Method for the quantitative determination of glycated proteins
BACKGROUND OF THE INVENTION 
     The invention concerns a method for the quantitative determination of glycated protein and especially haemoglobin (HbA1c), which is essentially composed of an enzymatic and a chromatographic method step. Subsequently the exact amount of HbA1c is determined by a differential measurement of for example glycated and non-glycated haemoglobin (HbA0). Alternatively the absolute content of glycated and non-glycated proteins can be determined by calibration with suitable standard materials. 
     There are numerous methods for determining glycated proteins. These can be basically divided into three groups depending on the way in which glycated and non-glycated protein components are separated and quantified (Clin. Chemistry 32 (1986), B64-B70). The first group consists of physicochemical methods based on the utilization of charge differences. These include the HPLC determination with cation exchanger columns such as Diamat, MonoS and PolyCat A (Bisse method) which are the most frequently used methods in clinical chemistry. In the case of haemoglobin for example the quantitative evaluation is carried out by a relative measurement of the HbA1c signal in relation to the total amount of Hb (% HbA1c). 
     Methods in the second group are those which utilize the different chemical reactivity of glycated and non-glycated protein. These include the thiobarbituric acid method in which for example the glucose bound to haemoglobin is converted into a yellow dye and measured photometrically and also affinity chromatography methods in which complex formation between the vicinal diol groups of the sugar residue and a boric acid group that is bound covalently to a support is used to separate glycated and non-glycated haemoglobin. The separated substance classes are quantified photometrically and the relative amount of glycated haemoglobin is calculated or, in the case of the thiobarbituric acid method quantified, as an absolute determination by calibration with suitable standard materials. 
     Thirdly, immunological methods may be mentioned. Specific antibodies are used in immunological methods. These recognize for example the structural unit at the N-terminal end of the β chain of the glycated haemoglobin molecule which is typical for HbA1c (e.g. Tina quant® HbA1c, Boehringer Mannheim). In the immunological methods the absolute content of the respective protein is determined. This necessitates the use of calibrators which have been assigned a target concentration by an independent method. The relative content of for example HbA1c cannot be obtained by a direct measurement. 
     The known methods are, however, associated with a number of disadvantages. Thus some of the physicochemical methods have a very poor selectivity since the measured signals of glycated protein are overlapped by the non-glycated variants. The shapes of the chromatographic peaks are often asymmetrical and difficult to integrate. The cation exchanger columns that are used are susceptible to small changes in the working conditions and to contamination. Due to the poor selectivity there is a high risk of measuring values that are too high (false positive values). 
     In the case of chemical methods it is difficult to standardize the procedure and interferences by other components containing sugar can only be avoided with a large amount of effort. It is not possible to differentiate between for example HbA1c and other glycated haemoglobin variants. 
     The immunological methods are characterized by a very high selectivity towards glycated protein variants. However, the quality of the results depends on the quality of the standard used for calibration. Suitable primary standards in an optimal quality are not at present available, in particular for HbA1c. Information on matrix dependencies cannot be obtained due to a lack of a suitable reference method. In this case false positive values are also frequently obtained (see for example J. Clin. Lab. Anal. 8, (1994), 128-134). 
     SUMMARY OF THE INVENTION 
     The object of the invention is therefore to provide a method which overcomes the disadvantages of the known methods. The object is achieved by firstly treating an aqueous sample containing a glycated protein or corresponding variants with a proteolytic enzyme and subsequently separating the resulting mixture containing the fragments by chromatography. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1 
     FIG. 1 shows the result of the method according to the invention (sample: whole blood haemolysate; enzymatic treatment and chromatograph (HPLC)). 
     FIGS. 2 and 3 
     FIGS. 2 and 3 show the result of the method according to the invention (sample: preparatively purified HbA1c; 2: cation exchanger; 3: cation exchanger and affinity chromatography). 
     FIG. 4 
     FIG. 4 shows the result of the method according to the invention (sample: preparatively purified HbA0 (cation exchanger)), 
     FIG. 5 
     FIG. 5 shows the result of the method according to the invention (sample: whole blood haemolysate, enzymatic treatment and chromatography (HPLC)). 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The sample material, in particular haemolysed whole blood, is incubated with a proteolytic enzyme in a suitable reaction buffer. Haemolysis is carried out by known methods. Proteases which are capable of cleaving a N-terminal peptide which is as short as possible from the β chain of haemoglobin have proven to be suitable in this case as the enzyme. Apart from trypsin, chymotrypsin and thermolysin in particular and especially endoproteinase Glu-C have proven to be suitable for this. The proteolytic enzymes are usually used at a concentration of 1/200 to 1/10 of the amount by weight of protein. 
     Examples of suitable reaction buffers are ammonium carbonate and/or sodium phosphate buffer which are usually used at concentrations of up to approximately 10 mM in a pH range of 3.5-8.5 and, depending on the respective protease, may be supplemented by further auxiliary substances such as activators and/or stabilizers such as e.g. sodium dodecyl sulphate, urea, guanidine hydrochloride or acetonitrile. 
     The incubation with the respective proteolytic enzyme is carried out between 20° C. and 40° C., preferably at ca. 25° C. and can be completed after several minutes to a maximum of 18 hours depending on the enzyme. 
     In addition it has proven to be particularly advantageous when the erythrocytes are separated before haemolysis and to carry out the haemolysis merely with water. 
     The solution obtained by enzyme treatment contains specific peptides for the proteins to be determined in each case in addition to other fragments. The peptide mixtures are subsequently separated in a HPLC process with good resolution (e.g. reversed phase materials) and detected photometrically. Such separation methods are generally known to a person skilled in the art (e.g. K. L. Stone, J. I. Elliot, G. Peterson, W. McMurray, K. R. Williams, &#34;Reversed-phase high performance liquid chromatography for fractionation of enzymatic digests and chemical cleavage products of proteins&#34;, Methods in Enzymology, 193 (1990), 389-412). Alternatively it is also possible to use other separation methods such as e.g. capillary electrophoresis (e.g. Capillary Electrophoresis Separations of Peptides: Practical Aspects and Applications, Chap. 9, p. 237-271 in Capillary Electrophoresis, Academic Press 1992). 
     The simultaneous selective determination of glycated and non-glycated protein is possible from the measured signal intensities of the glycated and the respective amount of non-glycated peptide. In the analytical evaluation the percentage of glycated protein in relation to non-glycated protein is obtained. The method is particularly advantageous for the determination of HbA1c in addition to non-glycated haemoglobin (HbA0). This method has proven to be highly specific and free from any type of interference, moreover the determination of the percentage content of HbA1c is possible without additional calibration or standardization. 
     The invention is elucidated further by the following examples. 
     EXAMPLE 1 
     The method was carried out using a whole blood haemolysate and HbA1c and HbA0 standard materials purified in different ways. In each case the N-terminal peptide with a length of twenty-two amino acids was used to quantify HbA1c and HbA0. 
     For purposes of comparison, the samples used were also measured with a HPLC method based on a cation exchanger (PolyCat A, Bisse method) without prior enzyme treatment. 
     A According to the invention (enzyme+HPLC) 
     A.1 Enzyme solution: 
     Endoproteinase Glu-C (sequencing grade Boehringer Mannheim; Cat. No. 1047817) 
     Protein concentration: ca. 1 mg/ml 
     Ratio protein: enzyme 20:1 
     25 mM ammonium acetate buffer containing 5% acetonitrile 
     pH 4.0 
     Enzymatic treatment, conditions: 18 h, 25° C. 
     A.2 HPLC step, conditions: 
     HPLC system HP 1090 
     Separation column Zorbax C8 stable bond 
     Gradient elution from water to acetonitrile 
     containing 0.1% trifluoroacetic acid (TFA) 
     Detection at 214 mm 
     B. HPLC step without prior enzymatic treatment 
     Column material: polyCatA 
     Procedure according to E. Bisse &amp; H. Wieland, 
     J. of Chromat. 434 (1988) 95-110 
     The results show that the selectivity of the cation exchanger chromatography alone does not meet the requirements; a false positive deviation of ca. 15% is found. The results of both methods are comparable in the case of sample material which was purified by affinity chromatography and no longer contains non-glycated haemoglobin since now the HbA1c signal in the cation exchanger chromatography can no longer be overlapped by non-glycated material. 
     
                                           TABLE 1                                 
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               % HbA1c  % HbA1c                                           
Sample         Glu-C + HPLC                                               
                        PolyCat A                                         
                               % deviation                                
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whole blood haemolysate                                                   
               5.5      6.5    15.4                                       
HbA1c purified preparatively                                              
               7.5      89     15.7                                       
cation exchanger                                                          
HbA1c purified preparatively                                              
               93       92     -1.1                                       
affinity chromatography                                                   
HbA0 purified preparatively                                               
               0        0.6                                               
cation exchanger                                                          
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     Table 1 shows a comparison between the method according to the invention and a pure HPLC method. 
     EXAMPLE 2 
     In order to check the reproducibility of the method, a whole blood sample was processed several times and, after the enzymatic treatment, the N-terminal peptide with a length of 6 amino acids is used to quantify HbA1c and HbA0 (cf. chromatogram FIG. 5). 
     Enzyme solution: 
     Endoproteinase Glu-C (sequencing grade Boehringer Mannhelm; Cat. No. 1047817) 
     Protein concentration: ca. 1 mg/ml 
     Ratio protein: enzyme 30:1 to 60:1 
     25 mM ammonium acetate buffer 
     pH 4.0 
     The results (Table 2) show that the method can be carried out very reproducibly. 
     
                       TABLE 2                                                     
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                  Ratio                                                   
                  Protein:  % HbA1c                                       
Sample            Enzyme    (Glu-C + HPLC)                                
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Whole blood haemolysate prep. 1                                           
                  43:1      5.35                                          
Whole blood haemolysate prep. 2                                           
                  43:1      5.38                                          
Whole blood haemolysate prep. 3                                           
                  43:1      5.48                                          
Whole blood haemolysate prep. 4                                           
                  43:1      5.25                                          
Whole blood haemolysate prep. 5                                           
                  43:1      5.44                                          
Whole blood haemolysate prep. 6                                           
                  34:1      5.39                                          
Whole blood haemolysate prep. 7                                           
                  51:1      5.50                                          
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     Table 2 shows the results of a reproducibility experiment using the HPLC step according to the invention; conditions: 
     HPLC system HP 1090 
     Separation column Zorbax SN-CN 
     Gradient elution from water to acetonitrile containing 0.1% trifluoroacetic acid 
     Detection at 214 nm. 
     FIG. 1 
     FIG. 1 shows the result of the method according to the invention (sample: whole blood haemolysate; enzymatic treatment and chromatography (HPLC)). 
     FIGS. 2 and 3 
     FIGS. 2 and 3 show the result of the method according to the invention (sample: preparatively purified HbA1c; 2: cation exchanger; 3: cation exchanger and affinity chromatography). 
     FIG. 4 
     FIG. 4 shows the result of the method according to the invention (sample: preparatively purified HbA0 (cation exchanger)). 
     FIG. 5 
     FIG. 5 shows the result of the method according to the invention (sample: whole blood haemolysate, enzymatic treatment and chromatography (HPLC)).