Evaluation of Plasma Total Antioxidant Capacity Levels and Osteocalcin in Prediabetes and Healthy Subjects

Introduction

Diabetes is a chronic metabolic disorder with a rapidly increasing prevalence highlighting the importance of continued research and the need for novel methods to both prevent and treat this pandemic.¹ Diabetes mellitus is a metabolic disease due to absolute or relative insulin deficiency. According to WHO report the prevalence of diabetes in adults worldwide has risen and the number will rise from 135 million in 1995 to 300 million by the year 2025. WHO estimates that India had 32 million diabetic subjects in the year 2000 and this number would increase to 80 million by the year 2030².

In diabetic patients, long-term damage, dysfunction, and failure of different organs,  especially the eyes (diabetic retinopathy), kidneys (diabetic nephropathy),  nerves (diabetic neuropathy), heart (myocardial infarction),  and blood vessels (atherosclerosis) are related to uncontrolled hyperglycemia. Generation of reactive oxygen species (ROS) increases in both types of diabetes and that the onset of diabetes is closely associated with oxidative stress. Convincing evidence has established the role of free radicals and oxidative stress in the pathogenesis and development of complications from DM, Levels of ROS are under tight control by the protective actions of antioxidant enzymes and nonenzymatic antioxidants in normal and healthy cells¹.

Diabetic patients from studies showed a decrease in antioxidant levels, low activity of antioxidant enzymes, and an increase in both reactive oxygen species (ROS) production and oxidative stress markers. Hyperglycemia can induce the overproduction of superoxide anions that affect several pathways that leads to diabetic complications².

Prediabetes can also be termed as intermediate or borderline hyperglycemia. The American Diabetes Association previously equated prediabetes with the WHO’s intermediate hyperglycemia. The Expert Committee on Diagnosis and Classification of Diabetes Mellitus identified an intermediate group of people with glucose levels that do not meet criteria for diabetes, yet having higher than normal. These individuals were defined as having impaired fasting glucose (IFG) [FPG levels 100 mg/dl (5.6 mmol/l) to 125 mg/dl (6.9 mmol/l)] or impaired glucose tolerance (IGT) [2 h values in the OGTT of 140 mg/dl (7.8 mmol/l) to 199 mg/dl (11.0 mmol/l)]. Individuals with IFG and/or IGT were referred as having prediabetes, showing the relatively high risk for the future development of diabetes. Globally, its prevalence has been found to be increasing, and by 2030, it can rise up to >470 million people suffering from it³.

Prediabetes is associated with dyslipidemia, endothelial dysfunction, obesity, dysglycemia, procoagulant state, insulin resistance, hypertension and inflammation placing individuals            with prediabetes at an increased risk of cardiovascular events. Prediabetes is associated with oxidative stress predisposing the patients to an increased risk of CVD⁴.

Osteocalcin (OC or bone Gla protein, BGP) and matrix Gla protein (MGP) were the first members of growing family of vitamin K-dependent carboxylated proteins synthesized outside the liver and found not to be involved in coagulation. Osteocalcin is released into the circulation when new bone is formed and is considered a marker of bone turnover. It was later identified as being secreted under normal, nonpathological conditions⁵.

A recent unexpected development of bone biology indicates that bone is an endocrine organ contributing to the regulation of a number of physiological processes. One of the functions regulated by bone through osteocalcin is glucose homeostasis.

Osteocalcin is an osteoblast specific protein that is secreted in large amounts in the bone extracellular matrix. Beyond the effects on bone remodelling, OCN regulates β-cell proliferation, insulin gene expression, and secretion in both mice and humans suggesting a potential role in glucose metabolism and in the pathogenesis of glucose alterations in T2DM physiopathology.¹⁷

Osteocalcin acts as a hormone in the body, causing beta cells in the pancreas to release more insulin, and at the same time directing fat cells to release the hormone, which increases sensitivity to insulin⁶.

Oxidative stress may contribute to the pathogenesis of diabetes mellitus through impairment of insulin action, injury to pancreatic β-cells, increased lipid peroxidation, and vascular endothelial damage⁷.

Oxidative stress has an inhibitory role on osteoblast. Generated superoxide from osteoclasts directly contributes to bone degradation⁸.

Albeit, there are several studies on oxidative stress in diabetes and osteocalcin in diabetes but there are limited studies pertaining to oxidative stress in prediabetes and also osteocalcin in prediabetes. Hence, this study is the first of its kind to find out the correlation between the levels of total antioxidant capacity and osteocalcin in prediabetes and thus the effect of free radicals on bone structure.

Methodology

Under aseptic precautions, 5ml of fasting venous blood sample was collected in fluoride vacutainer tubes for the estimation of plasma glucose, osteocalcin and total antioxidant capacity.

The subjects included in this study after taking a detailed clinical and drug history, physical examination, height and weight measured with the patient standing in light clothes and without shoes. Blood pressure was measured before the experimental procedure.  Body Mass Index (BMI) was calculated as body weight divided by height square (kilogramsper meter square).⁹

Estimation of plasma total antioxidant capacity was done by usingFerric reducing ability of plasma (FRAP) assay¹⁰. Based on the principle, at low pH when ferric tripyridyltriazine (Fe3+-TPTZ) complex is reduced to the ferrous form Fe2+, an intense blue color develops with absorption of maximum at 593nm.

Estimation of plasma osteocalcin levels by sandwich ELISA Technique, Kit procured fromWuhan Fine Biotech co.,Ltd, China¹¹.

Based on the principle of ELISA technique which measures human osteocalcin (1-49) and (1-43) in plasma sample.The assay utilizes the twosite “sandwich” technique with two selected antibodies that bind to different epitopes of human osteocalcin.

Results

The control and prediabetic subjects were males within in the age group of  30-70 years, BMI<25 (Table1). The mean value of FBS in healthy controls was 90.75±7.66 mg/dl and in prediabetics 112.07 ±7.97 mg/dl. FBS was high in prediabetes compared to healthy controls and the difference was statistically significant (P < 0.001). (Table 2 and 5).

The mean value of TAC in healthy controls was 268.50 ± 88.22 μmol/L and in prediabetics 421.60± 81.28 μmol/L. There was a significant decrease in TAC in prediabetics compared to healthy subjects. The difference was statistically ( P<0.001) depicted in table 3 and 5.

The mean value of osteocalcin level in controls was 5.91 ± 3.37 ng/ml and that of  prediabetics was  5.26 ± 2.59 ng/ml.The mean value of plasma osteocalcin level showed only minimal difference between controls and cases being slightly decreased in cases (P = 0.337) (Table 4).

Out of 40 controls less than 15 had 5ng/ml,  20 had 5-9 ng/ml and 5 had greater than 9ng/ml. Out of 40 cases, 17 had less than 5ng/ml , 22  had  5-9ng/ ml and only 1 had greater than 9ng/ml. (Table 4).

There was no significant correlation found between TAC and osteocalcin in healthy controls (r = -0.082, P=0.617) as well as in Prediabetics (r =0.153, P=0.617)  (Table 6).

Discussion

Diabetes mellitus is a global health problem affecting more than 6% of the world population and its prevalence is estimated to increase to about 552 million in 2030¹². These numbersdo not include the amount ofpeople with prediabetes, of which 90% is unaware of their situation¹³.

The prevalence of prediabetes is increasing worldwide to probably more than 400 million cases in 2030 and, if untreated, will progress to diabetes and the associated complications¹⁴.

Prediabetes is the asymptomatic stage of diabetes mellitus andmight be a useful target for early intervention therapies to prevent the development of this wide-spread disease and its associated complications like cardiovascular disease.

We focused on changes in total antioxidant capacity and osteocalcin levels, which can already be seen in the prediabetic state, to provide understanding about disease progression and possible tools for prevention and screening programs.

Based on the definition of prediabetes in this studyas having impaired fasting glucose (IFG) [FPG levels 100 mg/dl (5.6 mmol/l) to 125 mg/ dl (6.9 mmol/l)] or impaired glucose tolerance (IGT) [2 h values in the OGTT of 140 mg/dl (7.8 mmol/l) to 199 mg/dl (11.0mmol/l)] we expected to find adverse effects of mild hyperglycaemia manifested in impaired total antioxidant capacity and osteocalcin levels in the prediabetes group.

The plasma levels of TAC reflects both exogenous and endogenous antioxidants in plasma.In the present study we observed that the total antioxidant capacity was reduced (P<0.001)in prediabetics when compared to healthy controls15, 16.  This is in agreement with the earlier findings which showed that, presence of increased oxidative stress already at theprediabetes state, which is represented by a slight increase in GSSG levels together with a significant decrease in GSH/GSSG ratio in the prediabetes group.

This is consistent with earlier findings, which showed that changes in the antioxidant status, especially the glutathione system of the erythrocytes, characterize the initial phase of oxidative stress in diabetes mellitus progression and commence prior to the establishment of this disease¹⁶.

Osteocalcin is γ-carboxylated on the glutamic acids (GLU) 13, 17, and 20 of protein in mouse, and on GLU 17, 21, and 24 in humans.³⁰ The carboxylation of osteocalcin and other Gla proteins occurs in the lumen of the endoplasmic reticulum and involves 2 enzymes γ-glutamyl carboxylase and vitamin K epoxide reductase (VKORC1), which together constitute the vitamin K-dependent cycle.

This posttranslational modification increases the affinity of osteocalcin for Ca2+ and therefore for hydroxyapatite, the mineral component of bone extracellular matrix (ECM). The vast majority of osteocalcin secreted by osteoblasts gets trapped in bone ECM. In the serum, both the carboxylated and the undercarboxylated forms of osteocalcin are detected.

In this study, the levels of osteocalcin in prediabetics were slightly reduced when compared to healthy controls but the difference was not statistically significant, which is consistentwith the study conducted by Deidre Winnier  et al, in which the levels of osteocalin levels in IFG were similar to controls.¹⁷ However, there are studies which indicate thattotal osteocalcin levels decrease in Impaired glucose regulation as compared to normal glucose tolerance in particular in subjects with both impaired fasting glucose and impaired glucose tolerance.

Several studies have also reported low osteocalcin levels in patients with diabetes and metabolic syndrome and the correlations between and parameters of glucose metabolism have largely been consistent with animal models¹⁸.

Also osteocalcin levels were lower in diabetic compared to non-diabetic patients with metabolic syndrome19.This can be explained as Insulin, bone resorption, and osteocalcin activity are regulated by a feedforward loop, in which insulin signalling in osteoblasts decreases the expression of the gene that encodes the osteoprotegerin (Opg), thus increasing osteoclast mediated bone resorption which occurs at a pH of ^4.5. This acidic pH favours the decarboxylation of osteocalcin, releasing undercarboxylated osteoclacin into the systemic circulation. Thereafter, under carboxylated osteocalcin plays a regulatory role in glucose metabolism by promoting pancreatic insulin secretion and peripheral insulin sensitivity. Furthermore, leptin has been shown to modulate insulin sensitivity by reducing osteocalcin bioactivity in osteoblasts²⁰.

The oxidative stress blocks the activation of osteoblasts²¹. Osteocalcin is an osteoblastspecificprotein that is secreted in large amounts in the bone extracellular matrix¹⁷. Hence, this study was designed to find out the correlation between TAC and osteocalcin but there was no statistically significant correlation found between TAC and osteocalcin in healthy controls as well as in Prediabetes.

This might be due to the limitations in the study such as the patients were chosen according to their fasting plasma glucose levels where as their HbA1c results were not taken into consideration. The duration of prediabetic phase reflect the variation of biochemical parameters, hence follow up of study subjects need to be carried out.Inclusion of diabetic group into the study may indicate clearly the relationship between Diabetes mellitus and change in the biochemical parameters.

Conclusion

The minimal elevation of blood glucose levels in the prediabetic state may have a detectable influence on endothelial function. Increased oxidative stress as indicated by FRAP assay in this study is likely to be the link between the moderate hyperglycemia in prediabetes and pathological changes in endothelial function, which in the long-term may promote atherogenesis and result in the development of cardiovascular disease. In prediabetes, hormones like osteocalcin may affect glucose metabolism before overt T2DM occurs with tissue-specific mechanisms. Oxidative stress has an inhibitory effect on osteoblasts. Mody and colleagues have demonstrated that oxidative stressinhibits differentiation of M210B4, a marrow stromal cell line and MC3T3-E1, a preosteoblasticcell line, by measuring alkaline phosphatise (ALP) as an early differentiation marker.Furthermore, oxidative stress inhibits mineralization in these cell lines.Hence, this study indicates that early detection of prediabetes is essential to prevent  thedevelopment of DM and the its complications and a large population study is required to substantiate the results obtained in the present study.