Ethical Issues and Sample Collection

This study was conducted by the Genetic Engineering and Biotechnology Research Institute at the University of Sadat City, Egypt, in collaboration with the International Medical Center, from February to December 2024. The research received approval from both the medical ethics committee of the International Medical Center and the scientific ethics committee of the Genetic Engineering and Biotechnology Research Institute. The purpose of the study was thoroughly explained to all participants, who provided informed consent and understood the study’s rules and regulations. Key inclusion criteria were that samples were collected from both patients and healthy individuals with a mean age of 45 ± 10 years, prior to any exposure treatment. All patients were examined by a qualified stroke neurologist, and ischemic stroke was confirmed using CT scans and MRI. The exclusion criteria eliminated individuals with significant cardiac conditions, those younger than 35 or older than 55 years, and individuals with obesity, diabetes mellitus, tumors, renal or hepatic failure, or those taking medications that could influence levels of vitamin B12, folate, or zinc. Patients with thyroid or rheumatologic diseases, as well as those with a history of immunosuppressive or analgesic therapy, were also excluded. All participants underwent a comprehensive physical examination and standard biochemical testing, including a blood lipid profile. Clinical data collected from patients included hypertension, diabetes status, level of consciousness, swallowing difficulties, and smoking history. Control samples were collected from healthy individuals, comprising 50 males and 50 females within the study’s age range. These individuals had no history of clinical or radiological signs of cerebrovascular disease. Additionally, individuals who were smokers, underweight, suffering from cachexia, or had a history of eating disorders were excluded. A total of 100 blood samples were collected from patients diagnosed with thrombotic strokes, comprising 50 males and 50 females within the study’s specified age range. All stroke patients were accurately diagnosed and received medical care and supervision at the International Medical Center.

Homocysteine Determination

The quantitative determination of homocysteine levels in plasma samples was performed using a human ELISA Kit (CSB-E13814 h, CUSABIO, USA), which contains a specific antibody pre-coated onto a microplate. Plasma samples were collected using EDTA as an anticoagulant, following the manufacturer's protocol. The samples were centrifuged within 30 min of collection at 1000 × g for 15 min at 2–8 °C. The plasma was then aliquoted and stored at −20 °C for up to 5 days before testing. Prior to testing, all reagents were allowed to equilibrate at room temperature for 30 min, and the standard solution was freshly prepared through serial dilution. A total of 100 µL of both the standards and samples were carefully pipetted into the wells of the ELISA plate. The plate was covered with the provided adhesive strip and incubated for 2 h at 37 °C. Afterward, 100 µL of biotin-labeled antibody (1x) was added to each well and mixed gently. The plate was covered again with a new adhesive strip and incubated for an additional hour at 37 °C. Following incubation, the buffer was removed from each well, and the plate was washed twice with 200 µL of Wash Buffer. Then, 100 µL of HRP-avidin (1x) was added to each well, and the plate was covered with a fresh adhesive strip and incubated for 1 h at 37 °C. After incubation, the plate was washed twice as described previously. Finally, 50 µL of stop solution was added to each well, and the optical density was measured within 5 min using an ELISA reader set to 450 nm.

Erythrocyte Sedimentation Rate (ESR)

The Global Scientific Sedi-Rate™ (Autozero Westergren ESR System, USA) was used to measure the ESR in the collected samples. This system features a fibrous plug that prevents harmful substances from escaping through the top of the pipette. The polypropylene vial has a self-sealing stopper that can be easily pierced with a transfer pipette or piercing funnel, and the polystyrene pipette is graduated from 0 to 180 mm. To conduct the test, blood samples were drawn into a Westergren tube until the blood level reached 200 mm (mm). The tubes were then kept in a vertical position and stored at room temperature for one hour. The ESR was calculated by measuring the distance between the top of the blood mixture and the top of the sedimented red blood cells. The result, expressed in millimeters per hour (mm/h), indicates how quickly the red blood cells settle to the bottom of the test tube.

Low-Density Lipoprotein (LDL) Measurement

The LDL concentration in the obtained samples was determined using Beta-quantification analysis. Plasma aliquots with a density of 1.006 g/mL were subjected to ultracentrifugation, which resulted in the separation of VLDL and chylomicrons in the floating layer (d < 1.006 g/mL) above the infranatant containing IDL, LDL, and HDL (d > 1.006 g/mL). Cholesterol concentration in the bottom fraction was then measured after treating it with heparin and manganese chloride to precipitate ApoB, including IDL and LDL particles. Following centrifugation, the supernatant was discarded, and the HDL cholesterol concentration in the bottom fraction was determined. The LDL cholesterol concentration was calculated by subtracting the HDL concentration from the total cholesterol concentration in the bottom fraction containing IDL, LDL, and HDL (d > 1.006 g/mL). The concentration of VLDL-C in the ultracentrifugal supernatant was measured by subtracting the cholesterol concentration in the bottom fraction (d > 1.006 g/mL) from the total cholesterol concentration in the initial plasma aliquot before ultracentrifugation. VLDL-C = Total cholesterol – d > 1.006 g/mL cholesterol. It is preferred to calculate VLDL-C rather than directly measure it in the ultracentrifugal supernatant, as recovering cholesterol from this fraction, particularly with a high concentration of triglycerides, is challenging. The LDL-C concentration was calculated as [d > 1.006 g/mL cholesterol] – HDL cholesterol. In blood obtained samples, the cut-off level of LDL-C was considered as 70 mg/dL (Khalil et al. 2019).

Determination of Vitamin B12 and Folic Acid Levels

To measure vitamin B12 levels, 15 μL of serum was incubated for 9 min at 37 °C with vitamin B12 pretreatment reagents (pretreatment one and pretreatment two). Following this, the pretreated samples were incubated for another 9 min at 37 °C with a diluted (1:1000) solution of ruthenium-labeled vitamin B12 binding factor (REF 07212771190, Roche, Switzerland). This was followed by a further incubation for 9 min at 37 °C with diluted (1:1000) streptavidin-coated microparticles and biotin-labeled vitamin B12 (REF 07212771190, Roche, Switzerland). The reaction was then transferred into measuring cells, and the chemiluminescent emission generated was measured by a photomultiplier. The final values were calculated using a master curve provided via the reagent barcode. Similarly, folate levels in prepared serum were measured by incubating 25 μL of serum with folate pretreatment reagents (pretreatment one and two), a diluted (1:1000) solution of ruthenium-labeled folate binding protein, and biotin-labeled folate (REF 07559992190, Roche, Switzerland). Deficiency was defined as vitamin B12 levels < 203 pg/ml and folate levels < 3 ng/ml (ÖZKAN et al. 2007; Singh et al. 2017).

SNP Analysis in Stroke Patients Derived Blood Samples

The genotyping of six thrombosis-related SNPs (FV R506Q, H1299R, Y1702 C, FII G20210 A, and MTHFR C677 T, A1298 C) in blood samples from stroke patients was conducted using the CFX96™ Real-Time PCR System (C1000 Touch Thermal Cycler, Bio-Rad, UAS) program for SNP detection. In brief, genomic DNA was extracted from whole blood using silica-based spin columns, such as the Qiagen purification kit (USA). Genotyping of six SNPs (FV R506Q, H1299R, Y1702 C, FII G20210 A, and MTHFR C677 T, A1298 C) was performed using ready-to-use TaqMan assays from Thermo Fisher. For each assay, 20 ng of gDNA was amplified following the manufacturer's protocol, with a final reaction volume of 20 μL in a CFX96™ Real-Time PCR System thermal cycler. Each reaction was conducted in 96-well plates, using 8 μL reactions and the TaqMan® Genotyping Master Mix, as recommended by Thermo Fisher. PCR cycling was carried out on the Applied Biosystems® 7900 Real-Time PCR System according to the manufacturer's instructions, and the data were analyzed with SDS 2.4 software. Post-amplification products were examined on the Applied Biosystems® ViiA™ 7 Real-Time PCR System, and genotype calls were manually assigned by comparing with six No Template Controls.

DNA Purification and DNA Methylation Analysis

Genomic DNA was isolated from samples of healthy individuals and stroke patients using a DNA purification kit (Qiagen, USA), following the manufacturer's protocol. One microgram of purified DNA was treated with 1 M sodium bisulfite and 10 µL of DNA-protecting buffer in a total volume of 25 µL, using RNase- and DNase-free water. The mixture was initially denatured by incubating at 95ºC for 5 min, followed by a 7-h incubation at 50ºC to allow sodium bisulfite to convert unmethylated cytosines into uracil. To ensure complete conversion, this process was repeated three times using a thermal cycler (BIO-RAD, USA). The treated DNA was then used to amplify the promoter regions of the FV, F11, and MTHFR genes with specific primers listed in Table 1. Quantitative PCR (qRT-PCR) was performed using the QuantiTect SYBR Green PCR Kit (Qiagen, USA), with the GAPDH promoter region primer serving as a housekeeping gene for normalization. The qRT-PCR conditions were as follows: an initial denaturation at 95ºC for 5 min, followed by 35 cycles of 95ºC for 30 s, 60ºC for 30 s, and 72ºC for 30 s, with a final extension at 72ºC for 10 min to complete the amplification (Mohamed et al. 2022; Khalil et al. 2024). The methylation status within the promoter regions of the FV, F11, and MTHFR genes was determined by comparing fold changes in blood samples from stroke patients to those from healthy controls. To further assess the methylation status of the promoter regions in the specified genes, the genomic DNA was treated with a methylation-dependent restriction enzyme. A methylation-dependent enzyme, MspJI, which only cleaves the methylated cytosine in their restriction site (mCNNR(N)9). The digestion was carried out with 5 units of enzyme per 200 ng of DNA for 4 h at 37ºC. Post-digestion, the DNA was used for PCR amplification of the FV, F11, and MTHFR gene promoter regions, using the specific primers listed in Table 1. Conventional PCR conditions were as follows: an initial denaturation at 95ºC for 5 min, followed by 30 cycles of 95ºC for 30 s, 60ºC for 30 s, and 72ºC for 45 s. The PCR products were then resolved on a 1% agarose gel in 1X-TBE buffer and visualized under UV light (320 nm) using a gel documentation system (Elawdan et al. 2022; Guirgis et al. 2023). After amplification, methylated cytosines, cleaved by MspJI, were represented by two bands around with molecular wight of about 150 bp and 100 bp, while unmethylated fragments appeared as a single band at 250 bp.

Table 1 Oligonucleotides sequences used for promoter region of FV, FII, MTHFR, and GAPDH genesStatistical Analysis

Hardy–Weinberg equilibrium (HWE) for each SNP was assessed using Pearson’s χ2 tests. This test was also employed to evaluate differences in allelic and genotypic frequencies of between patients and controls (Graffelman 2010). Delta-delta Ct analysis was employed to assess relative gene expression, represented by fold changes in steady-state mRNA levels. Ct values for the housekeeping gene, GAPDH, were utilized for normalization. A two-tailed t-test was performed to evaluate differences in the data, with P < 0.05 indicating statistical significance (*) and P < 0.01 indicating high statistical significance (**) (Maher et al. 2020; Fekry et al. 2022).