Can Vitamin D Reduce Heart Muscle Damage After Bypass Surgery?

Vitamin D Treatment Attenuates Heart Apoptosis After Coronary Artery Bypass Surgery; A Double-Blind Randomized Placebo-Controlled Clinical Trial

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

Study Overview

• Status: Completed
• Conditions: Inflammation; Vitamin D Deficiency; Cardiomyopathies; Apoptosis
• Intervention / Treatment: Drug: Placebo; Drug: Vitamin D

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.

• Study Type: Interventional
• Enrollment (Actual): 70
• Phase: Phase 4

Contacts and Locations

Study Locations

• Iran, Islamic Republic of
  • Tehran, Iran, Islamic Republic of
    • Modarres Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years and older (Adult, Older Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

Description

Inclusion Criteria:

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

Exclusion Criteria:

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

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Prevention
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Quadruple

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: 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	Drug: Vitamin D; Participants undergoing coronary artery bypass graft (CABG) surgery are randomly allocated to group A (intervention), who receive 3 doses of vitamin D (50000 U) a day for 3 days before surgery
Placebo Comparator: control group; 35 patients that will receive placebo for 3 days and each day for 3 doses	Drug: Placebo; Placebo

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Caspase 2 Enzyme Level	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.	during the surgery, an average of 3 hours
Caspase 3 Enzyme Level, 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.	during the surgery, an average of 3 hours
Caspase 7 Enzyme Level	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.	during the surgery, an average of 3 hours
Interlukin-10 (IL-10) Serum Level	The concentration of IL-10 in the serum of patients was measured using a quantitative enzyme-linked immunosorbent assay (ELISA) kit .	Right before the intervention(3 days before surgery)
Interleukin-10 (IL-10) Serum Level	The concentration of IL-10 in the serum of patients was measured using a quantitative enzyme-linked immunosorbent assay (ELISA) kit .	procedure (before anesthesia induction)
Interleukin--10 (IL-10) Serum Level	The concentration of IL-10 in the serum of patients was measured using a quantitative enzyme-linked immunosorbent assay (ELISA) kit .	at the end of surgery after protamine reversal
Interleukin (IL-10) Serum Level	The concentration of IL-10 in the serum of patients was measured using a quantitative enzyme-linked immunosorbent assay (ELISA) kit .	the first postoperative day
Insulin Growth Factor	The concentration of the IGF-1 was also measured by a quantitative ELISA kit	Right before the intervention(3 days before surgery)
Insulin Growth Factor	The concentration of the IGF-1 was also measured by a quantitative ELISA kit	procedure (before anesthesia induction)
Insulin Growth Factor	The concentration of the IGF-1 was also measured by a quantitative ELISA kit	at the end of surgery after protamine reversal
Insulin Growth Factor	The concentration of the IGF-1 was also measured by a quantitative ELISA kit	the first postoperative day

Secondary Outcome Measures

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

Collaborators and Investigators

• Sponsor: Shahid Beheshti University of Medical Sciences
• Investigators: Principal Investigator: Erfan Tasdighi, MD, Shahid Beheshti University of Medical Science

Study record dates

Study Major Dates

• Study Start (Actual): September 1, 2017
• Primary Completion (Actual): December 1, 2018
• Study Completion (Actual): January 21, 2019

Study Registration Dates

• First Submitted: March 23, 2020
• First Submitted That Met QC Criteria: March 26, 2020
• First Posted (Actual): March 27, 2020

Study Record Updates

• Last Update Posted (Actual): October 19, 2021
• Last Update Submitted That Met QC Criteria: September 21, 2021
• Last Verified: September 1, 2021

More Information

Terms related to this study

• Keywords: Cardiovascular disease; Vitamin D; Coronary artery disease; Cardiopulmonary bypass; Apoptosis; Interleukin-10; Insulin-like growth factor-1
• Additional Relevant MeSH Terms: Pathologic Processes; Heart Diseases; Cardiovascular Diseases; Nutrition Disorders; Avitaminosis; Deficiency Diseases; Malnutrition; Inflammation; Vitamin D Deficiency; Cardiomyopathies; Physiological Effects of Drugs; Micronutrients; Vitamins; Bone Density Conservation Agents; Vitamin D
• Other Study ID Numbers: 13312; ISRCTN44896820 (Registry Identifier: ISRCTN); IRCT20180118038423N1 (Registry Identifier: IRCT)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: No

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No