Author's response to reviews

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1 Author's response to reviews Title:Placebo-Controlled Randomized Clinical Trial of Fish Oil's Impact on Fatigue, Quality of Life, and Disease Activity in Systemic Lupus Erythematosus Authors: Cristina Arriens Linda Hynan Robert Lerman David Karp Chandra Mohan Version:4Date:20 July 2015 Author's response to reviews: see over

2 July 6, 2015 Department of Internal Medicine Nehme Gabriel, Editor-in-Chief Nutrition Journal BioMed Central 236 Gray's Inn Road London WC1X 8HB United Kingdom Dear Dr. Gabriel and the Nutrition Journal editorial staff, Please find enclosed the edited version of an original, unpublished full-length research article entitled Placebo-Controlled Randomized Clinical Trial of Fish Oil s Impact on Fatigue, Quality of Life, and Disease Activity in Systemic Lupus Erythematosus by Cristina Arriens, Linda S. Hynan, Robert H. Lerman, David R. Karp, and Chandra Mohan. All authors have read and approved submission of the manuscript and meet criteria for authorship. Peer Reviewers: 1) Kevin Fritsche 2) Philippa Jackson Below we will respond to the reviewer comments. Thank you for reviewing our work and your consideration of the provided edits. Sincerely, Cristina Arriens and Chandra Mohan

3 Reviewer 1: First, as shown in Table 3, there are large and statistically SIGNIFICANT differences between the placebo and FO groups relative to the primary outcomes (e.g., energy/fatigue; quality of life) at the start of the intervention. It is unclear if these differences are a consequence of poor planning or bad luck relative to dropouts. We value the statements made by the reviewer. We did not attempt to conceal the baseline differences in Table 3. We discuss this shortcoming of the trial in the manuscript. Page 9 Inexplicably, the fish oil group reported lower scores at baseline for the RAND SF-36 and higher scores on the FSS, indicating worse quality of life and worse fatigue (Table 3). The radial diagrams representing all 8 subscales of the RAND SF-36 allow better visualization of the baseline differences as well as areas with greater improvement (Figure 3). Page 10 Due to the large difference in baseline values the results are somewhat difficult to interpret. Attempt to incorporate baseline values as a covariate resulted in loss of improvement signals in this small group of patients. (Also please see Table 3) Second, according to the data in Table 2, there appears to be a substantial difference between the placebo and FO groups in the proportion of subjects taking high-dose prednisone (i.e., >7.5 mg/d) at the end of the study (i.e., only 3 of 14 placebo subjects vs. 12 of 18 FO subjects). In this case, it appears that this was an unexpected consequence of dropouts. Regardless of the cause the authors need to do a better job explaining the potential confounding effects of high steroid use on the clinical and biochemical parameters assessed in this study. Similar concerns can be raised regarding statin use, which is much higher in the FO group relative to the placebo subjects. We have added a line to table 2 for the median and interquartile range for the prednisone dose for clarification. The change in statin use was not significant within groups (McNemar test). Statements have been added to the text to clarify the medication differences (in Results: Patient Population). Page 8 - For accuracy, change in prednisone dose was analyzed as a continuous variable to determine if significant changes in dose occurred between groups, with no significant difference being found (p=0.929). Hydroxychloroquine use was surprisingly near complete in the patients. Other immunosuppressant use did not vary significantly between groups. Statin use was similar at baseline between groups (p=0.235), but significantly higher in the fish oil patients at completion (p=0.0276). McNemar s test did not note a significant difference between baseline and completion of statin use for the fish oil group (p=0.248) or the placebo group (p>0.999). Also please see revised Table 2 Third, there exists a significant difference in the mean age and body mass index (BMI) between the placebo and FO treatment groups. That these differences existed at the start of the trial and persist throughout the study is problematic and should be addressed by the authors. This was previously noted in the text, and Table 1 has asterisks to note the statistical differences. We have added clarification statements to the text.

4 Page 7 Randomization resulted in two groups with similar demographics with the exception of higher age, body mass index (BMI), and hypertension comorbidity in the fish oil group compared to the placebo group (Table 1). Page 12 - Additionally, although randomization resulted in two similar groups based on demographics with the exception of older age and higher BMI in the fish oil group, the latter group had worse quality of life and fatigue at baseline. Age and BMI may have contributed to the disparity in baseline quality of life measures. Randomization is performed in order to match groups on both measured variables as well as unmeasured variables. In large studies randomization results in well matched groups, however in smaller studies random differences in measured variables are still possible. Fourth, the authors should have assessed the omega-3 status (e.g., omega index ) of these subjects at the start and completion of this study. This would allow the authors to verify compliance with the FO treatment independent of pill counts. Additionally, assessing changes in omega-3 status has been used to separate responders from non-responders in other RCTs involving FO supplementation. The authors could still have this analysis conducted, if they had RBCs from these subjects stored in a freezer. We appreciate the comment and would consider utilizing collection of cells in future trial designs. The omega index requires stored red blood cells to perform the laboratory study. Unfortunately, no cells were collected or stored on the patients in the trial; therefore it is not possible to perform this particular study for this trial. No change to manuscript see above reply. p. 2, line 4; p. 3, line 15: Omit the phrase/claim that omega-3 fatty acids are powerful antioxidants. The authors have mistakenly interpreted the drop in urinary 8-isoprostane from fish oil feeding (reported in citations #8&9) as an indication of a reduction in oxidative stress, when in fact, this phenomena is caused by the well-documented impact of FO feeding on tissue arachidonic acid, the immediate precursor of 8-isoprostane. We thank the reviewer for this insight. We have added a citation to the paper detailing studies describing the mechanistic process behind the anti-oxidant and some of the anti-inflammatory effects of omega-3 fatty acids. We have modified the text accordingly. Page 3 However, in another study, SLE patients given fish oil had improved flow-mediated dilation, disease activity, and 8-isoprostanes [8]. A study of EPA alone in six lupus nephritis patients noted significant reduction in urinary 8-isoprostane [9]. An elegant study by Groeger et al details a possible mechanism for omega-3 fatty acids anti-oxidant and anti-inflammatory effects [10]. p. 4, line 12: Define ACR (& all other abbreviations used throughout the paper) in a footnote to the title page. Based on discussion with the journal s editors, the house style is a list of abbreviations at the end of the manuscript as we have done. No changes have been made to the manuscript - please see above reply.

5 p. 4, line 22: Provide more detailed info about the placebo (i.e., purified olive oil). We have included more information to describe the placebo. Page 4 visually identical placebo (6 capsules/day purified [refined, not extra-virgin] olive oil Metagenics) p. 5, lines 23-24: The authors decision to present some treatment data as baseline minus 6-month treatment values is unfortunate, because this creates the situation where a negative change denotes IMPROVEMENT. This will create discord in the minds of most readers that is unnecessary and easily avoided. The authors are encouraged to re-express such data in a way that a positive treatment effect can be interpreted as BENEFICIAL. We have discussed this issue with the editors and others in the field. Although quality of life and disease activity measures easily identify improvement and worsening, the best way to present biomarker measures like cytokines is less clear. Given these ambiguities, we believe consistent methodology within the manuscript is absolutely essential. However, in response to reviewer 2 s comments, all measures have been changed to 6 month baseline which assures consistent methodology and many improvements are now positive. Page 5 [21-23]. For both disease activity measures, higher scores indicate worse disease activity with negative change from baseline (6 months baseline) indicating improvement. Page 5 and 6 Higher scores indicate a better quality of life and therefore a positive change from baseline denotes improvement. In addition to the Energy/fatigue subscale of the RAND SF-36, fatigue was assessed with the Fatigue Severity Scale (FSS) with lower scores indicating less fatigue and negative change from baseline denoting improvement [25]. Both scales have been utilized in prior evaluations of SLE patients [26]. The scores were all assessed by the same evaluator (unblinded) for consistency. Page 7 Difference scores were calculated for all continuous measures subtracting baseline measures from 6 month measures(end of study-baseline). Page 9 Comparison of score changes for the Energy/fatigue subscale of the Rand SF-36 utilizing the Mann-Whitney test yielded a trend in improvement for the fish oil group (median [IQR ]) compared to the placebo group (-2.50 [ ], p=0.092; Figure 2 A). Another related subscale, Emotional well-being also demonstrated a trend in improvement in the fish oil group (16.00 [ ] compared to the placebo group (4.00[ ], p=0.070; Figure 2 B). The FSS change scores were quite similar between the fish oil patients ( [ ] and placebo patients (0.222 [ ], p=0.350; Figure 2 C). Inexplicably, the fish oil group reported lower scores at baseline for the RAND SF-36 and higher scores on the FSS, indicating worse quality of life and worse fatigue (Table 3). The radial diagrams representing all 8 subscales of the RAND SF-36 allow better visualization of the baseline differences as well as areas with greater improvement (Figure 3). Disease Activity In addition to evaluating fatigue, we assessed disease activity using the PGA and SELENA-SLEDAI. The fish oil patients exhibited improvement in global disease activity ( [ ] compared to the placebo patients (0.50 [ ]) based on the PGA (p=0.015; Figure 4 A). The change in

6 SELENA-SLEDAI (fish oil [ ] and placebo 0.00 [ ]) and the change in renal SELENA- SLEDAI (24 lupus nephritis patients only; fish oil 0.00 [ ] and placebo 0.00 [ ]) scores did not indicate a significant difference between the two groups (p=0.417 and p=0.350, respectively; Figure 4 B and C). p. 7, lines 9-11: Reduce the number of significant figures reported for BMI down to a single number to the right of the decimal. We have made the change requested by the reviewer. Page 7. Median BMI was 29.3 (range: ), at the high end of being overweight. ALL TABLES AND FIGURES: Studies show that most readers expect the placebo or control group values to be shown/given first (i.e., on the left side of the figure), while treatment groups then follow. Please consider modifying your manuscript with the reader in mind. We have changed all figures and all tables as requested by the reviewer. Please see all figures and tables. Table 1: Avoid abbreviations, such as HTN, DM2, ACR or at least define them in a footnote to the table. The journal style is to present the abbreviation section after the conclusion according to the editors as stated above; therefore no change has been made. No changes made please see above comment. Table 2: Confirm that the relative proportions of subjects on high-dose prednisone or statins is NOT statistically 6 months between the placebo vs. FO treatment (i.e., report p-values). The change has been made in the text. We have added a line to the table with the prednisone dose listed as the median with interquartile range for clarification. The text has been updated to include the results of the statistical testing utilizing Mann-Whitney for baseline, 6 months, and change in dose between fish oil and placebo groups for prednisone. We have also reported results of statistical testing for statin use. Page 8 The unblinded evaluator did not alter medications for the duration of the study. For accuracy, change in prednisone dose was analyzed as a continuous variable to determine if significant changes in dose occurred between groups, with no significant difference being found (p=0.929). Hydroxychloroquine use was surprisingly near complete in the patients. Other immunosuppressant use did not vary significantly between groups. Statin use was similar at baseline between groups (p=0.235), but significantly higher in the fish oil patients at completion (p=0.0276). McNemar s test did not note a significant difference between baseline and completion of statin use for the fish oil group (p=0.248) or the placebo group (p>0.999). Table 3: Omit the lower third of this table since these data are displayed in Fig. 2 & 4.

7 We thank the reviewer for their comments; however we believe that providing the difference scores in Table 3 with median, interquartile range, and statistical testing is the most concise and comprehensive manner to deliver the complete data to the readers. Figure 2 (a and b) only highlights 2 subsets of the SF-36 data that showed improvement trends. Figure 4 displays the means of the data rather than medians for the purpose of visual display. No changes made please see above reponse. Figure 2 & 4: Provide the p-values somewhere within the figure instead of in the footnotes. Please convert the data to indicate that an increase is a BENEFIT. We agree with the reviewer that including p-values in the graphs would be an improvement and have made the requested changes. Please see all graph figures. Figure 5 is not needed since these same data are provided in Table 4. We thank the reviewer for the comment. Figure 5 includes ESR, IL-13, and IL-12 data, and Table 4 includes data on IL-13 and IL-12 (with other cytokines) but does not show ESR. Given the importance of the effect on ESR we have retained Figure 5. We have removed table 4 instead to eliminate the redundancy as requested by the reviewer. We have updated the text to reflect this change. Deleted Table 4 and text page 10 Additional soluble mediator results with between group difference signals are provided (Table 4).

8 Reviewer 2: Overall the report is written to a high standard and no major revisions are necessary. Minor essential revisions: - It is unclear if the SELENA-SLEDAI was also administered by the assessor who was not blind to the treatment. Please be explicit. We thank the reviewer for the comment. Text has been added to clearly state that the assessor was the same and was unblinded. Page 6 The scores were all assessed by the same evaluator (unblinded) for consistency. Pge 8 The unblinded evaluator did not alter medications for the duration of the study. Page Although the patients were blinded to treatment, the investigator was able to discern the treatment groups. Besides physician global assessment and selected components of the SLEDAI, all remaining measures were objective or patient-reported. - Include treatment compliance data We have added statements to the text at the request of the reviewer. Page 8 All completers consumed greater than 50% of their treatment. Fifteen of eighteen (83%) fish oil subjects and nine of fourteen (64%) placebo subjects consumed greater than 75% of their treatment. - Given that the randomization of subjects was ineffective (i.e. unbalanced groups on a number of baseline measures) please give more details as to how randomization was carried out. We have added statements for clarification to the text. Page 4-5 Patients were randomized using a simple block randomization 1:1 to receive fish oil (6 capsules/day equaling 2.25g EPA and 2.25g DHA Metagenics, Inc. Gig Harbor, WA) or visually identical placebo (6 capsules/day purified [refined, not extra-virgin] olive oil Metagenics) in addition to their background therapies, with treatment duration of 6 months. Page 12 Additionally, although randomization resulted in two similar groups based on demographics with the exception of older age and higher BMI in the fish oil group, the latter group had worse quality of life and fatigue at baseline. Age and BMI may have contributed to the disparity in baseline quality of life measures. Randomization is performed in order to match groups on both measured variables as well as unmeasured variables. In large studies randomization results in well matched groups, however in smaller studies random differences in measured variables are still possible. Discretionary revisions: - Change from baseline scores are more commonly calculated 6 month score baseline score. The fact that you calculated the opposite way round so that positive scoring items were negative was confusing. Consider reversing this calculation for the wider audience. The authors have changed all figures and tables to the requested format. Page 5 [21-23]. For both disease activity measures, higher scores indicate worse disease activity with negative change from baseline (6 months baseline) indicating improvement.

9 Page 5 and 6 Higher scores indicate a better quality of life and therefore a positive change from baseline denotes improvement. In addition to the Energy/fatigue subscale of the RAND SF-36, fatigue was assessed with the Fatigue Severity Scale (FSS) with lower scores indicating less fatigue and negative change from baseline denoting improvement [25]. Both scales have been utilized in prior evaluations of SLE patients [26]. The scores were all assessed by the same evaluator (unblinded) for consistency. Page 7 Difference scores were calculated for all continuous measures subtracting baseline measures from 6 month measures(end of study-baseline). Page 9 Comparison of score changes for the Energy/fatigue subscale of the Rand SF-36 utilizing the Mann-Whitney test yielded a trend in improvement for the fish oil group (median [IQR ]) compared to the placebo group (-2.50 [ ], p=0.092; Figure 2 A). Another related subscale, Emotional well-being also demonstrated a trend in improvement in the fish oil group (16.00 [ ] compared to the placebo group (4.00[ ], p=0.070; Figure 2 B). The FSS change scores were quite similar between the fish oil patients ( [ ] and placebo patients (0.222 [ ], p=0.350; Figure 2 C). Inexplicably, the fish oil group reported lower scores at baseline for the RAND SF-36 and higher scores on the FSS, indicating worse quality of life and worse fatigue (Table 3). The radial diagrams representing all 8 subscales of the RAND SF-36 allow better visualization of the baseline differences as well as areas with greater improvement (Figure 3). Disease Activity In addition to evaluating fatigue, we assessed disease activity using the PGA and SELENA-SLEDAI. The fish oil patients exhibited improvement in global disease activity ( [ ] compared to the placebo patients (0.50 [ ]) based on the PGA (p=0.015; Figure 4 A). The change in SELENA-SLEDAI (fish oil [ ] and placebo 0.00 [ ]) and the change in renal SELENA- SLEDAI (24 lupus nephritis patients only; fish oil 0.00 [ ] and placebo 0.00 [ ]) scores did not indicate a significant difference between the two groups (p=0.417 and p=0.350, respectively; Figure 4 B and C). All figures and tables as well. - Only a single sentence is included that considers the mechanisms that may underpin the reported effects. Consider expanding. We have expanded this and added a reference that was an extensive study of mechanistic effects of omega-3 fatty acids. Page 12 In a mouse model of SLE, MRL/lpr, fish oil supplementation was found to reduce IL-12 serum levels. Infectious challenge resulted in reduced IL-12 level in fish oil fed healthy mice [39, 40]. Omega-3 fatty acids have been noted to have anti-inflammatory properties including alteration to cytokine signaling and anti-oxidant effects in a recent mechanistic study [10]. Our findings of fish oil supplementation resulting in reduced ESR and IL-12, as well as increased IL-13 are in concordance with published human and animal studies. Importantly, these results add to the plausibility of fish oil resulting in reduced inflammation through multiple molecular mechanisms, including alterations in Th1/Th2 balance.

10 - As the authors note, there are a number of limitations of the study, to which end it would be better to label this trial as a pilot study. The authors note that published studies of fish oil that are similar in size have been presented as clinical trials in the literature, rather than pilot studies (Wright et al, Clark et al, Duffy et al, and Rondanelli et al). No changes to manuscript please see above response.