Clinical trial NCT04323852
Conditions: Vitamin D Deficiency Cardiomyopathies Apoptosis Inflammation
Keywords: Vitamin D Cardiovascular disease Apoptosis Insulin-like growth factor-1 Coronary artery disease Cardiopulmonary bypass Interleukin-10
Sponsor: Shahid Beheshti University of Medical Sciences
Start date: 2017-09-01; Completion date: 2019-01-21
Enrollment: 70 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

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.

Arms: arm 1: 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 arm 2: 35 patients that will receive placebo for 3 days and each day for 3 doses
Interventions: intervention 1: Vitamin D intervention 2: Placebo
Intervention descriptions: intervention 1: 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 intervention 2: Placebo

Protocol documents:
Prot_000.pdf: 
 
Can Vitamin D Reduce Heart Muscle Damage After Bypass Surgery? 
 
NCT04323852 
 
24/10/2020 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
Study protocol: 
 
Trial Design 
This randomized, double-blind, placebo-controlled study was conducted for 70 patients undergoing 
coronary surgery with CPB. The study was approved by the local Ethical Committee. The informed 
written consent was obtained from the patients prior to enrollment. Enrollment was started in December 
2018 and was accomplished in April 2019. The inclusion criteria were as follows: the patients who were 
referred for elective and isolated CABG using CPB with vitamin D deficiency (defined as 25- 
hydroxyvitamin D < 20 ng/mL) and normal kidney function (creatinine < 1.5 mg/dL). Vitamin D was 
measured by an 
immunoluminometric assay. The exclusion criteria were as follows: recent myocardial infarction, urgent 
CABG, nonisolated coronary surgery, redo surgery, malignant disease, the presence of acute or chronic 
inflammatory diseases, history of vitamin D treatment within previous 6 months, or unwillingness to 
participate. 
Intervention 
Following the informed consent, eligible study participants were randomly assigned (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 3 inactive medication tablets similar to those for other group, daily at the same time point 
(Figure 1). 
Except the pharmacists, all the investigators, patients, and the medical team were blinded to the group 
allocation. Coronary artery bypass was performed in the culprit lesions for both groups by one surgical 
team. The standard protocols for general anesthesia, surgical, and CPB management were 
performed for all patients and have already been described in detail. 
Outcome Measures 
The primary outcome was the degree of heart apoptosis by measuring the caspases 2, 3, and 7 activity 
from right atrial specimen with immunohistochemistry staining, the serum level 
of anti-inflammatory interleukin-10 (IL-10) and insulin-like growth factor 1 (IGF-1), and N-terminal 
(NT)-pro-brain natriuretic peptide (pro-BNP). The biopsy from right atrial appendage was taken at the 
end of surgery after removing the venous cannula in a nontraumatic manner and then was kept in formalin 
and parafinized in less than 24 hours. Blood samples were collected firstly at the baseline (by passing 24 
hours from hospital admission; T1), before anesthesia induction (T2), at the end of surgery after 
protamine reversal (T3), and also at the first postoperative day (T4) in order to measure the serum level of 
IGF-1, IL-10, and pro-BNP. The blood samples were centrifuged at 2500 rpm for 15 minutes within 1 
hour after blood sampling, and the serum was stored at 20C until assayed. 
Enzyme-Linked Immunosorbent Assay 
The concentration of IL-10 was measured using a quantitative enzyme-linked immunosorbent assay 
(ELISA) kit (Ref: BE53101, IBL International GmbH, Hamburg, Germany). The concentration of the 
IGF-1 was also measured by a quantitative ELISA kit (Ref: MD58011, IBL International GmbH). The 
proBNP measurement was done using a commercially available 2- site chemiluminescent immunometric 
assay (IMMULITE 2000 
NT-pro-BNP, Siemens Healthcare, Mannheim, Germany). Serum vitamin D was detected using the high-


performance liquid chromatography method (Agilent 1100 series HPLC, Germany). 
Immunohistochemistry Studies 
Immunohistochemical staining was performed on 5-mm thick sections. The slides were incubated at 37C 
for 24 hours and then deparaffinized in preheated xylene and rehydrated through descending grades of 
alcohol, and after that they were washed in distilled water. Heat-induced antigen retrieval was done by 
microwave oven with citrate buffer (pH 6.0) for anti-caspase 7 and Tris–EDTA (pH ¼ 8) for anticaspases 
2 and 3. Endoperoxidase blocking was done by adding hydrogen peroxide onto the sections. Then, the 
protein block was added for 5 minutes, and slides were washed in Tris-buffered saline (TBS). The 
primary 
antibody as anti-caspase 2 antibody, rabbit monoclonal (clone EPR16790, abcam), anti-caspase 3 
antibody, rabbit monoclonal (clone EPR16888, abcam), anti-caspase 7 antibody, and mouse monoclonal 
(clone 7-1-11, abcam) antibody were added and kept for 30 minutes, then they were washed in TBS. 
Mouse and Rabbit 
Specific HRP/DABIHC DetectionMicro-polymerKit (ab80436) were used and incubated for 15 minutes, 
and then were washed with TBS. 3,30-Diaminobenzidine (DAB) chromogen was added and kept for 5 
minutes. Slides were washed in distilled water, and counterwas stainedwith hematoxylin. Sections 
containing lymph 
node tissue were used as positive control. Negative control included primary antibody replaced with 
phosphate-buffered 

SAP_001.pdf: 
 
Can Vitamin D Reduce Heart Muscle Damage After Bypass Surgery? 
 
NCT04323852 
 
24/10/2020 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
Statistical analysis: 
 
The patients’ number (35 patients per group) was determined based on the 
previous trials investigating the caspase activity in the CABG setting.15,22,23 
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 were applied using the Bonferroni 
correction (type I error correction), to evaluate the change of measured 
inflammatory markers between the groups by over time. The Kolmogorov-
Smirnov test was performed for normality. Continuous variables and categorical 
variables were compared between groups using Student t test (or Mann-Whitney 
test for those meeting abnormal distribution) and w2, respectively. All the 
statistical analyses were performed using SPSS version 23 (SPSS, Chicago, 
Illinois). A P value <.05 was considered as significant.