Measurement of ammonia in blood
`
`Robert J Bamatn; MD
`
`The measurement of ammonia. now ltnown to be a normal constituent of all
`
`
`
`body fluids, is Fraught with problems. An elevated ammonia level in blood
`(100 y.molfL 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 of error are discussed. New tech»
`niques that permit continuous or real-time estimates of systemic ammonia
`levels over a broad range are also discussed. The aim should aiways 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:S1l-S20)
`
`Ammonia is novv considered a normal
`
`constituent of all body fluids, resulting
`from the metabolism of amino acids.
`
`However, doubts of the presence of
`Free ammonia in biologic solutions per-
`sisted until approximately 1960. Before
`this time the various methods available
`for the determination of ammonia con-
`
`tent in biologic fluids relied on the sep-
`aration or release :3? ammonia From the
`
`Fluid sample by volatilization after the
`addition ofan alkaline solution. With
`these methods, increased levels of am-
`
`monia had been reported in the blood
`of patients with severe hepatic Failure.
`Despite such reports. doubts about the
`presence of Free ammonia in blood con»
`tinued, mainly as a result of the con-
`cern that Free ammonia measured by
`these methods resulted From its libera-
`
`tion from labile amides in blood during
`incubation in alkaline solutions. Defin-
`
`itive proof was provided by studies
`showing that the enzyme glutamine de-
`hydrogenase specifically reacts with
`ammonia. (1.-ketoglutarate. and a coen-
`zyme. reduced nicotinamide-adenine
`dinucleotide, to Form glutamic acid.‘
`This reaction has become the basis for
`
`the most commonly used blood ammo-
`nia assay in clinical chemistry. One of
`the attractive features of this assay is
`that ammonia is determined directly at
`physiological pH without previous
`treatment of the sample with either an
`acid or a base.“
`
`Today, an elevated ammonia blood
`level is considered a strong indicator ol'
`an abnormality in nitrogen homeostasis.
`the flilost Cofllnlon related. to liver dy5'
`Function. In excess, ammonia is a potent
`toxin, principally of central nervous
`system limction. In the venous blood of
`healthy adults and children. blood am-
`
`ll‘ iimrnna Jafirrann ffnnrraiy, Pfiiirrflcfpbiir, Pann.:_u1mn.r'n.
`From the Dcpnrfnrcni agf Pat."£\I1.'irJg_y;
`Reprint requests: Robert J. Barsotti. PhD, Associate Professor, Deparrrnent of Pathology, Anato-
`my and Cell Biology. Thomas Jefferson Universiiy. 1020 Locust St, Philadelphia. PA 19107.
`Copyright @ 2001 by Mosby. Inc.
`0022-3476l20Gl)’$55.U{l + {J
`930/111852
`
`doi:lU.l057fn1pd.2U0l.l11332
`
`monia levels are approximately 30
`Llmol/L, and levels exceeding 1 mmolfl.
`occur under conditions of acute hyper-
`ammonemia.“ Over the past lU0years.
`numerous methods have been devel-
`
`oped to measure ammonia levels in var-
`ious body iluids including blood. plas-
`ma. erythrocytes, saliva, sweat. and
`urine.6 This briel’ review considers a
`Few of the most common methods cur-
`
`rently used to measure ammonia in
`blood: alkalization~diiFusion, enzymat-
`ic. ion exchange. and electrode. Sample
`collection, handling, storage. and some
`ol" the limitations and potential sources
`of errors associated with these methods
`
`are discussed. Finally, some promising
`novel techniques for the continuous
`monitoring of ammonia levels that may
`be used clinically in the near Future are
`also described.
`
`GLDH
`NADH
`
`Glmamine dehyclrogenase
`Reduced nicoiinamicle-adenine
`dlnudeotlde
`NADPH Reduced nicotiriamitle adenine
`dlnudeotlde phosphate
`Selected-Ion flow tube
`
`S|Fl'
`
`PREANALYTICAL
`
`METHOD Fon
`
`HANDLING BLOOD
`
`AMMoNIA
`
`DETERMINATION
`
`SAMPLES
`
`Most methods recommend collecting
`From‘ patients who have fasted for at
`least 6 hours with the use of a verified
`
`ammonia-Free heparin as an anticoagu-
`lant. Heparin is the preferred anticoag-
`ulant, because it has been shown to
`
`reduce red blood cell ammonia produc-
`
`SH
`
`LUPIN EX. 1015
`
`1of10
`
`1 of 10
`
`

`

`BARSOTTI
`
`pl-I Dependence of the Fraction of the ionized Fonn
`of Ammonia in Solution
`
`I
`
`.
`
`I.D|l
`
`PS
`
`P8
`
`PF’BS
`
`
`
`NH‘?NH,Ratio
`
`
` 7.0 8.!)
`SJ
`19.0
`11.U
`
`Fry 1'. Ratio between ionized and free gas form of ammonia in piasrna as function of pH.
`
`exclusively with blood specimens From
`healthy sul.>jects.8 Significantly more
`diliicult but much more constructive
`would be the establishment oilsimilar
`criteria for blood ammonia measure-
`
`ments lrom patients with metabolic or
`liver pathologic conditions. Standing
`blood or plasma samples From these
`patients Contain numerous elevated
`sources oi‘ ammonia. resulting in in-
`creases in the rate and amount ol‘ am-
`monia formed. Some of‘ these sources
`
`include elevated levels of circulating
`deaminase. Y-glutamyltransferase. an
`enzyme that may deaminate free amino
`acids. particularly glutamine in blood
`and plasma samples, resulting in over-
`estimates ol' 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 an-I-
`monia levels require the closest moni-
`toring. will continue to be the most un-
`reliable. To date, the easiest and most
`
`tion. EDTA can also be used. I)onors'
`
`arms should be as relaxed as possible.
`because muscle exertion may increase
`venous ammonia levels}? Blood is
`
`drawn into a chilled. heparinized vacu-
`um tube that is immediately placed on
`ice. and plasma is separated within 15
`minutes. It is crucial to keep blood
`samples cold alter collection, because
`the ammonia concentration olistanding
`blood and plasma increases sponta-
`neously. Nlost ol‘ this increase has been
`attributed to the generation and re~
`lease of‘ ammonia from red blood cells
`and the deamination of‘ amino acids,
`
`particularly glutamine.3‘“ Plasma am-
`monia levels OI‘ 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 ii‘
`
`stored froacen at —'20"C. To put the
`problem of‘ rising ammonia levels in
`perspective, the total nitrogen concen-
`tration in venous plasma oi‘ healthy
`adults exceeds l molfl. and represents
`a potential pool of free blood ammo-
`nia.'2 In normal healthy adults, homeo-
`stasis maintains lree ammonia levels at
`
`approximately 50 ttmol/L.
`It is important to note that the crite-
`ria For sample stability and the meth-
`ods i'or the measurement oi’ ammonia
`levels in blood were established almost
`
`Sl2
`
`THE JOURNAL OF PEDIATRICS
`JANUARY 200i
`
`ANALYTICAL METHODS
`
`FORTHE
`
`DETERMINATION OF
`
`AMMONIA
`
`Nlany oiithe procedures for ammonia
`determination involve 2 general steps:
`the release oi‘ ammonia gas or capture
`of ammonium ions from the sample
`and the quantitation ol’ the liberated
`gas or captured ions. Over the years
`the most common methods used to
`volatilize ammonia have been distilla-
`tion and aerationfmicrodil'l‘usion. ion-
`
`exchange chromatography, and blood
`or plasma deproteinization.
`
`Properttiea of/lnmwrtfa
`In attempting to understand the ra~
`tionale used to measure ammonia. it is
`
`important to review some oi‘ the physi-
`cal properties ol‘ the compound. Arn-
`monia (NH3)
`is a colorless. acrid-
`SITI{’.‘iiing gas at roof“ ‘l.'CTI"IpCratuT'C and
`pressure. It easily dissolves in water
`and ion izes to Form l\lH_,‘ as Follows:
`
`NH3 + H202 NH4'+ OH‘
`
`An increase in the pH or tempera-
`ture of the solution increases the level
`
`OF the ionized Form. Fig l shows the
`ratio that exists in plasma between the
`ionized or NH4' Form versus the
`gaseous or NH3 l‘orm as a liunction oi‘
`pH. Thus in plasma at pH 7.4.
`the
`NH4‘ Form represents approximately
`98% ofthe total ammonia. Many ofthe
`approaches used to estimate ammonia
`levels in body fluids involve volatili7.a-
`tion ol‘ the t\lH_1* ibrm oiiammonia into
`its gaseous iorm. NH5 ,by alitalization
`ofthe sample to a pH >10.
`
`Dtlrttfla tion
`
`One o|'the earliest techniques For the
`measurement oliammonia involves the
`addition ol"an all-taline buller to a sam-
`
`cost—ei‘l‘ective method is the stringent
`and diligent maintenance oi‘ blood
`samples on ice before. during, and
`alter plasma separation.
`
`ple oi‘ blood followed by in Vacuo dis-
`tillation. ’l‘he released ammonia gas is
`collected in at
`trap containing an
`aliquot ol-1 dilute acid. which converts
`
`2of10
`
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`
`

`

`THE ]OURNAL or PEDIATRICS
`VOLUME I38, NUMBER I
`
`the gas into the nonvolatile ammonium
`ion. This approach is rather cumber-
`some and slow. particularly when
`ulany Salrlples require analysis,
`3.!‘-
`though initially, speed was the primary
`advantage of this approach over the
`early microdilil-‘usion techniques de-
`scribed in the Following text. This ad-
`vantage, however. was temporary, and
`was lost with the development of
`smaller microdifliusion vessels. The last
`
`reported use of distillation was by
`Burg and Mock” in 1965.
`
`Aenzttion/Jficmfli radian
`Tecihniqued
`Another early technique that is still
`in use relies on liberation of Free am-
`
`monia by alkalization by the addition
`ofa 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 Berne” in 1953, 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
`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 of base 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»
`erl5"fi 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 5).
`Distillation and rnicrodiffusion both
`
`represent purification procedures that
`isolate ammonia From the many other
`constituents of blood and plasma, re»
`ducing the possible effects of other
`
`BARSOTTI
`
`Image available
`in print only
`
`Fly 2. Early miuodiffiision apparatus fordelrermination of blood ammonia. {Reproduced with per-
`mission from Conway E. Byrne A. An absorption apparatus for the micro-detennination of ammonia.
`Biochem J.
`|993‘,27:4 I 9-29. © ‘me Biochemical Society.)
`
`Current Micro-diffusion Apparatus
`
`Sample cornpertrnenl
`
`Tablet (Alkaline Buffer)
`
` Polyethylene fllm
`
`Illustration of microdiffusion technique used in Blood Ammonia Checker‘. (Reproduced with
`Fig}.
`permission fromTada K Okuda K.Watanabe K. et a|.A new method for screening for hyperam-
`monemia. Eur] Pediafl: |979:|30:|05-I0.)
`
`constituents and drugs on the ammo-
`nia assay. One main drawback of these
`procedures is that varying amounts of
`ammonia are liberated from the alka-
`
`line hydrolysis of proteins, especially
`hemoglobin, and labile amides. espe-
`cially glutarnine.'3'2"
`
`Ion-Excihange Clhmnzatagrapliy
`In this approach ammonia gas is not
`liberated from the sample. instead, a
`strongly acidic catiomexchange resin
`is used in batch mode to capture am-
`monium ions, NH4‘. The resin is
`added to and subsequently separated
`From the sample by centril'i.1gation. and
`the captured ammonium ions are then
`eluted From the resin by salt solutions
`or released as ammonia by the addi-
`tion of dilute alkali,” a technique de-
`scribed in detail more recently by
`Brusilowfl
`
`Deproteinézafion
`Whole blood or plasma proteins are
`precipitated by the addition of tri-
`chloroacetic or perchloric acids, and the
`ammonia is determined directly in the
`supernatant fluid after alltalization.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-
`clude titration, colorimetric;’fiuorimet-
`
`ric, electrode-baseds and enzymatic.
`
`Titration Memo?
`The ammonia liberated from the
`
`sample is trapped in an aliquot of di-
`
`SI3
`
`3of10
`
`3 of 10
`
`

`

`BARSOTTI
`
`THE JOURNAL or PEDIATRICS
`JANUARY 200|
`
`Temperature controlled
`
`Box \
`
`Gas Sensing Electrode
`ltnhranc: Elaclmde
`
`pl-I niaeirorln
`
` pt-I Eullar
`
`Helurence Buflel
`Gas Pcrrnefile Membrane
`[water imptmrlaua]
`
`
`
`4-“ Sample
`
`4— manna Buffer
`
`Sealed Mixing churn bar
`
`Fry -7. Sthematic 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
`
`by back-titration ol. the acid solution
`with a base while the pH is monitored
`with an indicator or electrode. The
`
`principal advantage oi‘ this approach is
`that it is inexpensive, requiring no spe-
`cialized equipment. The disadvan-
`tages. however. are signilicant. They
`include insensitivity, the requirement
`For large blood samples. and contami-
`nation by other volatile bases that may
`allect the final value. Overall. this pro-
`cedure is laborious and slow and there-
`
`fore is not used routinely.
`
`C0iarintetric/Fluort'ntetrt'c
`Reactiorw
`
`This method is based on the reaction
`
`of ammonia with a reagent to liorrn a
`colored complex that is measured by
`spectrometry or [‘luorometr_y. Among
`the first reactions used was the in-
`
`dophenol reaction, described by Berth-
`elot
`in l85923°:
`the lbrmation oF a
`
`bluish color by the reaction oi‘amrno~
`nia with phenol and hypochlorite. This
`method is commonly reFerred to as the
`phenol reaction. Another colorimetric
`reaction is the Nessler reaction,
`in
`
`which a brown»-orange color is formed
`by the reaction oiiamtnonia with mer-
`Cllry or potassium iodide in an alkaline
`solution. Some other colorimetric reac-
`
`tions include the use oli isocyanurate,
`cyanate, ninhydrin. or thymol hypo-
`bromite. Detection of‘ ammonia and
`
`primary amines down to the nanogram
`range is routinely performed with fluo-
`rescence derivatization reagents such
`
`as lluorescamine and o-phthalalcle-
`h_yde.2"'29 The principle advantages to
`this approach are speed, simplicity,
`specificity when used carei‘ull__v. and ex-
`cellent sensitivity. The disadvantage is
`that other substances in the blood ali-
`
`liect some reactions, For example. the
`Berthelot reaction is inhibited by ex‘-
`cess amino acids. creatine. glutamine.
`and some therapeutic agentsm
`
`Gad-aerwing Electronic
`With the introduction oi‘ the gas-
`sensitive electrode (cg. Orion. Nlodel
`951201), a number ol‘ reports have ap-
`peared describing the methods re-
`quired and the use olithe electrode in
`measuring ammonia levels in samples
`oi‘ blood. urine. cerebrospinal Fluid.
`and saliva over a broad range lirom lU
`p.mol/L to nearly 1 mmol/ L.
`When immersed in the sample or
`held closely above it.
`the dissolved
`gas oi‘ interest diliuses across a gas-
`permeable membrane into a small vol-
`ume ol' bullier. The reaction changes
`the pH oi’ the builier, which is sensed
`by an internal pH electrode or sensing
`electrode. The change in pH results in
`a change in the potential between the
`sensing electrode and a reference elec-
`trode immersed in a separate reference
`bul'l'er, all housed within the same elec-
`
`trode body. The arrangement is illus-
`trated in Fig 4. in which the scale oi‘
`some of‘ the com ponents is exaggerated
`For clarity.
`The main advantages oi‘ electrode-
`based ammonia assay are its cheap-
`
`ness. ease oi‘ use, and because it never
`
`comes in contact with the sample, its
`imperviousness to sample color, tur-
`bidity. viscosity. or the presence oi‘
`dI'ugs or other metabolites in the sam-
`pie. The electrode is best arranged
`above the surface 0}‘ the sample in a
`sealed environment
`(Fig 4). This
`avoids the accumulation oi‘ proteins
`and cell Fragments on the membrane
`surlaee that would normally occur
`during immersion into plasma or blood
`sample and minimizes the loss of am-
`monia Irom the sample away from the
`electrode
`during
`the measure-
`ment.22‘5"52 The disadvantages of the
`electrode-based system include the re-
`quirement oflarge sample volumes and
`slow sample reads. especially at low
`ammonia levels, requiring 10 to ]5
`minutes. In addition. rnaior dill'erences
`in either the osmolarity or temperature
`olithe sample and the sensing electrode
`bullet‘ must be avoided.
`
`Enzymatic Method
`The specificity oi‘ most methods For
`ammonia determination in biologic llu-
`ids relies on the physical separation ol-
`arnmonia from interfering substances
`by volatilization aFter alkalization. ln
`contrast,
`the most common method
`
`used in clinical laboratories is an en:r._y-
`matic method that measures ammonia
`
`directly. Thus sample preparation is
`relatively simple. because the previous
`liberation oiiammonia from the sample
`is not required. This assay is based on
`the reductive amination oi‘ 2-oxoglu~
`
`SI4
`
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`
`4 of 10
`
`

`

`THE JOURNAL OF PEDIATRICS
`VOLUME I38. NUMBER I
`
`h
`
`sou
`In
`'°°‘m.2$‘i‘lii.. "'°
`He carrier gas
`
`
`air {if bffllih SRITIDH
`
`BARSOTFI
`
`deleellon quadruple
`“mm mm” mass spectrometer
`fl|'|lI'I|'IBflI’0I'I
`IOI1 HEIEGIOF
`
`
`
`ion source
`
`9::
`(9.3. Arfl'|;D)
`
`
`
`Ion Inhcllnrt
`Ion injection
`orlllce
`OHIIBE
`
`lrqecltnn dtfluainn pump
`
`ijl
`10cm
`
`detection cltfltinlon pump
`
`I"i:q 5. Simplified schematic of selected-ion flcw ‘tube (S||'-T).
`
`tarate with glutamate dehydrogenase
`and reduced nicotinamide adenine di-
`
`nucleotide phosphate:
`
`2~Oxoglutarate + NH3 + NADPH
`TlGt.DH
`Glutamate + NADP
`
`The decrease in absorbance at 540
`the
`
`nm caused by
`oxidation of
`NADPH is proportional to plasma am-
`monia. GLDH is specific For ammonia
`and does not react with methylated
`amines. Early studies describing the
`enzymatic determination ol" ammonia
`used NADH as a coenavme. Because
`other NADH-consuming systems are
`present in blood, many of these reports
`overestimate the level of‘ Free ammonia,
`
`for example. iVtuting.53 The eI’l'ect oi‘
`these systems can be minimized. usual-
`ly by a 30-minute prcincubation peri-
`od. There are consideralily lewer
`NADPH—constiming sources in plas~
`ma. so the preincubation time can be
`reduced to a few minutes.5
`
`The assay can be used to measure am-
`monia levels over a broad range. from
`as low as 12 t.tmol.’L to as high as I
`rnrnol/L. The disadvantage of this ap-
`proach is the length and complexity oi‘
`the procedure. thereby enhancing the
`potential for variation in reported blood
`ammonia levels. llinot handled proper-
`ly, ammonia concentrations rise in
`standing blood or plasma. lndecd, be-
`cause standard proccdurcs for pre-
`analysis sample processing do not in-
`corporate any attempts to inhibit the
`
`continued liberation of ammonia from
`
`ods could alert medical stal‘l' to an im-
`
`the samples, except for lowering the
`temperature, ammonia levels in the
`sample will continue to increase during
`the assay and up to the time oiithc ini-
`tial readings. Thus overestimates oFam-
`monia levels are most likely in poorly
`handled samples containing elevated
`levels olltransaminases and alnino acids.
`
`Nor-natal Vafueafor B3003
`Amm.om'a Levels
`Table I lists selected blood ammonia
`
`levels l'or various blood sample types
`and assay methods from a number of
`studies. The average values l‘or arterial
`blood. plasma. venous blood. and plas-
`ma are 18, 23, 28, and 52 ].1molfl., re~
`spectively. The average value For ve-
`nous blood and plasma is 50 tlmol./L.
`
`FUTURE
`
`DEVELOPMENTS
`
`The major limitations ol‘convention-
`in vitro blood ammonia measure-
`
`al
`
`ments are the complexity involved in
`the proper drawing and handling oi‘
`the sample. the time allowed between
`drawing and assaying. and iinally. the
`assay itself. A consequence ol‘ these
`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
`oi‘ blood ammonia levels. Such meth-
`
`pending hyperammonemic condition
`and would more easily permit earlier
`selection and regulation oi‘ therapeutic
`interventions. Two promising methods
`that are under development and may
`eventually be used clinically are the se-
`lected-ion llow tube technique, which
`analyzes trace gases in breath. and a
`fiber-optic catheter tipped with an am-
`monia-sensitive indicator.
`
`Selected-for: Flow Tube
`
`Selected-ion Flow Tube is a quanti-
`tative method for the rapid. real-time
`analysis of the trace gas content of‘ at-
`mospheric air. It was originally devel-
`oped to study ionic reactions in the gas
`phase and is particularly valuable
`for providing ltinetics data on ion-
`molecule reactions. contributing to the
`current understanding oi‘ the chem-
`istry ollsomc low-temperature gaseous
`plasmas. especially interstellar clouds.
`The same technology is currently
`being developed to analyze trace gases
`in breath. Previous methods {or mea-
`surement oi‘ ammonia in breath have
`
`required large sampling Iiow rat'es3'l or
`long sampling times35 and are there-
`fore unsuitable For assessing the am-
`monia concentration from a single
`breath. A schematic oi" the SIFT appa-
`ratus is shown in Fig 5 talten from
`Smith and Spaniel.“ This technology is
`being llurthcr developed and may soon
`be a sensitive, quantitative method For
`the continuous real-time analysis ol'the
`trace—gas content ofhuman breath and
`
`S15
`
`5of10
`
`5 of 10
`
`

`

`Bansom
`
`THE _|ouaNAL or PEDIATRICS
`JANUARY 2001
`
`Tobie 1’. Reported assay techniques and blood arnrnonla levels in healthy subjects
`
`Reference
`
`Arterial blood
`
`.
`
`Hutchinson and Labby (1962)
`Gips and Wqbbens-Alberts (1968)
`Huizenga and Cips (1985)
`Huizeuga et al (1992)
`Huizenga et al (1992)
`Arterial plasma
`Crips and \«Vibbens-Alberts (1968)
`Huizenga and Gips (1935)
`Venous blood
`
`Dienst (1961)
`Forman (1964)
`Hutchinson and Labby (1962)
`Proelss and Wright (1973)
`Gips and Wibbens-Albefls (1963)
`McCullough (1967)
`Gangolli and Nicholson (1966)
`Sinniah et al (1970)
`Huizenga et al (1992)
`Huizenga et al (1992)
`Venous plasma
`Gerron et a1 (1976)
`Oberholzer et al (1976)
`Buttery et al (1982)
`Brusilow (1991)
`Cooke and Jensen (1985)
`Willems and Steenssens (1938)
`Spooner et al (1975)
`Seligson and Hirahara (1957)
`Mondzac et a1 (1965)
`Muting er al (1968)
`van Anken and Schiphorst (1974)
`Howanitz et al (1984)
`(:13 Fonseca-Wollheim (1990)
`
`42
`41
`15
`I6
`16
`
`41
`15
`
`2 l
`59
`42
`25
`41
`43
`40
`45
`16
`15
`
`9
`44
`33
`4
`32
`46
`27
`20
`2
`35
`5
`I1
`8
`
`.
`
`Ion-exchange/Nessler
`Supernatant/phenol
`N1icrodif1"usionfBAC I
`Microdiiiiusion/BAC II
`Enzymatic
`
`Supernatant/pl1enol
`Enzymatic
`
`1on—exchange/Nessler
`lon -exchange/phe no]
`lon-exchange/Nessler
`Supernatant/electrode
`Supernatant/phenol
`Supematantfphenol
`Supernatant/phenol
`Supernatant/phenol
`Microdiffixsion/BAC II
`Enzymatic
`
`lon-exchangefphenol
`lon-exchangefphenol
`Ion-exchange/phenol
`Ion-ext: han giefphenol
`Electrode
`Electrode
`Supernatantffluorometry
`Microdif1"usion!Nessler
`Enzymatic
`Enzymatic
`Enzymatic
`Enzymatic
`Enzymatic
`
`19
`22
`3
`21
`21
`
`23
`25
`
`15
`51
`21
`17
`22
`57
`50
`44
`21
`21
`
`19
`16
`21
`13
`44
`44
`52
`56
`30
`57
`22
`50
`29
`
`thus permit a continuous, noninvasive
`measure ofsystemic ammonia levels.
`This technique involves the genera-
`tion of positive ions that are created in
`a microwave discharge ion source,
`containing an appropriate gas mixture.
`In conventional mass spectrometry,
`ionization of the trace gas molecules is
`achieved by electron bombardment.
`resulting in molecular "cracking" and
`the production of complicated spectra.
`The SIFT technique uses a current ol‘
`precursor ions of a given mass-to»
`
`charge ratio. In the case of‘ ammonia
`detection, 1130* is extracted from this
`mixture of ions with a quadrupole
`mass filter. This current oF selected
`
`ions is then injected into a Fast-flowing
`inert carrier gas stream, usually heli-
`urn. The ions are carried along a 1»
`meter length flow tube and are sam-
`pled by a pinhole downstream of the
`injector. The sample of breath is intro-
`duced into the device by a sample port
`near the injector (Fig 5). Accurate
`trace gas analysis or quantification is
`
`possible because the reaction oF the
`primary ions, in this case H504. with
`ammonia is to Form ammonium ions
`
`and water is precisely defined in the
`SI PT. In general, the primary ions cho-
`sen must not react at significant rates
`with the rnaior components 0F the
`breath sample. oxygen, nitrogen,
`water, or carbon dioxide, because such
`
`reactions would saturate the primary
`ions. 111 turn, the primary ions must
`react eliiciently with the trace gases to
`be detected to Form identifiable prod»
`
`S16
`
`6of10
`
`6 of 10
`
`

`

`BARSOTFI
`
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`?I3 75 8-0 85 190 95
`15 20 25 30 35 9 -|I5< 55 BD,{>|
`aeetoasilrile
`almeliivmnim
`ta-tmatrmnmirua
`C3!-l13N
`N0}
`[M0]
`I200]
`[300]
`
`Molecular Weight
`
`Ffg 6. Sll-‘I’ spectrum obtained with H30" precursor ions of bmath sampled from patient with
`end-stage renal failure before hemodialysis treatment. Precursor ions and their respective isotopic
`variants are shown as open bars. and product ions are shown as ,f'lled bars. (Reproduced with permis-
`sion from Davies S. Spanel P. Smith D. Qtiantitafive analysis of ammonia on the breath of patients in
`end-stage renal failure. Kidney In‘-.. l997:52:223-28.)
`
`ic questions remain to be addressed
`before the ellicaqy of this approach is
`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
`indeed reflect the level in blood. Prelim-
`
`inary reports by Davies et all? show
`a correlation with the plasma urca lev-
`els alier ingestion of t1I'ca in normal
`volunteers and during hcmodialysis 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 and liver enzyme lev-
`els. This is primarily to minimize errors
`From contamination and Poor or im-
`
`proper sample handling and to im-
`prove overall expertise and thereby the
`reliability of the determinations.
`
`Sampife Praceaafrzg
`Nlore efforts must be made to reduce.
`
`the various
`or preferably eliminate.
`sources ofammonia formation in stand-
`
`ing blood or plasma. lfor example. an
`initial step incorporating an acid pre-
`cipitation with cold perchloric acid be-
`fore blood centrifugation should be fol-
`lowed by ion exchange method in batch
`mode to select and purify the ammoni-
`um ions from the few remaining ammo-
`nia-generat-ing systems in the depre-
`teinated plasma sample.
`
`Away Mateo?
`Multiple time points or serial mea-
`surements, perhaps 5 separate time in-
`tervals of ammonia content, should be
`
`made from the same sample of stand-
`ing plasma. If the increase in ammonia
`level is linear with time. that is, exhibit-
`
`ing zero order kinetics. then linear ex-
`trapolation of the time course to zero
`time provides an estimate ofthe ammo-
`nia level at the time ofcollection.
`
`SI‘!
`
`7of10
`
`THE JOURNAL OF PEDIATRICS
`VOLUME I38. NUMBER I
`
`uct ions. Thus the primary ions and the
`specificity of their interactions with the
`target trace gases in the breath sample
`is a determinant ofthe selectivity ofthe
`method. The identification and quanti-
`tation ofthe products formed are then
`detected by a mass spectrometer.
`Currentl_y, the technique can measure
`trace gases down to a partial pressure
`of approximately l0 ppb. The mean
`value of96U ppb for breath ammonia in
`normal adults was reported in Davies
`et alsf 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. H30‘
`(molecular weight = 19). and their
`water clusters with molecular weights
`of 57. 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 ofammonia and acetone are ele~
`
`vatcd compared with those of healthy
`adults, whereas the levels of ethanol,
`
`methanol, and propanol are normal.
`Amines are present‘ in this sample but
`not in normal breath. The presence of
`acetonitrilc is a clear indicator that the
`
`patient smokes. The technique is rapid
`enough to allow realtime. breath-to-
`breath quantitation of trace gas.
`However, a number oftcchnical hur-
`
`dles remain before the technique can be
`moved from the laboratory into the clin~
`ical setting. First. the equipment is still
`rather large. complicated. and cumber-
`some. Another problem is the preven-
`tion ofammonia loss in the condensate
`
`of respired air before it enters the appa-
`ratus.
`ifhere is no doubt that many
`of these technical problems will be
`overcome with further development.
`Nevertheless. several sig~nlficant' biolog-
`
`7 of 10
`
`

`

`BARSOTT1
`
`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 remains the
`main problem or source of "error" in
`accurate determinations of free ammo-
`
`nia levels in blood samples.
`However. whenever high blood am-
`monia levels are detected and consis-
`
`tent with the patients clinical status.
`an alternative method should be avail-
`able and used For verification.
`
`REFERENCES
`].
`
`Olson JA. Anfinsen CE. The crystal-
`lization and characterization of L-
`
`glutamic acid dehydrogenase. J Biol
`Chem l952il97:57-79.
`Mondzac A, Ehrlich GE. Seegmiller
`JE. An enzymatic determination of
`ammonia in biological fluids. J Lab
`Clin Med l965i55c526-3].
`van Anken HC. Schiphorst ME. A iti-
`netic determination of ammonia in plas-
`ma. Clin Chim Acta l974;55:]5l-7.
`. Brusilow SW Determination of urine
`orotate and orotidine and plasma am-
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`In: Hommes FA. editor.
`Techniques in diagnostic human bio-
`chemical: a laboratory manual. New
`York: Wiley-Liss; 199']. p. 345-7.
`Brusilow SW. Maestri NE. Urea cycle
`disorders: diagnosis, pathophysiology.
`and therapy. Adv Pediatr l996:‘i3:
`127-70.
`Huizenga JR. Tangerman A, Gips
`CH. Determination of ammonia in bio-
`
`iluids. Ann Clin Biochem
`logical
`l.99‘i;5l:529-‘l3.
`Lowenstein JM. Ammonia production
`in muscle and other tissues: the purine
`nucleon‘-:le cycle. Physiol Rev 1972;52:
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`da Fonseca-Wollheim F. Preanalytical
`increase OF ammonia in blood speci-
`mens From healthy subjects. Clin Chem
`l990:36:l‘l83-7.
`Gerron C-G, Ansley JD, Isaacs JW.
`Kutner MH. Rudman D. Technical
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`Howanitz JH. Howanitz PJ. Skrodz-
`ki CA, Iwanski JA. Influences of spec-
`imen processing and storage condi-
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`Clin Chem l93‘l;50:9U5~3.
`Lentner C. Geigy scientific tables. Vol
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`blood hematology. Somatometric data-
`blocd nitrogenous substances. Basel:
`Ciba-Ceigy Limited: 1984.
`Burg PVD. Moolc HW. A simple and
`rapid method For the determination of
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`l965;8:l6'2-4.
`Conway E, Byrne A. An absorption
`apparatus for the micro-determination
`of certain volatile substances. The
`micro-determination
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`ammonia.
`Biochem J l993';27:-1 19-29.
`Huizenga JR. Gips CH. Determina-
`tion oF blood ammonia using the Am-
`monia Checker. Iinn Clin Biochem
`l 935;2U: l 37-9.
`Huizenga J R. Tangerman A. Gips
`CH. A rapid method For blood ammo-
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`ammonia checker (BAC) II. Clin Chim
`Acta 1992;210:153-5.
`IoseFsohn M. Hicks J