Introduction

• The key roles which plasma proteins play in bodily function, together with the relative ease of assaying them, makes their determination a valuable diagnostic tool as well as a way to monitor clinical progress.

• In very general terms, variations in plasma protein concentrations can be due to any of three changes:

– rate of protein synthesis,

– rate of removal,

– the volume of distribution.

Proteins: Common properties• In spite of functional differences between the various

serum proteins, they have certain common biophysical and biochemical properties. These include:– a basic composition of carbon, hydrogen, nitrogen

and oxygen; – a backbone of covalent peptide bonds which join

the amino acid units together; and – absorption maxima in the ultraviolet region.

• Based on these properties, laboratory methods have been developed to determine the concentration of proteins in serum,

Serum Total Protein• Serum total protein, also called plasma total protein or total

protein, is a biochemical test for measuring the total amount of protein in blood plasma or serum.

• Protein in the plasma is made up of albumin and globulins.

• Note: the globulin in turn is made up of α1, α2, β, and γ globulins.

• These fractions can be quantitated using protein electrophoresis, but the total protein test is a faster and cheaper test that estimates the total of all fractions together.

• The traditional method for measuring total protein uses the biuret reagent, but other chemical methods are also available.

Methods of Total Protein Analysis

• Method 1: Kjeldahl; quantitative, protein nitrogen determination

• Method 2: Biuret; quantitative, increased absorption at 540 nm;

Specimen• Serum and plasma may be used, and all usually

yield comparable results, though, because of the presence of fibrinogen, plasma levels for total protein are 2 to 4 g/L higher than serum levels.

• A fasting specimen is not required but may be desirable to decrease lipemia.

• Total protein is stable in serum and plasma for – 1 week at room temperature, – and for at least 2 months at –20° C

Hypoproteinemia– Malnutrition and/or malabsorption– Excessive loss as in renal disease, GI leakage,– excessive bleeding, severe burns– Excessive catabolism– Liver disease

Hyperproteinemia– Dehydration– Monoclonal increases– Polyclonal increase

• Only disorders affecting the concentration of albumin and/or the immunoglobulins will give rise to abnormal total protein levels.

• Other serum proteins are never present in high enough concentrations for changes to have a significant overall effect.

The Biuret Method

• The Biuret reagent is made of (NaOH) and copper (II) sulfate (CuSO4), together with potassium sodium tartrate (KNaC4H4O6).

– A blue reagent which turns violet in the presence of proteins.

• The Sodium hydroxide does not participate in the reaction at all, but is merely there to provide an alkaline medium so that the reaction can take place.

Principle: Biuret Method

• Peptide bonds of proteins react with tartrate-complexed cupric ions in alkaline solutions to form a colored product.

• In a positive test, a copper(II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution.

• A violet color indicates the presence of proteins. • The intensity of the color, and hence the absorption at

540 nm, is directly proportional to the protein concentration, and can be determined spectrophotometrically at 540 nm.

Reference range

• Reference range for total proteins is 66.6 to 81.4 g/L• Results for males are approximately 1 g/L higher than results

for females; this difference is probably not of clinical significance.

• In newborns, the mean serum protein concentration is 57 g/L, increasing to 60 g/L by 6 months and to adult levels by about 3 years of age.

• Serum protein levels of premature infants can be much lower than that of full term infants, ranging from 36 to 60 g/L.

Albumin

• Albumin is the most abundant circulating plasma protein (40–60 % of the total)

• Playing important roles in the maintenance of the colloid osmotic pressure of the blood, in transport of various ions, acids, and hormones.

• It is a globular protein with a molecular weight of approximately 66,000 D and is unique among major plasma proteins in containing no carbohydrate.

• It has a relatively low content of tryptophan and is an anion at pH 7.4.

Analysis Methods• Method 1: Precipitation; quantitative

– Salt fractionation, Acid fractionation– Principle of analysis: Changes of net charge of protein

result in precipitation• Method 2: Tryptophan content; quantitative

– Principle of analysis: – Glyoxylic acid + tryptophan in globulin Purple chromogen

(Amax, 540 nm); Total protein – globulin = albumin.• Method 3: Electrophoresis; quantitative

– Principle of analysis: Albumin is separated from other proteins in electrical field; percent staining of albumin fraction multiplied by total protein value

• Method 4: Dye binding, quantitative– Methyl orange; BCG (bromcresol green); BCP (bromcresol

purple);

• Method 5: Dye binding; semiquantitative– Bromphenol blue in test strip changes color from yellow to

blue in presence of albumin most commonly used test for urine protein

• Specimen: Serum is the specimen of choice, but heparinized plasma can also be used if precautions are taken to prevent heparin interferences.

• Interfering Factors– Albumin is decreased in:

• Pregnancy (last trimester, owing to increased plasma volume)

• Oral birth control (estrogens) and other drugs. • Prolonged bed rest.• IV fluids, rapid hydration, overhydration.

Albumin Reference Interval for Serum

Age Men (g/L) Women (g/L)

21–44 33.3–61.2 27.8–56.5

Clinical Significance• Plasma albumin levels, although important for management

and follow-up, have very little value in clinical diagnosis. • Hyperalbuminemia is usually attributable to

• dehydration or hemoconcentration.• Hypoalbuminemia is usually the result of

• hemodilution, • a rate of synthesis less than the albumin loss, • diseases that cause a large albumin loss from urine,

skin, or intestine, • increased catabolism observed in fevers, untreated

diabetes mellitus, and hyperthyroidism.

Dye-binding Techniques

• Serum albumin is most often assayed using dye-binding techniques.

• Albumin preferentially binds to anionic dyes that do not attract globulins

• Bromcresol purple (BCP) and bromcresol green (BCG) are most commonly used

• The amount of light absorbed by the albumin –dye complex is proportional to the amount of albumin present