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

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139 Int J Res Med. 2016; 5(3); 139-145 e ISSN:2320-2742 p ISSN: 2320-2734

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

1

MD 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.

INTRODUCTION

Glucose molecules are joined to protein molecules through non-enzymatic glycation to form fructosamine and it reflects glycaemic control attained by diabetic patient over previous 2 to 3 weeks and is useful in monitoring the effectiveness of therapy in diabetes, in a manner analogous to the determination of glycated hemoglobin1. Other glycated

*Corresponding Author:

Dr. Tejaskumar R. Kalaria A/20, Sundarvan Society, New Sama Road, P.O.,

Chhani Road, Vadodara-390024, Contact No: +91 9998968153

E-mail address: tejaskalaria@gmail.com

serum proteins can also be used in similar manner depending on their half-lives, most widely evaluated of them is glycated albumin which is an indicator of glycaemic status over preceding 2-3 weeks. Due to 3-5 day circulating half-life of LDL, glycated LDL level reflects mean glycemia over the preceding week2. But, as serum total protein and serum albumin level tend to change widely in many conditions including diabetic nephropathy, corrected serum fructosamine and glycated albumin might be more useful than the uncorrected parameters3. Similarly as the value of LDL varies widely from person to person, biologically as well as due to treatment, the ratio of glycated β-lipoprotein to total β-β-lipoprotein might

ORIGINAL ARTICLE

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 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 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.

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140 Int J Res Med. 2016; 5(3); 139-145 e ISSN:2320-2742 p ISSN: 2320-2734

provide additional information as compared to glycated β-lipoprotein alone. The study was undertaken to assess levels of serum fructosamine corrected for serum total protein, serum glycated albumin corrected for serum albumin level and glycated lipoprotein to total β-lipoprotein ratio in type 2 diabetes mellitus patients with and without microvascular complications and to find out their correlation with the complications over and above the uncorrected parameters. MATERIAL AND METHODS

This was a hospital based cross-sectional study consisting of three groups and total 150 participants. Group 1 (control group, n=50) consisted of randomly selected age and sex matched non-diabetic subjects. They were free from any ailment which could affect the parameters under study (no clinical history or investigative result showing involvement of any organ system). They were selected from medical and paramedical staff, attendants of patients and persons coming to hospital for fitness purpose. Group 2 (type 2 diabetes mellitus patients without any microvascular complications, n=50) consisted of those with duration of diabetes 3 years or more, on life style modifications and oral anti-diabetic drugs and free from clinical or laboratory evidence of any microvascular complication of diabetes mellitus. Group 3 (type 2 diabetes mellitus patients with microvascular complications, n=50) consisted of those with duration of diabetes 3 years or more, on life style modifications, oral anti-diabetic drugs, insulin or combination of all three and diagnosed as having one or more microvascular complication of diabetes mellitus; either diabetic neuropathy (peripheral neuropathy diagnosed by 10 gm monofilament and reduced vibration perception using 128 Hz tuning fork), diabetic nephropathy (urine albumin ≥30 µg/ mg of creatinine with normal urine microscopy and not a known case of any other kidney disease) or diabetic retinopathy (diagnosed by direct ophthalmoscopy after mydriasis). Consecutive patients attending the medical

outpatient department were enrolled in groups 2 and 3 unless they met any of the exclusion criterions. Patients with type 1 diabetes mellitus, pregnant females and patients with diseases unrelated to diabetes which might significantly alter serum protein profile e.g. hepatic diseases, hematological malignancy, chronic infections and inflammations like tuberculosis, sarcoidosis, rheumatological diseases, infectious mononucleosis, AIDS were excluded from the study. Objectives of the study were explained to all subjects eligible for this study. Informed written consent of all the subjects was obtained for voluntary participation in study group, sample collection and for data utilisation for the purpose of publication. Data were recorded in a questionnaire designed for the study and it included socio-demographic data, presenting complains, detailed diabetes history including treatment and complications, history of other ailments, past, personal and family histories as well as findings of a thorough physical examination. For each subject, overnight fasting blood sample was collected in a fluoride vacutainer for FBS and in a plain vacutainer for other biochemical parameters. Urine sample was collected in universal container for urine creatinine and urine protein estimation. Post prandial (2 hour) sample was collected in a fluoride vacutainer for PP2BS estimation. Serum and plasma were separated within an hour of collection and stored at 2 to 8°C temperature till analyses were performed. Electrophoresis was performed within 5 days whereas all the other parameters were estimated on the same day. FBS and PP2BS were estimated by Glucose

Oxidase-Peroxidase (GOD-POD)

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estimated by modified Jaffe’s kinetic method, urinary protein was measured by Pyrogallol red end point method and urinary protein/creatinine ratio was calculated. All biochemical investigations were performed on a fully automated analyzer. Serum glycated β-lipoprotein was measured by agarose gel film electrophoresis at pH 8.6 followed by colour development with nitro-blue tetrazolium (NBT) by a modification of staining method adapted from Kunio Kobayashi et al [4]. Dried and compressed electrophoresis plate was immersed for 20 hours in glycated protein colour development reagent (5 mmol/l NBT in 50 mmol/l sodium carbonate buffer pH 10.3) at room temperature followed by two washes of stop solution (0.3% citric acid). NBT stained blue bands on the glycated protein film were scanned at 540 nm (Olympus glass filter plate) using a densitometric scanner (Nikon Super COOLSCAN 9000 ED). Image was analysed by free electrophoresis image analysis software GelAnalyzer 2010a. Proportions (%) of glycated albumin and glycated lipoprotein were represented as the percentage of total area under the scanned profile attributable to each peak. Concentration (in µmol/l) was calculated by multiplying percentage with serum fructosamine value. Staining in β region was considered to represent glycation of β-lipoproteins. Serum protein (stained with 0.5% acid blue 29 in 5% acetic acid) and lipoprotein (stained with 0.05% fat red 7B and 0.05% oil red O in methanol: water, 4:1) electrophoresis was performed simultaneously for all the samples to aid in quantitation by comparison of mobility of respective proteins (figure 1).

Figure 1: Correlation of serum

corrected fructosamine with corrected glycated albumin (n=150)

Corrected serum fructosamine (µmol/gm of total protein) was calculated by dividing serum fructosamine value (µmol/l) by serum total protein (gm/l). Corrected glycated albumin (µmol/gm of serum albumin) was calculated by dividing serum glycated albumin value (µmol/l) by serum albumin (gm/l). Similarly, glycated β-lipoprotein/ β-lipoprotein ratio was calculated by dividing glycated β-lipoprotein value (µmol/l) by serum LDL (after converting to gm/l). Data collected were recorded and analysed statistically to determine the significance of different parameters by MedCalc version 12.4. Statistical analysis was done by using unpaired t-test to find out significance of difference between two groups and correlation coefficient to find out statistical correlation between two variables and its significance. p value less than 0.05 was considered significant.

Results and discussion

Table 1: Comparison of study groups

Group 1 Group 2 Group 3 Number of participants 50 50 50 Sex (M/F) 58/42 54/46 48/52 Average age (years) 47±10 53±9 52±9 Average duration of

diabetes mellitus (in year)

- 6.3±2.8 8.2±3.8

FBS (mg/dl) 94±11 129±39 188±86 PP2BS (mg/dl) 119±11 184±57 271±109 Serum creatinine

(mg/dl)

0.89±0.20 0.96±0.19 1.03±0.29

Serum total protein (gm/dl)

6.8±0.6 6.8±0.5 6.7±0.5

Serum albumin (gm/dl) 4.3±0.3 4.4±0.3 4.3±0.3 Serum total cholesterol

(mg/dl)

174±35 177±37 184±43

Serum LDL cholesterol (mg/dl)

121±29 117±27 121±27

Urinary

protein/creatinine (mg/gm)

11±9 18±8 140±161

Serum fructosamine (µmol/l)

253±24 313±58 395±119

Corrected serum fructosamine (µmol/gm of serum total protein)

3.72±0.46 4.65±86 5.91±1.76

Serum glycated albumin (µmol/l)

136±26 162±42 202±78

Corrected serum glycated albumin (µmol/gm of serum albumin)

3.14±0.58 3.67±0.89 4.66±1.77

Serum glycated β-lipoprotein (µmol/l)

25.2±5.1 32.8±7.8 50.4±11.6

Glycated β-lipoprotein/ β-lipoprotein

(µmol/gm)

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Differences between the groups for serum corrected fructosamine value were statistically significant (table 2) and corrected fructosamine values correlated very well with both FBS and PP2BS in group 2 and 3, whereas the correlation was not significant in group 1 (table 3). Similarly, differences between these groups for mean corrected glycated albumin level were statistically significant (table 2). As in case with corrected fructosamine, corrected glycated albumin also correlated well with both FBS and PP2BS in group 2 and 3, whereas correlation was not observed in group 1 (table 3). Corrected glycated albumin also showed good correlation with corrected fructosamine across groups and correlation coefficient r was ≥0.88 in groups 2 and 3 (table 3). Such findings for these uncorrected parameters were reported elsewhere5. Neither corrected fructosamine nor corrected glycated albumin showed any significant difference from the uncorrected parameters in terms of either correlation coefficient or its significance with both FBS and PP2BS. Correlation between glycated albumin and fructosamine; and corrected glycated albumin and corrected fructosamine also did not show any differences. Both, whether corrected or not, correlated with each other very well in all the study groups (table 3, figure 1)5.

Table 2: Independent sample t-test: corrected fructosamine, corrected glycated albumin and glycated β-lipoprotein/ β-lipoprotein between study groups Study groups Corrected fructosamine Corrected glycated albumin

Glycated

β-lipoprotein/ β-lipoprotein ratio Test statistic t Two tailed Probability Test statistic t Two tailed Probabi -lity Test Statist -ic t Two tailed Probabi -lity 1 and 2 6.775 p<0.0001 3.492 p=0.00

08

5.057 p<0.0001 2 and 3 4.555 p<0.0001 3.540 p=0.00

07

5.985 p<0.0001 1 and 3 8.522 p<0.0001 5.744 p<0.00

01 10.78

2

p<0.0001 Table 3: Correlation of study

parameters:

Correlation between parameters Group 1 Group 2 Group 3 Serum corrected

fructosamine (µmol/gm) and FBS

(mg/dl)

r 0.1053 0.5730 0.7924

p 0.4669 <0.0001 <0.0001

Serum corrected fructosamine (µmol/gm) and PP2BS

(mg/dl)

r 0.0050 0.4411 0.8317

p 0.9727 0.0013 <0.0001

Serum corrected glycated albumin (µmol/gm) and FBS

(mg/dl)

r 0.0958 0.5548 0.7688

p 0.5079 <0.0001 <0.0001

Serum corrected glycated albumin (µmol/gm) and PP2BS

(mg/dl)

r -0.1259 0.4319 0.8056

p 0.3838 0.0017 <0.0001

Serum corrected glycated albumin (µmol/gm) and corrected fructosamine

(µmol/gm)

r 0.4384 0.8809 0.9342

p 0.0014 <0.0001 <0.0001

Serum glycated lipoprotein/ β-lipoprotein (µmol/gm)

and corrected fructosamine (µmol/gm)

r 0.1198 -0.0588 0.5238

p 0.4073 0.6849 0.0001

Serum glycated lipoprotein/ β-lipoprotein (µmol/gm) and corrected glycated albumin (µmol/gm)

r 0.1838 -0.1099 -0.1615

p 0.1877 0.4335 0.2480

r= correlation coefficient, p= probability Early studies showed that fructosamine measurement was independent of protein or albumin concentration as long as the concentrations of albumin and protein were within the reference range. However, other studies have found statistically significant relationships between fructosamine and either total protein or albumin concentrations and have recommended that fructosamine values be corrected for protein concentrations6-8. An argument for not routinely correcting fructosamine values for protein concentrations was proposed by Staley and contends that the molar concentration of serum protein and of reactive lysine groups will always be in excess. Therefore, the rate-limiting step will be the glucose concentration and not the serum protein concentration9. Schleicher et al. concluded that albumin concentration should not be used to correct fructosamine values because albumin concentration influences its own turnover, which in turn influences the amount of glycation10. Conversely, Lamb et al. concluded that correcting fructosamine values for serum albumin or total protein concentration is justifiable because the amount of fructosamine produced is in first-order

relation to albumin/protein

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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 adjustment by protein concentration12. Finally, Hill et al. concluded that while in a given population a relationship between serum fructosamine and protein may be apparent, the clinical utility of routine fructosamine correction has not been clearly established13. Probably a large-scale study is needed to resolve the debate about the need to correct fructosamine values for protein/albumin concentration. In the interim, as per majority sources, fructosamine can be reported as both corrected and uncorrected values6. Mean serum glycated β-lipoprotein/ β-lipoprotein ratio in group 1 was 21.6 µmol/gm, in group 2 was 29.7 µmol/gm and in group 3 was 43.7 µmol/gm. The differences between these groups were statistically significant (p<0.0001) (table 2). Irrespective of the groups, corrected glycated albumin showed excellent correlation with corrected fructosamine where as glycated lipoprotein/ β-lipoprotein ratio did not correlate well with either of fructosamine or glycated albumin (table 3, figures 1, 2 and 3).

Figure 2: Correlation of corrected serum fructosamine with glycated β-lipoprotein/ β-lipoprotein ratio (n=150)

Figure 3: Correlation of corrected glycated albumin with glycated β-lipoprotein/ β-lipoprotein ratio (n=150)

In this study glycated lipoprotein/ β-lipoprotein ratio is specifically elevated in diabetes patients with microvascular complications (group 3) and it also has larger inter-individual variation as indicated by wide SD values, this is the most significant feature of the study. The rise is disproportionate and not in line with rise in corrected glycated albumin; as indicated by comparing the groups (table 4). But again this holds true for uncorrected parameters as well and the glycated β-lipoprotein/ β-lipoprotein ratio does not provide any extra information5. Table 4: Change in parameters across groups in comparison to group 1 (considering group 1 values as 100%)

Parameter Group 1 Group 2 Group 3 Mean corrected

fructosamine

100 125 159

Mean corrected glycated albumin

100 117 148

Mean glycated β-lipoprotein/ β-lipoprotein ratio

100 138 202

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patients with microvascular complications and the rise was disproportionate to that of corrected glycated albumin. But, neither corrected fructosamine, corrected glycated albumin nor glycated lipoprotein/ β-lipoprotein ratio provided any additional information or strong correlation with any of study parameters as compared to the uncorrected parameters and thus such correction cannot be recommended routinely for reporting these parameters. REFERENCES

1. Armbruster DA. Fructosamine: structure, analysis, and clinical usefulness. Vol. 33, Clinical Chemistry. 1987. p. 2153–63.

2. Roohk HV, Zaidi AR. A review of glycated albumin as an intermediate glycation index for controlling diabetes. J Diabetes Sci Technol. 2008;2(6):1114–21.

3. Kunika K, Itakura M, Yamashita K. Correction of fructosamine value for serum albumin and globulin concentrations. Diabetes Res Clin Pract. 1991 Jan;13(1-2):37–44. 4. Kobayashi K, Ogasahara N,

Sakoguchi T, Kimura M, Taniuchi K,

Matsuoka A. Agarose gel

electrophoresis with nitroblue tetrazolium coloration for determination of glycated lipoproteins in serum from diabetics. Clin Chem. 1990 Jan;36(1):65–9.

5. Kalaria TR, Sirajwala HB, Gohel

MG. Evaluation of serum

fructosamine, serum glycated albumin and serum glycated β-lipoprotein in type 2 diabetes mellitus patients with

and without microvascular

complications. J Diabetes Metab Disord. 2016;In press.

6. Goldstein DE, Little RR, Lorenz RA, Malone JI, Nathan D, Peterson CM, Sacks DB. Tests of glycemia in diabetes. Vol. 27, Diabetes Care. 2004. p. 1761–73.

7. Van Dieijen-Visser MP, Seynaeve C, Brombacher PJ. Influence of variations in albumin or total-protein concentration on serum fructosamine concentration. Clin Chem. 1986 Aug;32(8):1610.

8. Oimomi M, Masuta S, Sakai M, Ohara T, Hata F, Baba S. Influence of serum protein levels on serum fructosamine levels. Jpn J Med. 28(3):312–5.

9. Staley M. Fructosamine and protein concentrations in serum. Clin Chem. 1987;33(12):2326–7.

10. Schleicher ED, Olgemöller B, Wiedenmann E, Gerbitz KD. Specific glycation of albumin depends on its half-life. Clin Chem. 1993 Apr;39(4):625–8.

11. Lamb E, Mainwaring-Burton R, Dawnay A. Effect of protein concentration on the formation of glycated albumin and fructosamine. Clin Chem. 1991 Dec;37(12):2138–9. 12. Henrichs HR, Lehmann P, Vorberg E. An Improved Fructosamine Assay for Monitoring Blood Glucose Control. Diabet Med. 1991 Jul;8(6):580–4. 13. Hill RP, Hindle EJ, Howey JE,

Lemon M, Lloyd DR.

Recommendations for adopting standard conditions and analytical procedures in the measurement of serum fructosamine concentration. Ann Clin Biochem. 1990 Sep;27 ( Pt 5):413–24.

14. Krauss RM. Lipids and lipoproteins in patients with type 2 diabetes. Diabetes Care. 2004 Jun;27(6):1496–504. 15. Mohan V, Deepa R, Velmurugan K,

Gokulakrishnan K. Association of small dense LDL with coronary artery disease and diabetes in urban Asian Indians - the Chennai Urban Rural Epidemiology Study (CURES-8). J Assoc Physicians India. 2005 Feb;53:95–100.

16. Younis N, Charlton-Menys V, Sharma R, Soran H, Durrington PN. Glycation of LDL in non-diabetic people: Small dense LDL is preferentially glycated both in vivo and in vitro. Atherosclerosis. 2009;202(1):162–8.

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diabetes. Clin Sci (Lond). 2013 Mar;124(5):343–9.

18. Cohen MP, Ziyadeh FN, Chen S. Amadori-modified glycated serum

proteins and accelerated

atherosclerosis in diabetes: pathogenic and therapeutic implications. J Lab Clin Med. 2006 May;147(5):211–9. 19. Cohen MP, Lautenslager G, Shea E.

Glycated LDL concentrations in non-diabetic and diabetic subjects

measured with monoclonal antibodies reactive with glycated apolipoprotein B epitopes. Eur J Clin Chem Clin Biochem. 1993 Nov;31(11):707–13. 20. Moro E, Alessandrini P, Zambon C,

Figure

Table 1: Comparison of study groups  Number of participants 50
Table 2: Independent sample t-test: corrected fructosamine, corrected glycated albumin and glycated β-lipoprotein/ β-lipoprotein between study groups
Figure 2: Correlation of corrected serum fructosamine with glycated β-lipoprotein/ β-lipoprotein ratio (n=150)

References

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