Pyridoxine-dependent seizures in Dutch patients: Diagnosis by elevated urinary alpha-aminoadipic semialdehyde levels

Article (PDF Available)inArchives of Disease in Childhood 92(8):687-9 · September 2007with28 Reads
DOI: 10.1136/adc.2006.103192 · Source: PubMed
Pyridoxine-dependent seizures (PDS) is a rare, autosomal recessively inherited disorder. Recently alpha-aminoadipic semialdehyde (alpha-AASA) dehydrogenase deficiency was identified as a major cause of PDS, which causes accumulation of both alpha-AASA and pipecolic acid (PA) in body fluids. We studied urinary and plasma alpha-AASA and PA levels in 12 Dutch clinically diagnosed patients with PDS. Alpha-AASA was elevated in both urine and plasma in 10 patients. In these patients plasma PA levels were also elevated but urinary PA levels were normal. In all patients with clinically definite PDS, and in most patients with probable or possible PDS, the clinical diagnosis of PDS could be confirmed at the metabolite level. Non-invasive urinary screening for alpha-AASA accumulation provides a reliable tool to diagnose PDS and can save these patients from the classical and potentially dangerous pyridoxine withdrawal test to prove PDS.
Pyridoxine-dependent seizures in Dutch patients: diagnosis by
elevated urinary alpha-aminoadipic semialdehyde levels
Levinus A Bok, Eduard Struys, Michel A A P Willemsen, Jasper V Been, Cornelis Jakobs
See end of article for
authors’ affiliations
Correspondence to:
Levinus A Bok, Maxima
Medical Center, Department
of Pediatrics, PO Box 7777,
5500 MB Veldhoven, The
Accepted 17 October 2006
Published Online First
6 November 2006
Arch Dis Child 2007;92:687–689. doi: 10.1136/adc.2006.103192
Background: Pyridoxine-dependent seizures (PDS) is a rare, autosomal recessively inherited disorder.
Recently a-aminoadipic semialdehyde (a-AASA) dehydrogenase deficiency was identified as a major cause
of PDS, which causes accumulation of both a-AASA and pipecolic acid (PA) in body fluids.
Methods: We studied urinary and plasma a-AASA and PA levels in 12 Dutch clinically diagnosed patients
with PDS.
Results: a-AASA was elevated in both urine and plasma in 10 patients. In these patients plasma PA levels
were also elevated but urinary PA levels were normal.
Discussion: In all patients with clinically definite PDS, and in most patients with probable or possible PDS, the
clinical diagnosis of PDS could be confirmed at the metabolite level. Non-invasive urinary screening for a-
AASA accumulation provides a reliable tool to diagnose PDS and can save these patients from the classical
and potentially dangerous pyridoxine withdrawal test to prove PDS.
yridoxine-dependent seizures (PDS) is a rare, autosomal
recessively inherited disorder usually presenting very
shortly after birth and in some cases in the womb. For
50 years PDS has been a clinical and biochemical conundrum
which has puzzled physicians and scientists.
Plecko et al and
Willemsen et al observed isolated pipecolic acid (PA) elevations
in the plasma and cerebrospinal fluid of PDS patients, yet the
biochemical relationship with pyridoxine metabolism remained
Recently, a-aminoadipic semialdehyde (a-AASA)
dehydrogenase deficiency due to pathogenic mutations in the
ALDH7A1 gene, was shown to be a major cause of PDS.
mammals, the essential amino acid L-lysine is degraded via PA
into the intermediate a-AASA, which is subsequently oxidised
to L-2-aminoadipic acid, a reaction catalysed by the enzyme a-
AASA dehydrogenase (EC, also named antiquitin)
(fig 1). In PDS patients, the lack of a-AASA dehydrogenase
leads to an accumulation of a-AASA and PA in body fluids. a-
AASA is in spontaneous reversible equilibrium with piper-
ideine-6-carboxylate (P6C) in the cytosol. Accumulated P6C
irreversibly reacts with active pyridoxine, ie, pyridoxal-5-
phosphate (P5P), by forming a Knoevenagel condensation
product. This irreversible reaction causes a secondary deficit of
P5P in affected children, which subsequently leads to epileptic
seizures. Restoration of the P5P pool can easily be achieved by
oral pyridoxine supplementation, which resolves the seizures.
Recently, we reported on the epidemiology and clinical
features of PDS in the Netherlands in this journal.
In that
paper the classical clinical criteria according to Baxter were
used to establish the diagnosis of PDS. We therefore re-
evaluated that series of PDS patients by measuring their levels
of a-AASA and PA in urine and plasma.
We re-evaluated all children (n = 11) with a diagnosis of
definite, probable or possible PDS from a recently described
Dutch cohort of 13 patients.
These patients and their parents
were invited to visit our hospital and were informed of the
novel insights into the pathophysiology of PDS. All except one
patient (patient 11) underwent further diagnostic work-up by
laboratory investigations of urine and blood. Furthermore, we
were able to include a recently born sibling of patient 10 in the
present study (patient 12).
a-AASA in urine and plasma was measured by liquid
chromatography-tandem mass spectrometry as previously
Quantitative determination of PA in urine and
plasma was performed by stable isotope dilution gas chroma-
tography-mass spectrometry.
Urine and/or blood samples were obtained from 11 patients.
The results of a-AASA and PA measurements are given in
table 1. a-AASA was elevated in the urine and plasma of 10
patients. PA in plasma was elevated in all patients with elevated
(plasma and urine) a-AASA, while urinary PA concentrations
were normal in all patients.
In this study, in all patients with a definite diagnosis of PDS
according to the criteria published by Baxter,
dehydrogenase deficiency could be proven at the metabolite
level by demonstrating elevated concentrations of a-AASA
(plasma and urine) and PA (plasma). The diagnosis was also
confirmed in two out of three patients with probable PDS and
in three out of four patients with possible PDS.
The diagnosis of probable PDS could not be confirmed in one
patient (patient 6). She is a younger sister of a girl with a
definite clinical diagnosis of PDS and a metabolically confirmed
diagnosis of a-AASA dehydrogenase deficiency (patient 4). She
had subtle neonatal seizures with only minimal epileptic
discharges on a 24-h EEG, which responded to 100 mg of
pyridoxine given intravenously. She is performing well at
school. Her normal development makes a diagnosis of PDS
unlikely since most PDS patients suffer from, at least a mild,
encephalopathy with learning difficulties.
However, the nature
of the neonatal seizure-like period remains unexplained. We
advised a trial period of pyridoxine withdrawal but could not
Abbreviations: a-AASA, a-aminoadipic semialdehyde; PA, pipecolic
acid; PDS, pyridoxine-dependent seizures; P5P, pyridoxal-5-phosphate;
P6C, piperideine-6-carboxylate
convince the parents to stop treatment. DNA analysis of
patients included in our study are pending.
In patient 11, originally diagnosed with possible PDS,
pyridoxine was recently withdrawn without recurrence of
seizures. We consider PDS a very unlikely diagnosis in this
patient because of the above observation and the fact that the
child is developing well. The parents did not want to cooperate
with further metabolic investigations.
In our first report,
we described two patients (patients 12
and 13 in that paper) who did not meet the criteria for definite,
probable or possible PDS. In both patients we have now
demonstrated normal a-AASA concentrations in plasma and
urine, as would be expected (data not shown).
This report is the first nationwide population-based study on
metabolically confirmed PDS. Our results show that at least 10
children with PDS were born in the Netherlands between
January 1991 and December 2004. As 2 764 697 children were
born in the Netherlands during this time (adapted from http://, the birth incidence of biochemically proven
PDS in the Netherlands is at least 1: 276 000 children. This
study further shows that most patients, namely nine out of 11
(82%), were diagnosed correctly using the criteria proposed by
Baxter. Thus, in circumstances where metabolic examination of
a-AASA and/or PA is not possible, applying the clinical criteria
proposed by Baxter seems a reliable method to establish a
diagnosis of PDS.
The concentrations of a-AASA and PA, in urine as well as in
plasma, vary considerably in patients with PDS. A remarkably
wide range of a-AASA and PA levels in patients has also been
found by Mills et al in their first report on a-AASA
dehydrogenase deficiency in PDS.
We have no clear explana-
tion for this wide range. Hypothetically it might reflect different
levels of a-AASA dehydrogenase residual activity, dietary
protein (L-lysine) intake, or the amount of supplemental
pyridoxine. It is tempting to speculate that optimum treatment
(ie, pyridoxine dosage) in PDS might be achieved by focusing
on the concentrations of a-AASA and PA.
Metabolic investigations of urinary concentrations of a-AASA
provide a reliable tool to prove PDS associated with a-AASA
dehydrogenase deficiency at the metabolite level. The poten-
tially dangerous trial of withdrawal of pyridoxine, classically
used to prove PDS, can now be avoided. The novel insights into
the pathophysiological processes that underlie PDS further
provide us with tools to better estimate the true incidence of
Figure 1 Metabolic pathway of L-lysine.
Table 1 Results of a-AASA and PA measurements
Patient Sibling Birth year
(Baxter criteria)
a-AASA urine
(mmol/mol cr)
PA urine
(mmol/mol cr)
PA plasma
PDS (confirmed
1 5 1991 Possible 16 8.0 0.11 6.5 Y
2 1992 Definite 4.7 0.9 0.27 5.8 Y
3 7 1992 Definite 29 5.7 0.00 4.6 Y
4 6 1992 Definite 4.0 1.1 0.02 7.0 Y
5 1 1993 Possible 24 5.0 0.11 5.0 Y
6 4 1994 Probable 0.2 ,0.2 0.02 2.2 N
7 3 1995 Probable 20 5.8 0.01 5.1 Y
8 1998 Possible 12 2.4 0.07 5.5 Y
9 2001 Definite 9.6 0.8 2.0 22 Y
10 12 2003 Probable 39 6.1 0.08 7.8 Y
11 2003 Possible NA NA NA NA N
12 10 2004 75 5.2 1.37 11 Y
N, no; NA, not available; Y, yes.
Control a-AASA concentrations are ,0.2 mmol/l for plasma and ,1 mmol/mol creatinine for urine.
Control values for PA in urine are 0.55–24.1 mmol/mol creatinine (,6 months of age) and 0.01–1.54 mmol/mol creatinine (.6 months of age).
For PA in plasma, control values are 3.75–10.8 mmol/l (,1 week of age), and 0.7–2.46 mmol/l (.1 week of age).
What is already known on this topic
PDS is a rare disease caused by alpha-aminoadipic
semialdehyde (a-AASA) dehydrogenase deficiency.
Epidemiological data on PDS are rare and are based on
clinical criteria for PDS.
What this study adds
Non-invasive, urinary screening for a-AASA accumula-
tion provides a reliable tool to diagnose PDS.
The birth incidence of metabolically confirmed PDS in this
nationwide Dutch study is estimated to be at least
1:276 000.
688 Bok,Struys,Willemsen,etal
PDS (at least 1:276 000 newborns in The Netherlands according
to this study) and will hopefully lead to an optimum treatment
regime for this serious neurometabolic disorder.
We would like to thank all replying clinicians for their cooperation, in
particular the following for kindly providing the patient data: Dr W
Baerts, Isala Klinieken, Zwolle; Dr F van Berkestijn, Universitair
Medisch Centrum, Utrecht; Dr AN Bosschaart and Dr RFHM
Tummers, Medisch Spectrum Twente, Enschede; Dr I de Coo,
Erasmus Medisch Centrum, Rotterdam; Dr GAPT Hurkx, Elkerliek
Ziekenhuis, Helmond; Dr R Kohl, Het Spittaal, Zuthpen; Dr LAEM
Laan, Leids Universitair Medisch Centrum, Leiden; Dr A van der Wagen,
Streekziekenhuis Midden-Twente, Hengelo.
Authors’ affiliations
Levinus A Bok, Department of Paediatrics, Ma´xima Medical Center,
Veldhoven, The Netherlands
Eduard Struys, Cornelis Jakobs, Metabolic Unit, Department of Clinical
Chemistry, VU University Medical Center, Amsterdam, The Netherlands
Michel A A P Willemsen, Department of Paediatric Neurology, University
Medical Center Nijmegen, Nijmegen, The Netherlands
Jasper V Been, Department of Paediatrics, Maastricht University Hospital,
Maastricht, The Netherlands
Competing interests: None.
1 Baxter P. Pyridoxine-dependent seizures: a clinical and biochemical conundrum.
Biochim Biophys Acta 2003;1647(1–2):36–41.
2 Plecko B, Hikel C, Korenke GC, et al. Pipecolic acid as a diagnostic marker of
pyridoxine-dependent epilepsy. Neuropediatrics 2005;36(3):200–5.
3 Plecko B, Stockler-Ipsiroglu S, Paschke E, et al. Pipecolic acid elevation in plasma
and cerebrospinal fluid of two patients with pyridoxine-dependent epilepsy. Ann
Neurol 2000;48(1):121–5.
4 Willemsen MA, Mavinkurve-Groothuis AM, Wevers RA, et al. Pipecolic acid: a
diagnostic marker in pyridoxine-dependent epilepsy. Ann Neurol
5 Mills PB, Struys E, Jakobs C, et al. Mutations in antiquitin in individuals with
pyridoxine-dependent seizures. Nat Med 2006;12(3):307–9.
6 Been JV, Bok LA, Andriessen P, et al. Epidemiology of pyridoxine dependent
seizures in the Netherlands. Arch Dis Child 2005;90(12):1293–6.
7 Kok RM, Kaster L, de Jong AP, et al. Stable isotope dilution analysis of pipecolic
acid in cerebrospinal fluid, plasma, urine and amniotic fluid using electron
capture negative ion mass fragmentography. Clin Chim Acta
8 Baxter P. Epidemiology of pyridoxine dependent and pyridoxine responsive
seizures in the UK. Arch Dis Child 1999;81(5):431–3.
Epoprostenol for severe pulmonary hypertension
verage survival time with untreated idiopathic pulmonary hypertension is 2.8 years in
adults and 10 months in children. Successful treatment with epoprostenol (prostacyclin)
was reported for adults in 1996 and for children in 1999. Now a report from the Great
Ormond Street Hospital for Children, London (Heart, published online 25 Oct 2006; doi 10.1136/
hrt.2006.096412) has provided details of all 39 children treated there with continuous
intravenous epoprostenol between 1997 and 2005.
The children (22 girls, 17 boys; age range 417 years, median 5.4 years) were all in WHO
functional classes III and IV. Twenty five had idiopathic pulmonary arterial hypertension and 14
pulmonary hypertension associated with congenital heart disease (6), cardiomyopathy (3),
connective tissue disease (2), chronic interstitial lung disease (2) and HIV infection (1).
Epoprostenol was started if oral therapies had failed or there were severe symptoms at
presentation. Parents were trained to prepare and administer the drug at home and training was
given to community health staff.
Over a mean follow-up of 27 months, seven children died and eight underwent transplanta-
tion (four double lung, three heart and lung, one heart only). Cumulative survival at 1, 2, and
3 years was 94%, 90% and 84%, respectively. WHO functional class improved and the mean
increase in 6-min walking distance was 77 m. Fourteen children needed additional drug therapy
(bosentan in eight, sildenafil in five, and both in one) and 17 with syncope or pre-syncope had
atrial septostomy. Epoprostenol therapy is effective in children.
Pyridoxine-dependent seizures in Dutch patients 689
    • "Leur prévalence est probablement sous-estimée puisque les marqueurs diagnostiques biochimiques ou génétiques ne sont disponibles que depuis peu. Elle a e ´té estimée a ` 1/276 000 naissances vivantes aux Pays-Bas [6]. Sur le plan clinique, on distingue des formes classiques dont le début est périnatal, et des formes tardives, plus rares [7,8]. "
    [Show abstract] [Hide abstract] ABSTRACT: Inborn error of metabolism may produce a complex clinical picture in which epilepsy is only one of the various neurologic manifestations including developmental delay/regression, mental retardation, and movement disorders. However, metabolic epilepsies may dominate the clinical presentation. A specific diagnosis of metabolic disorders in epileptic patients may provide the possibility of specific treatments that can improve seizures. In a few metabolic diseases such as vitamin-responsive epilepsies, epilepsy responds to specific treatments based on supplementation of cofactors. Certain rare vitamin-responsive inborn errors of metabolism may present as early encephalopathy with anticonvulsant-resistant seizures. These include pyridoxine-dependent seizures, pyridoxal-phosphate-dependent seizures, folinic acid-responsive seizures, and biotinidase deficiency. This review discusses our current understanding of these vitamin-responsive epilepsies. Copyright © 2013 Elsevier Masson SAS. All rights reserved.
    Full-text · Article · Sep 2013
    • "Although relatively rare, with an estimated incidence up to 1:276.000 births in The Netherlands (Bok et al. 2007), this condition is treatable and early diagnosis is of utmost importance. The current knowledge about the primary defect in PDS has provided new diagnostic biomarkers, i.e., α-AASA and P6C. "
    [Show abstract] [Hide abstract] ABSTRACT: The assessment of urinary α-aminoadipic semialdehyde (α-AASA) has become the diagnostic laboratory test for pyridoxine dependent seizures (PDS). α-AASA is in spontaneous equilibrium with its cyclic form Δ(1)-piperideine-6-carboxylate (P6C); a molecule with a heterocyclic ring structure. Ongoing diagnostic screening and monitoring revealed that in some individuals with milder ALDH7A1 variants, and patients co-treated with a lysine restricted diet, α-AASA was only modestly increased. This prompted us to investigate the diagnostic power and added value of the assessment of urinary P6C compared to α-AASA. Urine samples were diluted to a creatinine content of 0.1 mmol/L, followed by the addition of 0.01 nmol [(2)H(9)]pipecolic acid as internal standard (IS) and 5 μL was injected onto a Waters C(18) T3 HPLC column. Chromatography was performed using water/methanol 97/3 (v/v) including 0.03 % formic acid by volume with a flow rate of 150 μL/min and detection was accomplished in the multiple reaction monitoring mode: P6C m/z 128.1 > 82.1; [(2)H(9)]pipecolic acid m/z 139.1 > 93.1. Due to the dualistic nature of α-AASA/P6C, and the lack of a proper internal standard, the method is semi quantitative. The intra-assay CVs (n = 10) for two urine samples of proven PDS patients with only modest P6C increases were 4.7% and 8.1%, whereas their inter-assay CVs (n = 10) were 16 and 18% respectively. In all 40 urine samples from 35 individuals with proven PDS, we detected increased levels of P6C. Therefore, we conclude that the diagnostic power of the assessments of urinary P6C and α-AASA is comparable.
    Full-text · Article · Jan 2012
    • "After derivatization of the αAASA molecule with uorenylmethyloxycarbonyl chloride, the sample is directly analyzed by LC–MS/MS. Although this method is semiquantitative, it provides a sensitive and specic diagnostic tool [17,92,93]. Levels of urinary αAASA in normal individuals decline during the rst year of life: 0–0.5 years: b2 mmol/mol creatinine; 0.5–1 year: αAASA b 1 mmol/mol creatinine ; N1 year αAASA b 0.5 mmol/mol creatinine. "
    [Show abstract] [Hide abstract] ABSTRACT: Antiquitin (ATQ) deficiency is the main cause of pyridoxine dependent epilepsy characterized by early onset epileptic encephalopathy responsive to large dosages of pyridoxine. Despite seizure control most patients have intellectual disability. Folinic acid responsive seizures (FARS) are genetically identical to ATQ deficiency. ATQ functions as an aldehyde dehydrogenase (ALDH7A1) in the lysine degradation pathway. Its deficiency results in accumulation of α-aminoadipic semialdehyde (AASA), piperideine-6-carboxylate (P6C) and pipecolic acid, which serve as diagnostic markers in urine, plasma, and CSF.
    Full-text · Article · May 2011
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