KU Leuven Post-print
Reproducibility and validity of a food-frequency questionnaire to
assess usual intake of methyl-group donors.
Sara Pauwels, Ine Doperé, Inge Huybrechts, Lode Godderis, Gudrun Koppen, and
Greet Vansant.
N.B.: When citing this work, cite the original article
Original publication: Pauwels S., Doperé I., Huybrechts I., Godderis L. Koppen G.,
and Vansant G. Reproducibility and validity of a food-frequency questionnaire to
assess usual intake of methyl-group donors. Public Health Nutrition. 2015. Jan 14: 110.
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Reproducibility and validity of a food-frequency questionnaire to assess usual
intake of methyl-group donors
Sara Pauwels1,2*, I. Doperé1, I. Huybrechts3, L. Godderis1,4, G. Koppen2, and G. Vansant1
1
KU Leuven, Department of Public Health and Primary Care, Centre Environment & Health, Kapucijnenvoer 35 blok D
box 7001, 3000 Leuven, Belgium:2 Flemish Institute of Technological Research (VITO), Unit Environmental Risk and
Health, Vlasmeer 7, 2400 Mol, Belgium:3International Agency for Research on Cancer, Dietary Exposure Assessment
Group, 150 Cours Albert Thomas, 69372 Lyon CEDEX 08, France: 4IDEWE, External Service for Prevention and
Protection at work, Interleuvenlaan 58, 3001 Heverlee, Belgium
Short title: FFQ to assess intake of methyl-group donors
Acknowledgments: We acknowledge the women who volunteered to take part in this study.
Financial support: Funding for the present study was provided by a PhD grant (grant number
11B1812N) from The Research Foundation-Flanders (FWO) and the Flemish Institute of
Technological Research (VITO) for Sara Pauwels.
Conflict of interest: None
Authorship: S.P. and I. D. were responsible for the field work and data entry. I. H. helped with the
statistical analysis of the results. All authors helped in the evaluation of the results and the writing
of the manuscript. All authors have read and have approved the manuscripts before submission.
Approving ethics committee: UZ Leuven-Committee for Medical Ethics (reference number:
ML7975)
Key Words (3-4): FFQ: Dietary assessment: Validation: Methyl-group donors
*Corresponding author: Sara Pauwels, tel +32 16 377 768, fax +32 16 3 36997
email sara.pauwels@med.kuleuven.be
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Abstract
Objective: To develop and validate a novel ‘food-frequency questionnaire’ (FFQ) to assess the daily
intake of 4 methyl-group donors (methionine, choline, betaine, and folate).
Design: The relative validity of the FFQ was assessed by comparison with 7-day estimated diet
records (7d EDR, n80) and its reproducibility was evaluated by repeated administrations 6 weeks
apart (n92). Paired student’s t-tests were used to compare group means and de-attenuated Intraclass
correlations to investigate the ability of the FFQ to rank individuals according to their methyl-group
donor intake. De-attenuated Intraclass correlation coefficients were calculated between the test and
reference method for methionine, choline, betaine, folate, and the sum of methyl-group donors
(SUM). The weighted kappa (ƙ) was calculated as a measure of tertile agreement.
Setting: Flanders, Belgium.
Subjects: The FFQ was validated among Flemish women of reproductive age (18-35y).
Results: The questionnaire has an acceptable ranking ability (r0.32-0.68; weighted ƙ 0.10-0.35), but
overestimates the intake of folate (280.6μg vs. 212μg) and betaine (179.1mg vs. 147mg) compared
to the 7d EDR. Cross-classification analysis indicated that 20% (choline) of the subjects were
grossly misclassified in the validation study. The correlation between repeated administrations was
good (r0.62-0.83) with a maximal misclassification of 7% for betaine (weighted ƙ 0.44-0.66).
Conclusion: These results indicate that this newly developed FFQ is a reliable instrument with
acceptable validity for ranking individuals according to methyl-group donor intakes (except for a
poor agreement for choline (weighted ƙ 0.10) and a fair ranking ability for betaine (r0.32)) in
Flemish women of reproductive age.
Introduction
The impact of nutrition on the methylome has received much attention over the past years(1). The
methylome describes the distribution of methylated sequences within a given genome without
changes to the DNA sequence itself. In humans, DNA methylation takes place at the carbon-5
position of cytosine in CpG dinucleotides(1). One-carbon metabolism is central to the methylation of
DNA (Fig.1). The main nutrients implicated are methyl-group donors and cofactors including
methionine, choline, betaine, folate, vitamin B6, and vitamin B12 which are all derived from the diet.
Consequently, any dietary factor influencing this pathway may affect DNA methylation(2, 3). The
transfer of a methyl-group to the DNA depends on the availability of S-adenosylmethionine (SAM).
SAM is derived from methionine, which is de-methylated through the action of DNA
methyltransferases to S-adenosylhomocysteine (SAH)(4). SAH is hydrolyzed to homocysteine in a
reversible reaction that strongly favors SAH synthesis compared to hydrolysis. Homocysteine can
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leave the methionine cycle through the transsulfuration pathway or can be re-methylated back to
methionine with vitamin B12 acting as a cofactor and 5-methyltetrahydrofolate (5-methyl-THF) as
substrate(5).
In the context of a prospective observational study investigating the relation between the intake of
maternal methyl-group donors during each trimester of pregnancy and offspring DNA methylation,
an instrument assessing the daily intake of methyl-group donors was needed. At study onset, no
such tool was available since food composition databases for betaine and choline only recently
became available(6). It is preferred to use food-frequency questionnaires (FFQ) over diet records or
24-h recalls, because they have shown to be more suitable and accurate in assessing long-term or
habitual exposure to specific nutrients involved in one-carbon metabolism (e.g. folate)(7).
Studies on the effect of maternal diet and the methylome of the offspring are showing the
importance of maternal nutrition. Heijmans et al.(8) explained the long-term effects of prenatal
famine on the offspring methylome. They found an average decrease of 5.2% in DNA methylation
on the insulin like growth factor 2 (IGF2) gene locus, showing that maternal under-nutrition (low
intake of methyl-group donors) can indeed have persistent effects on the offspring throughout
adulthood. A similar study by Tobi et al.(9)suggested that famine exposure (in utero) only held a
long-term effect on the offspring IGF2 gene when the exposure was periconceptional. On the other
hand, Steegers-Theunissen et al.(10) found that also a periconceptional supplementation of mothers
with 400 μg of folic acid ( methyl-group donor) was associated with epigenetic changes (4.5%
higher DNA methylation) in the same IGF2 locus in the child.
A novel self-administrated semi-quantitative FFQ was designed to estimate the daily intake of
methionine, choline, betaine, folate, and the sum of methyl-group donors (SUM) in women of
reproductive age (18-35y). The aim of the present study was to assess the validity and
reproducibility of the FFQ using the 7-day estimated diet record (7d EDR) as reference method. The
reproducibility of the FFQ was determined by administrating the FFQ at two time points (6 weeks
apart) to the same group of women, so that the association between the two responses could be
assessed. Also the validity of the FFQ was determined to assess the degree to which the FFQ agrees
with the reference method (EDR). Even a subtle change in the design of the FFQ, or the use of the
FFQ in different demographic groups and cultures might affect its performance, so each new FFQ
should be validated(11).
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Materials and methods
Study design
A total of 296 women of reproductive age (18-35 years) were invited to participate in the study.
Female students or employees of the Catholic University of Leuven were asked to take part in the
study. An invitation letter, sent through mail or personally delivered, informed the participants
about the aim of the study. They were asked to return their first completed FFQ (FFQ1) and their
written consent in a sealed envelope. Six weeks later the second FFQ (FFQ2) together with the
EDR was distributed to only those participants from who we received FFQ1. Participants were
instructed to fill in the FFQ at day one, to start the EDR at day two, and to put the dates of
completion on the documents. Detailed instructions for filling in the FFQ and EDR were given. The
field work of the validation and reproducibility study was carried out from February to March 2012.
Of the 296 women invited to participate in the study, 161 women (54%) returned FFQ1. The 161
women were again invited to take part in the second phase of the study and of these 161 women 98
(61%) completed FFQ2 and the 7d EDR. Only FFQs without missing values were included in the
reproducibility study, so after exclusion of incomplete FFQs and outliers 92 women were included
in the analysis. Similarly, only complete 7d EDRs including 7 completed record days and
containing sufficiently detailed descriptions of the food products (including brand names, the food
type (e.g. the use of whole, semi-skimmed, or skimmed milk, the type of bread used, etc.)) and
portion sizes (expressed as household measures, standard units (e.g. a medium sized apple) or units
like grams or liters) consumed could be used in the validation study, so after exclusion of
incomplete EDRs 80 women were included in the analysis.
This study was conducted according to the guidelines laid down in the Declaration of Helsinki and
all procedures involving human subjects were approved by the [] (reference number: ML7975).
Written informed consent was obtained from all subjects.
Food-Frequency Questionnaire
The Flemish self-administrated semi-quantitative FFQ contained questions on the average
consumption of 51 food items during the past 3 months (Appendix 1). The time frame of 3 months
was chosen because this FFQ will be used to assess the intake of maternal methyl-group donors
during each trimester of pregnancy. The concept and structure of this FFQ was based on a
previously developed and validated FFQ estimating usual daily fat, fiber, alcohol, caffeine, and
theobromine intakes among Belgian post-menopausal women(12). However, the food list included in
our newly developed FFQ (51 food items) was adopted considering our study aims, namely to
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estimate the daily intake of methionine, choline, betaine, folate, and the sum of methyl-group
donors (SUM). Food items included in a food frequency questionnaire should be eaten reasonably
often by the population and/or contain a substantial amount of one or more nutrients of interest(11).
The 51 food items included in our FFQ were food items that are part of the Belgian diet (National
Food Consumption Survey) and/or had a high nutrient content (>P90) of one the 4 methyl-group
donors. All food items were listed according to their nutrient content (from high to low) and the top
10% food items were selected. Participants were asked to indicate their answers in a list of
frequencies: never or less than once a month; 1-3 d/month; 1 d/week; 2-4 d/week; 5-6 d/week; every
day(13). The FFQ also contained 3-5 daily portion size categories per food item and a list of common
standard measures as examples. For some food groups, additional questions were asked regarding
the type, such as specific type of fruit juice. All returned FFQs were reviewed for completeness and
written comments. Frequency data from the FFQ were converted to servings per day (e.g., 1
serving/week = 0.14 serving/day) to standardize the different frequency categories by means of a
common unit (servings per day). Portion size categories were converted to a portion (grams) per
serving by considering the mean of the portion size range that was chosen by the subject. The usual
food intakes were computed by multiplying the servings per day with the portion size per serving.
Usual nutrient (methionine, choline, betaine, and folate) intakes were calculated by multiplying the
usual food intakes with the nutritional value per 100g product. The food composition data were
based on the Dutch NEVO food composition database(14) for folate, the German BLS Nutrient
database(15) for methionine and the USDA database for the Choline Content of Common Foods(6) for
choline and betaine. The USDA database was also used for the nutrient content of folate and
methionine if not found in NEVO and BSL databases respectively. Databases of neighboring
countries or the USDA database for choline and betaine content were used since the Belgian food
composition database NUBEL(16) does not contain the four nutrients of interest.
Estimated diet record- reference method
In the present relative validation study the 7d EDR was chosen as the reference method. The
participants were given guidelines for filling in their diary and we also provided one correctly filled
in day as an example. The participants were asked to report all consumed foods and drinks over 7
consecutive days. In the 7d EDR, days are subdivided into 6 eating occasions (breakfast, morning
snacks, lunch, afternoon snacks, dinner, and evening snacks). Detailed information on the type
(including brand names, the food type (e.g. use of whole, semi-skimmed, or skimmed milk, the type
of bread used, etc.)) and portion size (expressed as household measures, standard units (e.g. a
medium sized apple) or units like grams or liters) of the foods consumed was collected using an
open entry format. Only complete food diaries, including 7 completed record days and containing
6
sufficiently detailed descriptions of the food products and portion sizes consumed, were taken into
consideration. In total 17 EDRs had to be excluded because of incomplete EDR. The complete
EDRs were coded and entered into a Diet Entry and Storage program (NUBEL Voedingsplanner(17))
using a manual on food portions and household measures(18). Methionine, choline, betaine, and
folate are not included in the Belgian food composition table Nubel(16), so the diet records were
linked to the same food composition databases as for the FFQ (Dutch NEVO food composition
database(14), the USDA database for the Choline Content of Common Foods(6), and the German
BLS Nutrient database(15)). Some typically Belgian food products like ‘speculaas’, a type of short
crust biscuit, are not included in the USDA database. For these products, the choline and betaine
content of the American variety was entered in the database. For example, a ginger biscuit was used
for the choline and betaine content of ‘speculaas’.
Statistical analysis
Statistical analyses were carried out using SPSS for windows version 20.0 (SPSS Inc., Chicago,
Illinois, USA). Results were considered statistically significant at an α two-tailed level of 0.05.
Tests for normality of the data were performed using the Kolmogorov-Smirnov test. Data were
normally distributed for the validation study and reproducibility study (after exclusion of 3 outliers
with unusual eating behaviors); consequently only parametric tests were used during analysis.
Means and standard deviations (SD) of the 4 methyl-group donating nutrients and the sum of all
(SUM) were estimated from the FFQs and EDRs. Paired Student’s t-test was used to determine
significant mean differences. Associations were described using Intraclass correlations. Correlations
ranging from 0.00 to 0.25 indicate weak or no relationship; those from 0.25 to 0.50 suggest a fair
degree of relationship; values of 0.50 to 0.75 are moderate to good; and values above 0.75 are
considered good to excellent(19). In the validity study, the de-attenuated Intraclass correlation
coefficients were calculated to correct for within-person variation in the EDR. This de-attenuated
correlation is calculated as the ratio of the inter-individual (true between subject variation in usual
intake) coefficient of variation over intra-individual (day by day variation in intake, day of the week
variation in females) coefficient of variation(20). Agreement between the EDR and the FFQ at an
individual level was assessed using mean difference, standard deviation and limits of agreement of
the difference, and visually represented in a Bland and Altman plot(21) (Fig.2). Individual results for
nutrient intake estimated by the EDR and FFQ were classified into tertiles to assess the
questionnaire’s ability to rank individuals according to the intake as in the EDR(13). The percentage
classified into the same and opposite tertile was calculated. Agreement between both methods was
assessed using weighted kappa (κ) statistics, calculated with a linear set of weights(22). The
measurement error of the FFQ was analyzed using ‘actual values for surrogate categories’(13). One7
way Anova test was used to determine significant differences in means between tertiles. Specificity
was defined as the proportion of those with a daily intake above the guidelines (23-25) on the basis of
the EDR who also fell above the guidelines on the FFQ (true negatives). Sensitivity was the
proportion of those with a daily intake below the guidelines on the basis of the EDR who also fell
below the guidelines on the FFQ (true positives). The positive predictive value was the proportion
of those identified by the FFQ as having an inadequate intake who actually had an inadequate intake
(according to the EDR). The negative predictive value was the proportion of those identified by the
FFQ as having and adequate intake who actually had an adequate intake (according to the EDR)
(Appendix 2).
Results
Validation study
In total, 80 women were included in the validation study. Table 1 presents the characteristics of the
study participants. Women included in the validation study had a mean age of 23 years (SD 4.1;
range 18-35), a mean weight of 60 kg (SD 9.6), a mean length of 1.68 m (SD 0.05), and a mean
BMI of 21.7 kg/m2 (SD 3.2). 26 women (32.5%) were employed at the time of the study, so 54
participants (67.5%) were students. Only 2 women (2.6%) smoked at the time of the study. Mean
intakes of methionine, choline, betaine, folate, and the sum of methyl-group donors (SUM)
estimated with the 7d EDR and the FFQ, mean differences, raw and de-attenuated Intraclass
correlation coefficients are presented in Table 2. The FFQ overestimates the intake of folate and
betaine in comparison with the EDR. The mean intakes of folate and betaine were significantly
different, but fair to good de-attenuated correlation coefficients ranged from 0.32 (betaine) to 0.68
(folate). The mean difference in methionine intake between FFQ1 and the EDR was -33.3 mg/d (SD
600.6), demonstrating that the FFQ underestimates methionine intake. The mean differences in
choline, betaine, folate, and SUM intakes between the two methods were 2.3 mg/d (SD 104.2), 32.1
mg/d (SD 78), 68.6 μg/d (SD 88), and 1.27 mg/d (SD 733) respectively, demonstrating that the FFQ
overestimates choline, betaine, folate, and SUM intake. This is graphically shown in Bland and
Altman plots (Fig 2.) The Bland and Altman plots show that outliers widened the limits of
agreement and made the plots more divergent. This divergent pattern could indicate increasing bias
with increasing intakes. Cross-classification analysis indicated that about half of the subjects (4054%) were classified correctly, while 9% (methionine) to 20% (choline) of the subjects were
grossly misclassified (Table 3). Results from the weighted κ statistics showed fair agreement
between the FFQ and EDR (0.21 for betaine to 0.35 for methionine and SUM), but a poor
agreement for choline (0.10). Actual values for surrogate FFQ tertiles showed a progressive
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increase in EDR intakes of methionine, betaine, folate, and the sum of methyl-group donors
between the first and third FFQ tertile (Table 4) with statistically significant differences in mean
intake between the tertiles for all nutrients except for choline. The specificity and sensitivity of the
FFQ, for indicating women with lower nutrient intake than the recommended daily intake, were 87
and 37% for folate, 33 and 97% for choline, and 100 and 100% for methionine respectively. The
positive and negative predictive value was 76% and 55% for folate, 97% and 33% for choline, and
100% and 100% for methionine respectively. The specificity and sensitivity of the FFQ for betaine
could not be calculated since there is no recommended daily intake for betaine.
Reproducibility study
In the reproducibility study, 92 women were included. Table 1 presents the characteristics of the
study participants. Women included in the reproducibility study had a mean age of 23 years (SD
3.9; range 18-35), a mean weight of 60.2 kg (SD 9.8), a mean length of 1.68 m (SD 0.06), and a
mean BMI of 21.7 kg/m2 (SD 3.1). 27 women (33.7%) were employed at the time of the study, so
53 participants (66.3%) were students. Only 2 women (2.1%) smoked at the time of the study. Mean
intakes of methionine, choline, betaine, folate, and the sum of methyl-group donors (SUM) from
FFQ1 and FFQ2 were significantly different and correlation coefficients ranged from 0.62 (betaine)
to 0.83 (folate) (Table 5). Cross-classification analysis indicated that 1% (methionine) to 7%
(betaine) of the subjects were grossly misclassified, while more than half of the subjects (57-71%)
were classified correctly (Table 6). Results from the weighted κ statistics showed moderate to good
agreement (0.44 for betaine to 0.66 for methionine).
Discussion
Validation study.
The FFQ has an acceptable ranking ability (r 0.32-0.68; weighted ƙ 0.10-0.35), but overestimates in
general the intake of folate (280.6μg vs. 212μg) and betaine (179.1mg vs. 147mg) compared to the
7d EDR. Cross-classification analysis indicated that 20% (choline) of the subjects were grossly
misclassified in the validation study.
Based on the comparison of the means, the FFQ seems to overestimate the mean intake of folate,
which has also been reported in previous studies(26-28), and betaine measured by the EDR.
Unfortunately, we could not determine whether this was due to over-reporting, because our FFQ
was not designed to estimate energy intake. A possible explanation is the contribution of vegetables
to total folate intake. In our FFQ, vegetables contributed 34% of total folate intake, which is
comparable to the 36% found in another FFQ(28). Fruit and vegetables are most often over-reported
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in FFQs(29), possibly because these are socially desirable foods(30). Another explanation for this
overestimation of folate is the long list of vegetables in the FFQ contributing to the discrepancy in
intake between the FFQ and EDR, since estimates of vegetable intake have been shown to be
related to the number of questions asked(31). A possible reason for the overestimation of betaine by
the FFQ is that the betaine values assigned to the food items in the EDR might be more accurate
(because of higher level of details in the food description) than the aggregated values assigned to
the food groups in the FFQ. Another possible explanation might be that some of the food groups
high in betaine have been overestimated by the subjects due to several possible reasons: difficulties
in assessing frequency of consumption of such large food groups, social desirable behavior, etc.
These possible reasons should be investigated further in future research.
Associations between dietary instruments (FFQ and EDR) measured by correlation coefficients
should be at least 0.40 and optimally in the range of 0.50-0.70 in order to reliably rank persons(13).
Because the de-attenuated correlation coefficients observed in the present study are between 0.400.68, we can conclude that the FFQ has a good ranking ability (except for betaine (0.32)). In the
present study we found a de-attenuated correlation of 0.68 for folate. Longnecker et al.(26) compared
nutrient intakes assessed with diet records and a 116 food item FFQ in 138 men and women. For
folate, they found a de-attenuated Pearson correlation coefficient of 0.42, which is lower than the
de-attenuated correlation coefficient found in the present study (0.68). Pufelette et al.(28) developed a
short FFQ to assess folate intake and validated it against a 7 day weighed diet record. For women,
the correlation coefficient between the two methods was 0.30. A higher de-attenuated Pearson
correlation coefficient for folate (0.77) was found by Sevak et al(27).They validated a 207 food item
FFQ against 24-hour-recalls in 100 women. No correlation coefficients for betaine and choline were
found in the literature, since a food composition database has only become available a few years
ago(6, 32). Also, no validation papers that assess the intake of methionine using an FFQ were found
in the literature.
The cross-classification analysis showed that the FFQ classified 40% for choline and 54% for SUM
in the same tertile and that there was a maximal misclassification of 20% for choline. Weighted κ
values showed a fair agreement between the FFQ and EDR, but a poor agreement for choline.
Sevak et al.(27) reported a moderate agreement (weighted κ of 0.44) for folate between the FFQ and
multiple 24-hour recalls. They found that the FFQ classified 41% of the women in the same quartile
for folate, as opposed to the 51% found in the present study. A lower misclassification of 5% (16%
in the present study) and similar classification of 45% (41% in the present study) in the same tertile
for folate (in women) was found by Pufuluete et al.(28) who developed a short FFQ to assess folate
intake (in men and women) and validated it against a 7 day weighed diet record.
10
Since the actual values for surrogate tertiles showed the expected significant stepwise increase in
nutrient intake, we concluded that the FFQ could reliably distinguish extreme nutrient intakes
(except for choline).
The Bland & Altman plots showed large limits of agreement (mean difference ± 2SD) between the
two methods, indicating the limited use of the FFQ to estimate nutrient intake for individuals. The
observed divergence in these plots suggests a greater difficulty in estimating nutrient intake with
higher mean nutrient intakes. This tool was developed to classify and rank subjects according to
their intake in an epidemiological setting, not to assess intake at an individual level.
Also, the FFQ is not a good tool to use at an individual level for estimating the intake of folate with
the Belgian Health Council guidelines(23) as reference values, because 33% of the women would
lose the possibility of receiving a required intervention (daily folate intake < 200μg). The specificity
and sensitivity of the other nutrients were more acceptable.
Reproducibility study.
The correlation between repeated administrations was good (r 0.62-0.83) with a maximal
misclassification of 7% for betaine (weighted ƙ 0.44-0.66).
Lower mean intakes of all the nutrients (7-12% lower) were observed in the second questionnaire
administration. Villamor et al.(33) found similar results when they assessed maternal intake of folate,
vitamin B12, choline, betaine, and methionine during first and second trimester of pregnancy. For
betaine and choline a lower mean intake was found during the second trimester of pregnancy, an
increase in mean intake was found for methionine, and no changes were found in mean intake for
folate. Seasonal variation cannot explain this difference because both FFQs were administrated
during spring. A possible explanation is that boredom was higher and motivation was lower during
the second administration.
In the present study, the intraclass correlation coefficients between the first and the second FFQ
range from 0.62 for betaine to 0.83 for folate. These good correlations between the repeated
administrations indicates that the random response error, sometimes due to lack of interest or
motivation of the respondent, is rather small. In reproducibility studies, the correlation coefficients
generally range from 0.5 to 0.7. Bidulesu et al.(34) reported correlations of 0.49 (folate), and 0.48
(choline) in men and women aged 45-64 years using a version of the Willett 61-item FFQ. A study
on intake of methyl-group donors during the first and second trimester of pregnancy using a 166
item FFQ showed correlations of 0.32, 0.55, 0.51, 0.43 for folate, choline, betaine, and methionine,
respectively(33). These correlations are lower than the correlations reported in the present study; 0.83
(folate), 0.80 (choline), 0.62 (betaine), and 0.78 (methionine). This lower correlation for folate
(0.55) was also found by Longnecker et al.(28). On the other hand, a high correlation for folate
11
(0.72) was also found by Pufulete et al.(26) who also assessed the intake of folate ( in women) with a
short newly developed FFQ.
The cross-classification analysis and weighted κ values indicated a moderate to good agreement
between the repeated administrations, with a maximal misclassification of 7% for betaine.
Strengths and limitations. To our knowledge this is the first validation study that assesses the intake
of methyl-group donors with a newly developed FFQ designed for these specific nutrients. This
FFQ will be used in an observational study, providing us with a convenient and reliable instrument
to rank individuals according to their intake of methyl-group donors(11). A key step in the validation
process is the selection of a reference method for the tool to be validated against. Since there is no
golden standard, it is important that the errors of both methods are as independent as possible. The
EDR was chosen as reference method in this study. Unlike the FFQ method, the EDR does not
depend on memory, is moreover open-ended, and involves direct estimation of portion size(11). The
EDR also takes into account the within-person variability in food intake, which is necessary
because in women there is a strong day-of-the-week effect(20). Therefore Intraclass correlations
were corrected for attenuation, which improved the correlations for all nutrients. Estimated diet
records (instead of weighed diet records) were used because they have the same order of accuracy
when ranking subjects and the respondent burden is lower(35). Food diaries with seven consecutive
recording days were used to deal with day-to-day variation and to cover all days of the week
equally. Seven consecutive dietary records might decrease the subject compliance and therefore decrease the
accuracy of the dietary records collected at the end of the 7-day period. Therefore, we compared the energy
intake calculated during the first 3 record days with those calculated during the last 3 record days.
There was no significant difference (p= 0.121) between the energy intake from the first 3 and last 3
days. Structured diaries as well as an example for filling in the EDR guide the women to report all
their consumptions, even for easily forgotten snacks for example soft drinks, candy, etc. In the
literature we find a wide range in sample sizes to assess validity and reproducibility, with a median
of 110 participants. A sample size of at least 50, and preferably 100 or more subjects, is
desirable(11). Because the performance of a dietary assessment instrument depends on the
characteristics of the study population and considering the target population in which the FFQ will
be used, women of reproductive age (18-35y) were recruited, leaving us with a large sample size
and homogenous group of women.
Limitations of this method is that it requires a high motivation of the participants (many recording
days), underreporting of intake and lack of adequate food description(13). In the present study, 61%
of the participants returned 2 FFQs and an EDR. This is a good response rate knowing how
intensive it is to fill in the 7d EDR. It was higher than a 15% response rate seen in other validation
12
studies(36). Women who filled in an EDR were promised nutritional advice in return, leading to
some selection bias of volunteers who are more concerned about health and diet. But forcing nonmotivated women to participate in the study might influence the quality of the data as well(37). A
limitation for the reproducibility study could be a possible memory effect during completion of the
second FFQ as women could possibly remember what they filled in six weeks ago. Furthermore,
because of dietary changes, for example food cravings and aversions, nausea, vomiting
(38, 39)
,
during pregnancy and even possible dietary restrictions (e.g. restrictions to avoid toxoplasmosis), it
might be more difficult to complete such FFQ during pregnancy. However, because of logistical
reasons it was not feasible to perform this validation study in a group of pregnant women.
One methyl-group donor, folic acid, derived from the diet is missing from our FFQ. In Belgium
many food items, for example breakfast cereals, are enriched with folic acid. At this time point
there is no list available of food items enriched with folic acid in Belgium, and folic acid is not
included in the Belgian food composition database. Food folate (without folic acid) was used to
calculate the daily folate intake, since the fortification of products is different in other countries.
Therefore the intake of folic acid could not be calculated. A last limitation is the use of food
composition databases from different countries. Differences between databases include description
of foods, calculation of nutrient content, recipe calculation, etc.(40).
Conclusion
The results found in this validation and reproducibility study indicate that the FFQ is a reliable
instrument with acceptable validity for ranking according to methyl-group donor intakes (except for
a poor agreement for choline (weighted ƙ 0.10) and a fair ranking ability for betaine (r 0.32)) in
Flemish women of reproductive age.
Financial support: Funding for the present study was provided by a PhD grant (grant number
11B1812N) from The Research Foundation-Flanders (FWO) and the Flemish Institute of
Technological Research (VITO). FWO and VITO had no role in the design, analysis or writing of
this article.
13
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15
16
Table 1 Characteristics of study participants
Unit/category
Age (years)
Weight (kg)
Length (m)
BMI (kg/m2)
Employment
Current smokers
Mean (SD)
Range
Mean (SD)
Mean ( SD)
Mean (SD)
%
%
Validation study
Reproducibility study
All women with dietary data
(n=80)
(n=92)
(n=161)
23 (4.1)
18-35
60 (9.6)
1.68 (0.05)
21.7 (3.2)
32.5
2.6
23 (3.9)
18-35
60.2 (9.8)
1.68 (0.06)
21.7 (3.1)
33.7
2.1
23 (4.2)
19-36
61.7 (9.4)
1.68 (0.06)
21.7 (3.0)
32.5
3.7
17
Table 2 Daily mean intake of methionine, choline, betaine, folate, and the sum of methyl-group donors (SUM) estimated with the 7-day estimated diet
record (7d EDR) and food-frequency questionnaire (FFQ1) (n 80)
7d EDR
Mean
SD
P*
FFQ1
Mean
SD
FFQ1-EDR
Mean
Correlation coefficient
SD
ICC
ICC**
P
Difference
Methionine (mg/d)
1732.4
388.9
1699.1
694.3
0.621
-33.3
600.6
0.43
0.62
<0.001
Choline (mg/d)
284.3
61.6
286.6
105.1
0.840
2.3
104.2
0.27
0.42
0.008
Betaine (mg/d)
147
51
179.1
73
<0.001
32.1
78
0.23
0.32
0.018
Folate (μg/d)
212
76.7
280.6
122.6
<0.001
68.6
88
0.58
0.68
<0.001
SUM (mg/d)
2163.9
455.3
2165.1
833.2
0.988
1.27
733
0.40
0.57
<0.001
7d EDR, 7-day estimated diet record; FFQ, food-frequency questionnaire; ICC, Intraclass correlation coefficient; SUM, sum of methyl-group donors
*
Paired student’s t-test was used to determine significant differences between means
**
De-attenuated Intraclass correlation coefficient
18
Table 3 Cross-classification analysis and weighted κ statistics for the 7-day estimated diet records (7d EDR) and food-frequency questionnaire (FFQ)
tertiles of usual daily intakes of methyl-group donors
Percentage classified in
Same tertile
Opposite tertile
Methionine
51
9
Choline
40
20
Betaine
44
14
Folate
51
16
SUM
54
11
Classification
33
22
by chance
CI, Confidence Interval; SUM, sum of methyl-group donors
Weighted kappa
(95% lower, upper CI)
0.35 (0.20,0.51)
0.10 (0.06,0.25)
0.21 (0.05,0.36)
0.27 (0.11,0.42)
0.35 (0.20,0.51)
19
Table 4 The use of actual values for surrogate tertiles to compare the usual daily intake of methyl-group donors of the food-frequency questionnaire
(FFQ) with the 7-day estimated diet record (7d EDR)
1st tertile
2nd tertile
3rd tertile
P*
Mean
SD
Mean
SD
Mean
SD
Methionine (mg/d)
1540.7
437
1742.6
322.2
1906.8
321.9
0.002
Choline (mg/d)
269.5
56.9
292
66
290.8
61.2
0.334
Betaine (mg/d)
128.4
38.9
146.8
50.3
165.1
56.9
0.030
Folate (μg/d)
192.2
56.1
197.3
34.2
245.8
109
0.017
SUM (mg/d)
1925
515.6
2213.8
342.6
2343.9
404.5
0.002
SUM, sum of methyl-group donors
*
One-way Anova test was used to determine significant differences in means between tertiles
20
Table 5 Mean intakes of methionine, choline, betaine, folate, and the sum of methyl-group donors (SUM) estimated with the first and second foodfrequency questionnaire (FFQ1 and FFQ2) (n 92)
FFQ1
P*
FFQ2
Mean
SD
Mean
SD
Methionine (mg/d)
1607.7
565.6
1489.3
472.3
Folate (μg/d)
265.6
105.6
243.7
Betaine (mg/d)
174.8
67.4
Choline (mg/d)
272.8
SUM (mg/d)
2055.6
FFQ1-FFQ2
Correlation coefficient
Mean difference
SD
ICC
P
<0.001
118.4
345
0.78
<0.001
90.1
<0.001
22
57.1
0.83
<0.001
155.5
52.6
<0.001
19.2
52.9
0.62
<0.001
88.5
255.3
76.2
<0.001
17.4
52.8
0.80
<0.001
677.2
1900.4
566.6
<0.001
155.1
415.6
0.78
<0.001
FFQ, food-frequency questionnaire; ICC, intraclass correlation coefficient; SUM, sum of methyl-group donors
*Paired student’s t-test was used to determine significant differences between means
21
Table 6 Cross-classification analysis and weighted κ statistics for the first and second food-frequency questionnaire (FFQ) tertiles of usual daily
intakes of methyl-group donors
Percentage classified in
Same tertile
71
Methionine
60
Choline
57
Betaine
70
Folate
71
SUM
33
Classification by
chance
SUM, sum of methyl-group donors
Weighted kappa
Opposite tertile
(95% lower, upper CI)
1
3
7
2
3
22
0.66 (0.51,0.80)
0.51 (0.36,0.65)
0.44 (0.29,0.58)
0.63 (0.49,0.78)
0.63 ( 0.49,0.78)
22
Figure legends
Figure 1- Simplified scheme of one-carbon metabolism. Nutrients included in this study appear in
bold.
5-methyl
THF,
5-methyl
tetrahydrofolate;
5,10-methylene
THF,
5,10-methylene
tetrahydrofolate; DHF, dihydrofolate; DMG, dimethylglycine; SAM, S-adenosylmethionine; SAH,
S-adenosylhomocysteine; THF, tetrahydrofolate.
Fig. 2 Bland & Altman plots. Difference between the mean methionine (A), choline (B), betaine
(C), folate (D), and the sum of methyl-group donors (SUM) (E) intake for the 7-day estimated diet
record (7d EDR) and food-frequency questionnaire (FFQ)
(Mean difference
and mean difference ± 2 SD ------)
Figures
Figure 1
Folate
Protein
DHF
Methionine
THF
SAM
DMG
DNA
methylation
Vit B12
Choline
5,10-methylene THF
Betaine
SAH
Homocysteine
ne
Vit B6
5-methyl THF
Cystathionine
Vit B6
Cysteine
23
Figure 2
A.
B.
Mean + 2SD
Mean + 2SD
Mean difference
Mean difference
Mean - 2SD
Mean - 2SD
D.
C.
Mean + 2SD
Mean difference
Mean - 2SD
Mean + 2SD
Mean difference
Mean - 2SD
E.
Mean + 2SD
Mean difference
Mean - 2SD
24
Appendix 1 Food-Frequency Questionnaire
Name:..................................
Date:
/
/
In this food-frequency questionnaire we inquire about your food habits of the past 3 months. In the
following table a variety of food products (food groups) is listed. Please describe (as exact as
possible) how often you eat or drink the listed products and indicate the average daily portion.
Consider also the meals taken away from home.
How often (frequency)?
In the column with the heading ‘How often do you consume the following product?’ there are 6
possible answers.
- Never or less than once per month
- 1-3 days per month
- 1 day per week
- 2-4 days per week
- 5-6 days per week
- Every day
Indicate your choice by filling in the circle near the answer that is most suitable for you.
How much?
In the column with the heading 'What is the average portion per day?' 3 to 5 portion size options are
given.
In the column with the heading 'Example portion sizes', a number of directive weights and measures
are given. These can help you to quantify the average portion sizes. Indicate your choice by filling
in the circle near the answer that is most suitable for you.
Which type do you usually use?
In the last column you should indicate for some food products (food groups) the type you usually
use. Please choose only one answer, unless options are equally frequent. In the latter case you may
indicate more options.
In case you would fill in the wrong option, you may cross it out and color another option. Please
indicate in such case the right answer with an arrow. Make sure you always fill in something, even
when you consume a product rarely or never. In such case, choose the option 'Never or less than
once per month' without indicating a portion size or type.
25
Food groups
How often do you
consume the
following product?
What is the average
portion per day?
Example portion
sizes
Which type do
you usually use?
Example
Consider the following example: a person eats 2 large slices of white bread every morning and 4
large slices for lunch and this for the past 3 months. At Sundays he/she eats 2 slices of sugar bread
instead of white bread. In the morning he/she drinks a glass of orange juice, however in the
weekends he/she drinks a cup of coffee.
Food groups
Fruit juice
Coffee/tea
Fantasy bread
(sugar bread,
raisin bread,
sandwich) and
Danish pastry
Types of white
bread (bread roll,
baguette) and
yeast bread
How often do you
consume the
following product?
○ Never or less than
once per month
○ 1-3 times per month
○ 1 time per week
○ 2-4 days per week
● 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 times per month
○ 1 time per week
● 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 times per month
● 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 times per month
○ 1 time per week
○ 2-4 days per week
● 5-6 days per week
○ Every day
What is the average
portion per day?
Example portion
sizes
Which type do you
usually use?
● 150 mL or less
○ 125 - 250 mL
○ 250 - 375 mL
○ 375 - 500 mL
○ 500 mL or more
1 glass = 150 mL
1 can = 200 mL
1 bottle (Looza)
= 200 mL
● Orange juice
○ Grapefruit juice
○ Other
○ 125 mL or less
● 125 - 250 mL
○ 375 - 500 mL
○ 500 mL or more
1 cup= 125 mL
1 beaker
= 225 mL
● Coffee
○ Tea
● 50 g or less
○ 50 - 100 g
○ 100 - 150 g
○ 150 g or more
Croissant = 50 g
1 Danish pastry
= 100 g
1 slice of bread
= 25 g
● Fantasy bread
○ Danish pastry
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
● 180 - 210 g
○ 210 g or more
½ baguette
= 120 g
1 slice of a big
bread = 30 g
1 slice of a small
bread = 20 g
1 slice of yeast
bread = 45 g
26
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Beer/wine
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Soup
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Fruit juice
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Vegetable juice ○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Softdrinks
○ Never or less than
(cola,
once per month
lemonade,...)
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Coffee/tea
○ 125 mL or less
○ 125 - 250 mL
○ 250 - 375 mL
○ 375 - 500 mL
○ 500 mL or more
1 cup = 125 mL
○ Coffee
1 beaker = 225 mL ○ Tea
○ 200 mL or less
○ 200 - 400 mL
○ 400 - 600 mL
○ 600 - 800 mL
○ 800 mL or more
1 bottle/glass beer
= 250 or 330 mL
1 can = 330 or
500 mL
1 glass of wine
= 125 mL
○ 200 mL or less
○ 200 - 400 mL
○ 400 - 600 mL
○ 600 mL or more
1 bowl = 250 mL
1 beaker = 225 mL
○ 150 mL or less
○ 150 - 300 mL
○ 300 - 450 mL
○ 450 mL or more
1 glass = 150 mL
1 carton = 200 mL
1 bottle (Looza)
= 200 mL
○ 150 mL or less
○ 150 - 300 mL
○ 300 - 450 mL
○ 450 mL or more
1 glass = 150 mL
1 carton (V8)
= 330 mL
1 bottle (Looza)
= 200 mL
○ 150 mL or less
○ 150 - 300 mL
○ 300 - 450 mL
○ 450 mL or more
1 glass = 150 mL
1 can = 330 mL
1 bottle = 200 mL
○ Alcohol free beer
○ Regular beer
( pils, Palm,...) or
strong beer (Duvel,
trapist,...)
○ Wine
○ Orange juice
○ Grapefruit juice
○ Other
○ Cola
○ Other
27
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Milk/
○ Never or less than
chocolate milk once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Yogurt, cottage ○ Never or less than
cheese, white
once per month
cheese, and
○ 1-3 time per month
curd
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Eggs
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Cheese spread ○ Never or less than
(type
once per month
Philadelphia)
○ 1-3 time per month
and melted
○ 1 time per week
cheese/cheese
○ 2-4 days per week
spread (Kiri,
○ 5-6 days per week
Ziz,...)
○ Every day
Feta, goat
○ Never or less than
cheese,
once per month
mozzarella, and ○ 1-3 time per month
Parmesan
○ 1 time per week
cheese
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Soy milk/
drinks and
yoghurt drinks
(Dan ’Up,
Fristi)
Other cheese
(Brie,
○ Never or less than
once per month
○ 125 mL or less
○ 125 - 250 mL
○ 250 - 375 mL
○ 375 mL or more
1 glass = 150 mL
1 beaker = 225 mL
1 soy drink
= 250 mL
1 bottle (Dan’Up)
= 600 mL
○ Soy milk/ soy
drinks
○ Yoghurt drinks
○ 125 mL or less
○ 125 - 250 mL
○ 250 - 375 mL
○ 375 mL or more
1 cup = 125 mL
1 beaker = 225 mL
1 bowl = 250 mL
1 glass = 150 mL
1 carton = 200 mL
○ 125 g or less
○ 125 - 250 g
○ 250 - 375 g
○ 375 g or more
1 pot yoghurt
= 125 g
1 pot cottage
cheese = 200 g
1 cup = 125 g
1 dish = 150 g
1 bowl = 250 g
○ Yogurt
○ Yogurt with fruit
○ Cottage cheese/
white cheese/curd
○ 15 g or less
○ 15 - 30 g
○ 30 - 45 g
○ 45 g or more
15 g per slice of a
big bread
10 g per slice of a
small bread
○ Cheese spread
type Philadelphia
○ Melted
cheese/cheese
spread
○ 15 g or less
○ 15 - 30 g
○ 30 - 45 g
○ 45 g or more
15 g goat cheese
per slice of a big
bread
1 small block of
feta = 5 g
1 Mozzarella ball
= 125 g
1 tablespoon of
grated Parmesan
= 10 g
○ Feta/goat cheese
○ Mozzarella/
Parmesan
○ 15 g or less
○ 15 - 30 g
15 g per slice of a
big bread
○ 1 piece or less
○ 2 pieces
○ 3 pieces or more
28
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ 30 - 45 g
○ 45 g or more
10 g per slice of a
small bread
○ 20 g or less
○ 20 - 40 g
○ 40 - 60 g
○ 60 g or more
1 slice of cheese
(10x10cm) = 25 g
○ 20 g or less
○ 20 - 40 g
○ 40 - 60 g
○ 60 g or more
20 g pâté per slice
of bread
○ 20 g or less
○ 20 - 40 g
○ 40 - 60 g
○ 60 g or more
15 g per slice of
bread
○ 50 g or less
○ 50 - 100 g
○ 100 - 150 g
○ 150 g or more
Breakfast
cereals: muesli
(normal and
crispy) and
oatmeal
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ 30 g or less
○ 30 - 60 g
○ 60 - 90 g
○ 90 g or more
1 spoon of canned
tuna/salmon = 25 g
1 slice of smoked
salmon = 30 g
1 young herring
= 50g
1 drained can of
mackerel/ sardines
= 120 g
1 bowl = 40 g
Breakfast
cereals: All
Bran (Kellogs)
○ Never or less than
once per month
○ 1-3 time per month
○ 30 g or less
○ 30 - 60 g
○ 60 - 90 g
Camembert,
Roquefort)
Hard cheese/
semi-hard
cheese
(Gouda,
Emmentaler,
Cheddar,
Gruyère,...)
Pâté/liver
sausage
Other meat
products (ham,
salami, chicken
ham,...)
Canned fish,
smoked fish,
and salted fish
1 bowl = 40 g
○ All Bran
○ Wheat Bran
29
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Breakfast
○ Never or less than
cereals: Special once per month
K and others
○ 1-3 time per month
(Type Smaks,
○ 1 time per week
Honey Pops,
○ 2-4 days per week
Frosties)
○ 5-6 days per week
○ Every day
Rusk, crisp
○ Never or less than
bread, and rice once per month
wafer
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Fantasy bread
○ Never or less than
(sugar bread,
once per month
raisin bread,
○ 1-3 time per month
brioche) and
○ 1 time per week
Danish pastry
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Types of white ○ Never or less than
bread (bread
once per month
roll, baguette)
○ 1-3 time per month
and yeast bread ○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
and wheat bran
Types of brown
bread (whole
grain,
multigrain, rye,
brown
baguette)
Dried fruit
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ 90 g or more
○ 30 g or less
○ 30 - 60 g
○ 60 - 90 g
○ 90 g or more
1 bowl = 40 g
○ 10 g or less
○ 10 - 20 g
○ 20 - 30 g
○ 30 g or more
1 piece = 8 g
○ 50 g or less
○ 50 - 100 g
○ 100 - 150 g
○ 150 g or more
Croissant = 50 g
1 Danish pastry
= 100 g
1 slice of bread
= 25 g
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 210 g
○ 210 g or more
½ baguette = 120 g
1 slice of a big
bread = 30 g
1 slice of a small
bread = 20 g
1 slice of yeast
bread = 45 g
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 210 g
○ 210 g or more
½ baguette = 120 g
1 slice of a big
bread = 30 g
1 slice of a small
bread = 20 g
○ 15 g or less
○ 15 - 30 g
○ 30 - 45 g
○ 45 g or more
1 prune, apricot,
date = 8 g
1 tablespoon raisins
= 12 g
○ Special K
○ Others
○ Fantasy bread
○ Danish pastry
30
Orange,
mandarin,
grapefruit,
kiwi,
strawberries,
and banana
Chocolate
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Nuts/seeds en
○ Never or less than
nut-seed-paste once per month
(peanut butter
○ 1-3 time per month
and sesame
○ 1 time per week
paste)
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Olives en
○ Never or less than
sundried
once per month
tomatoes
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Cereal bar,
○ Never or less than
granola bar,
once per month
dry biscuits and ○ 1-3 time per month
biscuits with
○ 1 time per week
chocolate
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Salty snacks
(crisps,
tortillas, salted
biscuits)
Cake/muffin
○ Never or less than
once per month
○ 100 g or less
○ 100 - 200 g
○ 200 - 300 g
○ 300 g or more
○ 25 g or less
○ 25 - 50 g
○ 50 - 75 g
○ 75 g or more
○ 20 g or less
○ 20 - 40 g
○ 40 - 60 g
○ 60 g or more
1 mandarin = 60 g
1 kiwi = 75 g
1 banana, orange
= 130 g
1 grapefruit
= 210 g
1 strawberry = 15g
1 Mignonette
= 10 g
1 individual bar of
chocolate = 50g
1 bar of a big
package = 25g
Easter egg or figure
in chocolate = 50 g
1 small packet of
crisps = 30 g
1 Tuc biscuit = 3 g
○ Orange,
mandarin, and
grapefruit
○ Kiwi
○ Banana
○ Strawberry
○ Crisps/tortilla
○ Salted biscuits
○ 15 g or less
○ 15 - 30 g
○ 30 - 45 g
○ 45 g or more
10 cashews = 20 g
1 tablespoon nuts
= 25g
1 tablespoon
peanut butter
= 15 g
○ 15 g or less
○ 15 - 30 g
○ 30 -45 g
○ 45 g or more
5 olives = 20 g
1 sundried tomato
= 12 g
○ Olives
○ Sundried
tomatoes
○ 20 g or less
○ 20 - 40 g
○ 40 - 60 g
○ 60 g or more
1 Special K bar
= 20 g
1 petit beurre
biscuit = 13 g
1 Evergreen cookie
= 40 g
1 chocolate chip
cookie = 20 g
○ Cereal bar,
granola bar
○ Biscuits without
chocolate
○ Biscuits with
chocolate
○ 20 g or less
○ 20 - 40 g
1 slice of cake
= 30 g
○ Cake
○ Muffin
31
Pancakes
Potatoes
(cooked,
steamed,
baked,
mashed...)
Rice and other
grains (bulgur,
quinoa,
couscous)
Pasta (penne,
spaghetti ,...)
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ 40 - 60 g
○ 60 g or more
1 cupcake = 30 g
1 muffin = 75 g
○ 60 g or less
○ 60 - 120 g
○ 120 - 240 g
○ 240 g or more
1 pancake = 60 g
○ 75 g or less
○ 75 - 150 g
○ 150 - 225 g
○ 225 - 300 g
○ 300 g or more
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ 25 g uncooked or less
= 62 g cooked or less
○ 25-50 g uncooked
= 62 -125 g cooked
○ 50 -75 g uncooked
= 125 - 187 g cooked
○ 75 - 100 g uncooked
= 187-250 g cooked
○ 100 g uncooked or more
= 250 g cooked or more
○ 40 g uncooked or less
= 100 g cooked or less
○ 40 - 80 g uncooked
= 100 - 200 g cooked
○ 80 - 120 g uncooked
= 200 - 300 g cooked
○ 120 - 160 g uncooked
= 300 - 400 g cooked
○ 160 g uncooked or more
= 400 g cooked or more
○ 50 g or less
○ 50 - 100 g
○ 100 - 150 g
○ 150 g or more
1 cooked potato
= 50 g
1 tablespoon of
mashed potatoes
= 50 g
20 French fries or
3-4 croquettes
= 100 g
60 g uncooked rice ○ White rice
= 150 g cooked rice ○ Brown rice
○ Other grains
1 tablespoon
cooked rice = 25 g
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Crustaceans,
shellfish
(scampi, crab,
shrimps,
lobster,
mussels, ...)
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Fish/fish sticks
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 240 g
1 bag of rice for 2
persons = 125 g
uncooked rice
50 g uncooked
pasta = 125 g
cooked pasta
○ White pasta
○ Wholemeal pasta
1 tablespoon
cooked pasta
= 25 g
1 scampi = 15 g
1 portion mussels
with shells = 1200g
1 fish stick = 30 g
1 serving of fish
= 175 g
○ Fish
○ Fish sticks
32
Beef (steak,
roast beef,…)
and veal (leg,
steak,…)
Pork (bacon,
pork chop,...)
and lamb (leg,
chop,...)
Other meat
(horse, rabbit,
wild,...)
Poultry
(chicken and
turkey)
Organ meats
(liver, kidney)
and tongue
Vegetarian
products (tofu,
quorn,
burgers)
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
NO vegetable
burgers,...
Raw vegetables ○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ 240 g or more
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 240 g
○ 240 g or more
1 steak = 175 g
1 slice of roast beef
= 40 g
1 vealsteak/leg
= 150 g
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 240 g
○ 240 g or more
1 portion of pork
belly = 75 g
1 hamburger
= 110 g
2 lamb chops
(boneless ) = 150 g
○ Pork
○ Lamb
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 240 g
○ 240 g or more
1 rabbit leg
(boneless) = 160 g
○ Horse
○ Other
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 240 g
○ 240 g or more
1 fillet = 160 g
1 chicken leg
= 160 g
1 chipolata = 70 g
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 240 g
○ 240 g or more
1 slice of foie gras
= 40 g
1 porcine kidney
= 140 g
○ 50 g or less
○ 50 - 100 g
○ 100 - 150 g
○ 150 g or more
1 large burger
= 95 g
1 small burger
= 55 g
1 box of quorn
minced meat
= 175 g
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 240 g
○ 240 g or more
1 serving of leafy
vegetables = 50 g
1 tablespoon
shredded carrots
= 20 g
1 tomato = 150 g
Dear steak = 100 g
○ Liver
○ Kidney
○ Tongue
33
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Cabbages
(Brussels
sprouts,
cauliflower,
red cabbage,
savoy) and
spinach
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
○ Never or less than
once per month
○ 1-3 time per month
○ 1 time per week
○ 2-4 days per week
○ 5-6 days per week
○ Every day
Other winter
vegetables
(Belgian
endive,
broccoli, beet,
leek, carrot,
turnip,...)
Summer/
Spring
vegetables
(corn, pepper,
soy beans,
asparagus,
mushroom,...)
Legumes
(chickpeas,
beans, green
beans, lentils,
peas,...)
Sauces (warm
and cold)
Lasagna,
spaghetti, and
pizza
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 240 g
○ 240 g or more
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 240 g
○ 240 g or more
1 tablespoon
Brussels sprouts
= 40 g
1 tablespoon red
cabbage = 25 g
1 tablespoon
cauliflower = 30 g
1 tablespoon
spinach = 45 g
1 chicory = 85 g
1 tablespoon
cooked vegetables
= 30 g
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 240 g
○ 240 g or more
1 asparagus = 50 g
1 tablespoon of
cooked vegetables
= 30 g
○ 60 g or less
○ 60 - 120 g
○ 120 - 180 g
○ 180 - 240 g
○ 240 g or more
1 tablespoon peas/
green beans = 25 g
1 tablespoon
chickpeas/lentils/
beans = 35 g
○ 12 g or less
○ 12 - 25 g
○ 25 - 50 g
○ 50 g or more
1 tablespoon
mayonnaise = 25 g
1 tablespoon gravy
= 12 g
1 tablespoon white
sauce = 20 g
○ 200 g of less
○ 200 - 400 g
○ 400 - 600 g
○ 600 g or more
1 frozen pizza
= 400 g
1 pre-packed
lasagna = 400 g
Do you occasionally use haddock liver, caviar or seaweed? ○ Yes
○ Cabbages
○ Spinach
○ No
In case your answer is yes, please clarify how much you use of these products.
-
Haddock liver:...................................................................................................
34
- Caviar:............................................................................................................
- Seeweed:..........................................................................................................
Do you use enriched products like breakfast cereals enriched with folic acid (see the package for
information).
○ Yes ○ No
In case your answer is yes, please specify the product:
...........................................................................................................
Do you use food supplements (vitamins and/or minerals)? Fe. Supradyn, 1 tablet/day
Fe. Omnibionta, 1 tablet/day
○ Yes ○ No
In case your answer is yes, leave the product name and your daily use:
.......................................................
35
Appendix 2 2x2 contingency table, specificity, sensitivity, and positive and negative predictive
values of the food-frequency questionnaire (FFQ) for estimating the daily methionine, choline, and
folate intake estimated, using the 7-day estimated diet record (7d EDR) as a reference.
Daily methionine intake (FFQ)
<10.4mg/kg
≥10.4mg/kg
Total
Daily methionine intake (EDR)
<10.4mg/kg
≥10.4mg/kg
Total
1
0
1
0
80
80
1
80
81
Daily choline intake (FFQ)
<425mg
≥425mg
Total
Daily choline intake (EDR)
<425mg
≥425mg
76
2
2
1
78
3
Total
78
3
81
Daily folate intake (FFQ)
<200µg
≥200µg
Total
Daily folate intake (EDR)
<200µg
≥200µg
16
5
27
33
43
38
Total
21
60
81
Specificity
Methionine (%)
Choline (%)
Folate (%)
100
33
87
Sensitivity
100
97
33
Predictive value
Positive
Negative
100
100
97
33
76
55
Guidelines
≥10.4mg/kg
≥425mg
≥200µg
36