J D England

Clinical Application of Glycosylated Hemoglobin Measurements

Glycosylated hemoglobin measurement has been shown to be a potentially useful tool for both a variety of research applications and for the management of patients with diabetes mellitus. None of the methods available to quantitate glycosylated hemoglobins is ideal. We have reviewed a number of critical methodologie considerations for Chromatographie procedures including the effects of sample storage under various conditions, and the importance of removing labile components prior to analyses. We have developed a method for the colorimetrie determination of glycosylated hemoglobins that is more rapid than methods reported previously, that correlates well with results using high-performance liquid chromatogra-phy, and that can he standardized between laboratories. We have reviewed our experience using glycosylated hemoglobin in a large population of diabetic youths. We have presented a method for developing realistic goals for glucose control using glycosylated hemoglobin and for using glycosylated hemoglobin as a patient education and care reinforcement tool.

Effects of Acute Changes in Blood Glucose on HbA1c

SUMMARY Although hemoglobin A1c (HbA1c) is generally considered to be an accurate index of long-term blood glucose regulation, several recent studies suggest that HbA1c may be acutely responsive to changes in blood glucose. We have examined the effects of acute changes in glucose concentration in vivo and in vitro on HbA1c. HbA1c was measured by a high-performance liquid chromatography (HPLC) method. HbA1c and plasma glucose were measured in seven diabetic patients and five control subjects before and 2 h after a standard breakfast. Only diabetic patients showed increases in HbA1c and plasma glucose values 2 h after the test meal (Δ HbA1c = 0.87 ± 0.24% and Δ glucose = 210 ± 0.33 mg/dl). The increment in HbA1c correlated significantly with the increment in plasma glucose (r = 0.73, P < 0.05). to examine the lability of these postmeal increments in HbA1c, erythrocytes from pre- and postmeal blood samples were incubated in 0.9% NaCl for 5 h at 37°C and HbA1c was re-measured. After saline incubations %HbA1c in pre- and postmeal blood samples decreased in both diabetics and controls, and from each subject time 0 and 2-h HbA1c values were nearly identical. HbA1c was then measured before and after incubations of erythrocytes from a larger group of diabetic patients (N = 55) and control subjects (N = 7). In both diabetic and control cells HbAlc decreased after saline incubations. Pre- and post-saline HbA1c values in diabetics were (means ± SEM) 10.07 ± 0.30% and 9.15 ± 0.27%, respectively; values in controls were 5.87 ± 0.11% and 5.46 ± 0.09%, respectively. The mean decrement in HbA1c was significantly greater in cells from diabetics than from controls (P < 0.001). In diabetics the HbA1c decrement correlated with both plasma glucose (r = 0.58, P < 0.001) and the preincubation HbA1c (r = 0.55, P < 0.001). After dialysis of hemolysates for 5 h at 37°C or 48–72 h at 4°C, HbA1c values showed decreases comparable to those after saline incubations of intact erythrocytes. However, decreases in HbA1c after dialysis were accompanied by increases in HbA1a+b, findings that were not observed after saline incubation. The results suggest that HbA1c exists in two chromatographically indistinguishable forms: one that represents the major portion of HbA1c in normals and diabetics, is not altered by short-term changes in plasma glucose, and can be estimated by measuring HbA1c after saline incubation of erythrocytes; and a second form that is responsive to short-term fluctuations of the blood glucose level. These labile and stable fractions may be identical to the labile Schiff-base and stable ketoamine forms of HbA1c, which have been previously described. For HbA1c to be an accurate indicator of long-term glucose control, saline incubation of erythrocytes or perhaps dialysis of hemolysates before HbA1c assay may be necessary. Otherwise the assay results will reflect recent changes in blood glucose levels.

Relationship of Glycosylated Hemoglobin to Oral Glucose Tolerance: Implications for Diabetes Screening

The oral glucose tolerance test (OGTT) for diagnosis of diabetes is inconvenient and requires a great deal of patient cooperation. Glycosylated hemoglobin (GHb), an index of long-term glycemic control, could offer several practical advantages over the OGTT for diabetes screening. We evaluated GHb as a screen for diabetes in 381 adults from a population with a high prevalence of non-insulin-dependent diabetes (Pima Indians). All individuals underwent a standard OGTT (75 g) and were separated into one of three groups: normal (N), impaired glucose tolerance (IGT), or diabetes mellitus (D) based on World Health Organization criteria. HbA1c, a GHb, was measured by highly precise high-performance liquid chromatography (interassay C.V. <4%). The normal range for HbA1c was 4.07–6.03% based on the 95% confidence interval for a nondiabetic, mostly Caucasian population. Compared with OGTT, HbA1c was highly specific (91%); an elevated HbA1c usually indicated D or IGT (sensitivity = 85 and 30%, respectively). A normal HbA1c did not, however, exclude a diagnosis of D or IGT. Based on previous epidemiological studies relating plasma glucose to chronic diabetic complications, GHb as measured in this study would properly identify the vast majority of subjects at risk. Long-term studies are necessary to determine the actual risk of complications in individuals with persistently normal HbA1c and D or IGT (based on OGTT).

The effect of elevated fetal hemoglobin on hemoglobin A1c results: five common hemoglobin A1c methods compared with the IFCC reference method.

Hemoglobin A1c (HbA1c) is an important indicator of risk for complications in patients with diabetes mellitus. Elevated fetal hemoglobin (HbF) levels have been reported to interfere with results of some HbA1c methods, but it has generally been assumed that HbA1c results from boronate-affinity methods are not affected by elevated HbF levels. None of the previous studies used the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method as the comparative HbA1c method. We, therefore, measured HbA1c in samples with normal and elevated HbF levels by several common assay methods and compared the results with those of the IFCC reference method.HbF levels of more than 20% artificially lowered HbA1c results from the Primus CLC 330/385 (Primus Diagnostics, Kansas City, MO), Siemens DCA2000 (Siemens Healthcare Diagnostics, Tarrytown, NY), and Tosoh 2.2+ (Tosoh Bioscience, South San Francisco, CA), but not the Bio-Rad Variant II (Bio-Rad Laboratories, Hercules, CA) and Tosoh G7. Physicians and laboratory professionals need to be aware of potential interference from elevated HbF levels that could affect HbA1c results, including those from boronate-affinity methods.

Hemoglobin A1c Values in Children with Overt Diabetes Maintained in Varying Degrees of Control

The concentration of hemoglobin A1c has been found to be elevated in patients with uncontrolled diabetes. HbA1c levels change at a slow rate so that a single measurement is believed to reflect the overall degree of control achieved by a patient for about the previous 2 mo. Using the Trivelli method, we determined HbA1c levels in 81 children with overt insulin-dependent diabetes and in 14 healthy children and young adults who served as controls. Five of the diabetic children with recent onset had sequential HbA1c determinations at 3–6-wk intervals. Each had a rapid decline in HbA1c level to near normal values after the patient was kept essentially euglycemic for about 60 days. Before the HbA1c determination, each diabetic child was given an overall control rating for the previous 2 mo or more based on criteria defined in our clinic. HbA1c values increased progressively as diabetic control declined. There was a statistically significant correlation between the clinical control ratings and HbA1c levels (P < 0.01 between all subgroups). Although there was a positive correlation between duration of diabetes and insulin requirement per kilogram of body weight in all groups combined, there was no significant difference when each subgroup was compared separately. These observations document the validity of our criteria for classifying patients in higher degrees of metabolic control.

A Prospective Study of Symptomatic Hypoglycemia in Young Diabetic Patients

The frequency of symptomatic hypoglycemia was determined prospectively over an 18-mo period in 147 children and adolescents with diabetes mellitus. All patients were treated with two daily injections of insulin. The data were analyzed to determine the relationship between episodes of symptomatic hypoglycemia and blood glucose control as assessed by hemoglobin A1c measurements. There were 542 patient visits during the study period. During each clinic visit, patients were separated into one of four hypoglycemic categories based on the medical history since the preceding visit. These categories were: no reactions, mild to occasional reactions, mild to frequent reactions, and severe reactions. Reactions were considered severe if they were characterized by altered central nervous system function or prolonged sympathetic nervous system symptoms. Forty-seven percent of the patients reported at least one reaction during the 18-mo study period, but only 4% (i.e., 6 out of 147 patients) reported severe reactions. The mean hemoglobin A1c level was significantly lower in patients who reported reactions than in patients who did not report reactions [hemoglobin A1c values (x¯ ± SEM) = 7.78 ± 0.1% vs. 9.48 ± 0.1%, respectively; P > 0.001]. The severity of hypoglycemia was inversely related to the degree of altered blood glucose control; episodes of frequent or severe symptomatic hypoglycemia occurred almost exclusively in patients with well-controlled diabetes as reflected in their near-normal hemoglobin A1c levels. There were no significant differences in mean insulin dose/kg, age, or duration of diabetes comparing patients in the four hypoglycemic categories. The results indicate that the hemoglobin A1c level can be a useful clinical guide to identify patients who are most likely to develop serious symptomatic hypoglycemia.

Glycated haemoglobin predicts progression to diabetes mellitus in Pima Indians with impaired glucose tolerance

SummaryGlycated haemoglobin could offer several practical advantages over the OGTT for assessing glucose metabolism. Initial cross-sectional studies (1983–1985) on 381 subjects (mostly Pima Indians) described the relationship between HbA1c (a specific glycated Hb) and the OGTT. We performed follow-up OGTTs and HbA1c measurements on 257 of these same subjects 1.6–6.1 years later. Subjects were again grouped according to both the result of the OGTT (normal, IGT or diabetes, by WHO criteria) and HbA1c result (normal or elevated based on mean ± 1.96 SD of normal). Of 66 subjects with IGT at baseline, 47 (71%) had normal HbA1c and 19 (29%) had elevated HbA1c. Twentysix (39%) of these subjects had diabetes at follow-up. Of these subjects with IGT, a significantly greater percentage of subjects with elevated HbA1c at baseline (68%) showed worsening to diabetes than those with a normal HbA1c (28%); (chi-square=7.8, df=1, p<0.01). Thus, in subjects with IGT, glycated Hb may be a useful predictor of progression to diabetes.

Hemoglobin A1c levels in children and adolescents with diabetes mellitus.

Hemoglobin A1c (HbA1c) was measured as an indicator of glucose control in 180 children and adolescents with diabetes mellitus who received two daily injections of insulin as part of a highly structured treatment program. A total of 426 HbA1c determinations was made in the group of 180 patients. HbA1c values were elevated in most patients despite the aggressive treatment. The HbA1c level was very elevated at diagnosis, fell to near normal after 60–90 days of insulin therapy, increased gradually, and reached a plateau after approximately 4 yr duration (at about twice the level in normal subjects) (mean ± SEM, 10.0 ± 0.2% and 5.34 ± 0.07%, respectively). Mean insulin dose (U/kg/24 h) paralleled both HbA1c and duration of diabetes. The relationship between endogenous insulin secretion and glucose control was examined in those patients with diabetes for longer than 5 yr. Patients were separated into three groups based on HbA1c levels: those with HbA1c less than 9% (N = 22), between 9 and 11% (N = 26), and greater than 11% (N = 28). Serum C-peptide and glucose concentrations were measured 2 h after a standard breakfast in those patients in the “low” and “high” HbA1c groups (mean HbA1c values 8.2% and 12.7%, respectively). C-peptide was detectable in all patients and the mean C-peptide levels did not differ significantly in the two groups, although postprandial glucose concentrations were significantly lower in the “low” HbA1c group (means ± SEM, 96 ± 11 and 211 ± 21 mg/dl, respectively; P < 0.001).

The excretion of tryptophan metabolitesfollowing deoxypyridoxine administration in mongoloid and nonmongoloid patients

No significant difference in the excretion of tryptophan metabolites was observedbetween mongoloid and nonmongoloid children after simple tryptophan loading. However, after the administration of deoxypyridoxine, a pyridoxine antagonist, a tryptophan load caused the mongoloid children to excrete significantly greater amounts of xanthurenic acid and 3-OH kynurenine than the controls. These results suggest that the mongol is more sensitive to pyridoxine deficiency than the normal child.

Recent advances in glycosylated hemoglobin measurements

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.