Krogholm, Kirstine Suszkiewicz

Copenhagen University Research Information System

Flavonoids as fruit and vegetable intake biomarkers:development, validation and application of flavonoid biomarkers in nutritional research

Adskillige valideringsstudier har vist, at fødevarefrekvensspørgeskemaer (FFQ) har lav præcision og akkuratesse, og det diskuteres til stadighed hvilken anvendelighed sådanne selvrapporterede data har med hensyn til at undersøge sammenhænge mellem kost og sygdom. Der er til dags dato dog ikke et bedre alternativ til de selvrapporterende metoder og derfor er en grundig validering af FFQ i forhold til én eller flere uafhængige referencemetoder, så som objektive biomarkører, meget aktuel og vigtig. Hvis to forskellige uafhængige referencemetoder er tilgængelige er det muligt at beregne triangulære validitets koefficienter (VCs) som et udtryk for det sande, men ukendte indtag. Hvis der kun indhentes information om én anden referencemetode beregnes normalt den bivariate korrelation mellem test og reference metode. I 2002 blev der publiceret et kostkontrolleret interventionsforsøg, der indikerede, at summen af 7 flavonoid aglyconer i 24t urin potentielt kunne anvendes som biomarkør for indtaget af frugt og grønt (Nielsen et al. 2002). Udgangspunktet for denne Ph.d. afhandling var at udvikle og validere denne potentielt nye frugt og grøntsags biomarkør yderligere og derefter at bruge den til validering af det selv-rapporterede indtag af frugt og grønt i interventions og kohorte studier.Denne Ph.d. Afhandling er baseret på fire videnskabelige artikler. Artikel Ibeskriver, at kontrollerede stigninger i indtaget af frugt og grønt medfører en lineær og sensitiv stigning i udskillelsen af de 7 flavonoider i 24t urin og underbygger dermed anvendeligheden af flavonoider i 24t urin som biomarkør for indtaget af frugt og grønt. Udskillelsen af de 7 flavonoider i morgen spoturin (dvs. alt urin udskilt mellem kl. 24:00 til og med første vandladning om morgenen) responderede også signifikant med indtaget af frugt og grønt. Sammenhængen var dog svagere end i 24t urin og indikerer dermed, at udskillelsen af de 7 flavonoider i 24t urin er en stærkere biomarkør for indtaget af frugt og grønt endudskillelsen af de 7 flavonoider i morgen spoturin.I Artikel II beskrives den biokinetiske profil af tre, fra kosten, vigtige flavonoider (quercetin, hesperetin og naringenin), da større forståelse af biotilgængeligheden og metabolismen af flavonoider er nødvendig for at kunne vurdere anvendeligheden af flavonoiderne som biomarkører. I Artikel I og II blev der observeret en høj inter-individuel variation i absorption og urinudskillelse af de undersøgte flavonoider, og det må derfor konkluderes, at udskillelsen af flavonoider i urin ikke er anvendelig som biomarkør på individniveau. Den intra-individuelle variation var til gengæld lav og dermed kan flavonoid biomarkører godt bruges til at validere en ændring i et individs indtag af frugt og grønt og tilat inddele en større gruppe af individer i forhold til indtag af frugt og grønt. I Artikel III bruges flavonoid biomarkøreren i 24t urin til at validere indtaget af frugt hos medarbejdere, der har deltaget i en ’gratis-frugt på arbejdspladsen’ intervention. Udskillelsen af flavonoider i 24t urin blev fundet at korrelere signifikant med indtaget af frugt estimeret ved 24t kosthistorisk interviews og indikerer dermed, at det anvendte 24t kosthistoriske interview giver et validt estimat af den sande effekt af ’gratis-frugt på arbejdspladsen’ interventionen.Ét af formålene med Artikel IV var at anvende den triangulære metode (Kaaks 1997, Ocké &amp; Kaaks 1997) til at validere det FFQ selvrapporterede indtag af frugt, grønt og drikkevarer med et højt indhold af flavonoider i en populationsbaseret kohorte i Danmark (omtales som `Inter99´ kohorten). Det andet formål med studiet omtalt i Artikel IV var, at undersøge om udskillelsen af flavonoider i morgen spoturin kunne substituere 24t urinopsamling og dermed gøre flavonoid biomarkøren mere anvendelig i større epidemiologiske undersøgelser. Forudsætningen for den triangulære metode er tre forskellige og uafhængige bestemmelser af frugt- og grønt-indtaget, og derfor blev også plasma koncentrationen af carotenoider, ud over FFQ, målt i tillæg til flavonoid biomarkøren. Den triangulære metode resulterede i VCs for FFQ, der var højere end bivariate korrelationskoefficienter mellem det totale indtag af frugt, grøntsager, juice og te estimeret ved FFQ og de enkelte biomarkører. De statistiske signifikante VCs for Inter99 FFQ og 2 uafhængige biomarkører indikerer, at Inter99 FFQ giver et validt estimat af indtaget af frugt, juice, te og grøntsager i Inter99 kohorten. VCs for FFQ var mellem 0.43-0.68 ved brug af flavonoid biomarkøreren i 24t urin og mellem 0.35-0.64 ved brug af flavonoid biomarkøreren i morgen spoturin. Dette indikerer at flavonoid biomarkøreren var i stand til at afspejle indtaget af frugt og grøntsager, te og juice i både 24t urin og i morgen spoturin i Inter99 kohorten. Opsamling af 24t urin er svært og tidskrævende og derfor vil brugen af flavonoider i morgen spoturin være en mere praktisk metode i forhold til 24t urinopsamling i valideringen af selvrapporterede indtag af frugt og grønt i større populationsstudier.Most validation studies show that the food frequency questionnaire (FFQ) is rather low in precision and accuracy, and there is an ongoing debate regarding the applicability of such self-reported data with regard to diet-disease relationships. However, no other method has so far been able to replace these dietary assessment questionnaires, and a thorough validation of the FFQ relative to one or two independent reference methods, such as objective biomarkers, is thus particularly important. If two independent reference methods are measured it is possible to use the method of triads for the validation, while calculation of the bivariate correlation coefficients is the common approach when using only one reference method. Back in 2002, a strictly controlled dietary intervention study indicated that the sum of 7 different flavonoid aglycones excreted in 24h urine samples potentially could be used as a biomarker of fruit and vegetable intake (Nielsen et al. 2002). The overall aim of the present Ph.D. thesis was to further develop and validate this potentially new fruit and vegetable biomarker and furthermore use it for the validation of self-reported dietary intake of fruits and vegetables in intervention and cohort studies. The Ph.D. thesis contains four scientific papers. Paper I provides evidence that the sum of 7 flavonoids in 24h urine respond in a linear and sensitive manner to moderate increases in the intake of fruits and vegetables, and thus consolidates that the flavonoids are a valid biomarker of fruit and vegetable intakes. In Paper I, the urinary recovery of the 7flavonoids in morning spot urine (i.e. all urine voids from midnight including the first morning void) was also found to respond to moderate increases in the intake of fruits and vegetables. However, the association was somewhat weaker than in 24h urine samples, indicating that the 24h urinary recovery of the 7 flavonoids is a stronger biomarker of the intake of fruit and vegetables than the urinary recovery of the 7 flavonoids in morning spot urine.In Paper II, the biokinetic profiles of some of the most important dietary flavonoids are described (quercetin, naringenin and hesperetin), since there was a need for improved understanding of bioavailability and metabolism of the flavonoids included in the flavonoid biomarker assay originally developed by Nielsen et al. (2000). In Paper I-II we observed a high degree of inter-individual variation in the absorption and urinary recovery of the flavonoids, and this makes it very difficult to separate individuals according to intake by use of the flavonoid biomarker in urine. The intra-individual variation was on the contrary low, and Paper II therefore supports the assumption, that 24h urinary recovery of flavonoids has great potential as a compliance biomarker and as a biomarker of both controlled and uncontrolled changes in the intake level of fruits and vegetables achieved by an intervention. In Paper III we use the flavonoid biomarker in 24h urine samples in a workplace-based intervention study providing free fruit or no free fruit to employees as validation of a 24h dietary recall. We found that the estimation of fruit intake by the 24h dietary recall was a valid estimate of the true effect of the free-fruit intervention programme, due to highly significant correlations between 24h urinary recovery of flavonoids and the selfreported intake of fruit. One of the aims of Paper IV was to use the method of triads (Kaaks 1997, Ocké &amp; Kaaks 1997) to validate the intake of fruits, vegetables and beverages rich in flavonoids in a population-based cohort in Denmark (referred to as the `Inter99’ cohort). The method of triads requires three different and independent measures of the variable of interest, and therefore, beside the FFQ, also the plasma concentration of carotenoids was measured in addition to the flavonoid biomarker. The second aim of Paper IV was to investigate whether the urinary recovery of flavonoids in morning spot urine could substitute the 24h urinary recovery of flavonoids as an alternative and more feasible biomarker of fruit and vegetable intake. Using the method of triads resulted in validity coefficients (VCs) for FFQ that were higher than the bivariate correlation coefficients between total fruit, juice, tea and vegetable intake estimated by FFQ and each of the biomarkers. This finding, of a statistically significant correlation between the Inter99 FFQ and two independent biomarkers indicates that the Inter99 FFQ provides a useful estimate of the overall dietary intake of fruits, juices, tea and vegetables in the Inter99 cohort. VCs for the FFQ ranged from 0.43-0.68 using 24h urine and from 0.35-0.64 using morning spot urine, indicating that the intake of fruits, juice, tea and vegetables was reflected by flavonoids both in morning spot and 24h urine. Collection of 24h urine is difficult and time consuming, and therefore morning spot urine may be a more convenient tool than 24h urine for validating the fruit and vegetable consumption in large population studies

Flavonoids as fruit and vegetable intake biomarkers:Development, validation and application of flavonoid biomarkers in nutritional research

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