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Molecular Diagnostics and Genetics |
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.
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Abstract |
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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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Introduction |
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AMP is usually deaminated in most tissues by AMP deaminase and, to a lesser extent, by the ADA degradation pathway. Unlike AMP, dAMP is not a substrate for AMP deaminase and must first be broken down to dAdo, a substrate for ADA (1).
Lymphoid tissues exhibit high ADA activity, suggesting that ADA is the focus of toxic deoxyribonucleotide catabolism in the immune system. In patients homozygous for ADA deficiency, very little dAdo accumulates during cell turnover, being reconverted to dATP by adenosine kinase (EC 2.7.1.20) and deoxyadenosine kinase (EC 2.7.1.76) (1).
The biochemical diagnosis of ADA is essentially based on dATP concentrations in erythrocytes. Traces of dAdo in plasma and urine are a characteristic feature of severely affected infants (2). Patients affected by SCID can exhibit a considerable heterogeneity in biochemical, clinical, and immunologic findings (3); a dramatic reduction of ADA activity and high dATP concentrations in erythrocytes, however, represent a universal finding for homozygous ADA deficiency (4).
Heterozygosity for the defect can be difficult to detect because many obligate heterozygotes have ADA activity at the lower limit of the reference interval (5); affected individuals, however, do not exhibit appreciable ADA activity. Prenatal diagnosis is possible by use of chorionic villus sampling (6) and enzyme and nucleotide assays in fetal blood (5) and cultured amniotic cells.
The defect is confirmed by the undetectable ADA activity in lysed red cells and lymphocytes. S-Adenosyl-L-homocysteine hydrolase (SAHH; EC 3.3.1.1) activity is also reduced in lysed red cells through dAdo competitive inhibition, and its evaluation provides further confirmation. Evaluation of purine nucleoside phosphorylase (PNP; EC 2.4.2.1) activity is also important to exclude another type of SCID attributable to PNP deficiency. Moreover, the evaluation of plasma ADA activity is fundamental during enzyme replacement therapy based on polyethylene glycol (PEG)-modified ADA administration.
Various HPLC methods have been developed for research and diagnostic purposes (6), but two-dimensional thin-layer chromatography (7) is also widely used.
Capillary electrophoretic separation techniques, with their high separation efficiency, flexibility, and high sample throughput, can potentially be useful for the diagnosis of inborn errors of purine and pyrimidine metabolism (8)(9)(10)(11). Capillary electrophoresis (CE) is powerful and inexpensive in the analysis of nucleotides, nucleosides, and bases (12) and could therefore be useful in the diagnosis and monitoring of SCID.
Here we report the development and testing of two CE methods for the determination of ADA, SAHH, and PNP activity and for the determination of toxic metabolites.
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Materials and Methods |
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purine metabolites
Sample preparation: RBCs.
We homogenized 15 µL of packed RBCs with five volumes of 0.4 mol/L perchloric acid (PCA) in 0.5-mL tubes, using a nylon motor pestle. Extracts were then centrifuged (12 000g for 10 min) in a cooled microcentrifuge. The supernatants were neutralized with 2.7 mol/L KOH. At such small sample volumes, correct neutralization is a critical step. Neutralized samples are stable at -80 °C for several weeks (CV <5%). Potassium perchlorate was precipitated by freezing-thawing and subsequent centrifugation at 12 000g for 5 min, and the supernatant was collected. Aliquots of the extracts were analyzed by CE.
Sample preparation: plasma and urine.
Aliquots of plasma were nitrogen-stream concentrated to 50% of the original volume and deproteinized with PCA (final concentration, 0.8 mol/L). Extracts were then centrifuged (12 000g for 10 min) in a cooled microcentrifuge, and supernatants were neutralized with 2.7 mol/L KOH. Potassium perchlorate was removed, and aliquots were analyzed as for RBCs. We processed 100-µL aliquots of untreated urine as reported above.
CE analysis of purine compounds (method 1).
Analysis of the purine compounds dAdo, Ado, AMP, ADP, ATP, dAMP, dADP, dATP, Ino, hypoxanthine (Hyp), and xanthine (Xan) was performed with a Bio-Rad Biofocus 3000 instrument (Bio-Rad Laboratories) fitted with a Supelco bare CEIect-Fs column [60 cm x 75 µm (i.d.); Supelco Inc.], with the window at a distance of 55.5 cm. Borate buffer (30 mmol/L) containing sodium dodecyl sulfate (10 mmol/L) was used. Conditions were pH 9.80, 18 kV, and 3 psi * s hydrodynamic load at 25 °C. The electric field was 300.00 V/cm with a current of 
90 µA. Electroosmotic flow velocity was 0.131 cm/s (calculated using methanol as a neutral marker) and did not differ significantly between calibrator and samples. The results were read over the range 190–300 nm and analyzed at 254 nm. Between runs, the capillary was washed with 0.1 mol/L NaOH for 30 s, followed by running buffer for 60 s.
determination of enzyme activities
ADA assay in lymphocytes, fibroblasts, and amniocytes.
We homogenized 1 x 106 cells in 25 µL of lysis buffer (0.25 mol/L HEPES, 0.025 mol/L CHAPS, and 0.025 mol/L dithiothreitol) and centrifuged the cell lysate at 12 000g for 7 min. Protein content in the supernatant was measured by the Coomassie brilliant blue binding procedure with Bio-Rad protein reagent and crystalline bovine serum albumin as the calibrator.
The enzyme activity was evaluated in a mixture containing 50 mmol/L Tris (pH 7.2) and 0.4 mmol/L adenosine as reported previously (14). A cell extract equivalent to 10 µg of protein was added to the mixture. The reaction was incubated at 37 °C, with aliquots withdrawn at 0 and 5 min (30 or 60 min in the case of ADA/SCID patients), stopped by addition of PCA (final concentration, 0.21 mmol/L), and neutralized with KOH (final concentration, 0.22 mmol/L). We followed the reaction time-course by analyzing aliquots from each time point by the CE method after Ino plus Hyp formation. Enzyme activity was expressed as nmol · s-1 · (kg protein)-1.
ADA assay in erythrocytes.
We homogenized 20 µL of packed RBCs in 20 µL of lysis buffer. The cell lysate was centrifuged at 12 000g for 5 min. To the same incubation mixture as for lymphocytes, we added 10 µL of supernatant. Aliquots were withdrawn, and the reaction was stopped at 0 and 10 min (30 or 60 min in the case of ADA/SCID patients). Enzyme activity was measured as reported above and expressed as nmol · s-1 · (L packed RBCs)-1.
ADA assay in plasma.
A 20-µL aliquot of untreated plasma was added to the same incubation mixture as for lymphocytes. The reaction was stopped at 0 and 30 min (5 min in the case of ADA/SCID patients during PEG-ADA therapy). The enzyme activity was evaluated as for lymphocytes and expressed as nmol · s-1 · (L plasma)-1.
PNP assay in erythrocytes.
PNP activity was determined as a control in RBCs in a mixture containing 50 mmol/L Tris (pH 7.2), 1 mmol/L EDTA, and 0.4 mmol/L Ino, as reported previously (14). We added 10 µL of cell extract obtained as for ADA activity to the mixture. The reaction was stopped at 0 and 30 min. The enzyme activity was evaluated by CE after Hyp formation and expressed as nmol · s-1 · (L packed RBCs)-1.
SAHH assay in erythrocytes.
SAHH activity was determined as a control in RBCs in a mixture containing 50 mmol/L HEPES (pH 7.3), 10 µmol/L erythro-9-(2-hydroxy-3-nonyl)-adenine, 0.1 mmol/L Ado, and 2 mmol/L homocysteine (15). We added 50 µL of RBC extract to the mixture. Aliquots were withdrawn, and the reaction was stopped at 0 and 30 min. The enzyme activity was evaluated after Ado consumption and expressed as nmol · s-1 · (L packed RBCs)-1.
CE analysis of enzyme substrates and products (method 2).
For the determination ADA, PNP, and SAHH activity, we separated Ado, Ino, Hyp, and S-adenosylhomocysteine (SAH) using a Supelco bare CEIect-Fs column [42 cm x 75 µm (i.d.); Supelco Inc.] with the window at a distance of 37.5 cm. The results were read and analyzed at 254 nm. Borate buffer (20 mmol/L) was used. The conditions were pH 10.00, 12 kV, and 2 psi s hydrodynamic load at 25 °C. The electric field was 285.71 V/cm with a current of 75 mA, and the electroosmotic flow velocity was 0.143 cm/s.
statistical analyses
Differences between groups were analyzed by the Kruskal–Wallis test. Correlation analysis was performed with the Spearman test. Significant differences were assumed for P <0.05 and are indicated in the tables and figures.
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Results |
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). Separation efficiency was >80 000 theoretical plates for all target compounds, except for Xan, which had an efficiency of 44 000 theoretical plates. Under these conditions the electric field was 300 V/cm. The increase in temperature reduced the migration time of compounds and increased peak symmetry but did not permit good resolution of dATP and AMP. Increasing the voltage to 20–22 kV did not affect selectivity or resolution but increased baseline noise, which was associated to a loss of sensitivity. Limits of detection of individual analytes were determined by injecting serial dilutions of stock solutions of known concentrations until the resulting electropherograms produced peaks with a signal-to-noise ratio of 2. Even at this low signal-to-noise ratio, the reproducibility showed a CV <6% for all peaks. There were no statistical differences between reproducibility evaluated at a signal-to noise ratio of 2 with respect to that evaluated at a signal-to noise ratio of 3.
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The range of concentrations was 2 µmol/L to 0.5 mmol/L for nucleotides, nucleosides, and bases. In this concentration range, the correlation coefficient (corrected peak area vs concentration) exceeded 0.99 for all compounds with a 3 psi * s injection (equivalent to 26.50 nL of aqueous solution injected, based on Poiseuille’s equation). Table 1
shows the regression equations and the limits of detection of individual analytes.
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The method had a very good reproducibility for migration times and individual peak areas in consecutive runs. Intraassay reproducibility was evaluated over 10 injections of a 0.05 mmol/L calibrator mixture; the highest CV for the peak areas was 2.7%, compared with an interassay CV of 3.6%.
The complete electropherograms of the intracellular nucleotides, deoxynucleotides, nucleosides, and bases in RBCs from healthy individuals and ADA/SCID patients are reported in Fig. 1
. The major intracellular compounds were identified by use of addition experiments, i.e., co-injecting reference nucleotides and observing their comigration. They were also identified by on-run spectral analysis; we found no interfering compounds.
As shown in Fig. 1C
, the method enabled evaluation of key diagnostic metabolites (dAMP, dADP, and dATP) in erythrocytes. Sampling variability was evaluated over eight acid extractions from the same ADA/SCID erythrocyte sample. Compounds with a concentration >30 µmol/L (ATP, ADP, dADP, dATP) had a CV in the range 7–16%. The other compounds had a CV in the range 11–18%. The CV of nucleotides were similar to those reported by other authors, using an HPLC method (16). Recovery of the compounds was measured by use of erythrocyte samples alone and samples supplemented with authentic standard (10 nmoles) followed by PCA extraction. The mean (SD) recovery was 94.21 (5.34) µmol/L. The order of migration was reproducible under the conditions described above. The limit of quantification was calculated as the minimum concentration in the sample that gave a signal at least two times higher than average noise, which represented the smallest peak that could be detected. For dATP, the key diagnostic metabolite, the limit of quantification corresponded to a concentration of 3.48 µmol/L of packed RBCs.
The method was also used to evaluate dAdo in plasma and urine of the patients. dAdo is readily detected together with major ultraviolet-absorbing compounds in the plasma and urine of affected individuals (Fig. 2
). There were some unidentified peaks that will be the subject of future study. The limits of quantification were 3.86 and 4.65 µmol/L in plasma and urine, respectively.
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Shown in Table 2
are the concentrations of the nucleotides, nucleosides, and bases and the corresponding deoxy forms per liter of packed erythrocytes in healthy individuals and ADA/SCID patients at diagnosis and during enzyme replacement therapy. We observed no significant differences in Ado nucleotides (AMP, ADP, ATP) or Ino, Hyp, and Xan. Ado was present in very high amounts in some patients at diagnosis or during therapy (300–400 µmol/L), and this accounted for the high SD reported. Deoxynucleotides are undetectable in healthy individuals, and fall almost to reference values in patients undergoing gene therapy and/or PEG-ADA treatment.
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More than 200 erythrocyte samples from the ADA/SCID patients were analyzed for deoxynucleotides during various therapy protocols. dATP, dADP, and dAMP concentrations may vary in relation to the degree of gene defect and PEG-ADA and/or gene therapy (17) (see also Figs. S-1A and S-1B in the Data Supplement that accompanies the online version of this article athttp://www.clinchem.org/content/vol49/issue11/). dAXP concentrations in ADA/SCID patients ranged from 450 µmol/L before therapy to almost undetectable after gene therapy (see Fig. S-1B in the online Data Supplement).
enzyme activities
ADA was evaluated as a marker of current pathology, whereas PNP activity was measured to exclude immunodeficiency attributable to PNP deficiency. SAHH activity was evaluated as a confirmatory test because it is inhibited by dAdo. These activities were determined on the basis of substrate consumption and product formation, with CE separation. We slightly modified our previous CE methods (18), shortening capillary length and lowering the voltage. Readings were made at 254 nm.
The method was linear from 2 µmol/L to 0.5 mmol/L. The nucleotides were identified by comparison of retention times with internal standards run simultaneously. Concentrations of compounds were determined based on peak areas of known compound concentrations. The method separated substrates and reaction products (Ado, Ino, Hyp, and SAH) in 8 min (Fig. 3
) with a separation efficiency >50 000 theoretical plates for the mixture of target compounds. Table 3
shows the linear regression curves and limits of detection for the analytes.
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The correlation coefficient (peak area vs concentration) exceeded 0.998 for all compounds over the range 2 µmol/L to 5 mmol/L for a 2 psi s injection (equivalent to 25.24 nL of aqueous solution injected, by Poiseuille’s equation). Intra- and interassay CV were lower than those for the method used for analysis of purine metabolites.
Xan did not form in the ADA and PNP assays (Fig. 3
). The linearity of reaction product formation with respect to incubation time was checked for all activities (see Fig. S-2A in the online Data Supplement) as was the linearity of total activity with respect to sample amounts in the incubation mixture (see Fig. S-2B in the online Data Supplement). The lower correlation coefficient was 0.9958, obtained for SAHH by plotting sample amounts in microliters of packed RBC vs total activity in pmol/s.
Blank controls were run for all activities to exclude spontaneous substrate degradation. Precision was also tested as reported in Table S-1 of the online Data Supplement. The minimum detectable enzyme activity in patients was 1.9 nmol · s-1 · L-1 for packed RBCs and 2.0 nmol · s-1 · kg-1 for peripheral blood lymphocytes obtained for ADA determinations. SAHH showed a minimum detectable activity of 5.6 nmol · s-1 · (L packed RBCs)-1.
The ADA, PNP, and SAHH activities in RBCs from ADA/SCID patients and healthy individuals, expressed in terms of reaction product formation, are shown in Table 4
. In healthy individuals we obtained activities similar to those obtained by other authors with different methods (19)(20)(21).
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In ADA/SCID patients, ADA activity was much reduced in RBCs at diagnosis, whereas PNP activity was within reference values. SAHH activity was as low as 4% relative to that of healthy individuals (Table 4
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ADA activity and dAXP concentrations in RBCs at diagnosis were in the range 0–138.8 nmol · s-1 · L-1 and 118–450 µmol/L, respectively. ADA activity in plasma ranged from 0 to 58.3 nmol · s-1 · L-1 (Table 5
). During enzyme replacement therapy with PEG-ADA, ADA activity ranged from undetectable to 14 555 nmol · s-1 · (L plasma)-1. In two patients (patients 1 and 2) who underwent stem-cell gene therapy, ADA activity in the plasma increased from 36.1 to 225.1 and from 30.5 to 83.3 nmol · s-1 · L-1, respectively. This increase was parallel to the decrease in toxic dAXP metabolites in RBCs (10% and 40% of the initial value for patients 1 and 2, respectively) down to values similar to those found in patients receiving successful bone marrow transplants (22). dAXP concentrations showed negative correlation with ADA activity in RBCs when the data for ADA/SCID patients were plotted (see Fig. S-1A in the online Data Supplements), but we found no correlation with AXP content (data not reported). Deoxynucleotides in RBCs decreased to almost undetectable concentrations when ADA activity approached 550 nmol · s-1 · L-1 (see Fig. S-1B in the online Data Supplement).
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Diagnosis can also be based on ADA activity in fibroblasts and peripheral blood lymphocytes (see Table S-2 in the online Data Supplement). Cultured amniocytes and chorionic villus can be analyzed for prenatal diagnosis. ADA activity in amniocytes of heterozygotes was 50% lower than that of healthy individuals, whereas in chorionic villus samples from ADA/SCID patient we found an activity of 55.9 nmol · s-1 · (kg protein)-1 vs 333.2 (12.5) nmol · s-1 · kg-1 for healthy individuals (see Table S-2 in the online Data Supplement), which is very close to the value of 358.8 (96.3) nmol · s-1 · kg-1 reported by other authors (23). Diagnoses based on biochemical data were confirmed by DNA analysis for all of the ADA/SCID patients in our study. In five patients with symptoms of immunodeficiency, biochemical and DNA analysis both excluded ADA/SCID.
When we measured ADA activity in various lymphocyte populations to determine the concentrations and stability of the vector ADA gene expression, unselected T-cell lines exhibited a mean (SD) value of 1112 (367) nmol · s-1 · kg-1 (n = 4), G418R T cells had a mean activity of 2694 (1322) nmol · s-1 · kg-1 (n = 3), and T-cell clones had a mean activity of 3527 (2352) nmol · s-1 · kg-1 (n = 7). In the healthy donor T-cell lines, it was 3000 (1890) nmol · s-1 · kg-1 (n = 4).
CE enabled complete screening of gene therapy protocols, revealing restoration of intracellular ADA activity in RBCs and various lymphocyte populations. ADA activity in RBCs rose from undetectable to 20–30% of the values in healthy controls as toxic deoxynucleotides fell (see Fig. S-1B and Table S-3 in the online Data Supplement).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5–15 µmol/L of packed erythrocytes or to 1% of the total erythrocyte adenine nucleotide (ATP + dATP) content. From these considerations arises the importance of having at hand fast and reliable analytical tools for evaluation of both toxic metabolites and enzyme activity. This is the first report on methodologies for complete testing of ADA-deficient patients, for diagnosis of ADA deficiency, and for monitoring of gene and enzyme replacement therapy. The CE methods described here were extremely adaptable for evaluation of nucleotide, nucleoside, and base concentrations in biological samples, making it possible to evaluate the corresponding enzyme activities. Our results indicate that these separation procedures offer the possibility of determining di- and triphosphate nucleosides, nucleosides, and their deoxynucleosides without any chemical manipulation of samples except PCA deproteinization. Hence the present CE assays minimize the risk of modification or loss of metabolites, making it possible to obtain the complete pattern of metabolites and related enzyme activities known to be markers of SCID that are also involved in other inherited disorders of purine metabolism, energy metabolism, and DNA and RNA synthesis. CE is also particularly versatile, allowing shifts from one separation method to another in a few minutes with limited reagent consumption (3 mL every 20–30 runs) and, theoretically, indefinite column life. After 550 runs with the same capillary for enzyme determinations, we found minimal variation in separation efficiency (CV <5%).
In conclusion, the present methodologies enable complete characterization of ADA deficiency/SCID for diagnostic purposes and could potentially be useful for monitoring enzyme-replacement and gene therapies. Moreover, these assays provide an additional tool to explore the transfection efficiency in the design of new retroviral vectors and to fully evaluate the ADA gene expression in transduced cells.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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evèík J, Adam T, Mazáová H. A fast and simple screening method for detection of 2,8-dihydroxyadenine urolithiasis by capillary zone electrophoresis. Clin Chim Acta 1996;245:85-92.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
evèík J, Adam T, Sázel V. A fast and simple screening method for detection of orotic aciduria by capillary zone electrophoresis. Clin Chim Acta 1997;259:73-81.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
X174 in patients with adenosine deaminase deficiency. Blood 1992;80:1163-1171.The following articles in journals at HighWire Press have cited this article:
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  B. Cassani, M. Mirolo, F. Cattaneo, U. Benninghoff, M. Hershfield, F. Carlucci, A. Tabucchi, C. Bordignon, M. G. Roncarolo, and A. Aiuti Altered intracellular and extracellular signaling leads to impaired T-cell functions in ADA-SCID patients Blood, April 15, 2008; 111(8): 4209 - 4219. [Abstract] [Full Text] [PDF]
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A. Mortellaro, R. J. Hernandez, M. M. Guerrini, F. Carlucci, A. Tabucchi, M. Ponzoni, F. Sanvito, C. Doglioni, C. D. Serio, L. Biasco, et al. Ex vivo gene therapy with lentiviral vectors rescues adenosine deaminase (ADA)-deficient mice and corrects their immune and metabolic defects Blood, November 1, 2006; 108(9): 2979 - 2988. [Abstract] [Full Text] [PDF]
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