Dr K D Wutzke

M Lotz

C Zipprich

The effect of pre-and probiotics on the colonic ammonia metabolism in humans as measured by lactose-[ 15 N 2 ]ureide

Keywords: Ammonia metabolism, human colon, lactose-[ 15 N 2 ]ureide, prebiotics, probiotics

Background:

The evaluation of ammonia detoxification by pre-and probiotics by means of colonic lactose-[ 15 N 2 ]ureide ( 15 N-LU) degradation is of great interest both scientifically and in terms of nutrition physiology.

Objective: Pre-and probiotics were supplemented in healthy adults to evaluate the effect of the ammonia metabolism in the human colon by means of 15 N-LU.

Methods: Fourteen subjects aged 20-28 years daily received a regular diet either without (no treatment) or with supplementation of 30 g fibre of potatoes (FP), 30 g wrinkle pea starch (WPS, resistant starch content: 12 and 70%, respectively), and 375 g Lactobacillus acidophilus yoghurt (LC1), over a 10-day period in randomised order.

After one week, 5.7 mg/kg body weight 15 N-LU was administered together with breakfast. A venous blood sample was taken after 6 h. Urine and faeces were collected over a period of 48 and 72 h, respectively. The 15 N-abundances were measured by isotope ratio mass spectrometry.

Results:

The mean renal 15 N-excretion differed significantly between the supplementation of FP and no treatment (32.5 vs 46.3%, P=0.034), FP and LC1 (32.5 vs 51.6%, P=0.001), and WPS and LC1 (38.5 vs 51.6%, P=0.048). The mean faecal 15 N-excretion amounted to 42.7% (no treatment), 59.7% (FP), 41.8% (WPS), and 44.0% (LC1). In comparison to no treatment the urinary 15 NH 3 -enrichment was significantly decreased 16 h after FP supplementation.

Conclusion:

The prebiotic intake of FP and WPS lowered the colonic generation and the renal excretion of toxic 15 NH 3 , respectively, when using 15 N-LU as a xenobiotic marker.

Introduction

In recent years, 15 N-labelled lactose ureide ( 15 N-LU) has been successfully used for studying colonic nitrogen and ammonia metabolism, respectively, to assess the effects of pre-and probiotics on the human intestinal flora (Cloetens et al., 2008a;De Preter et al., 2006, 2008;De Preter and Verbeke, 2006;[START_REF] Geboes | The influence of inulin on the absorption of nitrogen and the production of metabolites of protein fermentation in the colon[END_REF][START_REF] Jackson | Transfer of 15 N from oral lactose-ureide to lysine in normal adults[END_REF]. The molecular bond between the sugar moiety and urea of 15 N-LU has been shown to resist enzymatic degradation in the small bowel, but to be split by Clostridium innocuum in the caecum [START_REF] Heine | A novel stable isotope breath test: 13 C-labeled glycosylureides used as noninvasive markers of intestinal transit time[END_REF][START_REF] Mohr | Clostridium innocuum: a glucose ureide splitting inhabitant of the human microbial flora[END_REF][START_REF] Wutzke | The use of 13 C-labelled glycosyl ureides for evaluation of orocaecal transit time[END_REF][START_REF] Wutzke | The duration of enzyme induction in oroceacal transit time measuremenrts[END_REF]. 15 N-LU has the advantage of supplying both the nitrogen and the energy needed for microbial protein synthesis simultaneously and has therefore been used as a carrier for transporting 15 NH 3 (common abbreviation for the ammonia/ammonium equilibrium 15 NH 3 +H 3 O + ↔ 15 NH 4 + +H 2 O shifted to the ammonium side under physiological conditions) into the colon. Probiotics are defined as viable microorganisms that have beneficial effects in the prevention and treatment of specific pathologic conditions in the gut [START_REF] Fuller | Probiotics in man and animals[END_REF]. Human in vivo studies have proven that

Lactobacillus johnsonii formerly, known as Lactobacillus acidophilus (La1 and LC1, respectively), supports the physiological function of the gastrointestinal tract and stimulates the immune defence system [START_REF] Pfeiffer | Probiotics in alimentation: clinical evidence for their enhancement of the natural immunity of the gut[END_REF]. Prebiotics are defined as non-digestible food ingredients, mainly oligosaccharides, non-digestible polysaccharides, resistant starches, and others that support the growth of certain bacteria such as lactobacilli and bifidobacteria that have various beneficial effects on the gut, and, consequently, for the health of the host [START_REF] Gibson | Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics[END_REF].

Prebiotics have the ability of increasing the saccharolytic activity, but contrastingly to decrease the proteolytic activity of the microbiota of the gut [START_REF] Swanson | Fructooligosaccharides and Lactobacillus acidophilus modify gut microbial populations, total tract nutrient digestibilities and fecal protein catabolite concentrations in healthy adult dogs[END_REF]. The colonic microbiota is responsible for degrading proteins partly to potentially toxic metabolites such as ammonia, amines and others which are implicated in the pathogenesis of different diseases [START_REF] Smith | Enumeration of human colonic bacteria producing phenolic and indolic compounds: effects of pH, carbohydrate availability and retention time on dissimilatory aromatic amino acid metabolism[END_REF]. It is assumed that ammonia is assimilated systematically by the bacteria, resulting in reduced urinary excretion and corresponding increased faecal excretion. The scientific literature previously provided evidence that the consumption of probiotics and prebiotics containing resistant starch and other non-digestible carbohydrates may influence the bacterial production of toxic metabolic end products, particularly ammonia, in the human colon [START_REF] Cummings | Gastrointestinal effects of prebiotics[END_REF][START_REF] De Preter | The in vivo use of the stable isotope-labelled biomarkers lactose-[ 15 N]ureide and [ H 4 ]tyrosine to assess the effects of pro-and prebiotics on the intestinal flora of healthy human volunteers[END_REF][START_REF] Geboes | Validation of lactose[ 15 N, 15 N]ureide as a tool to study colonic nitrogen metabolism[END_REF].

Thus, the evaluation of ammonia detoxification by pro-and prebiotics, respectively, by means of bacterial 15 N-LU degradation to 15 NH 3 and its subsequent excretion and absorption is still of great interest scientifically, in terms of nutrition physiology, and, with respect to pre-and probiotics, of commercial interest. Therefore, the aim of the present study is to investigate further prebiotics in form of fibre of potatoes (FP) containing a type-1 resistant starch (RS1) of 12% and a total dietary fibre content of 60%, wrinkle pea starch (WPS) containing a type-2 resistant starch (RS2) of 70% and a total dietary fibre content of 1%, and more probiotics in form of L. acidophilus yoghurt (LC1), respectively, on the renal and faecal nitrogen excretion, the urinary 15 NH 3 -enrichment,

the absorption, and the potential incorporation in selected blood plasma fractions by using orally administered lactose-[ 15 N 2 ]ureide as a xenobiotic marker.

Methods

Tracer substance and supplements

15 N-LU was synthesized according to our method as described in detail [START_REF] Wutzke | Evaluation of oro-coecal transit time: a comparison of the lactose-[ 13 C, 15 N]ureide 13 CO 2 -and the lactulose H 2 -breath test in humans[END_REF]. Lactose was obtained from Merck Darmstadt Germany, whereas [ 15 N 2 ]urea (99 atom%) was obtained from Campro Scientific Berlin Germany. FP and WPS, with a RS1 content of 12% and a RS2 content of 70%, respectively, were provided from the manufacturer Emsland Group GmbH Emlichheim, Germany, whereas the LC1 yoghurt pure (colony forming units (cfu): 2.6x10 9 /100 g, calorific value: 75 kcal/100 g) was utilised from Nestlé AG Germany. Table 1 shows the detailed composition of both starch products as well as the LC1 yoghurt. Both FP and WPS are commercially available and are commonly used as supplements in bakery as well as in meat products.

Subjects

Fourteen healthy adults (9 female, 5 male, age: 20-28 years, body weight: 51.5-107.3 kg) volunteered for this study. They were in good health throughout the study. None of the subjects were receiving any medication or had a history of gastrointestinal diseases.

Furthermore, none of the subjects regurgitated after having received the pre-and

Study protocols

All volunteers received an individually standardised regular diet (carbohydrate, fat, and protein content 55%, 30%, and 15%, respectively) over a time span of 54 days.

According to the precursor studies of the Leuven group, no standard diets were imposed (Cloetens et al., 2008b;[START_REF] De Preter | The in vivo use of the stable isotope-labelled biomarkers lactose-[ 15 N]ureide and [ H 4 ]tyrosine to assess the effects of pro-and prebiotics on the intestinal flora of healthy human volunteers[END_REF]2006;2007;2008;De Preter and Verbeke, 2006). Nevertheless, the volunteers were urged to keep their usual dietary habits, with taken care that their diet remained as constant as possible within the 4 test phases. On the days of 15 N-LU administration the breakfast was identical in all 14 subjects. All volunteers received a continental breakfast made up of 1 wheat flour roll, 50 g jam, 50 g cheese, and 1 cup of tea at 7 45. No other food or drink was allowed until 6 h after breakfast. Blood, urine, and faeces samples were taken 30 min prior to the ingestion of 15 N-LU to determine the baseline abundance of 15 N. 15 N-LU was administered in a dosage of 5.7 mg/kg body weight, corresponding to a 15 N-excess dose of 0.4 mg/kg, at 8 00 as a single oral pulse strewn on a small area of the jam to ingest the label with one bite. A sample of venous blood was taken 6 h after 15 N-tracer administration at 14 00. On the 1 st and on the 2 nd day the urine was collected quantitatively in 2-and 4-, and in 6-h intervals, respectively, whereas faeces were collected quantitatively over 72 h. After a 4-day wash-out phase, 4 subjects each received either 10 g FP, 10 g WPS (dissolved in 100 ml tea), or 125 g LC1 (the equivalent of one yoghurt cup) together with breakfast, lunch, and dinner at 8 00, 13 00, and 19 00, respectively, in randomised order over a 10-day period. The 15 N-LU administration was repeated on the 23 rd day followed by the wash-out phase. On the 30 th day the randomised cross-over procedure was repeated with the next pre-or probiotic supplement followed by the 15 N-tracer administration on the 37 th day. After the wash out period the test was repeated with the remaining substance on the 44 th day followed by the final 15 N-LU administration (Figure 1). The procedure of 15 N-LU administration, blood, urine, and faeces collection was identical in all 4 tests. The 10day supplementation period includes the complete collection of urine and faeces, whereas 15 N-LU was administered on the 7 th day of the respective pre-or probiotic supplementation (Figure 1).

Sample preparation

The sample preparation prior to isotope ratio mass spectrometry (IRMS) analysis has been recently explained in detail [START_REF] Wutzke | 15 N-excretion of heat-killed Lactobacillus casei in humans[END_REF]. After centrifugation of the citrated blood sample, the plasma was treated with sulphuric Na 2 WO 4 . The soluble supernatant was removed by centrifugation and was stored at -20 °C until analysis [START_REF] Faust | Klinisch-chemische und isotopenanalytische Methoden zur Untersuchung des Stickstoff-Stoffwechsels mit N-15 beim Menschen[END_REF]. After defrosting, 0.5 ml supernatant was transferred to tin boats and was dried at 60 ºC for 2 h. Urine volumes and faecal masses scraped from polyvinyl film were homogenized, recorded and stored at -20 °C until analysis (Wutzke and Oetjens, 2005). After defrosting, 25 µl urine were transferred to tin boats, whereas 5 mg wet faeces were weighed on tin discs. Urinary ammonia was separated in vessels by microdiffusion, following alkalization of the urine with NaOH and trapped in boric acid [START_REF] Faust | Klinisch-chemische und isotopenanalytische Methoden zur Untersuchung des Stickstoff-Stoffwechsels mit N-15 beim Menschen[END_REF].

Analytical techniques

Supernatant, urine, and faeces samples were combusted in the elemental analyser SL TM (SerCon, Crewe, U.K.) using copper oxide at 900 °C and subsequently reduced to nitrogen gas using copper at 600 °C. Thereafter, the 15 N-abundances were measured by isotope ratio mass spectrometry (IRMS, Tracer mass 20-20 TM , SerCon, Crewe, U.K.)

as detailed previously [START_REF] Wutzke | Evaluation of oro-coecal transit time: a comparison of the lactose-[ 13 C, 15 N]ureide 13 CO 2 -and the lactulose H 2 -breath test in humans[END_REF]. A capillary interface is included for connecting the 20-20 TM with the SL TM . The precision (mean %RSD or CV) for 15 N is 0.2‰. The data were expressed either as 15 N-enrichment in atom percent excess (atom%exc), or as percentage cumulative urinary and faecal 15 N-excretion.

Percentage of cumulative urinary and faecal 15 N-excretion

The 15 ND = 0.0267 mmol 15 N/kg body weight.

Statistical method

The Kolmogorov-Smirnow test was used for testing the distribution normality. Then, the factorial one-and two-way analysis of variance (ANOVA) using a post hoc Bonferroni correction for multiple comparisons on program SPSS 15.0 for Windows were used for statistical analysis of the total N-, the faecal 15 N-, and the urinary 15 N-excretion, respectively, (Table 2-4) whereas the Kruskal-Wallis and the Mann-Whitney U-test were used for the urinary 15 NH 3 -enrichment (significance P≤0.01). All results are quoted as means ± s.d.

Results

The maximum 15 N-enrichments in urinary ammonia were reached 12 h after 15 N-LU administration in all 4 tests (no treatment, FP, WPS, and LC1 supplementation) averaging 0.027±0.019, 0.018±0.024, 0.023±0.019, and 0.029±0.018, atom%exc, respectively (Figure 2). After 48 h the mean renal percentage cumulative excretion amounted to 46.3±11.1, 32.5±8.0, 38.5±12.9, and 51.6±16.9% (Table 2), respectively, whereas after 72 h the mean total faecal excretion amounted to 42.7±20.6, 59.7±24.4, 41.8±26.9, and 44.0±21.7%, respectively (Table 3). The urine fractionation in intervals of 0-6 h, 6-24 h, and 24-48 h revealed that the major part of 15 N was excreted between 6-24 h (Table 2). The resulting total 15 N-exretion (urine + faeces) shows that 89.0%, 92.2, 80.2, and 95.6%, respectively, of the ingested dose were excreted. The measured 15 N-enrichments in the supernatant amounted to 0.036±0.015, 0.040±0.024, 0.041±0.017, and 0.036±0.020 atom%exc, respectively, whereas the respective numbers of the plasma protein ranged below the detection limit of the IRMS of 0.001 atom%. With the exception of the urinary 15 NH 3 -enrichment values, the Kolmogorov-Smirnow test showed that by the majority, the data were normally distributed. When using ANOVA, between no treatment and the 3 different supplementation regimes statistically significant differences of the total urinary 15 N-excretion (0-48 h) were observed: no treatment vs FP, FP vs LC1, and WPS vs LC1, respectively, (Table 2), whereas no significant differences could be found neither for the respective faecal 15 Nexcretion nor for the 15 N-enrichment of the supernatant of the blood plasma.

Furthermore, significant differences of the fractional urinary 15 N-excretion were observed between 6-24 h and 24-48 h as well as for the 15 NH 3 -enrichment between no treatment and FP supplementation (P=0.008) 16 h after 15 N-LU ingestion. The total urinary N-excretion measured over the 2-day period after 15 N-LU administration (25.1, 22.0, 23.9, and 29.4 g N/2 days) yielded no statistically significant differences between the 4 tests phases (Table 4).

Discussion

The primary metabolic function of the microbiota is the fermentation of non-digestible food ingredients that have passed the small bowel such as resistant starch, non-digestible polysaccharides, non-digestible oligosaccharides and proteins and others.

During this passage, these substrates are exposed to the differing conditions of the gastrointestinal tract and are then found nearly indigested in the faeces of the large bowel (Wutzke and Oetjens, 2005). The outcome of the subsequent complex metabolic activity of the colon is the supply of energy and the retrieval of reusable substrates for the microbiota itself, and, consequently, for the host. The protein degradation in the large bowel leads to the generation of amino acids (which can be further partly deaminated to ammonia) and of other potentially toxic metabolic end products such as phenols, indoles, amines and others [START_REF] Smith | Enumeration of human colonic bacteria producing phenolic and indolic compounds: effects of pH, carbohydrate availability and retention time on dissimilatory aromatic amino acid metabolism[END_REF]. In vivo studies have been shown that the ammonia generation and accumulation, respectively, can be reduced by lowering the protein supply and by the colonic fermentation of suitable nondigestible carbohydrates of the food [START_REF] Geboes | Validation of lactose[ 15 N, 15 N]ureide as a tool to study colonic nitrogen metabolism[END_REF]. In contrast to polysaccharides, preferably butyric acid is generated in the colon during the fermentation of resistant starch. However, the resulting generation of both short chain fatty acids (SCFA) and butyric acid lead to a decrease of the pH-value in the colon, and, consequently, to an inhibition of the protease activity, whereas the additional energy supply increases the total bacterial mass [START_REF] Macfarlane | The colonic flora, fermentation and large bowel digestive function[END_REF]. In combination, these effects lead to an increased incorporation of both amino acid nitrogen and ammonia by the microbiota. The Leuven group recently demonstrated that after 15 N-LU administration the supplementation of type-3 resistant starch (RS3) in comparison to baseline significantly reduced the renal 15 N-excretion (Cloetens et al., 2008b). Furthermore, the study revealed that the major part of 15 N was excreted between 6 and 24 h after 15 N-LU ingestion suggesting that the 15 NH 3 had not reached the distal colon, and, therefore, it only justifies the evaluation of the degradation processes in the proximal colon. When considering the results of the present study, our data are in the same order of magnitude of those of the Leuven studies and confirm the tendency of lowering the urinary 15 N-excretion after supplementation with prebiotics when using 15 N-LU (Cloetens et al., 2008b;[START_REF] De Preter | The in vivo use of the stable isotope-labelled biomarkers lactose-[ 15 N]ureide and [ H 4 ]tyrosine to assess the effects of pro-and prebiotics on the intestinal flora of healthy human volunteers[END_REF][START_REF] Geboes | Validation of lactose[ 15 N, 15 N]ureide as a tool to study colonic nitrogen metabolism[END_REF].

In the present study, the mean renal 15 N-excretion 48 h after 15 N-LU ingestion differed significantly between the supplementation of FP and baseline, FP and LC1, and WPS and LC1 (Table 2). Similar to the findings obtained after RS3 supplementation, we could confirm that the major part of 15 N was excreted in urine between 6 and 24 h after 15 N-LU ingestion, suggesting that the 15 NH 3 had not reached the distal colon (Cloetens et al., 2008b, Table 2). In contrast, the urinary 15 N-excretion between 0 and 6 h mainly reflects the glucose-[ 15 N 2 ]ureide absorption deriving from the enzymatic degradation of 15 N-LU by β-galactosidase in the small bowel [START_REF] Wutzke | The use of 13 C-labelled glycosyl ureides for evaluation of orocaecal transit time[END_REF]. RS3 is mainly retrograded amylose formed after cooking and cooling, whereas RS2 occurs relatively dehydrated in uncooked natural granular form [START_REF] Sajilata | Resistant starch -a review[END_REF]. When considering the results obtained after RS3 supplementation containing 54% RS3 and the data of our WPS supplementation containing 70% RS2 the outcome becomes obvious. In contrast to the findings of Cloetens et al., the decrease in the urinary 15 N-excretion in the present study was less-pronounced, indicating that, in comparison to RS3, less fermentable RS2 had reached the colon [START_REF] Heijnen | Consumption of retrograded (RS3) but not uncooked (RS2) resistant starch shifts nitrogen excretion from urine to feces in cannulated piglets[END_REF]. However, the corresponding findings after the supplementation of the mainly dietary fibre-containing FP were similar to those after WPS supplementation, indicating that the anaerobic bacterial degradation of cellulose to SCFA in the human colon is likewise responsible for decreasing the urinary 15 N-excretion (Figure 2 and Table 2). The observed significant difference of the urinary 15 NH 3 -enrichment between no treatment and FP, 16 h after 15 N-LU ingestion confirm the decreased urinary 15 N-excretion after FP supplementation in comparison to no treatment conditions and clearly reflect the microbial degradation of the 15 N-LU [START_REF] Birkett | Resistant starch lowers fecal concentrations of ammonia and phenols in humans[END_REF], Table 1). The observed significant 15 N-enrichments of the mainly urea-containing supernatant fraction of the blood plasma (0.035 atom%exc on average) 6 h after 15 N-LU ingestion clearly demonstrates the appearance of 15 N-urea and possibly of certain free 15 N-amino acids in the metabolic pool of the blood plasma indicating the release of 15 NH 3 after 15 N-LU degradation, its subsequent absorption and the following re-synthesis to 15 N-urea and partly to 15 N-amino acids. Nevertheless, the supernatant fraction of the blood plasma can also be potentially contaminated by the absorption of glucose-[ 15 N]ureide deriving from the enzymatic 15 N-LU degradation. The urinary 15 N-excretion observed within the first 6 hours after 15 N-LU administration could confirm this assumption. According to the kinetics of the urinary urea and ammonia enrichment after 15 N-LU administration described in an earlier published paper, the sample of venous blood was deliberately taken 6 h after 15 N-LU administration, since we assumed the highest 15 N-enrichment in the blood plasma fraction around this time point [START_REF] Wutzke | Evaluation of oro-coecal transit time: a comparison of the lactose-[ 13 C, 15 N]ureide 13 CO 2 -and the lactulose H 2 -breath test in humans[END_REF]. The significant 15 N-enrichments of the urinary ammonia fraction and of the supernatant of the blood plasma, respectively, indicate the immediate excretion, and, in particular, the absorption of 15 N deriving from the bacterial degradation of 15 N-LU in the human colon. However, the respective 15 N-abundances of the plasma protein precipitate ranged below the detection limit of the IRMS of 0.001 atom%. The measurement of the 15 N-enrichments in urinary ammonia as well as in the supernatant of the blood plasma when using 15 N-LU as a xenobiotic marker is a novelty.

The acidification of the colon caused by the fermentation to SCFA could reduce the enzymatic cleavage of 15 N-LU and the conversion of [ 15 N 2 ]urea to 15 NH 3 , respectively, resulting in an increased faecal 15 N-excretion. After supplementation of FP and LC1, the resulting faecal 15 N-excretions (59.7 and 44.0%, respectively) tend to result in an increase in comparison to no treatment (42.7%) but without a statistical significance.

The comparatively high standard deviations were presumably caused by an incomplete homogenization of the faeces prior to combustion in the SL TM . De Preter et al., 2004 observed that the supplementation of Lactobacillus casei to healthy adults in comparison to no treatment revealed no statistical significant differences of the urinary 15 N-excretion. Furthermore, a short term intake of Bifidobacterium breve and L. casei as well neither influenced the urinary nor the faecal 15 N-excretion, respectively [START_REF] De Preter | Effects of Lactobacillus casei Shirota, Bifidobacterium breve, and oligofructose-enriched inulin on colonic nitrogen-protein metabolism in healthy humans[END_REF]. Similar to these findings, we also did not observe a decrease of the urinary 15 N-excretion after the supplementation of LC1 yoghurt in comparison to no treatment.

We deliberately applied a portion of 3 yoghurt cups per day (375 g, equivalent to 9.8x10 9 cfu) to mimic common usage consumption [START_REF] Sanders | Bringing a probiotic-containing functional food on the market: microbiological, product, regulatory and labeling issues[END_REF].

The constant total urinary N-excretion over the 2-day period after 15 N-LU administration clearly argues for the ingestion of a standardised diet within the 4 test phases (Table 4).

We assume that the relatively short 10-day period of our LC1 supplementation and the 7-day period prior to 15 N-LU administration, respectively, is adequate for achieving steady state conditions. Nevertheless, our findings clearly demonstrate that commercially available LC1 yoghurt in the chosen dosage led to identical results as under no treatment conditions and has no immediate impact on the colonic ammonia metabolism when using 15 N-LU. When summarising our findings, we are convinced that lactose-[ 15 N 2 ]ureide is a valid tool for investigating nitrogen and ammonia metabolism in the colon. Furthermore, we suggest that the prebiotic intake lowered the colonic generation of 15 NH 3 and its corresponding urinary 15 N-excretion and tends to enhance the 15 NH 3 uptake by the microbiota. Seen in this light, the process of ammonia detoxification in the colon can be interpreted as an interplay of the microbiota with certain supplements establishing appropriate conditions for their growth and proliferation. In this sense, the lowering of the proteolytic generation and accumulation of ammonia in the colon and the resulting noxious effects for the host by suitable supplementations of pre-and probiotics, particularly in subjects suffering from certain diseases such as kidney insufficiency, hepatic encephalopathy, inflammatory bowel disease, and colon cancer is understandably of great interest for biochemists, physicians, dieticians and in particular the patients as well. No significant data were found.

  probiotics and the 15 N-LU, respectively. The volunteers were requested to avoid the additional consumption of pre-and probiotics within the study period. Written consent was obtained from all subjects. The testing protocol was approved by the Committee on Ethics of the Faculty of Medicine of the University of Rostock (Registration Number: II HV 11/2006).

  15 N-excess dose (D) of 15 N-LU is the product of the 15 N-enrichment in atom%exc (APE 15N-LU ) times the intake (I) times the number of labelled 15 N-atoms (n=2) divided by the molecular weight (MW=403.4 g/mol) and 100. D = APE 15N-LU • I • n / 100 • MW. The 15 N-excretion (E) in a specified urine or faeces sample is the product of the measured 15 N-enrichment in atom%exc (APE sample ) times the urine volume (vol) or the faecal mass (m) in ml or g, respectively, times the N-concentration (c N ) of the urine or faeces sample in mg/ml or mg/g, respectively, divided by 100. E = APE sample • vol(m) • c N / 100. The percentage of cumulative 15 N-excretion (PCE) is the product of the urinary or faecal 15 N-exretion (E) in mmol, respectively, times 100 divided by the 15 N-excess dose (D) in mmol, respectively (Wutzke and Oetjens, 2005). PCE = E • 100 / D.

Figure 1 :Figure 2 :

 12 Figure 1: Time frame of the study protocol Figure 2: Mean urinary 15 NH 3 -enrichment over a 24-h time span after the

  Figure 2

Table 1

 1 Percentage composition of fibre of potatoes, wrinkle pea starch, and Lactobacillus Mean percentage faecal 15 N-excretion 3 days after the supplementation of fibre of potatoes, wrinkle pea starch, Lactobacillus acidophilus, and no treatment when using

	acidophilus
	constituents	fibre of potatoes	wrinkle pea	L. acidophilus
			starch
	total starch	28 +	98 +	-
	RS1	12 -	-	-
	RS2 (amylose)	-	70 -	-
	total dietary fibre	60 -	> 1	-
	(cellulose)*
	protein	7 +	0.5 +	4.5
	carbohydrate	88	98	6.5
	fat	0.5 +	0.5 +	3.5
	moisture	10 +	14 +	85.5
	+ : maximal, -: minimal
	* minor amounts of hemicellulose, xylose, arabinose, pectin and lignin

Table 4

 4 Mean urinary total N-excretion in [g/2days] after the supplementation of fibre of potatoes, wrinkle pea starch, Lactobacillus acidophilus, and no treatment when using

	15 N-LU
	subjects	no treatment	fibre of	wrinkle pea	L. acidophilus
			potatoes	starch
	1	24.57	18.72	16.96	20.89
	2	37.36	28.03	47.22	61.31
	3	27.01	22.6	26.83	26.52
	4	18.45	14.33	14.56	18.68
	5	24.38	19.42	27.61	43.78
	6	32.15	28.63	27.6	22.9
	7	24.21	20.77	20.78	22.03
	8	19.09	21.92	19.32	24.74
	9	23.15	20.68	18.32	19.86
	10	25.11	17.57	23.8	21.09
	11	32.49	22.93	24.72	23.71
	12	22.32	23.66	16.52	20.73
	13	10.84	20.67	21.3	21.43
	14	30.32	27.92	28.79	64.4
	mean	25.07	22,0	23.88	29.4
	s.d.	6.7	4.1	8.1	15.4

Acknowledgements

This study was supported by Emsland Group GmbH Emlichheim, Germany.

Conflict of interest

The authors declare no conflict of interest.


Kirstine Suszkiewicz

Lea Bredsdorff

Sevil Alinia

Tue Christensen

SER Salka Elbøl Rasmussen

LOD Lars Ove Dragsted

Kirstine Suszkiewicz Krogholm

Free fruit at workplace intervention increases total fruit intake: a validation study using 24h dietary recall and urinary flavonoid excretion

Keywords: Validation, fruit, vegetables, biomarker, flavonoids, 24h dietary recall

Objective: To validate 24h dietary recall of fruit intake by measuring the total 24h excretion of ten different flavonoids in24h urine during an intervention with free fruit at workplaces.

Design and methods:

Employees at workplaces offering a free fruit program, consisting of daily free and easy access to fresh fruit, and controls employees at workplaces with no free fruit programme were enrolled in this validation study (n=103). Dietary intake was assessed by using a 24h dietary recall questionnaire at baseline and approximately 5 months later. Ten flavonoids, quercetin, isorhamnetin, tamarixetin, kaempferol, hesperetin, naringenin, eriodictyol, daidzein, genistein and phloretin, were measured using HPLC-electrospray ionisation-MS. Results: The 24h urinary excretion of total flavonoids and the estimated intake of fruits were significantly correlated (r s =0.31, p<0.01). The dietary intake of citrus fruits and citrus juices was significantly correlated with total excretion of citrus specific flavonoids (r s = 0.28, p<0.01), and orange was positively correlated with naringenin (r s =0.24, p<0.01) and hesperetin (r s =0.24, p<0.01).

Phloretin in urine was correlated with apple intake (r s =0.22, p<0.01) and also with overall estimated intake of fruit (r s =0.22, p<0.01). Conclusion: The present study shows that a 24h dietary recall can be used as a valid estimate of the intake of fruits in agreement with an objective biomarker of fruit intake in free fruit at workplace interventions.

INTRODUCTION

Meta-analyses of observational studies have repeatedly shown that a high fruit and vegetable intake is associated with a reduced risk of cardiovascular disease, even though causality and mechanism remain to be demonstrated (Riboli et al. 2003;[START_REF] Dauchet | Fruit and vegetable consumption and risk of coronary heart disease: a meta-analysis of cohort studies[END_REF][START_REF] He | Increased consumption of fruit and vegetables is related to a reduced risk of coronary heart disease: meta-analysis of cohort studies[END_REF][START_REF] Duijnhoven | Fruit, vegetables, and colorectal cancer risk: the European Prospective Investigation into Cancer and Nutrition[END_REF]. Based on these results, authorised food based dietary guidelines recommend to eat more fruits and vegetables, e.g. the '5 to 10 a day' programme in Canada, the 'go for 2&5' programme in Australia or the '6 a day' programme in Denmark. Fruit and vegetable intake is however still inadequate among the majority of the population in many countries (Ashfield-Wall et al. 2004;[START_REF] Lassen | Successful strategies to increase the consumption of fruits and vegetables: results from the Danish '6 a day' Work-site Canteen Model Study[END_REF]Guenter et al. 2006;Ovesen et al. 2007;Kimmens et al. 2009). The average intake of fruits and vegetables in Denmark is currently 434 g/day with only 16% of the population consuming an amount, corresponding to the Danish recommendations of 600 g fruits and vegetables per day for adults and children above 10 years old [START_REF] Astrup | Kostrådene[END_REF][START_REF] Fagt | Dietary habits of Denmark 1995-2006 Status and development with focus on fruits, vegetables and added sugar[END_REF].

It has been shown that simple provision of information is not enough in order to achieve significant changes in the dietary behaviour of a population [START_REF] Stockley | Toward public health nutrition strategies in the European Union to implement food based dietary guidelines and to enhance healthier lifestyles[END_REF][START_REF] Eertmans | Food likes and their relative importance in human eating behaviour: review and preliminary suggestions for health promotion[END_REF]. Free-fruit campaigns on worksites are therefore well suited as an environmental strategy with the potential for increasing the daily fruit intake in a given population. This study will investigate whether a 24h dietary recall is a valid tool to measure, if a free fruit intervention at workplace will increase fruit intake among employees.

Estimation of fruit and vegetable intake is complicated by a tendency to over-report intake of healthy foods (Gibson 1990). Furthermore food registration methods are often based on self-reported data, which implies the risk for subjective bias [START_REF] Kipnis | Empirical evidence of correlated biases in dietary assessment instruments and its implications[END_REF]. Biological markers of exposure as an alternative to the more traditional dietary assessment tools have been used for many years to provide semi-quantitative indexes of the exposure to individual food 5 constituents or food groups like fruits or vegetables [START_REF] Spencer | Biomarkers of the intake of dietary polyphenols: strengths, limitations and applications in nutrition research[END_REF]. A combination of several different flavonoids quantified in 24h urine samples has recently been shown to correlate more strongly to fruit intake, than plasma carotenoids. Therefore, at the present, a combination of urinary flavonoids seems to be the best performing objective biomarker of fruit intake [START_REF] Nielsen | Flavonoids in human urine as biomarkers for intake of fruits and vegetables[END_REF][START_REF] Brantsaeter | Urine flavonoids and plasma carotenoids in the validation of fruit, vegetable and tea intake during pregnancy in the Norwegian Mother and Child Cohort Study (MoBa)[END_REF][START_REF] Mikkelsen | Relative validity of fruit and vegetable intake estimated by the food frequenct questionnaire used in the Danish National Birth Cohort[END_REF]. This biomarker has therefore been used in a workplace based intervention study providing free fruit or no free fruit to employees as validation of a 24h dietary recall.

6

MATERIALS AND METHODS

Workplaces and subjects

Eight comparable workplaces in the Copenhagen area signed up for the study. Five were enrolled as intervention workplaces and the remaining three workplaces, without any actual consideration of free-fruit at the workplace, were enrolled as control workplaces. At each workplace, collaboration with a contact person was established. Recruitment of the employees, interested in participating in the study, occurred through this contact. Altogether 146 employees were enrolled at baseline in the free fruit at work intervention study. Of the 146 employees, a total of 103 accepted participation in the validation study. Pregnant and lactating women, and individuals, not expecting to be at the particular workplace 5 months later were excluded. The study protocol was accepted by the Ethics Committee of the Copenhagen and Frederiksberg municipalities (J. No. KA-20060047).

The intervention

Workplaces entered the study at distinct points in time, starting between June and September 2006. Assessments were made both at baseline and at termination approximately 5 months later. The intervention was a fruit program, consisting of a fruit basket placed where employees had free and easy access, i.e. in the reception or in a kitchenette. There was at least one piece of fruit per employee each day. The fruit intervention was not accompanied with informational, counselling or other health promotion activities in order to make a minimal difference to the controls.

Dietary assessment 7

Dietary intake was assessed by a 24h dietary recall questionnaire, which was a modified form of the dietary recall questionnaire from the Danish National Dietary Survey 2000-2002 [START_REF] Lyhne | Dietary habits in Denmark 2000-2002[END_REF] The recall was performed by trained interviewers both at baseline and after 5 months. The software program, General Intake Estimion System (GIES, version 1.0, Technical University of Denmark, National Food Institute, Division of Nutrition 2008), was used to calculate dietary intake.

Urine samples

Urine samples were collected by the employees through 24 hours at baseline and 5 months later in order to determine the concentration of flavonoids as an objective biomarker for fruit intake. The collection of the 24h urine samples started in the morning the day before completion of the 24h recall questionnaires and lasted until the next day at the same hour. Hence, the 24h urine sample and the 24h dietary recall questionnaire covered the same period of time.

All urine samples were collected as previously described by [START_REF] Krogholm | Urinary total flavonoid excretion but not 4-pyridoxic acid or potassium can be used as a biomarker for the intake of fruits and vegetables[END_REF]. The employees were instructed on how to collect the urine and in the importance of providing complete samples. Completeness of the urine collection was checked by oral questionnaires and study diaries. Anthropometric measurements (height, bodyweight, and waist circumference) were also performed, and will be published elsewhere.

Determination of urinary flavonoids

The flavonoids were determined by LC-MS after enzymatic hydrolysis of conjugates, as described by [START_REF] Nielsen | Identification and quantification of flavonoids in human urine samples by column-switching liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry[END_REF] with slight modifications. In short, 2 mL aliquots of each urine sample were initially added 500 ng of 13 C O-desmethylangolensin ( 13 C ODMA) internal standard (20 ng/μL DMSO) and enzymatically hydrolyzed. The hydrolyzed samples were evaporated to dryness under vacuum. The urine samples were then dissolved with 50 μL 100% methanol and 2.0 mL 0.5% formic acid and added on an Isolute SPE 100 column preconditioned with 2.0 mL 100% methanol and 2.0 mL 0.5% formic acid. The column was washed and the flavonoids eluted and evaporated as described in [START_REF] Nielsen | Identification and quantification of flavonoids in human urine samples by column-switching liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry[END_REF]. After evaporation, the samples were redissolved in 25 μL 100% methanol and 200 μL 0.5% formic acid and added 500 ng of 13 C-daidzein as an additional standard (20 ng/μL DMSO). The entire amount of supernatant was injected into the LC-MS system.

Reagents and standards

Enzymes used for the hydrolysis of the flavonoid glycosides in urine samples were arylsulphatase (Aerobacter aerogenes, 16.8 standard units/mL) from Sigma Chemicals Co. (St.

Louis, MO, USA) and β-glucoronidase (Escherichia coli, >200 standard units/mL) obtained from Boehringer Mannheim (Mannheim, Germany). Methanol and acetonitrile were of HPLC grade and obtained from Rathburne Ltd. (Walkerburn, United Kingdom). The flavonoid standards quercetin, kaempferol, isorhamnetin, tamarixetin and naringenin were obtained from Aldrich (Steinheim, Germany). Hesperetin, eriodictyol and phloretin was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Genistein and daidzein were obtained from LC Laboratories, (Woburn, MA). The isotopically labelled internal standards, 3 x 13 C O-9 desmethylangolensin ( 13 C ODMA), and 3 x 13 C daidzein were obtained from School of Chemistry, University of St. Andrews, UK. Dimethylsulfoxid (DMSO) (Urasol) was purchased from Merck & Co., Inc. The Isolute SPE 100 was purchased from Mikrolab, Aarhus, Denmark.

All standards were HPLC grade. The concentration of flavonoids in urine samples were determined as single determinations based on calibration curves generated by spiked blank urine samples as described by [START_REF] Nielsen | Identification and quantification of flavonoids in human urine samples by column-switching liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry[END_REF].

Statistical analysis

The concentration of flavonoids in urine and intake of fruit and flavonoid-rich foods did not have a normal distribution even after logarithmic transformation, so nonparametric tests were used in the statistical analysis of the data. Wilcoxon two related samples test were performed in the intervention (n=34) and control (n=45) group separately to evaluate changes from baseline to after 5 months in the urinary excretion of flavonoids and in intakes of fruit, and individual flavonoid-rich foods. The Mann-Whitney U tests were performed to compare the two groups (control and intervention) on one variable at the time. Spearman correlation (r s ) was used to examine associations between the intake of fruits, flavonoid-rich foods and the excretion of the flavonoid biomarkers in urine (n=158). The analyses were made using the SPSS package program version 14.0 (SPSS, Inc., Chicago, IL). P values < 0.05 (two-tailed) were regarded as statistically significant.

RESULTS

Participants and food intake 10 Altogether 103 employees were enrolled at baseline in the validation study. Of these, 99 employees did complete the 24h dietary recall and 24h urine collection at baseline. At the second measurement, after 5 months, the 24h dietary recall and the collection of 24h urine sample was completed by 79 out of the 99 employees. Main causes of drop-out or exclusion were unexpected termination of employment (16 individuals) and incomplete 24h urine collection.

Data from the 79 employees were analysed using Wilcoxon two related samples test and the Mann-Whitney U tests.

At baseline there were no significant differences between the control and intervention groups in the estimated dietary intakes of fruits, citrus fruits and citrus juices, vegetables, fruits and vegetables, apples and oranges based on 24h dietary recall questionnaires (see Table 1). After 5 months of intervention the total intake of fruits was higher in the group with free access to fruit at work every day, compared to the control group receiving no additional fruit (median 310 (15, 1312) g/day v. median 265 (0, 732) g/day, P<0.01) (see Figure 1). Also the overall change in fruit intake from baseline to after 5 months was significant higher in the intervention group (median 72 (310, 792) g/day, P<0.01) compared to the control group (median 2 (-308, 490) g/day). As seen in Table 1, the median intake of citrus fruits and juices increased from 0 g/day at baseline to 50 g/day after 5 months, and this change in intake in the intervention group was significant higher than in the control group (P<0.05). Also the intake of fruit in the control group tended to increase over the 5 months study period (from median 193 (2, 541) g/day to median 265 (0, 732) g/day), though the change was not statistically significant (see Figure 1).

Biomarkers of fruit intake

The median (P5, P95) 24h urinary excretion of total flavonoids (i.e. the sum of quercetin, isorhamnetin, tamarixetin, kaempferol, hesperetin, naringenin, eriodictyol, daidzein, 11 genistein and phloretin) was similar in the control and intervention groups at baseline (median 352 (66, 1506) µg v. median 175 (65, 2569) µg) (see Table 1). After 5 months of free access to fruit at workplace every day the 24h urinary excretion of total flavonoids, in the intervention group, had increased significantly from median 175 (65, 2569) µg to median 635 (78, 8262) µg.

Also the amount of total citrus flavonoids (i.e. the sum of hesperetin, naringenin and eriodictyol) increased significantly after 5 months in the intervention group (P<0.05). In the control group, the urinary excretion of total flavonoids and total citrus flavonoids was also increased significantly after 5 months (P<0.05) (see Table 1).

The quantitatively largest amounts of flavonoids excreted in urine were the citrus flavonoids hesperetin, naringenin and eriodictyol (see Table 1). Kaempferol, phloretin, quercetin, genistein, daidzein, isorhamnetin and tamarixetin were excreted in lower amounts and did not change significantly in any of the two groups over time (data not shown).

Correlations

In the present study we use the spearman correlations coefficients for validation of the self-reported fruit intake. Spearman correlations were calculated for the employees with complete 24h dietary recall and 24h urine collection at both baseline and after 5 months, corresponding to a total of 158 employees. Spearman correlations between dietary intake estimated by 24h dietary recall and 24h urinary excretion of different combinations of flavonoids showed that total flavonoids in urine was highly and significantly correlated (r s =0.31, P<0.01, n=158) with the total intake of fruits (see Table 2). Adding the intake of vegetables to the fruit intake did make the strength of the spearman correlation even stronger to total flavonoids in urine (r s =0.37, P<0.01). Urinary excretion of the combination of hesperetin, naringenin, phloretin and eriodictyol (referred to as the 'fruit marker'), all present in fruits or formed after fruit 12 consumption, were also positively correlated to intake of fruits (r s =0.29, P<0.01), but the correlation were stronger when including the isoflavonids, genistein and daidzein, and the flavonols quercetin, kaempferol, isorhamnetin and tamarixetin which all are present in both fruits and vegetables (r s =0.31, P<0.01) (see Table 2). Correlations between excretion of specific flavonoids and intake of particular foods were studied based on their known occurrence in these foods. The dietary intake of citrus fruits and citrus juices was significantly correlated with total citrus flavonoids (r s = 0.24, P<0.01), and oranges were positively correlated with naringenin (r s =0.24, P<0.01) and hesperetin (r s =0.24, P<0.01). Furthermore the total citrus flavonoids were significantly correlated to the intake of fruits in general (r s =0.28, P<0.01). Phloretin in urine was correlated with apple intake (r s =0.22, P<0.01) and also with overall estimated intake of fruit (r s =0.22, P<0.01). Finally, some highly significant correlations between individual flavonoids were observed: Urinary excretion of hesperetin was correlated with excretion of naringenin (r s =0.60, P< 0.0001) and eriodictyol (r s =0.58, P<0.0001), naringenin with eriodictyol (r s =0.52, P<0.0001) and phloretin (r s =0.23, P<0.001), quercetin with phloretin (r s =0.96, P<0.0001), isorhamnetin (r s =1, p<0.0001) and tamarixetin (r s =1, P<0.0001), and daidzein was correlated with genistein (r s =0.52, P<0.0001).

DISCUSSION

In the present study we used the combination of several different flavonoids excreted in 24h urine as a reference biomarker to the self-reported intake of fruits and flavonoidrich foods. The link between intake of fruits and vegetables and the urinary excretion of flavonoids has been well established in controlled intervention studies with specific foods [START_REF] Nielsen | Flavonoids in human urine as biomarkers for intake of fruits and vegetables[END_REF][START_REF] Krogholm | Urinary total flavonoid excretion but not 4-pyridoxic acid or potassium can be used as a biomarker for the intake of fruits and vegetables[END_REF]Brevik et al. 2005) and in free-living populations [START_REF] Nielsen | Flavonoids in human urine as biomarkers for intake of fruits and vegetables[END_REF][START_REF] Brantsaeter | Urine flavonoids and plasma carotenoids in the validation of fruit, vegetable and tea intake during pregnancy in the Norwegian Mother and Child Cohort Study (MoBa)[END_REF][START_REF] Mikkelsen | Relative validity of fruit and vegetable intake estimated by the food frequenct questionnaire used in the Danish National Birth Cohort[END_REF]. Therefore the significant 13 correlation between the urinary total flavonoids and fruit intake in the present study shows that the estimation of intake of fruits by the 24h dietary recall is a valid estimate of the true effect of the free fruit intervention programme. The correlation values and excretion levels in the present validation study are in the same range as previously reported [START_REF] Nielsen | Flavonoids in human urine as biomarkers for intake of fruits and vegetables[END_REF][START_REF] Krogholm | Urinary total flavonoid excretion but not 4-pyridoxic acid or potassium can be used as a biomarker for the intake of fruits and vegetables[END_REF]Brevik et al. 2005;[START_REF] Mikkelsen | Relative validity of fruit and vegetable intake estimated by the food frequenct questionnaire used in the Danish National Birth Cohort[END_REF]Brandsaetter et al. 2007).

In the present study, the quantitatively largest amounts of flavonoids excreted in urine were the citrus flavonoids (hesperetin, naringenin and eriodictyol). The main reasons for this is that the urinary excretion relative to the intake of the citrus flavonoids, hesperetin and naringenin, is roughly 10 times higher than for the flavonols [START_REF] Manach | Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies[END_REF][START_REF] Krogholm | Relative bioavailability of the flavonoids quercetin, hesperetin and naringenin given simultaneously through diet[END_REF]. Due to the high relative urinary excretion of citrus flavonoids, citrus fruit and citrus juice intake is more easily reflected by our flavonoid biomarker than the fruits containing only flavonols. This was for instance seen in the present study as highly significant correlations between intake of citrus fruits and citrus juices and total citrus flavonoids. Also the previously reported high correlation between phloretin in urine and intake of apples [START_REF] Mennen | Urinary flavonoids and phenolic acids as biomarkers of intake for polyphenol-rich foods[END_REF][START_REF] Brantsaeter | Urine flavonoids and plasma carotenoids in the validation of fruit, vegetable and tea intake during pregnancy in the Norwegian Mother and Child Cohort Study (MoBa)[END_REF] was confirmed in the present study (r s =0.22, p<0.01). Urinary phloretin was also correlated with overall estimated intake of fruits (r s =0.22, P<0.01), indicating that individuals consuming apples are 'high fruit consumers' in general. We could only observe weak correlations between the 24h urinary excretion of quercetin (r s =0.17, P<0.05) and intake of apple, including apple juice. Phloretin therefore appears to be a better biomarker for apple intake than quercetin, even though apples are also a dietary important source of quercetin. [START_REF] Ito | Polyphenol levels in human urine after intake of six different polyphenol-rich beverages[END_REF] has suggested, that phloretin is a metabolite of naringenin formed by reductive opening of the heterocyclic C-ring, and [START_REF] Mennen | Urinary flavonoids and phenolic acids as biomarkers of intake for polyphenol-rich foods[END_REF] has found high correlations between phloretin and naringenin (r s =0.51, P<0.0001). In the present study the correlation between phloretin and 14 naringenin was also significant (r s =0.23, P<0.01), although the association was weaker than in the study by [START_REF] Mennen | Urinary flavonoids and phenolic acids as biomarkers of intake for polyphenol-rich foods[END_REF].

Although the citrus flavonoids are dominating among the flavonoids measured in our biomarker, the present study consolidates the importance of including several different flavonoids and/or metabolites when using flavonoids as an overall biomarker for intake of fruits, since the combination of all the flavonoids measured in the present study, result in the strongest correlation to estimated intake of fruits (r s =0.31, P<0.01), with a 95% confidence interval from 0.16 to 0.45, which is acceptable when validation is the overall purpose. Adding more flavonoids and/or flavonoid metabolites present in fruits might further improve the accuracy of the flavonoid biomarker. Further research investigating this is needed. The magnitude of a given correlation between a biomarker and self-reported food intakes cannot be expected to be very high even if both methods is quite accurate, and there is at present no agreement in literature as to what constitutes a satisfactory level of correlation. The magnitude of the presented correlations between total flavonoid in urine and the estimated dietary intake of fruits, fruits and vegetables and citrus fruits and citrus juices, given the relatively small sample size and the rather narrow range in dietary intake in the present study, is rather high.

Being in a distinct protocol can result in changes in lifestyle and exercise, also referred to as the placebo effect. In the present study, this was reflected by an increased fruit intake in both the control and intervention group because volunteers in both groups were informed about the purpose of the study. Over the 5 months study period, the intake of fruits in the control group changed from median 193 (2, 541) g/day to median 265 (9, 732) g/day.

According to the 24h dietary recall, this increase was not significant, while the urinary total flavonoid responded significantly to this change in intake. This could indicate that the 24h dietary recall is less sensitive as a marker of fruit intake than our flavonoid biomarker. With a larger 15 sample size the power would increase and the 24h dietary recall would then be a cheap and apparently accurate marker of fruit intake. In conclusion, the present study shows highly significant correlations between intake of fruit, estimated by 24h dietary recall, and the reference biomarker for fruit intake, the 24h urinary excretion of total flavonoids. Citrus flavonoids' refers to the sum of hesperetin, naringenin and eriodictyol. Total flavonoids refer to the sum of quercetin, isorhamnetin, tamarixetin, kaempferol, hesperetin, naringenin, eriodictyol, daidzein, genistein and phloretin.

Acknowledgement

FIGURE

Median (95% CI) estimated intake of fruits (g/day) Control group (n=45)

Intervention group (n=34)

Baseline After 5 months Baseline After 5 months

  This study is part of the ISAFRUIT project, funded by the European commission under the Thematic Priority 5-Food Quality and Safety of the 6 th framework Programm of RTD (Contract no. FP6-FOOD-CT-2006-016279). The views and opinions expressed in this publication are purely those of the writers and may not in any circumstances be regarded as stating an official position of the European Commission. The authors are grateful for the dedication to this study from the employees who participated in this study. Furthermore the authors thank Anni Schou for skilful technical assistance. (1from baseline in same group (P<0.05, Wilcoxon). ** Significant different from baseline in same group (P<0.01, Wilcoxon). # Significant different from 'after 5 months' in control group (P<0.05, Mann-Whitney U test). ##Significant different from 'after 5 months' in control group (P<0.01, Mann-Whitney U test). § Significant different from change in control group (P<0.05, Mann-Whitney U test). § § Significant different from change in control group (P<0.01, Mann-Whitney U test).

19 TABLE 1: Median (P5, P95) dietary intake estimated by 24h dietary recall (g/day) and 24h urinary excretion of flavonoids (µg) in control and intervention group at baseline and after 5 months of a free-fruit at workplace campaign. Citrus flavonoids' refers to the sum of hesperetin, naringenin and eriodictyol. Total flavonoids refer to the sum of quercetin, isorhamnetin, tamarixetin, kaempferol, hesperetin, naringenin, eriodictyol, daidzein, genistein and phloretin, 'Fruit marker' refers to the sum of citrus flavonoids and phloretin.


MSc Jetta Tuokkola
email: jetta.tuokkola@iki.fifunding

Minna Kaila

MSc Carina Kronberg-Kippilä

MSc Harri K Sinkko

Timo Klaukka

Pirjo Pietinen

Riitta Veijola

O Simell

Jorma Ilonen

professor Mikael Knip

professor Suvi M Virtanen

Cow's milk allergy in children: adherence to a

Keywords: adherence, child, preschool, cow's milk allergy, elimination diet, infant

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Special infant formulas are used instead of cow's milk -based formulas in the diet of milkallergic infants if the infant is not fully breastfed. If cow's milk allergy (CMA) is diagnosed by a physician, children up to the age of 2 years receive reimbursement for their costs from the Finnish Social Insurance Institution, offering a great opportunity to study CMA. The costs of the special infant formulas are substantial to the society, but management will not be fully effective if the cow's milk free diet is not followed properly.

The risks of a cow's milk-free diet are well documented [START_REF] Hidvegi | Slight decrease in bone mineralization in cow milk-sensitive children[END_REF][START_REF] Jensen | Bone mineral status in children with cow milk allergy[END_REF][START_REF] Rockell | Two-year changes in bone and body composition in young children with a history of prolonged milk avoidance[END_REF]. The diet should be balanced, providing adequate amounts of calcium and vitamin D, either by using the correct substitute foods or by supplementation [START_REF] Grimshaw | Dietary management of food allergy in children[END_REF][START_REF] Vandenplas | Guidelines for the diagnosis and management of cow's milk protein allergy in infants[END_REF].

By asking parents about the level of the elimination, adherence to a therapeutic elimination diet in children has been assessed in three previous studies (Vlieg-Boerstra et al. 2006[START_REF] Eggesbo | Restricted diets in children with reactions to milk and egg perceived by their parents[END_REF][START_REF] Allen | Dietary advice, dietary adherence and the acquisition of tolerance in egg-allergic children: a 5-yr follow-up[END_REF]. These studies suggest problems in adhering to an elimination diet, whether warranted or not.

So far no study has objectively assessed the adherence to a therapeutic elimination diet in children with food allergy, such as in the form of food records. The aims of the present study were to analyse the diet of children following a cow's milk protein (CMP) -free diet, to study the reintroduction of milk products into children's diets, and to evaluate factors associated with the adherence and age of development of tolerance.

SUBJECTS AND METHODS

The Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study is a multidisciplinary prospective population-based cohort study [START_REF] Kupila | Feasibility of genetic and immunological prediction of type I diabetes in a population-based birth cohort[END_REF]. All newborns from the catchment areas of three university hospitals in Finland (Turku, Oulu, and Tampere) are screened for HLA-conferred susceptibility to type 1 diabetes from cord blood samples after parental informed consent. Infants with high and moderate risk for type 1 diabetes (15% of The presence of special infant formula reimbursement was checked from the Finnish Social Insurance Institution for children in the DIPP Nutrition Study for whom a personal identification code was known to the study (n = 6412). According to clinical practice guidelines, the diagnosis is based on improvement of symptoms on an elimination diet, followed by a positive food challenge outcome or, in rare cases, on a high IgE level and improvement of symptoms on an elimination diet [START_REF] Paediatric | Food allergy among children[END_REF]. For those 398 (6.2%) children who received the reimbursement, information on the month and year of the beginning of the reimbursement (in other words a diagnosed CMA) was obtained.

Three-day food records, completed blindly to the purpose of the present study, were collected at the ages of 3 and 6 months, and 1, 2 and 3 years [START_REF] Kyttälä | Foof consumption and nutrient intake in Finnish 1-6-year-old children[END_REF]. Parents were given written and oral instructions on how to record everything the child ate and drank during the recording period. Separate food record forms, which also comprised questions e.g. about the origin of the food, were provided for the day care. Three consecutive food record dates were assigned to the family in advance and contained two week and one week end day. Examples were given on how to report food consumption with household or weighed measures.

Vitamin and mineral supplement usage was also recorded. Food picture booklets were used to help the estimation of portion sizes [START_REF] Paturi | Picture book of food portion sizes[END_REF][START_REF] Ovaskainen | Accuracy in the estimation of food servings against the portions in food photographs[END_REF].

Trained research nurses checked the food records with the families, and nutrition researchers entered all the food consumption data using an in-house software into the Finnish national food composition databank Fineli (National Institute for Health and Welfare, Nutrition Unit 2009). Food consumption and nutrient intakes, as well as the amount of CMP were calculated as average daily intake using the database. When calculating the amount of CMP, its quantity in all foods was taken into account, except for sausages, fat spreads and commercial biscuits.

However, these were taken into account when classifying adherence. The recording of food records was checked for food consumption and nutrient intakes, and for sources of CMP, to ensure valid recording and the validity of the databank used.

Information on special diets was collected at the ages of 3 and 6 months, and at 1 and 2 years by open questions and at the age of 3 years by a structured, validated questionnaire [START_REF] Tuokkola | Agreement between parental reports and patient records in food allergies among infants and young children in Finland[END_REF]. Parental reports of food allergies were double-entered to ensure valid recording.

To study the adherence to the cow's milk elimination diet, the next food record within 6 months from receiving the special reimbursement was analysed for the level of the elimination. Food records were classified as containing milk products, containing hidden or small amounts of CMP, containing no CMP, and containing no obvious sources of CMP but having recipes for which there is some uncertainty on whether they contain CMP or not. The latter consists of food items from small local bakeries and meat product manufacturers for which the detailed composition could not be traced, and if a commercial product name was not identified in the food record. To study the reintroduction of milk products into the diet, subsequent food records either up to a food record containing milk products, up to the latest food record or up to 3 years of age were further analysed.

Background information (parental age and education, the number of siblings) was collected at the time of recruitment. Information on parental smoking, former childbirths of the mother, and place of residence were obtained from the Finnish Medical Birth Registry.

RESULTS

The adherence to the cow's milk elimination diet was studied in 267 children (67% of those with the reimbursement), from whom a food record within 6 months from receiving the reimbursement was available. Of these 267 families, 226 had followed the therapeutic elimination diet extremely accurately (Table 1). Small amounts of CMP were seen in the diet of 14 children, but only eight had used actual milk products. In addition, in 19 children there were no obvious sources of CMP, but food records included some recipes or foodstuffs for which there is uncertainty of them containing CMP. The parents of three children reported being in a process of an oral challenge test with milk and these children were classified adherent.

Those eight children whose parents reported consumption of milk products had a mean intake of 350 g (range 76 g -1136 g) of milk products during the 3 days. The amount of CMP ranged from 2.0 g to 36.5 g, with a mean intake of 8.5 g and median intake of 4.0 g of CMP.

Those 14 children whose parents reported consumption of foodstuffs classified as containing small amounts of CMP had a mean intake of 6.8 g (range 0.0 g -42.7 g) of milk products during the 3 days. The amount of CMP from these foods ranged from 0.0 g to 9.1 g, with a mean intake of 1.0 g and median intake of 0.02 g.

Four of the eight children who had used milk products had also used special infant formulas (Table 2). Mean intakes of calcium were below recommendations, but due to supplementation, mean intakes of vitamin D were sufficient (Table 3). In addition, the intakes of riboflavin and calcium were especially low in food records containing neither milk products nor a special infant formula (n = 89, data not shown).

Of the children with CMA, 59% were boys. The median duration of overall breastfeeding was 6 months and that of exclusive breastfeeding 2 months, and the reimbursement for the

DISCUSSION

The therapeutic elimination diet in children with a diagnosed CMA was very well adhered to.

As many as 85% of the families followed the diet extremely accurately, and an additional 7% had no obvious sources of CMP, but food records contained some foodstuffs with an unknown recipe. Small amounts of CMP were observed in the diet of 5% of the children, and only 3% had used milk products. Those who had consumed small amounts of CMP were older and more often monosensitised, but otherwise sociodemographic factors were not associated with the adherence. By the age of 3 years about a half of the children had begun to use milk products and a quarter of the children were still avoiding CMP. Initial adherence to the elimination diet was not associated with the age of ending the CMP-free diet.

Major strengths of the study are the large population studied, and that food records were collected for purposes other than to study the adherence to a CMP elimination diet, diminishing embellishment. On the other hand, there were some inexact elements in some food records: imprecise recording of food items consumed, or not having information detailed enough on products such as from small bakeries and meat product manufacturers dating years back. However, for 93% of the children consumption of foods containing even the smallest amounts of CMP could be accurately evaluated.

The limitations of the study lie within possible problems in diagnostics, in not knowing the children's' symptoms, in the cumbrousness of the database used for the purpose of the present study, and in the rather long intervals in which the diets of the children could be studied. Relying on registry data for the diagnosis of CMA may, in part, explain the rather high percentage (6.2%) of children with a diagnosed CMA, despite the national guidelines for the diagnosis (Finnish Paediatric Association 2004). In our previous validation study, we found that not all children underwent an oral challenge test before receiving the diagnosis, so overdiagnosis is possible [START_REF] Tuokkola | Agreement between parental reports and patient records in food allergies among infants and young children in Finland[END_REF]). In addition, the strong research in the field of food allergies in both university hospitals where the cohort was gathered may increase awareness of the disease and the tendency to seek for a diagnosis.

Two of the eight children with milk products in their diet had reportedly used a special infant formula only for 2 weeks, and these children had likely received the reimbursement on false grounds. There were four children, who consumed both a special infant formula, and some milk products. The costs of the use of special infant formulas to society are considerable, and unnecessary use should be avoided. Why eight children had consumed CMP-containing porridges, gruels and other ready-to-use baby foods, is not known.

Compared with previous studies addressing the adherence to a therapeutic elimination diet in children with food hypersensitivities, in which approximately one to two thirds of the populations were fully adherent, our study subjects show a much higher adherence (Vlieg-Boerstra et al. 2006[START_REF] Eggesbo | Restricted diets in children with reactions to milk and egg perceived by their parents[END_REF][START_REF] Allen | Dietary advice, dietary adherence and the acquisition of tolerance in egg-allergic children: a 5-yr follow-up[END_REF]. The reasons for this are most likely due to methodological differences (questionnaires vs. food records), as well as differences in the populations studied (age groups, the basis for diagnosis, and food allergies).

Adherence to dietary treatment of celiac disease shows variation between 30% to 95%, depending on the methods used and the age of the children; no studies report adherence in an age group comparable to the present cohort of children under the age of 3 years [START_REF] Hommel | Treatment regimen adherence in pediatric gastroenterology[END_REF]. Our results are based on 3-day food records, and we have no knowledge on the diets beyond those days, which is a limitation compared with the studies using questionnaires in defining the level of adherence. However, food records collected blindly to the purpose of the present study are presumed to give an otherwise reliable answer. The total number of days examined in our study amounts to 781. One particular reason for the high level of adherence in our study population could be the requirement for the food industry to mention CMP on food labels, making it easier to follow the elimination diet. Being aware of an increased genetic risk for type 1 diabetes may also increase the adherence to a dietary treatment.

In Finland those children who are allergic only to CMP do not often see a nutritionist, which may explain the association between monosensitisation and consuming CMP hidden in foodstuffs. The ingestion of these foods may be considered as accidents or as ignorance, and could be counteracted by dietary counselling, if necessary. Unfortunately, information on visits to a nutritionist was not available to us. On the other hand, monosensitised children may have a milder form of the disease, and actually tolerate small amounts of CMP. As in the study by [START_REF] Eggesbo | Restricted diets in children with reactions to milk and egg perceived by their parents[END_REF], maternal age and education were not associated with adherence to the elimination diet.

When studying the introduction of milk into the child's diet, our main hypothesis was that consuming small amounts of CMP would lead to a quicker recovery from CMA and reintroduction of milk products into the child's diet. However, in this group of children this was not the case; initial adherence was not associated with the age at reintroduction of milk products. Vlieg-Boerstra et al. (2006) reported similar findings: in their study unintentional consumption of an allergenic food did not predict the outcome of an oral challenge. Likewise, in the study by [START_REF] Allen | Dietary advice, dietary adherence and the acquisition of tolerance in egg-allergic children: a 5-yr follow-up[END_REF], adherence to an egg elimination diet did not predict the outcome of a challenge. In our study, the only background factor associated with an earlier introduction of milk products was maternal smoking during pregnancy. We assume that this association does not truly reflect a quicker recovery from CMA, but rather the motivation for following the prescribed diet. The question of whether ingesting small amounts of CMP in a home setting would induce tolerance should be addressed with more frequent follow-up. We could not find any such associations, but this could be due to the lengthy intervals of studying the inclusion of CMP into the diet. The age at recovery from CMA is earlier in the present study than that previously reported by [START_REF] Skripak | The natural history of IgE-mediated cow's milk allergy[END_REF], who studied children with an IgE-mediated CMA. The recovery rate in the present study follows more closely that presented by Vanto et al. (2004), who also included both IgE-mediated and non-IgE-mediated CMA in his study. The recovery rate may also be affected by the fact that the diagnosis is done in clinical practice and not by the researchers in the present study.

Children following a CMP-free diet require attention to fulfil their needs for calcium, vitamin D and riboflavin. In addition, children recovered from CMA consume less milk products than children on average do [START_REF] Kyttälä | Foof consumption and nutrient intake in Finnish 1-6-year-old children[END_REF]; the concern of receiving enough calcium and vitamin D is still present after the inclusion of milk into the diet.

In summary, adherence to a therapeutic elimination diet of children with diagnosed CMA was extremely good. Monosensitisation and older age are associated with sloppier adherence, possibly due the absence of nutritional information by a dietitian/nutritionist. Intakes of calcium, riboflavin and vitamin D are of concern in children who follow or have followed a CMP-free diet. This study does not show evidence that small amounts of CMP would lead to a quicker recovery from CMA in a real-life setting. 1 within 6 months from receiving a reimbursement of the special infant formulas 2 Estimated needs for energy at 3 months 2100 kJ for girls and 2200 kJ for boys, at 6 months for 2600 kJ for girls and 2700 kJ for boys, at 1 year 3400 kJ for girls and 3700 kJ for boys, and at the age of 2 years 4400 kJ for girls and 4700 kJ for boys.

3 Recommended intake 540 mg for 6 -11 months of age, 600 mg for 1-2 years of age 4 Recommended intake 10 µg for 6 -23 months of age, 7.5 µg for 2 years of age 5 Recommended intake 0.5 mg for 6 -11 months of age, 0.6 mg for 12-23 months of age, and 0.7 mg for 2 years of age

  those screened) are invited to participate in the study, excluding children with severe congenital abnormalities or diseases, or whose parents are of non-Caucasian origin or do not understand Finnish, Swedish or English. The DIPP Nutrition study is performed in the Oulu and Tampere University hospitals among children born since October 1996. The local Ethical Committees have approved the study. All families have given their written informed consent to participate in the DIPP study.

  SPSS software package 15.0(SPSS Inc, Chicago, IL, 2006) and R 2.7.0 (R Development CoreTeam, 2008) were used for statistical analyses. Multinomial logistic regression was applied to study the associations of adherence to the therapeutic elimination diet with background factors. Cox proportional hazards model with an accommodation to interval censored data was used to identify the factors associated with the age of recovery. 9 Tuokkola J

Figure 1 .

 1 Figure 1. Number of children, in whom cow's milk was reintroduced into the diet for the first

Table 1 .

 1 Adherence to cow's milk protein (CMP) elimination diet within 6 months after the diagnosis of cow's milk allergy, n = 267

	Child has consumed

Table 2 .

 2 Adherence to the cow's milk elimination diet: milks and special infant formulas that were used during the 3 study days 1 within 6 months from receiving a diagnosis of cow's milk allergy by age (each child may be accounted for more than once if having consumed more than one type of milk or drink; number of children = 264).

		3 months	6 months	1 year	2 years
		n = 48	n = 106	n = 106	n = 4
	Breast milk	26	41	17	0
	Amino-acid formula	6	26	25	0
	Hydrolysed formula	22	48	47	1
	Soy formula	3	7	24	2
	Cow's milk formula 2	1	6	1	0
	Cow's milk 2	0	0	1	0
	Soydrink	0	0	6	0
	Oatdrink	0	0	2	1
	Ricedrink	0	0	2	1

1 Those who had reported an ongoing challenge test with cow's milk are not included.

2

The reason for the consumption of cow's milk formula and cow's milk is not known.

Table 3 .

 3 Adherence to the cow's milk elimination diet: mean (SD) intakes of energy and certain nutrients, intake from foods and supplements together, of non-breastfed children by

	age 1
			3 months	6 months	1 year	2 years
			(n = 22)	(n = 67)	(n = 89)	(n = 4)
	Energy kJ 2		2430 (277)	2800 (554)	3910 (760)	4370 (325)
	Protein	% of energy	10.2 (0.8)	10.2 (1.6)	12.2 (2.7)	12.1 (2.9)
	Carbohydrates % of energy	40.7 (5.3)	50.8 (7.4)	55.0 (6.7)	56.1 (5.5)
	Fat	% of energy	49.0 (5.1)	38.7 (7.0)	32.4 (7.2)	31.2 (7.7)
	Calcium 3	mg	467 (60)	430 (130)	473 (191)	780 (248)
	Vitamin D 4	µg	14.7 (4.2)	14.1 (4.0)	13.3 (4.2)	12.3 (10.7)
	Riboflavin 5	mg	0.84 (0.26)	0.74 (0.25)	0.81 (0.38)	1.14 (0.55)

Meat and bakery products for which a recipe was not known, or if a commercial name of a meat or bakery product or a ready-to-use baby food was not specified

CMP in food items such as industrial baby-foods, bakery and meat products, or fat spreads containing CMP as an ingredient, cookies with milk chocolate, or a few cheese puffs.

Milk products used were cheese, ice cream, infant formulas, as well as milk-containing infant porridges, and gruels.

Acknowledgments

We are extremely grateful to all the families who took part in this study. We also acknowledge the excellent collaboration of the DIPP research nurses, doctors, nutritionists and laboratory staff over the years. The study was financially supported by the Academy of Finland (grants 63672, 79685, 79686, 80846, 201988, 210632, 129492, 126813), the Finnish Paediatric Research Foundation, the Juho Vainio Foundation, the Yrjö Jahnsson Foundation, Medical Research Funds of Turku, Oulu and Tampere University Hospitals, the Juvenile Diabetes Research Foundation (grants 197032, 4-1998-274, 4-1999-731, 4-2001-435), the Novo Nordisk Foundation and EU Biomed 2 (BMH4-CT98-3314), Doctoral Programs for Public Health, Foundation for Allergy Research, Research Foundation of Orion Corporation, Tampere Tuberculosis Foundation, and the Jalmari and Rauha Ahokas Foundation.

Vanto T, Helppila S, Juntunen-Backman K, Kalimo K, Klemola T, Korpela R et al. (2004).

Prediction of the development of tolerance to milk in children with cow's milk hypersensitivity. J Pediatr, 144, 218-222.

Vlieg-Boerstra BJ, van der Heide S, Bijleveld CM, Kukler J, Duiverman EJ, Wolt-Plompen SA et al. (2006). Dietary assessment in children adhering to a food allergen avoidance diet for allergy prevention. Eur J Clin Nutr 60, 1384-1390.

The study was financially supported by the Academy of Finland (grants 63672, 79685, 79686, 80846, 201988, 210632, 129492, 126813), the Finnish Paediatric Research Foundation, the Juho Vainio Foundation, the Yrjö Jahnsson Foundation, Competitive Research Funding of the Pirkanmaan Hospital District, Tampere University Hospital, Medical Research Funds of Turku and Oulu University Hospitals, the Juvenile Diabetes Research Foundation (grants 197032, 4-1998-274, 4-1999-731, 4-2001-435), the Novo Nordisk Foundation and EU Biomed 2 (BMH4-CT98-3314), Doctoral Programs for Public 3 Tuokkola J Health, Foundation for Allergy Research, Research Foundation of Orion Corporation, Tampere Tuberculosis Foundation, and the Jalmari and Rauha Ahokas Foundation. None of the authors had financial or personal interests in any company or organisation sponsoring the research at the time the research was done.

special infant formula was received at a median age of 5 months. Parental age and education, the number of siblings, gender, the duration of breastfeeding, urbanity of the place of living, and the study centre were not associated with the adherence to the therapeutic elimination diet. An older age of the child, as well as lack of other food allergies besides CMA, increased the risk of having small amounts of CMP in the diet (p < 0.001 for both; adjusted for maternal age and education, number of siblings, sex, duration of breastfeeding, and study centre).

The presence of CMP in the diet within 6 months from receiving the reimbursement did not predict the age at reintroduction of CMP in the diet, nor did place of residence, sex, or parental age or education level. The children of smoking mothers were reintroduced milk products at a younger age than the children of non-smoking mothers, when adjusted for the age and sex of the child, adherence to the elimination diet, maternal education and age, the duration of breastfeeding, and study centre (p < 0.05).

To study the reintroduction of milk products into the child's diet, subsequent food records were studied in children who initially had adhered to a CMP-free diet. Of the 259 children who had followed the CMP-free diet, subsequent food records were available from 215 children, who were observed further. Altogether 120 children had begun to use milk products during follow-up (Fig 1). At the age of 3 years, 55 children were still avoiding milk; 40 children had left the study before the age of 3 years and had not begun to use milk products up to the last available study point.

The main milk products used after the period of milk elimination were milk (87% of children), cheese (67% of children), and yoghurt (45% of children). However, amounts of milk products used were small, and only 17% of children consumed three glasses of milk, 23% used at least two slices of cheese and 15% used at least 1 dl of yoghurt daily.

Conflict of interest statement

The authors declare no conflict of interest.