PEER REVIEW HISTORY ARTICLE DETAILS TITLE (PROVISIONAL)

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1 PEER REVIEW HISTORY BMJ Paediatrics Open publishes all reviews undertaken for accepted manuscripts. Reviewers are asked to complete a checklist review form and are provided with free text boxes to elaborate on their assessment. These free text comments are reproduced below. This paper was submitted to a another journal from BMJ but declined for publication following peer review. The authors addressed the reviewers comments and submitted the revised paper to BMJ Paediatrics Open. The paper was subsequently accepted for publication at BMJ Paediatrics Open. TITLE (PROVISIONAL) AUTHORS REVIEWER Reviewer 1 REVIEW RETURNED 28-Feb-2017 GENERAL COMMENTS ARTICLE DETAILS Hyperglycaemic preterm neonates exhibit insulin resistance and low insulin production Salis, Emma; Reith, David; Wheeler, Benjamin; Broadbent, Roland; Medlicott, Natalie VERSION 1 - REVIEW This is an observational study of 10 hyperglycaemic preterm babies receiving insulin and 20 euglycaemic preterm controls. The authors have taken regular plasma samples for insulin and C-Peptide concentrations over a 24 hour period. Major concerns: 1. No attempt is made to address the fact that the two groups of babies may have been receiving different glucose intakes, the effect of sex on C-Peptide, and the insulin dose the babies were receiving 2. The authors assert on multiple occasions that they have shown these babies have increased insulin resistance as well as reduced insulin secretion. This needs to be justified in more detail. What do they mean by insulin resistance? How was insulin resistance calculated? How can they calculate this when all the babies are receiving exogenous insulin? 3. The two case studies do not add much to the paper. I would recommend taking these out. 4. The definition of euglycaemia is not given, but appears to be mm, which is a very broad range, and needs to be justified. 5. The euglycaemic babies did not have the samples taken at regular intervals, as the other group did. This is a limitation to the study and needs to be addressed in the Discussion. i.e. Euglycaemic babies may have had samples taken when they had a sepsis screen as they were unwell. 6. Figure 1 gives data for 9 of the 10 hyperglycaemic group, breaking them into high and low C-Peptide groups. Was this an a priori analysis plan? How were these two groups defined? What about the other baby in this group? How do these data relate to insulin/dextrose intake/sex? 7. The Discussion needs to be more in depth, and better referenced. 8. As all the hyperglycaemic babies, except the two case studies, were on insulin, it s impossible to know from this study what is the effect of hyperglycaemia, and what is the effect of insulin treatment. Minor: 1. The abstract has an intervention heading there wasn t an

2 REVIEWER REVIEW RETURNED GENERAL COMMENTS intervention 2. Give data, not just p values, in the abstract 3. Be clear every time they are mentioned that the insulin and C- peptide are plasma concentrations 4. The abstract says that exogenous insulin did not reduce insulin production; yet the babies on insulin had reduced C-peptide concentrations. 5. How much insulin were the babies receiving? 6. Table 1 mentions data is missing need more information on how much data is missing, and on which variables 7. Use weight z scores Sherwin, Catherine University of Utah School of Medicine, Pediatrics Conflict of Interest: I was the examiner for Dr Emma Salis thesis which was on this topic. I have no other conflicts to declare. 07-Apr-2017 The manuscript presented by Salis E et al. is clear and well presented. The topic is interesting and the method is well described. However, some comments deserve consideration. My major criticisms relates to the inclusion and exclusion criteria used in the design of the study: Hyperglycemia has been identified as a sign of sepsis in premature neonates. However, authors did not incorporate it as exclusion criteria. If not considered, could that be a cofounding factor? I would assume that euglycaemic preterm neonates group would have the same inclusion and exclusion criteria as hyperglycaemic premature neonates, except for blood glucose concentrations. Is that the case? It is not clear through the manuscript. Moreover, euglycaemic preterm neonates group is matched with hyperglycaemic based on PMA (less than 30 weeks), without considering GA. Why did the authors chose PMA instead of GA? Why did the authors chose the criteria of PMA lower than 30 weeks? Specific comments - P5-Materials and laboratory analyses: The last five sentences describe the use of a chemiluminescence detection reagent kit IV What is the purpose of that kit? - P5-Materials and laboratory analyses: How has blood glucose been measured (assay used)? - Table 1: The study mainly aims to investigate differences in blood glucose, insulin and C-peptide concentrations in two groups of preterm neonates only differing in their glycemic status. However, their prematurity is significantly different (both GA and PMA). Could that produce a bias on the results? - The number of preterm neonates included in the non-insulintreated group (n=20) is quite larger than the number included in the insulin-treated neonates (n=10). How could this difference affect the results? It should be clearly specified in some part of the manuscript. - P6-Results: Authors indicate that there are five neonates with low C-peptide concentrations and four neonates with high C-peptide concentrations. It is not clear if they belong to the hyperglycaemic or euglycaemic group. It should be clearly specified. - Figure 2: Y-label axis of graph (A) is incomplete.

3 Reviewer 1 VERSION 1 AUTHOR RESPONSE This is an observational study of 10 hyperglycaemic preterm babies receiving insulin and 20 euglycaemic preterm controls. The authors have taken regular plasma samples for insulin and C- Peptide concentrations over a 24 hour period. Major concerns: 1. No attempt is made to address the fact that the two groups of babies may have been receiving different glucose intakes, the effect of sex on C-Peptide, and the insulin dose the babies were receiving The neonates were on the standard regimen glucose rates for Dunedin Hospital NICU (dextrose 10%: 60mL/kg/day on day 1, 90 on day 2, 120 on day 3, 150 on day 4, 180 on day 5). Since the results are compared with PMA we believe the effects of the different timing and intakes when samples were obtained are already accounted for. We did look at the insulin doses that the babies were receiving but we have not included this in the paper, Hyperglycaemic neonates received insulin according to the following table: Sliding scale for insulin administration (Actrapid insulin neutral human) by continuous intravenous infusion at Dunedin Hospital NICU. Insulin infusion started at 0.5 ml/kg/hour (= 0.05 units/kg/hour) and adjusted using the sliding scale below based on subsequent blood glucose results Blood Glucose Dose Infusion Rate (0.1 unit/ml) Concentration (mmol/l) (units/kg/hour) (ml/kg/hour) > to to to <7 Stop infusion Stop infusion The following graph shows the different doses the neonates received:

4 Insulin dose (U/kg/h) We did however, show the insulin dose alongside BGC in Figure 2. We could potentially show all nine neonates as supplementary information and will leave that decision to the discretion of the Editor. With respect to the effect of sex on C-peptide, we believe the sample size was too small to be examining multiple covariates. There was no prior data or other reason to think there would be a difference. We found no difference in C-peptide according to sex in the euglycaemic group (see below). However, we did find a difference in the hyperglycaemic group but it is difficult to determine if this effect is due to sex because of the small sample size. There were only three males in the hyperglycaemic group and they were all also in the low C-peptide group. As there are multiple variables that could be affecting this it would difficult to confirm this effect is due to sex. Since there is no reason to think sex affects the results to this study we would rather not report on this. hyperglycaemic males C-peptide Ln_ C-peptide females C-peptide Ln_ C-peptide mean E-06 sd n Euglycaemic Time (hours) males C-peptide Ln_ C-peptide females C-peptide Ln_ C-peptide patient 1 patient 2 patient 3 patient 4 patient 5 patient 6 patient 7 patient 8 patient 9 ttest ttest

5 mean sd n The authors assert on multiple occasions that they have shown these babies have increased insulin resistance as well as reduced insulin secretion. This needs to be justified in more detail. What do they mean by insulin resistance? How was insulin resistance calculated? How can they calculate this when all the babies are receiving exogenous insulin? Insulin resistance as such was not calculated as insulin resistance is usually calculated using fasting insulin concentrations, it is impractical to measure fasting concentrations in neonates due to the risk of hypoglycaemia. Insulin resistance was determined by comparing insulin plasma concentrations, blood glucose concentrations, C-peptide plasma concentrations and I/CP ratios with PMA in both groups. All of these concentrations were significantly affected by PMA in both groups suggesting that all the neonates whether hyperglycaemic or not showed insulin resistance. Not all babies were receiving exogenous insulin, the twenty non-insulin treated neonates did not receive any insulin and as mentioned they too showed insulin resistance. This is discussed in the discussion but has been made clearer with the following paragraphs: Fasting insulin, homeostasis model assessment (HOMA), I/BGC ratio, and the Bennett index have all been used to predict insulin sensitivity [11]. However, in neonates it is impractical to measure fasting values because of the risk of hypoglycaemia. Therefore, for this study Insulin resistance was determined by comparing insulin plasma concentrations, blood glucose concentrations, C-peptide plasma concentrations and I/CP ratios with PMA in both groups. Preterm neonates have been shown to exhibit insulin resistance in the absence of hyperglycaemia. In a previous study, insulin and C-peptide concentrations were elevated in very preterm infants and decreased to term and this relationship persisted when blood glucose concentrations were accounted for.[5] Insulin resistance is most pronounced in extremely premature neonates, but reduces with increased gestation as shown by a change in I/CP at around 34 weeks.[5] The significant effect of PMA on insulin plasma concentrations, blood glucose concentrations, C-peptide plasma concentrations and I/CP ratios in this study indicate that hyperglycaemic neonates may also exhibit insulin resistance as a direct result to their prematurity. 3. The two case studies do not add much to the paper. I would recommend taking these out. We think the case study neonates add interest to the manuscript and gave evidence to support that hyperglycaemic neonates have insulin deficiency. We believe they would be of interest to clinical practitioners reading this paper. However, we are happy to leave this up to the discretion of the editor. 4. The definition of euglycaemia is not given, but appears to be mm, which is a very broad range, and needs to be justified. We thank the reviewer for bringing this to our attention and agree this was not clear. For the purpose of this paper euglycaemia was considered to be any neonate that was not hyperglycaemic or

6 hypoglycaemic as we were defining need for treatment. In Dunedin hospital NICU Hyperglycaemia is defined as a BGC of 10mmol/L or more, for two consecutive levels, four hours apart and hypoglycaemia is defined as a BGC <2.6 mmol/l. These are both defined in the paper however, we have added the following statement to the manuscript under Study population and design to make this clearer: For the purpose of this study euglycaemia was defined to be any neonate not requiring treatment for either hypo- or hyperglycaemia. 5. The euglycaemic babies did not have the samples taken at regular intervals, as the other group did. This is a limitation to the study and needs to be addressed in the Discussion. i.e. Euglycaemic babies may have had samples taken when they had a sepsis screen as they were unwell. None of these babies had sepsis, we have checked their CRP levels at the time of all the collected samples and none of the babies had a positive blood culture. However, we have added the following statement to the limitations section of the discussion: Another limitation of this study is that the euglycaemic babies did not have samples taken at regular intervals due to the opportunistic nature of sample collection. However, none of the neonates in this study showed any sign of illness such as sepsis that would affect the results and blood that was taken for culture remained sterile in all neonates. 6. Figure 1 gives data for 9 of the 10 hyperglycaemic group, breaking them into high and low C- Peptide groups. Was this an a priori analysis plan? How were these two groups defined? What about the other baby in this group? How do these data relate to insulin/dextrose intake/sex? These two groups were observed after samples had been analysed, they were not planned to fall into two groups. Because it is confusing having 10 hyperglycaemic babies in some places and 9 in others (this was due to one of the babies not having regular samples taken over 24 hours) we have removed the tenth baby as it does not change our results and conclusions. Figure 2 gives an example neonate of how the data relates to each other. 7. The Discussion needs to be more in depth, and better referenced. We feel the discussion is more in depth now after addressing the other comments of the reviewers. Also, there are a lack of studies in this area therefore there are few papers that can be referenced. This also indicates that our data will make an important contribution to a clinical issue that has received little attention. 8. As all the hyperglycaemic babies, except the two case studies, were on insulin, it s impossible to know from this study what is the effect of hyperglycaemia, and what is the effect of insulin treatment.

7 This is presented as our main limitation in the discussion of this paper. This is also why we think it is important to keep the two case study neonates in the paper. This is why we measured C-peptide rather than just insulin, to allow for the fact the hyperglycaemic babies were on insulin. Measurement of C-peptide clearance is proposed as a potential surrogate marker for endogenous insulin production. C-peptide can be used as a biomarker for insulin secretion as, unlike insulin, it is not hepatically cleared and has a longer plasma half-life. C-peptide is useful as an indicator of ß-cell function while on insulin treatment. This is because it is secreted in equimolar amounts to insulin and can therefore be used as a measure of insulin secretion rate, especially when exogenous insulin is being administered. Minor: 1. The abstract has an intervention heading there wasn t an intervention Thank you, the heading intervention has been removed. 2. Give data, not just p values, in the abstract Data has now been added to the results section of the abstract. 3. Be clear every time they are mentioned that the insulin and C-peptide are plasma concentrations Thank you, this has been added throughout the manuscript. 4. The abstract says that exogenous insulin did not reduce insulin production; yet the babies on insulin had reduced C-peptide concentrations. In the abstract it states: treatment with exogenous insulin did not appear to suppress insulin production this is discussed in the discussion of the manuscript. All the hyperglycaemic babies had lower C-peptide concentrations than the euglycaemic neonates including the two case study neonates when they were not receiving insulin. Therefore, we have gone with the theory of lower C-peptide concentrations being indicative of insulin deficiency, rather than exogenous insulin suppressing endogenous insulin. This is discussed in the following sentences from the discussion: The lower C- peptide plasma concentrations in the hyperglycaemic neonates are consistent with either suppression of insulin production when exogenous insulin is administered or that hyperglycaemic neonates produce less insulin indicating insulin deficiency. The case studies of two neonates who were initially euglycaemic, but became hyperglycaemic gave evidence to support the latter (i.e. insulin deficiency) as the C-peptide plasma concentrations did not decrease in these neonates with the introduction of insulin treatment (Figure 4). The hyperglycaemic neonates showed no apparent decrease in C- peptide plasma concentrations with increasing insulin dose, which is supported by the case study findings (Figure 2). We don t believe that we say it definitely does not suppress insulin we just say it did not appear to and this is also discussed in the limitations. If insulin suppression was occurring in these neonates we would ve expected the C-peptide concentrations to be overall lower, and higher in the two case study neonates prior to treatment.

8 5. How much insulin were the babies receiving? Different babies were on different doses of insulin according to the sliding scale. Sliding scale for insulin administration (Actrapid insulin neutral human) by continuous intravenous infusion at Dunedin Hospital NICU. Insulin infusion started at 0.5 ml/kg/hour (= 0.05 units/kg/hour) and adjusted using the sliding scale below based on subsequent blood glucose results Blood Glucose Dose Infusion Rate (0.1 unit/ml) Concentration (mmol/l) (units/kg/hour) (ml/kg/hour) > to to to <7 Stop infusion Stop infusion The following graph shows the different doses the neonates received: Insulin dose (U/kg/h) Time (hours) patient 1 patient 2 patient 3 patient 4 patient 5 patient 6 patient 7 patient 8 patient 9 6. Table 1 mentions data is missing need more information on how much data is missing, and on which variables The missing data is indicated by the different n values on the table. The variables with missing data are: weight, birth length and birth head circumference. We have clarified this by changing the footnote

9 of the table to: Note some data is missing where not recorded in patient notes as indicated by the different n values. 7. Use weight z scores We do not see the need to use weight z scores. In practice percentiles are used. Z scores assume that the weights are normally distributed (which they are not), whereas percentiles are nonparametric. However, all the babies in this study were within the normal weight-for-age range according to percentiles. They were also within the normal weight-for-age range according to their z- scores with a range of -1.5 to 1.7. We have added the footnote all neonates had appropriate weights for their gestational ages at the bottom of table 1. We think having the actual mean weight and SD has more meaning to the reader so would like to keep the weights in the paper and hope that the footnote on table 1 clarifies that the babies were appropriate sizes for their age. Therefore, we do not see the relevance of using either z scores or percentiles in this paper but are happy to leave this up to the discretion of the editor. Reviewer 2 Overall comments The manuscript presented by Salis E et al. is clear and well presented. The topic is interesting and the method is well described. However, some comments deserve consideration. My major criticisms relates to the inclusion and exclusion criteria used in the design of the study: Hyperglycemia has been identified as a sign of sepsis in premature neonates. However, authors did not incorporate it as exclusion criteria. If not considered, could that be a cofounding factor? None of the hypergycaemic (or euglycaemic) babies had sepsis at the time of this study. The following has been added to the limitations section of the discussion: However, none of the neonates in this study showed any sign of illness such as sepsis that would affect the results and blood that was taken for culture remained sterile in all neonates. I would assume that euglycaemic preterm neonates group would have the same inclusion and exclusion criteria as hyperglycaemic premature neonates, except for blood glucose concentrations. Is that the case? It is not clear through the manuscript. Moreover, euglycaemic preterm neonates group is matched with hyperglycaemic based on PMA (less than 30 weeks), without considering GA. Why did the authors chose PMA instead of GA? Why did the authors chose the criteria of PMA lower than 30 weeks? Yes that is correct, we have tried to make this clearer by adding the statement: Therefore the main difference in the inclusion and exclusion criteria of the two groups was the different BGC concentrations.

10 PMA was chosen as the main demographic for comparison as in previously published it produced the highest correlation coefficients with I/CP and I/BGC [5]. PMA also allows for the age at the time of the study rather than the gestation as birth. Postmenstrual age (PMA) was calculated for each neonate by correcting GA for the age at the time of the study (PMA = GA at birth + age from birth). A PMA of 30 weeks was chosen as all the insulin-treated neonates were less than 30 weeks and the non-insulin treated neonates were taken from a larger cohort and the cut-off of 30 weeks was a good age to achieve a ratio of 2:1 of non-insulin-treated to insulin treated neonates. Specific comments - P5-Materials and laboratory analyses: The last five sentences describe the use of a chemiluminescence detection reagent kit IV What is the purpose of that kit? The purpose of the detection reagent kit was to trigger the chemiluminescence reaction of the insulin and C-peptide chemiluminescent kits. This has been clarified in the Materials and laboratory analyses section. - P5-Materials and laboratory analyses: How has blood glucose been measured (assay used)? Blood glucose was measured with a Roche Cobas 8000 c702 analyser which is a UV test using an enzymatic reference method with hexokinase. The following has been added to the materials section Blood glucose was measured with a Roche Cobas 8000 c702 analyser. - Table 1: The study mainly aims to investigate differences in blood glucose, insulin and C-peptide concentrations in two groups of preterm neonates only differing in their glycemic status. However, their prematurity is significantly different (both GA and PMA). Could that produce a bias on the results? We agree with the reviewer that this might produce some bias. However, because we used gestational age categories when matching, and could not find a more appropriate method, there is the possibility that some smaller mismatches in gestational age may have arisen. The significant different in their prematurity is probably not unexpected with more premature neonates being more likely to develop hyperglycaemia. However, we have demonstrated that similar effects were seen in the two groups indicating that insulin resistance is a direct result of prematurity so for this study the difference in the prematurity of the two groups should not drastically affect the results. We have added this explanation to the limitations section of the discussion. - The number of preterm neonates included in the non-insulin-treated group (n=20) is quite larger than the number included in the insulin-treated neonates (n=10). How could this difference affect the results? It should be clearly specified in some part of the manuscript.

11 We chose a ratio of 2:1 to minimise bias and maximise the power of the study. A matching ratio of up to 4:1 can be used to achieve this and we found a ratio of 2:1 worked best with the control cases we had available. A study by Peter C. Austin, Statistical Criteria for Selecting the Optimal Number of Untreated Subjects Matched to Each Treated Subject When Using Many-to-One Matching on the Propensity Score, (doi: /aje/kwq224) justifies our reasoning of using this ratio and he found that increasing the number of controls matched to each case results in improved efficiency and that using 1:1 or 2:1 matching will result in optimal estimation of treatment effects, therefore our selection of 2:1 matches this nicely. The following statement has been added to the Patients and Methods section: A ratio of 2:1 of nontreated to treated was chosen to minimise bias and maximise the matched sample size. - P6-Results: Authors indicate that there are five neonates with low C-peptide concentrations and four neonates with high C-peptide concentrations. It is not clear if they belong to the hyperglycaemic or euglycaemic group. It should be clearly specified. These neonates all belong to the hyperglycaemic group, this has been made clearer with the statement: Of the insulin-treated neonates, five neonates had very low - Figure 2: Y-label axis of graph (A) is incomplete. Thank you, we can see how this is unclear and it has been changed to Insulin and C-peptide plasma concentrations (pmol/l). bmjpo: first published as /bmjpo on 5 September Downloaded from on 15 October 2018 by guest. Protected by copyright.