Determination of adenosine deaminase activity in dried blood spots by a nonradiochemical assay using reversed-phase high-performance liquid chromatography

Article abstract of DOI:10.1080/15257771003741406

Adenosine deaminase (ADA) deficiency is a rare metabolic disease causing severe combined immunodeficiency (SCID). An assay to determine ADA activity in dried blood spots was developed using reversed-phase HPLC. The assay was linear with reaction times up to at least 4 hours, and protein concentrations up to at least 2.2 mg/ml. The intra-assay CV and the inter-assay CV for the complete assay was 3.5 and 8.4%, respectively. The ADA activity in a control blood spot, stored at 4°C, remained stable for at least one year. Only a slightly decreased ADA activity (35 ± 13 nmol/mg/h, n = 4) was observed in heterozygotes for a c.704G > A mutation in the ADA gene when compared to that observed in controls (41 ± 13 nmol/mg/h, n = 108). In addition, increased ADA activity as found in a rare form of congenital anemia can be assessed, as observed in a bloodspot from a patient diagnosed with Diamond Blackfan anemia (ADA activity 150 nmol/mg/h). Copyright

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Determination of Adenosine Deaminase
Activity in Dried Blood Spots by a
Nonradiochemical Assay Using Reversed-
Phase High-Performance Liquid
Chromatography
A. B. P. van Kuilenburg a , L. Zoetekouw a , J. Meijer a & T. W.
Kuijpers b
a Academic Medical Center, Emma Children's Hospital , Laboratory
Genetic Metabolic Diseases , Amsterdam, the Netherlands
b Academic Medical Center, Emma Children's Hospital, Division
of Pediatric Hematology , Immunology and Infectious Diseases ,
Amsterdam, the Netherlands
Published online: 11 Jun 2010.
To cite this article: A. B. P. van Kuilenburg , L. Zoetekouw , J. Meijer & T. W. Kuijpers (2010)
Determination of Adenosine Deaminase Activity in Dried Blood Spots by a Nonradiochemical Assay
Using Reversed-Phase High-Performance Liquid Chromatography, Nucleosides, Nucleotides and Nucleic
Acids, 29:4-6, 461-465, DOI: 10.1080/15257771003741406
To link to this article: http://dx.doi.org/10.1080/15257771003741406
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Nucleosides, Nucleotides and Nucleic Acids, 29:461–465, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257771003741406
DETERMINATION OF ADENOSINE DEAMINASE ACTIVITY IN DRIED
BLOOD SPOTS BY A NONRADIOCHEMICAL ASSAY USING
REVERSED-PHASE HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY
A. B. P. van Kuilenburg,1 L. Zoetekouw,1 J. Meijer,1 and T. W. Kuijpers2
1Academic Medical Center, Emma Children’s Hospital, Laboratory Genetic Metabolic
Diseases, Amsterdam, the Netherlands
2Academic Medical Center, Emma Children’s Hospital, Division of Pediatric Hematology,
Immunology and Infectious Diseases, Amsterdam, the Netherlands
Adenosine deaminase (ADA) deficiency is a rare metabolic disease causing severe combined
2
immunodeficiency (SCID). An assay to determine ADA activity in dried blood spots was developed
using reversed-phase HPLC. The assay was linear with reaction times up to at least 4 hours, and
protein concentrations up to at least 2.2 mg/ml. The intra-assay CV and the inter-assay CV for
the complete assay was 3.5 and 8.4%, respectively. The ADA activity in a control blood spot,
stored at 4◦C, remained stable for at least one year. Only a slightly decreased ADA activity (35 13
nmol/mg/h, n = 4) was observed in heterozygotes for a c.704G > A mutation in the ADA gene when
compared to that observed in controls (41 13 nmol/mg/h, n = 108). In addition, increased ADA
activity as found in a rare form of congenital anemia can be assessed, as observed in a bloodspot
from a patient diagnosed with Diamond Blackfan anemia (ADA activity 150 nmol/mg/h).
Keywords Adenosine deaminase; blood spots; SCID; DBA; ADA
INTRODUCTION
Adenosine deaminase (ADA) is an enzyme of the purine salvage path-
way that catalyses the conversion of (deoxy)adenosine to (deoxy)inosine.
Patients with ADA deficiency suffer from severe combined immunodefi-
ciency (SCID) in which all lymphoid lineages are affected.[1,2] In addition
to the profound lymphopenia, the toxic levels of deoxyadenosine and dATP
can give rise to hepatic, skeletal, neurological and behavioral abnormalities.
ADA deficiency is diagnosed by low ADA activity in red blood cell lysates.
In contrast, patients with Diamond Blackfan anemia (DBA), which is a rare,
Address correspondence to A. B. P. van Kuilenburg, Academic Medical Center, Emma Children’s
Hospital, Laboratory Genetic Metabolic Diseases, P.O. Box 22700, 1100 DE, Amsterdam, the Netherlands.
E-mail: a.b.vankuilenburg@amc.uva.nl
461

462
A. B. P. Van Kuilenburg et al.
genetically and clinically heterogeneous, inherited red cell aplasia, present
with strongly elevated ADA activity in their erythrocytes.[3] In this article, we
developed a fast and sensitive assay to measure ADA activity in dried blood
spots using reversed-phase HPLC.
MATERIALS AND METHODS
The blood spot ( 30 µl) was soaked in 500 µl 50 mM potassium phos-
phate (pH 7.4) for 90 minutes and the eluate was used for analysis of ADA
activity. The reaction mixture contained an aliquot of cell sample (12–220
µg), 50 mM potassium phosphate (pH 7.4) and 500 µM adenosine in a total
volume of 100 µl. The reaction was started by the addition of the sample.
After 2 hours incubation at 37◦C, the reaction catalyzed by ADA was ter-
minated by the addition of 4 µl of ice-cold 8 M HCLO4 and kept on ice
for 10 minutes. After centrifugation, the resulting supernatant was saved for
analysis by reversed-phase HPLC. Protein concentration in the supernatant
was determined by the copper-reduction method using bicinchoninic acid,
as described by Smith et al.[4]
The supernatant (100 µl) was injected into the HPLC system and sep-
aration of adenosine from inosine and hypoxanthine was performed using
a gradient from buffer A [50 mM KH2PO4 (pH 6.5)] to buffer B [50 mM
KH2PO4 (pH 6.5) and 20% (v/v) methanol] at a flow rate of 0.6 ml/min by
HPLC on a reversed-phase column (Phenomenex, Torrance, CA, USA; C18
Gemini, 150 × 4.6 mm, 3 µm particle size) and a guard column with online
UV detection at 260 nm. Quantification of the amounts of inosine and hy-
poxanthine was performed by comparison with an external standard. The
amount of both inosine and hypoxanthine produced were added together
to determine the amount of ADA activity.
DNA was isolated from EDTA-blood using the NucleoSpin Tissue kit
(Macherey-Nagel, GmbH & Co. KG, Du¨ren, Germany). PCR amplification of
all 12 coding exons and flanking intronic regions of the ADA gene was carried
out using intronic primer sets. Sequence analysis of genomic fragments
amplified by PCR was carried out on an Applied Biosystems model 3730
automated DNA sequencer using the dye-terminator method.
RESULTS AND DISCUSSION
In this study, we developed an accurate assay to assess ADA activity in
blood spots. Figure 1 shows that a complete baseline separation was ob-
tained within 40 minutes for adenosine, inosine and hypoxanthine and that
the amount of inosine and hypoxanthine produced by ADA and purine
nucleoside phosphorylase, respectively, from a blood spot was readily de-
tectable. The detection limit of inosine and hypoxanthine in the HPLC

Adenosine Deaminase Activity in Blood Spots
463
FIGURE 1 HPLC elution profile. The elution profile was obtained of a reaction mixture after incubation
of a control sample for 2 hours at 37◦C.
system, defined as three times the value of the baseline noise, was approx-
imately 3.1 pmol and 0.5 pmol, respectively. Figure 2 shows that the ADA
activity from a blood spot increased linearly with protein concentrations up
to 2.2 mg/ml and reaction times up to at least 4 hours. The steady-state
kinetics of ADA with adenosine showed an apparent Km value of 32 µM
(Figure 3). The intra-assay CV and the inter-assay CV for the complete as-
say, HPLC detection and protein determination, were 3.5% (n = 10) and
8.4% (n = 10), respectively. The ADA activity (35.7
3.3 nmol/mg/h,
CV = 9.3%) in a control blood spot, stored at 4◦C, remained stable for at
least one year.
Homozygosity for the c.704G > A mutation (p.R235Q) in the ADA gene
resulted in a near complete ADA deficiency.[2] In carriers for the mutation,
FIGURE 2 Protein dependence and time dependence of the ADA reaction. A) The amount of product
(inosine + hypoxanthine) at various protein concentrations in the assay. The reaction was allowed to
proceed for 2 hours at 37◦C. Each data point represents the mean of three experiments SD. B) The
amount of product (inosine + hypoxanthine) produced by ADA at various time points. The ADA activity
was measured at a protein concentration of 0.25 mg/ml.

464
A. B. P. Van Kuilenburg et al.
FIGURE 3 Steady state kinetics of ADA. The insert shows the double reciprocal plot of the reaction
velocity versus the concentration of adenosine.
only a slightly reduced activity was observed when compared to control values
(41 13 nmol/mg/h, n = 108; Figure 4). Strongly increased ADA activity (150
nmol/mg/h) was observed in a bloodspot from a neonate (11 months old)
diagnosed with DBA, as reported previously for erythrocyte lysates.[3] Thus,
our results indicate that the analysis of the ADA activity in blood spots can
be used to accurately determine enzymatic activity for diagnostic purposes.
FIGURE 4 ADA activity in controls, carriers and a DBA patient. The ADA activity in controls is depicted
as a box plot. The whiskers on the bottom extend from the 2.5th percentile and top 97.5th percentile.
The circles represent outliers. The carriers were heterozygous for the c.704G > A mutation (p. R235Q)
in the ADA gene. DBA, Diamond Blackfan Anemia.

Adenosine Deaminase Activity in Blood Spots
465
REFERENCES
1. Hershfield, M.S.; Mitchell, B.S. Immunodeficiency diseases caused by adenosine deaminase deficiency
and purine nucleoside phosphorylase deficiency. In: The Metabolic and Molecular Basis of Inherited Disease
(Scriver, C.R., Beaudet, A.L., Sly, W.S., Valle, D., eds.). New York: McGraw-Hill; 2001, pp. 2585–2625.
2. Hershfield, M.S. Genotype is an important determinant of phenotype in adenosine deaminase defi-
ciency. Curr. Opin. Immunol. 2003, 15, 571–577.
3. Willig, T.N.; Pe´rignon, J.L.; Gustavsson, P.; Gane, P.; Draptchinskaya, N.; Testard, H.; Girot, R.; Debre´,
M.; Ste´phan, J.L.; Chenel, C.; Cartron, J.P.; Dahl, N.; Tchernia, G. High adenosine deaminase level
among healthy probands of Diamond Blackfan anemia (DBA) cosegregates with the DBA gene region
on chromosome 19q13. Blood 1998, 92, 4422–4427.
4. Smith, P.K.; Krohn, R.I.; Hermanson, G.T.; Mallia, A.K.; Gartner, F.H.; Provenzano, M.D.; Fujimoto,
E.K.; Goeke, N.M.; Olson, B.J.; Klenk, D.C. Measurement of protein using bicinchoninic acid. Anal.
Biochem. 1985, 150, 76–85.