Ammonia Measurement
Ammonia Measurement
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ANALYTICAL METHODS
FOR THE
DETERMINATION OF
AMMONIA
Many of the procedures for ammonia
determination involve 2 general steps:
the release of ammonia gas or capture
of ammonium ions from the sample
and the quantitation of the liberated
gas or captured ions. Over the years
the most common methods used to
volatilize ammonia have been distilla-
tion and aeration/microdiffusion, ion-
Fig 1. Ratio between ionized and free gas form of ammonia in plasma as function of pH. exchange chromatography, and blood
or plasma deproteinization.
Properties of Ammonia
tion. EDTA can also be used. Donors’ exclusively with blood specimens from In attempting to understand the ra-
arms should be as relaxed as possible, healthy subjects.8 Significantly more tionale used to measure ammonia, it is
because muscle exertion may increase difficult but much more constructive important to review some of the physi-
venous ammonia levels.7 Blood is would be the establishment of similar cal properties of the compound. Am-
drawn into a chilled, heparinized vacu- criteria for blood ammonia measure- monia (NH3) is a colorless, acrid-
um tube that is immediately placed on ments from patients with metabolic or smelling gas at room temperature and
ice, and plasma is separated within 15 liver pathologic conditions. Standing pressure. It easily dissolves in water
minutes. It is crucial to keep blood blood or plasma samples from these and ionizes to form NH4+ as follows:
samples cold after collection, because patients contain numerous elevated
the ammonia concentration of standing sources of ammonia, resulting in in- → NH ++ OH-
NH3 + H2O ← 4
blood and plasma increases sponta- creases in the rate and amount of am-
neously. Most of this increase has been monia formed. Some of these sources An increase in the pH or tempera-
attributed to the generation and re- include elevated levels of circulating ture of the solution increases the level
lease of ammonia from red blood cells deaminase, γ-glutamyltransferase, an of the ionized form. Fig 1 shows the
and the deamination of amino acids, enzyme that may deaminate free amino ratio that exists in plasma between the
particularly glutamine.8-11 Plasma am- acids, particularly glutamine in blood ionized or NH4+ form versus the
monia levels of whole blood main- and plasma samples, resulting in over- gaseous or NH3 form as a function of
tained at 4oC are stable for <1 hour. estimates of blood ammonia levels. pH. Thus in plasma at pH 7.4, the
When promptly separated from blood, Fast, careful handling and preparation NH4+ form represents approximately
plasma ammonia levels are stable at of blood samples is required, especially 98% of the total ammonia. Many of the
4oC for 4 hours and for 24 hours if from patients with metabolic or liver approaches used to estimate ammonia
stored frozen at –20oC. To put the pathologic conditions, to minimize pre- levels in body fluids involve volatiliza-
problem of rising ammonia levels in analysis increases in ammonia concen- tion of the NH4+ form of ammonia into
perspective, the total nitrogen concen- tration until other assay techniques or its gaseous form, NH3 ,by alkalization
tration in venous plasma of healthy methods are developed. Until this is of the sample to a pH >10.
adults exceeds 1 mol/L and represents accomplished, measurements in this
a potential pool of free blood ammo- patient population, in which blood am- Distillation
nia.12 In normal healthy adults, homeo- monia levels require the closest moni- One of the earliest techniques for the
stasis maintains free ammonia levels at toring, will continue to be the most un- measurement of ammonia involves the
approximately 30 µmol/L. reliable. To date, the easiest and most addition of an alkaline buffer to a sam-
It is important to note that the crite- cost-effective method is the stringent ple of blood followed by in vacuo dis-
ria for sample stability and the meth- and diligent maintenance of blood tillation. The released ammonia gas is
ods for the measurement of ammonia samples on ice before, during, and collected in a trap containing an
levels in blood were established almost after plasma separation. aliquot of dilute acid, which converts
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Aeration/Microdiffusion
Techniques Fig 2. Early microdiffusion apparatus for determination of blood ammonia. (Reproduced with per-
mission from Conway E, Byrne A. An absorption apparatus for the micro-determination of ammonia.
Another early technique that is still Biochem J. 1993;27:419-29. © the Biochemical Society.)
in use relies on liberation of free am-
monia by alkalization by the addition
of a strong base to the specimen. The
released ammonia diffuses through an
air- or nitrogen-filled gap and is
trapped in acid within the same appa-
ratus. This approach, introduced by
Conway and Berne14 in 1933, uses a
glass container resembling a Petri dish,
within which a smaller second cham-
ber is centered. The wall of this inner
chamber is approximately half of that Fig 3. Illustration of microdiffusion technique used in Blood Ammonia Checker. (Reproduced with
permission from Tada K, Okuda K,Watanabe K, et al. A new method for screening for hyperam-
of the outside wall. An aliquot of a
monemia. Eur J Pediatr. 1979;130:105-10.)
standard acid solution or ammonia in-
dicator such as bromocresol green is constituents and drugs on the ammo- Deproteinization
placed into the inner chamber, and the nia assay. One main drawback of these Whole blood or plasma proteins are
sample is added to the outer chamber procedures is that varying amounts of precipitated by the addition of tri-
(Fig 2). A measured quantity of base is ammonia are liberated from the alka- chloroacetic or perchloric acids, and the
added to the sample, and the “petri” line hydrolysis of proteins, especially ammonia is determined directly in the
dish is sealed and gently rotated to mix hemoglobin, and labile amides, espe- supernatant fluid after alkalization.22,23
the sample and base in the outer cham- cially glutamine.18-20
ber and then left at room temperature
for 90 minutes. This same principle is Ion-Exchange Chromatography QUANTITATION OF
used in the Blood Ammonia Check- In this approach ammonia gas is not AMMONIA
er15,16 and in the Kodak Ektachem liberated from the sample. Instead, a
dry-film method.17 In these systems strongly acidic cation-exchange resin After the release or capture of ammo-
the diffusion distance for ammonia is is used in batch mode to capture am- nia or ammonium ions, several methods
significantly reduced from those in the monium ions, NH4+. The resin is have been described to determine the
original diffusion apparatus of Conway added to and subsequently separated amount of ammonia present. The gen-
and Berne, requiring only 5 minutes to from the sample by centrifugation, and eral categories for these methods in-
complete (Fig 3). the captured ammonium ions are then clude titration, colorimetric/fluorimet-
Distillation and microdiffusion both eluted from the resin by salt solutions ric, electrode-based, and enzymatic.
represent purification procedures that or released as ammonia by the addi-
isolate ammonia from the many other tion of dilute alkali,21 a technique de- Titration Method
constituents of blood and plasma, re- scribed in detail more recently by The ammonia liberated from the
ducing the possible effects of other Brusilow.4 sample is trapped in an aliquot of di-
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Fig 4. Schematic of ion-selective electrode (left panel) and suggested arrangement for continuous-flow analysis of ammonia
in plasma samples (right panel).
lute acid, and the amount is measured as fluorescamine and o-phthalalde- ness, ease of use, and because it never
by back-titration of the acid solution hyde.24-29 The principle advantages to comes in contact with the sample, its
with a base while the pH is monitored this approach are speed, simplicity, imperviousness to sample color, tur-
with an indicator or electrode. The specificity when used carefully, and ex- bidity, viscosity, or the presence of
principal advantage of this approach is cellent sensitivity. The disadvantage is drugs or other metabolites in the sam-
that it is inexpensive, requiring no spe- that other substances in the blood af- ple. The electrode is best arranged
cialized equipment. The disadvan- fect some reactions, for example, the above the surface of the sample in a
tages, however, are significant. They Berthelot reaction is inhibited by ex- sealed environment (Fig 4). This
include insensitivity, the requirement cess amino acids, creatine, glutamine, avoids the accumulation of proteins
for large blood samples, and contami- and some therapeutic agents.30 and cell fragments on the membrane
nation by other volatile bases that may surface that would normally occur
affect the final value. Overall, this pro- Gas-sensing Electrode during immersion into plasma or blood
cedure is laborious and slow and there- With the introduction of the gas- sample and minimizes the loss of am-
fore is not used routinely. sensitive electrode (eg, Orion, Model monia from the sample away from the
951201), a number of reports have ap- electrode during the measure-
Colorimetric/Fluorimetric peared describing the methods re- ment.22,31,32 The disadvantages of the
Reactions quired and the use of the electrode in electrode-based system include the re-
This method is based on the reaction measuring ammonia levels in samples quirement of large sample volumes and
of ammonia with a reagent to form a of blood, urine, cerebrospinal fluid, slow sample reads, especially at low
colored complex that is measured by and saliva over a broad range from 10 ammonia levels, requiring 10 to 15
spectrometry or fluorometry. Among µmol/L to nearly 1 mmol/L. minutes. In addition, major differences
the first reactions used was the in- When immersed in the sample or in either the osmolarity or temperature
dophenol reaction, described by Berth- held closely above it, the dissolved of the sample and the sensing electrode
elot in 185923a: the formation of a gas of interest diffuses across a gas- buffer must be avoided.
bluish color by the reaction of ammo- permeable membrane into a small vol-
nia with phenol and hypochlorite. This ume of buffer. The reaction changes Enzymatic Method
method is commonly referred to as the the pH of the buffer, which is sensed The specificity of most methods for
phenol reaction. Another colorimetric by an internal pH electrode or sensing ammonia determination in biologic flu-
reaction is the Nessler reaction, in electrode. The change in pH results in ids relies on the physical separation of
which a brown-orange color is formed a change in the potential between the ammonia from interfering substances
by the reaction of ammonia with mer- sensing electrode and a reference elec- by volatilization after alkalization. In
cury or potassium iodide in an alkaline trode immersed in a separate reference contrast, the most common method
solution. Some other colorimetric reac- buffer, all housed within the same elec- used in clinical laboratories is an enzy-
tions include the use of isocyanurate, trode body. The arrangement is illus- matic method that measures ammonia
cyanate, ninhydrin, or thymol hypo- trated in Fig 4, in which the scale of directly. Thus sample preparation is
bromite. Detection of ammonia and some of the components is exaggerated relatively simple, because the previous
primary amines down to the nanogram for clarity. liberation of ammonia from the sample
range is routinely performed with fluo- The main advantages of electrode- is not required. This assay is based on
rescence derivatization reagents such based ammonia assay are its cheap- the reductive amination of 2-oxoglu-
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tarate with glutamate dehydrogenase continued liberation of ammonia from ods could alert medical staff to an im-
and reduced nicotinamide adenine di- the samples, except for lowering the pending hyperammonemic condition
nucleotide phosphate: temperature, ammonia levels in the and would more easily permit earlier
sample will continue to increase during selection and regulation of therapeutic
2-Oxoglutarate + NH3 + NADPH the assay and up to the time of the ini- interventions. Two promising methods
↑↓GLDH tial readings. Thus overestimates of am- that are under development and may
Glutamate + NADP monia levels are most likely in poorly eventually be used clinically are the se-
handled samples containing elevated lected-ion flow tube technique, which
The decrease in absorbance at 340 levels of transaminases and amino acids. analyzes trace gases in breath, and a
nm caused by the oxidation of fiber-optic catheter tipped with an am-
NADPH is proportional to plasma am- Normal Values for Blood monia-sensitive indicator.
monia. GLDH is specific for ammonia Ammonia Levels
and does not react with methylated Table I lists selected blood ammonia Selected-ion Flow Tube
amines. Early studies describing the levels for various blood sample types Selected-ion Flow Tube is a quanti-
enzymatic determination of ammonia and assay methods from a number of tative method for the rapid, real-time
used NADH as a coenzyme. Because studies. The average values for arterial analysis of the trace gas content of at-
other NADH-consuming systems are blood, plasma, venous blood, and plas- mospheric air. It was originally devel-
present in blood, many of these reports ma are 18, 23, 28, and 32 µmol/L, re- oped to study ionic reactions in the gas
overestimate the level of free ammonia, spectively. The average value for ve- phase and is particularly valuable
for example, Muting.33 The effect of nous blood and plasma is 30 µmol/L. for providing kinetics data on ion-
these systems can be minimized, usual- molecule reactions, contributing to the
ly by a 30-minute preincubation peri- current understanding of the chem-
od. There are considerably fewer FUTURE istry of some low-temperature gaseous
NADPH-consuming sources in plas- DEVELOPMENTS plasmas, especially interstellar clouds.
ma, so the preincubation time can be The same technology is currently
reduced to a few minutes.3 The major limitations of convention- being developed to analyze trace gases
The assay can be used to measure am- al in vitro blood ammonia measure- in breath. Previous methods for mea-
monia levels over a broad range, from ments are the complexity involved in surement of ammonia in breath have
as low as 12 µmol/L to as high as 1 the proper drawing and handling of required large sampling flow rates34 or
mmol/L. The disadvantage of this ap- the sample, the time allowed between long sampling times35 and are there-
proach is the length and complexity of drawing and assaying, and finally, the fore unsuitable for assessing the am-
the procedure, thereby enhancing the assay itself. A consequence of these monia concentration from a single
potential for variation in reported blood limitations is that blood ammonia mea- breath. A schematic of the SIFT appa-
ammonia levels. If not handled proper- surements are performed only a few ratus is shown in Fig 5 taken from
ly, ammonia concentrations rise in times each day. Alternative methods Smith and Spanel.36 This technology is
standing blood or plasma. Indeed, be- are still sought that are noninvasive or being further developed and may soon
cause standard procedures for pre- require a small catheter in a peripheral be a sensitive, quantitative method for
analysis sample processing do not in- vein but provide a continuous monitor the continuous real-time analysis of the
corporate any attempts to inhibit the of blood ammonia levels. Such meth- trace-gas content of human breath and
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Table I. Reported assay techniques and blood ammonia levels in healthy subjects
Reference
Reference number Method Value (µmol/L)
Arterial blood
Hutchinson and Labby (1962) 42 Ion-exchange/Nessler 19
Gips and Wibbens-Alberts (1968) 41 Supernatant/phenol 22
Huizenga and Gips (1983) 15 Microdiffusion/BAC I 8
Huizenga et al (1992) 16 Microdiffusion/BAC II 21
Huizenga et al (1992) 16 Enzymatic 21
Arterial plasma
Gips and Wibbens-Alberts (1968) 41 Supernatant/phenol 23
Huizenga and Gips (1983) 15 Enzymatic 23
Venous blood
Dienst (1961) 21 Ion-exchange/Nessler 15
Forman (1964) 39 Ion-exchange/phenol 31
Hutchinson and Labby (1962) 42 Ion-exchange/Nessler 21
Proelss and Wright (1973) 23 Supernatant/electrode 17
Gips and Wibbens-Alberts (1968) 41 Supernatant/phenol 22
McCullough (1967) 43 Supernatant/phenol 57
Gangolli and Nicholson (1966) 40 Supernatant/phenol 30
Sinniah et al (1970) 45 Supernatant/phenol 44
Huizenga et al (1992) 16 Microdiffusion/BAC II 21
Huizenga et al (1992) 16 Enzymatic 21
Venous plasma
Gerron et al (1976) 9 Ion-exchange/phenol 19
Oberholzer et al (1976) 44 Ion-exchange/phenol 16
Buttery et al (1982) 38 Ion-exchange/phenol 21
Brusilow (1991) 4 Ion-exchange/phenol 18
Cooke and Jensen (1983) 32 Electrode 44
Willems and Steenssens (1988) 46 Electrode 44
Spooner et al (1975) 27 Supernatant/fluorometry 32
Seligson and Hirahara (1957) 20 Microdiffusion/Nessler 56
Mondzac et al (1965) 2 Enzymatic 30
Muting et al (1968) 33 Enzymatic 57
van Anken and Schiphorst (1974) 3 Enzymatic 22
Howanitz et al (1984) 11 Enzymatic 30
da Fonseca-Wollheim (1990) 8 Enzymatic 29
thus permit a continuous, noninvasive charge ratio. In the case of ammonia possible because the reaction of the
measure of systemic ammonia levels. detection, H3O+ is extracted from this primary ions, in this case H3O+, with
This technique involves the genera- mixture of ions with a quadrupole ammonia is to form ammonium ions
tion of positive ions that are created in mass filter. This current of selected and water is precisely defined in the
a microwave discharge ion source, ions is then injected into a fast-flowing SIFT. In general, the primary ions cho-
containing an appropriate gas mixture. inert carrier gas stream, usually heli- sen must not react at significant rates
In conventional mass spectrometry, um. The ions are carried along a 1- with the major components of the
ionization of the trace gas molecules is meter length flow tube and are sam- breath sample, oxygen, nitrogen,
achieved by electron bombardment, pled by a pinhole downstream of the water, or carbon dioxide, because such
resulting in molecular “cracking” and injector. The sample of breath is intro- reactions would saturate the primary
the production of complicated spectra. duced into the device by a sample port ions. In turn, the primary ions must
The SIFT technique uses a current of near the injector (Fig 5). Accurate react efficiently with the trace gases to
precursor ions of a given mass-to- trace gas analysis or quantification is be detected to form identifiable prod-
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40. Gangolli S, Nicholson TF. The deter- would not be considered a significant Blood Sampling Protocol
mination of blood ammonia. Clin Chim problem, but there was no consensus A protocol for the handling of blood
Acta 1966;14:585-92.
on what level should prompt intra- samples for ammonia analysis should be
41. Gips CH, Wibbens-Alberts M. Ammo-
nia determination in blood using the venous therapy. The clinical status established in all centers where patients
TCA direct method. Clin Chim Acta should be helpful in this regard. with hyperammonemia are treated. A
1968;22:183-6. Many physicians have experienced minimum of 1 mL of blood is usually re-
42. Hutchinson JH, Labby DH. New the dilemma of aberrantly elevated plas- quired and should be placed on ice im-
method for microdetermination of
ma ammonia in patients in whom the mediately, and not allowed to stand for
blood ammonia by use of cation ex-
change resin. J Lab Clin Med 1962;60: clinical picture does not suggest hyper- more than 15 minutes, because this will
170-8. ammonemia. The reasons for these dis- increase the ammonia content of the
43. McCullough H. The determination of crepancies are not always known. Sam- sample. The blood, which can be arteri-
ammonia in whole blood by a direct pling techniques, storage and handling, al or venous, should be centrifuged as
colorimetric method. Clin Chim Acta
and delays in testing can all contribute soon as possible, after which the plasma
1967;17:297-304.
44. Oberholzer VG, Schwarz KB, Smith to an increase in the level of ammonia should be frozen at –70˚C if not ana-
CH, Dietzler DN, Hanna TL. Mi- within the sample. Thus the reported lyzed immediately. The use of capillary
croscale modification of a cation- level will be elevated and not truly re- blood should be avoided, because
exchange column procedure for plasma flect the ‘in vivo’ value. Conversely, and platelet aggregation and clotting leads
ammonia. Clin Chem 1976;22:1976-81.
for the same reasons, it is unlikely that a to elevated ammonia levels. For accu-
45. Sinniah D, Fulton TT, McCullough H.
Venous blood ammonium levels in con- normal blood ammonia value will be rate measurement of ammonia, ammo-
trol subjects and in patients with disor- aberrant, and thus a normal blood am- nia-free heparin or EDTA anticoagu-
ders of the liver. J Clin Pathol 1970; monia level will help to eliminate hyper- lant should be used. Education of
23:720-6. ammonemia from the differential diag- laboratory staff in the use of such a pro-
46. Willems D, Steenssens W. Ammonia
nosis of a comatose patient. tocol will go a long way to reducing the
determined in plasma with a selective
electrode. Clin Chem 1988;34:2372. numbers of factitious results.
Chronic Setting Measurements should be taken at
For the chronic treatment of a patient the same time of day and under the
AMMONIA with urea cycle disorder, it appears that same circumstances, because there is a
MEASUREMENT glutamine is not a good surrogate mark- diurnal variation in blood ammonia
DISCUSSION er for disease severity, because there ap- levels and the results may differ, for ex-
pears to be a poor correlation between ample, depending on whether the sam-
Acute Setting glutamine levels and outcomes. ple was obtained before or after a meal.
The prompt measurement of the In certain centers there has been a
plasma ammonia level is very impor- move away from using ammonia plas- Interpretation of Laboratory
tant in the acute management of hyper- ma levels in the chronic treatment of a Test Results
ammonemia, but the clinical status of a patient. This is partly due to the lack of The measurement of ammonia levels
patient’s brain function should out- concordance between plasma ammonia in blood is fraught with difficulty. In
weigh the importance of these test re- levels and the clinical status of the pa- most instances clearly aberrant values
sults. This is because ammonia levels tient and partly because elevated am- cannot be explained, although preana-
may not correlate with clinical status. monia can be intermittent and could be lytical artefacts are believed to account
When the ammonia level is low, pa- normal at the time of blood sampling, for many of these spurious results.
tients seem to be particularly sensitive although the patient is still metaboli- This argues for blood ammonia tests to
to small increases in the level, whereas cally unstable. be performed only by laboratories with
when the ammonia level is elevated, Is has been postulated that glutamine is experience in the procedure.
there seems to be an adjustment to the neurotoxin in the brain. It appears Plasma ammonia levels are known to
these high levels with fewer symptoms. that it is a harbinger of hyperammonemia increase with exercise, and this is one
This could explain why symptoms may and its blood level falls with the ammonia of many reasons why the correct inter-
precede significant rises in ammonia level. In the acute phase there may be pretation of ammonia levels in blood is
levels and high ammonia plasma levels some value to monitoring glutamine important. Squeezing a ball in the
can still be present when the patient is blood levels in addition to ammonia. hand for a few minutes has the effect of
clearly getting better. It was not agreed whether treatment raising blood ammonia levels to as high
A normal ammonia level in the new- is necessary for mild elevations of blood as 150 µmol/L.
born period is <50 µmol/L. A blood ammonia in a child during chronic man- Children do not like having blood
ammonia level of 70 to 80 µmol/L agement of the urea cycle disorder. taken from them, and most doctors will
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have had the situation where a child into hospital before the plasma ammo- dwelling catheter will increase the
fights the blood draw. When the labora- nia level has become significantly ele- amount of hemolysis caused by shear-
tory test comes back with a plasma am- vated. Paradoxically, some physicians ing, and therefore it is likely that the am-
monia level that is elevated, there is a have observed children with plasma am- monia level will be spuriously elevated.
dilemma in deciding whether the value monia levels in excess of 1,000 µmol/L A sample for blood ammonia determina-
is “real” or factitious because of exer- who, clinically, appear to be normal. tion should preferably be drawn from a
tion. In cases where elevated ammonia levels site other than an indwelling catheter.
Symptoms often precede a significant are unexpected, it may be useful to inquire How do we confirm that a raised am-
rise in plasma ammonia level. Parents of the parents how and when that blood monia level is not an artefact? The best
usually become very adept at recogniz- sample had been taken. This may aid in way is to repeat the measurement with
ing when their children are ill, and it is the interpretation of the laboratory result. another blood sample and fastidiously
common for a sick child to be brought Drawing up blood through a small in- following the guidelines on the collec-
tion, handling, and storage of samples.
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