Evaluation of biochemical and protein biomarkers analysis in type 2 diabetes mellitus

Kamal Uddin Zaidi; Firoz Naem Khan; Vijay Thawani; Richa Parmar

doi:10.4103/jmgims.jmgims_6_18

ORIGINAL ARTICLE

Year : 2019 | Volume : 24 | Issue : 1 | Page : 28-32

Evaluation of biochemical and protein biomarkers analysis in type 2 diabetes mellitus

Kamal Uddin Zaidi, Firoz Naem Khan, Vijay Thawani, Richa Parmar
Centre for Scientific Research and Development, Biotechnology Pharmacology Laboratory and Immunology Laboratory, People's University, Bhopal, Madhya Pradesh, India

Date of Web Publication

14-Mar-2019

Correspondence Address:
Dr. Kamal Uddin Zaidi
Centre for Scientific Research and Development, Biotechnology Pharmacology Laboratory and Immunology Laboratory, People's University, Bhopal - 462 037, Madhya Pradesh
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jmgims.jmgims_6_18

Abstract

Introduction: Diabetes mellitus (DM) type 2 (T2) is a metabolic disorder characterized by insulin resistance and affecting protein metabolism. There has been a great interest in the proteomic analysis of plasma and serum for the identification and characterization of protein biomarkers of different diseases. Materials and Methods: For the protein identification, one of the most important developments and technologies is the proteomics. In this work, the levels of protein biomarkers specific to T2 DM using sodium dodecyl sulfate polyacrylamide gel electrophoresis were identified and characterized. Results: Patients suffering from DM and normal healthy controls were recruited for this study. Biochemical and proteins biomarker assay were done. Some proteins were up and down-regulated in the samples of diabetes as compared to control. Conclusion: Assessment of the levels of biomarkers can aid early diagnosis and as well cure of T2DM.

Keywords: Biomarker, diabetes, insulin, protein

How to cite this article:
Zaidi KU, Khan FN, Thawani V, Parmar R. Evaluation of biochemical and protein biomarkers analysis in type 2 diabetes mellitus. J Mahatma Gandhi Inst Med Sci 2019;24:28-32

How to cite this URL:
Zaidi KU, Khan FN, Thawani V, Parmar R. Evaluation of biochemical and protein biomarkers analysis in type 2 diabetes mellitus. J Mahatma Gandhi Inst Med Sci [serial online] 2019 [cited 2023 Jan 28];24:28-32. Available from: https://www.jmgims.co.in/text.asp?2019/24/1/28/254136

Introduction

Proteomics is the study of protein with their structures, functions, and information coded in a cell which is expressed and regulated at the protein level to achieve the function of an organism.^[1] The protein biomarkers are helpful for predicting long-term mortality in patients with diabetes mellitus (DM), cancer, and coronary diseases. DM is a widely occurring disease whose global prevalence has risen. According to recent publications of the World Health Organization and International Diabetes Federation, DM is an epidemic and sixth leading cause of deaths worldwide. Humans are not the only species that develop DM. This disease occurs in dogs, cats, and other animals also. Type 2 (T2) DM is more common than T1DM and makes up about 90% of all cases of DM. It is also common in the developing economies. The disease is multiplying geometrically due to genetic and environmental factors.^[1] T2DM or non-insulin-dependent DM develops slowly. Initially, it begins with insulin resistance, which increases gradually until the body fails to maintain glucose homeostasis resulting in glucose intolerance with changes in biochemical processes.^[2] The T2DM can be only treated and has long-term complications. Prolonged high sugar levels in T2DM can affect the immune, cardiovascular, renal systems, and eye leading to complications such as neuropathy, peripheral vascular disease, renal disease, retinopathy, and coronary heart disease. It also affects salivary glands.^[3] The DM patients are highly predisposed to cardiovascular disease.^[4]

The DM is gaining the status of an epidemic in India with >62 million individuals who have been already diagnosed with the disease. In 2000, India with 31.7 million DM patients topped the world followed by China (20.8 million) and the United States (17.7 million) at the second and third position. Differential comparison between proteome of healthy and DM patients has been done to identify proteins that could be used as biomarkers. However, due to the emerging complexity of serum proteome, the occurrence of multiple isoforms raises the question of their potential use as biomarkers. The aim of this study was to evaluate the expression of protein biomarker in T2DM and normal individuals. For this purpose, to separate, compare, and identify proteins between DM patients and normal individuals, we combined Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) electrophoresis.

Materials and Methods

Sample collection

Fasting blood samples were collected from the OPD of People's College of Medical Science and Research Centre Hospital, of Bhopal, over a period of 1 month. About 2–3 ml of blood was withdrawn from the patients suffering from DM and healthy volunteers, under aseptic conditions, in sterile vials containing 1.5 mg/ml ethylenediaminetetraacetic acid. Samples were centrifuged at 2000 rpm for 5 min and the separated serum was stored at 20°C and used for further analysis.

Biochemical assays

Enzyme determination in sera was done by Autospan assay kits. Serum glucose, total protein, and α-amylase were determined spectrophotometrically. The serum glucose estimation was performed using glucose oxidase-peroxidase endpoint method. The serum amylase activity was determined by the kinetic enzyme assay kit. The ability of α-amylase to catalyze the hydrolysis of starch to maltose is the principle used to estimate amylase. Serum total protein was determined by Biuret method.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

Poly acrylamide gel electrophoresis in the presence of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to method of Laemmli.^[5],[6] The SDS-PAGE was performed using a 12% separating gel and 4% stacking gel.

Sample preparation

The samples for protein analysis were prepared by serum, mixed it with × 1 loading dye in 1:2 ratio and heated at 100°C for 10 min 7 then cooled for 5 min.

Procedure

Gel plates were cleaned, dried, and sealed with the help of silicon grease and gel assembly was set in gel caster. Separating gel (12%) was poured in between clamped glass plates. The gel was allowed to polymerize for 30 min. Then, the stacking gel (4%) was poured over it followed by comb insertion. On polymerization of the gel, comb was removed. Whole assemble was placed in buffer tank containing ×1 Tris-Glycein-SDS running buffer. The electrodes were connected and voltage was maintained at 100V. After completion, gel was carefully removed from glass plates and stained with staining solution for 2–3 h, and excess of stain was removed using de-staining solution.

Results

Blood glucose homeostasis indicates the balance of glucose ingestion and hepatic glucose production and peripheral glucose uptake and utilization. This equilibrium is maintained by complex interplay of several glucose elevating hormones such as glucagon, cortisol, growth hormone, catecholamines, glucose lowering hormone, and insulin. In this study, 30 samples were investigated of which 20 were DMT2 patients (9 men and 11 women-Groups I) and 10 were non diabetic controls (5 men and 5 women-Groups II). Thus, the men: women ratio was 9:11 for Group I and 1:1 for nondiabetics Group II. The age for both groups ranged from 38 to 70 years with mean age 52.32 ± 8.0 years for Group I and 50.43 ± 5.3 years for Group II [Table 1].

Table 1: Number of sample diabetics (Group I) and normal individuals (Group II)

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The amylase level was found to be higher in Group I ranging from 93.2 to 351.3 as compared to control where it was in the range of 24.3–85.2 [Figure 1]a and [Figure 1]b. The serum α-amylase levels of whole serum in this study showed significantly higher values in diabetics than in non diabetics.

Figure 1: Biochemical analysis: glucose concentration, amylase activities, and protein concentration (a) diabetics (b) controls

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The total proteins were estimated using Biuret method which revealed the difference in T2DM patients' protein concentration ranging from 8.8 to 15.1 mg/mL. In normal individuals, the protein concentration ranged from 5.8 to 8.16 [Figure 1]a and [Figure 1]b.

Protein profiling

The SDS-PAGE analysis of various samples revealed four types of banding patterns, with the number of bands ranging from 3 to 30. The maximum (35%) had a banding pattern with three bands [Table 2]. Proteins with molecular weight 24.0, 40.0, and 65.5 kDa were consistently present in the pattern 1. The pattern 2 showed that 20% of the sample had three proteins bands with molecular weights 11.5, 23, 90.5 kDa. The pattern 3 (30% of the sample) showed four protein bands with molecular weights 18.5, 23, 66, and 97 kDa. The minimum sample of 15% also showed four protein bands but with molecular weights 14, 18, 42, and 130 kDa which were consistently present in the in the pattern 1 [Table 2].

Table 2: Protein profile of type 2 diabetic patients

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The dendogram of T2DM showed that eight samples were grouped in two closely related clusters. The clusters of T2DM patients were significantly different and unrelated to that of the control group. It was also seen that sample from control tended to fall close together on cluster analysis. In our study, the SDS-PAGE pattern revealed several characteristic bands common to all samples. The sample under study was divided in two clusters [Table 3] with cluster one having eight samples and cluster two with 12 samples having a similarity of 79.3% and dissimilarity of 20.7% in the Jaccard's coefficient scale in the dendogram [Figure 2]. The cluster one was further divided into 1a and 1b with 3(DBT-18, DBT-14, DBT-08) and 5 (DBT-16, DBT-13, DBT-07, DBT-03, DBT-05) sample, respectively. The cluster 2 was also divided in two sub-clusters 2a (DBT-20, DBT-02, DBT-10) and 2b (DBT-17, DBT-15, DBT-12, DBT-04, DBT-05, DBT-19, DBT-11, DBT-01, DBT-09). Cluster 2a having three samples showed similarity of 89.5% and dissimilarity of 10.5% and 2b showed similarity of 87.3% and dissimilarity of 12.7%.

Table 3: Diversity class of type 2 diabetic patients based on the total protein profile

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Figure 2: DendroUPGMA: relationship among Type 2 diabetes mellitus based on their protein profiles by sodium dodecyl sulfate polyacrylamide gel electrophoresis

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The sub-cluster 2(b) was divided into sub cluster as 2b1 (DBT-17, DBT-15, DBT-12, DBT-04) showed similarity 87.9% and dissimilarity of 12.1% [Table 3]. Cluster as 2b2 (DBT-19, DBT-11, DBT-01, DBT-09) showed similarity 86.7% and dissimilarity of 13.3%. These differences being due to the difference in their protein profile which can be mediated due to the difference in the T2DM patient pattern to the anti-diabetic therapy. The study indicates that the profile of total protein from T2DM patients can be used for developing classification pattern.

Discussion

Significant correlation was found between both genders and two study groups. After a meal, the blood glucose concentration peaks, leading to insulin secretion from the pancreatic cells of healthy individuals. Within 10 min after a glucose load the blood insulin rises to maximum level, the so-called first or early phase of insulin release. If the blood sugar concentration remains high, the cells continue to release insulin, resulting in the second or late phase of insulin release.^[7],[8]

In the T2DM patients, the mean serum glucose value was found to be 211.50 ± 43.82 mg/dL. In nondiabetics, the mean serum glucose level was 88.81 ± 11.29 mg/dL [Figure 1]a and [Figure 1]b. Insulin lowers the blood glucose by acting on three main target tissues, namely, muscle, liver, and adipose tissue. First, the glucose uptake and utilization in muscle and adipose tissue is enhanced. In liver and muscle cells glycogen synthesis is enhanced, whereas break-down is suppressed, resulting in net storage of glycogen. Glucose release from liver is suppressed by inhibition of enzymes of the gluconeogenetic pathway.^[9] Opposite reaction happens in the fasting state, when blood glucose and insulin levels are low. Glucose production is then promoted by enhanced hepatic gluconeogenesis and glycogenolysis. At the same time, glycogen production and glucose-uptake in insulin-sensitive tissues is decreased, leading to elevation of the blood glucose level.^[9] Besides glucose, several other factors can promote insulin secretion, including amino acids, fatty acids, gastrointestinal peptides, and neuronal factors. The insulin deficiency in T1DM causes more disturbances in serum amylase than T2DM.^[9],[10] Some researchers found serum amylase level to be low and salivary amylase level to be higher in T2DM patients.^{[11],[12],[13],[14]}

López et al.^[15] found it to be lower while Tenovuo et al.^[16] reported it to be same in diabetics and nondiabetics. These differences may be attributed due to difference to stress levels, hormonal and metabolic changes in DM patients compared to nondiabetics. We found significantly higher levels of α-amylase in saliva than serum in diabetics and our results are in accordance with Malathi et al.^[17] who suggested that low-serum amylase levels in DM patients might be associated with an impaired insulin action due the insulin resistance and or inadequate insulin secretion.

Glycation of proteins, including hemoglobin and albumin have been implicated in complications of diabetes. The total proteins estimation revealed the difference in T2DM patients' protein concentration in a range of 8.8–15.1 mg/mL. In normal individuals the protein concentration ranged from 5.8–8.16. These findings are in agreement with those of Indira et al.^[18] who reported the assessment of salivary total protein in T2DM patients. On the other hand, total protein levels were increased from 6.8 ± 0.81 in controls to 7.31 ± 0.93. In case of serum globulin levels A/G ratio levels decreased from 2.31 ± 0.43 in controls to 1.84 ± 0.36.^[19]

The protein biomarkers are useful for diagnosis and prognosis of acute and chronic T2DM patients. In our investigations the minimal sample of 15% showed four proteins bands with molecular weights 14, 18, 42, and 130 kDa which were consistently present in the pattern 1. These findings are in agreement with those of Riaz^[1] for the identification of leptin, tumor necrosis factor-alpha, IL-6, monocyte chemoattractant protein-1, plasminogen activator inhibitor type 1 -1, Lipoprotein lipase, C-reactive protein and apolipoprotein C1, C2 protein biomarkers of DMT2 and therapy with Vitamin B1.

The cluster 1 was further divided into 1a and 1b with 3(DBT-18, DBT-14, DBT-08) and 5 (DBT-16, DBT-13, DBT-07, DBT-03, and DBT-05) sample, respectively. Similar findings have been reported by Zaidi et al.^[20] in the total protein profile and drug resistance in Candida albicans. The present findings of protein profiling in T2DM patients are in corroboration with the work of Riaz^[1] who reported the study of protein biomarkers of DMT2 and therapy with Vitamin B1.

Conclusion

This study of PAGE made a precise co-relation of the disease with the direct measurement of protein in a given sample, at a given time. The PAGE has become an important tool for assessing disorders for finding the association with proteins in evolving science of proteomics. Our research discovered and categorized the protein biomarkers for early diagnosis and cure of T2DM. These investigations would be supportive in measuring the biochemical changes in the patients of T2DM and will also assist in planning preventive and effective treatment strategies to decrease the disease load on health services and improving the quality of life of T2DM patients with better management. This research would also contribute to the identification of protein markers of T2DM, thus upgrading the diagnostic procedures for early detection of DM in our population.

Financial support and sponsorship

There is no funding

Conflicts of interest

There are no conflicts of interest.

References

1.	Riaz S. Study of protein biomarkers of diabetes mellitus type 2 and therapy with Vitamin B1. J Diabetes Res 2015;2015:150176.
2.	Al-Rawi NH. Oxidative stress, antioxidant status and lipid profile in the saliva of type 2 diabetics. Diab Vasc Dis Res 2011;8:22-8.
3.	Prathibha KM, Johnson P, Ganesh M, Subhashini AS. Evaluation of salivary profile among adult type 2 diabetes mellitus patients in South India. J Clin Diagn Res 2013;7:1592-5.
4.	Khatana SA, Taveira TH, Dooley AG, Wu WC. The association between C-reactive protein levels and insulin therapy in obese vs nonobese veterans with type 2 diabetes mellitus. J Clin Hypertens (Greenwich) 2010;12:462-8.
5.	Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.
6.	Zaidi KU, Ali AS, Ali SA. Purification and characterization of melanogenic enzyme tyrosinase from button mushroom. Enzyme Res 2014;2014:1-6.
7.	Grodsky GM. A new phase of insulin secretion. How will it contribute to our understanding of beta-cell function? Diabetes 1989;38:673-8.
8.	Del Prato S, Coppelli A, Tiengo A. Diabetes secondary to acquired disease to the pancreas. International Textbook of Diabetes Mellitus. Ch. 4. New York: Oxford University; 2004.
9.	Aronoff SL, Berkowitz K, Shreiner B, Want L. Glucose metabolism and regulation: beyond insulin and glucagon. Diabetes Spectr 2004;17;183-90.
10.	Ewadh MJ, Juda MT, Ali ZA, Ewadh MM. Evaluation of amylase activity in patients with type 2 diabetes mellitus. Am J Biosci 2014;2:171-4.
11.	de Almeida-Pititto B, Dias ML, de Moraes AC, Ferreira SR, Franco DR, Eliaschewitz FG, et al. Type 2 diabetes in Brazil: Epidemiology and management. Diabetes Metab Syndr Obes 2015;8:17-28.
12.	Jain R, Jain PK, Mangukiya K. Study of serum amylase in the patients of type 2 diabetes mellitus. Int J Sci Natl 2014;5:553-6.
13.	Amer S, Yousuf M, Siddqiui PQ, Alam J. Salivary glucose concentrations in patients with diabetes mellitus – A minimally invasive technique for monitoring blood glucose levels. Pak J Pharm Sci 2001;14:33-7.
14.	Meurman JH, Rantonen P, Pajukoski H, Sulkava R. Salivary albumin and other constituents and their relation to oral and general health in the elderly. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:432-8.
15.	López ME, Colloca ME, Páez RG, Schallmach JN, Koss MA, Chervonagura A, et al. Salivary characteristics of diabetic children. Braz Dent J 2003;14:26-31.
16.	Tenovuo J, Lehtonen OP, Viikari J, Larjava H, Vilja P, Tuohimaa P, et al. Immunoglobulins and innate antimicrobial factors in whole saliva of patients with insulin-dependent diabetes mellitus. J Dent Res 1986;65:62-6.
17.	Malathi L, Masthan KM, Balachander N, Babu NA, Rajesh E. Estimation of salivary amylase in diabetic patients and saliva as a diagnostic tool in early diabetic patients. J Clin Diagn Res 2013;7:2634-6.
18.	Indira M, Chandrashekar P, Kattappagari KK, Chandra LP, Chitturi RT, Bv RR. Evaluation of salivary glucose, amylase, and total protein in type 2 diabetes mellitus patients. Indian J Dent Res 2016;27:109. [PUBMED] [Full text]
19.	Nazki FA, Syyeda A, Mohammed S. Total proteins, albumin and HBA1c in type 2 diabetes mellitus. Int J Biochem 2017;3:40-2.
20.	Zaidi KU, Mani A, Thawani V, Mehra A. Total protein profile and drug resistance in Candida albicans isolated from clinical samples. Mol Biol Int 2016;2016:4982131.