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Home , Fructosamine, Impaired fasting glucose, Insulin resistance, Prediabetes

International J. of Healthcare and Biomedical Research, Volume: 08, Issue: 03, April 2020, 5- 10 DOI: 10.36848/IJHBR/2020/12100.51200

Original article: Assessment of levels and its utility in patients of

Manju Bala1, Meenakshi2*, Sameer Aggarwal3

1 Professor, Department of Biochemistry, Pt B D Sharma PGIMS, Rohtak, Haryana. 3 Ex-Professor, Department of Medicine, Pt B D Sharma PGIMS, Rohtak, Haryana. 2*Corresponding author: Meenakshi,Senior Resident, Department of Biochemistry, Lady Hardinge Medical College, New Delhi, India. ; E-mail- [email protected]

Abstract Background: Disordered leads to prediabetes which does not satisfy the diagnostic criteria of mellitus. Prediabetes is a risk factor for development of diabetes along with cardiovascular and microvascular complications. Fructosamine (FA) is a ketoamine formed when the carbonyl group of glucose reacts with an amino group of a forming glycated (mainly albumin). FA determination is the most widely used alternative to HbA1c. Objectives: To correlate fructosamine with blood glucose (FBG) & postprandial blood glucose (PPBG) in prediabetes. Materials and Methods: The present study was conducted in the Department of Biochemistry in collaboration with Department of Medicine, Pt B D Sharma PGIMS, Rohtak. Thirty patients of prediabetes diagnosed on the basis of HbA1c were enrolled as cases & 30 age matched healthy individuals were enrolled as control. Venous blood sample was withdrawn for estimation of

FBG, PPBG, HbA1c & FA levels.

Results and Conclusion: Mean ± SD of HbA1c in cases was 5.9 ± 0.2% & controls was 5.2 ± 0.3% (p value = 0.001). Mean ± SD of FA in cases was 256.2 ± 15.5 µmol/L and in controls was 236 ± 14.1 µmol/L (p value = 0.001). FA is positively correlated with FBG with r = 0.440 & p value = 0.015 and with PPBG with r = 0.596 & p value = 0.001. Thus, glycated protein i.e fructosamine correlates with glucose concentration and reflects the average glycemic status. Key words: Prediabetes, Fructosamine, Fasting blood glucose, Postprandial blood glucose.

Introduction Diabetes Mellitus (DM) is a complex polygenetic disorder which is associated with both heritable and environmental factors. resistance (IR) is a main pathophysiological factor in the development and progression of DM. DM has become more prevalent due to change in lifestyle leading to microvascular & macrovascular complications which reduces life expectancy for humans [1]. In 1979, National Diabetes Data Group (NDDG) introduced the concept of a intermediate metabolic state between normal glucose tolerance (NGT) and diabetes known as impaired glucose intolerance (IGT). Individuals with IGT does not meet the criteria of diabetes but there glucose levels are higher than normal [2]. So, IGT is an intermediate state in transition from NGT to . Subjects with IGT have normal blood glucose levels but have defect

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International J. of Healthcare and Biomedical Research, Volume: 08, Issue: 03, April 2020, 5- 10 DOI: 10.36848/IJHBR/2020/12100.51200 in insulin sensitivity along with impaired beta function which is characteristic feature of DM. As a result, subjects with IGT have higher risk for progression to type 2 diabetes with an annual conversion rate of 5–10% depending upon the ethnic group under study. In 1997, the American Diabetes Association (ADA) introduced another intermediate state known as (IFG) along with IGT in transition of glucose from NGT to type 2 diabetes. IFG is analogous to IGT having high risk of progression to type 2 diabetes [3]. In 1997, Expert Committee on the Diagnosis and Classification of Diabetes Mellitus extended the concept as prediabetes (preDM) by including IFG and IGT under it. So, preDM is a condition of which is at lower level than Diabetes [2]. According to ADA, diagnostic criteria for preDM is [4]:  Impaired fasting glucose (IFG) with fasting plasma glucose levels of 100 to 125 mg/dL (5.6 to 6.9 mmol/L).  Impaired glucose tolerance (IGT) with plasma glucose levels of 140 to 199 mg/dL (7.8 to 11.0 mmol/L) 2 hour postprandial.

(HbA1c) of 5.7 to 6.4%. According to ADA, Fasting plasma glucose (FPG) is the preferred test for diagnosing diabetes and preDM because of simplicity, acceptability and lower cost [2]. But FPG has a drawback as it reflects current glycemic status and requires fasting which is mostly done inappropriately by patients [5]. In case of suspicion, despite a normal FPG level, oral (OGTT) is a appropriate test for further diagnosis [2]. OGTT is the most valid first line screening tool for diagnosing diabetes. However, it is expensive, inconvenient and has weak reproducibility making it practically unacceptable for patients. It is a difficult and time consuming test to perform [5]. Because of these drawbacks, glycated hemoglobin (HbA1c) is a widely accepted test to estimate the degree of glycemic control as it can be measured irrespective of the food intake and is much more simple than FPG or OGTT. It reflects long term glucose concentration versus frequently changing glucose levels [6].

However, estimation of HbA1c is also affected by specific hemoglobinopathies which interferes with older HbA1c assays like fetal hemoglobin falsely increases and sickle cell hemoglobin & hemoglobin C lowers HbA1c levels. Turnover of red blood cells such as hemolysis, shortened erythrocyte life span, cirrhosis, acute or chronic blood loss or transfusions leads to abnormally low HbA1c level and false negative diagnosis. In contrast, subjects with iron deficiency or individuals genetically predisposed to greater levels of hemoglobin and elderly individual have falsely high values HbA1c levels leading to over diagnosis of diabetes [2].

Many proteins other than HbA1c, also undergo a nonenzymatic glycation process. Fructosamine (FA) is a ketoamine formed when the carbonyl group of glucose reacts with an amino group of a protein forming glycated serum proteins (mainly albumin). FA determination is the most widely used alternative to HbA1c. Since FA measures extracellular glycated protein, therefore its plasma levels are not related to extracellular & intracellular glucose dynamics [7]. Since albumin has a shorter half life (14–20 days) than hemoglobin, therefore both FA and glycated albumin provide information about blood glucose control over a shorter period than HbA1c [8]. However, FA assay is influenced by

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International J. of Healthcare and Biomedical Research, Volume: 08, Issue: 03, April 2020, 5- 10 DOI: 10.36848/IJHBR/2020/12100.51200 various factors like low plasma albumin concentration and increased albuminuria. But FA assay has some advantage like it is more readily automated, less labor intensive, quicker and cheaper than HbA1c assay [9]. Material and methods The present study was conducted in the Department of Biochemistry in collaboration with Department of Medicine, Pt. B. D. Sharma PGIMS, Rohtak. The study was approved by the Institutional Ethics Committee. In the present study, 30 patients of age group between 20-40 years were enrolled as cases based on HbA1c. 30 healthy and age matched individuals were enrolled as controls. Inclusion criteria: Patients of age group between 20-40 years satisfying the criteria of prediabetes on the basis of

HbA1c (5.7 to 6.4%) were included in the study. Exclusion criteria:  Patients with hemoglobin < 9 gm% and any history suggestive of hemoglobinopathies.  Patients with history suggestive of endocrine disorders like thyroid, adrenal and pituitary glands disorders.  Patients with history suggestive of any drug intake affecting glucose metabolism. Sample Collection After getting written consent from the cases and controls, detailed history was taken and recorded in their respective proforma. Eight mL of venous blood sample was taken from the antecubital vein aseptically, out of which:  For estimation of fasting blood glucose, 2 mL of blood was collected in sodium fluoride vacutainer after fasting of eight hours.  For estimation of postprandial blood glucose, 2 mL of blood was collected in sodium fluoride vacutainer. Sample was collected 2 hours after taking meals.

 For estimation of HbA1c, 2 mL of blood was collected in EDTA anticoagulant vacutainer.  For estimation of fructosamine, 2 mL of blood was collected in plain vacutainer. Samples were processed on the same day of collection. Serum from sodium fluoride and plain vacutainer was separated by centrifugation. The investigations were performed on the RANDOX (Randox Laboratories Limited, UK) by using standard Enzymatic kit methods [10]. HbA1c was determined by turbidimetric inhibition (TINIA) for hemolyzed whole blood [11]. Fructosamine was estimated by Colorimetric method by reaction with nitroblue tetrazolium (NBT) [12,13]. Statistical Analysis Primary outcome were calculated by applying Unpaired‘t’ test and secondary outcome were obtained by using two- tailed pearson correlation between variables of prediabetics cases and controls by using the statistical package (IBM SPSS 20). Data were considered to be significant if p < 0.05 and highly significant with p < 0.001. Result and Observations In the present study, it was observed that out of 30 cases, 21 (70%) had impaired fasting blood glucose while 25 (83.33%) had impaired postprandial blood glucose and 18 (60%) had both impaired fasting and postprandial blood glucose levels. Mean ± SD of fasting blood glucose levels of cases was 108.5 ± 10.5 mg/dL and controls was 89.4 ± 7.1 mg/dL with p value = 0.001. Mean ± SD of postprandial blood glucose levels of cases was 155.3 ± 13.2 mg/dL and controls was 121.2 ± 7.2 mg/dL with p value = 0.001. Mean ± SD of HbA1c in cases was 5.9 ± 0.21% and ISSN: 2319-7072 7

International J. of Healthcare and Biomedical Research, Volume: 08, Issue: 03, April 2020, 5- 10 DOI: 10.36848/IJHBR/2020/12100.51200 controls was 5.2 ± 0.32% with p value = 0.001. Mean ± SD of FA in cases was 256.2 ± 15.5µmol/L and controls was 236 ± 14.1 µmol/L with p value = 0.001 (Table 1). Two-tailed pearson’s correlation between parameters: In the present study, it was found that FA had positive correlation with both fasting and postprandial blood glucose (Table 2 & figure 1 and 2).

Figure 1: Graph showing correlation between FA & FBG

300

250

200

150

100 Fructosamine 50

0 0 50 100 150 Fasting blood glucose

Table 1: Analytical data of study groups S. No. Analytical data CASES CONTROLS p value (n = 30) (n = 30) Mean ± SD Mean ± SD 1. FBG (mg/dL) 108.5 ± 10.5 89.4 ± 7.1 0.001 2. PPBG (mg/dL) 155.3 ± 13.2 121.2 ± 7.2 0.001

3. HbA1c (%) 5.9 ± 0.2 5.2 ± 0.3 0.001 4. Fructosamine 256.2 ± 15.5 236 ± 14.1 0.001 (µmol/L)

Discussion In our study, there was statistically significant difference between mean of fructosamine in cases and controls. In contrast, MulaAbed et al observed during screening of diabetic patients that there was considerable overlap in FA values between diabetics and nondiabetics which limits its use as a diagnostic test and making it a complementary tool to differentiate between diabetics and nondiabetics. Plasma glucose had an established role in the diagnosis and assessment of short term glycemic control while FA mainly assess metabolic control [14].

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International J. of Healthcare and Biomedical Research, Volume: 08, Issue: 03, April 2020, 5- 10 DOI: 10.36848/IJHBR/2020/12100.51200

Fructosamine had positive correlation with fasting blood glucose and postprandial blood glucose (Table 2). Ko et al also observed a positive correlation of FBG and PPBG with fructosamine [9]. Rosediani et al also observed a positive correlation of FBG & PPBG with fructosamine in diabetics patients[15]. Koskinen et al also observed positive correlation of fasting blood glucose with fructosamine [16]. Fructosamine is influenced by the glycaemic control of short time period. The degree of protein glycation is a function of time and time dependant protein and glucose concentration. Thus, glycated protein i.e fructosamine correlates with glucose concentration and reflects the average glycemic situation [17]. Khan et al also observed positive correlation of FBG & PPBG with FA in diabetic patients. FA is a promising biomarker for 2-3 weeks status of blood sugar [18]. Selvin et al also observed that FA and glycated albumin have prognostic utility for the prediction of diabetes, chronic kidney and [19]. However, Manjrekar et al observed that FA predicts development of diabetes in high risk populations but is less sensitive in depicting chronic hyperglycemia [20]. Our study was in contrast to the study done by Vadodaria DV who observed that there was no significant association of fructosamine with fasting and postprandial blood glucose among prediabetic group [21]. Baker et al observed a positive correlation of FBG with fructosamine but postprandial hyperglycaemia has little effect because there was no correlation found between the FA concentration and glycemia two hours after a glucose load. So, FA estimation do not identify people with impaired glucose tolerance because many of them have normal fasting glucose concentrations at the time of testing [22]. Conclusion In conclusion, Fructosamine levels denote short interval changes in blood glucose levels. Fructosamine along with

HbA1c, fasting and postprandial blood glucose together serve as better tool to screen people for prediabetes.

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International J. of Healthcare and Biomedical Research, Volume: 08, Issue: 03, April 2020, 5- 10 DOI: 10.36848/IJHBR/2020/12100.51200

10. Sacks DB. Carbohydrates. In: Burtis CA, Ashwood ER, Bruns DE, editors. Teitz textbook of clinical chemistry and molecular diagnostics. 5th ed. Philadelphia: Elsevier; 2012. p.718-21. 11. Gene S, Omer B, Ustyol EA, Ince N, Bal F, Gurdol F. Evaluation of Turbidimetric Inhibition Immunoassay (TINIA) and HPLC methods for glycated haemoglobin determination. J Clin Lab Anal 2012;26:481-5. 12. Sacks DB. Diabetes Mellitus. In: Burtis CA, Ashwood ER, Bruns DE, editors. Teitz textbook of clinical chemistry and molecular diagnostics. 5th ed. Philadelphia: Elsevier; 2012. p.1448. 13. Schleicher ED, Vogt BW. Standardization of serum fructosamine assays. Clin Chem 1990;36:136-9. 14. MulaAbed WAS, Alnaemi AH. Performance indicators and validity of serum fructosamine assay as a diagnostic test in a screening program for diabetes mellitus. Saudi Med J 2003;24:477-84. 15. Rosediani M, Azidah AK, Mafauzy M. Correlation between fasting plasma glucose, postprandial glucose and glycated haemoglobin and fructosamine. Med J Malaysia 2006;61:68-71. 16. Koskinen P, Irjala K, Viikari J, Panulaontto R, Matikainen MT. Serum fructosamine in the assessment of glycaemic control in diabetes mellitus. Scand J Clin Lab Invest 1987;47:285-92. 17. Frandsen EK, Sabagh T, Bacchus RA. Serum fructosamine in diabetic pregnancy. Clin Chem 1988;34:316-9. 18. Khan S, Alam R, Khan MM. Association of Fructosamine and HbA1c in Newly Diagnosed Type 2 Diabetes Mellitus Patients. Indian J Basic Appl Med Res 2018;7:32-42. 19. Selvin E, Rawlings AM, Grams M, Klein R, Richey A, Sharrett, Steffes M, Coresh J.Prognostic utility of fructosamine and glycated albumin for incident diabetes and microvascular complications. Lancet Diabetes Endocrinol 2014;2:279–88. 20. Manjrekar PA, Hegde A, Shrilaxmi, d’Souza F, Kaveeshwar V, Jose A, Tasneem S, Shenoy R. Fructosamine in Non- diabetic First Degree Relatives of Type 2 Diabetes Patients: Risk Assessor. J Clin Diagn Res 2012;6:770-3. 21. Vadodaria DV. Fructosamine and Glycosylated Haemoglobin Levels in Patients with Different Glucose Status. Int J Innov Res Sci Eng Technol 2017;6:10186-93. 22. Baker JR, O’Connor JP, Metcalf PA, Lawson MR, Johnson RN. Clinical usefulness of estimation of serum fructosamine concentration as a screening for diabetes mellitus. Br Med J 1983;287:863-7.

Date of Submission: 24 February 2020 Date of Peer Review: 10 March 2020 Date of Acceptance: 21 April 2020 Date of Publishing: 30 April 2020 Author Declaration: Source of support: Nil , Conflict of interest: Nil Ethics Committee Approval obtained for this study? Yes Was informed consent obtained from the subjects involved in the study? Yes For any images presented appropriate consent has been obtained from the subjects: NA Plagiarism Checked: Urkund Software Author work published under a Creative Commons Attribution 4.0 International License

DOI: 10.36848/IJHBR/2020/12100.51200

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