People With Lab Experience Or Chem/biochem Majors, Please Help Me Figure This Out

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Pfff, I wish; then I wouldn't have to play the begging game like I am. I'm an undergrad 4+1 masters student, doing something called an REU; it's basically paid-for undergraduate research and since I'm the only person in the molecular sciences utilizing it, I get to actually choose a project.

I think if I do a thesis, it will be on something similar to Gilbert Ling's work in cell physiology. Speaking of, I had asked raypeatclips in PM, but does anybody know how Ling is doing? His website has been offline for a while. I know it happens, time to time, but it's been months. I thought to ask Dr. Peat what he knows, but again I hate the idea of bothering him with stuff.

Update about my prof: He finally understood about the ketoacid vs ketogenic acid thing, and actually linked me to a study that I had open already on ways to do it, haha: Estimation of α-keto acids in plant tissue: a critical study of various methods of extraction as applied to strawberry leaves, washed potato slices and peas

I say go for it :ss if the prof cares too little, then that's a bonus :ss
 

Ella

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Well, luckily I got a response from Dr. Peat and he linked me to this study: http://www.sciencedirect.com/science/article/pii/0003269768902947 which I of course immediately sent to my professor to delineate the difference. He also told me the "mystery reagent," which was DNPH. I wasn't so surprised to see he couldn't get that, because it's explosive; but he also said he couldn't get the straight ketoacids for comparison (which seems odd).

So, just waiting on prof's response.

That article is old and uses thin layer chromatography. Do you have access to HPLC or LC/MS detection by UV and fluorescence. You need to check out the chemistry lab or pharmacy labs and see what instrumentation is available to you. It is important that your project includes skills that are useful for your future career. I think even as an undergrad talk to professors of departments that have more sophisticated instrumentation. I did this and the professor (was not my supervisor) was so impressed with my aptitude with the instrumentation that if offered me a research position before I even graduated. Try to include as many different techniques into your project. IF you are looking to quantify this is what I would be looking at or even LC/MS/MS if it is available to you. You could do enzyme based assay. Getting pure standards which are expensive is important. It depends on how much money is available for your project. Keto-acids in fruits, juices and wines have been quantified which is why your professor is a little annoyed.

"Just by feeding keto acids, if they are the equivalent of the essential amino acids, you create them in your body by absorbing the ammonia and then making your own protein, and that was where I got the idea that some foods would contain natural keto acid equivalents and we did chromatography on potato juice and found that they are rich in the keto acids which will function as amino acids, even though they contain no ammonia, because they can absorb ammonia from your blood"

I would focus on orange juice, simply because many people freak out drinking fruit juice without the fibre. You want to be able to show reduced ammonia in urine, blood, feces in say diabetics or kidney impaired. You probably will not be able to use humans at undergrad level but could continue into Phd thesis layering more techniques such as protein expression studies, gene expression. You need to pick a disease condition, like diabetes, obesity (obesity would be good as these people are scared of fruit and sugar) or prehaps the aging as they have most problems with losing lean protein.
Pfff, I wish; then I wouldn't have to play the begging game like I am. I'm an undergrad 4+1 masters student, doing something called an REU; it's basically paid-for undergraduate research and since I'm the only person in the molecular sciences utilizing it, I get to actually choose a project.

I think if I do a thesis, it will be on something similar to Gilbert Ling's work in cell physiology. Speaking of, I had asked raypeatclips in PM, but does anybody know how Ling is doing? His website has been offline for a while. I know it happens, time to time, but it's been months. I thought to ask Dr. Peat what he knows, but again I hate the idea of bothering him with stuff.

Update about my prof: He finally understood about the ketoacid vs ketogenic acid thing, and actually linked me to a study that I had open already on ways to do it, haha: Estimation of α-keto acids in plant tissue: a critical study of various methods of extraction as applied to strawberry leaves, washed potato slices and peas
 

Ella

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That article is old and uses thin layer chromatography. Do you have access to HPLC or LC/MS detection by UV and fluorescence. You need to check out the chemistry lab or pharmacy labs and see what instrumentation is available to you. It is important that your project includes skills that are useful for your future career. I think even as an undergrad talk to professors of departments that have more sophisticated instrumentation. I did this and the professor (was not my supervisor) was so impressed with my aptitude with the instrumentation that if offered me a research position before I even graduated. Try to include as many different techniques into your project. IF you are looking to quantify this is what I would be looking at or even LC/MS/MS if it is available to you. You could do enzyme based assay. Getting pure standards which are expensive is important. It depends on how much money is available for your project. Keto-acids in fruits, juices and wines have been quantified which is why your professor is a little annoyed.

"Just by feeding keto acids, if they are the equivalent of the essential amino acids, you create them in your body by absorbing the ammonia and then making your own protein, and that was where I got the idea that some foods would contain natural keto acid equivalents and we did chromatography on potato juice and found that they are rich in the keto acids which will function as amino acids, even though they contain no ammonia, because they can absorb ammonia from your blood"

I would focus on orange juice, simply because many people freak out drinking fruit juice without the fibre. You want to be able to show reduced ammonia in urine, blood, feces in say diabetics or kidney impaired. You probably will not be able to use humans at undergrad level but could continue into Phd thesis layering more techniques such as protein expression studies, gene expression. You need to pick a disease condition, like diabetes, obesity (obesity would be good as these people are scared of fruit and sugar) or prehaps the aging as they have most problems with losing lean protein.
 
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explosionlord

explosionlord

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That article is old and uses thin layer chromatography. Do you have access to HPLC or LC/MS detection by UV and fluorescence. You need to check out the chemistry lab or pharmacy labs and see what instrumentation is available to you. It is important that your project includes skills that are useful for your future career. I think even as an undergrad talk to professors of departments that have more sophisticated instrumentation. I did this and the professor (was not my supervisor) was so impressed with my aptitude with the instrumentation that if offered me a research position before I even graduated. Try to include as many different techniques into your project. IF you are looking to quantify this is what I would be looking at or even LC/MS/MS if it is available to you. You could do enzyme based assay. Getting pure standards which are expensive is important. It depends on how much money is available for your project. Keto-acids in fruits, juices and wines have been quantified which is why your professor is a little annoyed.

"Just by feeding keto acids, if they are the equivalent of the essential amino acids, you create them in your body by absorbing the ammonia and then making your own protein, and that was where I got the idea that some foods would contain natural keto acid equivalents and we did chromatography on potato juice and found that they are rich in the keto acids which will function as amino acids, even though they contain no ammonia, because they can absorb ammonia from your blood"

I would focus on orange juice, simply because many people freak out drinking fruit juice without the fibre. You want to be able to show reduced ammonia in urine, blood, feces in say diabetics or kidney impaired. You probably will not be able to use humans at undergrad level but could continue into Phd thesis layering more techniques such as protein expression studies, gene expression. You need to pick a disease condition, like diabetes, obesity (obesity would be good as these people are scared of fruit and sugar) or prehaps the aging as they have most problems with losing lean protein.

Thanks for the thoughtful response!

Yes, both those methods are available (there's a whole facility for HRMS). I'm trying not to get too excited yet, as this all kind of rests on this professor's whim; but I fully intend to push it to the limit, even if I have to wait till summer semester to get unfettered access to some of these things (I think they take away funding if I wait too long, though). I don't think standards will be an issue, I know the DNPH won't, but I will [very nicely] insist on primaries. I hate to ask, but do you know any specific studies that quantify the keto-acids you mentioned? Even breaking the pay-wall with the university system, I found very little. If not, that's fine, but I would like to see something without dope-ishly asking the professor if he knows.

You're correct in assuming I can't work with humans, but the orange juice is a great consideration for now and the future (as you say).
 

Ella

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Pfff, I wish; then I wouldn't have to play the begging game like I am. I'm an undergrad 4+1 masters student, doing something called an REU; it's basically paid-for undergraduate research and since I'm the only person in the molecular sciences utilizing it, I get to actually choose a project.

I think if I do a thesis, it will be on something similar to Gilbert Ling's work in cell physiology. Speaking of, I had asked raypeatclips in PM, but does anybody know how Ling is doing? His website has been offline for a while. I know it happens, time to time, but it's been months. I thought to ask Dr. Peat what he knows, but again I hate the idea of bothering him with stuff.

Update about my prof: He finally understood about the ketoacid vs ketogenic acid thing, and actually linked me to a study that I had open already on ways to do it, haha: Estimation of α-keto acids in plant tissue: a critical study of various methods of extraction as applied to strawberry leaves, washed potato slices and peas

Here is another paper. Look at enzymatic determination of keto-acids. Contact the companies that produce the assays as these will be cheaper and easier if you don't have access to hplc or lc/ms. Are you allowed to incorporate feeding studies (humans) into your study. If you can wrangle it (need ethics committee approval). I know it's just urine and can be difficult to get approval. Talk to your professor if this can be included. Your time is precious. If you can determine amount of keto-acids fruit(s) of interest - you could include potato extract (this way you can compare the difference). If time permits, you could recruit some university students who are body builders or gym junkies. They will breaking down muscle tissue and you would expect to see more ammonia in their urine. Test amt of ammonia before and after keto-acid fruit juice/potato extract. This way you get experience in testing different matrices and molecules. Recruiting students or mates will be easier than nursing care patients or diabetics. It is only my suggestions, others may have better ideas. Perhaps you can recruit from the local gym.

You don't want to just determine the the amount of keto-acids unless you can come up with a novel, cheaper means of doing so. For example, developing a biosensor for keto-acids which can be used as a rapid test for industry - this will require more skills, time and supervisor who is skilled in the art. It sounds that your interest is more in the health benefits of keto-acids in the diet.

You also need to be clear which keto-acids you will be testing for. Are there specific ones which are more valuable than others? This would be useful to know. Ask Ray for advice here.

You need to think carefully about your future career going forward. If the results are favourable, you could approach industry, citrus, potato or juice company in sponsoring your research to take to the next level.

Branched-chain and aromatic amino acid catabolism into aroma volatiles in Cucumis melo L. fruit | Journal of Experimental Botany | Oxford Academic

Do a thorough search of food chemistry, biotechnology, analytical chemistry journals. If no comparison studies have been done between keto acids in fruit and potato juice, then this is a good start.

Development of an HPLC-fluorescence determination method for carboxylic acids related to the tricarboxylic acid cycle as a metabolome tool

Here's a paper using urine and blood via hplc chemiluinescence detection.

Determination of alpha-keto acids including phenylpyruvic acid in human plasma by high-performance liquid chromatography with chemiluminescence det... - PubMed - NCBI

The following is a recent lc/ms/ms technique which would be nice to do. Read the paper as it will have useful information on sample prep that you may not be aware of.

Development of a New LC-MS/MS Method for the Quantification of Keto Acids

I would love to help you out and it is frustrating I know. I have the equipment sitting in storage so I get a bit pissed off when universities have students incur huge debts however do not spend the money on lab equipment to do meaningful work. No-one uses TLC outside the university, so this would not be looked upon favourable on your resume. The universities would love to do away with the labs all together at the undergrad level and have them learn with simulation software. This is the reason science graduates lack laboratory skills. Chemistry is a dying art and chemists are being replaced by technicians that have been poorly trained.

Best of luck.
 
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explosionlord

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Here is another paper. Look at enzymatic determination of keto-acids. Contact the companies that produce the assays as these will be cheaper and easier if you don't have access to hplc or lc/ms. Are you allowed to incorporate feeding studies (humans) into your study. If you can wrangle it (need ethics committee approval). I know it's just urine and can be difficult to get approval. Talk to your professor if this can be included. Your time is precious. If you can determine amount of keto-acids fruit(s) of interest - you could include potato extract (this way you can compare the difference). If time permits, you could recruit some university students who are body builders or gym junkies. They will breaking down muscle tissue and you would expect to see more ammonia in their urine. Test amt of ammonia before and after keto-acid fruit juice/potato extract. This way you get experience in testing different matrices and molecules. Recruiting students or mates will be easier than nursing care patients or diabetics. It is only my suggestions, others may have better ideas. Perhaps you can recruit from the local gym.

You don't want to just determine the the amount of keto-acids unless you can come up with a novel, cheaper means of doing so. For example, developing a biosensor for keto-acids which can be used as a rapid test for industry - this will require more skills, time and supervisor who is skilled in the art. It sounds that your interest is more in the health benefits of keto-acids in the diet.

You also need to be clear which keto-acids you will be testing for. Are there specific ones which are more valuable than others? This would be useful to know. Ask Ray for advice here.

You need to think carefully about your future career going forward. If the results are favourable, you could approach industry, citrus, potato or juice company in sponsoring your research to take to the next level.

Branched-chain and aromatic amino acid catabolism into aroma volatiles in Cucumis melo L. fruit | Journal of Experimental Botany | Oxford Academic

Do a thorough search of food chemistry, biotechnology, analytical chemistry journals. If no comparison studies have been done between keto acids in fruit and potato juice, then this is a good start.

Development of an HPLC-fluorescence determination method for carboxylic acids related to the tricarboxylic acid cycle as a metabolome tool

Here's a paper using urine and blood via hplc chemiluinescence detection.

Determination of alpha-keto acids including phenylpyruvic acid in human plasma by high-performance liquid chromatography with chemiluminescence det... - PubMed - NCBI

The following is a recent lc/ms/ms technique which would be nice to do. Read the paper as it will have useful information on sample prep that you may not be aware of.

Development of a New LC-MS/MS Method for the Quantification of Keto Acids

I would love to help you out and it is frustrating I know. I have the equipment sitting in storage so I get a bit pissed off when universities have students incur huge debts however do not spend the money on lab equipment to do meaningful work. No-one uses TLC outside the university, so this would not be looked upon favourable on your resume. The universities would love to do away with the labs all together at the undergrad level and have them learn with simulation software. This is the reason science graduates lack laboratory skills. Chemistry is a dying art and chemists are being replaced by technicians that have been poorly trained.

Best of luck.

@Ella, meant to reply last night, was very tired. Thanks for all this encouragement, in both spirit and methodology. As I said, I'm lucky to have very good equipment to work with.

I wanted to let anyone interested know I'm breathing a sigh of relief, because my meeting with the supervisor on this program this afternoon went better than expected and it looks like I pretty much have carte blanche on this project. Also, it basically is going to commence when I'm ready to get started (figuring out the best way, securing reagents, et cetera; your input has already helped a lot, @Ella ) . I feel almost like this is too good to be true. Just glad there is not going to be any waiting around. I'll keep this updated as best I can, for those interested. Thoughts and ideas are welcome.
 

Ella

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Yes, both those methods are available (there's a whole facility for HRMS). I'm trying not to get too excited yet, as this all kind of rests on this professor's whim;

Don't worry too much about your professor. What does HRMIS stand for? I would get nice and chummy with whoever is in charge of that facility. I am so excited for you especially if you can get access to LC/MS/MS or HPLC/MS

I think if you contact the orange growers association or fruit producers; tell them what you are doing and see if they will support you in this research. Doesn't have to be a lot of money. They could provide you say the oranges needed for your experiment (point of harvest compared to point of sale), or pay for your reagents. I would get them interested from the start so they feel part of the journey with you. If you get good results from the oranges, they may jump to support your further in your research. See how your professor changes his stance and will grovel in order to help you. You never know, you may even get brownie points for showing initiative.

Find out the manufacturer of the instrument you will be using. They will have many methods that they have developed for different industries. See which method they have available on keto acids in fruit (wine industry is one that comes to mind) I have only used Agilent instruments and know they have lots of methods available. I would push for lc/ms or lc/ms/ms if you can get access, as these are state of the art. If you go this route, then you need to check whether the department has the spectra library that includes keto acids. If not, the following link which is free may have spectra data on keto acids. If not, they can help you and you could contribute to the database.

mzCloud – Partners
mzCloud – Advanced Mass Spectral Database

Again you need to be clear which keto acids you will be testing. Genova Diagnostics test organic acids via lc/ms/ms. I have attached their sample test which lists keto acids of interest. You need to isolate which ones are likely to be in your fruit of interest and how valuable are they are likely to be in metabolic processes.

You could ring their lab and see if they can offer advise on the methodology or guide to research papers on the methods.

Once you optimised the conditions for the instrument you are using, you can push through as many samples as you like. If you are not familiar in interpreting mass spectra. Find a good book in your library that teaches you. If you go down this route, then I will send you my special homemade marmalade. Remember Peat considers marmalade a superfood. No need to go to the Amazonian jungle to get our superfoods. You could compare the marmalade with store bought marmalade. This way you could see how the keto acids found in raw state change once subjected to high heat and perhaps you will find some interesting molecules produced from the cooking process.

Now wouldn't that be a hoot and give those sugar/fructose haters and paranoid fruit eaters out there a real jolt to their worldview.

You have me so excited. You go boy; the citrus/potato industry is going to love you. Orange and Potato juice will soon be the protein choice.

Hope it all falls into place for you.

Best,
 

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Don't worry too much about your professor. What does HRMIS stand for? I would get nice and chummy with whoever is in charge of that facility. I am so excited for you especially if you can get access to LC/MS/MS or HPLC/MS

I think if you contact the orange growers association or fruit producers; tell them what you are doing and see if they will support you in this research. Doesn't have to be a lot of money. They could provide you say the oranges needed for your experiment (point of harvest compared to point of sale), or pay for your reagents. I would get them interested from the start so they feel part of the journey with you. If you get good results from the oranges, they may jump to support your further in your research. See how your professor changes his stance and will grovel in order to help you. You never know, you may even get brownie points for showing initiative.

Find out the manufacturer of the instrument you will be using. They will have many methods that they have developed for different industries. See which method they have available on keto acids in fruit (wine industry is one that comes to mind) I have only used Agilent instruments and know they have lots of methods available. I would push for lc/ms or lc/ms/ms if you can get access, as these are state of the art. If you go this route, then you need to check whether the department has the spectra library that includes keto acids. If not, the following link which is free may have spectra data on keto acids. If not, they can help you and you could contribute to the database.

mzCloud – Partners
mzCloud – Advanced Mass Spectral Database

Again you need to be clear which keto acids you will be testing. Genova Diagnostics test organic acids via lc/ms/ms. I have attached their sample test which lists keto acids of interest. You need to isolate which ones are likely to be in your fruit of interest and how valuable are they are likely to be in metabolic processes.

You could ring their lab and see if they can offer advise on the methodology or guide to research papers on the methods.

Once you optimised the conditions for the instrument you are using, you can push through as many samples as you like. If you are not familiar in interpreting mass spectra. Find a good book in your library that teaches you. If you go down this route, then I will send you my special homemade marmalade. Remember Peat considers marmalade a superfood. No need to go to the Amazonian jungle to get our superfoods. You could compare the marmalade with store bought marmalade. This way you could see how the keto acids found in raw state change once subjected to high heat and perhaps you will find some interesting molecules produced from the cooking process.

Now wouldn't that be a hoot and give those sugar/fructose haters and paranoid fruit eaters out there a real jolt to their worldview.

You have me so excited. You go boy; the citrus/potato industry is going to love you. Orange and Potato juice will soon be the protein choice.

Hope it all falls into place for you.

Best,

:hearteyecat::hearteyecat:
 

Ella

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This method was done with rat plasma. Contains all the keto acids of interest, ketoacid nuances re. sample prep and derivatisation. You could contact the authors and get feedback on whether it is suitable for other matrices such as fruit juice. They may be able to provide valuable input if you needed to troubleshoot any problems when developing and optimising the method to your project. Start networking early in your career and you never know where you end up. Learn to ride on the shoulders of others before you so the journey is not too torturous.
 

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Waynish

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Yes, it's an important distinction. Unfortunately, most research in this sphere (e.g. that done on people with kidney problems) tends to use the blanket term "ketoacids" in reference to the ketogenic amino acids. So, trying to enunciate to my professor why I would like to investigate ketoacids is hard; I'm afraid he might ask me what link something like pyruvic acid has with protein synthesis, though I have mentioned the idea of bloodstream ammonia. He just e-mailed me after I inquired about fruit, and suggested maybe a ketogenic test on potatoes with differing nitrogen content, so this is kinda going in the wrong direction.

I went ahead and e-mailed Dr. Peat, hope he responds.

Honestly, if your enthusiasm is not enough to gain the support of your professor, then he isn't worth working under for one more moment. There are many profs out there in your field that will be happy to support your genuine research interests!
 

Ella

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http://www.jrnjournal.org/article/S1051-2276(09)00165-4/fulltext

I haven't read the full paper. Here's a review up to 2013.

https://bmcnephrol.biomedcentral.com/articles/10.1186/s12882-016-0278-7

Read the older research in this area. Ray and John McDougall would know the research well. Treatment for dialysis patients is low protein supplemented with a mix of keto acids plus amino acid supplementation. Amino acid to keto acids is reversible. It just may be that potato and fruits contain the perfect ratio of keto acids plus non-inflammatory amino acids that would make that KA/AA supplement redundant. Ray has often said that potatoes have a protein profile that is more valuable than milk. This is due to the keto acids component which are able to recycle amino acids from the breakdown of protein in the body, thus increasing the protein value of potatoes. So the reaction goes both ways. It is so frustrating when you have to educate your professor. Show him this paper on keto-analogues.

https://academic.oup.com/ndt/article-lookup/doi/10.1093/ndt/gft092

The concern is going too low protein and that people will not adhere to a vegetarian diet. Most people are lazy and stupid and they want everything in a pill. Can't imagine dialysis is much fun either. You need to find out whether potatoes were included in their vegetarian diet.

Here's a paper on amino acids in OJ

http://www.sciencedirect.com/science/article/pii/S0003267004010608

which contain many other beneficial molecules which may help in kidney function and much more delicious than a supplement.

Sorry if this is all over the place as I am rushing. Hope you can make sense to where it is leading.
 

Ella

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I think we need to be clear on the nomenclature here. I chased up the ketoacids in the supplement for kidney disease patients. Keto acids are carboxylic acid + a ketone, however, the supplement contains keto-amino acids:
The composition of one Cetolog tablet (mg) is: ketoisoleucine, L-ornithine: 153.24 ketovaline, L-ornithine: 72.67 ketoleucine, L-lysine: 161.78 ketovaline, L-lysine: 76.77 ketolucine, L-histidine, H20: 50.72 DL-hydroxymethioninate Ca: 28.30 L-tyrosine: 151.54 L-threonine: 74.68

which they refer to as ketoacids. So confusing. So when Peat is referring to keto acids which he detected in potato juice, which moeity does he mean? He did give you the strawberry paper which tested for keto acids. Your professor can be forgiven.
 

charlie

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explosionlord

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Don't worry too much about your professor. What does HRMIS stand for? I would get nice and chummy with whoever is in charge of that facility. I am so excited for you especially if you can get access to LC/MS/MS or HPLC/MS

I think if you contact the orange growers association or fruit producers; tell them what you are doing and see if they will support you in this research. Doesn't have to be a lot of money. They could provide you say the oranges needed for your experiment (point of harvest compared to point of sale), or pay for your reagents. I would get them interested from the start so they feel part of the journey with you. If you get good results from the oranges, they may jump to support your further in your research. See how your professor changes his stance and will grovel in order to help you. You never know, you may even get brownie points for showing initiative.

Find out the manufacturer of the instrument you will be using. They will have many methods that they have developed for different industries. See which method they have available on keto acids in fruit (wine industry is one that comes to mind) I have only used Agilent instruments and know they have lots of methods available. I would push for lc/ms or lc/ms/ms if you can get access, as these are state of the art. If you go this route, then you need to check whether the department has the spectra library that includes keto acids. If not, the following link which is free may have spectra data on keto acids. If not, they can help you and you could contribute to the database.

mzCloud – Partners
mzCloud – Advanced Mass Spectral Database

Again you need to be clear which keto acids you will be testing. Genova Diagnostics test organic acids via lc/ms/ms. I have attached their sample test which lists keto acids of interest. You need to isolate which ones are likely to be in your fruit of interest and how valuable are they are likely to be in metabolic processes.

You could ring their lab and see if they can offer advise on the methodology or guide to research papers on the methods.

Once you optimised the conditions for the instrument you are using, you can push through as many samples as you like. If you are not familiar in interpreting mass spectra. Find a good book in your library that teaches you. If you go down this route, then I will send you my special homemade marmalade. Remember Peat considers marmalade a superfood. No need to go to the Amazonian jungle to get our superfoods. You could compare the marmalade with store bought marmalade. This way you could see how the keto acids found in raw state change once subjected to high heat and perhaps you will find some interesting molecules produced from the cooking process.

Now wouldn't that be a hoot and give those sugar/fructose haters and paranoid fruit eaters out there a real jolt to their worldview.

You have me so excited. You go boy; the citrus/potato industry is going to love you. Orange and Potato juice will soon be the protein choice.

Hope it all falls into place for you.

Best,

HRMS is high-resolution mass spectrometry; the facility has LC/MS, not to worry.

This is all great stuff, thanks! I'm all about the orange growers idea, but I have to make sure things like that are acceptable per "the program" or what have you. I'm glad to see all this advice, since this is a big undertaking. I've already been studying about the LC/MS to surprise them with my skills, but I'm surely going to refer to these things you sent, very valuable! On the subject of oranges and what you said, I do wonder if there'd be a huge difference between organic oranges in one of these Whole Foods type/straight-from-the-farm, and "conventional" varieties at normal grocery. It's a consideration.

Thanks for all the papers, too, on progress so far. I agree with you about the nomenclature problem, and it really confounded my search until I talked with Dr. Peat. Still, pretty much every study does as you say, and really don't differentiate between the "ketos".

Honestly, if your enthusiasm is not enough to gain the support of your professor, then he isn't worth working under for one more moment. There are many profs out there in your field that will be happy to support your genuine research interests!

Maybe I misrepresented him; he actually was surprisingly enthusiastic, especially when he figured out I was talking about keto acids rather than ketogenic. The only problem was that I was referred by someone to him pretty randomly (as I have a different "main" professor; they are separated by their specific areas of study) and being one of these super-star professors, he has a lot on his plate and apparently just had a baby son, so I was happy to wait a bit if he was my only option. Luckily, one of my old chem profs was able to sign off on the deal and alleviate him so I can do this. He (superstar prof) might consult at some point, I think, but I think I'll mainly be dealing with me-myself-I and maybe some grad students and facility managers. So, yeah, I don't knock him for his inability to be as into it as he can.

Anyway, looks like I have phone calls to make, haha.
 

Philomath

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Ella

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Just by feeding keto acids, if they are the equivalent of the essential amino acids, you create them in your body by absorbing the ammonia and then making your own protein, and that was where I got the idea that some foods would contain natural keto acid equivalents and we did chromatography on potato juice and found that they are rich in the keto acids which will function as amino acids, even though they contain no ammonia, because they can absorb ammonia from your blood"

Yes, Ray was looking for the keto-acids .and not the keto-amino acids. Specifically,
glyoxylic acid, pyruvic acid, β-hydrixypyruvic acid, α-ketobutyric acid, oxalacetic acid, and α-ketoglutaric acid. Sorry for the confusion - trying to do a million things at once. Yes I was on the right track, we are looking for keto-acids as above, thus if you find appreciable amounts of α-ketoglutaric acid in your fruit, when taken into the body and there is lots of ammonia from breakdown of protein then the ammonium moiety is attached to the α-ketoglutaric acid and the body is provided with the glutamine which is rapidly loss under stress. Glutamate dehydrogenase is upregulated in the presence of toxic amounts of ammonia. If there is plenty of substrate α-ketoglutaric acid plus cofactor NAD+ then glutamine is replenished by the addition of the ammonium group. There are other transaminases involved. The reaction goes both ways; the focus is on the reverse reaction.

The kidney studies gave patients preformed keto amino acids + amino acids. Those studies di my head in, so your professor can be forgiven.

In my travels, I found a nice little gem. A lecture given by Sir Hans Kreb. Reading this paper would make one reconsider following a ketogenic diet. The ketogenic state was considered an unhealthy state that must be treated.
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