European Journal of Cardiovascular Medicine

Diabetes mellitus is a growing pandemic of unprecedented magnitude affecting one in ten individuals in all parts of the world. The risk of peripheral vascular diseases (PVD) in diabetic patients was found 3-5-fold higher compared to that in non-diabetics.  The microvascular and macrovascular complications in patients with type 2 diabetes is multifactorial and includes a very complex interaction including hyperglycaemia, hyperlipidaemia, oxidative stress, accelerated aging, hyperinsulinemia and/or hyperproinsulinaemia and alterations in coagulation and fibrinolysis.  A current hypothesis for the initial lesion in atherosclerosis involves changes in the endothelial cell function. Assessment of endothelial function, thus, can provide valuable insight into the pre-intrusive phase of atherosclerosis and can be used as an early marker of future atherosclerotic disease.  The invasive nature of the earlier available tests for assessment of endothelial function impeded their use in clinical practice. Development of non-invasive method of assessment of endothelial function by brachial artery flow mediated vasodilatation (FMD), as described by Celermajer, provided a valuable tool for cardiovascular research and for clinical application. This test is safe, non-invasive, repeatable and can be performed easily by experienced and qualified medical staff.  Among other factors, dyslipidaemia and increased levels of oxidized LDL are important pathogenic mechanisms of endothelial dysfunction in patients with diabetes. Since it has been proven that treatment of dyslipidemia with drugs improves endothelial function in non-diabetic, dyslipidaemia individuals, it becomes very tempting to extrapolate the conclusions from these findings to the diabetic population. 

In view of the above stated facts, the present study was undertaken to determine whether impairment in endothelial function occurs in patients with type 2 diabetes mellitus. It also aims to determine whether there is an increased dysfunction of the endothelium in the presence of diabetes mellitus with dyslipidaemia.

Study design: This is a case-control study.

Study setting: Departments of General Medicine and Endocrinology at Government General Hospital, Rajamahendravaram.

Study population: Inpatient and outpatient cases with diabetes mellitus We identified patients with diabetes mellitus between 30 and 70 years of age as cases. Controls included the patient attenders of the same age group and gender who are free from diabetes mellitus.

Study period: October 2022 to October 2023

Sampling technique and sample size calculation: All cases and controls were taken consecutively. To detect a statistically significant difference of approximately 4%–8% between cases and controls, 60 subjects were recruited in each group of same gender with a power of 90% and confidence interval of 95. The patients with diabetes were divided into 2 subgroups: those with dyslipidaemia (n=36) and those without dyslipidaemia (n=24).

Inclusion criteria:

1. Diagnosis of diabetes mellitus, based on ADA (American Diabetes Association) criteria ^[12]

• Fasting blood glucose ≥ 126 mg/dl (or)
• 2-hour post-prandial blood glucose ≥ 200 mg/dl (or)
• Random blood glucose ≥ 200 mg/dl with symptoms of hyperglycaemia (or)
• HbA1c ≥ 6.5%

2. Presence of dyslipidaemia, based on NCEP ATP – III (National Cholesterol Education Program Adult Treatment Panel) guidelines ^[13]

• LDL (Low density lipoprotein) – cholesterol ≥ 130 mg/dl (or)
• HDL (High density lipoprotein) – cholesterol ≤ 40 mg/dl in males and ≤ 50 mg/dl in females (or)
• TC (Total Cholesterol) ≥ 200 mg/dl

Exclusion criteria:

1. Presence of clinically evident microvascular or macrovascular complications of diabetes mellitus:

• Clinical or ECG evidence of CAD
• Past or present episodes of stroke and/or TIA
• Clinical evidence of peripheral vascular disease
• Features suggestive of diabetic retinopathy on fundus examination
• Presence of overt proteinuria or serum creatinine ≥ 2 mg/dl
• Clinical evidence of diabetic neuropathy

2. Associated co morbid illness which are likely to influence endothelial function:

• Smoking, Alcoholism, Hypertension, Congestive cardiac failure, Liver disease, Chronic infections, renal disease.

Method of data collection:

Patients and their attenders attending the medicine and endocrinology departments, during the period from October 2022 to October 2023, were included in the study. Informed consent was taken from all the study subjects. Clinical examination include blood pressure measurement, cardiovascular examination, and body-mass index (BMI) were done. Plasma glucose, serum cholesterol, serum triglycerides and high-density lipoprotein- cholesterol (HDL-C) were estimated. LDL-cholesterol (LDL-c) was calculated using the Friedewald formula.

The diameter of brachial artery was measured on B-mode ultrasound images. The right brachial artery was studied in all the subjects. Scan was done in resting phase, and the arterial flow velocity was measured with a pulsed Doppler signal at a 70⁰ angle to the vessel, with the range gate of 1.5mm in the center of the artery. Increased flow was then induced by the inflation of a sphygmomanometer cuff placed around the forearm (distal to the scanned part of the artery) to a pressure of 200 mm Hg for 4.5 min, followed by release. A second scan was performed continuously of 30 s before and 90 s after deflation of the cuff, including a repeated recording of flow velocity for the first 15 s after the cuff was released. Flow mediated dilatation (FMD) was calculated, and the average results of the two observations recorded. Flow-mediated dilatation was presented as the percent change from baseline to hyperaemia. Severe endothelial dysfunction was defined as FMD < 4.5%. ^[14]

Calculation of FMD:

FMD = (D2-D1) x 100/D1

Where, D1 is the brachial artery diameter at baseline and

D2 is the brachial artery diameter at 1 minute of cuff release

Reactive hyperaemia was established by an increase in peak systolic velocity (PSV) from baseline to immediately after cuff release.

Ethics approval:

Approval was obtained from the Institutional Ethics Committee, GSL Medical College, Rajahmahendravaram before commencement of the study.

Data analysis:

Data was entered in Microsoft Excel worksheet 2013 and Analysis was performed using SPSS software (Trial version 21). Descriptive statistical analysis has been carried out in the present study. Categorical variables were represented as proportions/percentages and quantitative variables were represented as mean and standard deviation. Unpaired student’s t test was used to compare the mean of continuous variables. P values ≤ 0.05 were considered statistically significant.

Figure 1: Normal dilatation of brachial artery from baseline to 90 sec after cuff release

Figure 2: Presence of reactive hyperaemia as shown by the increase in peak systolic velocity

Figure 3: Increased diameter of brachial artery from baseline to 90 sec after cuff release

Figure 4: Presence of reactive hyperaemia as shown by the increase in peak systolic velocity

Figure 5: Complete absence of dilatation of brachial artery from baseline to 90 sec after cuff release

Figure 6: Complete absence of dilatation of brachial artery from baseline to 90 sec after cuff release

Figure 7: Presence of reactive hyperaemia as shown by the increase in peak systolic velocity

In the present study 60 controls and 60 cases were taken. Cases were further stratified into two groups. Diabetics with dyslipidemia were one group (n=36), and those without dyslipidemia were the other group (n=24). Both cases and controls were age and gender-matched (frequency matching was done) as shown in Table 1.

Table 1: Age and gender distribution of the study participants

Table 2: Comparison of Baseline clinical characteristics of the study population

BMI= Body Mass Index, TC= Total Cholesterol, HDL= High Density Lipoprotein Cholesterol, LDL-c= Low Density Lipoprotein Cholesterol, TG= Triglycerides

Baseline comparison of lipid profile between the diabetes group and control group showed Elevated BMI, total cholesterol, LDL-C, TC/HDL and TG, Lowered HDL-C in the diabetes group. On comparison of the means of these variables using unpaired student’s t test, Total cholesterol, Low Density Lipoprotein Cholesterol and Total Cholesterol/ High Density Lipoprotein Cholesterol were found to have statistically significant difference amongst others. (Table 2)

Table 3: Comparison of lipid profile between diabetics with and without dyslipidaemia

BMI= Body Mass Index, TC= Total Cholesterol, HDL= High Density Lipoprotein, LDL-c= Low Density Lipoprotein Cholesterol, TG= Triglycerides

On comparison of the baseline variables between patients having diabetes with dyslipidaemia and dyslipidaemia, the following differences were observed:

1. The average age of the dyslipidemia group was lower
2. They had a higher body mass index
3. There was an elevated TC, elevated LDL-C, TG, TC/HDL-C and decreased HDL-C levels

All the values were found to be statistically significant using student’s t test.

Table 4: Comparison of FMD between cases and controls

Table 5: Comparison of FMD between diabetes with and without dyslipidaemia

The average FMD among controls was 15.42 ± 1.98 and among diabetics was 8.35 ± 4.45 demonstrating a significant impairment of flow-mediated dilatation (FMD) in diabetics when compared with normal subjects. Within the dyslipidemia sub group, FMD was markedly less with an average dilatation of 6.74 ± 4.75 than those without dyslipidaemia.

Table 6: Prevalence of endothelial dysfunction in the cases and controls

Severe endothelial dysfunction (FMD < 4.5%) was prevalent in 30% of the diabetics. Endothelial dysfunction could not be demonstrated in controls. Among the subgroups of cases, prevalence of severe ED was 44.5% in patients with dyslipidaemia, while 8.3% patients with normal lipid profile had severe ED.

Endothelial functional assessment using FMD technique is a simple, economical, non-invasive, widely available test; which can be used for both diagnostic and therapeutic purposes. The ultrasound assessment of arterial FMD responses provide valuable information about the risk stratification of dyslipidaemia Diabetes patients. In parallel, new developments in the field of DNA microarray technologies helps to identify target genes important for different phases of atherosclerosis. ^[15,16] These developments may replace mere angiographic detection of vascular disease to the more functional and genomic approach with the perspectives to evaluate new strategies in risk stratification and treatment. The present study demonstrated a significant impairment of flow-mediated dilatation (FMD) in diabetics when compared with normal subjects (8.35 ± 4.45 vs 15.42 ± 1.98, p value <0.0001). In the diabetic’s study group, patients with dyslipidaemia had significant impairment in FMD than patients without dyslipidaemia (6.74 ± 4.75 vs 9.82 ± 4.39, p value <0.02) and prevalence of severe ED was 44.5% in patients with dyslipidaemia, while 8.3% patients with normal lipid profile had severe ED.

Similar results were obtained in other studies. Bhargava K et al, in his study demonstrated a significant endothelial dysfunction in diabetes (5.51 ± 2.21%) when compared with normal study group (7.03 ± 2.87%). ^[17] They had shown that similar degree of endothelial dysfunction also occurred in patients with CAD without diabetes mellitus (4.56 ± 2.7%). In a study by Jadhav UM et al, diabetics showed significant impairment of endothelial function when compared with non-diabetics (p value <0.05). ^[18] They have also mentioned that the diabetic dyslipidaemia group showed more endothelial dysfunction but these values showed no statistical significance. Clarkson et al, have shown significant impairment of endothelial function in diabetics as compared to controls (5.0 ± 3.7 vs 9.3 ± 3.8%, p value <0.001). ^[19] Similarly Yu HI et al, have demonstrated a significant impairment of FMD of the brachial artery in diabetes. The impairment was more marked in the presence of peripheral arterial disease, dyslipidaemia and diabetic complications. ^[20] Yu Y et al study has demonstrated more prominent endothelial dysfunction in diabetics with microalbuminuria when compared with diabetics with normal albuminuria (8.0 ± 3.8% vs 9.7 + 4.3%, p value <0.05). ^[21] Antonaides C et al have shown that endothelial function was better in healthy controls when compared with diabetes patients and CAD (p value <0.001) or patients with diabetes alone (p value <0.001). They have also demonstrated the improved forearm vasodilatory response with usage of antioxidant vitamin C. ^[22]

Limitations of the study

This study being a case-control study revealed the results of only that part of the time and dis not include the follow up of study population, hence was not able to predict the future atherosclerotic events based on endothelial dysfunction. Some of the patients were on pharmacological treatment for diabetes and dyslipidemia with drugs that are proven to improve endothelial function (eg., Metformin, Statins). This could be a confounding factor in the present study.

Development of non-invasive method of endothelial function assessment by flow mediated vasodilatation (FMD) provided an extremely useful tool for cardiovascular research and for clinical application. A close correlation between the endothelial function in the human coronary and peripheral vasculature has been demonstrated. Ultrasonography is a reliable and accurate technique to determine flow-mediated dilatation in the superficial arteries. Reproducibility of flow mediated dilatation determination is best in the brachial artery in healthy subjects and in patients with atherosclerosis. B-mode ultrasound scan including brachial artery FMD may be of clinical value in the screening for endothelial dysfunction. Given the paucity of facilities and financial constraints for evaluation of endothelial function in India, measurement of endothelial dysfunction using a non-invasive method like brachial artery FMD acts as an attractive prospect. Long duration cohort studies involving a large study group is required to demonstrate the utility of this test as a predictor of atherosclerosis in diabetes in Indian population.

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