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Dietary biomarkers and compliance

Periodic Reporting for period 1 - Dietary biomarkers (Dietary biomarkers and compliance)

Reporting period: 2016-09-01 to 2018-08-31

Nutritional studies on the effect of a specific diet on physiological changes can be trusted only if the food consumption was done in a compliant manner and can be verified objectively by the scientists. In such a case the data would embody correct and objective information for further analysis and conclusions. The current dietary assessment instruments such as food frequency questionnaires, dietary records, recalls, etc. have been used for many years but they are flawed by their lack of objectivity. Development and implementation of objective tools for dietary assessment is therefore important for the nutrition research area.

This problem has at least two aspects. One is compliance, i.e. whether a test person was consuming what was required based on the study design. This is important only in nutritional trials. Volunteers reporting non–compliance are of large importance for the study since it allows handling data in a proper way. The second is the ability of the volunteers in a study to report accurately. This is an issue in all trials, observational as well as experimental. Forgetfulness, more or less deliberate skewing of records to make them fit anticipated health ideals, and lack of knowledge about foods are probably the most important factors affecting accuracy of objective assessments. Both aspects, i.e. compliance and reporting quality, might differ not only among studies, but also between individuals, cultures, gender, types of diet being examined. Therefore it is crucial to develop a tool which can accurately assess dietary compliance to ensure correctness of the study data.

The idea of the research project Dietary Biomarkers is to develop such a tool based on chemical analysis of biological samples, ideally urine, for the determination of food related biomarkers. Ideally, each consumed food component would leave a traceable unique biomarker of intake. A biomarker of food intake is a substance originating only from the food component in question or specifically produced during its metabolism. The biomarker should be quantifiable in a biological sample, e.g. blood, urine, faeces, etc. A biomarker analysis would then give the straight answer whether the food component was consumed or not. The project aims at selecting a set of biomarkers which would cover most of the commonly consumed food items and setting up an analytical method (liquid chromatography coupled with mass spectrometry, LC–MS) to determine their presence in biological samples collected during trials. The results of chemical analysis will be then compared with conventional instruments to assess agreement between the two methodologies. Discrepancies are of course expected as none of the tools are faultless.
"Suitable specific biomarkers were identified based on the literature research in order to cover some of the most commonly consumed foods. Afterwards, a state–of–the–art analytical method using liquid chromatography coupled with mass spectrometry to measure selected metabolites (food biomarkers) in urine has been developed. In addition, a novel sampling technique for collecting dried urine spots has been developed and tested. The tool has been developed with FabLab Copenhagen (an open access, non–profit user shared tool workshop). This invention has been submitted as a utility model application to Danish Patent Office.

The developed multimarker method has been first tested for compliance control on the MetAl project (study was registered on ID# NCT03384147) using urine samples. This study aimed at investigating short–term effects of moderate alcohol intake on biomarkers of intake and physiological responses. Compliance control, i.e. whether subjects were consuming alcohol on pre–defined basis, was successfully performed by measuring the level of specific alcohol intake related biomarkers.
The method has been also used for analysis of urine samples obtained from the NU–AGE project as part of an international collaboration with the Division of Human Nutrition, Wageningen University & Research, The Netherlands NU–AGE provided samples obtained from subjects who consumed various diets and information about foods consumed were provided within food records (i.e. the subjective diet assessment method). Results obtained by the analytical tool developed in this project were compared in order to assess the agreement between these two independent and substantially different compliance monitoring tools.

The main results can be summarized as follows:
A versatile analytical method which can semi–quantitatively analyze urine samples for specific food–related biomarkers has been established in order to control dietary compliance in human intervention studies.
Data were analyzed to check the validity of the dietary compliance assessment tool and recommendations for future developments.
A new prototype tool for collecting dried urine samples made of biodegradable poly–lactic acid by using a 3D–printer has been designed. This resulted in a utility model application which is currently under examination at a Danish Patent office. Preceding work using in house made kit was published in Bioanalysis Journal.
In total, the results have been disseminated in a publication, conference poster, utility model application and 2 internet communications. Two more publications will be considered depending on the further outcomes.