Metabolomics Study of Cereal Grains Reveals the Discriminative Metabolic Markers Associated with Anatomical Compartments
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PAPER METABOLOMICS STUDY OF CEREAL GRAINS REVEALS THE DISCRIMINATIVE METABOLIC MARKERS ASSOCIATED WITH ANATOMICAL COMPARTMENTS M. COULOMB1, A. GOMBERT1,2 and A.A. MOAZZAMI1,3* 1Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, P.O. Box 7015, SE 75007, Uppsala, Sweden 2Department of Food Science, Swedish University of Agricultural Sciences, P.O. Box 7051, SE 75007, Uppsala, Sweden 3NMR-metabolomics core facilities, Uppsala BioCentrum, Swedish University of Agricultural Sciences, Uppsala, Sweden *Corresponding author: Tel. +46 18672048, Fax +46 18672995, email: [email protected] ABSTRACT This study used NMR-based metabolomics to compare the metabolic profile of different ana- tomical compartments of cereal grains i.e. bran and endosperm in order to gain further insights into their possible role in the beneficial health effects of whole grain products (WG). Polar water- soluble metabolites in 64 bran and endosperm, samples from rye and wheat were observed using 600 MHz NMR. Bran samples had higher contents of 12 metabolites than endosperm samples. A comparative approach revealed higher contents of azelaic acid and sebacic acid in bran than in endosperm. In a pilot study, the consumption of WG rye bread (485 g) caused NMR signals in 24h urine corresponding to azelaic acid. The relatively high abundance, anatomical specificity, pat- tern of metabolism, urinary excretion in human, antibacterial, and anticancer activities suggest further studying of azelaic acid when exposure to WG or beneficial effects of WG are investigated. - Keywords: Metabolomics, NMR, wholegrain, rye, wheat, metabolites, biomarkers - 142 Ital. J. Food Sci., vol. 27 - 2015 INTRODUCTION MATERIALS AND METHODS Epidemiological studies have consistent- Serial sample collection and extraction ly shown that intake of whole grain (WG) can protect against the development of chronic dis- A total of 64 cereal samples, comprising 18 eases (SLAVIN et al. 2001), e.g. type 2 diabetes wheat endosperm, 24 wheat bran, 8 rye en- (T2D) (DE MUNTER et al. 2007, MURTAUGH et dosperm, and 14 rye bran were obtained from al. 2003), cardiovascular disease (CVD) (FLINT the HEALTHGRAIN (WARD et al. 2008) project or et al. 2009; JACOBS et al. 2007; MELLEN et al. from a local market. The endosperm and bran 2008), and certain cancers (CHAN et al. 2007, samples originated from HEALTHGRAIN projects HAAS et al. 2009; LARSSON et al. 2005; SCHATZ- were from the same grain sample material and KIN et al. 2008). The American Association of therefore were matched (Wheat samples n = Cereal Chemists provided the following scien- 18; and rye samples n = 8). The HEALTHGRAIN tific and botanical definition of WG in 1999: project rye varieties (and populations) included “whole grain shall consist of the intact, ground, potugaise-3, potugaise-6, Haute Loire, Gran- cracked or flaked caryopsis, whose principal drieu, Nikita, Rekrut, Dankowskie-Zlote, and anatomical component-the starchy endosperm, Lovaszpatonai-1. The details about rye va- germ and bran-are present in the same rela- rieties are given in NYSTROM et al. (2008). tive proportion as they exist in the intact cary- The HEALTHGRAIN project wheat varieties in- opsis” (International 1999). Whole grains are a cluded Disponent, Herzog, Tommi, Campari, rich source of fiber and bioactive compounds, Tremie, San Pastore, Gloria, Spartanka, Ava- including tocopherols, B vitamins, minerals, lon, Claire, Malacca, Maris Huntsman, Rial- phenolic acids, and phytoestrogens (FARDET, to, Riband, Obriy, CF99105, Chinese-Spring, 2010). It is generally recognized that the syn- and Cadenza. The details about wheat varie- ergistic action of compounds mainly present in ties are given by SHEWRY et al. (2010). All rye the bran and germ fractions of cereals accounts and wheat varieties were grown in the field at for the protective effects of WG products (FAR- Martonvasar, Hungary, in 2005. Full details DET, 2010; LIU, 2007). Recently, the composi- of the site including soil type, mineral compo- tion and the diversity of bioactive compounds sition, and weather condition has been given in different anatomical components of cereal by SHEWRY et al. (2010). grains have been systematically investigated in All samples were milled, and 0.5 g milled ma- a large number of different species and varie- terial was extracted in 5 mL Milli-Q water for 18 ties within the HEALTHGRAIN project (NYSTROM h. The samples were then centrifuged (5 min- et al. 2008; SHEWRY et al. 2010; WARD et al. 1500 g), and 2 mL supernatant was extracted, 2008). However, that project screened the cereal mixed with 8 mL ethanol and centrifuged (15 samples for bioactive compounds already doc- min-1,500 g) in order to precipitate the soluble umented in cereals using a targeted approach, viscose polymers. A 5 mL portion of the ethanol and made no comparison of the untagged pro- supernatant was dried using an evacuated cen- file of the metabolites in different compartments trifuge (Savant, SVC 100H, Savant Instrument of cereal grains. INC, NJ) and dissolved in phosphate buffer (280 Metabolomics is an untargeted approach in µL, 0.25 mol/L, pH 7.0), D2O (40 µL), and sodi- which the profile of metabolites in a biospeci- um-3-(trimethylsilyl)-2,2,3,3-tetradeuteriopropi- men is measured using high-throughput analyt- onate solution (TSP, 30 µL, 23.2 mmol/L) (Cam- ical methods, e.g. NMR and mass spectrometry bridge Isotope Laboratories, Andover, MA). The (LENZ and WILSON, 2007; NICHOLSON and WIL- mixture was then used for 1H NMR analysis. SON 2003). We have used this approach previ- An internal standard was added to the mixture ously to examine the complex physiological/bio- in order to ensure semi-quantitative measure- chemical effects of WG rye products in humans ments of metabolites captured by 1H NMR. For (MOAZZAMI et al. 2012; MOAZZAMI et al. 2014; 2D NMR analysis the mixture was freeze-dried MOAZZAMI et al. 2011). The aim of the present and dissolved in D2O before analysis. study was to search for the discriminative me- Human experiment and the preparation of tabolites in the two major anatomical compart- urine sample for NMR analysis ments in cereal grain, endosperm and bran, us- In a pilot study, a male subject (age 35; BMI ing an untargeted NMR-based metabolomics ap- = 23.4) consumed refined wheat bread 485 g proach and with the emphasis on wheat and rye for 6 days (breakfast 2 portions, lunch 1 por- to gain further insights into their possible role tion, dinner 1 portion). On day six, 24-hour in the beneficial health effects of whole grain urine was collected. On day seven, he substi- products. NMR analysis can potentially pro- tuted the 485 g refined wheat bread with 485 vide characteristic structural data, which can g of whole grain rye bread and the urine was be used for elucidation and eventual identifica- collected for 24 hours. During the seven days tion of unknown compounds found to discrimi- of experiment, any other cereal products were nate between the metabolic profiles of bran and avoided. The choice of consuming refined wheat endosperm in cereals. bread vs whole grain rye bread was made to Ital. J. Food Sci., vol. 27 - 2015 143 replicate the condition of previous human in- tegral regions (buckets) between 0.5-10 ppm, in terventions in which refined wheat bread was which area between 4.60-5.18 ppm containing used as the control diet (MOAZZAMI et al. 2011; residual water was removed. Each spectral re- MOAZZAMI et al. 2012; BONDIA-PONS et al. gion was then normalized to the intensity of in- 2013). The refined wheat bread was prepared ternal standard (TSP). from commercial refined wheat flour and whole grain rye bread was prepared from commercial Statistical analysis whole grain rye flour. The whole trial was re- peated twice, in two different times. This study Principal component analysis (PCA) and or- complied with the Helsinki Declaration, as re- thogonal partial least squares-discriminant vised in 1983. The urine samples were kept in analysis (OPLS-DA) were performed using SIM- -80°C freezers before analysis. The urine sam- CA-P+ 12.0.1 software (UMETRICS, Umeå, Swe- ples (500 µL) were mixed with phosphate buffer den) after centering and pareto-scaling of the (250 µL, 0.25 M, pH 7.0) containing 5 mmol/L data as previously described (MOAZZAMI et al. sodium-3-(trimethylsilyl)-2,2,3,3-tetradeuteri- 2011). The presence of outliers was investigat- opropionate (TSP) (Cambridge Isotope Labora- ed using PCA-Hotelling T2 Ellipse (95% CI) and tories, Andover, MA) as an internal standard. the normality of multivariate data was investi- Resulting solutions were centrifuged to remove gated using the normal probability plot of the particulate matter. The supernatant was then PCA model. Variable influences on projection transferred into 5-mm NMR tubes for 1H NMR (VIP) values of the OPLS-DA model were used analysis. For 2D-NMR analysis, 600 µL of the to determine the most important discrimina- supernatant was freeze-dried and dissolved in tive NMR bucket (signals). NMR buckets (sig- 600 µL D2O before 2D-NMR analysis. nals) with VIP > 1 for which the corresponding jack-knife-based confidence intervals were not NMR measurements close to or including zero were considered dis- and the identification of signals criminative. The significance of OPLS-DA mod- el was tested using cross-validated ANOVA (CV- The 1H NMR analyses (cereal extracts and ANOVA), which assesses the reliability of OPLS human urine) were performed on a Bruker models (CV-ANOVA p<0.05 means the OPLS- spectrometer operating at 600 MHz (Karlsruhe, DA model is reliable) (ERIKSSON et al. 2008). Germany). 1H NMR spectra were obtained us- The absolute concentrations of metabolites ing zgesgp pulse sequence (Bruker Spectro- with corresponding NMR signals that were found spin Ltd.) at 25°C with 128 scans and 65,536 to be discriminative in OPLS-DA were calculat- data points over a spectral width of 17942.58 ed from the NMR spectra using the NMR Suite Hz.