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Self Resveratrol Bioavailability Study


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#31 Hedgehog

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Posted 16 January 2008 - 08:11 PM

I finally got all my supplies! I'm going to dose at 5mg/kg. If it looks good I will add more people and spend more money on raw materials.

I'm simply going to follow the protocol that has been published and validated and is the same one that sirtris used.

As for me no eating any foods that could contain resveratrol!

Regards,

#32 tintinet

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Posted 16 January 2008 - 08:55 PM

I finally got all my supplies! I'm going to dose at 5mg/kg. If it looks good I will add more people and spend more money on raw materials.

I'm simply going to follow the protocol that has been published and validated and is the same one that sirtris used.

As for me no eating any foods that could contain resveratrol!

Regards,


Thanks. I await your results with anticip......ation! :)

I find it difficult to believe a few grapes, glasses of red wine and peanuts could significantly affect your results, though.

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#33 Hedgehog

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Posted 16 January 2008 - 09:13 PM

I agree but I have to follow the validated protocol. Plus the blank of just my blood plasma should varify that I don't have any resveratrol in my system.

So, I'm going to take the formulated resveratrol and I have another person taking just per trans-resveratrol. The following weekend I will flip it around and take just pure resveratrol with no formulation. If it works then I will add more people.

#34 malbecman

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Posted 17 January 2008 - 05:13 PM

I can't wait to see the results. I was considering doing the same type of study on myself but never got farther than that. Kudos to you.....

#35 lucid

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Posted 17 January 2008 - 05:29 PM

That seems like a low'ish dose (5mg/kg) for me that would only be .5g (104 kg x .005). The SRT501 studies used 5grams of micro-encapsulated small particle size resveratrol. So you might want to consider a higher dose. Look forward to seeing your results!

I would be interested in seeing results from the EtOH + a surfactant method (Peg3350 Tween or Lecithin). I would also be interested in trying out some mixing some emulsifiers in there. I am not very familiar with more high powered emulsifiers than lecithin but it would be interesting to test those out. Particularly I would love to see Max Watt's idea tested about using luteolin to prevent sulfonation of resveratrol. Sweet work hedgehog.

Edited by lucid, 17 January 2008 - 05:52 PM.


#36 Hedgehog

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Posted 17 January 2008 - 06:16 PM

That seems like a low'ish dose (5mg/kg) for me that would only be .5g (104 kg x .005). The SRT501 studies used 5grams of micro-encapsulated small particle size resveratrol. So you might want to consider a higher dose. Look forward to seeing your results!

I would be interested in seeing results from the EtOH + a surfactant method (Peg3350 Tween or Lecithin). I would also be interested in trying out some mixing some emulsifiers in there. I am not very familiar with more high powered emulsifiers than lecithin but it would be interesting to test those out. Particularly I would love to see Max Watt's idea tested about using luteolin to prevent sulfonation of resveratrol. Sweet work hedgehog.



SRT501, is a forumulation meaning it could only be 300mg of resveratrol and 2grams of carrier stuff. Unless you know that the SRT501 is just pure resveratrol.

I will use a higher dose if I don't get a good detection limit on my intrusment.

I bought 10mg of luteolin but it hasn't shown up. I'm afraid if I use to many compounds that absorb around 300nm my chromatograph would be very messy and the results would not be very good or even valid.

I might try lecithin if my current method doesn't work.

#37 maxwatt

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Posted 17 January 2008 - 06:18 PM

That seems like a low'ish dose (5mg/kg) for me that would only be .5g (104 kg x .005). The SRT501 studies used 5grams of micro-encapsulated small particle size resveratrol. So you might want to consider a higher dose. Look forward to seeing your results!

I would be interested in seeing results from the EtOH + a surfactant method (Peg3350 Tween or Lecithin). I would also be interested in trying out some mixing some emulsifiers in there. I am not very familiar with more high powered emulsifiers than lecithin but it would be interesting to test those out. Particularly I would love to see Max Watt's idea tested about using luteolin to prevent sulfonation of resveratrol. Sweet work hedgehog.


Caveat there. Luteolin differs from quercetin by one additional OH group, and I believe it to inhibits Sirt1. blocking resveratrol's activation. So though you might get higher serum levels with it, you are defeating the purpose by inactivating the pathway you want to activate.

#38 Hedgehog

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Posted 17 January 2008 - 06:27 PM

That seems like a low'ish dose (5mg/kg) for me that would only be .5g (104 kg x .005). The SRT501 studies used 5grams of micro-encapsulated small particle size resveratrol. So you might want to consider a higher dose. Look forward to seeing your results!

I would be interested in seeing results from the EtOH + a surfactant method (Peg3350 Tween or Lecithin). I would also be interested in trying out some mixing some emulsifiers in there. I am not very familiar with more high powered emulsifiers than lecithin but it would be interesting to test those out. Particularly I would love to see Max Watt's idea tested about using luteolin to prevent sulfonation of resveratrol. Sweet work hedgehog.


Caveat there. Luteolin differs from quercetin by one additional OH group, and I believe it to inhibits Sirt1. blocking resveratrol's activation. So though you might get higher serum levels with it, you are defeating the purpose by inactivating the pathway you want to activate.



Hi maxxwatt,

Do you think the quercetin or luteolin themselves inhibit SIRT1 or their metabolites? Or do think some sort of competitive binding to SIRT1? I guess you could do a test on whatever you think is and look at gene activation. Not sure how expensive this would be.. Maybe a student could do this.

#39 stephen_b

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Posted 17 January 2008 - 07:35 PM

Thanks for the legwork in putting together all of the responses. My back pain has gone away after going from 2 grams to 500 mg and now back to 1 g/day.

Stephen

#40 maxwatt

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Posted 17 January 2008 - 09:40 PM

That seems like a low'ish dose (5mg/kg) for me that would only be .5g (104 kg x .005). The SRT501 studies used 5grams of micro-encapsulated small particle size resveratrol. So you might want to consider a higher dose. Look forward to seeing your results!

I would be interested in seeing results from the EtOH + a surfactant method (Peg3350 Tween or Lecithin). I would also be interested in trying out some mixing some emulsifiers in there. I am not very familiar with more high powered emulsifiers than lecithin but it would be interesting to test those out. Particularly I would love to see Max Watt's idea tested about using luteolin to prevent sulfonation of resveratrol. Sweet work hedgehog.


Caveat there. Luteolin differs from quercetin by one additional OH group, and I believe it to inhibits Sirt1. blocking resveratrol's activation. So though you might get higher serum levels with it, you are defeating the purpose by inactivating the pathway you want to activate.



Hi maxxwatt,

Do you think the quercetin or luteolin themselves inhibit SIRT1 or their metabolites? Or do think some sort of competitive binding to SIRT1? I guess you could do a test on whatever you think is and look at gene activation. Not sure how expensive this would be.. Maybe a student could do this.


There are a couple of papers that have been referenced here over the past year stating that quercetin's major metabolite, quercetin 3-O-glucuronid, inhibits Sirt1. Quercetin does not extend the lifespan of C. elegans. Luteolin is so similar in structure I am sure its metabolite shares this characteristic.

ADDED REFERENCE

Edited by maxwatt, 17 January 2008 - 09:46 PM.


#41 krillin

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Posted 17 January 2008 - 09:46 PM

There are a couple of papers that have been referenced here over the past year stating that quercetin's major metabolite, inhibits Sirt1. Quercetin does not extend the lifespan of C. elegans. Luteolin is so similar in structure I am sure its metabolite shares this characteristic.


Quercetin and luteolin also induce glucuronidation enzymes after 3-4 days.

#42 Hedgehog

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Posted 17 January 2008 - 11:38 PM

There are a couple of papers that have been referenced here over the past year stating that quercetin's major metabolite, quercetin 3-O-glucuronid, inhibits Sirt1. Quercetin does not extend the lifespan of C. elegans. Luteolin is so similar in structure I am sure its metabolite shares this characteristic.

ADDED REFERENCE


Very true but if it potentially 'slightly' inhibits sirt1 but potently inhibits SULT1A1 (a sulfotransferase), i think you could use a very low dose of quercetin to have a synergestic effect with large doses of resveratrol?

There is also this report which conflicts the other C elegans study.

Comp Biochem Physiol B Biochem Mol Biol. 2008 Feb;149(2):314-23. Epub 2007 Oct 16.Posted Image <script language="JavaScript1.2">Links
Increase of stress resistance and lifespan of Caenorhabditis elegans by quercetin.
Kampkötter A, Timpel C, Zurawski RF, Ruhl S, Chovolou Y, Proksch P, Wätjen W.Institute of Toxicology, Heinrich-Heine University, Düsseldorf, Germany.

The health beneficial effects of a diet rich in fruits and vegetables are, at least in part, attributed to polyphenols that are present in many herbal edibles. Although many in vitro studies revealed a striking variety of biochemical and pharmacological properties data about the beneficial effects of polyphenols in whole organisms, especially with respect to ageing, are quite limited. We used the well established model organism Caenorhabditis elegans to elucidate the protective effects of quercetin, the main representative of the flavonol class of polyphenols, in vivo. Quercetin is taken up by the worms, enhanced the resistance to oxidative stress and prolonged the mean lifespan of C. elegans by 15%. Quercetin was shown to be a strong radical scavenger possibly explaining the observed down-regulation of mitochondrial manganese superoxide dismutase by a reduced need for this antioxidant enzyme for maintenance of cellular redox homeostasis. Quercetin treatment also led to a translocation of the C. elegans FoxO transcription factor DAF-16 into the nucleus, a state often correlated with stress response and longevity. According to our results we suggest that the protective and life prolonging action of quercetin is not only due to its strong antioxidant capacity but may also be mediated by modulation of signalling pathways.

PMID: 18024103 [PubMed - in



#43 maxwatt

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Posted 18 January 2008 - 02:54 AM

There are a couple of papers that have been referenced here over the past year stating that quercetin's major metabolite, quercetin 3-O-glucuronid, inhibits Sirt1. Quercetin does not extend the lifespan of C. elegans. Luteolin is so similar in structure I am sure its metabolite shares this characteristic.

ADDED REFERENCE


Very true but if it potentially 'slightly' inhibits sirt1 but potently inhibits SULT1A1 (a sulfotransferase), i think you could use a very low dose of quercetin to have a synergestic effect with large doses of resveratrol?

There is also this report which conflicts the other C elegans study.

Comp Biochem Physiol B Biochem Mol Biol. 2008 Feb;149(2):314-23. Epub 2007 Oct 16.Posted Image <script language="JavaScript1.2">Links
Increase of stress resistance and lifespan of Caenorhabditis elegans by quercetin.
Kampkötter A, Timpel C, Zurawski RF, Ruhl S, Chovolou Y, Proksch P, Wätjen W.Institute of Toxicology, Heinrich-Heine University, Düsseldorf, Germany.

The health beneficial effects of a diet rich in fruits and vegetables are, at least in part, attributed to polyphenols that are present in many herbal edibles. Although many in vitro studies revealed a striking variety of biochemical and pharmacological properties data about the beneficial effects of polyphenols in whole organisms, especially with respect to ageing, are quite limited. We used the well established model organism Caenorhabditis elegans to elucidate the protective effects of quercetin, the main representative of the flavonol class of polyphenols, in vivo. Quercetin is taken up by the worms, enhanced the resistance to oxidative stress and prolonged the mean lifespan of C. elegans by 15%. Quercetin was shown to be a strong radical scavenger possibly explaining the observed down-regulation of mitochondrial manganese superoxide dismutase by a reduced need for this antioxidant enzyme for maintenance of cellular redox homeostasis. Quercetin treatment also led to a translocation of the C. elegans FoxO transcription factor DAF-16 into the nucleus, a state often correlated with stress response and longevity. According to our results we suggest that the protective and life prolonging action of quercetin is not only due to its strong antioxidant capacity but may also be mediated by modulation of signalling pathways.

PMID: 18024103 [PubMed - in

It prolonged mean lifespan, not maximum lifespan.
Taking enough resveratrol will swamp the gucoridation and sulfonation enzymes. Based on references others have posted here, I think this occurs at about 500 to 800 mg. It would be interesting to do a dose-response graph, because you should then see a sharp increase in slope at this point.

Anyway, I prefer not to inhibt my Sirt1 even moderately. My experience with osteoarthritis (mostly in right big toe) indicates that the nfKappaB inhibition induced by Sirt1 activation, ceases after about 3 days of adding significant amounts of either quercetin or luteolin to my resveratrol supplementation.

If you google the imminst fora for "maxwatt big toe" you should find pictures of my big toe before and after taking high doses of resveratrol. The swelling is very noticeably less. The are pictures here IMAGES

Most of the pain is gone from the joint, all of the pain is gone from my other toes. After 3 days, quercetin and luteolin pain increases and comes comes back to my other toes, on both feet.

#44 Hedgehog

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Posted 18 January 2008 - 03:30 AM

Anyway, I prefer not to inhibt my Sirt1 even moderately. My experience with osteoarthritis (mostly in right big toe) indicates that the nfKappaB inhibition induced by Sirt1 activation, ceases after about 3 days of adding significant amounts of either quercetin or luteolin to my resveratrol supplementation.

If you google the imminst fora for "maxwatt big toe" you should find pictures of my big toe before and after taking high doses of resveratrol. The swelling is very noticeably less. The are pictures here IMAGES

Most of the pain is gone from the joint, all of the pain is gone from my other toes. After 3 days, quercetin and luteolin pain increases and comes comes back to my other toes, on both feet.


Maybe try a lower dose of quercetin just enough to inhibit the enzymes which is in the low nM and don't take it to activate or deactivate SIRT1 or enough for any sort of competitive competition for SiRt1 and Resveratrol. I would almost say 1-10mg of quercetin to 300mg of trans-resveratrol.

In my first test I won't be able to test this theory. Partly because I don't know if quercetin and resveratrol elute at the same time with the HPLC method. However, I will be testing the elution time with a run of quercetin and resveratrol to see if my method will separate both compounds. Another thing, quercetin and resveratrol absorb around the same wavelength so if it is my plasma I should be able to see it. However, if I only take 10mg I doubt I will see it at all. Will save this experiment until next time.



Do you think only 1-10mg of quercetin would cause your toe to swell up again?

#45 niner

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Posted 18 January 2008 - 03:38 AM

Anyway, I prefer not to inhibt my Sirt1 even moderately. My experience with osteoarthritis (mostly in right big toe) indicates that the nfKappaB inhibition induced by Sirt1 activation, ceases after about 3 days of adding significant amounts of either quercetin or luteolin to my resveratrol supplementation.

If you google the imminst fora for "maxwatt big toe" you should find pictures of my big toe before and after taking high doses of resveratrol. The swelling is very noticeably less. The are pictures here IMAGES

Most of the pain is gone from the joint, all of the pain is gone from my other toes. After 3 days, quercetin and luteolin pain increases and comes comes back to my other toes, on both feet.


Maybe try a lower dose of quercetin just enough to inhibit the enzymes which is in the low nM and don't take it to activate or deactivate SIRT1 or enough for any sort of competitive competition for SiRt1 and Resveratrol. I would almost say 1-10mg of quercetin to 300mg of trans-resveratrol.

In my first test I won't be able to test this theory. Partly because I don't know if quercetin and resveratrol elute at the same time with the HPLC method. However, I will be testing the elution time with a run of quercetin and resveratrol to see if my method will separate both compounds. Another thing, quercetin and resveratrol absorb around the same wavelength so if it is my plasma I should be able to see it. However, if I only take 10mg I doubt I will see it at all. Will save this experiment until next time.

Do you think only 1-10mg of quercetin would cause your toe to swell up again?

1-10mg of quercetin is within the amount that you would get from a varied diet. Apples and onions are a couple sources. The problem with quercetin is that, like resveratrol, it gets conjugated all to hell. The ratio of free quercetin to quercetin conjugates is probably on par with that of resveratrol, if not significantly worse. This changes the calculation of how much you'll want to take in order to inhibit sulfotransferases, but it's hard to say what the number is.

#46 Hedgehog

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Posted 18 January 2008 - 03:49 AM

1-10mg of quercetin is within the amount that you would get from a varied diet. Apples and onions are a couple sources. The problem with quercetin is that, like resveratrol, it gets conjugated all to hell. The ratio of free quercetin to quercetin conjugates is probably on par with that of resveratrol, if not significantly worse. This changes the calculation of how much you'll want to take in order to inhibit sulfotransferases, but it's hard to say what the number is.


I'm guessing if/when I try this my spectrum is going to be super messy and won't even give good/valid results. However, I will use 10mg of piperine in this weekends experiment. It appears from the attached spectrum that there might be a 75-25 sulfate to glucose metabolites based on area?? However, this is a urine sample... In any case if piperine has any effect we might see a lower B and D peak compared to the control.

Attached Files

  • Attached File  HPLC.JPG   32.64KB   42 downloads

Edited by hedgehog_info, 18 January 2008 - 03:56 AM.


#47 maxwatt

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Posted 18 January 2008 - 04:05 AM

Anyway, I prefer not to inhibt my Sirt1 even moderately. My experience with osteoarthritis (mostly in right big toe) indicates that the nfKappaB inhibition induced by Sirt1 activation, ceases after about 3 days of adding significant amounts of either quercetin or luteolin to my resveratrol supplementation.

If you google the imminst fora for "maxwatt big toe" you should find pictures of my big toe before and after taking high doses of resveratrol. The swelling is very noticeably less. The are pictures here IMAGES

Most of the pain is gone from the joint, all of the pain is gone from my other toes. After 3 days, quercetin and luteolin pain increases and comes comes back to my other toes, on both feet.


Maybe try a lower dose of quercetin just enough to inhibit the enzymes which is in the low nM and don't take it to activate or deactivate SIRT1 or enough for any sort of competitive competition for SiRt1 and Resveratrol. I would almost say 1-10mg of quercetin to 300mg of trans-resveratrol.

In my first test I won't be able to test this theory. Partly because I don't know if quercetin and resveratrol elute at the same time with the HPLC method. However, I will be testing the elution time with a run of quercetin and resveratrol to see if my method will separate both compounds. Another thing, quercetin and resveratrol absorb around the same wavelength so if it is my plasma I should be able to see it. However, if I only take 10mg I doubt I will see it at all. Will save this experiment until next time.



Do you think only 1-10mg of quercetin would cause your toe to swell up again?

No, there's that much in OJ or grapefruit juice. 250 mg definitely did. And 100 mg of luteolin/day.

#48 Hedgehog

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Posted 18 January 2008 - 10:33 PM

Two subjects will be tested. one with a formulation and the other with pure resveratrol.

Dosing:
6mg/kg (a total of about 400mg)
Time Points: 0, 7, 15, 25, 35, 50, 1hr 20min, 1hr 50min, 3hrs, 4hrs, 5hrs 20min
No. Standards is 7 ranging from: 1600nM - 16nM
No. of injections is 32 at 30mins per injection leaves about 16hrs for everything to be completed

Start drawing blood tomorrow around 8-9am (Stanford Hospital), then do A LOT of prep work to get the samples ready, so 6hrs of testing + 3-4hrs of lab work, then another 16hrs of HPLC run time. Then I have to process the data!

Probably won't have data until Sunday night.

Next weekend I will flip the subjects around and one will get the formulation and the other will get pure resveratrol. Maybe one of us has a faster metabolism. If it looks good then I will order more raw materials and test more subjects.

Any suggestions?

#49 ilanso

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Posted 19 January 2008 - 09:43 AM

Next weekend I will flip the subjects around and one will get the formulation and the other will get pure resveratrol. Maybe one of us has a faster metabolism. If it looks good then I will order more raw materials and test more subjects.

Any suggestions?


Other than good luck and have a relaxing weekend (oops, I wish they were mutually compatible)?
Oh yeah, it would help if neither subject touched any recreational RSV for a week prior to each experiment.
And loads of thanks from all of us!

#50 Hedgehog

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Posted 20 January 2008 - 08:19 AM

Well good news and bad news.



Good news is that I can easily detect Trans-Resveratrol at 16 nano-molar concentrations.

The nurse would only allow one of us the draw blood because they said one of us had to drive home. After being famished and taking about 30mL of blood I got really light headed I honestly couldn't take any more. Light headed and dizzy. All I wanted to do is sleep, I didn't feel sick just like I wanted to sleep. I have a history of getting light headed when not eating very much… So I had to stop at 2hrs 16min.



After I ate something I started to feel ok. However, it still took about 3hrs to feel normal again.



Also two of my time points one of them being critical didn't centrifuge well and wasn't able to get a big enough aliquot to analyze. I still attempted to get some results.



I used a C-18 solid phase cartridge as published in the literature to separate hydrophobic compounds in the blood from polar compounds in the blood. This appeared to work well, however, I ran a little experiment within the plasma test. I wanted to make sure the C-18 filter was working correctly. So I ran a normal standard on the HPLC with a known 320nM Trans-resveratrol and then used the C-18 filter version of the same solution. A good filter should give >90% recovery. Will the lowest was 27% fuc$ing percent recovery and varied among other standards. In other words, my sample probably got eluted at a different time during the extraction process. This could also be due to column over loading of sample or to fast of an elution flow rate. Sometimes protocols are like an art form. Obviously I haven't mastered this one. So next time I'm going to try to do a liquid-liquid extraction and hopefully precipitate the blood proteins and obtain my resveratrol that way w/o having to-do a solid phase extraction.



So basically this was a great LONG learning experiment and know what I will do next time.



Sorry to disappoint, but to be honest I was happy to even see resveratrol in my blood plasma and ID it. Pretty freaking cool if you ask me. If my results are valid I had a [plasma] of 60nM at 2hrs 16min. Anyways…..



Things to do next time. Different extraction method (liquid-liquid), use a different HPLC column, find a different type of centrifuge container.



I will try again in the near future.



Cheers,


#51 tintinet

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Posted 20 January 2008 - 03:20 PM

Well good news and bad news.



Good news is that I can easily detect Trans-Resveratrol at 16 nano-molar concentrations.

The nurse would only allow one of us the draw blood because they said one of us had to drive home. After being famished and taking about 30mL of blood I got really light headed I honestly couldn't take any more. Light headed and dizzy. All I wanted to do is sleep, I didn't feel sick just like I wanted to sleep. I have a history of getting light headed when not eating very much… So I had to stop at 2hrs 16min.



After I ate something I started to feel ok. However, it still took about 3hrs to feel normal again.



Also two of my time points one of them being critical didn't centrifuge well and wasn't able to get a big enough aliquot to analyze. I still attempted to get some results.



I used a C-18 solid phase cartridge as published in the literature to separate hydrophobic compounds in the blood from polar compounds in the blood. This appeared to work well, however, I ran a little experiment within the plasma test. I wanted to make sure the C-18 filter was working correctly. So I ran a normal standard on the HPLC with a known 320nM Trans-resveratrol and then used the C-18 filter version of the same solution. A good filter should give >90% recovery. Will the lowest was 27% fuc$ing percent recovery and varied among other standards. In other words, my sample probably got eluted at a different time during the extraction process. This could also be due to column over loading of sample or to fast of an elution flow rate. Sometimes protocols are like an art form. Obviously I haven't mastered this one. So next time I'm going to try to do a liquid-liquid extraction and hopefully precipitate the blood proteins and obtain my resveratrol that way w/o having to-do a solid phase extraction.



So basically this was a great LONG learning experiment and know what I will do next time.



Sorry to disappoint, but to be honest I was happy to even see resveratrol in my blood plasma and ID it. Pretty freaking cool if you ask me. If my results are valid I had a [plasma] of 60nM at 2hrs 16min. Anyways…..



Things to do next time. Different extraction method (liquid-liquid), use a different HPLC column, find a different type of centrifuge container.



I will try again in the near future.



Cheers,


Nice effort! Thanks!

#52 Hedgehog

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Posted 20 January 2008 - 04:36 PM

Nice effort! Thanks!


No Problem. Sometimes it is very hard to reproduce results in a paper or even a method for that much. I think I know what went wrong and think I can fix it. At least I know that I can detect resveratrol at a very low level and was able to see a resveratrol peaks in all of my samples. This tells us that at least the method sorta worked. I'm also not a C-18 solid phase extraction expert. I understand the theory but don't have that much working knowledge of it.

Hopefully this other method will take less time to prepare the samples.

So I haven't given up! :-D

Edited by hedgehog_info, 20 January 2008 - 07:14 PM.


#53 inawe

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Posted 20 January 2008 - 08:35 PM

Hedgehog,
Sorry for my ignorance. But, why do you have to fast during the experiment? You might eat something that will add stuff other than RSV to your blood. Or it might contain minute quantities of RSV which wont much change the outcome. Just don't eat peanuts or blueberries, drink wine or chew on polygonum cuspidatum.

#54 Hedgehog

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Posted 20 January 2008 - 10:31 PM

Hedgehog,
Sorry for my ignorance. But, why do you have to fast during the experiment? You might eat something that will add stuff other than RSV to your blood. Or it might contain minute quantities of RSV which wont much change the outcome. Just don't eat peanuts or blueberries, drink wine or chew on polygonum cuspidatum.

Its a good question.

Well I'm trying to get reproducible results. Food adds another variable to the experiment. True some foods might aid in the bioavailability. But if you just eat at random times and take resveratrol and do an experiment with it your resveratrol concentrations is going to vary all over the place. Which means your results are not reproducible. If you can control absorption to a certain degree by not eating you can add one or more known things to a formulation and see if it works.

Also, another thing to think about besides that is if you have many compounds in your blood and analyze it you are going to have a messy chromatograph. Which means that potential your results will not be valid because you have interfering peaks.

Hope this helps.

#55 sUper GeNius

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Posted 21 January 2008 - 12:02 AM

Hedgehog,
Sorry for my ignorance. But, why do you have to fast during the experiment? You might eat something that will add stuff other than RSV to your blood. Or it might contain minute quantities of RSV which wont much change the outcome. Just don't eat peanuts or blueberries, drink wine or chew on polygonum cuspidatum.

Its a good question.

Well I'm trying to get reproducible results. Food adds another variable to the experiment. True some foods might aid in the bioavailability. But if you just eat at random times and take resveratrol and do an experiment with it your resveratrol concentrations is going to vary all over the place. Which means your results are not reproducible. If you can control absorption to a certain degree by not eating you can add one or more known things to a formulation and see if it works.

Also, another thing to think about besides that is if you have many compounds in your blood and analyze it you are going to have a messy chromatograph. Which means that potential your results will not be valid because you have interfering peaks.

Hope this helps.


HH.
What are the ramifications, if any, of your 60nM blood level.

#56 Hedgehog

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Posted 21 January 2008 - 01:04 AM

Hedgehog,
Sorry for my ignorance. But, why do you have to fast during the experiment? You might eat something that will add stuff other than RSV to your blood. Or it might contain minute quantities of RSV which wont much change the outcome. Just don't eat peanuts or blueberries, drink wine or chew on polygonum cuspidatum.

Its a good question.

Well I'm trying to get reproducible results. Food adds another variable to the experiment. True some foods might aid in the bioavailability. But if you just eat at random times and take resveratrol and do an experiment with it your resveratrol concentrations is going to vary all over the place. Which means your results are not reproducible. If you can control absorption to a certain degree by not eating you can add one or more known things to a formulation and see if it works.

Also, another thing to think about besides that is if you have many compounds in your blood and analyze it you are going to have a messy chromatograph. Which means that potential your results will not be valid because you have interfering peaks.

Hope this helps.



HH.
What are the ramifications, if any, of your 60nM blood level.


Well I think in the low nM level you achieve neuronal protection. However, I expect my results are not really quantitative rather I can only say I saw resveratrol in my blood.

#57 malbecman

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Posted 22 January 2008 - 05:19 PM

Nice effort, Hedgehog_info, thanks!

I did a fair amount of metabolism studies in grad school and I always did liquid extractions of the serum and microsomal incubations I did. Basically, if you add >50% organic, you will usually precipitate all/most proteins and then you just need to spin the sample down, filter it and inject on the HPLC (sometimes I wouldnt even filter it if I was in a hurry but make sure you use a guard column if you go that route). Adding the organic does dilute your sample (whereas the SPE columns can concentrate samples for you) but you can always speed-vac them a little if need be......

Good luck with the next round......



Nice effort! Thanks!


No Problem. Sometimes it is very hard to reproduce results in a paper or even a method for that much. I think I know what went wrong and think I can fix it. At least I know that I can detect resveratrol at a very low level and was able to see a resveratrol peaks in all of my samples. This tells us that at least the method sorta worked. I'm also not a C-18 solid phase extraction expert. I understand the theory but don't have that much working knowledge of it.

Hopefully this other method will take less time to prepare the samples.

So I haven't given up! :-D



#58 Hedgehog

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Posted 22 January 2008 - 05:33 PM

Nice effort, Hedgehog_info, thanks!

I did a fair amount of metabolism studies in grad school and I always did liquid extractions of the serum and microsomal incubations I did. Basically, if you add >50% organic, you will usually precipitate all/most proteins and then you just need to spin the sample down, filter it and inject on the HPLC (sometimes I wouldnt even filter it if I was in a hurry but make sure you use a guard column if you go that route). Adding the organic does dilute your sample (whereas the SPE columns can concentrate samples for you) but you can always speed-vac them a little if need be......

Good luck with the next round......


Thanks, the other method I'm going to try uses 50:50 MeOH and acidified Plasma. Spin it a couple of times and chill it. The final volume is about 0.5mL, I have a 0.2um PVDF filter but 0.5mL would pratically be the void volume. Any suggestions...

At the moment I don't have a guard column but have looked into getting one.

The last method I used a roto-vap to concentrate my samples...

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#59 malbecman

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Posted 22 January 2008 - 06:27 PM

Well, if you spin the sample down real hard, you can usually get away with just doing a straight injection (as long as the supernatent looks clear). You will be putting some minor material on your column which will eventually degrade its performance but that can always be cleaned off with TFE (or just buy a new column). 0.2uM is pretty small pore size, you could get away with 0.45uM ,IMHO, and it would filter more easily.......


Nice effort, Hedgehog_info, thanks!

I did a fair amount of metabolism studies in grad school and I always did liquid extractions of the serum and microsomal incubations I did. Basically, if you add >50% organic, you will usually precipitate all/most proteins and then you just need to spin the sample down, filter it and inject on the HPLC (sometimes I wouldnt even filter it if I was in a hurry but make sure you use a guard column if you go that route). Adding the organic does dilute your sample (whereas the SPE columns can concentrate samples for you) but you can always speed-vac them a little if need be......

Good luck with the next round......


Thanks, the other method I'm going to try uses 50:50 MeOH and acidified Plasma. Spin it a couple of times and chill it. The final volume is about 0.5mL, I have a 0.2um PVDF filter but 0.5mL would pratically be the void volume. Any suggestions...

At the moment I don't have a guard column but have looked into getting one.

The last method I used a roto-vap to concentrate my samples...



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