Protective Effects of Tocotrienols in Cerebral and Myocardial Ischemia-Reperfusion Injury: A Systematic Review

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In this systematic review, Dr. Ramli and colleagues explored to potential of tocotrienols in both cerebral and myocardial I/R injury and from the reported studies, it seems that tocotrienols consistently showed a beneficial effect on such pathological conditions. Overall, this is a nicely written and clearly described manuscript. I do have some suggestions to incorporate:

So far, the authors only "touched" the effects of tocotrienols qualitatively. They showed that the substance alters structural, functional and even biochemical properties of the brain and heart. However, it remains unclear whether this effect was attained only in a supraphysiological dose/concentration or even in the daily consumption dose. I think it is necessary to have an extensive discussion (or even a subsection) regarding the dose since all studies showed a broad range of doses. 

Thank you very much for your valuable feedback. We have added a paragraph to describe the dosage aspect of the included studies.

Discussion, page no. 12, line no. 740-753.

“Interestingly, the dose range for both cardioprotective and neuroprotective effects are distinct and broad. The studies involving the myocardial I/R injury model utilised long-term individual tocotrienols (α, γ, ?) with a dose between 0.02 and 5 mg/kg/day [10,11,14,15]. This dose range is substantially lower than the studies in the cerebral I/R injury model that used either combined tocotrienols and α-tocopherol (200 mg/kg) [17,21], mixed tocotrienols (400 mg/day) [20], or α-tocotrienol (50 mg/kg/day) [19]. The human equivalent dose (HED) calculated (based on a 60-kg human) [40] for cardioprotective effects ranged from 0.2 to 48 mg/day [9,10,22,30], while the dose range for the neuroprotective effects was 240-960 mg/day [17,19,21]. The question of the tolerability of the dose might arise as the upper limit of the dose required is remarkably high. In an open-label, randomised study, Qureshi, et al. [41] had reported that 750-1000-mg tocotrienols given orally were well-tolerated with a similar pharmacokinetic profile compared to lower doses. Furthermore, no adverse effects were reported [41]. This study indicates that the HED derived from the studies is safe to be consumed.”

From Table 1 for example, when I saw the doses used in those studies, I was wondering why the dose required in the brain I/R was much higher than the myocardial I/R? Can the authors speculate on the cause? It is related to the blood brain barrier? Please also discuss this important issue.

Thank you very much for your valuable feedback. We have added a paragraph that discussed the higher dose requirement of tocotrienol in brain I/R injury.

Discussion, page no. 12-13, line no. 754-775.

“The reason for the higher dose requirement for neuroprotection is obscure, but is probably due to the different pharmacokinetic properties of tocotrienols. α-tocopherol transfer protein (α-TTP) plays a pivotal role in the incorporation of α-tocopherol into very-low-density lipoprotein (VLDL) before its release into the circulation or other cells [42,43]. Although the liver is the major site of α-TTP, other organs have been shown to express low levels of α-TTP. Hosomi, et al. [42] had detected the presence of α-TTP mRNA in the brain. This protein has a 100% binding affinity towards α-tocopherol but variable affinity towards other tocopherol isomers and tocotrienols [43]. Lower binding affinity may partly explain why the brain requires a higher dose of tocotrienols to increase their transportation into the brain. For instance, Hansen, et al. [44] reported a remarkable accumulation of vitamin E (α-tocopherol, γ-tocotrienol, ?-tocotrienol) in the brain of the hen fed with high doses of tocopherol and/or annatto tocotrienols.”

Please improve the presentation and visibility of the flowchart in Figure 2. Make sure to adhere with PRISMA guideline in creating the flowchart and mention specifically in the title of the figure that it is a PRISMA flowchart.

Thank you very much for your valuable feedback. We have modified our table based on the PRISMA 2020 flow diagram.

Results, page no. 4, line no. 369.

“PRISMA flow diagram for identification of studies via databases”

For Figure 1, kindly refer to the manuscript.

Please add the PRISMA checklist as a supplement and mention clearly in the text that this study was done in accordance with PRISMA guidelines. 

Thank you very much for your valuable feedback.

We already added the statement regarding PRISMA.  

Methods, page no. 2, line no. 128-130.

“The study was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [22], except we did not register our protocol in any database.”

Also, we have added PRISMA checklist as a supplement file.

Line 30: I don't think it is needed to mention about the funding in the abstract. There is a designated place to declare the funding in the end of the manuscript.

Thank you very much for your valuable feedback. We have removed the funding statement.

Lines 73-75: Please speculate on the cause of declining research on tocotrienols or just remove the statement. In the current form, it is not useful.

Thank you very much for your valuable feedback. We decided to remove the sentence as suggested.

Line 85: please clarify whether "models" should be "animal models" or the authors included other form of models in this study?

Thank you very much for your valuable feedback. We included in/ex vivo or in vitro model. We have added the terms in the statement.

Methods, page no. 2, line no. 133-134.

“…the I/R injury models (P) (in/ex vivo or in vitro) using tocotrienol…”

Line 90: "article published in other languages". In fact, the authors did not mention "English" in the inclusion criteria. Please add the opted language in the inclusions.

Thank you very much for your valuable feedback. We have added the word ‘English’ in the statement.

Methods, page no. 2, line no. 138.

“…articles published in languages other than English.”

Please review the writing style used when mentioning the authors of certain papers. For example "Mishima, Tanaka, Pu, Egashira, Iwasaki, Hidaka, 141 Matsunaga, Takata, Karube and Fujiwara". There is a general consensus that if there are more than 3 authors in the list, they has to be abbreviated to "et al." or "and colleagues / co-authors" and even more, in the text, the authors can only include the first author of the paper, continued with those abbreviations. Mentioning the whole list of authors is very distracting and not necessary. Please revise.

Thank you very much for your valuable feedback. We have already changed according to your suggestions.

Table 1: although the brain part is clearly presented, the heart part is very messy. Please describe the effects of tocotrienols in each study (one-by-one) as done in the brain section. At the moment, it is not clear which effect belongs to which study.

Thank you very much for your valuable feedback.

We constructed this table similar to table 2. It seemed messy because there was no visible line that separates between rows. This is the journal format that we need to adhere to. We have edited the table by shifting the row of Das et al. (11) to the bottom of the table because this row has the most extensive parameters.

Kindly refer to Results, Table 3, page no. 9-10.

Also, please change "comments" to "effects" in the heading of Table 1.

Thank you very much for your valuable feedback. We have changed the term “comments” to “effects”.

Kindly refer to Results, Table 1, page no. 5.

I probably miss this but what are those superscripted greek alphabets in Table 3? I somehow could not find the meaning of those characters in the table legend.

Thank you very much for your valuable feedback. We have added the description regarding all the superscripts and symbols used.

Kindly refer to Results, Table 3, page no. 10, line no. 497-498.

“↓ reduced; ↑ increased; > higher than

α alpha; ? delta; γ gamma; F: female; M: male;…”