Assessment of protein carbonyl in type II diabetes mellitus in a tertiary care hospital

Abstract               Background: Diabetes mellitus (DM) is an endocrine dysfunction, which is related with metabolic disorder. Hyperglycemia occurs primarily due to the insufficiency of secretion or action of endogenous insulin. It is accelerated by reduced glycemic control which increases free radicals causing oxidative stress. Materials and Methods: Overall 180 subjects were involved in the study. The cases comprised of 45 each of male female diabetics. 45 non diabetic males and 45 non diabetic females were selected as controls. Protein carbonylation in T2DM (Type 2 diabetes mellitus) subjects was assessed by DNPH method. In our study the mean and SD values of protein carbonyl were considered for all cases and control of both sexes of three different age groups. Results: Both diabetic males and females had elevated Protein carbonyl (PC) level as compared to controls (p value 0.05). The present study shows that with raise in free radicals in T2DM causes oxidation of protein which in term increases a level of PC and also support an involvement between these protein oxidation and type 2 diabetes as well as their positive association with different age groups The stronger response observed in serum samples from patients with higher patients’ age groups compared with lower age groups. Conclusion:  our findings suggest that protein carbonyl contents may be useful in evaluating the progression of diabetes mellitus type 2 and in elucidating the mechanisms of disease pathogenesis

Key Word: diabetes mellitus.

INTRODUCTION

Type 2 diabetes mellitus and it’s associated to resultant complications are rapidly becoming the world’s most considerable cause of morbidity and mortality¹.Diabetes mellitus is mainly a metabolic disorder characterized by hyperglycaemia and deficiency of secretion or action of endogenous insulin^1,2. Although the etiology of diabetes mellitus is not completely defined but environmental factors have been concerned¹. Oxidative stress plays a fundamental role in the onset of diabetes mellitus as well as its related complications. When the stability between the free radicals and antioxidant activity disrupt, subsequently oxidative stress arise³. Number of studies indicates a major part for amplified in protein oxidation as a key source of cellular dysfunction observed in several disorders⁴. Alterations of structural protein be able to lead to loss of function⁵ Protein plays a especially significant function in biochemistry. It is a essential bio-molecule of the cells. Proteins are the possible target of ROS, Protein glycation in diabetes mellitus patients is mainly an end result of glucose auto-oxidation, which is a process initiated generally by ROS and RNS⁶. Suitable to several oxidative modification reactions, carbonyls groups in proteins such as aldehyde/ ketonesappear. Carbonylated proteins are hydrophobic in nature and resistant to proteolysis⁵. Protein carbonyl is the regularly used indicator of protein oxidation. There is an emergence of carbonyl groups such as aldehyde/ ketone groups of proteins which are caused because of oxidative modification. Oxidative modification of proteins can occur in two pathways – 1. α-amidation pathways, 2. Oxidation of glutamyl side chains (6,7). Content of protein carbonyl is more different cells and plasma of diabetic patients. Modification of protein function outcome is cellular dysfunction and tissue damage. Assessment of ROS is not a simple method so protein oxidation can be assessed in terms of protein carbonyl which is a constant result of irreversible non enzymatic oxidation. Our study has been formulated in order to estimate carbonyl stress among diabetics of various age groups.

MATERIALS AND METHODS

The study was conducted in the Department of Biochemistry Mamatha academy of Medical Sciences hospital, Bachupally, Hyderabad, a tertiary care center in the duration of June 2018- December 2018. Total 180 subjects of age between 20 to 50 years included in the study population and the study group included 90 diabetes mellitus type 2 patients (45male and 45 female).  The control group comprised 90 non diabetic subjects (45 male and 45 female). The mean ages of all studied patients and controls were not significantly different between the groups (p>0.05). The subjects of both the sexes were categorized under three age groups such as 20 to 30 years, 31 to 40 years and 41 to 50 years. In females samples are collected in between 1-4 days of menstrual cycle. The research protocol was approved by ethics committee of the Institution.  Both cases and control subjects were subjected to estimation of biochemical parameters. Venous blood samples were collected after taking aseptic precautions from the study subjects. 3mL of blood was collected in plain red colored Vacutainer tubes; Samples were left for 20 minutes at room temperature and centrifuged at 3000 rpm for 4 to 5 minutes. Serum protein carbonylation was measured by DNPH method. FBS (Fasting Blood Sugar) and PPBS (Postpondrial Blood Sugar) were done by GOD/POD method (7). Data generated were analyzed using openepi online statistical tool and Microsoft Excel 2007. Comparison mean and standard deviation values were made for the various parameters for test and control subjects using independent samples t test. Results were considered statistically significant 95%confidence interval (p<0.05).

RESULTS

Protein oxidation characteristically results in an increase in carbonyl contents; therefore, increase in protein carbonylation is measured to be a most consistent biomarker of protein oxidation (8). Results showed significant increase in serum protein carbonyl contents in 90 diabetes mellitus patients compared with 90 normal human subjects (p0.05) The average carbonyl contents (mean±SD) in studied subjects of Diabetes mellitus type2 serum proteins and Normal Human (NH) proteins of different age groups were 20-30 years (302.12±21.53), 31-40years (311.12±49.16), 41-50years (419.69±94.47) of cases, and 20- 30 years(205.31±52.22), 31-40years (212.15±13.35), 41-50years (306.29±60.69) of controls in males(table1). Whereas20-30years (346.43±62.12), 31-40 years (429.22±43.41), 41-50years (457.92±21.75) of cases, and 20-30years (227.72±34.53), 31-40years (242.89±34.27), 41-50 years (289±15.79) in female population(table2). Our novel data showed that, protein carbonyl content was considerably higher in diabetes mellitus type 2 patients compared with non-Diabetic subjects (p<0.05).

Table 1: Evaluation between Age And Sex and correlated levels for Protein carbonyl (PC) and Fasting Blood Sugar (FBS) and Postpondrial Blood Sugar (PPBS) among cases and controls of male population.

Table 2: Evaluation between Age And Sex and correlated levels for Protein carbonyl(PC) and Fasting Blood Sugar(FBS) and Postpondrial Blood Sugar(PPBS) among cases and controls of female population.

DISCUSSION

Diabetes mellitus type 2 is the most common form of diabetes, which is a complex and multifactorial chronic metabolic disorder and its prevalence has been increasing gradually in all over the world. Since of this tendency, it is fast suitable an epidemic in all over the world with the number of people affected predictable to double in the next 10 years due to amplifying in aging population^1-3. Till date no cure has yet been found. In the assessment of Protein carbonyl, it was experiential that the mean and SD values of male and female cases of three different age (20- 30 years, 31-40 years, 41-50years) were elevated than that of controls and the p value was statistically significant(P≤0.05). This indicates that free radicals are additional in cases due to oxidative stress and the activity of antioxidant decreases. Our results do have the same opinion with Konukoğlu D et al⁸ and Kalaivanam et al⁹. Increased glycation of proteins occur due to carbonyl stress in T2DM patient¹⁰. Deprived glycemic control is one of the factors in production of amplified protein oxidation in diabetic patients. With increase in activity of ROS and superoxide anions, hyperglycemia occurs. This decreases the level of antioxidants as a result of which level of protein carbonyl increases. Due to raise in plasma protein carbonyl the diabetic complications will also develop¹¹. Oxidation of protein carbonyl leads the protein resistant to hydrolysis. Products obtain due to carbonylation of protein affects cellular physiology unfavorably.

CONCLUSION

Our outcome reveals that the role of protein carbonyl formation in type 2 diabetes patients of different age groups which might play an active part in the development of disease. Our findings support an involvement between these protein oxidation and type 2 diabetes as well as their positive association with different age groups The stronger response observed in serum samples from patients with higher patients’ age groups compared with lower age groups and it suggest that protein carbonyl contents may be useful in evaluating the progression of diabetes mellitus type 2 and in elucidating the mechanisms of disease pathogenesis.

REFERENCES

1. Pirola L, Balcerczyk A, Okabe J, El-Osta A. Epigenetic phenomena linked to diabetic complications. Nat Rev Endocrinol. 2010;6(12):665-75.
2. Olokoba AB, Obateru OA, Olokoba LB. Type 2 diabetes mellitus: a review of current trends. Oman Med J. 2012;27(4):269-73.
3. Prerana Deokar, Abhay Jagtap, Chaitanya Yerawar, Correlation of protein carbonyl and MDA in diabetes and its Complications, Indian Journal of Basic and Applied Medical Research; March 2016: Vol.-5, Issue- 2, P. 284-289 OBES SURG (2009) 19:321–326 DOI 10.1007/s11695-008- 9691-8).
4. Shacter E. Quantification and significance of protein oxidation in biological samples. Drug Metab Rev. 2000;32(3-4):307-26
5. Chakravarti B, Chakravarti DN. Oxidative modification of proteins: age-related changes. Gerontology. 2007;53(3):128-39.
6. Chetyrkin S, Mathis M, Pedchenko V, Sanchez OA, McDonald WH, Hachey DL, et al. Glucose autoxidation induces functional damage to proteins via modification of critical arginine residues. Biochemistry. 2011;50(27):6102-12 [6] Origin Dubourg L, de Precigout V, Aparicio M, Clerk M: Evaluation and significance of “carbonyl stress” in long-term uremic complications. Kidney Int.1999; 55:389–399.
7. KaplanL. A. Clinical Chemistry. The. C. V. Mosby Co.St.Louis. Baltimore. Philadelphia Toronto. 1989, 854-856.
8. Konukoglu D, Kemerli GD, Sabuncu T, Hatemi HH. Protein carbonyl content in erythrocyte membranes in type 2 diabetic patients. Horm Metab Res. 2002 Jul; 34(7): 367-70. PubMed PMID: 12189583.
9. Kalaivanam. K n, santhosh kumar., bheemasen. R, Balu mahendran.k, role of protein oxidation and lipid peroxidation Markers status in niddm patients, world journal of pharmacy and pharmaceutical sciences, volume 3, issue 6, 1510-1520.
10. Muronetz VI, Melnikova AK, Seferbekova ZN, Barinova KV, Schmalhausen EV. Glycation, Glycolysis, and Neurodegenerative Diseases: Is There Any Connection? Biochemistry (Mosc). 2017 Aug;82(8):874-886. doi: 10.1134/S0006297917080028. PMID: 28941455.
11. Babu CK, Khanna SK, Das M. Antioxidant status of erythrocytes and their response to oxidative challenge in humans with argemone oil poisoning. Toxicol Appl Pharmacol 2008;230: 304-11.