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Letters to the Editor |
1 Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital and, Palacky University Olomouc, Olomouc, Czech Republic
2 Department of Analytical Chemistry, Palacky University Olomouc, Olomouc, Czech Republic
aAddress correspondence to this author at: Laboratory for Inherited Metabolic Disorders, University Hospital, I. P. Pavlova 6, 775 20 Olomouc, Czech Republic. Fax 420-5-88442509; e-mail david.friedecky{at}gmail.com.
To the Editor:
Shipkova et al. (1) recently reported in Clinical Chemistry that liquid chromatography can be used to determine the activity of inosine triphosphate pyrophosphohydrolase (ITPase) in erythrocytes. We find that ITPase can also be measured by capillary electrophoresis.
We performed capillary electrophoresis on a Beckman P/ACE 5510 with a diode array detector. Electrophoretic separations were carried out in an uncoated silica capillary (20 cm effective/27 cm total length, 50 µm internal diameter; Polymicro CE and CEC Technologies) at a constant voltage of –30 kV (1111 V/cm). We set the data rate of the detector at 16 Hz. Samples were loaded by a low-pressure injection (0.5 psi, 6 s). Ultraviolet detection was performed at 250 nm.
We prepared buffer containing citric acid (40 mmol/L) and cetyltrimethylammonium bromide (0.8 mmol/L), adjusted to pH 4.4 with 
-aminobutyric acid (2), filtered and sonicated for 0.5 min before use. At the beginning of each working day, the capillary was washed for 2 min with water and separation buffer, and also washed between runs for 1 min with separation buffer. We prepared a 100 µmol/L aqueous mixture of inosine monophosphate (IMP) and inosine triphosphate (ITP). The compounds could be separated within 0.8 min with separation efficiency up to 1 200 000 theoretical plates/m in a mixture and 300 000 theoretical plates/m in biological samples (Fig. 1
).
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We collected blood samples in EDTA tubes from healthy blood donors and patients undergoing azathioprine therapy (who gave informed consent). Erythrocytes were separated by centrifugation (1200g, 5 min) and washed twice with 3 volumes of NaCl, 9 g/L. We lysed 200 µL of erythrocytes with 1 mL of ice-cold distilled water, centrifuged the lysates at 5000g for 10 min, and mixed 25 µL of supernatant with 100 mmol/L Tris buffer (150µL, pH 9.0), 10 mmol/L dithiothreitol (10 µL), and 1 mol/L MgCl2 (10 µL). The mixture was preincubated for 5 min at 37 °C, after which 40 mmol/L ITP (10 µL) was added and incubated for 15 min at 37 °C (2). The samples were deproteinized with 20 µL of trichloroacetic acid, 1 mol/L, sonicated (30 s), and centrifuged at 5000g for 1 min. The supernatant was injected into the capillary or stored at –50 °C. We measured hemoglobin (Hb) in the lysate with a Radiometer ABL 725 (Diamond Diagnostics).
The signal-to-noise ratio was >6 at 2.0 nkat/g Hb [7.2 µmol of IMP/(g Hb · h)]. The calibration curve was linear from 0.01 to 10 mmol/L (y = 11.3x – 0.6 nkat/g Hb; r = 0.9974). We evaluated recovery and imprecision by assaying erythrocytes with the added mixture of IMP. Recoveries were 85%, 83%, 76%, 75%, and 80% for 0.06, 0.25, 0.54, 1.80, and 3.00 mmol/L added IMP (n = 6), respectively. The lower recoveries reflect coprecipitation of IMP with proteins, which agrees with previously published findings (1). Imprecision values (as CV, n = 10) were 2.1%, 1.2%, and 1.0% (within-day CV) and 4.2%, 3.2%, and 2.4% (between-day CV) for 0.06, 0.54, and 3.00 mmol/L additions of IMP, respectively. The reproducibility values (CV) of migration times for 10 samples from healthy volunteers were 0.92%, 2.8%, and 2.5% for run-to-run, sample-to-sample, and between-day measurements (n = 10), respectively. Because of the use of acidic separation medium, we observed no interference during the analysis of samples from 80 healthy blood donors and 20 patients undergoing azathioprine therapy.
With this simple capillary electrophoresis method, we estimated a reference interval (n = 80, 38 males and 42 females) for healthy white individuals of 13.3–112.2 nkat/g Hb (5%–95%), with a median of 57.8 nkat/g Hb, which agrees with the previously published data (3).
Availability of alternative methods is important because available analytical equipment varies among laboratories. Measurements of ITPase are important because ITPase deficiency may alter 6-mercaptopurine (azathioprine) metabolism, leading to adverse reactions (3), and the deficiency affects >10% of the population (4).
Acknowledgments
Grant/funding support: This study was supported by Grant MSM 6198959205 from the Ministry of Education, Youth and Sports (Czech Republic).
Financial disclosures: None declared.
References
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