Glycosylated haemoglobin as an alternative to the glucose tolerance test for the diagnosis of diabetes mellitus

Transcription

1 Ann Clin Blochem 1985; 22: Glycosylated haemoglobin as an alternative to the glucose tolerance test for the diagnosis of diabetes mellitus EVA LESTER, A D FRAZER, CAROLE A SHEPHERD and F J WOOD ROFFE From the DepartmentofChemical Pathology and Diabetic Clinic, North Middlesex Hospital, Sterling Way, London N181QX, UK SUMMARY. A comparison was made between the results of a standard 75 g oral glucose tolerance test and total haemoglobin Al in 168 subjects referred by their general practitioners for the diagnosis of diabetes mellitus. The subjects were classified as having normal, impaired or diabetic glucose tolerance using WHO criteria. Of 8 subjects with normal glucose tolerance only three had haemoglobin Al concentrations over 8%. Of with impaired glucose tolerance two had haemoglobin Al concentrations over 8%. Of 47 with diabetic responses, eight had haemoglobin Al concentrations below 9%. There was a significant difference between the haemoglobin Al concentration when fasting and that 2 hours after the glucose load in the subjects with a diabetic glucose tolerance response, but no significant difference between the two values in the other subjects. The earliest description of blood sugar response to the ingestion of glucose dates back 7 years' when the first clinically applicable method for the quantitative estimation of sugar in the blood was introduced.f Since then the glucose tolerance test has been modified constantly. Different glucose loads, different analytical methods and different criteria for the interpretation of the results have been employed. Because diabetes mellitus is defined in terms of these blood-glucose levels, the test almost always gives an apparently clear-cut definition. However, there are many problems inherent in the test itself and in the way it is performed, most of which have been apparent for a very long time. They include the effects of age, previous diet, exercise, stress, gastro-intestinal emptying, whether capillary or venous blood or plasma are used, and the method of glucose estimation. The test requires prior co-operation from the patient and half a day of his time. In 1958 Allen et al. 3 identified a minor haemoglobin fraction that eluted ahead of the main haemoglobin A fraction on cationexchange chromatography. He called the fast fraction haemoglobin Ale' In 1968 Rahbar" Correspondence: Dr Eva Lester 74 reported that this fast haemoglobin fraction was elevated in patients with diabetes mellitus. Trivelli et al. 5 found similar increases in the fast haemoglobin fraction in the study of a hundred patients with diabetes mellitus. Since then there have been many studies of glycosylated haemoglobin in relation to the monitoring of diabetic control and diabetic complications. A correlation has been established between glycosylated haemoglobin and blood glucose profiles taken between 4 and 12 weeks previously.p: 7 In view of the increasing familiarity of clinicians with glycosylated haemoglobin values we have investigated whether it would be possible to replace the glucose tolerance test with haemoglobin Al measurements for the diagnosis of diabetes mellitus. Subjects and methods One hundred and sixty-eight subjects were studied. Almost all of these were referred by their general practitioners directly to the Chemical Pathology department. A small number were referred from the Diabetic Clinic where a random blood sugar was not diagnostic. There were 89 men and 79 women and their ages

2 Diagnosis of diabetes mellitus-s-hba, vs. glucose tolerance test 75 ranged from 21 to 8 years. Patients who telephoned the department to make an appointment were given verbal instructions about taking a 'normal' diet for 3 days before the test and the overnight fast from midnight. Some were also given written instructions issued by us to their GPs. All tests were performed in the morning. A basal venous blood sample was collected for the estimation of glucose and haemoglobin AI' Each patient was then given an oral dose of 75 g of glucose (Hycal) and venous blood taken for glucose estimation 1 h later and for glucose and haemoglobin Al 2 h later. Blood glucose was estimated using venous plasma by the glucose oxidase/4 aminophenozone and phenol method of Trinder" on a Technicon AA2 analyser. Using the WHO criteria? for the interpretation of glucose tolerance test results the patients were divided into those with a normal test, those with diabetes mellitus and those with impaired glucose tolerance. Glycosylated haemoglobin was measured by the Corning 'Glytrac' electrophoretic procedure (Coming Medical & Scientific) which is based on the alteration of the electrophoretic mobility on agar gel of haemoglobin by its glycosylation.? It involves the quantitation of total haemoglobin and haemoglobin AI by automatic scanning densitometry and has been found to correlate well with results obtained by high-pressure liquid chromatography.l'' Blood was collected into EDTA bottles and lysed immediately. The haemolysate was stored for no longer than 7 days at 4 C. Experiments, showed that the haemolysates were stable for 14 days at 4 C. Precision experiments showed that in our hands the within-batch coefficient of variation was 3 2% and the between-batch coefficient of variation was 4 5%. The glycosylated haemoglobin concentrations in samples taken when fasting and 2 h after the glucose load were compared for each group using the paired t-test. The correlation between the total areas under the glucose tolerance test curve and the glycosylated haemoglobin concentration was examined in the three groups of subjects. Results Of the 168 patients, 8 had normal responses, 47 had diabetic responses, and had impaired glucose tolerance. Three patients had a lag storage type curve with normal fasting and 2 hour values and a l-hour value between 11 and 12 mmol/l, Table 1 summarises the glycosylated haemoglobin values before and after the glucose load in the three groups of subjects. In the normal subjects the mean fasting haemoglobin Al was 5 92% and the mean 2-hour haemoglobin Al level was 5 86%. There was no significant difference between these values. The actual range of values was 4 1 to 8 8% but only three subjects had values over 8 %. No significant correlation was found between the area under the glucose tolerance curve and the fasting or 2-hour glycosylated haemoglobin level. In the subjects with impaired glucose tolerance the mean fasting haemoglobin AI was 7 41% and the mean 2-hour haemoglobin AI was 7 46%. There was no significant differences between the values. The actual range was 4 2 to 11 5%. Two subjects had values over 8% both before and after the glucose load. There was no correlation between the area under the glucose tolerance test curve and the fasting or 2-hour glycosylated haemoglobin level. The three subjects with lag storage type curves had glycosylated haemoglobin levels of 4 6, 6 6 and 7 5% respectively. All five subjects with normal or impaired glucose tolerance whose glycosylated haemoglobin concentration was high, had values over 8% both before and after the glucose load. In the subjects with a diabetic response the mean fasting haemoglobin Al was 11 69% and the mean 2-hour haemoglobin AI was 12 9%. The difference between these two results was significant at the 5% level. The actual range of values was 8 to 16 % and eight subjects had TABLE 1. tolerance Glycosylated haemoglobin and plasma glucose in subjects with normal. impaired and diabetic glucose Mean glycosylated haemoglobin % Mean plasma glucose (mmoi/l) Glucose tolerance Number Fasting 2 h after 75 s glucose Fasting 1 h after 75 g glucose Normal ±1 O5 5 86±O 93 4 R9±O 6(J 6 64± 1 73 Impaired 7 41± ±2 6 2±1 Q3 12-lJ6±1 67 Diabetic ± ± ±4 IR 2lJ 34±4 6

3 ,76 Lester et al. values below 9%. Fig. 1 shows the distribution of the 2-hour glycosylated haemoglobin values in the three groups of subjects. In these subjects also there was a significant correlation between the area under the glucose tolerance curve and both the fasting haemoglobin Al level (r = 736) and the 2-hour haemoglobin AI level (r =,732) as shown in Fig. 2. The mean glucose concentration in the normal subjects rose from 4 89 mmol/l in the ~ fasting state to 6 64 mmolll 1 hour after!!... glucose, while the diabetics showed a much ~ 12 greater mean rise from mmol/l to 2 34 ~ mmol/l which was sufficient to cause a measur- ~ able increase of a total glycosylated haemo- ~ globin presumably due to an increase in the..r:: Schiff base. In this series of 47 diabetic subjects : there were three whose glycosylated haemoglo- ị. o u, ~!! c :c E s: ' ~.,,.. u,.. 9 Cl 8., "":!!r" Normal Impaired Glucose tolerance tests Diabetic FIG. 1. Distribution of glycosylated haemoglobin values 2 h after a 75 g glucose load in subjects with normal, impaired and diabetic glucose tolerance. FIG Area under glucose tolerance curve Relationship between the area under the glucose tolerance curve and the glycosylated haemoglobin value 2 h after a glucose load in diabetic subjects (r = 732; y = 2 x ). bin was below 9% before the glucose load and rose above 9% after it. Discussion The two tests we have compared measure different aspects of glucose homeostasis. The glucose tolerance test examines the response of blood glucose and therefore insulin reserve to a glucose challenge while glycosylated haemoglobin is a measure of the day-to-day control of blood glucose. In almost all cases of diabetes mellitus the diagnosis can be made on the basis of symptoms, glycosuria and an unequivocally raised blood-glucose concentration. However in some patients a single fasting, random, or postprandial blood glucose measurement falls into

4 Diagnosis of diabetes mellitus-s-hba, vs. glucose tolerance test 77 the equivocal range. Many general practitioners when they refer the patient to hospital for a blood test would prefer to have a definite answer rather than risk having to ask the patient to return for a further test. For this reason many so-called 'unnecessary' glucose tolerance tests are requested. We have therefore compared glycosylated haemoglobin with a glucose tolerance test, even though in most cases neither is necessary to make a diagnosis. Our results showed that very few subjects with normal tolerance of 75 g glucose load have elevated glycosylated haemoglobin values. Of the 8 subjects with normal glucose tolerance, only three (2'8%) had glycosylated haemoglobin values over 8 % and in only one of these was it over 8 5%. Of the 47 subjects with diabetic glucose tolerance curves, eight (17%) had glycosylated haemoglobin values below 9 % and of these four (8'5%) were between 8 and 8 5%. Only two of the subjects with impaired glucose tolerance had high glycosylated haemoglobin values. Without the unphysiological stimulation of the 75 g glucose load it might be expected that the blood glucose concentration of this group would remain within the normal limits formost of the time. This might also explain the normal glycosylated haemoglobin values in some of the less severe but frank diabetics. However, it is also reasonable to ask whether the two subjects with glycosylated haemoglobin levels of 9 3% and 11 5% are actually diabetic in spite of their glucose curves which showed only impaired tolerance. Dods and Bolney!' in a study of 44 diabetic subjects using a g glucose load and six different sets of criteria for the classification of diabetes mellitus found that between 16 7% (one out of six) and 64 3% (nine out of 14) diabetics had normal glycosylated haemoglobin values. However there was a variance of up to 25% between the different scoring systems in the definition of diabetes mellitus so that many of the subjects presumably had borderline diabetes. Dunn et al.12 in a study of 228 subjects found that only six of 159 (3'8%) with normal glucose tolerance tests had high haemoglobin Al but 23 of 63 (37%) diabetics had normal haemoglobin AI' Again they used g glucose load and three different sets of criteria for the definition of diabetes and had no impaired glucose tolerance group so that it is difficult to make direct comparisons with our results. Our findings of a rise in mean glycosylated haemoglobin in the diabetic subjects are in line with those of a recent study where glucose was infused into normal fasting volunteers to raise their blood glucose to a mean level of 16 6 mmolll and the mean haemoglobin Al level rose by 1 3% at 6 min. 12 In an earlier study of six women, no change was found in sequential undialysed glycosylated haemoglobin during a 3 h g oral glucose tolerance test in which the highest level of plasma glucose reached was 11 7 mmoul. 14 In our subjects the rise in measured glycosylated haemoglobin could have been avoided by incubating the red cells in saline to remove the short-term, rapidly formed haemoglobin Al component before preparation of the haernolysate.p In relation to the diagnosis of diabetes mellitus in individual patients our findings indicate that since there was no change in glycosylated haemoglobin after a glucose load in the non-diabetic subjects and since in three of the diabetic subjects glucose loads caused their glycosylated haemoglobin to rise from the equivocal to the clearly diabetic range, glycosylated haemoglobin after a meal may be more helpful than a fasting level. We found the 'Glytrac' method to be simple and robust although relatively expensive. The reagent costs were roughly 1 per test but the method had the advantage that it can be used by the staff in a routine Chemical Pathology department. In conclusion we would suggest that using our method for haemoglobin Al measurement a single random estimation giving a result below 8% excludes diabetes mellitus, one above 9% confirms diabetes mellitus and patients with results between 8 and 9% require further investigation. Unfortunately there is as yet no diagnostic method which indicates the prognosis or even the need for treatment in individual asymptomatic non-insulin-dependent diabetics. What is now required is a long term study of glycosylated haemoglobin on the lines of the Bedford population study." References 1 Jacobsen ATB. Untersuchung uber den Einfluss verschiedener Nahrungsmittel anf den Blutzucker bei normalen, zuckerkranken und graviden Personen. Biochemische Zeitschrift 1913; 56: Bang I. In: Bergman JF, ed. Der Blutzucker. Wiesbaden, 1913; Allen DW, Schroeder, WA, Balog J. Observations on the chromatographic heterogeneity of normal adult and foetal haemoglobin: a study of the effect of crystallization and chromatography on the heterogeneity and isoleuline content. JAm Chem Soc 1958; 8:

5 78 Lester et at. 4 Rahbar S. An abnormal hemoglobin in the cells of diabetics. Clin Chim Acta 1968; 22: Trivelli LH, Ranney HM, Lai H-T. Hemoglobin components in patients with diabetes mellitus. N Eng/ J Med 1971; 284: Paisey RB, Macfarlane DB, Sherriff RS, et at. The relationship between blood glycosylated haemoglobin and home capillary blood levels in diabetics. Diabetologia 198; 19: Dunn PJ, Cole RA, Soeldner JS, et at. Temporal relationship of glycosylated haemoglobin concentrations to glucose control in diabetics. Diabetologia 1979; 17: Trinder P. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann C/in Biochem 1969; 6: WHO Expert Committee on Diabetes Mellitus, Second Report. Technical Report Series No Geneva: WHO, 198. Menard L, Dempsey ME, Blankstein LA, et at. Ouantitative determination of glycosylated haemoglobin AI by agar gel electrophoresis. C/in Chem 198; 26: Dods RF, Bolney C. Glycosylated haemoglobin assay and oral glucose tolerance compared for detection of diabetes mellitus. C/in Chem.1979; 25: Dunn PJ, Cole RA, Soeldner JS, et at. Reproducibility of hemoglobin Ale" and sensitivity to various degrees of glucose intolerance. Ann Intern Med 1979; 91: Goebel FD, Fuessl H. Changes in glycosylated haemoglobin after oral glucose load. Br Med J 1982; 284: Widness JA, RogIer-Brown TL, McCormick KL, et at. Rapid fluctuations in glycohemoglobin (haemoglobin Ale> related to acute changes in glucose. J Lab Clin Med 198; 95: Svendsen PA, Christiansen JS, Soegaard U, et al. Rapid changes in chromatographically deter: mined haemoglobin Ale" induced by short-term changes in glucose concentration. Diabetologia 198; 19: Butterfield WJH. Summary of the results of the Bedford Survey. Proc R Soc Med 1964; 57: 196. Accepted for publication / June 1984