Impaired hydroxylation of tyrosine and tryptophan due to deficiency of BH4 reduces the formation of catecholamine and serotonin. The measurement of CSF neurotransmitter metabolites is of capital importance for clinical and treatment follow-up, as it allows to separate typical from atypical cases of BH4 deficiency (the latter are phenotypically defined by the absence of clinical signs and symptoms and do not need any treatment for biogenic amine deficiency).

Metabolism of tyrosine and tryptophan:

Following the hydroxylation of tyrosine and tryptophan to L-DOPA and 5-hydroxytryptophan, these products are decarboxylated by the aromatic amino acid decarboxylase to form the active neurotransmitters dopamine and serotonin. Dopamine is further hydroxylated by b-hydroxylase to noradrenaline, which can be methylated to form adrenaline. The main routes of catabolism of the catecholamines and serotonin involve either methylation by O-methyltransferase or formation of homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5HIAA) through the action of aldehyde dehydrogenase and monoamine oxidase. Depending on the type of HPA, the turnover of tyrosine and tryptophan can be influenced by different factors: e.g. actual plasma and CSF phenylalanine levels, transport of aromatic amino acids across the blood-brain barrier, residual activity of the defective enzyme in the brain, or gene dosage effects.

Metabolites:

In order to distinguish between the different variants of BH4 deficiency, i.e. typical (severe, general) and atypical (mild, peripheral, partial) forms, quantification of the neurotransmitter metabolites, 5HIAA and HVA, is essential. Quantification is easily performed using HPLC with electrochemical detection. Simultaneously, 3-O-methyl-DOPA, a metabolite of L-DOPA, and a marker of decarboxylation, can be measured.

Differential diagnosis:

Except for the atypical (mild, peripheral, partial) forms of GTPCH, PTPS, and PCD deficiencies, 5HIAA and HVA are dramatically decreased in the CSF of patients with the severe form of BH4 deficiency. The levels of both metabolites decrease with age in all BH4 deficiencies and a similar correlation was found in control children. In the mild forms of DHPR deficiency, 5HIAA is usually decreased, while HVA levels are normal.

In addition to BH4 deficiencies, neurotransmitter metabolites analysis is essential in disorders presenting without hyperphenylalaninemia:

• Dopa-responsive dystonia; (DRD, Segawa disease), dominant GTPCH def.; OMIM#600225
• Sepiapterinreductase (SR) deficiency (no OMIM)
• Tyrosine hydroxylase (TH) deficiency; OMIM#191290
• Aromatic L-amino acid decarboxylase (AADC) deficiency; OMIM#107930
• Dopamine b-hydroxylase (DbH) deficiency; OMIM#223360

Collection of CSF specimen:

Discard the first 0.5 ml of CSF and collect the next 1-2 ml in a small tube. Keep immediately frozen at -70°C. There is no need for any additives or to collect additional CSF fractions.

Preconditions:

Before medication, preferably between 8 and 10 h in the morning.

Pitfalls:

Unstable in blood-contaminated samples (centrifuge immediately). Interference by some drugs in some HPLC systems (e.g., Panadol, ...). Reference values are age dependent. Our and most other reference values are established for the first 1-3 ml of CSF. Some laboratories use higher CSF fractions and other reference values which may lead to misinterpretation of results.

Reference and pathological values

Neurotransmitter metabolites and folates in CSF (click on image for larger view)

5HIAA = 5-hydroxyindoleacetic acid; HVA = homovanillic acid; 3OMD = 3-O-methyldopa; MHPG = 3-methoxy-4-hydroxyphenylglycol; 5MTHF = 5-methyltetrahydrofolic acid

• Hyland K, Biaggioni I, Elpeleg ON, Nyggard TG, Gibson KM. Disorders of neurotransmitter metabolism. In: Blau N, Duran M, Blaskovics M, eds. Physician's Guide to the Laboratory Diagnosis of Metabolic Diseases. London: Chapman & Hall, 1996: 79-98.
   
• Blau N, Barnes I, Dhondt JL. International database of tetrahydrobiopterin deficiencies. J Inherit Metab Dis 1996;19:8-14.
   
• Blau N, Blaskovics M. Hyperphenylalaninemia. In: Blau N, Duran M, Blaskovics M, eds. Physician's Guide to the Laboratory Diagnosis of Metabolic Diseases. London: Chapman & Hall, 1996: 65-78.

REFERENCES

• Blau N, Thöny B, Cotton RGH, Hyland K. Disorders of tetrahydrobiopterin and related biogenic amines. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Vogelstein B, eds. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. New York: McGraw-Hill, 2001: 1725-1776.
   
• Hyland K, Biaggio I, Elpeleg ON, Nyggard TG, Gibson KM. Disorders of neurotransmitter metabolism. In: Blau N, Duran M, Blaskovics M, eds. Physician's Guide to the Laboratory Diagnosis of Metabolic Diseases. London: Chapman & Hall, 1996: 79-98.
   
• Blau N, Blaskovics M. Hyperphenylalaninemia. In: Blau N, Duran M, Blaskovics M, eds. Physician's Guide to the Laboratory Diagnosis of Metabolic Diseases. London: Chapman & Hall, 1996: 65-78.
   
• Bonafé, L., B. Thöny, W. Leimbacher, L. Kierat and N. Blau (2001). "Diagnosis of Dopa-responsive dystonia and other tetrahydrobiopterin disorders by the study of biopterin metabolism in fibroblasts." Clin Chem 47: 477-485.
   
• Blau, N., L. Bonafé and B. Thöny (2001). "Tetrahydrobiopterin deficiencies without hyperphenylalaninemia: Diagnosis and genetics of Dopa-responsive dystonia and sepiapterin reductase deficiency." Mol Genet Metab 72: 172-185.