Interferences in the Assay of Serum Albumin Using the Bromcresol Green Indicator - Lab Report Example

Interferences in the Assay of Serum Albumin Using the Bromcresol Green Indicator Performing a simple experiment using the Bromcresol-Green(BCG) method for Albumin concentration determination in human serum an observation was made that upon the addition of protein globulins an interference occurs raising the result significantly. This finding concurs with a literature review that was performed. Albumin's molecular structure was reported from research in the past 30 years. Albumin's clinical importance was discussed, explaining that the test for Albumin is not a marker for any specific disease, while it is an important indicator for the physiological condition of the body. It was explained that an abnormally high result for Albumin needs to resolved and at face-value is considered a spurious result. The cause of elevated albumin levels are usually a result of accompanying serum proteins, acute phase infections, elevated immunoglobulins(found in multiple myeloma), or autoimmune issues. The BCG method was thoroughly researched. It is a valuable test for screening, when performing mass analysis of blood work in large clinical laboratories. It has fallen out of favor with laboratory clinicians for fear that the test can mask hypoalbunemia and low readings. BCG is an acid-base indicator, its acid to base color change is from yellow to blue, with a range in pH from 4.2-5.6. The detection of hypoalbunemia is always important to a physician. Low Albumin readings are indicative of a deteriorating condition in the body. An alternative to the BCG indicator is Bromcresol Purple(BCP), also an acid-base indicator. A comparison studies have been performed of the two methods. When compared to a benchmark analytic method Capillary Zone Electrophoresis(CZE), the positive bias for BCG was 3.77, while BCP faired much better with 0.67. Our experiment produced linear curves needed in detection and quantification of albumin concentrations. We demonstrated the effect on accompanying globulins elevating the result in BCG determinations of albumin. We also showed that time, reagent concentration and wavelength of absorbance significantly affect albumin readings. We bring to consideration for further testing the affect of pH on the test. Background and Literature review In clinical medical laboratories the testing for serum albumin, globulins and total protein are standard procedures, performed daily. Theses assays may also be requested STAT or on an emergency basis for patients suffering a crisis situation. The techniques for determining albumin and total protein are numerous and varied. Most clinical laboratories perform three routine three routine examinations; 1) Total Protein(TP) 2) Serum Albumin(Alb) 3) Serum Globulins Human blood contains both a cellular and liquid portion. Albumin is the most abundant protein in the human circulatory system1 and contributes 80% to providing colloid osmotic pressure2. Albumin is chiefly responsible for maintenance of the blood's acid-base balance, commonly referred to as pH3. In mammals it is synthesized by the liver as a preproalbumin. It undergoes a two cleavage processes before release into the body's circulation in its final form. It has been determined that the half-life of a albumin molecule is 19 days4.. Figure 1: Molecular Cleavage of albumin from preproalbumin->proalbumin->albumin Figure 2: The classical perception of the Albumin molecule Peters, T., Jr. (1985). Serum Albumin. Adv. Protein Chem.37; 161-2455 Figure 3: Primary, secondary and tertiary structure (Carter and Ho, 1994) . 6 This picture shows the bovine albumin amino acid sequence. The BSA molecule is made up of three homologous domains (I, II, III) which are divided into nine loops (L1-L9) by 17 disulphide bonds. The loops in each domain are made up of a sequence of large-small-large loops forming a triplet. Each domain in turn is the product of two subdomains (IA, IB, etc.). The primary structure of albumin is unusual among extracellular proteins in possessing a single sulfhydryl (Cys-34) group. In the light of new information i.e., x-ray crystallographic data, albumin structure is predominantly alpha-helical (67%) with the remaining polypeptide occurring in turns and extended or flexible regions between subdomains with no beta-sheets Physical-Chemical Properties The albumin molecule is not uniformly charged within the primary structure. At neutral pH, calculated a net charge of -10, -8, and 0 for domains I, II, and III for bovine serum albumin. The surface charge distribution is shown in: Figure 4:. AB CD Space filling model of serum albumin molecule with basic residues coloured in blue, acidic residues in red, and neutral ones in yellow. (A) Front view, (B) back view, (C) left side, and (D) right side (Carter and Ho, 1994)6.. Unlike the asymmetric charge distribution on the primary structure, the distribution on the tertiary structure seems fairly uniform. Figure 5: Ball-Stick model (Carter and Ho 1994)7 The most outstanding property of albumin is its ability to bind reversibly an incredible variety of ligands. BSA is the principal carrier of fatty acids that are otherwise insoluble in circulating plasma. It also performs many other functions such as, sequestering oxygen free radicals and inactivating various toxic lipophilic metabolites such as bilirubin (Emerson, 1989)8 Albumin has a high affinity for fatty acids, hematin, bilirubin and a broad affinity for small negatively charged aromatic compounds. It forms covalent adducts with pyridoxyl phosphate, cysteine, glutathione, and various metals, such as Cu (II), Ni (II), Hg (II), Ag (II), and Au (I). As a multifunctional transport protein, albumin is the key carrier or reservoir of nitric oxide, which has been implicated in a number of important physiological processes, including neurotransmission (Stamler et al., 1992)9.. It also belongs to a multigene family of proteins that include alpha-fetoprotein (AFP) and vitamin D-binding protein (VDP), which is also known as G complement (GC) protein. The Clinical significance of Albumin Two popular screening techniques use Bromcresol-Green and Bromcresol-Purple indicators, these methods tend to overestimate albumin concentration when the serum albumin is low especially when levels of alpha and beta globulins are elevated. Clinical studies have now demonstrated that bromcresol-purple is more sensitive and gives results in closer accordance to the most accurate electrophoretic methods. Causes of a decreased plasma albumin include: 1. Decreased Synthesis 2. Increased Catabolism 3. Increased Loss Nephrotic Syndrome Exudate loss in Burns Bleeding Gut Loss 4. Redistribution Hemodilution Increased capillary permeability Decreased Lymph Clearance Other conditions and diseases where a low albumin reading is expected include, hepatic failure, renal disease, burns, trauma and sepsis. Low serum albumin is a non specific marker of disease. A fall in the albumin concentration reflects a deterioration and a rise recovery. Malnutrition is not a significant cause of low albumin levels. The body maintains serum albumin at the expense of of muscle protein12 Experimental Design Using standard spectrophotometric light absorbance technique and Bromcresol-Green(BCG) as our indicator we measured absorbance of Bovine Serum Albumin(BSA), Gamma Globulin Bovine blood(B-Gamma Glob) and a mixture of the BSA and B-Gamma Glob. We were attempting to observe under controlled conditions the affect of accompanying serum proteins on the determination of serum levels Albumin. Methods Using a standard solution of BSA(10g/l) dilutions were made obtaining samples 10g/L, 20g/L, 30g/L, 40g/L, 50g/L and 60g/L. Included was a blank of de-ionized water(DDH2O). A given BCG was used as an indicator and diluted 1:7.5(8ml in 52ml DDH2O). Tubes were mixed well and incubated 10 minutes at room temperature. At 630nm wavelength light absorbance was measured. This procedure was repeated for B-Gamma Glob added too varying concentrations of BSA to test the affect of interfering proteins. Absorbance curves were obtained. A fortress sample was also tested for absorbance at 630nm wavelength absorbance and the result for BSA determined. Second Experiment A parallel experiment was performed changing the following conditions. The concentration of BCG was doubled by using 16 ml in 44ml of DDH2O and shortening incubation time from 10 minutes to 5 minutes. Results Procedure for Bovine Serum Albumin and BCG Dilution of Standard 0, Standard 1, Standard 2 , Standard 3, Standard 4, Standard 5 , Standard 6 Standard 6 Standard 5 Standard 4 Standard 3 Standard 2 Standard 1 Standard 0 DDH2O (ml) 0.00 0.1 0.2 0.3 0.4 0.5 0.6 Standard (ml) 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Concentration(mg/ml) 6 4.17 2.67 1.5 0.67 0.17 0.00 For colour development 8 ml of Bromocresol green BCG was Diluted by adding 52 mls of DDH20 Blank Standard 0 Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Fort.Standard DDH2O (ml) 0.01 ------ ------- ---------- --------- --------- --------- --------- Diluted Standard (ml) ---- 0.01 0.01 0.01 0.01 0.01 0.01 ------ Diluted Sample ----- --------- -------- -------- ---------- ----------- ---- 0.01 Reagent BCG(ml) 3 3 3 3 3 3 3 3 Tube were mixed well and incubated for 10 minutes at room temperature Manual Spectrophotometric Procedure: Wavelength Temperature Cuvette Measurment 630nm 20-25 C 1 cm light path Against reagent Blank Absorbance Results [BSA]=mg/ml Abs Duplicate Abs 0 ----------- ---------- Standard 1 0.17 0.073 0.069 2 0.67 0.162 0.159 3 1.50 0.25 0.24 4 2.67 0.317 0.328 5 4.17 0.42 0.388 6 6.00 0.492 0.497 Globulin gamma bovine blood Abs Albumin+globulin Abs duplicate Albumin+globulin 0.157 0.163 0.187 0.187 0.277 0.265 0.369 0.346 0.468 0.473 0.551 0.553 0.648 0.651 Absorbance obtained with BSA and Globulin mixture Dilution of Standard 0, Standard 1, Standard 2 , Standard 3, Standard 4, Standard 5 , Standard 6 Standard 0 Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Bovine Serum albumin 0.00 0.1 0.2 0.3 0.4 0.5 Globulin 0.06 0.06 0.06 0.06 0.06 0.06 DDH2O (ml) 0.54 0.44 0.34 0.24 0.14 0.04 Concentration 0 0.17 0.67 1.5 2.67 4.17 BCG and manual spectrophotometric readings were prepared and performed as in previous procedure. A curve obtained and is superimposed on the BSA concentration curve obtained in the previous procedure and seen in the above graph Second experiment Procedure for Bovine Serum Albumin and BCG Dilution of Standard 0, Standard 1, Standard 2 , Standard 3, Standard 4, Standard 5 , Standard 6 Standard 6 Standard 5 Standard 4 Standard 3 Standard 2 Standard 1 Standard 0 DDH2O (ml) 0.00 0.1 0.2 0.3 0.4 0.5 0.6 Standard (ml) 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Concentration 6 4.17 2.77 1.5 0.67 0.17 0.00 Mix the tubes well For colour development -16 ml of Bromocresol green BCG was Diluted by adding 44 mls of DDH20 Blank Standard 0 Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Fort.Standard DDH2O (ml) 0.01 ------ ------- ---------- --------- --------- --------- --------- Diluted Standard (ml) ---- 0.01 0.01 0.01 0.01 0.01 0.01 ------ Diluted Sample ----- --------- -------- -------- ---------- ----------- ---- 0.01 Reagent BCG 3 3 3 3 3 3 3 3 Mix the tubes well, Leave for 5 min at 20- 25 C Measure the absorbance of the calibrate and sample against the reagent blank with spectrophotometry at wavelength 660nm Manual Procedure Wavelength Temperature Cuvette Measurment 660nm 20-25 C 1 cm light path Against reagent Blank Albumin @ g/l Abs Albumin Abs duplicate Albumin Abs Albumin + globulin Abs duplicate Albumin + Globulin 0 0 0 0.186 0.183 10 0.048 0.041 0.199 0.216 20 0.105 0.106 0.207 0.221 30 0.168 0.189 0.246 0.247 40 0.222 0.23 0.285 0.301 50 0.319 0.31 0.348 0.349 60 0.379 0.389 0.398 0.401 Discussion These experiments using the BCG method correlate to some interesting material discussed by professional researchers in the literature review. Modern analyzers use the BCG method purely as screening method for determining albumin levels. It has been long known that the BCG reaction is two-step, quickly reacting with albumin and then in step two reacting with other serum proteins in lower concentrations. Robertson in 1981 reports "Dye concentration and reaction time are the two most critical factors in determining serum albumin usung BCG"10. Our experiment tested both a decreased reaction time and increase dye concentration. We clearly see that the globulin blank is reading an absorbance of 0.17 and elevating our result with a parallel curve above the BSA readings. We also saw a decrease of absorbance in our standards with the lowering of incubation time and change in wavelength from 630nm to 660nm. For nearly 40 years BCG has been considered fast and reliable for initial screening of serum albumin the test is performed in a succinate buffer at pH 4.2,then adding the sample starts the reaction. 13. BromCresol Green is an acid base indicator its acid-base color transformation is from YELLOW=BLUE pH range 4.0-5.611 Chemical Mechanism Add Serum BCG(in succinate buffer)pH 4.2------------------- Formation Alkaline Products pH >5.6 Yellow Solution function time Blue Solution Adding serum to the BCG reagent starts the reaction and modern autoanalysers convert the time of reaction to a quantitative value. The bottom line is the laboratory world is switching from BromCresol Green to BromCresol Purple due to reports like these. When BCG albumin testing was compared to capillary zone electrophoresis; a positive bias of 3.77- 5.26 g/l n=72 and 57 samples showed a difference greater or equal to 5 grams. Bromcresol Purple faired significantly better 0.67g/l bias,-4.37 to 3.06. Bromcresol Green though still extensively used is now considered an inferior test by most authorities in the lab world. Conclusions Albumin the major serum protein fulfills multiple roles in the physiologic processes of the human body, including maintaining blood pH balance, transport, fluid and electrolyte balance. Methods for precision testing of albumin concentration include electrophoresis, and immunossays. These procedures are cumbersome and require highly trained technical staff to perform. Clinical laboratories therefore fallback to faster more efficient and cheaper techniques to do these tests on a daily basis as they are requested. Modern laboratories now use fast batch autoanalysers to perform chemistry that include up to 20 parameters of tests to screen for disease, Albumin and Total protein are always included on these screens which use the acid base indicators Bromcresol Green and Bromcresol Purple as the principal reagent. In the last 15 years it has been recognized that a positive Bias for the Bromcresol Green method can lead to the masking of a hypoalbumenia condition. Hypoalbumenia though not a marker for specific disease does alert a physician that a critical degenerative condition exists. True hyperalbumenia is rare, again these results are usually resolved with the finding that an immunoglobulin elevation exists for numerous possible reasons, ie. Acute infection, autoimmune disregulations or multiple myeloma. The four essential factors causing interference in performing the Albumin screening are: 1. Accompanying serum proteins reacting with reagent 2. pH maintenance of the Test 3. Concentration of the buffer 4. Time of reaction We tested for three of these factors and it may be interesting rerun the experiment in a buffered solution and test the effect of pH. Please note normal values for Albumin vary from lab and analyzer and method performed. For this reason they have not been included in this report. References 1http://www.friedli.com/research/PhD/chapter5a.html Albumin Research Natural and Homeopathic Healing 15 March 2009 web site 2 Carter, D. C. and Ho, J. X. (1994). Structure of Serum Albumin. Adv Protein Chem. 45; 153-203 3.Figge, J., Rossing, T. H. and Fencl, V. (1991). The Role of serum-proteins in Acid-Base Equilibria. J. Lab. Clin. Med. 117; 453-467 4. Waldmann, T. A. (1977). In: "Albumin Structure, Function and Uses". (Eds. V. M. Rosenoer, M. Oratz, and M. A. Rothschild ), pp. 255-273 Pergamon, Oxford 5. Peters, T., Jr. (1985). Serum Albumin. Adv. Protein Chem.37; 161-245 6 Carter, D. C. and Ho, J. X. (1994). Structure of Serum Albumin. Adv. Protein Chem. 45; 153-203 7. Carter, D. C. and Ho, J. X. (1994). Structure of Serum Albumin. Adv. Protein Chem. 45; 153-203. 8. Emerson, T. E., Jr. (1989). Unique features of albumin: A brief review. CRC Crit. Care Med. 17; 690-694. 9. Stamler, J. S., Singel, D. J., and Loscalzo, J. (1992). Biochemistry of Nitric Oxide and its Redox-Activated Forms. Science 258; 1898-1902 10. http://www.clinchem.org/cgi/reprint/27/1/144.pdfClin. Chem 27/1 144-146 Robertson (1981) web site 16 March 2009 11. http://chemistry.about.com/library/weekly/aa112201a.htmAcid-Base indicators. About Chemistry web site 15 March 2009 12. http://www.ccmtutorials.com/misc/albumin/keypoints.htmCritical Care Tutorials Kenneth Neligan University of Pensylvania web site 16 March 2009 13. http://www.sciencedirect.com/science Albumin standards and the measurement of serum albumin with bromcresol greenBasil T. Doumas*, W. Ard Watson and Homer G. BiggsDepartment of Clinical Pathology, School of Medicine, University of Alabama in Birmingham, Alabama 35233, U.S.A. Received 29 June 1970 web site 15 March 2009 Read More