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Association of glycated minor hemoglobin fraction of A1 with microalbuminuria and glycemic status in diabetic subjects

Muhammad Saiedullah*, Shoma Hayat, Syed Muhammad Kamaluddin, Shahnaj Begum
Dept. of Biochemistry & Cell Biology, Bangladesh University of Health Sciences, Mirpur, Dhaka, Bangladesh

Abstract Background: Glycated hemoglobin A1c (HbA1c) is the well characterized amadori product and the role of HbA1c in the management of diabetes mellitus (DM) has been established. Clinical significance of glycated minor hemoglobin sub-fractions is less studied. Objective: In this context the present cross-sectional study was carried out to explore the association of glycated minor hemoglobin sub-fraction of A1 with microalbuminuria and glycemic status in diabetic subjects. Methods: A total of 150 confirmed diabetic subjects were included. Blood specimens were collected for the analyses of hemoglobin sub-fractions by ion-exchange high performance chromatography and spot urine specimens were collected for the estimation of urine albumin creatinine ratio (ACR). Results were expressed as mean±SD and data were analyzed by appropriate statistical tests. Results: The mean HbA0, HbA1a, HbA1b and HbA1c levels were 82.79±3.27, 0.91±0.18, 1.76±0.43 and 8.86±2.13% respectively. The mean ACR was 38.3±37.9 mg/g. The correlation coefficient of ACR was -0.1151 (p=0.1609) for HbA0, 0.06989 (p=0.4022) for HbA1a, 0.1678 (p=0.0401) for HbA1b, 0.1130 (p=0.1685) for HbA1c and 0.2194 (p=0.0070) for age. Among the minor sub-fractions, HbA1b showed significant positive association with ACR (β=0.1183, p=0.0211) and the β value of HbA1b was 0.1946 (p=0.0211) for HbA1c. Conclusion: This study revealed a positive association of glycated minor hemoglobin sub-fraction HbA1b with renal function and glycemic status, while HbA1a did not show such association. Keywords: Diabetes mellitus, Glycated hemoglobin, Glycated minor hemoglobin fractions Originally published in Diab Endocr J 2012;40(1):14-16 * Corresponding author: md.saiedullah@gmail.com

Introduction Diabetes mellitus (DM) is a chronic metabolic disorder characterized by rise in blood glucose level and derangement in protein and fat metabolism.1 The formation of advanced glycation end products (AGEs) plays an important role for the development and progression of the long term complications of DM.2 The first step of AGE formation is known as aldimine or Schiff base formation by an enzyme independent reversible reaction. In the second step, by an enzyme dependent irreversible reaction, stable adducts of glucose or fructose with amino group of proteins are formed. The adduct formation may enhance protein cross-link, protein oxidation which are linked to the loss of function. Glycated hemoglobin A1c (HbA1c) is the well characterized amadori product, produced in the early stage of AGE formation and the role of glycated hemoglobin A1c (HbA1c) in the management of DM has been established.3-5 Depending on the site of glycation of hemoglobin chain and their elution properties during ion-exchange high performance chromatographic (HPLC) separation, beside the native and major hemoglobin A0 and HbA1c, minor sub-fractions eg., HbA1a, HbA1b, HbA1d etc are also found. HbA1a and HbA1b have shown to be associated with fetal-type erythropoiesis6 and renal function.7,8 but not associated with HbA1c.6 But another study showed significant positive correlation with HbA1c for HbA1a and HbA1b.9 As there is a controversy regarding the association of HbA1a and HbA1b with HbA1c, this study was undertaken to explore the association of renal function (microalbuminuria) and glycemic status (HbA1c) with HbA minor sub-fractions by adjusting confounders. Materials and methods One hundred and fifty specimens were collected from confirmed diabetic subjects during July 2011 to June 2012. Blood specimens were collected in blood collection tubes (BD vacutainer® containing 3.6 mg K2EDTA; BD, Franklin Lakes, NJ USA) and spot urine specimens were collected in clean glass tubes. HbA0 HbA1a, HbA1b and HbA1c were measured by high-pressure liquid chromatography (HPLC) method using D-10™ glycosylated hemoglobin testing system (Bio-Rad Laboratories, Inc., Hercules, CA, 94547, USA). Urine microalbumin level was measured by a particle-enhanced turbidimetric inhibition immunoassay method using Dimension RxL max automated analyzer (Siemens Healthcare Diagnostics Ltd.) and urine creatinine concentration was measured by modified Jaffe´’s method using Dimension RxL max automated analyzer. Urine ACR was calculated as ratio of microalbumin concentration and creatinine concentration in urine and results were expressed in mg/g unit. Results are expressed as

mean±SD and the Pearson correlation coefficient of ACR with HbA0, HbA1a, HbA1b and HbA1c, age and serum creatinine was determined by linear regression analysis. To assess the significance of association of ACR with HbA sub-fractions, multivariate linear regression model was used. Statistical analyses were performed by using STATISTICA version 8 for Windows.

Results The mean age of the total subjects was 50±12.7 years and of the total subjects, 49% were male and 51% were female. The mean HbA0, HbA1a, HbA1b and HbA1c levels were 82.79±3.27, 0.91±0.18, 1.76±0.43 and 8.86±2.13% respectively. Twenty two percent (22%) of the total subjects had HbA1c≤7.0%, 33% had HbA1c between 7.0 to 9.0% and 45% had HbA1c>9.0%. The mean ACR was 38.3±37.9 mg/g. Sixty one percent (61%) of the study subjects had ACR≤30.0 mg/g and 39% had ACR>30.0 mg/g. The correlation coefficient of ACR was -0.1151 (p=0.1609) for HbA0, 0.06989 (p=0.4022) for HbA1a, 0.1678 (p=0.0401) for HbA1b, 0.1130 (p= 0.1685) for HbA1c and 0.2194 (p=0.0070) for age. Multivariate linear regression analyses considering ACR as dependent variable and HbA0, HbA1a, HbA1b, HbA1c as independent variables and sex as categorical variable showed that the β value was 0.1373 (p=0.9883) for HbA0, 0.0457 (p=0.6196) for HbA1a, 0.1183 (p=0.0211) for HbA1b, 0.1373 (p=0.3997) for HbA1c and 0.2754 (p=0.0011) for age and -0.0714 (p=0.3882) for male. On the other hand, multivariate linear regression analyses considering HbA1b as dependent variable and HbA0, HbA1a, HbA1c as independent variables and sex as categorical variable showed that the β value was 0.3981 (p=0.0209) for HbA0, -0.1399 (p=0.1389) for HbA1a, -0.3312 (p=0.0473) for HbA1c and -0.0509 (p=0.5522) for age and -0.0524 (p=0.5384) for male. The significant association between HbA1b and HbA1c remains statistically significant when further adjusted for ACR (β=0.1946, p=0.0211). Multivariate regression analyses also showed that the β value of HbA1a was 0.0096 (p=0.9478) for HbA1c adjusted for HbA0, HbA1b, age and sex.

Discussion In this study, we observed significant positive association of HbA1b with ACR in unadjusted or adjusted linear regression model. This result is inconsistent with the findings of Choi et al6 and Smith et al.7 Unlike the results of Choi et al,6 we found significant positive association between HbA1b and HbA1c. But this is consistent with the study of Saiedullah et al.9 Furthermore, HbA1a showed no significant association with ACR and HbA1c. These discrepancies may depend on the severity of the disease and the presence of renal impairment among subjects of these studies. Urine albumin creatinine ratio is the early detection marker of renal dysfunction and is affected by factors such as hypertension, duration of diabetes, smoking, body mass index, exercise, oxidized LDL etc. HbA1c reflects the glycemic status of the previous three months. In the case of overt hyperglycemia for a long time, basement membrane proteins are glycosylated with the loss of charge selectivity and increased glomerular perfusion and filtration.10,11,12 This may be associated with the glycation that mimics HbA1b. Since, ion-exchange chromatography produces the values of minor sub-fractions of HbA1 along with HbA1c, the evaluation of elevated HbA1b sub-fractions may provide additional information regarding early renal impairment in diabetic subjects. Conclusion Glycated minor hemoglobin sub-fraction HbA1b is positively associated with renal function and glycemic status, while HbA1a does not show such association. References 1. Vinik A and Flemmer M. Diabetes and macrovascular disease. Journal of diabetes and its complications, 2002;16:235-245. 2. Gugliucci A. Glycation as the glucose link to diabetic complications. J Am Osteopath Assoc 2000;100: 621-634. 3. Jeffcoate SL. Diabetes control and complications: the role of glycated haemoglobin, 25 years on. Diabet Med 2004; 21 (7): 657-665. 4. Bunn HF, Gabbay KH, Gallop PM. The glycosylation of hemoglobin: relevance to diabetes mellitus. Science 1978; 200 (4337): 21-27.

5. Kasezawa N, Kiyose H, Ito K, Iwatsuka T, Kawai H, Goto Y, et al. Criteria for screening diabetes mellitus using serum fructosamine level and fasting plasma glucose level. The Japanese Society of Multiphasic Health Testing and Services (JMHT), Fructosamine Working Committee. Methods Inf Med 1993; 32 (3): 237-340. 6. Choi JW and Lee MH. Fetal-type erythropoiesis is more closely linked to glycated minor hemoglobin fractions than hemoglobin A1c in diabetics. Acta Haematol 2012;127:205–207. 7. Smith WG, Holden M, Benton M, Brown CB. Glycosylated and carbamylated haemoglobin in uraemia. Nephrol Dial Transplant 1989; 4: 96–100. 8. Oimomi M, Yoshimura Y, Kubota S, Kawasaki T, Takagi K, Baba S. Hemoglobin A 1 properties of diabetic and uremic patients. Diabetes Care 1981;4:484–486. 9. Saiedullah M, Ferdoush M, Begum S, Rahman MR, Sarkar A, Ahmad A. Studies on subfractions of hemoglobin A1 in diabetic subjects. Diab Endocr J 2009;38(Suppl 1):20. 10. Tarsio JF, Reger LA, Furcht LT. Molecular mechanisms in basement membrane complications of diabetes: alterations in heparin, laminin, and type IV collagen association. Diabetes 1988;37:532–9. 11. Nelson RG, Bennett PH, Beck GJ, Tan M, Knowler WC, Mitch WE, et al. Development and progression of renal disease in Pima Indians with noninsulin-dependent diabetes mellitus: Diabetic Renal Disease Study Group. N Engl J Med 1996;335:1636–42. 12. Fujita H, Narita T, Ito S. Abnormality in urinary protein excretion in Japanese men with impaired glucose tolerance. Diabetes Care 1999;22:823–6.