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
- Developing and validating an analytic procedure
- Collecting reference data from healthy subjects of different ages
- 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 days |
|5–30 days |
|1–12 months |
|1–8 years |
|8–18 years |
|18–51 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 |
|Gender||Variant 1/ |
|Age at |
|3-OMD nmol/L |
|1||5||Female||p. Ser250Phe |
|4||6||Male||p. Cys281Trp |
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
- Di Carlo E, et al. J Chromatogr B Analyt Technol Biomed Life Sci. 2021;1185:122999.
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