Glycated hemoglobin: methodologies and clinical applications.
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Biomedical subjects
Publications and source records attributed to J D England.
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We present data on a filter-paper collection and assay method for measurement of glycated hemoglobin (gHb). Onto filter paper dipped into a solution of glucose oxidase (EC 1.1.3.4) and allowed to dry, approximately 20 microL of capillary blood was spotted. For analysis, we eluted the dried blood spot from the paper by soaking in water for 1 h, then measured the gHb in the eluate by affinity chromatography. Because the gHb significantly increased from day 1 to day 14 of storage, it was necessary to standardize the day of elution from the paper. We found a high correlation between gHb measured from samples stored for 14 days on treated paper and gHb measured by affinity chromatography from frozen whole blood or hemolysates (r = 0.96). This method is convenient, requiring small amounts of blood and little sample handling and assay time, and may be particularly useful in certain situations such as large-scale screening for diabetes.
As the clinical utility of glycated hemoglobin (gHb) measurement increases, so does the need for standardization of values between different methods and different laboratories. Using three different methods, we examined the feasibility of interlaboratory standardization of gHb measurement. A liquid-chromatographic (HPLC) system from our research laboratory was designated the reference method. For gHb standards we used erythrocyte hemolysates prepared from blood samples from nondiabetic and diabetic subjects. Values assigned to each standard were based on the mean of multiple gHb determinations by the HPLC method. A clinical laboratory routinely prepared hemolysates and assayed gHb by commercially available ion-exchange ("mini column") and affinity chromatographic methods. For each assay a standard curve was constructed and gHb values were derived from these curves. Samples analyzed in the clinical laboratory were also analyzed in the research laboratory and the curve-derived values were compared with the HPLC-measured values, to determine the accuracy of our interlaboratory standardization procedure. Correlations were excellent (r = 0.99). The lack of significant differences between calculated and HPLC-measured values indicates that interlaboratory standardization is feasible.
We present data on the use of filter-paper blood collection for measurement of glycosylated whole-blood proteins (gWB) (hemoglobin and plasma proteins). A capillary blood sample, obtained by fingerprick, is spotted directly onto filter paper (Schleicher & Schuell 903). The blood spot is washed briefly with alcohol (ethanol or isopropanol) to remove free glucose and dried before shipment to the laboratory. In the laboratory, the blood is eluted from the paper and analyzed for gWB by a colorimetric method. The gWB is primarily a measure of glycosylated hemoglobin (gHb) with a small contribution from glycosylated plasma protein. Concentrations of gWB and gHb are highly correlated (r = 0.91). The filter-paper method offers advantages over currently available methods for quantifying gHb and may be particularly useful in screening for diabetes and for assessing glycemic control in patients from remote areas.
A 33-year-old homosexual man with symptoms and signs of a focal brain process was subsequently found to have an acquired immune deficiency syndrome (AIDS) with biopsy-proven progressive multifocal leukoencephalopathy. This report reemphasizes the association of progressive multifocal leukoencephalopathy with AIDS and probably is best viewed as another example of an opportunistic CNS infection complicating deficient cell-mediated immunity.
Glycosylated hemoglobins have gained wide acceptance as an accurate index of long-term blood glucose control in diabetes mellitus. A variety of glycosylated hemoglobin assays is available. There is a high degree of correlation between results determined by these assays. The ideal laboratory method for measuring glycosylated hemoglobin in the diabetic should be accurate, precise, easily standardized, inexpensive, and rapidly performed. Unfortunately, none of the currently used methods meet all of the criteria necessary to be considered the ideal laboratory method. The most widely used methods for quantitating glycosylated hemoglobins--including ion exchange chromatography, electrophoresis, isoelectric focusing, thiobarbituric acid colorimetry, and affinity chromatography--are reviewed with respect to the important advantages and disadvantages of each method for the clinical laboratory. Techniques for quantitating glycosylated proteins other than hemoglobins, such as albumin, are also discussed.
Under proper conditions, whole blood can be stored at room temperature for as long as 21 days before measurement of glycosylated hemoglobin by affinity chromatography. Whole blood (anticoagulated with EDTA or heparin) was placed in capillary tubes, which were then sealed at both ends and stored at room temperature. Just before assay, whole blood was rinsed from the tubes and diluted 10-fold with water. Samples of each patient's blood were assayed as whole-blood hemolysates by affinity chromatography after zero, seven, 14, and 21 days of storage. Values for glycosylated hemoglobin did not change over 21 days of storage and values for each storage day correlated well (r = 0.97, p less than .0001) with hemoglobin A1C measured in fresh erythrocyte hemolysates by "high-performance" liquid ion-exchange chromatography.
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.
Twenty-eight patients with mild essential hypertension were treated with placebo for six weeks and then with active medication for a further 32 weeks. Twelve patients were well-controlled with 160-320 mg/day of slow release oxprenolol alone, 12 required oxprenolol and chlorthalidone, and four also required hydrallazine. beta-adrenergic blockade reduced the basal level of plasma free fatty acid (FFA) by 41%. A sub-maximal exercise test reduced the level of FFA by 34% during placebo treatment. The same exercise test during beta-blockade reduced the already lowered basal FFA level by a further 45%. Only female subjects experienced exercise-induced leg fatigue during beta-blockade. They always had higher FFA levels than males, but the relative changes in FFA concentration were similar in both sexes. Exercise testing induced a 52 mmHg rise in systolic blood pressure, but this was reduced to only 14 mmHg during treatment. Patients controlled on oxprenolol alone showed no significant change in plasma lipid and lipoprotein levels. Those patients ultimately requiring combination drug therapy experienced a statistically significant rise in plasma triglycerides and a significant fall in high density lipoprotein cholesterol. The biological importance of these changes is uncertain.
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Glycosylated hemoglobin was measured in Mystromys albicaudatus, a rodent model of diabetes mellitus, using a newly developed colorimetric assay. The mean glycosylated hemoglobin for non-diabetic Mystromys (n = 321) was 14.8 nmol hydroxymethylfurfural / 10 mg hemoglobin which is similar to that obtained in non-diabetic humans. Animals with a glycosylated hemoglobin value greater than 19 nmol hydroxymethylfurfural / 10 mg hemoglobin were characterized as diabetics. Significant correlations were found between glycosylated hemoglobin and both plasma glucose and urine glucose levels.
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.
Atenolol, Pindolol and Propranolol in single daily doses administered to 18 selected patients with mild essential hypertension achieved adequate control of blood pressure. Chlorothiazide had been initially administered twice a day without full control of blood pressure and this diuretic therapy was continued unaltered throughout the study. Methacholine challenge testing of respiratory function was performed during the placebo phase and with each beta adrenoreceptor-blocking drug. In the 18 non-asthmatic patients, the reduction in FEV1, was significant only for propranolol therapy when compared to placebo. Each beta adrenoreceptor-blocking drug was associated with small, but significant, increases in fasting plasma triglyceride concentrations and suppression of fasting immuno-reactive glucagon concentrations.
This colorimetric assay for glycosylated hemoglobin can be performed in 120 min with equipment available in most clinical laboratories. The glucose moiety of glycosylated hemoglobin is converted to 5-hydroxymethylfurfural by heating with oxalic acid for 60 min is an autoclave at 124 degrees C and 124 kPa (18 lb/in.2). The adduct formed by reacting 2-thiobarbituric acid with hydroxymethylfurfural is measured photometrically and results are expressed either as nanomoles of hydroxymethylfurfural or as fructose equivalents. Within-assay and between-assay coefficients of variation were less than 2% and less than 3%, respectively. Comparison of results for 50 patients' specimens as measured by the present assay and as analyzed for hemoglobin A1c by liquid chromatography showed excellent correlation (r = 0.98).
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1. Chlorothiazide twice a day plus atenolol, metoprolol, pindolol and propranolol in single daily doses administered to patients with essential hypertension achieved effective control of blood pressure. 2. Each beta-adrenoreceptor-blocking drug was associated with small, but significant, increases in plasma triglyceride concentrations and suppression of fasting immuno-reactive glucagon concentrations.