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.     

Elevation rate of glycosylated hemoglobins in dogs after induction of experimental diabetes mellitus

The glycosylated hemoglobin, hemoglobin A_1c (HbA_1c), which reflects average plasma glucose of the previous few weeks, has recently been used to monitor humans with diabetes mellitus. Further understanding of the HbA_1c elevation rate would improve interpretation of HbA_1c. We determined glycosylated hemoglobin elevation rates in 5 dogs with induced diabetes. Hemoglobin A_1c was determined by an established column chromatographic technique; plasma glucose by glucose oxidase. Values were determined on 13 normal dogs and compared with values obtained weekly from surgically or chemically-induced diabetic dogs. Hemoglobin A_1c increased in a fashion that could be predicted by modelling. The model predicts that large changes in Hb A_1c will occur within the first few weeks of a sudden change in glucose and that a new plateau will be reached at a time equal to the erythrocyte life span. In the present experiment abnormally elevated HbA_1c occurred after 2 wk of hyperglycemia. The results should approximate the elevation rate after acute onset and sustained severe hyperglycemia in humans, because humans and canines are hematologically similar and thus extend previously reported studies on human out-patient diabetics.     

A rapid micro-scale method for the measurement of haemoglobin A1(a+b+c)

Summary A rapid method is described for the measurement of total glycosylated haemoglobins (HbA_1(a+b+c). The procedure utilizes 0.05 ml of blood and takes forty minutes to complete manually. Eighty blood samples can be analysed without automation by one person in a day. Each analysis uses less than 2 mg of potassium cyanide, resulting in a method that is both safe and rapid for routine hospital laboratories. The inter-assay coefficient of variation was 4% and that for intra-assay measurements 3%, over the range 5–20% HbA_1(a+b+c). The method confirmed that the level of HbA_1(a+b+c) is elevated in imperfectly controlled diabetics. Amongst patients with blood glucose levels of less than 10 mmol/l the mean level of HbA_1(a+b+c) was found to be 8.5%; samples from 14 known diabetics gave a mean value of 10.9%, whereas 17 known non-diabetic samples gave a mean value of 8.3%. In the group of samples from 27 diabetic individuals with blood glucose levels above 10 mmol/l the mean level of HbA_1(a+b+c) was found to be 13.5%.     

Glycosylated haemoglobin and steady-state mean blood glucose concentration in type 1 (insulin-dependent) diabetes

Summary Since glucose control and glycosylated haemoglobin varies asyncroneously, we have studied the steady-state relationship between these two factors. In Type 1 (insulin-dependent) diabetic patients with a constant haemoglobin A_1c during the preceding 2 years, 15 ambulatory blood glucose profiles during a 5-week period showed a constant glucose level and provided a precise estimate of the mean blood glucose concentration. In addition, we studied 15 non-diabetic subjects who provided three glucose profiles and had one haemoglobin A_1c determination performed. A good correlation was found for a curvilinear relationship (haemoglobin A_1c=2.07 x mean blood glucose^0.596, r=0.98). This close relationship indicates that glycosylated haemoglobin is a valuable, but not very sensitive, index of glucose control.     

Glycosylated hemoglobin in patients with newly discovered juvenile-onset diabetes mellitus in the days immediately following the beginning of insulin treatment

Summary We studied the behavior of fast hemoglobin fractions in newly discovered diabetic patients, before and in the 10 days immediately following the beginning of insulin therapy, in order to verify whether or not the rapid improvement of glycemic control involved a rapid reduction of total HbA₁ and of its fractions. We observed a rapid and highly significant fall of HbA_1(a+b+c) and HbA_1c levels after only 1 or 2 days of insulin therapy, followed by a slower decrement in the other 3–10 days. HbA_1(a+b) showed a slower decrement trend, reaching levels significantly below baseline values only after 7–10 days. These results suggest that rapid changes occurring in glycosylated hemoglobin levels after the beginning of insulin treatment in newly discovered diabetic patients involve mainly HbA_1c. The kinetics of glycosylated hemoglobin reduction, with a first rapid decrement followed by a slower one, may suggest the hypothesis that rapid changes are due to reversible Schiff base de-glycosylation, the ketoamine linkage being the true index of long term glycemic control.     

Does ethanol intake interfere with the evaluation of glycated hemoglobins?

Summary Ethanol and/or its metabolites interfere with the chromatographic assay of glycated hemoglobins. Fasting plasma glucose, blood ethanol, HbA₁, HbA_a1+b, MCV and GGT were determined in 22 control subjects, 22 alcoholics, 22 diabetic patients and 22 alcoholic diabetic patients. Fasting plasma glucose and all hemoglobin fractions were lower in alcoholic subjects and, except for HbA_1a+b, higher in diabetic patients and in alcoholic diabetic patients. HbA₁ and HbA_1c correlated well with plasma glucose but not with blood ethanol, MCV and GGT. Glycated hemoglobin was not found to be a useful marker for alcohol abuse. With the chromatographic method we used, the evaluation of glycated hemoglobin fractions, chiefly HbA_1c, confirms its usefulness in monitoring the metabolic control of diabetic subjects, even in case of ethanol abuse.     

Serum adiponectin concentrations are not related to glycosylated hemoglobin levels (HbA1c) in obese diabetic and non-diabetic caucasians.

Although circulating adiponectin has been inversely correlated with obesity, type 2 diabetes and serum glycosylated hemoglobin (HbA1c) in humans, contradictory reports on that subject exist. In this study, serum concentrations of adiponectin in obese non-diabetic and diabetic humans were measured to examine whether they were associated with levels of HbA1c. The WHO definitions of obesity and diabetes were used. One hundred and five obese euglycemic subjects and 49 obese diabetics (aged 51+/-6.9, and 52+/-6.7 years, respectively) were studied. Their BMI, HbA1c and % of body fat were measured. Adiponectin was determined by an enzyme-linked immunosorbent assay. Although the serum adiponectin concentrations differed between diabetics and non-diabetics ( P<0.01), they were not correlated with HbA1c (r=-0.0814; P=0.5823, and r=-0.1861; P=0.1099, for diabetics and non-diabetics, respectively). Both diabetics and non-diabetics were segregated into tertiles according to their HbA1c levels. Plasma adiponectin did not differ significantly between the high (H), intermediate (I), and low (L) HbA1c tertiles. CONCLUSION: Concentrations of adiponectin were not correlated with levels of glycosylated hemoglobin in the diabetic and non-diabetic subjects examined.     

Level of serum fructosamine in Saudi diabetic patients

Summary Fructosamine, a compound used to measure serum glycosylated proteins was assayed in 105 Saudi diabetic subjects and 54 healthy non-diabetic Saudi subjects. Fructosamine concentrations in diabetics were significantly higher than in healthy controls (p<0.0005). Fructosamine concentrations correlated significantly with fasting blood glucose and HbA₁ in diabetics (r=0.677, p<0.0005, and r=0.598, p<0.0005, respectively). The correlation between fructosamine and HbA₁ was significant in the oral hypoglycemic-treated diabetics and poor in the insulin-treated diabetic group (r=0.568, p<0.0005, and r=0.526, p=0.01). Fructosamine concentrations correlated poorly with the duration of diabetes (r=0.221, p<0.05).     

Modification of Hemoglobin by Acetaldehyde: A Time Course Study by High Pressure Liquid Chromatography

Acetaldehyde forms stable adducts with proteins, and rapidly eluting hemoglobins on cation exchange chromatography have been found to be elevated in persons consuming excess alcohol. Incubation of hemoglobin hemolysate with 5 mM acetaldehyde at 37°C for various time intervals resulted in linear increases in the amounts of hemoglobin (Hb)A1a+b and HbA1c fractions determined by cation exchange high pressure liquid chromatography. The rate of formation of the HbA1c fraction was significantly higher (p < 0.001) than that of the HbA1a+b fraction. No increases in the amounts of minor hemoglobins were observed when hemoglobin was incubated with 0.05 mM acetaldehyde. Incubation of hemoglobin with 5 m acetaldehyde followed by reduction with sodium borohydride (NaBH₄.) resulted in a significant increase in both HbA1a+b and HbA1c fractions. The rate of formation of the HbA1c fraction was again significantly faster than that of HbA1a+b. Dialysis of nonreduced acetaldehyde- modified hemoglobin had no effect on the amounts of the two minor hemoglobin fractions. Dialysis of NaBH₄-reduced acetaldehyde-modified hemoglobin resulted in decreased amounts of the HbA1a+b fractions but no changes in the HbAlc fractions. Incubation with sodium cyanoborohydride led to minimal changes in chromatographic properties of hemoglobin. The clinical utility of acetaldehyde-modified hemoglobin eluting in the HbA1c fraction in the detection of excess alcohol consumption appears to be limited by the high concentration of acetaldehyde required. Furthermore, attempts to stabilize acetaldehyde-Schiff base adducts of hemoglobin with reducing agents must include appropriate controls, since the reductive step alone may lead to changes in the chromatographic properties of hemoglobin.     

Hemoglobin A1C separation by isoelectric focusing

A modified type of isoelectric focusing has been applied successfully to the separation of hemoglobin A_1C (HbA_1C) from HbA in normal and diabetic cell lysates. It consists of transforming a linear pH gradient into a nonlinear one, by the addition of an amphoteric substance (“separator” or “pH gradient modifier”) with an isoelectric point (pI) close to the pI's of the two hemoglobins. Among the “modifiers” tested, histidine, proline, threonine, β-alanine, 6-amino caproic acid, and 5-amino valeric acid are not useful in the hemoglobin pI range (pH 6.9–7.0). The dipeptide histidyl-glycine (pI = 6.8; pI ?pK₁ = 1) is very efficient in flattening the pH gradient, in the hemoglobin region, even when added in low concentrations (10–100 mM), thus affording full resolution of the two hemoglobin species.     

Synthesis of glycosylated haemoglobin in vivo

Summary The synthesis of glycosylated haemoglobins in vivo was measured during 24 h of controlled hyperglycaemia in seven insulin dependent diabetics. The mean blood glucose concentration was 22 mmol/l, while electrolytes and other metabolites were kept normal by infusion of 4–23 IU of insulin during hyperglycaemia. The study confirmed the velocity and magnitude of unstable HbA_1c formation previously found in vitro. The stable HbA_1c formed in 24 h was on average 0.006% of total haemoglobin/ mmol glucose. This compares well with the rate of HbA_1c synthesis reported in normal subjects using ⁵⁹Fe-kinetic measurements, and is in accordance with the concept of slow changes in stable HbA_1c with time and glucose concentration. To investigate the possibility that the rate of HbA_1c synthesis varies with erythrocyte age, glycosylated haemoglobins were measured in erythrocyte fractions after density separation on Percoll-Albumin gradients. We found both in normal subjects and in insulin treated diabetics that the 5% least dense cells contained 70%–80% of whole blood HbA_1c. Assuming the least dense cells to be the youngest erythrocytes, this observation is inconsistent with a slow linear increase in HbA_1c. Similar results were obtained in six newly diagnosed insulin dependent diabetic patients both before and after the first 30 days of insulin treatment, even though a marked decrease in young cell HbA_1c would be expected with the improved glucose control observed. We therefore conclude that density separation of erythrocytes is an inadequate technique to study age related HbA_1c synthesis.     

Rapid changes in chromatographically determined haemoglobin A1c induced by short-term changes in glucose concentration

Summary Chromatographically determined haemoglobin A_1c concentration was measured during short-term (1–24 h) changes in glucose concentration in vitro and in vivo. In vitro at 37 °C the HbA_1c concentration increased with glucose concentration and time both in normal and diabetic erythrocytes. In normal erythrocytes incubated in 20–100 mmol/l glucose, the increases in the HbA_1c concentration were maximal after 4–6 h and then stable for the next 18–20 h. During the first hour, increases in the HbA_1c concentration were linear with time and on average 0.034% HbA_1c × h^–1 × mmol/l glucose^–1. In erythrocytes, after a rapidly produced increase (2h), HbA_1c decreased to preincubation concentrations during a further incubation of the erythrocytes in a glucose-free medium at 37 °C for 4–6 h. The mean rate of linear decrease was 0.017% × h^–1 × mmol/l glucose^–1. After incubation of erythrocytes in 100 mmol/l glucose for 24 h, 1.3% HbA_1c remained stable for 6 h in saline. The rapid increase in HbA_1c concentration, as determined by chromatography, was not due to stable HbA_1c (ketoamine linked glucose) as no increase was found in the HbA_1c concentrations determined by the thiobarbiturate method. In juvenile diabetics controlled by an artificial beta-cell, rapid changes of blood glucose concentration (up to 20 mmol/l) resulted in increases in HbA_1c concentration of as much as 1.9% within 12 h (mean 1.1%). Rapid in vivo increases in HbA_1c concentration were reversible by normalization of the blood glucose concentration. That rapid changes in HbA_1c may occur in daily diabetic life was evidenced by differences in HbA_1c concentration between blood samples from out-patient diabetics incubated in saline for 16 hours at 4 °C and 37 °C (range of differences 0.2–1.4% HbA_1c). The differences correlated to the blood glucose concentration at the time of sampling blood for HbA_1c determination. Thus, incubation of blood at a low glucose concentration prior to determination of the glycosylated haemoglobin concentration may overcome interference from rapidly produced HbA_1c.     

Comparison of the BioRad Variant and Primus Ultra2 high‐pressure liquid chromatography (HPLC) instruments for the detection of variant hemoglobins

Introduction: Hemoglobin variants are a result of genetic changes resulting in abnormal or dys‐synchronous hemoglobin chain production (thalassemia) or the generation of hemoglobin chain variants such as hemoglobin S. Automated high‐pressure liquid chromatography (HPLC) systems have become the method of choice for the evaluation of patients suspected with hemoglobinopathies. Methods: In this study, we evaluated the performance of two HPLC methods used in the detection of common hemoglobin variants: Variant and Ultra2. Results: There were 377 samples tested, 26% (99/377) with HbS, 8.5% (32/377) with HbC, 20.7% (78/377) with other hemoglobin variant or thalassemia, and 2.9% with increased hemoglobin A₁c. The interpretations of each chromatograph were compared. There were no differences noted for hemoglobins A₀, S, or C. There were significant differences between HPLC methods for hemoglobins F, A₂, and A₁c. However, there was good concordance between normal and abnormal interpretations (97.9% and 96.2%, respectively). Conclusion: Both Variant and Ultra2 HPLC methods were able to detect most common hemoglobin variants. There was better discrimination for fast hemoglobins, between hemoglobins E and A₂, and between hemoglobins S and F using the Ultra2 HPLC method.     

Plasma lipoproteins and apolipoproteins in insulin-dependent and young non-insulin-dependent Arab women

Summary Plasma lipids, lipoproteins and apolipoproteins (apo) were analysed in 30 young Arab IDDM and 50 young insulin-requiring NIDDM women. The mean age of IDDM and NIDDM groups was 20.2 and 34.5 years, and mean duration of diabetes was 5.7 and 4.6 years, respectively. Two groups of 40 and 60 healthy women (matched for age and BMI) provided corresponding control groups. In comparison with control subjects, diabetics showed marked increases in the following parameters: total cholesterol (TC), low density lipoprotein (LDL) cholesterol, total triglycerides (TG), very low density lipoprotein (VLDL) triglycerides, phospholipids, apoB, LDL apoB, glucose and glycosylated hemoglobin (HbA_1c) as well as the ratios of total cholesterol/high density lipoprotein (HDL) cholesterol, LDL-cholesterol/HDL-cholesterol, LDL cholesterol/high density lipoprotein 2 (HDL₂) cholesterol and apoB/apoAI. Plasma LCAT activity, concentrations of HDL₃ apoAI and apoAII in plasma and lipoprotein fractions were normal in both the diabetic groups. Levels of C-peptide, HDL, HDL₂ and HDL₃ cholesterol, plasma apoAI, HDL apoAI and HDL₂ apoAI were markedly decreased in the diabetic groups as compared to their corresponding controls. There was no significant correlation between fasting glucose or HbA_1c and any of the above parameters. Despite insulin therapy in both the diabetic groups studied, abnormalities in lipids, apoB and apoAI still persisted. Our data suggest a possible higher risk of atherosclerosis in these patients.     

The measurement of haemoglobin A1c by isoelectric focussing in diabetic patients

Summary Incubation in vitro of human red cells in increasing glucose concentrations results in a rise in both haemoglobin A_1c levels and an intermediate band when measured by an isoelectric focussing method. There are strong correlations between blood glucose levels, levels of haemoglobin A_1c and the intermediate band both in vitro and in blood samples taken from diabetic patients. As the intermediate band is also included in the measurement of haemoglobin A_1c by the usual analytical methods, this may lead to inaccurate results.     

Risks of Diabetic Nephropathy with Variation in Hemoglobin A1c and Fasting Plasma Glucose

Background

This study examined whether annual variation in glycosylated hemoglobin A_1c (HbA_1c) and fasting plasma glucose (FPG), as represented by the coefficient of variation (CV), can predict diabetic nephropathy independently of mean FPG, mean HbA_1c, and other risk factors in patients with type 2 diabetes.

Methods

A computerized database of patients with type 2 diabetes aged ≥30 years and free of diabetic nephropathy (n = 3220) who were enrolled in the Diabetes Care Management Program of China Medical University Hospital before 2007 was used in a time-dependent Cox proportional hazards regression model.

Results

The incidence rates of diabetic nephropathy were 16.11, 22.95, and 28.86 per 1000 person-years in the first, second, and third tertiles of baseline HbA_1c-CV, respectively; the corresponding incidence rates for FPG-CV were 9.46, 21.23, and 37.51 per 1000 person-years, respectively. After multivariate adjustment, the corresponding hazard ratios for the second and third tertiles versus the first tertile of annual HbA_1c-CV were 1.18 (95% confidence interval [CI], 0.88-1.58) and 1.58 (95% CI, 1.19-2.11), respectively, and 1.55 (95% CI, 0.99-2.41) and 4.75 (95% CI, 3.22-7.01) for FPG-CV, respectively. The risks of diabetic nephropathy for HbA_1c-CV and FPG-CV stratified according to age, gender, renal function, and hypertension status were provided.

Conclusions

Annual FPG and HbA_1c variations have a strong association with diabetic nephropathy in patients with type 2 diabetes. Whether intervention for reducing glucose variation should be administered needs to be examined in a future study.     

Insights into the Progression of Labile Hb A1c to Stable Hb A1c via a Mechanistic Assessment of 2,3-Bisphosphoglycerate Facilitation of the Slow Nonenzymatic Glycation Process

Nonenzymatic glycation (NEG) of human hemoglobin (Hb A) consists of initial non covalent, reversible steps involving glucose and amino acid residues, which may also involve effector reagent(s) in the formation of labile Hb A_1c (the conjugate acid of the Schiff base). Labile Hb A_1c can then undergo slow, largely irreversible, formation of stable Hb A_1c (the Amadori product). Stable Hb A_1c is measured to assess diabetic progression after labile Hb A_1c removal. This study aimed to increase the understanding of the distinctions between labile and stable Hb A_1c from a mechanistic perspective in the presence of 2,3-bisphosphoglycerate (2,3-BPG). 2,3-Bisphosphoglycerate is an effector reagent that reversibly binds in the Hb A_1c pocket and modestly enhances overall NEG rate. The deprotonation of C2 on labile Hb A_1c in the formation of the Amadori product was previously proposed to be rate-limiting. Computational chemistry was used here to identify the mechanism(s) by which 2,3-BPG facilitates the deprotonation of C2 on labile Hb A_1c. 2,3-Bisphosphoglycerate is capable of abstracting protons on C2 and the α-nitrogen of labile Hb A_1c and can also deprotonate water and/or amino acid residues, therefore preparing these secondary reagents to deprotonate labile Hb A_1c. Parallel reactions not leading to an Amadori product were found that include formation of the neutral Schiff base, dissociation of glucose from the protein, and cyclic glycosylamine formation. These heretofore under appreciated parallel reactions may help explain both the selective removal of labile from stable Hb A_1c and the slow rate of NEG.     

Specific radioimmunochemical identification and quantitation of hemoglobins a2 and f

Hyperimmune antisera to chromatographically purified hemoglobins F and A₂, were produced in rabbits and made specific for the immunogen by adsorption with normal human hemoglobin A conjugated to cyanogen bromide-activated agarose. A radioimmunoassay was established that permitted identification and quantitation of each of these two minor hemoglobins in hemolysates containing other hemoglobin components. The quantities of hemoglobins A₂, and/or F present in hemolysates of individuals with β-thalassemia, sickle cell anemia, Hb-C disease, and other hematological disorders were determined immunochemically, and the results were compared to values obtained by microcolumn chromatography for the measurement of Hb-A₂ or with the alkali denaturation technique in quantitating Hb-F. The immunoassay procedure has a greater sensitivity than other commonly employed techniques and can detect as little as 0.05 μg of these hemoglobins.     

Genetic Determinants of Variability in Glycated Hemoglobin (HbA<Subscript>1c</Subscript>) in Humans: Review of Recent Progress and Prospects for Use in Diabetes Care

Glycated hemoglobin A_1c (HbA_1c) indicates the percentage of total hemoglobin that is bound by glucose, produced from the nonenzymatic chemical modification by glucose of hemoglobin molecules carried in erythrocytes. HbA_1c represents a surrogate marker of average blood glucose concentration over the previous 8 to 12 weeks, or the average lifespan of the erythrocyte, and thus represents a more stable indicator of glycemic status compared with fasting glucose. HbA_1c levels are genetically determined, with heritability of 47% to 59%. Over the past few years, inroads into understanding genetic predisposition by glycemic and nonglycemic factors have been achieved through genome-wide analyses. Here I review current research aimed at discovering genetic determinants of HbA_1c levels, discussing insights into biologic factors influencing variability in the general and diabetic population, and across different ethnicities. Furthermore, I discuss briefly the relevance of findings for diabetes monitoring and diagnosis.     

Effect of sulfonylurea therapy and plasma glucose levels on hemoglobin A1c in type II diabetes mellitus

It is well known that hemoglobin A_1c reflects plasma glucose concentrations in patients with diabetes mellitus. To examine hemoglobin A_1c and plasma glucose relationships in sulfonylurea-treated patients, 25 patients with well-controlled type II diabetes (fasting plasma glucose 128 ± 6 mg/dl, hemoglobin A_1c 7.6 ± 0.5 percent) were evaluated in a double-blind study. This study was divided into two phases (periods I and II). During period I each patient was given a diet plus a placebo and was followed every two weeks until the mean of two consecutive plasma glucose determinations was more than 50 mg/dl above the initial plasma glucose concentration obtained while the patient was taking sulfonylurea. At that point each patient entered period II. At the beginning of period II each patient was switched in a double-blind fashion to either diet plus a placebo or diet plus tolazamide. Fasting plasma glucose concentrations increased to 178 ± 9 mg/dl (p < 0.005) for all patients by week 2 of period I. The increase in hemoglobin A_1c concentration was seen to lag behind the increasing fasting plasma glucose concentration by four to six weeks. Fasting plasma glucose and hemoglobin A_1c concentrations returned to values indistinguishable from initial values in patients who were given tolazamide and who responded to it. A positive correlation was noted when the hemoglobin A_1c concentration was compared with the fasting plasma glucose concentration measured four to six weeks previously.