Can Vitamin D Reduce Heart Muscle Damage After Bypass Surgery?

Phase 4

Completed

Conditions: Inflammation
Vitamin D Deficiency
Cardiomyopathies
Apoptosis

Interventions: Drug: Placebo
Drug: Vitamin D

Registration Number: NCT04323852

Lead Sponsor: Shahid Beheshti University of Medical Sciences

Brief Summary: Background and study aim:

Heart diseases are among the most common causes of death worldwide. A large proportion of deaths are caused by heart attacks (myocardial infarction), where blood flow to the heart is reduced resulting in damage to the heart muscle. If the arteries supplying blood to the heart start to become blocked, Coronary Artery Bypass Grafting (CABG) surgery is a treatment to replace the blocked sections of artery can reduce angina (chest pain). However, CABG surgery has complications, including an increased risk of heart attack. Vitamin D deficiency is thought to be linked to poorer recovery from heart attack and CABG surgery. This study aims to investigate if vitamin D supplementation can reduce injury to the heart following CABG surgery.

Who can participate? Adults with vitamin D deficiency undergoing CABG

What does the study involve? Participants are randomly allocated to one of two groups. Those in the first group receive vitamin D at 3 doses per day for 3 days before surgery. The second group will receive a dummy pill (placebo). Both groups will have standard CABG surgery.

What are the possible benefits and risks of participating? Those in the vitamin D group might benefit from its effects. Vitamin D has few side effects, especially when taken for only a few days.

Where is the study run from? Shahid Modarres Hospital (Iran)

When is the study starting and how long is it expected to run for? September 2017 to January 2019

Who is funding the study? Deputy of Research of Shahid Beheshti School of Medicine

Who is the main contact? Dr Erfan Tasdighi erfan.tasdighi@gmail.com

Detailed Description: 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.

Recruitment & Eligibility

Status: COMPLETED

Sex: All

Target Recruitment: 70

Inclusion Criteria

Candidate for first-time elective CABG surgery for coronary artery disease (CAD)
Coronary artery surgery only (i.e. no valvular surgery)
Cardiopulmonary pump used during surgery
Vitamin D level below 30 ng/ml

Exclusion Criteria

Renal failure or creatinine level >1.5 mg/dl
Previous use of vitamin D supplement

Study & Design

Study Type: INTERVENTIONAL

Study Design: PARALLEL

Arm && Interventions

Group	Intervention	Description
control group	Placebo	35 patients that will receive placebo for 3 days and each day for 3 doses
vitamin D	Vitamin D	35 patients that pass the inclusion criteria and do not have exclusion criteria that will receive Vitamin D for 3 days and each time 3 doses of 50000 units

Primary Outcome Measures

Name	Time	Method
Interleukin--10 (IL-10) Serum Level	at the end of surgery after protamine reversal	The concentration of IL-10 in the serum of patients was measured using a quantitative enzyme-linked immunosorbent assay (ELISA) kit .
Caspase 2 Enzyme Level	during the surgery, an average of 3 hours	Caspase 2 enzyme activity in right atrial specimen measured by with immunohistochemistry staining The positive control and negative control were taken from human lymph node tissue. as for the control tissue a humane lymph node was used. Positivity for active caspase is shown using cytoplasmic staining . the number of apoptotic cells was measure per each high power filed (HPF) under light microscopy.
Caspase 3 Enzyme Level, an Average of 3 Hours	during the surgery, an average of 3 hours	Caspase 3 enzyme activity in right atrial specimen measured by with immunohistochemistry staining The positive control and negative control were taken from human lymph node tissue. as for the control tissue a humane lymph node was used. Positivity for active caspase is shown using cytoplasmic staining . the number of apoptotic cells was measure per each high power filed (HPF) under light microscopy.
Caspase 7 Enzyme Level	during the surgery, an average of 3 hours	Caspase 7 enzyme activity in right atrial specimen measured by with immunohistochemistry staining The positive control and negative control were taken from human lymph node tissue. as for the control tissue a humane lymph node was used. Positivity for active caspase is shown using cytoplasmic staining . the number of apoptotic cells was measure per each high power filed (HPF) under light microscopy.
Interlukin-10 (IL-10) Serum Level	Right before the intervention(3 days before surgery)	The concentration of IL-10 in the serum of patients was measured using a quantitative enzyme-linked immunosorbent assay (ELISA) kit .
Interleukin (IL-10) Serum Level	the first postoperative day	The concentration of IL-10 in the serum of patients was measured using a quantitative enzyme-linked immunosorbent assay (ELISA) kit .
Insulin Growth Factor	the first postoperative day	The concentration of the IGF-1 was also measured by a quantitative ELISA kit
Interleukin-10 (IL-10) Serum Level	procedure (before anesthesia induction)	The concentration of IL-10 in the serum of patients was measured using a quantitative enzyme-linked immunosorbent assay (ELISA) kit .

Secondary Outcome Measures

Name	Time	Method
Blood Units Usage	discharge 1 day	number of pack cell that was administered for the patient
Ventilator Application	discharge 1 day	period of time that the patient was on ventilator
Hemorrhage After Surgery	immediately after surgery	after the surgery all the hemorrhage of patients was collected by suctioning and the exact amount of bleeding has been reported.
Creatinine Level	immediately after surgery	The serum level of Creatinine in the patients which is a measurement of the kidney function