yop
`
`Laboratory
`Diagnosis
`of Metabolic
`Diseases
`
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`Editors
`
`@5
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` LUPIN
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`Physician’s Guide
`to the Laboratory Diagnosis
`of Metabolic Diseases
`
`with CD-ROM
`
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`ew WN)
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`eh Saitoh
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`elay and mistakes in the diagnosisofinherited
`metabolic diseases may have devastating con-
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`ae
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`of Adobe Acrobat Readerat: «
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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
`
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` LEC Cal
`
`Nenad Blau
`Division of Clinical Chemistry
`and Biochemistry
`) University Children’s Hospital
`Steinwiesstrasse 75
`8032 Ziirich
`Switzerland
`e-mail: blau@access.unizh.ch
`
`Milan E. Blaskovics
`3639 Amesbury Road
`Los Angeles, CA 90027
`USA
`e-mail: blaskk@earthlink.net
`
`Marinus Duran
`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 & 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
`© 1996 Chapman & Hall
`
`Library of Congress Cataloging-in-Publication Data
`Physician's guide to the laboratory diagnosis of metabolic diseases /[edited by] N. Blau
`..
`fet al.].- 2nd ed.
`ps cm.
`Includes bibliographical references and index.
`ISBN 3-540-42542-X (hd.: alk.paper)
`L. Metabolism - Disorders - Diagnosis - Handbooks, manuals, etc. 2. Metabolism,
`Inborn errors of - Diagnosis - Handbooks, manuals, etc. 3. Diagnosis,
`Laboratory — Handbooks, manuals, etc. I. Blau, N. (Nenad), 1946 -
`[DNLM: 1. Metabolism, Inborn Errors - diagnosis. 2. Diagnosis, Differential.
`3, Laboratory Techniques and Procedures. WD 205 P578 2002]
`RB147.P476 2002
`616.3'°9075-de21
`2002021642
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`
`Inherited Hyperammonemias
`CLAUDE BACHMANN
`
`11.1
`Introduction
`Hyperammonemia (systemic venous or arterial plasma ammonia >80 in
`newborns or >50 pmol/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. Amongthe 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 1:30000 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 Syndromeis dis-
`appearing.
`The actual enzymeactivity 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 endogenousbal-
`ance or imbalance between protein synthesis and catabolism. The clinical
`heterogeneity of the disorders and any prognostic predictions will
`thus
`only partly depend onthe genetic backgroundif residual protein is present.
`“Mild” leaky variants (unstable enzymes in vitro or residual enzyme activ-
`
`261
`
`~
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`ovaeinLURI
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`
`262 Inherited Hyperammonemias
`
`the hyperammonemia, Because of the expertise and experience
`
`ity) may lead to severe hyperammonemiccrises if protein catabolism pre-
`dominates (e.g. major weight loss in newborns, viral infections etc.).
`Hyperammonemia is toxic to the brain. It exerts reversible (mostly sero-
`toninergic) and irreversible effects. Blood ammonia (NH3) concentrations
`exceeding 180 «mol/L, or a coma lasting more than 2-3 days appear 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 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 madeas rapidly as possible and not later than 12-24 h
`in orderto initiate specific treatment. Among the non-artefactual hyperam-
`monemias, 2/3 are due to urea cycle defects, and 1/3 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 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 re-
`striction of natural nutrients or due to the enzyme block, These would be
`arginine orcitrulline, 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 termside 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 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
`
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`
`
`263
`
`Introduction
`
`needed in managing patients with UCD’s, the transfer of patients into a
`center with experiencedclinicians and a laboratoryis recommended.
`Brain ammonia toxicity depends upon the level of blood ammonia,
`which crosses membranesin 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 in-
`creased serotonin secretion [2]. The increased glutamate released by neu-
`rons and its decreased re-uptake is probably exotoxic. The accumulation of
`glutaminein astrocytes has been shown, under extreme conditions, to lead
`to astrocytic swelling which may be responsible for terminal brain edema.
`The mechanismsaffecting 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 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 uponthe arginine in the cyto-
`sol originating from protein catabolism, urea cycle synthesis, and therapeu-
`tic applications.
`
`
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`264 Inherited Hyperammonemias
`
`*
`
`’
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`synthetase (CPS 1)
`
`Xp21.1
`
`311250
`
`215700
`
`11.5 Arginase |
`
`6q23
`
`207800
`
`11.2 Nomenclature
`
`No. Disorder
`Expression
`Chrome-
`MIM
`some
`
`11.1 Carbamylphosphate Mitochondrial: liver (periportal >
`2q35
`237300
`pericentral; all urea cycle enzymes)
`and much less intestine (enzyme not
`expressed in red or white blood cells
`or fibroblasts)
`11.2 Ornithine transcar- Mitochondrial: liver and muchless
`intestine. Mosaicism in heterozygote
`bamylase
`(OTC, OCT)
`females (not expressed in red or white
`blood cells or fibroblasts)
`11.3 Argininosuccinate
`Cytosolic: Liver, kidney (cortical prox- 9q34
`imal tubule); intestine, myenteric neu-
`synthetase (AS);
`Citrullinemia
`rons, ileal and colonic muscles; CNS:
`Type |
`not in astrocytes (selective neurons in
`neocortex, and midbrain), brainstem,
`diencephalon,cerebellar molecular
`and granularlayer; eye. Fibroblasts
`Cytosolic: Liver; kidney (cortical prox- 7cen-ql1.2 207900
`11.4 Argininosuccinate
`imal tubule); intestine, myenteric neu-
`lyase (AL); Argini-
`nosuccinic aciduria 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
`11.6 N-Acetylglutamate Mitochondrial: liver > intestine
`synthetase (NAGS) > spleen. Intestine (enzyme not ex-
`pressed in red or white blood cells or
`fibroblasts)
`Solute carrier fami- Basolateral membrane
`ly 7 A7 (SLC7A7);
`Liver, intestine, kidney
`Lysinuric protein
`intolerance
`11.8 Solute carrier fami- Mitochondrial membrane: Fibroblasts
`ly 25 ALS
`(SLC25.A15,
`ORNT1); Hyperor-
`nithinemia-hyper-
`ammonemia-homo-
`citrullinuria (HHH)
`syndrome
`11.9 A‘-Pyrroline-5-car- Mitochondrial
`boxylate synthe-
`tase, PYCS
`
`11.7.
`
`unknown
`
`237310
`
`14qil.2
`
`222700
`
`13q14
`
`238970
`
`10q24.3
`
`138250
`
`
`
`8 of 20
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`

`

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`
`
`
`No. Disorder
`MIM
`Chromo-
`Expression
`some
`
`
`Metabolic Pathway
`
`265
`
`10q23.3
`
`138130
`
`11.10 Glutamate dehydro- Mitochondrial
`genase 1, GTP
`binding site muta-
`tions (GLUD1);
`Hyperinsulinism-
`Hyperammonemia
`syndrome
`11.11 Citrullinemia type Mitochondrial membrane,liver (not
`2 (SLC25A13 gene) kidney) with secondary AS deficiency
`Citrin deficiency
`
`—_7q21.3
`
`603471
`
`SPENTeT
`ten
`
`
`
`;
`je
`
`TEATS
`arithee
`
`11.3. Metabolic Pathway
`
` Cytosol
`
`
`
`
`GLUD1
`
`11.10
`NAG9 Wd
`
`NAGS
`
`11.6
`
`
`reels
`AcCoA
`ORN
`4
`
`AcCarnitine
`
`OAT
`
`
`
`¥GLU-SA “byes” A,P.sC «—» PRO
`me SLC7A7
`
`
`11.7
`
`oa
`
`FUM
`
`AL
`M4
`no
`h
`<~——+—._ ARG
`UREA 7
`
`A
`RGI or 2
`% |
`cAT
`Dibasic AA
`
`ORN
`
`ORNT1
`118
`
`GLU
`
`GLN
`
`Mitochondrion
`
`Glutamine synthetase
`
`NH;
`CPS
`
`Carb.-P
`
`orc
`
`CIT
`
`CT
`
`
`
`
`Orotic acid
`“
`OMP —» Orotidine
`
`UMP ~, Uridine
`Uracil
`
`
`
`Fig. 11.1. Metabolites: GLU, glutamate; 2-Oxo-Glut, 2-oxoglutarate; NAG, N-acetylgluta-
`mate; NH;, ammonia; Carb.-P, carbamylphosphate; ORN, ornithine; CIT, citrulline; ASP,
`aspartate; ASA, argininosuccinate; FUM, fumarate; ARG, arginine; Dibasic AA, dibasic
`amino acids
`(lysine, ornithine, arginine); yGlu-SA, gammaglutamyl
`semialdehyde;
`AIP5C, pyrroline-5-carboxylate; PRO, proline; GLN, glutamine; OMP, orotidine 5’-mono-
`phosphate; UMP, uridine 5’-monophosphate. Enzymes: OAT, ornithine-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 aspartate/
`glutamatecarriers (citrin).
`
`
`
`
`9 of 20
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`

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

`

`267
`Signs and Symptoms
`
`
`H Disease “Specific” Signs and Symptoms
`
`Disease
`
`Cholestatic jaundice, hepatic steatosis and siderosis
`Citrullinemia type 2
`Hyperammonemia not obligate
`(decreased AS acitivity,
`citrin deficiency)
`Argininosuccinic aciduria
`
`Facies with epicanthic fold, depressed nasal bridge
`(saddle nose) as newborn
`Nervous system:increasedirritability 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-
`histiocytic autophagocytosis
`Kidney: glomerulonephritis
`Bones: osteopenia
`Immunity: decreased responseto varicella immunization
`Neurological: pyramidal signs in absence of decerebration
`(C. Dionisi-Vici, personal communication)
`Hematology: factor VII & X deficiency
`Eye: cataracts
`Bone and skin: hyperlaxicity and increased skin elasticity
`Hyperammonemia (mild) only preprandial!
`
`Argininemia
`
`Lysinuric protein
`intolerance
`
`HHHsyndrome
`
`Pyrroline 5 carboxylate
`synthetase deficiency
`(few patients, possibly
`ascertainment bias):
`Glutamate dehydrogenase
`mutations
`
`Fasting hypoglycemias noticed mostly in infants orlater
`(hyperinsulinism)
`Growthfailure, variable mental retardation
`
`
`
`
`
`
`11 of 20
`
`11 of 20
`
`

`

`Inherited Hyperammonemias
`268
`
`
`
`
`11.5 Reference Values
`
`
`
`
`
`
`
`Analyte
`<28 days
`4 months
`1-12
`2-14 years Adult
`Adult
`Remarks
`mature
`months
`men
`women
` Ammonia (P) en- <80
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`0.035-0.26 Increase in 6h
`
`
`urine after protein
`
`
`load of 1 g/kg:
`
`
`<0.7 mmol/mol 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 the testing laboratory which should be contacted for correct sampling and transport”
`conditions.
`3
`
`
`55-120 (55-420) 28-150 (40-125)
`110-290
`60-230 (75-275)
`(115-330)
`380-660
`(380-710)
`200-490
`(185-645)
`120-260
`(130-310)
`
`zymatic (j1mol/L)
`
`Ammonia (P)
`microdiffusion
`(mol/l)
`Amino acids (P)
`(umol/I)
`
`Arginine
`Arginino-
`succinate
`Citrulline
`
`Ornithine
`Lysine
`
`Glutamine
`
`Alanine
`
`Proline
`
`Amino acids (U)
`(fractional tubular
`reabsorption, %)
`Lysine
`Ornithine &
`Arginine
`Orotate (mmol/
`mol creatinine)
`
`>95%
`
`0.7-3.3
`
`
`
`
`
`<50
`
`21-95
`
`18-74
`
`17-68
`
`21-71
`
`19-63
`
`Range: sam-
`pling >3.5h
`after end of last
`feed (p.p.: 1-
`3.5h after feed)
`77-165 (65-200) 41-190 (60-190)
`<2
`
`10-65
`
`35-140
`
`25-125
`
`17-41 (13-45)
`
`11-32 (8-36)
`
`10-30
`
`20-55
`
`15-55
`
`10-110
`45-145
`
`60-470
`
`100-310
`
`50-190
`
`200-720
`(265-650)
`110-480
`(190-550)
`64-272
`(120-260)
`
`30-100
`135-260
`
`20-90
`115-250
`
`550-830
`
`440-810
`
`240-600
`
`200-550
`
`100-380
`
`70-270
`
`0.2-3.8
`
`0,08-0.44
`
`postprandially
`30-60 pmol/! high-
`er depending on
`time and N load
`
`In contrast to
`other amino acids
`Citrulline is higher
`pre- than post-
`prandially
`
`12 of 20
`
`12 of 20
`
`

`

`
`
`Pathological Values/Differential Diagnosis
`
`269
`
`11.6 Pathological Values/Differential Diagnosis
`
`No.
`
`The extent of increase varies widely depending on mutation and especially
`internal and external nitrogen load.
`
`NH,g
`Arg
`ASA Cit
`OROT Homo- Pro
`FTR
`Gln/
`Orn (P)
`(P)
`(UU)
`(Pfu)
`(Uv)
`citrul-
`(P)
`DibAA NH?
`line (U)
`
`lll CPS ildef.
` f-TT
`{-n
`nd
`l-n
`{-n
`n-f
`no
`>1.6
`(tT)
`11.2 OTC def.
`n-TT
`l
`nd
`|-n
`t-TT
`n-f
`on
`>1.6
`
`11.3 Citrulline-=fT Ll nd ttt t-TT T n-f n
`
`
`
`
`
`
`mia I
`Citrulline-
`mia IT
`
`Tf
`
`n-T
`
`Tf
`
`tT
`
`n
`
`T-TT
`
`I
`
`ttt
`
`=T
`
`n-ff
`
`>1.6
`
`11.4 Arginino-
`succinic
`aciduria
` Argininemia n-T
`
`11.5
`
`Tit
`
`T
`
`T
`
`t-TT
`
`1
`
`>1.6
`
`
`
`Tt
`
`Exclude
`Arg load
`11.6 NAGS def. nd=|-n{-TT l {-n nd
`
`
`
`
`17 LPI
`fit
`|
`ond
`f
`1-11
`t
`ou
`|
`11.8 HHH
`T-TT
`n
`n
`T
`{ (not
`syndrome
`in neo-
`nates)
`
`
`
`7?
`f-f®
`
`n
`
`nd
`
`|-n
`on
`
`n
`
`|
`
`PYCS def.
`11.9
`11.10 Hyperinsu-
`linism-hy-
`perammone-
`mia (HIHA)
`syndrome
`
`* Only fasting not postprandial,
`e Unchanged by protein load orrestriction. Non-responsiveness to benzoate or phenylbutyrate treatment.
`FTR, fractional tubular reabsorption of dibasic amino acids comparedto creatinine; n, normal; nd, not detectable;
`ASA, argininosuccinate; OROT, orotic acid.
`
`Exclude
`canned
`food/milk
`
`<1.6 DD
`Bypass
`of liver
`
`Hypo-
`glycemia
`
`
`
`
`
`13 of 20
`
`13 of 20
`
`

`

`
`
`270~—sInherited Hyperammonemias
`
`
`
`Cnesaadanonianaebasoncaer
`
`11.7 Loading Tests
`
`The goal of loading tests is to unmask a functional defect where there is re-
`sidual activity.
`In heterozygotic females with OTC deficiency (x-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 intake regime can be calculated prior to the load. Post-
`prandial hyperammonemialevels 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 carbamyl-
`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
`
`q 12,
`
`a
`o_o a
`--O--
`“0
`-00—40
`
`O27,
`13
`14
`#45
`146
`és
`
`i
`3
`4
`4
`=
`2
`a
`2
`4
`3a
`
`eeso
`—- © Basal
` & Peak
`es
`
`a
`k
`ee
`*
`-
`.
`;
`ips
`
`.
`a
`:
`+A
`__
`o
`
`
`
`100
`
`10
`
`1
`
`0.1
`
`oO
`
`&=
`
`= 3§
`
`2
`£
`3
`=
`E
`a
`2
`2
`o
`
`Age (years)
`
`the variation of orotate after allopurinol
`Fig. 11.2. Basal orotate decreases with age:
`challenge is shown (adapted fromBurlina etal. [10])
`
`
`
`14 of 20
`
`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 y: 13.0 mmol/mol Cr; 6-10 y: 9.3 mmol/mol Cr; 10-17 y: 10.2 mmol/
`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 inaself
`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 allopurinoltest, 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
`and/or 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 HPLCafter alkalinisation.
`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/molcreatinine.
`
`(Inrrr
`
`15 of 20
`
`15 of 20
`
`

`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`P-Pro & Orn,
`
`
`
`
`v
`
`{pyrroline § Carboxylate Synthetase deficiency
`
`
`Organic Acidurias including
`Valproate or Pivalateester treatment
`
`
`
`Defects of Fatty Acid Beta-oxidation
`ar of Carnitine metabolism
`
`
`
`
`
`
`
`
`
`
`
`
`Transient Hyperammonomenia of the
` molar
`Premature, open Ductus venosus;
`
`
`P-GIn/NHF
`+ Hypoglycemia (Hyperinsulinism):
`"
`
`<1.6
`|
`Glutamate Dehydrogenase mutation
`
`
`
`
`deficiency
`
`decr.
`Carbamoylphosphate Synthetase (CPS) or
`
`N-Acetylglutamate Synthetase (NAGS)
`CPS, NAGS
`
`
`Liver,
`
`
`
`Hepatic Insufficiency,Blind loop syndrome or
`
`neurogenic bladderinfected with Urease cont.
`aaa| bacteria; Asparaginase therapy; Malnutrition
`(intrauterine, postnatal
`or iatrogenic Arginine deficiency)
`
`
`
`
`
`
`
`
`
`272
`~—‘Inherited Hyperammonemias
`
`11.8 Diagnostic Flow Chart
`
`P-Arginine
`incr.
`
`
`¥
`
`-
`
`
`
`
`Argininemia (Arginase | Deficiency)
`or Arginine-HCl load
`
`| N
`
`N
`
`
`at
`
`Argininosuccinic Aciduria
`(Argininosuccinate Lyase deficiency)
`
`¥
`
`
`U-Orotate
`
`incr.
`
`
`N
`eee eee Noll. 7
`
`U-Orotate
`iner.
`
`
`
`
`
`Citrullinemia
`(Argininosuccinate Synthetase deficiency)
`
`Pyruvate Carboxylase deficiency
`Renalinsufficiency
`
`Homocitrullinuria-Hyperornithinemia-
`Hyperammonemia (HHH) Syndrome
`| Artefact (milk processing) or neonatal HHH
`Lysinuric Protein Intolerance
`
`Ornithine Transcarbamylase deficiency
`
`Fig. 11.3. Top d
`rithm for the di
`
`diagnosis of hy.
`nemia based on
`of amino acids
`nine (P+U), U-
`organic acids, ¢
`acylcarnitines ¢
`chain fatty acid
`ma. Abbreviatic
`increased; decr.
`Homocit., hom¢
`FTR, fractional
`absorption (1-|
`nine * U-amino
`(U-creatinine —
`acid)]; MCFA,1
`chain fatty acid
`breviations as i
`
`16 of 20
`
`16 of 20
`
`

`

`
`
` Specimen Collection
`273
`
`
`
`11.9 Specimen Collection
`
`
`
`Precondition
`Material
`Handling Pitfalls
`
`
`
`
`
`
`
`
`
`
`At least 4h after end of the
`last meal or stopping intra-
`venous AA supply from a
`central vein or artery
`
`EDTA blood
`
`Immediately put on ice and Capillary sampling increases
`centrifuge (4°C) notlater
`ammonia concentrations
`than 15 min after sampling,
`plasma decanted and frozen
`at -20°C
`
`Stable at -20°C up to 48h Muscle hyperactivity liber-
`ates ammonia as does a
`prolonged garrot or hemoly-
`sis
`
`High ALT or yGT increase
`Avoid any dilution of sam-
`ple before assay (as distilled ammonia
`water, or other acid solution
`contain ammonia trapped
`from air)
`
`Plasma
`
`Centrifuge within 15 min
`
`Urine 5-10
`ml
`
`Use HPLC methodoriso-
`tope dilution only.
`Orotidine seems to have a
`
`
`
`
`
`
`
`
`
`Benzoate,
`. phenylace-
`tate, phenyl-
`butyrate
`24 hour
`Hippurate,|No change of dose during
`Cumulatively frozen
`For checking therapeutic
`urine collec-
`phenylacetyl-
`the last 5 days
`compliance
`tion
`Indicate total 24 h urine vol-
`glutamine,
`phenylacetate
`
`~
`
`Lithium
`
`heparinate
`plasma
`
`limited stability
`
`ume and total daily dose of
`benzoate, phenylacetate or
`
`phenylbutyrate
`
`
`
`17 of 20
`
`False low values are found
`with pyruvate concentra-
`tions >200 LM
`False high values in micro-
`diffusion methods by
`osmotic hemolysis and
`glutamine breakdown
`Contamination by intracel-
`lular fluid (capillary blood).
`Urine (spot)
`Deproteinize with sulfosa-|Glutamine release by muscle
`licylic acid
`activity (e.g. seizures)
`Keep frozen and at pH 2 for
`accurate glutamine results
`Freeze rapidly to avoid bac-
`terial interference
`Store at -20°C
`
`
`
`
`
`
`
`
`
`“ Amino acids
`
`_ Orotate
`
`
`
`17 of 20
`
`

`

`274
`
`Inherited Hyperam monemias
`
`
`Pitfalls
`Material
`Handling
`Test
`Precondition
`
`NAGS, CPS,
`OTC
`
`Specify appr. protein intake
`in the last 3 days
`
`Liver biopsy
`(30 mg)
`
`Blot and freeze in liquid
`nitrogen, store immediately
`at -80°C, send with ample
`dry ice
`
`HHH,
`ORNT1
`
`Fibroblasts
`
`Activity dependent on pro-
`tein intake. Only optimized
`NAGS assays (with arginine)
`should be used.
`Gene expressionof the urea
`cycle enzymes in liver is
`down regulated to 10% by
`lipopolysaccharides
`Frozen material cannot be
`used for ornithine incor-
`poration assay
`
`
` 4
`
`
`
`
`
`11.10 Prenatal Diagnosis
`
`The laboratory should be contacted before collecting/sending specimens.
`
`Comment
`Metabolite
`DNA
`Protein (activity)
`(amniotic fluid)
`
`NAGSdef.
`CPS def.
`
`OTC def.
`
`AS (citrul-
`linemia 1)
`AL (ASA-uria)
`
`Argininemia |
`LPI
`HHH
`
`Gene (RELP/mutation)
`Not feasible
`Not inall instances, only
`known mutations
`Manyprivate mutations
`
`Notfeasible
`Fetal liver *
`
`Fetal liver*
`
`Not feasible
`Not informative
`
`Not informative
`
`Not predictive in female
`fetus
`
`Manyprivate mutations
`
`Amniocytes/chorionic villi Not informative
`
`Many private mutations
`
`Amniocytes/chorionic villi ASA (AF)
`
`Fetal red cells
`Amniocytes/chorionic villi
`Ammniocytes
`
`Enzyme activity and ASA
`concentration should both
`be assayed
`
`Assay in cultured, not fro-
`zen sample
`
`(week 16-17) have been performed, but are not withoutrisk of
`® Varies with gestational age. Intrauterine liver biopsies
`lly required (instability of enzyme and transport cond
`fetal liver hemorrhage. A simultaneous control sample is usua
`tions!).
`
`°
`
`4 s
`
`sAiha
`
`11.11 DNA Analysis
`Can be performed in some CPS deficiency (microsatellite analysis) most
`OTC deficiencies (RFLP rarely mutation analysis asfirst step), AS, AL, argi-
`nase deficiency, LPI], HHH and HIHA syndrome, not in NAGSdeficiency.
`
`
`
`18 of 20
`
`18 of 20
`
`

`

`275
`Summary/Comments
`
`The performing laboratory sho