HPLC with electrochemical and fluorescence detection procedures for the diagnosis of inborn errors of biogenic amines and pterins
Introduction
Catecholamines and serotonin are biogenic amines (BA) related with central and autonomous vegetative nervous system function. They are mainly involved in movement, sleep, thermoregulation, learning, memory, emotional behaviour, as well as other functions (Grace et al., 1998). Several inborn errors of BA metabolism have recently been established (Blau et al., 2001). The first step in the diagnosis requires the analysis of catecholamines and serotonin metabolites in cerebrospinal fluid (CSF), since blood and urine analysis of these compounds may be uninformative for the identification of these disorders (Hyland et al., 1993).
Several enzymatic defects have been related with primary BA deficiencies. Tyrosine hydroxylase deficiency (OMIM: 191290) causes a dopamine defect; it is diagnosed by low 3-ortomethyldopa (3-OMD), 3-methoxy-4-hydroxyphenylglycol (MHPG) and homovanilic acid (HVA) concentrations in CSF (Hoffmann et al., 1998). Aromatic l-amino acid descarboxylase deficiency (OMIM: 107930) causes a reduction of both dopaminergic and serotoninergic metabolites; it is diagnosed by high concentrations of 5-hydroxytryptophan (5-OHtrp) and 3-OMD together with low 5-hydroxiindolacetic acid (5-HIAA) and HVA values in CSF (Blau et al., 2001, Hoffmann et al., 1998). Dopamine β-hydroxylase deficiency (OMIM: 223360) causes norepinephrine deficiency; it is diagnosed by low plasma norepinephrine/dopamine ratio (Timmers et al., 2004). High CSF HVA with low MHPG concentrations may be observed, causing high HVA/MHPG ratio values (Blau et al., 2001). Furthermore, a possible tryptophan hydroxylase (EC 1.14.16.4) dysfunction causing low 5-OHtrp and 5-HIAA values has recently been reported, although no molecular identification of the primary defect is available for this disorder (Ramaekers et al., 2001).
Tetrahydrobiopterin (BH4) is the main cofactor for tryptophan, tyrosine and phenylalanine hydroxylases. Defects of this cofactor affect both serotonin and dopamine biosynthesis and are usually associated with hyperphenylalaninemia (HPA) (Blau et al., 2001). However, at least two BH4 deficiencies, GTP cyclohydroxylase-1 autosomal-dominant (OMIM: 600225) and sepiapterin reductase (OMIM: 182125), may occur without HPA. The biochemical diagnosis of these disorders must be performed through the analysis of pterins (biopterin (BP) and neopterin (NP)) in CSF (Bonafé et al., 2001).
HPLC with electrochemical and fluorescence detection procedures have been applied for CSF BA and pterin analysis, respectively. These procedures are sensitive and specific, permit rapid and reproducible analysis with minimal sample preparation, and may be available in clinical laboratories (Schmidt et al., 1990). The BA and pterin analysis in CSF is important for the early diagnosis of these disorders in the paediatric age, since the potential benefits of specific treatment of some of these disorders have been demonstrated (Hyland, 2003). However, there are few reports concerning detailed analytical procedures for the diagnosis of these congenital disorders. Moreover, data concerning reference values in a paediatric population have scarcely been reported (Hyland et al., 1993, Komori et al., 1999), to our knowledge.
Our aim was to standardize previously reported HPLC procedures for the analysis of BA and pterins in CSF and to establish reference values for a paediatric population.
Section snippets
Subjects
Reference values were established in 127 subjects (age range 11 days to 16 years; average 3.83; sex: 71 males and 56 females) whose CSF samples were submitted to our laboratory under suspicion of viral or bacterial meningitis, encephalitis, or other neurological conditions of non-metabolic origin. Exclusion criteria were: disturbance of the biogenic amines or pterin metabolism, inborn errors of intermediary and energy metabolism, movement disorders and neuroimaging abnormalities, and diagnosis
Results
Within-run and between-run imprecision data and analytical interval for BA and pterin metabolites are reported in Table 1. Recovery was evaluated by adding, 3-OMD, MHPG, 5-OHTrp, 5-HIAA, HVA, NP and BP to a CSF sample to obtain final concentrations of 216, 124, 70, 494, 316, 25.1 and 27.9 nmol/l, respectively. Samples obtained were analysed in triplicate, and the range of the mean recoveries was 96–104%.
In the whole group of controls, a negative correlation was observed between all the variables
Discussion
HPLC with electrochemical and fluorescence detection offers enough sensitivity and quality for the analysis of BA and pterins in CSF in order to diagnose inborn errors of metabolism of these compounds (Schmidt et al., 1990). To date several HPLC procedures have been published for BA (Mena et al., 1984, Schmidt et al., 1990, Yi et al., 1994) and pterin analysis (Blau et al., 1992), with slight differences among them. We present here two modified HPLC procedures for BA and pterin analysis, as
Acknowledgements
This work was supported by the FIS grants (REDEMETH G03/054 and INERGEN C03/05). Aida Ormazabal is the recipient of a grant from the Generalitat de Catalunya (2001SCR 00417).
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