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Quantitative Determination of Albumin in Urine

By Kong Lee Fall 2010 Chem 4101. Quantitative Determination of Albumin in Urine. Albumin. Albumin is a protein produced by the liver . It can be detected in urine. It has the ability to bind different hydrophobic anions and fatty acids. Mass of 66438.0 Da. 3. (9). Problem.

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Quantitative Determination of Albumin in Urine

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  1. By Kong Lee Fall 2010 Chem 4101 Quantitative Determination of Albumin in Urine

  2. Albumin • Albumin is a protein produced by the liver. It can be detected in urine. • It has the ability to bind different hydrophobic anions and fatty acids. • Mass of 66438.0 Da 3 (9)

  3. Problem • Normal albumin levels in urine is 15–150 mg/day, less than 10 mg/dL. • Increased excretion of protein in urine is a sign of kidney disease. •  There have been research done that shows a high protein diet may lead to kidney damage. • Problem: Will prolong addition of supplemental protein such as whey and soy proteins cause kidney damage? • Hypothesis: Adding a protein supplement to meals twice daily over a period of time will cause kidney damage. 3

  4. Technique • The amount of albumin in urine can be measured by Capillary Electrophoresis. • Mass spectrometer will be used to detect eluent. • CE bufferis borate buffer of .01M with a pH of 10.2. 1 (8)

  5. 1 (7) Instrumentation • NANOFOR®01 – Spectrophotometric detector. • Wavelength range: 200-700 nm. • Fluctuation noise: 4·10 -5 AU. • High –voltage power supply: Voltage: ±30 k V; current: 1-150 mkA; instability: 0.03 % max • Sample injection: Electrokinetic, vacuum, pressure • Sample and buffer trays: 96-well plate for samples, 24 vials for buffers • Capillary: Fused silica 30–80 м i.d., 360 м o.d., up to 1000 m m long 1

  6. 5 Figure of Merit • Mass resolving power: 10^2 – 10^4 • Mass accuracy: 100 ppm • Mass range: 10^4 • Linear dynamic range: 10^7 • Precision: 0.1-5% • Abundance sensitivity: 10^4 – 10^6 • Efficiency (transmission x duty cycle) <1-95% • Speed: 1-20 Hz • Compatibility with ionizer continuous • Cost: relatively low • Size/weight/utility requirements: bench top

  7. Procedures • Collect urine from 2 group of individuals. The control group without a protein supplement and the variable group that intakes the protein supplement. Collect samples on monthly basis. • Urine will be purified by ultrafiltration. • After filtration, sample will be introduced to CE/MS. • Ionization will be done by electrospray ionization. • MS will use single quadrupole for detection of albumin. 4

  8. Data Analysis • Compare the protein intake of the control and variable group. • Compare the amount of albumin found in their urine. • After several months, try to find a correlation between amount of protein intake and kidney disease.

  9. Example Results • 1-g-globulin(15.35 %); 2-b-globulin(8.15 %); 3-a2-globulin(9.3 %); 4- a1-globulin (4.9 %); 5-albumin (60.5 %); 6- low-molecular fraction (1.8 %) 7 (7)

  10. Other Methods 6

  11. Conclusion • CE with MS detection is a fast and efficient way to quantitatively detect albumin in urine. • Drawbacks of CE/MS are: • Low pH may cause proteins to precipitate. • Low volume of sample injected causing low sensitivity. • Price range is high.

  12. References • 1) Bessonova EA, Kartsova LA, Shmukov AU. Electrophoretic determination of albumin in urine using on-line concentration techniques. J Chromatogram A. 2007 May 25;1150(1-2):332-8. Epub 2006 Nov 28. • 2) Hortin, Glen L. Sviridov, Denis. Analysis of molecular forms of albumin in urine. Proteomics Clin. Appl. 2008, 2. 950-955 • 3) Jenkins, Margaret A. Urine Proteins. Method in Molecular Medicine. Human Press Inc. Vol 27. 20 – 28 • 4) Liu, Cheng-ming (Yorba Linda, CA), Wang, Hann-ping (Yorba Linda, CA) 1996. Method of sample preparation for urine protein analysis with capillary electrophoresis. United States. Beckman Instruments, Inc. (Fullerton, CA). 5492834 http://www.freepatentsonline.com/5492834.html • 5) McLucky, Scott A. Wells, Mitchell J. Mass Analysis at the Advent of the 21st Century. Purdue University. Chem. Rev. 2001, 101, 571 – 606. • 6) McQueen M. J. Challenges for International Standardization of Microalbumin in Urine. McMaster University and Hamilton Regional Laboratory Medicine Program. Hamilton, Ontario, Canada. Oct 16, 2007. • 7) Institution of Analytical Instrumentation, Russian Academy if Sciences, Saint-Petersburg, Russia. • 8) Skoog Douglas A. Holler James F. Crouch Stanley R. Principles of Instrumental Analysis. Thomson Brookscole. 2007.p.867 – 889 • 9) Hamilton James A. Albumin. Access science from McGraw Hill. University of Minnesota Library Twin Cities. http://www.accessscience.com.floyd.lib.umn.edu/content.aspx?searchStr=albumin&id=021100

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