Different metabolic and absorption patterns of betaine in response to dietary intake of whole-wheat...

ORIGINAL PAPER

Different metabolic and absorption patterns of betaine in responseto dietary intake of whole-wheat grain, wheat aleurone or ryealeurone in catheterized pigs

Christian Clement Yde • Jeroen Jasper Jansen •

Peter Kappel Theil • Hanne Christine Bertram •

Knud Erik Bach Knudsen

Received: 14 February 2012 / Revised: 28 August 2012 / Accepted: 5 September 2012 / Published online: 19 September 2012

� Springer-Verlag 2012

Abstract In the present study, a novel method of mea-

suring the uptake of nutrients by 1H NMR spectroscopy

was performed to determine dietary effects. Six pigs

catheterized in the portal vein and mesenteric artery were

fed breads made from whole-wheat grain (WWG), wheat

aleurone flour (WAF; 19 % aleurone of bread) or rye

aleurone flour (RAF; 15 % aleurone of bread) in a repeated

3 9 3 crossover design. Three meals were provided daily

(at 0, 5 and 10 h), and each period comprised of 1 week.

Portal and arterial blood samples were collected at fasting

(-30 min) on day 4–7, and on day 7, pooled blood samples

were collected at 0–2.5, 2.5–5, 5–7.5 and 7.5–10 h after

first (0 h) daily meal. Using the arterial-venous difference

and ANOVA–simultaneous component analysis, plasma

betaine was found to accumulate during the experimental

period. On day 7, plasma betaine concentrations was

20–40 % higher than when feeding the WAF diet com-

pared with RAF and WWG (P = 0.001), whereas the

content of betaine in the WAF diet was about twice as

high. The arterial-venous difference showed a tendency

(P = 0.078) for the diet–time interaction of betaine

absorption in the absorption phases but there was no diet

effect (P = 0.31), which indicates a steady-state absorption

has been reached on day 7. Furthermore, high plasma

betaine levels correlated with low plasma creatine of

endogenous origin. In conclusion, the explorative method

revealed that the plasma concentration and absorption of

betaine were differentially modified by the diets.

Keywords Absorption � Aleurone � ASCA � Betaine �Metabolomics � NMR

Introduction

Wholegrain cereal products have been extensively studied

in recent years, because epidemiological studies have

indicated that its consumption protects against a number of

diseases associated with the Western lifestyle such as

obesity, cardiovascular disease, certain types of cancer

and type 2 diabetes [1, 2]. The physiological effects of

wholegrain cereal are however unclear, since the beneficial

effects of wholegrain cereals can most likely be assigned to

a complex mixture of bioactive compounds, many of which

associated with dietary fibre (DF) [3]. A better under-

standing of the mechanisms behind the protective effects of

wholegrain cereals may lead to the development of new

cereal products with health-promoting effects. Methods

for isolation of the aleurone layer from grain have, for

C. C. Yde � P. K. Theil � K. E. B. Knudsen

Department of Animal Science, Faculty of Science

and Technology, Aarhus University,

Blichers Alle 20, 8830 Tjele, Denmark

C. C. Yde (&) � H. C. Bertram

Department of Food Science, Faculty of Science

and Technology, Aarhus University,

Kirstinebjergvej 10, 5792 Aarslev, Denmark

e-mail: [email protected]

J. J. Jansen

Biosystems Data Analysis, Faculty of Sciences,

University of Amsterdam, P.O. Box 94215,

1090 GE Amsterdam, The Netherlands

J. J. Jansen

Netherlands Metabolomics Centre, Einsteinweg 55,

2333 CC Leiden, The Netherlands

J. J. Jansen

Institute for Molecules and Materials, Analytical Chemistry,

Radboud University Nijmegen, Heyendaalseweg 135,

6525 AJ Nijmegen, The Netherlands

123

Eur Food Res Technol (2012) 235:939–949

DOI 10.1007/s00217-012-1825-5

example, already been developed [4, 5]. There has been a

growing interest in the nutritional and health-related

properties of wheat aleurone fraction because the aleurone

layer contains a high amount of micronutrients, phyto-

chemicals and DF [6]. A 4-week human intervention study

by Price et al. [7] suggested that consumption of wheat

aleurone–enriched bread resulted in increased plasma

betaine and a lower total homocysteine and LDL choles-

terol. Betaine is an osmolyte and also donates a methyl in

the transmethylation process in the methionine cycle,

important for decreasing plasma homocysteine and syn-

thesizing S-adenosylmethionine [8, 9]. Homocysteine is a

risk factor for cardiovascular disease [10], and S-adeno-

sylmethionine is a direct methyl donor in many important

pathways [11]. However, other studies indicate that betaine

may be harmful by increasing serum low-density lipopro-

tein concentration [10, 12], such that the health-related

significance of betaine intake is ambiguous.

Proton (1H) NMR spectroscopy detects all mobile pro-

ton-containing metabolites down to the low lmol level and

is therefore a suitable technique for explorative studies of

biofluids. The metabolic response of either wholegrain rye

and bran or wheat wholegrain has previously been studied

by 1H NMR-based metabolomics, and these studies suggest

that a wholegrain diet elevates betaine levels in plasma

[13–15] and liver [16]. Furthermore, NMR measurements

of wheat showed higher concentration of betaine in the

aleurone layer, intermediate in the bran fraction and lowest

in the flour fraction [17].

In this study, we applied ANOVA–simultaneous com-

ponent analysis (ASCA) to select biomarkers to analyse

data [18, 19]. This is a multivariate extension of ANOVA

that takes both the covariance between variables and the

design of the experiment into account, which separates

metabolic variation associated with time, how diet

affects the dynamic response and the residual variation

between individuals. In this method, the spectral differ-

ences between simultaneously collected portal and arte-

rial samples of catheterized sows, representing the net

absorption and metabolism of nutrients [20], allows more

dedicated exploration of the diet effects than a study of

the measured spectra themselves with standard chemo-

metric methods like principal component analysis. A pre-

vious experiment on ileal cannulated pigs and fed the

same experimental diets studied the degradation of

nutrients in the small and large intestine [21, 22]. The

insulin response of the pigs in the present study has been

published before [23]. The present paper explores the

metabolic effect and absorption dynamics, when changing

the feeding from a reference refined wheat flour diet to a

whole-wheat grain, a wheat aleurone flour (19 % aleurone

of bread) or a rye aleurone flour diet (15 % aleurone

of bread).

Materials and methods

Diets

Breads were made of standard white wheat flour (WFL) for

the washout diet and whole-wheat grain (WWG), wheat

aleurone–rich flour (WAF) and rye aleurone–rich flour

(RAF) for the experimental diets. The ingredients for the

WFL bread were white wheat flour (Triticum aestivum L.

cv. Tiger), purified wheat fibres (Vitacel R200; J. Rette-

nmaier and Sohne GmbH, Rosenberg, Germany), rapeseed

oil, sugar, salt, wheat gluten (LCH A/S, Frederiksberg,

Denmark) and yeast, while in WWG, WAF and RAF

breads, wholegrain wheat (BFEL, Karlsruhe, Germany),

wheat aleurone–rich flour (Buhler AG, Uzwil, Switzerland)

and rye aleurone–rich flour (Raisio plc, Raisio, Finland)

replaced the white wheat flour and the Vitacel fibre.

Information on the chemical analysis of the breads can be

found in [21, 23]. After production, the breads were cut

into pieces and mixed. After weighing, meal portions were

frozen at -20 �C and thawed immediately before con-

sumption. The four breads were balanced with regard to

contents of starch, protein, fat, energy and DF, but they

varied in DF characteristics (see Tables 1, 2 for dietary

ingredients and chemical composition). Estimates of the

content of aleurone in the WAF and RAF breads were

based on an assumption of approximately 90 % purity of

the wheat aleurone fraction and 7 % aleurone content in the

wheat aleurone kernel [24, 25] and that a high proportion

of grain phosphorous is associated with the aleurone cells.

Based on the analysis of phosphorous in the whole kernel

and aleurone fraction, it was estimated that the aleurone

cells contributed with approximately 54 % of total phos-

phorous. This information was then used to estimate the

contribution of aleurone in the rye aleurone fraction and

diets (Table 1) taking into account that the aleurone layer

in rye account approximately 9 % of the total grain [24].

Experimental design and animals

Experiments were in compliance with the guidelines of the

Danish Ministry of Justice and regulations for humane care

and use of animals in research [The Danish Ministry of

Justice, Animal Testing Act (Consolidation Act no. 726 of

9 September 1993 as amended by Act no. 1081 of 20

December 1995)]. Daily feed allowance was 2.7 times of

maintenance [2.7 9 460.2 kJ (110 kcal) of DE/kg 0�75;

NRC, 1998], which supplied 210 g DF/d. The pigs were

fed three times daily at 09.00 (breakfast), 14.00 (lunch) and

19.00 h (dinner) with an amount of 40, 40 and 20 % of the

daily supply, thereby mimicking the diurnal variation in

cereal intake experienced by humans. The pigs used in the

study were from the swineherd at Aarhus University,

940 Eur Food Res Technol (2012) 235:939–949

123

Faculty of Agricultural Sciences, Foulum, Denmark. Six

female pigs (Landrace 9 Yorkshire) with a bodyweight of

56.5 kg (±1.8 kg) were included in the experiment, which

was designed as a repeated 3 9 3 crossover design. The

pigs were adapted to the pen for 5 days, and then the

animals were surgically fitted with a flow probe (Transonic,

20A probe, 20 mm; Transonic System, Inc., Ithaca, NY,

USA) around the portal vein, a catheter in the portal vein

and a catheter in the mesenteric artery [26]. After the

surgery, the animals were allowed a 5–7-day recovery

period before entering a 21-day experimental period (three

consecutive experimental weeks). In each experimental

week, the pigs were fed a washout diet (WFL) on days 1–3,

and then the pigs were fed one of the three experimental

breads on days 4–7. The pigs had access to water ad libi-

tum, whereas no straw was supplied. Fasting blood samples

(30 min before the first daily meal) were collected from the

portal vein and the mesenteric artery on day 4–7. On day 7,

consecutive blood samples were drawn simultaneously

from the portal vein and the mesenteric artery during

0–10 h after the first daily meal (breakfast). Four artery and

four venous plasma samples were pooled to represent the

mean content of metabolites in plasma for the postprandial

period relative to the first daily meal: 0–2.5 (P1), 2.5–5

(P2), 5–7.5 (P3), 7.5–10 (P4) h after first daily meal. Blood

samples were collected in 9-mL Na-heparinized vacutain-

ers, and plasma was harvested. The pooled plasma samples

were stored at -80 �C until analysis.

NMR measurements

NMR measurements were carried out on a Bruker Avance

III 600 MHz NMR spectrometer (Bruker BioSpin, Rhein-

stetten, Germany) operating at a frequency of 600.13 MHz

and equipped with a 5-mm TXI probe. Two hundred

microlitre aliquots of the plasma samples was mixed with a

solution of 400 lL 0.9 % saline (NaCl) and 20 % D2O. 1H

NMR spectra were obtained using a Carr-Purcell-

Meiboom-Gill (CPMG) pulse sequence [27] with water

suppression to attenuate signals from macromolecules. The

total spin–spin relaxation delay was 100 ms, the spin-echo

delay was 1 ms, and the recycle delay was 2 s. The spectra

were acquired by 64 scans into 32 k data points on a

spectral width of 17.34 ppm at a temperature of 310 K.

A fixed receiver-gain value was used for recording all

samples. An exponential line-broadening of 0.3 Hz was

applied prior to the Fourier transformation. Each spectrum

was manually phased, baseline corrected and referenced to

the glucose anomeric doublet at 5.23 ppm. The arterial-

venous difference between simultaneous collected plasma

Table 1 Dietary ingredients of experimental diets

Washout diet Experimental diets

WFL WWG WAF RAF

Ingredients (g/kg, as-fed basis)

Vitacel R200 (99.5 % cellulose)a 69

Standard wheat flour 711

Wheat whole grain 813

Wheat aleurone 2b 214

Rye flour (3–3.5 % ash)c 365

Wheat starchd 516 359

Wheat glutene 59 36 116 116

Rapeseed oil 86 76 79 85

Sugar 15 15 15 15

Baker’s yeast 15 15 15 15

Vitamin–mineral mixturef 4 4 4 4

Aleurone contribution to diet (%) 19 15

a Vitacel R 200, J. Rettenmaier and Sohne GmbH, Rosenberg, Germanyb Leuron� ASP02, Buhler AG, Uzwil, Switzerlandc Fibre-rich rye flour with 3–3.5 % ash, Raisio plc., P.O. Box 101, 21201 Raisio, Finlandd Wheat starch ‘‘foodstar’’, LCH A/S, Peter Bangs Vej 33, DK-2000 Frederiksberge Vital wheat gluten, LCH A/S, Peter Bangs Vej 33, DK-2000 Frederiksbergf Supplying per kg diet: retinol 660 lg, cholecalciferol 12.5 lg, a-tocopherol 30 mg, menadione 11 mg, thiamine 1 mg, riboflavin 2 mg,

d-pantothenic acid 5.5 mg, niacin 11 mg (available), biotin 27.5 lg, cyanocobalamin 11 lg, pyridoxine 1.65 mg, Fe 25 mg, Cu 10 mg, Zn

40 mg, Mn 13.9 mg, Co 0.15 mg, I 0.01 mg, Se 0.15 mg and maize Ca2(PO4)3, K2PO3, NaCl, CaCO3 as a carrier (Solivit Mikro 106, Løvens

Kemiske Fabrik, Vejen, Denmark)

Eur Food Res Technol (2012) 235:939–949 941

123

samples from the portal vein and the mesenteric artery of

each pig was calculated by subtracting the spectral mes-

enteric artery data points from the corresponding portal

vein data points (DAV difference). The DAV difference

was multiplied by the portal flow measurements to mimic

the net absorption as described by Rerat et al. [28]. The

NMR spectra and the DAV difference from 9.0–5.0 to

4.4–0.5 ppm were binned into 0.013 ppm regions and

integrated using Matlab (version 2009a, The MathWorks,

USA).

Prior to analysis, diet extracts were made of the washout

diet (WFL) and the three experimental diets (WWG, WAF

and RAF) in triplicates by adding 1 mL D2O and 500 lL

H2O to 100 mg of the diet. The solutions were sonicated

for 10 min and afterwards mixed for 1 h and centrifuged

for 5 min at 14,000 g. 545 lL extract were added 55 lL

D2O with 0.05 % w/w TSP as an intern standard. Standard1H NMR spectra were acquired using 90� pulse experiment

and presaturation of the water signal on at a temperature of

310 K with 64 scans and 32 k data points over a spectral

width of 12.15 ppm. The relaxation delay was 5 s, and the

acquisition time was 2.25 s. An exponential line-broaden-

ing of 0.3 Hz was applied prior to the Fourier transfor-

mation. The betaine signal at 3.26 ppm and the TSP peak

were manually integrated, and the concentration of betaine

in the extracts was calculated from these two integrals.

Assignment was achieved by the use of 2D 1H–1H

correlation spectroscopy (COSY) with double quantum

filter (DQF) and 2D 13C–1H heteronuclear single quantum

coherence (HSQC) experiments performed on representa-

tive venous plasma (see Fig. 1; Table 3) and diet extract

samples using water suppression. The COSY spectra were

acquired with a spectral width of 6,127 Hz in both

dimensions, 4 K data points, 512 increments with 64

transients per increment and a recycle delay of 1.5 s. The

HSQC spectra were acquired with a spectral width of

6,250 Hz in the 1H dimension and 21,128 Hz in the 13C

dimension, a data matrix with a size of 2,048 9 512 data

points, 256 transients per increment and a recycle delay of

2 s. The 13C chemical shift was referenced to the a-glucose

anomeric carbon at 94.9 ppm. As discussed in a previous

paper [20], the betaine signal at 3.26 ppm in the NMR

spectra of plasma samples is overlapped with a downfield

line of a doublet of doublets from b-glucose at 3.25 ppm.

However, in this study, the variation of the betaine signal is

only slightly affected by variation of the glucose signal

because the diets were formulated to contain a balanced

starch content.

Table 2 Chemical composition of the experimental diets

Washout diet Experimental diets

WFL WWG WAF RAF

Chemical composition (g/kg DM)

DM (g/kg as is) 639 654 704 691

Ash 37 43 58 41

Protein (N 9 6.25) 168 173 177 173

Fat 128 137 134 146

Carbohydrates

Sugars

Glucose 2 5 3 4

Fructose 8 12 10 10

Sucrose 1 2 1 3

Starch 513 506 470 505

Total non-starch polysaccharides 117 (16) 105 (26) 105 (15) 104 (39)

Fructans 2 3 2 10

Cellulose 66 17 14 12

Non-cellulosic polysaccharides 49 (14) 85 (23) 89 (13) 82 (29)

b-Glucans 1 (0) 4 (1) 8 (0) 9 (2)

AX 26 (11) 61 (17) 62 (9) 50 (19)

Klason lignin 10 22 23 26

Total dietary fibre 127 127 127 130

Gross energy (MJ/kg DM) 20.56 20.35 20.35 20.43

Betaine content (lg/g DM) 114 ± 7 253 ± 9 518 ± 13 283 ± 5

Values in brackets are soluble non-starch polysaccharides


123

Data analysis

Prior to the data analysis, the data were divided into two

subsets. First, the fasting samples from day 4–7 were

grouped as the adaption dataset (data_AD). To use the

information that all periods starts with a washout diet

(WFL), the day 4 sample was subtracted from days 4–7

samples for each pig and experimental week combination,

an operation familiar from Geometric trajectory analysis

[29]. Second, the data from the fasting sample and the

pooled samples from day 7 were grouped as the profile

dataset (data_Pro). The average of all day 7 fasting samples

for each pig (i.e. for all three breads) was subtracted from

all samples belonging to the same pig. The indices used for

data_AD: i = 1,…, 6 for the pigs; j is the bin for each of

the biomarkers or their DAV difference; k = 1,…, 4 for the

sampling points (days 4–7); l = 1, 2, 3 for the breads

(WWG, WAF, RAF); and m = 1, 2 for the blood com-

partments (portal, arterial).

The plasma data and the DAV difference were mean-

centred and Pareto-scaled by dividing each variable by the

square root of its standard deviation. To remove individual

differences between the pigs that were present already

before diet change for each individual, the sample collected

at t = 0 was removed from all samples of that individual in

the analysis of the 4-day study. Biomarker selection was

performed using ANOVA–simultaneous component anal-

ysis (ASCA) [18, 19]. The ASCA model for time point

k and diet l is given as:

Xkl ¼ 1ItTl;kPT

l þ 1ItTll;klP

Tll þ Tlll;klP

Tlll þ Ekl

where 1I is a length I column vector, Xkl is the (I 9 J)

matrix of pre-processed data, tl,k is the length Rl vector of

submodel I scores for time point k, tll,kl are the scores on

submodel II for time point k and diet h, which is a length

R2 and Tlll,kl is the (I 9 Rlll) matrix of submodel III scores;

Rl, Rll, and Rlll indicate the number of components for each

submodel and Pl, Pll, and Plll contain the corresponding

loadings; Ekl is the (I 9 J) matrix of model residuals. The

ASCA model subdivides the total variation in the data into

three submodels. The first submodel I describing the

‘Time’ factor, that is, the dynamic variation similar to all

diets. Submodel II is of prime interest to this study,

because it shows how each diet differentially modifies the

metabolic concentration or DAV difference over time. The

third submodel describes the natural variation among

biological replicates and is of minor interest to the

current experimental questions. The ASCA methodology

that is currently available in the literature does not allow

for assessment of the magnitude of the effects observed in

submodel II. This information is provided by the submodel

III scores, but these cannot be directly compared with the

scores of submodel II, because they are expressed on

different loadings. Such a comparison can be obtained by

expressing the full pre-processed data onto the submodel II

loadings, through orthogonal projection. This can be done

by:

T�ll;11

..

.

T�ll;KL

264

375 ¼

X11

..

.

XKL

264

375Pll

where T�ll;11 is the (I 9 Rll) matrix of biological replicate

scores expressed on submodel II. The T�ll;kl for each diet–

time point combination can now serve to compare the

magnitude of the difference between two time points

Fig. 1 Representative 1H NMR spectrum of a venous plasma sample

when feeding the wholegrain (WG) diet, postprandial from 5 to 7.5 h

after first daily meal. The assignment of important metabolites

are shown as: 1 betaine, 2 creatine, 3 glucose, 4 lactate, 5 lipid,

6 N-acetyl glycoprotein

Table 3 Assignment of important bins found in the multivariate data

analysis of the plasma samples

Metabolites Chemical shift (1H/ppm) Chemical shift

(13C/ppm)

Betaine 3.26 (s, N(CH3)3),

3.88 (s, CH2)

56.0 N(CH3)3,

69.2 (CH2)

Creatine 3.05 (s, CH3 (creatine/

creatinine)),

3.92 (s, CH2)

39.5 (CH3), 56.2 (CH2)

Glucose 5.23 (d, a-glucose CH1),

many signals in the

region 3.22–3.90

94.4 (a-glucose CH1),

many signals in the

region 62.8–78.6

Glycoprotein 2.03 (s, N-acetyl groups) 24.7 (N-acetyl groups)

Lactate 1.33 (d, CH3),

4.11 (q, CH)

22.8 (CH3), 71.1 (CH)

Lipid 0.79–0.93 (m, CH3),

1.16–1.34 (m, CH2)

0.79–0.93 (m, CH3),

1.16–1.34 (m, CH2)

Multiplicity: s singlet, d doublet, q quartet, m multiplicity


123

and/or diet in tll,kl to that between the biological replicates

in both groups.

The selected plasma biomarkers (important bins) were

analysed as repeated measurements using the MIXED

procedure of Statistical Analysis System software (SAS

Institute Inc., Cary, NC, USA) as described by Littell et al.

[30] to obtain least square mean values of the diet and time

effects and the diet 9 time interaction. First, model (1)

including the blood compartment (arterial or venous) as a

fixed effect was performed on all samples for all the

selected biomarkers, which tested significant differences

between arterial and venous samples (i.e. net absorption).

Second, model (2) was used to analyse the portal levels of

all selected biomarkers, and if model (1) showed a sig-

nificant difference between portal and arterial samples,

then the DAV difference was also analysed. The ANOVA

models where Xijklm represents the NMR signal is

Xijklm ¼ lj þ ajk þ bjl þ cjm þ abjkl þ eijklm; ð1Þ

Xijkl ¼ lj þ ajk þ bjl þ abjkl þ eijkl; ð2Þ

where lj is the overall offset for the bin j, ajk is the overall

time effect, bjl is the diet effect, cjm is the blood com-

partment, abjkl is 2-factorial interaction between time and

diet and eijklm is the error term. Random effects for both

models were pig and the interaction of diet, time and pig.

Least square means were compared by applying the prob-

ability of difference (PDIFF) option in the MIXED pro-

cedure of SAS. The level of significance was set at

P \ 0.05, while a tendency was reported when P \ 0.10.

The multivariate data analysis with ASCA and

MLPLSDA was performed using Matlab (version 2009a,

The MathWorks, USA) and in-house MATLAB rou-

tines. These routines are available on the Internet at

http://www.bdagroup.nl/. The univariate data analysis was

performed using SAS statistical software package version

9.1 (SAS Institute, Cary, NC, USA).

Results

Metabolic changes at fasting

Figure 2a shows the scores for the diet 9 time interaction

from the ASCA model of the day 4–7 venous samples

(adaption dataset), which contributed 13.5 % to the total

variance (12.9 % in submodel I; 73.6 % in submodel III).

The trajectories start at -0.5 h on day 4 where all the pigs

have been fed the same washout diet (WFL) for 3 days.

The WAF diet followed a distinctive path, whereas the

RAF and WWG diets showed similar trajectories from days

4–6. On day 7, the scores of RAF and WWG showed the

most variation. The loadings in Fig. 2b indicated that high

betaine content was especially correlated with the WAF

trajectory. Signals from glucose, glycoprotein, lactate and

lipid were also found to vary between the metabolic tra-

jectories. However, only the diet 9 time interaction for

betaine (P = 0.002) and N-acetyl glycoprotein (P = 0.046)

was significant in the ANOVA model (Table 4; Fig. 3), and

the diet effect for glucose, lactate and lipid signals was

non-significant. Both betaine (P = 0.43) and glycopro-

tein (P = 0.53) showed no arterial-venous difference, and

therefore, no enrichment in the plasma after passing the

gastrointestinal tract was observed. The betaine levels

Betaine

Glucose signals Lipid and

lactate

N-acetyl glycoprotein

(A)

(B)

Fig. 2 Anova–simultaneous component analysis (ASCA) scores

a and loadings b for the diet 9 time interaction from trajectory

analysis of the venous samples from day 4 to 7. The ten variables with

the highest absolute values of the loadings in component one and two

have been assigned


123

http://www.bdagroup.nl/

increased gradually from day 4 to 7 where all diets were

significantly different, although most pronounced when

feeding the WAF diet (P = 0.0001). The venous plasma

levels of glycoprotein were highest when feeding the RAF

diet and lowest for the WWG diet. An ASCA model using

the DAV difference resulted in high spread between the

samples of the diet 9 time interaction, which indicated the

pigs did not significantly adapt their metabolome to the new

diet by any common mechanism (data not shown).

Postprandial absorption profiles

The diet 9 time interaction explained 17.1 % of the vari-

ance in the ASCA model of the DAV difference (profile

dataset). The scores in Fig. 4a for component 1 revealed

that the DAV difference for the pigs fed the WAF diet

differed from the RAF and WWG diets in P1, P2 and P4,

corresponding to 0–150, 150–300 and 450–600 min after

first daily meal, respectively. Overall, the scores indicated

no effect of diet on the DAV difference at fasting; in P1,

the scores for WAF was higher than for RAF; in P2 in

particular, the WAF and WWG diets deviated; in P3, the

scores approached each other; and in P4, the scores for the

WAF diet were higher than for both the WWG and RAF

diets. No difference between the diets were visible in the

scores for component 2 (contributed to 6 % of the variance;

data not shown), and therefore, the analysis of DAV dif-

ference was restricted to score component 1. The ten most

important bins in the first component of the loadings can be

ascribed to betaine, glucose or lactate (Fig. 4b). Table 5

shows mean values and P values for the different effects

as elucidated by the ANOVA model. A tendency for a

diet 9 time interaction on betaine DAV difference was

observed (P = 0.078). The venous betaine level was high

for the WAF diet compared with RAF and WWG diets

(P \ 0.0001; Fig. 5); however, the DAV difference was

similar for the three experimental diets (P = 0.31). Venous

lactate (P = 0.033) and glucose (P = 0.008) concentra-

tions were found to be high when feeding the RAF and

WWG diets and low for the WAF diet. A diet effect was

observed for plasma creatine showing lower creatine

when feeding WAF compared with RAF and WWG diets

(P = 0.043).

Table 4 The relative intensities from day 4–7 of selected venous

plasma metabolites in comparison with the total amount calculated

(the intensity of a specific bin, diet and day divided by the sum of all

diets and days of that bin)

Metabolites Betaine Glycoprotein

Bin (ppm) 3.253 2.034

WWG

Day 4 0.06fgh 0.08ab

Day 5 0.08def 0.08ab

Day 6 0.08de 0.08ab

Day 7 0.09cd 0.08b

WAF

Day 4 0.05h 0.09ab

Day 5 0.10bc 0.09ab

Day 6 0.10bc 0.08ab

Day 7 0.13a 0.08ab

RAF

Day 4 0.06gh 0.08b

Day 5 0.07efg 0.08b

Day 6 0.07ef 0.08b

Day 7 0.11b 0.09a

SEM 0.01 0.00

P values

Absorption 0.43 0.53

Diet 0.001 0.86

Time \0.0001 0.71

Diet 9 time 0.002 0.046

Means along column (metabolite) not sharing the same superscript are

significant different (P \ 0.05)

0.04

0.05

0.06

0.07

0.08

0.09

0.1

0.11

0.12

0.13

0.14

7654

Rel

ativ

e in

ten

sity

Day (Fasting)

Betaine levels (venous)

WWG WAF RAF

Fig. 3 The betaine levels of the

venous samples from day 4 to 7

as determined by ANOVA.

Values are means, and bars

show standard error mean


123

Discussion

The present study explored the metabolic effects and

absorption dynamics of nutrients from breads made of rye

or wheat aleurone fractions or whole-wheat grain using 1H

NMR-based metabonomics. The ASCA scores showed a

distinctive trajectory of the WAF bread from day 4 to 6,

which indicated that the metabolic changes at fasting

separated the WAF bread from the RAF and WWG breads.

These differences of feeding the WAF bread can mainly be

ascribed to venous betaine. This is in good agreement with

the high plasma betaine concentrations found in humans

after consumption of wheat aleurone [7] and that wheat

aleurone has shown a high content of betaine [17]. The

betaine plasma levels rose from day 4 to 7 for all diets and

this accumulation of betaine on day 7 reflected the content

of betaine in the experimental diets (WAF [ RAF [WWG) and the washout diet (WFL). The betaine content in

WAF compared with RAF and WWG was about twice as

high, which leads to 20–40 % higher plasma betaine con-

centration when feeding WAF. Thus, our results show a

good agreement between betaine intake and plasma betaine

concentration. We estimated the content of aleurone in

experimental breads to be approximately 30 % higher in

WAF compared with RAF, so the results should probably

not be ascribed to the difference between the wheat and rye

kernel. The question remains whether plasma betaine

reaches a plateau on day 7 (3 days from washout diet) or

whether such a maximum would be reached if the pigs

were kept on the experimental diets for a longer period of

time. Furthermore, it may be questioned if the WWG and

RAF diets after longer exposure will reach the levels of

WAF. Schwab et al. [31] have shown that following a rapid

increase in betaine intake in humans steady-state plasma

betaine concentrations are reached after a few weeks. The

results also pointed to glycoprotein being important and

variable metabolite responsible for the interaction of diet

and time going from low plasma glycoprotein (day 4–7) to

high plasma glycoprotein (day 7). However, due to the

many functions of glycoprotein as secreted proteins, it is

difficult to address the effect on the RAF diet with any

specific physiological processes. A poor model for the

DAV difference for the days 4–7 data is in agreement with

small arterial-venous differences due to limited absorption

from the gastrointestinal tract at fasting and that is in turn

difficult to detect.

The absorption profile using fasting samples (30 min

before first daily meal) and four consecutive pooled sam-

ples of 2.5-h intervals represented the postprandial phase

following the first two daily meals. The data suggested that

the most characteristic absorption profile was obtained

when feeding WAF in comparison with the WWG and

RAF diets. Assigning these differences in the absorption

profiles, however, can be more challenging. In spite of

plasma betaine concentration being highest when feeding

WAF, no significant DAV difference between the diets was

observed in the postprandial phase. This finding suggests

that the absorption has reached a steady-state level on day 7

(3 days after feeding the experimental diets) by homoeo-

stasis of betaine regardless of the differences of betaine

content in the diets. This can explain the time profile of

plasma betaine with a maximum concentration just after

lunch (P3 samples). Other studies have found betaine

absorbed into the portal vein in both chicken [32, 33] and

pregnant sows [20]. Pharmacokinetic studies of betaine by

Schwab et al. [34] in humans showed a rapid absorption

(maximum at 0.90 h) after ingestion of oral doses of

betaine. Our study also differed from studies feeding a high

betaine diet [35] or a wholegrain wheat cereal [36] in

humans by having a later postprandial plasma betaine peak.

Urine samples from the same experiment pointed at low

creatinine excretion when feeding the WAF diet (data not

Fig. 4 ASCA diet 9 time interaction scores (a) and loadings b for

the first component of the DAV difference from the profile on day 7.

a shows both the scores for the mean trajectories and for each specific

sample. The variables have been ascribed to: 1 glucose, 2 lactate,

3 betaine (the variable in brackets is due to glucose and betaine

signals), 4 glucose region


123

shown), which was consistent with a concomitant low

venous plasma creatine. The lack of an DAV difference for

creatine indicated that the diet effect in plasma creatine is

of endogenous origin. This is inconsistent with a study with

non-gestating sows by Yde et al. [20, 37], who found a

positive relationship between betaine intake and elevated

endogenous creatine plasma concentrations. It is also

known that creatine synthesis depends on remethylation of

Table 5 The relative intensities of selected venous samples and their DAV difference on day 7 (the intensity of a specific bin, diet and day

divided by the sum of all diets and days of that bin)

Metabolites

Bin (ppm)

Betaine

3.253

Creatine

3.928

Glucose

5.226

Lactate

4.113

Type Venous DAV difference Venous Venous DAV difference Venous DAV difference

WWG

Fasting 0.05 -0.05 0.07 0.05 -0.01 0.04 -0.01cd

P1 0.06 0.03 0.08 0.08 0.09 0.08 0.10abcd

P2 0.07 0.34 0.08 0.08 0.13 0.08 0.18ab

P3 0.06 0.05 0.08 0.08 0.07 0.08 0.00cd

P4 0.06 0.01 0.07 0.06 0.07 0.07 0.11abcd

WAF

Fasting 0.07 0.07 0.06 0.05 0.03 0.05 0.06bcd

P1 0.07 -0.06 0.05 0.06 0.04 0.07 -0.01cd

P2 0.08 -0.05 0.05 0.06 0.05 0.06 0.03bcd

P3 0.09 0.23 0.07 0.08 0.09 0.07 0.12abc

P4 0.08 -0.29 0.04 0.05 0.01 0.05 -0.04d

RAF

Fasting 0.06 -0.02 0.07 0.05 0.00 0.05 -0.02cd

P1 0.07 0.42 0.08 0.08 0.15 0.09 0.24a

P2 0.06 0.06 0.07 0.07 0.07 0.07 0.07bcd

P3 0.06 0.16 0.08 0.08 0.11 0.08 0.11abcd

P4 0.06 0.08 0.07 0.07 0.07 0.07 0.06bcd

SEM 0.00 0.13 0.01 0.01 0.03 0.01 0.06

P values

Absorption 0.001 0.76 \0.0001 \0.0001

Diet \0.0001 0.31 0.043 0.008 0.29 0.033 0.41

Time \0.0001 0.15 0.0013 \0.0001 0.002 \0.0001 0.24

Diet 9 time 0.09 0.078 0.15 0.08 0.19 0.14 0.041

0.04

0.05

0.06

0.07

0.08

0.09

0.1

Fasting 0-2.5 (P1) 2.5-5 (P2) 5-7.5 (P3) 7.5-10 (P4)

Rel

ativ

e in

ten

sity

Time after first feeding (h)

Betaine levels (venous)

WWG WAF RAF

Fig. 5 The betaine levels of the

venous samples on day 7 as

determined by ANOVA. Values

are means, and bars show

standard error mean


123

homocysteine by betaine [38]. Further studies are needed to

determine if there is a link between the metabolic and

absorption variations found for betaine, glucose, glycopro-

tein, lactate and lipid. It would be interesting to examine the

relationship between circulating betaine and creatine in

tissue by measurements of betaine-homocysteine methyl-

transferase and guanidinoacetate methyltransferase activi-

ties that catalyses betaine and creatine, respectively.

Concluding remarks

1H NMR-based metabolomics in combination with mea-

surements of the DAV difference and data analysing using

ASCA enabled us to elucidate how the diets differentially

modified the metabolic concentration or absorption in time.

Plasma betaine was found to accumulate when changing

the feeding from a refined wheat flour diet to the experi-

mental diets and was higher when feeding the WAF diet

(estimated 19 % aleurone) compared with RAF (estimated

10 % aleurone) and WWG diets (20–40 % higher at day 7)

compared with RAF and WWG, whereas the content of

betaine in the WAF diet was about twice as high. A ten-

dency was found for the diet–time interaction of betaine

absorption in the absorption phases, but the effect of diet

was non-significant. This shows that a steady state of

betaine absorption has been reached on day 7. In addition,

high plasma betaine when feeding the WAF diet corre-

sponded to low plasma creatine of endogenous origin.

Acknowledgments The European Commission in the Communities

Sixth Framework Programme, Project HEALTHGRAIN (FOOD-CT-

2005-514008) is greatly acknowledged for financial support. The

Danish Agency for Science, Technology and Innovation is thanked

for co-funding the PhD project for Christian Clement Yde. Jeroen

Jasper Jansen was financed by the Netherlands Metabolomics Centre

(NMC), which is a part of the Netherlands Genomics Initiative/

Netherlands Organisation for Scientific Research. There is no conflict

of interest.

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