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Technical Briefs |
1 Department of Clinical Pathology and
2
Clinical Laboratory, Osaka Medical College, 2-7, Daigaku-machi, Takatsuki, Osaka 569-8686, Japan
aaddress correspondence to this author at: Department of Clinical Pathology, Osaka Medical College, 2-7, Daigaku-machi, Takatsuki, Osaka 569-8686, Japan; fax 81-726-84-6548, e-mail shimizu{at}poh.osaka-med.ac.jp
Hemoglobin A1c (HbA1c), which is defined as Hb that is irreversibly glycated at the N-terminal valine of the ß-chain, is an important index in the monitoring of glucose control in patients with diabetes (1). Considerable discrepancies in the measured values of HbA1c have been observed among methods and among laboratories (2)(3). To standardize HbA1c methods, Kobold et al. (4) proposed a high-level reference method based on liquid chromatography combined with electrospray ionization mass spectrometry (ESI-MS) analysis of the glycated and nonglycated N-terminal hexapeptides of the Hb ß-chains, which are released by enzymatic cleavage of the intact Hb molecule by endoproteinase Glu-C. Their method is now the officially approved IFCC reference method (5).
In our laboratory, Hb variants have been measured by ESI-MS (6)(7). Specimens containing Hb variants showed unexpected HbA1c values, especially when analyzed by HPLC. To correctly estimate the degree of Hb glycation in such specimens, we have applied the peptide method using ESI-MS as proposed by Kobold et al. (4), but the ion signals of peptides occasionally fluctuate, depending on the MS conditions in our laboratory.
We devised a method with a stable-isotope-labeled internal standard to obtain more reproducible values in laboratories where mass spectrometers are used for multiple purposes. We also used synthetic nonlabeled peptides to calibrate the measurement, as reported previously (6)(7). For HbA1c standardization, we used the calibrators provided by the IFCC working group. The calibrators were prepared by mixing isolated HbA0 and HbA1c (8).
Four hexapeptides containing the six N-terminal amino acids of the Hb ß-chain were chemically synthesized by Peptide Institute Inc. (Osaka, Japan): a nonglycated, unlabeled hexapeptide (Val-His-Leu-Thr-Pro-Glu; HD0, lot no. 749-901201), 1-deoxyfructosyl-hexapeptide (GD0; lot no. 480709), hexapeptide labeled with isopropyl-d7 leucine (HD7; lot no. 740-101295), and 1-deoxyfructosyl-hexapeptide labeled with the same deuterated amino acid (GD7; lot no. 740-102051). The purity of the peptides was ascertained by HPLC as 99.1%, 99.3%, 99.4%, and 99.5%, respectively. We weighed 
3 mg of each peptide and dissolved it in distilled water to adjust the concentration to 90 mmol/L. The peptides were mixed in the desired molar ratios. Endoproteinase Glu-C (lot no. PBIO 160-016), an enzyme that specifically cleaves the carboxyl side of glutamic acid residues, was purchased from PE Biosystems. Other reagents (spectrophotometric grade) were purchased from Nacalai Tesque and used without further purification.
The MS system was a TSQ7000 triple-stage quadruple mass spectrometer with a conventional electrospray ion source (ThermoQuest). The HPLC system was an Ultrafast Microprotein Analyzer (Michrom BioResources) with a reversed-phase microcolumn [ZORBAX-SB-CN; 150 x 0.5 mm (i.d.); 5 µm bead size]. The electrospray ion source was run with nitrogen at 0.4 MPa. Sheath gas and nitrogen auxiliary gas were used at an HPLC flow rate of 40 µL/min. The spray voltage was 4.5 KV, and the transfer capillary temperature was 200 °C. The mass spectrometer was tuned and calibrated with Met-Arg-Phe-Ala and horse muscle apomyoglobin mixture.
Univalent ions for the four peptides were selected for ion monitoring: m/z 695.4 for the HD0 hexapeptide, m/z 702.4 for the HD7 hexapeptide, m/z 857.4 for the GD0 hexapeptide, and m/z 864.4 for the GD7 hexapeptide. We chose univalent ions for monitoring because signal noise was lower for monitoring with univalent ions than with divalent ions. The signal intensities of the same deuterium-labeled and unlabeled peptides in the mixture were equivalent, and the ratios of signal intensities were identical to the ratio of molar concentrations of each peptide in the mixture. As internal standards, we added a mixture of labeled glycated and labeled nonglycated peptides in which the molar ratio of labeled glycated to labeled nonglycated peptides was 1:10 (10 µL containing 20 µmoles of labeled glycated peptide and 200 µmoles of labeled nonglycated peptide) to test samples.
To construct a calibration curve for the glycated hexapeptide, we added the internal standard to solutions containing various ratios of unlabeled glycated and nonglycated peptides. We analyzed 100 µL of the solutions, which contained 200 pmoles of nonglycated hexapeptide and 0, 5.0, 9.5, 14.0, and 17.5 pmoles of glycated hexapeptide. To construct a calibration curve for HbA1c, we added the internal standard (mixture of labeled glycated and nonglycated peptides in a 1:10 molar ratio) to a solution of the calibrators provided by the IFCC Working Group for HbA1c Standardization after digestion with endoproteinase Glu-C. The digestion was performed according to the method of Kobold et al. (4). Two routine samples were measured and compared with both calibration curves to test the intra- and interassay variability of MS analyses. Analyses were repeated 10 times continuously in a day and 5 times on different days. The internal standard containing labeled glycated and nonglycated peptides (1:10 molar ratio) was added to the solution after digestion with endoproteinase Glu-C.
Using selected-ion monitoring of the column eluate, we measured peak areas at m/z 695.4 for HD0, m/z 702.4 for HD7, m/z 857.4 for GD0, and m/z 864.4 for GD7 (all ions were univalent) and calculated the peak intensity ratio of glycated to nonglycated peptides by the following equation:
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, we obtained a highly linear calibration curve for the glycated hexapeptides over a wide concentration range (0–20%). We also obtained a highly linear calibration curve for HbA1c over a wide concentration range (Fig. 1B
). As shown in Table 1
, the peak intensity values for the two samples used to assess assay variability were highly reproducible (CVs 
2%). The percentages of glycated peptide and HbA1c calculated on the basis of their respective calibration curves were similar. The causes of the small discrepancies between the values obtained with the two calibration curves should be identified by further experiments. Probable causes may include the different susceptibilities of glycated and nonglycated Hb to the enzyme, errors in mixing glycated and nonglycated materials, and impurities in the calibrators.
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We previously reported a method that involved monitoring of both univalent and divalent ions (6)(7), whereas Kobold et al. (4) measured only divalent ions, and in the present report, we measured only univalent ions. Use of the expression (0.5 x peak area of doubly protonated ions + 1 x peak area of univalent ions) for each peptide to calculate the ratio of the peak intensities for both peptides improved the reproducibility of the ratio over that calculated with use of divalent ions only. However, in repeated experiments, the values obtained without use of labeled internal standards varied, depending on instrument conditions. The values reported here were fairly constant among assays on different days. Daily calibration is not necessary because the peak intensity ratios for labeled glycated and nonglycated peptides were highly reproducible. The peak intensity ratio for glycated/nonglycated peptides measured with the standard material, in which the concentration ratio of glycated/nonglycated was 1:10, remained constant with a mean (SD) value of 0.189 (0.007) and a CV of 1.2%.
We have used the proposed method to assess the HbA1c values in specimens that contain abnormal Hb. The method is convenient and reliable for determining HbA1c in such specimens.
Acknowledgments
We thank the IFCC Working Group for HbA1c Standardization for providing the calibrators.
References
The following articles in journals at HighWire Press have cited this article:
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  D. B. Sacks Hemoglobin Variants and Hemoglobin A1c Analysis: Problem Solved? Clin. Chem., August 1, 2003; 49(8): 1245 - 1247. [Full Text] [PDF]
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