LUPIN
`EX. 1006
`
` i
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`1 of 20
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`

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` ii
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`2 of 20
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`
`N. Blau - M. Duran
`
`M. E. Blaskovics — K. M. Gibson
`
`(Eds.)
`
`Physician's Guide
`to the Laboratory Diagnosis
`of Metabolic Diseases
`
`Second Edition
`
`Foreword by C.R. Scriver
`
`With 100 Figures and 270 Tables
`
` iii
`
`3 of 20
`
`

`
`Nenad Blau
`
`Marinus Duran
`
`Division of Clinical Chemistry
`and Biochemistry
`University Children’s Hospital
`Steinwiesstrasse 75
`8032 Ziirich
`Switzerland
`e-mail: l)lau@access.unizh.ch
`
`Milan E. Blaskovics
`
`3639 Amesbury Road
`Los Angeles, CA 90027
`USA
`e-mail: blaskk@earthlink.net
`
`Academic Medical Center
`Dept. of Pediatrics
`and Clinical Chemistry
`Meibergdreef 9
`1105 AZ Amsterdam
`The Netherlands
`e-mail: m.duran@amc.uva.nl
`
`K. Michael Gibson
`
`Biochemical Genetics Laboratory
`Dept. of Molecular and Med. Genetics
`Oregon Health 8: Science University
`2525 SW 3rd Avenue, Suite 350
`Portland, Oregon 97201, USA
`e-mail: gibsonm@ohsu.edu
`
`ISBN 3-540-42542—X 2nd Edition Springer-Verlag Berlin Heidelberg New York
`Title of the 1st Edition:
`N. Blau, M. Duran, M.E.. Blaskovics
`Physician’s Guide to the Laboratory Diagnosis of Metabolic Diseases
`(:3 1996 Chapman 3: l-[all
`
`Library of Congress Cataloging—in—Publication Data
`Physician’s guide to the laboratory diagnosis of metabolic diseases! [edited by] N. Blau
`..
`[et al.]. — 2nd ed.
`pa, cm.
`Includes bibliographical references and index.
`ISBN 3-540—42542—X {hd.: allcpaper)
`I. Metabolism — Disorders — Diagnosis — Handbooks, manuals, etc. 2. Metabolism,
`Inborn errors of— Diagnosis — Handbooks. manuals. etc. 3. Diagnosis.
`Laboratory — Handbooks. manuals, etc. 1. Blau. N. (NenadJ, 1946 —
`lDNLM: 1. Metabolism. inborn Errors — diagnosis. 2. Diagnosis, Differential.
`3. Laboratory Techniques and Procedures. WD 205 P573 2002]
`RB}-&?.P476 2002
`6l6.3'9075—dc2l
`2002021642
`
`This work is subject to copyright. All rights are reserved, whether the whole or part of the material is
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`UN|VEFiSlT\r
`OF
`PENNSYLVAM
`LIBFIAPIEE
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`.-._._..._...............~..-.._.-....._......_._._,._._.._-._._...._.,_._...___
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`i-
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`isS,
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`_.
`
`g
`
`11
`
`Inherited Hyperammonemias
`CLAUDE BACI-I MANN
`
`11.1
`
`Introduction
`
`Hyperammonemia (systemic venous or arterial plasma ammonia >80 in
`newborns or >50 t1mol.’L after 28 days postnatally) is due either to an in-
`creased production exceeding the capacity to detoxify (as in colonization
`with urease containing microorganisms in an intestinal loop, a neurogenic
`bladder or with a ureterosigmoidostomy), or to a decreased detoxification
`capacity. Among the latter causes are primary or secondary defects of en-
`zymes involved in ammonia detoxification or a deficiency of intermediates
`needed as substrates for a functional urea cycle, such as a nutritional, en-
`zyme, or transport defect, or to interference with portal circulation so that
`portal blood does not reach the hepatocytes (a portacaval bypass or a pat-
`ent ductus), which can cause “transient hyperammonemia of the prema-
`ture”. Ammonia detoxification is reduced in deficiencies of urea cycle en-
`zymes, transport proteins [estimated incidence 1130000 newborns, [1]) in
`conditions where glutamate or acetyl Cori is decreased (valproate therapy
`and organic acidurias), with carnitine and CoA (sequestered by pathologi-
`cal acyl moieties) and defects of mitochondrial beta-oxidation or carnitine
`metabolism. These lead to a deficient
`formation of N—acetylgiutarnate
`(NAG), an obligate activator of the first step of ammonia detoxification,
`and thus to a functional NAGS deficiency. An acetyl CoA deficiency further
`reduces pyruvate carboxylase, which blocks gluconeogenesis. These two ef-
`fects of an acetyl CoA deficiency lead to a Reye syndrome. Today, because
`more specific etiological diagnoses can be made, the Reye Syndrome is dis-
`appearing.
`The actual enzyme activity in urea cycle disorders (_UCD) in vivo is only
`partially assessed by in vitro assays (artificial conditions]. It is a problem
`and must always be viewed in respect to the nitrogen load entering the
`pathway (Fig. 11.3). This depends as well on the exogenous nut1'itionai sup-
`ply or bacterial ammonia production in the gut as on the endogenous bal-
`ance or imbalance between protein synthesis and catabolism. The clinical
`heterogeneity of the disorders and any prognostic predictions will
`thus
`only partly depend on the genetic background if residual protein is present.
`“Mild" leaky variants [unstable enzymes in vitro or residual enzyme activ-
`
`261
`
`-
`
`-— -
`
`--
`
`-
`
`-
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`-
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`5 of 20
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`

`
`252
`
`Inherited I-lyperaininonemias
`
`ity) may lead to severe hyperammonemic crises if protein catabolisin pre-
`dominates (e.g. major weight loss in newborns, vi1'al infections etc.).
`Hyperainmonemia is toxic to the brain. It exerts reversible [mostly sero-
`toninergic) and irreversible effects. Blood ammonia (NH3) concentrations
`exceeding 180 tt1nol.’L, or a coma lasting more than 2-3 days appeal’ to be
`associated with irreversible defects which worsen with the duration of the
`
`coma. Thus, ammonia should be assayed in any sick newborn as a “stat”
`analysis together with a sepsis work-up, or with the suspicion of an intra-
`cerebral hemorrhage which is not confirmed. If hyperammonernia is found,
`it should be confirmed by a second “stat" assay with samples obtained for
`a complete laboratory evaluation (plasma and simultaneous spot urine). A
`diagnosis should be made as rapidly as possible and not later than 12-2411
`in order to initiate specific treatment. Among the non-artefactual hyperam-
`monemias, 2;'3 are due to urea cycle defects, and U3 to organic acidemias
`and other defects which can not be distinguished by the extent of the hy-
`perammonemia. Blood gas analyses and anion gap determinations are of-
`ten not helpful since secondary lactic acidosis is often present in UCD pa-
`tients with circulatory failure. Since the specific treatment used for a UCD
`can be deleterious to patients with organic acidurias [e.g., amino acid mix-
`tures containing high isoleucine or valine and to some extent benzoate and
`phenylbutyrate, especially in an MCAD clehydrogenase deficiency and vice
`versa), a rapid diagnosis is necessary. If a decision to treat is made, emer-
`gency therapy (see below) prolonged beyond 24 hours will lead to low es-
`sential amino acids and impaired protein synthesis with all
`the ensuing
`risks and complications (coagulation problems, hemodialysis and or hemo-
`filtration). This can be avoided or minimized by a rapid and complete labo-
`ratory evaluation. The laboratory workload should not be underestimated.
`Besides the initial diagnostic studies, frequent monitoring is required dur-
`ing treatment. In a UCD, treatment consists of measures for reducing the
`nitrogen load (restricting natural protein intake, gut acidification with lac-
`tulose) and providing the substrates which are rate limiting due to the re-
`striction of natural nutrients or due to the enzyme block. These would be
`arginine or citrulline, citric acid in the case of ASA, essential amino acids
`in calculated amounts, and adequate calcium, phosphate, iron,
`trace ele-
`ments and vitamins. Also if needed, substrates for alternate pathways such
`
`as benzoate, with proper controls in neonates. One must be very cautious
`with the chronic use of phenylbutyrate because of it‘s long term side ef-
`fects, which include its interference with cell replication and farnesylation.
`The above treatment should only be instituted after a definite diagnosis is
`made and it would be contraindicated in a11 organic aciduria. Whatever
`treatment variation is used, it must be carefully controlled, especially in or-
`der to avoid chronic malnutrition due to a deficiency of essential amino
`acids. Dietary management
`is actually more of a challenge,
`in practice.
`than is
`the hyperammonemia. Because of the expertise and experience
`
`6 of 20
`
`

`
`Introduction
`
`263
`
`needed in managing patients with UCD’s, the transfer of patients into a
`center with experienced clinicians and a laboratory is recommended.
`Brain ammonia toxicity depends upon the level of blood ammonia,
`which crosses membranes in its unciissociated form (p1( 9.05 at 37 °C). In-
`creased brain ammonia is considered to augment the synthesis of gluta-
`mate and giutamine,
`the intercellular transport moiety. This in turn in-
`creases the transport capacity of large neutral amino acids [including tryp-
`tophan) at
`the blood brain barrier {yGT dependant) and elicits an in-
`creased serotonin secretion [2]. The increased glutamate released by neu-
`rons and its decreased re—uptake is probably exotoxic. The accumulation of
`glutamine in astrocytes has been shown, under extreme conditions, to lead
`to astrocytic swelling which may be responsible for terminal brain edema.
`The mechanisms affecting the energy pathways in brain are still controver-
`sial. Since the major portion of brain glutamate is synthesized within the
`brain, it is not at all clear if plasma glutamine plays any pathogenic role in
`the brain’s toxicity. It
`is, however, an indicator of ammonia detoxification
`in the peri-central part of the hepatic lobules (urea cycle enzymes are peri-
`portal), or of its release by muscle or other tissues. When managing pa-
`tients, one must also know that arginine, a semi—essential amino acid,
`is
`mainly synthesized in the kidneys from citrulline, which in turn is formed
`predominantly in the intestine (see Table 11.2]. Argininosuccinate synthe-
`tase and lyase and the arginine transporters (CAT), additionally, play a role
`in the recycling of citrulline to arginine (e.g. for NO synthesis in kidneys,
`intestine and brain [3].
`The inherited enzyme deficiencies listed in Table 11.2 lead to the accu-
`mulation of substrates and deficiencies of products. For correct interpreta-
`tion of laboratory results, one need be aware that substrate accumulation
`can affect the prior enzyme in the pathway (e.g. increased carbamyl phos-
`phate inhibits CPS}. A deficiency of urea cycle intermediates (transport or
`enzyme products or dietary substances) e.g. arginine or ornithine, is often
`rate limiting. It can initiate a vicious cycle, which worsens the urea syn-
`thetic capacity in the cytosol (e.g. by limiting protein synthesis), or in the
`mitochondria (deficient stimulation of NAGS and of substrate for OTC}.
`Measured plasma values reflect cytosolic metabolite concentrations, not
`those of mitochondria. Protein catabolisni contributes to the plasma amino
`acid values. Thus, the interpretation of results for plasma arginine, proline
`and lysine must be done within the context of the pattern found for all of
`the amino acids. Urea concentrations depend upon the arginine in the cyto-
`sol originating from protein catabolism, urea cycle synthesis, and therapeu-
`tic applications.
`
`7 of 20
`
`

`
`264
`
`inherited 1-lypcrammonemias
`
`11.2 Nomenclature
`
`No. Disorder
`
`Expression
`
`11.1 Cmbamylphospliate
`syntlietase (CPS 1)
`
`Ornitliine transcar-
`barnylase
`(OTC, OCT)
`
`Argininosuccinate
`syntlietase (AS);
`Citrullinemia
`Type 1
`
`Argininosuccinate
`lyase (AL); Argini—
`nosuccinic aciduria
`
`Arginase 1
`
`N-Acetylglutarnate
`synthetase (N{\GS}
`
`Solute carrier fami-
`ly ? A7 (SLC7A7)-,
`Lysinuric protein
`intolerance
`Solute carrier fami-
`ly 25 A15
`[SLC25Ai5,
`ORNT1); Hyperm-
`nitl1inemia—hyper—
`ammonernia—homo—
`cilrullinuria (HHH)
`syndrome
`i_\'-Pyrroline—5-car-
`boxylate synthe-
`tase, PYCS
`
`Mitocliondrial: liver (periportal —>
`pericenlral; all urea cycle enzymes)
`and much less intestine (enzyme not
`expressed in red or white blood cells
`or fibroblasts)
`Mitocl1ond1'ial: liver and much less
`intestine. Mosaicisin in hetcrozygote
`females (not expressed in red or white
`blood cells or fibroblasts)
`
`Cytosolic: Liver, kidney (cortical prox-
`imal tubule); intestine, myenteric neu-
`rons, ileal and colonic muscles; CNS:
`not in astrocytes (selective neurons in
`neocorlex, and midbrain), brainstern.
`diencephalon, cerebellar molecular
`and granular layer; eye. Fibroblasts
`Cylosolic: Liver; kidney (cortical prox-
`imal tubule); intestine, rnyenteric neu-
`rons, ileal and colonic muscles; CNS:
`cerebrum ubiquitous, cerebellum (not
`in cerebellar white matter); eye: Red
`cells, fibroblasts
`Arginase 1 cytosol (liver)
`Arginase 2 (mitochondria: small intes-
`tine, ltidney [outer medulla and partly
`cortex), CNS: ubiquitous; eye: solely
`retina. Red cells, Fibroblasts
`Mitochondrial: liver >> intestine
`>> spleen. Intestine (enzyme not ex-
`pressed in red or white blood cells or
`fibroblasts)
`Basolateral mernbrane
`Liver, intestine, kidney
`
`MIM
`
`23?3UU
`
`21 57900
`
`7'cen—q1l.2
`
`207300
`
`unknown
`
`l4q1l..2
`
`222700
`
`Mitochondrial membrane: Fibroblasts
`
`l3ql4
`
`238970
`
`Mitochondrial
`
`lDq24.3
`
`133250
`
`8 of 20
`
`

`
`Metabolic Pathway
`
`265
`
`Disorder
`
`Expression
`
`Chrorno-
`some
`
`MIM
`
`Glutamate dehydro- Mitochondrial
`genase 1, GT?
`binding site muta—
`tions (GLUDI);
`Hyperinsu1inism-
`Hyperamrnonernia
`syndrome
`Citrullinemia type Mitochondrial membrane, liver (not
`2 (SLC25At3 gene) kidney) with secondary AS deficiency
`Citrin deficiency
`
`10q23.3
`
`133130
`
`7q2t.3
`
`6lJ34?1
`
`11.3 Metabolic Pathway
`
`Mitochondrion
`
`omfic acid
`J;OMP —- Orotidine
`
`l
`UMP «EL Uridine
`Uracil
`
`ASA
`
`FUM
`
`N0
`\
`
`AcCarnitine
`
`fiL
`‘I1.-1
`
`A RG
`.161 or 2
`”‘5
`I
`car
`Dihasic AA
`
`YGLU-SA W .ATP5C -1——I-v PRO
`“.9
`
`SLCYA7
`11.7
`
`Fig. 11.1. Metabolites: GLU, glutamate; 2—Oxo—Glut, 2-oxoglutarate; NAG, N—acetylgluta-
`mate; NH3, ammonia; Carb.—P, carbamylphosphate; ORN, ornithine; CIT, citrulline; ASP,
`aspartate; ASA. arginiriosuccinate; FUM, fumarate; ARG. arginine; Ditmsic AA, dibasic
`amino acids
`(lysine, ornithine, arginine);
`}rGt':r—3A, gammaglutamyl
`semialdehyde;
`AIPEC, p1,'rrolineA5-carboxylate; PRO, proline; GLN, glutamine; OMP, orotidine 5'—mono-
`phosphate; UMP, uridine 5'-monophosphate. Enzymes: OAT, orniLhine—oxoacid amino-
`transferase; NOS, nitric oxide synthetases; CAT, cationic amino acid transporters (y+);
`others as listed in the table in Sect. 11.2. Glutamate and 2—oxoglutarate are key metabo-
`lites for the interconnection of the Krebs cycle [shown as cycle) and the urea cycle; they
`are also important substrates for transamination reactions (e.g. ASAT, ALAT) including
`mitochondrial aspartate synthesis which is transported to the cytosol by the aspartatel
`glutamate carriers (citrin).
`
`9 of 20
`
`

`
`266
`
`Inherited Hyperarnrnonemias
`
`11.4 Signs and Symptoms
`(Common to all urea cycle disorders except argininemia)
`
`Central nervous system
`
`Eye
`
`Circulation
`Kidney
`Lung
`Liver
`
`Hair 8: skin
`
`Loss of appetite, vomiting
`Aversion of high protein con-
`taining food (Protein intoler-
`ance) with consequent
`malnutrition
`Lethargy, somnolence, coma
`Seizures (neonates, infants)
`Hyperpnea (up to 6 months)
`Hypmlhyperthermia (new-
`borns)
`Muscular l1ypo—!l1ypertonus
`Ataxia, irritability. sleep dis-
`turbance (children)
`Asterixis, delusions. psychotic
`behavior (>10 years)
`Mental retardation
`Scotornas, visual hallucina-
`tions
`
`Circulatory failure
`Renal failure
`
`Hemorrhage
`Hepatomegaly
`
`Fibrosis, cirrhosis (chronic)
`Fragility of hairftricliorrhexis
`nodosa
`
`Metabolic alkalosis
`Growth retardation, osteo-
`porosis, Vit B12, Zn defi-
`ciency
`
`Terminally; cerebral edema
`Increased glutamine release
`Respiratory alkalosis
`
`Papilledema
`
`Metabolic acidosis
`Metabolic acidosis
`
`Cytoiysis (ALAT, ASAT in-
`creased]; reduced protein
`synthesis: coagulation de-
`fect
`
`Low urea (P) (not always]
`Arginine deficiency
`(including iatrogenic)
`
`10 of 20
`
`

`
`Signs and Symptoms
`
`267
`
`I Disease “Specific” Signs and Symptoms
`
`Disease
`
`Citrullinemia type 2
`(decreased AS acitivity,
`citrin deficiency)
`Argininosuccinic aciduria
`
`Argininemia
`
`Lysinuric protein
`intolerance
`
`HI-II-I syndrome
`
`Pyrroline 5 carboxylate
`synthetase deficiency
`(few patients. possibly
`ascertainment bias):
`Glutamate dehydrogenase
`mutations
`
`Cholestatic jaundice, hepatic steatosis and siderosis
`i-iyperammonernia not obligate
`
`Facies with epicamhic fold, depressed nasal bridge
`(saddle nose) as newborn
`Nervous system: increased irritability and muscle tone.
`Progressive loss of motor and mental skills and increasing
`spasticity of the lower extremities. Seizures; ataxia, atl1eto—
`sis, dysarthria
`Lungs: proteinosis, interstitial pneumonia
`(white lung disease)
`Hematology: hemolysis (increased LDH, ferritin), lympho-
`liistiocytic autopiiagocytosis
`Kidney: glomeruionephritis
`Bones: osteopenia
`immunity: decreased response to varicella immunization
`Neurological: pyramidal signs in absence of decerebration
`(C. Dionisi-Vici, personal communication)
`Hematology: factor VII 5: X deficiency
`Eye: cataracts
`Bone and skin: hyperlaxicity and increased skin elasticity
`I-lyperamrnoneinia (mild) only preprandial!
`
`Fasting hypoglycemias noticed mostly in infants or later
`(liyperinsulinism)
`Growth failure, variable mental retardation
`
`11 of 20
`
`

`
`263
`
`Inherited Hyperamrnonemias
`
`11.5 Reference Values
`
`Analyte
`
`<28 days
`mature
`
`4 months
`
`1-12
`months
`
`2-14 years Adult
`men
`
`Adult
`women
`
`Remarks
`
`Ammonia (P) en- <80
`Z)'H1atiC 11117101!’0
`
`Ammonia (P)
`microdiffusion
`(prnolfl)
`Amino acids (P)
`(pmolll)
`
`Arginine
`Arginino-
`succinate
`
`1844
`
`IT-63
`
`Range: sam-
`pling >3.5 h
`after end of last
`feed (p.p.: I-
`35 h after feed)
`77-165 (65-200) 41-190 (60-190)
`<2
`
`10-65
`
`Citr1J.11it'tE‘.
`
`17-41 [13-45}
`
`11-32 (0-36)
`
`10-30
`
`postprandially
`30-60 urnolil high-
`er depending on
`time and N load
`
`I
`
`In contrast to
`other amino acids
`
`Citrulline is higher
`pre- than post-
`prandially
`
`_
`
`'
`
`55-120 (55-420) 28-150 (40-125)
`110-290
`60-230 (75-275)
`[1 1 5-330)
`380-660
`(380-710}
`200-490
`(185-645)
`120-260
`(130-310)
`
`200-720
`[265-650]
`110-480
`(190-550]
`64-272
`(120-260)
`
`10-110
`45-145
`
`60-470
`
`100-310
`
`50-190
`
`30-100
`135-260
`
`20-90
`115-250
`
`550-830
`
`440-810
`
`240-600
`
`200-550
`
`100-380
`
`70-270
`
`Ornithine
`Lysine
`
`Glutamine
`
`Alanine
`
`Proline
`
`Amino acids (U)
`(fractional tubular
`reabsorption, %)
`Lysine
`Ornithine 8:
`Arginine
`Orotate (mrnoll
`mol creatinine)
`
`0.2-3.8
`
`0.08-0.44
`
`0.035-0.26 Increase in 6 11
`urine after protein _
`load of 1 gikg:
`<05.’ rnmolfmol cr
`
`No reference values can be given for enzyme assays, since the results depend upon the method used. The reference:
`ranges must be obtained from Ihe testing laboratory which should be contacted for correct sampling and transport
`conditions.
`"
`
`12 of 20
`
`

`
`Pathological Valuesmifferential Diagnosis
`
`269
`
`11.6 Pathological Values/Differential Diagnosis
`
`The extent of increase varies widely depending on mutation and especially
`internal and external nitrogen load.
`
`ASA Cit
`
`Arg
`(P)
`
`OROT
`(U)
`
`H0l'J.'10- Pro
`citrul-
`(P)
`line (U)
`
`Glnl
`NH;
`
`Om (P)
`
`—
`—
`
`(T)
`T
`
`.
`
`Exclude
`Arg load
`
`Exclude
`canned
`food.’milk
`
`Hypo-
`glycemia
`
`CPS 1 def.
`
`OTC def.
`Citrulline—
`rnia I
`Citrulline—
`mia II
`
`Arginino—
`succinic
`aciduria
`
`Argininemia
`
`NAGS def.
`
`—
`
`LPI
`HHH
`syndrome
`
`T— T I
`T-Tl
`
`T“
`PYCS def.
`Hyperinsu— T—Tb
`linism—hy-
`peram mone-
`rnia (HIHAJI
`syndrome
`
`‘ Only fasting not postprandial.
`'’ Unchanged by protein load or restriction. Non-responsiveness to henzoate or phenylbutyrate treatment.
`FTR, fractional tubular reabsorption of dibasic amino acids compared to creatinine; :1, normal; nd, not detectable;
`ASA, argininosuccinate; OROT. orotic acid.
`
`13 of 20
`
`

`
`ETD
`
`Inherited Hyperammonemias
`
`11.7 Loading Tests
`
`The goal of loading tests is to unrnaslt a functional defect where there is re-
`sidual activity.
`In heterozygotic females with OTC deficiency (x—chron1o—
`somal) it can only be used to confirm, but never to exclude a carrier status
`because of rnosaicisni {Lyon hypothesis) which might be strongly skewed
`towards the wild type and thus overlap normal values.
`Protein loading tests bear the risk of eliciting hyperamrnonemia. How-
`ever, the loading dose can be adapted. Regardless, these tests should only
`be used when a diagnosis is uncertain and after establishing a daily ammo-
`nia profile (preprandial and 1 and 2 hours postprandially) so that a safe
`tolerated protein intalce regime can be calculated prior to the load. Post-
`prandial hyperarnmonernia levels determined during the profile allows one
`to estimate the risk of a protein loading dose.
`The popular Allopurinol test does not take into account variations of the
`flux through the pyrimidine synthetic pathway, be it from the carbaInyl-
`phosphate load, due either to endogenous protein breakdown or exogenous
`protein and nucleotides or to tissue regeneration (CPSIIJ. Furthermore, the
`regulation of the first multifunctional enzyme is generally ignored. A phos-
`phoribosylpyrophosphate deficiency (as in the Lesch-Nyhan syndrome) also
`leads to increased orotate. The interpretation of results is not as straightfor-
`ward as one would wish. False positive and false negative tests have been
`described. The diagnostic value of orotate [orotic acid) vs an orotidine as-
`say is an ongoing debate.
`Procedure: After collecting a baseline urine sample, urine is collected in
`4 sequential 6 hour periods and stored frozen after the oral administration
`
`'63‘
`EC
`‘5n:u._
`'5.)
`73
`E2-o
`.:1:.4'3u
`
`EE
`
`9O
`
`Age (years)
`
`1?
`
`the variation of orotate after allopurinol
`Fig. 11.2. Basal orotate decreases with age:
`challenge is shown (adapted from Burlina et al. [1U])
`
`14 of 20
`
`

`
`Loading Tests
`
`271
`
`of a single dose of allopurinol {children >6 y: 100 mg, 6-10 y: 200 mg,
`>10 y: 300 mg).
`The upper limits or maximal orotate excretion after a load is [10]: 6 mo
`-6 yr 13.0 mmolimol Cr; 6-10 y: 9.3 mmol/mol Cr; 10-]? y: 10.2 mmol.’
`mol Cr [see Fig. 11.7). For orotidine,
`the limit of decision is 8 mrnol/mol
`creatinine [11].
`
`Protein load: A protein load is done when a diagnosis is unclear or for
`heterozygote detection in OTC deficiencies. After one has determined a dai-
`ly profile for pre- and postprandial ammonia and the amino acids in a self
`chosen diet, the protein content should be estimated per meal. The patient
`should not be in a catabolic but steady state for at least 4 days. For women,
`the test should be avoided around the period of n1enst1'uation. The protein
`load is, in contrast to the allopurinol test, also useful for assessing protein
`tolerance. False negatives have been described in conjunction with OTC
`heterozygote testing; skewed toward a predominance of wild-type OTC.
`Procedure: After a breakfast of mainly carbohydrates, a baseline urine
`(approximately 4 hours) is collected. After the last voiding, a high protein
`meal
`(lean meat, poultry, cottage cheese, etc.) containing 1 g/kg body
`weight (bw) is given as “a load”. The dose should be reduced, if, by history
`andior experience, a protein intolerance to such a dose is suspected. Urine
`is quantitatively collected during the 6 hours after the end of the meal and
`cumulatively frozen. It is assayed for orotate by HPLC after allcalinisation.
`In adult women, the upper limit of normal for orotate 6 hours post load,
`after a 1 gfkg bw load, is 0.7 mmol/mol creatinine.
`
`15 of 20
`
`

`
`272
`
`Inherited Hyperammoncmias
`
`11.8 Diagnostic Flow Chart
`
`P—Arg i ni ne
`in r_r.
`
`in
`
`U-Orotate
`incr.
`
`P-Ginfwi-If
`< 1.6
`
`de cr.
`CPS, MAGS
`Liver
`
`Fig. 11.3. Top d
`rithm for the di
`
`diagnosis of h1,'_
`nemia based on
`of amino acids
`nine (P+U), U-
`organic acids. g
`acylcarnitines (
`chain fatty acid
`ma. Abbreviatic
`increased; dear.
`Hamocit., harm
`FTR, fractional
`absorption (l—[
`nine *U~amin0
`(U-creatinine -
`acid}]; MCF/1,:
`chain fatty acid
`breviations as i
`
`Algininemia inrginasel Deficiency)
`or nrginine-HCI Eoad
`
`Argininosuccinic Aciduria
`iflrgininosuccinale Lyase deficiency]
`
`Citrullinemia
`ifugininosuccinate Symhelase deficiency)
`
`Pyruvate Carboxylase deficiency
`Renal insufficiency
`
`Homociirullinuria~Hyperorniihinemia—
`I-Eyperamrnanemia {HHHJ Syndrome
`
`Lysinuric Protein intolerance
`
`|
`
`Ornithine Transcarbarnylase deficiency
`
`I-I Pyrroline 5 Carboxylate Synthetase deficiency
`
`Organic Acidurias including
`Valproate or Pivalateester treatment
`
`Defects of Fatty Acid Beta—o><idation
`or of Carniline metabolism
`
`Transieni Hyperamrnonomenia of the
`Premature, open Ductus venosus;
`+ Hypoglycemia iflyperinsuiinisrn):
`Glutamate Dehydrogenase mutation
`
`|
`
`Carbamoylphosphale Synthetase (CPS) or
`N-Acelylglutarnate Synthetase ENAGS)
`deficiency
`
`Hepatic insufficiency, Blind loop syndrome or
`neurogenic bladder infected with Urease cont.
`‘ ‘ ' ‘ ' ' ' '3’ bacteria; Asparaginase rherapy;Ma1nuirition
`iintrauterine, postnatai
`or iatrogenic Arginine deficiency:
`
`16 of 20
`
`

`
`11.9 Specimen Collection
`
`Precondition
`
`Material
`
`Handling
`
`Pitfalls
`
`Specimen Collection
`
`273
`
`At least 4 h after end of the EDTA blood
`last meal or stopping intra~
`venous AA supply from a
`central vein or artery
`
`Immediately put on ice and
`centrifuge (4 °C] not later
`than 15 min after sampling,
`plasma decanted and frozen
`at -20 "C
`
`Stable at -20 “C up to 43 h
`
`Avoid any dilution of sam-
`ple before assay (as distilled
`water, or other acid solution
`contain ammonia trapped
`from air)
`
`if: Amino acids
`
`Plasma
`
`Centrifuge within 15 min
`
`Urine (spot)
`
`Urine 5-10
`ml
`
`Lithium
`
`‘neparinate
`plasma
`
`24 hour
`urine collec-
`tion
`
`Deproteinize with sulfosa-
`licylic acid
`Keep frozen and at pH 2 for
`accurate glutamine results
`Freeze rapidly to avoid bac-
`terial interference
`Store at —20°C
`
`Cumulatively frozen
`
`'
`
`Benzoate,
`. phei1ylace-
`tare, phenyl-
`butyrate
`Hippurate,
`No change of dose during
`phenylacetyh the last 5 days
`glutamine,
`phenylacetate
`
`Capillary sampling increases
`ammonia concentrations
`
`Muscle hyperactivity liber-
`ates ammonia as does a
`prolonged garrot or hemoly-
`sis
`
`High ALT or yGT increase
`ammonia
`
`False low values are found
`with pyruvate concentra-
`tions >2{}0 p.tM
`False high values in micro-
`diffusion methods by
`osmotic hemolysis and
`glutamine breakdown
`Contamination by intracel-
`lular fluid (capillary blood).
`Glutamine release by muscle
`activity (e. g. seizures)
`
`Use HPLC method or iso-
`
`tope dilution only:
`Orotidine seems to have a
`
`limited stability
`
`For checking therapeutic
`compliance
`Indicate total 24 h urine vol-
`
`ume and total daily dose of
`benzoate, phenylacetate or
`phenylbutyrate
`
`17 of 20
`
`

`
`274
`
`inherited I-lyperammonctnias
`
`Test
`NAGS, CPS,
`OTC
`
`Precondition
`Specify appr. protein intake
`in the last 3 days
`
`Material
`Handling
`Liver biopsy Blot and freeze in liquid
`(30 mg)
`nitrogen. store immediately
`at -80 °C, senrl with ample
`dry ice
`
`Fibroblasts
`
`Pitfalls
`Activity dependent on pro-
`tein intake. Only optimized
`NAGS assays (with arginine)
`should be used.
`Gene expression of the urea
`cycle enzymes in liver is
`down regulated to 10% by
`lipopolysaccharides
`Frozen material cannot be
`used for ornithine incor-
`poration assay
`
`11.10 Prenatal Diagnosis
`
`The laboratory should be contacted before collectinglsending specimens.
`
`DNA
`
`Protein (activity)
`
`Metabolite
`(amniotic fluid)
`
`Comment
`
`NAGS def.
`CPS clef.
`
`OTC def.
`
`Gene (RFLPJ'mutation)
`Not feasible
`Not feasible
`Not in all instances, only Fetal liver“
`known mutations
`Many private mutations
`
`Fetal liver“
`
`Not feasible
`Not informative
`
`Not informative
`
`Not predictive in female
`fetus
`
`AS (citrui—
`Many private mutations Amniocytesichorionic villi Not informative
`linemia 1)
`AL (ASA—uria) Many private mutations Arnniocyteslchorionic villi ASA (AF)
`
`Arginineniia 1
`LPI
`1-IHH
`
`Fetal red cells
`Aniniocytesfcliorionic villi
`Arnniocytes
`
`Enzyme activity and ASA
`concentration should both
`be assayed
`
`Assay in cultured, not fro-
`zen sample
`
`3 Varies with gestational age. Intrauterine liver biopsies (week 1647) have been performed, but are not without risk of ",
`fetal liver hemorrhage. A simultaneous control sample is usually required (instability of enzyme and transport condia
`tionsi).
`’
`
`11.11 DNA Analysis
`
`Can be performed in some CPS deficiency {microsatellite analysis) most
`OTC deficiencies (RFLP rarely mutation analysis as first step), AS, AL, argi—
`nase deficiency, LPI, HI-IH and HIHA synclrome, not in NAGS deficiency.
`
`18 of 20
`
`

`
`Surnmaryfcomrnents
`
`2395
`
`The performing laboratory should be contacted before sample collection
`if possible; otherwise EDTA blood or tissue samples should be collected for
`DNA extraction.
`
`11.12 Initial Treatment (Management while awaiting results}
`
`As stated in the introduction, a rapid diagnosis is required in all instances,
`with collections of blood and simultaneous spot urines as outlined above.
`Before initiating any emergency treatment, one must ask the question
`whether treatment is desirable, if at all, especially in newborns where the
`prognosis is still reserved (e.g. in known male OTC deficiencies, except for
`the milder variants, which in a few instances can present with hyperarnmo—
`nemia at a few days of life).
`The emergency treatment aims at stopping the endogenous and exoge-
`nous protein supply, at supplementing the missing arginine and at giving
`excess carnitine in order to replenish its free stores and trigger the urinary
`excretion of pathologic acylcarnitines in organic acidurias.
`I Stop per oral protein supply or i.v. amino acid preparations.
`0 Glucose 8-10 mglkg per minute i.v. (with insulin if needed); check plas-
`ma lactate 2 hours after start!
`Arginine I-1C1 i.v. 2 mmolikg b.w. as priming dose in 2 hours and then
`2 rnmolflcg per 24 h.
`Carnitine i.v. 50 mmolfltg b.w. as priming dose in hours and then
`300 mgfkg b.w. per 24 11. Stop when organic aciduria has been excluded.
`
`11.13 Summarylcomments
`
`For improving the prognosis of inherited hyperammonemias, a major pre-
`condition is a timely and rapid accurate diagnosis in order to avoid irrever-
`sible damage to the patients brain. This is a motivating challenge to the
`technicians of well trained and experienced centers in close collaboration
`with clinical dieticians and other personnel which give guidance and sup-
`port to the parents. A well equipped laboratory with validated methods and
`quality assurance is needed and must be prepared to work in emergency
`situations. The burden continues after a diagnosis is made according to the
`algorithm presented (without short-cuts) because long term therapy must
`be adapted to the individual patient with his individual ammonia detoxify-
`ing capacity and nitrogen load [13]. Overtreatment with excessively re-
`stricted essential amino acids (especially plasma isoleucine <25 umolr’L) is
`a major problem with inexperienced teams, who focus primarily on the am-
`monia levels. An understanding of the biochemical pathways and their
`
`19 of 20
`
`

`
`276
`
`Inherited tlyperatnmonemias
`
`complexity is needed for adequate interpretation, for which the profes-
`sionals in the laboratory can be of great help to the clinician.
`
`References
`
`. Summar M, Tuchni-an M. Proceiedings of a consensus conference for the manage-
`ment of patients with urea cycle disorders. jaurmtl of Pediatrics 2001; 138(1): 36-
`510.
`
`transferase deficiency:
`. Bachrnann C. Ornithine carbamoyi
`problems. J’ l::i1er'irMet‘rtb Dis 1992; 15(4): 578-591
`. Braissant O, Gotoh T. Loop M, Mori M. Bachmann C. L-arginine uptake, the citrul~
`line-NO—cycle and arginase II in the rat brain: an in situ hybridisation study. Molec-
`:ilrtrBr.1tiit Research 1999; 70(2): 231-241.
`
`findings, models and
`
`. Aral B, Schlenzig IS, Liu S, Kamoun P. Database cloning human delta 1-pyrroline-
`5-carboxylate syntltetase (PSCS) cDNA:
`:1 bifunctionel enzyme catalyzing the first 2
`steps in proline biosynthesis. Comptes Remius de l’ Acaderttie des Sciences. Serie ill,
`Sciences ole La Vie 1996; 319(3): l7l—1}'8
`. Stanley CA, Fang 1', Kutyna K et al. Molecular basis and characterization of the hy-
`perinsulinisnilliypcranimonemia syndrome: predotninance of mutations in exons 11
`and 12 of the glutamate dchyclrogenase gene. Diabetes 2000; 49(4): 667-6