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Affinity chromatography method for the measurement of glycosylated haemoglobin: comparison with two methods in routine use
257
Clinica Chimica Acta, 136 (1984) 257-262
Elsevier
CCA 02766
Brief technical note
Affinity chromatography method for the measurement of glycosylated haemoglobin: comparison with two methods in routine use W.G. John a**,E.C. Albutt b, G. Handley ’ and R.W. Richardson a LIDepartment of Biochemistry Coventry and Warwickshire Hospital, Stoney Stanton Road, Coventry (UK), b Department
of Clinical Chemistry, General Hospital, Steelhouse Lane, Birmingham
’ Department
of Biochemistry, Pathology Laboratory,
L.akin Road
(UK) and
Warwick (UK)
(Received July 1st; revision October 3rd, 1983) Key words: Affinity chromatography; ion
Hemoglobin, glycosylated; Agar electrophoresis; Mini -column
-exchangechromatography
Introduction Blood glucose reacts with haemoglobin to form an unstable Schiffs base which is then converted by Amadori rearrangement to a stable keotamine [l]. This glycosylated haemoglobin (G Hb), formed over the life span of the erythrocyte, can be used as an indicator of long-term control in diabetic patients [2]. The glycosylation of haemoglobin and its clinical usefulness have been the subject of a number of reviews [3,4]. The methods available for the estimation of G Hb either measure the total HbAl (i.e. HbAla + b + c), or measure the main stable glycosylated fraction HbAlc after isolation from the other fractions. The former methods (including electrophoresis and mini-column ion-exchange chromatography) are more appropriate for use in routine clinical laboratories handling large work loads from diabetic clinics. However, agar electrophoresis [5] using commercially prepared agar plates and scanning densitometers is expensive although technically simple, and separation of G Hb by mini-column ion-exchange chromatography [6] is sensitive to pH and ionic strength of buffer and operating temperature. Affinity chromatography methods are claimed to be less sensitive to these factors [7]. We have investigated the affinity chromatographic medium Glycogel B (Pierce and Warriner, Chester, Cheshire, UK) and compared the results with those from commercially available kits based on agar electrophoresis and mini-column ion-exchange chromatography being used routinely in two other hospital laboratories.
* Correspondence and reprint requests to W.G. John, address as above. 0009-8981/84/$03.00
0 1984 Elsevier Science Publishers B.V.
258
Methods Affinity chromatography
Blood samples (in fluoride-oxalate or EDTA) were centrifuged, 100 ~1 of packed red cells were haemolysed in 2 ml distilled water on a vortex mixer. 100 ~1 of haemolysate were added to a mini-column containing 1 ml Glycogel B, which had previously been equilibrated with 10 ml wash buffer (250 mmol/l ammonium acetate, 50 mmol/l magnesium chloride and 3 mmol/l sodium azide; the pH was adjusted to 8.6 f 0.1). The haemolysate was washed into the gel with 1 ml wash buffer and when this had drained into the gel, a further 5 ml wash buffer was added. The entire 6 ml eluate, containing the non-glycosylated Hb, was collected and the volume adjusted to 15 ml with water. 3 ml sorbitol buffer (200 mmol/l sorbitol, 50 mmol/l EDTA and 3 mmol/l sodium azide in 100 mmol/l tris(hydroxymethyl)aminomethane buffer; the pH adjusted to 8.6 f 0.1) was added to the column to dissociate and elute bound G Hb, and the eluate collected. The absorbance of each fraction was measured at 414 nm. Percentage G Hb was calculated as: A414(bound) A414(bound) + (5 X A414(non-bound))
xlOO=%GHb
The columns were regenerated using 5 ml distilled water followed by 5 ml 0.1 mol/l acetic acid (in which the columns were stored at 4°C) and equilibrated with 5 ml wash buffer before use. Agar electrophoresis *
Procedure, reagents and equipment were as provided by Corning Medical Ltd. (Halsted, Essex, UK). Mini-column ion-exchange chromatography **
Procedure and reagents were as provided by BioRad Laboratories Hertfordshire, UK).
Ltd. (Watford,
Results and discussion
The affinity chromatography method described for the measurement of G Hb is precise (Table I), dependent only on the adjustment of the unbound fraction to 15 ml and accurate pipetting of 3 ml sorbitol buffer, and is simple to perform; the flow of buffer ceases when its level reaches the top of the gel. The column therefore does not require close attention as the gel will not dry out, and this also allows the collection of two distinct fractions. * Clinical Chemistry, General Hospital, Birmingham, UK. ** Biochemistry Department, Pathology Laboratory, Warwick,
UK.
259
TABLE Precision
I for G Hb using affinity
chromatography
Within-batch _F(SD)%GHb cv (%) F(SD)%GHb GV (%)
TABLE
(n = 10)
Between-batch
18.45 (0.19) 1.0
18.70 (0.28) 1.5
5.79 (0.16) 2.0
6.00 (0.12) 2.0
(n = 15)
11
Effect of increased
glucose
concentration
on the binding
of haemoglobin
Glucose concentration of haemolysate (g/l)
%GHb non-diabetic
diabetic
0
6.6 6.5 6.6 6.2
15.2 15.3 15.3 14.9
0.1 0.5 1.0
TABLE
III
Regeneration Number
0 5 11 12 15 16
of the affinity
of regenerations
column
-
affect on the % G Hb result %GHb non-diabetic
diabetic
5.5 5.4 5.9 5.3 5.5 5.4
20.0 20.5 20.0 20.3 20.5 20.3
The binding of G Hb to the column is not affected by increased glucose concentrations in the haemolysate (Table II), even at levels which, due to the dilution, would not be encountered during normal separation; the red cells do not therefore need to be washed. The haemolysate showed a significant increase (p < 0.001) in measured % G Hb after one month’s storage at 4”C, the results indicate that separation should be performed within 1 week of haemolysate preparation. The time for a single chromatographic run is about 40 min, and increases slightly as the column ages; and regeneration time is about 1 h. Because the columns can be regenerated at least 16 times (Table III), the cost of each G Hb estimation is about lop, making this the cheapest commercially available separation medium. The range of G Hb in healthy hospital personel was shown to be 5.0%8.5% which agrees with other workers [8,9], but unlike Klenk et al [9] we found the distribution to be log/normal and not Gaussian. Blood can be collected into
260
fluoride-oxalate anti-coagulated tubes, and therefore has the advantage that blood glucose estimations can be performed on the same specimen. G Hb showed good correlation with fasting (r = 0.92) and random (r = 0.75) blood glucose. In routine use, over a number of weeks, the affinity method correlated well with both agar electrophoresis (Fig. 1) and mini-column ion-exchange chromatography (Fig. 2). The higher values found for f% G Hb by the affinity method in comparison to these other methods may be due to the inclusion of a minor G Hb fraction separating with HbAo on agar electrophoresis. Bunn et al [lo] have also suggested that G Hb is present in the HbAo fraction. They have identified structural heterogeneity in HbAo showing that glucose binds covalently to a number of different amino acids on the (Y and p chains, but only one modification, at the /3 chain NH, terminus, results in the formation of a electrophoretically distinct minor haemoglobin. The poorer correlation with mini-column ion-exchange chromatography may have been due to the smaller number of comparisons (all of which were 27’
26.
24.
22.
6.
Or
2
4
6
8
%HbAl
Agar
10
12
14
16
16
20
22
electropharesis
Fig. 1. Correlation between % HbAl measured by agar electrophoresis chromatography. Measurements performed over a 2-month period.
and W G Hb measured by affinity
261 26,
24. n=34 m:1.67 22. c: -3.41 r : 0.83 20-
P’ 0.001
18-
16.
8-
6.
4.
0
2
4 %HbAl
6 Ion exchange
8
10
12
14
16
chromatography
Fig. 2. Correlation between %I HbAl measured by mini-column ion-exchange chromatography (results corrected for temperature using a calibrant) and % G Hb measured by affinity chromatography. Measurements performed over a 6-week period.
within the diabetic range). The poor precision of the ion-exchange method [ll] may also have contributed. The affinity method has been shown to provide a simple and reliable method for the routine measurement of 4%G Hb with no need for the purchase of expensive equipment; also the principle involved in separation may provide scope for extension to the measurement of other glycosylated proteins.
262
References 1 Bunn HF, Heney DN, Kamin S, Gabbay KH, Gallop PM. The biosynthesis of human hemoglobin Ale. J Clin Invest 1976; 57: 1652-1659. 2 Gabbay KH, Hasty K, Breslow JL, Ellison RC, Bunn HF, Gallop PM. Glycosylated hemoglobins and long term blood glucose control in diabetes mellitus. J Clin Endocrinol Metab 1977; 44: 859-864. 3 Garel MC, Blouquist Y, Molko F, Rosa J. HbAlc: a review on its structure, biosynthesis, clinical significance, and methods of assay. Biomedicine 1979; 30: 230-240. 4 Mayer TK, Freedman ZR. Protein glycosylation in diabetes mellitus: a review of laboratory measurements and their clinical utility. Clin Chim Acta 1983; 127: 147-184. 5 Menard L, Dempsey ME, Blankstein LA, Aleyassine H, Wacks M, Soeldner JS. Quantitative determination of glycosylated hemoglobin Al by agar gel electrophoresis. Clin Chem 1980; 26: 1598-1602. 6 Welch SG, Boucher BJ. A rapid micro-scale method for the measurement of haemoglobin Al(a + b + c). Diabetologia 1978; 14: 209-211. 7 Mallia AK, Hermanson GT, Krohn RI, Fujimoto EK, Smith PK. Preparation and use of a boronic acid affinity support for separation and quantitation of glycosylated hemoglobins. Anal Lett 1981; 14: 649-661. 8 Gould BJ, Hall PM, Cook JGH. Measurement of glycosylated haemoglobins using an affinity chromatography method. Clin Chim Acta 1982; 125: 41-48. 9 Klenk DC, Hermanson GT, Krohn RI et al. Determination of glycosylated hemoglobin by affinity chromatography: comparison with calorimetric and ion-exchange methods, and effects of common interference. Clin Chem 1982; 28: 2088-2094. 10 Bunn HF, Shapiro R, McManus M et al. Structural heterogeneity of human hemoglobin A due to non-enzymatic glycosylation. J Biol Chem 1979; 254: 3892-3898. 11 Hammons GT, Junger K, McDonald JM, Ladenson JH. Evaluation of three minicolumn procedures for measuring hemoglobin Al. Clin Chem 1982; 28: 1775-1778.
Clinica Chimica Acta, 136 (1984) 257-262
Elsevier
CCA 02766
Brief technical note
Affinity chromatography method for the measurement of glycosylated haemoglobin: comparison with two methods in routine use W.G. John a**,E.C. Albutt b, G. Handley ’ and R.W. Richardson a LIDepartment of Biochemistry Coventry and Warwickshire Hospital, Stoney Stanton Road, Coventry (UK), b Department
of Clinical Chemistry, General Hospital, Steelhouse Lane, Birmingham
’ Department
of Biochemistry, Pathology Laboratory,
L.akin Road
(UK) and
Warwick (UK)
(Received July 1st; revision October 3rd, 1983) Key words: Affinity chromatography; ion
Hemoglobin, glycosylated; Agar electrophoresis; Mini -column
-exchangechromatography
Introduction Blood glucose reacts with haemoglobin to form an unstable Schiffs base which is then converted by Amadori rearrangement to a stable keotamine [l]. This glycosylated haemoglobin (G Hb), formed over the life span of the erythrocyte, can be used as an indicator of long-term control in diabetic patients [2]. The glycosylation of haemoglobin and its clinical usefulness have been the subject of a number of reviews [3,4]. The methods available for the estimation of G Hb either measure the total HbAl (i.e. HbAla + b + c), or measure the main stable glycosylated fraction HbAlc after isolation from the other fractions. The former methods (including electrophoresis and mini-column ion-exchange chromatography) are more appropriate for use in routine clinical laboratories handling large work loads from diabetic clinics. However, agar electrophoresis [5] using commercially prepared agar plates and scanning densitometers is expensive although technically simple, and separation of G Hb by mini-column ion-exchange chromatography [6] is sensitive to pH and ionic strength of buffer and operating temperature. Affinity chromatography methods are claimed to be less sensitive to these factors [7]. We have investigated the affinity chromatographic medium Glycogel B (Pierce and Warriner, Chester, Cheshire, UK) and compared the results with those from commercially available kits based on agar electrophoresis and mini-column ion-exchange chromatography being used routinely in two other hospital laboratories.
* Correspondence and reprint requests to W.G. John, address as above. 0009-8981/84/$03.00
0 1984 Elsevier Science Publishers B.V.
258
Methods Affinity chromatography
Blood samples (in fluoride-oxalate or EDTA) were centrifuged, 100 ~1 of packed red cells were haemolysed in 2 ml distilled water on a vortex mixer. 100 ~1 of haemolysate were added to a mini-column containing 1 ml Glycogel B, which had previously been equilibrated with 10 ml wash buffer (250 mmol/l ammonium acetate, 50 mmol/l magnesium chloride and 3 mmol/l sodium azide; the pH was adjusted to 8.6 f 0.1). The haemolysate was washed into the gel with 1 ml wash buffer and when this had drained into the gel, a further 5 ml wash buffer was added. The entire 6 ml eluate, containing the non-glycosylated Hb, was collected and the volume adjusted to 15 ml with water. 3 ml sorbitol buffer (200 mmol/l sorbitol, 50 mmol/l EDTA and 3 mmol/l sodium azide in 100 mmol/l tris(hydroxymethyl)aminomethane buffer; the pH adjusted to 8.6 f 0.1) was added to the column to dissociate and elute bound G Hb, and the eluate collected. The absorbance of each fraction was measured at 414 nm. Percentage G Hb was calculated as: A414(bound) A414(bound) + (5 X A414(non-bound))
xlOO=%GHb
The columns were regenerated using 5 ml distilled water followed by 5 ml 0.1 mol/l acetic acid (in which the columns were stored at 4°C) and equilibrated with 5 ml wash buffer before use. Agar electrophoresis *
Procedure, reagents and equipment were as provided by Corning Medical Ltd. (Halsted, Essex, UK). Mini-column ion-exchange chromatography **
Procedure and reagents were as provided by BioRad Laboratories Hertfordshire, UK).
Ltd. (Watford,
Results and discussion
The affinity chromatography method described for the measurement of G Hb is precise (Table I), dependent only on the adjustment of the unbound fraction to 15 ml and accurate pipetting of 3 ml sorbitol buffer, and is simple to perform; the flow of buffer ceases when its level reaches the top of the gel. The column therefore does not require close attention as the gel will not dry out, and this also allows the collection of two distinct fractions. * Clinical Chemistry, General Hospital, Birmingham, UK. ** Biochemistry Department, Pathology Laboratory, Warwick,
UK.
259
TABLE Precision
I for G Hb using affinity
chromatography
Within-batch _F(SD)%GHb cv (%) F(SD)%GHb GV (%)
TABLE
(n = 10)
Between-batch
18.45 (0.19) 1.0
18.70 (0.28) 1.5
5.79 (0.16) 2.0
6.00 (0.12) 2.0
(n = 15)
11
Effect of increased
glucose
concentration
on the binding
of haemoglobin
Glucose concentration of haemolysate (g/l)
%GHb non-diabetic
diabetic
0
6.6 6.5 6.6 6.2
15.2 15.3 15.3 14.9
0.1 0.5 1.0
TABLE
III
Regeneration Number
0 5 11 12 15 16
of the affinity
of regenerations
column
-
affect on the % G Hb result %GHb non-diabetic
diabetic
5.5 5.4 5.9 5.3 5.5 5.4
20.0 20.5 20.0 20.3 20.5 20.3
The binding of G Hb to the column is not affected by increased glucose concentrations in the haemolysate (Table II), even at levels which, due to the dilution, would not be encountered during normal separation; the red cells do not therefore need to be washed. The haemolysate showed a significant increase (p < 0.001) in measured % G Hb after one month’s storage at 4”C, the results indicate that separation should be performed within 1 week of haemolysate preparation. The time for a single chromatographic run is about 40 min, and increases slightly as the column ages; and regeneration time is about 1 h. Because the columns can be regenerated at least 16 times (Table III), the cost of each G Hb estimation is about lop, making this the cheapest commercially available separation medium. The range of G Hb in healthy hospital personel was shown to be 5.0%8.5% which agrees with other workers [8,9], but unlike Klenk et al [9] we found the distribution to be log/normal and not Gaussian. Blood can be collected into
260
fluoride-oxalate anti-coagulated tubes, and therefore has the advantage that blood glucose estimations can be performed on the same specimen. G Hb showed good correlation with fasting (r = 0.92) and random (r = 0.75) blood glucose. In routine use, over a number of weeks, the affinity method correlated well with both agar electrophoresis (Fig. 1) and mini-column ion-exchange chromatography (Fig. 2). The higher values found for f% G Hb by the affinity method in comparison to these other methods may be due to the inclusion of a minor G Hb fraction separating with HbAo on agar electrophoresis. Bunn et al [lo] have also suggested that G Hb is present in the HbAo fraction. They have identified structural heterogeneity in HbAo showing that glucose binds covalently to a number of different amino acids on the (Y and p chains, but only one modification, at the /3 chain NH, terminus, results in the formation of a electrophoretically distinct minor haemoglobin. The poorer correlation with mini-column ion-exchange chromatography may have been due to the smaller number of comparisons (all of which were 27’
26.
24.
22.
6.
Or
2
4
6
8
%HbAl
Agar
10
12
14
16
16
20
22
electropharesis
Fig. 1. Correlation between % HbAl measured by agar electrophoresis chromatography. Measurements performed over a 2-month period.
and W G Hb measured by affinity
261 26,
24. n=34 m:1.67 22. c: -3.41 r : 0.83 20-
P’ 0.001
18-
16.
8-
6.
4.
0
2
4 %HbAl
6 Ion exchange
8
10
12
14
16
chromatography
Fig. 2. Correlation between %I HbAl measured by mini-column ion-exchange chromatography (results corrected for temperature using a calibrant) and % G Hb measured by affinity chromatography. Measurements performed over a 6-week period.
within the diabetic range). The poor precision of the ion-exchange method [ll] may also have contributed. The affinity method has been shown to provide a simple and reliable method for the routine measurement of 4%G Hb with no need for the purchase of expensive equipment; also the principle involved in separation may provide scope for extension to the measurement of other glycosylated proteins.
262
References 1 Bunn HF, Heney DN, Kamin S, Gabbay KH, Gallop PM. The biosynthesis of human hemoglobin Ale. J Clin Invest 1976; 57: 1652-1659. 2 Gabbay KH, Hasty K, Breslow JL, Ellison RC, Bunn HF, Gallop PM. Glycosylated hemoglobins and long term blood glucose control in diabetes mellitus. J Clin Endocrinol Metab 1977; 44: 859-864. 3 Garel MC, Blouquist Y, Molko F, Rosa J. HbAlc: a review on its structure, biosynthesis, clinical significance, and methods of assay. Biomedicine 1979; 30: 230-240. 4 Mayer TK, Freedman ZR. Protein glycosylation in diabetes mellitus: a review of laboratory measurements and their clinical utility. Clin Chim Acta 1983; 127: 147-184. 5 Menard L, Dempsey ME, Blankstein LA, Aleyassine H, Wacks M, Soeldner JS. Quantitative determination of glycosylated hemoglobin Al by agar gel electrophoresis. Clin Chem 1980; 26: 1598-1602. 6 Welch SG, Boucher BJ. A rapid micro-scale method for the measurement of haemoglobin Al(a + b + c). Diabetologia 1978; 14: 209-211. 7 Mallia AK, Hermanson GT, Krohn RI, Fujimoto EK, Smith PK. Preparation and use of a boronic acid affinity support for separation and quantitation of glycosylated hemoglobins. Anal Lett 1981; 14: 649-661. 8 Gould BJ, Hall PM, Cook JGH. Measurement of glycosylated haemoglobins using an affinity chromatography method. Clin Chim Acta 1982; 125: 41-48. 9 Klenk DC, Hermanson GT, Krohn RI et al. Determination of glycosylated hemoglobin by affinity chromatography: comparison with calorimetric and ion-exchange methods, and effects of common interference. Clin Chem 1982; 28: 2088-2094. 10 Bunn HF, Shapiro R, McManus M et al. Structural heterogeneity of human hemoglobin A due to non-enzymatic glycosylation. J Biol Chem 1979; 254: 3892-3898. 11 Hammons GT, Junger K, McDonald JM, Ladenson JH. Evaluation of three minicolumn procedures for measuring hemoglobin Al. Clin Chem 1982; 28: 1775-1778.