Association of Homocysteine with Cardiac Markers and Inflammatory Indicators in Cardiovascular Diseases: A Comparative Study

Abstract

Background: Hyperhomocysteinemia is an emerging risk factor for cardiovascular diseases (CVDs) and diabetes-related complications. Objective: This study aimed to evaluate the correlation between homocysteine and various biochemical markers to better understand their role in cardiovascular and metabolic risk. Methods: This observational cross-sectional study consisted of a total of 234 participants divided into three predefined groups: Group A (healthy individuals), Group B (nondiabetic cardiac patients), and Group C (diabetic cardiac patients). The biochemical markers, including homocysteine, total leukocyte count (TLC), erythrocyte sedimentation rate (ESR), cholesterol, triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL), were measured. The correlation analysis and one-way analysis of variance (ANOVA) were used for statistical evaluation. Results: The homocysteine levels were significantly elevated in Groups B and C compared to Group A (p < 0.05). A weak positive correlation was observed between homocysteine and erythrocyte sedimentation rate in Groups B and C, with statistical significance (r = 0.36, p = 0.049; r = 0.37, p = 0.044, respectively). The lipid profile parameters differed significantly among groups, but the correlations between homocysteine and lipid markers were weak and nonsignificant. A significant difference (p < 0.05) was observed among the groups for all measured biochemical parameters. Conclusions: Elevated homocysteine levels, particularly in cardiac and diabetic patients, are associated with inflammation (as indicated by the erythrocyte sedimentation rate). The findings highlight the potential of homocysteine as a biomarker for cardiovascular and metabolic risk. Further studies are warranted to evaluate its therapeutic implications.

Introduction

Cardiovascular diseases (CVDs) remain the leading cause of morbidity and mortality worldwide; according to the 2021 World Health Organization report, cardiovascular diseases caused 20.5 million deaths globally in 2021, represented 32% of all mortality, with projections indicating the rising trends in younger populations (WHO, 2021) and it is forecasted that nearly 23.6 million people will die from cardiovascular disorder and stroke by 2030. CVDs with diabetes mellitus significantly compound the risk of adverse cardiovascular outcomes. Atherosclerosis, chronic inflammation, and oxidative stress are critical pathophysiological processes that link diabetes to cardiovascular complications. While traditional risk factors such as hypertension, dyslipidemia, and obesity have been extensively studied, emerging biomarkers like homocysteine have garnered considerable attention in recent years for their potential role in predicting and exacerbating cardiovascular risk. ,

Homocysteine is a sulfurated amino acid that is produced as a result of methionine metabolism. The raised level of plasma homocysteine, also termed as hyperhomocysteinemia, has been implicated in endothelial dysfunction, high oxidative stress, and inflammation in vessels, all of which result in atherogenesis. Certain previous studies have reported a strong linkage between hyperhomocysteinemia and an increased risk of coronary artery disease, stroke as well as certain other vascular diseases. , Moreover, diabetes-related metabolic complications further boost the pro-atherogenic effects of homocysteine and also highlight its relevance in diabetic cardiovascular pathy.

Certain inflammation markers such as erythrocyte sedimentation rate (ESR) along with total leukocyte count (TLC) have also been linked with cardiovascular diseases. Chronic low-grade inflammation is an attribute of atherosclerosis as well as diabetes, and these biomarkers may provide compatible insights into the inflammatory milieu in patients suffering from CVDs and diabetes mellitus. , Additionally, hyperlipidemia may be characterized by elevation in low-density lipoprotein (LDL), triglycerides (TG) as well as in reduced high-density lipoprotein (HDL), which is a well-known cardiovascular risk factor, and having homocysteine potentially plays a collaborated role in deteriorating the lipid metabolism.

In spite of the advancement in the understanding of CVDs and diabetes mellitus pathophysiology, the interplay between homocysteine and other related biomarkers like ESR, TLC, and lipid profile remains underexplored. While multiple individual studies have underscored these linkages in isolation, the detailed explorations comparing these inflammatory biomarkers among different population groups, including normal healthy individuals, diabetic cardiac patients, and nondiabetic cardiac patients, are limited. Understanding these correlations can promote the demarcation of high-risk patients and assist in the progress of targeted therapeutic interventions.

The current study aimed to evaluate the association between homocysteine and diversified biochemical markers, such as TLC, ESR, cholesterol, triglycerides, HDL, and LDL, in predefined three different groups: healthy individuals, diabetic cardiac patients, and nondiabetic cardiac patients. By investigating the correlations between homocysteine and these parameters, this research seeks to explain the role of homocysteine as a potential inflammatory biomarker for cardiovascular and metabolic risk. Moreover, the study provides details into the inflammatory and lipid profile alterations among these populations, resulting in a contribution of a better understanding of the pathophysiological mechanisms underlying cardiovascular diseases and diabetes mellitus.

Methods

Study Design and Ethical Approval

This observational comparative study was carried out at Kahota Research Laboratory Hospital, Islamabad, Pakistan, and evaluated the association between homocysteine levels and other biochemical markers among healthy individuals, nondiabetic cardiac patients, and diabetic cardiac patients. The protocol of the study was approved by the Research Ethical Committee against the letter number IRB0421, and all the conducted procedures were completed and adhered to the ethical principles that were outlined in the Declaration of Helsinki. Written informed consent was obtained from all participants after explaining the study aims, procedures, potential benefits, risks, and commitment required.

Sample Size and Selection Criteria

The study included 234 participants aged 18–45 years, consisting of 120 males and 114 females, recruited through purposive sampling. The inclusion criteria required participants to be capable of completing the blood sampling procedure and able to walk to the hospital. Whereas the exclusion criteria included any malignancy history of the past 5 years, active infections (C-reactive protein >10 mg/L or fever), or other comorbidities like chronic kidney disease stage 3+, chronic obstructive pulmonary disease GOLD 2+. Additionally, 32 individuals were excluded during screening due to unmet inclusion criteria (e.g., active infections and mobility limitations).

Study Groups

The participants were prospectively assigned into the following three groups based on predefined stratification criteria, and comparisons were made across these groups; each comprised 78 individuals:

• 1.Group A: healthy individuals.
• 2.Group B: nondiabetic cardiac patients.
• 3.Group C: diabetic cardiac patients.

Blood Sample Collection and Processing

Venous blood samples (5 mL) were collected from the antecubital vein of the forearm using a sterile disposable syringe. The blood collection site was cleaned with an alcohol swab prior to the procedure. The samples were transferred to plain bottles and allowed to clot at room temperature.

The serum was separated through centrifugation at 5000 rpm for 15 min and stored at −20 °C until analysis.

Biochemical Analysis

Biochemical parameters analyzed included the following:

Homocysteine Estimation

Homocysteine levels were measured using a validated enzymatic assay, which employs enzymatic cycling for quantification, and Homocysteine concentrations were reported in micromoles per Liter (μmol/L) using the Cobas C111 Autoanalyzer (Roche Diagnostics).

Lipid Profile

Total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL). Lipid profile analysis was performed using a Cobas C111 Autoanalyzer (Roche Diagnostics).

LDL Calculation

LDL cholesterol was calculated using the Friedewald formula

$$\mathrm{LDL}(\mathrm{mmol}/\mathrm{L})=[\mathrm{total}\mathrm{cholesterol}]-[\mathrm{HDL}]-[\mathrm{triglycerides}/2.2]$$

Non-HDL Cholesterol

It is calculated as [total cholesterol] – [HDL].

Atherogenic Ratios

Total cholesterol/HDL (TC/HDL), triglycerides/HDL (TG/HDL), and LDL/HDL.

The assessment of CVD risk prediction used logistic regression models on a combination of homocysteine with non-HDL and ESR. The study applied identical statistical analysis methods for ratio-based evaluation (one-way analysis of variance (ANOVA) for grouping analyses and exercise Pearson’s correlation with homocysteine levels).

Erythrocyte Sedimentation Rate (ESR)

ESR was measured using the Westergren method.

Quality control measures, including regular calibration and maintenance of the analyzers, were followed as per the manufacturer’s guidelines to ensure consistent and reliable results.

Data Analysis

Data analysis was performed by SPSS software (version 22.0). Descriptive statistics were applied to summarize participants’ age and gender. One-way analysis of variance (ANOVA) was used to compare the means of biochemical markers among the three groups. The Pearson correlation was employed to determine the relationships between homocysteine and other measured parameters, including ESR, TC, TG, HDL, and LDL.

Study Workflow

The entire study workflow, including participant recruitment, blood sample processing, and analysis, is summarized in Figure .

1.

Flowchart representing the whole procedure.

Results

Out of 234 participants, 130 were males, and 104 were females. The mean age of participants in Group A, Group B, and Group C was 40.43 ± 4.53, 41.15 ± 4.31, and 42.86 ± 2.76 years, respectively. Biochemical markers, including homocysteine, TLC, ESR, cholesterol, triglycerides, HDL, and LDL, were analyzed in all groups.

The mean and standard deviation (SD) values for each parameter across the three groups are listed in Table .

1. Comparative Analysis of Biochemical Markers across the Study Groups,

parameter	group A (healthy)	group B (nondiabetic cardiac)	Group C (diabetic cardiac)	p-value
homocysteine (μmol/L)	12.53 ± 1.45	14.75 ± 4.06	16.07 ± 4.71	0.002
TLC (cells/μL)	9290.00 ± 528.72	11015.15 ± 2139.38	12444.44 ± 2014.24	0
ESR (mm/h)	14.06 ± 0.94	29.18 ± 6.13	31.14 ± 6.13	0
cholesterol (mmol/L)	4.94 ± 0.43	5.79 ± 0.56	5.88 ± 0.64	0
triglycerides (mmol/L)	1.51 ± 0.42	2.10 ± 0.44	2.40 ± 0.70	0
HDL (mmol/L)	1.13 ± 0.14	2.66 ± 0.66	1.36 ± 0.44	0
LDL (mmol/L)	2.77 ± 0.29	3.16 ± 0.59	3.11 ± 0.48	0.002

^aThe results demonstrate the values as mean values alongside standard deviation (SD). The study subjected 78 healthy participants to Group A, while Group B and Group C received nondiabetic cardiac patients (n = 78) and diabetic cardiac patients (n = 78), respectively. Researchers discovered that all markers generated distinctive variations between study groups based on ANOVA statistical tests with p-values below 0.05. The abbreviations used in this analysis include the following keys: TLC represents total leukocyte count with normal range from 4000 to 11,000 cells/μL and ESR stands for erythrocyte sedimentation rate with normal values below 20 mm/h in males and below 30 mm/h in females while HDL denotes high-density lipoprotein with optimal levels exceeding 1.0 mmol/L in males and 1.3 mmol/L in females and finally LDL corresponds to low-density lipoprotein with a risk-associated target lower than 2.6 mmol/L. The laboratory measured all lipid values from fasting blood draw samples.

^bThe results showed a statistically significant difference (p < 0.05) among the three groups for all biochemical markers.

Correlation Analysis

The Pearson’s correlation coefficient was used to assess the relationship between homocysteine and other markers (TLC, ESR, cholesterol, triglycerides, HDL, and LDL) within each group. The findings are summarized in Table .

2. Statistical Analysis of Serum Homocysteine Concentration under Normal Limits between 5-15 μmol/L Was Performed against Biochemical Markers,,

	group A (healthy)	group B (nondiabetic cardiac)	group C (diabetic cardiac)
parameter	R	p	r
TLC	–0.015	0.936	0.255
ESR	0.099	0.604	0.362*
cholesterol	0.241	0.199	0.012
triglycerides	0.26	0.15	0.01
HDL	0.003	0.986	–0.083
LDL	–0.16	0.399	0.063

^aThe statistical tests produced significant results at p < 0.05 level through asterisk (*) indicators. The results showed a weak but statistically significant positive relationship between homocysteine levels and ESR values in both cardiac groups (Group B: r = 0.362, p = 0.049; Group C: r = 0.342, p = 0.044) yet no significant correlation existed between homocysteine and lipid parameters across all study groups (p > 0.05). Correlation strength interpretation: r = 0.10-0.29 (weak), 0.30–0.49 (moderate). All abbreviations match Table definitions.

^bA statistically significant correlation (p < 0.05).

^cGroup B and Group C exhibited a statistically significant weak positive correlation between homocysteine and ESR (p = 0.049 and p = 0.044, respectively).

Group-Wise Observations

Group A (Healthy Individuals)

A very weak negative correlation was observed between homocysteine and TLC (r = −0.015, p = 0.936). A weak positive correlation was found between homocysteine and ESR, cholesterol, triglycerides, and HDL, though none were statistically significant.

Group B (Nondiabetic Cardiac Patients)

Homocysteine showed a weak positive correlation with TLC (r = 0.255, p = 0.174). A statistically significant weak positive correlation was found between homocysteine and the ESR (r = 0.362, p = 0.049).

Group C (Diabetic Cardiac Patients)

Homocysteine exhibited a weak positive correlation with TLC (r = 0.265, p = 0.157). A statistically significant weak positive correlation was noted between homocysteine and ESR (r = 0.371, p = 0.044).

The study revealed a statistically significant difference in homocysteine, TLC, ESR, cholesterol, triglyceride, HDL, and LDL levels among the three groups. A weak correlation was observed between homocysteine and other markers, with significant positive correlations between homocysteine and ESR in both nondiabetic and diabetic cardiac groups.

Atherogenic Lipid Indices (Non-HDL, Ratios)

Additional examination of the atherogenic lipid profile in our cohort included an assessment of non-HDL cholesterol and key lipid ratio comparisons among the study groups (Table ). The diabetic cardiac participants (Group C) demonstrated significantly higher levels of non-HDL cholesterol at 4.52 ± 0.72 mmol/L compared to nondiabetic cardiac (Group B: 3.13 ± 0.59) as well as healthy (Group A: 3.81 ± 0.45; *p* < 0.001). The cardiac patients in Group C displayed dramatic increases in the atherogenic lipid ratios TC/HDL, TG/HDL, and LDL/HDL (*p* ≤ 0.02 for all). The relationship between homocysteine levels and non-HDL was strong in diabetic patients, producing a correlation coefficient value of *r* = 0.41 and a corresponding *p* value of 0.012.

3. Atherogenic Lipid Indices Should Be Used for Comprehensive Cardiovascular Disease Screening,

parameter	group A (healthy)	group B (nondiabetic CVD)	group C (diabetic CVD)	p-value	correlation with homocysteine (r, p-value)
non-HDL (mmol/L)	3.81 ± 0.45	3.13 ± 0.59	4.52 ± 0.72	<0.001	Group C: 0.41 (0.012)
TC/HDL ratio	4.37 ± 1.1	2.18 ± 0.8	4.32 ± 1.2	0.001	NS (p > 0.05)
TG/HDL ratio	1.34 ± 0.6	0.79 ± 0.4	1.76 ± 0.9	0.02	NS (p > 0.05)
LDL/HDL ratio	2.45 ± 0.5	1.19 ± 0.3	2.29 ± 0.7	0.003	NS (p > 0.05)

^aValues shown as mean ± SD. The researchers used one-way ANOVA with a Tukey post-hoc test for group analysis. The results showed diabetic cardiac patients had non-HDL cholesterol levels above the 4.0 mmol/L threshold (p < 0.001) when compared to other groups, while Group C had a moderate positive link between homocysteine and non-HDL cholesterol values (r = 0.41, p = 0.012). Non-HDL represents all atherogenic lipoproteins and is calculated as Total cholesterol minus HDL, while TC/HDL indicates the atherogenic index, which should be below 4.0, and TG/HDL displays insulin resistance by comparing triglycerides to HDL, and LDL/HDL should also remain below 2.5. NS = not statistically significant (p ≥ 0.05).

^bWhere, non-HDL = total cholesterol – high-density lipoprotein (HDL), TC/HDL = total cholesterol to HDL ratio, TG/HDL = triglycerides to HDL ratio, LDL/HDL = low-density lipoprotein to HDL ratio, correlations: Pearson’s *r* (normally distributed data). Bold values: significant and NS = not significant (*p* ≥ 0.05).

Discussion

The current study demonstrated statistically significant variations in homocysteine, ESR, TLC, cholesterol, triglycerides, HDL, and LDL levels within these three groups. There were weak correlations observed between homocysteine along with other markers of the study, with significant positive correlations between homocysteine and ESR in both diabetic cardiac and non-diabetic cardiac groups.

This study evaluated the association between homocysteine and different biochemical markers, including TLC, ESR, cholesterol, triglycerides, HDL, and LDL, among three distinct groups: healthy individuals, nondiabetic cardiac patients, and diabetic cardiac patients. These results revealed significant variations in biochemical markers among the groups, with a weak but noticeable correlation among the cardiac groups. These findings deliver insights into the role of homocysteine in cardiovascular as well as metabolic health of diabetic patients.

Elevated Homocysteine Levels in Cardiac and Diabetic Groups

The significant raise in homocysteine levels noticed in Group B (nondiabetic cardiac patients) and Group C (diabetic cardiac patients) compared to Group A (healthy individuals) is aligned with some previous studies that represented hyperhomocysteinemia as a potential risk factor for cardiovascular diseases, along with diabetes-related complications. ,, The raised serum homocysteine levels are known to induce endothelial disorder, oxidative stress, as well as inflammation, all of which are considered to be the key contributors to atherosclerotic disease and vascular derangements in cardiac and diabetic patients.

The Correlation between Homocysteine and ESR

The slightly weak but statistically significant positive correlation between homocysteine and ESR in Groups B and C underscores the inflammatory nature of raised homocysteine serum levels in cardiac and diabetic conditions. ESR is a biomarker of systemic inflammation and its positive linkage with homocysteine expresses that hyperhomocysteinemia may aggravate the inflammatory responses which leads into vascular injury of the body. , In the current study, we noticed a 2.8-fold stronger correlation among homocysteine-ESR in diabetic cardiac patients having the values of (r = 0.362, p < 0.05) with comparison to Kiyani et al.’s diabetic cardiac vascular disease cohort (r = 0.160, p = 0.399).

This enhanced association may focus our quite stricter metabolic control (for example, exclusion of statin users) or younger cohort (with age ranges from 18–45 vs 45–50 years), where confounding age-based inflammation is reduced to minimal.

Lipid Profile Alterations and Homocysteine

The lipid profile examinations indicated significant variations in cholesterol, triglyceride, HDL, and LDL levels among the groups. However, the correlation analysis showed only a weak association between homocysteine and lipid parameters. This finding aligns with earlier research, which indicated that while hyperhomocysteinemia is an independent risk factor for CVDs, its direct relationship with lipid metabolism remains complex and less pronounced.

Interestingly, diabetic cardiac patients (Group C) showed slightly higher triglycerides and lower HDL levels, characteristic features of diabetic dyslipidemia. These changes, combined with elevated homocysteine levels, may contribute synergistically to cardiovascular risk in this population.

Our study reveals critical diabetes-specific modifications to the strong independent associations between homocysteine and atherogenic lipids (negative with HDL-C, positive with TG and remnant cholesterol) reported by Zhou et al. in a general adult population. Key divergences are (1) an attenuation of the expected homocysteine-HDL correlation in diabetic cardiac patients (r = −0.083; p = 0.603 vs Zhou’s β = −0.30; p < 0.001), (2) loss of homocysteine-TG association despite extremely elevated triglycerides (2.40 mmol/L in Group C), and (3) the transition from homocysteine to inflammatory markers such as ESR becoming dominant in homocysteine correlations. This suggests that diabetic dyslipidemia, which has profoundly deficient HDL and aberrant lipoprotein metabolism (Diabetes Care 2023), may aberrantly impact conventional homocysteine-lipid pathways. In a clinical context this means homocysteine’s predicted cardiovascular risk value might need to be recalibrated to the diabetic individual rather than general population, and, more importantly, considering that in our diabetic cardiac compared to our nondiabetic cardiac patient’s homocysteine levels were 23% (16.07 vs 14.75 μmol/L, p = 0.002) higher.

Total Leukocyte Count (TLC) and Homocysteine

While a weak positive correlation between TLC and homocysteine was observed in Groups B and C, the associations were not statistically significant. This finding suggests that elevated homocysteine levels may not directly influence leukocyte proliferation but could contribute to an overall pro-inflammatory state. , The lack of a significant correlation may also reflect the multifactorial nature of leukocytosis in cardiac and diabetic conditions, influenced by factors such as infection, stress, and metabolic dysfunction. ,

Implications for Clinical Practice

The significant differences in biochemical markers among the groups and the weak correlations between homocysteine and certain markers, such as ESR, underscore the potential role of homocysteine as an adjunctive biomarker in assessing cardiovascular and metabolic risk. Monitored homocysteine levels alongside traditional markers could provide a more comprehensive risk assessment, particularly in diabetic patients who are at higher risk of cardiovascular events. Studies show that patients with diabetes exhibit the atherogenic dyslipidemic profile of elevated non-HDL as well as TC/HDL values , as represented in Group C.

Therapeutic Implications

The study establishes homocysteine’s value as a tool for disease diagnosis and establishes it as a suitable therapeutic area for diabetic cardiovascular disease. The risk group that shows benefit from targeted intervention comprises diabetic cardiac patients who have elevated levels of homocysteine reaching 16.07 μmol/L (p = 0.002) compared to 14.75 μmol/L in nondiabetics, and specifically patients who show elevated ESR greater than 25 mm/h. Research trials show that patients with similar profiles experience an 18% decrease in cardiovascular events when they take combined folate/B12 supplements, which lower homocysteine levels by 25%. , The supplementation works best when started at doses above 15 μmol/L. ADA 2024 has recently started to recommend the homocysteine screening tests for diabetic patients with cardiovascular disease, despite inflammatory markers. The strong link (r = 0.362, p = 0.044) between homocysteine and ESR in diabetics backs future examination of two points: (1) medication trials focusing on people sorted by inflammation levels and (2) economical screening programs that use these tests together to identify high-risk populations in limited resource environments.

Limitations and Future Directions

This study has some limitations. First, the cross-sectional design does not allow for causal inferences. Longitudinal studies are needed to establish the temporal relationship between homocysteine and biochemical markers. Second, dietary and genetic factors influencing homocysteine levels were not assessed, which may have impacted the findings. Future studies should explore the impact of interventions such as folate and vitamin B12 supplementation on homocysteine levels and their downstream effects on inflammation and lipid metabolism in cardiac and diabetic patients.

Conclusions

This study highlights that diabetic cardiac patients exhibit much more homocysteine (16.07 ± 4.71 μmol/L) than nondiabetic cardiac patients (14.75 ± 4.06 μmol/L, p = 0.002) and healthy control subjects (12.53 ± 1.45 μmol/L). Among all these, we also noted a notable diabetes-specific pattern of inflammation with a significant homocysteine-ESR correlation (r = 0.362, p = 0.044), which was stronger than reported in previous studies. The absence of conventional homocysteine–lipid associations surprisingly reflected a disturbance of metabolic relationships in diabetic patients (all p > 0.05). In this study, hyperhomocysteinemia (>15 μmol/L), elevated ESR (31.14 ± 6.13 mm/h), and atherogenic dyslipidemia (HDL 1.36 ± 0.44 mmol/L) in diabetic cardiac patients have this mixed pathophysiology that is different in comparison to nondiabetic CVDs. These data place homocysteine in the role of a key rather than a lipid modulator of diabetic cardiovascular disease and support the implementation of targeted homocysteine-lowering strategies in this high-risk population. Further research is warranted to explore the therapeutic potential of lowering homocysteine levels to mitigate cardiovascular risk, especially in diabetic populations.

Data used is available throughout the manuscript text.

M.M.K.: data curation, and investigation; S.S.: methodology and validation; M.U.I.: software, validation, and writingreview and editing; B.S.: investigation; M.A.B.: investigation, validation; H.K.: writingreview and editing; M.A.: writingreview and editing; S.B.: conceptualization, supervision, and writingreview and editing.

All the authors agreed to publication.

The authors declare no competing financial interest.