Amino acids: continuation

Methods of Analysis:
1. Total Nitrogen
- A total nitrogen determination measures all chemically bound nitrogen in
the sample. The method can be applied to various biologic samples,
including plasma and urine. In plasma, both the total protein and
nonprotein nitrogenous compounds, such as urea and creatinine, are
measured. The analysis of total nitrogen level is useful in assessing
nitrogen balance. Monitoring the nitrogen nutritional status is particularly
important in patients receiving total parenteral nutrition, such as
individuals with neurologic injuries who are sustained on intravenous
fluids for an extended period.
- The method for total nitrogen analysis uses chemiluminescence
o The sample, in the presence of oxygen, is heated to a high
temperature (1100 20*C). Any chemically bound nitrogen is
oxidized to nitric oxide. The nitric oxide is then mixed with ozone,
nitrogen dioxide molecule (NO2). When this molecule decays to the
ground state, it emits chemiluminescent light, which is detected,
amplified and converted to an electronic signal, proportional to the
total nitrogen content in sample. This chemiluminescence signal is
compared with that of a standard for quantitation
Total Proteins
-

The specimen most often used to determine the total protein is serum rather
than plasma. A fasting specimen is not needed. Interferences in some of the
methods occur in the presence of lipemia; hemolysis falsely elevates the total
protein result because of the release of RBC proteins into the serum
The reference interval for serum total protein is 6.5-8.3 g/dL (65-83 g/L) for
ambulatory adults. In the recumbent ratio, the serum total protein
concentration is 6.0-7.8 g/dL (60-78 g/L). This lower normal range is a result
of shifts in water distribution in the extracellular compartments. The total
protein concentration is lower at birth, reaching adult levels by age 3 years.
There is a slight decrease with age. Lower total protein levels are also seen in
pregnancy

2. Kjeldahl
- The classic method for quantitation of total protein which determines
nitrogen
- In this method, an average of 16% nitrogen mass in protein is assumed to
calculate the protein concentration
- The actual nitrogen content of serum proteins varies from 15.1% to 16.8%.
Therefore, error is introduced if a protein standard (calibrated with the

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Kjeldahl method) is used that differs in composition from the serum specimen
to be analyzed, because the percentage of nitrogen will not be the same.
The method also requires the assumption that no proteins of significant
concentration in the unknown specimen are lost in the precipitation step.
The serum proteins are precipitated with an organic acid such as TCA
(trichloroacetic acid) or tungstic acid
The nonprotein nitrogen is removed with the supernatant
The protein pellet is digested in H2SO4 with heat (340-360*C) and a catalyst,
such as cupric sulfate, to speed the reaction. Potassium sulfate is also
introduced to increase the boiling point to improve the efficiency of digestion.
The H2SO4 oxidizes the C, H and S in protein to CO2, H2O and SO2. The
nitrogen in the protein is converted to ammonium bisulfite (NH4HSO4)
measured by adding alkali and distilling the ammonia into a standard boric
acid solution. The ammonium borate (NH4HBO3) formed is then titrated with
a standard solution of HCl to determine the amount of nitrogen in the original
protein solution

3. Refractometry
- Useful when a rapid, easy method that requires a small volume of serum is
needed
- The velocity of light is changed as it passes the boundary between two
transparent layers (air and water), causing the light to be refracted (bent).
When a solute is added to the water, the refractive index at 20*C of 1.330 for
pure water is increased by an amount proportional to the concentration of the
solute in solution. Because the majority of the solids dissolved in serum are
protein, the refractive index reflects the concentration of protein
- Practical for many purposes to estimate total serum protein by refractomeric
methods
- In addition to protein, serum contains several nonprotein solids, such as
electrolytes, urea and glucose that contribute to the refractive index of
serum. Therefore, the built-in scale in the refractometer must be calibrated
with a serum if a known protein concentration that also has the nonprotein
constituents present. An assumption is made that the test samples contain
these other solutes in nearly the same concentration as in the calibrating
serum
- Error is introduced when these substances are increased or when the sample
is icteric, lipemic or hemolyzed. The refractive index is also temperature
dependent and some refractometers incorporate a built-in temperature
correction. The total protein is commonly measured with a hand-held
refractometer. A drop of serum is placed by capillary action between a
coverglass and the prism. The refractometer is held so that light is refracted
through the serum layer. The refracted rays cause part of the field of view to
be light, producing a point at which there is a sharp line between light and
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dark. The number of grams per liter at this line on the internal scale is read.
The temperature is corrected in the TS meter by a liquid crystal system

4. Biuret
- Most widely used method and the one recommended by the International
Federation of Clincal Chemistry expert panel for the determination of total
protein
- In this reaction, cupric ions (Cu2) complex with the groups involved in the
peptide bond. In an alkaline medium and in the presence of at least two
peptide bonds, a violet-colored chelate (abundant metal in complex) is
formed.
- The reagent also contains sodium potassium tartrate, to complex cupric ions
to prevent their precipitation in the alkaline solution and potassium iodide,
which acts as an antioxidant
- The absorbance of the colored chelate formed is measured at 540 nm
- Small peptides react, the color of the chelate produced has a different shade
than that seen with larger peptides. The color varies from a pink to a reddish
violet
The color that is formed is proportional to the number of peptide bonds
present and reflects the in addition to the NHCO group that occurs in the
peptide bond, cupric ions will react with any compound that has two or more
of the following groups: NHCH2 and NHCS
- The method was named because a substance called biuret
(NH2CONHCONH2) reacted with cupric ions in the same manner. There must
be a minimum of two of the reactive groups; therefore, amino acids and
dipeptides will not react total protein level. However, in the presence of
abnormally small proteins, such as those seen in multiple myeloma, the Cprotein concentration is underestimated due to the lighter shade of color
produced. Lipemia in the sample is an interferent.

5. Dye Binding
- Based on the ability of most proteins in serum to bind dyes, although the
affinity with which they bind may vary
- Bromphenol blue, Ponceau S, amido black 10B, lissamine green and
Coomasie brilliant blue have been used to stain protein bands after
electrophoresis
- A dye-binding method, Coomassie brilliant blue 250, relies on the binding of
Coomassie brilliant blue 250 to protein causing a shift in the absorbance
maximum of the dye from 465 to 595 nm
- The increase in absorbance at 595 is used to determine the protein
concentration

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-

Although the method is simple and fast, the unequal dye-binding responses
of individual proteins require caution when applying this test to the complex
mixture of protein found in serum

6. Fractionation, Identification and Quantitation of Specific Proteins

- In the assay of total serum proteins, useful diagnostic information can be
obtained by determining the albumin fraction and the globulins
- A reversal or significant change in the ration of albumin and total globulin,
the albumin/globulin (A/G) ratio, is found in diseases of the kidney and liver
- To determine the A/G ratio, total protein and albumin are measured and
globulins are calculated by subtracting the albumin from the total protein
(total protein/albumin globulins)
The following are methods for measuring protein fractions:
-

Salt fractionation
o Fractionation of protein is done using precipitation
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Globulins are separated from albumin by salting out, using sodium salt
to cause precipitation of the globulins
o The albumin that remains in solution in the supernatant is then
measured by any of the routine total protein methods
o Salting out is not used today because direct methods are available that
react specifically with albumin in a mixture of proteins
Albumin
o The most widely used methods for determining albumin are dyebinding procedures
o The pH of the solution is adjusted so that albumin is positively charged
o The albumin is attracted to and binds to an anionic dye by electrostatic
forces. When bounds to albumin, the dye has a different absorption
maximum than the free dye. The amount of albumin is calculated by
measurement of the absorbance of the albumin-dye complex
o A variety of dyes have been used, including methyl orange, 2,42hydroxy-azobenzene-benzoic acid (HABA), bromcresol green (BCG) and
bromcresol purple (BCP)
o Methyl orange is nonspecific for albumin; g-lipoproteins and some a1
and a2-globulins also will bind to this dye
o HABA has a low sensitivity but is more specific for albumin
o In addition, several compounds, such as salicylates, penicillin,
conjugated bilirubin and sulfonamides, interfere with the binding if
albumin to HABA
o BCG is not affected by interfering substances such as bilirubin and
salicylates; however, hemoglobin binds to BCG. For every 100 mg/dL of
hemoglobin, albumin is increased by 0.1 g/dL
o The specificity of the reaction for albumin can be improved by taking
absorbance readings within a standardized short interval (15 minutes
due to a-globulins contributing significantly to the absorbance at long
incubation) after mixing
o BCP is an alternative dye used for albumin determinations, binding
specifically to albumin
o BCP is not subject to most interferences, is precise and exhibits
excellent correlation with immunodiffusion reference methods
o

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Total Globulins
o Another approach to fractionation of proteins is the measurement of
total globulins
o Albumin can then be calculated by subtraction of the globulin from the
total protein
o The total globulin level in serum is determined by a direct colorimetric
method using glyoxylic acid. Glyoxylic acid, in the presence of Cu2 and
in an acid medium (acetic acid and H2SO4), condenses with
tryptophan found in globulins to produce a purple color
o Albumin has approximately 0.2% tryptophan, compared with 2%-3%
for the serum globulins
o When calibrated using a serum of known albumin and globulin
concentrations, the total globulins can be determined
o The measurement of globulins based on their tryptophan content has
never come into common use because of the ease and simplicity of the
dye-binding methods for albumin
Electrophoresis
o When an abnormality is found in the total protein or albumin, an
electrophoresis is usually performed
o If an abnormality is seen on the electrophoretic pattern, an analysis of
the individual proteins within the area of abnormality is made
Serum Protein Electrophoresis
High-Resolution Protein Electrophoresis
Capillary Electrophoresis
Isoelectric Focusing
Immunochemical methods reaction of the protein (antigen) and its antibody
is measured
o Methods using various modifications of this principle include:
Radial immunodiffusion (RID)
Immunoelectrophoresis (IEP)
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Immunofixationelectrophoresis (IFE)
Electroimmunodiffusion
Immunoturbidimetry, and
Immunonephelometry

Proteins in other body fluids

-

Peritoneal, pleural, seminal, vaginal fluids and tears

Most often urine and CSF

Urinary protein
1. Qualitative tests reagent test strip (common)
2. Quantitative assays
-

12 or 24-hour urine The results are reported generally in terms of weight of

protein per 24 hours by calculating the amount of protein present in the total
volume of urine collected during that time

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Cerebrospinal Fluid Proteins

-

CSF is formed in the choroid plexus of the ventricles of the brain by

ultrafiltration of the blood plasma
Protein measurement is one test that is usually requested on CSF, in addition
to glucose level and differential cell count, culture and sensitivity
The accepted reference interval for patients between 10 and 40 years of age
is 15-45 mg/dL of CSF protein
Increased total CSF proteins there is increased permeability of the capillary
endothelial barriers
o Bacterial, viral and fungal meningitis; traumatic tap; multiple sclerosis;
obstruction; neoplasm; disk herniation; and cerebral infarction
o The degree of permeability can be evaluated by measuring the CSF
albumin and comparing it with the serum albumin. Albumin is usually
used as the brain barrier
Low CSF protein values
o Hyperthyroidism and when fluid is leaking from the CNS

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