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Chemistry (including Biochemistry)/microneedle biopsy, measure total atp content, measure total magnesium content

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Expert: - 1/30/2012


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QUESTION: three ways to obtain muscle specimen i read are open excision, bergstrom needle, and microneedle biopsy. would microneedle biopsy provide enough specimen to measure total magnesium content? and would miconeedle biopsy provide enough specimen to measure total atp content? also what are the techniques called?,used for measurement of total magnesium content and total atp content in muscle biopsy that you are refering to in your answer to my question? i've read about some of the different techniques but wanted to know if we're talking about the same thing. thank you

ANSWER: Hello Nha!

Microneedle biopsy will get you about 7-70 milligrams of cells; that's not a lot. Given the limited amount of sample, you'll want to think hard about what kind of assay you're going to use.

If you're trying to design a research experiment I'd reccomend looking at the different ways to detect ATP or Mg that your laboratory is capable of. Talk to labmates and people in other labs to see if anyone has done experiments like this. They'll be an invaluable resource in getting your assay working before you start using limited amounts of patient samples.

That said, a lot of biochemistry of this type is done using kits of one form or another. A couple luciferase assays to measure ATP would be here. You'd need to establish some standard curves to ascertain how many cells gives you a reproducible result.

http://www.promega.com/resources/product-guides-and-selectors/protocols-and-appl
http://www.nature.com/app_notes/nmeth/2006/060516/full/an1705.html

As for magnesium content, you have a couple strategies available to you.
Fluorescence: You could go through the Molecular Probes catalog (always a good resource) and talk to one of their sales representatives. You're looking for a dye that fluoresces when bound to Mg++. Start here.

http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbo

Alternatively if you have access to a flame ionization spectrometer you could try that; it's very sensitive, and may be in the sample ranges you need.
(http://en.wikipedia.org/wiki/Magnesium_in_biology#Measuring_magnesium_in_biologi)

Note that all of these techniques are destructive, so you'll have to talk to people and plan your experiments carefully. It may be that microneedle biopsy does not provide enough sample for you to do all of these tests, in which case you'll need to pick what tests will give you the most important information.

As always, design your experiment and run it by your principal investigator or experienced lab manager. They should be able to tell you what has worked in the lab before.

Good luck!

---------- FOLLOW-UP ----------

QUESTION: would the 7mg-70mg  provide enough sample for the luciferase assay for atp? this would only measure atp-mg right? it wouldn't be able to  detect atp not bound to magnesium right? would 7mg-70mg  provide enough sample to measure total magnesium content using the flame atomic absorption spectroscopy technique(AAS)? wikipedia magnesium biology)would 7mg-70mg provide enough sample to measure total magnesium using(also in wiki inductively coupled plasma mass spectrometry or atomic emission spectroscopy )? would 7mg-70mg be enough to allow BOTH tests (luciferase for atp and flame atomic absorption spectroscopy for total magnesium content)? thanks the answers were very informative

ANSWER: Hello Nha!

As far as I know, each test I described would only detect either ATP or Mg, but not both.

Unfortunately I can't answer if 70 mg of cells would be enough. That's something you'd have to try with 'dummy' (i.e. non patient) cells in the equipment you have and see. There's so much variability between equipment and detectors that unless you have labmates who can tell you for certain what they have done in the past, we can't rightly say from just a theoretical discussion. ESPECIALLY if the first trial doesn't work - you want to have enough patient sample that you can do the experimental run at least twice, ideally three times.

At this point I'd stop talking to me and go start talking to people in your department. :)  Good luck!

---------- FOLLOW-UP ----------

QUESTION: what is the normal levels of total atp content and total magnesium content found in normal muscle from muscle biopsy? i've searched online but couldn't find an answer. thanks

Answer
Hi Nha!

Here's what Google Scholar gives me.

If we approximate normal ATP content of human skeletal muscle is at equilibrium with the surrounding blood, it would be 0.60 ± 0.17 μM/L.
http://www.biomedcentral.com/1472-6793/9/24

If we're looking for something in terms of weight of total human skeletal muscle, 20.1 ± 1.1 mmol/kg dry wt is the amount according to these folks.
http://ajpendo.physiology.org/content/274/4/E600.full


Other interesting reading.

Here's a review on human plasma ATP levels.
http://www.clinchem.org/content/53/2/318.full.pdf

You might want to see if you can buy access to this article. It looks relevant to your research. (I don't have it.)
http://onlinelibrary.wiley.com/doi/10.1002/mrm.1910220258/abstract

This one correlates muscle ATP usage with magnesium in the diet of older persons.
http://www.ncbi.nlm.nih.gov/pubmed/16895893

Good luck!

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Trista Robichaud, PhD

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No homework questions, especially ones copied and pasted from textbooks. I will answer questions about principles or give hints, but I do not do other's homework. I'm comfortable answering basic biochemistry, chemistry, and biology questions up to and including an undergraduate level of understanding. This includes molecular biology, protein purification, and genetics. My training/inclination is primarily in structural biology, or how the shapes of things affect their function. Other interests include protein design, protein engineering, enzyme kinetics, and metabolic diseases such as cancer, atherosclerosis, and diabetes. My chemistry weaknesses are that I do not know organic or inorganic synthesis well, nor am I familiar with advanced inorganic reactions. I will attempt quantum mechanics and thermodynamics questions, but primarily as they relate to biological systems. Furthermore, I cannot tell you if a skin photograph is cancerous, or otherwise diagnose any disease. I can tell you how we currently understand the basic science behind a disease state, but I cannot recommend treatment in any way. Please direct such questions to your medical professional.

Experience

I hold a PhD in Biomedical Science from the University of Massachusetts Medical School in Worcester. I specialize in Biochemistry, with a focus on protein chemistry. My thesis work involved the structure and functions of the human glucose transporter 1. (hGLUT1) Currently I am a postdoc working in peptide (mini-protein) design and enzymology at the University of Texas Health Science Center in San Antonio, Texas. I am in Bjorn Steffensen's lab (PhD, DDS), studying gelatinase A and oral carcinoma.

Organizations
2001 American Association for the Advancement of Science
2007 American Chemical Society
2007 Protein Society
2011 UTHSCSA Women’s Faculty Association


Publications
Levine KB, Robichaud TK, Hamill S, Sultzman LA, Carruthers A. Properties of the human erythrocyte glucose transport protein are determined by cellular context. Biochemistry 44(15):5606-16, 2005. (PMID 15823019)
Robichaud TK, Appleyard AN, Herbert RB, Henderson PJ, Carruthers A “Determinants of ligand binding affinity and cooperativity at the GLUT1 endofacial site” Biochemistry 50(15):3137-48, 2011. (PMID 21384913)
Xu X, Mikhailova M, Chen Z, Pal S, Robichaud TK, Lafer EM, Baber S, Steffensen B. “Peptide from the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) inhibits membrane activation of matrix metalloproteinase-2 (MMP-2)” Matrix Biol. 2011 Sep;30(7-8):404-12. (PMID: 21839835)
Robichaud TK, Steffensen B, Fields GB. Exosite interactions impact matrix metalloproteinase collagen specificities. J Biol Chem. 2011 Oct 28;286(43):37535-42 (PMID: 21896477)

Poster Abstracts:
Robichaud TK, Carruthers. A "Mutagenesis of the Human type 1 glucose transporter exit site: A functional study." ACS 234th Meeting, Boston MA. Division of Biological Chemistry, 2007
Robichaud TK, Bhowmick M, Tokmina-Roszyk D, Fields GB “Synthesis and Analysis of MT1-MMP Peptide Inhibitors” Biological Chemistry Division of the Protein Society Meeting, San Diego CA 2010
Robichaud TK; Tokmina-Roszyk D; Steffensen B and Fields GB “Catalytic Domain Exosites Contribute to Determining Matrix Metalloproteinase Triple Helical Collagen Specificities” Dental Science Symposium. UTHSCSA 2011
Robichaud TK; Tokmina-Roszyk D; Steffensen B and Fields GB “Exosite Interactions Determine Matrix Metalloproteinase Specificities” Gordon Research Conference on Matrix Metalloproteinase Biology, Bristol RI 2011


Education/Credentials
Oakland University, Auburn Hills MI BS, Biochemistry 1998
University of Massachusetts Medical School, Worcester MA PhD, Biochemistry & Molecular Pharmacology 2001-2008
University of Texas Health Science Center, San Antonio TX Postdoc, Biochemistry 2009-Present


Awards and Honors
1998 Honors College Graduate, Oakland University
2009 Institutional National Research Service Award, Pathobiology of Occlusive Vascular Disease T32 HL07446
2011 1st Place, Best Postdoctoral Poster, Dental Science Symposium, UTHSCSA, April 2011


Past/Present Clients
Invited Seminars:
Robichaud TK, Fields GB. “Synthesis and Analysis of MTI-MMP Triple Helical Peptide Inhibitors” Pathology Research Conference, University of Texas Health Science Center San Antonio Pathology Department (June 18th, 2010)
Robichaud TK & Hill, B “How To Give A Great Scientific Talk” Invited Lecture, Pathobiology of Occlusive Vascular Disease Seminars, UTHSCSA (Nov 11th 2010), Cardiology Seminar Series, Texas Research Park (Feb 21st, 2011)
Robichaud TK; Tokmina-Roszyk D; Steffensen B and Fields GB “Exosite Interactions Determine Matrix Metalloproteinase Specificities” Gordon-Keenan Research Seminar “Everything You Wanted to Know About Matrix Metalloproteinases But Were Afraid to Ask” Bristol, RI (Aug 6th, 2011)

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