1. By Kong Lee Fall 2010 Chem 4101

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 buffer is borate buffer of.01M with a pH of 10.2. 1 (8)

5. 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 (7)1 1

6. 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 5

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 MethodProsCons Reverse phase HPLCGreat sensitivity.Time consuming. LC-MSFinger printing, high selectivity. Limiting detection limit due to interference. Low sensitivity. ImmunoassayHigh sensitivity, high selectivity. Modified forms of protein not detected. DipstickCheap and time efficientUrine discoloration may create difficulty in visually interpreting the test results 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 21 st 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