Capillary electrophoresis in diagnosis and monitoring of adenosine deaminase deficiency.
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 degrees 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 degrees 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 micro mol/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.
Dipartimento di Medicina Interna, Scienze Endocrino-Metaboliche e Biochimica, Università degli Studi di Siena, 53100 Siena, Italy.
Filippo
F
Carlucci
Antonella
A
Tabucchi
Alessandro
A
Aiuti
Francesca
F
Rosi
Federica
F
Floccari
Roberto
R
Pagani
Enrico
E
Marinello
D000243:Adenosine Deaminase / Q000097:blood / Q000172:deficiency* / Q000652:urine; D043383:Adenosylhomocysteinase / Q000097:blood / Q000652:urine; D000328:Adult; D002648:Child; D003839:Deoxyadenosines / Q000097:blood / Q000378:metabolism / Q000652:urine; D019075:Electrophoresis, Capillary; D006801:Humans; D011683:Purine-Nucleoside Phosphorylase / Q000097:blood / Q000652:urine
D003839:Deoxyadenosines; D011683:Purine-Nucleoside Phosphorylase; D043383:Adenosylhomocysteinase; D000243:Adenosine Deaminase