58 replies to this topic

[Hedgehog]

Hi All,

I'm trying to design an experiment of my res plasma concentration. I have access to HPLC instruments but wanted to see what other people thought about my experimental design.

First I we sort of already know that Res has very low bioavailability from other animal and human published studies.


1^st off typical bioavailability calculations use a formula that requires an intravenous application of the drug and an oral administration. Well I'm not going to do IV infusion of Res. Sorry…

So I thought if I make a linear standard curve of Res then hopefully my plasma concentration will fit some where on that curve. If it does I can extrapolate how much actually got into my blood stream.

I would be following this method.

Determination of trans-Resveratrol in Plasma by

HPLC

Anal. Chem. 1999, 71, 747-750




My idea is the Piperine has showed to facilitate bioavailability of other compounds but I can't find any data for Piperine and Res?



On a fasting stomach I would take 10mg of Bioperine wait for 30mins for it to inhibit the degradation pathways of res. After 30mins or T=0 for Res administration I would take a sample of my blood as a blank for the HPLC run. This will insure there are no interfering peaks. I will attempt to take samples at T=10,20,30,45,60,120min. I need to have some blood left J



Hopefully I will get an absorbance that will fit on the linear curve. Also the time points will allow use to so if Bioperine stably inhibits the degradation of res.



Your thoughts are more then welcome.

[health_nutty]

This is truly amazing. Please do this experiment and let us know the results. It would also be very interesting to do other studies such as with a combination of oil, lecithin whey protein, luteolin, 50% extract versus 98%, etc.

[malbecman]

Timepoints look good although if you can, you might want to add one out like at 3 or 4 hours. This will help define the tail end of your curve and what I remember from silymarin oral dosing, you won't have very rapid uptake (as is typical w/ oral dosing) and the plasma conc. curve will go out pretty far. Does the Analy. Chem. paper have the half-life, t 1/2?

[ilanso]

I would first run this experiment without the piperine, as a pure Rsv benchmark.

[Hedgehog]

This is truly amazing. Please do this experiment and let us know the results. It would also be very interesting to do other studies such as with a combination of oil, lecithin whey protein, luteolin, 50% extract versus 98%, etc.

That would be nice but there is only one of me which makes it hard to test all those plus I don't want to take my blood to often. If Piperine doesn't work then I might try another method.

[Hedgehog]

Timepoints look good although if you can, you might want to add one out like at 3 or 4 hours. This will help define the tail end of your curve and what I remember from silymarin oral dosing, you won't have very rapid uptake (as is typical w/ oral dosing) and the plasma conc. curve will go out pretty far. Does the Analy. Chem. paper have the half-life, t 1/2?

humm good point but doesn't Res have a fast absorbtion rate and also fast degradation rate?

I will add a 3 to 4hr time point just to get a good idea of T=1/2 The paper doesn't talk about T=1/2 but hopefully I will see linear decay and we could do our best guess?

[Hedgehog]

How do you think I should take the Res? I have to get an exact amount which means I have to weigh it out. I was thinking of just doing a EtOH shot with I dunno 300mg of Res? Is that to low or to high? Should I add Vitamin C to help stabilize? This also has to be in pure form. As in it can't be in a pill with other stuff.



To ilanso:

If I had more subjects I would definitely take Res first w/o Piperine. However, there is a lot of literature on this compound having low bioavailability. I think if I case I can see any differences compared to these studies we might be on to something. I thought long and hard about that. I was even taking of taking Res doing the HPLC to see concentration, wait for 2hr take a 10mg piperine, wait another 30min and then complete the tests I layed out above. However, this would allow for too many variables?


I would be interested in doing some test of pills from different vendors. Such as test called "content uniformity" which looks at strength of tablets. However, I probably won't' have time for that...

I hope to start my personal bioavailability test some time mid-late-Jan. But in the mean time I need to collect as much information and obtain all the proper protocols and reagents.

[malbecman]

I've seen a half-life of 15 minutes and one of 2 hours in rats that were orally admin. resveratrol in 2 different papers. Still, its your blood. You should only need a few mls at each timept, right (from my back of the envelope calculations....)?

Timepoints look good although if you can, you might want to add one out like at 3 or 4 hours. This will help define the tail end of your curve and what I remember from silymarin oral dosing, you won't have very rapid uptake (as is typical w/ oral dosing) and the plasma conc. curve will go out pretty far. Does the Analy. Chem. paper have the half-life, t 1/2?

humm good point but doesn't Res have a fast absorbtion rate and also fast degradation rate?

I will add a 3 to 4hr time point just to get a good idea of T=1/2 The paper doesn't talk about T=1/2 but hopefully I will see linear decay and we could do our best guess?

[zoolander]

Hedgehog what makes you believe that this study is worth doing? I don't see how you could possibly conduct a study (n=1) looking into the bioavailability of resveratrol in humans and get results that would even be worth considering seriously. You plan to supplement yourself, take your own blood and inject the processed samples into HPLC.

There is considerable interest in resveratrol and it's ability to effect sirtuins. So don't you think that people have been working on trying to increase resveratrols bioavailabilty in humans?? There are most likely teams of people working on this problem. Teams that have decades of experience that know how to design and control studies properly.

What do you plan to do with the results?

[Hedgehog]

Hedgehog what makes you believe that this study is worth doing? I don't see how you could possibly conduct a study (n=1) looking into the bioavailability of resveratrol in humans and get results that would even be worth considering seriously. You plan to supplement yourself, take your own blood and inject the processed samples into HPLC.

There is considerable interest in resveratrol and it's ability to effect sirtuins. So don't you think that people have been working on trying to increase resveratrols bioavailabilty in humans?? There are most likely teams of people working on this problem. Teams that have decades of experience that know how to design and control studies properly.

What do you plan to do with the results?

Well yes n=1 is meaningless statistically. The experiment should only take well 30mins after the last time point. Plus solvent prep.

If there were teams of people then I would have expected to see at least 1 paper that address res + piperine and bioavailability. Having said that usually experiments that fail don't get published. However, looking at the bioavailability from a biochemist aspect this is a straight forward approach to addressing the problem. If this was a funded study then yes I would implement controls and obviously have a group that does take piperine and one that doesn't.

I think the methodology is pretty straight forward. If I get a some absorbance at the right time in the chromatogram then I should be able to quantify it, pending it is within my standard linear range.

IMO:

• worst case, I don't see any Res (maybe below my HPLC's detection limits which is a very high probability)
• Best case I can detect some Res and my personal bioavailability will be lower, same or higher compared to other studies.

[ilanso]

To ilanso:

If I had more subjects I would definitely take Res first w/o Piperine. However, there is a lot of literature on this compound having low bioavailability

I am saying do the benchmark only to control for your own personal bioavailability, with and without the piperine.
Think of it as calibration.

[niner]

Hedgehog what makes you believe that this study is worth doing? I don't see how you could possibly conduct a study (n=1) looking into the bioavailability of resveratrol in humans and get results that would even be worth considering seriously. You plan to supplement yourself, take your own blood and inject the processed samples into HPLC.

There is considerable interest in resveratrol and it's ability to effect sirtuins. So don't you think that people have been working on trying to increase resveratrols bioavailabilty in humans?? There are most likely teams of people working on this problem. Teams that have decades of experience that know how to design and control studies properly.

What do you plan to do with the results?

Zoo, there are lots of pharmacokinetic studies published with n=1. The standard deviations between subjects are significant, but they are not enormous. N=1 is WAY better than n=0. Probably the most valuable thing that hedghog_info could learn would have to do with different dosing schemes. Here it's even less bad to have n=1 because individual variation has been removed. Probably the best way to stay consistent from run to run would be to maintain a close to identical diet for 24 hours before the test.

There may well be other people who are looking at human studies, but they will probably be interested in proprietary formulations, a la Biotivia. If Hedgehog has the tools, maybe we could come up with some volunteers...

edit: Hey, this is my thousandth post! WooHoo! And a worthy occasion it is!

Edited by niner, 21 December 2007 - 09:24 PM.

[Hedgehog]

If Hedgehog has the tools, maybe we could come up with some volunteers...

Well I honestly thought about it but 1st I don't want ppl at my work. If Res is stable in plasma that it might be an option for ppl to send samples under ice but I haven't seen any stability data of Res in blood plasma.

[Hedgehog]

To ilanso:

If I had more subjects I would definitely take Res first w/o Piperine. However, there is a lot of literature on this compound having low bioavailability

I am saying do the benchmark only to control for your own personal bioavailability, with and without the piperine.
Think of it as calibration.

Sounds good, I will try two-three time points the day before along with the standards.

[Hedgehog]

If Hedgehog has the tools, maybe we could come up with some volunteers...

Well I honestly thought about it but 1st I don't want ppl at my work. If Res is stable in plasma that it might be an option for ppl to send samples under ice but I haven't seen any stability data of Res in blood plasma.

I will keep my plasma samples for a week and see how much they degrade over time stored in a dark cold fridge.

[maxwatt]

If Hedgehog has the tools, maybe we could come up with some volunteers...

Well I honestly thought about it but 1st I don't want ppl at my work. If Res is stable in plasma that it might be an option for ppl to send samples under ice but I haven't seen any stability data of Res in blood plasma.

I will keep my plasma samples for a week and see how much they degrade over time stored in a dark cold fridge.

If you can do multiple tests with different protocols, can you test co-administration with luteolin? There is reason to believe it is a strong inhibitor of liver sulfonation which should result in higher levels of resveratrol, probably more so than bioperin or quercetin.

(PM me if you need a small amount of luteolin, or order some from beyond-a-century.)

[MP11]

Great that you're doing this.

What brand resv are you using?

[Hedgehog]

Great that you're doing this.

What brand resv are you using?

Well I actually haven't tried Res yet. I just become curious about it and saw all the problems with the breakdown of it. What do you suggest? I bought some 50% Cis:Trans Res, but for my studies Bio-Availability studies I will be testing myself on 100% T Res. Where is the best place to get high quality stuff? >98%? I think I need about 1g for my 1st study. However for my daily Res I would like to develop a method a entantiomeric test method to determine if what I'm buying is actually 50% trans Res.

To Maxwatt: Can you send me or post some info about luteolin and Res breakdown. That would be an interesting study.

My first test will be to administor Piperine + Res, and obtain results.
The next test will be just Res with no Piperine.
Then luteolin and or querctin.

[zoolander]

Having said that usually experiments that fail don't get published.

What are you suggesting here? Are you suggesting that studies with good results only get published? I think that's what you are suggesting. Well that's shonky research if you ask me. The sort of shonky research you see with commercially funded studies conducted by teams of sell out lackies. Good or bad. Positive or negative. Results are results and should be put forward. The only "failed" experiment, IMO, is a badly designed experiment.

If this was a funded study then yes I would implement controls and obviously have a group that does take piperine and one that doesn't.

You don't necessarily need funds to control a study. You need knowledge and understanding. There's no use conducting a study that is compounded and uncontrolled.

worst case, I don't see any Res (maybe below my HPLC's detection limits which is a very high probability)

HPLC is very sensitive and should be able to detect resveratrol. I know this for a fact because my secondary supervisors lab uses HPLC to detect resveratrol. You should know that there is more than just sensitivity with HPLC. There is the run time of mobile phases, pH, solvent suitability, making sure you have the right column and so on.

Zoo, there are lots of pharmacokinetic studies published with n=1. The standard deviations between subjects are significant, but they are not enormous. N=1 is WAY better than n=0.

Hang on a second niner. Re-read what you just wrote. You can't tell whether the SD is significant or not because there is only one subject. Unless you test for that significance between people you cannot state whether it is significant or not. How can you relate this one subject to the rest of the population? Some people simply do not respond to treatments.

We don't know of hedgehogs medical records. Even if we (and perhaps he) do not know if there are any unknown metabolic/pathological conditions. Hedgehog may be able to tell us all that he has a clean bill of health but I would have done the same a few months back. I wouldn't now because a fluke test diagnosed me with severe sleep apnea which means my immune system is most likely compromised, iron metabolism is shot and so on.

Just for the record when you use a capital N you are refering to the number of studies and not subjects. A lower case 'n' of course refers to the number of subjects in a study (N).

Anyhow.....there is nothing stopping hedgehog from conducting his own experiment however the results of this experiment should not be taken very seriously because who knows what went on. None of us know hedgehog from a bar of soap but it appears that some of us are ready to seriously consider the results of his little experiment as valid

[niner]

Having said that usually experiments that fail don't get published.

What are you suggesting here? Are you suggesting that studies with good results only get published? I think that's what you are suggesting. Well that's shonky research if you ask me. The sort of shonky research you see with commercially funded studies conducted by teams of sell out lackies. Good or bad. Positive or negative. Results are results and should be put forward. The only "failed" experiment, IMO, is a badly designed experiment.

Yes, you and I both agree on this, but there is still a publication bias for positive results. It is partially due to the experimenters, who just aren't that excited about the negative results, and partly due to the reviewers and editors, who are often also not that excited about negative results. Of course, not all negative results are equally negative. Some are very interesting in their negativity ("Sun fails to rise...") but overall the bias is there.

Zoo, there are lots of pharmacokinetic studies published with n=1. The standard deviations between subjects are significant, but they are not enormous. N=1 is WAY better than n=0.

Hang on a second niner. Re-read what you just wrote. You can't tell whether the SD is significant or not because there is only one subject. Unless you test for that significance between people you cannot state whether it is significant or not. How can you relate this one subject to the rest of the population? Some people simply do not respond to treatments.

Right, that was really unclear. What I meant was that in studies I've seen with multiple subjects, the SD was generally not so large as to render an n=1 experiment meaningless.

We don't know of hedgehogs medical records. Even if we (and perhaps he) do not know if there are any unknown metabolic/pathological conditions. Hedgehog may be able to tell us all that he has a clean bill of health but I would have done the same a few months back. I wouldn't now because a fluke test diagnosed me with severe sleep apnea which means my immune system is most likely compromised, iron metabolism is shot and so on.

Yet I bet if you were a subject in a resveratrol PK study, you would be within one SD of the mean. Considering the alternative metrics that we currently have, Maxwatt's big toe or some guy's hives, hedgehog's offer of actual plasma levels is an extraordinary improvement.

[Hedgehog]

You don't necessarily need funds to control a study. You need knowledge and understanding. There's no use conducting a study that is compounded and uncontrolled.

Well res in plasma has already been studied so the knowledge and understanding is already there based on a few papers. I'm not trying to invent the wheel but just add a new variable.

HPLC is very sensitive and should be able to detect resveratrol. I know this for a fact because my secondary supervisors lab uses HPLC to detect resveratrol. You should know that there is more than just sensitivity with HPLC. There is the run time of mobile phases, pH, solvent suitability, making sure you have the right column and so on.

Why does he detect resveratrol? Is it blood plasma concentration? I'm using a C-18 column, Phase A is acetic acid in H20, and think phase B is MeOH or ACN with a flow rate of 1.5. Run time is around 19mins I think. I would have to double check but it is all laid out in the paper that I posted in the first message. I would be very curious to see how you are detecting Res and what method you are following and what problems you have encountered so I can have a heads up.The only reason why I'm crossing my figures about the detection limits is because most of the experiments that look at plasma Res concentrations use HPLC-MS which is VERY sensitive as you probably know. I do have access to one of these but I don't want to use it unless I have too.

Happy Holidays.

[niner]

Great that you're doing this.

What brand resv are you using?

Well I actually haven't tried Res yet. I just become curious about it and saw all the problems with the breakdown of it. What do you suggest? I bought some 50% Cis:Trans Res, but for my studies Bio-Availability studies I will be testing myself on 100% T Res. Where is the best place to get high quality stuff? >98%? I think I need about 1g for my 1st study. However for my daily Res I would like to develop a method a entantiomeric test method to determine if what I'm buying is actually 50% trans Res.

To Maxwatt: Can you send me or post some info about luteolin and Res breakdown. That would be an interesting study.

My first test will be to administor Piperine + Res, and obtain results.
The next test will be just Res with no Piperine.
Then luteolin and or querctin.

I don't think the brand matters that much. The 50% extracts are not racemic mixtures, they are 50% t-resveratrol, very little cis, and 50% other stuff. For a PK study, I'd suggest using 98 or 99% resveratrol. Anthony Loera's company, Revgenetics, could supply it, along with Relentless Improvement, Biotivia, or some other company whose name I've forgotten. (MegaResveratrtol?) Anthony has a program where he will provide free materials for academic research. I don't know if this would qualify, but it might. As for the experiments, I like what you've chosen so far. I'd do the straight resveratrol first so you won't need as long of a washout. I'd be interested in seeing a test of the protocol that some of us are using: A solution phase micronization in PEG: Prepare a solution of resveratrol in 95% ethanol (Most use Everclear for this- apr. 12.5 ml/gm resv). Prepare a second solution of Miralax (a medium MW PEG sold cheaply OTC in drug stores as a laxative) in water. I use 2-3 grams in about 135 ml water, but others may differ. Pour the ethanolic solution into the PEG solution, with stirring; an immediate very fine white precipitate develops. Add a mixer, (I use clear fruit juice) stir and chug. (If you don't mind the taste, you could skip the mixer.) I do this on an empty or mostly empty stomach.

I'm sure that you'd prefer to avoid as many needle sticks as possible. Do you have access to the supplies you'd need to set up a temporary catheter? I was a subject in a PK study once and that's how they did it.

[Hedgehog]

I don't think the brand matters that much. The 50% extracts are not racemic mixtures, they are 50% t-resveratrol, very little cis, and 50% other stuff. For a PK study, I'd suggest using 98 or 99% resveratrol. Anthony Loera's company, Revgenetics, could supply it, along with Relentless Improvement, Biotivia, or some other company whose name I've forgotten. (MegaResveratrtol?) Anthony has a program where he will provide free materials for academic research.

Your probably right about the mixtures not being 50% of each cis/trans. I'm actually going to test the sample I will be getting.
I will contact Anthony about my study. Maybe I could get a discount for what I need.

I'm sure that you'd prefer to avoid as many needle sticks as possible. Do you have access to the supplies you'd need to set up a temporary catheter? I was a subject in a PK study once and that's how they did it.

well I just need a few needle sticks. My GF is a nurse and hopefully we are having a good day when she draws my blood. he he he. I might get her and a few of my friends to participate to increase my sample size.

Edited by hedgehog_info, 24 December 2007 - 04:07 AM.

[maxwatt]

Great that you're doing this.

What brand resv are you using?

Well I actually haven't tried Res yet. I just become curious about it and saw all the problems with the breakdown of it. What do you suggest? I bought some 50% Cis:Trans Res, but for my studies Bio-Availability studies I will be testing myself on 100% T Res. Where is the best place to get high quality stuff? >98%? I think I need about 1g for my 1st study. However for my daily Res I would like to develop a method a entantiomeric test method to determine if what I'm buying is actually 50% trans Res.

To Maxwatt: Can you send me or post some info about luteolin and Res breakdown. That would be an interesting study.

My first test will be to administor Piperine + Res, and obtain results.
The next test will be just Res with no Piperine.
Then luteolin and or querctin.

This might help:

Planta Med. 2006 Jun;72(7):596-603. Epub 2006 May 29. Links
Influence of biotransformation of luteolin, luteolin 7-O-glucoside, 3',4'-dihydroxyflavone and apigenin by cultured rat hepatocytes on antioxidative capacity and inhibition of EGF receptor tyrosine kinase activity.Schlupper D, Giesa S, Gebhardt R.
Institute of Biochemistry, Medical Faculty, University of Leipzig, Leipzig, Germany.

Flavonoids are known as biologically active compounds. Although this has been shown by several in vivo studies, it is still elusive whether their metabolites exert similar activities. Herein we investigated the biotransformation of four different flavonoids, 3',4'-dihydroxyflavone, apigenin, luteolin and luteolin 7-O-glucoside, by cultured rat hepatocytes using a combination of enzymatic deconjugation, HPLC separation and high-resolution mass spectrometry. These flavonoids were chosen because they are active components of many plants, e. g., artichokes. All flavonoids showed rather complex metabolite patterns dominated by phase II metabolites, mainly sulfates, methyl sulfates and methyl glucuronides, but also of combined glucuronide and sulfate conjugates. Phase I metabolism by hydroxylation was rendered likely only for apigenin to form luteolin. When culture media containing the flavonoids and their metabolites were assayed for antioxidative capacity by the DPPH assay, only compounds with hydroxy groups in position 3' and 4' of the B ring were active. Thus, during metabolism of (inactive) apigenin a strong increase in the antioxidative effect was observed while that of the other three flavonoids decreased with time. Determination of EGF receptor tyrosine kinase activity likewise revealed strong inhibition in the presence of a catechol group at ring B. However, in this case the situation was much more complex resulting in a significant increase of the inhibitory activity of 3',4'-dihydroxyflavone and apigenin, but not of luteolin and luteolin 7-O-glucoside during 22 h of incubation. These results show that the biotransformation of flavonoids is very complex and may result not only in a loss but also in a gain of biological activity depending on the individual structural features.

PMID: 16732514 [PubMed - indexed for MEDLINE]

J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Apr 1;848(2):182-7. Epub 2006 Nov 9. Links
Quantitation of trans-resveratrol and detection of its metabolites in human plasma and urine by high performance liquid chromatography.Boocock DJ, Patel KR, Faust GE, Normolle DP, Marczylo TH, Crowell JA, Brenner DE, Booth TD, Gescher A, Steward WP.
Department of Cancer Studies & Molecular Medicine, Cancer Biomarkers & Prevention Group, 5th Floor RKCSB, Leicester Royal Infirmary, Leicester LE2 7LX, UK. djb27@le.ac.uk

We describe a reversed-phase HPLC method that uses gradient elution and UV detection (325 nm) to determine levels of resveratrol and identify six major conjugated metabolites in the plasma and urine of human volunteers after administration of a single oral dose of 1g. Waters Atlantis C18 3 microm served as the stationary phase. The gradient was formed using ammonium acetate and methanol, containing 2% propan-2-ol. Detection is linear between 5 ng/mL and 500 ng/mL in plasma (5-1000 ng/mL in urine). The coefficient of variation for intra- and inter-day variation is <10%. The average recovery of resveratrol from plasma and urine is 58+/-3%. The data presented in this report demonstrate a rapid, sensitive and accurate method for the analysis of resveratrol and its metabolites in human plasma and urine for pharmacokinetic studies.

PMID: 17097357
1: Pharm Res. 2006 Sep;23(9):2107-15. Epub 2006 Aug 9. Links
Increased transport of resveratrol across monolayers of the human intestinal Caco-2 cells is mediated by inhibition and saturation of metabolites.Maier-Salamon A, Hagenauer B, Wirth M, Gabor F, Szekeres T, Jäger W.
Department of Clinical Pharmacy and Diagnostics, University of Vienna, A-1090, Vienna, Austria.

PURPOSE: The study's aim was to investigate the dose-dependent effect of sulfation and glucuronidation on intestinal absorption of resveratrol, a dietary constituent found in grapes and various medical plants. MATERIALS AND METHODS: The intestinal epithelial membrane transport kinetics and metabolism of resveratrol (10-200 microM) was studied using Caco-2 monolayers cultured in Transwells. RESULTS: Along with resveratrol it was possible to identify three metabolites, namely, resveratrol-4'-O-glucuronide (M1), resveratrol 3-O-gucuronide (M2), and resveratrol-3-O-sulfate (M3) by LC/MS and NMR. Efflux of the glucuronides M1 and M2 followed Michaelis-Menten kinetics significantly favouring basolateral efflux. The predominant metabolite was the monosulfate M3, however, its formation was strongly inhibited at higher resveratrol concentrations. As biotransformation was either inhibited or saturated, total amount of resveratrol transported across the Caco-2 monolayers increased as much as 3.5-fold at 200 microM resveratrol. This value might be even higher when taking into account the high intracellular concentration of resveratrol, which accounted for up to 61% of the applied dose. CONCLUSIONS: Our data demonstrate a concentration-dependent biotransformation of resveratrol in Caco-2 cells, which may also apply to human enterocytes affecting oral bioavailability.

PMID: 16952002

The above study indicates that sulfonation of resveratrol is inhibited by hight doses of resveratrol; I found dramatically increased effects at doses above 500 mg. I've been experimenting with luteolin for its sulfonation inhibition to achieve the same end. As luteolin is cheaper than resveratrol this would have some advantage. However, luteolin is also active, as a sirtuin activator and anti-inflammatory via nfKappa-B inhibition.

[ilanso]

zoolander: Anyhow.....there is nothing stopping hedgehog from conducting his own experiment however the results of this experiment should not be taken very seriously because who knows what went on. None of us know hedgehog from a bar of soap but it appears that some of us are ready to seriously consider the results of his little experiment as valid

This might be the case if we were looking for absolute results. What I am looking to take away from hedgehog's n=1 study is the relative shape of the Rsv-alone and Rsv+various curves.
It's like I trust my dachshund's weight a lot more when I subtract just mine from both mine and her combined (regardless of the scale). Scales and people are more consistent at their delta performance. Whence the increased confidence when using identical twins in controlled studies.

[Hedgehog]

Zoo, there are lots of pharmacokinetic studies published with n=1. The standard deviations between subjects are significant, but they are not enormous. N=1 is WAY better than n=0.

Hang on a second niner. Re-read what you just wrote. You can't tell whether the SD is significant or not because there is only one subject. Unless you test for that significance between people you cannot state whether it is significant or not. How can you relate this one subject to the rest of the population? Some people simply do not respond to treatments.

We don't know of hedgehogs medical records. Even if we (and perhaps he) do not know if there are any unknown metabolic/pathological conditions. Hedgehog may be able to tell us all that he has a clean bill of health but I would have done the same a few months back. I wouldn't now because a fluke test diagnosed me with severe sleep apnea which means my immune system is most likely compromised, iron metabolism is shot and so on.

Just for the record when you use a capital N you are refering to the number of studies and not subjects. A lower case 'n' of course refers to the number of subjects in a study (N).

Anyhow.....there is nothing stopping hedgehog from conducting his own experiment however the results of this experiment should not be taken very seriously because who knows what went on. None of us know hedgehog from a bar of soap but it appears that some of us are ready to seriously consider the results of his little experiment as valid

Well with the study i'm just trying to see if there is a difference compared to other published studies. If there is then I will make my sample size larger and maybe conduct different experiments to determine the best way to administer T-res. The T-res methods have already been published and validated so all I have to do is try different things and see which works best for my bioavailability.

I will give you all my data and notes of what went on if you want. I have no reason to lie or give false information. I just thought it would be fun and interesting and posted on this forum to get everybodys idea of what I should do and/or how to do it.

[Anthony_Loera]

Sounds interesting.

Why not? PM me and I could send you a few capsules of X1000 you can play with, they are 1000mg 99% t-res.

A

Zoo, there are lots of pharmacokinetic studies published with n=1. The standard deviations between subjects are significant, but they are not enormous. N=1 is WAY better than n=0.

Hang on a second niner. Re-read what you just wrote. You can't tell whether the SD is significant or not because there is only one subject. Unless you test for that significance between people you cannot state whether it is significant or not. How can you relate this one subject to the rest of the population? Some people simply do not respond to treatments.

We don't know of hedgehogs medical records. Even if we (and perhaps he) do not know if there are any unknown metabolic/pathological conditions. Hedgehog may be able to tell us all that he has a clean bill of health but I would have done the same a few months back. I wouldn't now because a fluke test diagnosed me with severe sleep apnea which means my immune system is most likely compromised, iron metabolism is shot and so on.

Just for the record when you use a capital N you are refering to the number of studies and not subjects. A lower case 'n' of course refers to the number of subjects in a study (N).

Anyhow.....there is nothing stopping hedgehog from conducting his own experiment however the results of this experiment should not be taken very seriously because who knows what went on. None of us know hedgehog from a bar of soap but it appears that some of us are ready to seriously consider the results of his little experiment as valid

Well with the study i'm just trying to see if there is a difference compared to other published studies. If there is then I will make my sample size larger and maybe conduct different experiments to determine the best way to administer T-res. The T-res methods have already been published and validated so all I have to do is try different things and see which works best for my bioavailability.

I will give you all my data and notes of what went on if you want. I have no reason to lie or give false information. I just thought it would be fun and interesting and posted on this forum to get everybodys idea of what I should do and/or how to do it.

[tintinet]

Why not?

So, thus far, what do you plan to test?

Methods proposed include:

1. empty stomach

2. with lipids (e.g., olive oil or cod liver oil, etc.)

3. with milk or whey

4. with quercetin

5. with luteolin

6. with ETOH +/- sublingual/transmucosal

7. with DMSO transdermal.

Any others?

Zoo, there are lots of pharmacokinetic studies published with n=1. The standard deviations between subjects are significant, but they are not enormous. N=1 is WAY better than n=0.

Hang on a second niner. Re-read what you just wrote. You can't tell whether the SD is significant or not because there is only one subject. Unless you test for that significance between people you cannot state whether it is significant or not. How can you relate this one subject to the rest of the population? Some people simply do not respond to treatments.

We don't know of hedgehogs medical records. Even if we (and perhaps he) do not know if there are any unknown metabolic/pathological conditions. Hedgehog may be able to tell us all that he has a clean bill of health but I would have done the same a few months back. I wouldn't now because a fluke test diagnosed me with severe sleep apnea which means my immune system is most likely compromised, iron metabolism is shot and so on.

Just for the record when you use a capital N you are refering to the number of studies and not subjects. A lower case 'n' of course refers to the number of subjects in a study (N).

Anyhow.....there is nothing stopping hedgehog from conducting his own experiment however the results of this experiment should not be taken very seriously because who knows what went on. None of us know hedgehog from a bar of soap but it appears that some of us are ready to seriously consider the results of his little experiment as valid

Well with the study i'm just trying to see if there is a difference compared to other published studies. If there is then I will make my sample size larger and maybe conduct different experiments to determine the best way to administer T-res. The T-res methods have already been published and validated so all I have to do is try different things and see which works best for my bioavailability.

I will give you all my data and notes of what went on if you want. I have no reason to lie or give false information. I just thought it would be fun and interesting and posted on this forum to get everybodys idea of what I should do and/or how to do it.

[meat250]

interesting post, please keep us posted as to the results!!!!

Meat

[Hedgehog]

I decided to actually test blood plasma and urine samples. This will give us more info about the breakdown of Resveratrol. I have also added Luteolin to the protocol. I will have to do a couple of ID tests to determine if the same HPLC method will work. Because luteolin has a similar structure I hope the HPLC method will pick it up.

I have ordered a luteolin standard, this will server for ID and quantization purposes. Apigenin will be used in the experiment and hopefully we will see how much luteolin will be formed in the plasma.

If the HPLC method picks up luteolin then we can see both luteolin and Resveratrol in the blood stream and see how fast it degrads w/ piperine + another type inhibitor.

I also found in the literature that BOTH forms of resveratrol CIS and TRANS inhibit EGFR activation via PKC inhibition (I think i got that right I would have to go dig up the article correct me if i'm wrong). I guess if you take a mixture of both the CIS and TRANS form can still inhibit some natural functions in the body. However, it seems like people still take megadoses and nothing seems to happen? Probably because it is degraded so fast in the blood and doesn't ever achieve the concentration needed inhibit PKC.