The cross-sectional study was conducted at the Department of Biochemistry, Jawaharlal Nehru Medical College, KLES Dr Prabhakar Kore Hospital & Medical Research Centre, Belagavi, Karnataka, India from January 2018 to December 2018. Before commencement of study, ethical clearance was obtained from Institutional Ethics Committee on Human Subject’s Research, Jawaharlal Nehru Medical College, Belagavi (MDC/DOME/94). After obtaining written consent, 100 (50 male and 50 female) (10% CI) patients were recruited for the study.

Clinically diagnosed cases of T2DM (aged 20 years or above) as per ADA guidelines were selected [3]. Patients on oral hypoglycaemic agents and insulin treatment were also included.

Critically-ill patients admitted in the Intensive Care Unit (ICU), patients below the age of 20 years, pregnant women, active smokers, alcoholic individuals, patients with a history of chronic illness such as hypertension, inflammatory diseases, cardiac or renal disorder, Type 1 DM, and patients on long term meditation such as antioxidant supplementation or lipid lowering drugs were excluded from the study.

The HbA1c and FBS reports along with serum sample of OPD and IPD patients enrolled in the study were collected from the Biochemistry Laboratory.

Overnight fasting venous blood samples (5 mL) were collected, and serum obtained by centrifugation was stored at -4°C until further analysis.

Reference value: (as per ADA guidelines) [4].

4.3%-5.6%=Non-Diabetic/Normal

5.7%-6.4%=Increased Risk for Diabetes/Prediabetic

>6.4%=Diabetes

Data analysis was done by using Statistical Package for the Social Sciences (SPSS) Software version 16. Data was analysed using independent t-test within the groups and Karl Pearson Correlation Coefficient in between the groups. The p-value <0.05 was considered statistically significant.

Clinically diagnosed 50 T2DM men and 50 women were categorised into 5 age groups. The minimum number of subjects (n=8) belonged to 31-40 years age group and the maximum number of subjects (n=33) to 51-60 years age group. The mean and standard deviation age range was 53.22±12.81 years [Table/Fig-1].

Gender-wise analysis showed Mean±SD in men was 52.24±13.51 years, 21-30 years age group had the least prevalence of n=5 (10%) and the highest prevalence (n=16 (32%)) was found in ≥61 years. Mean±SD in women was 54.22±12.13 years, 31-40 years group had the least prevalence of n=2 (4%) and the highest prevalence (n=20 (40%)) was found in 51-60 years group [Table/Fig-1].

Comparison of FBS, HbA1c, and NO was done between the genders using an independent t-test. However, no clinically or statistical significant results were obtained [Table/Fig-2].

Highly significant positive correlation was observed between FBS and HbA1c, FBS and NO and HbA1c and NO [Table/Fig-3].

DM is generally associated with increased risk of cardiovascular disorders and other co-morbidities throughout the world. The vascular complication of diabetes such as micro and macrovascular depends on hyperglycaemia. These complications were developed due to production of Reactive Oxygen Species (ROS) via various pathways. The ROS accumulation and activation of stress-induced intercellular signal pathways induces cellular damage, while the disease progresses due to inappropriate endogenous antioxidant defence mechanism [9]. In the present study, mean±SD FBS in males and females was found to be 154.20±45.49 mg/dL and 162.90±60.87 mg/dL.

NO is lipid soluble gas released from the vascular endothelium and inflammatory mediator. It plays a vital role in vascular tone and inhibition of cell proliferation. It is an effective inflammatory mediator because of its strong reactivity with oxygen, superoxide and iron-containing compounds [6]. Currently, various studies on glucose-dependent increase in NO production in case and control groups are available but none had compared in between the genders. Therefore, the present study is first of its kind in which the levels of FBS, HbA1c and NO among males and females belonging to different age groups were compared. However, no significant difference was observed in between the genders.

In the present study, statistically significant positive correlation was observed between FBS and HbA1c levels. These findings were in accordance with Monnier L et al., and Shrestha L et al., studies, which reported the relative contribution of fasting glucose increased gradually with increasing levels of HbA1c: 30.3% in the lowest vs. 69.5% in the highest (p<0.001), and both postprandial and FBS were significantly correlated with HbA1c [10,11]. Postprandial glucose showed better correlation to HbA1c than fasting blood glucose (r=0.630, p=0.05 vs r=0.452, p=<0.001) [11].

In the present study, statistically significant positive correlation was observed between HbA1c and NO levels. Similar findings were reported by various other studies [12-15]. The reason for increased NO levels in Type 2DM is oxidative stress and inflammation. Endothelium releases NO in response to different inflammatory mediators and it results in vasodilation. Such increase of NO can be a compensatory mechanism or defensive response [14]. Another theory for increased NO levels in Type 2DM is chronic hyperglycaemia, which leads to overflow of polyol pathway products along with depletion in the NADPH. NADPH plays a vital role for the enzymes in the metabolism of the ROS by acting as a co-factor. Increased inflammation and endothelial injury in diabetes, lead to increased synthesis of NO through inducible nitric oxide synthase enzyme in endothelium [15]. On the contrary, Hsu YC et al., and Kumar S et al., reported a decrease of NO [16,17]. These variations observed can be accounted to differences in the duration of diabetes. Initially, endothelium produces NO in higher amount to protect the organism from oxidative stress and inflammation. But after exhaustion, NO decreases in later stages.

Previous studies have correlated FBS, HbA1c and NO with each other in all subjects as one group, but in the present study FBS, HbA1c and NO were compared separately for both the gender. While there was significant increase in FBS, HbA1c and NO levels in both males and females, no statistical significance or clinical relevant results were obtained.

Serum NO was correlated with FBS and HbA1c in both males and females indicating that hyperglycaemia increases NO levels in type 2 DM patients. Since abnormal NO metabolism is associated with diabetes macrovascular and microvascular complications, early assessment of NO levels can prevent development and progression of diabetes related complications.

Serum NO levels were estimated by manual Classical Griess Reaction method, which can be replaced by fully automated methods like HPLC for more accuracy. There are lacunae of studies about the level of NO in medication adherent and non-adherent type 2 DM. Further cohort studies required to assess the effect of treatment on nitric oxide level in type 2 DM.