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`EX. 1006
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`
`N. Blau - M. Duran
`
`M. E. Blaskovics v K. M. Gibson
`
`(Eds.)
`
`Physician’s Guide
`to the Laboratory Diagnosis
`of Metabolic Diseases
`
`Springer
`
`Second Edition
`
`Foreword by C.R. Scriver
`
`With 100 Figures and 270 Tables
`
`. ét.
`
` 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: blau@access.unizh.ch
`
`Academic Medical Center
`Dept. of Pediatrics
`and Clinical Chemistry
`Meibergdreef 9
`1105 AZ Amsterdam
`The Netherlands
`e-mail: m.duran@amc.uva.nl
`
`Milan E. Blaskovics
`
`K. Michael Gibson
`
`3639 Amesbury Road
`Los Angeles, CA 90027
`USA
`e-mail: blaskk@earthlink.net
`
`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@oh8u.edu
`
`ISBN 3-540-42542—X 2nd Edition Springer-Verlag Berlin Heidelberg New York
`Title of the ist Edition:
`N. Blau, M. Duran, ME. Blaskovics
`Physician’s Guide to the Laboratory Diagnosis of Metabolic Diseases
`(:3 1996 Chapman & Hall
`
`Library of Congress Catalogingeinrpublication Data
`Physician’s guide to the laboratory diagnosis of metabolic diseases! [edited by! N. Blau
`..
`[et al.]. — 2nd ed.
`p.; cm.
`Includes bibliographical references and index.
`lSBN 3-540—42542-X (lid; alkpaper)
`i. Metabolism - Disorders — Diagnosis ~ Handbooks, manuals, etc. 2. Metabolism,
`Inborn errors of— Diagnosis — Handbooks. manuals. etc. 3. Diagnosis,
`Laboratory — Handbooks. manuals, etc. ]. Blau. N. (Nenad), 1946 —
`EDNLM: 1. Metabolism. Inborn Errors — diagnosis. 2. Diagnosis, Differential.
`3. Laboratory Techniques and Procedures. WD 205 P578 2002]
`RB}4T.P476 2002
`616.3'9075—dc21
`2002021642
`
`This work is subject to copyright. All rights are reserved, whether the whole or part of the material is
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`11
`
`Inherited Hyperammonemias
`CLAUDE BACI—I MANN
`
`
`
`.-hm...—qu-—i—.—y.
`
`}.
`
`iii9g
`
`11.1
`
`Introduction
`
`Hyperammonemia (systemic venous or arterial plasma ammonia >80 in
`newborns or >50 nmolfL 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 premai
`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 CoA 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—acetylglutamate
`(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 nutritional 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
`
`'
`
`-— -
`
`--
`
`-
`
`-
`
`-
`
`5 of 20
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`

`ity) may lead to severe hyperammonemic crises if protein catabolism pref
`dominates (eg. major weight loss in newborns, viral infections eta).
`Hyperammonemia is toxic to the brain. It exerts reversible (mostly sero—
`toninergic) and irreversible effects. Blood ammonia (NI-l3) concentrations
`exceeding 180 tunolfL, or a coma lasting more than 2‘3 days appear to be
`associated with irreversible defects which worsen with the duration of the
`
`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—
`facts, 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 an 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
`
`
`
`262 Inherited I-lyperamuionemias
`
`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 hyperammonemia 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—24 h
`in order to initiate specific treatment. Among the non-artefactual hyperam—
`monemias, 21'3 are due to urea cycle defects, and 113 to organic acidemias
`and other defects which can not be distinguished by the extent of the hy—
`perarnmonemia. 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 dehydrogenase 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 ref
`striction of natural nutrients or due to the enzyme block. These would be
`arginine or citruliine, 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
`
`the hyperammonemia. Because of the expertise and experience
`
`6 of 20
`
`

`

`263
`Introduction
`
`
`tic applications.
`
`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 undissociated form (pK 9.05 at 37 °C).
`In—
`creased brain ammonia is considered to augment the synthesis of gluta-
`mate and glutamine,
`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 ins
`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 (eg. 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 (eg. 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 catabolism 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—
`
`7 of 20
`
`

`

`264
`Inherited l-Iyperammonemias
`
`11.2 Nomenclature
`
`No. Disorder
`
`MIM
`Expression
`
`23??)00
`
`11.1 Cai‘bamylphosplralc
`synthetase (CPS 1)
`
`Ornitliine transcar-
`bamylase
`(OTC, OCT)
`
`Argininosuccinate
`synthetase (AS);
`Citrullinemia
`Type 1
`
`Argininosuccinate
`lyase (AL); Argini-
`nosuccinic aciduria
`
`Arginase l
`
`N-Acetylglutamate
`synthetase (NAGS)
`
`Solute carrier fami—
`ly ? A? (SLCZ’AT);
`Lysinuric protein
`intolerance
`Solute carrier famlr
`1y 25 A15
`[SLC25A15,
`ORNTI); Hyperor—
`nitliinemia—hyper—
`ammonemia-homoe
`cilrullinuria (HHH)
`syndrome
`A'-Pyrroline—5-car—
`boxylate synthe-
`tase, PYCS
`
`Mitochondrial: liver (periportal —»
`pericenlral; all urea cycle enzymes)
`and much less intestine (enzyme not
`expressed in red or white blood cells
`or fibroblasts)
`Mitochondrial: liver and much less
`intestine. Mosaicism in heterozygote
`females (not expressed in red or white
`blood cells or fibroblasts)
`
`21 5700
`
`207300
`
`ken—QUE
`
`unknown
`
`14q11.2
`
`222200
`
`Mitochondrial membrane: Fibroblasts
`
`13ql4
`
`2389M
`
`Mitochondrial
`
`10q24.3
`
`133250
`
`Cytosolic: Liver, kidney (cortical prox-
`imal tubule); intestine, myenteric neu-
`rons, ileal and colonic muscles; CNS:
`not in astrocytes (selective neurons in
`neocortex, and rnidbrain), brainstem.
`diencephalon, cerebellar molecular
`and granular layer; eye. Fibroblasts
`Cytosolic: Liver; kidney (cortical prox-
`imal tubule); intestine, myenteric 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, kidney [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 membrane
`Liver, intestine, kidney’
`
`
`
`8 of 20
`
`

`

`Metabolic Pathway 265
`
`
`
`MIM
`Chromo—
`Expression
`Disorder
`some
`
`
`10(1233
`
`138130
`
`Glutamate dehydro- Mitochondrial
`genase 1, GT?
`binding site rnutaw
`tions (GLUDI);
`Hyperinsulinism-
`Hyperammonemia
`syndrome
`Citrullinemia type Mitochondrial membrane, liver (not
`2 (SLC25A13 gene) kidney) with secondary AS deficiency
`Citr'm deficiency
`
`7q21.3
`
`603471
`
`11.3 Metabolic Pathway
`
`
`Cytosol
`
`Mitochondrion
`
`
`
`Fig. 11.1. Metabolites: GLU, glutamate; 2—Oxo—Glut, 2—oxoglutarate; NAG. N—acetylgluta—
`mate; NH3, ammonia; Cara—P. carbamylphosphate; ORN, ornithine; CIT, citrulline; ASP,
`aspartate; ASA. argininosuccinate; FUM, fumarate', ARG. arginine; Dibnsic AA, dibasic
`amino acids
`(lysine, ornithine, arginine);
`yGlzt—SA, gammaglutamyl
`semialdehyde;
`AIPEC, pyrrolineAS-carboxylate; PRO, proline; GLN, glutamine; 0MP, orotidlne 5'—mono-
`phosphate; UMR uridine 5'-monophosphate. Enzymes: OAT, orniLhineeoxoacid amino—
`transferaso; NOS, nitric oxide synthetases; CAT, cationic amino acid transporters (y+);
`others as listed in the table in Sect. 11.2. Glutamate and Zeoxoglutarate 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 (eg. ASAT, ALAT) including
`mitochondrial aspartate synthesis which is transported to the cytosol by the aspartatel'
`glutamate carriers (citrin).
`
`erotic acid
`l0MP —- Orotidine
`
`
`
`AcCarnitine
`
`l
`UMP m1 Uridine
`Uracil
`
`ASA
`
`AL
`11.4
`N0
`_‘.\._
`NOS
`
`FUM
`
`ARE
`
`can
`
`UREA c—j I
`ARG] or 2
`“‘5
`I
`Dihasic AA
`
`CilfiT
`
`TGLU-SA W lflTF'EC 4——p PRO
`“.9
`11.7
`SLCYA?
`
`9 of 20
`
`

`

`266
`
`Inherited Hyperammonemias
`
`
`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)
`Hypo—[hyperthermia (new-
`barns)
`Muscular ltyponlhypertonus
`Ataxia, irritability. sleep dis-
`turbance (children)
`Asterixis, delusions. psychotic
`behavior (>10 years)
`Mental retardation
`Scotomas, visual hallucina»
`tions
`
`Circulatory failure
`Renal failure
`
`Hemorrhage
`Hepatomegaly
`
`Fibrosis, cirrhosis (chronic)
`Fragility of hairltrichorrhexis
`nodosa
`
`Eye
`
`Circulation
`Kidney
`Lung
`Liver
`
`Hair 8t skin
`
`Terminally: cerebral edema
`Increased glutamine releasa
`Respiratory alkalosis
`
`Papilledema
`
`Metabolic acidosis
`Metabolic acidosis
`
`Cytoiysis (ALAT, ASAT in-
`creased]; reduced protein
`synthesis: coagulation de—
`feet
`
`Low urea (P) (not always)
`Arginine deficiency
`
`11.4 Signs and Symptoms
`(Common to all urea cycle disorders except argininemia)
`
`Central nervous system
`Metabolic alkalosis
`Growth retardation, osteo—
`porosis, Vit 1312, Zn defi-
`ciency
`
`(including iatrogenic)
`
`10 of 20
`
`

`

`267
`Signs and Symptoms
`
`
`l Disease “Specific” Signs and Symptoms
`
`Disease
`
`Choiestatic jaundice, hepatic steatosis and siderosis
`Citrullinemia type 2
`Hyperammonemia not obligate
`(decreased AS acitivity,
`citrin deficiency]
`Argininosuccinic aciduria
`
`Argininemia
`
`Lysinuric protein
`intolerance
`
`Facies with epicanthic 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, atheto—
`sis, dysarthria
`Lungs: proteinosis, interstitial pneumonia
`(white lung disease)
`Hematology: hemolysis (increased LDH, ferritin), lympho—
`liistiocytic antop‘nagocytosis
`Kidney: glomerulonephritis
`Bones: osteopenia
`immunity: decreased response to varicella immunization
`Neurological: pyramidal signs in absence of decerebration
`(C. Dionisi—Vici, personal communication)
`Hematology: factor VII Br X deficiency
`Eye: cataracts
`Bone and skin: hyperlaxicity and increased skin elasticity
`Hyperammonemia (mild) only preprandial!
`
`
`
`HHI—I syndrome
`
`Pyrroline 5 carboxylate
`synthetase deficiency
`(few patients. possibly
`ascertainment bias):
`Glutamate dehydrogenase
`mutations
`
`Fasting hypoglycemias noticed mostly in infants or later
`(hyperinsulinism)
`Growth failure, variable mental retardation
`
`
`
`11 of 20
`
`

`

`268
`
`Inherited Hyperammonemias
`
`11.5 Reference Values
`
`
`
`Remarks
`Adult
`2~14 years Adult
`1—12
`4 months
`<28 clays
`Analyte
`women
`men
`months
`mature
`
`
`Ammonia (P) en- <80
`zymatic [meUD
`
`Ammonia (P)
`microdiffusion
`(umolfl)
`Amino acids (P)
`(umolll)
`
`Arginine
`Arginino-
`succinate
`
`1844
`
`1?—68
`
`Range: sam—
`pling >15 h
`after end of last
`feed (p.p.: 1~
`3.5 h after feed)
`77—165 (65—200) 417190 (60—190)
`<2
`
`10-65
`
`Citrulline
`
`17—41 (13—45}
`
`11-32 (8—36)
`
`10—30
`
`Ornitlline
`Lysine
`
`Glutamine
`
`Alanine
`
`Proline
`
`55—120 (55-420) 28—150 (40—125)
`110~290
`60-230 (75—235)
`[1 15—330)
`380—660
`(3804'10)
`200—490
`(185—645)
`120—260
`(130-310)
`
`200—720
`[265-650]
`110—480
`{1907550)
`64—272
`(120—260)
`
`No reference values can be given for enzyme assays, since the results depend upon the method used. The reference:
`ranges must be obtained from file testing laboratory which should be contacted for correct sampling and transport
`conditions.
`
`pestprandially
`30-60 umolil high-
`er depending on
`time and N load
`
`6
`
`In contrast to
`other amino acids
`
`Citrulline is higher
`pre- than post-
`prandially
`
`_
`
`'
`
`30—100
`135—260
`
`20—90
`115—250
`
`550~830
`
`440—810
`
`240—600
`
`200—550
`
`100—380
`
`?07270
`
`10-110
`45714-5
`
`50—470
`
`100—3 10
`
`50—190
`
`0.2—3.8
`
`03870.44
`
`'-
`
`Amino acids (U)
`(fractional tubular
`reabsorption, %)
`Lysine
`Ornithine &
`Arginine
`Orotate (mmol!
`0335—026 Increase in 6 h
`mol creatinine)
`urine after protein _
`load of 1 gikg:
`<05? rnmolfmol cr
`
`12 of 20
`
`

`

`—
`—
`
`(T )
`T
`
`on: def.
`Citrulline—
`mia I
`Citrulline-
`mia II
`
`Argininor
`succinic
`aciduria
`
`Argininemia
`
`NAGS def.
`
`—
`
`T—TT
`T-Tl
`
`.
`
`Exclude
`Arg load
`
`Exclude
`canned
`foodlmilk
`
`Hypo-
`glycemia
`
`
`
`Pathological Valueleifferential Diagnosis 269
`
`11.6 Pathological Values/Differential Diagnosis
`
`The extent of increase varies widely depending on mutation and especially
`internal and external nitrogen load.
`
`Arg
`ASA Cit
`OROT Homo- Pro
`Glnl
`Orn (P)
`(P)
`(U)
`citrul-
`(P)
`NH.“L
`line (U)
`
`CPS 1 def.
`
`
`
`LPI
`HHH
`s ndrome
`Y
`T“
`PYCS def.
`Hyperinsu— T—Tb
`linismehy-
`perammone—
`mia (HIHA)
`syndrome
`
`‘ Only fasting not postprandial.
`b Unchanged by protein load or restriction. Non-responsiveness to benzoate or phenylbuty'rate treatment.
`FTR, fractional tubular reabsorption of dibasic amino acids compared to creatinine; n. normal; nd, not detectable;
`ASA, argininosuccinate; OROT. orotic acid.
`
`13 of 20
`
`

`

`2TB
`
`Inherited Hyperaminonemias
`
`11.7 Loading Tests
`
`
`
`
`
`
`
`
`
`Age (years)
`
`14
`
`15
`
`16
`
`1?
`
`.mump
`
`9C
`
`)
`
`The goal of loading tests is to unmask a functional defect where there is re—
`sidual activity.
`In heterozygotic females with OTC deficiency (it—chromo—
`somal) it can only be used to confirm, but never to exclude a carrier status
`because of mosaicism {Lyon hypothesis) which might be strongly skewed
`towards the wild type and thus overlap normal values.
`Protein loading tests bear the risk of eliciting hyperammonemia. 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 carbarnyl-
`phosphate load, due either to endogenous protein breakdown or exogenous
`protein and nucleotides or to tissue regeneration (CPSII). 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
`
`challenge is shown (adapted from Burlina et al. [10])
`
`'63
`Ec
`‘5muu
`
`U T
`
`)E2oEE
`
`Fig. 31.2. Basal orotate decreases with age:
`
`the variation of orotate after allopurinol
`
`14 of 20
`
`

`

`
`Loading Tests
`271
`
`of a single dose of allopurinol {children >6 y: 100 mg, 6—10y: 200 mg,
`>10 y: 300 mg).
`The upper limits or maximal orotate excretion after a load is [10]: 6 mo
`-6 y: 13.0 mmolimol Cr; 6—10 y: 9.3 mmol/rnol Cr; 10—]? y: 10.2 mmol.Ir
`mol Cr [see Fig. 11.7). For orotidine,
`the limit of decision is 8 mmol/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 menstruation. 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
`andlor 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 g/kg bw load, is 0.7 mmol/mol creatinine.
`
`15 of 20
`
`

`

`
`272
`Inherited Hyperammonemias
`
`11.8 Diagnostic Flow Chart
`
`
`
`Algininemia [Nginasel Deficiency)
`or Arginine-HCI toad
`
`Argininosuccinic Aciduria
`iArgininosuccinate Lyase deficiency]
`
`Citrullinemia
`[Argininosuccinate Synthetase deficiency)
`
`Pyruvate Carboxylase deficiency
`Renal insufficiency
`
`Homociiiullinuria-Hyperornithinemia—
`Hyperammonemia {HHH} Syndrome
`
`
`
`Lysinuric Protein intolerance
`
`
`
`
`
`U—Orotate
`incr.
`
`
`pl Pyrroline 5 Carboxylate Synthetase deficiency
`
`I
`
`Ornithine Transcarbarnylase deficiency
`
`Fig. 11.3. Top d
`rithm {or the di
`
`diagnosis of by
`nemia based on
`of amino acids
`nine {P+U), U-
`organic acids. 3
`acylcarnitines (
`chain fatty acid
`ma. Abbreviatic
`increased; dear.
`Homocit., hom(
`FTR, fractional
`absorption (1-[
`nine *U—amino
`(U-creatinine -
`acid}]; MCFA,1
`chain fatty acic‘
`
`l
`
`
`
`
`
`breviations as i
`
`P—Arg i ni ne
`in cr.
`
`in
`
`U-Orotate
`incr.
`
`P-Pro 84 Orn,
`
`P-Ginfl-li-I;
`< 1.6
`
`de cr.
`CPS, MAGS
`Liver
`
`
`
`
`
`Organic Acidurias including
`Valproate or Pivalateester treatment
`
`Defects of Fattyr Acid Beta-oxidation
`or at Camitine metabolism
`
`Transient Hyperammonomenia of the
`Premature, open Ductus vennsus;
`+ Hypoglycemia (Hyperinsuiinism):
`Glutamate Dehydrogenase mutation
`
`
`
`
`
`
`Carbamoylphosphate Synthetase (CPS) or
`N-Acelylglutamate Synthetase (MAGS)
`deficiency
`
`Hepatic insufficiency, Blind loop syndrome or
`neurogenic bladder infected with Urease cont.
`_ _ _ _ . . . -b— bacteria; Asparaginase lhetapy; Malnuttition
`lintrauterine, postnatal
`or iatrogenic Arginine deficiency:
`
`16 of 20
`
`

`

`
`
`Specimen Collection 273
`
`11.9 Specimen Collection
`
`
`Material
`Precondition
`Pitfalls
`Handling
`
`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 “CJ not later
`than 15 min after sampling,
`plasma decanted and frozen
`at —20 c'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)
`
`':' Amino acids
`
`Plasma
`
`Centrifuge within 15 min
`
`phenylbutyrate
`
`Benzoate,
`. phenylace—
`tare, phenyl-
`bulyrate
`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 hemnly-
`sis
`
`High ALT or yGT increase
`ammonia
`
`False low values are found
`with pyruvate concentra-
`tions >230 uM
`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, phenylacetale or
`
`_ Drotate
`
`Urine (spot)
`
`Urine 5-10
`ml
`
`Lithium
`
`heparinate
`plasma
`
`24 hour
`urine collec—
`tion
`
`Deproteinize with sulfosae
`licylic acid
`Keep frozen and at pH 2 for
`accurate glutamine results
`Freeze rapidly to avoid bac—
`terial interference
`Store at —20°C
`
`Cumulativer frozen
`
`'
`
`17 of 20
`
`

`

`
`
`Fibroblasts
`
`
`
`11.10 Prenatal Diagnosis
`
`NAGS def.
`CPS def.
`
`OTC def.
`
`2T4 Inherited l-lyperammonemias
`
`Test
`Precondition
`Material
`Handling
`Pitfalls
`
`NAGS, CPS,
`Specify appr. protein intake
`Liver biopsy Blot and freeze in liquid
`Activity dependent on pro-
`OTC
`in the last 3 days
`(30 mg)
`nitrogen. store immediately
`tein intake. Only optimized
`at —80 DC, send Willi ample
`NAGS assays (with arginine)
`dry ice
`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 ornitbine incor—
`poration assay
`
`
`nase deficiency, LPI, HHH and HIHA syndrome, not in NAGS deficiency.
`
`The laboratory should be contacted before coilectingisending specimens.
`
`DNA
`Protein (activity)
`Metabolite
`Comment
`(amniotic fluid)
`
`Gene (RFLP/mutation)
`Not feasible
`Not feasible
`Not in all instances, only Fetal livera
`known mutations
`Many private mutations
`
`Not feasible
`Not informative
`
`Fetal liver“
`
`Not informative
`
`Not predictive in female
`fetus
`
`AS (citruir
`Many private mutations
`Amniocytesi‘chorionic villi Not informative
`linemia 1)
`AL (ASA—ui'ia) Many private mutations Amniocytesichorionic villi ASA (AF)
`
`Argininemia 1
`LPI
`HHH
`
`Fetal red celis
`Amniocytesi’chorionic villi
`Amniocytes
`
`Enzyme activity and ASA
`concentration should both
`be assayed
`
`Assay in cultured. not fro-
`zen sample
`
`a'
`
`
`3 Varies with gestational age. intrauterine liver biopsies (week 1671?) 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—
`
`18 of 20
`
`

`

`2?5
`Summary/{Comments
`
`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 hyperammo—
`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.
`- Stop per oral protein supply or i.v. amino acid preparations.
`- Glucose 8-10 mglkg per minute i.v. (with insulin if needed); check plasw
`ma lactate 2 hours after start!
`Arginine HCl i.v. 2 mmolikg b.w. as priming dose in 2 hours and then
`2 mmoll‘kg per 24 h.
`Carnitine i.v.
`5t] mmolflcg 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 Summary/Comments
`
`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 timolfL) 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 tlyperarnmonemias
`
`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, Tuchruan M. Proceedings of a consensus conference for the manage-
`ment of patients with urea cycle disorders. journal of Pediatrics 2001; 138(1): 36-
`510.
`
`transferase deficiency:
`. Bachmann C, Ornithine carbanioyi
`problems. J Inherit Mel‘er 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-
`ular Brain Research 1999; 70(2): 231—241.
`
`findings, models and
`
`. Aral B, Schlenzig IS, Liu S, Kamoun P. DatabaSe cloning human delta l-pyrroline-
`S-carboxylate synthetase (PSCS) cDNA: a bifunctional enzyme catalyzing the first 2
`steps in proline biosynlhesis. Comptes Readies de l’ Acade’mie des Sciences. Serie ill,
`Sciences de La Vie 1996; 319(3): Uleli'B
`. Stanley CA, Fang l, Kutyna K et al. Molecular basis and characterization of the hy—
`perinsulinisniihypcrammonemia syndrome: predominance of mutations in e