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Clinical Chemistry 49: 1830-1838, 2003; 10.1373/clinchem.2003.021576
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(Clinical Chemistry. 2003;49:1830-1838.)
© 2003 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Capillary Electrophoresis in Diagnosis and Monitoring of Adenosine Deaminase Deficiency

Filippo Carlucci1,a, Antonella Tabucchi1, Alessandro Aiuti2, Francesca Rosi1, Federica Floccari1, Roberto Pagani1 and Enrico Marinello1

1 Dipartimento di Medicina Interna, Scienze Endocrino-Metaboliche e Biochimica Università degli Studi di Siena, 53100 Siena, Italy.

2 TIGET Scientific Institute H.S. Raffaele, 201 Milano, Italy.

aAddress correspondence to this author at: Dipartimento di Medicina Interna, Scienze Endocrino-Metaboliche e Biochimica. University of Siena, Nuovi Istituti Biologici, Via Aldo Moro, 53100 Siena, Italy. Fax 39-0577-234285.

Background: The diagnosis and monitoring of severe combined immunodeficiency disease (SCID) attributable to adenosine deaminase (ADA) deficiency requires measurements of ADA, purine nucleoside phosphorylase (PNP), and S-adenosyl-L-homocysteine-hydrolase (SAHH) activity and of deoxyadenosine metabolites. We developed capillary electrophoresis (CE) methods for the detection of key diagnostic metabolites and evaluation of enzyme activities.

Methods: Deoxyadenosine metabolites were separated in 30 mmol/L sodium borate–10 mmol/L sodium dodecyl sulfate (pH 9.80) at 25 °C on a 60-cm uncoated capillary. For determination of enzyme activities, substrate–product separation and measurements were carried out in 20 mmol/L sodium borate (pH 10.00) at 25 °C on a 42-cm uncoated capillary.

Results: Deoxynucleotides and deoxyadenosine were readily detectable in erythrocytes and urine, respectively. Both methods were linear in the range 2–500 µmol/L (r >0.99). Intra- and interassay CV were <4%. Enzyme activities were linear with respect to sample amounts in the incubation mixture and to incubation time (r >0.99 for both). In erythrocytes from healthy individuals, mean (SD) ADA activity was 5619 (2584) nmol/s per liter of packed cells. In erythrocytes of SCID patients at diagnosis, ADA activity was 56.9 (48.3) nmol/s per liter of packed cells; SAHH activity was also much reduced. PNP activity was similar in patients and controls.

Conclusions: CE can be used to test ADA deficiency and enables rapid assessment of ADA expression in hematopoietic cells of SCID patients during therapy.




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