Author's response to reviews

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1 Author's response to reviews Title: Use of food frequency questionnaire to assess relationships between dietary habits and cardiovascular risk factors in NESCAV study: validation with biomarkers Authors: Nicolas Sauvageot Alaa Alkerwi Adelin Albert Michèle Guillaume Version: 4 Date: 13 September 2013 Author's response to reviews: see over

2 Competence center in methodology and statistics nicolas.sauvageot@crp-sante.lu th September 2013 Paper No.: Dear Sir, We thank the two reviewers for their valuable comments which have helped improve the paper. We are pleased to return you the new version revised in regard to the reviewers comments and suggestions. Our point-to-point responses to all issues raised by the reviewers are extensively prepared, and revisions are highlighted in the text in yellow. We hope that the revised version is now suitable for publication in your Journal. Yours sincerely, Nicolas Sauvageot, MSc Centre de Recherche Public Santé, Competence center in methodology and statistics, Grand-Duchy of Luxembourg.

3 Point-to-point responses Manuscript title: Use of food frequency questionnaire to assess relationships between dietary habits and cardiovascular risk factors in NESCAV study: validation with biomarkers (Manuscript Number: MS: ) Responses to reviewer (Ulla Toft ): My main concern is related to the use and interpretation of most of the biomarkers used in this study. Many of the included biomarkers are not direct biomarkers of the dietary intake and much better biomarkers exists than those used in the study. Thus for example serum lipids are used as biomarkers for fiber, carbohydrate intake etc. But the validity of these as biomarkers is low because blood lipids are affected by many different factors, and can only be used on very controlled conditions. Thus the authors for example conclude that the FFQ appears to be useful for assessing total fiber intake based on a significant correlation with serum cholesterol, and with serum triglyceride for women. But this conclusion can t be made based using biomarkers that are affected by several other dietary factors (and other factors) than fiber intake. In contrast for example plasma alkylresorcinols have been found to be a good direct biomarker of wholegrain consumption, serum pentadecanoic acid and heptadecanoic acid are biomarkers of the intake of dairy fat, serum vitamin C and carotenoids are widely used as biomarkers for the intake of fruit and vegetables etc. The value of this study would improve by included less but more valid biomarkers. We do agree with the reviewer that nutritional biomarkers should have a strong direct relationship with dietary intakes for the validation of a dietary instrument as the FFQ. However, such biomarkers (recovery and predictive biomarkers) are rare and expensive. Most others nutritional biomarkers (concentration biomarkers) are indicative measures of the nutritional status, which not only reflect intake but also the amount available after absorption and metabolism. Therefore, correlations of such biomarkers with estimates of dietary intake are often weak. In this study, we used many of those affordable biomarkers to assess the validity of several micro-nutrients intakes. However, due to high measurement cost of these concentration biomarkers to assess macro-nutrients, no other biomarkers were measured (1, 2). Although, the literature has shown significant associations between these macro-nutrients intake such as fat, carbohydrates, dietary cholesterol and blood lipids(3), many different factors may affect their validity. The use of HDL, LDL and TG measurements, as dietary biomarkers still requires much validation(3). In the revised version of our article, all presented associations between macro-nutrients estimates and blood lipids have been removed, in order to avoid the limitations raised by blood lipids as biomarkers. Additionally, we used the plasma β-carotene, which is a good biomarker of fruit and vegetable intakes (3-6). Associations of this biomarker with estimated intakes of fruit and vegetables and vegetables alone and fruits alone have been documented. Although others authors reported higher correlation (7-10), significant positive correlations were found between plasma β-carotene concentration and estimated intakes of fruits and vegetables (r=0.17 in men and r = 0.1 in women), vegetables alone (r = 0.18 in men and r = 0.11 in women) and fruits alone (r = 0.11 in men and r = 0.1 in women). These results have been added in the new version of the article.

4 Except for fruits and vegetables intakes, this biomarker-validation study focuses mainly on the validity of several micro-nutrients intakes. As a complementary approach, macro-nutrients estimates and others micro-nutrients have been assessed in the second study (already published) in which their estimates from FFQ and a 3-day dietary record (DR) were compared(11). The main findings of this previous study have also been included in the revised version of the present paper. The results from this study alone actually indicate a rather low validity of the food frequency questionnaire and it is a major drawback of this study that the results from the dietary records are not included or published, so that it would be possible to the readers to evaluate the overall validity and agreement between the FFQ and the dietary records, combined with the results from the biomarkeranalyses. Based on the results in this study the conclusions should be modified as the validity of the food frequency questionnaire is overestimated as it is now. The author should consider to wait for the publication of the paper including the dietary records to give a better picture of the validity of the FFQ. Or consider to combine the two papers. Furthermore the authors should consider using the method of triad in their validation of the FFQ We thank the reviewer for this relevant comment. In fact, the second FFQ validation study, based on the comparison of FFQ dietary intakes against 3-day diet records (DR) has recently been published in the Journal of Nutrition and Food Sciences (11). Thus, our current findings from the biomarkers study supported by the validation study against DR permit to evaluate the overall validity of our FFQ. Globally, in the study comparing FFQ with DR, the relationship of macro- and micro-nutrients was satisfactory although, results for proteins, cholesterol, starch, vitamins A, E and B12 need to be interpreted with caution. In the present biomarker-validation study, it has been shown that our FFQ performed well in ranking most of micro-nutrient and fruits and vegetables intakes. Interestingly, despite of no agreement for vitamin B12 in the comparison with DR, significant correlations were observed between FFQ estimates and plasma concentrations. As suggested by the reviewer, our results interpretation and conclusion have been modified in the new version of the manuscript. Concerning the suggestion of the reviewer to use the method of triad in the validation of the FFQ, it is not feasible in our study. In fact, this method requires information about the dietary intake for the three methods, namely FFQ, DR and biomarkers, collected from the same sample, which are not available for us. Therefore, the overall validity of the FFQ has been done in two complimentary steps: A dietary study comprising a comparison of the FFQ against DR in a conventional sample of adult women. A biomarker study comprising a comparison of FFQ estimates against nutritional biomarkers concentrations in a subsample of the NESCAV study participants. Owing to the multiple statistical approaches used to analyse the data and the important quantity of findings derived from both studies, we opted to split the overall validation of the FFQ in two sub studies. We hope that this answer corresponds to the reviewer s expectations. Minor comments The authors choose to exclude individuals above the 99th percentile or below the first percentile. However, there are no arguments that these should be less valid data just because the data are in the highest or lowest part of the population. The author however could consider using the Goldberg method to identify extreme misreports according to the reported energy intake In fact, we did not exclude individuals above the 99 th percentile or below the first percentile. As explained in the data-cleaning part, the first step was to replace the outliers values of nutrient intakes (those lying above the 99 th percentile or below the 1 st percentile) by missing values (as extreme values could potentially influence the results). Then, participants with more than 10 missing values of nutrient intakes were excluded. This method led to the elimination of 40 (4.1%) out of 984

5 individuals. Since our methodology of excluding some extreme individuals is debatable, we performed the statistical analyses on both the entire sample and after exclusion of individuals according to the Goldberg method (as suggested by the reviewer). Since 62 individuals were excluded because of lack of information on micro-nutrients use, analyses on the whole sample were performed on 922 participants. No significant changes in the results were noticed. The Goldberg method was then applied to identify misreport according to the reported energy intake (12). This led to the exclusion of 48/922 individuals. Again, no significant changes in the results were observed. Since the conclusions were the same regardless of excluding potential misreports, the results computed on the entire sample have been presented in the revised article. Responses to reviewer (Junko Ishihara): Major Compulsory Revisions Pg. 5 Methods, Nutritional biomarkers Authors should include detail about specimen collection and analyses. OK. Detail about specimen collection and analyses has been added in the paragraph Nutritional biomarkers Were the participants fasted at the blood collection? All participants but 21 (2.3%) were fasted at the blood collection. Since the biomarkers considered in the revised article are not affected by the fast status, we did not exclude those individuals from the analyses. Was urine 24-hour urine or spot-urine? It was a morning spot-urine. What kind of analytical procedures were done for each biomarker. Further detailed information about specimen collection and laboratory analyses has been added in supplementary table 1. Pg. 11 Conclusion Authors need to include that sodium intake also should be interpreted with caution. OK. This information has been added to the revised article in the discussion and conclusion. Minor Essential Revisions Pg.9 Discussion, 4th paragraph For Vitamin B6 intake, we used... Shouldn t it be vitamin B9 or folate intake? We thank the reviewer and apologize for this mistake. It has been corrected in the new version of the manuscript. 1. V. E. Hedrick, A. M. Dietrich, P. A. Estabrooks, J. Savla, E. Serrano, B. M. Davy, Dietary biomarkers: advances, limitations and future directions. Nutrition journal 11, 109 (2012) / ). 2. A. M. Leufkens, F. J. van Duijnhoven, S. H. Woudt, P. D. Siersema, M. Jenab, E. H. Jansen, T. Pischon, A. Tjonneland, A. Olsen, K. Overvad, M. C. Boutron-Ruault, F. Clavel-Chapelon, S. Morois, D. Palli, V. Pala, R. Tumino, P. Vineis, S. Panico, R. Kaaks, A. Lukanova, H. Boeing, K. Aleksandrova, A. Trichopoulou, D. Trichopoulos, V. Dilis, P. H. Peeters, G. Skeie, C. A.

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