Measurement of ammonia in blood
`Robert J Barsottt, Php
`
`
`
`The measurement of ammonia, now known to be a normal constituent of all
`bodyfluids, is franght with problems. An elevated ammonialevelin blood
`(100 pmol/L or higher) is an indicator of an abnormality in nitrogen home-
`ostasis. The collection, handling, storage, and analysis of blood samples,
`their limitations, and potential sources oferror are discussed. Newtech-
`niques that permit continuousorreal-time estimates of systemic ammonia
`levels over a broad range are also discussed. The aim should always be to
`minimize the release of ammonia from the collected sample before analysis.
`Recommendations are made on the collection and processing of blood sam-
`ples, for it is by standardization and rigid adherence to these techniques
`that the reliability of the test results will be improved. (J Pediatr 2001;
`138:311-S20)
`
`monia levels are approximately 30
`Limol/L, and levels exceeding ] mmol/L
`occur under conditions of acute hyper-
`ammonemia.*° Overthe past 100 years,
`numerous methods have been devel-
`oped to measure ammonia levels in var-
`ious body fluids including blood, plas-
`ma, erythrocytes, saliva, sweat, and
`urine.© This brief review considers a
`fewof the most common methods cur-
`rently used to measure ammonia in
`blood: alkalization-diffusion, enzymat-
`ic, ion exchange, and electrode. Sample
`collection, handling, storage, and some
`ofthe limitations and potential sources
`oferrors associated with these methods
`are discussed. Finally, some promising
`novel
`techniques for the continuous
`monitoring of ammonialevels that may
`be used clinically in the near future are
`also described.
`
`Ammonia is nowconsidered a normal
`itive proof was provided bystudies
`showing that the enzyme glutamine de-
`constituent ofall body fluids, resulting
`from the metabolism of amino acids.
`hydrogenase specifically reacts with
`ammonia, Ot-ketoglutarate, and a coen-
`However, doubts of the presence of
`zyme, reduced nicotinamide-adenine
`free ammoniain biologic solutions per-
`dinucleotide, to form glutamic acid.!
`sisted until approximately 1960, Before
`This reaction has becomethe basis for
`this time the various methods available
`Glutamine dehydrogenase
`GLDH
`for the determination of ammonia con-
`the most commonly used blood ammo-
`NADH—Reduced nicotinamide-adenine
`dinucleotide
`tent in biologic fluids relied on the sep-
`nia assayin clinical chemistry. One of
`NADPH Reduced nicotinamide adenine
`aration or release of ammonia from the
`the attractive features ofthis assayis
`dinucleotide phosphate
`fluid sample byvolatilization after the
`that ammoniais determined directlyat
`Selected-ion flow tube
`addition of an alkaline solution. With
`physiological pH without previous
`these methods, increased levels of am-
`treatment of the sample with either an
`acid or a base.”"5
`monia had been reported in the blood
`of patients with severe hepaticfailure.
`Today, an elevated ammonia blood
`Despite such reports, doubts about the
`level is considered a strong indicator of
`presence offree ammonia in blood con-
`an abnormality in nitrogen homeostasis,
`the most commonrelated to liver dys-
`tinued, mainly as a result of the con-
`cern that free ammonia measured by
`function. In excess, ammoniais a potent
`these methods resulted from its libera-
`toxin, principally of central nervous
`tion from labile amides in blood during
`system function. In the venous bloodof
`incubation in alkaline solutions. Defin-
`healthy adults and children, blood am-
`
`SIFT
`
`PREANALYTICAL
`METHOD FoR
`HANDLING BLOOD
`AMMONIA
`DETERMINATION
`SAMPLES
`
`Front the Department of Pathology, Ubomae Jefferson University, Philadelphia, Pennayloania,
`Reprint requests: Robert J. Barsotti, PhD, Associate Professor, Department of Pathology, Anato-
`my and Cell Biology, Thomas Jefferson University, 1020 Locust St, Philadelphia, PA 19107.
`Copyright © 2001 by Mosby, Inc.
`0022-3476/2001/$35.00 + 0
`9/0/111832
`
`doi:10.1067/mpd.2001.111832
`
`Most methods recommendcollecting
`from patients who havefasted for at
`least 6 hours with the use ofa verified
`ammonia-free heparin as an anticoagu-
`lant. Heparinis the preferred anticoag-
`ulant, because it has been shown to
`reduce redbloodcell ammonia produc-
`
`LUPIN EX. 1015
`
`1 of 10
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`1 of 10
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`

`THE JOURNAL OF PEDIATRICS
`JANUARY 2001
`
`ANALYTICAL METHODS
`FOR THE
`DETERMINATION OF
`AMMONIA
`
`Manyofthe procedures for ammonia
`determination involve 2 general steps:
`the release of ammonia gas or capture
`of ammonium ions from the sample
`and the quantitation ofthe liberated
`gas or captured ions. Over the years
`the most common methods used to
`volatilize ammonia have been distilla-
`tion and aeration/microdiffusion, ion-
`exchange chromatography, and blood
`or plasma deproteinization.
`
`Properties ofAmmonia
`In attempting to understand the ra-
`tionale used to measure ammonia,it is
`important to review some ofthe physi-
`cal properties of the compound. Am-
`monia (NH;) is a colorless, acrid-
`smelling gas at room temperature and
`pressure. It easily dissolves in water
`andionizes to form NH,’ asfollows:
`
`NH, + H,O @ NH,'+ OH"
`
`An increase in the pH or tempera-
`ture of the solution increases the level
`of the ionized form. Fig 1 shows the
`ratio that exists in plasma between the
`ionized or NH,'
`form versus the
`gaseous or NH, form as a function of
`pH. Thus in plasma at pH 7.4,
`the
`NH,' form represents approximately
`98%of the total ammonia. Manyofthe
`approaches used to estimate ammonia
`levels in body fluids involve volatiliza-
`tion of the NH,’ form of ammonia into
`its gaseous form, NH, »byalkalization
`of the sample to a pH +10.
`
`Distillation
`
`Oneofthe earliest techniques forthe
`measurement of ammonia involves the
`addition ofan alkaline buffer to a sam-
`ple of blood followed by in vacuo dis-
`tillation. The released ammonia gasis
`collected in a trap containing an
`aliquot ofdilute acid, which converts
`
`2 of 10
`
`BARSOTTI
`
`pH Dependenceofthe Fraction of the Ionized Form
`of Ammonia in Solution
`
`0.60
`
`=Ss
`
`
`
`NH,'/NH,Ratio é
`
`8 T
`
`a
`
`8.0
`
`10.0
`
`11.0
`
`9.0
`pH
`
`Fig 1, Ratio between ionized and free gas form of ammonia in plasma as function of pH.
`
`tion. EDTA can also be used. Donors’
`arms should be as relaxed as possible,
`because muscle exertion mayincrease
`venous ammonia levels.’ Blood is
`drawn into a chilled, heparinized vacu-
`um tube that is immediately placed on
`ice, and plasmais separated within 15
`minutes. It is crucial to keep blood
`samples cold after collection, because
`the ammonia concentration of standing
`blood and plasma increases sponta-
`neously. Most ofthis increase has been
`attributed to the generation and re-
`lease of ammonia from red bloodcells
`and the deamination of amino acids,
`particularly glutamine.*!! Plasma am-
`monia levels of whole blood main-
`tained at 4°C are stable for <1 hour.
`When promptly separated from blood,
`plasma ammonia levels are stable at
`4°C for 4 hours and for 24 hours if
`stored frozen at —20°C. To put the
`problem ofrising ammonia levels in
`perspective, the total nitrogen concen-
`tration in venous plasma ofhealthy
`adults exceeds | mol/L and represents
`a potential pool of free blood ammo-
`nia.'* In normal healthy adults, homeo-
`stasis maintains free ammonia levels at
`approximately 30 Umol/L.
`It is important to note that the crite-
`ria for sample stability and the meth-
`ods for the measurement of ammonia
`levels in blood were established almost
`
`exclusively with blood specimens from
`healthy subjects.® Significantly more
`difficult but much more constructive
`would be the establishment ofsimilar
`criteria for blood ammonia measure-
`ments from patients with metabolic or
`liver pathologic conditions. Standing
`blood or plasma samples from these
`patients contain numerous elevated
`sources of ammonia, resulting in in-
`creases in the rate and amount of am-
`monia formed. Some of these sources
`include elevated levels of circulating
`deaminase, y-glutamyltransferase, an
`enzymethat may deaminate free amino
`acids, particularly glutamine in blood
`and plasma samples, resulting in over-
`estimates of blood ammonia levels.
`Fast, careful handling and preparation
`of blood samples is required, especially
`from patients with metabolic or liver
`pathologic conditions, to minimize pre-
`analysis increases In ammonia concen-
`tration until other assay techniques or
`methods are developed. Until this is
`accomplished, measurements in this
`patient population, in which blood am-
`monia levels require the closest moni-
`toring, will continue to be the most un-
`reliable. To date, the easiest and most
`cost-effective method is the stringent
`and diligent maintenance of blood
`samples on ice before, during, and
`after plasma separation.
`
`$12
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`THE JOURNAL OF PEDIATRICS
`Votume 138, Numer |
`
`the gas into the nonvolatile ammonium
`ion. This approach is rather cumber-
`some and slow, particularly when
`many samples require analysis, al-
`thoughinitially, speed was the primary
`advantage of this approach over the
`early microdiffusion techniques de-
`scribed in the following text. This ad-
`vantage, however, was temporary, and
`was lost with the development of
`smaller microdiffusion vessels. The last
`reported use ofdistillation was by
`Burg and Mook!* in 1963.
`
`Aeration/Microdiffusion
`Techniques
`Another early techniquethatis still
`in use relies on liberation of free am-
`monia byalkalization by the addition
`of a strong base to the specimen. The
`released ammoniadiffuses through an
`air- or nitrogen-lilled gap and is
`trapped in acid within the same appa-
`ratus. This approach, introduced by
`Conway and Berne!4 in 1933, uses a
`glass container resembling a Petri dish,
`within which a smaller second cham-
`ber is centered. The wall of this inner
`chamberis approximately half ofthat
`of the outside wall. An aliquot of a
`standard acid solution or ammonia in-
`dicator such as bromocresol green is
`placed into the inner chamber, and the
`sample is added to the outer chamber
`(Fig 2). A measured quantity ofbase is
`added to the sample, and the “petri”
`dish is sealed and gently rotated to mix
`the sample and base in the outer cham-
`ber and then left at room temperature
`for 90 minutes. This same principle is
`used in the Blood Ammonia Check-
`er!>!® and in the Kodak Ektachem
`dry-film method.'” In these systems
`the diffusion distance for ammonia is
`significantly reduced from those in the
`original diffusion apparatus of Conway
`and Berne, requiring only 5 minutes to
`complete (Fig 3).
`Distillation and microdiffusion both
`represent purification procedures that
`isolate ammonia from the manyother
`constituents of blood and plasma, re-
`ducing the possible effects of other
`
`BARSOTTI
`
`Image available
`in print only
`
`Fig 2. Early microdiffusion apparatus for determination of blood ammonia. (Reproducedwith per-
`mission from Conway E, Byrne A. An absorption apparatus for the micro-determination of ammonia.
`Biochem J. 1993;27:419-29. © the Biochemical Society.)
`
`Current Micro-diffusion Apparatus
`
`Sample compartment
`
`Tablet (Alkaline Buffer)
`Cover
`
`Polyethylene film
`
`Color pHindicator
`
`ea
`Polyethylene spacer
`Window
`
`Probe Light Beam
`
`Detector
`
`Illustration of microdiffusion technique used in Blood Ammonia Checker (Reproduced with
`‘ig 3.
`permission from Tada K, Okuda K, Watanabe kK, et al.A new method for screening for hyperam-
`monemia. Eur | Pediatr. 1979;| 30:105-10.)
`
`constituents and drugs on the ammo-
`nia assay. One main drawbackofthese
`procedures is that varying amounts of
`ammonia are liberated from the alka-
`line hydrolysis of proteins, especially
`hemoglobin, and labile amides, espe-
`cially glutamine.!*29
`
`Ton-Exchange Chromatography
`In this approach ammoniagasis not
`liberated from the sample. Instead, a
`strongly acidic cation-exchangeresin
`is used in batch mode to capture am-
`monium ions, NH,*. The resin is
`added to and subsequently separated
`from the sample by centrifugation, and
`the captured ammonium ionsare then
`eluted from the resin bysalt solutions
`or released as ammonia bythe addi-
`tion ofdilute alkali,?! a technique de-
`scribed in detail more recently by
`Brusilow.4
`
`Deproteinization
`Whole blood or plasma proteins are
`precipitated by the addition oftri-
`chloroacetic or perchloric acids, and the
`ammoniais determined directly in the
`supernatantfluid afteral kalization.22-25
`
`QUANTITATION OF
`AMMONIA
`
`After the release or capture of ammo-
`nia or ammonium ions, several methods
`have been described to determine the
`amount of ammonia present. The gen-
`eral categories for these methods in-
`cludetitration, colorimetric/fluorimet-
`ric, electrode-based, and enzymatic.
`
`Titration Method
`The ammonia liberated from the
`sample is trapped in an aliquotofdi-
`
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`BARSOTTI
`
`THE JOURNAL OF PEDIATRICS
`JANUARY 2001
`
`Gas Sensing Electrode
`
`PH electrode Reference Electrode
`
`Reference Buffer
`Gas Permeable Membrane
`(water impervious)
`
`Temperature Controlled
`
`
`
`Sealed Mixing Chamber
`
`Box Ss
`
`(_)<— Sample
`
`)<— Alkaline Butter
`
`Fig 4. Schematic of ion-selective electrode(left pane!) and suggested arrangement for continuous-flow analysis of ammonia
`in plasma samples (right panel).
`
`lute acid, and the amount is measured
`by back-titration of the acid solution
`with a base while the pH is monitored
`with an indicator or electrode. The
`principal advantage ofthis approach is
`thatit is inexpensive, requiring no spe-
`cialized equipment. The disadvan-
`tages, however, are significant. They
`include insensitivity, the requirement
`for large blood samples, and contami-
`nation byother volatile bases that may
`affect the final value. Overall, this pro-
`cedureis laborious and slow andthere-
`fore is not used routinely.
`
`Colorimetric/Fluorimetric
`Reactions
`
`This method is based on the reaction
`of ammonia with a reagent to forma
`colored complex that is measured by
`spectrometryor fluorometry. Among
`the first reactions used was the in-
`dophenol reaction, described by Berth-
`elot
`in 1859254:
`the formation of a
`bluish color by the reaction of ammo-
`nia with phenol and hypochlorite. This
`method is commonlyreferred to as the
`phenol reaction. Another colorimetric
`reaction is the Nessler reaction,
`in
`which a brown-orange color is formed
`by the reaction of ammonia with mer-
`cury or potassium iodide in an alkaline
`solution. Some othercolorimetric reac-
`tions include the use ofisocyanurate,
`cyanate, ninhydrin, or thymol hypo-
`bromite. Detection of ammonia and
`primary amines down to the nanogram
`rangeis routinely performed with fluo-
`rescence derivatization reagents such
`
`as fluorescamine and o-phthalalde-
`hyde.**-?? The principle advantages to
`this approach are speed, simplicity,
`specificity when used carefully, and ex-
`cellent sensitivity. The disadvantageis
`that other substances in the blood af-
`fect some reactions, for example, the
`Berthelot reaction is inhibited by ex-
`cess amino acids, creatine, glutamine,
`and sometherapeutic agents.*”
`
`Gas-sensing Electrode
`With the introduction of the gas-
`sensitive electrode (eg, Orion, Model
`951201), a numberofreports have ap-
`peared describing the methods re-
`quired and the use ofthe electrode in
`measuring ammonialevels in samples
`of blood, urine, cerebrospinal fluid,
`andsaliva over a broad range from 10
`[tmol/L to nearly 1 mmol/L.
`When immersed in the sample or
`held closely above it,
`the dissolved
`gas ofinterest diffuses across a gas-
`permeable membrane into a small vol-
`ume of buffer. The reaction changes
`the pH ofthe buffer, which is sensed
`by an internal pH electrode or sensing
`electrode. The change in pH results in
`a changein the potential between the
`sensing electrode and a reference elec-
`trode immersed in a separate reference
`buffer, all housed within the same elec-
`trode body. The arrangementis illus-
`trated in Fig 4, in which the scale of
`some of the components is exaggerated
`for clarity.
`The main advantages of electrode-
`based ammonia assay are its cheap-
`
`ness, ease ofuse, and because it never
`comesin contact with the sample, its
`imperviousness to sample color, tur-
`bidity, viscosity, or the presence of
`drugs or other metabolites in the sam-
`ple. The electrode is best arranged
`above the surface of the sample in a
`sealed environment
`(Fig 4). This
`avoids the accumulation of proteins
`and cell fragments on the membrane
`surface that would normally occur
`during immersion into plasma or blood
`sample and minimizes the loss of am-
`monia from the sample away from the
`measure-
`electrode
`during
`the
`men
`2 The disadvantagesof the
`£22.31
`electrode-based system includethe re-
`quirementoflarge sample volumes and
`slow sample reads, especially at low
`ammonia levels, requiring 10 to 15
`minutes. In addition, major differences
`in either the osmolarity or temperature
`ofthe sample and the sensing electrode
`buffer must be avoided.
`
`Enzymatic Method
`The specificity of most methods for
`ammonia determination in biologic flu-
`ids relies on the physical separation of
`ammonia from interfering substances
`byvolatilization after alkalization. In
`contrast,
`the most common method
`usedin clinical laboratories is an enzy-
`matic method that measures ammonia
`directly. Thus sample preparation is
`relatively simple, because the previous
`liberation of ammonia from the sample
`is not required. This assay is based on
`the reductive amination of 2-oxoglu-
`
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`THE JOURNAL OF PEDIATRICS
`Vo.ume 138, Numer|
`
`BARSOTTI
`
`injection quadruple
`detection quadruple
`mass filter
`mass spectrometer
`Roots pump
`air or breath sample
`
`He carrier gas
`
`
`channeltron
`lon detector
`
`
`
`carrier gas
`flow
`
`
`ion source
`ion injection
`ion injection
`orifice
`
`orifice
`gas
`
`
`(e.g. Ar/H20)
`
` 41
`
`injection diffusion pump
`
`0cm
`
`detection diffusion pump
`
`Fig 5. Simplified schematic of selected-ion flow tube (SIFT).
`
`tarate with glutamate dehydrogenase
`and reduced nicotinamide adeninedi-
`nucleotide phosphate:
`
`2-Oxoglutarate + NH; + NADPH
`NWGLDH
`Glutamate + NADP
`
`The decrease in absorbance at 340
`nm caused by
`the oxidation of
`NADPH is proportional to plasma am-
`monia. GLDHis specific for ammonia
`and does not react with methylated
`amines. Early studies describing the
`enzymatic determination of ammonia
`used NADH as a coenzyme. Because
`other NADH-consuming systems are
`present in blood, manyofthese reports
`overestimate the level of free ammonia,
`for example, Muting.® The effect of
`these systems can be minimized, usual-
`ly by a 30-minute preincubation peri-
`od. There are considerably fewer
`NADPH-consuming sourcesin plas-
`ma, so the preincubation time can be
`reduced to a few minutes.*
`The assay can be used to measure am-
`monialevels over a broad range, from
`as low as 12 Umol/L to as high as |
`mmol/L. The disadvantage ofthis ap-
`proach is the length and complexity of
`the procedure, thereby enhancing the
`potential for variation in reported blood
`ammonialevels. If not handled proper-
`ly, ammonia concentrations rise in
`standing blood or plasma. Indeed, be-
`cause standard procedures for pre-
`analysis sample processing do not in-
`corporate any attempts to inhibit the
`
`continued liberation of ammonia from
`the samples, except for lowering the
`temperature, ammonia levels in the
`sample will continue to increase during
`the assay and up to the timeofthe ini-
`tial readings. Thus overestimates of am-
`monia levels are most likely in poorly
`handled samples containing elevated
`levels oftransaminases and amino acids.
`
`Normal Valuesfor Blood
`Ammonia Levels
`Table I lists selected blood ammonia
`levels for various blood sample types
`and assay methods from a number of
`studies. The average values for arterial
`blood, plasma, venous blood, and plas-
`ma are 18, 23, 28, and 32 Umol/L, re-
`spectively. The average value for ve-
`nous blood and plasmais 30 [Lmol/L.
`
`FUTURE
`DEVELOPMENTS
`
`The majorlimitations of convention-
`in vitro blood ammonia measure-
`al
`ments are the complexity involved in
`the proper drawing and handling of
`the sample, the time allowed between
`drawing andassaying, and finally, the
`assay itself. A consequence ofthese
`limitations is that blood ammonia mea-
`surements are performed only a few
`times each day. Alternative methods
`are still sought that are noninvasive or
`require a small catheter in a peripheral
`vein but provide a continuous monitor
`of blood ammonia levels. Such meth-
`
`ods could alert medical stalf to an im-
`pending hyperammonemic condition
`and would more easily permit earlier
`selection and regulation of therapeutic
`interventions. Two promising methods
`that are under development and may
`eventually be used clinically are the se-
`lected-ion flow tube technique, which
`analyzes trace gases in breath, and a
`fiber-optic catheter tipped with an am-
`monia-sensitive indicator.
`
`Selected-ion Flow Tube
`Selected-ion Flow Tube is a quanti-
`tative method forthe rapid, real-time
`analysis of the trace gas contentofat-
`mospherieair. It was originally devel-
`oped to studyionic reactions in the gas
`phase and is particularly valuable
`for providing kinetics data on ion-
`molecule reactions, contributing to the
`current understanding of the chem-
`istry of some low-temperature gaseous
`plasmas, especially interstellar clouds.
`The same technology is currently
`being developed to analyze trace gases
`in breath. Previous methods for mea-
`surement of ammonia in breath have
`required large sampling flow rates™ or
`long sampling times® and are there-
`fore unsuitable for assessing the am-
`monia concentration from a single
`breath. A schematic of the SIFT appa-
`ratus is shown in Fig 5 taken from
`Smith and Spanel.*° This technology is
`being further developed and may soon
`be a sensitive, quantitative method for
`the continuousreal-time analysis ofthe
`trace-gas content of human breath and
`
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`BARSOTTI
`
`THE JOURNAL OF PEDIATRICS
`JANUARY 2001
`
`Table I, Reported assay techniques and blood ammonialevels in healthy subjects
`
`Reference
`Reference
`ROPECEementsUMEennOTNeeennnnnneeValue(mol/L)
`Arterial blood
`
`Hutchinson and Labby(1962)
`Gips and Wibbens-Alberts (1968)
`Huizenga and Gips (1983)
`Huizengaet al (1992)
`Huizengaet al (1992)
`Arterial plasma
`Gips and Wibbens-Alberts (1968)
`Huizenga and Gips (1983)
`Venous blood
`
`Dienst (1961)
`Forman (1964)
`Hutchinson and Labby(1962)
`Proelss and Wright (1973)
`Gips and Wibbens-Alberts (1968)
`McCullough (1967)
`Gangolli and Nicholson (1966)
`Sinniahet al (1970)
`Huizengaet al (1992)
`Huizengaet al (1992)
`Venous plasma
`Gerronet al (1976)
`Oberholzeret al (1976)
`Buttery et al (1982)
`Brusilow (1991)
`Cooke and Jensen (1983)
`Willems and Steenssens (1988)
`Spooneret al (1975)
`Seligson and Hirahara (1957)
`Mondzacet al (1965)
`Mutinget al (1968)
`van Ankenand Schiphorst (1974)
`Howanitz et al (1984)
`da Fonseca-Wollheim (1990)
`
`42
`4]
`15
`16
`
`16
`
`4]
`15
`
`21
`59
`42
`25
`4]
`43
`40
`45
`16
`
`16
`
`9
`44
`38
`4
`52
`46
`27
`20
`
`2
`33
`3
`11
`8
`
`lon-exchange/Nessler
`Supernatant/phenol
`Microdittusion/BAC |]
`Microdittusion/BAC II
`
`Enzymatic
`
`Supernatant/phenol
`Enzymatic
`
`lon-exchange/Nessler
`lon-exchange/phenol
`lon-exchange/Nessler
`Supernatant/electrode
`Supernatant/phenol
`Supernatant/phenol
`Supernatant/phenol
`Supernatant/phenol
`Microdittusion/BAC II
`
`Enzymatic
`
`lon-exchange/phenol
`lon-exchange/phenol
`lon-exchange/phenol
`lon-exchange/phenol
`Electrode
`Electrode
`Supernatant/fluorometry
`Microdittusion/Nessler
`
`Enzymatic
`Enzymatic
`Enzymatic
`Enzymatic
`Enzymatic
`
`19
`22
`8
`21
`
`21
`
`23
`23
`
`15
`31
`21
`17
`22
`57
`30
`44
`21
`
`21
`
`19
`16
`21
`18
`44
`44
`32
`56
`
`30
`57
`22
`30
`29
`
`thus permit a continuous, noninvasive
`measure ofsystemic ammonialevels.
`This technique involves the genera-
`tion ofpositive ions that are created in
`a microwave discharge ion source,
`containing an appropriate gas mixture.
`In conventional mass spectrometry,
`ionization ofthe trace gas molecules is
`achieved by electron bombardment,
`resulting in molecular “cracking” and
`the production of complicated spectra.
`The SIFT technique uses a current of
`precursor ions of a given mass-to-
`
`charge ratio. In the case of ammonia
`detection, HO? is extracted from this
`mixture of ions with a quadrupole
`mass filter. This current of selected
`ionsis then injected into a fast-flowing
`inert carrier gas stream, usually heli-
`um. The ions are carried along a 1-
`meter length flow tube and are sam-
`pled by a pinhole downstream ofthe
`injector. The sample ofbreath is intro-
`ducedinto the device by a sample port
`near the injector (Fig 5). Accurate
`trace gas analysis or quantificationis
`
`possible because the reaction of the
`primaryions, in this case HO", with
`ammonia is to form ammoniumtons
`and water is precisely defined in the
`SIFT. In general, the primary ions cho-
`sen must not react at significant rates
`with the major components of the
`breath sample, oxygen,
`nitrogen,
`water, or carbon dioxide, because such
`reactions would saturate the primary
`ions. In turn, the primary ions must
`reactefficiently with the trace gases to
`be detected to form identifiable prod-
`
`$16
`
`6 of 10
`
`6 of 10
`
`

`

`THE JOURNAL OF PEDIATRICS
`Vo.ume 138, Numer|
`
`BARSOTTI
`
`|| |
`
`Isoprene
`(320)
`
`7
`
`~
`phenol
`(610)
`
`3
`
`a3
`
`a1
`
`-
`
`
`
`precursor lons
`BIR ae
`a on
`
`
`methanol
`(400)
`
`104
`
`108
`
`Counts/sec
`
`10!
`
`1
`
`
`
`uct ions. Thus the primaryions and the
`specificityoftheir interactions with the
`target trace gases in the breath sample
`is a determinantofthe selectivity of the
`method. The identification and quanti-
`tation of the products formed are then
`detected by a mass spectrometer.
`Currently, the technique can measure
`102
`us
`4
`trace gases downtoapartial pressure
`8895
`of approximately 10 ppb. The mean
`value of 960 ppb for breath ammoniain
`normal adults was reported in Davies
`et al*” and thus is well above the mini-
`mum sensitivity of the apparatus. Fig 6
`shows a spectrum obtained from a
`breath sample taken from a uremic pa-
`tient (end-stage renal failure) before
`hemodialysis. The sample was taken
`and stored in a Teflar bag and then
`transferred to the laboratory for analy-
`sis by SIFT. The molecular weight (x-
`axis) for each molecule identified is
`shown at the top of each bar. Open
`bars represent precursor ions, H,O"'
`(molecular weight = 19), and their
`water clusters with molecular weights
`of 37, 55, and 73. The solid bars show
`the counts for each trace gas, ammoni-
`um ions (molecular weight = 18), and
`their water clusters, with molecular
`weights of 36 and 54. The partial pres-
`sures of ammonia andacetone are ele-
`vated compared with those of healthy
`adults, whereas the levels of ethanol,
`methanol, and propanol are normal.
`Aminesare presentin this sample but
`not in normal breath. The presence of
`acetonitrile is a clear indicator that the
`patient smokes. The techniqueis rapid
`enough to allow realtime, breath-to-
`breath quantitation oftrace gas.
`However, a numberoftechnical hur-
`dles remain before the technique can be
`movedfrom the laboratoryinto the clin-
`ical setting. First, the equipmentisstill
`ratherlarge, complicated, and cumber-
`some. Another problem is the preven-
`tion of ammonia loss in the condensate
`ofrespired air before it enters the appa-
`ratus. There is no doubt that many
`of these technical problems will be
`overcome with further development.
`Nevertheless, several significant biolog-
`
`15 20 25 30 35 4
`
`45,50 55
`
`60_ 65
`70 75 80 85 " 95
`~<trimethylamine
`CgHygN
`(200)
`(200)
`Molecular Weight
`Fig 6. SIFT spectrum obtained with H,0° precursor ions of breath sampled from patient with
`end-stage renalfailure before hemodialysis treatment. Precursor ions and their respective isotopic
`variants are shown as oben bars, and product ions are shownas filled bars. (Reproduced with permis-
`sion from Davies 5, Spanel P Smith D, Quantitative analysis of ammonia on the breath of patients in
`end-stage renalfailure. Kidney Int,
`|1997;52:223-28.)
`
`acetonitrile
`(90)
`
`dimethylamine
`
`ic questions remain to be addressed
`before the efficacy of this approach ts
`established, including the correlation
`between breath ammonia levels and
`blood levels and the factors determining
`breath ammonia levels. One concern is
`whether breath ammonia is determined
`predominantly by urea hydrolysis with-
`in the oral cavity, or whether it does
`indeedreflect the level in blood. Prelim-
`inary reports by Davies et al*” show
`a correlation with the plasma urea lev-
`els after ingestion of urea in normal
`volunteers and during hemodialysis of
`patients with end-stage renal failure,
`suggesting that elevated breath ammo-
`nia levels are generated systemically.
`
`RECOMMENDATIONS
`
`In facilities treating large numbers of
`patients with liver or metabolic disor-
`ders, the establishment ofa separate
`facility is recommended for the han-
`dling, monitoring, and measuring of
`blood ammonia andliver enzymelev-
`els. This is primarily to minimize errors
`from contamination and poor or im-
`
`proper sample handling and to im-
`prove overall expertise and therebythe
`reliability of the determinations.
`
`Sample Processing
`Moreefforts must be madeto reduce,
`or preferably eliminate,
`the various
`sources of ammonia formation in stand-
`ing blood or plasma. For example, an
`initial step incorporating an acid pre-
`cipitation with cold perchloric acid be-
`fore blood centrifugation should befol-
`lowed byion exchange methodin batch
`modeto select and purify the ammoni-
`um ions from the few remaining ammo-
`nia-generating systems in the depro-
`teinated plasma sample.
`
`Assay Method
`Multiple time points or serial mea-
`surements, perhaps 6 separate time in-
`tervals of ammonia content, should be
`made from the same sample ofstand-
`ing plasma. If the increase in ammonia
`level is linear with time, that is, exhibit-
`ing zero order kinetics, then linear ex-
`trapolation ofthe time course to zero
`time provides an estimate of the ammo-
`nia level at the time ofcollection.
`
`$|7
`
`7 of 10
`
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`
`

`

`BARSOTTI
`
`CONCLUDING REMARKS
`
`The choice of technique for ammonia
`determination depends predominantly
`on the available equipment. Preanaly-
`sis handling including contamination
`and improper or inadequate proce-
`dures to limit ammonia formation in
`the sample before analysis remainsthe
`main problem or source of“error” in
`accurate determinations offree ammo-
`nia levels in blood samples.
`However, whenever high blood am-
`monia levels are detected and consis-
`tent with the patient's clinical status,
`an alternative method should be avail-
`able and used for verification.
`
`REFERENCES
`
`Olson JA, Anfinsen CB. The crystal-
`lization and characterization of L-
`glutamic acid dehydrogenase. J Biol
`Chem 1952;197:67-79.
`Mondzac A, Ehrlich GE, Seegmiller
`JE. An enzymatic determination of
`ammonia in biological fluids. J Lab
`Clin Med 1965;66:526-31.
`van Anken HC, Schiphorst ME. Aki-
`netic determination of ammoniainplas-
`ma. Clin Chim Acta 1974;56:151-7.
`Brusilow SW. Determination of urine
`orotate and orotidine and plasma am-
`In:
`monium.
`Hommes FA, editor.
`Techniques in diagnostic human bio-
`chemical: a laboratory manual. New
`York: Wiley-Liss; 1991. p. 345-7.
`Brusilow SW, Maestri NE. Urea cycle
`disorders: diagnosis, pathophysiology,
`and therapy. Adv Pediatr 1996;43:
`127-70.
`Huizenga JR, Tangerman A, Gips
`CH. Determination of ammoniain bio-
`logical
`fluids. Ann Clin Biochem
`1994;31:529-43.
`Lowenstein JM. Ammonia production
`in muscle and othertissues: the purine
`nucleotide cycle. Physiol Rev 1972;52:
`582-414,
`da Fonseca-Wollheim F, Preanalytical
`increase of ammonia in blood speci-
`mens from healthy subjects. Clin Chem
`1990;36:1483-7.
`Gerron GG, Ansley JD, Isaacs JW,
`Kutner MH, Rudman D. Technical
`pitfalls in measurement of venous plas-
`ma NH3 concentration. Clin Chem
`1976;22:665-6.
`. Glasgow AM. Clinical application of
`
`“I
`
`$18
`
`blood ammonia determination. Lab
`Med 1981;12:151-7.
`. Howanitz JH, Howanitz PJ, Skrodz-
`ki CA, Iwanski JA. Influences of spec-
`imen processing and storage condi-
`tions on results for plasma ammonia.
`Clin Chem 1984;30:906-8.
`. Lentner C. Geigyscientific tables. Vol
`3. Physical chemistry composition of
`blood hematology. Somatometric data-
`blood nitrogenous substances. Basel:
`Ciba-Geigy Limited; 1984.
`. Burg PVD, Mook HW. A simple and
`rapid method for the determination of
`ammonia in blood. Clin Chim Acta
`1963;8: 162-4.
`. Conway E, Byrne A. An absorption
`apparatus for the micro-determination
`of certain volatile substances. The
`micro-determination
`of
`ammonia.
`Biochem J 1993;27:419-29.
`. Huizenga JR, Gips CH. Determina-
`tion of blood ammonia using the Am-
`monia Checker. Ann Clin Biochem
`1983;20:187-9.
`Huizenga JR, Tangerman A, Gips
`CH. A rapid method for blood ammo-
`nia determination using the newblood
`ammonia checker (BAC) IT. Clin Chim
`Acta 1992;210:153-5.
`losefsohn M, Hicks JM. Ektachem
`multiplayer dry-film assay for ammo-
`nia evaluated. Clin Chem 1985;31:
`2012-4,
`Jacquez JA, Jeltsch R, Hood M. The
`measurement of ammonia in plasma
`and blood. J Lab Clin Med 1959;
`53:942-50.
`. Reif AE. The ammonia content of
`blood and plasma. Anal Biochem 1960;
`1:351-9.
`. Seligson D, Hurahara K. The mea-
`surement of ammonia in whole blood
`erythrocytes and plasma. J Lab Clin
`Med 1957;49:962-74.
`. Dienst SG. An ion exchange method
`for plasma ammonia concentration. J
`Lab Clin Med 1961;58:149-55.
`Moses