data for Cmax data in rats. I'd hoped it would prove better than the M^23 or M3/4 approximations that have been used, as these do not allow for other metabolic differences. If 5 mg/kg is acceptable in rats, but 25mg/kg weakens the heart as shown when their isolated hearts are tested, what would be the equivalent doses in humans?

[niner]

For a given mode of administration (eg, oral with chow, or by gavage) I'd expect AUC to correlate with Cmax. Sirtris seems to aim fr a high Cmax with their resveratrol formulations; do they think a 'threshold' level is necessary for their formuations to show effectiveness, I wonder?

Having food in the system slows down and "spreads out" the drug, while being fasted is more of a bolus. This affects the Cmax quite a lot, but not so much the AUC. At the extremes of very high and very low bioavailability, the Cmax and AUC correlate strongly, but in the middle there is more room for them to vary. I'm going on memory for this, but didn't Sirtris' HPMC formulation have a lower Cmax but higher AUC than the earlier Tween formulation? I think that there might well be a threshold phenomenon with resveratrol activity, but I'm starting to think that the threshold that matters is obtaining a higher AUC. The Cmax is very transient, and is a plasma phenomenon. A large AUC is going to do more to raise tissue levels than the Cmax, essentially by definition. Flux of the drug from plasma to tissue depends on concentration gradient and time. While Cmax has a high concentration gradient in its favor, the time is incredibly short.

[TianZi]

As someone who's been taking 2 grams of 99% purity resv powder twice daily on an empty stomach for more than two years, I'm concerned by what I think I'm reading here. But as a layman, I'm not sure if my understanding is correct.

From previous studies, I'd concluded that there were no negative side effects of resveratrol in mice until a very high dosage--the equivalent of more than 20 grams (20,000 mg) for a 150 lb. human--was reached.

Indeed, the landmark "low dose resveratrol study" finding resv to be a partial mimetic for CR used a dosage that was about the equivalent of 800 mg for a 150 lb human, and per the results of that study the strongest positive effects were found in opposing harmful age-related changes in the cardiovascular system. No negative side-effects from the resv dosage were found.

Yet here we have a study finding harmful effects on the -->heart <-- at an equivalent human dose of 2 grams for an 80 kg human (80 x 25; I'm not sure how to factor in the differences in metabolisms between humans and rats)? In this respect, the concluding line in the abstract stated in pertinent part, "... at higher doses (>25 mg/kg), it potentiates a death signal by down-regulating redox proteins and up-regulating pro-apoptotic proteins."

Granted, it's a higher dose than used in the study I mentioned above, but not by THAT much.

Is this study an outlier? A someone else noted above, it seems to directly contradict the results in the extremely high dose mouse study that transformed mice into "super-athletes". And I believe the "olympic mouse" study was itself replicated in part or whole by other researchers, and that similar high doses in other studies were delivered and only positive effects observed. Further, the Spanish study mentioned by Maxwatt above specifically used rats (the same strain of rats, I wonder?), and found no harmful effects at high doses. Question: should the adverse results noted in Das's study have been detected in the Spanish study. which focused on studying hematologic and biochemical markers? I'd think signs of damage to the heart would be something you'd look for.

What other studies support the conclusions of this one, and which contradict it? The Sirtris diabetes trials are using pretty high does of resv, and I'm unaware of any negative side effects on the human trial participants. Is it the case that "high" doses of resv may positively influence the cardiovascular system in some ways, but impact it negatively in others?

Also, to what extent can these differences be explained by the use of particular strains of mice in most studies, and a particular strain of rats in this one? Which more resembles the human heart--the hearts of the mice used in the other studies, or the rats in Das's study, paying attention to the particular lab strains of each that were used? As I understand that pig hearts are much closer to a human's than a mouse's heart is, I'd be interested to see what research if any has been done using similar doses of resveratrol in swine.

Edited by TianZi, 27 July 2009 - 06:57 AM.

[maxwatt]

It is hard to say what relevance a reperfusion study of isolated rat hearts has to in vivo oral administration.

The rat hearts were cut out of the rats, but kept beating in a medium, and subject to ischemia (lack of oxygen) for 30 minutes followed by reperfusion (oxygen supplied) for 2 hours. Ischemia causes damage to the cells, and cell-death (apoptosis) typically occurs to damaged cells on reperfusion, This may have relevance to what might happen in case of a heart-attack, or heart-stoppage by electric shock. Perhaps the resveratrol-treated hearts with more mitochondria had higher energy/oxygen requirements, and so were more prone to damage and subsequent apoptosis. Maybe.

I do not have access to Das' paper, and have not seen the methods section. I believe resveratrol was administered by gavage (liquid forced into the rats stomachs.) In other studies his lab has administered resveratrol with cyclodextrin, which makes it water-soluble and increases serum levels several times for a given dose. As gavage uses liquid, I suspect cyclodextrin was used here. A typical rat weighs 200 grams, a mouse 20 grams. Using the M/^2/3 approximation 25mg/kg becomes 116 mg/kg in a mouse; Auwerx mice received 400 mg/kg without apparent ill-effect, though he did not isolate their still-beating hearts and subject them to reperfusion. For a 70 kg man, the M^2/3 approximation of a rat's dose of 25 mg becomes 50 mg/kg; 50*70 = 3.5 grams. As rats do not glucuronate or sulfonate resveratrol as efficiently as humans, their serum levels will be much higher from the equivalent dose, and the human dose may be higher by a factor of two or more. In short, the human equivalent to Das' rats' 25mg/kg is over 3.5 grams, and probably twice that or more.

Unfortunately Das does not give us the blood serum levels in his abstract, and he probably did not measure them. Were I judging the paper, I would have asked they measure blood serum levels before accepting it for publication. I've not found good data for rats' serum levels, though I've looked. The mice in Auwerx' study that received 400 mg/kg had blood serum levels equivalent to what Boocock measured in humans from a 1 gram dose. Auwerx mice were given resveratrol in food, not by gavage. I suspect Das' rats were getting the equivalent of a higher dose than 25 mg/kg due to the method of administration, but this is speculation. The methods Das' used were certainly extreme, and it is possible too that his results are an artifact of this methodology.

In short, Das' paper indicates that there is an inverted U-shaped curve for resveratrol; beyond some optimum amount, there can be deleterious effects despite the beneficial effects seen on the rising side of the curve. But I think we already knew that would be true. Unfortunately, the paper does not tell us what that optimum amount might be for oral doses in humans. I think most of us have been assuming up to 5 grams to be safe, as that was the dose used in Sirtris Pharmaceuticals' clinical trials. But the trials have lasted only a few months with a few hundred subjects, and they did not isolate their human subjects' hearts and subject them to reperfusion. The only down-side reported in this forum has been joint pain or tendinitis, and this is not that common and was not seen in Sirtris' subjects. There were no heart attacks reported in the studies, and no deleterious effects in animal studies with doses less than 1000 mg/kg. But there are no long-term studies in humans, and will not be for years to come.

For myself, I still take between 400 mg and a gram of resveratrol. I find the benefits immediate and obvious at my age, with arthritic joints and athletic predilections. I found larger doses were no more helpful for my needs after the first year or so of use, and smaller doses have not been helpful. YMMV.

[TianZi]

It is hard to say what relevance a reperfusion study of isolated rat hearts has to in vivo oral administration. [... BIG snip by MR]

Thanks very much Maxwatt, your reply was very helpful to me.

Edited by Michael, 01 August 2009 - 11:42 AM.
Trim quote

[Anthony_Loera]

I believe Gedarkstorm had commented on this previously as well:

The study is interesting. Rats have a heart rate around 300 bpm, they are fed resveratrol, dissected, and their hearts are used isolated from the body for the study (might have been cut out). The hearts were then left without adequate blood supply for 30 minutes (ischemia). That's about 9000 heartbeats, and for a person that would equal ~128 minutes. I don't know if heart attacks last 128 minutes, all the medical stuff I've seen says the actual attack lasts only a few minutes at most.

But, considering mice given 400 mg/kg of resveratrol, rather than the lower 50 mg/kg in this study, are completely fine heart wise and show markably increased health of arteries.. probably don't have to worry about having a heart attack in the first place. And if your heart stopped long enough to simulate an ischemia equal to the rats, then, well, your brain would be quite dead, so the effects of a high dose of resveratrol hurting your heart a little bit would become moot.

That's just how it seems to me, and I won't pass it off as anything but my opinion.

A

Edited by Anthony_Loera, 28 July 2009 - 12:27 PM.

[Michael]

All:

This paper [(1) below] found negative effects from resveratrol in high doses in rats.

Das found that 5 mg/kg administered by gavage exerts a survival signal by up-regulating anti-apoptotic and redox proteins Akt and Bcl-2, while at higher doses (>25 mg/kg), it potentiates a death signal by down-regulating redox proteins and up-regulating pro-apoptotic proteins. (After 14 days, isolated working hearts were prepared from both experimental and control animals, and the hearts were subjected to 30-min global ischemia followed by 2 h of reperfusion.)

I found this curious, as Lagouge [2] administered 400mg/kg to mice with only positive effect. 400 mg is the dose that made athletes out of couch potato rats. [sic: mice -MR]

The metabolic difference between mice and rats is such that 400 mg/day resulted in a plasma level of 120 ng/ml (Lagouge) albeit in chow, vs by gavage in the rats.

ISTM that, rather than looking at the pharmacokinetic and possible interspecies differences that have dominated discussion of this study, there are 2 much more important differences between it and the Auwerx group's report to take into account.

First, the mice in Auwerx's study were morbidly obese and eating the high-sucrose, high-saturated-fat D12327 research diet. As we know from the Sinclair/de Cabo report (3), high-dose resveratrol does help to partially normalize the lives of such animals -- but it doesn't do much -- and, in terms of mortality, doesn't do anything -- for normally-fed. normal-weight mice. Just as a drug that does wonders for a person with metabolic syndrome (say, a high-dose statin) could well be expected to be largely deleterious to a person with a healthy metabolism, it's not necessarily surprising if there was a negative effect of even a more moderate dose of resveratrol given to normally-fed. normal-weight rats. It's even possible, in principle, that the lack of an overall effect on mortality in the normal mice in (3), in the face of the apparent benefits on some health parameters, reflects a compensatory increase in mortality from other causes of death, though there was certainly no clear evidence of such from the limited pathology reports).

Second, the two studies weren't even evaluating the same parameters. Auwerx's group tested aerobic function and parameters of the metabolic syndrome and mitochondrial biogenesis, but they didn't induce experimental ischemia on their hearts, nor evaluate cardiac apoptotic signaling; the global gene expression data were done in gastrocnemius muscle, but only of a few proteins involved in mitochondrial biogenesis were examined in the heart (which FWIW was unaffected: "PGC-1a expression, PGC-1a acetylation, and heart function [BP, systolic and diastolic function, heart rate] were not altered by [resveratrol]") -- and these parameters were tested in anaesthetized mice, not even during or immediately after forced exercise, which might have been expected to have stressed the heart. Of course, if you don't test for something, you don't see it. Again, statins may save the lives of many overweight, hypercholesterolemic humans, but that doesn't necessarily mean that they don't also damage their mitochondria -- just that on balance, it's a better tradeoff than waiting for the ongoing atherosclerotic plaque deposits to develop into ischemic heart disease and cardiac arrest.

-Michael

1. Resveratrol, a unique phytoalexin present in red wine, delivers either survival signal or death signal to the ischemic myocardium depending on dose
Jocelyn Dudley, Samarjit Das, Subhendu Mukherjee, Dipak K. Das
J Nutr Biochem. 2009 Jun;20(6):443-52. PMID: 18789672

2: Lagouge M, Argmann C, Gerhart-Hines Z, Meziane H, Lerin C, Daussin F, Messadeq N, Milne J, Lambert P, Elliott P, Geny B, Laakso M, Puigserver P, Auwerx J. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell. 2006 Dec 15;127(6):1109-22. Epub 2006 Nov 16. PubMed PMID: 17112576.

3. Pearson KJ, Baur JA, Lewis KN, Peshkin L, Price NL, Labinskyy N, Swindell WR, Kamara D, Minor RK, Perez E, Jamieson HA, Zhang Y, Dunn SR, Sharma K, Pleshko N, Woollett LA, Csiszar A, Ikeno Y, Le Couteur D, Elliott PJ, Becker KG, Navas P, Ingram DK, Wolf NS, Ungvari Z, Sinclair DA, de Cabo R.
Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span.
Cell Metab. 2008 Aug;8(2):157-68.
PMID: 18599363 [PubMed - as supplied by publisher]

[niner]

Just as a drug that does wonders for a person with metabolic syndrome (say, a high-dose statin) could well be expected to be largely deleterious to a person with a healthy metabolism, it's not necessarily surprising if there was a negative effect of even a more moderate dose of resveratrol given to normally-fed. normal-weight rats.

I don't see how we can say "could well be expected to be largely deleterious". It might be deleterious (or not). It really depends on the MOA. Bear in mind that the animals in question had their hearts removed and deprived of oxygen for half an hour. After that there were negative effects, but that situation is really not terrifically analogous to anything a (surviving) human is going to experience.

[Anthony_Loera]

niner,

After readiing the last sentence in your post, I can't seem to get the image of
A certain poor fellow getting his heart pulled out of his chest by a witch doctor
While Dr. Jones and shorty watch in disbelief...

A

[niner]

After readiing the last sentence in your post, I can't seem to get the image of
A certain poor fellow getting his heart pulled out of his chest by a witch doctor
While Dr. Jones and shorty watch in disbelief...

The scene in my head is the Terminator in that biker bar...

[maxwatt]

After readiing the last sentence in your post, I can't seem to get the image of
A certain poor fellow getting his heart pulled out of his chest by a witch doctor
While Dr. Jones and shorty watch in disbelief...

The scene in my head is the Terminator in that biker bar...

The Island of Doctor Moreau?

Isolated working rat heart preparation
Twenty-four hours after the last dose of resveratrol administered by gavaging, the rats were anesthetized with sodium pentobarbital (80 mg/kg body weight ip injection) (Abbott Laboratories, North Chicago, IL, USA) and anticoagulated with heparin sodium (500 IU/kg body weight ip injection) (Elkin-Sinn, Inc., Cherry Hill, NJ, USA). After an adequate period of anesthesia, thoracotomy was conducted, and the hearts were perfused with KHB buffer (118 mM sodium chloride, 4.7 mM potassium chloride, 1.7 mM calcium chloride, 25 mM sodium bicarbonate, 0.36mMpotassium biphosphate, 1.2mMmagnesium sulfate and 10mMglucose) in retrograde Langendorff mode at 37°C at a constant perfusion pressure of 100 cm of water (10 kPa) in the aorta for a 15-min washout and equilibration period [30]. After the Langendorff mode, the heart was switched to the working mode (antegrade perfusion) at a constant perfusion pressure of 17 cm of water (1.7 kPa) in the left atrium for 10 min. The baseline functional parameters were collected after steady-state cardiac function was established. For the control group, the hearts were perfused continuously in antegrade mode and cardiac function measurements for heart rate, coronary flow, aortic flow, left ventricular enddiastolic pressure, left ventricular developed pressure and its first derivative were done at 0 (baseline), 10, 30, 60, 90 and 120 min during the reperfusion. After the stabilization period in the antegrade mode, the ischemia reperfusion effects consisted of closing the antegrade perfusion line and subjecting the heart to 30 min of ischemia. The heart was then switched to the retrograde mode for 5 min to avoid the development of high incidence ventricular fibrillation and switched to the working heart antegrade mode where the cardiac function parameters were measured as described previously [30]. All hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. Any heart that showed any cardiac disturbance (ventricle arrhythmia and fibrillation) during the entire experiment was excluded from this study.

It is hard to say what such extreme procedures tell us, and then only after the rats are already dead. Resveratrol is known to induce apoptosis in damaged cells (as in cancer cells) and ischemia damages cells. Under these conditions, it would not be unexpected for higher doses to do this more effectively,as this study showed.

Edited by Michael, 01 May 2010 - 10:15 PM.
Paper extract readability

[maxwatt]

... As we know from the Sinclair/de Cabo report (3), high-dose resveratrol does help to partially normalize the lives of such animals -- but it doesn't do much -- and, in terms of mortality, doesn't do anything -- for normally-fed. normal-weight mice. ...

Just what did the de Cabo paper show about the effects of resveratrol? It delayed functional decline, shown in increased bone density, slowed cataract formation, improved vascular function, improved locomotor function (balance and coordinatin). Even slight improvements in these parameters can make a big difference in quality of life.

What did the mice die of? "...the vast majority of these cases were lymphomas, a tumor type for which the efficacy of resveratrol has not been thoroughly assessed, and that is thought to be triggered mainly by endogenous retroviruses in mice..." The C57B mice used in de Cabos study are infected with an endogenous retrovirus. If the lab had burned down in a fire, one could as well declare resveratrol did not extend life-span because the mice all died in the fire.

So many substances have been tested for life-extension using C57B mice, and have come up short. It looks to me the reason is that unless the substance protects against lymphoma, the mice will die of that before other life-extending effects are manifest. We have a flawed model for testing life-extension.


* Pearson KJ, Baur JA, Lewis KN, Peshkin L, Price NL, Labinskyy N, Swindell WR, Kamara D, Minor RK, Perez E, Jamieson HA, Zhang Y, Dunn SR, Sharma K, Pleshko N, Woollett LA, Csiszar A, Ikeno Y, Le Couteur D, Elliott PJ, Becker KG, Navas P, Ingram DK, Wolf NS, Ungvari Z, Sinclair DA, de Cabo R.
Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span.
Cell Metab. 2008 Aug;8(2):157-68.
PMID: 18599363 [PubMed - as supplied by publisher]

N.B. CR (including EOD feeding) is known to protect against lymphomas. Resveratrol does not. De Cabo found EOD feeding plus resveratrol mice lived longer than the EOD alone group. It is possible that EOD protected against lymphoma in these groups, and the addition of resveratrol extended life-span beyond what EOD alone did by other mechanisms.

[kismet]

I don't see how we can say "could well be expected to be largely deleterious". It might be deleterious (or not).

It's biology 101 (or my understanding thereof). And you know the saying: it's easier to screw up than to improve a healthy system. I don't know any metabolic intervention which does not involved trade-offs and I know even less such interventions which are worth partaking despite those trade-offs.
maxwatt, what about rapamycin actually increasing lymphoma incidence but still extending lifespan of heterogenous mice (some of which have a C57BL background)? I'm not sure if the heterogenous NIA mice are superior to C57BL standard strains, but we'll see how Resv fares when it's tested in the upcoming ITP cohort of heterogenous mice. Until then I can understand all the scepticism. We have not enough meaningful data - a hypothesis why resveratrol failed + studies in short lived animals are not what I'd call "proof" in the scientific sense. It's only a starting point to gather real, meaningful data. But I certainly like your hypothesis on the face of it.

And it is not just C57BL, most substances tested in other long lived strains e.g. Wistar rats always seem to come up short too. Remember deprenyl? All the credible studies show modest average life span extension in long lived rats but only at the right dose. While based on some early data one might jump to unjustified conclusions (max life span extension in hamsters sounds good, or does it?) Premature data is premature. Premature can be dangerous.

Edited by kismet, 30 July 2009 - 02:21 PM.

[Michael]

... As we know from the Sinclair/de Cabo report (3), high-dose resveratrol does help to partially normalize the lives of such animals -- but it doesn't do much -- and, in terms of mortality, doesn't do anything -- for normally-fed. normal-weight mice. ...

What did the mice die of? "...the vast majority of these cases were lymphomas, a tumor type for which the efficacy of resveratrol has not been thoroughly assessed, and that is thought to be triggered mainly by endogenous retroviruses in mice..." The C57B mice used in de Cabos study are infected with an endogenous retrovirus. If the lab had burned down in a fire, one could as well declare resveratrol did not extend life-span because the mice all died in the fire.

Max, did you read the full paragraph from which you're quoting?

Histopathology
Blinded postmortem histopathology for disease or predisease states was performed on visceral organs including the heart, kidneys, liver, spleen, lungs, and pancreas (Table S3). Resveratrol treatment did not significantly alter the distribution of pathologies in SD groups. This included neoplasias, despite the potency of resveratrol against implanted or chemically induced tumors, recently reviewed elsewhere (Baur and Sinclair, 2006). This may be related to the fact that the vast majority of these cases were lymphomas, a tumor type for which the efficacy of resveratrol has not been thoroughly assessed, and that is thought to be triggered mainly by endogenous retroviruses in mice (Kaplan, 1967; Risser et al., 1983). ((1); my emphasis)

Above, you seem to be mistaking them to mean that .the vast majority of deaths in C57Bl/6 mice are caused by lymphomas, which if you read that quote in context, it's clear that they aren't saying (and in any case isn't true). Rather, "these cases" refers to neoplasia. They're saying that the majority of the cancers in their study were lymphomas, of which the majority in turn are thought to be caused by ERVs. If you look at their Table S3, you'll see that "only" about half of the mice died from any form of cancer, which isn't much lower than the rate in humans (and remember that mice don't naturally get heart disease) -- and some proportion of these in turn are lymphomas (they don't break this down).

The mice died of all kinds of things, IOW (notably, other cancers, kidney disease, and (in the obese, high-sucrose-high-saturated-fat-fed mice (the ones that first raised the hype factor on resverarol) fatty liver), not just lymphomas -- it's just that lymphoma is the single biggest cause of cancer, which latter is the single biggest cause of death.

Note also that resveratrol didn't just fail to protect them from lymphomas, but that "Resveratrol treatment did not significantly alter the distribution of " "histopathology for disease or predisease states ... [in] visceral organs including the heart, kidneys, liver, spleen, lungs, and pancreas (Table S3)" in the animals fed a normal diet.

[Continued below -- stupid block-quoting limits ... !!]

Edited by Michael, 29 August 2009 - 01:31 PM.

[Michael]

[Continued from previous post ...]

The C57B mice used in de Cabos study are infected with an endogenous retrovirus. If the lab had burned down in a fire, one could as well declare resveratrol did not extend life-span because the mice all died in the fire.

Do remember that every mammal on the planet is infected with an endogenous retrovirus . "Endogenous retroviruses (ERVs) constitute approximately 8-10% of the human and mouse genome." (2)

So many substances have been tested for life-extension using C57B mice, and have come up short. It looks to me the reason is that unless the substance protects against lymphoma, the mice will die of that before other life-extending effects are manifest. We have a flawed model for testing life-extension.

Well, I do agree with this to a point -- but anything short of human testing is always flawed in some sense. The question isn't whether these mice die of something more often than something else (this is true of any model!): the question is whether they die early in some artificial way. The C57Bl/6 is used so often in large part because for a mouse it is very long-lived: C57Bl/6 live longer than other strains precisely because they don't die earlier of other causes. You've gotta die of something ...

While (6) wasn't a lifespan study, it used the more genetically-diverse hybrid of C57BL/6×C3H/HeF1, and still found "no decrease in spontaneous tumors at the time of sacrifice (Supplemental Table S2). In particular, spontaneous liver tumors were abundant in mice fed the control diet or resveratrol, but rare in CR mice."

All that said:

N.B. CR (including EOD feeding) is known to protect against lymphomas. Resveratrol does not. De Cabo found EOD feeding plus resveratrol mice lived longer than the EOD alone group. It is possible that EOD protected against lymphoma in these groups, and the addition of resveratrol extended life-span beyond what EOD alone did by other mechanisms.

Yup, an entirely plausible hypothesis -- although it's worth noting that nominally, at least, more of the EOD-fed animals died of cancer than AL animals did (though the difference (60% vs 50%) wasn't significantly different, and again, we don't have the breakdown on lymphomas specifically). The major reductions in death were in kidney disease and in "other." The EOD-plus-lower-dose-resveratrol group had about the cancer incidence of AL animals -- it's just that they took longer to develop it.

Again, you've gotta die of something.

Despite what I say above and elsewhere, I am still following the resveratrol story, and am glad to know that NIA's ITP is also testing the stuff, and doing so in a more genetically-heterogeneous stock. This has its pros and cons, but these mice do have a nice, long life and a diversity of causes of death. (Unfortunately, AFAICS, they aren't repeating the combined EOD-plus-res part of (1), nor, as would IMO be better yet, combining it with a higher, but submaximal, level of CR).

But to those taking more supplemental resveratrol than is present a moderate intake of wine a day right now, before such studies are completed, I ask: where is the better evidence on which you're gambling your long-term health? We have no evidence of extension of life in any mammal -- just the near-normalization of premature mortality in animals rendered obese and diabetic because of a life gorging on a high-sucrose, high-saturated-fat diet. Of note, (8) found (similarly to Auwerx and de Cabo) that resveratrol gave significant protection to obese, high-fat-diet fed Wistar rats (not mice, let alone C57Bl/6) against "hyperglycaemia, dyslipidemia, increased serum oxidized-LDL (ox-LDL) and hepatic oxidative stress," it gave no such benefits to normal-weight, normal-diet rats: in fact, resveratrol supplementation of "standard-fed-rats reduced glutathione-antioxidant defense system and enhanced hepatic lipid hydroperoxide [my emphasis]." Above, remember, just such a diabetic, obese, junkfood-died rodent model study (Auwerx) was favorably referenced as evidence of the safety of resveratrol!

In mice, resveratrol doesn't fully replicate CR-induced gene expression changes (6) nor induce some demonstrably important CR-induced metabolic changes (7). Heck, it's not even clear that the stuff does any good in yeast (where the whole hype got started) (3); nor in Drosophila elegans (note that Partridge's group is possibly the most experienced lab in the world in doing lifespan studies in Drosophila) (4); nor in another (tephritid) species of fruit flies ((5): there may have been a marginal effect in flies fed 1 of the 24 different diet combinations, but I'm skeptical of that result, and IAC there was no effect in the other 23 diets); nor in C. elegans (4) -- and in mammals, again, the evidence to date is against it, except as a complementary intervention in animals also on mild EOD CR.

There are no studies on the safety of resveratrol in humans lasting even a few months, and there what appear to me to be a disturbing number of independent reports of joint pain. These are anecdotal, of course, and could be coincidental, but they aren't the standard headaches-and-upset-tummies stuff, and there seem to be an awful lot of them. Even if you aren't getting this pain, if it's real, the unknown mechanism leaves open the question of unknown other, more subtle or long-term effects in humans.

Again, I challenge you folks to seriously ask: on what evidential basis are you gambling your health on this stuff?

-Michael

1. Pearson KJ, Baur JA, Lewis KN, Peshkin L, Price NL, Labinskyy N, Swindell WR, Kamara D, Minor RK, Perez E, Jamieson HA, Zhang Y, Dunn SR, Sharma K, Pleshko N, Woollett LA, Csiszar A, Ikeno Y, Le Couteur D, Elliott PJ, Becker KG, Navas P, Ingram DK, Wolf NS, Ungvari Z, Sinclair DA, de Cabo R.
Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span.
Cell Metab. 2008 Aug;8(2):157-68.
PMID: 18599363 [PubMed - as supplied by publisher]

2. Lee YK, Chew A, Phan H, Greenhalgh DG, Cho K. Genome-wide expression profiles of endogenous retroviruses in lymphoid tissues and their biological properties. Virology. 2008 Apr 10;373(2):263-73. Epub 2008 Jan 9. PubMed PMID: 18187179; PubMed Central PMCID: PMC2427371.

3. Kaeberlein M, McDonagh T, Heltweg B, Hixon J, Westman EA, Caldwell S, Napper A, Curtis R, Distefano PS, Fields S, Bedalov A, Kennedy BK.
Substrate specific activation fo sirtuins by resveratrol.
J Biol Chem. 2005 Jan 31; [Epub ahead of print]
PMID: 15684413

4. Bass TM, Weinkove D, Houthoofd K, Gems D, Partridge L.
Effects of resveratrol on lifespan in Drosophila melanogaster and Caenorhabditis elegans.
Mech Ageing Dev. 2007 Aug 14; [Epub ahead of print]
PMID: 17875315 [PubMed - as supplied by publisher]

5. The Prolongevity Effect of Resveratrol Depends on Dietary Composition and Calorie Intake in a Tephritid Fruit Fly.
Zou S, Carey JR, Liedo P, Ingram DK, Müller HG, Wang JL, Yao F, Yu B, Zhou A.
Exp Gerontol. 2009 Mar 2. [Epub ahead of print]
PMID: 19264118

6. Barger JL, Kayo T, Vann JM, Arias EB, Wang J, Hacker TA, Wang Y, Raederstorff D, Morrow JD, Leeuwenburgh C, Allison DB, Saupe KW, Cartee GD, Weindruch R, Prolla TA.
A low dose of dietary resveratrol partially mimics caloric restriction and retards aging parameters in mice.
PLoS ONE. 2008 Jun 4;3(6):e2264.
PMID: 18523577 [PubMed - in process]

7. Resveratrol treatment in mice does not elicit the bradycardia and hypothermia associated with calorie restriction.
Mayers JR, Iliff BW, Swoap SJ.
FASEB J. 2008 Dec 4. [Epub ahead of print]
PMID: 19056839

8. Resveratrol toxicity: effects on risk factors for atherosclerosis and hepatic oxidative stress in standard and high-fat diets.
Rocha KK, Souza GA, Ebaid GX, Seiva FR, Cataneo AC, Novelli EL.
Food Chem Toxicol. 2009 Mar 16. [Epub ahead of print]
PMID: 19298841

Edited by Michael, 30 July 2009 - 04:19 PM.
Clarification

[Anthony_Loera]

Micheal,

I really do like your posts, believe it or not.

Although you are great at nailing down studies to provide evidence for most of your arguments, the independent reports you mention regarding issues don't seem to be from any study up to this point. Because of this I think I can insert a wrench into it since we are all talking about what other people think... and not a true study regarding arthritis or joint pain. I believe I can mention an independent email sent to me stating that the issue maybe in Oxalic acid that maybe found in the 50% resveratrol. I have to say, that it was an interesting item I did not think about until this email came to my inbox.

resveratrol doesn't fully replicate CR-induced gene expression changes

In regards to CR, it is pretty interesting for practicioners like yourself. and although longevity is attributed, it also increases susceptibility to infections (see below): http://health-studie...riction-cr.html
So I think, it maybe debatable if we need to have CR fully replicated specially because of the down side, as it appears that straight CR has a downside. Then again, we obviously need more research to understand the benefits on humans:

I'll keep researching the Oxalic acid issue, and maybe start testing for it as well, although I don't think I will find much in high purity (98%, 99%) resveratrol.

Cheers
A

Edited by Anthony_Loera, 30 July 2009 - 05:17 PM.

[tunt01]

think michael makes some good points. but resveratrol isn't exactly a NCE. some chemist in pfizer didn't just dream up this molecule. riskiness of resveratrol is most likely a function of dose, which is very difficult to know.

dose at the lower end is likely safest. it may make the most sense to just take some procyanidin/OPC pills w/ some resv content, rather than resv alone.

[Michael]

Although you are great at nailing down studies to provide evidence for most of your arguments, the independent reports you mention regarding issues don't seem to be from any study up to this point.

I did actually specify that in my post. Certainly, you wouldn't expect those kinds of effects from the small, very short-term human studies (none of them controlled IAC) that have been done to date.

Because of this I think I can insert a wrench into it since we are all talking about what other people think...

Not clear what you mean here.

I believe I can mention an independent email sent to me stating that the issue maybe in Oxalic acid that maybe found in the 50% resveratrol. I have to say, that it was an interesting item I did not think about until this email came to my inbox. [...] I'll keep researching the Oxalic acid issue, and maybe start testing for it as well, although I don't think I will find much in high purity (98%, 99%) resveratrol.

I don't think oxalic acid can credibly explain the problem. First, oxalic acid is asserted to aggravate rheumatoid arthritis by various 'alternative health' sources, but I could barely find anything about this being a problem in the medical literature, and nothing authoritative or from a formal study. Second, nearly none of the anecdotal reports are from people who had an existing joint problem. Third, I doubt there's enough oxalic acid present in 50% resveratrol products to have an effect: I dug 'round a bit and couldn't find any data (have you analyzed your or anyone else's 50% extracts, Anthony?), but even if the whole other 50% were oxalic acid (and we know it's not, of course: there's the other stilbenes and polyphenols, including the emodin and other anthraquinones, for starters), that would still put a 500 mg pill in the same ballpark as a small serving of many vegetables.

And, finally, while most reports of resveratrol-associated joint pain come from people who either used 50% extracts or never specified what exactly they were using, there are several reports of joint pain in people using 98% pure resveratrol (also here, and see here ([Edit: this was a link to a report in someone using Vital Prime, which I seem to've dropped and can't now find again] this brand didn't match label claim when you (Anthony) had it tested, but was still 87% resveratrol -- not much room for oxalates), apparently here, and possibly here

think michael makes some good points. but resveratrol isn't exactly a NCE. some chemist in pfizer didn't just dream up this molecule.

Kinda like morphine, colchichine, hemlock, atropine, nutmeg, reserpine, digoxin, scopolamine, taxol, ...?

-Michael

Edited by Michael, 29 August 2009 - 07:27 PM.
Explaining missing link (!)

[tunt01]

While (6) wasn't a lifespan study, it used the more genetically-diverse hybrid of C57BL/6×C3H/HeF1, and still found "no decrease in spontaneous tumors at the time of sacrifice (Supplemental Table S2). In particular, spontaneous liver tumors were abundant in mice fed the control diet or resveratrol, but rare in CR mice."

The study also notes in the very next sentence:

Thus, although a low dose of resveratrol can improve quality of life by retarding aging parameters such as cardiac dysfunction, a nutritional or pharmaceutical strategy to alsoincrease lifespan in mice will likely require blockage of the IGF-1 axis or its targets.

I believe Maxwatt takes luteolin which lowers IGF-1 in combnation with resveratrol, which could be a very sound approach. I don't think anyone is equating resveratrol directly to CR or suggesting resveratrol is the single solution in extending life. I think Maxwatt has stated previously that he thinks it is part of the equation, which is exactly what the authors concluded in A low dose of dietary resveratrol partially mimics caloric restriction and retards aging parameters in mice.


When looking at the incidence rate of liver tumors in the S2 section provided by the study, the data is as follows:



So while there wasn't as dramatic a reduction in cancer in the resv population relative to CR, I don't exactly find it discouraging in light of the positive findings of improved cardiac biomarkers. To me, it is only further confirmation that a low dose of resveratrol is ideal for a daily diet. It's possible that a "polypill" type approach (like longevenix -- something above and beyond a basic multi-vitamin) could make sense as a life extension aid.

I am a bit surprised how low this went in dose. On a human equivalent dose basis, this is about 24mg for a 160 lbs. male., which is a bit lower than I thought would make sense last time I looked at this issue.

Kinda like morphine, colchichine, hemlock, atropine, nutmeg, reserpine, digoxin, scopolamine, taxol, ...?

a little too draconian, i think....

[maxwatt]

...

I am a bit surprised how low this went in dose. On a human equivalent dose basis, this is about 24mg for a 160 lbs. male., which is a bit lower than I thought would make sense last time I looked at this issue.

No doubt there is an inverted U-shaped dose-response curve for resveratrol. But at what point is less no longer more, and more less?

[niner]

I am a bit surprised how low this went in dose. On a human equivalent dose basis, this is about 24mg for a 160 lbs. male., which is a bit lower than I thought would make sense last time I looked at this issue.

The Human Equivalent Dose is generally employed for first-time-in-humans toxicity studies, not for dose translation for equivalent pharmacodynamic effect. I think that's why it looks so low. A reasonable equivalent for a human is probably a lot more than this, though not a multi-gram megadose. Based on PK data from humans and rats, I once computed (here)that humans needed 1.3 times the dose (in mg/kg) to get the same plasma levels of resveratrol, using similar but not equivalent formulations. (The rats had the better formulation, though not wildly so, so 1.3 may be an underestimate.)

Edited by niner, 31 August 2009 - 05:06 PM.

[tunt01]

idk, it still seemed extremely low to me. they used 4.9 mg/kg in mice. so that is 0.39729 mg per kg in human. so 28.6 mg for a 160 lbs adult. guess my math was a little light..

[niner]

idk, it still seemed extremely low to me. they used 4.9 mg/kg in mice. so that is 0.39729 mg per kg in human. so 28.6 mg for a 160 lbs adult. guess my math was a little light..

By my reckoning, assuming you want to see the same plasma levels in humans as in rats, you'd need 4.9 * 1.3 = 6.37mg/kg.

160 lb * (1kg/2.2lb) * 6.37mg/kg = 463mg. That sounds about right, imho.

The factor of 1.3 is only ballpark, but c'mon, 29mg isn't going to do much. You are using the HED calc in a way that it isn't really intended to function.

Edited by niner, 31 August 2009 - 07:47 PM.

[tunt01]

By my reckoning, assuming you want to see the same plasma levels in humans as in rats, you'd need 4.9 * 1.3 = 6.37mg/kg.

160 lb * (1kg/2.2lb) * 6.37mg/kg = 463mg.

I used the BSA equivalent calc based on http://www.fasebj.or...fasebj;22/3/659 . I think the lifegen/weindruch paper that is discussed previously in the thread using mice @ 4.9 mg/kg, mentions that a low dose turns on a certain set of longevity genes (it discusses overlap of gene expression between CR and resveratrol, etc.).

But I *think* there is a comment in that paper mentioning that a low dose turns on a high % of these genes expressed in CR, and that a higher dose may not be ideal because it would change the expression profile or increase expression in genes not desired (if i recall properly, dont have the paper atm i am on the road).


why wouldn't you want to equate a low dose strategy to the BSA-HED used in this paper? i realize what you are trying to do in terms of plasma level, but what is the differential between human/mice such that the HED-BSA calc is invalid for resv.

Edited by Michael, 29 October 2009 - 07:55 PM.
Trim quotes

[maxwatt]

I used the BSA equivalent calc based on http://www.fasebj.or...fasebj;22/3/659 . I think the lifegen/weindruch paper that is discussed previously in the thread using mice @ 4.9 mg/kg, mentions that a low dose turns on a certain set of longevity genes [...] But I *think* there is a comment in that paper [...] that a higher dose may not be ideal because it would change the expression profile or increase expression in genes not desired [...]

why wouldn't you want to equate a low dose strategy to the BSA-HED used in this paper? i realize what you are trying to do in terms of plasma level, but what is the differential between human/mice such that the HED-BSA calc is invalid for resv.

Humans have more efficient enzymes WRT sulfonation and such; our livers eliminate it faster, more efficiently than mice. We need more to see the same blood serum levels as mice than the BSA calc would estimate.

Interesting to note: the Auwerx study use 400 mg/kg, and measured mouse blood levels. Boocock measured approximately the same blood levels in humans with a one gram dose, if I remember correctly. I posted about it last month.

Edited by Michael, 29 October 2009 - 07:57 PM.
Trim quotes