Can Vitamin D Reduce Heart Muscle Damage After Bypass Surgery?

Enrollment started in June 2018 and was completed in December 2018. The inclusion criteria were as following: the patients referred for elective and isolated Coronary Artery Bypass Graft (CABG) using Cardiopulmonary Bypass (CPB) with vitamin D deficiency (defined as 25-hydroxyvitamin D [25(OH) D] < 20 ng/mL) and normal kidney function (creatinine <1.5mg/dL). The exclusion criteria were: recent myocardial infarction, urgent CABG, non-isolated coronary surgery, redo surgery, malignant disease, presence of acute or chronic inflammatory diseases, history of vitamin D treatment within previous 6 months, or unwillingness to participate.

Intervention Following informed consent, eligible study participants were randomly assigned (by using a computer- generated random code) in a 1:1 ratio to receive either placebo or a total of 450,000 international units (IU) vitamin D3 (three 50,000 IU of vitamin D3 tablet daily for 3 days) before operation. The placebo group received three inactive medication tablets daily at the same time point. With the exception of the pharmacists, all the investigators, patients and the medical team were blinded to the group allocation.

Coronary artery bypass was done in the culprit lesions for both groups by one surgical team. The standard protocol for general anesthesia, surgical and CPB management were performed for all patients and have already been described in detail [16].

Outcome measures The primary outcome was the degree of heart apoptosis by measurement of caspase 2, 3 and 7 activity from right atrial specimen with immunohistochemistry staining, and the serum level of anti-inflammatory interleukin-10 (IL-10) and insulin- like growth factor (IGF-1), and N-terminal pro v-type Brain Natriuretic Peptide (nt-pro BNP). The biopsy from right atrial appendage was taken at the end of surgery after venous cannula removal in a nontraumatic fashion, kept into formalin and in less than 24h parafinized. Blood samples were collected at the baseline (T1), before anesthesia induction (T2), at the end of surgery after protamine reversal (T3) and the first postoperative day (T4) to measure the serum level of IGF-1, IL-10 and pro BNP. The blood samples were centrifuged at 2500 rpm for 15 min within one hour after blood sampling, and the serum was stored at -20°C until assayed.

Enzyme-linked immunosorbent assay The concentration of IL-10 was measured by a quantitative ELISA kit . The concentration of the IGF- 1 was measured by a quantitative ELISA kit .

Serum vitamin D was detected by using the high performance liquid chromatography method . The pro BNP measurement was done using a commercially available two- site chemiluminescent immunometric assay .

Immunohistochemistry studies Immunohistochemical staining was performed on 5-micrometer thick sections. The slides were incubated at 37°C for 24 hours and de-paraffinized in pre-heated xylene and rehydrated through descending grades of alcohol, washed in distilled water. Heat induced antigen retrieval was done by microwave oven with citrate buffer (pH 6.0) for anti-caspase-7 and Ethylenediamine Tetraacetic Acid; buffered solution (Tris-EDTA) (pH=8) for anti-caspase-2 and 3. endoperoxidase blocking was done by adding hydrogen peroxide on the sections. The protein block then added for 5 minutes, slides were washed in Tris-Buffered Saline (TBS). .The primary antibody as anti-caspase-2 antibody, rabbit monoclonal , anti-caspase-3 antibody, rabbit monoclonal , anti-caspase-7 antibody, and mouse monoclonal (clone 7-1-11 , abcam) antibody were added and kept for 30 minutes, washed in TBS. Mouse and Rabbit Specific horseradish peroxidase/Diaminobenzidine (HRP/DAB) immunohistochemistry (IHC) Detection Micro-polymer Kit were used and incubated for 15 minutes then washed with tris-buffered saline (TBS). Diaminobenzidine (DAB) chromogen was added and kept for 5 minutes. Slides washed in distilled water and counter stained with hematoxylin. Sections containing lymph node tissue were used as positive control. Negative control included primary antibody replaced with phosphate buffered saline (PBS). Immunostained sections were reviewed for cytoplasmic expression of anti-caspase-2, anti-caspase-3 and anti-caspase-7. The number of immune-reactive cells per High Power Field (HPF) (X 400) was counted. For this purpose at least 10 HPF were assessed and the average of positive cells was recorded.

Sample size and statistical methods The determination of the patient number (30 patients per group) was based on previous trials investigating the caspase activity in the CABG setting .

Categorical variables were reported as numbers and percentages, whereas mean± standard deviation was expressed for continuous variables. Repeated measures of analysis of variance and multiple comparisons using the Bonferroni correction (type I error correction) were applied for evaluating the change of measured inflammatory markers between the groups over time. The Kolmogorov-Smirnov test for normality was performed. Continuous variables and categorical variables were compared between groups using Student's t test (or Mann-Whitney test for those meeting abnormal distribution) and Chi-square, respectively. All the statistical analyses were performed using SPSS version 23 (SPSS, Chicago, IL, USA). A p values <0.05 was considered to be significant.