Amino Acid Analysis

Blood samples for amino acid analysis should be drawn after at least a 6- to
8-hour fast to avoid the effect of absorbed amino acids originating from
dietary proteins. The sample is collected in heparin, and the plasma is
promptly removed from the cells, taking care not to aspirate the layer of
platelets and leukocytes. If this step is not performed with caution, the plasma
becomes contaminated with platelet or leukocyte amino acids, in which the
contents of aspartic acid and glutamic acid, for example, are about 100-fold
higher than those in plasma. Hemolysis should be avoided for the same
reason. Deproteinization should be performed within 30 minutes of
sample collection, and analysis should be performed immediately or the
sample should be stored at -20°C to -40°C.

Urinary amino acid analysis can be performed on a random specimen for

screening purposes; however, for quantitation, a 24-hour urine preserved with
thymol or organic solvents is required. Amniotic fluid also may be analyzed.

For preliminary screening, the method of choice is thin-layer

chromatography. The application of either one- or two-dimensional
separations depends on the purpose of the analysis. If one is searching for
a particular category of amino acids, such as branched-chain amino
acids, or even a single amino acid, usually one dimensional separations
are sufficient. Separation conditions can be selected in such a way as to
offer good resolution of the amino acid in question.

For more general screening, two-dimensional maps are essential. In two-

dimensional chromatography, the amino acids are allowed to migrate
along one solvent front, and then the chromatogram is rotated 90° and a
second solvent migration occurs. A variety of solvents have been used,
including butanol-acetic acid-water and ethanol-ammonia-water mixtures.
The chromatogram is visualized by staining with ninhydrin, which gives
most amino acids a blue color.

When the possibility of an amino acid disorder has been indicated in the
preliminary steps, amino acids can be separated and quantitated by cation-
exchange chromatography using a gradient buffer elution, a HPLC reversed-
phase system equipped with fluorescence detection, or capillary
electrophoresis. Another technique that provides a highly specific and
sensitive method for the measurement of amino acids is tandem mass
spectrometry.

Methods of Analysis of Proteins

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 non
protein 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. 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 (O 3) to form an excited nitrogen
dioxide molecule (NO 2*). When this molecule decays to the ground
state, it emits a photon of light. The amount of light emitted is
proportional to the concentration of nitrogen in the 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. The specimen need not be collected when the patient
is fasting, although interferences in some of the methods occur with the
presence of lipemia. Hemolysis will falsely elevate the total protein result
because of the release of RBC proteins into the serum. Clear serum
samples, tightly stoppered, are stable for a week or longer at room
temperature, for a month at 2°-4°C, and for at least 2 months at -20°C .
The reference interval for serum total protein is 6.5-8.3 g/dL (65-83
g/L) for ambulatory adults. In the recumbent position, 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.

Kjeldahl.
The classic method for quantitation of total protein is the Kjeldahl
method. Because it is precise and accurate, it is used as a standard by
which other methods are compared. In this method, nitrogen is
determined; an average of 16% nitrogen mass in protein is assumed to
calculate the protein concentration.

The serum proteins are precipitated with an organic acid such as TCA 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 cata-
lyst, 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 CO 2 CO, H2O,
and SO2. The nitrogen in the protein is converted to ammonium bisulfite
(NH4HSO4), which is then measured by adding alkali and distilling the
ammonia into a standard boric acid solution. The ammonium borate
(NH4H,BO3) formed is then titrated with a standard solution of HCL to
determine the amount of nitrogen in the original protein solution.

This method is not used in the clinical laboratory because it is time

consuming and too tedious for routine use. The nitrogen content of each
individual protein may differ from the 16% assumed in the Kjeldahl
calculation described. The actual nitrogen content of serum proteins varies
15.1-16.8%. Thus, if one uses a protein standard (calibrated with the
Kjeldahl) that differs in composition from the serum specimen to be
analyzed, an error is introduced because the percentage of nitrogen will
not be the same. It is also necessary to assume that no proteins of
significant concentration in the unknown specimen are lost in the
precipitation step. Despite these assumptions, the Kjeldahl method is still
considered by some to be the reference method for proteins.

Refractometry.
Refractometry is useful when a rapid 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 (i.e. air and water), causing the
light to be bent (refracted). 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. This propor-
tionality holds fairly well over a 2- to 3-fold increase in concentration
(i.e. from 520 g/dL). Because the majority of the solids dissolved in serum
are protein, the refractive index reflects the concentration of protein.

However, 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 of 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 serum is
pigmented (from bilirubin), 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 handheld 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 dark.
The number of grams per liter at this line on the internal scale is read. The
temperature is corrected in the TS meter (American Optical Corp, Scientific
Instruments Division; Buffalo, NY) by a liquid crystal system.
The measurement of total protein by refractometry is easy and fast. The
accuracy is acceptable, with a reported agreement of ±3% with the biuret
method, but it is subject to false-positive interferences.

Biuret.
The biuret procedure is the most widely used method and the one
recommended by the International Federation of Clinical 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 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.
When 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. However, there is no discernible difference in the
reaction given by the proteins normally seen in plasma. Over a wide range
of concentrations, therefore, the color that is formed is proportional to the
number of peptide bonds present and reflects the total protein level.
However, in the presence of abnormally small proteins, such as those seen
in multiple myeloma, the concentration of the protein would be
underestimated due to the lighter shade of color produced. If lipemic
sera must be analyzed, a method for overcoming this problem is available .

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.

Dye Binding.
The dye-binding methods are 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
Coomassie brilliant blue have been used to stain protein bands after
electrophoresis. Additionally a dye-binding method for the determination
of total protein using Coomassie brilliant blue 250 has been described.
The binding of Coomassie brilliant blue 250 to protein causes 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.
Although the method is simple and fast, the unequal dye-binding
responses of individual proteins has prompted a recommendation for
caution when applying this test to the complex mixture of protein found
in serum.

Ultraviolet Absorption.
Serum proteins also have been estimated by the use of ultraviolet
spectrophotometry. Proteins absorb light at 280 nm and at 210 nm. The
absorptivity (absorbance of a 1% solution in a 1-cm light path) at 280 nm
is related to the absorbance of tyrosine, tryptophan, and phenylalanine
amino acids in the protein. Human albumin has only one tryptophan
residue in the molecule and has an absorptivity of 5.31 compared with
fibrinogen, which has 55 tryptophan residues and an absorptivity of 15.1.

The absorbance of proteins at 210 nm is a result of the absorbance of the

peptide bond at that wavelength. The wavelength at which maximal
absorbance occurs depends to a small degree on the conformation of the
protein. These methods have rarely been used in clinical laboratories but
are used routinely in research laboratories to monitor eluates of protein
separations from columns. To use these methods, the assumptions must
remain that the composition of the unknown serum specimen is near that
of the calibrating solution.

Determination of Total Globulins.

Another approach to fractionation of proteins is the measurement of total
globulins. Albumin can then be calculated by subtraction of the globulin
from total protein. 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 H 2SO4), condenses with
tryptophan found in globulins to produce a purple color. Albumin has
approximately 0.2% tryptophan, compared with 2-3% for the serum globu-
lins. When calibrated using a serum of known albumin and globulin
concentrations, the total globulins can be determined. 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.