This document discusses several methods for determining total protein concentration and specifically measuring albumin concentration in biological fluids. It provides details on the Kjeldahl method, Biuret method, direct optical methods, Lowry (Folin-Ciocalteu) method, turbidimetric and nephelometric methods for measuring total protein. For albumin, it describes its structure and function, clinical significance of increased and decreased plasma concentrations, and dye-binding methods for quantifying albumin concentration.

1. PROTEIN ESTIMATION
Determination of Total Protein
Plasma normally contains about 6.5 to 8.5 g/dL protein, and serum about 4% less.
Determination of total protein in biological fluids in some respects represents a
greater challenge than analysis of a specific protein, because variable protein
composition of biological fluids leads to variable carbohydrate composition,
charge, and physical characteristics of proteins in the mixture. Many methods of
protein analysis respond differentially to different proteins and present problems
when applied to specimens of varying protein composition.
Kjeldahl Method. This method is rarely used in clinical laboratories but is of
historical importance and is sometimes used as a reference method. Protein
nitrogen is converted to ammonium ion by heating with sulfuric acid in the
presence of a catalyst. Ammonium ion is measured by alkalinization, distillation,
and acid titration, or by Nessler’s reagent. Protein is estimated to contain 16%
nitrogen (multiply N by 6.25). Errors in protein estimation occur if the amino acid
composition is unusual, and if nitrogen content differs from 16%. Nonprotein N,
such as from urea and amino acids, also is measured, so a protein precipitation step
may be required.
Biuret Method. Under strongly alkaline conditions, Cu2+ ions form multivalent
complexes with peptide bonds in proteins. Binding shifts the absorption spectrum
of Cu2+ ions to shorter wavelengths, leading to a color change from blue to violet
that has been termed the biuret reaction. Binding of Cu2+ ions to the organic
compound biuret yields a similar color change, hence the name. Absorbance
change from protein addition is measured spectrophotometrically at ≈540 nm,
and this serves as a relatively simple method for quantifying protein. Absorbance
changes at 540 nm also result from binding of Cu2+ ions by many compounds that
can form chelates with five- or six-member rings, where amino, amide, or hydroxyl
groups bind to Cu2+ ions. Such compounds include (1) serine, (2) asparagine, (3)
ethanolamine, (4) tris(hydroxymethyl)aminomethane, and (5) many others.

2. Direct Optical Methods. Absorbance of UV light at 200 to 225 nm and 270 to 290
nm have been used to measure protein concentrations and is commonly applied to
monitor chromatographic separations of peptides and proteins. Absorbance at 280
nm depends primarily on the tryptophan and tyrosine content of a protein. This
technique works best for purified proteins with known absorptivity. For complex
mixtures, accuracy and specificity suffer from variable content of tryptophan and
tyrosine and from absorbance of low molecular weight compounds such as free
amino acids, uric acid, and bilirubin. At 200 to 225 nm, peptide bonds are chiefly
responsible for UV absorbance (70% at A205); specific absorption by proteins at
these shorter wavelengths is 10 to 30 times greater than at 280 nm.70 Many low
molecular weight compounds such as urea also have absorbance at wavelengths
below 220 nm. Accurate measurement of proteins by this method may require
removal of low molecular weight molecules before absorbance measurements are
performed.
Lowry (Folin-Ciocalteu) Method. The detection limit of the Lowry method is
about100 times lower than that of the biuret method. Specimens are mixed with an
alkaline copper solution followed by addition of the Folin-Ciocalteu reagent.
Phosphotungstic acid and phosphomolybdic acid are reduced to tungsten blue and
molybdenum blue by copper complexed with peptide and by tyrosine and
tryptophan residues. Absorbanceof products is measured at 650 to 750 nm.
Reactivity of proteins varies with the content of tyrosine and tryptophan. Low
molecularweightcompoundsincluding tryptophan and tyrosine and drugs such
as salicylates, chlorpromazine, tetracyclines, and some sulfa drugs also give
positive interference. Analysis of a fluid such as urine with high concentrations of
phenolic compounds requires removal of low molecular weight substances before
protein is measured.70
Turbidimetric and Nephelometric Methods. Many different reagents have been
used to aggregate protein for turbidimetric or nephelometric assays, including (1)
trichloroacetic acid, (2) sulfosalicylic acid, (3) sulfosalicylic acid combined with
sodium sulfate, and (4) benzethonium chloride or (5) benzalkonium salts under
alkaline conditions. Precipitation methods for total protein assay depend on
forming a suspension of uniform, insoluble protein particles, which scatter incident
light. Albumin and globulins often give different reactivities in precipitation
methods.

3. ALBUMIN
The name albumin (L. albus = white) originated from the white precipitate formed
during the boiling of acidic urine from patients with proteinuria. Normally,
albumin is the most abundant plasma protein from the fetal period onward,
accounting for about half of the plasma protein mass. It is a major component of
most body fluids, including interstitial fluid, CSF, urine, and amniotic fluid. More
than half of the total pool of albumin is in the extravascular space.
Biochemistry of Albumin. Albumin has a nonglycosylated polypeptide chain of
585 amino acids and a calculated molecular weight of 66,438 Da. It has a heart-
shaped threedimensional structure stabilized by 17 intrachain S-S bonds.121 It is a
relatively stable protein, resisting denaturation up to higher temperatures than most
plasma proteins. Albumin is synthesized by hepatocytes. The synthetic The normal
plasma half-life of albumin is 15 to 19 days.reserve of the liver is substantial; in
nephrotic syndrome, albumin synthesis can increase threefold above normal.
Function of Albumin
1 serving as the major component of colloid osmotic pressure
2 serving as a transporter for a diverse range of substances, including fatty acids
and other lipids, bilirubin, foreign substances such as drugs, thiol-containing amino
acids, tryptophan, calcium, and metals.
Clinical Significance of Albumin
Increased Plasma Concentrations. Increased concentrations of albumin occur
with dehydration or artifactually from prolonged tourniquet time or specimen
evaporation prior to analysis. High albumin concentrations therefore suggest
problems with hydration or specimen handling.

4. Decreased Plasma Concentrations
Analbuminemia. Although affected individuals have plasma albumin
concentrations <0.5 g/L (≤1% of normal), symptoms often are absent or consist
ofmild edema, lipodystrophy, and dyslipidemia. No increased risk for
atherosclerosis has been noted. The plasma half-life of infused albumin in affected
individuals is prolonged to 50 to 60 days.
Inflammation.
Hepatic Disease. The liver has synthetic capacity to maintain albumin
concentrations until parenchymal damage or loss is severe, with loss of more than
50% of function
Urinary Loss/Kidney Disease. Normally, the glomerular filtration barrier
efficiently prevents entry into the urinary ultrafiltrate by proteins the size of
albumin or larger. Usually, only 1 to 2 g/d of albumin passes through the
glomerular barrier, and 99.9% of albumin in the glomerular ultrafiltrate is taken up
by proximal tubules of the kidney and degraded. Only about 10 mg/d of albumin is
normally excreted in urine. Small increases and urine albumin excretion to >30
mg/d are indicators of early stages of glomerular or tubular injury.105 This has
been termed microalbuminuria (“micro” refers to excretion of small amounts, not a
smaller form, of albumin).
Gastrointestinal Loss. Protein-losing enteropathy may result in losses as great as
those seen in the nephrotic syndrome.
Protein-Calorie Malnutrition. Albumin concentrations serve as a means of
detecting and monitoring protein-caloriemalnutrition,
Burn Injury. Patients with burn injury can experience severe losses of albumin
from wounds
Edema and Ascites. Edema and ascites rarely are the result of decreased plasma
albumin concentrations per se.

5. Dye-Binding Methods.
Dye-binding methods depend on shifts in the absorbance spectra of dyes when
they bind to albumin. bromcresol green (BCG) or purple (BCP) upon albumin
binding. The affinity of these dyes is higher for albumin than for other proteins,
providing some specificity for albumin. BCP generally is slightly more specific for
albumin and yields lower values than BCG This dye binds to polypeptide chains
under acidic conditions, resulting in decreased absorbance at 465 nm and increased
bsorbance at 595/620 nm. Dye-binding methods offer good sensitivity, and
pyrogallol red has become one of the most commonly used dyes for analysis of
fluids with lower protein concentrations such as urine and CSF. for maintain in
acidic ph succinate buffer is used