A-red
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Making Artificial Liver Bile

Postby A-red » Tue Feb 03, 2009 7:38 pm

I'm trying to make artificial liver bile using a composition list that I found from a book on Medical Physiology. It states that bile is made up of:
water
bile salts (bile acids)
bilirubin
cholesterol
fatty acids
lecithin
and some electrolytes. Such as, Na(1+), K(1+), Ca(1+), Cl(1-), HCO3(1-)

Some problems that I'm encountering with this are that (1) I'm trying to find some kind of scientific recipe that can essentially give me the moles/amounts of these composites. I do have the amounts of each of these parts of bile, but I'd like to see how scientists go about using them. (2) Bile acids are mostly made up of cholic acid, and chenodeoxycholic acid. Thing is, bile acids are kept at "intestinal pH", so if I do manage to mix all of my things up, how can I make sure that the pH can be similar to that in the intestine. (3) I'm aware that by mixing these things together, some of them might have reactions which can change the pH of the whole mixture, and cholesterol can be easily affected by that. Would I need some kind of buffer in this experiment?

Thanks.

barretttomlinson
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Re: Making Artificial Liver Bile

Postby barretttomlinson » Wed Feb 04, 2009 2:28 am

Hi,

This paper may give you a little more information of the composition of bile:

http://www.pubmedcentral.nih.gov/pagere ... dex=1#page

The physiologic pH you are looking for appears to be between 7.1 and 8.5 (slightly basic):

http://www3.interscience.wiley.com/jour ... 1&SRETRY=0

http://books.google.com/books?id=eMRJYO ... &ct=result

It is possible you might have to adjust the pH.

Best of luck on the project!

Barrett Tomlinson

A-red
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Re: Making Artificial Liver Bile

Postby A-red » Wed Feb 04, 2009 7:57 pm

Hi Barrett,

Thanks for the links! I looked at the information in them. The second link that you mentioned does not load... Other than my research on bile, I'm having a few more problems.

(1) I'm not quite sure where I could get bile salts (which are mostly made up of cholic acid and chenodeoxycholic acid, I've learnt), bilirubin (which is made up of decomposed hemoglobin molecules, and so it's hard to obtain, if at all), cholesterol, lecithin, or the fatty acid (which I'm assuming must be a simple chain of fatty acid, but I don't know which one...)
The electrolytes can easily be provided by my school, and I know I can maintain the acidity between 7.1 and 8.5 (which is the level that the bile salts should be at). It's just the other things that I can't obtain easily, which are the key ingredients too! Are there any alternatives to the above ingredients? Like, are there other chemicals that might have the same functions or similar structures?

(2) I also learnt recently that the sensitive materials, like cholesterol, should be added last... Where can I find out which ones are sensitive, and then in which order should I mix the sensitive ones in?

I'm running into a lot of problems... :cry: Please help me!!

Thanks again,
A-red

HeatherL
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Re: Making Artificial Liver Bile

Postby HeatherL » Thu Feb 05, 2009 11:47 pm

Hi A-red,

This is an interesting project, and I'm sorry to see you running into so many problems.

One question I have is, what is your ultimate goal? Are you just trying to make bile, or are you planning to use the bile to conduct further experiments? If it's the latter, perhaps you can obtain bile samples from a laboratory to use for your experiments. Could you give us a little more insight into your ultimate objectives?

Best wishes,
Heather

A-red
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Re: Making Artificial Liver Bile

Postby A-red » Fri Feb 06, 2009 11:24 am

Hi Heather,

Actually, I'm making the bile for a kind of experiment later on. I'm going to be adding it to oil, to watch how it physically breaks it down. Then I'm going to add acetaminophen (Tylenol) to the oil and bile, and see how the function is affected. Then I will add echinacea instead of Tylenol and observe and compare the effects that has on the ability of my 'bile' to break the fat down. I talked this over with my Biology teacher too, and he suggests that I should combine the ingredients that I are accessible to me (I can get cholesterol by the way). Then I can try to maintain the pH levels between 7.1 and 8.5 using NaOH and HCl, depending on whether I want to increase or decrease the levels.

I'm essentially trying to make something that mimics the function of bile. I know this bile isn't going to be as good as natural bile... which is why I'm a little concerned. But I'm figuring that since my project is a study, whatever I can accomplish in the tests I described would be beneficial to my whole project. But if on the other hand, I can't get the bile to break down anything then it shouldn't affect my project. Because my main focus is the research that I'm putting into drug metabolism and the function that bile plays in that.

The science fair is actually next Friday (Feb 13) so I'm getting a little panicky.... I'll try to see if my prepared bile can mimic the natural functions, and if it doesn't then that's okay, because I can fall back on my research, which is quite extensive thankfully. If I can make it to the next level, I'll get a bit more time inbetween to either make better bile, or get some samples from a laboratory like you suggested.

I'd be really grateful for whatever help I can still get in terms of preparing bile, or any research resources that might aide me in my study.

Thanks,
A-red

A-red
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Re: Making Artificial Liver Bile

Postby A-red » Sat Feb 07, 2009 12:22 am

Hi,

I'd like to give y'all a quick update on my project. While I was trying to prepare bile today at school (which was honestly turning out to be a much more complicated task than I'd thought), I went to up this teacher for some help. And guess what? She just happened to have DRIED BILE! Just like you suggested getting samples, Heather!! I am so excited now, because I can actually go on to my tests now, instead of focussing on making the bile!

So, I now have to figure out how much water I should add to it so that I get a good, natural consistency. There's another tiny problem with that... she could only give me 0.62 grams, which was all that was left of the 3 grams she initially had. So what I need to do is figure out what maximum amount of water I can add to the dried bile, so that I can get a bit more to work with.

My new plan is as follows: (1) find out what maximum amount of water I can add to the dried bile, (2) divide the bile solutions into four separate samples: one as the control sample to observe its function with the fat I use, and three for the different kinds and mixtures of drugs that I'm going to be putting in, (3) find out how much acetaminophen or echinacea would be naturally metabolized by the amount of bile that I can prepare, (4) when I'm taking my observations, I'll time the reactions, and then also check the pH levels after the fat has disintegrated enough that it's not visible. I'm guessing that acetaminophen will make the solution more acidic (it's an acid after all).

So that's what I'm doing tomorrow (Sat). I'm going to find answers to the questions I listed above, and if you have any ideas, I'd be extremely grateful!

Thanks and if I run into any other problems, I'll let you know!

A-red

A-red
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Re: Making Artificial Liver Bile

Postby A-red » Sun Feb 08, 2009 1:15 pm

Hey there,

Update: I figured out how much water I need to add to the sample of dried bile that I have. Yay! :D And I've typed up my procedure and everything. But I'm just having a bit of trouble right now with the amount of Tylonel that I should use... I have those Regular Strength 325mg tablets. I'm sure I need to grind up one tablet, or two maximum, and then... what? I know I'd have to dissolve the Tylenol in some water (I'm using distilled water for all of my tests by the way), but I'm not sure how much Tylenol would end up in the liver when one takes a regular dose (325mg)? And then in the liver, how much of it would go into the bile? Does all of it go there? Probably not, because the Tylenol would go into the blood stream to help the nerves, like when we have a headache or a muscle cramp. How can I figure that out? :?

Thanks,
A-red

HeatherL
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Re: Making Artificial Liver Bile

Postby HeatherL » Sun Feb 08, 2009 5:32 pm

Hi A-red,

Thanks for the update! I'm glad to hear that you were able to figure out how much water to add to the bile to conduct your experiment. :)

I'm a bit confused about your next step. :? I thought that you would be looking at the effects of Tylenol on the ability of bile to break up oil. I know you've done a lot of research on your topic, but to help you focus your efforts, let's review how bile works:

Bile is an "amphipathic" molecule (having both polar and nonpolar parts) that is used to aid in lipid (fat) digestion. It is produced in the liver, then stored and concentrated in the gall bladder. When fats from a meal move from the stomach to the beginning of the small intestine (called the duodenum), the gall bladder sends bile to the duodenum. There, the bile acts to break up the fats into smaller pieces so that the digestive enzymes can better break down those fats. I like to think of it working the way soap works. When you get oil on your hands, it doesn't come off with just water. But if you put soap on your hands, the soap molecules break the oil into smaller pieces that you can then wash away with water. Here is the Wikipedia article on bile: http://en.wikipedia.org/wiki/Bile

My point is that I don't think you should be worried about how much Tylenol makes it to the liver. Instead, you would want to estimate how much of the Tylenol makes it to the duodenum of the small intestine. Some of the Tylenol may be absorbed across the stomach lining (into the blood), so you could reduce the amount compared to an entire pill. However, most absorption occurs in the small intestine, so I don't think using one pill's worth will be that inaccurate for your experiment.

I hope that helps. Please continue to keep us posted, and let us know if you have further questions.

Best wishes,
Heather

A-red
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Re: Making Artificial Liver Bile

Postby A-red » Sun Feb 08, 2009 5:43 pm

Hi Heather!!

Thanks soooooooooooooo much!! That helps a LOT!!! :D :D :D I was about to conduct my experiment just now, and decided to check on my post before I did so. I was getting worried too... But you totally saved my day!! Alright!! :lol:

Okay, so I'll do my experiment, using almost all of the Tylenol pill and then I'll record my observations and everything. I'll let you know if anything else comes up!

Cheers,
A-red

A-red
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Liver Damage

Postby A-red » Tue Feb 17, 2009 11:01 pm

Hi there,

Thanks for all of the help y'all gave me with my project, because guess what? I'm going into my city's District Science Fair!!! :D :D

My project in the end was really good. I had been able to do my experiment with the bile, and I got really great results. See when I added 3 drops of cooking oil, as a fat, in the centre of a pure bile sample, it disintegrated gradually but quickly, within 10 seconds. But then when I added ground-up Tylenol to the bile, and put in 3 drops of oil, it simply floated on top as a big blob! The same happened when I added Echinacea to the bile, and when I added Tylenol and Echinacea together.

After talking with the judges about my project, and getting really good feedback, I have a few ideas about how I can make my project more valid for answering my question. My overall question is whether Tylenol and Echinacea actually damage our liver if taken together. So in my next experiments I'm going to be testing why the Tylenol and Echinacea stopped the bile from performing its functions. Is it because acetaminophen's polarity counteracted with the bile's? Is acetaminophen in the same chemical structure by the time it gets to the liver, or has it been transformed into one of its metabolites by then? When food is digested, does any of it go into the bile at all? Questions like that. And I mentioned these questions to my judges, and, fortunately, they were impressed by the fact that I'm not stopping where I am right now!!

So first off, I'm going get more bile (at least 20g I hope!!!), so that I can conduct more trials. Obviously I can't just pass off my results so far as being the final thing... So any idea where I could buy more bile, or dried bile? And if you have any suggestions on what else I could work on to prove or disprove that Tylenol and Echinacea together are harmful for the liver, I would love to hear them!!! :)

Cheers,
A-red

A-red
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Re: Making Artificial Liver Bile

Postby A-red » Wed Mar 04, 2009 9:19 pm

Hi,

I'm wondering if there is a way for me to measure the amount of bilirubin in dead liver cells?? I have designed an experiment in which I'm testing the affects of acetaminophen and echinacea on cow liver cells under a microscope. Is there any way at all that would allow me to measure toxic substances in the cells, via staining etc?

Also, I've worked a lot on understanding metabolism of drugs (especially acetaminophen and echinacea) and I have also studied how either or both of these drugs can affect bile's function, as well as a liver cell's structure (cell lining, nucleus, etc.). I don't have just one experiment that sums it all up... because my question can't be answered that way. If you recall my question was "How and why does the interaction of Echinacea and Acetaminophen damage the liver?" I decided on just testing as many things as I can, and then concluding with a possible answer to that question, which would initially be a hypothesis for an experiment that I want to do, if I had the equipment and the permission to use living samples. Is that okay? Would judges mark me down for just giving them a hypothesis as the end result of my study, in place of a conclusion?

Thanks,
A-red

amyc
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Re: Making Artificial Liver Bile

Postby amyc » Mon Mar 09, 2009 6:28 am

A-Red - Congratulations on the success of the project and on moving to the next level! I am working to get an expert to address your questions here (and I know you also have a new thread, too).

Amy
Science Buddies

Craig_Bridge
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Re: Making Artificial Liver Bile

Postby Craig_Bridge » Mon Mar 09, 2009 6:55 am

Congratulations. My wife was a pharmaceutical toxicologist and did her thesis on using liver tissue culture to perform initial screening of substances. From what I remember, if you lyse the liver cells and then centrifuge them, any bilirubin will end up in the solute. You can then normalize the pH and run this through the same testing you would do with blood plasma to detect bilirubin. You need to do a little research on your specific bilirubin testing method to determine if there is anything left over from they lysing process that could interfere with the assay so there might be some other pre-processing needed to remove the interfering chemical.

The typical way toxins are measured is via High Pressure Liquid Chromatography and Gas Chromatography sometime used in tandem with HPLC feeding GC.

The only thing that you can see with a microscope are any leasions or other changes in the liver tissue itself. The toxins would be way too small to see. Even if you used an electon microscope, you might see shadows of the elements in the chemicals but you wouldn't be able to figure out what the chemicals were.
-Craig

A-red
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Re: Making Artificial Liver Bile

Postby A-red » Mon Mar 30, 2009 10:53 am

Hi there,

I researched a bit on the centrifugation idea that you gave me Craig, and I've decided to go along that route. But I realized quickly that separating bilirubin or albumin (a protein that binds to bilirubin for the excretory process) from a batch of liver cells can get very complicated. Instead, I'm planning on simply centrifuging samples of liver tissue with and without acetaminophen and/or Echinacea, and comparing the amounts of the separations on a gradient. I'm going to do this by measuring the amounts in the test tube with a ruler, because I think there would be apparent lines between the separations.

What I'm having some trouble with now is how I'm going to prepare the liver tissue for centrifugation... That is, how should I lyse the liver cells (soap detergent and alcohol??)? I'm guessing I'd have to break down the liver tissue in a blender, and then lyse the cells. I'm also confused as to how the tissue might be centrifuged.... because I'm sure I'd need some kind of solution first, and if it's all solid after the blending process, should I add water to this?

Please reply as soon as you can, because I'm planning on doing my experiment tomorrow!!

Thanks,
A-red
Last edited by A-red on Mon Mar 30, 2009 9:43 pm, edited 1 time in total.

A-red
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Re: Making Artificial Liver Bile

Postby A-red » Mon Mar 30, 2009 9:43 pm

Hi,

I just want to make sure you understand the urgency of my question... the science fair is this Thursday, and I've been trying to patch up my areas of confusion with no luck. This isn't an integral part of my project, but it is definitely going to give it a boost, seeing as it is a study.

Thanks again,
A-red


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