Diabetic retinopathy (DR) remains one of the most common microvascular complications of diabetes mellitus, affecting nearly one-third of the diabetic population worldwide and standing as the leading cause of vision loss among working-age adults [1,2]. Its progression from non-proliferative diabetic retinopathy (NPDR) to proliferative diabetic retinopathy (PDR) reflects a complex interplay between hyperglycemia-induced oxidative stress, inflammation, and endothelial dysfunction [3]. Traditional risk factors such as glycemic control, duration of diabetes, hypertension, and dyslipidemia have been well established. However, the identification of novel biomarkers associated with DR could enhance early diagnosis and improve patient outcomes [4].

Homocysteine, a sulfur-containing amino acid derived from methionine metabolism, has garnered attention due to its potential role in vascular injury through oxidative stress, endothelial dysfunction, and inflammation [5]. Elevated homocysteine levels, termed hyperhomocysteinemia, have been implicated in cardiovascular and cerebrovascular diseases and have recently been explored in the context of diabetic microvascular complications [6]. Several studies have reported that homocysteine may play a critical role in retinal capillary damage through oxidative injury and pro-thrombotic states, thereby contributing to the pathogenesis of DR [7,8].

Studies have indicated that homocysteine levels are frequently elevated in patients with diabetes, potentially due to impaired renal function, vitamin B12 or folate deficiencies, or increased oxidative stress associated with chronic hyperglycemia [9]. However, data linking homocysteine directly with the development and progression of DR remain inconsistent across different populations and study designs [10].

This study seeks to evaluate the relationship between serum homocysteine levels and the presence and severity of diabetic retinopathy in patients with type 2 diabetes attending a tertiary care hospital. By identifying whether elevated homocysteine serves as an independent biomarker for DR, we aim to contribute to the growing understanding of the biochemical pathways underlying diabetic complications and offer a potential avenue for risk stratification and therapeutic intervention.

A cross-sectional observational study was conducted over a period of 12 months in the Department of  Ophthalmology at a tertiary care teaching hospital. A total of 120 patients with confirmed type 2 diabetes mellitus (T2DM), aged between 40–70 years, were recruited. Informed consent was obtained from all participants.

Inclusion criteria were patients with history of at least 5 years of type 2 diabetes mellitus. Patients with other causes of retinopathy (e.g., hypertensive or retinal vein occlusion), chronic kidney disease (CKD stage 3 and above), current vitamin B12 or folate supplementation, or recent infection or surgery were excluded.

All subjects underwent a detailed history, systemic examination, and laboratory tests, including fasting plasma glucose, HbA1c, lipid profile, and serum homocysteine levels measured using chemiluminescent immunoassay. Fundoscopic evaluation was done by a retinal specialist using slit-lamp biomicroscopy after mydriasis. Digital fundus photography was also performed in all the cases. DR was classified into three groups: No DR, Non-Proliferative DR (NPDR), and Proliferative DR (PDR) based on Early Treatment Diabetic Retinopathy Study (ETDRS) guidelines.

Statistical analysis was conducted using SPSS version 25. Continuous variables were expressed as mean ± SD, while categorical variables were presented as frequencies and percentages. ANOVA and chi-square tests were used to assess differences between groups. Pearson’s correlation coefficient was applied to determine the association between homocysteine levels and DR severity. A p-value < 0.05 was considered statistically significant.

The demographic and clinical characteristics of the 120 enrolled participants revealed a mean age of 56.2 ± 8.4 years, with a slight male predominance (68 males and 52 females). The average duration of diabetes was 10.3 ± 4.1 years, and the mean HbA1c level was 8.4 ± 1.2%, suggesting poor glycemic control in a significant portion of the cohort. These baseline values provide a representative profile of middle-aged patients with longstanding type 2 diabetes attending a tertiary care hospital [Table 1].

Upon fundoscopic evaluation, 50 patients (41.7%) showed no signs of diabetic retinopathy, 45 (37.5%) had non-proliferative diabetic retinopathy (NPDR), and 25 (20.8%) had progressed to proliferative diabetic retinopathy (PDR). The distribution pattern underscores a substantial burden of retinopathy, with nearly 60% of patients exhibiting retinal changes, highlighting the importance of early ophthalmologic screening in diabetic care [Table 2].

The categorization of patients based on serum homocysteine levels showed a distinct gradient across DR groups. Among patients without DR, most had homocysteine levels below 10 µmol/L. In contrast, those with NPDR and PDR had progressively higher homocysteine levels, with the majority of PDR cases falling in the >15 µmol/L range. This trend indicated a statistically significant association between elevated homocysteine and DR severity [Table 3].

A correlation analysis demonstrated a strong positive relationship between serum homocysteine levels and the presence and severity of DR (r = 0.52, p < 0.001). Duration of diabetes and HbA1c levels also showed moderate positive correlations with DR (r = 0.39 and 0.44, respectively), both statistically significant. These findings support the hypothesis that homocysteine acts independently alongside known risk factors to exacerbate microvascular complications in diabetic patients [Table 4].

Table 1: Baseline Characteristics of Study Participants

Table 2: Distribution of Diabetic Retinopathy (DR) Stages

Table 3: Distribution of Homocysteine Levels by DR Status

Table 4: Correlation between Variables and DR Presence

This study aimed to elucidate the association between serum homocysteine levels and the development and progression of diabetic retinopathy in T2DM patients. Our findings demonstrate a statistically significant positive correlation between elevated homocysteine levels and both the presence and severity of DR.

The pathophysiological mechanisms linking homocysteine to microvascular complications are multifaceted. Homocysteine induces oxidative stress through the generation of reactive oxygen species (ROS), disrupts endothelial nitric oxide pathways, and promotes inflammation and thrombogenesis—all contributing to capillary occlusion and neovascularization observed in DR [11,12]. The strong positive correlation (r = 0.52) identified in our study aligns with previous research where they also found hyperhomocysteinemia to be a significant risk factor for DR [13].

Our findings are consistent with a previous studies in South India, where elevated plasma homocysteine levels were independently associated with the presence of DR, even after adjusting for age, gender, duration of diabetes, and HbA1c [14]. However, some studies have yielded conflicting results. Some researchers did not find a significant relationship between homocysteine and DR in a multiethnic population, possibly due to differences in dietary intake, renal function status, and genetic polymorphisms affecting homocysteine metabolism [15,16].

It is important to note that homocysteine levels can be influenced by folate and vitamin B12 status. While we excluded patients on supplementation, subclinical deficiencies may still confound our results. Future studies should include measurement of these vitamins to refine the association further.

Duration of diabetes and poor glycemic control (as reflected by HbA1c) were also significantly associated with DR, reinforcing existing literature [17,18]. The independent correlation of homocysteine with DR, however, highlights its potential as a novel biomarker for early screening and risk stratification.

From a clinical standpoint, screening for hyperhomocysteinemia in diabetic patients, especially those with prolonged disease duration or poor glycemic control, could allow early intervention strategies. These may include folic acid and B-vitamin supplementation, dietary modifications, or even pharmacologic lowering of homocysteine levels, which may hypothetically delay or reduce DR progression [19].

Limitations of this study include its cross-sectional design, which precludes causal inferences, and the relatively small sample size. Also, renal function markers were not extensively explored, though patients with CKD stage ≥3 were excluded. Furthermore, inter-laboratory variation in homocysteine measurement could affect reproducibility.

Despite these limitations, our study contributes significantly to the evidence base by reinforcing the association between hyperhomocysteinemia and DR in a South Asian diabetic population. Given the growing diabetes burden in developing countries, low-cost biomarkers such as homocysteine could be integrated into routine diabetic care to identify high-risk individuals.

Future longitudinal studies with larger sample sizes, and inclusion of nutritional and renal parameters, are warranted to confirm these associations and determine whether lowering homocysteine can influence DR outcomes.

This study demonstrates a significant positive association between elevated serum homocysteine levels and the presence and severity of diabetic retinopathy in patients with type 2 diabetes. Hyperhomocysteinemia could serve as a novel and cost-effective biomarker for identifying individuals at higher risk for retinopathy. Incorporating homocysteine level screening into standard diabetes management protocols, along with traditional risk factors, may improve early detection and intervention strategies. Further prospective studies are needed to validate these findings and explore therapeutic implications.

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