European Journal of Cardiovascular Medicine

Diabetes is a metabolic disorder characterized by elevated blood sugar that results from defects in insulin production and/or insulin action, and impaired function in the metabolism of carbohydrates, lipids and proteins which leads to macro and microvascular complications. [1] There are emerging evidences that oxidative stress makes significant contribution to the progression of diabetes and its associated complications. Type 1 diabetes is a chronic illness, usually caused by an 

autoimmune destruction of insulin-producing β-cells in the islets of the pancreas. As a consequence, the body produces none to very little insulin resulting in a relative or absolute deficiency of insulin. T2DM also referred as Non-Insulin Dependent Diabetes Mellitus (NIDDM) or adult-onset diabetes, includes patients who have insulin resistance and usually have relative insulin deficiency [2] However, these patients do not need insulin administration for their treatment and survival of the patient. Majority of the patients with T2DM are obese, and obesity itself further contributes to insulin resistance to some extent. The non-obese individuals with T2DM may have an increased percentage of body fat distributed predominantly in the abdominal region. According to American Diabetes Association (ADA), T2DM accounts for ~90-95 % of all diabetes mellitus patients worldwide. [3]

Fatty liver disease is a broad term for the buildup of triglyceride fats in the liver. This condition is common, and most people show no signs or symptoms and do not experience any adverse effects. However, fatty liver is a progressive disease and as it advances in severity it can cause irritation, inflammation and scarring known as fibrosis, when fat content of the liver increases to greater than 5-10%, its function can become significantly impaired. There are two types of fatty liver disease which have different causes but result in similar liver injuries. The first is Alcoholic Fatty Liver Disease (AFLD) and is caused by high alcohol consumption (>20 grams/20 milliliters per day). It is the earliest stage of alcoholic liver disease which may progress to the most severe stage, cirrhosis. The second type is Non-Alcoholic Fatty Liver Disease (NAFLD) and occurs for reasons other than alcohol consumption. It is often associated with symptoms of insulin resistance (a condition affecting fat metabolism) including obesity, Type 2 diabetes, hypertension and dyslipidemia.

In addition to the association with disordered glucose metabolism, most patients with FLD have other clinical characteristics that qualify them for the diagnosis of metabolic syndrome. A common operative definition of the metabolic syndrome is the presence of any three of the five abnormalities: abdominal obesity (increased waist size or waist/hip ratio), impaired glucose tolerance or overt diabetes, elevated triglycerides, low high-density lipoprotein cholesterol, and elevated blood pressure [4]. In addition to disordered glucose metabolism, represent risk factors for cardiovascular disease, and some of these abnormalities, such as hypertension, may also contribute to micro-vascular complications in diabetes.

Aims & Objective:

The assessment of the oxidative stress and inflammatory marker in Diabetic alcoholic fatty liver disease and compare with control group.

This descriptive study was conducted in Department of Biochemistry, Index Medical College & Hospital, and Research centre. In this study 200 samples selected from OPD, clinical and laboratory diagnosed with T2DM with fatty liver disease with the age group of 30 to 60 years who were attending the Medicine OPD & IPD at Index Medical College & Hospital. In this study sample divided into two groups, Group 1consist 100 patients had T2DM with fatty liver disease and group 2, 100 healthy individual acts as a control group. In this study those patients were excluded who were suffering from infectious disease, malignancy, congenital liver disease; drug induced hepatitis and pregnant patients. After completion of history and followed all precaution, 5 ml of venous blood drawn using disposable syringe and collected blood in sterile clot activator vial and proceed for laboratory test according to study in which included Blood sugar level that’s estimated by GOP-POD end point colorimetric method (Fasting, PP and HbA1c), Oxidative stress biomarkers (LPO, MDA, SOD, Catalase) and Inflammatory markers (CRP, IL6, TNF α) and reading was tabulated for statistical analysis.

Statistical Analysis: Statistical evaluation was performed using the SPSS-24 software. Data obtained from the study groups were compared using the Student-t test. The results were expressed as mean ± standard deviation and the p< 0.001 value was considered statistically significant.

In the present study, a total of 200 adult age group 30-70 years of both genders were taken for the present study in which 30% female and 70% male, 100 case of USG diagnosed T2DM with fatty liver were taken in the study and compared with 100 healthy matched controls.

Lipid biomarkers, oxidative stress, and inflammatory markers were compared between, T2DM with FLD and healthy control and found the association of oxidative stress, and inflammatory markers with diabetic parameters.

Table 1: Defining characteristics of individuals participating in the study

Values are given as mean ± SD from 100 subjects in each group.

As showing in above table, total number of sample is 200 in which 100 subjects in control group (healthy individuals) and study group (T2DM with FLD). Mean age of control group has 52.90±10.66 and study group has mean age 53.18±10.98 with male female ratio 70/30 and 68/38 respectively in control and study group. Mean weight 78.30±13.62 in control group and 75.99±15.11 in study group found statistically followed by mean height 172.65±12.72 and 169.32±12.82 respectively in control and study group.

Fatty liver disease FLD is a condition in which lipids deposit in the hepatocytes liver, in which little or no inflammation or liver cell damage. Simple fatty liver generally does not get bad enough to damage liver or its complications. The study is a comparative study of 200 patients in which 100 patients T2DM with Fatty liver disease and 100 subjects taken as a healthy control group.

In our study the mean FBS value of the T2DM patients was 186.94±52.90 well above the ADA criteria to diagnose diabetes and the PPBS which was higher than upper limit (272.18±83.46) cut off value of 140 mg/dl, whereas control group had blood glucose value as 105.07±24.17 and 148.02±16.35 for FBS and PPBS respectively suggestive of normoglycemia. These values correlate well with clinical diagnosis.

The present study was evaluated the oxidative stress profile regarding antioxidant enzymes (SOD and Catalase), and oxidative cell damage (LPO and MDA) in patients with T2DM with FLD and control. The observation shows significantly increase in LPO and MDA in study groups. In other studies, changes of oxidative stress marker are followed by the decreasing of the hepatic content of reduced glutathione (GSH) and vitamin E along with the abnormal activity of antioxidant enzymes including GSH peroxidase, superoxide dismutase, and catalase [5]. When changes of OS markers correlate with the clinical severity of the disease suggested that oxidative damages can involved to development of T2DM with FLD[5].

Lipid Peroxidation (LPO) is initiated through free radical or non-radical hydrogen from polyunsaturated fatty acids (PUFAs), resulting in decreased membrane fluidity, increased membrane permeability, and membrane-bound protein function are lost [6]. A carbon-centered lipid radical generation is a initiatial step, these radicals and oxygen reacts together to form a lipid peroxyl radical. This lipid peroxyl radical produced another electron from a nearby lipid to generate a new lipid radical and lipid hydroperoxide, termed the propagation step, result in hundreds of lipid peroxidation products (LPP) generated from a single initiation event[7]. The termination this step of lipid peroxidation involves the self-quenching of the peroxy radical or through antioxidants like vitamin E, vitamin C, SOD, catalase, and peroxidase. Hydroperoxide degradation occurs through two-electron reduction, carried out primarily by glutathione peroxidases and selenoprotein P, or through one-electron reduction, which leads to continued lipid peroxidation [6]. The current study has also shown LPO, MDA, SOD and Catalase are significantly high in T2DM with FLD patients.

Pro-inflammatory cytokines like tumour necrosis factor (TNF-α), various interleukins (IL), interferon and high sensitivity C-reactive protein also play an important role in the pathogenesis of liver disease [9]. Furthermore, excessive accumulation of lipid in hepatic cell gives a source of oxidative stress by generating an excess of reactive oxygen species (ROS), which leads to lipid peroxidation of hepatic cell, cytokine production and hepatic inflammation [10]. 

Storage of triglycerides in hepatocytes causes oxidative stress, lipid peroxidation, and pro-inflammatory cytokines [e.g. tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6)]. Some animal studies have shown elevated fatty acids in the liver, and it may cause higher levels of TNF-α. When hepatocytes get damaged, liver-specific macrophages (Kupffer cells) are activated and produce more TNF-α and IL-6 into the blood stream, which leads to the production of the acute phase protein high-sensitivity CRP.[8] In our study, we found elevated CRP levels were significant associated with T2DM with FLD compared with control group, and our findings are similar to Nigam et al [8]. Few studies have found elevated CRP levels are a useful diagnostic marker for T2DM with FLD. The disequilibrium between pro-inflammatory and anti-inflammatory cytokines aggravates the complications related to T2DM [10]. In the present study level, inflammatory markers (CRP, IL6, and TNF-α) were significantly higher in the T2DM with FLD comparison to the control group (P<0.001). Malik A et al [11] reported the levels of pro-inflammatory cytokines such as TNF-α, IL-6 and anti-inflammatory cytokine IL-10 were significantly higher in T2DM individuals compared to controls in their study. Similar results have been reported by Darko SN et a[12], Abbatecola AM et al[13], Hu FB et al[14], Al-Shukaili A et al[15], and Rodrigues KF et al[16] in their respective study. Lemieux I et al[17] study reported on the correlation between fasting insulin levels and C-reactive protein levels in the plasma of diabetics showing that insulin resistance and inflammatory processes are linked. It has been observed that adipose tissue can synthesize main pro-inflammatory cytokines, tumor necrosis factor, and interleukin-1 and -6 and that inflammatory biomarkers are linked with body fat mass, suggesting that activated innate immunity and inflammation are important biological factors in the pathogenesis of diabetes mellitus and the complications of type 2 diabetes mellitus[18]. Furthermore, soluble and immobilized CRPs have been demonstrated to mediate the uptake of native low-density lipoprotein (LDL) into macrophages [19]. CRP may also function as a substrate for membrane-associated neutrophil serine protease that cannot be up-regulated. Studies on inflammatory markers have noted that participants with a combined elevation of both IL-6 and IL-1β had about a three-fold increase in the risk of developing diabetes, whereas low levels of IL-1β alone demonstrated no substantial increase in risk [20]. Significant results were obtained with IL-6/IL-10 and IL-6/IL1β. Studies reviewing IL-6 and tumor necrosis factor–α (TNF-α) have established roles in the regulation of APRs with other investigations providing a possible link between these two biomarkers and cardiovascular events [21]. Additionally, an association with TNFα receptor 2, IL-6, and CRP has been demonstrated, with elevated levels of CRP noted as a strong independent predictor of T2DM. So our findings support the above pathology and noted the significantly higher inflammatory markers in diabetes patients with respect to normal healthy subjects. The current study has also shown higher CRP levels are higher in T2DM patients compare with control group.

In conclusion, it could be concluded that the levels of inflammatory cytokines and Oxidative stress was higher in T2DM compare with healthy control group. These findings demonstrate a significant relationship between oxidative stress and T2DM with Fatty liver disease but no significant difference in inflammatory markers has been observed. Since, increased inflammatory marker in fatty liver disease is indicative of liver injury, due importance should be given to assessment of inflammatory markers in the management of patients with fatty liver disease Antioxidants of interest in clinical and pre-clinical assessment of T2DM with FLD include GSH, SOD, CAT, and GPx, which appear to be most reliably detected in liver samples.

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