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.
https://pubmed.ncbi.nlm.nih.gov/34715572/
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 (mean) | N | 3-OMD [nmol/L] (min–max) 2.5–97.5 percentiles | 5-HTP [nmol/L] (min–max) 2.5–97.5 percentiles |
| 2–5 days (59 hours) | 345 | 501.9±98.1 (262.9–842.1) 340.8–724.8 | 27.9±9.1 (7.7–53.6) 11.5–45.9 |
| 5–30 days (12 days) | 81 | 541.5±105.9 (267.3–783.9) 347.1–760.0 | 35.5±8.8* (12.3–57.2) 19.5–50.3 |
| 1–12 months (4 months) | 54 | 364.9±132.7* (136.1–727.7) 198.6–652.1 | 34.5±11.0 (14.1–69.0) 19.6–53.6 |
| 1–8 years (4 years) | 91 | 140.4±35.2* (79.8–241.6) 86.4–218.6 | 27.1±8.1* (11.1–48.4) 20.0–40.6 |
| 8–18 years (12 years) | 38 | 109.8±20.0* (77.3–151.7) 79.2–143.7 | 28.0±8.2 (11.5–46.2) 11.7–42.8 |
| 18–51 years (34 years) | 68 | 107.3±19.1 (67.8–159.4) 71.0–144.9 | 27.4±8.4 (15.2–44.1) 15.2–42.8 |
*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) | Gender | Variant 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) |
| 1 | 5 | Female | p. Ser250Phe (c.749C>T)/ p. Ser250Phe (c.749C>T) | 24 | 2993.1 | 124.1 |
| 2 | 23 | Female | p.Tyr37Thrfs*5 (c.105delC)/ p.F237S (c.710 T>C) | 30 | 1702.8 | 79.8 |
| 3 | 33 | Female | p.Tyr37Thrfs*5 (c.105delC)/ p.F237S (c.710 T>C) | 39 | 1211.3 | 103.8 |
| 4 | 6 | Male | p. Cys281Trp (c.843C>G)/ p. Met362Thr (c.1085T>C) | 18 | 1961.7 | 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
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