Metabolic syndrome (syndrome X, insulin resistance syndrome) consists of a group of metabolic abnormalities that confer increased risk of atherosclerotic cardiovascular disease (ASCVD) and diabetes mellitus. Evolution of the criteria for the metabolic syndrome since the original definition by the World Health Organization (WHO) in 1998 reflects growing clinical evidence and analysis by a variety of consensus conferences and professional organizations that led to the increasing understanding of this syndrome. The major features of the metabolic syndrome include central obesity, hypertriglyceridemia, lower levels of high-density lipoprotein cholesterol (HDL-C), hyperglycemia, and hypertension and insulin resistance.1

The concept of interrelated metabolic disturbances was first introduced 80 years ago as a constellation of hypertension, hyperglycemia and gout (Eckel et al., 2005)2. Ferrannini et al. stated in 1987 that essential hypertension correlates with impaired insulin-mediated glucose uptake.3 One year later, Gerald Reaven proposed that insulin resistance and its compensatory hyperinsulinemia predisposed individuals to hypertension, dyslipidemia (high plasma levels of triglycerides and low levels of high-density lipoprotein particles) and diabetes thus being the underlying cause of CVD.1 Since then the concept has progressively emerged into clinical practice and several definitions have been proposed to describe MetS as discussed in more detail later.

In 1999, the World Health Organization published a definition of the MetS (MetS/WHO) for research purposes with the aim to improve the recognition and comparability of the MetS. MetS/WHO was the first widely accepted definition of the MetS with specific thresholds including insulin resistance, obesity, dyslipidemia, hypertension and microalbuminuria.4

Gamma-glutamyl transferase (GGT) has been regarded as a biomarker of hepatobiliary disease and alcohol abuse.5 GGT is secreted by extrahepatic tissues, including kidney, epididymis, fibroblasts, lymphocytes, and lung.6 GGT has a vital role in the extracellular catabolism of glutathione, the principal thiol antioxidant in humans. GGT enhances the availability of cysteine to promote intracellular glutathione (GSH) resynthesis, thereby counteracting oxidant stress.7  It is expressed in the atheromatous core of coronary plaques where it colocalizes with oxidized low density lipoprotein (LDL) and foam cells.8 GGT can be a proinflammatory marker as it mediates the interconversion of the glutathione-containing inflammatory mediator leukotriene C4 into leukotriene D4.9 In a review article by Malnick Set al. GGT was concluded as a predictive marker for MetS, CVD, and heart failure.

Ferritin plays a crucial role in the regulation of iron homeostasis and is an accepted biomarker to evaluate body iron stores. Elevated serum ferritin levels have been demonstrated to predict type 2 diabetes mellitus in several studies independently. In cross-sectional studies, high ferritin levels have been associated with hypertension, Dyslipidemia, elevated fasting insulin and blood glucose levels, and central adiposity. Lianlong Yet al. in their study found that serum ferritin was positively correlated with different indices of MetS except for high-density lipoprotein cholesterol (HDL-C).

‘The decreased ability of insulin to act effectively on target tissues is called as insulin resistance’. The exact molecular mechanism of insulin resistance is not completely understood. It is probably thought to be due to defects in insulin signal transduction pathways leading to reduced expression of GLUT4 receptors. This leads to defective glucose uptake by target tissues.10

Central obesity is associated with insulin resistance. Increased adipocyte mass releases free fatty acids and a number of adipokines. Increased FFA and adipokines impair glucose utilization in skeletal muscles, increase hepatic glucose production and affect β-cell function.

Aims and objectives of the study

Aims

 The aim of the present study is to investigate the correlation of ‘association of serum ferritin and GGT levels with metabolic syndrome and insulin resistance’

Objectives of the study

1. To investigate if elevated serum ferritin levels are associated with the development of the metabolic syndrome.
2. To investigate the relationship between metabolic syndrome and high blood levels of GGT.
3. To study the correlation of insulin resistance with serum ferritin levels with serum GGT levels in metabolic syndrome.

Study Design: Comparative Descriptive Cross - Sectional Study. Patients attending outpatient clinic (OPD) of Department of General Medicine at Hassan Institute of Medical sciences Hassan, between March 2023 and September 2024. Age and sex matched healthy volunteers or patient attenders constituted the control group. 18 months (March-2023 to September-2024). There were a total of 100 participants in the study: 50 individuals with Metabolic Syndrome and 50 healthy controls of the comparable age and sex. Sample size was calculated using the formula Where, Zα : alpha level of significance at 95% CI = 1.96 Z1-β : For Power of 90% = 1.28 σ : Pooled standard deviation SDPooled = √([SD12 + SD22]/2) = 63.03 d =Effect size = (Mean of Group 1 – Mean of Group 2) ÷ Pooled SD = 1.0026 Thus, each group must include at least 22 samples. Hence the sample size of 50 Metabolic Syndrome patients for MS group and 50 age and gender matched controls was justified. Inclusion criteria constituted of the following: a. Age ≥ 18 years b. Participating patients who provide their informed consent. c. Patients in Group A have three or more of the criteria of Metabolic Syndrome as outlined by the Adult Treatment Panel III of the National Cholesterol Education Program (ATP III). d. A waist measurement of 102 centimeters or more for men or 88 centimeters or more for women is considered to be obese. e. Hypertriglyceridemia is indicated by serum triglycerides more than 150 mg/dL. f. Men must have an HDL cholesterol level of 40 mg/dl or lower, while women must have a level of 50 mg/dl or fewer. g. Patients who have arterial hypertension (BP 130/85 mm Hg) or who take blood pressure medications h. Diagnosis of diabetes when fasting plasma glucose levels are over 100 mg/dl. Exclusion criteria constituted of the following • Patients with existing conditions such as heart disease, severe renal disease, cirrhosis, pregnancy, or acute or chronic hepatitis. • Thyroid problems and other endocrinological irregularities. • Serum ferritin >500 ng/ml; hemochromatosis predisposition or clinical evidence. • Patients using oral contraceptives or hypolipidemic medications. • Abuse of alcohol or drugs suspected. Methodology The study was conducted after obtaining Institutional Ethics Clearance. All the participants who met the eligibility criteria were included in the study after obtaining informed consent. Information regarding socio-demographic profile, clinical history was noted down in a preformed semi-structured study proforma. Detailed clinical examination was conducted. Measurements of participant’s height, weight, waist circumference (WC), Blood Pressure were be done. WC was measured at the midpoint between the lower margin of the least palpable rib and the top of the iliac crest, using a stretch resistant tape (Intra individual Measurement Error ±1.31 cm). Blood pressure was measured from the left arm in the sitting position placing BP apparatus (Omron HEM-8712) at the level of the heart, after 10 minutes of resting. Group A had 50 patients of Metabolic syndrome visiting outpatient department Gen. Medicine, of the hospital. Metabolic syndrome is defined in accordance with National Cholesterol Education Program Adult Treatment Panel III (ATP III) criteria if at least three of the following components were observed: 1. Central obesity: Waist circumference >102cm for men or >88cm for women. 2. Hypertriglyceridemia: Serum triglycerides >150 mg/dl. 3. Low HDL cholesterol <40 mg/dl for men or<50 mg/dl for women. 4. Arterial hypertension >130/85mmHg or patients on antihypertensive treatment. 5. Hyperglycemia: Fasting plasma glucose ≥110 mg/dl. Group B consisted of 50 normal age and sex matched group of people without Metabolic syndrome. These individuals was selected from those attending the hospital a routine check‑up or attendants of patients who were willing to be part of this research. After explaining the procedure to the study participants, 5 ml of fasting venous blood was obtained by veni-puncture under aseptic conditions and divided into 2 parts, first part of blood was taken in a sterile EDTA tube and used for measuring HbA1c. Second part in a plain tube, centrifuged and separated serum was used for measuring serum insulin, serum ferritin and other parameters. About 2 ml of blood was collected in plain tube after 2 hour of food which was centrifuged and serum was separated. Then, serum was used for the estimation of biochemical parameters using Johnson and Johnson Vitros 250 dry chemistry auto-analyzer. The blood Glucose estimation was done by Glucose Oxidase Peroxidase method (GOD-POD), Urea by Urease method, Creatinine by enzymatic kinetic method, Total Cholesterol by cholesterol oxidase method, TG by Enzymatic colorimetric test- GPO. HDL-C by Direct Enzymatic colorimetric, LDL-C calculated by Friedwald’s formula. GGT was estimated by G-glutamyl-p-nitroanilide method [16]. Estimation of serum ferritin and insulin was done by using automated Chemiluminescence Immunoassay system (CLIA). ESTIMATION OF INSULIN RESISTANCE (IR) USING HOMA MODEL (Homeostatic Model Assessment) IR = (Fasting plasma glucose in mg/dl x Fasting serum insulin in μIU/ml) ÷ 405 The patient was considered to have insulin resistance if HOMA1-IR value was more than 2.7. : C) PARAMETERS ESTIMATED IN THE STUDY PARTICIPANTS: ANTHROPOMETRIC PARAMETERS a) BMI (Kg/m2) b) Height, Weight, Waist Circumferences c) Blood Pressure (mm of Hg) GLYCEMIC PARAMETERS a. Fasting Blood Glucose b. Post Prandial Glucose c. HbA1c (Glycated Hemoglobin) d. Serum insulin (μIU/ml) e. Insulin resistance by HOMA-IR f. = [Fasting serum glucose (mg/dl) x Fasting serum insulin (μIU/ml)] /405. IRON PARAMETERS Serum ferritin (ng/ml) Other Biochemical parameters a. Serum GGT b. Fasting Lipid profile, c. Serum Creatinine and Uric acid level d. HsCRP level STATISTICAL ANALYSIS OF DATA Data was compiled in MS excel spread-sheet and analysed using SPSS software version 20. The results of the categorical variable were presented using proportion and analysed using chi-square test and results of continuous variables were presented as mean and SD and analysed using T-test and one way ANOVA and other appropriate statistical tests. Associations with p value of < 0.05 were considered as statistically significant.

Table 1: Distribution Of Biochemical Parameters Among Cases And Control Groups

• The mean blood urea levels of Metabolic syndrome group patients (23.9±8.3mg/dl) was not significantly associated with that of control group (20±14.5mg/dl).
• The mean fasting blood sugar (FBS) levels of Metabolic syndrome group patients (180.3±66.1mg/dl) was significantly higher compared to that of control group (82.7±9.0mg/dl).
• The mean post-prandial blood sugar (PPBS) levels of Metabolic syndrome group patients (298.4±86.1mg/dl) was significantly higher compared to that of control group (134.7±21.6mg/dl).
• The mean HbA₁C levels of Metabolic syndrome group patients (8.9±3.1%) was significantly higher compared to that of control group (5.1±0.5%).
• The mean GGT levels of Metabolic syndrome group patients (64.7±47.3U/L) was significantly higher compared to that of control group (24.1±5.4U/L).
• The mean HsCRP levels of Metabolic syndrome group patients (15.1±9.3mg/L) was significantly higher compared to that of control group (7.7±4.5mg/L).
• The mean serum ferritin levels of Metabolic syndrome group patients (164.5±134.9ng/ml) was significantly higher compared to that of control group (120.9±49.9 ng/ml).
• The mean serum fasting insulin levels of Metabolic syndrome group patients (15.9±8.4µIU/ml) was significantly higher compared to that of control group (10.1±6.3 µIU/ml)).
• The mean insulin resistance of Metabolic syndrome group patients (6.6±5.1) was significantly higher compared to that of control group (1.6±0.7).

Table 2: Distribution of Biochemical parameters among cases and control groups

• The distribution of Biochemical parameters among cases and control groups
• The mean serum creatinine levels of Metabolic syndrome group (0.8±0.2mg/dl) showed no significance when compared to that of control group (0.8±0.2mg/dl).

Table 3: Correlation of Insulin Resistance With Biochemical Parameters Among The Metabolic Syndrome Patient Group.

Table 4: Binary Logistic Regression Analysis to estimate the association of biochemical parameters with Insulin Resistance (HOMA-IR) among Metabolic Syndrome Patient Group

The results of binary univariate regression analysis performed for metabolic syndrome cases to identify biochemical risk factors for Insulin Resistance. It was found that higher levels of Body Mass Index, Fasting Blood sugar, PPBS and HBA1c were found to be significant risk factors for development of insulin resistance

Over the last decade there has been a global epidemiological transition in the disease pattern. The relative impact of infectious diseases as well chronic diseases like cardiovascular disease (CVD) and diabetes are increasing.11 It has been estimated that the incidence of DM and CVDs will double by 2025. The metabolic syndrome (MetS, Syndrome X, Insulin resistance syndrome, IRS) is a constellation of several cardiovascular risk factors promoting atherosclerotic cardiovascular disease (ASCVD).12 It consists of an atherogenic dyslipidemia (i.e. elevated triglycerides, low high density lipoprotein cholesterol (HDL-C)), elevation of blood pressure and glucose, prothrombotic and proinflammatory states. Metabolic syndrome is a complex web of metabolic factors that are associated with a 2-fold risk of CVD and a 5-fold risk of diabetes.123 Asian Indians are at a high risk of developing diabetes and CVD as the number of cases are consistently increasing. Recent data show that about one third of the urban population in India’s major cities has metabolic syndrome.13 Iron has important role in the normal physiological functions of the human body. Serum ferritin, one of the key proteins in regulating iron homeostasis, is a widely available clinical biomarker to evaluate iron status and especially important for detecting iron deficiency. However, growing evidence has shown that even moderately increased iron stores represented by high-normal ferritin concentrations are associated with diabetes.14

In our study, table 7 depicts that fasting blood sugar levels, post prandial blood sugar levels and HbA1C levels among MetS patients are significantly higher compared to control group. Our findings of a steadily increasing trend of diabetes risk with the number of MetS components for both IFG and non-IFG levels are true to those of Japanese population-based studies conducted.[130,131]  As expected, those with IFG had a much higher risk of diabetes than those without IFG for a given number of MetS components. These observations suggest that increased fasting glucose levels is an important contributing factor for metabolic syndrome.15

Metabolic syndrome is a state of chronic low grade inflammation caused due to systemic oxidative stress induced by obesity and insulin resistance with increased activation of downstream signalling cascades that cause atherogenesis and tissue fibrosis. A rise in inflammatory markers has been seen in MetS. Serum GGT and serum ferritin concentrations were significantly higher in MetS patients compared to controls. In subclinical inflammation, GGT could be elevated because of its role in glutathione homeostasis and oxidant stress.133 Ferritin being an acute phase reactant, thus elevated serum ferritin levels might show systemic inflammation besides increased body iron stores. It is known that inflammation regulates the expression of ferritin mRNA and protein levels, and its secretion.109 Excessive iron deposition leads to production of hydroxyl radicals, which cause lipid peroxidation. Various studies have shown the same findings as that of our present study.16

Wei D et al. in their research on the Chinese population, a total of 1288 individuals, aged 20–74 years, participated in their survey. 1024 individuals (84.0%), including 436 men and 588 women, were eligible subjects whose data was proceeded to final analysis. Serum GGT and serum ferritin levels were significantly higher in subjects with metabolic syndrome compared to those without metabolic syndrome in both genders. (Serum GGT: p = 0.000 according to the revised NCEP-ATP III and CDS definitions in females; p = 0.003 according the revised NCEP-ATP III definition and p = 0.000 according the CDS definition in males. Serum ferritin: p = 0.000 according to the revised NCEP-ATP III and CDS definitions in females; p = 0.003 according the revised NCEP-ATP III definition and p = 0.000 according the CDS definition in males). GGT is the principal enzyme that influences the extracellular hydrolysis of glutathione (GSH). Ferritin affects the catalytic activities of GGT. The reactive products generated from GGT mediated cleavage of GSH may cause the reduction of ferric iron to ferrous iron. Elevated levels of GGT and ferritin then result in increased production of reactive oxygen species (ROS), aggravating oxidative stress, and leading to peroxidation of lipids by highly reactive free radicals. The adverse effects of ferritin overload and increased GGT mutually reinforce each other, ultimately leading to tissue injury and increased risk of MetS and its consequences. In our study, significantly higher positive association was found between serum GGT, serum ferritin in MetS patients when compared to controls. These observations suggest that increased GGT levels, serum ferritin levels are an important contributing factor for metabolic syndrome.17

The present study suggests that on linear regression fasting blood sugar (FBS) levels, post-prandial blood sugar (PPBS) levels, fasting insulin levels, high sensitive C-reactive protein levels were found to be significantly associated with homeostasis model assessment-insulin resistance (HOMA-IR). The present study also showed raised serum GGT and serum ferritin levels in cases of metabolic syndrome in comparison to controls. The significant association between GGT and ferritin suggests their role towards the increasing oxidant stress and inflammatory load in MetS which is an inflammatory condition. Hence, these parameters could be used as markers for early diagnosis of MetS

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