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Articles |
1
Boehringer Mannheim GmbH Lab Diagnostics, Research Center Tutzing, Bahnhofstr. 9-15, D-82327 Tutzing, Germany.
2
Department of Clinical Chemistry, University of Lund,
Malmö University Hospital, S-20502 Malmö, Sweden.
3
De Weezenlanden Ziekenhuis, Groot Wezenland 20, NL-8000
GM Zwolle, The Netherlands.
a Author for correspondence. Fax 49-8158-224055; e-mail uwe_kobold{at}bmg.boehringer-mannheim.com
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many different routine methods (>20) claiming to measure Hb A1c are currently used by the clinical laboratories. The methods either exploit the charge differences existing between Hb A1c and Hb A0 (ion-exchange chromatography, electrophoresis, isoelectric focusing) or are immunological methods that use poly- or monoclonal antibodies directed towards the glycated N-terminal group of the ß-chain of Hb. At present, no internationally accepted reference system, to which the routine assays could be adjusted (7), exists nor do any internationally approved primary or secondary reference materials or a reference method. As a result, values differ in a clinically unacceptable magnitude between methods and laboratories as has been demonstrated in several trials (8)(9)(10)(11)(12). To overcome this problem, national standardization activities have been initiated in some countries to achieve standardization on a national scale, but these activities are independent of each other and tend to use different approaches.
To achieve a uniform international standardization, the IFCC has established a working group on Hb A1c standardization that coordinates activities worldwide. The working group is developing a reference system that will be the basis for the international standardization (13). The first step was a clear definition of the analyte Hb A1c on the basis of its molecular structure. Historically Hb A1c was defined as a certain peak in an HPLC system, but this is no longer scientifically acceptable. To overcome this problem, one suggested definition for Hb A1c was Hb that is glycated at the N-terminal of the ß-chain [ß-(N-deoxyfructosyl)hemoglobin] because this is the major compound in the HPLC peaks and the major form of all glycohemoglobins; however, the issue of potential double glycation at both ß-chains or an additional glycation at any lysine residue was not discussed explicitly (2). To make this clear, the IFCC group has now defined Hb A1c as Hb that is irreversibly glycated at one or both N-terminal valines of the ß-chains. This also covers Hb that is additionally glycated at any lysine residue in the ß-chain. Hb that is solely glycated at a lysine residue is not considered to be Hb A1c.
The second step of the standardization process is the development of a
reference method that can measure Hb A1c specifically
according to this molecular definition. At this time, such a method is
not yet available. HPLC methods, which have often been used as
reference methods for standardization of routine tests, are not really
reference methods in the scientific sense. They provide precision and
long-term stability but lack specificity. Different values for Hb
A1c in the same blood are obtained depending on the
conditions used, because the Hb A1c peaks contain different
kinds and amounts of substances that are not Hb A1c[14–19]. The differences between certain methods can be rather
large. Between the Bio-Rex 70 HPLC method used as a designated
comparison method for the internal standardization of the DCCT study
(2) and the Mono S ion-exchange chromatography method
(20)(21), differences as large as 20% were
found at the reference range concentrations, despite both methods'
claiming to measure Hb A1c [22]. Although the
Mono S system is more specific than the Bio-Rex 70 method, it cannot be
accepted as a reference method because carbamylated Hb and dimers of
glycated 
- and nonglycated ß-chains coelute with Hb
A1c in this system (19)(23).
Because none of the currently available methods for Hb A1c
determination shows sufficient specificity to be used as a reference
method, we have developed one that distinguishes the smaller N-terminal
parts of the ß-chains of Hb A1c from those of the Hb
A0 molecules, thus avoiding the heterogeneity created by
modifications of other glycation sites of the Hb molecule.
The most specific Reference Methods in clinical chemistry available today are based on mass spectrometric detection (gas chromatography–mass spectrometry with isotopic dilution). Most were developed in the 1980s, e.g., for cholesterol, progesterone, testosterone, cortisol, estradiol, or thyroxine (24). We have applied electrospray ionization (ESI-MS), which is a relatively new mass spectrometric technique suited for the analysis of polar biomolecules of low and high molecular mass (25). The process of ESI is characterized by formation of polyprotonated or polydeprotonated ions of intact analyte molecules that are produced from a fine spray of an aqueous solution of the analyte, assisted by a strong electrical field at atmospheric pressure. The ions have low internal energy and are not prone to fragmentation. The measurement of mass-to-charge values of these multiple charged ions gives the inherent molecular mass of the analyte molecules. The ion currents produced by ESI are dependent on the analyte concentration. The ESI technique has improved dramatically within the last 5 years, and detection limits in the lower picomole to upper femtomole range are now available. ESI-MS is ideally suited for an on-line coupling with HPLC, and the combination of these techniques might become a powerful instrument for the development of new Reference Methods for a variety of analytes.
Considering the high cost of ESI-MS equipment, we concurrently investigated the possibility of developing and providing another method based on capillary electrophoresis (CE), a technique that is more generally available in clinical laboratories.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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enzymatic cleavage of hemoglobin
Endoproteinase Glu-C sequencing grade (Boehringer Mannheim; cat.
no. 1047817) was used for cleavage of Hb. An aliquot of 1 mg of Hb from
the patients' samples or the primary calibrators was transferred to a
1-mL crimp cap glass vial, mixed with 20 µL of a solution of 50 µg
of Glu-C in 250 µL of water and diluted with 25 mmol/L ammonium
acetate buffer (pH 4.0) up to a total volume of 500 µL. Vials were
carefully closed with crimp caps and incubated with gently shaking at
37 °C for 18 h. Digestion was stopped by freezing at
-20 °C. For the cleavage experiments with trypsin an Hb solution
consisting of 2 mg of Hb in 300 µL of distilled water was used. After
3 min of boiling, 200 µL of ammonium bicarbonate (0.5 mol/L, pH 8.2)
was added. The solution was incubated for 2 h at 37 °C with 20
µL of N-tosyl-L-phenylalanyl chloromethyl
ketone-treated trypsin (Sigma, 5 mg/mL in 1 mmol/L HCl and 12 mmol/L
CaCl2). The digestion was stopped by adding 2 drops of 6
mol/L HCl.
calibrators
Human blood from healthy, nondiabetic volunteers was used to
isolate Hb A0 and Hb A1c for the
preparation of primary calibrators. Erythrocytes were sedimented by
centrifugation, washed with sodium chloride, hemolyzed with water, and
stabilized in an EDTA/KCN solution to isolate hemoglobins. Purification
was achieved by three chromatographic steps: cation-exchange
chromatography on SP-Sepharose HP, affinity chromatography on
GLYCO-GEL® II Boronate Affinity Gel, and cation-exchange
chromatography on SP-Sepharose HP again. Purified Hb A0 and
Hb A1c were stored stabilized by KCN in an
EDTA/2-(N-morpholino)propanesulfonic acid buffer system.
Purified Hb A1c and Hb A0 were analytically
characterized by Mono S cation-exchange chromatography, capillary
isoelectric focusing, ESI-MS, and quantification of ß-N-terminal
peptides by enzymatic cleavage and reversed-phase HPLC. Total Hb was
determined by the Hb cyanide method (26). Details of the
preparation and characterization are described elsewhere
(23). Calibrators were prepared by mixing Hb
A1c and Hb A0 primary materials. Mixing of
calibrators was done with calibrated pipettes, on the basis of weight
according to the total Hb concentration and the degree of Hb
A0 impurity in the Hb A1c preparation. For
calibration of the analytical systems described below, a set of 6
calibrators covering the range of 0–15% Hb A1c (relative
to total Hb) was used.
method a. hplc separation and on-line esi-ms detection
Samples prepared as described above were analyzed in a combined
HPLC-ESI-MS system. The HPLC system consisted of an HP 1090 liquid
chromatograph (Hewlett-Packard) with a DR 5 solvent-delivery system, a
thermostat-equipped autosampler, an autoinjector, a Rheodyne no. 7010P
(ERC) column-switching valve, a 0.159-cm Swagelok no. SS-100-3
(B.E.S.T.) T-piece, a relay box for control of pneumatic valves
(Festo), a Kratos Programmable Absorbance Detector Spectroflow 783
(Bioanalytische Instrumente) with 2.4-µL cell volume, and a 2.1
x 150 mm analytical HPLC column (ZORBAX SB-CN, 5 µm, no. AS-RT-1245;
P/N:883700.905; Axel Semrau).
Flow rate was set to 300 µL/min, column temperature to 50 °C. Injection volume was 50 µL of digest. A gradient elution was performed with eluent A (0.25 mL/L trifluoroacetic acid in water) and eluent B (0.23 mL/L trifluoroacetic acid in acetonitrile): 0 min, 0% B; 30 min, 15% B; 31 min, 100% B; 34 min, 100% B; 35 min, 0% B; 39 min, stop run). For quantitative determinations a column switching valve was positioned after the HPLC column, and only the fraction between the 5-min and 20-min elution times was allowed to enter the detection system to avoid contamination of the electrospray ion source. The photometric detector was set to 214 nm and was used for control purposes only.
The mass spectrometric system was an SSQ700 single-stage quadrupole mass spectrometer with an electrospray ion source (Finnigan MAT). The HPLC system was connected on-line with the photometric detector and the electrospray ion source by 0.12-mm (i.d.) steel capillaries. The electrospray ion source was run with 414 kPa nitrogen sheath gas and nitrogen auxiliary gas at an HPLC flow rate of 300 µL/min. Spray voltage was 4.5 kV, transfer capillary temperature 200 °C. The mass spectrometer was tuned and calibrated with tetrapeptide MRFA, myoglobin mixture; resolution was set to 0.7 amu peak half-width and the electron multiplier to 13 kV. Acquisition mode was set to centroid, multiple ion detection at m/z 348.2 and 429.2 for the double-protonated ions of nonglycated and glycated N-terminal hexapeptides of the hemoglobin ß-chain.
For measurements a sequence was set up for calibration bracketing: primary calibrators–samples–primary calibrators. Four injections were made for each vial. The ion chromatograms for m/z 348.2 and 429.2 were recorded, the peak areas integrated, and the ratios of area m/z 429.2 vs m/z 348.2 calculated. The mean values of four injections at a time per vial were calculated. The ratio of peak areas was a linear function of the percent Hb A1c for a set of primary calibrators. With the data set of primary calibrators a calibration function was calculated by linear regression of the measured ratio against the default values of percent Hb A1c. This calibration function was used to determine the percent Hb A1c values of the measured patient samples.
method b: hplc and ce
Samples prepared as described above were separated by
reversed-phase chromatography on a C18 column (PepRPC
HR 5/5; Pharmacia). HPLC separation was done as described earlier
(27) with the following modifications: an HPLC system,
including two P-500 pumps, a GP-250 gradient programmer, and a UV
detector 2141 (LKB-Pharmacia); flow rate was 1 mL/min, gradient elution
was performed with eluent A (1 mL/L trifluoroacetic acid in water) and
eluent B (1 mL/L trifluoroacetic acid in acetonitrile): 0 min, 0% B;
40 min, 18% B; 41 min, 100% B; 43 min, 100% B; 44 min, 0% B; 52
min, stop run. Absorbance was monitored at 214 and 280 nm. The actual
fraction, the first dominating peak, was identified by amino acid
analysis, collected, and lyophilized. The material was dissolved in 40
µL of 0.1 mL/L trifluoroacetic acid in water immediately before
electrophoresis. In a second step the mixture of glycated and
nonglycated ß-N-terminal hexapeptides was separated by CE performed
on a Beckman P/ACE system 5000 equipped with System GoldTM
version 8.1 software. Fused-silica capillaries, 50 µm (i.d) x 67 cm
(no. 338472; Beckman Instruments), were used. New capillaries were
conditioned by rinsing with 0.1 mol/L NaOH for 10 min and buffer for
another 10 min. A typical run would be 100 mmol/L phosphate buffer, pH
2.5, 15-s pressure injection, 25-kV voltage, temperature at 20 °C,
and absorbance monitored at 214 nm. Calibration was carried out as
described in method A.
hplc comparison method
Mono S ion-exchange chromatography, a reliable and
well-established routine method, was chosen for a comparison with the
newly developed methods. In this method Hb A1c is
separated from other hemoglobins Hb A1a, Hb
A1b, Hb F, acetylated Hb, -chain dimers, the glutathione
adduct Hb A3, and Hb A0 by a salt gradient in
malonate buffer at pH 5.7 on a Mono STM HR 5/5 column
(Pharmacia) as described earlier (20)(21). The
pre-Hb A1c is eliminated by incubation of samples in a
citrate/phosphate buffer, pH 5.4, for 30 min at 37 °C
(21).
method comparison studies
Two sets of patient samples were measured on different occasions
with the HPLC-MS procedure, the HPLC-CE procedure, and Mono S
ion-exchange chromatography. The results were statistically evaluated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). For the final methods we used 18-h digestion at 37 °C.
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method a: hplc separation and on-line esi-ms detection
The HPLC separation has been optimized for the HPLC-ESI-MS system
to achieve good resolution between glycated and nonglycated
ß-N-terminal hexapeptides and good separation from all other peptide
fragments. Mass spectra from synthetic glycated and nonglycated
ß-N-terminal hexapeptide calibrators are shown in Fig. 2
. The spectra show both single- and double-protonated ions, and
no fragmentation is observed. For quantitative measurements the
double-protonated ions were chosen because of their better response
behavior. Resolution of the mass analyzer was set to 1 Da. The very
high specificity of the mass spectrometric detection is shown in Fig. 3
. The chromatogram recorded in a scan mode, which is similar to
a photometric detection at 215 nm, is compared with the multiple-ion
detection mode for the doubly protonated ions at m/z 348.2
and 429.2, which represent the hexapeptides released from Hb
A1c and Hb A0. This comparison clearly shows
the superior specificity of the MS detection. The detection limit of
the analytical system at multiple-ion detection mode is sufficient to
get superior signal-to-noise ratios. By simple least-squares regression
a linear calibration function was generated (Fig. 4
). The range over which the response (ratio of glycated
hexapeptide to nonglycated hexapeptide) is linearly proportional to the
percent Hb A1c was evaluated by measuring a set of six
primary calibrators covering the range 0–15% Hb A1c. The
stability and reproducibility of the total analytical system were
proven by repeated measurement of three patients' samples (~4%,
5%, and 7.5% Hb A1c) within a period of 4 weeks
(duplicates on 4 different days). The three samples were run together
with a larger set of patients' samples. Including all calibration and
test assays, >250 injections were done during this period, in
addition to the routine use of the mass spectrometer for
qualitative peptide and protein analysis. The overall CVs for Hb
A1c amounts of 4.09%, 5.37%, and 7.91% were 2.0%,
1.2%, and 2.5%, respectively. This demonstrates a very good
reproducibility of the system. The overall procedure of the complete
method is shown in the flow chart in Fig. 5
.
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method b: hplc separation and ce
We also studied a second independent multidimensional approach to
quantify the ratio of glycated to nonglycated ß-N-terminal
hexapeptides. This was done by reversed-phase HPLC combined with
off-line CE and photometric detection. Glycated and nonglycated
ß-N-terminal hexapeptides coeluted together on the
C18 column used. This step was used for an enrichment
of these peptides. In a second step, CE, which separated the
C18 fraction into two peaks, was introduced (Fig. 6
). By comparing synthetic glycated and nonglycated
ß-N-terminal hexapeptides, it was obvious that the peptides showed
different absorbance values at 214 nm. The system was therefore
calibrated in the same way as the ESI-MS method with the same
calibrators (area % used for calculations). The within-day CVs of the
HPLC-CE system for Hb A1c at 3.86%, 6.36%, and 12.0%
were 1.92%, 1.58%, and 1.62%, respectively. The overall procedure of
the complete method is shown in the flow chart in Fig. 5
.
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method comparison studies
These studies were carried out to compare the two newly developed
methods and also to compare the new methods with a reliable
well-established routine HPLC method, the Mono S method. The comparison
between the HPLC-CE and HPLC-MS methods (Fig. 7
) shows an excellent agreement. The comparisons of the Mono S
method against HPLC-MS also demonstrate good correlations (Fig. 8
). Comparison of Mono S vs HPLC-CE is similar (y
= 0.836 x + 1.35, r = 0.994). The
remarkable slope is expected because of the theoretical considerations
that Mono S gives higher Hb A1c values in the lower
concentration range, because of impurities that coelute with the Hb
A1c peak, and gives lower values in the upper concentration
range, because of the additional glycation of 
-amino groups of
lysine that will not coelute with the original Hb A1c peak,
thus resulting in a reduction of the Hb A1c signal. Because
all ion-exchange chromatographic systems are traceable to each other
(9)(10), there will be traceability between
currently used ion-exchange systems and the described candidate
Reference Methods.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Trypsin, which cleaves peptide bonds with lysine and arginine at the
C-terminal side, is a commonly used enzyme for peptide mapping in
hemoglobinopathies. We were able to show that the lysine residues at
position 8 in the ß-chain are glycated in samples with increased Hb
A1c concentrations but not in those with normal
concentrations. Thus the use of trypsin to release N-terminal
octapeptides for quantification purposes would include the risk of
getting doubly or singly glycated octapeptides at the Lys-8 position.
Therefore trypsin cleavage was not usable. Endoproteinase Glu-C cleaves
the N-terminal part of the ß-chain between the two glutamic acid
residues at positions 6 and 7. The resulting fragments contain only a
single glycation site at the N-terminal valine and can thus be used to
separate Hb A1c. The actual cleaving site is easily exposed
to the enzyme under mild denaturing conditions at pH 4.0. Complete
denaturation before digestion exposes additional substrates to the
enzyme and yields a more complex peptide mixture. With the modern
multidimensional analytical techniques of on-line HPLC and ESI-MS or
the off-line system of HPLC and CE, the two ß-N-terminal hexapeptides
of Hb A1c and Hb A0 could be separated and
quantified with the necessary analytical performance. By analyzing the
mixture of peptide fragments resulting from the endoproteinase Glu-C
digestion of whole-blood samples, we obtained high specificity and a
low detection limit. The approach excludes the interference of
carbamylated and acetylated N-terminal species as well as other
N-terminal adducts or nonglycated ß-chains from the dimer of
glycated -chain–nonglycated ß-chain (which coelute for example
with Hb A1c in the Mono S system).
We have developed two quantitative analytical methods that specifically measure the ratio of the N-terminal hexapeptides of Hb A1c and Hb A0. The calibration of both methods was done with primary calibrators that are mixtures of Hb A1c and Hb A0 primary calibrators. The CE method is a simple and robust technique but requires an off-line preparation of samples. The ESI-MS method requires advanced equipment but permits highly specific detection like the classical Reference Methods in clinical chemistry and has the practical advantage of on-line coupling without time-consuming sample processing. In our method, MS has been used for the first time in a Reference Method for proteins or peptides. The method is a good example of how to adapt the well-known concept of Reference Methods for low-Mr analytes to high-Mr proteins. From a theoretical point of view HPLC-ESI-MS offers higher specificity than HPLC-CE. This motivates its position as the most advanced candidate for the forthcoming Hb A1c Reference Method. However, with the observed analytical performance, the HPLC-CE method could also serve as a Reference Method. According to the NCCLS definition of analytical methods, the HPLC-CE system could be classified as a Reference Method while the HPLC-ESI-MS method has the potential to be a Definitive Method. Both methods will now be evaluated further in an international network of reference laboratories organized by the IFCC Working Group on Hb A1c Standardization.
Clearly, however, the Hb A1c values found with the new Reference Methods will be considerably lower than those found with many current routine methods, especially when they are calibrated against the DCCT method (3). The designated comparison method in that study was a cation-exchange HPLC method with Bio-Rex 70 as resin. Although the method was very stable during the 9-year period of the DCCT study, it has become evident that the Hb A1c result by this method is only 60% specific, due to ß-N-deoxyfructosyl Hb (17). More sophisticated assays with other resins, e.g., Mono S, yield lower Hb A1c results, but still show a perfect correlation with the Bio-Rex 70 method (19)(20). The new Reference Methods will solve this lack of specificity. For this reason lower values of Hb A1c will be expected in nondiabetic persons as well as in diabetic patients. Therefore, reference ranges from nondiabetics and recommended values for optimal therapy in diabetic patients will have to be revised and adjusted to the new accuracy. Preliminary experiments with more specific methods, e.g., the Mono S method (18), have already shown that translation of the Hb A1c values found in the DCCT study into values based on these more specific methods is feasible. The translation of Hb A1c values found in the DCCT and other important clinical trials into values that are based on the new reference system will be a task of the IFCC Working Group on Hb A1c Standardization (13) because the previous clinical experience must be maintained.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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A H Berg and D B Sacks Haemoglobin A1c analysis in the management of patients with diabetes: from chaos to harmony J. Clin. Pathol., September 1, 2008; 61(9): 983 - 987. [Abstract] [Full Text] [PDF]
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R. Johnson, J.-O. Jeppsson, U. Kobold, W. Hoelzel, A. Finke, and K. Miedema Standardization of Hemoglobin A1c The authors of the reference cited above respond: Clin. Chem., May 1, 1998; 44(5): 1068 - 1069. [Full Text] [PDF]
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A. Mosca, R. Paleari, A. Made, C. Ferrero, M. Locatelli, and F. Ceriotti Commutability of control materials in glycohemoglobin determinations Clin. Chem., March 1, 1998; 44(3): 632 - 638. [Abstract] [Full Text] [PDF]
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D. B. Sacks Implications of the Revised Criteria for Diagnosis and Classification of Diabetes Mellitus Clin. Chem., December 1, 1997; 43(12): 2230 - 2232. [Full Text] [PDF]
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