Total Protein Measurement methods

Human proteins – More than 50,000 Within one cell 3000 to 5000 Serum – More than 1400 different proteins

Interim Consensus Reference Intervals for 14 Plasma Proteins In Human Serum *Values are slightly lower in fresh samples (assayed <8 hr after draw)

Distribution – Vascular,extravascular space – Kind and proportions of individual proteins Molecular size Specificity of some of their transport mechanisms Disease

Plasma proteins Alterations of plasma proteins – Genetic origin – Physiological – Pathological Clinical findings Technical

Plasma proteins Patient ’s endocrine status – Rates of hepatic synthesis – Steroid hormones Inflammatory acute-phase reaction Mask effect

Increased Plasma Levels – acute dehydration no clinical utility Synthetic rate and intravascular—extravascular shifts Decreased Plasma Levels – Decreased synthesis Primary or genetic – Analbuminemia Acquired – Inflammatory processes – Increased catabolism Utilization or loss

ANALYSIS OF PROTEINS Methods Specific quantitative assays (Individual proteins) – Immunochemical methods Nephelometry Turbidimetry RID Electroimmunoassay RIA or enzyme immunoassay (EIA) – very low concentrations Detection and identification – Electrophoresis Quantitative measurements of total protein

ANALYSIS OF PROTEINS Methods Nephelometric and turbidimetric – Speed and ease – Formation of Ag-Ab complex – Light absorption & scattering

Assay Characteristics Limit of Detection – nephelometric methods 10 µg/mL – turbidimetric methods 20 to 30 µg/mL – RID methods 10 to 20 µg/mL – RIA methods nanograms per milliliter

Assay Characteristics Precision – Nephelometry and turbidimetry within-run coefficients of variation (CVs) of less than 5% equilibrium methods – less precise than the kinetic methods – RID and EIA systems 5 to 1 5% – RIA 5 to 10%

Assay Characteristics Turnaround Time – Nephelometric and turbidimetric Kinetic methods – Fast,within minutes Equilibrium methods – Up to 1 h – RID 24 to 48 h of incubation – RIA Several hours

Electrophoresis, Laboratory Considerations Buffer – Barbital ionic strength of 0.05 pH 8.6 Sample is 3 to 5 µL Support medium – 1.5 mA per 2-cm width of cellulose acetate – 10 mA per 1-cm width of agarose medium

Usually five bands (albumin, α 1, α 2, β and y) – a sixth band Serum is fresh buffer containing Ca 2+ ions Densitometry – quantification of individual bands Stain poorly – high proportions of lipid – Carbohydrate

Mask effect – Too low concentrations – over- shadowed Visualize bands – Amido black and Ponceau S – Coomassie brilliant blue Dried, record Lipoproteins – Migrate variable Normal control serum

Hyperproteinemia – Dehydration Inadequate water intake Excessive water loss – Vomiting, diarrhea, Addison’s disease, or diabetic acidosis Hypoproteinemia – Hemodilution Recumbent position – Decreases total protein concentration by 0.3 to 0.5 gIdL All the individual plasma proteins to the same degree Intravenous infusions

Mild hyperproteinemia – Increases in APR and polyclonal immunoglobulins Marked hyperproteinemia – high levels of the monoclonal immunoglobulins Multiple myeloma

Methods (total protein) Biuret Method Peptide bonds react with Cu 2+ ions in alkaline solutions to form a colored product – Presence of peptide bonds Tri-, oligo-, and polypeptides react – By spectrophotometry at 540 nm – The intensity of the color produced is proportional to the number of peptide bonds Interference – Small peptides – Ammonium ions

Methods Biuret Method Detection limit – 1 and 15 mg of protein Simple, sufficiently precise A fasting serum or plasma – to decrease lipemia Hemolysis should be avoided

Direct Photometric Methods – Absorption of ultraviolet (UV) light – at 200 to 225 nm – at 270 to 290 nm Limitations – Uneven distribution aromatic ring – Free tyrosine and tryptophan – Uric acid – Bilirubin

At 200 to 225 nm – Peptide bonds are chiefly responsible for UV absorption Removal of small interfering molecules – by dilution – gel filtration

Dye-Binding Methods CBB binds to protonated amine groups of amino acid simple Fast, & linear up to 150 mg/dl Absorbance at 595 nm Limitation – Unequal affinities for dyes – Binding capacities of individual proteins – Inability to define a consistent material for use as a calibrator

Positive interferences – Tolbutamide, high concentrations of urea Negative interferences – Very high concentrations of NaCl – Hydrogen chloride (HCl)

FoIin-Ciocalteu (Lowry) Method Reaction with Cu 2+ in alkaline solution to form copper—peptide bond protein complexes Folin-Ciocalteu reagent – Phosphotungstic-phosphomolybdic acid Tyrosine or tryptophan Reduce Cu 2+ Reduced Cu 2+ form complex with Folin-Ciocalteu reagent – Measurement at nm Detection limit – 1 0—60  g/mL

FoIin-Ciocalteu (Lowry) Method Measuring total protein in urine or CSF Limitation – Positive interference Drugs such as salicylates, chlorpromazine, tetracyclines, and some sulfa drugs – Removal of interferences Gel filtration

KjeldahI’s Method Acid digestion – Convert nitrogen in the protein to ammonium ion Concentration of ammonia nitrogen – Double iodides (potassium and mercuric) form a colored complex with ammonia Limitation – Time consuming Impractical for wide spread routine use

Precipitation methods Precipitation of protein – Sulfosalicylic acid – Trichloroacetic acid (TCA) Scatter incident Light TCA precipitation ( Another approach ) – Addition of biuret reagent to the precipitate Suitable for a fairly large volume of specimen(urine)

Comments Specimen Collection and Storage – Test specimens must be – Nonhemolyzed – Cell-free – Lipemic sera should not be assayed – Test tubes must remain covered Dust and dirt particle contamination – Storage conditions – Use of outdated reagents

Calibration of Total Protein Methods Reference material – Bovine or human albumin Biuret method – Serum (or serum pool) with a normal albumin/globulin ratio Precipitation methods Dye-binding methods Calculations – Calibration curve consisting of 8 to 15 points