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Clinical Chemistry 52: 240-247, 2006. First published December 29, 2005; 10.1373/clinchem.2005.059501
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(Clinical Chemistry. 2006;52:240-247.)
© 2006 American Association for Clinical Chemistry, Inc.

Drug Monitoring and Toxicology

Measurement of Erythrocyte Inosine Triphosphate Pyrophosphohydrolase (ITPA) Activity by HPLC and Correlation of ITPA Genotype-Phenotype in a Caucasian Population

Maria Shipkova1,a, Kristin Lorenz1, Michael Oellerich2, Eberhard Wieland1 and Nicolas von Ahsen2

1 Central Institute of Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Stuttgart, Germany.
2 Department of Clinical Chemistry, Georg-August-University, Göttingen, Germany.

aAddress correspondence to this author at: Central Institute of Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Kriegsbergstrasse 60, D-70174 Stuttgart, Germany. Fax 49-278-4809; e-mail m.shipkova{at}klinikum-stuttgart.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Inosine triphosphate (ITP) pyrophosphohydrolase (ITPA) catalyzes the pyrophosphohydrolysis of ITP/dITP and xanthosine triphosphate to prevent incorporation of unusual nucleotides into RNA and DNA. Important mutations leading to enzyme deficiency are 94C>A and IVS2 + 21A>C. An association between ITPA 94C>A and adverse reactions during azathioprine treatment has been shown. To investigate the ITPA phenotype, an HPLC procedure was developed and phenotype-genotype correlations were assessed.

Methods: The enzymatic conversion of ITP to inosine monophosphate (IMP) was terminated by perchloric acid and saturated dipotassium hydrogen phosphate. We quantified the IMP at 262 nm after separation on an Aqua perfect C18 column using 20 mmol/L phosphate buffer, pH 2.5. We also genotyped samples for ITPA 94C>A and IVS2 + 21A>C by real-time fluorescence PCR.

Results: The assay was linear to 3 mmol/L IMP [~500 µmol/(g Hb · h)] with a lower limit of quantification of 4 µmol/L [~0.5 µmol/(g Hb · h)]. With IMP-enriched samples, within- and between-day imprecision was ≤3.6% and ≤4.9%, respectively, and the inaccuracy was ≤5.2%. With pooled erythrocytes, within- and between-day imprecision was 3.8% and 7.5%, respectively. ITPA activity in 130 healthy controls was between <0.5 and 408 µmol IMP/(g Hb · h). Mutant allele frequencies were 0.062 (94C>A) and 0.131 (IVS2 + 21A>C). When we used a cutoff of 125 µmol IMP/(g Hb · h), phenotyping detected all 94C>A mutant cases, all 94C>A and IVS2 + 21A>C compound heterozygotes, all IVS2 + 21A>C homozygotes, and 6 of 24 IVS2 + 21A>C heterozygote-only cases. A novel IVS2 + 68T>C mutation was also found.

Conclusions: The HPLC procedure provides an excellent ITPA phenotype-genotype correlation and led to the discovery of a novel IVS2 + 68T>C mutation. The method could facilitate investigation of the role of ITPA activity for drug toxicity during thiopurine therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The thiopurine drugs 6-mercaptopurine and azathioprine (AZA) 1 are widely used in the treatment of patients suffering from various diseases, including childhood acute leukemia, inflammatory bowel disease, severe rheumatoid arthritis, and autoimmune hepatitis, as well as for the prevention of acute rejection after organ transplantation (1)(2). A large body of evidence suggests that intolerance to therapy is mediated in part by pharmacogenetically relevant polymorphisms in thiopurine S-methyltransferase (TPMT; EC 2.1.1.67), an enzyme that catalyzes the conversion of thiopurines to nontoxic or less toxic methylated compounds, as opposed to their metabolic activation to cytotoxic 6-thiopurine nucleotides (see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.content/vol52/issue2) (2)(3)(4)(5)(6). Individuals with low or undetectable TPMT activity are at high risk for developing life-threatening myelosuppression at standard doses of thiopurine drugs (7)(8). Patients with high TPMT activity may experience treatment failure (9)(10) or hepatotoxicity(11), but the situation is much less clear than for the TPMT deficiencies (12)(13)(14). In addition, an estimated 10% to 30% of patients cannot tolerate AZA therapy because of nonmyelosuppression adverse reactions, such as influenza-like symptoms, nausea, vomiting, hepatotoxicity, pancreatitis, fever, or rash (1)(12)(15)(16). Two recent studies with patients suffering from chronic inflammatory disease, a retrospective study by Marinaki et al. (17) and a prospective one by von Ahsen et al. (16), have provided evidence that deficiency in another polymorphic enzyme—inosine triphosphate (ITP) pyrophosphohydrolase (ITPA; EC 3.6.1.19)—could represent a further pathomechanism for thiopurine side effects in addition to TPMT. ITPA, a cytosolic enzyme present in many tissues (e.g., erythrocytes, leukocytes, heart, liver, endocrine glands), catalyzes the pyrophosphohydrolysis of ITP/dITP and xanthosine triphosphate, thereby preventing accumulation of unusual nucleotides in cells and incorporation into RNA and DNA. The most important mutations leading to enzyme deficiency are 94C>A and IVS2 + 21A>C (18). The ITPA 94C>A allele frequency varies between 0.01 and 0.19 in different world ethnic populations and is ~0.07 among Caucasians (19)(20). Individuals who are homozygous for this polymorphism are completely deficient of erythrocyte ITPA activity, whereas heterozygotes have a mean ITPA activity that is 22.5% of the "normal" mean activity, consistent with a dimeric structure of the enzyme (18). ITPA activity in heterozygous carriers of the intron IVS2 + 21A>C mutation averages 60% of the control mean, whereas homozygous carriers of this mutation have activity similar to that in heterozygous carriers of the ITPA 94C>A polymorphism. The IVS2 + 21A>C mutation was shown to act on splicing efficiency (21). Compound heterozygotes for 94C>A and IVS2 + 21A>C have 10% remaining ITPA activity (18). The frequency of the intron mutation (~0.13) is higher than that of the 94C>A polymorphism, and compound heterozygotes would be expected to occur with a higher frequency than 94C>A homozygotes (18). It has been suggested that ITPA deficiency may have pharmacogenomic implications, and the abnormal metabolism of 6-mercaptopurine and AZA in affected patients may lead to toxicity (18)(22)(23). Indeed, as mentioned above, Marinaki et al. (17) found that the ITPA 94C>A polymorphism was significantly associated with flu-like symptoms, rash, and pancreatitis under AZA therapy. Using data from a prospective study, von Ahsen et al. (16) reported that drop-outs during the first 2 weeks of AZA therapy because of side effects were significantly more frequent in carriers of this mutant allele. In contrast, other case–control studies could not confirm these findings in different settings (24)(25)(26). All of these studies involved only genotyping. Functional studies to confirm that patients suffering from adverse effects indeed have decreased ITPA activity might be useful to investigate the relevance of introducing ITPA testing into clinical practice.


Figure 1
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  		Figure 1. Representative chromatograms.

(A), reagent blank run (without cell lysate); (B), erythrocyte lysate with ITPA activity of 229 µmol IMP/(g Hb · h); (C), respective sample blank run (without ITP).

To enable such studies, and to investigate the role of ITPA testing for clinical laboratory practice, we developed and validated a new HPLC procedure for the determination of the enzyme activity in isolated erythrocytes. The method uses fast sample preparation and a short run time, which are important advantages when large batches of samples must be analyzed. A phenotype-genotype correlation study was carried out with 130 samples from local (Caucasian) blood donors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
study samples
Anonymous EDTA-anticoagulated blood samples from local Caucasian blood donors (65 female, 65 male) were kindly provided by the local blood bank service. This study was approved by the Institutional Review Board (no. 13/10/04, medical faculty, University Göttingen).

reagents and chemicals
ITP, D,L-dithiothreitol (DTT), and Hanks balanced salt solution were supplied by Sigma. IMP was obtained from Fluka. Acetonitrile (HPLC grade), phosphoric acid, Tris, KH2PO4, K2HPO4, MgCl2, and perchloric acid were purchased from Merck.

Stock solutions of ITP (40 mmol/L) and IMP (18 mmol/L) were prepared in distilled water. Both solutions were stored at –20 °C. The solutions of Tris (100 mmol/L, adjusted to pH 9.0 with 1 mmol/L hydrochloric acid), MgCl2 (1 mol/L), and perchloric acid (4 mol/L) were stored at 4–8 °C, whereas the saturated K2HPO4 solution was kept at ambient temperature. The DTT solution (10 mmol/L) and the DTT–MgCl2–Tris mixture (4.5, 0.3, and 0.3 mL, respectively, prepared from the solutions described above) were always prepared immediately before use.

preparation of erythrocyte lysates
Blood samples (5 mL) were collected in EDTA tubes. Erythrocytes were isolated as described previously (27). After determination of the hemoglobin concentration (K4500; Sysmex), we portioned the isolated erythrocytes into 200-µL aliquots and stored them at –20 °C until analysis. Erythrocytes obtained from venous blood of healthy volunteers were used for preparation of calibrator and quality-control samples. For this purpose, only anonymous excess material from blood samples sent to our laboratory for routine analysis was used to perform all investigations; therefore, according to the guidelines of the local ethics committee, approval of the Institutional Review Board and/or donor written informed consent were not required.

incubation conditions for ITPA phenotyping
For our new method to assess ITPA activity, we adopted the incubation conditions previously described by Duley et al. (28), but we used a new, faster, and simpler procedure to stop the incubations. For analysis, erythrocyte aliquots of 200 µL were resuspended in 5 volumes (6-fold dilution) of ice-cold distilled water (this step led to the lysis of the erythrocytes). The lysate was centrifuged (13 000g for 10 min), and 25 µL of supernatant was placed in a 1.5-mL polypropylene tube with a mixture of 170 µL of DTT–MgCl2–Tris (see above). After vortex-mixing, the tubes were preincubated for 5 min at 37 °C. The reaction was then started by addition of 10 µL of ITP solution (40 mmol/L). Thus, the incubation mixture consisted of erythrocyte lysate as well as ~1.95 mmol/L ITP, 7.32 mmol/L DTT, 48.8 mmol/L MgCl2, and 4.88 mmol/L Tris (final concentrations). The tubes were incubated for 15 min at 37 °C, and the ITPA reaction was stopped by addition of 20 µL of 4 mol/L perchloric acid and incubation of the samples on ice for 10 min after vortex-mixing. This step was followed by addition of 30 µL of saturated dipotassium hydrogen phosphate solution to neutralize the perchloric acid. After vortex-mixing and centrifugation at 13 000g for 10 min, 200 µL of the supernatant was transferred into a 0.5-mL tube, and 20 µL was injected into the HPLC system. A sample blank containing all reagents except ITP was run for each sample. The sample blank was treated with perchloric acid and dipotassium hydrogen phosphate solution immediately after the preincubation step. In addition, a reagent blank containing all reagents, but no sample, was incubated with each batch. This was done to verify the quality of the ITP solution, which is known to possess a limited stability (29). ITPA activity was expressed as µmol of IMP formed per gram of hemoglobin per hour [µmol/(g Hb·h)] after correction for the actual sample hemoglobin concentration and the incubation time.

linearity of the enzyme reaction
To confirm the linearity of the enzyme reaction under the incubation conditions described above, IMP formation was measured in the presence of various concentrations of ITP (0.0025–2 mmol/L). Michaelis–Menten constants (Km) and maximum formation rates (Vmax) were determined by Lineweaver–Burk plot analysis. In addition, the linearity of the reaction was studied at 5 time points from 0 to 30 min, using 25 µL of lysate (6-fold sample dilution), and at a fixed time of 15 min with various enzyme concentrations (3- to 30-fold dilutions of the isolated erythrocyte samples). All experiments were performed in duplicate.

chromatographic conditions
The HPLC system (Dionex) consisted of a chromatographic pump (Model P680), an automatic injector (ASI 100), a diode array detector (UVD 340U), and a computer interface system controller linked to a PC. The HPLC separation was performed on a C18 Aqua perfect column (5 µm; 4.0 x 250 mm; MZ Analysentechnik), protected by a Merck Li Chrospher 60 RP-select B guard column. The column was maintained at 35 °C to improve separation. The mobile phases consisted of solution A (20 mmol/L phosphate buffer, pH 2.5) and solution B [700 mL of acetonitrile and 300 mL of phosphate buffer, pH 2.5 (20 mmol/L final concentration)], which formed the following gradient: 0–5.0 min, 0% B; 5.0–5.1 min, 0%–100% B; 5.1–8.1 min, 100% B; 8.1–8.2 min, 100%–0% B. The flow rate was 1.1 mL/min. Solution A was used for chromatographic separation, whereas solution B served as the wash step. The detection wavelength was set at 262 nm. Chromeleon software, Ver. 6.5 (Dionex-Gynkotek), was used for recording and calculating the data and also for recording the ultraviolet spectra when required. Calculations were made in the external standard mode, using peak-area ratios.

calibration and control samples
Calibration and quality-control samples for the HPLC procedure for IMP determination were prepared by adding an IMP stock solution (18 mmol/L) to erythrocyte lysates. The concentration of the calibrator was 0.9 mmol/L IMP [ITPA activity ~140 µmol/(g Hb · h)], whereas the concentrations of the control samples were 0.063, 0.54, and 1.8 mmol/L IMP [~10, 90, and 300 µmol/(g Hb · h)], respectively. The values were corrected for endogenous IMP. No ITP was added to these samples, and they were treated with perchloric acid and dipotassium hydrogen phosphate solution without incubation. For control of the complete procedure (enzyme reaction plus stopping step), 1 erythrocyte pool with an ITPA activity of 211 µmol/(g Hb · h) was included in every incubation batch.

assessment of performance characteristics
The linearity of the method was assessed by use of erythrocyte lysates with IMP added to yield concentrations between 4 µmol/L and 3 mmol/L (7 different concentrations; n = 3 for each concentration), equivalent to an ITPA activity range of ~0.5 to 500 µmol/(g Hb · h). The detection limit was calculated using a signal-to noise ratio of 3, the lower limit of quantification using a signal-to noise ratio of 6. For this purpose, the noise signal of the baseline was obtained from segments of the chromatogram that preceded the IMP peak. The within-run and between-run imprecisions were studied for erythrocyte lysates to which IMP had been added to yield concentrations of 0.063, 0.54, and 1.8 mmol/L, and for the erythrocyte pool with a mean ITPA activity of 211 µmol/(g Hb · h), as described above. Extraction efficiency was calculated by comparing the peak areas obtained from erythrocyte lysates with added IMP (7 different concentrations over the range between 4 µmol/L and 3 mmol/L; n = 3 for each concentration) to the peak areas obtained in solutions containing the same amount IMP in mobile phase A, which were directly injected on the column without further sample preparation. To obtain information on the accuracy of the method, we calculated the recovery by comparing the measured concentrations with the expected concentrations using the 3 IMP-enriched quality-control samples described above.

stability of ITPA activity in erythrocytes
To investigate the stability of ITPA activity in frozen erythrocytes (–20 °C) over time, 3 erythrocyte pools were prepared. Each pool was divided into 5 aliquots, 4 of which were stored at –20 °C. The first aliquot was investigated on day 1 (reference activity), the other aliquots were analyzed on day 15 and at month 1, month 3, and month 4. In addition, to obtain information about storage conditions, which must be considered if transport of the sample is needed before ITPA analysis, each of 3 additional EDTA-blood pools were divided into 2 aliquots, one of which was used immediately for erythrocyte isolation and refrigeration, whereas the second was used after 3 days of storage at room temperature. The samples were assayed in duplicate.

ITPA genotyping
Genotyping of ITPA mutations 94C>A and IVS2 + 21A>C was performed as described previously (21) with some modifications. To include the IVS2 + 68 polymorphic site, new amplification primers were used in an asymmetric PCR [Exon2_for (0.2 µM), 5'-GTG GCA CAG AAA ATT GAC CG-3'; Exon3_rev (0.5 µM), 5'-CGG CTC CCC CTG GTA CTC C-3'], which gave a 134-bp product. In addition to the previously described IVS2 + 21 detection probe and anchor (21), we added a single detection probe [IVS2 + 68_WT (0.2 µM), 5'-CTG TGA CCT GAC TTT CTG TGT GTC TGT TT-3'-fluorescein]. The detection probe uses guanosine quenching and functions without an adjacent anchor (30). The IVS2 detection probes were multiplexed in a single PCR reaction and gave melting points at 48 °C (IVS2 + 21 WT), 55 °C (IVS2 + 21C), and 66 °C (IVS2 + 68 WT). Samples displaying additional melting peaks were cloned into the pGEM-T vector (Promega), and plasmid DNA from several clones was sequenced (fmol DNA Cycle SequencingSystem; Promega) on an automated DNA sequencer (Licor).

statistics
Using Lineweaver–Burk plots, we estimated graphically the apparent Michaelis–Menten constants, maximum velocity (Vmax), and affinity constant (Km). We used the D’Agostini–Pearson test to assess deviation from the gaussian distribution (MedCalc Software). Between-group comparisons (Mann–Whitney U-test) and the Jonckheere–Terpstra test for trend-by-genotype were carried out with SPSS 12 for Windows. The 95% confidence interval (CI) for the single binomial parameter was calculated according to Wilson (31).

To establish the stability of ITPA activity in erythrocytes, we calculated critical differences (dk) according to the formula dk = 2 x {surd}2 x S, with S being the standard deviation of the method from day to day. Values were considered significantly different if the absolute difference between 2 values, x1 and x2, was greater than dk |x1 - x2| > dk) (32).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Representative chromatograms of a reagent blank run, an erythrocyte lysate with ITPA activity of 229 µmol/(g Hb · h), and the respective sample blank run are shown in panels A, B, and C, respectively, of Fig. 1Up . The IMP was eluted from the column as a baseline-separated symmetric peak with a retention time of 6.0 min, which facilitated its subsequent quantification. No chromatographic interference was seen during the analysis of samples from 130 healthy blood donors or during the analysis of samples from 111 patients under AZA therapy (data not shown). A wash step using a second mobile phase containing 700 mL/L acetonitrile was included to achieve a short chromatography time and to avoid interference from elution of late peaks originating from the preceding chromatographic run. Thus, the full chromatographic run, including reequilibration, was completed within 11 min.

The assay was linear up to an IMP concentration of 3000 µmol/L [r >0.999, Sy|x = 0.0498; y = 11.52 (0.018)x + 0.0044 (0.020), where y is the peak area and x is the IMP concentration (values in parentheses are the SE)], which is approximately equivalent to an ITPA activity of 500 µmol/(g Hb · h). The detection limit was 2 µmol/L IMP, the lower limit of quantification was 4 µmol/L IMP [ITPA activity ~0.5 µmol/(g Hb · h)], and the median extraction efficiency of the compound was 85% (range, 73%–92%). The within- and between-run imprecision and the analytical recovery data are given in Table 1 .


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  		Table 1. Imprecision and analytical recovery of the HPLC assay for the quantification of ITPA activity in erythrocyte lysate.

We assessed the influence of the substrate concentration on ITPA activity, using 7 concentrations of ITP (0.0025–2 mmol/L). The apparent Km was 175 µmol/L, and the maximum velocity (Vmax) was 9.1 µmol/L · min. Therefore, the saturating concentration of ITP used (>10 times the Km) in all subsequent experiments was 1.95 mmol/L (see Materials and Methods). The rate of ITPA reaction was linear (r = 0.985; P <0.001) up to 20 min: y = 0.0844x – 0.0408, where y is the IMP concentration and x is time in minutes. With an incubation time fixed at 15 min, the reaction rate was linearly (r = 0.992; P <0.001) associated with the enzyme concentration (3- to 30-fold sample dilution): y = 1.3194x + 0.0254, where y is the IMP concentration and x is the enzyme concentration.

We observed that precipitated and neutralized supernatants were stable up to 10 h at room temperature on the auto sampler (data not shown). More than 800 chromatographic runs were achieved with a single analytical column and without any deterioration of the separation performance.

There was no significant decrease in the ITPA activities of all 3 erythrocyte pools studied [initial values, 169.6, 143.0, and 217.0 µmol/(g Hb · h)] when they were stored at –20 °C for up to 4 months [final values, 155.0, 135.9, and 188.7 µmol/(g Hb · h)]. In addition, storage of EDTA-blood samples at room temperature for 3 days before erythrocyte isolation did not lead to significant changes in the ITPA activity measured, when compared with the isolation procedure performed immediately after the blood collection (activity changes ≤3.5%).

The method was used to analyze ITPA activity in blood samples collected from 130 Caucasian blood donors (Table 2 ; 65 female, 65 male). The median activity was significantly lower in females [187.4 µmol IMP/(g Hb · h)] compared with males [255.6 µmol IMP/(g Hb · h); P = 0.002, Mann–Whitney U-test]. The frequency distribution of ITPA activity is shown in Fig. 2 . The values determined ranged between <0.5 and 408.3 µmol IMP/(g Hb · h), with a median of 219.1 µmol IMP/(g Hb · h). The histogram gives the impression of a bimodal or even trimodal distribution. When assessed by the D’Agostini–Pearson test, the activity distribution for the whole population was not significantly different (P = 0.130) from the gaussian distribution or from the female subgroup (P = 0.653). The male subgroup distribution was almost nongaussian (P = 0.057).


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  		Table 2. Median (range) ITPA activities observed in healthy blood donors stratified by genotype and sex.1


Figure 2
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  		Figure 2. Frequency distribution plot of erythrocyte ITPA activity in 130 Caucasian blood donors stratified according to sex.

On stratification according to ITPA 94C>A and IVS2 + 21A>C genotypes, we observed an excellent phenotype-genotype correlation (Fig. 3A ). The Jonckheere–Terpstra test was highly significant (P <0.001) for a trend by genotype. A single case displayed a suspicious broadened melting transition at 64 °C with the IVS2 + 68 detection probe. Sequencing revealed the presence of a novel IVS2 + 68T>C mutation (Fig. 3B ). The case was heterozygous for this mutation and wild type for ITPA 94C>A and IVS2 + 21A>C. The corresponding ITPA activity was 110.7 µmol IMP/(g Hb · h), ~60% of the median female wild-type ITPA activity. In the remaining 129 cases, we observed 114 ITPA 94C>A wild-type, 14 heterozygous, and 1 homozygous individual. The latter had an ITPA activity below the assay limit of detection. The ITPA 94C>A genotypes were in Hardy–Weinberg equilibrium (P = 0.448). We observed 98 ITPA IVS2 + 21A>C wild-type, 28 heterozygous, and 3 homozygous cases. The ITPA IVS2 + 21A>C genotypes were also in Hardy–Weinberg equilibrium (P = 0.559).


Figure 3
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  		Figure 3. Genotype-phenotype results stratified by ITPA 94C>A and IVS2 + 21A>C genotypes.

(A), genotype-phenotype correlation for 130 Caucasian blood donors. The gray arrowhead indicates a single case with a novel ITPA IVS2 + 68T>C mutation. The gray line represents the suggested cutoff activity of 125 µmol/(g Hb · h). (B), sequencing of plasmid DNA with the novel mutation indicated by a gray arrowhead. Black arrow, direction of sequencing.

Heterozygous ITPA IVS2 + 21A>C carriers had ITPA activities in the low normal range, but they could not be differentiated by the HPLC activity assay (Fig. 3BUp and Table 2Up ). ITPA IVS2 + 21A>C homozygous cases (n = 3) had ITPA activities identical to that of heterozygous ITPA 94C>A mutation carriers, whereas compound heterozygotes for 94C>A and IVS2 + 21A>C had even lower activities. Table 2Up presents a detailed analysis of ITPA activity by genotype. When we used a cutoff of 125 µmol IMP/(g Hb · h), the activity assay detected all 94C>A mutant cases, all compound heterozygotes for 94C>A and IVS2 + 21A>C, all IVS2 + 21A>C homozygotes, 6 of 24 IVS2 + 21A>C heterozygote-only cases, and the single heterozygous case with the novel IVS2 + 68T>C mutation.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we describe a new validated HPLC-based method for the determination of ITPA activity in isolated erythrocytes, which enables ITPA activity testing in clinical practice. In addition, using the new procedure, we were able to observe an excellent phenotype-genotype concordance.

The simple and rapid sample treatment procedure using perchloric acid/saturated dipotassium hydrogen phosphate ensures cessation of the enzymatic reaction and at the same time yields a clean extract that can be directly analyzed by HPLC. The product of the chemical reaction between perchloric acid and dipotassium hydrogen phosphate—potassium perchlorate—is insoluble in water and therefore easily removed by centrifugation. This step gives a pH of the supernatant that ensures both stability of the analyte IMP (nucleotides are instable at acidic pH) and a long life of the chromatographic column. Thus, the new procedure eliminates the need for additional organic liquid-liquid back-extraction (up to 5-fold) used by previous methods (28)(33). Omitting such a tedious back-extraction step is advantageous to improve the precision of the method. As shown in Table 1Up , both favorable analytical precision and accuracy were achieved.

The chromatographic separation contributes to a high analytical specificity, which is advantageous compared with former colorimetric procedures based on the quantification of inorganic phosphate after treatment with inorganic pyrophosphatase (23)(29)(34). In contrast to other preliminary HPLC assays to measure ITPA activity, which were embedded in clinical reports, our validated assay uses reversed-phase chromatography instead of anion-exchange separation. Such an approach is generally more robust. The single chromatographic run time of 11 min (including the wash step) in our protocol is favorable for an HPLC method and enables its use in routine practice.

ITPA activity in erythrocyte samples was stable for up to 4 months when stored at –20 °C as well as up to 3 days when whole blood was stored at room temperature before isolation of erythrocytes. This shows that blood samples for investigation of ITPA activity can be shipped without cooling to specialized laboratories.

The new HPLC-based method for the determination of the ITPA phenotype was used to analyze blood samples collected from 130 Caucasian blood donors. All IMP concentrations measured were within the working range of the method. Comparing only wild-type allele carriers, males had a significant 20% higher median ITPA activity than females. An ITPA activity of 125 µmol IMP/(g Hb · h) appeared to be a suitable cutoff to differentiate between the wild type and all observed ITPA mutations, except for the heterozygous IVS2 + 21A>C genotype. The activity of the latter significantly overlaps with the lower range of activity of wild-type allele carriers. The median activity of IVS2 + 21A>C heterozygotes was 61% of the median wild-type control activity, whereas that of homozygotes was 30%. Our findings compared favorably with those reported previously by Sumi et al. (18). However, note that the abstract of the report by Sumi et al. (18) wrongly assigns 60% activity to IVS2 + 21A>C homozygotes instead of heterozygotes, an error that is unfortunately beginning to perpetuate [see Refs. ( (26)(35))]. The ITPA 94C>A mutant allele frequency was 0.062 (95% CI, 0.038–0.098), and the ITPA IVS2 + 21A>C mutant allele frequency was 0.131 (95% CI, 0.095–0.177), which is identical to the report by Sumi et al. (18). A single case had an ITPA activity of 110.7 µmol IMP/(g Hb · h) and was wild type for 94C>A and IVS2 + 21A>C. Sequencing revealed a novel IVS2 + 68T>C mutation that led to ~60% remaining enzyme activity. In a Japanese population, a single IVS2 + 68T>G mutation was described that led to ~30% remaining enzyme activity (35). We suspect that both intronic mutations of this triallelic polymorphism (IVS2 + 68T>C and IVS2 + 68T>G) are functional, because linkage is not a likely explanation if different mutations at the same site affect the phenotype. The mutations are sited close to a putative branch point at IVS2 + 64 (CTGaCT). We have previously shown that IVS2 + 21A>C is functional because of an effect on splicing efficiency (21), and the same could be true for mutations at IVS2 + 68.

Although with some contradictions (24)(26), there is now good evidence for an association of ITPA 94C>A with nonmyelosuppression adverse drug reactions to AZA (16)(17)(36). Because the 94C>A mutation consistently shows activities <125 µmol IMP/(g Hb · h), we believe that other ITPA genotypes that also have activities below the cutoff (Fig. 3AUp ) confer a comparable risk for thiopurine-related adverse drug reactions. In studies with only genotyping, there was no association between heterozygous carrier status of the IVS2 + 21A>C mutation and adverse events (16)(17)(25). In this context, it is a plea for phenotyping, as some female IVS2 + 21A>C heterozygotes had activities <125 µmol IMP/(g Hb · h). In analogy to TPMT and under the same caveat (previous blood transfusion), phenotyping will detect ITPA activity deficiency independent of whether it is caused by novel mutations or by other factors influencing the activity. We found no evidence for altered ITPA activity under thiopurine therapy in a preliminary comparison between 111 patients undergoing therapy [mainly AZA; median activity, 197.8 µmol IMP/(g Hb · h)] and our control group (n = 130; P = 0.414, Mann–Whitney U-test).


   Acknowledgments
 
We gratefully acknowledge the excellent technical assistance of Sandra Hartung and Elena Hekeler. We thank MZ-Analysetechnik for technical support.


   Footnotes
 
1 Nonstandard abbreviations: AZA, azathioprine; TPMT, thiopurine methyltransferase; ITP, inosine triphosphate; ITPA, inosine triphosphate pyrophosphohydrolase; IMP, inosine monophosphate; DTT, dithiothreitol; and CI, confidence interval.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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