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WEEK 5 - Proteins.pptx

This document provides information about proteins in the blood. It discusses that proteins are composed of amino acids and are involved in many cellular processes. There are two major groups of serum proteins - albumins and globulins. Albumins comprise 60% of serum proteins and are made in the liver, serving as transport proteins. Globulins comprise the remaining 40% and include antibodies and proteins involved in blood coagulation and iron transport. The document also provides details on methods for measuring total proteins, albumin, and globulins in serum and considerations that can affect protein tests.

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PROTEINS Prepared by: UGEE BON S. FERRANCULLO,RMT Faculty Member RCI College of Medical Laboratory Science
INTRODUCTION: Proteins are essential components of cells and body fluids. They are formed from chains of amino acids. Some amino acids are made by the body, whereas others must be provided bydietary protein.Proteins are synthesized in the liver and secreted by the hepatocyte into the circulation except immunoglobulins which takes place in the plasma cell. These are macromolecules composed of polymers of covalently linked amino acids that are involved in every cellular process. It repairs body tissue, transports metabolic substances; maintain osmotic pressure and blood pH and acts as biocatalyst.
INTRODUCTION: Two major groups of serum proteins are the albumins and the globulins. ALBUMIN GLOBULIN • Albumins comprise approximately 60% of total serum proteins • The albumins are made in the liver and are homogeneous in structure. They serve as transport proteins and help maintain fluid balance in the body. • Globulins comprise the 40% of total serum protein. • The globulins are a heterogeneous group of molecules. Antibodies, blood coagulation proteins, enzymes, and proteins that transport iron are all serum globulins.
ALBUMIN: Made by the liver and makes up about 60% of the total protein. Albumin keeps fluid from leaking out of blood vessels, nourishes tissues, and transports hormones, vitamins, drugs, and substances like calciumthroughout the body. The reference range for serum albumin is 3.8 to 5.0 g/dL (38–50 g/L).
ALBUMIN Concentration of this protein is inversely proportional to the severity of the liver disease thus classified as negative acute phase reactant. In hepatic circulatorydisorder,its concentration reflects the shift of protein and fluid into ascites and its important contribution to the plasma oncoticpressure. Measurement for albumin is done to correlate its result to its clinical significance. Causes of hypoalbuminemia(decreased levels of albumin) and hyperalbuminemia (increased levels of albumin) corresponds with clinical diseases accordingly.
GLOBULINS Globulins make up the remaining 40% of proteins in the blood. The globulins are a varied group of proteins, some produced by the liver and some by the immune system. They help fight infection and transport nutrients. It is usually measured by subtracting the value of serum albumin from the total protein concentration. It consist of α1, α2, β, µ fractions, each fraction consists of a number of different proteins with different functions. These globulin fractions, together with albumin are the major proteins contribute to serumelectrophoresis. Globulin = Total Protein- Albumin ReferenceValue:2.3-3.5 g/dL (23-35 g/L)
SERUM ELECTROPHORESIS:
SERUM ELECTROPHORESIS: Abnormal Serum ElectrophoresisPattern: 1) Gamma Spike Multiple Myeloma 2) Beta-Gamma Bridging Hepatic Cirrhosis 3) Alpha 2- GlobulinBand Spike Nephrotic Syndrome 4) Alpha 1- GlobulinFlat Curve- JuvenileCirrhosis 5) Spikes of alpha1, alpha2and beta globulinband- Inflammation
Albumin/Globulin ratio is determined to validate if globulin is higher than albumin. If globulin is greater than albumin it is known as inverted A/G ratio seen in some diseases. ALBUMIN/GLOBULIN RATIO
Total Protein Determination: 1. Kjeldahl Method: - reference method but not routinely used. - it is based on measurement of nitrogen contentof protein. -Acid digestion with H2SO4 in the presence of a catalyst releasing ammonium ions from nitrogen containing compounds. The ammonium ions are then quantified by: • Titration w/ alkali • Nesslerization • Berthelot Reaction - end product:Ammonia
Total Protein Determination: 2. Colorimetric Methods: • Biuret Method: - most widely used method - it requires 2 peptide bonds and an alkaline medium. - end product: Violet-ColoredChelate • Folin-Ciocalteu Reagent - Involves oxidation of phenolic compound to give a deep blue color • Coomasie Brilliant Blue Dye - Sensitive for detection down to 1 ug of protein • Ninhydrin - Produces a violet color by reacting to primary amines • Greenberg -Measures TP and Albumin using the Folin- Ciocalteu Reagent which oxidizes phenolic compounds such as tyrosine, tryptophan, phenylalanine and histidine to give deep blue color. • Looney-Walsch - Measures TP and Globulin which precipitates protein using SAA and TCA.
Total Protein Determination: 3. Refractive Index • Accurate for measuring serum protein concentrationas dissolved for solute for levels above 2.5 g/dL 4. Specific Gravity • Simple, used widely as a screening test forprotein 5. Turbidimetric • Measure protein in CSF and urine with additional of SSA and TCA
Principles of the Test Method: Total Protein: Biuret Method Cupric ions complex the groups involved in the peptide bond forming a violet colored- chelate which is proportional to the number of peptide bonds present and reflects the total protein level at 545nm. Reference Value: Adult: 6.5-8.3 g/dL(65-83 g/L) Conversion Factor:10  It requires at least 2 peptide bonds and an alkaline medium.  Reagents:  Rochelle Salt  Alkaline Copper Sulfate  NaOH  Potassium (K) Iodide
Biuret Method: Blank standard Sample Blank 20 ul Standard 20 ul Sample (serum) 20 ul Reagent 1000 ul 1000 ul 1000 ul
Violet colored- chelate Mix and incubate for 5 minutes at 25 ◦C and read absorbance at 540- 545 nm within 60 minutes.
Special Consideration for ProteinTest: • In samples collected during the morning, the proteins concentrations are 3 % higher than in samples collected during the afternoon. • Changing the patient position from standing up to lying down, induce a passage of liquid from extravascular to intra-vascular compartment that may reduce the relative concentrations of proteins and of the analytes related to the proteins up to 10%. • During the pregnancy, due to the rise of intra-vascular liquid, the total protein decreases significantly, up to 14% reduction..
Special Consideration for ProteinTest: • Tourniquet use for more than 3 minutes increases total protein concentration in blood up to 5%. Physical exercises increase the proteinsconcentrations. • Bilirubinup to 32 mg/dL, hemoglobin up to 130 mg/dL and triglycerides up to 500 mg/dL do not interferesignificantly • Triglycerides values between 500 and 1100 mg/dL provide positive interferences which can be minimized by using the blank of the sample using 0.85% NaCl as diluent, or photometry of bichromatic reading with primary filter at 545 nm and secondary filter at 700 nm. • Hemoglobinvalues over 130 mg/dL provide positive interferences which can not be minimized by using the blank of the sample.
Special Consideration for ProteinTest: Linearity: Ranging from 1.0 to 14.0 g/dL. If total proteins concentration exceeds 14.0 g/dL, the sample must be diluted with 0.85% NaCl. Multiply the result by the appropriatedilution factor. Calculation: Total Proteins = Abs unknown/ Abs. standardx 4 (g/dl)
Test Methods for Albumin:
Principle of the test method for Albumin: Salt precipitation method  Globulinsare precipitated in high salt concentrations;albumin in supernatant is quantitatedby biuret reaction. Dye Binding Method  Albumin binds to dyecauses shift in absorption maximum Dyes used formeasurement: • Bromocresol green (most commonly used dye; used extensively in automatic analyzers) • Bromocresol purple (most specific) • Methyl Orange • HABA (2,4’-hydroxyazobenzene-benzoicacid)
Bromocresol Green Dye Method Blank standard Sample Blank 10 ul Standard 10 ul Sample 10 ul Reagent 1000 ul 1000 ul 1000 ul Mix and incubate for 10 minutes at 25 ◦C and read absorbance at 540 nm within 60 minutes ReferenceValue:Adult 3.8-5.3 g/dL (38-53 g/L) Conversion Factor:10
References: Bishop, Michael L, Foody, Edward P .and Schoeff, Larry E. Clinical Chemistry Principles, Techniques, and Correlations 7th edition Rodriguez, Maria Teresa T. Clinical Chemistry Review Handbook for Medical Technologist .
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