A comparative study on fatty acid profile in selected vessels of coronary artery bypass graft (CABG).

Atherosclerosis is one of the leading non-communicable diseases in Sri Lanka. Analysis of fatty acid composition in blood vessels is important in understanding the development of atherosclerosis. Here, analyses of fatty acid profiles in major arteries which are commonly used in Coronary Artery Bypass Graft surgery (CABG) were subjected to investigation. Patients (n = 27) undergoing elective CABG were enrolled in the study. A small biopsy segment of the saphenous vein (SV), radial artery (RA), and left internal mammary artery (LIMA) of patients was obtained during the surgery. The fatty acid (FA) profile of tissue samples was analyzed using Gas Chromatography-Mass Spectroscopy (GCMS). Among the different arteries tested, palmitic acid and stearic acid were the predominant fatty acids. As far as monounsaturated FA (MUFA) are concerned, oleic acid was found to be the most abundant MUFA in vessels. The FA profile of LIMA samples had a higher SFA percentage and lower unsaturated FA percentage compared to other vessels. Furthermore, the vessel samples of RA indicated the highest percentage of pro-inflammatory ω -6 polyunsaturated fatty acids (PUFA) as well as a higher percentage ratio between ω -6: ω -3 PUFA. The fatty acid composition and ω -6: ω -3 PUFA ratio suggests that LIMA graft is preferred for CABG over RA and SV.

Introduction

Ischemic heart disease is the leading cause of death worldwide [1]. The success of coronary artery bypass grafting (CABG) which is one of the common surgical techniques used to improve the blood flow in the heart is dependent on the long-term patency of arterial and venous grafts [2]. The majority of patients receive left internal mammary artery (LIMA) grafts to the left anterior descending (LAD) coronary artery compared to other grafting options such as saphenous vein (SV) grafts or radial artery (RA) [3]. According to literature, the LIMA is considered a superior graft for long-term patency when compared to a vein graft [4]. Both histological and chemical composition of grafts could have influences on early proatherogenic changes and late graft failure 3–5 years of surgery [5, 6].

Fatty acid composition of vessel walls is an important modulator in vascular function [7]. Changes in fatty acid composition can modify the properties of cell membranes including fluidity and permeability, either directly via their insertion in the membrane or indirectly through their effects on lipid metabolism [8]. Hence, several studies have observed the influence of the vessel wall fatty acid composition in atherosclerosis [9, 10].

It has been found that inflammation present in advanced plaques can lead to critical cardiovascular events, thus fatty acid composition and inflammatory cells in vessel walls are key to plaque stability and morphology [11]. Furthermore, contribution to inflammation from eicosanoids originating from arachidonic acid can be controlled by increasing the levels of EPA and DHA in cells of vessel walls [11]. Therefore, the ratio of such ω-6 fatty acids to ω-3 fatty acids is important in the determination of inflammation [12]. Though there are abundant studies correlating atherosclerosis with plasma fatty acid composition [13, 14], only limited studies have been conducted on the analysis of the vessel wall fatty acid compositions. A comparative study conducted on aorta and LIMA wall fatty acid profiles have found differences in the fatty acid profile of aorta and LIMA and suggest individual fatty acids as a contributing factor for atherogenesis by observing lower levels of unsaturated fatty acids and higher amounts of saturated fatty acids in the atherosclerotic aorta [10]. Similarly, high levels of polyunsaturated fatty acids have been observed in SV compared to aortic tissue [15].

These findings emphasize the importance of fatty acid profiles in atherosclerotic tissues and their role in atherogenesis and coronary artery disease (CAD). Even though the fatty acid profile of SV, and LIMA had been studied before [10, 15], there was no information on the comparison of these profiles with radial arteries. Therefore, this study aims to compare the fatty acid profile of common vessels used in CABG. Such comparison of fatty acids present in different vessels will aid in the understanding the association between fatty acid profile and atherosclerotic plaque redevelopment.

Materials and methods

Selection of CAD patients

Twenty-seven angiographically proven coronary artery disease patients were enrolled in this study. The sample size was calculated using a single group standard deviation of Docosahexaenoic acid level of the internal mammary artery [10]. The subjects were accepted for elective CABG at Nawaloka Hospital, Colombo, Sri Lanka. Biopsy samples (1–2) g of LIMA (12 samples), SV (20 samples), and RA (9 samples) were obtained during surgery (Total 41 samples). Ethical clearance (Ref. 34/19) was obtained from the ethics review committee of the Faculty of Medical Sciences, University of Sri Jayewardenepura. The study was conducted according to the guidelines set by the Declaration of Helsinki. Informed written consent from all participants was obtained prior to recruitment to the study.

Fatty acid analysis

After sampling the specimens were preserved in methanol and kept in a refrigerator (2–8°C) until analysis. The fatty acid extraction was mainly based on the Folch method [16]. Each tissue sample was homogenized with chloroform (SRL, reagent grade): methanol (SRL, reagent grade) mixture (2:1 v/v) to a final 20-fold dilution and methanol used in preservation was used here. The sample was washed with 0.9% NaCl solution and was followed by centrifugation. The upper phase was discarded and the lower layer which contains the fat extract was evaporated to near dryness. The extracted fatty acids were methylated in the presence of the base catalyst KOH (Sigma Aldrich) in methanol. The Fatty acid methyl esters were then extracted to hexane (SRL, HPLC grade) by centrifuging. Gas Chromatography-Mass Spectroscopy (GCMS) analysis was performed on Agilent 7890A GC coupled to 5975C MS with Triple-Axis Detector using a split /splitless inlet with He as the carrier gas with 1 mL/min rate. The HP 5MS column with dimensions 30m, 0.25mm, 0.25um was used for analysis. The oven temperature program was as follows: the column was initially held at 90 ⁰C for 1 min after injection, then increased to 150 ⁰C with a 12 ⁰C/min heating ramp for 5 min. Then increased to 180 ⁰C with a 5 ⁰C/min heating ramp for 6 min and increased to 210 ⁰C with a 3 ⁰C/min heating ramp for 10 min and finally to a temperature of 250 ⁰C with a rate of 10 ⁰C/min for 4 min. The total run time was 39 min. The injector port temperature was held at 270 ⁰C. MS source temperature was 230 ⁰C and MS Quadrupole temperature was at 150 ⁰C and mass range analysis was at 50–550 m/z. The compound identification was done comparing MS results with NIST database Chemstation® software. The percentage of individual fatty acids was calculated as the area percentage of each fatty acid to the total area of all fatty acids detected.

Statistical analysis

The data was analyzed using SPSS version 25. Since the Kolmogorov–Smirnov test reported that the variables are not normally distributed, Mann–Whitney U-tests were used to compare the fatty acid percentage of LIMA, SV, and RA. A p-value of < 0.05 was considered to be significant. A linear regression model was developed to determine the association of confounding variables with fatty acid levels. The results were reported as mean ± standard deviation (SD).

Results

The demographic and clinical characteristics of the study subjects are shown in Table 1. The study included 27 patients with an average age of 58 years and 85% of the study population were male. Almost half of the patients recruited were diabetic (53%), 74.1% were hypertensive and 84.1% were diagnosed as hyperlipidemic. The average lipid profile parameters of the study group were within normal reference ranges. More than 2/3 of individuals had myocardial infarction prior to CABG. Out of 23 males, 20 individuals had history of smoking and 05 of them are still continuing occasionally. The estimated 10-year primary risk of atherosclerotic cardiovascular disease (ASCVD) among patients was estimated and was found to be > 7.5% for all. Out of sample, 20 individuals were in high-risk group and 07 in the intermediate-risk group [17].

Table 1

Demographic and clinical characteristics of patients in the study.

CAD patients (n = 27)
Age (y)	57.9 ± 8.3
Gender (n)
Male	23
Female	4
Hypertension (%)	74.1%
Diabetes (%)	55.5%
Hyperlipidemia	81.4%
Total Cholesterol (mg/dL)	162 ± 36
LDL	104 ± 40
HDL	36 ± 8
Triglycerides	145 ± 40
Statin use Atorvastatin 40 mg daily dose	92.6%
Atorvastatin 40 mg daily + Ezetimibe 10 mg	7.4%
Family history of CAD	59.3%
Prior Myocardial Infarction	70.3%
History of Smoking (n = 20/27)	74.0%
Previously smoked (n = 15/20)	75%
Currently smoking (n = 5/20)	25%
ASCVD risk estimation (range %)
Low (< 5%)	-
Borderline (5–7.4%)	-
Intermediate (7.5–19.9%) n = 7	8.5–18.2%
High risk (> 20%) n = 20	25–47%

The percentage of individual fatty acids is given in Table 2 and fatty acid profiles of the three samples LIMA, SV, and RA have been compared. The percentages of fatty acids are given in S1 Table for LIMA, S2 Table for SV, and S3 Table for RA.

Table 2

Fatty acid composition (Mean%±SD) of LIMA, SV, and radial artery in patients (n = 27) with CAD.

	Segment
Fatty acid	LIMA	SV	RA
12:0 (Lauric acid)	6.49 ±3.40*	3.41 ± 4.12	3.32 ± 2.43
14:0 (Myristic acid)	9.18 ±4.67	7.15 ± 10.14	7.33 ± 2.79
16:0 (Palmitic acid)	36.04 ± 13.57	30.44± 11.42	31.84 ±12.20
16:1 (Palmitoleic acid)	6.66 ± 5.76	7.61 ± 4.37	5.39 ±2.43
18:0 (Stearic acid)	6.45 ± 4.95	9.62 ± 11.10	5.78 ±2.19
18:1 (Oleic acid)	31.76 ± 13.96	40.30± 20.61	40.59 ±8.25
18:2, ω-6 (Linoleic acid)	5.96 ± 4.08	3.78 ± 2.44	5.79 ±2.79
20:4, ω-6 (Arachidonic acid)	0.49 ± 0.42*	4.44 ± 3.69	1.72 ±0.92
22:6, ω-3 (Docosahexaenoic acid)	0.41 ± 0.29	1.66 ±1.24	0.27 ±0.06

* p<0.05, mean values are significantly different compared to SV and RA

Palmitic acid (16:0) was the major fatty acid in all the tissues followed by oleic acid (18:1) and stearic acid (18:0). The percentage concentration of the monounsaturated fatty acid, oleic acid (18:1) was comparatively higher in SV and RA than LIMA.

The comparison of the fatty acid profile of LIMA with other vessels indicates a significantly high percentage of lauric acid (C12:0) compared to SV (p = 0.001) and RA (p = 0.036). A significantly lower arachidonic acid (AA) percentage was observed in LIMA compared to SV (p = 0.018) and RA (p = 0.019). The only ω-3, PUFA reported was Docosahexaenoic acid (DHA), with the lowest percentage being found in RA.

In order to obtain a summary of the above variations, the levels of saturated (SFA) and unsaturated fatty acids(USFA) were analyzed and the results are given in Table 3. In LIMA samples, SFA content was higher than the USFA. In contrast, SV and RA contained a higher proportion of USFA than SFA. The Ratio of AA, ω-6/ DHA, ω-3 was significantly higher in RA compared to SV and LIMA.

Table 3

Summary of main categories of fatty acids in LIMA, SV and RA.

	Segment
Fatty acid	LIMA (Mean%±SD)	SV (Mean%±SD)	RA (Mean%±SD)
Saturated FA (SFA)	54.63± 12.8	45.65±16.06	48.27± 9.5
Unsaturated FA (MUFA + PUFA)	45.37± 12.8	54.35± 16.0	51.73± 9.5
USFA: SFA	0.95± 0.65	1.8 ± 2.4	1.16 ± 0.53
PUFA, ω-6	5.2 ± 2.3	5.7 ± 4.2	6.3 ± 3.5
AA, ω-6/ DHA, ω-3	1.8 ± 0.57	2.9 ± 0.9	5.4 ± 0.6*

*p<0.05, mean value is significantly different compared to SV and LIMA.

The linear regression models were developed for fatty acid with significant variance to determine the effect of confounding factors and the results are as given in S4 Table. The factors considered were age, gender, and presence of diabetes mellitus, hypertension, hyperlipidemia, family history of CAD, smoking, and lipid profile parameters. The results revealed that family history of CAD was significantly associated with a lauric acid level of SV (B = 1.922, p = 0.05, 95% CI; -0.12–3.85). Family history of CAD (B = 2.996, p = 0.026, 95% CI; 0.447–5.54) and smoking (B = 3.642, p = 0.039, 95% CI; 0.225–7.06) were significant factors in associated with arachidonic acid level of saphenous vein. Further, gender was a significant (B = 0.409, p = 0.044, 95% CI; 0.013–0.804) confounding factor for increased arachidonic acid level in LIMA.

Discussion

The present study reports the comparison of the fatty acid composition of three biopsy specimens of three important vessels collected during CABG, namely LIMA, RA, and SV for the first time in literature. The FA profile of vessels that are implanted during CABG can have an effect on long-term stability. The results were compared with similar studies found in the literature where two vessels have been compared.

Studies conducted by Oskouei and coworkers have compared the fatty acid composition of aorta and SV and indicated that SV had a higher percentage of PUFA [15]. The percentages of major FA in aortic samples indicated higher amounts of palmitic acid and lower amounts of oleic acid compared to SV. The fatty acid profiles of LIMA and aorta were compared by Bahrami et al and the results indicate higher SFA content in the aorta than the LIMA with palmitic acid and stearic acid as the major SFA [10]. Therefore, FA profiles of SV and LIMA indicate lower SFA content than aorta in literature. According to observations in this study, SV contained a high percentage of USFA and a low percentage of SFA compared to both RA and LIMA. Although the SFA content was high for RA than the SV yet it was lower than in LIMA. Since palmitic acid percentages are almost the same, higher stearic acid content observed in RA results in a higher percentage of SFA. The opposite trend was observed for USFA in RA.

When considering the long-term success of the grafted vessels, it is important to prevent the redevelopment of atherosclerotic plaque [18]. Plaque develops as a consequence of endothelial injury and activation, monocyte accumulation and formation of foam cells, lipid accumulation and smooth muscle proliferation (atherogenesis), extreme inflammation. Plaque progression and plaque rupture ultimately cause thrombogenesis and obstruct the blood flow to cardiac muscles [19]. Therefore, it is worth considering the role of ω-3 and ω-6 fatty acid levels in the redevelopment of atherosclerotic plaque [20].

Several studies have reported the effects of ω−3 fatty acids on the improvement of vascular structure and function. Reduced LDL uptake in the arterial wall is a reported function of ω−3 fatty acids which is achieved by reducing expression of lipoprotein lipase [21]. The effect of ω−3 fatty acids on smooth muscle cells has been studied in cell lines and human coronary arteries. Smooth muscle cell proliferation has been suppressed through inhibition of DNA synthesis and replication in ω−3 fatty acid (DHA and EPA) incorporated cell lines [22]. Similar observations are also reported in human coronary arteries after consumption of fish oils [23]. The favorable effects of DHA on reducing arterial stiffness are also observed through direct vasodilation and inhibition of vasoconstrictor response in human studies [24]. In addition to these favorable structural effects of ω−3 fatty acids on the vascular system, there are some favorable functions as well. Improved endothelial-dependent dilation is one of the important vessel wall functions of ω−3 fatty acids which is achieved by increasing NO production [25] and decreasing oxidative stress [26].

It has been observed that ω−3 fatty acids are independently associated with a lower level of pro-inflammatory markers [IL-6, IL-1ra, tumor necrosis factor-a (TNFa) and CRP] and a higher level of anti-inflammatory markers [soluble IL-6r, IL-10, transforming growth factor-a (TGFa)] [27]. Hence, the presence of ω−3 fatty acids is beneficial in inflammatory-related diseases like atherosclerosis.

On the contrary, ω-6 fatty acids such as AA metabolites (prostaglandin 2, thromboxane 2, and leukotriene 4) promote prothrombotic and proinflammatory markers (IL-1, IL-6, NFKB, and TNF). Thus leading to inflammation, [28] thrombosis, atherosclerotic properties and contributing to the development of inflammatory disorders and excessive cell proliferation [29]. Therefore, levels of ω-6 fatty acids in vessels could be detrimental to health. In the present study, the percentage of the pro-inflammatory, arachidonic acid levels was significantly low in LIMA, compared to SV and RA suggesting a possible higher success rate in LIMA grafts.

The level of ω-3 fatty acid (Docosatetraenoic acid) observed in this study did not indicate a significant variation among the vessel types. However, in most of the studies, the ratio between ω-6 to ω-3 has been reported as an indication of the risk of atherosclerosis [12]. The observed ratio in the present study indicates a significantly low value for LIMA compared to the other two vessels. When the ratio of ω-6: ω -3 PUFA, increases it could potentiate inflammatory processes and consequently predispose to inflammatory diseases including atherosclerosis [20]. Therefore, the fatty acid profiles studied in LIMA, SV, and RA in the present study highlight the favorable fatty acid distribution of LIMA compared to RA and SV for CABG.

Limitations

The number of specimens collected varies with the surgical procedure; hence in some patients, all three different samples could not be collected.

Conclusions

There are significant differences observed in the percentage of fatty acids present in LIMA, SV, and RA biopsy samples obtained from CABG patients. Our results suggest that the risk of the redevelopment of atherosclerosis is less with LIMA when compared to SV and RA based on fatty acid composition.

Percentages fatty acids in LIMA.

(DOCX)

Click here for additional data file.

Percentages fatty acids in SV.

(DOCX)

Click here for additional data file.

Percentages fatty acids in RA.

(DOCX)

Click here for additional data file.

Linear regression analysis.

(DOCX)

Click here for additional data file.

29 Jul 2021

PONE-D-21-17715

A comparative study on fatty acid profile in selected vessels of coronary artery bypass graft (CABG)

PLOS ONE

Dear Dr. Wanniarachchi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

All reviewers and editors found that this manuscript is interesting and addresses a significant clinical problem, but the reviewers identified some important issues. These comments include small sample number, methodological problems, and English writing. The editors concur. Suggestions are all good ones and very constructive. Most of them can be addressed without additional analyses. The editor suggests that the authors should discuss some of the issues as limitations and de-emphasize the authors’ conclusion. If you are able to respond adequately to the points by the reviewers and editors, we are willing to reassess a revised manuscript; however, we cannot commit ourselves at this time to its publication.

Please submit your revised manuscript by Sep 11 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see:  http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at  https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Michinari Nakamura, MD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure:

The work was supported by an institutional research grant from the University of Sri Jayewardenepura (Grant number ASP/01/RE/SCI/2019/67) awarded to D.D.C. de S.Wanniarachchi and funding received to E.M.S.Bandara from Nawaloka Hospital (PLC) Sri Lanka

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript describes that the analyses of fatty acid profile composition in major arteries which are commonly used in Coronary Artery Bypass Graft surgery (CABG) by GC-MS approach. This work provides valuable clinical information about the fatty acid profile composition on saphenous vein, radial artery and left internal mammary artery from patients undergoing elective CABG.

My comments are as follows:

1. More effort needs to be put on English editing.

2. How the percentage of each individual fatty acid was calculated? More detail descriptions are needed. How the samples are normalized?

3. Are the size numbers for all LIMA, SV and RA samples 27?

Reviewer #2: The present study reports comparison of fatty acid composition of three biopsy specimens of blood

vessels collected during CABG that include LIMA, RA, and SV vessels. The studies are well conducted albeit limited samples . They report the presence of free cholesterol and cholesterol esters in human atherosclerotic plaques . The findings suggest that ( based on fatty acid composition ) RA samples have more atherogenic activity compared to the SV and LIMA. These results may suggest that the risk of redevelopment of atherosclerosis is less with LIMA when compared to SV and RA (based on the fatty acid composition) for future CABG treatments.

The study is interesting and highly relevant to CABG treatment despite some limitations. The study is sound but it is unclear if the limited number of patient samples can allow them to reach definite conclusions .

Some grammatical errors do appear in the manuscript which needs attention.

Reviewer #3: In the study titled, "A comparative study on fatty acid profile in selected vessels of coronary artery bypass graft (CABG)," the investigators set out to compare the fatty acid composition of saphenous veins, radial arteries, and left internal mammary arteries that are being prepared as grafts for CABG. There are a number of major concerns with the structure of this manuscript in its current format. Logic is extremely hard to follow throughout.

1) The authors draw a conclusion that fatty acid composition of the LIMA makes it the best graft used for CABG, yet they provide no data on the relationship between fatty acid composition of a vessel and its ability to maintain graft patency. Nor do they provide any mechanism to support why fatty acid composition would influence graft patency.

2) Introduction: 3rd sentence structure is confusing as written.

3) Introduction: The logic is difficult to follow throughout the introduction. For example, the second paragraph attempts to introduce the concept that fatty acid composition is important to vessels, but concludes with a non sequitur about endothelial dysfunction as an indicator of atherosclerosis.

4) Introduction: Several component statements lack appropriate ciations.

a. “Fatty acid composition of vessel walls is an important modulator in vascular function.”

b. “Changes in fatty acids composition can modify the properties of cell membranes including fluidity and permeability, either directly via their insertion in the membrane or indirectly through their effects on lipid metabolism.”

5) Introduction: The third paragraph is unclear as written. Are the authors trying to say that saturated fats are good or bad? Are the authors trying to say thay poly-unsaturated fats are good or bad? There is no clear logic path in the way this paragraph is written.

6) Introduction: “Even though the fatty acid profile of SV, and LIMA had been studied before, there was no information on comparison of these profiles with radial artery.” Citations?

7) Introduction: Overall, does not clearly lay out the knowledge gap or what the goals of this study are.

8) Methodology: The statistical approach is not clearly written. Exactly what statistical methods were used to employ comparison between the three groups (LIMA, RA, and SV)? How were results adjusted for confounding variables?

9) Methodology: Numbers were small. How was this study powered?

10) Results: Table 1 is insufficient in terms of the data required for demographics and clinical characteristics. Need more than just Age, Sex, Hypertension, and Diabetes. Need information on: Age, BMI, Sex, DM, Hypertension, Hyperlipidemia, Cholesterol levels, Statin use, Smoking history, Family history, CAD, prior MI, GFR, ASCVD risk score, CVA.

11) Results: Table 2: what statistical test(s) were used to compare the 3 group and derive the P values?

12) Results: Table 3: what statistical test(s) were used to compare the 3 group and derive the P values?

13) The conclusion: “The fatty acid composition of RA indicates more atherogenic activity compared to the SV and LIMA. Our results suggest that the risk of redevelopment of atherosclerosis is less with LIMA when compared to SV and RA based on the fatty acid composition.” - The authors do not present any data to support this conclusion. This is not a study of graft patency or mechanisms of graft patency.

14) The study discussion indicates that the investigators assume the fatty acids in the vessels all come from atherosclerotic plaques within the vessel walls, yet there is no data to support these vessels, being used for bypass, had atherosclerosis.

15) There is no structure to the discussion; the rationale is difficult to follow.

16) The discussion section is missing a careful assessment of the study limitations.

17) Frequent English grammatical errors throughout.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Muthu Periasamy

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

11 Sep 2021

PlosOne - Reviewer comments

Reviewer Comment Revision

General comments 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Check ed.

2. Thank you for stating the following financial disclosure:

The work was supported by an institutional research grant from the University of Sri Jayewardenepura (Grant number ASP/01/RE/SCI/2019/67) awarded to D.D.C. de S.Wanniarachchi and funding received to E.M.S.Bandara from Nawaloka Hospital (PLC) Sri Lanka.

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Funding statement is amended to include The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. Ethics statement is only written under the methods section

Reviewer 1 This manuscript describes that the analyses of fatty acid profile composition in major arteries which are commonly used in Coronary Artery Bypass Graft surgery (CABG) by GC-MS approach. This work provides valuable clinical information about the fatty acid profile composition on saphenous vein, radial artery and left internal mammary artery from patients undergoing elective CABG.

Comment 1 More effort needs to be put on English editing Grammar checked and revised.

Comment 2 How the percentage of each individual fatty acid was calculated? More detail descriptions are needed. How the samples are normalized? The percentage of individual fatty acids was calculated as the area percentage of each fatty acid to the total area of fatty acids detected.

Comment 3 Are the size numbers for all LIMA, SV and RA samples 27? Grafting vessels selected from patients vary depending on the surgical procedure. Hence two or three different vessel samples were obtained from a patient. Therefore, among 27 patients selected in the study LIMA = 12, SV = 20 RA = 9 samples were analyzed.

Reviewer 2 The present study reports comparison of fatty acid composition of three biopsy specimens of blood

vessels collected during CABG that include LIMA, RA, and SV vessels. The studies are well conducted albeit limited samples . They report the presence of free cholesterol and cholesterol esters in human atherosclerotic plaques . The findings suggest that ( based on fatty acid composition ) RA samples have more atherogenic activity compared to the SV and LIMA. These results may suggest that the risk of redevelopment of atherosclerosis is less with LIMA when compared to SV and RA (based on the fatty acid composition) for future CABG treatments.

.

Comment 1 The study is interesting and highly relevant to CABG treatment despite some limitations. The study is sound but it is unclear if the limited number of patient samples can allow them to reach definite conclusions The sample size was estimated with single group mean (N = (Zα/2)2 s2 / d2) .

Considering the standard deviation of Docosahexaenoic acid level of internal mammary artery (reference number 8).

Hence, Zα/2 = 1.96, s= 2.5, d=1

N=24

The corrected sample size after 10% allowance of missing is 27.

Comment 2 Some grammatical errors do appear in the manuscript which needs attention. Grammar revised. and corrections are highlighted

Reviewer 3 In the study titled, "A comparative study on fatty acid profile in selected vessels of coronary artery bypass graft (CABG)," the investigators set out to compare the fatty acid composition of saphenous veins, radial arteries, and left internal mammary arteries that are being prepared as grafts for CABG. There are a number of major concerns with the structure of this manuscript in its current format. Logic is extremely hard to follow throughout.

Comment 1 The authors draw a conclusion that fatty acid composition of the LIMA makes it the best graft used for CABG, yet they provide no data on the relationship between fatty acid composition of a vessel and its ability to maintain graft patency. Nor do they provide any mechanism to support why fatty acid composition would influence graft patency. The graft patency depends on multiple parameters where fatty acid of composition of grafting blood vessels is also an important parameter. Therefore the study is focused on analysis fatty acid composition since there are limited studies conducted. Support by literature (Petruzzo

et al., 2001). Included in the discussion.

Comment 2 Introduction: 3rd sentence structure is confusing as written. Corrected as “The majority of patients receive left internal mammary artery (LIMA) grafts to the left anterior descending (LAD) coronary artery compared to other grafting options such as saphenous vein (SV) grafts or radial artery (RA)”.

Comment 3 Introduction: The logic is difficult to follow throughout the introduction. For example, the second paragraph attempts to introduce the concept that fatty acid composition is important to vessels, but concludes with a non sequitur about endothelial dysfunction as an indicator of atherosclerosis. Corrected as “Hence, several studies has observed the influence of the vessel wall fatty acid composition in atherosclerosis”.

Comment 4 Introduction: Several component statements lack appropriate ciations.

a. “Fatty acid composition of vessel walls is an important modulator in vascular function.”

b. “Changes in fatty acids composition can modify the properties of cell membranes including fluidity and permeability, either directly via their insertion in the membrane or indirectly through their effects on lipid metabolism.” References are included in the paper.

Comment 5 Introduction: The third paragraph is unclear as written. Are the authors trying to say that saturated fats are good or bad? Are the authors trying to say thay poly-unsaturated fats are good or bad? There is no clear logic path in the way this paragraph is written. The third paragraph is revised to emphasis the role of fatty acids in cell walls in developing atherosclerosis.

Comment 6 Introduction: “Even though the fatty acid profile of SV, and LIMA had been studied before, there was no information on comparison of these profiles with radial artery.” Citations? References included.

Comment 7 Introduction: Overall, does not clearly lay out the knowledge gap or what the goals of this study are. The introduction section is revised to emphasize the knowledge gap and highlighting the goal of the study.

Comment 8 Methodology: The statistical approach is not clearly written. Exactly what statistical methods were used to employ comparison between the three groups (LIMA, RA, and SV)? How were results adjusted for confounding variables? The data was analyzed using SPSS version 25. Since the Kolmogorov–Smirnov test reported the variables are not normally distributed, Mann–Whitney U-tests were used to compare the fatty acid percentage of LIMA, SV and RA. A p value of <0.05 was considered to be significant.

A linear regression models were developed to determine the association of confounding variable with fatty acid level. The results included in the manuscript and detail analysis were included in the supporting information.

Comment 9 Methodology: Numbers were small. How was this study powered? The sample size was estimated with single group mean (N = (Zα/2)2 s2 / d2) .

Considering the standard deviation of Docosahexaenoic acid level of internal mammary artery (reference number 8).

Hence, Zα/2 = 1.96, s= 2.5, d=1

N=24

The corrected sample size after 10% allowance of missing is 27.

Comment 10 Results: Table 1 is insufficient in terms of the data required for demographics and clinical characteristics. Need more than just Age, Sex, Hypertension, and Diabetes. Need information on: Age, BMI, Sex, DM, Hypertension, Hyperlipidemia, Cholesterol levels, Statin use, Smoking history, Family history, CAD, prior MI, GFR, ASCVD risk score, CVA. Table 1 is revised to include further information suggested.

Comment 11 Results: Table 2: what statistical test(s) were used to compare the 3 group and derive the P values? Mann–Whitney U-tests were used to compare the fatty acid percentage of LIMA, SV and RA. A p value of <0.05 was considered to be significant.

Comment 12 Results: Table 3: what statistical test(s) were used to compare the 3 group and derive the P values? Mann–Whitney U-tests were used to compare the fatty acid percentage of LIMA, SV and RA. A p value of <0.05 was considered to be significant.

Comment 13 The conclusion: “The fatty acid composition of RA indicates more atherogenic activity compared to the SV and LIMA. Our results suggest that the risk of redevelopment of atherosclerosis is less with LIMA when compared to SV and RA based on the fatty acid composition.” - The authors do not present any data to support this conclusion. This is not a study of graft patency or mechanisms of graft patency. The main focus of the paper is to find the presence of fatty acids which would attribute to the development of plaque. It is not a study of graft patency. The conclusions were drawn from the perspective of fatty acid composition.

Comment 14 The study discussion indicates that the investigators assume the fatty acids in the vessels all come from atherosclerotic plaques within the vessel walls, yet there is no data to support these vessels, being used for bypass, had atherosclerosis. The study samples of SV,RA,and LIMA are left over parts of vessels used in CABG during surgery. These do not contained plaques.

Comment 15 There is no structure to the discussion; the rationale is difficult to follow. Discussion section was revised

Comment 16 The discussion section is missing a careful assessment of the study limitations. Study limitations were mentioned.

Comment 17 Frequent English grammatical errors throughout. Grammar revised.

17 Nov 2021

A comparative study on fatty acid profile in selected vessels of coronary artery bypass graft (CABG)

PONE-D-21-17715R1

Dear Dr. Wanniarachchi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Michinari Nakamura, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Majority questions were addressed and the work also provides valuable clinical information. However, the quality of English is not good enough for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

1 Dec 2021

PONE-D-21-17715R1

A comparative study on fatty acid profile in selected vessels of coronary artery bypass graft (CABG)

Dear Dr. Wanniarachchi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Michinari Nakamura

Academic Editor

PLOS ONE