Kaplan: Clinical Chemistry, 5th Edition

Clinical References - Methods of Analysis

Amino Acid Screening

Kevin Carpenter i

Name: Amino acid screening

Clinical significance: click here

Refer to Chapter 52, Diseases of Genetic Origin, in the 5th edition of Clinical Chemistry:
Theory, Analysis, Correlation.

Students’ Quick Hyperlink Review

• Principles of analysis and current usage
• Reference and preferred methods
• Specimen
• Interferences
• Interpretation
• Performance goals
• References
• Details of plasma and urine amino acid methods

Principles of Analysis and Current Usage

Inborn errors of metabolism affecting amino acid metabolism were among the first to be
described, and from a historical perspective, testing for inborn errors was often limited to
qualitative tests for amino acids in plasma or urine. While there is no doubt the amino acid
disorders are now only a small part of the inherited metabolic disease spectrum, there is still a
place for initial screening tests which, although rarely diagnostic, may give insight into the
confirmatory investigations required. Amino acid screens are usually run in conjunction with
organic acids, and interpretation of both requires considerable experience and is usually confined
to laboratories with a special interest in inborn errors of metabolism.

i
Amino acid screening
Previous and current authors of this method:
First edition: Zulfikarali H. Verjee
Methods edition: Not updated
Second edition: Not updated
Third edition: Not updated
Fourth edition: Not updated
Fifth edition: Kevin Carpenter

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Quantitative analysis of amino acids in physiological fluids is a specialist investigation and
outside the scope of this chapter, but it is essential to confirm findings from screening tests and
to monitor treatment.

One of the reasons amino acid disorders figured so prominently in the early descriptions of
inborn errors of metabolism was the availability of a suitable locating reagent for various
chromatographic techniques. Ninhydrin (triketohydrindene hydrate), has been in use for over 90
years and reacts with primary or secondary amines through a complex process to produce
Ruhemann’s purple. All α-amino acids will react with Ninhydrin at room temperature, and
heating to 105°C will allow all compounds containing a primary or secondary amine to react.
Although the basic reaction results in production of a purple color, there are many subtle
variations in the colors different amino acids yield, which may be of value in identifying poorly
separated compounds. The amino acids proline and hydroxyproline, for example, react in a
different manner to produce a characteristic yellow color. Ninhydrin staining may be
conveniently achieved for chromatography, simply by running the chromatography plate or
paper a second time in a solvent containing Ninhydrin. Alternatively, spraying or dipping in the
stain dissolved in acetone can be used for chromatography or electrophoresis on any support
media.

Different stains, often used in conjunction with Ninhydrin, can enhance detection of specific
species, and platonic iodide reagent can be used as an overstain after reading the Ninhydrin stain
to locate sulfur-containing amino acids, which appear white or cream against a pink background.

Location of amino acids by Ninhydrin may be almost universal, but the range of techniques
available to effect separation of the amino acids in physiological fluids is wide. There are two
main categories of separation techniques: chromatography and electrophoresis. These may be
used singly in one or two dimensions or in combination as a two-dimensional separation.

Chromatography of amino acids was originally performed on paper, commonly using n-

butanol/acetic acid/water solvent systems running overnight [1]. Enhanced separation may be
achieved by running in a second solvent at 90 degrees to the original, using t-butanol/methyl
ethyl ketone/ammonia solvents for the second separation. Resolution on paper can be remarkably
good, but the medium has been largely superseded by the use of commercially available thin-
layer cellulose plates which afford excellent and reproducible separations but may require “de-
salting” of urine samples prior to analysis. Multiple samples per sheet can be applied if only a
single dimensional separation is run.

The major disadvantage of chromatographic separation is the time required to obtain a result.
Typical separations are run overnight, and if a rapid result is required, this can be an issue. A
more rapid alternative to paper or thin-layer chromatography is electrophoresis. Cellulose acetate
electrophoresis using a formic acid/acetic acid buffer at pH 1.9 can give reasonable separation in
20 to 30 minutes at voltages of 200-300 V [2].

Even better separations can be achieved using paper as the support media and operating at high
voltages [3]. This method generates considerable heat and requires instrumentation incorporating
a cooling plate and suitable safeguards against electric shock. However, modern ceramic cooling

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

plates linked to a recirculating coolant reservoir all but eliminate problems of the paper drying
out during the run, and excellent results are obtained using 2500 V, 20-minute separation on 265
× 200 mm paper sheets which can comfortably fit up to 9 urine samples per sheet.

For all electrophoresis methods, samples are applied close to the anode end with the dibasic
amino acids, ornithine, lysine, and arginine migrating the furthest, and acidic compounds taurine
and phosphoethanolamine remaining close to the origin. S-sulfocysteine (a marker for sulfite
oxidase deficiency) migrates slightly towards the anode, so samples are applied a few cm above
the anode position to allow for this.

Electrophoresis does not completely separate amino acids with similar isoelectric points, but
when used in combination with chromatography run at 90 degrees to the electrophoresis, the
techniques are complimentary, enabling separation of amino acids neither technique in isolation
can achieve [4]. The downside to the enhanced separation is that only one sample per sheet can
be applied, and therefore throughput is limited.

Reference and Preferred Methods

There are no reference methods for amino acid screening.

The American College of Medical Genetics publishes Standards and Guidelines for Clinical
Genetics Laboratories [5]. The 2006 edition states, “Qualitative amino acid analysis must
reliably detect conditions in which there are either gross or modest elevations of specific amino
acids in blood and/or urine” but warns “Qualitative amino acid analysis by thin-layer
chromatography (TLC) is suitable only for the detection of gross abnormalities. As some
disorders may be missed by this method, its use for the purpose of evaluating high-risk patients
should be discouraged.”

However, TLC is widely used for initial amino acid screening, and provided the limitations are
understood, it can still yield valuable information. For laboratories setting up amino acid
screening for the first time, TLC will give reliable results in most hands, whereas the enhanced
resolution achieved by high-voltage electrophoresis requires more specialized equipment and
considerable skill to get consistent results.

Specimen
Plasma or urine can be used for amino acid screens, and many labs request both. This can be
useful where increased concentrations of particular amino acids in urine may be due to their
accumulation in blood or as a result of impaired renal reabsorption. Comparison of the patterns
obtained in the two samples will reveal the source of the increase. Plasma alone will not be
informative for detection of renal transport defects such as cystinuria or detection of generalized
aminoaciduria such as is seen in Fanconi syndrome. Plasma requires deproteinization prior to
running, and urine samples may need removal of inorganic salts by ion exchange resin if TLC is
used.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Samples should be stored and transported at or below −20°C to prevent bacterial degradation.
Under such conditions, most amino acids are stable for many years. Typical volumes required for
chromatographic or electrophoretic techniques are only a few hundred microliters. Urine samples
should be loaded or diluted according to creatinine concentration.

Interferences
Ninhydrin will react with a number of exogenous compounds that may interfere with
identification and interpretation. Penicillin-containing antibiotics produce a number of
Ninhydrin-positive metabolites. Other antibiotics may also produce abnormal spots, as will
acetaminophen, L-dopa, & GABA analogues.

Diet influences the pattern of amino acids seen, with a generalized increase in excretion
postprandially and specific increases in carnosine, anserine, and 1-methylhistidine following
white poultry meat ingestion. Catabolism will result in a decrease in alanine concentration and an
increase in β-aminoisobutyrate concentration.

Decreased levels of serine and glutamine may be seen in bacterially contaminated urine samples.

Interpretation
Interpretation of the profiles obtained in both urine and plasma is a highly skilled job requiring
experience of what to expect in normal and disease states, and the information given here should
be considered a very basic guide only. It must also be noted that not all aminoacidurias result in
clinical disease [6].

In normal plasma, the most prominent amino acids are glutamine and alanine, with lesser
amounts of glycine, valine, lysine, leucine, serine, and threonine usually visible. The aromatic
amino acids phenylalanine and tyrosine give quite faint bands, as do the remainder of the
physiological amino acids.

In normal urine, age is an important factor in the amino acid excretion pattern. Babies often show
an immaturity of renal reabsorption mechanisms, resulting in an increased excretion of proline,
hydroxyproline, glycine, and sometimes cystine and lysine. Taurine is also prominent at birth but
declines rapidly. Generally “heavy” patterns (nonspecific increases in all amino acids) are seen
in the neonatal period, gradually declining to adult-type patterns during childhood and
adolescence.

A normal urine pattern is dominated by glycine, with lesser and similar-intensity bands seen for
alanine, serine, and glutamine. Lysine, cystine, and the aromatic amino acids produce only very
faint bands in the healthy individual.

Inborn errors of amino acid metabolism will usually result in accumulation of the amino acid
proximal to the defect in the synthetic pathway in both plasma and urine. Thus phenylketonuria
gives elevated phenylalanine, nonketotic hyperglycinemia gives elevated glycine, and maple-
syrup urine disease shows increases in valine, leucine, and isoleucine. Urea-cycle defects will all

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

give elevated glutamine, but argininosuccinate synthetase deficiency will also result in elevated
citrulline, and argininosuccinate lyase deficiency will show the presence of argininosuccinate
and its anhydride.

In all instances, interpretation should be performed in light of the clinical condition of the patient
and preferably in conjunction with organic acid profile results.

Amino Acid Performance Goals

The key determinant of analytical acceptability for amino acid screening is the ability to detect
inherited metabolic disease if present. Since the majority of the methods described here allow
several samples to be run on a single plate or sheet, the simplest way to ensure reliability may be
to run one or more positive controls on each plate. However, since this limits the analytes being
controlled to those elevated in the control sample chosen, it may be useful to supplement the
sample with additions of other key amino acids at concentrations typical of those found in
disease. In this way, one can be sure an elevation in a particular amino acid can be reliably
detected, and it also serves as a rough comparator for grading the extent of the increase in a
patient sample.

References
1 Smith I, Ersser RS. Amino acids and Related Compounds. In: Smith I and Seakins J W T.
Chromatographic and Electrophoretic Techniques. 4 ed. London: William Heinemann
Medical Books; 1976. 75-121.
2 Kohn J. Cellulose Acetate Electrophoresis. In: Smith I. Chromatographic and
Electrophoretic Techniques. 4 ed. London: William Heinemann Medical Books Ltd;
1976. 90-137.
3 Beale D, Smith I. High Voltage Paper Electrophoresis. In: Smith I. Chromatographic and
Electrophoretic Techniques. 4 ed. London: William Heinemann Medical Books Ltd;
1976. 31-65.
4 Shih VE, Mandell R, Sheinhait I. General Metabolic Screening Tests. In: Hommes FA.
Techniques in Diagnostic Human Biochemical Genetics. New York: Wiley-Liss; 1991.
45-68.
5 American College of Medical Genetics. Standards and Guidelines for Clinical Genetics
Laboratories. 2006 Edition. http://www.acmg.net/Pages/ACMG_Activities/stds-
2002/f.htm Accessed: 2007-08-09
6 Shih VE. Detection of hereditary metabolic disorders involving amino acids and organic
acids. Clin Biochem 1991; 24: 301-309.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Procedure 1: Plasma Amino Acids by Thin-Layer Chromatography
Principle
Plasma samples are deproteinized by ethanol precipitation and applied to cellulose TLC plates.
The plates are run twice in an n-butanol/acetone/acetic acid/water solvent, the second run
incorporating Ninhydrin into the solvent to locate the amino acids.
Reagents
1. Absolute Ethanol
2. TLC Solvent 1 (tank 1). In a fume hood, measure out: 21 mL of acetone, 21 mL n-
butanol, 6 mL of glacial acetic acid, and 12 mL of water. Pour into a TLC tank and cover
with a glass lid. Allow 30 min for equilibration before use. Make fresh solutions every 2
days.
3. Ninhydrin 0.2 % w/v. Weigh out 0.5 g of Ninhydrin. Dissolve in 250 mL acetone. Store
in a brown stoppered bottle, stable for 1 month.
4. Locating Solvent 2 (tank 2). In a fume hood, measure out: 21 mL of the 0.2% Ninhydrin
solution, 21 mL n-butanol, 6 mL of glacial acetic acid, and 12 mL of water. Pour into a
TLC tank, and cover with a glass lid. Allow 30 min for equilibration before use. Make
fresh solutions every 2 days.
5. Commercial amino acid standard mixture. Example: Sigma A2161 diluted 1:8 with
ethanol to give equivalent of 125 µmol/L concentrations in plasma. Stored at −80°C.
Equipment:
• Cellulose TLC plates 10 × 20 cm (Merck # 5730)
• TLC tanks
• Micropipette for delivery of sample to TLC plate

Assay

1. Samples are deproteinized by mixing with ethanol. In a microcentrifuge tube add 200 µL
of ethanol to 50 µL of plasma. Centrifuge for 2 min at 10,000 g.
2. Transfer supernatant to a clean tube.
3. Mark a cellulose thin-layer plate with lines 1.5 cm long and 1 cm apart, 1.5 cm from the
bottom edge of the plate, using a soft pencil. NOTE: Use gloves and take care not to touch
the plate; fingerprints contain enough amino acids to stain the plate.
4. Apply 15 µL of the plasma extract in 3 lots of 5 µL, allowing the extract to completely
dry between applications (warm air may be used to aid drying between applications). The
sample is applied using a 5 µL positive-displacement micropipette as a streak 1-cm long
between the lines drawn on the plate.
5. A maximum of 7 samples can be run on a single plate. One track is reserved for the
amino acid standard mixture applied as per plasma samples.
6. Place the TLC plate in tank 1, and develop until the solvent reaches the top of the plate
(approximately 20 to 30 min).
7. Remove the plate, and allow to dry completely in a fume hood.
8. Place the plate in tank 2, and develop until solvent reaches the top of plate.
9. Remove the plate, and allow to dry overnight. Color will develop at room temperature,
but if an urgent result is required, plate may be heated at 100°C to aid drying and
development.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

 10. Comparison of individual tracks with standard mixture allows qualitative scoring of
amino acids, but gross elevations should always be followed up by quantitative analysis
in a specialist laboratory.

Procedure 2: Urine Amino Acids by High-Voltage Electrophoresis

Principle
High-voltage electrophoresis (HVE) is a very rapid and efficient method for separating small
molecules. At pH 1.9, the basic amino acids lysine, arginine, and histidine become positively
charged and travel rapidly towards the cathode (−), whereas the acidic amino acid cysteic acid is
negatively charged and remains near the origin close to the anode (+).
A standardized volume of urine (100 nmol creatinine) is applied as a narrow band onto a paper
sheet. Electrophoresis is performed for 24 min at 2500 V. Staining the electrophoretogram with
Ninhydrin localizes the amino acids. The profiles are interpreted, and then the sheet is
overstained with iodoplatinate reagent that enables visualization of sulfur-containing compounds,
including homocystine, methionine, cystine, and S-sulfocysteine.

NOTE: plasma may be run on the same method but must be deproteinized before application to
the paper.

Reagents
1. Formic acid/acetic acid buffer pH 1.9. Add 37.5 mL formic acid and 150 mL glacial
acetic acid slowly to approx. 1500 mL of Milli-Q water in fume hood. Make up to 2 L
with Milli-Q water. Store in brown glass bottle at room temperature. Stable for 1 month.
2. Ninhydrin 0.2 % w/v. Weigh out 0.5 g of Ninhydrin. Dissolve in 250 mL acetone. Store
in a brown stoppered bottle, stable for 1 month.
3. Chloroplatinic acid, 1 g/500 mL. Weigh out 1g of chloroplatinic acid (CAUTION:
highly toxic, wear mask and gloves). Make up to 500 mL with water.
4. Hydrochloric Acid (HCl) 6M.
5. Potassium iodide, 167 g/L. Weigh 41.75 g potassium iodide, make up to 250 mL with
water; replace approx. every 12 months. Store in brown glass bottle.
6. Iodoplatinate (IP) stain. Make up the stain in the fume hood immediately prior to
staining. Mix 5 mL of chloroplatinic acid, 0.5 mL potassium iodide, 1 mL 6M HCl, and
42.5 mL acetone.

Equipment:
REQUIRED: High-voltage electrophoresis equipment capable of running at 2500 V, with
integrated cooling system. A typical system would be:
• Pharmacia HVE system:
o Hoefer PS3000 DC power supply
o Multiphor II electrophoresis unit comprising:
o Buffer tank
o Ceramic cooling plate
o Electrode holder and electrodes
o Safety lid
o Multi Temp III cooling unit.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

 • Electrophoresis paper, Machery Nagel MN 214, 200 × 265 mm
• IEF Electrode strips (Pharmacia Cat. # 18-1004-40)
• Wash bottle with a fine jet
• Large glass dish
• Glass plate 195 mm square
• Blotting paper, 445 × 570 mm, 135 gsm, APPM
• Oven set at 100°C ± 2°C, for drying and 125°C ± 2°C for Ninhydrin stain development
• Micropipette for delivery of sample

Assay
Electrophoresis

1. Calculate volume of urine in µL to be applied by dividing100 by creatinine concentration

in mmol/L.
2. Turn on recirculating cooler and set to 4°C
3. In lead pencil, mark out electrophoresis paper 6 cm from one end for 9 samples. These
should be 1.0 cm long, 1.5 cm from both edges, and 1.0 cm apart.
4. Apply sample as a narrow band in small volumes, drying between applications using hot
air.
5. When all samples have been applied, carefully wet the sheet with buffer, avoiding
wetting the origin directly and allowing buffer to run up to the origin from both sides.
6. Gently blot the sheet between a folded sheet of filter paper and place on the ceramic
cooling plate, origin at anode end.
7. Wet the IEF electrode strips with buffer, and blot gently before applying to the sheet 1cm
from each end.
8. Place the glass plate on the sheet to maintain good contact with cooling plate.
9. Position IEF electrodes over electrode strips and connect electrodes. Fit safety lid.
10. Turn on power supply, and run at constant voltage 2500 V (approximately 50-60 mA) for
25 min.
11. Turn off power supply. Remove sheet and hang to dry in oven at 100°C for 5 min.

Ninhydrin and iodoplatinate staining

1. Stain sheet by rapidly dipping through Ninhydrin reagent in a staining tray. Dry briefly in
fume hood before transferring to oven at 120°C for 3 min.
2. Interpretation of Ninhydrin staining requires experience and knowledge of changing
excretion patterns with age. Follow-up of abnormal patterns requires quantitation in a
specialist centre.
3. If required, following interpretation of the Ninhydrin stain, the sheet can be overstained
with iodoplatinate reagent to highlight the presence of sulfur-containing amino acids.
4. Ensure that the staining dish and paper are thoroughly dry, then add a volume of the IP
stain.
5. Quickly and evenly dip the paper through the stain.
6. Hold the papers in the fume hood briefly to remove any excess acetone. Paper clips are
used to hold the papers in a cylindrical shape. These cylinders are stood on their end in a
glass tank in the fume hood overnight at room temperature. The Ninhydrin positive bands

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Clinical References - Methods of Analysis 12-9

7. The iodoplatinate stain will give a pale pink/peach background with sulfur-containing
amino acids, homocystine, methionine, cystathionine, cystine, and S-sulfocysteine,
having a cream or white color.
8. Several drugs and other compounds will give cream bands, and interpretation again
requires considerable experience.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.