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

Ella

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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.

There are studies that check keto-acids during the different seasons. I may have included the paper which deals with virus infected plants. Basically, sun-ripened fruits will have more keto-acids. Location where grown will be an important factor; sunny California compared to cooler areas. Now it would be interesting to know whether organically grown have increased levels. You would think by providing the plants with inputs high in the amino acids would increase the keto-acids. I think the melon studies subjected melon cubes to amino acids to increase aroma volatiles. As organically grown fruit have different inputs than conventional, you may see a difference here. Nice to know; not that I want to pay extra for my oranges. Useful for growers to know that certain inputs can increase the levels of keto acids be they organic or conventional grown.

Glad that you are using lc/ms. Now that we are clear on which keto acids you are looking for; you need to ensure that you select the appropriate column for the method. The lab may already have one. You don't want an old one. Test the column beforehand ensuring there are no issues with resolution. You don't want an old column that has not been properly maintained. Columns are expensive, so determine which one you require so that it can be ordered before you start and not during your experiment. As mentioned previously, the manufacturer of the instrument can provide advice on column selection for the method you are using.

Find out who the guru in lc/ms is in the lab. You will need assistance and guidance, as setting up the right conditions for your sample can take time, to get just right. You want to do this as efficiently as practical otherwise you will run out of time. It is one thing learning the theory however totally new ball game when it is hands on. You will need someone knowledgeable in the art to help you troubleshoot.
 
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explosionlord

explosionlord

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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.
images

Oh, did Dr. Peat tell you those keto-acids? I was dreading having to bother him again, ha ha.

You're quite right about finding a guru; even theoretically, LC/MS is difficult because of so many variables (proprietary software, not to mention the hardware like you mentioned). Can we just do electrophoresis? (JUST KIDDING) In any case, I won't give up. I like the challenge, but the time constraints are daunting.

Again, I really want to thank you for all this. It's like Holmes throwing Lestrade bones.
 

Ella

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LC/MS is a breeze once you have optimised the conditions. I have only ever used Agilent, which are a dream to use. I can't vouch for other makes. You don't want to spend time trying to learn the software so find that guru who can guide you on how it works.

I have attached recent paper by the Russians on GlutamateDehydrogenase. Very nice review on this enzyme. It behaves differently in different cells and tissue sites.

"The prevalent direction of the GDH reaction is determined by cell- and tissue-specific metabolic networks. In rat brain, the oxidative deamination of glutamate by GDH is favored [7–14]. However, neuronal GDH activity is two to five times lower than it is in astrocytes, where it must compete with highly expressed glutamine synthase that transforms glutamate to glutamine [15]. In liver, the glutamate dehydrogenase reaction is in equilibrium, whereas in pancreas and kidney, the enzyme catalyzes the oxidative deamination of glutamate to form 2-oxoglutarate [16–18]."

I had problems cutting & pasting the following section. It makes the distinction how GDH in plants under stress catalyses the backward rxn.

Indeed, this activity enase trisubstrate (backward) reaction for nitrogen assimilation [19]. Indeed, this activity of plant GDH is elevated at increased concentrations of ammonia. The latter often occurs under stressful conditions, such as high temperatures, the absence of light, pollution and aging [20,21]. Under normal metabolic conditions, plants also use GDH in the bisubstrate (forward) reaction. Under these conditions, nitrogen assimilation is performed by the two enzymes catalyzing the glutamine: 2-oxoglutarate amidotransferase reaction using ferredoxin or NADH.

You could include electrophoresis if you have time. isolating the enzymes that catalyse the reaction and while you are at it isolate the genes. The Russian paper includes kinetic studies with GDH from peas. There is loads of good stuff in this one. So the controlling factors that determine which way the reaction is going to proceed is high levels of ammonia and stress in plants, thus can we expect the same occurs in humans? The references should provide plenty of food for thought. I wish I had time to ready them:(

The following is of interest:

"GDH is also detected in bovine thyroid gland cytosol, where it is thought to be involved in the synthesis of thyroid hormones [36]."

The keto acids where in that paper Peat gave you. Do you have access to the full paper? You will need to research the health benefits of each one along with the ketoamino acids.
The following is a primer.
Amino acid metabolism

You have much territory to cover.
 

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explosionlord

explosionlord

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LC/MS is a breeze once you have optimised the conditions. I have only ever used Agilent, which are a dream to use. I can't vouch for other makes. You don't want to spend time trying to learn the software so find that guru who can guide you on how it works.

I have attached recent paper by the Russians on GlutamateDehydrogenase. Very nice review on this enzyme. It behaves differently in different cells and tissue sites.

"The prevalent direction of the GDH reaction is determined by cell- and tissue-specific metabolic networks. In rat brain, the oxidative deamination of glutamate by GDH is favored [7–14]. However, neuronal GDH activity is two to five times lower than it is in astrocytes, where it must compete with highly expressed glutamine synthase that transforms glutamate to glutamine [15]. In liver, the glutamate dehydrogenase reaction is in equilibrium, whereas in pancreas and kidney, the enzyme catalyzes the oxidative deamination of glutamate to form 2-oxoglutarate [16–18]."

I had problems cutting & pasting the following section. It makes the distinction how GDH in plants under stress catalyses the backward rxn.

Indeed, this activity enase trisubstrate (backward) reaction for nitrogen assimilation [19]. Indeed, this activity of plant GDH is elevated at increased concentrations of ammonia. The latter often occurs under stressful conditions, such as high temperatures, the absence of light, pollution and aging [20,21]. Under normal metabolic conditions, plants also use GDH in the bisubstrate (forward) reaction. Under these conditions, nitrogen assimilation is performed by the two enzymes catalyzing the glutamine: 2-oxoglutarate amidotransferase reaction using ferredoxin or NADH.

You could include electrophoresis if you have time. isolating the enzymes that catalyse the reaction and while you are at it isolate the genes. The Russian paper includes kinetic studies with GDH from peas. There is loads of good stuff in this one. So the controlling factors that determine which way the reaction is going to proceed is high levels of ammonia and stress in plants, thus can we expect the same occurs in humans? The references should provide plenty of food for thought. I wish I had time to ready them:(

The following is of interest:

"GDH is also detected in bovine thyroid gland cytosol, where it is thought to be involved in the synthesis of thyroid hormones [36]."

The keto acids where in that paper Peat gave you. Do you have access to the full paper? You will need to research the health benefits of each one along with the ketoamino acids.
The following is a primer.
Amino acid metabolism

You have much territory to cover.

Wow, yes, this is immensely interesting. It's amazing how the pieces seem to come together, but the clues are distributed all over time and space so nobody has really unified all of it; science, I guess.

No, I could not gain access to the paper despite having access to Science Direct through school. I will try a library computer, Science Direct sometimes lets me see things from home, sometimes it puts me in a "log in via your institution" loop.
 

Ella

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the clues are distributed all over time and space so nobody has really unified all of it

Yes,my luv:) That is your job as a budding scientist, like Sherlock, leaving no stone unturned and filling in the missing pieces of the puzzle. The following is advice Ray gave me after I had gone down many rabbit holes and dead ends in my own research. The research literature was filled with vested interests, myths, deception and lies and I found myself in a very lonely place.

I think it is necessary to look at these established ideas, which are absurd and unfounded, because we are forced to wallow in them every day, until people have come to treat them as if they were intelligent, intellectually responsible, descriptions of reality; if we realize that silly ideas and connotations have been built into our language, then we can begin to give serious attention to the "anomalies" of science.

"Anomalies" are facts that don't obey the law, and so they are of special importance for anyone who cares about understanding things. If you have some parts left over after you have assembled a puzzle, maybe the puzzle wasn't correctly assembled. So-called anomalies are often the most productive places to look for solutions.” RP

I would not be too concerned about contacting Peat. You need an expert in the field to guide you in your project. I am sure Peat would be thrilled to help you out knowing that you are continuing where he left off. There are still many pieces to this puzzle. So OK, we establish that fruits have abundant keto acids but how does the mechanism work once we eat the fruit. Is there going to be a forward or backward reaction? Will the reaction be different in the different compartments of the body? How do we know whether these will be beneficial or deleterious reactions? Recycling ammonia would be good, but what if the reaction increases glutamate which feeds tumor cells? So it would be pertinent to know the abundance of each keto acid. So many questions that need to be answered. Peat may already have a good idea. Glutamate is a nonessential AA and Peat specifically states the essential Amino acids.

Glutamate and its receptors in cancer
 

haidut

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I wanted to chime in on the ketoacids. I did some research on that back in 2013 and I think strawberry leaves are the richest source known. Another user on the forum posted a similar study.
Protein Vs Keto Acids

Not sure if sourcing and boiling strawberry leaves is very feasible for most but it would be very cheap at least.
 
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explosionlord

explosionlord

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I wanted to chime in on the ketoacids. I did some research on that back in 2013 and I think strawberry leaves are the richest source known. Another user on the forum posted a similar study.
Protein Vs Keto Acids

Not sure if sourcing and boiling strawberry leaves is very feasible for most but it would be very cheap at least.

Hey! Yeah, that study you cited was really one of my first clues on how to start the process of delineating. I'm not sure yet what kind of leeway I'll have as far as testing time and trials, but if I can fit strawberry leaves in, I certainly will. It should be interesting to try the leaves with the method done with LC/MS/MS in the Japanese study posted by @Ella to compare results with the 60 year-old study.
 

haidut

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Hey! Yeah, that study you cited was really one of my first clues on how to start the process of delineating. I'm not sure yet what kind of leeway I'll have as far as testing time and trials, but if I can fit strawberry leaves in, I certainly will. It should be interesting to try the leaves with the method done with LC/MS/MS in the Japanese study posted by @Ella to compare results with the 60 year-old study.

You can also drink some strawberry leaves tea or potato juice soup and test for blood (or breath) ammonia 30min later. But the full analysis is good to have as otherwise we don't know how much leaves to consume to get the equivalent of say 1 potato.
 
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explosionlord

explosionlord

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You can also drink some strawberry leaves tea or potato juice soup and test for blood (or breath) ammonia 30min later. But the full analysis is good to have as otherwise we don't know how much leaves to consume to get the equivalent of say 1 potato.

That's a capital idea. I'm technically not allowed to test on people, but doing so on myself and reporting here will be great.
 
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explosionlord

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Just wanted to post here and let those interested know this is still in progress; having to wait on approval for the funding for the standards. I'm going for the "good stuff," as suggested by Ella, so I'm leery they might turn me down/try to force me to go with >=98% solutions and stuff like that. I have a meeting on Wednesday with the "supervisor" where I'm going to attempt to get approval for outside funding sources, just in case something like that happens. I'm going to go for the outside funding anyway; but just so I don't ruffle any feathers needlessly, I'm going to ask first and see what she says.

So, just a waiting game at the moment.
 
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explosionlord

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Ha ha.

Anyway, to anyone at all interested in this:

I figured I'd update, though there's unfortunately been little progress. I have obtained all chemicals and the column I need, but due to being a lowly undergraduate and the scheduling of these very good LC/MS/MS machines, the first opening is June. It may be longer since my supervisor on the project, who *has* to be with me when I do the experiment, is likely to not be available during summer. So, just so you know I'm in no way abandoning this project. It'll probably be a while, is all.
 
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Ha ha.

Anyway, to anyone at all interested in this:

I figured I'd update, though there's unfortunately been little progress. I have obtained all chemicals and the column I need, but due to being a lowly undergraduate and the scheduling of these very good LC/MS/MS machines, the first opening is June. It may be longer since my supervisor on the project, who *has* to be with me when I do the experiment, is likely to not be available during summer. So, just so you know I'm in no way abandoning this project. It'll probably be a while, is all.

Won't you be in graduate school by then?
 
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explosionlord

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How much do the pure standards cost?

I'm honestly not sure how much they paid for them; I'd have to look at the Sigma site to see what their basic price is, again. I know that the right phenyl column was upwards of $700, which surprised me that they went for it. Also, Ella, I'll say that I am doing what I can to jockey for the June position no matter what, not just resting on my laurels; but I can only force it so hard, these Biodesign people are super-busy. Still, I'm trying to maybe get them to take responsibility rather than the REU director-person.

Won't you be in graduate school by then?

Ha, well I already am technically (I guess, though not treated that way). Doing a 4+1 masters in Med. Biochem and I've already done 1 seminar and one class for the masters.
 

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