European Journal of Cardiovascular Medicine

Diabetes Mellitus is most common endocrine disease. It is a group of metabolic disease which is characterized by hyperglycaemia, various clinical manifestations and systemic complications and is caused by either deficiency in the secretion or action of insulin or both. The metabolic derangement is frequently associated with permanent and irreversible functional and structural changes in the cells of the body, those of the vascular system, being particularly most susceptible. The chronic hyperglycaemia of diabetes is associated with long term damage, dysfunction and failure of different organs, and these changes in turn lead to development of well-defined clinical entities, the so-called complications, which may affect especially the eyes, kidneys, heart, blood vessels, the skin and the nervous system.¹ Interest in trace elements has been steadily increasing over the last 25 years.

Trace elements are accepted as essential substances for optimum human health, because of their diverse metabolic characteristics and functions. They serve a variety of catalytic, structural and regulatory functions in which they interact with macromolecules such as enzymes, pro hormones, pre secretory granules and biological membranes.² Direct association of minerals, trace elements and vitamins in the pathogenesis and natural course of both type 1 and 2 diabetes mellitus has been observed in many research studies. An alteration in the metabolism of these minerals and vitamins has been demonstrated. Diabetes mellitus is a heterogeneous disease associated with an absolute or relative deficiency of minerals as well as insulin resistance.3

Some trace elements act as antioxidants, prevent membrane peroxidation while others act directly on glucose metabolism. It is generally agreed that disturbed concentration of Zinc (Zn) and Magnesium (Mg) in the body are often found in patients of diabetes mellitus. Among the trace elements Copper (Co) and Zn are of particular interest.⁴ In subjects with Insulin Dependent Diabetes Mellitus (IDDM), Zn concentrations have been demonstrated to be lower in leucocytes and erythrocytes than in serum, while no such alteration has been found with copper.5 Cu is involved in oxidation – reduction and has a dominant role in diverse proteins such as cytochrome oxidase and cytoplasmic superoxide dismutase. Zinc another essential trace element, is a component of many enzymes, and plays an important role in the maintenance of several tissue functions including the synthesis, storage and release of insulin.^6,7 Zn plays an important role in glucose metabolism.⁸

The aim of the present study was to assess the relationship between serum Zinc level and HbA1C level in newly diagnosed type 2 diabetes mellitus and to compare serum Zinc level with its risk factors.

The study was conducted at Sri Guru Ram Das University of Health Sciences, Sri Amritsar. Patient presenting in OPD/IPD of Medicine department who are known case recently diagnosed type 2 Diabetes Mellitus were recruited to current study after taking written and informed consent from January 1,2023 to March 31,2024. The present study included a total of 175 cases, comprising recently diagnosed type 2 DM patients, and 100 age and gender-matched healthy controls without any comorbidities.

A complete history of the patients was collected using a predefined proforma. All patients were subjected to thorough general and systemic examinations. Under strict aseptic conditions blood sample was collected from all the patients and the serum was investigated for creatinine, urea, zinc, HbA1c, cholesterol, triglycerides and fasting blood glucose. Urine samples were collected and urine proteins were determined by turbidimetric method using 3% sulphosalicylic acid.

Inclusion criteria

All type 2 Diabetes Mellitus patients age more than 40 years, willing to participate in the study

Exclusion Criteria:

• Type 1 Diabetes Mellitus
• Sepsis
• Pregnant female
• Gestational diabetes
• Patient on Zinc supplementation /OCPs/ hormonal therapy / steroid therapy
• Chronic kidney disease stage 4/5
• Chronic liver disease
• Any Malignancy
• Malnutrition
• Diarrhoea
• H/o of Psychiatric illnessExclusion criteria

Study patients who is taking zinc supplementation or drugs that interfere with zinc absorption, patients with chronic disease and pregnancy, diabetic related complications and patients on oral hypoglycemics or insulin.

METHODOLOGY

All the patients of type 2 diabetes mellitus (newly diagnosed or old patients) were included in the current study after applying inclusion and exclusion criteria and after taking written and informed consent.

A detailed history about type 2 diabetes mellitus (duration of diabetes, treatment, level of glycemic control) was obtained from the patients with special reference to some clinical points related to the diabetes and its complication (dimness of vision, numbness, burning sensation of handing feet, non-healing ulcer). After detailed history, a thorough clinical examination was conducted to find out any clinical evidence of microvascular or macrovascular complications (neurological examination to find out any sign of sensory, motor or autonomic neuropathy, peripheral pulsation was checked to rule out any atherosclerotic changes, fundus examination to rule out any retinopathy) and relevant laboratory investigation were done as per the protocol. Blood samples were collected and sent to laboratory for measurement of FBS/RBS, HbA1c, RFT and serum zinc levels and then correlation of serum zinc levels in type 2 DM and its complication was established. Healthy controls were age and gender matched individuals coming to OPD/admitted in hospital for other non- related illnesses. Written and informed consent was taken from controls and they were also subjected to serum zinc level.

Laboratory Investigations:

All blood specimens were drawn with minimal stasis from antecubital vein using a dry sterile disposable syringe and needle and the specimen were labelled with subjects age, sex and identification number EDTA samples (for HbA1C) were kept at room temperature until processed within 4 hours of collection HbA1C was estimated using the HPLC method 2 ml of peripheral venous blood was withdrawn from each individual and serum was stored at 2-8 °C for sérum zinc detection. Serum zinc levels was assayed with the zinc kit using colorimetric method.

Statistical analysis

The data from the present study was systematically collected and compiled in Microsoft Excel, then statistically analysed using the Statistical Package for the Social Sciences (SPSS) 26 to draw relevant conclusions. Categorical data was presented as numbers and percentages, while parametric data was presented as mean±standard deviation (SD). The student’s t-test and ANOVA was applied to analyse continuous variables. Categorical data was analysed using the Chi-square test or Fisher’s exact test, as appropriate. A significance level of p<0.05 was considered significant, and p<0.01 was considered highly significant.

Table 1: Baseline characteristics

Among individuals aged 41-50 years, 27.4% belong to the case group, while 38% are in the control group. In the 51-60 age group, 22.9% are cases and 22% are controls. For individuals aged 61-70 years, the case group comprises 36.0%, while the control group constitutes 27%. In the 71-80 age group, 11.4% are cases, with controls at 12%. Finally, among individuals over 80 years old, 2.3% are cases, and 1% are controls. Among cases 49.1% are females and 50.9% are male, and in controls 59% are females and 41 % are male. The distribution of BMI among the case and control groups indicates that 33.7% of cases and 34% of controls fall into the normal weight. Overweight represents 48.0% of cases and 43% of controls. For Obesity Class I, 14.9% of cases and 18% of controls are observed, while Obesity Class II accounts for 3.4% of cases and 5% of controls.

Table 2: Distribution of cases according to HbA1c and duration of diabetes mellites

The distribution of HbA1c levels among the observed population is 15.4% of patients have HbA1c levels <7.0, 11.4% fall within the range of 7.1-8.0, 17.7% have HbA1c levels between 8.1 and 9.0, 13.7% fall within the range of 9.1-10.0, and the highest proportion, 41.7%, have HbA1c levels greater than 10.0. Among the studied population, 12.0% are newly diagnosed with diabetes, while 20.0% have been diagnosed for less than 5 years. The majority, comprising 47.4% of individuals, have a duration of DM ranging from 5 to 10  years.  Additionally,  20.6%  have  suffered  DM  for  over  10  years.

Table 3: Comparison of serum zinc levels in both the groups

A significant difference in mean serum zinc levels between groups, with a p-value of <0.001. Cases exhibits lower mean serum zinc levels (58.46µg/dL±16.03) compared to the controls (77.02 µg/dL±17.44). The majority of patients in the Cases have serum zinc levels ≤60µg/dL (57.9%), while in the Controls primarily comprises patients with serum zinc levels between 61-80 µg/dL (63.0%).

Table 4: Association of mean serum zinc levels with HbA1c levels

The association between zinc levels and HbA1c levels discloses a significant correlation, revealing zinc's role in glycemic control among patients with diabetes. Lower mean serum zinc levels are consistently observed with increasing HbA1c levels, with patients having HbA1c levels >10 exhibiting the lowest mean serum zinc level of 52.0±13.6µg/dL. Conversely, those with HbA1c levels ≤7 demonstrate the highest mean serum zinc level of 66.0±18.3µg/dL.

Table 5: Association of serum zinc levels with coronary artery disease

Patients with serum zinc levels falling within the 41-60 µg/dL range exhibit the highest percentage of CAD cases at 53.3%. Conversely, patients with serum zinc levels in the range of 81-100 µg/dL exhibit the lowest percentage of CAD cases at 6.7%. However, there are no CAD cases observed among patients with serum zinc levels >100 µg/dL. The p-value of 0.469 suggests that these differences in CAD percentages across serum zinc level groups are statistically insignificant.

Table 6: Association of serum zinc levels with peripheral artery disease

The patients with serum zinc levels falling within the range of ≤40 µg/dL and 41-60 µg/dL demonstrate equal percentages of PAD cases at 37.5%. Additionally, patients with serum zinc levels in the range of 61-80 µg/dL exhibit a lower percentage of PAD cases at 25.0%. However, there are no PAD cases observed among patients with serum zinc levels of 81-100 µg/dL or >100 µg/dL. However, the p-value of 0.067 indicates that this association is statistically insignificant.

Table 7: Association of serum zinc levels with cerebrovascular accident

In our study about 85.7% had serum zinc level below <60 µg/dL demonstrated p-value of 0.017, indicating a statistically significant association between serum zinc levels and CVA occurrence.

Diabetes, a metabolic disorder, leads to significant morbidity and mortality, primarily due to macrovascular complications and microvascular complications. The World Health Organization (WHO) predicted that 3.4 million people died from the consequences of high fasting blood sugar in 2010. Currently, 347 million adults worldwide have diabetes, and its prevalence continues to rise, with expectations that it will become the 7th top cause of death by 2030.⁹ Persistent hyperglycemia in uncontrolled diabetes mellitus leads to a spectrum of complications both acute and chronic, which significantly impact health and quality of life. Diabetes mellitus stands as one of the primary contributors to CVD, blindness, kidney failure, and lower limb amputation. Acute complications include diabetic ketoacidosis, hypoglycemia, hyperglycemic hyperosmolar state, and hyperglycemic diabetic coma.¹⁰

Chronic complications encompass both microvascular and macrovascular complications. Microvascular complications affect small blood vessels, include nephropathy, neuropathy, and retinopathy. On the other hand, macrovascular complications affect large vessels of blood and include coronary artery disease (CAD), peripheral artery disease (PAD), and cerebrovascular disease. It's alarming that every year, a significant percentage of middle-aged individuals with diabetes experience a cardiovascular event. The estimated rate ranges from 1.4% to 4.7%, highlighting the substantial burden of CVD in diabetic populations. Effective management and control of diabetes are crucial in preventing or delaying the onset of these debilitating complications and improving overall health outcomes.¹¹

The distribution of individuals by age groups within the case and control shows notable differences. Among individuals aged 41-50 years, 27.4% are cases, while 38% are controls. In the 51-60 age group, 19% are cases, contrasting with 22% controls. Moving to the 61-70 age bracket, 36.0% are cases, while 27% are controls. Within the 71-80 age range, 11.4% are cases, slightly less than the 12% observed in controls. Lastly, among individuals over 80 years old, 2.3% are cases, with 1% being controls. These disparities across age groups suggest potential age-related implications on the relationship between serum zinc levels and type 2 Diabetes Mellitus and its complications, warranting further exploration. BMI among both case and control groups suggests a relatively similar pattern, with a slightly higher prevalence of overweight in the case group compared to the control group. Specifically, 33.7% of cases and 34% of controls fall into the normal weight, while 48.0% of cases and 43% of controls are classified as overweight. Additionally, 14.9% of cases and 18% of controls are categorized as Obesity Class I, and 3.4% of cases and 5% of controls fall into Obesity Class II. Despite these variations, the statistical analysis reveals a non-significant difference in BMI distribution between the two groups, as evidenced by the p-value of 0.769. The observed trend in serum zinc levels across different durations of DM underscores a significant association between disease duration and zinc status. Notably, as the duration of DM increases, there is a consistent decline in mean serum zinc levels. Patients diagnosed with DM for over 10 years exhibit the lowest mean serum zinc level of 48.9 µg/dL, suggesting a potential depletion of zinc reserves over time. In contrast, newly diagnosed patients and those with a DM duration of less than 5 years demonstrate relatively higher mean serum zinc levels of 63.2 µg/dL and 62.4 µg/dL, respectively. These findings are aligned with Bhiwgade et al.¹² This inverse relationship between DM duration and serum zinc levels suggests a progressive impact of the disease on zinc metabolism. Our finding reveals weak inverse relationship between duration of diabetes and Serum Zinc levels in diabetic patients.

The notable difference in mean serum zinc levels between cases (58.46 µg/dL±16.03) and controls (77.02µg/dL±17.44), with a p-value of <0.001, suggests a potential association between zinc deficiency and type 2 Diabetes Mellitus (DM). The predominance of lower serum zinc levels in cases (≤40 µg/dL, 38.9%) compared to controls (41-60 µg/dL, 63.0%) underscores the relevance of zinc status in DM pathogenesis. These results were consistent with a previous study done by Saharia et al¹³ and Farooq et al.¹⁴  The association between serum zinc levels and HbA1c levels highlights a significant correlation, indicating zinc's potential role in glycemic control among diabetic patients. Lower mean serum zinc levels are consistently observed with higher HbA1c levels, suggesting a link between zinc status and glucose regulation. Patients with HbA1c levels >10 have the lowest mean serum zinc level of 52.0 µg/dL, indicating potential zinc depletion in poorly controlled diabetes. Conversely, those with HbA1c levels ≤7 exhibit the highest mean serum zinc level of 66.0 µg/dL, suggesting a positive association between optimal glycemic control and sufficient zinc levels. Seetharaman et al¹⁵ inclused in their study that Zinc, an essential element is useful in the synthesis, storage and secretion of insulin. Estimating the level of serum zinc becomes important to know the status of insulin resistance in diabetic patients. The findings regarding the association between serum zinc levels and HbA1c levels align with those reported by Bhiwgade et al¹² suggesting a consistent correlation between zinc status and glycemic control among patients with diabetes.

Patients with serum zinc levels between 41-60 µg/dL exhibit the highest percentage of CAD cases, while those with levels between 81-100 µg/dL show the lowest percentage. Interestingly, no CAD cases are observed among patients with serum zinc levels exceeding 100 µg/dL. However, the p-value of 0.469 suggests that these differences in CAD percentages across serum zinc level groups are statistically insignificant. The association between Zn levels and CAD has been clarified. The findings of a meta- analysis suggest that relatively low levels of Zn might have a potential role in the pathogenesis of CAD. Recently, Sanghani et al¹⁶ has underscored the association between zinc deficiency and diabetic patients who are prone to CAD. Patients with serum zinc levels falling within the range of ≤40 µg/dL and 41-60 µg/dL demonstrate equal percentages of PAD cases. However, there are no PAD cases observed among patients with serum zinc levels of 81-100 µg/dL or >100 µg/dL. Despite these trends, the p-value of 0.067 indicates that this association is statistically insignificant. Patients with a DM duration of 5-10 years exhibit the highest percentage of PAD cases, followed by those with a duration >10 years. Conversely, patients newly diagnosed with DM or with a duration of less than 5 years exhibit lower percentages of PAD cases. However, the p-value of 0.121 suggests that there may not be a statistically significant association between the duration of DM and the presence of PAD. This implies that while there may be trends in PAD prevalence across different DM durations, they are statistically insignificant. Sharma et al¹⁷ also quoted in their study that duration of diabetes were significant predictors of PAD, which also aligned with findings from previous studies.

In conclusion, our study highlights the complex relationship between serum zinc levels, glycemic control, duration of DM, and the prevalence of various diabetic complications. While we observed significant associations between serum zinc levels and the severity of nephropathy, retinopathy, and neuropathy, as well as with the occurrence of CVA, the findings regarding PAD, CAD were less conclusive. The lower serum zinc levels were consistently linked to more severe forms of nephropathy, retinopathy, and neuropathy, highlighting the role of zinc in diabetic complications. However, while higher HbA1c levels and longer DM durations exhibited trends towards increased prevalence of complications, these associations were not always statistically significant, indicating the influence of other contributing factors.

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