New Method for Diagnosing AADC Detects 2 Biomarkers in Blood Spots

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by Steve Bryson PhD |

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A new method has been developed that detects two biomarkers associated with aromatic l-amino acid decarboxylase (AADC) deficiency in dried blood spots at the same time, a study reveals.

This method for simultaneously identifying these separate disease biomarkers was found to be accurate and precise — and can be used as a non-invasive tool to help better diagnose AADC deficiency, the scientists said.

The study, “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,” was published in the Journal of Chromatography B.

AADC deficiency is caused by a mutation in the DDC gene, which contains the instructions for the AADC enzyme. AADC is necessary for the production of the chemicals — or neurotransmitters — dopamine and serotonin in the brain, which are essential for nerve cell communication.

Without a functional AADC enzyme, dopamine precursors like L-Dopa accumulate inside cells, and are then broken down and chemically transformed into 3-O-methyldopa, known as 3-OMD. This metabolite, therefore, becomes elevated in patients with the genetic disorder, as is the serotonin precursor 5-hydroxytryptophan, or 5-HTP.

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Diagnosing AADC deficiency currently relies on measures of AADC activity and on the biochemical profile of a patient’s cerebrospinal fluid or CSF, the liquid that surrounds the brain and spinal cord. To collect this liquid for testing requires an invasive lumbar puncture, also known as a spinal tap. The CSF sample also must be examined for disease biomarkers in a specialized lab.

In recent studies, researchers have shown that 3-OMD can be found in other body fluids, like urine, plasma, and dried blood spots. These “spots” are, essentially, drops of blood collected on filter paper. This technique is commonly used in newborn screenings in hospitals.

Measuring 3-OMD in dry blood spots has been established as a less invasive way to help diagnose AADC deficiency. Notably, however, other conditions associated with high 3-OMD levels still must be ruled out.

Thus, detecting both 5-HTP and 3-OMD in dried blood spots, at the same time, would further simplify diagnoses in symptomatic AADC patients, as well as support newborn screening programs.

Now, scientists based at the University of Rome, in Italy, developed a method for detecting 5-HTP and 3-OMD in dried blood spots simultaneously.

The team applied a technique called ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and optimized the approach to be as accurate as possible at diagnosing AADC patients, with as few as possible false-positives.

First, the team established reference values for healthy individuals. Blood was collected from 345 infants during routine newborn screening programs. The babies — 175 females and 170 males —ranged in age from 2 to 5 days.

Blood samples also were collected, during routine clinical care, from 81 infants, ages 5 to 29 days, 183 children and adolescents, ages 1 month to 17 years, and 68 adults, ranging from 18 to 51 years. None of these individuals were receiving medicinal therapy.

These reference participants were then divided into six age groups, to account for age-related changes in metabolite levels. Notably, while 5-HTP remained overall stable over the years, 3-OMD levels decreased markedly from 1 month to 3 years, and remained low after that age.

After establishing the reference values for each age group, the researchers examined dry blood spot samples from four AADC deficiency patients to validate their method. These patients were 18 to 39 years old, and three were female.

Results showed that all patients had higher-than-normal levels of both biomarkers. The mean value for 3-OMD was 1,780 nanomols per litre (nmol/L) – reference value 1-144.9 nmol/L – and  94.8 nmol/L for 5-HTP (reference 15.2–42.8 nmol/L).

For comparison, the investigators examined three additional samples from patients (ages 16–17) with other neurological conditions also treated with dopamine-related medications. In these controls, the levels of 3-OMD also were markedly increased, with a mean value of 6,159.6 nmol/L, but levels of 5-HTP were within normal range.

“A UPLC-MS/MS method was developed and validated for the simultaneous determination of 3OMD and 5HTP in [dry blood spots],” the team concluded. “The method is accurate and precise, and it was successfully applied to the diagnosis of AADC deficiency.”