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Year : 2021  |  Volume : 11  |  Issue : 1  |  Page : 88-93

Biochemical Assessment of Hyperhomocysteinemia-Mediated Oxidative Stress in Coronary Artery Disease Patients: A Hospital-Based Cross-Sectional Study

1 Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Al-Khoud 123, Muscat, Oman
2 Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Oman
3 Cardiology Unit, Department of Medicine, College of Medicine and Health Sciences, Sultan Qaboos University, Al-Khoud 123, Muscat, Oman
4 School of Engineering, University of Guelph, Albert A. Thornbrough Building, 50 Stone Road East, Guelph, Ontario, Canada

Date of Submission08-Jul-2020
Date of Decision15-Jul-2020
Date of Acceptance29-Jul-2020
Date of Web Publication01-Oct-2020

Correspondence Address:
Mostafa I Waly
Associate Professor, Food Science and Nutrition Department CAMS, Sultan Qaboos University, Muscat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijnpnd.ijnpnd_71_20

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This study aimed to assess the status of B-vitamins (folate, vitamin B6, and B12) and homocysteine (HCY) in the sera of Omani coronary artery disease (CAD) patients. Sixteen Omani patients (10 males and 6 females) gave consent for blood sampling and were enrolled in the study on voluntary basis. All patients were evaluated for their anthropometric and biochemical measurements of B-vitamins, glutathione (reduced and oxidized), HCY, and quantification of N-homocysteinylated albumin protein. It was observed that both male and female patients had a comparable age (57.64 ±9.86, 56.5 ±10.04 years, respectively) with no significant difference, P = 0.69 and both genders were obese based on their body mass index (31.22 ± 8.17 kg/m2 for males and 30.26 ± 4.70 kg/m2for females). Serum levels of folate, vitamins B6, and B12 were lower than the normal reference values in all the study participants. There was depletion in glutathione levels (higher level of oxidized glutathione versus lower level of reduced glutathione) in the sera of all study participants. High serum HCY levels in both males and females (75.81±9.21 and 68.66±8.1 μmol/L, respectively) suggest that both males and females had hyperhomocysteinemia. Correlation coefficient analysis revealed that the serum HCY levels were negatively correlated with serum reduced glutathione, folic acid, vitamins B6, and B12 levels in both male and female study participants. The serum HCY level was positively correlated with age, body mass index, and serum oxidized glutathione. Proteomic measurements of N-homocysteinylation in serum albumin revealed that N-homocysteinylated albumin was present in all the assayed serum samples of study participants. The results suggest that low serum status of B-vitamins might act as a metabolic trigger for the observed hyperhomocysteinemia, oxidative stress, and pathological formation of N-homocysteinylated albumin protein, which collectively aggravates the CAD risk in the studied Omani patients.

Keywords: B-vitamins, coronary artery disease, hyperhomocysteinemia, oxidative stress

How to cite this article:
Al-Alawi KS, Waly MI, Sadiq MA, Al Balushi RM, Ali A. Biochemical Assessment of Hyperhomocysteinemia-Mediated Oxidative Stress in Coronary Artery Disease Patients: A Hospital-Based Cross-Sectional Study. Int J Nutr Pharmacol Neurol Dis 2021;11:88-93

How to cite this URL:
Al-Alawi KS, Waly MI, Sadiq MA, Al Balushi RM, Ali A. Biochemical Assessment of Hyperhomocysteinemia-Mediated Oxidative Stress in Coronary Artery Disease Patients: A Hospital-Based Cross-Sectional Study. Int J Nutr Pharmacol Neurol Dis [serial online] 2021 [cited 2022 Aug 18];11:88-93. Available from:

   Introduction Top

Westernization of food choices and sedentary lifestyle, during the last three decades, in Omani population has led to an increasing rate of non-communicable diseases, including cardiovascular diseases.[1],[2] In Oman, coronary artery disease (CAD) is the most common type of cardiovascular diseases and accounted for 29.4% of hospital deaths in 2010 and 32.5% in 2012[3],[4]. The CAD risk factors include both modifiable and non-modifiable risk factors, and clinical studies continue to support the notion that CAD is a lifestyle-related syndrome in which oxidative stress is a cofactor.[5] Hyperhomocysteinemia, elevated serum homocysteine levels, is involved in the pathogenesis of oxidative stress.[5] There are multifactorial causes of hyperhomocysteinemia including the nutritional deficiencies of certain B-vitamins (folate, riboflavin, vitamin B6 and B12).[1] Nutritional deficiencies of B-vitamins and hyperhomocysteinemia have been reported to be involved in the pathogenesis of CAD in Omani patients with CAD.[5]

Adequate dietary intake of B-vitamins is crucial for ensuring a threshold blood level of these nutrients in supporting the homocysteine (HCY)-mediated trans-methylation and trans-sulfuration pathways [Figure 1].[5] Under conditions of low folate, vitamin B6, or vitamin B12; HCY is accumulated and leads to its associated hyperhomocysteinemia and low biosynthesis of glutathione, the major intracellular antioxidant.[5] In healthy cells, more than 90% of the total glutathione pool is in the reduced form, and less than 10% exists in the disulfide form, and a low ratio of reduced glutathione/oxidized glutathione is an indication of oxidative stress.[6],[7]
Figure 1 Simplified schematic of the folate-dependent methionine cycle.

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Oxidative stress is a metabolic condition under which cellular antioxidant capacity is not counterbalancing the oxidative damage induced by various insults such as, free radicals and environmental toxins.[8] Depletion of glutathione and inhibition of antioxidant enzymes (glutathione peroxidase, superoxide dismutase, and catalase) are the biochemical indicators for the cellular oxidative damage and might be considered as metabolic triggers for cardiovascular diseases.[9]

Another proposed mechanism of HCY-mediated pathogenesis of CAD is the ensuing-alteration of circulating protein structure and loss of this protein function. HCY is covalently attached to proteins, including S-homocysteinylation by forming mixed disulfides with cysteine residues and N-homocysteinylation by forming homocystamides.[10] Formation of N-HCY proteins is an important contributor to the pathological consequences of hyperhomocysteinemia.[11],[12]

CAD constitutes a major drain on Oman’s human and financial resources, threatening the advances in health and longevity achieved over the past four decades.[13] This research aims to assess the status of B-vitamins (folate, vitamin B6, and B12) and homocysteine (HCY) in the sera of Omani coronary artery disease (CAD) patients. Conducting such study in Omani patients with CAD represents a strategy for the secondary prevention of CAD among these patients and is very important to prevent major post-infarction adverse events. To the best of our knowledge, this is the first study of its kind in Oman.

   Subjects and Methods Top

This study was conducted at a territory care hospital that treats Omani CAD patients from all over the country with inpatient and outpatient services. The study design was a cross-sectional study and was conducted from January 1st to August 30th, 2019. The study participants were known CAD patients from both genders with regular cardiology clinic follow up visits. They were enrolled on voluntary basis and were informed that they have the right to withdraw from the research study at any time without penalty or loss of benefits to which they are otherwise entitled. Before starting the study interview, signed consent forms were obtained after informing the study participants about the purpose of the study, and confidentiality was assured. All the enrolled study participants were non-smokers, free of chronic diseases (cancer, gastrointestinal diseases, atherosclerosis, chronic liver diseases, kidney diseases), endocrine disorders, and with no history of any genetic disorders. This is a hospital-based cross-sectional study and the sample size was based on the participants enrollment on voluntary basis. Based on the inclusion and exclusion criteria, 16 Omani patients (10 males and 6 females) were recruited in this study. Ethical approval was obtained from the Research Ethics Committee of the College of Medicine and Health Sciences (EC/250/17, MREC#1629). A study questionnaire was used to collect the relevant information (age and anthropometry) from all study participants, who were personally interviewed at the cardiology clinic.

The weight in kilograms (kg) and height in meters (m) were measured by the attending staff nurse at the cardiology clinic These anthropometric measurements were based on the standard protocol at the hospital. Body mass index (BMI) for every study participant was calculated as weight in kilograms divided by height in meters squared (kg/m2) and was categorized as normal, overweight and obese based on the following cut-off values.[5],[14]: Underweight: <18.5 kg/m2, normal weight:18.5–24.9 kg/m2, over weight:25.0–29.9 kg/m2, obese: 30.0–39.9 kg/m2, and morbid Obese: ≥ 40.0 kg/m2.

The blood samples were drawn according to the standard hospital procedures, fasting blood samples (10 ml of venous blood) were drawn by venipuncture into a plain tube by trained staff nurses at the phlebotomy department, Following centrifugation of the plain tubes, the sera was separated to Eppendorf tubes and was stored at −80°C for subsequent analyses. All the diagnostic tests used have high precision and have been validated by running positive and negative quality controls before running the assayed samples. The biochemical parameters analyzed in serum samples were: (a) folate, vitamins B6 and B12 by using an automated random-access immunoassay system (Siemens Medical Solutions Diagnostics, ADVIA Centaur Chemistry Analyzer, Bohemia, NY, USA), (b) homocysteine level was determined by the Immulite 2000 Homocysteine Analyzer,[14] (c) oxidative stress indices: reduced glutathione and oxidized glutathione were assayed by Glutathione Detection Assay (BioVision, Inc., CA, USA, Kit number: K264), and (d) serum identification and quantification of N-Homocysteinaled albumin (N-HCY albumin) were determined by enrichment of N-HCY albumin in the serum samples (protein digests) using aldehyde resin coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. This assay was conducted as previously reported and validated by Zang et al., 2016 [10].

The collected data were expressed as mean ± standard deviation (SD) for the continuous variables or percentages for the categorical variables. The Statistical Package GraphPad Prism Software (version 5.0) was used to analyze unpaired Student’s t-test. The unpaired t-test was used to compare the means of both the groups in order to establish the statistical evidence whether the population means differed significantly between groups. It is a parametric test that is used to compare the grouping variables. Pearson correlation coefficients (r) were quantified for determining the correlations between homocysteine and different study variables. P-value < 0.05 is considered to be significantly different. The value of the correlation coefficient (r) varied between +1 and −1. A value of ± 1 indicates a perfect degree of association between the two variables. As the correlation coefficient value goes towards 0, the relationship between the two variables will become weaker. The direction of the relationship is indicated by the sign of the coefficient; a positive sign indicates a positive relationship and a negative sign indicates a negative relationship.

   Results Top

Sixteen Omani cardiac patients (10 males and 6 females) from the outpatient cardiology clinic were included in the study. The average age for males and females were 57.64 ±9.86, 56.5 ±10.04 years, respectively with no significant difference, P > 0.05. The body mass index (BMI) of both male and female patients was comparable and was not significantly different, P = 0.71 [Table 1]. The biochemical measurements of serum levels of B-vitamins (folate, vitamin B6, and B12) are summarized in [Table 2]. It is notable that both males and females had lower serum levels of these micronutrients as compared to the reference values. As illustrated in [Figure 2], the serum levels of reduced glutathione were comparable among both males and females with no significant differences (8.17±0.91 and 7.89±0.53 μmol/L, respectively, t=0.682, df=14, P=0.51).
Table 1 Anthropometric assessment of the study participants

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Table 2 Serum measurements of folate, vitamin B6, and vitamin B12

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Figure 2 Serum glutathione levels in males and females study participants. (A) Reduced glutathione, (B) oxidized glutathione.

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Higher values for serum oxidized glutathione were detected with no significant differences between the two genders (27.81±1.21 and 28.31±1.14 μmol/L, respectively, t=0.8168 df=14, respectively, P=0.43). Both males and females had hyperhomocysteinemia as shown in [Figure 3]. The serum HCY levels in males and females were more than the normal homocysteine value, and comparable with no significant difference (75.81±9.21and 68.66±8.1 μmol/L, respectively, t=1.569, df=14, P = 0.14) [Figure 3]. As shown in [Table 3] the serum HCY was negatively correlated with serum reduced glutathione, folic acid, vitamins B6, and B12 levels in males and females. The serum HCY was positively correlated with age, body mass index, and oxidized glutathione. [Table 4] presents the proteomic measurements of N-homocysteinylation (N-HCY) in serum albumin, and it was observed that N-HCY albumin proteins were detectable in all of the assayed serum samples from both male and female patients. Detection of N-HCY albumin protein indicated the modification of albumin protein in relation to hyperhomocysteinemia.
Figure 3 Serum homocysteine (HCY) levels in males and females study participants. Normal HCY level (4–14.99 μmol/L). Hyperhomocysteinemia is divided into: Mild (15–29.99 μmol/L), intermediate (30–99.99 μmol/L), severe (≥100 μmol/L).

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Table 3 Correlations coefficients between hyperhomocysteinemia and study variables

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Table 4 Quantification of N-HCY albumin in the sera of the study participants

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   Discussion Top

The data in this study revealed that hyperhomocysteinemia and low serum levels of B vitamins (folate, vitamins B6 and B12) were associated with hyperhomocysteinemia-mediated oxidative stress as indicated by glutathione depletion (lower level of reduced glutathione and higher level of oxidized glutathione), as well as the pathological formation of N-HCY albumin. Secondary prevention of CAD is a multifactorial approach including lifestyle adjustments (diet and physical activity) and pharmacological intervention.[15],[16] Other approaches for improving long-term clinical outcomes after the surgery include the management of hypertension and diabetes mellitus, smoking cessation, weight loss, and cardiac rehabilitation.[17] Our study highlights the importance of biochemical measurements of serum B-vitamins, HCY and glutathione in order to ensure adequate intake of B-vitamins among CAD patients. This is a remarkable recommendation as it may be a missing component in secondary prevention guidelines of the American Heart Association, American College of Cardiology, the European Society of Cardiology, and the National Heart Foundation of Australian Societies.[18]

The elevated BMI among the study participants indicated the prevalence of obesity, a common risk factor of CAD. B-vitamins (folate, vitamin B6, and B12) are involved in normal HCY metabolism. In the presence of insufficient B-vitamins, HCY levels can increase due to inadequate function of HCY-dependent methylation and transsulfuration reactions.[19],[20] Results from several randomized controlled trials indicate that combinations of vitamins B12 and folic acid supplements with or without vitamin B6 decrease the homocysteine levels in patients with cardiovascular diseases.[21],[22] The data in our study revealed that serum HCY is negatively correlated with serum reduced glutathione, folic acid, vitamins B6, and B12 levels in both males and females study participants. Meanwhile the serum HCY is positively correlated with age, body mass index, and oxidized glutathione. Our data also revealed that the pathological formation of N-HCY serum albumin protein was higher than the normal human serum levels, which was previously reported to contain up to 0.3% N-HCY albumin.[23] The main limitation of this study was the limited sample number based on the enrollment rate for the study participants which was entirely on volunteer basis.

   Conclusion Top

The results of the present study illustrate that lower serum levels of B-vitamins might be a trigger for hyperhomocysteinemia and its associated biochemical modifications in serum glutathione and the formation of N-Homocysteinylated albumin protein. Oxidative stress was evidenced by the observed higher level of oxidized glutathione as compared to its reduced form, which might be considered as a biochemical sequential of hyperhomocysteinemia, the typical risk factor for CAD. This study signifies the importance of biochemical assessment of B-vitamins, HCY, and glutathione for patients with CAD. We advocate for a similar study with a larger sample size to confirm our preliminary findings.

   Acknowledgements Top

We thank all study participates who participated in this study.

Author Contributions

KSA personally interviewed the study participants, supervised the anthropometry measurements, and collected the serum samples. MIW performed the biochemical assays. MIW, MAS, AA, and RMA contributed equally in the study design and made critical revisions and arguments of the final manuscript. MIW, MAS, AA, and RA performed data analysis and provided results interpretation and review of literature. All authors have read and approved the final manuscript.

Financial support and sponsorship

We would like to acknowledge the generous support of the Food Science and Nutrition Department at Sultan Qaboos University for providing all needed resources to carry out this research project.Conflicts of Interest

There are no conflicts of interest.

   References Top

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3], [Table 4]

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