Measurement of ammonia in blood
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`Robert J Barsottt, PhD
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`The measurement of ammonia, now known to be a normal constituent of all
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`bodyfluids, is franght with problems. An elevated ammonialevelin blood
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`(100 pmol/L or higher)is an indicator of an abnormality in nitrogen home-
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`ostasis. The collection, handling, storage, and analysis of blood samples,
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`their limitations, and potential sources oferror are discussed. Newtech-
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`niques that permit continuousorreal-time estimates of systemic ammonia
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`levels over a broad rangeare also discussed. The aim should always be to
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`minimize the release of ammonia from the collected sample before analysis.
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`Recommendations are made on the collection and processing of blood sam-
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`ples, for it is by standardization and rigid adherence to these techniques
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`that the reliability of the test results will be improved. (J Pediatr 2001;
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`138:511-S20)
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`monia levels are approximately 30
`Umol/L, and levels exceeding 1 mmol/L
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`occur underconditions of acute hyper-
`ammonemia.”° Overthe past 100years,
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`numerous methods have been devel-
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`oped to measure ammonialevels in var-
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`ious bodyfluids including blood,plas-
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`ma, erythrocytes, saliva, sweat, and
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`urine.® This brief review considers a
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`few of the most common methods cur-
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`rently used to measure ammonia in
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`blood: alkalization-diffusion, enzymat-
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`ic, ion exchange, and electrode. Sample
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`collection, handling, storage, and some
`ofthe limitations and potential sources
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`oferrors associated with these methods
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`are discussed. Finally, some promising
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`novel
`techniques for the continuous
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`monitoring of ammonialevels that may
`be used clinically in the near future are
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`also described.
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`SIFT
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`PREANALYTICAL
`METHOD For
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`HANDLING BLOOD
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`AMMONIA
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`DETERMINATION
`SAMPLES
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`Ammonia is nowconsidered a normal
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`itive proof was provided by studies
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`showing that the enzyme glutaminede-
`constituentofall body fluids, resulting
`from the metabolism of amino acids.
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`hydrogenase specifically reacts with
`ammonia, O-ketoglutarate, and a coen-
`However, doubts of the presence of
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`zyme, reduced nicotinamide-adenine
`free ammoniain biologic solutions per-
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`dinucleotide, to form glutamic acid, !
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`sisted until approximately 1960, Before
`This reaction has becomethe basis for
`this time the various methods available
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`GLDH=Glutamine dehydrogenase
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`for the determination of ammonia con-
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`the most commonly used blood ammo-
`NADH—Reduced nicotinamide-adenine
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`dinucleotide
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`tent in biologic fluids relied on the sep-
`nia assayin clinical chemistry. One of
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`NADPH Reduced nicotinamide adenine
`aration or release of ammonia from the
`the attractive features ofthis assay is
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`dinucleotide phosphate
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`that ammoniais determined directly at
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`fluid sample byvolatilization alter the
`Selected-ion flow tube
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`addition ofan alkaline solution. With
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`physiological pH without previous
`these methods, increased levels of am-
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`treatment of the sample with either an
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`acid or a base.”"5
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`monia had been reported in the blood
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`of patients with severe hepaticfailure.
`Today, an elevated ammonia blood
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`Despite such reports, doubts about the
`level is considered a strong indicator of
`presenceoffree ammoniain blood con-
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`an abnormality in nitrogen homeostasis,
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`the most commonrelated to liver dys-
`tinued, mainly as a result of the con-
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`cern that free ammonia measured by
`function. In excess, ammoniais a potent
`these methods resulted from its libera-
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`toxin, principally of central nervous
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`tion from labile amides in blood during
`system function. In the venous blood of
`incubation in alkaline solutions. Defin-
`healthy adults and children, blood am-
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`Frant the Department of Pathology, Thomas Jefferson Univeraity, Philadelphia, Pennayloanta.
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`Reprint requests: Robert J. Barsotti, PhD, Associate Professor, Department of Pathology, Anato-
`myand Cell Biology, Thomas Jefferson University, 1020 Locust St, Philadelphia, PA 19107.
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`Copyright © 2001 by Mosby,Inc.
`0022-3476/2001/335.00 + 0 9/0/111832
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`doi:10,.1067/mpd.2001.111832
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`Most methods recommendcollecting
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`from patients who havefasted for at
`least 6 hours with the use of a verified
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`ammonia-free heparin as an anticoagu-
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`lant. Heparin is the preferred anticoag-
`ulant, because it has been shown to
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`reduce red bloodcell ammonia produc-
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`SII
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`Par Pharmaceutical, Inc. Ex. 1022
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 1 of 10
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`BARSOTTI
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`pH Dependenceof the Fraction of the Ionized Form
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`of Ammonia in Solution
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`0.60
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`e3
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`NH,"/NH,Ratio é
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`8 7
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`.0
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`8.0
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`10.0
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`11.0
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`9.0
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`pH
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`THE JOURNAL OF PEDIATRICS
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`JANUARY 2001
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`ANALYTICAL METHODS
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`FOR THE
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`DETERMINATION OF
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`AMMONIA
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`Manyof the procedures for ammonia
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`determination involve 2 general steps:
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`the release of ammonia gas or capture
`of ammonium ions from the sample
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`and the quantitation ofthe liberated
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`gas or captured ions. Over the years
`the most common methods used to
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`volatilize ammonia have been distilla-
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`tion and aeration/microdiffusion, ion-
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`exchange chromatography, and blood
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`or plasma deproteinization.
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`Properties ofAmmonia
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`In attempting to understand the ra-
`tionale used to measure ammonia,it is
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`important to review some ofthe physi-
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`cal properties of the compound. Am-
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`monia (NH;) is a colorless, acrid-
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`smelling gas at room temperature and
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`pressure. It easily dissolves in water
`andionizes to form NH," asfollows:
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`NH, + H,O @ NH,‘+ OH"
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`An increase in the pH or tempera-
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`ture ofthe solution increases the level
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`of the ionized form. Fig 1 shows the
`ratio that exists in plasma between the
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`ionized or NH,‘
`form versus the
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`gaseous or NH, form as a function of
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`pH. Thus in plasma at pH 7.4,
`the
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`NH,* form represents approximately
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`98%of the total ammonia. Manyofthe
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`approaches used to estimate ammonia
`levels in body fluids involve volatiliza-
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`tion of the NH,’ form of ammonia into
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`its gaseous form, NH, ,byalkalization
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`of the sample to a pH +10.
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`Distillation
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`Oneofthe earliest techniques for the
`measurement of ammonia involves the
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`addition olan alkaline buller to a sam-
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`ple of blood followed by in vacuo dis-
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`tillation. The released ammonia gasis
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`collected in
`trap containing an
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`aliquot ofdilute acid, which converts
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`Fig 1. Ratic between ionized and free gas form of ammonia in plasma as function of pH.
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`tion. EDTA can also be used. Donors’
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`arms should be as relaxedas possible,
`because muscle exertion may increase
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`venous ammonia levels.’
`Blood is
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`drawn into a chilled, heparinized vacu-
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`um tube that is immediately placed on
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`ice, and plasmais separated within 15
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`minutes. It
`is crucial to keep blood
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`the ammonia concentration ofstanding
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`blood and plasma increases sponta-
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`neously. Most ofthis increase has been
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`attributed to the generation and re-
`lease of ammonia from red bloodcells
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`and the deamination of amino acids,
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`particularly glutamine.®!! Plasma am-
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`monia levels of whole blood main-
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`tained at 4°Care stable for <1 hour.
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`When promptly separated from blood,
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`plasma ammonia levels are stable at
`4°C for 4 hours and for 24 hours if
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`stored frozen at —20°C. To put the
`problem ofrising ammonia levels in
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`perspective, the total nitrogen concen-
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`tration in venous plasma ofhealthy
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`adults exceeds ] mol/L and represents
`a potential pool of free blood ammo-
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`nia.'* In normal healthy adults, homeo-
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`stasis maintains free ammonia levels at
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`approximately 30 [mol/L.
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`It is important to note that the crite-
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`ods for the measurement of ammonia
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`levels in blood were established almost
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`exclusively with blood specimens from
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`healthy subjects.® Significantly more
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`difficult but much more constructive
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`would be the establishment ofsimilar
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`criteria for blood ammonia measure-
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`ments from patients with metabolic or
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`liver pathologic conditions. Standing
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`blood or plasma samples from these
`patients contain numerous elevated
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`sources of ammonia, resulting in in-
`creases in the rate and amount of am-
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`monia formed. Some ofthese sources
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`include elevated levels of circulating
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`deaminase, Y-glutamyltransferase, an
`enzyme that may deaminate [ree amino
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`acids, particularly glutamine in blood
`and plasma samples, resulting in over-
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`estimates of blood ammonia levels.
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`Fast, careful handling and preparation
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`of blood samples is required, especially
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`from patients with metabolic or liver
`pathologic conditions, to minimize pre-
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`analysis increases in ammonia concen-
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`tration until other assay techniques or
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`methods are developed. Until this is
`accomplished, measurements in this
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`patient population, in which blood am-
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`monia levels require the closest moni-
`toring, will continue to be the most un-
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`reliable. To date, the easiest and most
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`cost-effective methodis the stringent
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`and diligent maintenance of blood
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`samples on ice before, during, and
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`alter plasma separation.
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`$12
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`Par Pharmaceutical, Inc. Ex. 1022
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 2 of 10
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`

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`THE JOURNAL OF PEDIATRICS
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`Votume 138, Numeer |
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`the gas into the nonvolatile ammonium
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`ion. This approach is rather cumber-
`some and slow, particularly when
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`many samples require analysis, al-
`thoughinitially, speed was the primary
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`advantage of this approach over the
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`early microdiffusion techniques de-
`scribed in the following text. This ad-
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`vantage, however, was temporary, and
`was lost with the development of
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`smaller microdiffusion vessels. The last
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`reported use ofdistillation was by
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`Burg and Mook! in 1963.
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`BARSOTTI
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`Image available
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`in print only
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`Aeration/Microdiffusion
`Techniques
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`Anotherearly technique thatis still
`in use relies on liberation of free am-
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`monia byalkalization by the addition
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`of a strong base to the specimen. The
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`released ammonia diffuses through an
`air- or nitrogen-filled gap and is
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`trapped in acid within the same appa-
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`ratus. This approach, introduced by
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`Conwayand Berne!* in 1933, uses a
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`glass container resembling a Petri dish,
`within which a smaller second cham-
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`ber is centered. The wall of this inner
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`chamber is approximately half ofthat
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`of the outside wall. An aliquot ofa
`standard acid solution or ammonia in-
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`dicator such as bromocresol green is
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`placed into the inner chamber, and the
`sample is added to the outer chamber
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`(Fig 2). A measured quantityofbaseis
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`added to the sample, and the “petri”
`dish is sealed and gently rotated to mix
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`the sample and base in the outer cham-
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`ber and then left at room temperature
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`for 90 minutes. This same principle is
`used in the Blood Ammonia Check-
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`er!®!® and in the Kodak Ektachem
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`dry-film method.'” In these systems
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`the diffusion distance for ammonia is
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`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-
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`
`
`ducing the possible effects of other
`
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`
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`Fig 2. Early microdiffusion apparatus for determination of blood ammonia. (Reproduced with per-
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`mission from Conway E, Byme A. An absorption apparatus for the micro-determination of ammonia.
`
`
`
`
`
`Biochern J. 1993:27:4 19-29, © the Biochemical Society,)
`
`
`
`
`Current Micro-diffusion Apparatus
`
`Sample compartment
`
`
`
`
`Polyethylenefilm
`
`
`
`
`
`
`
`
`
`
`
`
`
`Tablet (Alkaline Buffer)
`Cc
`over
`
`
`>~—Polyethylene spacer
`
`Color pHindicator es
`ii—Window
`
`
`
`
`
`
`
`
`
`
`
`
`Probe Light Beam
`
`Detector
`
`
`
`
`
`
`\\lustration of micrediffusion technique used in Blood Ammonia Checker (Reproduced with
`Fig 3.
`
`
`
`
`
`
`
`
`
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`
`
`
`
`permission from Tada Kk, Okuda K,Watanabe Kk, et al. A new method for screening for hyperam-
`
`
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`
`
`
`
`
`
`
`
`monemia. Eur | Pediatr: 1979;| 30;105-10.)
`
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`
`
`
`
`
`constituents and drugs on the ammo-
`
`
`
`
`
`
`
`nia assay. One main drawback ofthese
`procedures is that varying amounts of
`
`
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`
`
`
`ammonia are liberated from the alka-
`
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`
`
`
`
`
`
`line hydrolysis of proteins, especially
`
`
`
`
`hemoglobin, and labile amides, espe-
`
`cially glutamine.!*29
`
`
`
`
`
`
`Ton-Exchange Chromatography
`
`
`
`
`
`
`
`
`In this approach ammonia gasis not
`
`
`
`
`
`liberated from the sample. Instead, a
`strongly acidic cation-exchange resin
`
`
`
`
`
`
`
`
`
`
`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
`
`
`
`
`
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`the captured ammoniumionsare then
`eluted from the resin bysalt solutions
`
`
`
`
`
`
`
`
`
`
`
`
`
`or released as ammonia bythe addi-
`
`tion ofdilute alkali,?! a technique de-
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`
`
`
`
`
`
`scribed in detail more recently by
`
`Brusilow.
`
`
`
`
`Deproteinization
`
`
`
`
`
`
`Whole blood or plasma proteins are
`precipitated by the addition oftri-
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`
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`
`
`
`
`
`
`chloroacetic or perchloric acids, and the
`
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`
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`ammoniais determined directly in the
`supernatant fluid after alkalization Pad
`
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`
`
`
`QUANTITATION OF
`
`AMMONIA
`
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`
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`After the release or capture of ammo-
`nia or ammonium ions, several methods
`
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`have been described to determine the
`
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`
`
`
`
`
`
`
`
`amount of ammonia present. The gen-
`
`
`
`
`
`eral categories for these methods in-
`clude titration, colorimetric/fluorimet-
`
`
`
`
`
`
`ric, electrode-based, and enzymatic.
`
`
`
`Titration Method
`The ammonia liberated from the
`
`
`
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`
`
`
`
`
`
`sample is trapped in an aliquotofdi-
`
`
`
`$13
`
`
`
`Par Pharmaceutical, Inc. Ex. 1022
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 3 of 10
`
`

`

`BARSOTTI
`
`
`THE JOURNAL OF PEDIATRICS
`
`
`
`
`
`JANUARY 2001
`
`
`
`
`Gas Sensing Electrode
`
`
`
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`
`PHelectrode Reference Electrode
`
`
`
`
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`Reference Buffer
`
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`Gas Permeable Membrane
`
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`(water impervious)
`
`
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`Temperature Controlled
`
`
`
`
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`Both
`
`(_)<— Sample
`
`
`
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`(-) 4— Alkaline Butfer
`
`Sealed Mixing Chamber
`
`
`
`
`Fig 4. Schematic of ion-selective electrode(left pane!) and suggested arrangement for continuous-flow analysis of ammonia
`
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`in plasma samples (right panel).
`
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`lute acid, and the amount is measured
`
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`by back-titration of the acid solution
`
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`with a base while the pH is monitored
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`with an indicator or electrode. The
`
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`principal advantage ofthis approachis
`thatit is inexpensive, requiring no spe-
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`
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`cialized equipment. The disadvan-
`
`
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`
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`tages, however, are significant. They
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`include insensitivity, the requirement
`
`
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`for large blood samples, and contami-
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`
`
`
`
`
`nation byother volatile bases that may
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`
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`alfect the final value. Overall, this pro-
`cedureis laborious and slow andthere-
`
`
`
`
`
`
`fore is not used routinely.
`
`
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`Colorimetric/Fluorimetric
`Reactions
`
`This method is based on the reaction
`
`
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`
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`
`
`
`
`of ammonia with a reagent to form a
`colored complex that is measured by
`
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`
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`
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`spectrometry or fluorometry. Among
`
`
`
`
`the first reactions used was the in-
`
`
`
`
`
`
`
`
`
`dophenolreaction, described by Berth-
`the formation of a
`in 18597":
`elot
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`
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`
`
`
`bluish color by the reaction of ammo-
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`
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`
`
`nia with phenol and hypochlorite. This
`method is commonlyreferred to as the
`
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`
`
`
`
`
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`
`
`
`phenol reaction. Another colorimetric
`
`
`
`
`
`
`reaction is the Nessler reaction,
`in
`
`
`
`
`
`
`which a brown-orange color is formed
`
`
`
`
`
`
`by the reaction of ammonia with mer-
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`
`
`cury or potassiumiodide in an alkaline
`solution. Some othercolorimetric reac-
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`
`
`
`
`
`tions include the use ofisocyanurate,
`
`
`
`
`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 fluorescamine and o-phthalalde-
`hyde.?4-?? The principle advantages to
`
`
`
`
`this approach are speed, simplicity,
`
`
`
`
`
`
`
`
`
`specificity when used carefully, and ex-
`
`
`
`
`
`cellent sensitivity. The disadvantage is
`that other substances in the bloodaf-
`
`
`
`
`
`
`
`
`
`
`
`fect some reactions, for example, the
`Berthelot reaction is inhibited by ex-
`
`
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`
`
`
`
`
`
`cess amino acids, creatine, glutamine,
`
`and sometherapeutic agents.*”
`
`
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`
`
`
`
`
`Gas-sensing Electrode
`
`
`
`
`
`
`
`With the introduction of the gas-
`sensitive electrode (eg, Orion, Model
`
`
`
`
`
`
`
`951201), a number ofreports have ap-
`
`
`
`
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`
`
`peared describing the methods re-
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`
`
`
`
`quired and the use ofthe electrede in
`
`
`
`
`
`measuring ammonia levels in samples
`
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`of blood, urine, cerebrospinal fluid,
`
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`
`
`and saliva over a broad range from 10
`
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`
`
`
`Lmol/Lto nearly 1 mmol/L.
`
`
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`
`
`When immersed in the sample or
`
`
`
`
`
`held closely above it,
`the dissolved
`
`
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`
`
`gas ofinterest diffuses across a gas-
`
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`
`
`permeable membrane into a small vol-
`
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`ume olfbuffer. The reaction changes
`
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`the pH ofthe buffer, which is sensed
`
`
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`by an internal pH electrode or sensing
`
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`
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`
`
`electrode. The change in pH results in
`
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`
`
`a change in the potential between the
`
`
`
`
`
`sensing electrode and a reference elec-
`
`
`
`
`
`trode immersed in a separate reference
`buffer, all housed within the same elec-
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`
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`
`
`
`
`
`
`
`
`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 ofelectrode-
`
`
`
`
`
`
`
`
`
`based ammonia assay are its cheap-
`
`
`
`
`
`
`
`
`ness, ease of use, and because it never
`
`
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`
`
`
`
`
`
`
`
`comes in contact with the sample, its
`imperviousness to sample color, tur-
`
`
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`
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`
`bidity, viscosity, or the presence of
`
`
`
`
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`
`drugs or other metabolites in the sam-
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`
`
`
`ple. The electrode is best arranged
`
`
`
`
`
`
`
`
`above the surface of the sample in a
`
`
`
`
`
`sealed environment
`(Fig 4). This
`avoids the accumulation of proteins
`
`
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`
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`
`
`
`
`and cell fragments on the membrane
`
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`
`
`
`surface that would normally occur
`
`
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`
`during immersion into plasma or blood
`
`
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`
`
`
`sample and minimizes the loss of am-
`monia from the sample away from the
`
`
`
`
`
`
`
`measure-
`the
`electrode
`during
`
`
`
`men
`52 The disadvantages of the
`{2251
`
`
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`
`
`electrode-based system include the re-
`
`
`
`
`
`
`quirementoflarge sample volumes and
`
`
`
`
`
`
`slow sample reads, especially at low
`
`ammonia levels, requiring 10 to 15
`
`
`
`
`
`minutes. In addition, major differences
`
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`
`
`
`in either the osmolarity or temperature
`ofthe sample and the sensing electrode
`
`
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`buffer must be avoided.
`
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`
`
`Enzymatic Method
`
`
`The specificity of most methods for
`
`
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`
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`
`
`
`ammonia determination in biologic flu-
`ids relies on the physical separation of
`
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`
`
`ammoniafrom 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-
`
`Sl4
`
`
`
`Par Pharmaceutical, Inc. Ex. 1022
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 4 of 10
`
`

`

`THE JOURNAL OF PEDIATRICS
`Votume 138, Numer |
`
`BARSOTTI
`
`injection quadru
`detection quadruple
`mass filter
`mass spectrometer
`Roots pump
`air or breath sample
`
`He carrier gas
`
`
`
`
`
`=> H30* or 0+
`
`
`ion source
`ion injection
`
`orifice
`orifice
`gas
`
`(e.g. AifH20)
`
`injection diffusion pump
`
`——
`10cm
`
`detection diffusion pump
`
`Fig 5. Simplified schematic of selected-ion flow tube (SIFT).
`
`tarate with glutamate dehydrogenase
`and reduced nicotinamideadenine di-
`nucleotide phosphate:
`
`2-Oxoglutarate + NH, + NADPH
`NGLDH
`Glutamate + NADP
`
`The decrease in absorbance at 340
`nm caused by the 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 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 sources in plas-
`ma, so the preincubation time can be
`reduced to a few minutes.*
`The assaycan be used to measure am-
`monia levels over a broad range, from
`as low as 12 [mol/L to as high as 1
`mmol/L. The disadvantage ofthis ap-
`proachis 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 time ofthe ini-
`tial readings. Thus overestimates of am-
`monia levels are most likely in poorly
`handled samples containing elevated
`levels oftransaminases and aminoacids.
`
`NormalValuesfor Blood
`Ammonia Levels
`Table | lists selected blood ammonia
`levels for various blood sample types
`and assay methods from a numberof
`studies. The average values for arterial
`blood, plasma, venous blood, and plas-
`ma are 18, 23, 28, and 32 |tmol/L, re-
`spectively. The average value for ve-
`nous blood and plasmais 30 [tmol/L.
`
`FUTURE
`DEVELOPMENTS
`
`The major limitations 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 and assaying, andfinally, the
`assayitself. 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 staff 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 flowtube 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 for the rapid, real-time
`analysis ofthe trace gas content ofat-
`mospheric air. It was originally devel-
`opedto 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 technologyis
`being further developed and may soon
`be a sensitive, quantitative method for
`the continuousreal-time analysis ofthe
`trace-gas content of human breath and
`
`SI5
`
`Par Pharmaceutical, Inc. Ex. 1022
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 5 of 10
`
`

`

`BARSOTTI
`
`
`
`THE JOURNAL OF PEDIATRICS
`
`
`
`
`
`JANUARY 2001
`
`
`
`
`
`
`
`
`
`
`
`
`
`Table I, Reported assay techniques and blood ammonia levels in healthy subjects
`
`
`
`Reference
`
`Reference
`
`RSETEN nnennnnnennntnmnnnnenmneUMEeenONOeneanmnmnneevalue(Amol/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)
`
`
`
`Oberholzer et al (1976)
`
`
`
`
`Butteryet al (1982)
`
`
`Brusilow (1991)
`
`
`
`
`Cooke and Jensen (1983)
`
`
`
`
`Willems and Steenssens (1988)
`
`
`
`
`Spooneret al (1975)
`
`
`
`Seligson and Hirahara (1957)
`
`
`
`
`Mondzacet al (1965)
`
`
`
`
`Muting etal (1968)
`
`
`
`
`van Anken and Schiphorst (1974)
`
`
`
`
`Howanitz et al (1984)
`
`
`
`da Fonseca-Wollheim (1990)
`
`
`21
`
`59
`
`42
`
`23
`
`41
`43
`40
`45
`16
`
`
`
`
`
`
`
`16
`
`
`
`
`lon-exchange/Nessler
`
`
`lon-exchange/phenol
`
`
`lon-exchange/Nessler
`
`Supernatant/electrode
`
`Supernatant/phenol
`
`Supernatant/phenol
`
`Supernatant/phenol
`
`Supernatant/phenol
`Microdiffusion/BAC I]
`
`
`
`
`Enzymatic
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`42
`
`4]
`15
`
`16
`
`
`
`16
`
`
`lon-exchange/Nessler
`
`Supernatant/phenol
`Microdiftusion/BAC I
`
`Microdiftusion/BAC II
`
`
`
`Enzymatic
`
`
`41
`
`15
`
`Supernatant/phenol
`
`Enzymatic
`
`
`
`
`9
`
`44
`38
`
`
`4
`52
`
`46
`
`
`27
`20
`
`
`2
`
`33
`
`S
`
`11
`
`8
`
`
`
`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
`
`2]
`
`
`
`21
`
`23
`mas
`
`
`
`
`
`15
`
`31
`
`21
`
`17
`
`22
`
`57
`
`30
`
`44
`21
`
`
`
`21
`
`
`19
`
`16
`
`21
`
`18
`44
`
`44
`
`
`52
`56
`
`
`30
`
`57
`
`22
`
`30
`
`29
`
`
`
`
`
`
`thus permit a continuous, noninvasive
`
`
`
`
`
`measure of systemic 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 moleculesis
`
`
`
`
`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 masgs-to-
`
`charge ratio. In the case of ammonia
`
`
`
`
`
`
`
`
`
`
`
`
`detection, H,O* 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 ofbreathis 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 ammoniumions
`
`
`
`
`
`
`
`
`
`
`
`
`
`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
`
`
`
`
`
`
`react efficiently with the trace gases to
`
`
`
`
`
`be detected to form identifiable prod-
`
`
`
`
`$16
`
`
`Par Pharmaceutical, Inc. Ex. 1022
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 6 of 10
`
`

`

`BARSOTTI
`
`
`
`peeeames
`
`C3HygN
`(200)
`
` —
`
`trimethylamine
`(209)
`
`own 8 ee “pe %
`oakJs<"
`Molecular Weight
`
`{“co
`
`104
`
`oO
`oO
`A 108
`io)~
`
`c3O
`
`o 1
`O
`
`401
`
`THE JOURNAL OF PEDIATRICS
`Votume 138, Numer |
`
`uct ions. Thus the primaryions and the
`specificity of their interactions with the
`target trace gases in the breath sample
`is a determinant ofthe selectivityofthe
`method. The identification and quanti-
`tation of the products formed are then
`detected by a mass spectrometer.
`Currently, the technique can measure
`trace gases downtoa partial pressure
`of approximately 10 ppb. The mean
`value of 960 ppb for breath ammonia in
`normaladults was reported in Davies
`et al®” and thus is well above the mini-
`mum sensitivity ol 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 laboratoryfor analy-
`sis by SIFT. The molecular weight (x-
`axis) for each molecule identilied 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 3