UNIT: Proteins 16tproteins.wpd Task Determination of Total Protein, Albumin and Globulins Objectives Upon completion of this exercise, the student will be able to: 1. Explain the ratio of albumin and globulin in total protein. 2. Determine the total protein by Biuret Method. 3. Determine albumin by the BCG Method. 4. Determine globulin content. 5. Explain the clinical significance of abnormal protein values. Introduction Blood is a complex mixture of cells suspended in a fluid medium, plasma. 92-93% of this fluid medium is water and the remaining 8% is dissolved proteins, minerals, glucose, etc. By far, the largest amount of the total solutes are the plasma proteins, collectively referred to as TOTAL PROTEIN. In serum, one of the plasma proteins, FIBRINOGEN, is missing, but it only constitutes 3-6% of the total protein in plasma. To standardize normal values, measurement of total protein has been confined primarily to serum samples. On the basis of their solubility properties, the serum proteins can be divided into two major fractions, ALBUMIN and GLOBULINS. The globulins can be further subfractionated into four major fractions: 1 globulin, 2 globulins, globulins, and globulins. Serum proteins serve a number of different functions. They constitute a portion of the amino acid pool of the body. They can be de-aminated to give ketoacids which can provide caloric energy, or be transformed into carbohydrates and lipids. Some serum proteins are also transport agents, carrying many vital metabolites, metal ions, carbohydrates and lipids. Enzymes, antibodies, and certain hormones are also proteins. Serum proteins are important in maintaining the osmotic pressure and the ph of the blood. Liver produced fibrinogen and prothrombin proteins play a major role in coagulation. Fibrinogen constitutes 0.2-0.4 gm/dl of the protein in plasma. There is a small diurnal variation of total plasma protein of about 0.5 gm/dl. Increased protein concentration: dehydration all fractions affected equally multiple myeloma (and related diseases) one fraction greatly increased, usually gamma Decreased protein concentration: kidney damage extensive bleeding severe burns inadequate intake deficient absorption Albumin makes up about 60% of the total plasma proteins. It has a relatively small size (MW = 69,000). It is synthesized exclusively in the liver and acts as a carrier for many ions and watersoluble substances (calcium, bilirubin, fatty acids). It also acts to maintain osmotic pressure. MLAB 2401 - Clinical Chemistry Lab Manual H 137
Albumin is increased in dehydration. It is decreased in renal disease (nephrotic syndrome with protein lost in urine), liver insufficiency with decreased albumin synthesis, severe malnutrition, acute and chronic inflammation, malignancy, pregnancy, and burns. The globulin fraction of protein can be obtained from electrophoresis or calculated by subtracting the albumin from the total protein. The quantity of the globulin fraction is required to calculate the A/G ratio. Methods of Determination 1. TOTAL PROTEIN a. Kjeldahl Method In this determination, the total nitrogen (protein nitrogen plus nonprotein nitrogen) is determined in the serum by Nessler's reaction after the proteins are converted to ammonium sulfate. Calculations are needed to compensate for nonprotein nitrogen measured and to convert nitrogen measurement to protein concentration. Each gm of nitrogen represents 6.25 grams of protein.) b. Folin-Ciocalteu In this reaction, a deep blue colored compound is formed when a phenol reagent is added to alkalinized protein solution. c. Refractometer The refractive index of an aqueous solution increases directly with an increase in solute concentration. Even though changes in concentration of different substances may affect the refractive index unequally, the concentrations of electrolytes and small organic molecules in serum usually do not vary greatly; hence, a change in refractive index is indicative of a change in protein concentration. A rapid and direct way of measuring the serum total proteins requiring only one drop of serum. Values obtained by refractive index correlate well with those found by the Biuret method, but discrepancies should be expected when abnormal serum proteins (paraproteins) are present or in specimens that are lipemic, icteric, or hemolyzed. d. Biuret Method All soluble proteins, on addition of a copper salt in alkaline solution (NaOH), give purple colored complex which is generally known as biuret. This method uses a standardized alkaline copper tartrate solution. The reaction occurs in the peptide bonds of tripeptides or larger. 2. ALBUMIN a. Electrophoresis b. Biuret Method The globulin in serum is precipitated by the addition of 23% sodium sulfate (salting out). Ethyl-ether is added to help separate the precipitated globulin by centrifugation. The chemical determination of albumin is based on the formation of a purple colored complex (biuret) between alkaline copper sulfate and the albumin. c. Direct Dye-Binding In recent years, the ability of albumin to combine with dyes has been utilized. Methyl orange, bromcresol green (BCG) and 2,4-hydroxyazobenzene benzoic acid (HABA) are the colored dyes most commonly used. Under ideal conditions, the dye binds to serum albumin, but not to other proteins in serum. H 138 MLAB - Clinical Chemistry 2401 Lab Manual
2. GLOBULIN - Globulin is calculated by subtracting the measured albumin from the measured total protein. Globulin = Total Protein - Albumin Equipment 1. Spectrophotometer 4. Albumin Color Reagent 2. Cuvettes & Pipets 5. NaCl solution (0.85%) 3. Biuret Reagent References Kaplan, Alex. Clinical Chemistry. Sigma Technical Bulletins, #540, #630 Bishop. Clinical Chemistry, pages 180-184. Procedure Total Protein - Sigma 541-2 Reagents Biuret Reagent - A standardized alkaline copper tartrate solution (copper sulfate and sodium hydroxide) Specimen Fresh serum Steps 1. Label test tubes for blank, standard(s), controll(s) and patients. 2. Add 0.05 ml sample to appropriate tubes. 3. Add 2.5 ml Biuret reagent to each tube. 4. Allow to stand 10-15 minutes at room temperature. 5. Read absorbance at 540 nm using blank to zero the spectrophotometer. Results 1. If a series of standards are analyzed, a standard curve can be used to determine the concentration of the unknowns. 2. But if only one standard is analyzed in the test run, the Total Protein is calculated as follows: MLAB 2401 - Clinical Chemistry Lab Manual H 139
Expected Values Adult normal = 6.0-8.2 g/dl Albumin Reagents Albumin reagent bromcresol green Specimen Fresh serum Steps 1. Make 1:5 dilution of each standard, control, and sample using 0.85% NaCl (normal saline) solution. 2. Label test tubes for blank standard, control and tests. 3. To each tube, add 3.0 ml albumin color reagent. 4. Add 0.1 ml of diluted sample to appropriate tubes. To blank tube, add 0.1 ml NaCl. 5. Mix well and allow to stand at room temperature for 10 minutes. 6. Read absorbance of each test at 630 nm using blank to zero the spectrophotometer. Results Expected Values Normal Range = 3.8-5.1 g/dl H 140 MLAB - Clinical Chemistry 2401 Lab Manual
Protein Worksheet Name Date I. Total Protein Wavelength Linearity Spectrophotometer Used Identification Absorbance Concentration (units) Refractometer Reading Control 1 Control 2 Calculation formula(s) and examples Total Protein Quality Control Your Results Controls range of expected results. In control? Yes / No Level 1 ID Level 2ID Accepting Patient Results? Reason MLAB 2401 - Clinical Chemistry Lab Manual H 141
II. Albumin Wavelength Linearity Spectrophotometer Used Identification Absorbance Concentration (units) Control 1 Control 2 Calculation formula(s) and examples Level 1 ID Level 2ID Albumin Quality Control Your Results Controls range of expected results. In control? Yes / No Accepting Patient Results? Reason III. Globulin Identification Concentration (units) Control 1 Control 2 Calculation formula(s) and examples H 142 MLAB - Clinical Chemistry 2401 Lab Manual
Study Questions Name Instructions: Based on information presented in lecture and lab notes, answer the following questions. Legibly write your answers in the space provided. Unless otherwise indicated, each question is worth one point. Date 1. Plasma proteins are synthesized in the. 2. What are the two major fractions of serum proteins? 3. Explain why serum rather than plasma is the preferred specimen for total protein determinations. 4. Which blood protein group is the smallest in molecular weight? 5. The biuret method measures the presence in the protein molecule of A. nitrogen B. peptide bonds C. tyrosine D. tryptophane 6. Common ingredients of all biuret reagents are A. sodium potassium tartrate and NaOH B. sodium tungstate and sulfuric acid C. copper sulfate and sulfuric acid D. copper sulfate and sodium hydroxide E. cuprous oxide and sodium hydroxide 7. What is the physiological role of albumin? 8. The globulin concentration of a patient with a total protein of 6.9 gm/dl and an albumin of 3.4 gm/dl is. 9. In the Kjeldahl Method, what is actually being measured? MLAB 2401 - Clinical Chemistry Lab Manual H 143
10. List three (3) dyes that have been commonly used in the quantitation of albumin. 11. The absorbance of the patient's sample in an albumin procedure is 0.376. The 5.0 gm/dl standard gave an absorbance of 0.355. What should be reported to the doctor? 12. Briefly explain why normal saline rather than distilled water is used for protein or albumin dilutions. H 144 MLAB - Clinical Chemistry 2401 Lab Manual