Determination of glycosylated hemoglobin by affinity chromatography: comparison with colorimetric and ion-exchange methods, and effects of common interferences.

1982 ◽  
Vol 28 (10) ◽  
pp. 2088-2094 ◽  
Author(s):  
D C Klenk ◽  
G T Hermanson ◽  
R I Krohn ◽  
E K Fujimoto ◽  
A K Mallia ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Affinity Chromatography ◽  
Glycosylated Hemoglobin ◽  
Isotonic Saline ◽  
Thiobarbituric Acid ◽  
Reference Interval ◽  
Average Error ◽  
Affinity Separation ◽  
Wash Buffer

Abstract An affinity-chromatographic method for determination of glycosylated hemoglobin (Anal. Lett. 14: 649-661, 1981) is compared with the thiobarbituric acid colorimetric (I) (Clin. Chem. 27: 669-672, 1981) and the ion-exchange liquid-chromatographic (II) (Diabetes 29: 623-628, 1980) methods. A correlation of 0.98 was obtained for the affinity method vs II and 0.97 for affinity vs I (n = 51). The within-run CV was 1.9% for specimens from non-diabetic individuals and 1.0% for those from diabetics. The respective between-run CVs were 3.4% and 2.4%. Failure to remove "labile" glucose adducts by 5-h incubation of erythrocytes in isotonic saline (37 degrees C) contributed an average error of 13.1% for II, 5.4% for I, and 1.6% for the affinity method. Affinity chromatography gave a decrease of 0.1-0.2% glycosylated hemoglobin for each 1.0 degree C temperature increase between 18 and 27 degrees C. Varying the pH of the wash buffer used in the affinity procedure from 7.75 to 8.25 (pH 8.0 optimum) produced at net change of 0.5% in glycosylated hemoglobin with one diabetic specimen. Using the affinity method, we determined the reference interval for glycosylated hemoglobin in 124 apparently healthy individuals to be 5.3 to 7.5% (mean 6.36%, SD 0.55%). Rechromatography by II and isoelectric focusing analysis of the fractions obtained by the affinity separation revealed a substantial population of glycosylated hemoglobins not measured by II. The affinity method offers a rapid, simple, precise, and accurate alternative to methods currently in use and gives substantial freedom from many common interferences.

Effects of whole blood storage on results for glycosylated hemoglobin as measured by ion-exchange chromatography, affinity chromatography, and colorimetry.

1983 ◽  
Vol 29 (6) ◽  
pp. 1113-1115 ◽  
Author(s):  
R R Little ◽  
J D England ◽  
H M Wiedmeyer ◽  
D E Goldstein
Keyword(s):  
Ion Exchange ◽  
Affinity Chromatography ◽  
Whole Blood ◽  
High Performance ◽  
Glycosylated Hemoglobin ◽  
Thiobarbituric Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sample Collection ◽  
Sample Stability

Abstract After storage of whole blood at either 4 or 20 degrees C, results for glycosylated hemoglobin by ion-exchange chromatography ("high-performance" liquid and mini-column chromatography), thiobarbituric acid colorimetry, and affinity chromatography were compared. At 4 degrees C, all methods gave acceptable results for samples stored for as long as a week. At 20 degrees C, the colorimetric and affinity methods also showed sample stability for a week or more. The ion-exchange methods were associated with a marked increase in values for glycosylated hemoglobin after a few days of storage. Evidently, care in details of sample collection and handling is especially important for ion-exchange methods, and the colorimetric and affinity methods have advantages over ion exchange in situations where long delays between sample collection and assay are unavoidable.

Evaluation of a colorimetric method for determination of glycosylated hemoglobin.

1983 ◽  
Vol 29 (1) ◽  
pp. 135-140 ◽  
Author(s):  
J C Standefer ◽  
R P Eaton
Keyword(s):  
Blood Glucose Concentration ◽  
Colorimetric Assay ◽  
Glycosylated Hemoglobin ◽  
Colorimetric Method ◽  
Hemoglobin Concentration ◽  
Reference Interval ◽  
Diabetic Patients ◽  
Analytical Recovery ◽  
Wide Range

Abstract We evaluated a colorimetric assay for glycosylated hemoglobin to determine the effects of several variables --oxalic acid concentration, extraneous glucose, hemoglobin concentration, hydrolysis interval, and 5-hydroxymethylfurfural destruction--and the precision. The interference seen when the blood glucose concentration exceeds 2.0 g/L (11 mmol/L) can be eliminated by washing the erythrocytes with 9 g/L saline. The accuracy of this assay is not influenced by hemoglobin concentrations from 80 to 150 g/L. The background nonspecific color, although substantial, is similar from sample to sample. After a 5-h hydrolysis at 100 degrees C, about 85% of the hexose is released, and the analytical recovery of 5-hydroxymethylfurfural is constant over a wide range of glycosylated hemoglobin concentrations. The 5th to 95th percentile reference interval for a population of 65 nondiabetic individuals was 4.6 to 6.1 mol per 100 mol of total hemoglobin. The range of values for a population of 85 diabetic patients was 6.9 to 20.4 mol per 100 mol.

Aminophenylboronic acid affinity chromatography and thiobarbituric acid colorimetry compared for measuring glycated albumin.

1985 ◽  
Vol 31 (2) ◽  
pp. 229-234 ◽  
Author(s):  
M Rendell ◽  
G Kao ◽  
P Mecherikunnel ◽  
B Petersen ◽  
R Duhaney ◽  
...  
Keyword(s):  
Glycated Hemoglobin ◽  
Glycosylated Hemoglobin ◽  
Glycated Albumin ◽  
Thiobarbituric Acid ◽  
Reactive Material ◽  
Prolonged Incubation ◽  
Diabetic Status ◽  
Chromatographic Procedure ◽  
Aminophenylboronic Acid

Abstract Two techniques originally developed for measurement of glycated ("glycosylated") hemoglobin but also applicable to determination of glycated albumin are the thiobarbituric acid colorimetric technique (I) and the aminophenylboronic acid affinity chromatographic procedure (II). The latter reliably distinguishes diabetics from nondiabetics, and concentrations of glycated hemoglobin and glycated albumin are linearly correlated. I is nonspecific; it neither correlates with diabetic status nor with values derived via the affinity technique. Most of the chromogenic material is present in the fraction of albumin that does not bind to aminophenylboronic acid. Glucose interferes significantly with I but only slightly with II. Prolonged incubation of plasma with glucose dramatically increases the II-determined glycated albumin. Reactivity with thiobarbituric acid increases much less, and mainly in the II-bound fraction. This fraction contains a high proportion of nonspecifically reactive material. The percentage of glycated albumin determined in crude plasma samples by II differs only slightly from the value determined by purifying the albumin from the plasma. This technique appears more promising than I for eventual clinical applications.

scholarly journals Comparison of Three Different Assay Procedures for the Determination of HbA1 with Special Attention to the Influence of Pre-HbA1c, Temperature and Haemoglobin Concentration

1985 ◽  
Vol 22 (3) ◽  
pp. 261-268 ◽  
Author(s):  
W Kortlandt ◽  
H J M Van Rijn ◽  
J O O Hoeke ◽  
J H H Thijssen
Keyword(s):  
Ion Exchange ◽  
Affinity Chromatography ◽  
Protein Concentration ◽  
Haemoglobin Concentration ◽  
Glycated Haemoglobin ◽  
Chromatography Method ◽  
Electrophoresis Method ◽  
Agar Gel Electrophoresis ◽  
Significant Interference

Three different methods for the measurement of glycated haemoglobin or HbA1, based on agar gel electrophoresis, ion-exchange and affinity chromatography were compared. All three showed acceptable precision (overall CV being less than 5%) and correlated well with each other ( r> 0·945). The ion-exchange and affinity chromatography method proved to be independent of the amount of pre-HbA1c present. The electrophoresis method was independent of temperature in contrast to the other two methods, which shewed a strong and comparable temperature dependency. All three methods were dependent on the haemoglobin concentration and/or protein content of the haemolysate. Both ion-exchange and electrophoresis showed significant interference by changes in haemoglobin concentrations, whereas the protein concentration significantly biased the affinity chromatography figures. Taking into account their specific merits all three methods are acceptable for routine use.

Quantitative Assessment of Soluble Fibrin in Plasma by Affinity Chromatography – A Comparative Study with desAA-Fibrin, desAABB-Fibrin and Fibrinogen

1985 ◽  
Vol 54 (02) ◽  
pp. 533-538 ◽  
Author(s):  
Wilfried Thiel ◽  
Ulrich Delvos ◽  
Gert Müller-Berghaus
Keyword(s):  
Affinity Chromatography ◽  
Calcium Ions ◽  
Quantitative Assessment ◽  
Fluid Phase ◽  
Soluble Fibrin ◽  
Binding Characteristics ◽  
Different Temperatures ◽  
Immobilized Proteins ◽  
Sepharose 4B

SummaryA quantitative determination of soluble fibrin in plasma was carried out by affinity chromatography. For this purpose, desAA-fibrin and fibrinogen immobilized on Sepharose 4B were used at the stationary side whereas batroxobin-induced 125I-desAA-fibrin or thrombin-induced 125I-desAABB-fibrin mixed with plasma containing 131I-fibrinogen represented the fluid phase. The binding characteristics of these mixtures to the immobilized proteins were compared at 20° C and 37° C. Complete binding of both types of fibrin to the immobilized desAA-fibrin was always seen at 20° C as well as at 37° C. However, binding of soluble fibrin was accompanied by substantial binding of fibrinogen that was more pronounced at 20° C. Striking differences depending on the temperature at which the affinity chromatography was carried out, were documented for the fibrinogen-fibrin interaction. At 20° C more than 90% of the applied desAA-fibrin was bound to the immobilized fibrinogen whereas at 37° C only a mean of 17% were retained at the fibrinogen-Sepharose column. An opposite finding with regard to the tested temperature was made with the desAABB-fibrin. Nearly complete binding to insolubilized fibrinogen was found at 37° C (95%) but only 58% of the desAABB-fibrin were bound at 20° C. The binding patterns did not change when the experiments were performed in the presence of calcium ions. The opposite behaviour of the two types of soluble fibrin to immobilized fibrinogen at the different temperatures, together with the substantial binding of fibrinogen in the presence of soluble fibrin to insolubilized fibrin in every setting tested, devaluates affinity chromatography as a tool in the quantitative assessment of soluble fibrin in patients’ plasma.

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