Decreased peroxidase activity of glycated hemoglobin inversely correlated with levels of glycated hemoglobin in type 2 diabetes patients

Background and objective: Diabetes mellitus is a metabolic disorder with diverse etiological factors. Imbalances between oxidative stress and antioxidant defenses play critical roles in the pathogenesis and progression of diabetes. Peroxidase, an antioxidant enzyme similar to superoxide dismutase and catalase, is involved in the etiology and progression of diabetes. This study aimed to elucidate the relationship between peroxidase activity of hemoglobin and glycated hemoglobin with fasting blood glucose levels in patients with type 2 diabetes mellitus. Methods: Sixty-four patients with type 2 diabetes and an equal number of healthy controls were recruited for the study. Peroxidase activity of hemoglobin and glycated further modifications and standardization.

Background and objective: Diabetes mellitus is a metabolic disorder with diverse etiological factors.Imbalances between oxidative stress and antioxidant defenses play critical roles in the pathogenesis and progression of diabetes.Peroxidase, an antioxidant enzyme similar to superoxide dismutase and catalase, is involved in the etiology and progression of diabetes.This study aimed to elucidate the relationship between peroxidase activity of hemoglobin and glycated hemoglobin with fasting blood glucose levels in patients with type 2 diabetes mellitus.
Methods: Sixty-four patients with type 2 diabetes and an equal number of healthy controls were recruited for the study.Peroxidase activity of hemoglobin and glycated further modifications and standardization.patients with type 2 diabetes compared to healthy controls, as was peroxidase activity of glycated hemoglobin (p<0.001).In both patients and controls, peroxidase activity of glycated hemoglobin was significantly higher than normal hemoglobin.In patients with type 2 diabetes, glycated hemoglobin showed a significant negative correlation (r=-0.308,p=0.013) with its peroxidase activity, indicating increased activity is associated with poorer glycemic control.Furthermore, peroxidase activity of normal hemoglobin positively correlated with peroxidase activity of glycated hemoglobin (mainly HbA1c) (r=0.305,p=0.014).
Conclusions: Peroxidase activity of glycated hemoglobin increases with glycated hemoglobin levels, indicating deteriorating glycemic control in patients with type 2 diabetes.

INTRODUCTION
Diabetes mellitus is a metabolic disorder of multiple etiologies leading to impaired normal glucose metabolism, resulting in the common feature of hyperglycemia.While type 1 diabetes results from an absolute deficiency of insulin, type 2 diabetes is characterized by a marked decrease in insulin function, primarily manifested as insulin resistance along with defects in glucose utilization.In type 2 diabetes, insulin resistance increases either due to receptor or post-receptor cascade defects, as target tissues gradually become insen-sitive to insulin, resulting in chronic high plasma glucose and insulin levels. 1 In both types of diabetes, oxidative stress is generated during disease development, and as a compensatory mechanism, antioxidant enzymes such as catalase, peroxidase, superoxide dismutase, and glutathione peroxidase are utilized to neutralize the oxidizing effects of reactive oxygen species. 2,3Even patients suffering from diabetes for a short period, and yet to develop complications, exhibit reduced antioxidant plasma capacity and uric acid levels, indicating that oxidative stress occurs early in diabetes. 4moglobin is a hemoprotein that can act as an enzyme peroxidase with high oxidizing potential when present with oxidizing equivalents like hydrogen peroxide (H2O2). 5In red blood cells, this dangerous oxidizing tendency is regulated by the reducing environment, primarily reduced glutathione and the deficiency of oxidizing equivalents.Methemoglobin is the ferric form of hemoglobin and is short-lived, being effectively converted back to ferro-Hb (deoxy-Hb) by methemoglobin reductase. 2,68][9] For Hb appearing outside RBCs in hemolysate, the intracellular protective phenomenon is lost, and Hb exhibits higher peroxidase activity.This is significantly increased in inflammation (e.g., sepsis, diabetes) by the generation of superoxide radicals.The superoxide radicals may be transformed into hydrogen peroxide (H2O2), which fuels Hb peroxidase activity. 10roxidases in the body can be either: (1) true peroxidases, which generate free radicals in the peroxidase cycle like myeloperoxidase, eosinophil peroxidase, and lactoperoxidase; or (2) pseudo-peroxidases, enzymes that behave like peroxidases under external conditions, such as hemoglobin, myoglobin, and cytochrome C. 11 Enhanced peroxidase activity has been observed in oxidative stress and may initiate various human diseases like diabetes mel-litus. 11Therefore, regulating peroxidase activity is important.Not only do structurally dif-ferent peroxidases differ in properties and location, but there are also specific mechanisms to control peroxidase activity for different hemoproteins. 11he study aims to determine if there is any relationship between the peroxidase activity of hemoglobin and glycated hemoglobin in patients with type 2 diabetes mellitus.

Participants
Sixty-four patients with type 2 diabetes (30 males and 34 females) and a similar number of healthy controls (35 males and 29 females) from the same age group were selected for the study.Patients aged between 25 to 75 years with type 2 diabetes mellitus diagnosed and confirmed by clinical and biochemical tests were included.However, the following patients were excluded: those with other endocrine disorders, type 1 diabetes, pregnancy, polycystic ovarian disease, renal failure, malignant diseases, or hemolytic anemias like thalassemia.The study was undertaken in 2014-2015 after approval by the Institutional Ethics Committee of N.R.S. Medical College, Kolkata, India.

Peroxidase activity
There are various methods to assay the peroxidase enzyme, with the pyrogallol method being especially notable.In the past, peroxidase activity of a cell was expressed in units based on the oxidation rate of pyrogallol, a method introduced by Willstatter and Stoll in 1917.This is not in accordance with later studies (Maehly and Chance 1954).Willstatter and Heiss (1923) studied the method of oxidizing pyrogallol to purpurogallin.According to their assay: Thus, the formation of 1 mg purpurogallin should yield 102µl of CO2 (J.Ettori, 1948).Sumner and Gjessing (1943) devised a new method conducting the reaction in 20 ml phosphate buffer.They found the resulting peroxidase activity values were somewhat higher than Willstatter's method.Later, the effects of dilution and buffer concentrations on enzyme activity were investigated for different preparations, comparing Willstatter's method to Sumner and Gjessing's.Colorimetric assay methods have also been compared to the manomet-ric assay based on CO2 formation when pyrogallol was oxidized with a catalyst.Various hydrogen donor molecules have been used to assay peroxidase, including potentially carcinogenic compounds like o-dianisidine.Some researchers report improved assays using 4-aminoantipyrine as the hydrogen donor (Trinder 1966).Reaction rate is determined by measuring increased absorbance at 505 nm from hydrogen peroxide decomposition. 12ased on Goth's serum catalase assay method, a modified method to estimate hemoglobin peroxidase activity was developed. 13In the original Goth method, serum was used to measure serum catalase activity.In this method, diluted whole blood containing only red blood cells was used to assess hemoglobin peroxidase activity.
The hemolysate prepared from patients' whole blood contains the enzyme peroxidase and supernatant of the resin tube used to estimate glycated hemoglobin.Added hydrogen peroxide acts as the substrate.The enzymatic reaction was stopped with 1.0 ml of 32.4 mmol/l ammonium molybdate ((NH4)6Mo7O24, 4H2O heptamolybdate tetrahydrate), and the yellow molybdate-hydrogen peroxide complex was measured at 405 nm against B3.

Preparation of buffers and reagents (check carefull)
Buffer for peroxidase assay: 60mmol/L Sodium/Potassium Phosphate Buffer, pH 7.4 32.4mmol/L ammonium molybdate solution 60mmol/L hydrogen peroxide in 60mmol/L Na-K-Phosphate Buffer, pH 7.4 Normal saline

Procedure
Hemolysate was prepared by mixing 20 microliters of ethylene diamine tetra acetic acid (EDTA) blood with 180 microliters of normal saline. 1 milliliter of substrate (hydrogen peroxide) was added to 0.02 ml of hemolysate containing hemoglobin peroxidase, and the reaction was stopped by adding 1 ml of ammonium molybdate.Absorbance (B1) was measured spectrophotometrically at 405nm.Two blanks were calculated -substrate blank (B2) and true blank (B3).The substrate blank (B2) was determined spectrophotometrically by taking the absorbance of 1 ml of substrate and 0.02 ml of buffer with 1 ml of ammonium molybdate.The true blank (B3) was calculated from the absorbance of 1 ml buffer, 1 ml ammonium molybdate, and 0.02 ml of buffer at the same wavelength. 13molysate peroxidase For glycated hemoglobin, the resin tube supernatant was used instead of hemolysate, and B1 was calculated with dilution factors adjusted in both cases.

Standardization of peroxidase activity assay
Different volumes of pooled hemolysate were used to construct a curve relating absorbance (B1) at 405nm (Figure 1 ).

Other parameter estimation
Fasting blood glucose was estimated by the glucose oxidase peroxidase (GOD-POD) method.Glycated hemoglobin was measured by ion-exchange chromatography with standardized kits.Total hemoglobin was estimated by the cyanmethemoglobin method using Drabkin's reagent.

Statistical Analysis
Data was entered into MS Excel and cleansed.It was then transferred to IBM SPSS version 20 and analyzed.Data normality was checked and descriptive statistics like mean and standard deviation were generated.Analytical statistics like the student's t-test were used for comparisons.A p-value <0.05 was considered significant.

RESULTS
In this case control study, it was found that peroxidase activity of hemoglobin was significantly decreased (p<0.0001) in patients with type 2 diabetes compared to healthy controls, as was peroxidase activity of glycated hemoglobin in these patients (p<0.001,Table 1).Additionally, peroxidase activity of glycated hemoglobin was significantly higher than peroxidase activity of normal hemoglobin in both patients and controls (Figure 2).Glycated hemoglobin showed a significant negative correlation (r= -0.308, p= 0.013) with its peroxidase activity in patients with type 2 diabetes (Figure 3 ), indicating that increased activity is associated with poorer glycemic control.Furthermore, peroxidase activity of normal hemoglobin positively correlated with peroxidase activity of glycated hemoglobin (mainly HbA1c) (r= 0.305, p= 0.014).

DISCUSSION
][16] In our study, peroxidase activity of both hemoglobin and glycated hemoglobin was significantly reduced in patients with type 2 diabetes compared to healthy controls.In an experimental study with diabetic rats, Wu et al. (1999) reported that in diabetic rat kidney tissue, superoxide dismutase, peroxidase and catalase activities significantly declined at weeks 8 and 16. 17 In 2014, Kresyun et al. reported that after modeling streptozotocin-induced diabetes in Wistar rats, antioxidant enzyme activities of superoxide dismutase and catalase decreased. 18In previous studies, it has been reported that peroxidase activity in plasma is decreased in patients with chronic diseases like type 2 diabetes, where increased oxidative stress plays an important role from the early stages.In one similar study, glutathione peroxidase levels decreased in patients with type 2 diabetes with or without complications like coronary artery atherosclerosis, which supports our findings. 19This decrease in the peroxidase antioxidant enzyme has been attributed to poor glycemic control (decreased glycated hemoglobin).The peroxidase activity of hemoglobin depends on the pH of the solution. 20owever, at the same pH, the peroxidase activity of glycated hemoglobin is higher than that of normal hemoglobin for individual subjects in our study.
There are certain limitations in this study.As a pilot study, this case control study had a small sample size.In the future, the researchers plan to recruit more patients with type 2 diabetes as cases, and more healthy volunteers as controls.In this study, there is no scale exhibiting the increase in peroxidase activity of glycated hemoglobin corresponding to the percentage increase in glycated hemoglobin.This again requires recruiting a large number of study subjects.

CONCLUSIONS
The peroxidase activity of glycated hemoglobin increases with levels of glycated hemoglobin, indicating deteriorating glycemic control in patients.If spectrophotometric assays of glycated hemoglobin peroxidase activity using inexpensive reagents could be developed, this may allow assessment of patients' glycemic control.

ABBREVIATIONS
A-Z list is needed.

Figure 2
Figure 2 Comparison of mean peroxidase activity of hemoglobin and glycated hemoglobin in patients with type 2 diabetes and healthy controls.

Figure 3
Figure 3 Correlation scatterplot of HbA1c and peroxidase activity of glycated hemoglobin in patients with type 2 diabetes.