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A useful tool for the diagnosis of L-amino acid decarboxylase deficiency

Simultaneous determination of 5-hydroxytryptophan and 3-O-methyldopa in dried blood spot by UPLC-MS/MS: A useful tool for the diagnosis of L-amino acid decarboxylase deficiency

Di Carlo E, et al. J Chromatogr B Analyt Technol Biomed Life Sci. 2021;1185:122999.

Publication Date | October 2021
Authors | Di Carlo E, Santagata S, Sauro L, Tolve M, Manti F, Leuzzi V, Angeloni A, Carducci C.
Citation | J Chromatogr B Analyt Technol Biomed Life Sci. 2021;1185:122999.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare, inherited disorder of neurotransmitter synthesis.1,2 Caused by variants in the dopa decarboxylase (DCC) gene, AADC deficiency results in the depletion of serotonin and dopamine, and the accumulation of 3-O-methyldopa (3-OMD) and 5-hydroxytryptophan (5-HTP), in the central nervous system.1,3 If left untreated, AADC deficiency results in severely disabling neurological impairment.1

The current gold standard for AADC deficiency diagnosis is high-performance liquid chromatography analysis of neurotransmitter metabolites in cerebrospinal fluid; showing a relevant increase in 3-OMD and 5-HTP.1 More recently, dried blood spot (DBS) testing has been used to successfully detect elevated 3-OMD as a diagnostic biomarker in affected patients, showing the potential to be used in newborn screening programmes.1,4–6 However, without the concurrent measurement of 5-HTP, other conditions resulting in increased 3-OMD need to be excluded.1

Di Carlo, et al. aimed to establish a reliable method to assess both metabolites in DBS through ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technology, to create a fast and specific diagnostic tool to detect 5-HTP alongside 3-OMD.1

The study involved 3 steps:1

  1. Developing and validating an analytic procedure
  2. Collecting reference data from healthy subjects of different ages
  3. Collecting pathological data from patients affected by AADC deficiency and subjects affected by levodopa-responsive movement disorders of different aetiologies


After extraction from DBS, 3-OMD and 5-HTP were separated by UPLC and detected by MS/MS.1 Mass spectrometric parameters were optimised to obtain the highest sensitivity and specificity for 3-OMD and 5-HTP determination.1 Chromatographic separation was accomplished in 13 minutes, with the limit of detection being 2.4 and 1.4 nmol/L of blood for 3-OMD and 5-HTP, respectively.1

Reference values of 3-OMD and 5-HTP in newborns, children, and adults were assessed in 677 subjects (age range 48 hours–51 years) who were divided into 6 age groups.1 No significant 3-OMD and 5-HTP differences were observed between females and males in the control population.1 However, a marked influence of age was observed for 3-OMD concentration, with a remarkable continuous reduction in 3-OMD concentration observed after the first month of life, stabilising at 8 years of age.1 Age had a less evident effect on 5-HTP, showing only a slight decrease with age after the first week of life (Table 1).1

Table 1: Reference values for 3-OMD and 5-HTP in DBS (mean and range), according to reference population age1

Age range
N 3-OMD [nmol/L]
2.5–97.5 percentiles
5-HTP [nmol/L]
2.5–97.5 percentiles
2–5 days
(59 hours)
345 501.9±98.1
5–30 days
(12 days)
1–12 months
(4 months)
1–8 years
(4 years)
8–18 years
(12 years)
18–51 years
(34 years)

*P<0.001 in a Wilcoxon test between the age group and the previous one.

DBS samples were obtained from 4 patients (3 females aged 18–39 years) affected by AADC deficiency (Table 2).1 For these 4 patients, both 3-OMD and 5-HTP concentrations were increased in DBS; mean and SD values were 1780.6±773.1 nmol/L (rv 71.0–144.9) and 94.8±19.0 nmol/L (rv 15.2–42.8) for 3-OMD and 5-HTP, respectively.1

Table 2: 3-OMD and 5-HTP concentration in DBS of patients affected by AADC deficiency1

Patient Age at
diagnosis (years)
GenderVariant 1/
Variant 2
Age at
sampling (years)
3-OMD nmol/L
(rv 1.0–144.9)
5-HTP nmol/L
(rv 15.2–42.8)
15Femalep. Ser250Phe
p. Ser250Phe
242993.1 124.1
(c.710 T>C)
301702.8 79.8
(c.710 T>C)
391211.3 103.8
46Malep. Cys281Trp
p. Met362Thr
181961.7 81.2

In 3 subjects affected by levodopa-responsive movement disorders that required L-dopa/carbidopa supplementation, analysis showed a marked increase in 3-OMD (6159.6±3449.1 nmol/L, rv 73.2–192.2), with normal 5-HTP in 2 subjects and a very slight increase in only 1 subject (41.5±4.6 nmol/L, rv 11.4–42.3).1

Overall, these results show that simultaneous measurement of 5-HTP and 3-OMD in DBS leads to an improvement in specificity and sensitivity for the biochemical diagnosis of AADC deficiency.1

  1. Di Carlo E, et al. J Chromatogr B Analyt Technol Biomed Life Sci. 2021;1185:122999.
  2. Himmelreich N, et al. Mol Genet Metab. 2019;127(1):12–22.
  3. Hyland K, et al. Pediatr Neurol. 2020;106:38–42.
  4. Brennenstuhl H, et al. J Inherit Metab Dis. 2020;43(3):602–610.
  5. Chen PW, et al. Clin Chim Acta. 2014;431:19–22.
  6. Burlina A, et al. Mol Genet Metab. 2021;133(1):56–62.


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