Hemoglobin A1a Research Articles

Global Harmonization of Hemoglobin A1c

Measurement of glycohemoglobin (GHb) is widely used in patients with diabetes mellitus as a monitor of long-term glycemic control (1)(2)(3). In addition, prospective randomized clinical trials, most notably the Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS), have demonstrated that GHb is a measure of the risk for the development of diabetes complications (4)(5). GHb is therefore an integral component of the management of patients with diabetes. GHb comprises several different hemoglobin-glucose adducts, including hemoglobin A1a (HbA1a), HbA1b, and HbA1c. More than 30 different methods are commercially available to measure GHb. Together these factors have led to considerable variation in reference intervals and results reported by different laboratories. When the DCCT was published in 1993, the lack of standardization of GHb methods produced very wide variability among methods, with values ranging from 4.0% to 8.1% on the same blood sample (6). In the United States, the NGSP (previously known as the National Glycohemoglobin Standardization Program) has reduced interlaboratory variation (7). Using a standardization process based on the DCCT reference method, the NGSP has promoted a dramatic improvement in comparability of GHb values among laboratories (3). Data from the 2003 GH2 survey from the College of American Pathologists indicated that ≥98% of participating laboratories use NGSP-certified methods and report results as HbA1c or HbA1c equivalents (3). Analogous standardization programs in Sweden and Japan (8)(9), established to harmonize GHb results, have also reduced variability among GHb results. More recently, the IFCC Working Group on HbA1c Standardization prepared primary reference materials of pure HbA1c and HbA0 and developed a reference method for HbA1c (10). They defined HbA1c as the stable adduct of glucose to the N-terminal valine of the β-chain of hemoglobin. In the reference method, hemoglobin is cleaved …

Improved rapid procedure for simultaneous determinations of hemoglobins A1a, A1b, A1c, F, C, and S, with indication for acetylation or carbamylation by cation-exchange liquid chromatography

Although the clinical utility of glycohemoglobin (HbA1c) as an indicator of blood glucose control is generally accepted, the use of cation-exchange liquid chromatography for its quantification is controversial, given numerous studies that show no correlation between HbA1c and other indicators of blood glucose control in uremic subjects. We report a specific cation-exchange liquid-chromatographic method that measures simultaneously HbA1a, HbA1b, HbA1c, HbF, HbA0, HbC, and HbS quickly and reproducibly. Carbamylation or acetylation is indicated by extra peaks. Carbamylated hemoglobin increased the result for HbA1c by as much as 1% above the nonuremic value. The method is a fast (approximately 15 min per sample), easy to perform, reproducible, and sensitive screening method for abnormal hemoglobins. Within-assay CVs were < 0.7% in all cases and between-assay CVs were < 1.4%.

Alcoholic cerebellar degeneration is not a dose-dependent phenomenon.

Eleven alcoholics with cerebellar degeneration (eight with computerized tomography confirmation of cerebellar atrophy) were matched with nonataxic alcoholics and nonalcoholics. There were no laboratory or physiological markers for ataxia, including hemoglobin A1a + b, red blood cell transketolase, liver function enzymes, and measures of reaction time and hand-eye coordination. Acetaldehyde-modified hemoglobin levels (as hemoglobin A1a + b) did not, as previously reported, distinguish between alcoholics and nonalcoholics. There was 24% less annual alcohol consumption in ataxic alcoholics compared with nonataxic alcoholics, 9% less lifetime consumption in ataxic alcoholics, and 33% less maximal daily intake. The finding that ataxic alcoholics do not have higher alcohol consumption than nonataxic alcoholics suggests that alcoholic cerebellar degeneration is not a dose-dependent phenomenon, and that alcoholics with cerebellar degeneration may have an idiosyncratic sensitivity to the neuronal effects of alcohol.

Glycosylation of human hemoglobin A Kinetics and mechanisms studied by isoelectric focusing

The reaction between glucose and hemoglobin A (HbA) leading to hemoglobin A 1c through a labile intermediate, designated anodal glycobemoglobin A, was studied in vitro using an isoelectric focusing method. Studies were performed on the kinetics of the formation and breakdown of the labile intermediate. The reactions of HbA with various aldohexoses and of hemoglobin A 1c and anodal glycohemoglobin A with phenylhydrazine were studied. It is suggested that the glucose in the anodal glycohemoglobin A band is in the pyranose form of a Schiff base formed between the N-terminal amino group of the protein and d-glucose, while the fructose in hemoglobin A 1c is in the pyranose form of the ketimine comprising also the N-terminal of the group. Elution peaks from ion-exchange chromatography of the hemoglobins termed HbA 1a, HbA 1b, HbA 1c and HbA studied by isoelectric focusing revealed that: HbA 1a formed two bands and HbA 1b one band far toward the anode; both contained minor fractions of HbA 1c and HbA; HbA 1c appeared as a single band, while HbA was contaminated with some HbA 1c. Hemoglobin A 1c purified by isoelectric focusing eluted as one peak when analysed by ion-exchange chromatography, while anodal glycohemoglobin A co-eluted with the first HbA 1c fractions in the chromatogram. From kinetic studies it appeared that the rate constant for formation ( frsolk 1) of anodal glyeohemoglobin A was 0.096 · 10 −3 1/ mol · s −1 at 37° C. The constant for dissociation ( k −1) was 0.10 · 10 −3 s −1. From these an equilibrium constant K of 0.96 1/mol was calculated. The apparent Arrbenius activation energies for the ( k 1) and ( k −1) reactions were 42.5 and 47.5 kJ/degree, respectively. Consequently the equilibrium constant K is predicted to be nearly temperature-independent within the temperature range investigated. This was fully substantiated by experiments conducted at different temperatures. Furthermore, the values of the apparent Arrhenius activation energies allows the values of the rate constants to be calculated at any temperature within the experimental temperature range. This information is of importance for a closer understanding of the mechanisms of glycohemoglobin accumulation in red blood cells.

Reactions of Biologic Aldehydes with Proteins

The amino functions of proteins are susceptible to modification by the carbonyl moieties of aldehydes that are found in vivo, such as formaldehyde, glyceraldehyde, acetaldehyde, and glucose. Formaldehyde reacts fairly nonspecifically with proteins. However, under controlled conditions, it may be used as a structural probe to supply the carbonyl component for the reductive alkylation of proteins. Glyceraldehyde reacts less extensively with proteins, as exemplified by its reaction with hemoglobin, and has therapeutic potential as an antisickling agent. Recent work has focused on the modification of hemoglobin by acetaldehyde. This primary metabolite of ethanol metabolism is elevated in alcoholics. Clinical studies on fast-chromatographing hemoglobins (hemoglobin A1a–c) have suggested that a novel adduct may be formed in persons abusing alcohol. Alcoholic patients have an elevated concentration of minor hemoglobins but normal or subnormal amounts of glycosylated hemoglobin (hemoglobin A1c) as determined by radioimmunoassay. Acetaldehyde was found to form adducts with hemoglobin (HbAAA), both in vitro and in vivo, leading to a change in Chromatographic properties. Browning occurred with higher concentrations of acetaldehyde. HbAA formation thus provides a potential marker for alcoholism in addition to providing a biochemical hypothesis regarding the secondary sequelae of alcohol intake.

The clinical utility of glycosylated hemoglobin

Measurement of glycosylated hemoglobins in diabetic patients has been available to clinicians for about five years. Such measurements correlate with mean serum glucose determinations over time; therefore, they have stimulated a number of studies to determine (1) if these assays are useful in diagnosing diabetes, (2) the clinical utility of determinations of minor hemoglobins in monitoring diabetic control, and (3) the relationship of glucose “control” (as indicated by concentrations of glycosylated hemoglobins) to abnormalities or “sequelae” of the diabetic state. High concentrations of glycosylated hemoglobins are highly specific for diabetes, and positive findings provide a useful diagnostic test. However, this measurement is less sensitive than a glucose tolerance test. As a clinical tool, these hemoglobins are most useful in labile diabetes, i.e., juvenile-onset diabetes and diabetes in pregnancy. In adult-onset diabetes, the fasting serum glucose concentration is apt to correlate well with the concentration of hemoglobins A1a−c. A correlation between several abnormalities associated with diabetes mellitus and concentrations of hemoglobins A1a−c have been reported. These abnormalities include abnormalities of the erythrocyte, leukocyte, platelet, and coagulation cascade and hormonal profiles in juvenile-onset diabetes and diabetes in pregnancy. In addition, correlation have been reported between certain risk factors or abnormalities associated with vascular disease and concentrations of minor hemoglobins, including lipid profiles, microvascular disease as reflected by retinal changes and quadriceps capillary basement membrane thickening, and macrovascular disease as reflected by pulse volume recordings. These studies have led to a reevaluation of the role of glucose “control” in contributing to diabetic sequelae, and, thus, have stimulated new approaches to the management of diabetes.

Clinical evaluation of measuring glycosylated hemoglobin levels for assessing the long-term blood glucose control in diabetics.

The glycosylated hemoglobin levels were determined in the hemolysates obtained from 14 normal subjects and 67 patients with adult-onset diabetes mellitus, using the column chromatographic procedure of Trivelli et al. The levels of hemoglobin A1a+b (HbA1a+b) and HbA1c and the sum of HbA1a+b and HbA1c (HbA1a+b+HbA1c) in normal subjects averaged 2.3 +/- 0.4 (SD)%, 5.3 +/- 0.8% and 7.6 +/- 1.0%, respectively. Although a slight increase in HbA1a+b was found in patients with diabetes mellitus (mean +/- SD=2.8 +/- 0.7%), it was not significantly different from that in normal subjects. Despite the wide range, HbA1c and HbA1a+b+HbA1c were significantly increased in patients with diabetes mellitus (6.9 +/- 1.8% for HbA1c, p < 0.01 and 9.7 +/- 2.2% for HbA1a+b+HbA1c, p < 0.01). A significant correlation existed between the glycosylated hemoglobins and plasma glucose levels determined in the same blood (r = +0.57, p < 0.001). Moreover, the glycosylated hemoglobin levels correlated significantly with the average glucose levels for several months preceding the hemoglobin measurements. In particular, a striking correlation was evident in the plots of HbA1a+b+HbA1c against the mean plasma glucose for 3 months prior to the hemoglobin measurements, which had a correlation coefficient of 0.79 (p < 0.001). The present findings revealed that the glycosylated hemoglobins reflect the time-averaged blood glucose levels in diabetics for during approximately the proceeding 3 months, indicating the usefulness of measuring the glycosylated hemoglobins in assessing the long-term blood glucose control in diabetics.

Quantitation of hemoglobin A1a+b and hemoglobin A1c by automated "high-performance" liquid chromatography.

The glycosylated hemoglobins A1a+b and A1c have been rapidly and precisely quantitated in 5-micro L samples of human blood hemolysate (approximately 240 micrograms of hemoglobin) by cation-exchange column chromatography. Total chromatographic is 22.0 min. Proportions of Hb A1c range from 3.85 to 6.71% in normal individuals and from 4.23 to 19.90% in diabetic subjects. Within-day variation was 1.58 and 1.10% for mean Hb A1c proportions of 4.92 and 10.32%, respectively. Hb A1c and Hb A1 are stable in hemolysates stored at 4 degrees C for as long as seven days, and indefinitely under liquid nitrogen.

A simple microchromatographic column for determination of hemoglobins A1a + b and A1c.

A simple microchromatographic homemade column was devised for the measurement of glycosylated minor hemoglobin fractions. The mean and one standard deviation of hemoglobins A1a + b, A1c and A1a + b + c determined by the homemade column in 150 nondiabetic controls and 56 juvenile onset insulin-dependent diabetics were 2.6 +/- 0.5%, 6.0 +/- 1.0%, 8.6 +/- 1.1%, 3.3 +/- 0.9%, 13.7 +/- 2.2% and 16.9 +/- 2.8% respectively. A twofold increase in hemoglobins A1c and A1a + b + c levels was observed in the diabetics as compared to the non-diabetic controls suggesting that the homemade column is valid for the measurement of all three glycosylated hemoglobin fractions and assessment of blood glucose control in diabetic patients. The homemade column procedure yields accurate and reproducible results, is simple to perform, inexpensive, relatively rapid and may be used in the routine clinical laboratory. Hemoglobin A1a + b + c levels measured by a commercial column in 35 non-diabetic controls and in 56 diabetics showed good correlation (R = 0.91) with hemoglobin A1a + b + c levels determined by the homemade column. The commercial column is valid for the measurement of the combined glycosylated hemoglobin A1a + b + c fraction and may be used in the routine clinical laboratory to assess diabetic control.

The quantities of various minor hemoglobin components in old and young human red blood cells

The red blood cells from several normal healthy adult individuals have been separated into young and old cells using a density layer centrifugation technique. The quantities of the following minor hemoglobin components were determined: the hemoglobins A1a, A1b and A1c by Amberlite IRC-50 chromatography; the Hb-A 2 by DEAE-cellulose chromatography; and Hb-F by an alkali denaturation procedure. No differences were observed in the percentages of each of these hemoglobin fractions as present in the two types of cell layers. It has, therefore, been concluded that differences in oxygen affinities of the two cell types are not due to differences in the quantities of minor hemoglobin fractions but should probably be sought in salt-labile changes in the hemoglobin molecules.