Microvascular complications of type 2 diabetes mellitus

ORIGINAL ARTICLE Corrected fructosamine, corrected glycated albumin and glycated β-lipoprotein/ β-lipoprotein ratio in patients with microvascular complications of type 2 diabetes mellitus

Tejaskumar R. Kalaria1*, Vijay Vaidya2, Roma Shah3, Habibunnisha B. Sirajwala4

1MD MRCP (UK), Consultant diabetologist, Schvijk Hospital and Research Centre, Vadodara,2MD, Associate Professor, Department of Radiology, GMERS Medical College & Hospital, Gotri, Vadodara, 3MD, Consultant Anaesthesiologist, Freelancer, Vadodara, 4MD, Associate Professor, Department of Biochemistry, Government Medical College, Vadodara.

ABSTRACT BACKGROUND AND OBJECTIVES: It has been shown in studies that there are significant variations in glycation of various serum proteins in type 2 diabetes mellitus patients with and without microvascular complications. Present study aimed to evaluate whether correcting serum fructosamine for serum total protein level, serum glycated albumin for serum albumin level and deriving ratio of glycated β-lipoprotein to total β- lipoprotein enhances their significance and correlation over and above serum fructosamine, glycated albumin and glycated β-lipoprotein respectively in type 2 diabetes mellitus patients with and without microvascular complications. METHODS: This was a cross sectional study involving 150 individuals at a tertiary care hospital in western India. 50 participants were healthy controls (group 1), 50 were type 2 diabetes patients without any evident microvascular complication (group 2) and 50 were type 2 diabetes patients with one or more microvascular complications (group 3). Serum fructosamine, FBS, PP2BS and other biochemical parameters were measured. Glycated albumin and glycated β-lipoprotein were measured by agarose gel electrophoresis followed by NBT staining. Corrected serum fructosamine, corrected glycated albumin and glycated β-lipoprotein/ β-lipoprotein ratio were calculated. Unpaired t-test was used to find out significance of difference between two groups and correlation coefficient to find out statistical correlation between two variables. RESULTS: Differences between the groups for corrected fructosamine, corrected glycated albumin and glycated β-lipoprotein/ β-lipoprotein ratio were significant (p<0.001). Corrected glycated albumin and corrected fructosamine correlated with FBS, PP2BS and with each other in all diabetic patients (groups 2 and 3). Glycated β-lipoprotein/ β-lipoprotein ratio was remarkably elevated in group 3 and it did not correlate either with corrected fructosamine or corrected glycated albumin. CONCLUSION: Glycated β-lipoprotein/ β- lipoprotein ratio was markedly elevated in type 2 diabetes patients with microvascular compilations but neither of corrected fructosamine, corrected glycated albumin or glycated β-lipoprotein/ β-lipoprotein ratio provided any information in addition to that provided by the uncorrected parameters.

Key-words: Type 2 diabetes mellitus, microvascular complications, corrected fructosamine, corrected glycated albumin, corrected glycated β-lipoprotein

INTRODUCTION serum proteins can also be used in similar Glucose molecules are joined to protein manner depending on their half-lives, most molecules through non-enzymatic widely evaluated of them is glycated glycation to form fructosamine and it albumin which is an indicator of reflects glycaemic control attained by glycaemic status over preceding 2-3 diabetic patient over previous 2 to 3 weeks weeks. Due to 3-5 day circulating half-life and is useful in monitoring the of LDL, glycated LDL level reflects mean effectiveness of therapy in diabetes, in a glycemia over the preceding week2. But, manner analogous to the determination of as serum total protein and serum albumin glycated hemoglobin1. Other glycated level tend to change widely in many conditions including diabetic nephropathy, *Corresponding Author: corrected serum fructosamine and glycated Dr. Tejaskumar R. Kalaria albumin might be more useful than the A/20, Sundarvan Society, uncorrected parameters3. Similarly as the New Sama Road, P.O., value of LDL varies widely from person to Chhani Road, Vadodara-390024, person, biologically as well as due to Contact No: +91 9998968153 treatment, the ratio of glycated β- E-mail address: [email protected] lipoprotein to total β-lipoprotein might 139 Int J Res Med. 2016; 5(3); 139-145 e ISSN:2320-2742 p ISSN: 2320-2734

Microvascular complications of type 2 diabetes mellitus provide additional information as outpatient department were enrolled in compared to glycated β-lipoprotein alone. groups 2 and 3 unless they met any of the The study was undertaken to assess levels exclusion criterions. Patients with type 1 of serum fructosamine corrected for serum diabetes mellitus, pregnant females and total protein, serum glycated albumin patients with diseases unrelated to diabetes corrected for serum albumin level and which might significantly alter serum glycated β-lipoprotein to total β- protein profile e.g. hepatic diseases, lipoprotein ratio in type 2 diabetes mellitus hematological malignancy, chronic patients with and without microvascular infections and inflammations like complications and to find out their tuberculosis, sarcoidosis, rheumatological correlation with the complications over diseases, infectious mononucleosis, AIDS and above the uncorrected parameters. were excluded from the study. Objectives MATERIAL AND METHODS of the study were explained to all subjects This was a hospital based cross-sectional eligible for this study. Informed written study consisting of three groups and total consent of all the subjects was obtained for 150 participants. Group 1 (control group, voluntary participation in study group, n=50) consisted of randomly selected age sample collection and for data utilisation and sex matched non-diabetic subjects. for the purpose of publication. Data were They were free from any ailment which recorded in a questionnaire designed for could affect the parameters under study the study and it included socio- (no clinical history or investigative result demographic data, presenting complains, showing involvement of any organ detailed diabetes history including system). They were selected from medical treatment and complications, history of and paramedical staff, attendants of other ailments, past, personal and family patients and persons coming to hospital for histories as well as findings of a thorough fitness purpose. Group 2 (type 2 diabetes physical examination. For each subject, mellitus patients without any overnight fasting blood sample was microvascular complications, n=50) collected in a fluoride vacutainer for FBS consisted of those with duration of and in a plain vacutainer for other diabetes 3 years or more, on life style biochemical parameters. Urine sample was modifications and oral anti-diabetic drugs collected in universal container for urine and free from clinical or laboratory creatinine and urine protein estimation. evidence of any microvascular Post prandial (2 hour) sample was complication of diabetes mellitus. Group 3 collected in a fluoride vacutainer for (type 2 diabetes mellitus patients with PP2BS estimation. Serum and plasma microvascular complications, n=50) were separated within an hour of consisted of those with duration of collection and stored at 2 to 8°C diabetes 3 years or more, on life style temperature till analyses were performed. modifications, oral anti-diabetic drugs, Electrophoresis was performed within 5 insulin or combination of all three and days whereas all the other parameters were diagnosed as having one or more estimated on the same day. FBS and microvascular complication of diabetes PP2BS were estimated by Glucose mellitus; either diabetic neuropathy Oxidase-Peroxidase (GOD-POD) (peripheral neuropathy diagnosed by 10 enzymatic end point method. Serum gm monofilament and reduced vibration fructosamine was estimated by NBT perception using 128 Hz tuning fork), colorimetric kinetic assay. Serum diabetic nephropathy (urine albumin ≥30 Creatinine concentration was measured by µg/ mg of creatinine with normal urine modified Jaffe’s kinetic method. Serum microscopy and not a known case of any total cholesterol was estimated by other kidney disease) or diabetic Cholesterol Oxidase-Peroxidase (CHOD- retinopathy (diagnosed by direct PAP) enzymatic end point method. Serum ophthalmoscopy after mydriasis). LDL cholesterol was measured by direct Consecutive patients attending the medical enzymatic method. Urinary creatinine was

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Microvascular complications of type 2 diabetes mellitus estimated by modified Jaffe’s kinetic Corrected serum fructosamine (µmol/gm method, urinary protein was measured by of total protein) was calculated by dividing Pyrogallol red end point method and serum fructosamine value (µmol/l) by urinary protein/creatinine ratio was serum total protein (gm/l). Corrected calculated. All biochemical investigations glycated albumin (µmol/gm of serum were performed on a fully automated albumin) was calculated by dividing serum analyzer. Serum glycated β-lipoprotein glycated albumin value (µmol/l) by serum was measured by agarose gel film albumin (gm/l). Similarly, glycated β- electrophoresis at pH 8.6 followed by lipoprotein/ β-lipoprotein ratio was colour development with nitro-blue calculated by dividing glycated β- tetrazolium (NBT) by a modification of lipoprotein value (µmol/l) by serum LDL staining method adapted from Kunio (after converting to gm/l). Data collected Kobayashi et al [4]. Dried and compressed were recorded and analysed statistically to electrophoresis plate was immersed for 20 determine the significance of different hours in glycated protein colour parameters by MedCalc version 12.4. development reagent (5 mmol/l NBT in 50 Statistical analysis was done by using mmol/l sodium carbonate buffer pH 10.3) unpaired t-test to find out significance of at room temperature followed by two difference between two groups and washes of stop solution (0.3% citric acid). correlation coefficient to find out statistical NBT stained blue bands on the glycated correlation between two variables and its protein film were scanned at 540 nm significance. p value less than 0.05 was (Olympus glass filter plate) using a considered significant. densitometric scanner (Nikon Super Results and discussion COOLSCAN 9000 ED). Image was Table 1: Comparison of study groups analysed by free electrophoresis image Group 1 Group 2 Group 3 analysis software GelAnalyzer 2010a. Number of participants 50 50 50 Sex (M/F) 58/42 54/46 48/52 Proportions (%) of glycated albumin and Average age (years) 47±10 53±9 52±9 glycated lipoprotein were represented as Average duration of - 6.3±2.8 8.2±3.8 the percentage of total area under the diabetes mellitus (in year) scanned profile attributable to each peak. FBS (mg/dl) 94±11 129±39 188±86 Concentration (in µmol/l) was calculated PP2BS (mg/dl) 119±11 184±57 271±109 by multiplying percentage with serum Serum creatinine 0.89±0.20 0.96±0.19 1.03±0.29 fructosamine value. Staining in β region (mg/dl) Serum total protein 6.8±0.6 6.8±0.5 6.7±0.5 was considered to represent glycation of β- (gm/dl) lipoproteins. Serum protein (stained with Serum albumin (gm/dl) 4.3±0.3 4.4±0.3 4.3±0.3 0.5% acid blue 29 in 5% acetic acid) and Serum total cholesterol 174±35 177±37 184±43 (mg/dl) lipoprotein (stained with 0.05% fat red 7B Serum LDL cholesterol 121±29 117±27 121±27 and 0.05% oil red O in methanol: water, (mg/dl) 4:1) electrophoresis was performed Urinary 11±9 18±8 140±161 protein/creatinine simultaneously for all the samples to aid in (mg/gm) quantitation by comparison of mobility of Serum fructosamine 253±24 313±58 395±119 respective proteins (figure 1). (µmol/l) Corrected serum 3.72±0.46 4.65±86 5.91±1.76 Figure 1: Correlation of serum fructosamine (µmol/gm corrected fructosamine with corrected of serum total protein) glycated albumin (n=150) Serum glycated 136±26 162±42 202±78 albumin (µmol/l) Corrected serum 3.14±0.58 3.67±0.89 4.66±1.77 glycated albumin (µmol/gm of serum albumin) Serum glycated β- 25.2±5.1 32.8±7.8 50.4±11.6 lipoprotein (µmol/l) Glycated β-lipoprotein/ 21.6±5.5 29.7±9.8 43.7±13.4 β-lipoprotein (µmol/gm)

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Microvascular complications of type 2 diabetes mellitus Differences between the groups for serum Serum corrected r 0.0050 0.4411 0.8317 fructosamine p 0.9727 0.0013 <0.0001 corrected fructosamine value were (µmol/gm) and PP2BS statistically significant (table 2) and (mg/dl) Serum corrected r 0.0958 0.5548 0.7688 corrected fructosamine values correlated glycated albumin p 0.5079 <0.0001 <0.0001 very well with both FBS and PP2BS in (µmol/gm) and FBS (mg/dl) group 2 and 3, whereas the correlation was Serum corrected r -0.1259 0.4319 0.8056 not significant in group 1 (table 3). glycated albumin p 0.3838 0.0017 <0.0001 (µmol/gm) and PP2BS Similarly, differences between these (mg/dl) groups for mean corrected glycated Serum corrected r 0.4384 0.8809 0.9342 glycated albumin p 0.0014 <0.0001 <0.0001 albumin level were statistically significant (µmol/gm) and (table 2). As in case with corrected corrected fructosamine (µmol/gm) fructosamine, corrected glycated albumin Serum glycated β- r 0.1198 -0.0588 0.5238 also correlated well with both FBS and lipoprotein/ β- p 0.4073 0.6849 0.0001 lipoprotein (µmol/gm) PP2BS in group 2 and 3, whereas and corrected correlation was not observed in group 1 fructosamine (µmol/gm) (table 3). Corrected glycated albumin also Serum glycated β- r 0.1838 -0.1099 -0.1615 showed good correlation with corrected lipoprotein/ β- p 0.1877 0.4335 0.2480 lipoprotein (µmol/gm) fructosamine across groups and correlation and corrected glycated coefficient r was ≥0.88 in groups 2 and 3 albumin (µmol/gm) (table 3). Such findings for these r= correlation coefficient, p= probability uncorrected parameters were reported Early studies showed that fructosamine elsewhere5. Neither corrected fructosamine measurement was independent of protein nor corrected glycated albumin showed or albumin concentration as long as the any significant difference from the concentrations of albumin and protein uncorrected parameters in terms of either were within the reference range. However, correlation coefficient or its significance other studies have found statistically with both FBS and PP2BS. Correlation significant relationships between between glycated albumin and fructosamine and either total protein or fructosamine; and corrected glycated albumin concentrations and have albumin and corrected fructosamine also recommended that fructosamine values be did not show any differences. Both, corrected for protein concentrations6-8. An whether corrected or not, correlated with argument for not routinely correcting each other very well in all the study groups fructosamine values for protein (table 3, figure 1)5. concentrations was proposed by Staley and Table 2: Independent sample t-test: contends that the molar concentration of corrected fructosamine, corrected serum protein and of reactive lysine glycated albumin and glycated β- groups will always be in excess. lipoprotein/ β-lipoprotein between study Therefore, the rate-limiting step will be the groups glucose concentration and not the serum Study Corrected Corrected Glycated β- protein concentration9. Schleicher et al. groups fructosamine glycated lipoprotein/ β- albumin lipoprotein ratio concluded that albumin concentration Test Two tailed Test Two Test Two should not be used to correct fructosamine statistic Probability statistic tailed Statist tailed t t Probabi -ic t Probabi values because albumin concentration -lity -lity influences its own turnover, which in turn 1 and 2 6.775 p<0.0001 3.492 p=0.00 5.057 p<0.0001 10 08 influences the amount of glycation . 2 and 3 4.555 p<0.0001 3.540 p=0.00 5.985 p<0.0001 Conversely, Lamb et al. concluded that 07 1 and 3 8.522 p<0.0001 5.744 p<0.00 10.78 p<0.0001 correcting fructosamine values for serum 01 2 albumin or total protein concentration is Table 3: Correlation of study justifiable because the amount of parameters: fructosamine produced is in first-order Correlation between parameters Group 1 Group 2 Group 3 relation to albumin/protein Serum corrected r 0.1053 0.5730 0.7924 11 fructosamine p 0.4669 <0.0001 <0.0001 concentration . Henrichs et al. warned (µmol/gm) and FBS that if fructosamine values are corrected (mg/dl)

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Microvascular complications of type 2 diabetes mellitus for protein concentration, overall precision may be reduced by the imprecision of the total protein determinations. Furthermore, even when the total protein concentration is normal, dysproteinemias (i.e., qualitative changes in serum protein composition) may affect fructosamine values; this cannot be corrected by simple 12 adjustment by protein concentration . In this study glycated β-lipoprotein/ β- Finally, Hill et al. concluded that while in lipoprotein ratio is specifically elevated in a given population a relationship between diabetes patients with microvascular serum fructosamine and protein may be complications (group 3) and it also has apparent, the clinical utility of routine larger inter-individual variation as fructosamine correction has not been 13 indicated by wide SD values, this is the clearly established . Probably a large- most significant feature of the study. The scale study is needed to resolve the debate rise is disproportionate and not in line with about the need to correct fructosamine rise in corrected glycated albumin; as values for protein/albumin concentration. indicated by comparing the groups (table In the interim, as per majority sources, 4). But again this holds true for fructosamine can be reported as both 6 uncorrected parameters as well and the corrected and uncorrected values . Mean glycated β-lipoprotein/ β-lipoprotein ratio serum glycated β-lipoprotein/ β- does not provide any extra information5. lipoprotein ratio in group 1 was 21.6 Table 4: Change in parameters across µmol/gm, in group 2 was 29.7 µmol/gm groups in comparison to group 1 and in group 3 was 43.7 µmol/gm. The (considering group 1 values as 100%) differences between these groups were Parameter Group 1 Group 2 Group 3 statistically significant (p<0.0001) (table Mean corrected 100 125 159 2). Irrespective of the groups, corrected fructosamine Mean corrected glycated 100 117 148 glycated albumin showed excellent albumin correlation with corrected fructosamine Mean glycated β- 100 138 202 where as glycated β-lipoprotein/ β- lipoprotein/ β-lipoprotein lipoprotein ratio did not correlate well with ratio Precise mechanisms underlying the either of fructosamine or glycated albumin disproportionate rise of glycated β- (table 3, figures 1, 2 and 3). lipoprotein/ β-lipoprotein ratio in diabetes Figure 2: Correlation of corrected patients with microvascular complications serum fructosamine with glycated β- compared to those without them are yet to lipoprotein/ β-lipoprotein ratio (n=150) be understood but the most conceivable explanation is elevated small dense LDL in diabetes which is more susceptible to glycation than more buoyant LDL14-17. Glycated and glycoxidized LDL has been associated with enhanced tissue responses i-n many studies and they might play an important role in development of microvascular complications.18-20 To conclude, not only serum fructosamine and glycated albumin but also corrected

Figure 3: Correlation of corrected fructosamine and corrected glycated glycated albumin with glycated β- albumin can be used as markers of short to lipoprotein/ β-lipoprotein ratio (n=150) medium term glycemic control and they correlate well with FBS and PP2BS. Also glycated β-lipoprotein/ β-lipoprotein ratio was markedly elevated in type 2 diabetes 143 Int J Res Med. 2016; 5(3); 139-145 e ISSN:2320-2742 p ISSN: 2320-2734

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