Examination of Postmortem β-Hydroxybutyrate Increase in Forensic Autopsy Cases

Round 1

Reviewer 1 Report

Presented manuscript No. applsci-1773497 describes quantiication of BXB level in postmortem cases to estimate PMI. All data obtained were properly compared by statistical tests. The manuscript is very interesting and its scientific value is high. Its readerships can be various scientis, such as toxicologists and doctors. The manuscript is properly constructed using well English. I appreciate the authors' contribution to research and data analysis; it was done properly and carefully. I have no comments, beside lines 78 and 79. In these lines, obvious and well-known general statements were presented. these lines can be deleted.

Author Response

Reviewer 1: Presented manuscript No. applsci-1773497 describes quantiication of BXB level in postmortem cases to estimate PMI. All data obtained were properly compared by statistical tests. The manuscript is very interesting and its scientific value is high. Its readerships can be various scientis, such as toxicologists and doctors. The manuscript is properly constructed using well English. I appreciate the authors' contribution to research and data analysis; it was done properly and carefully. I have no comments, beside lines 78 and 79. In these lines, obvious and well-known general statements were presented. these lines can be deleted.

 

Our response: Thank you for your polite review and feedback. As per your suggestion, we have deleted lines 78 and 79.

Reviewer 2 Report

Dear Authors

 

I have read the article in great detail and have a few comments. 

First: the subject of numerous studies is to estimate the time since death based on laboratory tests. Therefore, each such attempt is interesting, although most likely, it will have too many limitations in common forensics practice. 

Second: the "Characteristics of the patients" should be transferred to the material and methods part.

Third and the most important: I have reservations about the studied groups. The causes and mechanisms of death are too varied. Maybe you should check only the early postmortem stage (max. two days after death in smaller periods, for example, 0, 3, 6, 9, 12, 18, 24, 36, and 48 hours after death). What were the conditions of the body after that, whether decomposition or other postmortem changes affecting metabolism were evident?

I also do not see the pieces of information about: 

• Where were the bodies between death and postmortem examination (at the place of death, in the morgue?);
• What were the conditions in the morgue?
• Were the corpses at the place of death for several days, or not, and if so, under what external conditions? 

So as You see, the material and methods should include clear information about the external factors (temperature, humidity, etc.), at the place of death and in the morgue; when the bodies were transferred to the morgue?; how long the body was at the place of death, and others critical information.

In such rare studies, the test group must be homogeneous, and changes must be assessed under standardized conditions because only such results can be representative.

   

 

Sincerely

Reviewer

 

Author Response

Reviewer 2: First: the subject of numerous studies is to estimate the time since death based on laboratory tests. Therefore, each such attempt is interesting, although most likely, it will have too many limitations in common forensics practice.

 

Our response: Thank you for your feedback. We agree with your opinion that estimating the time since death has too many limitations. Therefore, the purpose of our study was to examine whether BHB values could be affected by the postmortem interval and not to estimate the time of death with these values. Our study merely suggests that BHB values can be affected by the postmortem interval.

 

Reviewer 2: Second: the "Characteristics of the patients" should be transferred to the material and methods part.

 

Our response: Thank you for your helpful suggestion. We have moved the “Characteristics of the patients” section to the Materials and Methods.

 

Reviewer 2: Third and the most important: I have reservations about the studied groups. The causes and mechanisms of death are too varied. Maybe you should check only the early postmortem stage (max. two days after death in smaller periods, for example, 0, 3, 6, 9, 12, 18, 24, 36, and 48 hours after death). What were the conditions of the body after that, whether decomposition or other postmortem changes affecting metabolism were evident?

 

Our response: Thank you for your pertinent feedback. As you have pointed out, the causes and mechanisms of death are varied. Further, we should have analyzed only the early postmortem stages with further subdivided groups. However, when cadavers are subdivided into smaller groups, such as grouping according to 3-h increments of the postmortem interval, we cannot be certain of enough sample size for each group. Therefore, we divided the cadavers into four groups.

To the best of our knowledge, no studies have examined the effect of postmortem change, including decomposition, on the serum BHB values.

 

Reviewer 2: I also do not see the pieces of information about:

Where were the bodies between death and postmortem examination (at the place of death, in the morgue?);

What were the conditions in the morgue?

Were the corpses at the place of death for several days, or not, and if so, under what external conditions?

 

Our response: Thank you for your feedback. We have added all the known information regarding the cadavers in the Material and Methods section.

We apologize for not being able to answer your queries completely. Certainly, to estimate the time of death from laboratory examination, forensic scientists must consider various aspects, such as the cause of death, environment in which the cadavers were exposed, and situation in which the cadavers were preserved. This would be a very difficult task.

However, the aim of our study was not to estimate the time of death, but rather to examine whether a postmortem interval could affect serum BHB values. We considered the possibility that several factors, as you have rightly stated, such as the external factors (including temperature and humidity) at the place of death and in the morgue, time the bodies were transferred to the morgue, and duration of the body at the place of death, could all affect serum BHB values. Further, we considered the postmortem interval to be a factor that affected the BHB values. In the present study, we sought to only demonstrate the relationship between the postmortem interval and serum BHB values.

Reviewer 3 Report

Review on the manuscript of Kondou H et al.,: “Examination of postmortem β-hydroxybutyrate increase in forensic autopsy cases”.

In this manuscript, the authors explored the relationship between the blood levels of β-hydroxybutyrate (BHB) and the postmortem interval (PMI). The authors found significantly lower levels of BHB in patients with a PMI < 24 h, as compared to patients with a PMI ≤ 72 h or a PMI > 72 h. Based on this, the authors suggest that the levels of BHB may increase overtime after death, and, therefore, they may not be unequivocal parameter to diagnose ketoacidosis as a cause of death.

The data shown in the manuscript seem to be clear, and, in general, well described. After a careful observation of the work, some issues that arise in the paper are listed below for the consideration of the authors.

1 – The patients used in the study were divided in 4 groups. Based on the information provided in the results’ section - “The PMI ranged from 12 h to 16 d” – the patients included in group 4 (PMI > 72 h) had a PMI between 3 and 16 days? How do the BHB levels vary in this group? Do patients with PMI of 16 days show higher levels of BHB than patients with a PMI of 4 days, for example? If the authors want to make the point that the levels of BHB may increase overtime after death, I recommend the authors to explore in more detail this group of patients and show the conclusions they can take out of it (not just to refer it as a limitation of the study).

2 – For the data included in table 1, the authors show the standard deviation. However, the graphs shown in figures 1 and 2 represent the average +/- standard error. For a question of consistency, I would recommend the authors to uniformize this discrepancy, by always using or the standard deviation or the standard error.

3 – From the data shown in figure 2, I conclude that BHB levels does not significantly increase after death. Based on this, is it relevant to take in consideration the PMI while diagnosing ketoacidosis as a cause of death? In practice, this contradicts the main point of the manuscript. Although the authors discuss this question (“Although the antemortem and postmortem values were compared as an additional examination, the sample size was insufficient, specifically for groups with prolonged PMI. However, the small sample analysis revealed a tendency for the postmortem values to be greater than the antemortem values.”), the authors should consider to remove some data from the manuscript (or increase the number of patients) in order to avoid misinterpretations.

Author Response

Reviewer 3: 1 – The patients used in the study were divided in 4 groups. Based on the information provided in the results’ section - “The PMI ranged from 12 h to 16 d” – the patients included in group 4 (PMI > 72 h) had a PMI between 3 and 16 days? How do the BHB levels vary in this group? Do patients with PMI of 16 days show higher levels of BHB than patients with a PMI of 4 days, for example? If the authors want to make the point that the levels of BHB may increase overtime after death, I recommend the authors to explore in more detail this group of patients and show the conclusions they can take out of it (not just to refer it as a limitation of the study).

 

Our response: Thank you for your helpful suggestion. We have divided Group 4 into two groups, PMI < 1 week and PMI > 1 week. We have compared the BHB values between these groups and have added the result of this analysis in the Result section.

 

Reviewer 3: 2 – For the data included in table 1, the authors show the standard deviation. However, the graphs shown in figures 1 and 2 represent the average +/- standard error. For a question of consistency, I would recommend the authors to uniformize this discrepancy, by always using or the standard deviation or the standard error.

 

Our response: Thank you for your helpful suggestion. We have uniformed the data to standard error.

 

Reviewer 3: 3 – From the data shown in figure 2, I conclude that BHB levels does not significantly increase after death. Based on this, is it relevant to take in consideration the PMI while diagnosing ketoacidosis as a cause of death? In practice, this contradicts the main point of the manuscript. Although the authors discuss this question (“Although the antemortem and postmortem values were compared as an additional examination, the sample size was insufficient, specifically for groups with prolonged PMI. However, the small sample analysis revealed a tendency for the postmortem values to be greater than the antemortem values.”), the authors should consider to remove some data from the manuscript (or increase the number of patients) in order to avoid misinterpretations.

 

Our response: Thank you for your helpful suggestion. Since increasing the sample size was difficult, we have deleted the antemortem and postmortem comparisons. Accordingly, we have made several changes to the Discussion section.

Round 2

Reviewer 2 Report

Dear Authors

As a forensic pathologist, I am aware of the numerous limitations visible in the research. I still think research groups should be more homogeneous. However, a paper should be published in this form due to the rarely foreseen research results in this area. 

 

Sincerely Reviewer  

Author Response

Reviewer 2: As a forensic pathologist, I am aware of the numerous limitations visible in the research. I still think research groups should be more homogeneous. However, a paper should be published in this form due to the rarely foreseen research results in this area.

 

Our response: Thank you for your comment. As indicated, our study has various limitations. In the future, we plan to perform a more detailed study with sufficient number of samples.

Reviewer 3 Report

Second review on the manuscript of Kondou H et al.,: “Examination of postmortem β-hydroxybutyrate increase in forensic autopsy cases”.

In this manuscript, the authors explored the relationship between the blood levels of β-hydroxybutyrate (BHB) and the postmortem interval (PMI). The authors found significantly lower levels of BHB in patients with a PMI < 24 h, as compared to patients with 24 h < PMI ≤ 72 h or a PMI > 72 h. In addition, the authors found median BHB values of 406.5 and 1235 in patients with a PMI < 1-week and a PMI > 1-week, respectively. Based on this, the authors suggest that the levels of BHB may increase overtime after death, and, therefore, they may not be unequivocal parameter to diagnose ketoacidosis as a cause of death.

In the current version of the manuscript the authors tried to improve its overall quality, based on the reviewers’ comments. The conclusions are now supported by the data shown in the manuscript. I would just recommend the authors to mention the results they found for the PMI < 1-week and PMI > 1-week groups, since the differences were very striking, thus supporting the main conclusion of the work. 

Author Response

In the current version of the manuscript the authors tried to improve its overall quality, based on the reviewers’ comments. The conclusions are now supported by the data shown in the manuscript. I would just recommend the authors to mention the results they found for the PMI < 1-week and PMI > 1-week groups, since the differences were very striking, thus supporting the main conclusion of the work.

 

Our response: Thank you for your feedback. We have added the explanation for the Wilcoxon’s rank sum test of Group 4 in the Discussion section.

 

Page 5, lines 156-159

 

Additionally, the result of the Wilcoxon’s rank sum test of Group 4 revealed that the BHB values in the PMI > 1 week group were higher than those in the PMI < 1 week group. This corroborated with the previous findings, which showed that postmortem BHB values were associated with PMI.