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, α1, α2, β and y)– a sixth band • Serum is fresh • buffer containing Ca2+ 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 Cu2+ 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 Cu2+ in alkaline solution to form copper—peptide bond protein complexes

• Folin-Ciocalteu reagent– Phosphotungstic-phosphomolybdic acid

• Tyrosine or tryptophan Reduce Cu2+ • Reduced Cu2+ form complex with Folin-Ciocalteu

reagent– Measurement at 650 - 750 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