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`monia lovels are approximately 50
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`pmol/L. and levels exceeding 1 mmol/L
`
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`occur under conditions of acute hyper-
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`amrnonemia.‘l'5 Over the past 1 00 years.
`numerous methods have been devel-
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`oped to measure ammonia levels in var-
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`ious body fluids including blood. plas»
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`ma. erythrocytes, saliva. sweat, and
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`urine.6 This brief review considers a
`Few ol'the most common methods cur-
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`rently used to measure ammonia in
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`blood: alkalizatiomdil‘limion. enzymat-
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`ic. ion exchange. and electrode. Sample
`
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`collection. handling. storage. and some
`
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`
`of the limitations and potential sources
`of errors 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 ammonia levels that may
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`be used clinically in the near Future are
`also described.
`
`
`
`GLDH
`
`NADH
`
`
`
`SIFF
`
`
`Glmzmine dehydrognnase
`
`
`Reduced nicofimmide-admlne
`
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`'dtnudeotlde
`
`NADFH Redacted murmnideadenine
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`dlnudeotide phosphane
`
`Selected-Ibo flow cube
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`
`P REANALYTICAL
`
`
`METHOD FOR
`
`HANDLING BLOOD
`
`DETERMINATION
`
`
`AMMONIA
`
`SAMPLES
`
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`
`Measurement of ammonia in blood
`
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`Roller! JBaraotti, Plat)
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`The measurement of ammonia. now known to be a normal constituent of all
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`body fluids, is Fraught with problems. An elevated ammonia level in blood
`
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`(100 umolf 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 of error are discussed. New tech-
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`niques that permit continuous or real-time estimates of systemic ammonia
`
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`levels over a broad range are 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:
`
`133:511-520)
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`
`Ammonia is now considered a normal
`
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`
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`constituent of all body fluids, resulting
`from the metabolism of amino acids.
`
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`
`
`However. doubts of the presence of
`
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`Free ammonia in biologic solutions per-
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`
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`sisted until approximately 1960. Before
`this time the various methods available
`
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`
`
`for the determination of ammonia con-
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`tent in biologic fluids relied on the sep-
`aration or release 0“ ammonia From- the
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`
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`fluid sample by volatilizat'ion after the
`addition of an alkaline solution. With
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`these methods, increased levels of am-
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`monia had been reported in the blood
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`of patients with severe hepatic Failure.
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`Despite such reports. doubts about the
`
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`
`presence of Free ammonia in blood con—
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`tinued. mainly as a result of the con-
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`
`
`cern that Free ammonia measured by
`these methods resulted From its libera-
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`
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`tion From labile amides in blood during
`incubation in alkaline solutions. Delin-
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`itive proof was provided by studies
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`showing that the enzyme glutamine de-
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`
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`hydrogenase specifically reacts with
`
`
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`ammonia, (L—ketog'lutarate. and a .coen-
`
`
`zyme. reduced nicotinamide-adeninc
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`
`
`
`dinucleotide. to Form glutamic acid.1
`Th is reaction has become the basis for
`
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`the most commonly used blood ammo-
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`nia assay in clinical chemistry. One of
`
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`
`
`the attractive features of this assay is
`
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`
`that ammonia is determined directly at
`
`
`
`
`physiological pH without previous
`
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`
`
`treatment of the sample with either an
`
`acid or a base?5
`
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`
`Today. an elevated ammonia blood
`
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`level is considered a strong indicator of
`
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`an abnormality in nitrogen homeostasis.
`
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`the most Common related to liver dys-
`
`
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`t'unction. In excess. ammonia is a potent
`
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`toxin, principally of central nervous
`
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`
`
`system Function. In the venous blood of
`
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`
`
`healthy adults'and children. blood am—
`
`f‘i‘om (be DWl‘fmtfli sf Padding”; mm» ngfl'rrmm Under-airy. Pathddpbiir, Pennsylmmh.
`
`
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`
`Reprint requests: Robert J. Barsotti. PhD, Associate Professor. Department of Pathology, Anato-
`
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`my and Cell Biology. Thomas Jeli'erson University. 1020 [.oeust St. Philadelphia. PA 1910?.
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`
`Copyright Q 2001 by Mushy. Inc.
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`002234792001135530 + 0 9101311352
`
`
`
`doi:l0.1067lmpd.2001.111832
`
`
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`
`Most methods recommend collecting
`
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`
`
`from patients who have lasted 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 blood cell ammonia produo»
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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 of10
`
`

`

`BARSOTTI
`
`
`THE JOURNAL OF PEDIATRICS
`
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`
`
`jANUARY 200!
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`pH Dependence of the Fraction of the Ionized Form
`of Ammonia in Solution
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`1.00
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`'3'ss-
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`8.0
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`s.B
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`0.00
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`m
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`m 11.0
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`u
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`NHflNI-l,Ratio
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`
`ANALYTICAL METHODS
`
`
`FORTHE
`
`DETERMINATION OF
`
`
`AMMONIA
`
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`
`
`Many of the procedures For ammonia
`
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`
`determination involve 2 general steps:
`
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`the release of ammonia gas or capture
`
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`of ammonium ions from the sampie
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`
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`and the quantitation of the liberated
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`gas or captured ions. Over the years
`the most Common methods used to
`
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`
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`volatilize ammonia have been distill-a»
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`
`tion and aeration/microdifl‘usion. ion-
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`exchange chromatography, and blood
`
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`
`or plasma deproteinization.
`
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`
`Properties afAntnwnia
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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 of the physi-
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`cal properties of the compound. Am-
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`
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`monia (N H3)
`is a colorless, acrid-
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`smelling gas at room temperature and
`
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`
`
`pressure. It easily dissolves in water
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`and ioni'zes to form NH 4'as l'ol lows:
`
`
`N113 + H20 2 NH4*+ ()H'
`
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`
`
`An increase in the pH or tempera-
`ture of the solution increases the level
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`
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`of the ionized Form. Fig 1 shows the
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`ratio that exists in plasma between the
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`
`ionized or NH4‘ Form warms the
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`gaseous or NH?5 form as a Function of
`
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`pH. Thus in plasma at pH 7.4. the
`
`
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`NH 4‘ form represents approximater
`
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`
`
`98% of the total ammonia. Many of the
`
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`
`
`approaches used to estimate ammonia
`
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`
`
`levels in body Fluids-involve volatiliza.»
`
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`
`
`
`tion of the NFL"r Form of ammonia into
`
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`
`
`its gaseous form. NH3 ,by alkalization
`
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`ot‘the sample to a pH >10.
`
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`Distiflattbn
`
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`
`
`One of the earliest techniques For the
`measurement of ammonia inv'olvres the
`
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`
`addition of an alkaline buffer to a sam-
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`ple of blood followed by in vacuo dis-
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`tillation. The released ammonia gas is
`
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`collected in a trap containing an
`
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`aliquot ol'dilute acid. which converts
`
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`Fig 1. Ratio 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 reiaxed as possible.
`
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`because muscle exertion may increase
`venous ammonia leveisf 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 plasma is separated within 15
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`
`
`minutes. It is crucial to keep blood
`
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`samples cold after collection, because
`
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`
`
`the ammonia concentration of standing
`
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`
`
`blood and plasma increases sponta-
`
`
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`
`
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`neously. Nlost of this increase has been
`
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`
`
`attributed to the generation and re-
`lease of ammonia From red blood cells
`
`
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`
`
`and the deamination of amino acids,
`
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`
`
`
`particularly glutaminefi" I Plasma am-
`monia levels ol~ whole blood main-
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`tained at 4“C are 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 it'
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`stored frozen at —20°C. To put the
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`
`problem of rising ammonia levels in
`
`
`
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`perspective. the total nitrogen concen-
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`
`
`tration in venous plasma of healthy
`
`
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`
`
`adults exceeds 1 mo]!L and represents
`
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`
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`a potential pool of free blood ammo-
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`
`
`nia. '2 In normal healthy adults, homeo~
`stasis maintains free ammonia levels at
`
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`
`
`approximately 50 pmoUL.
`
`
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`
`
`It is important to note that the crite-
`
`
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`
`
`
`ria For sample stability and the meth-
`ods For the measurement or ammonia
`
`
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`
`
`levels in blood were established almost
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`
`exclusively with blood specimens From
`
`
`
`
`healthy subjects.8 Significantly more
`difficult but much more constructive
`
`
`
`
`
`would be the establishment of similar
`
`
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`
`
`
`criteria for blood ammonia measure-
`
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`
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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 ol‘am-
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`
`monia Formed. Some of these sources
`
`
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`
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`
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`
`
`include elevated levels of circulating
`
`
`
`deaminase. ’Y-glutamyltransferase. an
`
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`
`
`enzyme that may deaminate free amino
`
`
`
`
`
`acids. particularly glutamine in blood
`
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`
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`and plasma samples, resulting in over-
`estimates of blood ammonia levels.
`
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`Fast, careful handling and preparation
`
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`
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`ofblood samples is required, especially
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`From patients with metabolic or liver
`
`
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`pathologic conditions, to minimize pre-
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`
`analysis increases in ammonia concen-
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`
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`tration until other assay techniques or
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`
`
`methods are developed. Until this is
`
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`
`accomplished. measurements in this
`
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`patient population, in which blood ams
`
`
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`
`
`monia levels require the closest moni-
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`
`
`tori ng, will continue to be the most un-
`reliable. To date, the easiest and most
`
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`
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`cost—effective method is the stringent
`
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`
`and diligent maintenance of blood
`
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`samples on ice before. during, and
`
`
`
`after plasma separation.
`
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`
`
`SIZ
`
`
`
`Par Pharmaceutical, Inc. Ex. 1022
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 2 of10
`
`

`

`THE jOURNAL or PEDIATRICS
`
`
`
`
`VOLUME I38. NUMBER |
`
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`the gas into the nonvolatile ammonium
`
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`
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`ion. This approach is rather cumber—
`
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`
`
`some and slow. particularly when
`
`
`
`
`many samples require analysis. al-
`
`
`
`
`
`
`though initially, speed was the primary
`
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`
`
`advantage of this approach over the
`
`
`
`early microdifi‘usion techniques de-
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`
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`scribed in the Following text. This ad-
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`
`vantage, however. was temporary. and
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`
`was lost with the development of
`smaller microdiflilsion vessels. The last
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`reported use of distillation was by
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`Burg and Mock” in 1965.
`
`
`
`Aeration/Miami?! edict:
`
`Tocéniquea
`
`
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`
`
`
`Another early technique that is still
`in use relies on liberation of Free am-
`
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`
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`
`
`monia by alkalization by the addition
`
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`
`ol'a strong base to the specimen. The
`
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`
`
`
`released ammonia diFFuses through an
`
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`
`
`air- or nitrogen-filled gap and is
`
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`
`
`trapped in acid within the same appa»
`
`
`
`
`
`ratus. This approach, introduced by
`
`
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`
`
`
`
`Conway and BerneH in l935, uses a
`
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`
`
`glass container resembling a Petri dish.
`within which a smaller second cham-
`
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`
`
`her is centered. The wall of this inner
`
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`chamber is approximately half of that
`
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`of the outside wall. An aliquot of a
`standard acid solution or ammonia in-
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`
`
`
`
`
`
`dicator such as bromocresol green is
`
`
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`
`
`
`
`piaced into the inner chamber, and the
`
`
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`
`
`sample is added to the outer chamber
`
`
`
`
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`
`
`
`(Fig 2). A measured quantity of base is
`
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`
`
`
`added to the sample, and the I'petri"
`
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`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—
`
`
`
`
`
`erls'lfi and in the Kodak Elttachem
`
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`
`dry-film method.” In these systems
`the diffusion distance For ammonia is
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`significantly reduced From those in the
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`original diFFusion apparatus of Conway
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`and Borne, requiring only 5 minutes to
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`complete (Fig 5).
`Distillatidn and microdil'fusion both
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`represent purification procedures that
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`isolate ammonia From the many other
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`constituents of blood and plasma, re»
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`ducing the possible effects of other
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`BARSOTTI
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`Image available
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`in print only
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`2. Early microdifi‘usion apparatus for determination of blood ammonia {Reproduced with per-
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`mission from Conway E, Byme AArI absorption apparatus for the micro-determination of ammonia.
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`Biochem j.
`|993;2714 | 9-29. ('3 the Biochemical Society.)
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`Current Micro-diffusion Apparatus
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`Sample compartment
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`Color pl'l indicator
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`Polyethylene fllln
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`22am
`—m
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`mm (Alkaline Bullet]
`CW9?
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`Poiyefliylsna spacer
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`m I
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`/\
`Probe Light Beam
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`Detector
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`Illustration of microdifi'usion technique used in Blood Ammonia Checker. (Reproduced with
`F133.
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`permission fromTada K Okuda K.Watanabe K. et aJ.A new medical forscr‘eening for hyperam»
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`monemia. Eur] Pediatr. |979:| 30:|05-|0.)
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`constituents and drugs on the ammo-
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`nia assay. One main drawback of these
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`procedures is that varying amounts of
`ammonia are liberated from the alka—
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`line hydrolysis of proteins. especially
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`hemoglobin. and labile amides, espe-
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`cially glutamine. '3'?“
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`Ion-Exchange Cfimmgrapby
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`In this approach ammonia gas is not
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`liberated From the sample. Instead, a
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`strOngly acidic cation-exchange resin
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`is used in batch mode to capture am-
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`monium ions. NH4‘. The resin is
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`added to and subsequently separated
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`from the sample by centriFugation. and
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`the captured ammonium ions are then
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`eluted From the resin by salt solutions
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`or released as ammonia by the addi-
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`tion ol" dilute alkali.” a technique de-
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`scribed in detail more recently by
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`Brusilow.‘1
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`Depraneimizahbn
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`Whole blood or plasma proteins are
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`precipitated by the addition of tri-
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`chloroacetic or perchloric acids. and the
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`ammonia is determined directly in the
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`supernatant fluid after alkalizati0n_22..25
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`QUANTITATION OF
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`AMMONIA
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`After the release or capture of ammo-
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`nia or ammonium ions. several methods
`have been described to determine the
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`amount of ammonia present. The gen-
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`eral categories for these methods in-
`clude titration, colorimetricffluorimet-
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`ric. electrode-based. and enzymatic.
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`Htmtiau Media?
`The ammonia liberated From the
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`sample is trapped in an aliquot of di»
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`SIS
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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 3 of10
`
`

`

`BARSOTTI
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`Gas Sensing Electrode
`Hakim Electrode
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`pH nlnelroda
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`Fulcrum Buller
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`Gan Permewle Membrane
`{water impervleua]
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`THE JOURNAL or PEDIATRICS
`
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`JANUARY 200|
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`Temperatwe Conme
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`Bou\
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`4—- Sample
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`Sealed Mixing Chamber
`
`. <— Altattm Bumr
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`Fig 4. Schematic of ion-selective electrode {left panel] and suggested arrangement for continuous-lion.r 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 ol' the acid solution
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`with a base while the pH is monitored
`with an indicator or electrode. The
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`principal advantage ol‘this approach is
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`that it is inexpensive, requiring no spe-
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`cialized equipment. The disadvam
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`tages. however. are signilicant. They
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`include insensitivity. the requirement
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`for large blood samples. and contami-
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`nation by other volatile bases that may
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`affect the linal value. Overall. this pro-
`cedure is laborious and slow and there-
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`fore is not used routinely.
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`Colart'metric/Fluart'metn'c
`Rfdfl'iflfld
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`This method is based on the reactiOn
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`of ammonia with a reagent to form a
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`colored complex that is measured by
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`spectrometry or litiorometry. Among
`the lirst reactions used was the in-
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`dophenol reaction. described by Berth-
`the Formation of a
`in 1859233:
`elot
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`bluish color by the reaction of ammo-
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`nia with phenol and h‘ypochlorite. This
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`method is commonly referred to as the
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`phenol reaction. Another colorimetric
`reaction is [he Nessler reaction,
`in
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`which a brown—orange color is Formed
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`by the reaction ol‘ammonia with mer—
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`cury or potassium iodide in an alkaline
`solution. Some other colorimelric reac-
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`tions include the use of isocyanurate,
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`cyanate, ninhydrin. or thymol hypo-
`bromite. Detection of ammonia and
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`primary amines down to the nanogram
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`range is routinely performed with line-
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`rescence derivatization reagents such
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`as l'luorescamine and o-phthalaltle~
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`hydc.2'l'2q The principle advantages to
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`this approach are speed, simplicity.
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`
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`specificity when used carefully. and ex-
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`
`cellent sensitivity. The disadvantage is
`that other substances in the blood at1
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`l‘ect some reactions. For exam ple, the
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`Berthelot reaction is inhibited by ex-
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`cess amino acids. creatine, glutamine,
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`and some therapeutic agents.30
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`Gad-(tending flier-trade
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`With the introduction of the gas—
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`sensitive electrode (cg. Orion. .Model
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`951201). a number of reports have ap—
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`peared describing the methods re-
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`quired and the use ol‘the electrode in
`
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`measuring ammonia levels in samples
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`liuid.
`of blood. urine. cerebrospinal
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`and saliva over a broad range from 10
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`pmol/L to nearly 1 mmolr’L.
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`When im merscd in the sample or
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`held closely above it.
`the dissolved
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`gas of interest dill‘uses across a gas
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`permeable membrane into a small vol-
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`ume of boiler. The reaction changes
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`the pH ol’ the buffer, which is sensed
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`by an internal pH electrode or sensing
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`electrode. The change in pH results in
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`a change in the potential between the
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`sensing electrode and a reference elec-
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`trode immersed in a separate reference
`buli'er. all housed within the same elec-
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`tl‘ode body. The arrangement is illus-
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`trated in Fig 4.
`in which the scale ol'
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`some of the components is exaggerated
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`For clarity.
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`The main advantages ol' electrode—
`
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`based ammonia assay are its cheap—
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`ness. ease of use. and because it never
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`comes in contact with the sample. its
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`imperviousness to sample color, tur-
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`bidity, viscosity. or the presence ol-
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`drugs or other metabolites in the sani-
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`ple. The electrode is best arranged
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`above the surl’ace ol‘ the sample in a
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`sealed environment
`( ‘ig 4). This
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`avoids the accumulation ol‘ proteins
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`and cell Fragments on the membrane
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`surl‘ace 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-
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`monia Item the sample away li‘om the
`measure—
`the
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`
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`electrode
`during
`22.3.4.5
`ment.
`
`
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`
`2 The disadvantages oil the
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`electrode-based system include the re-
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`
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`quirement ol‘large sample volumes and
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`slow sample reads. especially at low
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`ammonia levels, requiring ID to 15
`minutes. In addition. maior dili'erences
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`in either the osmolarity or temperature
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`ol'the sample and the sensing electrode
`bull's-r must be avoided.
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`
`Enzymetic Method
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`The specificity ol‘ most methods For
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`ammonia determination in biologic liu-
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`ids relies on the physical separation of
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`ammonia from interfering substances
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`by volatilization alter alltalization. In
`contrast,
`the most common method
`
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`used in clinical laboratories is an enzy-
`matic method [hat measures ammonia
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`directly. Thus sample preparation is
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`relatively simple. because the previous
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`liberation ol‘ammonia from the sample
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`
`
`is not required. This assay is based on
`
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`the reductive amination ol‘ 2~oxoglu~
`
`SH
`
`
`
`Par Pharmaceutical, Inc. Ex. 1022
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 4 of10
`
`

`

`THE JOURNAL or PEDIATRICS
`VOLUME I38. NUMBER l
`
`BARSOTTI
`
`Inlectkm alt-ample
`mu "not
`Emil-Ill)
`at or breath sample
`He carrier gas
`
`cnnnnoltron
`1
`mlcmnve
`Ion detect»
`
`
`nun-tor
`
`
`. ".09 a 0;
`
`
`Ion Douro:
`Ion Injection
`coma
`ounce
`gas
`
`(O-I‘ AM)
`
`
`detectlon quadruple
`mu spectrometer
`
`e—
`
`Injection until-ton pump
`
`l—l
`10cm
`
`detection dim-Ion pump
`
`Fig 5. Simplified schematic of selected-ion flow tube tSer).
`
`taratc with glutamate dchydrogenase
`and reduced nicotinamide adenine di-
`
`nucleotide phosphate:
`
`'2-0xoglutarate + NH3 + NADPH
`TiCLDH
`Glutamate + NADP
`
`The decrease in absorbancc at 340
`
`nm caused by the oxidation ol‘
`NADPH is proportional to plasma am—
`monia. CLDH is specilic 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. many ol'these reports
`overestimate the level of Free ammonia,
`
`for example, Nluting.33 rl‘he 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 low minutes.3
`
`The assay can be used to measure am-
`monia levels over a broad range. from
`as low as 12 pmol/l. to as high as l
`mmol/|.. The disadvantage of this ap-
`proach is the length and complexity of
`the procedure. thereby enhancing the
`potential for variation in reported blood
`ammonia levels. ll' 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 ol‘ 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 ol'the ini-
`tial readings. Thus overestimates ol‘am—
`monia levels are most likely in poorly
`handled samples containing elevated
`levels ol'transaminascs and amino acids.
`
`Normal VII/[1&1for Blank)
`Ammonia Lew/.1
`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 timol/L re-
`spectively. The average value for ve-
`nous blood and plasma is 30 pmol/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 or
`the sample. the time allowed bchvccn
`drawing and assaying. and linally. the
`assay itsell‘.
`/\ consequence of 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
`ol'blood ammonia levels. Such meth-
`
`pending hyperammoncmic condition
`and would more easily permit earlier
`selection and regulation ol‘ therapeutic
`interventions. 'l‘vvo promising methods
`that are under development and may
`eventually be used clinically are the se-
`lected-ion l'low tube technique, which
`analyzes trace gases in breath. and a
`fiber-optic catheter tipped with an am—
`monia—sensitive indicator.
`
`Selected-inn Flam Tube
`
`Selected~ion Flow Tube is a quanti-
`tative method l‘or the rapid, real-time
`ana ysis of the trace gas content ol‘ at-
`mospheric air. It was originally devel-
`oped to study ionic reactions in the gas
`phase and is particularly valuable
`for providing kinetics data on ion-
`molecule reaL‘tions. contributing to the
`current understanding ol‘ 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 ol‘ammonia in breath have
`
`required large sampling “ow rates'H or
`long sampling times” and are there-
`lbre unsuitable For assessing the am-
`monia concentration from a single
`breath. A schematic ol‘thc SIFT appa-
`ratus is shown in Fig 5 taken from
`Smith and Spanelfi6 This technology is
`being further developed and may soon
`be a sensitive. quantitative method for
`the continuous real~time analysis ol‘ the
`trace-gas content ol‘ human breath and
`
`SIS
`
`Par Pharmaceutical, Inc. Ex. 1022
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 5 of10
`
`

`

`BARSUI’T]
`
`
`
`THE joumm. or PEDIATRICS
`
`
`
`
`
`jANUARY 200i
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`To“: 1. Reported assay techniques and blood ammonia levels in healthy subjects
`
`..
`
`
`
`
`
`
`
`
`Arteriai blood
`
`
`
`
`
`
`Hutchinson and Lobby (I962)
`
`
`
`
`Gips and Mbbe‘ns—Alberts (1968)
`
`
`
`
`Huizenga and Gips (1985)
`
`
`
`
`Huizenga et a] (1992)
`
`
`
`
`
`Huizenga et a1 (1992)
`
`
`Arterial plasma
`
`
`
`
`Gips and \«VibbensAlberts (1968')
`
`
`
`
`Huizenga and Gips (1985)
`Venous bIood
`
`
`
`
`Die net (1961)
`
`
`Forman (1964)
`
`
`
`
`Hutchinson and Labby (1962)
`
`
`
`
`Proelss and Wright (1973)
`
`
`
`
`Gips and Wibbens—Alberts (1968)
`
`
`McCulldugh (1967)
`
`
`
`Gangoili and Nicholson (1966)
`
`
`
`
`Sinniah et al (1970)
`
`
`
`Huizenga er a1 (1992)
`
`
`
`Huizenga et a1 (1992)
`
`
`Venous plasma
`
`
`
`
`
`German et a1 (1976)
`
`
`
`Oberholzer et a1 (1976)
`
`
`
`
`Buttery et al (1982)
`
`
`Brusilow (1991)
`
`
`
`
`Cooke and Jensen (1985)
`
`
`
`
`Mliems and Steenssens (1938)
`
`
`
`
`Spoons-r et al (1975)
`
`
`
`Seligson and Hirahar'a (1957')
`
`
`
`
`Mondzac et a1 (1965)
`
`
`
`
`Mating et a1 (1968)
`
`
`
`
`Van Anken and Schiphorst (1974)
`
`
`
`
`Howanitz et a1 (1984')
`da Fonseca-Wollheim (1990)
`
`
`
`
`
`
`
`
`
`
`Reference
`
`
`
`
`. ..
`
`
`
`
`
`..
`
`
`
`
`
`
`
`42
`
`41
`
`15
`
`16
`
`16
`
`
`41
`
`15
`
`
`2 l
`
`59
`
`42
`
`23
`
`41
`
`45
`
`40
`
`45
`
`16
`
`16'
`
`
`9
`
`44
`
`53
`
`4
`
`32
`
`46
`
`27
`
`20,
`
`2
`
`35
`
`5
`
`I 1
`8
`
`
`
`Ion-exchange/Nessler
`
`Supernatant/phenol
`
`Microdif'fizsionfBAC 1
`
`Microdifthsiou/BAC II
`
`Enzymatic
`
`
`Supernatant/phenol
`
`Enzymatic
`
`
`
`
`Ion-exchangefNessler
`
`
`Ion-exchangefphenol
`
`
`Ion-exchange/Nessler
`
`Supernatant/electrode
`
`Supernatant/phenol
`
`Supernatantfphenol
`
`Supernatant/phenol
`
`supernatantfphenol
`
`
`Microdifiiss‘i‘onfBAC II
`
`Enzymatic
`
`
`
`lon-exchangez’phenol
`
`Ion-exchangef-pl'lenol
`
`
`Ion-exchange/phenol
`
`
`Ion -exc han gek'ph cool
`
`Electrode
`
`Electrode
`Supematantffluoromeny
`
`Microdil'i'ilsion/Nessler
`
`Enzymatic
`
`Enzymatic
`
`Engrmatic
`
`Enzymatic
`
`Enzymatic
`
`
`
`
`19
`
`22'
`8
`
`
`21
`
`21
`
`
`23
`
`'25
`
`
`15
`
`51
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`2-1
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`17
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`22
`
`57
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`50
`
`44
`
`21
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`21
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`l9
`
`16
`
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`18
`
`.44
`
`44
`
`5'2
`
`56
`3D
`
`
`57
`
`22
`50
`
`'29
`
`
`
`
`
`
`
`thus permit a continuous, noninvasive
`
`
`
`
`
`measure of systemic ammonia levels.
`
`
`
`
`This technique involves the genera-
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`
`
`
`
`
`
`tion 0F positive ions that are created in
`
`
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`
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`a microwave discharge ion source,
`
`
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`
`
`containing an appropriate gas mixture.
`
`
`
`
`In conventional mass spectrometry,
`
`
`
`
`
`
`
`ionization of the trace gas molecules is
`
`
`
`
`achieved by electron bombardment.
`
`
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`
`
`resulting in molecular “cracking” and
`
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`
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`the production of complicated spectra.
`
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`
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`The SIF‘T technique uses a current of
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`precursor ions of a given mass~to~
`
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`
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`charge ratio. in the case of ammonia
`
`
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`
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`detection, HEOJr is extracted from this
`
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`
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`mixture of ions with a quadrupole
`mass filter. This current oF selected
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`ions is then injected into a Fast-flowing
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`inert carrier gas stream, usually heli-
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`um. The ions are carried along a 1»
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`meter length flow tube and are sam-
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`pled by a pinhole dovvnstream of the
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`injector. The sample of breath is intro-
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`duced into the define by a sample port
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`near the injector (Fig 5). Accurate
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`trace gas analysis or quantification is
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`possible because the reaction of the
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`primary ions, in this case 1150+, with
`ammonia is to form ammonium ions
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`and water is precisely defined in the
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`SI F’l‘. In general, the primary ions cho-
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`sen must not react at significant rates
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`with the major components of the
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`nitrogen,
`breath sample, oxygen,
`water. or carbon dioxide, because such
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`reactions would saturate the primary
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`ions. In turn. the primary ions must
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`react efficiently with the trace gases to
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`be detected to Form identifiable prod-
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`SIG
`
`
`Par Pharmaceutical, Inc. Ex. 1022
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 6 of10
`
`

`

`BARSOTTI
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`(610)
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`Molecular Weight
`F114] 6. SlFT spectrum obtained with H3O' precursor ions of breath sampled from patient with
`end-stage renal failure before hemodialysis treatment. Precursor ions and their respective isotopic
`variants are shown as open bars. and productions are shown as filled bars. (Reproduced with permis-
`sion from Davies S. Spanel P, Smith D. Quantitative analysis of ammonia on The breath of patients in
`end-stage renal failure. Kidney Int.
`|997z52z223~28.)
`
`ic questions remain to be addressed
`before the efficacy of this approach is
`established. including the correlation
`between breath ammonia levels and
`
`blood levels and the factors determining
`
`breath ammonia love 3. 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 al57 Show
`a correlation with the plasma urea lev-
`els a